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the amount of microorganisms in the oral fluid (saliva) ranges from 4 millions to 5 billions per 1 milliliter, and in dental plaque between 10 and 1000 billions per 1 gram. their resistance to non - specific oral protective factors and environmental factors (e.g., antiseptic solutions) depends on the cellular membrane, a thick layer of mucus, a capsule, or other biological characteristics. for this reason, standard bacterial cultures with differing biological characteristics were selected for this study : staphylococcus aureus are gram - positive cocci found in the nostrils of up to 60% of humans ; they form microcapsules, and are resistant to high temperatures (up to 70c), disinfectant, and antiseptic solutions, and cause suppurative inflammatory processes. staphylococcus aureus is an important pathogen because of its virulence, antimicrobial resistance, and association with various diseases, including fatal systemic infections, intoxication, cutaneous infectious, and opportunistic diseases. klebsiella pneumoniae are capsule - forming bacteria found in the respiratory tract and are capable of causing atypical pneumonia. candida albicans is a fungus and is part of the normal microflora of the oral cavity, but it can cause an opportunistic infection in immuno - depressed subjects. escherichia coli is part of the normal microflora of the large intestine ; however, due to poor hygiene it can enter the oral cavity and cause suppurative inflammatory processes. the procedures that a prosthodontist performs may cause damage to the patient s oral mucosa and gingiva. the saliva mixes with blood. during the manufacturing of the dental impressions, blood and saliva enter the setting impression material, and it becomes contaminated with bacteria and viruses [47 ]. for this reason, according to general hygiene norms, such impressions should be disinfected ; yet disinfection does not always yield the desirable results. when producing gypsum casts using non - disinfected or incompletely disinfected dental impressions, the microorganisms travel from the surface of the impression material into the cast. the infected material then is taken to the manufacturing facilities and may pose threat to dental technicians. dental casts are subject to several forms of contamination, ranging from direct contact with a patient s saliva, to procedures involving measurement, planning, manufacture, laboratory shipping, and storage. dental technicians touch the impressions or the casts with their hands, and their skin is frequently damaged due to their work. when cutting the dental casts, bacteria, fungi, or particles of the disinfectant may travel with the plaster dust from the material to the respiratory tract of personnel, causing infections, and may also accumulate on the clothing or open surfaces in the room. the aim of study was to evaluate the survival of staphylococcus aureus, klebsiella pneumoniae, and escherichia coli bacteria, and candida albicans fungus in non - disinfected dental casts. the results of the study could be used to determine how long dental casts may pose threat to the health of the personnel and the patients. perhaps disinfection of dental impressions would be sufficient, skipping additional disinfection of the dental casts in order to reduce exposure of the personnel to possible allergens the residues of disinfectant solutions in the plaster. although extensive research has been carried out, there are no published studies in which similar issues were addressed. during this experimental study we investigated the survival of the selected bacteria and fungi in dental casts. the study was conducted using standard bacterial (staphylococcus aureus atcc 25928, klebsiella pneumoniae atcc 13883, and escherichia coli atcc 25922) and fungal (candida albicans atcc 60193) cultures. saline suspensions (mcfarland turbidity standard 0.5) were prepared using standard bacterial cultures cultivated in trypticase soy agar for 24 hours at 3537c, and candida albicans culture cultivated in sabouraud agar for 3 days. we evaluated and compared changes in the number of the microorganisms in gypsum casts at different time intervals. gypsum casts (an equivalent to dental cast) were manufactured in a sterile laminar - flow hood under sterile conditions, using standard alabaster plaster class 2 moldabaster the plaster was sterilized ; therefore, there was no need to form a control group. the dental casts were prepared by pouring 65 g sterile plaster powder, 5 ml sterile distilled water, and 1 ml of a specific bacterial or fungal suspension (mcfarland turbidity standard 0.5) into a sterile mixing container. a sterile spatula was used to stir the contents until an integral mass was formed, which was subsequently placed into a sterile closed container. during the test period the closed containers with the samples were placed on a dental lab shelf to simulate casual cast storage conditions. the number of the aforementioned standard microorganisms per 1 g of the substance was evaluated after 1, 2, 24, 48, 72, 96, and 120 hours. sterile clamps were used in the making of each sample. the sample a piece of the gypsum cast of a known weight (ca. 1.0 g) was ground to powder in a sterile mortar, and then placed into a sterile glass with 5 ml of sterile saline solution. the suspension of plaster powder was diluted at the ratio of 1:5, 1:25, 1:125, and 1:625. one ml of the suspension from each test tube with different dilution ratios was poured into 10 ml of 45c trypticase soy agar (or sabouraud agar, in the case of candida albicans) in 2 petri dishes that were then stored at 3537c (in case of candida albicans, sabouraud agar was stored at 2025c). after the cultivation of the indicated duration, colonies of the respective bacterium or fungus in the 2 petri dishes were evaluated, calculating the mean number of the colonies ; multiplying this number by the degree of dilution revealed the number of bacteria or fungus per 1 g of plaster. the data were considered to be statistically significant if the probability of an error was p<0.05. the study showed that k. pneumoniae survived the longest in gypsum casts up to 4 days compared to the other studied microorganisms (figure 1). a statistically significant reduction in the number of k. pneumoniae (from 837911935 to 44120845) in gypsum casts was observed after 1 day (p<0.05). the number of k. pneumoniae colonies was also the highest, compared to other bacteria. after the first and the second hour in the gypsum cast, the number of bacteria remained nearly unchanged. it was only after 2 days that a statistically significant difference was detected (a reduction from 59154676 to 9285102, p<0.05). gram - positive bacteria (staphylococci) remained viable for up to 3 days in the gypsum casts, presumably due to their cell membrane composed of a thick peptidoglycan layer, a microcapsule (a thin layer of mucus over the cell membrane), and high resistance to environmental factors (figure 2). no gram - negative bacteria (e.g., e. coli) were found in gypsum casts after 2 days, and a statistically significant reduction in their numbers was observed just after 2 hours (from 19483336 to 470574, p<0.05) (figure 3). microorganisms with a eukaryote structure (e.g., the fungus c. albicans) died within 2 days, and a statistically significant reduction was observed after 1 day (from 31 3662759 to 12 2911234, p<0.05) (figure 4). figure 5 presents the comparison of the standard microorganisms amounts (the bacteria and the fungi) at different time intervals, and the quantitative reduction in their numbers. the quantitative evaluation of the viability of standard microorganisms in gypsum casts showed that physical - chemical characteristics of gypsum casts have different effects on the survival of the standard microorganisms. structural and physiological characteristics of k. pneumoniae and s. aureus affect their survival (their quantity) with respect to that of e. coli and c. albicans. the findings of the study showed that the microorganisms did not multiply in the gypsum casts ; instead, their numbers significantly dropped. the study was conducted using the most common oral bacteria and fungus : staphylococcus aureus, klebsiella pneumoniae, candida albicans, and escherichia coli. studies performed in japan showed that all the microorganisms used in our study staphylococcus aureus, klebsiella pneumoniae, and escherichia coli were detected in the oral cavities of 13.6% of patients with no health complaints. according to other authors, staphylococcus aureus is detected in the oral cavities of 15.8% of patients, and candida albicans in 63.0% of patients. the findings of our study showed that the duration of the survival of microorganisms in gypsum casts varied depending on the types of the studied cultures. these bacteria have a thick layer of peptidoglycan and a microcapsule, which is a mucous layer of polysaccharide origin. the gram - negative bacteria klebsiella pneumoniae and escherichia coli differ from staphylococci in their cell membrane structure. however, klebsiella bacteria have a thick mucous layer of polysaccharide origin a capsule that ensured their longest survival in gypsum casts compared to other microorganisms. escherichia coli have no capsule and thus are more susceptible to unfavorable environmental conditions, thus these bacteria survived in gypsum casts for only 12 days. the fungus candida albicans, although it has a eukaryote cell structure, survived in gypsum casts longer than escherichia coli did because fungi have a chitin membrane. the oral cavity may contain pathogenic as well as relatively pathogenic microorganisms that can be transferred into the environment through impressions. in patients with damaged oral mucosa, staphylococcus aureus, klebsiella pneumoniae, and escherichia coli may cause suppurative inflammatory processes. escherichia coli may cause not only a local inflammatory process, but bacteremia as well. candida albicans, as part of the normal oral microflora, is found in 4060% of individuals ; however, this fungus can cause candidiasis in immuno - compromised states or in cases of antibiotic overuse. most commonly, alginate and silicone dental impressions are used in clinical practice. due to their composition, structure, and the hydrophilic setting mechanism, alginate impressions are easily infected by oral microorganisms. a more monolithic structure of the silicone impression mass and its non - hydrophilic setting mechanism significantly reduce the attachment of microorganisms to the impression surface. selecting a suitable impression mass may reduce the spreading of the infective agents as early as the first stages of prosthesis manufacturing. alginate impressions are more frequently contaminated with oral bacteria and fungi, compared to silicone impressions, and they are also more difficult to disinfect. alginate impressions pose a significantly higher threat of contaminating dental casts with bacteria and fungi, compared to silicone impressions, thus silicone impressions are safer with respect to the transmission of the infective agents. previous studies have shown that dental impressions delivered at dental laboratories contain bacteria irrespectively of whether they were disinfected or washed under running water. this suggests that dental casts produced using well - disinfected impressions require no additional disinfection. if the dental casts are stored under normal conditions of a dental technician s office, the number of microorganisms should change more rapidly than it did during our study due to frequently changing temperature and humidity, and because of the plaster ph of 6.1. an additional disinfection of dental casts would not yield significant results, compared to the risk that the dental technicians would face because of exposure to a potential allergen the disinfectant on the surface of the dental cast. although studies have shown that the effect of the disinfectant on the precision of the dental casts is not significant, the risk remains that the disinfectant may alter the physical characteristics of the dental cast surface, making it more porous, brittle, and more susceptible to wear and tear. rinsing of the oral cavity with an antiseptic solution prior to odontological procedures may reduce the presence of microorganisms in the oral cavity. the most popular oral antiseptic, chlorhexidine, is not always effective against fungi or bacterial spores. thus, mouth rinsing with an oral antiseptic prior to taking impressions followed by full disinfection of the impressions would reliably reduce the contamination of dental casts with microorganisms. as our results show if the impression is disinfected and pre - impression mouth rinsing is used consistently, the dental casts would pose less threat because of the poor survivability of bacteria and fungi in gypsum. gram - positive bacteria staphylococcus aureus and gram - negative bacteria klebsiella pneumoniae demonstrated the longest survival in dental casts (up to 4 days), compared to escherichia coli and candida albicans, which remained viable during the first 2 days). dental casts are not a suitable medium for the multiplication of the colonizing microorganisms. individual protective measures (gloves, protective goggles, and respiratory protection measures) should be used in order to avoid possible contamination of dental impressions and casts with microorganisms, and to prevent their transmission and spread in environments such as dental laboratories.	backgroundthis study evaluated the survival of the most prevalent oral bacteria and fungi (staphylococcus aureus, klebsiella pneumoniae, escherichia coli, and candida albicans) in dental casts, and compared changes in the amounts of these microorganisms at different time intervals to determine how long dental casts may pose threat to the health of dental personnel and patients.material/methodswhen manufacturing the casts, regular water was replaced with sterile distilled water, where suspensions of the studied bacteria or the fungus at certain concentrations were prepared. when the dental casts were fully set (solidified), plaster shavings were examined immediately after the contact of the studied microorganism with the plaster, as well as after 1, 2, 24, 48, 72, 96, and 120 hours. following that, we measured how the amount of the studied bacteria and fungi in 1 gram of the plaster changed within the studied period of time.resultsklebsiella pneumoniae survived in plaster for up to 4 days, and the reduction in the number of these bacteria became statistically significant after 1 day (p<0.05). staphylococcus aureus remained viable in plaster for up to 4 days, and the number of these bacteria dropped after 1 day (p<0.05). escherichia coli disappeared after 2 days, and a reduction was already observed after 2 hours (p<0.05). candida albicans in plaster models died within 2 days, and a reduction in their number was observed after 1 day (p<0.05).conclusionsthe microorganisms did not multiply in the gypsum casts and their number significantly dropped instead of increasing.
peripheral arterial disease (pad) in people with type 2 diabetes mellitus (t2 dm) exhibits broad clinical characteristics and various consequences and is known as one of the major macrovascular complications of t2 dm, the prevalence of which is on the rise. atherosclerosis is recognized as the most direct and important cause of pad, but acute or chronic limb ischemia may be induced by various risk factors. due to the increasing number of patients who undergo peripheral vascular procedures due to the aging of the overall population, the worldwide increase in t2 dm prevalence, and developments in medical technologies, the number of subjects who are exposed to the risks for pad and related complications is on the rise. in this review, we will discuss the clinical and epidemiologic characteristics of pad, as well as the clinical significance of pad in t2 dm subjects. pad is a general term for all vascular diseases that lead to abnormal function and structure of the aorta, its branches, and the lower - limb arteries secondary to atherosclerosis and thromboembolism - related pathophysiologies. typically, atherosclerosis decreases the size of the vascular lumen, leading to deficiencies in arterial perfusion without noticeable symptoms. however, depending on the degree of obstruction, various symptoms, including claudication, resting pain, ulceration, or gangrene, may occur during exercise. the fontaine and rutherford classifications are the most well - known systems, under which increased stage describes severe symptoms (table 1). depletion of effective vascular volume by thrombosis, embolism, dehydration, and shock may be a cause of acute exacerbation of pad. this acute arterial occlusion is a fatal disease that requires emergent manipulations, such as amputation. but, most studies on the prevalence of pad among general populations used a low ankle - brachial index (abi, 0.9) as a surrogate for pad. from the analyses of national health and nutrition examination survey 1999 to 2004 in the united states, prevalence of low abi was 5.9%, corresponding 7.1 million adults with pad. excluding subjects with known cardiovascular disease, prevalence was 4.7%. similarly, a total of 5.8% of the united states population age 40 years had a low abi or history of lower - extremity revascularization, representing 6.8 million people. prevalence of pad was known diverse by ethnicity, with black people having the highest age - adjusted prevalence of low abi. prevalence of low abi is known to increase with age. in one study, prevalence of low abi was 1.9% in the age group of 40 to 59 years, 8.1% in the group of 60 to 74 years, and 17.5% in the group of 75 years. although pad is presumed to be more common in men, the prevalence of low abi does not estimated to vary significantly by gender. the pad prevalence in t2 dm subjects is unclear, but in the framingham heart study, 20% of symptomatic pad subjects also exhibited diabetes. according to the the prevention of progression of arterial disease and diabetes (popadad) study, 20.1% of 40-year - old patients with diabetes were associated symptoms exhibited pad. however, given that a large number of pad patients are asymptomatic, it is assumed that more subjects with diabetes exhibit pad. in the study conducted with 6,880 german people whose age 65 years, the prevalence of pad by low abi in diabetes subjects was 26.3% ; whereas, prevalence of pad in non - diabetic subjects was 15.3%. in korea, there has been no epidemiologic study on the prevalence of pad in the total population, although a study was conducted in 14 university hospitals with approximately 1,400 50-year - old t2 dm subjects exhibiting risk factors for pad. in this study, 11.9% of the subjects, there were some epidemiologic evidences which have investigated mainly in the university hospitals in korea. in the previously diagnosed patients with coronary artery disease or cerebrovascular disease, prevalence of pad by low abi was estimated to be 7.8%. in the prospective study among subjects with acute ischemic stroke or transient additionally, prevalence of pad by low abi was 12.8% in the patients with previous percutaneous coronary intervention. dyslipidemia, smoking, hypertension, and diabetes are known risk factors of pad, similar to coronary artery disease. these risks increase in patients who are 70-year - old ; in those who are 50- to 69-year - old with a history of diabetes or smoking ; and in patients 40- to 49-year - old with diabetes and one or more atherosclerosis - related risk factor(s), intermittent claudication, abnormalities in pulse palpation of the lower limb or atherosclerosis in non - peripheral arteries (e.g., coronary, carotid, and renal artery). when a symptom of claudication appears, the symptom becomes aggravated in approximately 10% to 20% of patients in 5 years and advances to critical limb ischemia (cli) in 1% to 2% of patients. the clinical progression of pad patients with diabetes is worse than that of those who are not. lesions occur over a wider range of vasculature ; the frequency of amputation was higher ; and there was a significant difference in mortality. in the results from multicenter study conducted by asian subjects with type 2 diabetes, obesity, smoking, duration of diabetes and previous cardiovascular disease were identified as independent risk factors of pad. non - fatal myocardial infarction (mi) or stroke occurs in 20% of patients, and 15% to 30% of patients were died. in the results from domestic study, subjects with previous history of coronary artery disease or cardiovascular disease had significantly higher prevalence of diabetes, dyslipidemia, renal insufficiency, and pad with claudication. cli occurs in both lower limbs in 50% of patients at 1-year follow - up. cli is accompanied by lower limb amputation in 25% and by cardiovascular disease in 25%. the abi is the standard for the diagnosis of pad in clinical treatment or epidemiologic studies and represents a useful resource for the salvage of the lower limb, wound healing and survival prediction of patients. abi is calculated as the ratio of systolic blood pressure in the brachial artery to that in the posterior tibial artery after resting in a supine position, which is considered abnormal when it is below 0.90. when the diagnostic threshold was set to 0.90, the sensitivity of abi for the diagnosis of pad with > 50% stenosis was 95%, and its specificity was 100%. an abi between 0.41 and 0.90 is evaluated as a mildly to moderately decreased blood flow, and an abi < 0.40 is evaluated as severely decreased blood flow. in the recent meta - analysis, the pooled sensitivity and specificity of abi 0.90 for pad diagnosis were 75% and 86% and the pooled platelet lymphocyte ratio and neutrophil lymphocyte ratio were 4.18 and 0.29, respectively. these results may means that abi is useful for identifying serious stenosis in clinical practice. however, in patients with diabetes, patients with distal lesions in elderly patients, and patients with mild stenosis less than 75%, diagnostic value of abi was decreased. angiography is the most accurate test to examine vascular anatomy and pathology and is, considered an essential evaluation tool prior to surgical management. however, because angiography is an invasive procedure, risks such as bleeding, infection, and rupture as well as side effects associated with contrast media, such as hypersensitivity reaction and nephrotoxicity, may occur. in recent years, non - invasive imaging technologies have been developed, and ultrasound, multidetector computed tomography (mdct) and magnetic resonance (mr) angiography have been widely used. such non - invasive imaging modalities had reported comparable diagnostic and prognostic accuracy to conventional angiography. in meta - analyses, sensitivity for detecting more than 50% stenosis or occlusion in mdct was estimated 95%, and its specificity was 96%, and sensitivity for diagnosing segmental steno - occlusions for mr angiography was estimated 94.7% and specificity was 95.6%. however, several limitations were also reported. we could observe decreased accuracy by intestinal gas, and decreased sensitivity in multivessel lesion in ultrasound imaging. in mdct images, the risk of contrast media induced nephrotoxicity is well known in the subjects with chronic kidney disease. moreover, utility of mr angiography is limited for detection of in - stent restenosis, and risk of gadolinium induced nephrogenic systemic fibrosis is also concerned in the subjects with chronic kidney disease. the diagnosis of pad necessitates the regulation of associated risk factors, medical treatment, and intervention of lesions in the lower limb. the management of risk factors of pad is similar to the regulation of risk factors of disease in the coronary artery. exercise is known to be effective for the improvement of symptoms of claudication, the extension of walking distance to the aggravation of pain and the promotion of maximum oxygen consumption. walking exercise is typically recommended, and patients are asked to partake in this exercise at least three times a week for 30 to 45 minutes for 12 weeks or more. a systematic review identified that compared exercise with usual care or placebo, exercise improved walking ability from 50% to 200%, and significantly improved maximal walking time, pain - free walking distance, and maximum walking distance. the cumulative proportions with mis after 10 years were 11% and 53% ; the cumulative rates of cardiac deaths 6% and 43% ; and the 10-year survival 82% and 46% among non - smokers and smokers, respectively. for patients who have difficulty in stopping smoking on their own, varenicline, bupropion, and nicotine replacement therapy the management of associated diseases, such as diabetes, high blood pressure, and dyslipidemia, has an important impact on the clinical progress of pad. in particular, meticulous foot care to prevent aggravation, crucial for clinical progress in diabetic patients, is recommended. in diabetic patients, the general goal of blood glucose regulation is to achieve glycosylated hemoglobin < 7%, but the goal of treatment is individualized for each patient 's characteristics. no controlled trials have directly evaluated the effects of glucose lowering on the natural history of pad. the goal hypertension therapy is to achieve blood pressure below 140/90 mm hg, and the use of angiotensin - converting enzyme inhibitor is preferentially considered. there are no data evaluating whether treatment of hypertension alters aggravation in patients with pad. nevertheless, hypertension should be controlled in such patients to reduce morbidity from cardiovascular disease. the use of statins for dyslipidemia regulation is preferentially considered, which may be combined with fibric acid derivatives. in a systemic review, specifically evaluated patients with lower limb pad concluded that lipid - lowering therapy reduced disease progression, helped alleviate symptoms, and improved total walking times and pain - free walking distance. there is an insufficient evidence to suggest a direct relationship between the use of antiplatelet agents and increases in pain or walking distance, but such agent have been proven to be significantly effective in decreasing the incidence of mi, stroke, or cardiovascular death ; thus, low - dose aspirin (75 to 325 mg / day) is typically recommended. a systematic review found a similar reduction in mortality for patients with claudication treated with antiplatelet therapy compared with placebo. clopidogrel is a secondary drug recommended if aspirin can not be used ; 75 mg / day is recommended. the clopidogrel versus aspirin in patients at risk of ischaemic events (caprie) trial found that clopidogrel 75 mg / day had a modest, but significant advantage over aspirin 325 mg / day for the prevention of stroke, mi, and pad in subjects with a recent stroke, mi, or pad. cilostazol is a phosphodiesterase inhibitor that impedes platelet aggregation and exhibits vasodilatory effect and improves dyslipidemia. treatment with cilostazol for 24 weeks reportedly improved walking distance without claudication by approximately 54% on average compared with a placebo group or a group to which pentoxifylline was administered. in a meta - analysis, the administration of 200 mg / day cilostazol for 12 to 24 weeks increased the maximum walking distance without claudication by up to 50% or 67%. if patient symptoms do not improve despite proper drug treatment and rehabilitation, invasive procedures should be considered. if normal activities are not possible due to claudication and if the lesions exhibit minimal surgery - related risks and the intervention exhibits a high probability of success, re - perfusion should be attempted through a transcutaneous procedure or operation. early intervention is recommended when resting pain, ischemic ulceration, or necrosis is present. balloon angioplasty and stent insertion are known to be effective in the alleviation of symptoms in many pad patients. in the past, the transcutaneous vascular procedure was used restrictively to cause stenosis of short lesions or occlusive lesions, but with advances in technology, transcutaneous vascular intervention has been widely applied prior to the surgical procedure and widely attempted in patients in whom it is difficult to perform the surgical treatment. for the long - term success of transcutaneous vascular intervention, the position and length of the lesions are important, and surgical treatment is preferred if the lesions are long, if there is stenosis in multiple areas, if the lesion is occlusive and if the lesion is calcified and stenotic. experimental or investigational agents for pad include prostaglandins, naftidrofuryl, propionyl levocarnitine, defibrotide, ginkgo biloba, hyperbaric oxygen, and angiogenic growth factors. naftidrofuryl can be used for treatment of claudication, and it has known to have fewer side effects than cilostazol. however, there had not been any large scale, randomized trial for relevant treatment, and more clear evidences are required for extensive clinical application. as pad becomes an increasingly important complication in t2 dm subjects, a deeper understanding of the related diseases and the establishment of a standardized method of treatment is particularly important. indeed, pad confers poorer prognosis in t2 dm patients compared with nondiabetic patients, and early diagnosis, management and positive treatment are necessary. however, with the exception of a few epidemiological studies, there is a lack of data concerning korean type 2 diabetes patients, and more strategies for the diagnosis and treatment of pad based on empirical evidence are needed.	peripheral arterial disease (pad) in patients with type 2 diabetes mellitus (t2 dm) exhibits broad clinical characteristics and various consequences and is known as one of the major macrovascular complications of t2 dm. atherosclerosis is recognized as the most direct and important cause of pad, but acute or chronic limb ischemia may be the result of various risk factors. in light of the increasing number of patients who undergo peripheral vascular procedures, the number of subjects who are exposed to the risks for pad and related complications is increasing. in this review, we will discuss the clinical and epidemiological characteristics of pad, as well as the clinical significance of pad in t2 dm subjects.
superficial siderosis of the central nervous system due to chronic, recurrent subarachnoid hemorrhage is a rare and potentially debilitating disorder. the classic manifestation is progressive bilateral sensorineural hearing loss, although ataxia and pyramidal signs also are common. in the largest review of literature to date, though the age of presentation ranges from 15 to 78 years, it is seen more commonly in patients above 40 years.. we hereby present a young boy with this disorder to emphasize its occurrence in children. a twelve - year - old boy presented to the neurology outpatient clinic with a history of intermittent holocranial headaches since two years and recent - onset blanking spells. the headaches were moderately severe, with no diurnal variation, occurring once in two weeks with no particular aggravating factor and subsiding with analgesic medication. however, at two years of age, he had developed sudden - onset vomiting, irritability, and imbalance which was progressive, not associated with fever, trauma, or varicella infection. over the subsequent month, he developed a divergent squint. a computed tomography of the brain revealed a mass lesion in the cerebellar vermis with supratentorial hydrocephalus. he underwent near - total excision of the tumor, with a thin rim of the tumor left behind. for the residual rim of tumor margin, he was subjected to external radiotherapy to the posterior fossa with 6 mv photons (total dose being 5040cgy/28fr/54 days) and was declared free of the disease subsequently. a magnetic resonance imaging (mri) of the brain was done which was normal except for postoperative changes ; hence, the headaches were treated with plain analgesics. over the next two years, he developed intermittent brief episodes of blanking spells, when he was referred to our center. a thorough neurological assessment revealed nystagmus, ataxia, and dysarthria with a normal tone. an electroencephalogram done for the blanking spells showed mild epileptiform abnormality and he was put on oxcarbamazepine. a repeat mri revealed cystic space in the posterior fossa (as a result of the previous surgery) and hypointensities around the cerebellar folia [figure 1a ]. assessment of brainstem auditory evoked potentials confirmed bilateral mild sensorineural hearing loss. a repeat clinical assessment after six months showed significant deterioration in dysarthria and ataxia. a repeat mri after six months showed progression in the deposition of hemosiderin in the cerebellar folia from the previous one [figure 1b ]. the clinical picture of a progressive worsening ataxia with sensorineural hearing loss along with the neuroimaging findings of the deposition of hemosiderin in the cerebellum clinches a diagnosis of superficial siderosis in this child, secondary to the surgery of the posterior fossa done in early childhood. (a) susceptibility weighted images showing hypointensities around the cerebellar folia, representing deposition of hemosiderin (b) imaging done after six months showed progressively increased deposition of hemosiderin nevertheless, the origin of superficial siderosis of the central nervous system remains undetermined in most cases. highly vascular spinal tumors, vascular abnormalities of the central nervous system, and surgical procedures of the posterior fossa are the most commonly identified sources of chronic bleeding. a past history of trauma and prior intradural surgery may be further risk factors. it is a distinct clinical syndrome characterized by sensorineural deafness (95%), cerebellar ataxia (88%), and pyramidal signs (76%). other features include dementia (24%), bladder disturbance (24%), anosmia (at least 17%), anisocoria (at least 10%), and sensory signs (13%). less frequent features are extraocular motor palsies, pain in the neck or back, bilateral sciatica, and lower motor neuron signs (5 - 10% each). in a case report by sevki., headache was also quoted to be a presenting symptom. in our patient, headache was the main presenting complaint followed later by progressive ataxia and hearing loss. the pathogenesis of superficial siderosis involves recurrent subarachnoid hemorrhage resulting in the prolonged contact of these tissues with iron. within the cerebellum, the microglia as well as bergmann glia are uniquely sensitive to iron - mediated cell damage. the terminal processes of bergmann glia that interface with the subarachnoid space mediate iron uptake from the cerebrospinal fluid (csf), inducing the synthesis of ferritin within these cells. because ferritin sequesters iron and is thus thought to play a role in iron detoxification, intracellular iron may not cause toxicity until ferritin biosynthesis is overwhelmed by a large iron load. excess free iron may then stimulate lipid peroxidation, leading to localized tissue necrosis. in the past, superficial siderosis was diagnosed almost exclusively at autopsy. with the advent of mr imaging mri has identified siderosis in reportedly asymptomatic patients. in one series, which examined 8,843 consecutive mri studies, however, he had clear signs of a progressive deficit on serial assessments. in most cases, the use of various therapies has been reported without a clearly discernible benefit, namely, steroids, iron chelators, selegiline, vitamin c, and other antioxidants. in cases where a source of chronic subarachnoid bleeding is identified, however, in about 25 - 40% of the patients, a source of siderosis is not identified, as in our patient, and treatment remains essentially symptomatic. to conclude, our case is unusual as the classical syndrome of superficial siderosis is seen at a young age, secondary to an early surgery of the posterior fossa.	superficial siderosis of the central nervous system results from deposition of hemosiderin in the subpial layers of the brain and spinal cord. patients usually present after 40 years of age with progressive ataxia and sensorineural hearing impairment. we present the case of a twelve - year - old boy who had a surgery of the posterior fossa at the age of two years and then developed recurrent headaches, instability of gait, and hearing deficit at around ten years of age. clinical examination revealed progressive ataxia and mild sensorineural hearing loss. he also had infrequent seizures with mild electroencephalographic abnormality. his serial magnetic resonance imaging (mris) showed a progressive deposition of hemosiderin in the cerebellar folia and around the brainstem, confirming a diagnosis of superficial siderosis. this case report draws attention to this rare condition, usually seen in adults, even though rarely it can be seen in children as a chronic sequela of surgery of the posterior fossa.
a significant proportion of chemical reactions involving proteins are mediated through electrostatic interactions of their ionizable residues (1). such residues greatly influence the conformation of a protein and therefore its function (2,3), as demonstrated by their folding mechanisms (46), enzyme catalysis and protein protein interactions (7). with respect to enzyme catalysis, residues can act as proton donors and acceptors within the catalytic site and help stabilize transition states, with a concomitant influence on the rate of reaction (8,9). the dissociation constant (ka) is a measure of the acidity of a compound, i.e. its ability to donate a proton. ka values range widely from 10 for the strongest acids, such as sulphuric, to 10 for the weakest, such as methane. therefore a negative logarithmic scale is usually applied (pka = log10 ka), whereby ka values for sulphuric acid and methane would become pka values of 10 and 50, respectively. generally, more negative pka values correspond to stronger acids. the pka values of individual amino acid residues in proteins are determined by the ionization of their side - chain groups. for the 20 natural amino acids, pka values range from 4.0 for the side - chain carboxyl of aspartate to 12.0 for the side - chain guanididium group of arginine. main - chain groups are not ionizable, although two additional ionizable groups exist at the n- and c - termini. residues within proteins have pka values that are moderated by their micro - environments, the nature of their near neighbours, the extent of hydrogen bonding and so on and can take on a range of values different from that of a model residue. nmr spectroscopy is the most widely used method for determining the pka values of individual residues, with an accuracy of 0.1 ph units. although many nmr methods are available, most entries in the protein pka database (ppd) are derived using h, c and n experiments. inaccuracies in nmr experiments stem from the range of ph values tested, variations in ionic strength and the reversibility of the titration (10). in light of this, new combination methods are being used based on nmr spectroscopy coupled with site - directed mutagenesis, which leads to more accurate pka values (10,11). the functional importance of ionizable residues has led to numerous attempts to predict individual residue - specific pka values (1216). molecular dynamic simulations have also been used for such predictions, although this only gives rise to a marginal increase in accuracy (17). as only a small handful of reviews have attempted to compile residue - specific protein pka values (10,18,19), it was decided to develop a database that would serve as a standard compendium against which to compare new experimental or theoretical results. the ppd v1.0 contains > 1400 amino acid pka values, sourced from experimental data. cross - references to several external databases the protein data bank (pdb) (20), the enzyme nomenclature and classification database (21) and the national center for biotechnology information (ncbi) entrez - protein have also been incorporated into the database. ppd v1.0 has been implemented using a postgresql relational database, which provides an appropriate infrastructure for all foreseeable future developments of the archive. the data were initially compiled in a microsoft access database after exhaustive searching of the primary literature, which included using keyword searches of the ncbi pubmed database (). the postgresql database is structured into seven normalized tables, populated from a flat - file export of the access database using perl scripts integrated with sql. as data are continually accumulating, archiving data is an on - going process : automatic, periodic updates will be made to the postgresql database. these forms target either a perl / sql script or a cgi script which in turn queries the database. the bespoke search engine facilitates fast, efficient and flexible data retrieval (searching the database). the data within ppd was sourced from the primary literature to give > 1400 entries, containing pka values for > 160 proteins (table 1). the database contains pka values for amino acid side - chains, as well as the n- and c - termini. however most entries focus on glutamate, lysine, histidine and aspartate, which together account for > 75% of the data. as these four are all key ionizable residues, the apparent bias is not driven by our selection, but by the available experimental data. very little data are currently available for arginine : its pka value (12) essentially precludes measurement by titration as proteins will denature at such a high basic ph. these provide links to the protein sequence, using ncbi entrez - protein, and any relevant protein structure in the pdb (20). if applicable, the enzyme classification is also given, with links to the enzyme nomenclature and classification database, developed in line with the international union of biochemistry and molecular biology (21), providing details of the enzyme reactions. in addition, a link is given to the original literature reference via the ncbi pubmed journals database. the ability to carry out accurate predictions of pka values depends on having access to a high quality source of data ; a principal aim of ppd is to provide such a source. only experimentally determined pka values are cited in ppd ; predicted pka values are not included. the quality of data contained in ppd v1.0 is largely dependent upon the accuracy of each experimental determination, thus it contains only values from certain selected techniques : nmr spectroscopy, raman difference spectroscopy and uv spectroscopy. intrinsic factors include invariant properties of the protein investigated, such as sequence and structure. extrinsic factors include the experimental conditions used, such as the temperature, the range of ph tested, protein concentrations as well as the experimental method. as logistic considerations preclude us from undertaking independent verification of the data, we are obliged to trust the values reported in the literature. it should be noted that the phenomenon of cooperative deprotonation can create circumstances under which pka values can not be used as a parameter that describes the ionization behaviour of the corresponding group (2224). two methods to search ppd are available : an amino acid query - based interface (figure 1) and a blast (25) interface. the implementation of a bespoke search system allows the user to perform extensive or focussed searches from a single user interface. the simplest search, using the amino acid query interface, would specify one amino acid residue only. a complex search would accommodate up to four amino acids and pka ranges, along with experimental method, protein name and species. the default option returns amino acids and their associated properties (figure 1b) ; while the second option returns proteins which contain the specified amino acids (figure 1c). a local database of protein sequences found in ppd was compiled from swissprot (26) and an additional postgresql table was created to hold this data. the local database is searched using the ncbi blastp and blastx programs (25), allowing input of either protein or nucleotide sequences. the html front - end connects to a web server - based pl / cgi script which interacts with the blastp or blastx programs. the output contains links to ppd entries, which are created using swissprot (26) accession codes. there is an obvious need to extend the number of entries through continuous addition of data from new, and newly - identified, publications. the database also needs to be maintained, ensuring links to external databases remain current. initially, as with all databases, random errors will occur owing to human error during data acquisition or will be extant within the original experimental data. the database will be assessed for errors and inconsistencies, thus maintaining, as far as possible, the overall veracity of our data. as mentioned, we have tried to maintain a high degree of accuracy, through rigorous data selection ; however, user feedback will foment improvements. moreover, feedback focussing on the search interfaces and the general infrastructure will allow us to develop appropriately both the database and its interface in an efficient and ergonomic manner. the ppd is a unique compilation of protein pka values sourced from experimental data only. ppd is novel : no database of its kind currently exists. compared with other post - genomic databases, the size of ppd is limited, but this reflects its highly focused nature : the burgeoning of such focussed databases is a continuing trend in modern bioinformatics (27,28). access to ppd data is given through an interface available via the world wide web and includes both a blast search and an amino acid query search system. the blast search, which is linked to pka entries and external databases, allows ppd to be a cohesive and integrated source of protein information. ppd facilitates data - driven in silico prediction methods addressing the relationship between ionizable groups and protein function, be that protein protein interaction, protein folding or enzyme catalysis. a brief summary of pka data for each amino acid is shown in table 3, which also includes both the mean and sd of the corresponding measured pka values. from the ppd data, we have shown the distribution of pka values for the six most frequent residues : glutamic acid, lysine, tyrosine, aspartic acid, histidine and cysteine (figure 2). certain residues (aspartate, glutamate, lysine and histidine) have pka values which show relatively narrow distributions, while other residues (cysteine and tyrosine) show a wider dispersion of values ; however, this may only be a reflection of the amount of data available for these residues. while it is clear that mean values approximate closely model values, the corresponding sds are high, reflecting the wide distribution of ionization states in actual proteins. aspartate, for example, has a mean pka of 3.6 versus a model value of 4.0, yet the sd is 1.4. as the data for each residue increases, trends in residue - specific pka data will become more evident and more certain. in recent years, there has been an impetus to accumulate data on all scales from the atomic to the genomic ; this has led to a rapid increase in the number of databases. databases are increasingly forming the backbone of science in general and post - genomic biology in particular. ppd v1.0 was developed to provide an easily accessible compilation of protein pka values. despite the small size of ppd, the data it contains has utility throughout many different disciplines and, we may hope, the database will grow, through time, into a comprehensive protein pka resource. (b) shows the default result presentation, from which the pka data (d) for the specified residues can be accessed. (c) shows the alternative presentation, with the display of proteins containing the nominated amino acid(s). each column represents a count of pka values for the specified amino acid and pka.	the protein pka database (ppd) v1.0 provides a compendium of protein residue - specific ionization equilibria (pka values), as collated from the primary literature, in the form of a web - accessible postgresql relational database. ionizable residues play key roles in the molecular mechanisms that underlie many biological phenomena, including protein folding and enzyme catalysis. the ppd serves as a general protein pka archive and as a source of data that allows for the development and improvement of pka prediction systems. the database is accessed through an html interface, which offers two fast, efficient search methods : an amino acid - based query and a basic local alignment search tool search. entries also give details of experimental techniques and links to other key databases, such as national center for biotechnology information and the protein data bank, providing the user with considerable background information. the database can be found at the following url :.
symmetry has long fascinated the great minds in many disciplines from art to theoretical physics. in the field of medicine the circulation system is powered mainly by cardiac muscle and valves activities, which are specifically the coherent dilation and contraction of right and left ventricles. today, left ventricular systolic function evaluation has become routine in most clinical settings ; left ventricular diastolic function measurement is getting more and more attentions ; unfortunately, satisfactory assessment of right ventricular function is still a dream by clinicians due to lack of available reliable indexes. a number of new indexes introduced in this paper as a result of the symmetry concept will enable us to obtain a much better picture of cardiac function. this is easy to understand with many cardiac clinicians / researchers having already taken advantage of it. the second level of symmetry is systole and early diastole, which is based on the fact that both systolic and early diastolic processes are active. this will lead to the finding of counterparts of indexes used only in either systolic or early diastolic process. however, there is no farther endeavor to systemically explore how far and deep this theory can go. at the beginning of human development, both the ventricles developed from two adjacent segments of the primary heart tube. the left ventricle will pump up the systemic circulation, while the right ventricle needs to provide energy to the pulmonary circulation. in the pulmonary system, the circulation resistance is approximately a third of that in the systemic circulation, which makes the right ventricle wall thickness roughly half of its left neighbor. since the left ventricle is much stronger, the interventricular septum is captured in the regular daily task of left ventricular contraction and relaxation. in terms of shape and power, both ventricles are asymmetric. with our interests focusing on the behavior of contraction and relaxation echocardiography branches from medical imaging where ultrasound waves are used to exam the structure and function of the heart. here, we are interested in what the symmetry principle will bring us in this rapidly evolving field. symmetry of both left and right ventricular activity with regard to behavior is supported by some indexes describing both ventricles. in 1989, bargiggia,. introduced dp / dt to describe left ventricular systolic function, ten years later, oh,. pushed it on the right side of the heart. another example is the tei index,, which is also developed to evaluate both ventricular functions. symmetry of both diastolic and systolic processes is suggested by the fact that both are active. due to left ventricular relaxation is active, left ventricular diastolic time constant is discovered and well accepted to be the most established index to describe left ventricular diastolic function. since left ventricular contraction is also an active process ; it is reasonable for us to think that left ventricular systolic time constant exists. this idea can be tested with a similar experiment conducted by weiss,. except focusing on the systolic process. interestingly, based on the developed computational and mathematical methods, we can prove the symmetry between left ventricular systole and diastole. to introduce the proof, we first examine the calculation of tau, which is : tau = 1.2 (t1 t3), (t1 t3) is the time interval from 3 m / s to 1 ms on the descending branch of continuous wave doppler mitral regurgitation spectrum. this means, (t1 t3) is the core to evaluate left ventricular diastolic function. in terms of left ventricular systolic function evaluation by dp / dt, we can find that dp / dt = 32/(t3 t1), (t3 t1) is the time interval from 1 m / s to 3 m / s on the ascending branch of continuous wave doppler mitral regurgitation spectrum. since only (t3 t1) is a variable, it is the key to evaluate left ventricular systolic function. it is interesting to ponder upon the question as to how much this symmetry of systole and diastole principal can cover. the systole includes isovolumic contraction and ejection phases. however, there are four phases in diastole : isovolumic relaxation, rapid inflow, diastasis and atrial systole. from the above analysis, we come to a conclusion that there could be symmetric tau and dp / dt, which reveals the symmetry exists between isovolumic contraction and isovolumic relaxation. it deserves to mention that during isovolumic periods, both the aortic and mitral valve are closed, which makes it the best time to evaluate cardiac function due to relative independence of foreload and afterload. since the active cardiac muscle movement is still a driving force of these periods, especially in the early stage of the phase, symmetry should still apply. for example, the color m - mode flow propagation velocity could be measured in both phases. in the late stage of the phases, the active systolic contraction / diastolic relaxation comes to an end, which shows that the symmetry is ready to phase out. the last two phases of diastole, diastasis and atrial systole, can not be found in systole. so far, there are two levels of symmetry in cardiac function evaluation, one is the symmetry of left and right ventricles, and the other is the symmetry between systole and early diastole. under this principal, we introduce a list of new indexes that can be valuable in cardiac function evaluation the dp / dt is used to describe both left and right ventricular systolic function (figure 1a, c). today, dp / dt is integrated into many echo machines on the market for evaluation of left and right ventricular systolic function. with the symmetric contraction and relaxation on mind, we can introduce two new indexes to evaluate left and right ventricular diastolic function from dp / dt. this will be of great interest to clinicians and/or researchers, who have been enthusiastic with the systolic dp / dt (s). the same measurement can be done on the other branch of the regurgitation spectrum (figure 1a, c). with regards to the methodology, dp / dt measurement can also be done on aortic or pulmonary regurgitation spectrum (figure 1b, d). the left ventricular diastolic time constant, tau is the most established index to describe left ventricular diastolic function. with increasing interests focusing on the non - invasive endeavors in echo lab, and blessed by the fast digital technology progress, tau could be measured in our local clinic in the near future. the non - invasive calculation of tau is based on the measurement performed on either the descending branch of the continuous - wave doppler mitral regurgitation spectrum, or the ascending branch of continuous - wave doppler aortic regurgitation spectrum (figure 1a, b). suggested by the symmetry principle, the following three types of missing tau will come into light : (1) left ventricular systolic time constant ; (2) right ventricular diastolic time constant ; and (3) right ventricular systolic time constant. ai : aortic insufficiency ; lv : left ventricle ; mr : mitral regurgitation ; pi : pulmonary regurgitation ; rv : right ventricle ; tr : tricuspid regurgitation. left ventricular diastolic time constant is the most established index to describe left ventricular diastolic function. right ventricular tau(s) are even more pragmatic since there are more pulmonary and tricuspid regurgitations, compared with aortic and mitral valve regurgitations. furthermore, right ventricular tau(s) are more valuable since few satisfactory indexes exist to describe right ventricular function due to its irregular shape (dr sherif nagueh, personal communication). ai : aortic insufficiency ; lv : left ventricle ; mr : mitral regurgitation ; pi : pulmonary regurgitation ; rv : right ventricle ; tr : tricuspid regurgitation. ai : aortic insufficiency ; lv : left ventricle ; mr : mitral regurgitation ; pi : pulmonary regurgitation ; rv : right ventricle ; tr : tricuspid regurgitation. myocardial performance index or tei index is used to evaluate both the left and right ventricular function., both the tei indexes focus on systolic elements. following the two level symmetry principle, there should be two more tei indexes focusing on diastolic elements. this index is measured with color doppler m - mode when the mitral valve opens from the four - chamber view. the m - mode cursor is placed in the center of the column of mitral valve. color m - mode flow propagation velocity is used to evaluate left ventricular diastolic function. by applying symmetry principle, we will get three new indexes : (1) the first one can be used to evaluate left ventricular systolic function with the color doppler m - mode cursor placed in the center of left ventricular outflow tract. (2) the second one can be used to evaluate right ventricular diastolic function with the color doppler m - mode cursor placed in the center of the column of tricuspid valve. (3) the third one can be used to evaluate right ventricular systolic function with the color doppler m - mode cursor placed in the center of the right ventricular outflow tract. all about color m - mode flow propagation velocity so far, only four doppler major indexes in echocardiography have been analyzed based on the two level symmetry principle. we are showing another seventeen (question marks in the tables) indexes or measurement approaches under symmetry principle in this paper. clinicians and/or researchers working with other cardiac imaging facilities may try this symmetry principal in their own field as well. the idea of symmetry in cardiac function assessment will fly when all indexes in the tables are tested one by one in the future. lv : left ventricle ; lvot : left ventricular outflow tract ; mv : mitral valve ; rv : right ventricle ; rvot : right ventricular outflow tract ; tv : tricuspid valve. the dp / dt is used to describe both left and right ventricular systolic function (figure 1a, c). today, dp / dt is integrated into many echo machines on the market for evaluation of left and right ventricular systolic function. with the symmetric contraction and relaxation on mind, we can introduce two new indexes to evaluate left and right ventricular diastolic function from dp / dt. this will be of great interest to clinicians and/or researchers, who have been enthusiastic with the systolic dp / dt (s). the same measurement can be done on the other branch of the regurgitation spectrum (figure 1a, c). with regards to the methodology, dp / dt measurement can also be done on aortic or pulmonary regurgitation spectrum (figure 1b, d). the left ventricular diastolic time constant, tau is the most established index to describe left ventricular diastolic function. with increasing interests focusing on the non - invasive endeavors in echo lab, and blessed by the fast digital technology progress, tau could be measured in our local clinic in the near future. the non - invasive calculation of tau is based on the measurement performed on either the descending branch of the continuous - wave doppler mitral regurgitation spectrum, or the ascending branch of continuous - wave doppler aortic regurgitation spectrum (figure 1a, b). suggested by the symmetry principle, the following three types of missing tau will come into light : (1) left ventricular systolic time constant ; (2) right ventricular diastolic time constant ; and (3) right ventricular systolic time constant. ai : aortic insufficiency ; lv : left ventricle ; mr : mitral regurgitation ; pi : pulmonary regurgitation ; rv : right ventricle ; tr : tricuspid regurgitation. left ventricular diastolic time constant is the most established index to describe left ventricular diastolic function. right ventricular tau(s) are even more pragmatic since there are more pulmonary and tricuspid regurgitations, compared with aortic and mitral valve regurgitations. furthermore, right ventricular tau(s) are more valuable since few satisfactory indexes exist to describe right ventricular function due to its irregular shape (dr sherif nagueh, personal communication). ai : aortic insufficiency ; lv : left ventricle ; mr : mitral regurgitation ; pi : pulmonary regurgitation ; rv : right ventricle ; tr : tricuspid regurgitation. ai : aortic insufficiency ; lv : left ventricle ; mr : mitral regurgitation ; pi : pulmonary regurgitation ; rv : right ventricle ; tr : tricuspid regurgitation. myocardial performance index or tei index is used to evaluate both the left and right ventricular function., both the tei indexes focus on systolic elements. following the two level symmetry principle, there should be two more tei indexes focusing on diastolic elements. this index is measured with color doppler m - mode when the mitral valve opens from the four - chamber view. the m - mode cursor is placed in the center of the column of mitral valve. color m - mode flow propagation velocity is used to evaluate left ventricular diastolic function. by applying symmetry principle, we will get three new indexes : (1) the first one can be used to evaluate left ventricular systolic function with the color doppler m - mode cursor placed in the center of left ventricular outflow tract. (2) the second one can be used to evaluate right ventricular diastolic function with the color doppler m - mode cursor placed in the center of the column of tricuspid valve. (3) the third one can be used to evaluate right ventricular systolic function with the color doppler m - mode cursor placed in the center of the right ventricular outflow tract. all about so far, only four doppler major indexes in echocardiography have been analyzed based on the two level symmetry principle. we are showing another seventeen (question marks in the tables) indexes or measurement approaches under symmetry principle in this paper. clinicians and/or researchers working with other cardiac imaging facilities may try this symmetry principal in their own field as well. the idea of symmetry in cardiac function assessment will fly when all indexes in the tables are tested one by one in the future. lv : left ventricle ; lvot : left ventricular outflow tract ; mv : mitral valve ; rv : right ventricle ; rvot : right ventricular outflow tract ; tv : tricuspid valve.	both right and left ventricles are developed from two adjacent segments of the primary heart tube. though they are different with regard to shape and power, they mirror each other in terms of behavior. this is the first level of symmetry in cardiac function assessment. both cardiac muscle contraction and relaxation are active. this constructs the second level of symmetry in cardiac function assessment. combination of the two levels will help to find some hidden indexes or approaches to evaluate cardiac function. in this article, four major indexes from echocardiography were analyzed under this principal, another seventeen indexes or measurement approaches came out of the shadow, which is very helpful in the assessment of cardiac function, especially for the right cardiac function and diastolic cardiac function.
recent proposals for treatment of advanced cancer of the larynx emphasize more conservative approaches and make the definition of treatment an even more complex1. however, total laryngectomy is still frequently adopted in such cases and questionnaires on health conditions and quality of life have been recommended as key promoters of success of treatment planning2. after total laryngectomy, oropharyngeal dysphagia can compromise the quality of life by requiring the modification of eating habits, affect socialization, and lead to a degree of isolation in activities with family members3. oropharyngeal dysphagia is a common symptom in patients with tumors in the head and neck regions4 and its etiology may be related to how surgical technique would be used, adjuvant treatments such as radiotherapy and chemotherapy, and comorbidities such as advanced age and depression5 6. the impact of difficulty in deglutition in quality of life of total laryngectomized has been assessed using generic instruments3 7 8 9 or instruments specific to this function2 10 11. the results of these studies indicate that the overall quality of life after total laryngectomy is approaching the standard of the general population, as opposed to specific domains such as deglutition, which often appear associated with negative aspects8. nevertheless, the results are still preliminary, especially owing to the multiple number of existing instruments and because dysphagia is still underdiagnosed in this group of patients5. the aim of this study was to describe the effect of deglutition in quality of life of patients undergoing total laryngectomy. the participants included patients undergoing treatment for esophageal speech acquisition at the department of speech therapy in a cancer referral center located in pernambuco, northeastern part of brazil. we included patients who underwent total laryngectomy with neck dissection and postoperative radiotherapy, with completion of treatment for at least 3 months. we excluded patients with neurological disorders and head and neck disease and those subjected to other procedures in the head and neck. the quality of life related to deglutition was assessed using the swallowing quality of life questionnaire (swal - qol), which has been validated for brazilian portuguese subjects12. the questionnaire comprises 44 questions that assess 11 domains related to quality of life (burden, feed duration, desire, symptoms frequency, food selection, communication, fear, mental health, social function, sleep, and fatigue). the answers were converted into scores ranging from 0 to 100, divided into quintiles (0 as a minimum score and 100 as a maximum positive score). in each domain, the score values regarding responses were summed and the result was divided by the number of questions in the domain, reaching the end score. the scores 049 were interpreted as a severe impact, 5070 as a moderate impact, and 71100 slight impact or no impact10. the analysis was performed using descriptive statistics. due to the final conversion of the swal - qol results into categories (ordinal categorical variable), we used the median as a measure of central tendency and the minimum and maximum as a measure of dispersion. among the issues addressed by the additional instrument, we herein highlight the general health status and self - reported and food consistencies accepted by the volunteer. the research was submitted to the ethics and human research committee and approved under the number 67/2010. the volunteers who agreed to participate in the study signed a free consent term, in accordance to resolution 196/96 of the national research ethics counsel. a sample of 15 volunteers had a mean age of 63 9.3 years and the following profile : male (86.7%), married (53.3%) and uncompleted elementary education (60%). the self - reported condition general health was rated as good by a significant percentage of volunteers (graphic 1). graphic 2 shows that most participants described the consistency of the food consumed in the last week as difficult to chew. table 1 shows the distribution of swal - qol domains, according to the median. it was observed that the communication and fear domains were those with the lowest scores, indicating severe impact on quality of life related to deglutition. the feeding duration the descriptive analysis (chart 1) of data revealed that items with higher absolute and relative frequency of responses with scores between 0 and 50 were longer time required to eat (53.3% ; feeding duration domain), cough to remove the liquid or food out of the mouth when they are standing (40% and 46.7%, respectively, frequency of symptoms domain), difficult to understand (46.7% ; communication domain), and fear choking and having pneumonia (40% and 53.3%, respectively ; fear domain). in brazil, a group of researchers published 2 studies in which the swal - qol questionnaire was used in patients after a total laryngectomy in so paulo ; however, unlike the present study, they found only a moderate impact of deglutition on quality of life10 11. in one of these studies, the domains that resulted in the lowest median values were communication and desire for food, whose impact was moderate on the quality of life. domain was not considered in this study. in the other work with a sample of the same health service, the lowest median found in the feeding duration and communication domains, with moderate impact on quality of life11. it is noticed that the communication domain is always present and associated with negative aspects. the permanent loss of laryngeal voice and the difficulties of adapting to alternative communication can support this result13. the fact that the volunteers of this research are not yet fully adapted to esophageal speech may have influenced the result. in the institution where the collection was made, the esophageal voice was the method of communication rehabilitation, since the other possibilities of more sophisticated vocal rehabilitation (tracheoesophageal and laryngeal electronic prostheses) require costs that are not compatible with the profile of low - income users of this service. we emphasize that the individual variations in relation to culture, beliefs, religions, social, economic, professional, and family situations10 can exert a strong influence on the perception that the individuals have of their quality of life. thus, the differences and similarities between our study and others may possibly be explained by the variable aspects of social determinants, whose importance should be further explored in future research. the swal - qol is a protocol that considers a specific functional domain ; however, there are protocols that assess the overall quality of life and include deglutition3 9. an australian study3 investigated the effect of dysphagia on quality of life of 110 patients after total laryngectomy. this study used the world health organization quality of life - brief (whoqol - brief) and the university of washington qol (uw - qol). there was no difference between the findings of the subjects with and without dysphagia ; however, total laryngectomy with dysphagia had more functional impairment, reduced social participation, and higher levels of depression and anxiety. the authors conclude that although dysphagia does not directly determine the quality of life after total laryngectomy, it can have a negative impact on functional and psychological well - being of the patient. in another study8 in patients with total laryngectomy with more than 2 years of completion of treatment, the overall quality of life did not differ from that of the general population, but there was adherence to specific scales of the physical domain, which is influenced by age, sex, radiotherapy, and chemotherapy. in the sample of these authors, because of our small sample size, we did not compare the results by gender. when considering the patient 's perspective regarding the impact of total laryngectomy on quality of life, a qualitative study15 received reports of psychological and functional problems, including dysphagia. the authors highlight the high number of difficulties reported even after end of treatment and reinforce the need for maintenance of the monitoring team of multidisciplinary rehabilitation for longer periods after surgery. about this, the literature says that the longer survival is not an accurate reflection of the success of treatment and does not necessarily indicate better quality of life2. therefore, there is a need to stimulate a long - term care to these individuals and to promote the application of more reliable instruments that can capture the impression of the subject with respect to their quality of life. for longer - term evaluations, the performance status scale for head and neck cancer patients (pss - hn) was used to assess the degree of dysphagia before and after total laryngectomy in 20 patients9. the result reinforces the remarkable social impact that the deglutition difficulty has after total laryngectomy3, even after a long period after treatment. moreover, it agrees with the results of our research that revealed a moderate impact on however, this same work9, differs from ours with respect to food consistency, but not to other studies using swal - qol. the percentage of subjects with restriction for solids is comparable to that found in a previous study with the same number of individuals (26.3%)11. in another study with 12 patients, 4 had restriction10, but these had the lowest scores, which suggests that the consistency of food interferes with quality of life10. when considering the small number of volunteers, we decided not to make this comparison for believing that the subgroups would be uneven and the result would not be representative of the entire population. despite the severe and moderate impact found in some domains of swal - qol, no volunteers rated their general health less than satisfactory, and previous studies have also found the same pattern10 11. this finding can be explained by time to clinical stability at the time of answering the questionnaire. it also reveals that, despite the impact caused by deglutition difficulties, this does not interfere negatively in the context of general health from the perspective of the respondent10 11. the frequency of deglutition difficulties is greater when surgery is combined with radiotherapy, as observed in the treatment profile of all of our subjects. researchers recruited 26 patients who underwent only surgery and 95 who underwent surgery combined with radiotherapy. it was found that the deglutition was better in the group whose treatment was surgical alone14. unlike our current findings, those of our previous research2 did not detect a negative impression of the patients in relation to deglutition after treatment. in that study, another specific instrument was used for assessment of dysphagia, the md anderson dysphagia inventory. this shows that the choice of the questionnaire to be applied must be judicious as well as the interpretation of data by the multidisciplinary team. after total laryngectomy, deglutition exerted a severe impact on quality of life in terms of communication and fear domains and a moderate impact on the feeding duration domain. the authors thank the national council for technology and scientific development (cnpq), which provided financial support through the notice mct / cnpq / health - ct / ms / sctie / decit n 67/2009 - rebrats.	summary introduction : total laryngectomy creates deglutition disorders and causes a decrease in quality of life aim : to describe the impact of swallowing and quality of life of patients after total laryngectomy. method : a case series study. patients completed a swallowing and quality of life questionnaire composed of 44 questions assessing 11 domains related to quality of life (burden, eating duration, eating desire, frequency of symptoms, food selection, communication, fear, mental health, social functioning, sleep, and fatigue). the analysis was performed using descriptive statistics, including measures of central tendency and variability. results : the sample comprised 15 patients who underwent total laryngectomy and adjuvant radiotherapy. of these, 66.7% classified their health as good and 73% reported no restrictions on food consistency. the domains communication and fear represented severe impact and eating duration represented moderate impact on quality of life. the items with lower scores were : longer time to eat than others (domain eating duration), cough and cough to remove the liquid or food of the mouth when they are stopped (domain symptom frequency), difficulties in understanding (domain communication) and fear of choking and having pneumonia (domain fear). conclusion : after total laryngectomy, patients report that swallowing issues have moderate to severe impact in communication, fear, and eating duration domains.
the gibbon genome contains all previously described classes of transposable elements that are mostly shared with the other primates. one exceptional addition is the lava element, a novel retrotransposon that emerged exclusively in gibbons and has a composite structure comprised of portions of other repeats (3- l1-alus - vntr - alu - like -5) (fig. searches of nleu1.0 retrieved 1,797 lava insertions, 1,256 of which were 3-intact elements, many carrying signs of target - primed reverse transcription (tprt). the distribution of 3-intact lava elements uncovered a significant overlap with genes (pearson chi - squared, p=0.017) and gene ontology (go) analyses using the database for annotation, visualization, and integrated discovery (david) showed a significant functional enrichment exclusive to the microtubule cytoskeleton category (fdr=0.031, p - value=0.001) (supplementary information s7 and file 6) (edf 3). additional analyses with meta - pathway database tools refined this enrichment to pathways related to chromosome segregation, including establishment of sister chromatid cohesion and mitotic metaphase and anaphase (table st7.3). genes with lava insertions include proteins that function as checkpoints for cell division and for spindle integrity / architecture (e.g., map4, cep164, bub1b), participate in kinetochore assembly and attachment to the spindle (e.g., mad1l1, clasp2), and play a role in chromosome segregation during cell division (e.g., kifap3, kif27) (edt 1). intragenic lava insertions were skewed toward introns (pearson chi - squared, p=0.0001) and were less frequent than expected when within < 1 kbp of the nearest exon junction (edf 3). the majority (74%) of intronic lava elements were found in the antisense orientation. we hypothesized that intronic antisense lava insertions may cause early transcription termination (ett) by providing a polyadenylation site in antisense orientation, as previously described for l1 elements (edf 3). indeed, we found 84.1% of the 3-intact lava elements encoded a perfect polyadenylation signal at their 3-end in antisense orientation. to obtain experimental evidence that lava elements disrupt transcription, we performed a reporter assay in which the 3-end of a luciferase gene construct lacking a transcriptional termination site was fused to the 3-terminal fragments of lava_e and lava_f elements, mimicking the arrangement observed in gibbon genes (fig. luciferase activity exceeding background level by ~50% was observed from the lava_f reporter construct (fig. further, 3 rapid amplification of cdna ends (race) experiments confirmed that the transcription termination site had been supplied from the lava element (edf 3). thus antisense intronic lava insertions can cause ett with some variability possibly due to the genomic context of the polyadenylation site, which explained the difference between the two reporter constructs. we also investigated lava induced ett in vivo by analyzing rna - seq data generated for asia (table st2.4). specifically, we looked for paired - end reads only partially aligning to an antisense lava element due to untemplated residues and then identified cases for which presence of a poly(a) tail was preventing full - length alignment. this analysis revealed that elements from a variety of sub - families have the potential to cause ett, including those identified for lava elements inserted in the microtubule cytoskeleton genes (e.g. b2r2, c4b, b1r2) (edt 1). of note, we observed that ett occurred at relatively low levels as we identified a significant number of read pairs indicative of normal transcription and splicing for lava - terminated genes (table st7.5). this is to be expected, as full inactivation of many of these genes would be incompatible with life. on the other hand, as alternative splicing and rna - pol ii transcript termination/ polyadenylation are tightly coupled processes, lava - mediated ett could also act by differently affecting distinct isoforms and/or influence the ratio between isoforms. finally, lava insertions may also impact gene expression by functioning as exon traps, as shown for sva elements. one putative example of an exon trapping event was identified for hormad2, a gene that monitors the formation of synapsis during crossover (supplementary information s7, table st7. since genome reshuffling began in the common ancestor of all extant gibbon species, lava insertions must have occurred in key genes before the four genera diverged. we experimentally confirmed the mode and tempo of all 23 lava insertions in genes from the microtubule cytoskeleton category using both site - specific pcr and in silico methods (edf 4) and found that most of the insertions (15/23) were shared by the four gibbon genera (supplementary file 6). eleven of the genes match the structural requirements for ett and five of them are also shared. these genes include map4, involved in spindle architecture, and cep164, a g2/m checkpoint whose inactivation results in an aberrant spindle during cell division (edt 1). we explored the relationship between lava family expansion and evolution of the gibbon lineage and, through analyses of diagnostic mutations, identified 22 lava subfamilies (fig. 3c). in addition, we tested for presence / absence of 200 lava loci from among the evolutionarily youngest elements in each subfamily (edf 4) across 17 unrelated gibbon individuals and found that 52% of loci were shared among all four genera, whereas 27% were nomascus - specific. the remaining lava insertions showed a variety of confounding phylogenetic relationships consistent with incomplete lineage sorting (ils) of ancestral polymorphisms, perhaps as a result of a rapid radiation of gibbon genera (supplementary information s7 ; table st7.1 - 2). we used a maximum likelihood method to obtain age estimates for the 22 lava subfamilies. in the case of the two oldest subfamilies, lava_a1 and lava_a2, we obtained estimates of ~18 mya and ~17 mya, respectively (table st7.3). a coalescent - based methodology implemented in the software g - phoscs using nleu1.0 estimated a gibbon - great ape population divergence time of ~16.8 mya (95% ci : 15.9 - 17.6 mya) assuming a split time with macaque of 29 mya (supplementary information s4). hence, the lava element likely originated around the time of the divergence of gibbons from the ancestral great ape / human lineage. the evolutionary history of the gibbon lineage and, in particular, the timing and order of splitting among the four genera, is still a subject of debate. to address this issue we generated medium coverage (mean ~15x) wgs short read data for two individuals from each of the four genera, including two different hylobates species (h. moloch and h. pileatus) (table st2.1 - 2). while phylogenetic analysis of assembled whole mitochondrial dna genomes using beast strongly supported monophyletic groupings for each gibbon genus, the branching order of the four genera remained unresolved (fig. neighbor joining trees constructed from pairwise sequence divergence, k, across ~11,000 genic (200 bp) and ~12,000 non - genic (1 kb) autosomal loci supported a supermatrix sequence topology of (((siamang (ssy), hoolock (hle)), nomascus (nle)), (h. pileatus (hpl)), h. moloch (hmo)) (fig. 4a), though bootstrap confidence for the node separating nle and hylobates was low (~52%). this topology was also the most frequently observed when constructing k - based unweighted pair group method with arithmetic mean (upgma) trees along the genome using non - overlapping 100 kbp sliding windows. however all 15 possible rooted topologies for the four genera were observed at considerable frequencies (edf 5), consistent with the extensive ils observed in the lava element analysis. in order to infer the most likely bifurcating species topology amongst the four genera while taking into account ils, we employed a novel coalescent - based abc methodology using the autosomal nongenic and genic loci (veeramah. the topology described above had the highest combined posterior probability, though support was relatively low (p(model)=17%) and other topologies, including one with nle and hylobates interchanged as the most external taxa, had comparable probabilities (fig. the estimated internal branch lengths under the best species topology using our abc framework and g - phocs were very short, supporting a rapid speciation process for the four gibbon genera (fig 4b - right). given this observation and uncertainty in the best topology, we also estimated parameters under an instantaneous speciation model (fig. assuming an overall autosomal mutation rate of 1 x 10/site / year, we placed the beginning of the speciation process at ~5 mya under both models, with the two hylobates species diverging ~1.5 mya. consistent with the abc analysis, ssy and hle share the largest number of alleles across the whole genome (table st8.5). however, nle and the two hylobates samples are both significantly closer to ssy than hle as assessed by the d - statistic. this result could be explained by two independent gene flow events between ssy and both nle and hylobates. however fertile intergenic hybrids have yet to be observed either in the wild or captivity ; an alternative explanation would be long - term population structure in the gibbon ancestral population. both the abc and g - phocs analyses suggest that the ancestral gibbon effective population size (ne) was large (80,000 - 130,000) but neither of these frameworks can distinguish this from a structured ancestral population. the coalescent - based analysis (fig 4a), along with estimates of genome - wide heterozygosity (fig st8.2), suggests a larger long - term ne for both n. leucogenys and h. moloch compared to the other species. analysis using the pairwise sequentially markovian coalescent (psmc) model indicates that these two species underwent an increase in ne during the late pleistocene era (500 - 100 thousand years ago (kya) followed by a subsequent decrease in ne 100 - 50 kya (fig. it is important to point out that fluctuation in ne could result from changes in the actual number of individuals in the population, changes in population structure, and/or variable gene flow. accelerated substitution rates are a hallmark of adaptive evolution, and genomic regions with excess lineage - specific substitutions have been found to have functional roles. we identified 240 short (153 bp median length) regions with accelerated substitution rates in the gibbon lineage (gibars). we observed that gibars were primarily intergenic (66%) and tended to co - localize near the same genes as lava elements (p - value=81e-06 ; odds ratio of 2.74 (1.794.07, 95% ci)). consistent with this finding, a go enrichment test for genes within + /100 kbp of each gibar (in comparison with background genes) revealed enrichment for the chromosome organization category (benjamini - hochberg fdr < 5%) (edf 6). given evidence of functional roles gathered for human accelerated regions, we speculate that the gibars may create functional elements (e.g., enhancer, protein - binding domains) to modulate the transcriptional effect of local lava insertions (supplementary information s12 and file 9). we assessed the potential presence of positive selection in 13,638 human genes with one - to - one orthologs in gibbon using a branch - site likelihood ratio test (supplementary information s10). one of the most striking features of gibbons is their use of brachiation (i.e., arboreal locomotion using only the arms). we uncovered evidence related to traits possibly associated with this adaptation such as the gibbon 's longer arms, more powerful shoulder flexors, rotator muscles, and elbow flexors. first, some genes whose functions relate to these anatomical specializations appear to have undergone positive selection in gibbons. they include tbx5 (p - value=0.00015), required for the development of all forelimb elements ; col1a1 (pro - alpha1 chains of type i collagen) (p - value=3.39e-11), the fibril - forming collagen main protein of bones, tendons, and teeth ; and chrna1 (acetylcholine receptor subunit alpha precursor) (p - value=0.00039), involved in skeletal muscle contraction. these genes have not been identified as positively selected in other primates to date. we also observed that some genes involved in chondrogenesis (snx19, id2, and ext1) were associated with gibars. finally, the chondroadherin gene (chad) coding for a cartilage matrix protein is specifically duplicated in all gibbon genera (edf 2). our sequencing, assembling, and analysis of the gibbon genome has provided numerous insights into the accelerated evolution of the gibbon karyotype and identified genetic signatures related to gibbon biology. first, sds and repetitive sequences were the best predictors of gibbon - human breakpoints, although we excluded a causal role given the predominance of non - homology - based repair signatures. furthermore, accelerated rearrangement was confined to large - scale chromosomal events, pointing to a mechanism responsible for causing gross chromosomal changes, rather than global genomic instability. this is in line with our hypothesis that the high rate of chromosomal rearrangements may have been due to lava - induced premature transcription termination of chromosome segregation genes. this effect may have occurred at a low enough level to be compatible with life but sufficient to increase the frequency of chromosome segregation errors. of note, the link between erroneous chromosome segregation and increased chromosomal rearrangement has been recently demonstrated by others through in vitro experiments. the question remains how such a high number of chromosomal rearrangements could become fixed in such a relatively short time. one possibility is that a combination of geographic isolation and post - mating reproductive barriers accelerated the radiation of the four gibbon genera. our estimates dated the lineage - splitting event to the miocene - pliocene transition, when major changes in the distribution of tropical and subtropical forests were caused by the elevation of the yunnan plateau and rise in sea levels. furthermore, fluctuation in sea levels beginning in the early pliocene appears to have brought about cycles of forest fragmentation and amalgamation, leading to alternating range compression and expansion for many mammalian groups. together these results advance our knowledge of the unique traits of the small apes and highlight the complex evolutionary history of these species. moreover, our analyses of the shattered gibbon genome helped gain insight into the mechanisms of chromosome evolution and uncovered a novel source of genome plasticity. sanger - based whole - genome sequencing was performed as described for other species. the genome assembly was generated using the arachne genome assembler assisted with alignment data from the human genome (supplementary information s1). the source dna for the sequencing was derived from a single female (asia ; studbook no. short - read libraries were constructed at the oregon health & science university (ohsu) following standard illumina protocols and sequenced on an illumina hiseq 2000. a, the table compares the gibbon assembly statistics to those of other primates sequenced with a similar strategy. b, the plot represents the percentage of the 10,734 single - copy gene hmms (hidden markov models) for which just one gene (blue) is found in the different mammalian genomes in ensembl 70. the missing hmms (cyan) either do not match any protein or the score is within the range of what can be expected for unrelated proteins. the remaining category (green) represents hmms for which the best matching gene scores better than unrelated proteins but not as well as expected. a, the image shows a representative gibbon - only wssd (whole - genome shotgun sequence detection) call by sanger read depth. the duplication identified in this case overlaps with the gene chad that codes for a cartilage matrix protein. b, examples of fish hybridizations on gibbon metaphases using duplicated human fosmid clones that were identified by the (wgs) detection strategy (red signals). c) intrachromosomal tandem duplication confirmed using cohybridization with a single control probe (blue signals). c, megabases of lineage - specific and shared duplications for primates based on grchr37 read depth analysis. moreover, insertions are significantly enriched in introns and depleted in exons, most likely as a result of selection against insertions in exons. b, schematic representation of the mechanism through which lava intronic insertions in anti - sense orientation might cause early termination of transcription : a) truncated transcript ; b) normal transcript (pa = polyadenylation site). c, we calculated the distance to the nearest exon for each intronic lava and compared this to what would be expected for random insertions (i.e., background). we found fewer insertions than expected by chance within 1 kbp of the nearest exon. lava_f 3 tsd is highlighted by dark background ; the major antisense lava_f polyadenylation signal (maps) is in red. a, screenshots from the integrative genomics viewer (igv) browser for loci map4, rabgap1, and bbs9. each column shows portions of the igv visualization of a lava insertion locus identified in nleu1.0 and its flanking sequence. read pairs are colored in red when their insert size is larger - than - expected, indicating the presence of a lava insertion. map4 is a shared lava insertion, while rabgap1 and bbs9 are nomascus - specific. b, lava elements containing at least 300 bp of the la section of lava elements were selected and reanalyzed using repeatmasker to determine subfamily affiliation and divergence from the consensus sequence. lava elements are grouped based upon their subfamily affiliations (see legend top right). the x - axis shows the percent divergence from the respective consensus sequence, and the y - axis shows the number of elements with a certain percent divergence from the consensus sequence. a, neighbor - joining trees for gibbons using non - genic loci. b, upgma trees for 100 kbp nonoverlapping sliding windows moving along the gibbon genome reporting the top 15 topologies (see also supplementary table st8.3). different sequence types are shown on the x - axis, and the y - axis displays the fraction of gibars and candidate regions annotated to the respective class. gibars are significantly enriched in intergenic regions (p = 4.7e-6) and significantly depleted in exons (p = 7.3e-6). p - values for each class were calculated with the fisher 's exact test. introns are comparably prevalent in candidates and gibars, while in utr and flanking region counts are too low to draw meaningful conclusions (data not shown). b, treemap from revigo for goslim biological process terms with a benjamini - hochberg fdr of 5%. each rectangle is a cluster representative ; larger rectangles represent superclusters including loosely related terms. microtubule cytoskeleton go category with lava insertions genes highlighted in gray carry lava insertions that are shared, antisense, and carry a perfect antisense polyadenylation site.	gibbons are small arboreal apes that display an accelerated rate of evolutionary chromosomal rearrangement and occupy a key node in the primate phylogeny between old world monkeys and great apes. here we present the assembly and analysis of a northern white - cheeked gibbon (nomascus leucogenys) genome. we describe the propensity for a gibbon - specific retrotransposon (lava) to insert into chromosome segregation genes and alter transcription by providing a premature termination site, suggesting a possible molecular mechanism for the genome plasticity of the gibbon lineage. we further show that the gibbon genera (nomascus, hylobates, hoolock and symphalangus) experienced a near - instantaneous radiation ~5 million years ago, coincident with major geographical changes in southeast asia that caused cycles of habitat compression and expansion. finally, we identify signatures of positive selection in genes important for forelimb development (tbx5) and connective tissues (col1a1) that may have been involved in the adaptation of gibbons to their arboreal habitat.
the patient was a 3-year - old boy with no known underlying medical history who was identified in 2003 as having clinical verruga peruana by the classic appearance of the eruptive nodular rash (figure 1). he and his family lived in a rural setting near the town of caraz, ancash region, peru. he lived in close proximity to numerous pets and farm animals and had experienced insect bites around the time the eruptive rash developed. his rash had been present for 30 days, and he had no fevers, chills, or arthralgias. baseline laboratory studies included complete blood counts and bacterial culture for bartonella species, using methods previously described (5,6). briefly, the media was biphasic, consisting of bacto agar with proteose peptone no. 3 (becton, dickinson and co., sparks, md, usa), dextrose, sodium chloride and 10% defibrinated sheep s blood, and rpmi supplemented with 10% inactivated fetal bovine serum. clinical presentation of verruga peruana in 3-year - old boy, peru, 2003. the physical examination revealed that the child had 56 lesions, distributed mainly on the extremities. laboratory values were the following : hemoglobin level 12.8 mg / dl (reference range 1113 g / dl), hematocrit 39% (reference range 31%43%), and platelet count of 300,000/l (reference range 15,000400,000) ; his leukocyte count was elevated at 28,000/l (reference range 4,10010,900) with 51% eosinophils, for which he was referred for further evaluation. the peripheral blood smear was negative for intracellular organisms, but blood culture was positive for a bartonella species. this species was further studied and found to be novel on the basis of genetic sequencing of the isolate obtained from the standard blood culture (7). his condition was treated with azithromycin, and the rash fully resolved (8,9). to confirm the identity of the isolate from this patient, in 20112012 we conducted molecular analyses (including pcr, nested pcr [npcr ], and sequencing) on the whole blood culture isolate, bartonella species no. initial denaturation was at 95c for 1 min, followed by 45 cycles of denaturation at 95c for 30 s, annealing at 56c for 30 s, and elongation at 68c for 90 s for pcr and 70 s for npcr, and then by a final extension step at 72c for 7 min. for glta95c, denaturation was for 1 min, followed by 45 cycles of denaturation at 95c for 30 s, annealing at 51c for 30 s, and elongation at 68c for 60 s, and then by the final extension step at 72c for 7 min. for rpob, primers were selected from the conserved regions of rna polymerase -subunit encoding gene (rpob) after alignment of the rpob from b. quintana and b. vinsonni for pcr and npcr. pcr and npcr were carried out by using conditions identical to those described for glta (table). pcr products were purified by using the qiaquick pcr purification kit (qiagen, valencia, ca, usa) before sequencing. pcr products were sequenced in both directions by using the bigdye terminator v3.1 cycle sequencing kit (life technologies, carlsbad, ca, usa) and run on an automated 3130xl gene analyzer (life technologies). the sequence analysis was performed by using bioedit version 7.1.3 (ibis biosciences, carlsbad, ca, usa). the multiple sequence alignments were performed with the clustalw multiple alignment application also in bioedit version 7.1.3. phylogenetic trees were created with mega5 software using the neighbor - joining tree method with 1,000 bootstrap replicates (11). the 1,351-bp sequence of the rrs fragment was found to be 99.0% similar to the rrs fragment of b. bacilliformis. the 312- and 589-bp fragments of glta and rpob, respectively, were found to be most similar to their counterparts of b. bovis at 89.4% (glta) and 85.9% (rpob), respectively (technical appendix). the sequence similarity ranges for the rrs, glta, and rpob for recognized bartonella species are 97.7%99.8%, 83.4%96.1%, and 85.9% 96%, respectively (12). in addition, rpob and glta are believed to have the best discriminating power for bartonella species (13). la scola. proposed that a new species be designated if the sequence similarities are < 96% and < 95.4% for a 327-bp fragment of glta and a 825-bp fragment of rpob, respectively (13). the sequence similarities for candidatus bartonella ancashi 20.00 to other known bartonella species fall well below these suggested values, providing more evidence that this agent is unique. phylogenetic analysis of the rrs, glta, and rpob, gene fragments provide additional evidence for identification of a unique bartonella agent. the concatenated sequence of glta and rpob gene fragments placed the new bartonella isolate in an exclusive clade that is most closely aligned with b. bacilliformis (figure 2). the results from the phylogenetic analysis combined with the sequence similarity data provide evidence that this isolate, candidatus bartonella ancashi 20.00, is unique (12,13). phylogeny for concatenated sequences of novel bartonella isolate (boldface), including a 312-character fragment of glta and a 589-character fragment of rpob. the neighbor - joining tree method (1,000 bootstrap replicates) was employed using mega5 software (11), and the distances were calculated by using the jukes - cantor method, in which units are calculated as the number of base pair substitutions per site (10). the variety of bacteria that have been implicated in the clinical spectrum of bartonellosis is increasing as molecular methods are applied to isolates that previously were identified by using clinical criteria or biochemical testing. the novel bacterium may have similar epidemiologic, clinical, and microbiologic properties to b. bacilliformis, but without relating these data to a full molecular characterization, that assumption is precarious. to address this public health deficiency, a new diagnostic paradigm this is particularly true for areas with high biodiversity, a point identified by other investigators who have termed these regions hot zones for emerging infectious diseases (14). unfortunately, tools such as high - throughput sequencing are rare in developing settings where risk for novel pathogen emergence is highest. investment in advanced molecular diagnostic platforms in the developing setting will be an essential tool for expanding pathogen discovery ; of course, this should be accompanied by parallel investments in training in molecular laboratory techniques and analysis for resident scientists. opportunities for grants and stable faculty positions must also be supported to encourage qualified scientists to remain in the developing setting. finally, evidence indicates that humans contract bartonellosis only once and that lifelong immunity results from that primary infection (15). because of this circumstance, and the inability to identify an animal reservoir of b. bacilliformis, peruvian scientists and others have identified bartonellosis as a disease that may be eradicated in the andean region through development of a vaccine against b. bacilliformis, targeted treatment of patients, and vector control programs (15) further molecular and immunologic studies should be undertaken if this disease is to be targeted for eradication. the 312- and 589-bp fragments of glta and rpob, respectively, were found to be most similar to their counterparts of bartonella bovis at 89.4% (glta) and 85.9% (rpob).	while studying chronic verruga peruana infections in peru from 2003, we isolated a novel bartonella agent, which we propose be named candidatus bartonella ancashi. this case reveals the inherent weakness of relying solely on clinical syndromes for diagnosis and underscores the need for a new diagnostic paradigm in developing settings.
in recent times, hyperinsulinemia has been proposed not only to be associated with obesity but also to be a cause of obesity (reviewed in the study by shanik.). dopamine neurons of the ventral tegmental area (vta) have been implicated in the incentive, reinforcing and motivational aspects of food intake. we have recently demonstrated that insulin either applied exogenously or elevated by a sweetened high - fat meal can induce long - term depression (ltd) at excitatory synapses onto dopamine neurons. this reduction of synaptic efficacy in the vta is linked to a reduced anticipatory activity or preference for palatable food. thus, under physiological circumstances, insulin action in the vta has a vital role in regulating food intake by decreasing salience for food - related cues after a meal. however, it is unknown how insulin regulates dopamine neurons of the vta under pathological circumstances such as hyperinsulinemia. btbr t itpr3/j (btbr) mice constitute a hyperinsulinemic mouse strain that is predisposed to obesity. these mice have a mutation in the itpr3 gene at the tufted locus, which gives rise to tufted hair in older mice as well as to different taste perceptions due to alterations in the taste receptor inositol 1,4,5-trisphosphate receptor, type 3. furthermore, the btbr mouse strain has elevated plasma insulin levels relative to the c57bl/6 j strain and has a higher fat mass than many other inbred mouse strains. therefore, we hypothesized that insulin - induced ltd of vta neurons of btbr mice would be disrupted. all protocols were in accordance with the ethical guidelines established by the canadian council for animal care and were approved by the university of british columbia or university of calgary animal care committees. c57bl/6 j mice were obtained from the university of british columbia breeding facility or jackson laboratories (sacramento, ca, usa). btbr mice were obtained from the jackson laboratory and bred in the ubc facility. both strains were fed chow ad libitum (teklad 2918). all electrophysiological recordings were performed in male mice ranging from p19 to p30 as per. slices in the recording chamber were superfused with bicarbonate - buffered solution (artificial cerebrospinal fluid) saturated with 95% o2/5% co2 and containing (in mm) 126 nacl, 1.6 kcl, 1.1 nah2po4, 1.4 mgcl2, 2.4 cacl2, 26 nahco3 and 11 glucose as well as picrotoxin (100 m) (at 3234 c). electrodes (34.5 m) contained (in mm) 117 cesium methansulfonate, 20 hepes, 0.4 egta, 2.8 nacl, 5 tea - cl, 2.5 mgatp and 0.25 nagtp (ph 7.27.3, 270285 mosm). series resistance (1025 m) and input resistance were monitored online with a 10 mv depolarizing step (400 ms) given before every afferent stimulus. dopamine neurons were identified by the presence of a hyperpolarizing cation current (ih), which is a good predictor of tyrosine hydroxylase (th) neurons in mice. a bipolar stimulating electrode was placed 100300 m rostral to the recording electrode and was used to stimulate excitatory afferents at 0.1 hz. coronal brain sections (30 m) were fixed in 4% paraformaldehyde overnight, blocked with 5% goat serum/0.3% triton x-100/0.2% bovine serum albumin in phosphate - buffered saline (ph 7.4) for 2 h at room temperature and then incubated for 48 h at 4 c with mouse th (1:1000) and rabbit insulin receptor (1:100) monoclonal antibodies. the sections were then washed and incubated for 2 h at room temperature with donkey anti - mouse texas red (1:50) and goat anti - rabbit fitc (1:50) secondary antibodies. slices were washed and mounted onto slides and coverslipped (fluromount, sigma, oakville, on, canada). immunofluorescent images were captured using an fv10i olympus confocal laser scanning microscope with a 60 phase - contrast oil - immersion objective / na 1.35. all protocols were in accordance with the ethical guidelines established by the canadian council for animal care and were approved by the university of british columbia or university of calgary animal care committees. c57bl/6 j mice were obtained from the university of british columbia breeding facility or jackson laboratories (sacramento, ca, usa). btbr mice were obtained from the jackson laboratory and bred in the ubc facility. both strains were fed chow ad libitum (teklad 2918). all electrophysiological recordings were performed in male mice ranging from p19 to p30 as per. slices in the recording chamber were superfused with bicarbonate - buffered solution (artificial cerebrospinal fluid) saturated with 95% o2/5% co2 and containing (in mm) 126 nacl, 1.6 kcl, 1.1 nah2po4, 1.4 mgcl2, 2.4 cacl2, 26 nahco3 and 11 glucose as well as picrotoxin (100 m) (at 3234 c). electrodes (34.5 m) contained (in mm) 117 cesium methansulfonate, 20 hepes, 0.4 egta, 2.8 nacl, 5 tea - cl, 2.5 mgatp and 0.25 nagtp (ph 7.27.3, 270285 mosm). series resistance (1025 m) and input resistance were monitored online with a 10 mv depolarizing step (400 ms) given before every afferent stimulus. dopamine neurons were identified by the presence of a hyperpolarizing cation current (ih), which is a good predictor of tyrosine hydroxylase (th) neurons in mice. a bipolar stimulating electrode was placed 100300 m rostral to the recording electrode and was used to stimulate excitatory afferents at 0.1 hz. coronal brain sections (30 m) were fixed in 4% paraformaldehyde overnight, blocked with 5% goat serum/0.3% triton x-100/0.2% bovine serum albumin in phosphate - buffered saline (ph 7.4) for 2 h at room temperature and then incubated for 48 h at 4 c with mouse th (1:1000) and rabbit insulin receptor (1:100) monoclonal antibodies. the sections were then washed and incubated for 2 h at room temperature with donkey anti - mouse texas red (1:50) and goat anti - rabbit fitc (1:50) secondary antibodies. slices were washed and mounted onto slides and coverslipped (fluromount, sigma, oakville, on, canada). immunofluorescent images were captured using an fv10i olympus confocal laser scanning microscope with a 60 phase - contrast oil - immersion objective / na 1.35. consistent with previous reports, fasted plasma insulin concentrations (measured as in were significantly higher in btbr mice than in c57bl/6j mice (btbr : 2.90.6 ng ml, n=8 ; c57bl/6 j : 1.30.3 ng ml, n=7 ; p0.05, t - test, t=0.42, d.f.=38 ; figure 1e). to determine whether insulin - induced ltd in btbr mice was deficient because of the inability to induce a synaptic depression in vta dopamine neurons, we tested whether synapses could undergo ltd induced by low - frequency stimulation (40 mv, 6 min, 1 hz stimulation). low - frequency stimulation induced a significant ltd at excitatory synapses of vta dopamine neurons in btbr mice (665% of baseline at 30 min, figures 2a and b, p0.05, t - test, t=0.40, d.f.=7). insulin - induced ltd required synthesis of endocannabinoids that act retrogradely at cannabinoid-1 receptors to depress glutamate release onto vta dopamine neurons. to determine whether the inability of insulin to induce ltd in the vta of hyperinsulinemic mice was due to insufficient cb1r - dependent synaptic depression, we bath applied the cb1r agonist win55232 - 2 (1 m) for 5 min to vta slices. win55232 - 2 significantly decreased ampar epscs (554% figures 2c and d ; n=5, p0.05, t - test, t=0., these data suggest that, whereas regulation of synaptic efficacy at vta synapses of hyperinsulinemic btbr mice is normal, insulin - induced ltd is deficient. several possible explanations exist for the inability of insulin to induce an ltd at excitatory synapses of dopamine neurons. in physiological situations, insulin gains access to the brain by active transport across the blood brain barrier and can mediate its effects by signaling through insulin receptors expressed throughout the brain. here, we observed a similar expression pattern of insulin receptors in th neurons in btbr mice as in c57bl/6j mice, suggesting that disrupted insulin - induced ltd in btbr mice is not due to reduced insulin receptor expression. btbr mice have higher fat mass than c57bl/6 j mice likely resulting in higher circulating leptin. as leptin decreases glutamatergic release onto dopamine neurons, it is possible that insulin - induced endocannabinoids acting presynaptically at cb1 receptors to inhibit glutamate release have a blunted effect because of high leptin levels lowering the release probability. however, this is unlikely as a cb1 receptor agonist induced a similar ltd in btbr or c57bl/6 j mice. alternatively, disrupted insulin - induced ltd may be due to insulin resistance because of impaired signaling at the level of the insulin receptor or its downstream effectors, due to genetic differences in insulin receptors, cognate signaling pathways or hyperinsulinemia. indeed, others have demonstrated that diet - induced hyperinsulinemia can induce impaired insulin signaling in hippocampal neurons. for example, ltd was not present and ltp was reduced along with a reduction in spine density in hippocampal neurons from high - calorie - diet - fed mice compared with controls. in contrast, others have observed that hyperinsulinemia caused no change in plasticity at the hippocampal synapses but a reduction in the ability of insulin to induce ltd in ca1 neurons. consistent with this, we found that hyperinsulinemia did not alter the plasticity of excitatory synapses onto vta neurons but reduced insulin - induced ltd. insulin action in the vta decreases opioid - stimulated food intake, food anticipatory behavior, preference for food and palatable food intake when sated. enhanced synaptic transmission in the vta has been associated with learning about cues that predict food delivery. therefore, one may speculate that a suppression of excitatory synaptic transmission in the vta by post - ingestive insulin release makes food - predicting cues less salient. an inability of insulin to dampen salience to food - predicting cues may lead to increased caloric intake, even when sated. in summary, our results suggest that, in hyperinsulinemic mice, insulin in the vta does not cause an ltd of excitatory inputs to dopamine neurons, and thus information relayed to the vta about cues predicting food is not suppressed after feeding when brain insulin levels are normally elevated.	obesity has drastically increased over the last few decades. obesity is associated with elevated insulin levels, which can gain access to the brain, including into dopamine neurons of the ventral tegmental area (vta), a brain region critical for mediating reward - seeking behavior. synaptic plasticity of vta dopamine neurons is associated with altered motivation to obtain reinforcing substances such as food and drugs of abuse. under physiological circumstances, insulin in the vta can suppress excitatory synaptic transmission onto vta dopamine neurons and reduce aspects of palatable feeding behavior. however, it is unknown how insulin modulates excitatory synaptic transmission in pathological circumstances such as hyperinsulinemia. using patch - clamp electrophysiology, we demonstrate that, in a hyperinsulinemic mouse model, insulin has reduced capacity to cause a synaptic depression of vta dopamine neurons, although both low - frequency stimulation - induced long - term depression and cannabinoid - induced depression were normal. these results suggest that insulin action in the vta during pathological hyperinsulinemia is disrupted and may lead to increased feeding behavior.
vascular malformations (vms) of the hand are rare and can have a variable clinical progression. such lesions may expand with the growth of the patient or progressive ectasia, but they do not proliferate. similarly, they do not undergo spontaneous resolution. they can be classified as simple (capillary, lymphatic, venous, or arteriovenous), combined (defined as two or more vms found in one lesion), or in association with other anomalies. arteriovenous malformations (avms), including arteriovenous fistulas (avfs), can present as congenital vascular lesions arising from errors in embryonic vasculogenesis or acquired in life secondary to trauma. avms are characterized by high - velocity turbulent blood flow from an artery to a vein, bypassing a normal capillary bed and can be clinically staged according to shobinger. high - flow avms of the upper limb can be classified as type a : anomalies with either single or multiple avfs, aneurysms, or ectasias of the arterial side of the circulation ; type b : anomalies with micro- or macro - fistulas that were localized to a single axial artery of the limb, hand, or digit ; type c : diffuse arteriovenous anomalies with macro- and micro - fistulous connections involving all tissues of the limb. angiography is the current gold standard imaging modality and in majority of avms displays a central core, or nidus, of abnormal connections from enlarged feeding arteries to draining veins. surgical or endovascular treatment can be complex with risk of uncontrollable bleeding, incomplete resection, and recurrence including rapid collateralization of adjacent arteries. this may be complicated by skin necrosis leading to amputation and associated loss of function. thermal imaging is a non - invasive, non - contact technology that does not require intravenous contrast agents. it has been used experimentally in various areas of plastic surgery including free - flap monitoring, perforator and perforasome mapping, and burn depth assessment. the infrared radiation that is emitted is a surrogate measure of blood flow and can be objectively quantified. the majority of thermal imaging cameras measure infrared radiation in the 115 m range, with most clinical applications focusing on the long - wave infrared from 7 to 14 m. recent smartphone - based systems have brought low - cost thermal imaging to the clinic as high - resolution industrial systems may be cost - prohibitive. we present the case of a patient with an avm affecting the hand in which thermal imaging has been used as an adjunct to capture baseline images to allow monitoring of progression. a 39-year - old female was referred to our regional peripheral vascular anomalies clinic with a 24-year history of a lesion affecting the left nondominant hand which started as a pea size mass, progressing to involve the palm, digits, and forearm. she underwent a surgical debulking procedure at the age of 17, but the lesion continued to expand. schooling and work were unaffected till the age of 21 ; however, due to pain, weakness, and coordination difficulties, she was not able to return to employment. on examination, an extensive vascular anomaly was noted to involve the palm, index and middle fingers, and distal forearm and wrist [figure 1a ], with bruit and a palpable thrill. an ultrasound doppler scan and magnetic resonance angiography confirmed the diagnosis of a high - flow avm, and digital subtraction angiography was used to define the anatomy of the malformation and shows features of a type - a avm [figure 1b ]. nerve conduction studies showed no evidence of compression neuropathy but some axonal loss, presumably due to mild ischemic changes secondary to the avm. 39-year - old female with a 24-year history of a lesion affecting the left nondominant hand diagnosed as a congenital arteriovenous malformation of the left hand and wrist. (a) photograph of the hand shows the arteriovenous malformation visibly involves the radial aspect of the palm and index finger and (b) digital subtraction angiography confirms a type - a arteriovenous malformation of the radial artery, palmar arch, and digital arteries of the index and middle fingers. there is minimal contrast agent seen in the thumb, ring or little finger digital arteries (black arrow). steal affecting the thumb, ring and little fingers (white arrows), and (d) thermal image of the unaffected right hand for comparison. to monitor the steal phenomenon affecting the thumb and ulnar - sided digits, a thermal image was recorded in the clinic using an flir sc660 thermal imaging camera (flir systems inc., images were recorded at 22c and 50% relative humidity at a distance of 70 cm after exposure of the hand for 3 min to allow surface cooling of the skin. baseline images of the affected [figure 1c ] and unaffected hand [figure 1d ] were recorded for future monitoring of the avm with regards to progression of the lesion or of the steal phenomenon. to date, the patient has declined any radiological or surgical intervention to date and is managed with a compression garment and simple analgesia for pain relief. avms of the hand are more likely to cause functional impairment than those at other sites, and surgical and/or embolization may have catastrophic consequences to the digits. a comprehensive review by upton., noted that only 12% of the upper limb vms were avms (4 type a, 15 type b, and 14 type c). although avms are rare, they exhibit more aggressive and unpredictable clinical behavior than other forms of vms. the fast turbulent flow created between the directly connected arterial and venous systems is responsible for a destructive hemodynamic effect that determines clinical symptoms and potentially high recurrence rates. complete surgical excision in conjunction with embolization sometimes may be impossible without threatening the viability of the digits, and limited debulking may lead to recurrence. avms can be located in various anatomic areas and sometimes occur simultaneously in several regions. in a study by vainyte., extracranial avms were analyzed, of which 80.4% were located in the extremities and the gluteal region, allowing easy access for thermal imaging. at present, progression of avms there is no non - invasive objective test that can determine the evolution or expansion of the lesion, or associated distal vascular steal that may occur in the extremities. although upton., included distal vascular steal in the type - c avm classification, we have shown that this may still occur in type a lesions and could lead to symptoms requiring surgical intervention. although no skin or trophic changes have been noted in the reported case, a combination of angiography and thermal imaging has indicated the presence of markedly reduced distal perfusion, which may be subclinical at present. the natural history of avms of the hand can be variable, and thermal imaging provides an objective measure of progression that can determine the optimum time for surgery and/or embolization, balancing the risk of iatrogenic digital ischemia versus inherent vascular steal. we suggest that thermal imaging is an adjunct that can be used in addition to clinical examination and/or angiography for the diagnosis and follow - up of arteriovenous malformations, to monitor progression or vascular steal, and also for recording recurrence after surgical excision. with the advantage of being a non - invasive imaging modality that does not require intravenous contrast, or ionizing radiation exposure	vascular malformations of the hand are rare. angiography is the current gold standard imaging modality. thermal imaging is an emerging noninvasive, noncontact technology that does not require intravenous contrast agents. we present the case of a patient with an arteriovenous malformation affecting the hand in which thermal imaging has been used as an adjunct to capture baseline images to allow monitoring of progression. we suggest that thermal imaging provides an adjunct that can be used in addition to clinical examination and/or angiography for the diagnosis and routine follow - up of conservatively managed arteriovenous malformations, to monitor progression or vascular steal, and also for recording recurrence after surgical excision for which there is known to be a significant incidence. with the benefit of being a noninvasive imaging modality that does not require intravenous contrast, or ionizing radiation exposure, office - based thermal imaging may become commonplace.
bacterial strains and plasmids plpq2 (12) is a psc101-derived plasmid in which expression of the e. coli phop - phoq operon is driven by the lacuv5 promoter. pnl3 (12) is a pbr322-derived reporter plasmid for assaying phop - mediated transcriptional activation that contains the phon promoter fused to lacz. paed4q (12) is a puc19-derived plasmid in which the phoq sensor domain (residues 43190) is expressed from the phage t7 10 promoter (13). e. coli strain csh26q has an internal deletion in the chromosomal phoq gene, which results in a null phenotype (12). e. coli strains bl21(de3) and b834(de3) were used to express phoq for purification. -galactosidase assays-galactosidase assays were performed as described (14) in n - media with additions of mgcl2 as indicated using strain csh26q / f lacikan / pnl3 containing derivatives of plpq2 with wild - type phoq or mutant variants. variants of phoq in which single amino acids at residues 50, 54, or 179 were individually substituted were constructed by site - directed pcr mutagenesis. primers were synthesized in which the appropriate codons were substituted to produce mutant phoq variants as indicated, and the resulting amplified phoq fragments were then cloned into a plasmid vector using nearby restriction enzyme sites. / f lacikan / pnl3 strains containing derivatives of plpq2 with wild - type phoq or mutant variants were grown to mid - log phase in n - media with appropriate antibiotics as described previously (14) and then induced for 2 h at 37 c with the addition of 1 mm isopropyl--d - thiogalactopyranoside. cells were harvested by centrifugation, resuspended in resuspension buffer (20 mm tris - hcl, ph 8.0, 100 mm nacl), and lysed by sonication on ice. cell membranes were recovered from the supernatant by centrifugation at 100,000 g for 15 min and were washed once with resuspension buffer. proteins from equal amounts of membrane were separated by sds - page and were subsequently transferred to immobilon - p transfer membrane (millipore). western analysis was performed using an antibody with specificity for the e. coli phoq cytoplasmic domain as described previously (14). the e. coli phoq wild - type sensor domain construct (residues 43190), and the acid mutant construct (residues 43190) in which residues 148154 were changed from eddddae to qnnnnaq were expressed and purified from e. coli strain x90(de3) as previously described (12). selenomethionyl (semet)5 phoq 43190 (wild type) was expressed from cells grown in selenomethionine minimal media, and native phoq 43190 (acid mutant) was expressed from cells grown in luria - bertani (lb) media. crystals of semet phoq 43190 (wild - type) protein were grown by the hanging drop, vapor diffusion method in a buffer containing polyethylene glycol 3400, tris (ph 8.5), sodium acetate, and 5 mm nicl2. crystals of the native phoq 43190 (acid mutant) protein were grown by the same method in a buffer containing 1.8 m ammonium sulfate, polyethylene glycol 4000, and sodium acetate. data collection and structure determination data from a four - wavelength multiwavelength anomalous dispersion experiment at the selenium k - edge were collected from a single frozen semet wild - type phoq 43190 crystal at beamline x4a of the national synchrotron light source at brookhaven national laboratory. diffraction to bragg spacings of 2.5 (280-mm detector distance) was collected using 1.5 rotations. data from a single frozen crystal of native acid mutant phoq 43190 were collected at a home cuk radiation source with a raxis - iv detector. the native crystals diffracted to bragg spacings of 2.0 (140-mm detector distance), and data were collected using 1 rotations. denzo and scalepack of the hkl program suite (15) were used to index and merge all data sets. selenium sites were found from the multiwavelength anomalous dispersion data after inspection of harker sections of bijvoet- and dispersive - difference patterson maps generated using ccp4 (16), and they were refined in sharp (17) in space group p21. a partial model of semet phoq 43190 (wild type) was generated by automatic model building using arp / warp (18), and this model was used in a molecular replacement search to determine phases of the native phoq 43190 (acid mutant) in space group p41. the rest of the native phoq 43190 (acid mutant) model was built manually in o (19), and the completed model was refined to a resolution of 2.0 using cns (20) for iterative cycles of simulated annealing, conjugate gradient energy minimization, temperature - factor refinement, and manual rebuilding. an additional density feature was found to be best fit with a sulfate ion, and this was included in the model and refined as well. the complete native phoq 43190 (acid mutant) model aided the manual rebuilding of the semet phoq 43190 (wild type) model, which was then refined by the same methods as before to a resolution of 2.5 against the low energy remote dataset. three locations of additional electron density could be best fit with one acetate and two nickel ions, and these were added to the model and included in the refinement. data collection and refinement statistics for both structures are listed in tables 1 and 2. the atomic coordinates and structure factors for both wild - type and acid mutant e. coli phoq sensor domains have been deposited with the protein data bank as accession codes 3bq8 and 3bqa, respectively. 2.5 0.9879 (low remote) 10480 3.8 18.6 95.2 (93.2) 6.1 (26.5) sephoq wt 2 2.5 0.9793 (edge) 10401 3.7 19.4 94.6 (92.6) 6.2 (25.4) sephoq wt 3 2.5 0.9790 (peak) 10460 3.7 18.6 95.0 (93.3) 7.0 (27.9) sephoq wt 4 2.5 0.9641 (high remote) 10539 3.7 19.7 95.4 (94.3) 6.5 (23.4) phoq mutant 2.0 1.5418 19615 3.3 22.4 99.0 (99.3) 3.5 (8.9) avalues in outermost shell are given in parenthesesbrmerge = (\|ii ii\|)/\|ii\|, where ii is the integrated intensity of a given reflection phoq 43190 diffraction data values in outermost shell are given in parentheses rmerge = (\|ii ii\|)/\|ii\|, where ii is the integrated intensity of a given reflection table 2phoq 43190 refinement statisticsparameterwild - type phoqacidic mutant phoq bragg spacings () 20 - 2.5 20 - 2.0 space group p21 p41 cell parameters 33.59, 113.96, 45.53 44.42, 44.42, 151.86 a, b, c ()/,, () 90, 109.46, 90 90, 90, 90 r 20.3% 16.8% rfree 30.1% 23.0% number of reflections 10,255 19,573 number of total atoms (non - hydrogen) 2,421 2,829 number of protein atoms 2,233 2,488 number of ligand atoms 6 (1 acetate molecule, 2 nickel ions) 5 (1 sulfate ion) number of waters 184 336 average b factor 44.8 27.3 r.m.s. bonds () angles () 1.4 1.3 ar = (\|\|fo\| \|fc\|\|)/\|fo\|, where fo and fc denote observed and calculated structure factors, respectivelybrfree was calculated using 5% of data excluded from refinement phoq 43190 refinement statistics r = (\|\|fo\| \|fc\|\|)/\|fo\|, where fo and fc denote observed and calculated structure factors, respectively rfree was calculated using 5% of data excluded from refinement 1) was determined by multiwavelength anomalous dispersion from a single semet crystal at the selenium k - edge. two molecules were found forming an apparent dimer in the asymmetric unit in space group p21. clear electron density allowed residues to be traced in for residues 45134 and 137188 of molecule a and for residues 4575, 83135, and 138186 of molecule b. missing residues correspond to n- and c - terminal segments and to loops. a total of 275 ordered residues, 184 water molecules, 2 nickel ions, and 1 acetate ion was refined against the low remote data to a resolution of 2.5 with r and rfree values of 20.3% and 30.1%, respectively (tables 1 and 2). a, the overall structure in complex with nickel is shown as a ribbon diagram. the arg-50 asp-179 salt bridge is shown with residue side chains depicted in a stick representation (carbon colored yellow, nitrogen colored blue, and oxygen colored red). a line of black dots marks interacting atoms of the salt bridge. colored dots are drawn to represent a hypothetical path of the protein backbone through disordered regions corresponding to residues 135 and 136 of molecule a (light purple), and residues 136 and 137 of molecule b (light red). b, the structure of one subunit (molecule a) of the wild - type phoq 43190 dimer is shown from a 90 rotation about the vertical axis of the dimer as shown in a. secondary structure elements are labeled in black. a, the overall structure in complex with nickel is shown as a ribbon diagram. the arg-50 asp-179 salt bridge is shown with residue side chains depicted in a stick representation (carbon colored yellow, nitrogen colored blue, and oxygen colored red). colored dots are drawn to represent a hypothetical path of the protein backbone through disordered regions corresponding to residues 135 and 136 of molecule a (light purple), and residues 136 and 137 of molecule b (light red). b, the structure of one subunit (molecule a) of the wild - type phoq 43190 dimer is shown from a 90 rotation about the vertical axis of the dimer as shown in a. secondary structure elements are labeled in black. 2) was determined by molecular replacement starting from a partial model of wild - type phoq 43190. two molecules were found in the asymmetric unit in space group p41. as for the wild - type structure, these molecules are related by a quasi - diad axis of symmetry, but the arrangement is very different in this case (see below). residues at positions 43188 and 45190 of molecule a and b, respectively, were found to be ordered. a total of 292 residues, 336 water molecules, and 1 sulfate ion was refined to r and rfree values of 16.8% and 23.0%, respectively, at a resolution of 2.0 against data collected from a single native crystal (tables 1 and 2). both the wild - type and acidic cluster mutant phoq 43190 structures adopt a mixed /-fold containing a central five - stranded anti - parallel -sheet flanked by a long n - terminal -helix and additional loops and helices on each side. this type of fold has been reported previously in the structures of other histidine kinase sensor domains such as dcus (21) and cita (22) in addition to the s. typhimurium phoq sensor domain (9). pairwise superimposition of all molecules using lsqman (23), based on a structurally invariant core of 47 c positions determined at the 5 level of escet (24, 25) comparison, yields r.m.s.d. values ranging from 0.25 to 0.42. small differences in the rest of the structure may be attributed to differences in crystal packing and a certain amount of intrinsic structural lability that may be related to protein function. the e. coli and s. typhimurium sensor domains are very similar in sequence (81.3% identity), and their structures are also similar. pairwise superimposition of the wild - type e. coli molecules with those of the s. typhimurium phoq yield r.m.s.d. values ranging from 0.89 to 0.97 over an average of 129 c positions, and matches for acidic cluster mutant phoq likewise yield r.m.s.d. because the acidic cluster mutant phoq 43190 was refined to a higher resolution and contains a greater number of ordered residues, we define the secondary structure elements based on dssp (26) assignments of molecule a of this structure. a, the overall structure of the acid mutant phoq 43190 (molecule a) is shown as a ribbon diagram with secondary structure elements labeled in black. b, a stereo plot of the c trace of the same acid mutant phoq 43190 molecule. a, the overall structure of the acid mutant phoq 43190 (molecule a) is shown as a ribbon diagram with secondary structure elements labeled in black. b, a stereo plot of the c trace of the same acid mutant phoq 43190 molecule. 2, taking the acidic cluster mutant phoq 43190 structure for reference, and secondary structure assignments are specified in fig. 3. the phoq sensor domain begins with n - terminal helix h1 (residues 4662), located on the backside of the central -sheet. helix h1 connects up to a pair of short antiparallel -strands s (residues 6466) and s (residues 6971) that loop above the sheet. the peptide backbone then runs back down through helix h2 (residues 7679) and connects into the first strand s1 (residues 8589) of the sheet. edge strand s2 (residues 9598) follows s1 and connects into helices h3a (residues 103108) and h3b (residues 111113), which cross over the front of the central sheet to join the other edge strand, s3 (residues 118125). strand s3 runs down the far side of the sheet and leads to helices h4 (residues 126133) and h5 (residues 137148), which are angled below the sheet. strand s4 (residues 154164), the longest strand in the structure, follows h5, and runs up the length of the central sheet. the peptide backbone then loops back down into the middle strand s5 (residues 173178) of the sheet, and the structure ends with helix h6 (residues 183185), which follows s5 and continues to the c - terminal end. electron - density features corresponding to non - protein moieties were found in both the wild - type and acidic cluster mutant phoq 43190 structures. in the wild - type structure, one nickel ion is coordinated by carboxylate oxygen atoms of asp-151 and asp-152 from the acidic cluster of subunit a and by asp-128 from symmetry mate b, and the other nickel ion is coordinated by asp-151 and asp-152 of b and asp-128 of a. asp-128 is located on h4, and asp-151 and asp-152 are located in the eddddae acidic cluster in the loop between h5 and s4, which had previously been implicated in divalent cation binding (12). the presence of nickel at the two sites was confirmed in a bijvoet difference fourier synthesis generated from a dataset collected above the nickel k - edge but below the selenium k - edge. an additional feature of nonprotein density was found in a pocket formed between the two molecules of the asymmetric unit ; this was modeled as an acetate ion coordinated by side chains of residues gln-81 of molecule a, and arg-53 and asn-117 of molecule b. arg-53, gln-81, and asn-117 are found in h1, in the loop between h2 and s1, and in the loop between h3b and s3, respectively. nickel was a necessary component in the crystallization buffer to obtain crystals of wild - type phoq 43190, and the addition of acetate was found to improve crystal size and appearance. in the acidic cluster mutant structure, a single region of unexpected electron density was modeled as a sulfate ion, which is complexed with water molecules coordinated by guanidinium groups of arg-50 and arg-53 from h1 of molecule a, and arg-169 from the loop between s4 to s5 of molecule b. ammonium sulfate was the main precipitant in the crystal buffer used to grow the acidic cluster mutant phoq 43190 crystals. secondary structure elements of e. coli phoq are also shown and labeled above the alignments, with helices in red and strands in blue.inthe top set, the pdc sensor domains phoq and cita are aligned with the pyp pas domain. regions in cita and pyp having c positions structurally aligned with those in e. coli phoq are shaded in light gray.inthe bottom set, the pas domains clk-6, fixl, and ncoa-1 are aligned with pyp. regions in clk-6, fixl, and ncoa-1 having c positions structurally aligned with those of pyp are shaded in light gray. organism names are abbreviated in italics (ec for e. coli, st for s. typhimurium, kp for klebsiella pneumoniae, eh for ectothiorhodospira halophila, dm for drosophila melanogaster, bj for bradyrhizobium japonicum, and mm for mus musculus). secondary structure elements of e. coli phoq are also shown and labeled above the alignments, with helices in red and strands in blue.inthe top set, the pdc sensor domains phoq and cita are aligned with the pyp pas domain. regions in cita and pyp having c positions structurally aligned with those in e. coli phoq are shaded in light gray.inthe bottom set, the pas domains clk-6, fixl, and ncoa-1 are aligned with pyp. regions in clk-6, fixl, and ncoa-1 having c positions structurally aligned with those of pyp are shaded in light gray. organism names are abbreviated in italics (ec for e. coli, st for s. typhimurium, kp for klebsiella pneumoniae, eh for ectothiorhodospira halophila, dm for drosophila melanogaster, bj for bradyrhizobium japonicum, and mm for mus musculus). comparison between pdc sensor domains and pas domains a dali (27) search comparing phoq 43190 to proteins in the pdb structural data base reveals great similarity to other histidine kinase sensor domains such as s. typhimurium phoq (9), luxq (28, 29), and dcus (21) (z scores = 22.3, 6.3, and 6.2, respectively ; r.m.s.d. these periplasmic sensor domains have been described by others as belonging to the pas (per - arnt - sim) domain superfamily (30), and dcus and cita have been directly compared with pyp (31). pas domains are found in a wide range of organisms and include a diversity of intracellular signaling domains, including fixl, nifl, ntry, yntc, ntrb, and nifu (32). dali (27) also finds similarities between e. coli phoq 43190 and pas domains such as the transcriptional coactivator ncoa-1 (33) and the drosophila clock protein clk-6 (34), although these matches score lower than those with other sensor domains (z scores = 5.2 and 2.5, respectively ; r.m.s.d. values = 2.6 and 4.4, respectively), and pyp is not listed. a structure - based alignment of the phoq and cita sensor domains with the pyp, clk-6, fixl, and ncoa-1 pas domains illustrates the similarities and several important differences between these classes of proteins (fig. we have defined the criteria for the alignments such that corresponding structurally aligned segments have at least three contiguous c positions lying within 2.5 of each other. pairwise sequence identities between phoq and these pas domains range from 6% to 8%, and structural alignments are very restricted (3039 c positions, table 3). in all cases, more residues can be superimposed in alignments between different sensor domains or between different pas domains than in alignments between sensor domains and pas domains. only the -sheets are in common between pas domains and the / sensor domain ; differences primarily lie in residues between the second and third -strands and in the n - terminal helix, present only in sensor domains. these structural differences distinguish these sensor domains from pas domains and have led us to term this the pdc (phoq - dcus - cita) domain. thus, pdc proteins are distinguishable both from pas domains and from sensor domains that form four helical bundles such as tar (35) and narx.6 phoq is distinguished from dcus and cita in having the h4-h5 helix pair and acid cluster as an insertion. values of pdc and pas domain superimpositionspypphoq cita 1.46 over 49 c pyp 1.02 over 39 c clk-6 1.34 over 65 c 1.21 over 37 c fixl 1.07 over 66 c 1.01 over 36 c ncoa1 1.22 over 41 c 1.28 over 30 c r.m.s.d. values of pdc and pas domain superimpositions dimerization of phoq 43190the wild - type phoq 43190 dimer (rotation = 158.6, screw translation t = -3.11) (36) buries 1528 of total accessible surface area between the two subunits. the dimer is quasi - symmetric about an interface formed primarily by interactions between the h1 helices of each protomer that include hydrophobic contacts, hydrogen bonding, and electrostatic interactions. there are also additional interactions between residues on helix h1 and residues in the loops between s4 and s5, h2 and s1, h3b and s3, and h2 and s1. the structure reveals a salt bridge formed between asp-179 of molecule a and arg-50 of molecule b in which the corresponding c positions are separated by 11.3. a symmetrically related salt bridge between arg-50 of molecule a and asp-179 of molecule b is not formed due to the asymmetric nature of the dimer, which places those c positions 16.9 apart. the orientation of the wild - type phoq monomers is such that both the n - terminal h1 and c - terminal h6 helices are aligned in the same direction in a manner where the ends would be poised to enter into the transmembrane segments tm1 and tm2 of a full - length molecule. the roughly parallel arrangement of the n- and c - terminal helices in the wild - type phoq 43190 dimer is similar to that observed in the dimeric structure of the aspartate receptor tar (35). two acid mutant molecules interact in their crystal environment through similar surfaces as those in the wild - type dimer, and they bury a substantial interface (2350), but orientations are radically different. this seems to be a non - physiological arrangement, because n- and c - terminal helices point in opposite directions and the arg-50 asp-179 salt bridge, seen below to be functionally important, does not form. measurements of activity in vivo to examine the importance of the dimer interface, and in particular the arg-50 asp-179 salt bridge, to the function of phoq, r50d and d179r single mutants, which would disrupt the salt bridge, and an r50d / d179r double mutant, which could recreate the salt bridge in the opposite direction, were constructed. an additional g54d mutant was constructed in which we reasoned the insertion of a charged residue in the hydrophobic interface would be disruptive. the abilities of such mutants to activate phop - mediated transcription of a phon - lacz reporter gene in vivo were compared with the wild - type protein. as shown in fig. 4a, phon - lacz expression is induced to roughly 3400 units by mgcl2 limitation when wild - type phoq protein is present, but it is only induced to only 900 and 245 units, respectively, when the phoq - r50d and phoq - d179r mutants are present. the effect of the double mutant is indeed compensatory rather than additive ; reporter expression is activated to 2225 units for phoq - r50d / d179r, > 50% of the wild - type level. the effect of the dimer - disrupting phoq - g54d mutant results in reporter gene induction to roughly 142 units in the magnesium limiting condition, an effect that most closely matches that of the phoq - d179r mutant. these results indicate that the salt bridge observed in the crystal structure is physiologically important in the function of the protein and suggest that the integrity of the interface is crucial for the formation of the active state. a, e. coli strain csh26q / flacikan, containing the pnl3 reporter plasmid and plpq2 for expressing the wild - type and mutant phoq proteins, were assayed for -galactosidase activity following growth in n - media (0 m mgcl2) or as supplemented with 40 m or 10 mm mgcl2. standard deviations are < 10% of the mean, calculated from at least three individual experiments. b, the relative expression levels of the wild - type and mutant phoq proteins are shown by western blotting, probed using an antibody specific for the cytoplasmic domain of phoq. plpq2 vector lacking the phop - phoq operon. in vivo activity and relative expression levels of phoq. a, e. coli strain csh26q / flacikan, containing the pnl3 reporter plasmid and plpq2 for expressing the wild - type and mutant phoq proteins, were assayed for -galactosidase activity following growth in n - media (0 m mgcl2) or as supplemented with 40 m or 10 mm mgcl2. standard deviations are < 10% of the mean, calculated from at least three individual experiments. b, the relative expression levels of the wild - type and mutant phoq proteins are shown by western blotting, probed using an antibody specific for the cytoplasmic domain of phoq. the sensor domain of phoq is clearly similar in structure to dcus (21) and cita (22) sensor domains despite being very dissimilar in sequence (7.4% and 4.5% identity, respectively). this phoq - dcus - cita (pdc) fold represents a different structural class (/) of sensor domains from the all - helical sensor domain classes such as tar (35) and narx. the pdc domain fold characteristically begins with a long n - terminal -helix that leads into a central -sheet core flanked by short helices on both sides. the central five - stranded antiparallel -sheet has the same topology as that of pas domains, and this feature dominates in structure similarity searches. this, in turn, has led others to identify pdc sensor domains as pas domains ; however, there are no actual similarities outside the -sheet, whereas pdc and pas domains each have their own distinguishing characteristics. the functionally important n - terminal helix is unique to pdc domains, and the segment connecting the two -strands at the edges of the sheet is distinctive for pdc and pas domains. in pdc domains, the residues between the second and third strands transverse across the front of the sheet in a single span, often helical. phoq residues in this region align with those in cita and in other x - ray structures of pdc sensor domains, such as dcus, lisk, and dctb. in pas domains, the segment between the second and third strands takes a longer and more tortuous path than in pdc domains, comprising multiple helices and typically ending in a helix running antiparallel to the third strand. such differences between pdc domains (dcus and cita) and pas domains were also noticed previously (37). certain sequence similarities have been established among pas domain family members (30), but pas - conserved sequences are not found in the phoq sensor domain. it appears that, despite sharing topologically identical five - stranded -sheets, pdc and pas domains do nevertheless belong to separate protein superfamilies. in the wild - type structure, the n - terminal helices that form the dimer interface lie in a parallel orientation, and we believe that the dimer interface observed in the wild - type phoq structure is biologically relevant. the orientation of the phoq protomers allows for the formation of the arg-50 asp-179 salt bridge, and the physiological importance of the salt bridge to function was confirmed by our mutagenesis studies (see above). other studies characterizing phoq mutants (d179a or d179l) are also consistent with importance of the salt bridge to phoq function (38) ; however, the difference in phenotypes obtained between the two sets of mutational studies on the same location may be a result of differences in charge and size of the mutant residues. such results suggest that normal phoq function is dependent and sensitive to the energetics of the dimer interface. it is believed that histidine kinase transmembrane regions form four - helical bundles as observed in the structure of the natronobacterium pharaonis phototaxis srii - htrii complex (39). the structures of the narx sensor domain and the aspartate receptor tar (35) have parallel n- and c - terminal helical regions that would be compatible with connection to such membrane - spanning regions in an intact molecule. the arrangement of the wild - type phoq protomers orients the n- and c - terminal helices in a parallel manner also suitable for connection with a four - helical bundle membrane - spanning region, and a similar orientation of terminal helices has also been observed in a dcus dimer. it must be noted that, in contrast to the wild - type e. coli phoq sensor domain, the s. typhimurium phoq sensor (9) shows a different putative dimeric arrangement. distances between the two n- and c - terminal residues within the s. typhimurium phoq dimer are reported to be 27 and 55, respectively (9), whereas the distances between n- and c - terminal residues within the wild - type e. coli phoq are each 18 apart, as measured between c positions corresponding to residues 45 and 186 in each protomer. we believe that the dimeric arrangement of protomers in the e. coli phoq structure places the n- and c - terminal ends in a position more suitable for connection into a putative four - helical bundle arrangement of transmembrane helices. the physiologically relevant arg-50 asp-179 salt bridge observed in the wild - type phoq dimer is not present in the s. typhimurium phoq dimer, which places the c positions of residues arg-50 and asp-179, and arg-50 and asp-179, 28.1 and 27.5 apart, respectively. in order for the salt bridge to form, a large rearrangement would be required at the dimer interface of the s. typhimurium phoq structure. asp-179 in the structure of the s. typhimurium phoq (9) sensor domain participates in hydrogen bonding with the side chains of thr-48 and lys-186 (9), and mutations of asp-179 and lys-186 result in impaired phoq function (9). equivalent residues in the e. coli phoq sensor domain are within weak van der vaals contact distance but are not hydrogen - bonded. it is possible that these differing arrangements of residues along the interface between the c - terminal helix and the central sheet in the two phoq structures may reflect different conformational signaling states. such residues may be involved in mediating conformational changes between regions of the protein involved with ligand binding to the membrane - spanning helices. additional experiments will be required to address such possibilities. in an attempt to learn more about the role of the acidic cluster in phoq function, we solved the phoq 43190 structure containing the eddddae to qnnnnaq mutations (amino acid residues 148154), which render the protein defective in divalent cation binding and activation. these crystals were obtained in a buffer that lacked divalent cations, and nothing is bound to the replacement sequence, although it adopts essentially the same conformation as for the wild - type structure. the acidic cluster mutant also forms a quasi - diad dimer in the crystal ; however, unlike in the wild - type dimer, here the n - terminal helices lie in an antiparallel orientation at the dimer interface and the arg-50 asp-179 salt bridge does not form. we believe this arrangement to be non - physiological, because the n- and c - terminal pairs are too far apart to connect to the putative membrane - spanning four - helix bundle. self - association propensities of membrane proteins are diminished by orders of magnitude when freed from membrane tethering (40). thus, even though sensor domains are expected to be functionally dimerized in situ on the membrane, they may exhibit quite low dimer affinity when isolated in solution. the e. coli phoq sensor domain has been shown to be monomeric in solution by analytical ultracentrifugation with a lower limit of 600 m measured for the kd of dimerization (12), and likewise both the cita and dcus sensor domains have also been reported to be mostly monomeric in solution (21, 41). it follows that a certain lability may exist in the manner by which sensor domains self - associate, and therefore dimeric interactions within in a crystal - lattice environment may not necessarily be biologically relevant. such lability has been observed in studies involving the cita sensor domain (22) and in our unpublished studies of the dctb and lisk sensor domains. in such cases, non - physiological interactions between sensor domain protomers can be found in the crystal lattice, sometimes mediated by required ions in crystallization buffers. we suggest that the dimers formed in the e. coli acid mutant phoq and s. typhimurium phoq structures may simply be the result of crystal packing interactions that have overridden the relatively weak affinity for the formation of a physiologically relevant dimer. does suggest that alteration of the acidic cluster by mutagenesis, and perhaps by divalent cation binding as well, might also affect the energetics of the dimer interface. if such effects are applicable to the intact membrane - bound molecule, then signal transduction mediated by cation binding at the acidic cluster would seem to employ a mechanism that involves a dynamic dimer interface and may not require quaternary interactions with additional proteins or with the membrane. mg and ca are thought to act in part as physiologically relevant signaling ligands by directly binding the phoq sensor domain and producing a conformational change that influences the enzymatic activities of the cytoplasmic domain (68). although we were unable to obtain suitable crystals in the presence of either mg or ca ions, the crystallization buffer contained 5 mm nicl2, which was required for the formation of crystals. the structure of wild - type phoq 43190 contains two ni ions, each associated with one of the two molecules in the asymmetric unit, forming lattice contacts between the side chains of asp-151 and asp-152, and asp-128 of a symmetry mate. asp-151 and asp-152 are part of the acidic cluster (eddddae) that had previously been implicated in divalent cation binding (12). divalent cations (ca) are also associated with acid clusters of two of four salmonella phoq sensor domains (9), but that site is displaced from our ni site and coordinated peripherally by asp-149. there do not seem to be ion - associated conformational distinctions in these two structures. divalent cations thermodynamically stabilize the sensor domain fragment to denaturation in a fashion expected for a direct binding model, and this mode of binding requires the presence of the acidic cluster. in addition, substitution of the cluster with uncharged isosteres (qnnnnaq) results in a protein that is defective for in vivo activation when extracellular mg concentrations are low ; however, substitution of the cluster with alanine residues (aaaaaaa) results in a protein that functions normally in response to extracellular mg.7 one possible explanation for these paradoxical results is that the sensor domain possesses one or more additional ligand binding sites, and neutralization of the acidic cluster site by conservative substitution with non - charged isosteres mimics divalent cation binding at this site to favor formation of the repressed state. when the acidic cluster site is substituted with alanines, the side chains necessary to form the repressed state are absent, and the protein can respond normally to binding at the other site(s). interaction between a ni ion and asp-128 in the crystal structure suggests that this residue might be part of such an additional physiologically relevant divalent - cation binding site. but the d128a mutation, both in the context of the wild - type acidic cluster and the alanine - substituted cluster, results in a protein that still responds normally to extracellular mg and ca (data not shown). perhaps asp-128 is not absolutely necessary for binding at the second site, asp-128 either may not interact with mg or ca, or such interactions do not affect protein function. close inspection of the structure does not reveal any other potential divalent cation binding sites. the acidic cluster resides in the lobe of phoq formed by the connection from h5 to s4. alignment of phoq from various organisms indicates that the cluster and lobe are present in the enteric versions of phoq but not in non - enteric versions (42), suggesting that the acidic cluster may play a specialized role in enteric bacteria. the position of the lobe with respect to the dimer interface and the rest of the structure suggests that it might lie very close to the membrane in the intact molecule. it has been suggested that from the s. typhimurium phoq structure that acidic regions may interact with the membrane phospholipids in a manner to allow chelation of divalent ions such as ca (9) ; however, such a mechanism has not been established in vivo. further research may be required to elucidate the specific role of the acidic cluster in its relation to divalent binding and signaling, and any involvement of membrane phospholipids in biological function.	the phop - phoq two - component system is a well studied bacterial signaling system that regulates virulence and stress response. catalytic activity of the histidine kinase sensor protein phoq is activated by low extracellular concentrations of divalent cations such as mg2 +, and subsequently the response regulator phop is activated in turn through a classic phosphotransfer pathway that is typical in such systems. the phoq sensor domains of enteric bacteria contain an acidic cluster of residues (eddddae) that has been implicated in direct binding to divalent cations. we have determined crystal structures of the wild - type escherichia coli phoq periplasmic sensor domain and of a mutant variant in which the acidic cluster was neutralized to conservative uncharged residues (qnnnnaq). the phoq domain structure is similar to that of dcus and cita sensor domains, and this phoq - dcus - cita (pdc) sensor fold is seen to be distinct from the superficially similar pas domain fold. analysis of the wild - type structure reveals a dimer that allows for the formation of a salt bridge across the dimer interface between arg-50 and asp-179 and with nickel ions bound to aspartate residues in the acidic cluster. the physiological importance of the salt bridge to in vivo phoq function has been confirmed by mutagenesis. the mutant structure has an alternative, non - physiological dimeric association.
mesothelin (msln) is a 40-kda cell differentiation - associated glycoprotein appearing with carcinogenesis. msln was found as an antigen recognized by the monoclonal antibody (mab), k1, generated by immunization of mice with the human ovarian carcinoma cell line, ovcar-3. the protein has been named as msln because the expression of msln in normal tissue was limited to mesothelial cells lining the pleura, pericardium, and peritoneum. on the contrary, msln is widely expressed in human cancers, for example, the majority of ovarian cancers and pancreatic adenocarcinomas, and in 100% of epithelial mesotheliomas. recent studies showed that it is also found in lung adenocarcinomas, gastric cancers, triple - negative breast cancers, uterine serous carcinoma, acute myeloid leukemia, and cholangiocarcinoma [213 ]. because of its limited distribution in normal tissues and elevated expression in cancers, msln has the potential to become a suitable target for a wide range of cancer diagnosis and therapy by using its specific antibodies. a precursor of msln is encoded as a 622-amino acid glycoprotein and cleaved by furin into a membrane - attached 40-kda form (msln) and a 31-kda - shed protein, megakaryocyte potentiating factor (mpf). the physiological function of msln is not fully elucidated as msln - deficient mice are fertile and do not exhibit any apparent phenotype. however, recent studies indicate that msln may play an important role in cell adherence, cell survival / proliferation, tumor progression, and chemoresistance. msln may aid in the peritoneal implantation and metastasis of tumors through its interaction with ca125 (also known as muc16), an ovarian cancer antigen [1618 ]. msln overexpression promotes cancer cell invasion by inducing matrix metalloproteases 7 and 9 [19, 20 ]. overexpression of msln constitutively activates nf-b and it leads to higher interleukin-6 production and induces tumorigenesis. further, the msln expression promotes resistance to certain chemotherapy drugs, such as tnf-, paclitaxel, and a combination of platinum and cyclophosphamide [23, 24 ]. since msln is overexpressed in a variety of malignancies, it is a good target for anti - msln antibody - based diagnosis and therapy. a number of anti - msln mabs have been developed, and morab-009 (amatuximab) is a chimeric (mouse / human) antibody. hn1 was isolated from a human scfv phage display library and converted into a fully intact human igg. as trace amount of msln can be detected in the blood of some patients with msln - positive cancers, in vitro diagnostic tests have been developed not only for diagnosis but also for following the course of some of these patients. a murine mab against msln, clone 11 - 25, was established by immunizing mice with recombinant human msln. the 11 - 25 mab was utilized in a sandwich elisa for detecting soluble form of msln in sera of patients with mesothelioma. the 11 - 25 mab binds to msln in soluble form(s) and to a membrane - attached form. because the soluble form(s) of msln is present in very small amount (1.43.8 nmol / l), it should not interfere with antibody - based therapies that target the msln antigen on cancer cells. positron emission tomography (pet) is a noninvasive, highly sensitive, and a quantitative tomographic imaging modality. it is clinically important as an imaging tool in cancer diagnosis and staging for a number of malignancies. the antibody - based pet technology is an attractive method for noninvasive tumor detection since this strategy combines the high sensitivity of pet with the high antigen specificity of mabs. cu (t1/2 = 12.7 h) is the widely used isotope for antibody - based pet, partly due to its wide availability, low cost, and versatile chemistry. as msln is overexpressed in a wide range of cancers, anti - msln 11 - 25 mab has the potential to become a pet imaging agent for detecting various kinds of msln - expressing cancers. in this study, we performed in vitro and in vivo investigations of anti - msln (11 - 25) mab to evaluate its utility as an imaging probe for detecting msln - expressing tumors. to apply to pet imaging, we labeled dota - conjugated 11 - 25 mab with positron - emitting cu and monitored in vivo distribution through pet imaging of human pancreatic cancer xenografts in nude mice. mono - n - hydroxysuccinimide ester 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (dota - mono - nhs ester) was purchased from the macrocyclics (dallas, tx). amicon ultra 0.5 centrifugal filter units were purchased from merck millipore (billerica, ma). anti - msln mab 11 - 25 (igg2b,) was prepared from the culture media of hybridoma clone 11 - 25, which was previously generated by immunizing mice with a recombinant human msln protein, as described. briefly, balb / c mice were immunized with recombinant msln (10571908 bp region of nm_005823 : secretory extracellular domain), mixed with freund 's complete adjuvant for first immunization, and boosted 4 times with the same antigen emulsified with the incomplete adjuvant. the msln - specific antibodies were purified on a protein a column from the culture fluid of hybridoma cells. among the purified mabs, 11 - 25 mab showed the highest reactivity in immunohistochemistry and flow cytometry. as igg2b isotype - matched control, mouse mab against keyhole limpet hemocyanin (klh) was purchased from r&d systems (minneapolis, mn). human pancreatic cancer cell lines, bxpc-3, cfpac-1, and panc-1, human lung cancer cell lines, nci - h226, msto-211h, and nci - h520, and a human epidermoid carcinoma, a-431, were purchased from american type culture collection (atcc, rockville, md) and were maintained in rpmi-1640 (for bxpc-3, h226, 211h, and h520), iscove 's modified dulbecco 's medium (imdm ; for cfpac-1), and dulbecco 's modified eagle medium (dmem ; for panc-1), containing 10% fetal bovine serum, 1% penicillin / streptomycin, in a humidified incubator maintained at 37c with 5% co2. the concentration of the soluble form of msln, in conditioned culture media of cancer cell lines, was determined by a sandwich elisa in a similar way, as previously reported. ninety - six - well microtiter plates (maxisorp, thermo fisher scientific inc., waltham, ma) were coated with anti - msln mab, 1430 (5 g / ml) at 4c overnight. each cell line was cultured for 5 days in their appropriate culture medium and the conditioned medium was collected by centrifugation. the conditioned medium was diluted five- and tenfold with pbs containing 1% bsa, 0.1% tween-20, and added to the wells. after washing with pbs containing 0.1% tween-20, the wells were incubated for 1 hour with biotinylated mab, 11 - 25, and subsequently reacted for 30 minutes with avidin - conjugated peroxidase (dako, glostrup, denmark) diluted to 1 : 20,000. followed by four washes with pbs, 100 l / well tetramethylbenzidine (tmb) (moss inc., soluble msln concentration was calculated by referring to a standard curve using serially diluted recombinant msln. western blot analysis was performed with cancer cells, that is, bxpc-3, cfpac-1, panc-1, and a-431. from each cancer cell line, total proteins were extracted with ripa buffer (25 mm tris - hcl ph 7.6, 1% np-40, 0.1% doc, 0.1% sds, 0.15 m nacl, 1 mm edta, 10 mg / l leupeptin, and 1 mm pmsf) and electrophoresed on 415% sds - polyacrylamide gradient gel (bio - rad). 5% skimmed milk in 20 mm tris - hcl ph 7.5, 500 mm nacl, 0.05% (v / v) tween-20 (tbs - t) for 1 hour, the blot membrane was incubated with 10 g / ml of 11 - 25 mab or mouse anti- action mab, ac-15 (sigma), at 4c for 18 hours. the membrane was then incubated with peroxidase - labeled anti - mouse igg f(ab)2 (rockland, gilbertsville, pa) for 12 hours at 4c. after washing with tbs - t buffer, the color was developed with dab. the area of each band was measured with the imagej software (national institutes of health, bethesda, md). semiquantitative reverse transcription pcr was performed to analyze the expression of msln in bxpc-3, cfpac-1, panc-1, and a-431 cells. total rna was extracted from cancer cell lines using trizol reagent (life technologies corporation, carlsbad, ca) after standard protocol. msln- and glyceraldehyde-3-phosphate dehydrogenase- (gapdh-) mrna were first reverse - transcribed to cdna. semiquantitative pcr was carried out using the lightcycler real - time detection system (roche diagnostics, mannheim, germany), according to the manufacturer 's instructions, for 50 cycles of 10 seconds (s) at 95c, 2 s at 68c, and 10 s at 72c. cultures of bxpc-3, cfpac-1, panc-1, and a-431 cells were harvested into single - cell suspension by treatment with pbs containing 0.1% collagenase type iv (invitrogen) and 1 mm edta. the 1 10 cells were washed once with cold pbs and incubated for 1 hour on ice in the presence of the 11 - 25 mab at 5 g / ml in pbs containing 2% fbs and 1 mm edta. as control, cells were treated with isotype control (igg2b) at 4 g / ml. then, the cells were washed twice and incubated with goat anti - mouse igg labeled with alexa fluor 488 for 1 hour on ice. the cells were washed twice and suspended in 0.5 ml pbs containing 2 g / ml propidium iodide and 1 mm edta before analysis with a facsaria flow cytometer (bd biosciences). bxpc-3, cfpac-1, and panc-1 cells were plated onto 8-well chamber slides at a concentration of 2 10 cells/0.2 ml / well and incubated for 3 days at 37c with 5% co2. the cells were washed three times with serum - free medium and fixed with 4% formaldehyde in pbs at room temperature (rt) for 10 minutes. the cells were washed with pbs containing 10 mm glycine and blocked with dako protein block for 10 minutes. then, the cells were treated with 5 g / ml anti - msln mab overnight at 4c. the cells were washed with pbs and incubated with fitc - labeled anti - mouse igg (life technologies), alexa fluor 594-labeled wheat germ agglutinin (wga) (life technologies), and 4, 6-diamidino-2-phenylindole dihydrochloride (dapi) (dojindo, kumamoto, japan) for 30 minutes. cells were imaged using a confocal laser scanning microscope, lsm510 (zeiss, oberkochen, germany). for staining of cancer xenografts, the cancer cells were inoculated to nude mice, as described in the animal model section, and the resulting tumors were taken from the mice, soaked in oct compound, and frozen. the frozen sections were prepared with cryostat and stained with alexa fluor 488-labeled 11 - 25 mab, alexa fluor 594-labeled wga, and dapi. all animal experiments were conducted in accordance with guidelines of okayama university and approved by university 's animal care and use committee (oku-2013098). five - week - old male balb / c nude mice were purchased from charles river (tokyo, japan) and were maintained under specific pathogen - free conditions at okayama university before use. for in vivo fluorescent imaging, male balb / c nu / nu mice (8 - 9 weeks old) were inoculated subcutaneously with panc-1 (1 10 cells) in the right thigh and bxpc-3 (5 10 cells) in the left thigh. for pet imaging, male balb / c nu / nu mice (9 - 10 weeks old) were inoculated with cfpac-1 (3 10 cells) in the right shoulder as well as panc-1 in the right thigh and bxpc-3 in the left thigh. the imaging experiments were performed when the tumors became about 8 mm in diameter. the 11 - 25 mab was labeled to alexa fluor 750 dye, according to the manufacturer 's protocols (molecular probes inc., briefly, the antibody (1 mg / ml) in 0.1 m carbonate buffer (ph 8.8) was reacted with alexa fluor 750 for 1 hour in the dark. unlabeled dye was removed by gel filtration on a sephadex g-25 (ge healthcare, uppsala, sweden) column. purified alexa fluor 750-labeled antibody was filtrated through a 0.2 m syringe filter and stored at 4c before use. the absorbance at 280 nm and 752 nm was measured and the degree of labeling (dol) was calculated after the manufacturer 's instructions. alexa fluor 750-labeled 11 - 25 mab (90 g / mouse) was administered via tail vein of mice bearing both bxpc-3 and panc-1 (n = 3). fluorescence from the labeled alexa fluor 750 was then monitored by ivis-200 imaging system (xenogen, alameda, ca) with emission at 680 nm and excitation at 780 nm 24, 48, and 72 hours after intravenous (i.v.) dota conjugation to mabs was carried out by incubation in pbs ph 7.0 for 3 hours at a ratio of dota - nhs : 11 - 25 mab, or control anti - klh mab (igg2b,), being 100 : 1. the number of conjugated dota molecules per molecule of igg was calculated with maldi - tof ms (4800 plus maldi tof / tof analyzer, ab sciex, framingham, ma) by comparison of average molecular mass of untreated and dota - conjugated antibodies. cu was produced by a cyclotron (hm-12 cyclotron, sumitomo heavy industries, ltd., tokyo, japan) and purified, according to the previously reported method. for radiolabeling, cucl2 (480550 mbq) was diluted with 0.1 m sodium acetate buffer (ph 6.5) and added to 0.51 mg of dota - conjugated 11 - 25 mab or 0.48 mg of dota - anti - klh mab, respectively. radiochemical purity was determined by a combination of thin - layer chromatography and autoradiography (tlc - arg) and hplc (lc-20, shimadzu co., kyoto, japan). for tlc - arg, samples were spotted on silica gel plates (silica gel, 60 rp-18 f254s, millipore) and developed using 50 mm edta (ph 8.0)/methanol = 1/2 as the mobile phase. radioactivity derived from antibody - bound cu stays at the origin and unbound cu goes to the top of the chromatogram. hplc was performed on tskgel super sw3000 column (4.6 mm 30 cm, tosoh corp., tokyo, japan) with 10 mm phosphate 300 mm nacl (ph 7.0) as a mobile phase at a flow rate of 0.35 ml / min. in vitro stability of the radiolabeled mabs after 24 and 48 hours of incubation in mouse plasma at 37c was also analyzed. cu - dota-11 - 25 mab or anti - klh mab (50 l) was added to 450 l of mouse plasma. after 24- and 48-hour incubation, aliquots of the incubated mixture of radiolabeled mab with the plasma were injected to hplc with the super sw3000 column, the eluate was fractionated, and their radioactivity was counted by a -counter (accuflex 7001, hitachi aloka medical). titers of 11 - 25 mab, dota - conjugated 11 - 25 mab, and cu - dota-11 - 25 mab were tested by elisa. the wells of a 96-well microtiter plate were coated with 100 l of the purified msln protein at 1 g / ml and incubated overnight at 4c. wells were blocked with pbs containing 1% bsa for 2 hours. then serially diluted antibody solution was added and incubated for 1 hour. after three washes with 250 l pbs containing 0.05% tween-20, the wells were treated with peroxidase - conjugated goat anti - mouse igg (life technologies) for 1 hour. after washes, 100 l of tmb - us (moss inc., pasadena, md) was added and the color was developed. then, the reaction was stopped by adding 100 l of 2 n h2so4. the absorbance at 450 nm was measured with sunrise - basic plate reader (tecan, grodig, austria). assay was performed with live cells using a modification of the method of li.. bxpc-3 cells were transferred to 24-well plates at 5 10 cells / well / ml and cultured for 4 days in a co2 incubator at 37c. five to eight hundred nm of cu - dota-11 - 25 mab was incubated with the cell monolayers in triplicate for 2 hours on ice in a complete growth medium (rpmi-1640 medium containing 10% fetal bovine serum). the cells were washed twice with 1 ml of ice - cold pbs and lysed by incubation at 37c in 0.3 ml of 0.2 m sodium hydroxide for 30 min. aliquots of each sample were analyzed for protein by a bicinchoninic acid assay (pierce). dissociation constant in nm was estimated by nonlinear fitting of the specific binding versus the concentration of cu - dota-11 - 25 mab using prism software (graphpad software, inc., la jolla, ca). each mouse bearing bxpc-3, cfpac-1, and panc tumor was anesthetized by inhalation of isoflurane and injected with approximately 11 mbq of cu - dota-11 - 25 mab (15 g mass) (n = 3) or cu - dota - anti - klh mab (11 mbq in 14 g protein) (n = 3) via the tail vein and 30-minute (at 0 and 24 hours) or 60-minute (at 48 hours) static pet scans were performed using a small animal pet scanner (clairvivo pet, shimadzu, kyoto, japan) and the images were reconstructed using the 3d - drama method. after the pet scans, ct data were acquired at 80 kv and 450 a with slice thickness of 90 m by using explore ct (ge healthcare japan, tokyo, japan). pet and ct images were converted into dicom format, fused, and analyzed using the pmod software version 3.3 (pmod technologies ltd., following the terminal pet scans at 48 hours after injection, all mice were euthanized for biodistribution studies. in addition, separate groups of mice were injected with 11 mbq of cu - dota-11 - 25 mab or with cu - dota - anti - klh mab and euthanized at 24 or 48 hours after injection for biodistribution studies. the data were expressed as percentage of injected dose per gram of tissues (% id / g). in vivo stability was measured by tlc. when the mice were sacrificed for biodistribution analysis 24 and 48 hours after injection, an aliquot of the blood was taken and centrifuged at 5,000 g for 10 min and 1 l of the plasma was spotted on the tlc. also an aliquot of liver and kidney was weighed and three volumes (v / w) of pbs were added and homogenized. the homogenate was centrifuged at 5,000 g for 10 min and 1 l of the supernatant was spotted to the tlc plate. statistical analysis was performed using a nonpaired student t - test for comparison of 2 groups. table 1 shows the result of soluble msln determination in the culture media of cancer cells, determined by a sandwich elisa. pancreatic adenocarcinoma cells, cfpac-1 and bxpc-3, and lung mesothelioma cells, msto-211h and nci - h226, were positive for soluble msln. on the other hand, pancreatic carcinoma cells, panc-1, and lung squamous carcinoma cells, nci - h520, were negative. to determine the expression of the msln protein on the pancreatic carcinoma cells, figure 1(a) shows that bxpc-3 and cfpac-1 cells expressed the msln protein but panc-1 and a-431 cells did not. a-431 is human epidermal carcinoma cell line and is known as msln - negative. figure 1(c) shows the results of flow cytometric analysis of the four cancer cells with anti - msln mab, 11 - 25. the antibody reacted with 50.9% of bxpc-3 cells, 31.4% of cfpac-1 cells, 0.6% of panc-1 cells, and 4.4% of a-431 cells. next, the expression of msln mrna on the four cell lines was investigated by semi - quantitative pcr (figures 1(d) and 1(e)). the binding of 11 - 25 mab to the fixed cultured pancreatic carcinoma cells and xenografts in mice was examined by fluorescent immunohistochemical staining. the antibody showed specific binding to both cultured cells and xenografts of cfpac-1 and bxpc-3 cells, but not to those of panc-1 cells (figures 2(a) and 2(b)). to analyze the in vivo distribution of 11 - 25 mab by fluorescence imaging, alexa fluor 750-labeled 11 - 25 mab was administered to mice bearing bxpc-3 and panc-1 tumors, and in vivo imaging was conducted with ivis 200. figure 3 shows the result of nirf imaging. at 24 hours after injection, fluorescence from alexa fluor 750-labeled 11 - 25 mab was detected throughout the body of the animal ; however, msln - positive bxpc-3 xenograft gave off brighter fluorescence than its neighborhood did. furthermore, time - dependent accumulation of fluorescence to bxpc-3 tumor xenografts was observed and fluorescence in other parts was cleared gradually. at 72 hours after injection, significant fluorescence strength was detected mainly in the bxpc-3 tumor xenograft. figure 3(b) shows the ex vivo image of the dissected tumor xenograft of another mouse treated in the same way at 24 hours after injection. dota - conjugated 11 - 25 mab or anti - klh mab was purified by column chromatography with pd-10. the average molecular weights of 11 - 25 mab and dota-11 - 25 mab determined by maldi - tof - ms were 151,599 da and 153,038 da, respectively. the average number of dota molecules conjugated per 11 - 25 mab molecule was 3.7, as determined by dividing the mass difference between dota-11 - 25 mab and 11 - 25 mab by the molecular weight of dota (386 da). in the same way, the average number of dota molecules per anti - klh mab was determined to be 4.8. binding of dota - conjugated 11 - 25 mab to the msln protein showed that 11 - 25 mab had a similar affinity to the msln after conjugation with dota (figure 4). dota - conjugated 11 - 25 mab and anti - klh mab were incubated with cu and purified as described in section 2. the radiochemical purity of resultant cu - dota - mabs determined by hplc was 97.9% for 11 - 25 mab and 100% for anti - klh mab. the specific activities of cu - dota-11 - 25 mab and cu - dota - aklh mab were 0.74 and 0.82 mbq/g, respectively. the immunoreactivity retention after radiolabeling of 11 - 25 mab against msln protein was measured by elisa (figure 4). at the concentration of 1000 ng / ml, the od value of cu - dota-11 - 25 mab was 66.0% compared to that of native 11 - 25 mab. in vitro stability in plasma after 24 and 48 hours of incubation analyzed by hplc was 78.0% and 76.9% for cu - dota-11 - 25 mab and 82.8% and 77.9% for cu - dota - anti - klh mab. the same preparations were also analyzed by tlc arg (figure 5(a)) and the results tended to be higher (94.3% and 92.6% for cu - dota-11 - 25 mab) than those analyzed by hplc. for in vitro experiment, cu - dota-11 - 25 mab was prepared and a binding study of cu - dota-11 - 25 mab with alive bxpc-3 cells was performed at 4c. bxpc-3 cells exhibited saturable cu - dota-11 - 25 mab binding (figure 6). the kd of cu - dota-11 - 25 mab binding for the msln on bxpc-3 cells was 353 63 nm. we performed pet imaging of cu - dota-11 - 25 mab in mice bearing bxpc-3, cfpac-1, and panc-1 tumors at 0, 24, and 48 hours after the intravenous injection. figures 5(b) and 5(c) show the in vivo stability of cu - dota-11 - 25 mab and cu - dota - anti - klh mab in the plasma, liver, and kidney at 24 hours and 48 hours after injection. both of cu - dota-11 - 25 mab and cu - dota - klh mab were stable in the blood until 48 hours after injection, and they were partly metabolized in liver and kidney extracts. figure 7(a) is the 3d volume rendering image of ct of the mouse and shows the position of xenografts and the position of the cross section. in the upper cross section images of figure 7(b), high accumulation of cu - dota-11 - 25 mab was observed in the cfpac-1 xenograft (red arrowhead) at 24 and 48 hours. in the lower cross section images, cu - dota-11 - 25 accumulation was observed in the bxpc-3 xenograft (yellow arrowhead) but not in the panc-1 xenograft (white arrowhead). on the other hand, the accumulation of cu - dota - anti - klh mab in cfpac-1 and bxpc-3 xenografts (figure 7(c)) was lower than those of cu - dota-11 - 25 mab. in the biodistribution study, relatively high accumulation of cu - dota-11 - 25 mab was observed in the blood, liver, and msln - positive tumors (figure 8). in cfpac-1 xenografts, the accumulation of cu - dota-11 - 25 mab was significantly higher than that of cu - dota - anti - klh mab at both time points. in bxpc-3 xenografts, the accumulation of cu - dota-11 - 25 mab was significantly higher than that of cu - dota - anti - klh mab at 48 hours after injection (figures 8(a) and 8(b)). at both 24 and 48 hours after injection, the tumor to blood ratio and tumor to muscle ratio of cu - dota-11 - 25 mab in bxpc-3 tumor and in cfpac-1 tumor were significantly higher than those in panc-1 tumor (p < 0.05 and p < 0.01, resp., figures 8(c)8(f)). the average weight of individual panc-1, bxpc-3, or cfpac-1 tumor was 77 49 mg, 50 38/mg, or 207 78 mg, respectively. in vivo stability of cu - dota-11 - 25 mab and cu - dota - aklh mab was measured by tlc. the membrane - bound form of msln is present on a wide range of cancer cells [213 ] and its expression in normal tissues is relatively limited in the mesothelial cells. soluble form of msln is found in the circulation in some cancer patients and monitoring the level of soluble msln is useful for diagnosis of malignant pleural mesothelioma [26, 32 ]. but the levels are too low (1.43.8 nmol / l) to affect the in vivo targeting by anti - msln antibodies. with these properties of msln expression, msln can be a promising targeting molecule for a diverse range of cancers. our aim is to develop a specific antibody - based pet probe that is available for detecting such a wide range of cancers. for that purpose, we selected 11 - 25 mab as a candidate and confirmed that 11 - 25 mab was available for detecting msln in western blotting, flow cytometry, immunohistochemistry, and in vivo nirf imaging. the high mortality rate of the pancreatic cancer is due to the high incidence of metastatic disease at initial diagnosis, the aggressive clinical course, and the lack of adequate systemic therapies. however, patients diagnosed at an early stage have the potential for therapeutic approach for prolonged survival. there is a need to develop a highly specific technique for early diagnosing. accumulating evidence has shown that msln is overexpressed in various cancers, including pancreatic adenocarcinoma, ovarian cancer, and mesothelioma. msln is an attractive candidate as a molecular target for pancreatic cancer marker - specific imaging or immunotherapy. so, we selected pancreatic adenocarcinoma cells, bxpc-3 and cfpac-1, as an example set of target cells. we first confirmed that 11 - 25 mab can detect the soluble form of msln in the conditioned medium of pancreatic adenocarcinoma and mesothelioma (table 1). pancreatic adenocarcinoma cells, cfpac-1 and bxpc-3, excreted soluble msln but epithelioid carcinoma, panc-1, did not. then we confirmed the expression of msln protein in these cells by western blot and flow cytometry using 11 - 25 mab and the expression of mrna by semiquantitative reverse transcription pcr (figure 1). the results were msln - positive for cfpac-1 and bxpc-3 and negative for panc-1, which were consistent with the soluble msln levels and previous reports [32, 33 ]. the immunohistochemistry results of these cell lines detected by 11 - 25 mab showed that msln localized on the membrane and in the cytoplasm (figure 2). on the culture slides or dishes, we observed that only a part of bxpc-3 cells, that is, the cells at the edge of cell clusters, was stained with 11 - 25 mab. at the edge of the cluster it was reported that msln plays a role in cell adhesion, migration, and proliferation [3436 ]. this tendency is similar to the previous immunohistochemistry reports that the expression of msln was strongly observed in the invasive component of pancreatic cancer, but not in the noninvasive component even within the same specimen [4, 37, 38 ]. on the contrary, the xenografts of bxpc-3 and cfpac-1 in mice were uniformly stained with 11 - 25 mab (figure 2(b)). part of the reason is supposed to be that the human carcinoma cells were invading in mouse tissue and overexpressed msln. as shown in figure 3, we confirmed the specific accumulation of nirf - conjugated 11 - 25 mab in msln - positive bxpc-3 xenograft by in vivo fluorescent imaging. as the blood clearance of alexa fluor 750-labeled 11 - 25 mab was slow, the contrast between msln - positive tumors and normal tissues was better at 48 hours after injection than at 24 hours after injection. nirf has excellent optical characteristics in terms of stability, high sensitivity, and low autofluorescence. although tissue penetration of nirf is limited (up to several centimeters in tissues), nirf - labeled antibody could be used for image - guided surgical resection of tumors in combination with preoperative pet imaging to detect the tumors. for the detection of cancer, high - sensitivity imaging in deep tissue pet imaging has the potential to meet the required sensitivity for tumors ; besides it is noninvasive and is directed to the whole body. the radioactive metals are widely used for labeling peptides and antibodies by conjugation of metal chelators. cu has a half - life of 12.7 hours and decays by 17.9% by + decay to ni, 39.0% by decay to zn, 43.1% by electron capture to ni, and 0.475% -radiation / internal conversion. these emissions are 0.579 and 0.653 mev for and positron, respectively, and 1.36 mev for. cu has been widely used for labeling antibodies for pet [4042 ] and several clinical studies revealed that cu - labeled igg can detect tumors within 48 hours after injection [43, 44 ]. we used mono - n - hydroxysuccinimide ester 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (dota - mono - nhs ester) as a chelating agent for cu. although dota does not form very stable complex with cu, several clinical studies revealed that cu - labeled dota - igg is adequately stable for pet imaging to detect lesions even around the liver. many structures, reported to form cu - complexes with improved stability, such as teta, te2a, and cb - te2a, require harsher reaction conditions to incorporate cu. in addition, although dota - conjugated therapeutic and diagnostic agents, such as y - dota - toc, are clinically approved, any other chelating agents which form cu - complex are not. the dota-11 - 25 mab had similar reactivity to antigen msln, as compared with native 11 - 25 mab. further, cu - dota-11 - 25 mab kept the antigen specificity and bound to bxpc-3 cells in vitro with dissociation constant (kd) at 353 63 nm (figure 5). cu - dota-11 - 25 mab enabled visualization of msln - positive tumors by pet imaging. high accumulation of cu - dota-11 - 25 mab in msln - positive tumors was observed compared to that in the msln - negative tumor. as the tumor size of cfpac-1 tumor was larger than the others, nonspecific accumulation of igg probably by enhanced permeability and retention effect in the cfpac-1 tumor was observed. the biodistribution studies showed that the accumulation of cu - dota-11 - 25 mab in bxpc-3 and cfpac-1 tumors was significantly higher than that in panc-1 cells (figure 8). however, the clearance of cu - labeled igg in the blood was very slow in general. reported the biodistribution of in (t1/2 = 2.8 day)-k1 antibody in athymic nude mice bearing a-431-k5 (a-431 cells transfected with mesothelin gene) xenografts. the peak uptake of in - k1 by a-431-k5 tumors was at 72 hours after injection. as the half - life of cu is shorter than that of in, we only investigated the biodistribution at 24 and 48 hours after injection. although the tumor to blood ratio was no more than 1, the ratio of cu - dota - mab in msln - positive tumors was significantly higher than that in msln - negative panc-1 tumor. the accumulation of cu - dota-11 - 25 mab in the liver was relatively high, as it was previously reported for cu - labeled igg. the presence of fc receptor might have led to the nonspecific binding of igg into the liver. transchelation of cu to superoxide dismutase and metallothionein could also be a part of the reason for the accumulation in the liver. as the distribution of cu - dota-11 - 25 mab in the pancreas or stomach was of very low level (figure 7), msln - positive pancreatic cancer or gastric cancer could be visualized in high contrast by in vivo pet imaging with cu - dota-11 - 25 mab. antibodies against human epidermal growth factor receptor 2 (her2) have been approved for the treatment of her2-positive breast cancer and they are used for companion diagnostic testing for targeted cancer therapies, as her2-positive breast cancer is about 30%. on the contrary, msln overexpression has been observed in a wide range of cancers ; diagnostics with pet probe made from anti - msln antibody has the potential to be applicable to a broad spectrum of cancers. development of fragmented antibody derivatives such as fab and single - chain fv should be useful to achieve faster blood clearance and better target - to - non - target contrast at an early time point after administration relative to that of igg 11 - 25 mab. in conclusion, 11 - 25 mab specifically detected msln both in soluble form by elisa and in cell - attached form by flow cytometry / immunohistochemistry and in vivo nirf imaging. further, we successfully labeled 11 - 25 mab with cu, with preservation of immunoreactivity. we evaluated the affinity of cu - dota-11 - 25 mab in vitro and used it as an in vivo pet imaging probe to detect msln - expressed pancreatic cancers. it was confirmed that cu - labeled 11 - 25 mab significantly accumulated in msln - expressing tumors compared to msln - negative tumor by pet imaging and biodistribution studies. as msln is highly expressed in various human tumors, our findings suggest that msln - specific imaging using cu - labeled 11 - 25 mab has the potential to be widely used in the diagnosis of patients with msln - positive cancers such as malignant mesotheliomas and ovarian cancers and pancreatic cancers.	mesothelin (msln) is a 40-kda cell differentiation - associated glycoprotein appearing with carcinogenesis and is highly expressed in many human cancers, including the majority of pancreatic adenocarcinomas, ovarian cancers, and mesotheliomas, while its expression in normal tissue is limited to mesothelial cells lining the pleura, pericardium, and peritoneum. clone 11 - 25 is a murine hybridoma secreting monoclonal antibody (mab) against human msln. in this study, we applied the 11 - 25 mab to in vivo imaging to detect msln - expressing tumors. in in vitro and ex vivo immunochemical studies, we demonstrated specificity of 11 - 25 mab to membranous msln expressed on several pancreatic cancer cells. we showed the accumulation of alexa fluor 750-labeled 11 - 25 mab in msln - expressing tumor xenografts in athymic nude mice. then, 11 - 25 mab was labeled with 64cu via a chelating agent dota and was used in both in vitro cell binding assay and in vivo positron emission tomography (pet) imaging in the tumor - bearing mice. we confirmed that 64cu - labeled 11 - 25 mab highly accumulated in msln - expressing tumors as compared to msln - negative ones. the 64cu - labeled 11 - 25 mab is potentially useful as a pet probe capable of being used for wide range of tumors, rather than 18f - fdg that occasionally provides nonspecific accumulation into the inflammatory lesions.
chronic diseases represent the leading cause of death and disability and a major challenge facing today 's healthcare system.1 approximately half of canadians and americans suffer from a chronic illness.1 2 comorbidity of chronic diseases is increasingly common among adults and is more prevalent with advancing age. for example, 13% of canadians aged 2039 years, 71% of those aged 6079 years, and 82% of those 80 years and older, report multiple health problems.3 individuals with comorbid mental and physical illness experience worse outcomes, inadequate care and increased mortality relative to those without mental illness.46 mental disorder is elevated in people with any chronic disease,79 specifically among those with diabetes,10 chronic obstructive pulmonary disorder (copd),11 and asthma.12 patten13 found that the annual cumulative incidence of depression was higher among individuals with any long - term medical condition relative to those without such problems. other research has found that patients with copd were significantly more likely to be diagnosed with depression than people with diabetes or controls.14 various pathways between chronic pain and depression have been postulated. people with multiple pains experience more depression than those with a single pain source.15 pain can affect sleep patterns and increase emotional distress. the experience of frequent pain might also be a signal for depression.16 disability from chronic illness can also be a risk factor for depression.17 other debilitative effects of some illnesses (eg, changes in physical appearance, decreased functioning) can heighten the risk of depression.7 18 these pathways may also differ depending on the type of physical illness experienced. a small number of epidemiological studies have investigated gender differences in onset of mental illness secondary to a chronic physical illness. one study showed higher prevalence of mental disorder / distress among women with chronic disease relative to men.7 schneider found an increased risk of depression among patients with copd. he also found that lifetime prevalence of depression was twofold among women compared to men. with copd duration moreover, his findings showed that more patients with depression and copd died within a year of the index date ; notably, the gender difference in mortality was greater among men.19 a national health study on asthma showed that gender differences in depression decrease with advancing age, but depression remains higher among those with asthma relative to those without the illness.8 patten found that hypertension was associated with an elevated risk of depression, but only among men.20 given the equivocal nature of the findings, current evidence does not provide a clear picture of the gendered nature of the relationship between mental and physical disorders. using a unique dataset comprised of medical and national survey data, the primary objective of this cohort study was to examine : (1) whether gender was an important risk factor when considering whether those with baseline physical illness (pi asthma, chronic obstructive pulmonary disease, type ii diabetes and hypertension) were more likely to use medical services for mental illness (mi) relative to a comparison group without baseline pi and (2) whether there are gender differences in onset of usage of medical services for mi. ethical approval was obtained from st michael 's hospital and sunnybrook health sciences centre research ethics review boards. all permanent residents of ontario are eligible for healthcare without deductibles or copayments.21 adults were eligible for inclusion in this study if they had a valid health card number at the index date of the study (20002001) and the date of death occurred after the study start date. the methodology adopted in this study is similarly described in a paper by the same research team in their investigation of the relationship between baseline mi and the onset of pi.22 the institute for clinical evaluative sciences (ices) provided the data for this study. physical and mental health data and other health indicators were obtained from the ontario linking file of the canadian community health survey (cchs) 1.1 (cycle 20002001).23 the cchs is a nationally representative and cross - sectional survey which collected data from non - institutionalised canadians over the age of 12 years. respondents in the linking file agreed to have their responses linked to medical data for research purposes. the response rate was 82.0% (39 278), and 90.8% agreed to linkage ; 92% of those who agreed were successfully linked providing a sample of 32 848. we excluded 5% of cases with missing values on variables other than income, and restricted the sample to those aged 1874 years. the final sample was 17 050 (6213 in the physical illness cohort and 10 837 in the control cohort). ontario health insurance plan (ohip) data contains physician visits and procedure claims which include service and diagnostic codes, fees for service provided, and date of service. the canadian institutes for health information (cihi) discharge abstract database (dad) contains up to 16 diagnoses and 12 procedures for acute care hospitalisations and day procedures across ontario. the ontario mental health reporting system (omhrs) provides data on patients in adult designated inpatient mental health beds, including beds in general, provincial psychiatric, and specialty psychiatric facilities. we determined eligibility for health services and death date from the registered person data base (rpdb) ; ontario 's healthcare registry that provides information on age, sex, postal code, eligibility and death data. the physical illness (pi) cohort was defined using the cchs and medical records, and included respondents in the linked cchs who self - reported at least one pi (asthma, copd, hypertension, or type ii diabetes). people not self - reporting pi, but who met the criteria for any of these conditions in the validated medical registries (eg, a respondent saw a physician for physical health reasons) were also included in the pi cohort (n=6213), and were subject to the same inclusion criteria as respondents in the cchs. similar to our previous study,22 we used four disease - specific ices - validated cohort databases to identify physical illness in the 10-year follow - up period. patient records for those with any of the four physical health diagnoses are retained in disease - specific registries at ices. people with asthma had one hospital admission or two ohip claims with asthma diagnosis within 2 years (sensitivity : 84% ; specificity : 76%).24 those with copd had one copd diagnosis in ohip or cihi - dad (sensitivity : 85% ; specificity : 78.4%).25 hypertensive patients had one hospital admission with a hypertension diagnosis, or an ohip claim with a hypertension diagnosis followed within 2 years by either an ohip claim or a hospital admission with a hypertension diagnosis (sensitivity : 72% ; specificity : 95%).26 people with type ii diabetes had at least one hospitalisation or at least two claims for physicians services within 2 years (sensitivity : 86% ; specificity : 97%).27 the administrative data was assessed for accuracy using abstracted medical records (primary care charts). in the case of hypertension, additional validation was conducted using self - reported survey data from a national census. to ensure that mi usage did not occur prior to or concurrently with pi, we excluded anyone with the following : (1) self - reported depression, (2) consulted a mental health professional, (3) felt they needed treatment for an emotional problem, but did not receive care, (4) who had physician or er visits or hospitalisations for mental health reasons during the two years prior to the cchs interview. in the cchs, self - reported depression was assessed using the composite international diagnostic interview short form for major depression (cidi - sfmd). the cidi - sf is a validated epidemiological tool to identify depression used in non - clinical settings. in this study, major depression was defined as those reporting four or more depressive symptoms ; meaning that 81.25% of cases meeting these criteria would have major depression according to the full cidi.28 the prevalence of pi conditions in the cchs was as follows : asthma (9.0%) ; copd (4.2%) ; type ii diabetes (5.5%) ; hypertension (16.6%). a control cohort (n=10 837) included respondents who did not meet the criteria for inclusion in the pi cohort, and those who did not have a mi based on self - report and medical data. onset of mi usage in the 10-year follow - period was defined using a combination of service and diagnostic codes used by ontario physicians. when mental health services are provided, physicians may assign either a mental health - specific service code (when a visit exceeds 20 min in length) or a general service code (for shorter visits), which is a 3-digit shortened version of the codes provided in the international classification of diseases and reflects the most responsible reason for any visit.29 30 diagnostic codes for mental health usage were derived from a validation paper.29 the list of conditions includes diagnostic codes for psychotic disorders, non - psychotic disorders (including anxiety and depression), substance abuse disorders and social problems. the study compared patient surveys and administrative claims, and established that claims data are reasonable to identify patients who had received mental healthcare (sensitivity : 80.7% ; specificity : 90.7%). mental illness usage was defined as at least one visit to a physician or specialist for mental health reasons. the event or first episode was defined as the first usage for any of the co - occurring health conditions in either omhrs, ohip or the dad. we controlled for several health indicators known to be associated with gender differences in health31 32 : socioeconomic and demographic characteristics (highest level of education attainment, annual household income, age, marital status, ethnoracial origin) ; and health behaviours (perceived stress and activity level, smoking and drinking status). leisure - time physical activity was calculated based on the frequency and duration of each activity within the 3 months prior to being surveyed (energy expenditure ee) and its value of metabolic energy cost. we used predefined categories from statistics canada for physically inactive, moderately active, and active,23 combining moderately active and active for this analysis. heavy drinking was defined as 14 plus weekly drinks for men and 10 plus weekly drinks for women. respondents were classified as having high and low stress based on their responses to the question : thinking about the amount of stress in your life, would you say that most days are (not at all stressful, not very stressful, a bit stressful, quite a bit stressful, or extremely stressful)? high stress reflected responses of quite a bit stressful or extremely stressful. the remaining responses were considered low stress. we adjusted for disease comorbidity using three measures : restriction of activities, resource utilization bands (rubs) and aggregated diagnosis groups (adgs). rubs and adgs were generated using the johns hopkins adjusted clinical groups (acg) system and represent morbidity burden of populations.33 rubs are constructed using age, sex and mixtures of diagnoses associated with expected intensity of resource use with values categorised as low (0, 1), intermediate (2 and 3), and high (4, 5) burden of illness on the healthcare system. adgs represent a collection of diagnostic codes similar in severity and expected persistence over time. adgs reflect comorbidity and additional health burden on top of that reflected by rubs and were categorised as low (01), moderate (25) and high (634). restriction of activities was self - reported : do you have any difficulty hearing, seeing, communicating, walking, climbing stairs, bending, learning or doing any similar activities? and coded sometimes, often and never. we provide unweighted sample numbers and weighted proportions generated using the survey weights provided by statistics canada.34 cox proportional hazard regression was used to estimate the effect of gender and pi status on mi while adjusting for the effects of other covariates. this models the length of time until the occurrence of an event onset of mi during the 10-year follow - up. survival curves for time to first occurrence of mi were obtained with the kaplan meier method. tests for proportionality and interactions with time were not significant (eg, plotted survival function vs log - log survival function ; tests for interactions of each variable with time). sas v.9.2 (sas institute, cary, north carolina, usa) was used for data manipulation and statistical analysis. the institute for clinical evaluative sciences (ices) provided the data for this study. physical and mental health data and other health indicators were obtained from the ontario linking file of the canadian community health survey (cchs) 1.1 (cycle 20002001).23 the cchs is a nationally representative and cross - sectional survey which collected data from non - institutionalised canadians over the age of 12 years. respondents in the linking file agreed to have their responses linked to medical data for research purposes. the response rate was 82.0% (39 278), and 90.8% agreed to linkage ; 92% of those who agreed were successfully linked providing a sample of 32 848. we excluded 5% of cases with missing values on variables other than income, and restricted the sample to those aged 1874 years. the final sample was 17 050 (6213 in the physical illness cohort and 10 837 in the control cohort). ontario health insurance plan (ohip) data contains physician visits and procedure claims which include service and diagnostic codes, fees for service provided, and date of service. the canadian institutes for health information (cihi) discharge abstract database (dad) contains up to 16 diagnoses and 12 procedures for acute care hospitalisations and day procedures across ontario. the ontario mental health reporting system (omhrs) provides data on patients in adult designated inpatient mental health beds, including beds in general, provincial psychiatric, and specialty psychiatric facilities. we determined eligibility for health services and death date from the registered person data base (rpdb) ; ontario 's healthcare registry that provides information on age, sex, postal code, eligibility and death data. the physical illness (pi) cohort was defined using the cchs and medical records, and included respondents in the linked cchs who self - reported at least one pi (asthma, copd, hypertension, or type ii diabetes). people not self - reporting pi, but who met the criteria for any of these conditions in the validated medical registries (eg, a respondent saw a physician for physical health reasons) were also included in the pi cohort (n=6213), and were subject to the same inclusion criteria as respondents in the cchs. similar to our previous study,22 we used four disease - specific ices - validated cohort databases to identify physical illness in the 10-year follow - up period. patient records for those with any of the four physical health diagnoses are retained in disease - specific registries at ices. people with asthma had one hospital admission or two ohip claims with asthma diagnosis within 2 years (sensitivity : 84% ; specificity : 76%).24 those with copd had one copd diagnosis in ohip or cihi - dad (sensitivity : 85% ; specificity : 78.4%).25 hypertensive patients had one hospital admission with a hypertension diagnosis, or an ohip claim with a hypertension diagnosis followed within 2 years by either an ohip claim or a hospital admission with a hypertension diagnosis (sensitivity : 72% ; specificity : 95%).26 people with type ii diabetes had at least one hospitalisation or at least two claims for physicians services within 2 years (sensitivity : 86% ; specificity : 97%).27 the administrative data was assessed for accuracy using abstracted medical records (primary care charts). in the case of hypertension, additional validation was conducted using self - reported survey data from a national census. to ensure that mi usage did not occur prior to or concurrently with pi, we excluded anyone with the following : (1) self - reported depression, (2) consulted a mental health professional, (3) felt they needed treatment for an emotional problem, but did not receive care, (4) who had physician or er visits or hospitalisations for mental health reasons during the two years prior to the cchs interview. in the cchs, self - reported depression was assessed using the composite international diagnostic interview short form for major depression (cidi - sfmd). the cidi - sf is a validated epidemiological tool to identify depression used in non - clinical settings. in this study, major depression was defined as those reporting four or more depressive symptoms ; meaning that 81.25% of cases meeting these criteria would have major depression according to the full cidi.28 the prevalence of pi conditions in the cchs was as follows : asthma (9.0%) ; copd (4.2%) ; type ii diabetes (5.5%) ; hypertension (16.6%). a control cohort (n=10 837) included respondents who did not meet the criteria for inclusion in the pi cohort, and those who did not have a mi based on self - report and medical data. onset of mi usage in the 10-year follow - period was defined using a combination of service and diagnostic codes used by ontario physicians. when mental health services are provided, physicians may assign either a mental health - specific service code (when a visit exceeds 20 min in length) or a general service code (for shorter visits), which is a 3-digit shortened version of the codes provided in the international classification of diseases and reflects the most responsible reason for any visit.29 30 diagnostic codes for mental health usage were derived from a validation paper.29 the list of conditions includes diagnostic codes for psychotic disorders, non - psychotic disorders (including anxiety and depression), substance abuse disorders and social problems. the study compared patient surveys and administrative claims, and established that claims data are reasonable to identify patients who had received mental healthcare (sensitivity : 80.7% ; specificity : 90.7%). mental illness usage was defined as at least one visit to a physician or specialist for mental health reasons. the event or first episode was defined as the first usage for any of the co - occurring health conditions in either omhrs, ohip or the dad. we controlled for several health indicators known to be associated with gender differences in health31 32 : socioeconomic and demographic characteristics (highest level of education attainment, annual household income, age, marital status, ethnoracial origin) ; and health behaviours (perceived stress and activity level, smoking and drinking status). leisure - time physical activity was calculated based on the frequency and duration of each activity within the 3 months prior to being surveyed (energy expenditure ee) and its value of metabolic energy cost. we used predefined categories from statistics canada for physically inactive, moderately active, and active,23 combining moderately active and active for this analysis. heavy drinking was defined as 14 plus weekly drinks for men and 10 plus weekly drinks for women. respondents were classified as having high and low stress based on their responses to the question : thinking about the amount of stress in your life, would you say that most days are (not at all stressful, not very stressful, a bit stressful, quite a bit stressful, or extremely stressful)? high stress reflected responses of quite a bit stressful or extremely stressful. the remaining responses were considered low stress. we adjusted for disease comorbidity using three measures : restriction of activities, resource utilization bands (rubs) and aggregated diagnosis groups (adgs). rubs and adgs were generated using the johns hopkins adjusted clinical groups (acg) system and represent morbidity burden of populations.33 rubs are constructed using age, sex and mixtures of diagnoses associated with expected intensity of resource use with values categorised as low (0, 1), intermediate (2 and 3), and high (4, 5) burden of illness on the healthcare system. adgs represent a collection of diagnostic codes similar in severity and expected persistence over time. adgs reflect comorbidity and additional health burden on top of that reflected by rubs and were categorised as low (01), moderate (25) and high (634). restriction of activities was self - reported : do you have any difficulty hearing, seeing, communicating, walking, climbing stairs, bending, learning or doing any similar activities? and coded sometimes, often and never. we provide unweighted sample numbers and weighted proportions generated using the survey weights provided by statistics canada.34 cox proportional hazard regression was used to estimate the effect of gender and pi status on mi while adjusting for the effects of other covariates. this models the length of time until the occurrence of an event onset of mi during the 10-year follow - up. survival curves for time to first occurrence of mi were obtained with the kaplan meier method. tests for proportionality and interactions with time were not significant (eg, plotted survival function vs log - log survival function ; tests for interactions of each variable with time). sas v.9.2 (sas institute, cary, north carolina, usa) was used for data manipulation and statistical analysis. the pi cohort had an equal gender distribution ; there were slightly more men than women in the control cohort (54.4%). more respondents in the pi cohort reported no high school diploma (25.4%) relative to those in the control group (12.7%). twice as many respondents in the pi cohort (8.7%) reported income under can$20 000 in comparison with controls (4.4%). more people in the pi cohort were aged 50 + years (54.2% vs 21.9%), and reported often or some activity restriction versus controls (33.7% and 16.3, respectively), and higher morbidities relative to controls (40.2% and 19.8% ; 79.0% and 49.6% ; respectively). descriptive statistics for the physical illness and control cohorts by sociodemographic characteristics and health indicators weighted percentages and unweighted ns are shown. overall, the prevalence of usage of health services for mi in the sample was 44.6% (41.9% in the controls and 50.2% in the pi cohort ; p=0.001). half (49.7%) the people in the control cohort reported having used medical services for a mh problem and pi at follow - up. table 2 shows the percentage / number of respondents in each cohort that subsequently used medical services for mi by the end of the 10-year follow - up period (columns 2/3). more men and women in the pi cohort used services for a subsequent mental illness than their counterpart controls (women : 55.6% vs 48.1% ; men : 44.7% vs 36.7%, respectively ; p=0.0001). the relative risk (rr) of usage among women in the pi group relative to women controls was 1.16. respondents in the pi cohort consistently reported higher usage for mi than controls across all risk factors with at least a 10% or greater difference for several variables, including age, education, income, stress, disability and rural location. adjusted hrs for onset of mental health utilisation for the physical illness and control cohorts adjusting for sociodemographic characteristics and health indicators (n=17 050) the median onset of mi usage for the entire sample was 39.3 months. there was a modest difference in median onset of usage of medical services for mi between the pi (36.7 months or 3.05 years) and control (40.9 months or 3.41 years ; p=0.0059) cohorts. for men with pi, the median time to onset of usage for mi was 40.3 versus 45.6 months (3.4 years and 3.8 years, respectively ; p=0.0059) among controls. for women in the pi cohort, the median time to event was 33.9 versus 37.5 months among controls (2.8 and 3.1 years, respectively ; p=0.0172). based on bivariate logistic regressions (not shown), women were 1.45 times more likely to use medical services for mi compared with men (hr=1.45, ci 1.35 to 1.55). respondents with a pre - existing pi were 1.32 times more likely to use medical services for mi (hr=1.32, ci 1.23 to 1.42). female (hr=1.28, ci 1.19 to 1.37) and pre - existing pi (hr=1.14, ci 1.05 to 1.23) remain statistically significant after adjusting for other risk factors. to further explore gender differences in risk of onset of usage of medical services for mi, we examined the adjusted probability of onset of the event by gender and cohort. as figure 1 shows, the probability of onset of usage of medical services for mi differed across the groups. while risk increased over time for all groups, women with pi were at greatest risk of onset of usage of medical services for mi, followed by women controls, men with pi and men controls. we also tested for an interaction between gender and pi status which was not significant (hr=0.99, ci 0.85 to 1.13). we conducted condition - specific cohort analyses to test if gender differences were masked by combining the physical illnesses. the gender by condition - specific cohort interactions were not significant (data not shown). the p values ranged from 0.31 for copd to 0.96 for diabetes. in table 3, we present the hrs for the adjusted main effects models showing the hrs and p values for female and cohort by each physical illness condition. the hrs for females were highly similar across the health conditions and to the combined hr shown in table 2 (hr=1.28). the condition - specific cohort hrs were also very similar to the combined hr (hr=1.14) in table 2 ; hypertension (hr=1.16, ci 1.04 to 1.29) ; copd (hr=1.19 ; ci 1.01 to 1.37) ; diabetes (hr=0.96, ci 0.81 to 1.12) ; asthma (hr=1.16, ci 1.04 to 1.28). adjusted hrs for onset of mental health utilisation for gender and cohort by physical illness conditions (n=17 050) we also conducted age - stratified analyses to determine if the gender effect was similar across age categories. we found that the gender differences were very similar across the younger age groups (1829, 3039, 449, 5059) with hrs ranging from 1.26 to 1.39. these are also similar to the hrs for females, shown in table 2 (hr=1.28). the gender difference was smaller and not statistically significant in the oldest age group (6075 years). the purpose of this study was two - fold : to examine whether people with chronic pi were more likely to begin using medical services for mi over a 10-year period ; and, to examine if men and women with / without chronic pi have similar onset of usage of medical services for mi. pre - existing, available, validated cohorts were used for all illnesses to ensure accuracy of diagnoses. at the end of the follow - up period, the overall prevalence of medical service usage for mi was 44.6% (controls : 41.9% ; pi cohort : 50.2%). the finding that pi was associated with greater risk of onset of usage of medical services for mi replicates earlier studies.35 36 in fact, we found that within the pi cohort, the onset of medical service usage for mi was 10% higher among women. women with pi also experienced slightly faster onset of medical service usage for mi than men with pi, about 6.4 months earlier. this suggests that within a 3-year window (sample median of 39.3 months), women use mental health services 6 months earlier than men. a recent study examined onset of pi among men and women with mi and found that women developed pi about one year earlier than their male counterparts.22 there are several potential implications of the gender difference in onset in medical service usage. if women begin to use services for mental illness 6 months earlier than men, this could be positive suggesting that women seek help earlier which could lead to better mental health outcomes ; and, it might be perceived as negative for men who defer care for a number of months. alternatively, it could mean that symptoms are worse among women, hence the reason they seek care earlier than men. with further respect to gender differences, women were at greater risk of earlier onset of medical service usage for mi than men after adjusting for covariates in the survival model. this gender difference is reflected in previous research.7 19 risk of onset of usage of health services for mi over the 10-year period differed by gender - cohort groups. women with pi were at greatest risk of usage of health services for mi, followed by women without pi. the next group most at risk was men with pi followed by men without pi. a previous study found that women with chronic somatic disease were at greater risk of developing a mental disorder than men.7 we also found that the gender difference in onset of mental health usage was similar for the younger age categories and was smaller and non - significant for older adults (6075 years). this difference in help - seeking behaviour between older and younger adults seems to have some support in the literature. for example, mackenzie examined attitudes in help - seeking and found that older adults had more positive attitudes towards help - seeking than young people. available research is not clear on the gendered nature of the relationship between pi and mi or on the nature of the onset of usage of medical services for mi. this study is unique, exploring onset of mental health service usage among those with and without physical illness who were also free of previous mental illness at the study start date. while one study found a higher risk of depression among men with hypertension,20 others have shown that women who live with pi are at greater risk of mi.8 19 part of the lack of clarity in the field is that studies use different definitions of pi and mi, and use different data sources (eg, survey vs administrative data ; self - report vs medical records). the high prevalence of medical service usage for mi in this sample may reflect our definition of usage, which is broader in scope than specific diagnoses like depression or anxiety disorder. our definition is based on a chart validation study of the specific medical data examined in this study.29 the nature of the data limited our ability to examine specific mental health disorders. for example, the ontario physician fee code structure allows flexibility with respect to what fee code a physician can use for a mental health visit. this can result in a single diagnoses (eg, depression) being assigned to different fee codes. research suggests that the true prevalence of pi may be underestimated in self - reported survey data,38 however, the cchs sample is representative of the canadian population. the study and its findings are restricted to respondents in ontario and, therefore, are only generalisable to the ontario population. we restricted our analyses to definitions of pi diagnoses and mi usage from validated registries. future research might consider other chronic conditions to understand gender differences in onset of usage of medical services for mi, with the latter clearly defined with respect to diagnoses. under - sampling of ethnic groups in the cchs limited our ability to explore mi by ethnic groupings, as we were only able to look at white versus others and 10-year - recent immigrants. this area of research would benefit from analyses that explore gender differences among those with and without pi by ethnicity. in the administrative data, the physician fee codes do not capture alternate payment plans. as a result, physicians in community health centres are absent from the data, but non - fee - for - service usage represents a small minority of primary care visits and very few specialists visits (less than 6%).39 it is possible that increased access to a physician to treat a chronic physical health condition (eg, who have regular contact with their family doctor) may afford a person increased opportunities to consult on comorbid health conditions like mi. if this were the case, then higher usage of medical services for mi might reflect different consulting patterns between the pi and control groups rather than the impact of pi itself. while this is possible, we know that the control group itself is not entirely free of chronic illness (even though we adjust for multiple health comorbidities using acgs / rubs and restriction of activities). additional research is necessary to untangle the relationship between pi and usage of services for mi. the high prevalence of medical service usage for mi in this sample may reflect our definition of usage, which is broader in scope than specific diagnoses like depression or anxiety disorder. our definition is based on a chart validation study of the specific medical data examined in this study.29 the nature of the data limited our ability to examine specific mental health disorders. for example, the ontario physician fee code structure allows flexibility with respect to what fee code a physician can use for a mental health visit. this can result in a single diagnoses (eg, depression) being assigned to different fee codes. research suggests that the true prevalence of pi may be underestimated in self - reported survey data,38 however, the cchs sample is representative of the canadian population. the study and its findings are restricted to respondents in ontario and, therefore, are only generalisable to the ontario population. we restricted our analyses to definitions of pi diagnoses and mi usage from validated registries. future research might consider other chronic conditions to understand gender differences in onset of usage of medical services for mi, with the latter clearly defined with respect to diagnoses. ethnicity is a complicating factor in this field of research. under - sampling of ethnic groups in the cchs limited our ability to explore mi by ethnic groupings, as we were only able to look at white versus others and 10-year - recent immigrants. this area of research would benefit from analyses that explore gender differences among those with and without pi by ethnicity. in the administrative data, the physician fee codes do not capture alternate payment plans. as a result, physicians in community health centres are absent from the data, but non - fee - for - service usage represents a small minority of primary care visits and very few specialists visits (less than 6%).39 it is possible that increased access to a physician to treat a chronic physical health condition (eg, who have regular contact with their family doctor) may afford a person increased opportunities to consult on comorbid health conditions like mi. if this were the case, then higher usage of medical services for mi might reflect different consulting patterns between the pi and control groups rather than the impact of pi itself. while this is possible, we know that the control group itself is not entirely free of chronic illness (even though we adjust for multiple health comorbidities using acgs / rubs and restriction of activities). additional research is necessary to untangle the relationship between pi and usage of services for mi. understanding pathways from pi to mi medical service usage is important for health interventions. for example, goldberg40 highlights three ways that chronic pi can lead to depression. first, people with multiple pains associated with pi are at higher risk for depression. for example, dworkin found that primary care patients with a single pain were not at increased risk, but those with three or more were five times more likely to experience depression. pain itself can cause emotional distress and poor sleep.16 moreover, the frequent report of pain might itself be a signal for depression. second, adults with pi may develop a disability which can bring on depression for those who were healthy before onset of the pi.17 third, physical changes associated with some diseases can increase the allostatic load on the patient and heighten risk of depression.18 in fact, verhaak suggest that the negative characteristics of the disease throughout its life course, whether progressive or episodic, and other stressful consequences, such as reduced physical abilities, pain, changes in physical appearance, can be strong contributors to developing mental illness. disability related to chronic illnesses like copd can include fatigue, shortness of breath, pain, and reduced ability to function normally, and can affect mental health. with this in mind, it is reasonable to assume that the mechanisms that explain the association between chronic disease and mental illness will differ by the type of illness ; an important consideration for clinicians helping their patients adapt to the changes brought on by chronic illness. while studies have found that prolonged episode and increased incidence heighten major depression in people who have medical conditions, we lack knowledge of these pathways in relation to gendered experiences of illness. further research with an eye to gender analysis and focus on onset of mental illness in cohorts with and without chronic health problems is necessary to understand the types of disparities and inequalities that individuals experience, and to guide health system changes surrounding healthcare quality, delivery and public health policy. women, by contrast with men, are more likely to seek medical services for mental illness. people with a pre - existing chronic physical illness are more likely to use medical services for mental illness. current evidence on gender differences in onset of usage of medical services for mental illness for those with and without physical illness is not definitive. findings from this study suggest that women with physical illness were at greatest risk of using medical services for mental illness followed by women without physical illness, men with physical illness and men without physical illness. within any 3-year window, women with physical illness used medical services for mental illness 6.4 months earlier than men.	backgroundpeople with comorbid mental and physical illness (pi) experience worse health, inadequate care and increased mortality relative to those without mental illness (mi). the role of gender in this relationship is not fully understood. this study examined gender differences in onset of mental health service usage among people with physical illness (copd, asthma, hypertension and type ii diabetes) compared with a control cohort.methodswe used a unique linked dataset consisting of the 20002001 canadian community health survey and medical records (n=17 050) to examine risk of onset of mi among those with and without pi among ontario residents (1874 years old) over a 10-year period (20022011). adjusted cox proportional survival analysis was conducted.resultsunadjusted use of mi medical services in the pi cohort was 55.6% among women and 44.7% (p=0.0001) among men ; among controls 48.1% of the women and 36.7% of the men used mi medical services (p=0.0001). the relative risk of usage among women in the pi group relative to controls was 1.16. among men, the relative risk was 1.22. women were 1.45 times more likely to use mi medical services relative to men (hr=1.45, ci 1.35 to 1.55). respondents in the pi cohort were 1.32 times more likely to use mi medical services (hr=1.32, ci 1.23 to 1.42) relative to controls. women in the pi cohort used mi medical services 6.4 months earlier than pi males (p=0.0059). in the adjusted model, women with pi were most likely to use mi medical services, followed by women controls, men with pi and men controls. there was no significant interaction between gender and pi cohort.conclusionsfurther, gender - based research focusing on onset of usage of mi services among those with and without chronic health problems will enable better understanding of gender - based health disparities to improve healthcare quality, delivery and public health policy.
malignant lymphoma is a heterogeneous group of diseases with a wide spectrum of histological subtypes. traditionally, malignant lymphomas have been divided into hodgkin lymphomas and non - hodgkin lymphomas. more than 40 subgroups are defined ; both indolent and aggressive variants exist, and a wide range of treatment is used. the danish national lymphoma registry (lyfo) was established in 1982, covering the western part of denmark (jutland and funen), including patients with non - hodgkin lymphoma. in 1998, the lyfo aims to register the clinical and paraclinical characteristics of patients diagnosed with malignant lymphoma in denmark. the lyfo aims to monitor the clinical course of the disease as well as to monitor and improve the quality of treatment nationwide by benchmarking departments and patient risk groups. the lyfo includes all patients diagnosed with malignant lymphoma in denmark, referred to one of the ten departments of hematology. patients with cutaneous lymphomas, some hiv+-associated lymphomas, and some lymphomas where treatment is futile are not referred to hematological departments and hence not registered in the lyfo. patients diagnosed and treated outside of denmark are not registered ; according to the database registration, it is two patients per year. patients who relapse where the initial lymphoma diagnosis was prior to start of the registry in that region are not registered. by the end of 2014, ~23,000 patients were registered since 1982. since it is a national quality database, all departments of hematology are obligated to register patients in the lyfo at the time of diagnosis, at the end of first - line treatment, at relapse, and at the end of follow - up or death. standardized forms are used to collect data on lymphoma patients (table 1). since 2005, the registration is submitted electronically through a secure internet - based database system, and all information is saved electronically. for each patient, four forms are available in a consecutive manner (figure 1). at the time of diagnosis the department responsible for the treatment initiation and evaluation has the obligation to enter the form. the diagnosis is coded according to the who classification of tumours of haematopoietic and lymphoid tissues.1 since a minor subset has a discordant diagnose, this is also registered. ann arbor stage, b - symptoms, largest tumor diameter, eastern cooperative oncology group performance status, concomitant neoplastic diseases, and treatment strategy are required in the form. in addition to a number of laboratory test results (table 1), information of each nodal and extranodal involvement sites is required. the treatment form consists of detailed information on the first - line treatment : chemotherapy regimens, treatment with monoclonal antibodies, radiotherapy, radioimmunotherapy, major surgery, and date and type of eventually autologous stem - cell therapy. a response assessment is required and has been adjusted to the different versions of the international response criteria for malignant lymphoma.2 furthermore, toxicity assessment is recorded for patients experiencing toxicity grades iii and iv.3 in the case of clinical or histologically confirmed relapse or refractory disease, defined as progression within 3 months after termination of first - line treatment, a relapse form is requested. this form is also completed for all patients who were initially observed without need of treatment. the relapse form requires date of relapse, lymphoma histology at relapse, treatment information of second - line treatment, and response assessment identical to the treatment form. a follow - up form is requested at death or termination of outpatient follow - up. this form includes information on vital status, date of follow - up or death, and disease remission status. the four schemes in the database are merged to an analyzable dataset, where new data fields are derived from the entered values in the schemes. this process utilizes calculation of a number of prognostic indexes such as the international prognostic index (ipi)4 and the follicular lymphoma international prognostic index5 and a number of nodal and extranodal sites, in addition to calculation of both result and process quality indicators. result quality indicators are mortality 30 and 180 days after diagnosis for patients receiving treatment, response to first - line treatment and kaplan - meier survival estimates 1, 3, and 5 years after the time of diagnosis. the process quality indicators include time from diagnosis to start of treatment, fulfillment of entered data used in the international prognostic index, and the inclusion rate in clinical trials. to ensure high registry completeness, the lyfo is cross - referenced with the danish national patient registry and the national pathology registry. since all patients with a hospital admission, both as inpatient and outpatient, are registered in the danish national patient registry with diagnosis and date of contact, all patients referred to a hematological department are identified by this cross - reference.6 departments can instantly retrieve lists of patients with missing registration in the database, both at time of diagnosis and time of an eventual relapse. all departments register in the lyfo and fulfill the demand of coverage of at least 90%. the department responsible for the treatment initiation and evaluation has the obligation to enter the form. the diagnosis is coded according to the who classification of tumours of haematopoietic and lymphoid tissues.1 since a minor subset has a discordant diagnose, this is also registered. ann arbor stage, b - symptoms, largest tumor diameter, eastern cooperative oncology group performance status, concomitant neoplastic diseases, and treatment strategy are required in the form. in addition to a number of laboratory test results (table 1), information of each nodal and extranodal involvement sites is required. the treatment form consists of detailed information on the first - line treatment : chemotherapy regimens, treatment with monoclonal antibodies, radiotherapy, radioimmunotherapy, major surgery, and date and type of eventually autologous stem - cell therapy. a response assessment is required and has been adjusted to the different versions of the international response criteria for malignant lymphoma.2 furthermore, toxicity assessment is recorded for patients experiencing toxicity grades iii and iv.3 in the case of clinical or histologically confirmed relapse or refractory disease, defined as progression within 3 months after termination of first - line treatment, a relapse form is requested. this form is also completed for all patients who were initially observed without need of treatment. the relapse form requires date of relapse, lymphoma histology at relapse, treatment information of second - line treatment, and response assessment identical to the treatment form. a follow - up form is requested at death or termination of outpatient follow - up. this form includes information on vital status, date of follow - up or death, and disease remission status. the four schemes in the database are merged to an analyzable dataset, where new data fields are derived from the entered values in the schemes. this process utilizes calculation of a number of prognostic indexes such as the international prognostic index (ipi)4 and the follicular lymphoma international prognostic index5 and a number of nodal and extranodal sites, in addition to calculation of both result and process quality indicators. result quality indicators are mortality 30 and 180 days after diagnosis for patients receiving treatment, response to first - line treatment and kaplan - meier survival estimates 1, 3, and 5 years after the time of diagnosis. the process quality indicators include time from diagnosis to start of treatment, fulfillment of entered data used in the international prognostic index, and the inclusion rate in clinical trials. to ensure high registry completeness, the lyfo is cross - referenced with the danish national patient registry and the national pathology registry. since all patients with a hospital admission, both as inpatient and outpatient, are registered in the danish national patient registry with diagnosis and date of contact, all patients referred to a hematological department are identified by this cross - reference.6 departments can instantly retrieve lists of patients with missing registration in the database, both at time of diagnosis and time of an eventual relapse. all departments register in the lyfo and fulfill the demand of coverage of at least 90%. in addition to the follow - up form, which is completed at the time of termination of outpatient follow - up or death, whichever occurs first, a linkage to the danish civil registration system secures that date of death is available.7 thereby, no patient is lost to follow - up. data from the lyfo has for decades been used for both clinical epidemiological research and for monitoring the treatment of lymphoma patients in denmark.8 the danish lymphoma group, who runs the database, publishes annually a report based on data from the lyfo in collaboration with registry support center of epidemiology and biostatistics (east).9 the quality indicators are presented for each department by year together with comments from clinicians and epidemiologists. the reports have shown improved 180 days mortality for patients with diffuse large b - cell lymphoma (dlbcl), improving from 12% to 10% during the past 4 years. the annual report is publically available and is used by both clinicians and for administrative purposes. in case of one department having significant worse outcome than the others, the administrative staff will examine the numbers and differences and draw the clinicians attention to the differences in order to standardize treatment and outcome. as an example, the survival for patients with dlbcl was significantly lower in two out of ten departments in 20012007. the survival has increased, but is now at the same level for all departments due to publication of the survival results (figure 2). a substantial number of papers originated from the lyfo have been published in the past decade in international journals, and collaboration with other national databases has resulted in unique publications.913 in 2014, a publication of mantle cell lymphoma showed male sex to be an independent negative prognostic factor, and that both rituximab and autologous stem - cell transplant were independently associated with better outcome.11 a study on routine imaging in the follow - up setting after treatment for dlbcl compared the widespread use of routine imaging in denmark with the more restrictive use in sweden. no impact was found on survival, and the study concludes that routine imaging for dlbcl in the first complete remission is not recommended.13 data quality in the lyfo has recently been measured through a validation process, and the data quality and coverage was found to be very high with positive predictive values for individual variables ranging from 87% to 100% using individual medical records as reference and a registration completeness of 94.9% using danish national patient registry (dnpr) as reference.14,15 health care professionals (eg, medical doctors, study nurses) report to the database, and consent is not required from patients due to the danish legislation. registration in the lyfo is approved by the national board of health and the danish data protection agency (2006 - 54 - 2093). a data entry manual is publically available, and training is performed locally. prior to submission of a form, an internal validation of the data fields is performed and the clinician has to clarify open issues (eg, date of diagnosis, lymphoma subtype) before submission of the form can be performed. funding from the danish regions allows the maintenance and development of the database and the database office. registry support center of epidemiology and biostatistics east is responsible for it support as well as statistical and epidemiological support. the lyfo is a nationwide registry, established in 1982, with extensive information on all lymphoma patients treated at a department of hematology in denmark. the database is used for quality assurance, clinical epidemiological research, and administrative purposes.	aim of databasethe danish national lymphoma registry (lyfo) was established in order to monitor and improve the diagnostic evaluation and the quality of treatment of all lymphoma patients in denmark.study populationthe lyfo database was established in 1982 as a seminational database including all lymphoma patients referred to the departments of hematology. the database became nationwide on january 1, 2000.main variablesthe main variables include both clinical and paraclinical variables as well as details of treatment and treatment evaluation. up to four forms are completed for each patient : a primary registration form, a treatment form, a relapse form, and a follow - up form. variables are used to calculate six result quality indicators (mortality 30 and 180 days after diagnosis, response to first - line treatment, and survival estimates 1, 3, and 5 years after the time of diagnosis), and three process quality indicators (time from diagnosis until the start of treatment, the presence of relevant diagnostic markers, and inclusion rate in clinical protocols).descriptive dataapproximately 23,000 patients were registered in the period 19822014 with a median age of 65 years (range : 16100 years) and a male / female ratio of 1.23:1. patients can be registered with any of 42 different subtypes according to the world health organization classifications.conclusionlyfo is a nationwide database for all lymphoma patients in denmark and includes detailed information. this information is used for both epidemiological research and clinical follow - up as well as for administrative purposes.
micrornas are small endogenous rna molecules that affect many biological processes by regulating gene expression in a post - transcriptional way. they are 2122 nt in length whose primary role is to regulate gene expression through translational repression and/or mrna degradation (1). the first mirna molecules and mirna targets were identified in 1993 via classical genetic techniques in caenorhabditis elegans (2). since then, there has been a dramatic increase in the number of mirnas registered in mirbase (3). in parallel, the development of the first computational target prediction programs (47) led to the experimental identification of dozens of mirna targets (8), and emphasized the need to provide mirna target predictions in an efficient way to assist biologists in experimental design. the previous version of the diana - microt web server (9) presented extensive information for predicted mirna target gene interactions in a user - friendly interface. it offers links to nomenclature, sequence and protein databases, information for experimentally verified targets through tarbase (8) and targets predicted by pictar (6) or targetscan (10). also, users are facilitated by being able to search for targeted genes using different names or functional features. here, we describe an extensive update of this web server with several important improvements : (i) an advanced bibliographic analysis which correlates mirnas to diseases, (ii) support for two additional species, (iii) a graphical display with all relevant functional information from the ucsc genome browser, (iv) tracking of changes in mirna nomenclature and (v) user personalized sessions allowing personal query history and bookmarks. diana microt web server provides functional analysis of mirnas that reaches beyond a simple listing of mirna targets through integration of knowledge extracted from bibliography and known biological pathways. in the previous version of the web server, bibliographic integration considered automated searches in pubmed providing publications related to a mirna, each target gene or combination of the two. related diseases has been added that directly associates a mirna to publications connected to one or several diseases. this feature is based on information included in the title or the abstract of publications found in pubmed. all abstracts associated with a mirna are retrieved from pubmed, based on the presence of the name of the mirna or a member of its family, as defined by mirbase, in the title or abstract of the publication. the retrieved publications are associated with medical subject headings (mesh), the national library of medicine 's controlled vocabulary thesaurus, through their metadata. all disease associated mesh terms for a mirna are counted and visualized through a tag cloud (figure 1), where mesh terms appear in a size proportional to the number of publications reporting this mirna - disease association. the mesh terms of the tag cloud also serve as hyperlinks to the relevant publications. for example, in figure 2 mir-455-star (mir-455) has been associated with a publication indicating that lower expression of this mirna correlates with poor overall survival in endometrial serous adenocarcinoma (11). figure 1.an example of a tag cloud for hsa - mir-1 showing relevant disease associated mesh terms. figure 2.example of a diana - microt web server results page. related diseases tag cloud contains links to pubmed and specifies all papers which associate the particular disease with the corresponding mirna. pubmed links provides automated bibliography searches based only on the name of mirnas, protein coding genes or the combination of both. ucsc graphic link presents the predicted binding sites in a ucsc genome browser window along with tracks such as snps and repeat elements. the left side of the page is devoted to the administration of the user personal space and reports their latest searches and bookmarks. an example of a tag cloud for hsa - mir-1 showing relevant disease associated mesh terms. related diseases tag cloud contains links to pubmed and specifies all papers which associate the particular disease with the corresponding mirna. pubmed links provides automated bibliography searches based only on the name of mirnas, protein coding genes or the combination of both. ucsc graphic link presents the predicted binding sites in a ucsc genome browser window along with tracks such as snps and repeat elements. the left side of the page is devoted to the administration of the user personal space and reports their latest searches and bookmarks. the feature that allowed the user to filter the targets of a mirna based on kegg pathways (12) is now integrated in the initial input query form. the user has now the option to enter a mirna together with names of pathways of interest and provided immediately with only the mirna target genes found in the pathways of interest. for example, a query for hsa - mir-221 would return 113 predicted targets at a score threshold of 0.5 while the combined search of the mirna with the term mapk - signaling - pathway would filter the results to 21 relevant targets. in addition, the positions of the binding sites on the mrna of the target gene are graphically presented through the ucsc genome browser. this automatic upload can be used to provide information in comparison to other tracks of interest such as single nucleotide polymorphisms (snps), repeat elements and alternative 3-utr splice forms. the relation of mirnas to function and diseases described above is based on mirna nomenclature. since mirna biology is still a field in flux, it can occur that a mirna may change name between two successive versions of mirbase. due to such changes, researchers may lose track of a mirna full history and related literature searches will remain incomplete. to address this issue, an extended analysis on 13 versions of mirbase (versions 7.1. to 14) is performed, and the nomenclature history of each mirna is extracted. this version includes 1884 mature mirnas for the four species supported in the web server. mimat id and one associated mirna specific name. among versions, changes are found in 77 mimat ids (38 in human, 37 in mouse and 2 in drosophila) and 151 mirna names (76 in human, 71 in mouse and 4 in drosophila). this indicates that name changes are more frequent than changes in mimat ids. to keep track of these changes, its name was later changed to mmu - mir-455 - 5 p in version 8.2 and later appeared as mmu - mir-455 in version 10.0 (figure 3). this information can be retrieved from the mirna history, but is also used to extend the association of mmu - mir-455 with endometrial serous adenocarcinoma in the publication where it is referred to as mir-455 - 5 p (figure 2). figure 3.graphic presentation of the changes involved in the history of mirna mmu - mir-455. initially, mimat0003485 was presented in version 8.1 as mmu - mir-455 but its name changed consecutively to mmu - mir-455 - 5 p in version 8.2 and mmu - mir-455-star (mmu - mir-455) in version 10.0. similarly, mimat0003742 was first presented in version 8.2 as mmu - mir-455 - 3 p, while in version 10.0, its sequence changed and it was renamed to mmu - mir-455. initially, mimat0003485 was presented in version 8.1 as mmu - mir-455 but its name changed consecutively to mmu - mir-455 - 5 p in version 8.2 and mmu - mir-455-star (mmu - mir-455) in version 10.0. similarly, mimat0003742 was first presented in version 8.2 as mmu - mir-455 - 3 p, while in version 10.0, its sequence changed and it was renamed to mmu - mir-455. the first version of diana - microt web server was designed to support the functional analysis of human and mouse mirnas. now the server has been updated with predictions for two additional species and newer versions of mirbase (mirbase 13) and ensembl (ensembl 54) (13). in total predictions for 723 new mirnas have been added, out of which 147 correspond to drosophila melanogaster, 154 to c. elegans and the rest being new homo sapiens and mus musculus mirnas. this results in an approximately doubled number of predicted targets in comparison to the previous version, totaling to more than six million predicted target genes. the web server can support different prediction algorithms and currently provides the targets of an updated version of the previously used algorithm (14). while microt - v3.0 is based on features separating real and mock (shuffled) mirnas the current version, microt - v4.0, uses high throughput experimental data for the same purpose (15). to allow users to take full advantage of the web server 's functions, the most important is an integrated personal user space in which users can easily save important searches and results that they wish to keep for future analysis. in particular, the system keeps the most recent user searches, giving the opportunity to repeat searches. a bookmarking mechanism provides the opportunity to save interesting results along with user comments. the personal space provides usage statistics regarding the most recent searches, thus, enabling them to keep track of their latest findings. it is noted that researchers may use any feature of the web server irrespectively of the personal user space feature. finally, special attention has been given to the web server documentation introducing hovering help notes for important fields. diana microt web server provides functional analysis of mirnas that reaches beyond a simple listing of mirna targets through integration of knowledge extracted from bibliography and known biological pathways. in the previous version of the web server, bibliographic integration considered automated searches in pubmed providing publications related to a mirna, each target gene or combination of the two. related diseases has been added that directly associates a mirna to publications connected to one or several diseases. this feature is based on information included in the title or the abstract of publications found in pubmed. all abstracts associated with a mirna are retrieved from pubmed, based on the presence of the name of the mirna or a member of its family, as defined by mirbase, in the title or abstract of the publication. the retrieved publications are associated with medical subject headings (mesh), the national library of medicine 's controlled vocabulary thesaurus, through their metadata. all disease associated mesh terms for a mirna are counted and visualized through a tag cloud (figure 1), where mesh terms appear in a size proportional to the number of publications reporting this mirna - disease association. the mesh terms of the tag cloud also serve as hyperlinks to the relevant publications. for example, in figure 2 mir-455-star (mir-455) has been associated with a publication indicating that lower expression of this mirna correlates with poor overall survival in endometrial serous adenocarcinoma (11). figure 1.an example of a tag cloud for hsa - mir-1 showing relevant disease associated mesh terms. figure 2.example of a diana - microt web server results page. related diseases tag cloud contains links to pubmed and specifies all papers which associate the particular disease with the corresponding mirna. pubmed links provides automated bibliography searches based only on the name of mirnas, protein coding genes or the combination of both. ucsc graphic link presents the predicted binding sites in a ucsc genome browser window along with tracks such as snps and repeat elements. the left side of the page is devoted to the administration of the user personal space and reports their latest searches and bookmarks. an example of a tag cloud for hsa - mir-1 showing relevant disease associated mesh terms. related diseases tag cloud contains links to pubmed and specifies all papers which associate the particular disease with the corresponding mirna. pubmed links provides automated bibliography searches based only on the name of mirnas, protein coding genes or the combination of both. ucsc graphic link presents the predicted binding sites in a ucsc genome browser window along with tracks such as snps and repeat elements. the left side of the page is devoted to the administration of the user personal space and reports their latest searches and bookmarks. the feature that allowed the user to filter the targets of a mirna based on kegg pathways (12) is now integrated in the initial input query form. the user has now the option to enter a mirna together with names of pathways of interest and provided immediately with only the mirna target genes found in the pathways of interest. for example, a query for hsa - mir-221 would return 113 predicted targets at a score threshold of 0.5 while the combined search of the mirna with the term mapk - signaling - pathway would filter the results to 21 relevant targets. in addition, the positions of the binding sites on the mrna of the target gene are graphically presented through the ucsc genome browser. this automatic upload can be used to provide information in comparison to other tracks of interest such as single nucleotide polymorphisms (snps), repeat elements and alternative 3-utr splice forms. the relation of mirnas to function and diseases described above is based on mirna nomenclature. since mirna biology is still a field in flux, it can occur that a mirna may change name between two successive versions of mirbase. due to such changes, researchers may lose track of a mirna full history and related literature searches will remain incomplete. to address this issue, an extended analysis on 13 versions of mirbase (versions 7.1. to 14) this version includes 1884 mature mirnas for the four species supported in the web server. mimat id and one associated mirna specific name. among versions, changes are found in 77 mimat ids (38 in human, 37 in mouse and 2 in drosophila) and 151 mirna names (76 in human, 71 in mouse and 4 in drosophila). this indicates that name changes are more frequent than changes in mimat ids. to keep track of these changes, its name was later changed to mmu - mir-455 - 5 p in version 8.2 and later appeared as mmu - mir-455 in version 10.0 (figure 3). this information can be retrieved from the mirna history, but is also used to extend the association of mmu - mir-455 with endometrial serous adenocarcinoma in the publication where it is referred to as mir-455 - 5 p (figure 2). figure 3.graphic presentation of the changes involved in the history of mirna mmu - mir-455. initially, mimat0003485 was presented in version 8.1 as mmu - mir-455 but its name changed consecutively to mmu - mir-455 - 5 p in version 8.2 and mmu - mir-455-star (mmu - mir-455) in version 10.0. similarly, mimat0003742 was first presented in version 8.2 as mmu - mir-455 - 3 p, while in version 10.0, its sequence changed and it was renamed to mmu - mir-455. initially, mimat0003485 was presented in version 8.1 as mmu - mir-455 but its name changed consecutively to mmu - mir-455 - 5 p in version 8.2 and mmu - mir-455-star (mmu - mir-455) in version 10.0. similarly, mimat0003742 was first presented in version 8.2 as mmu - mir-455 - 3 p, while in version 10.0, its sequence changed and it was renamed to mmu - mir-455. the first version of diana - microt web server was designed to support the functional analysis of human and mouse mirnas. now the server has been updated with predictions for two additional species and newer versions of mirbase (mirbase 13) and ensembl (ensembl 54) (13). in total predictions for 723 new mirnas have been added, out of which 147 correspond to drosophila melanogaster, 154 to c. elegans and the rest being new homo sapiens and mus musculus mirnas. this results in an approximately doubled number of predicted targets in comparison to the previous version, totaling to more than six million predicted target genes. the web server can support different prediction algorithms and currently provides the targets of an updated version of the previously used algorithm (14). while microt - v3.0 is based on features separating real and mock (shuffled) mirnas the current version, microt - v4.0, uses high throughput experimental data for the same purpose (15). to allow users to take full advantage of the web server 's functions, several functional improvements have been implemented (figure 2). the most important is an integrated personal user space in which users can easily save important searches and results that they wish to keep for future analysis. in particular, the system keeps the most recent user searches, giving the opportunity to repeat searches. a bookmarking mechanism provides the opportunity to save interesting results along with user comments. the personal space provides usage statistics regarding the most recent searches, thus, enabling them to keep track of their latest findings. it is noted that researchers may use any feature of the web server irrespectively of the personal user space feature. finally, special attention has been given to the web server documentation introducing hovering help notes for important fields. in recent years it has become apparent that web applications are an essential tool for researchers to decipher complex biological processes. as one of these, the diana microt web server has been updated to integrate different additional resources in order to offer insight for putative mirna involvement in biological processes, and to allow researchers to supplement their knowledge with already published scientific material. performing an extended analysis on the versioning pattern of mirbase the history of each mirna has been extracted, offering a unique feature in computational mirna analysis. we believe that the current web server update provides important tools for biological analysis and improves interaction with the complicated interconnections regarding mirna functionality. the hellenic republic, general secretariat for research and technology (09syn-13 - 1055). funding for open access charge : 09syn-13 - 1055.	micrornas (mirnas) are small endogenous rna molecules that are implicated in many biological processes through post - transcriptional regulation of gene expression. the diana - microt web server provides a user - friendly interface for comprehensive computational analysis of mirna targets in human and mouse. the server has now been extended to support predictions for two widely studied species : drosophila melanogaster and caenorhabditis elegans. in the updated version, the web server enables the association of mirnas to diseases through bibliographic analysis and provides insights for the potential involvement of mirnas in biological processes. the nomenclature used to describe mature mirnas along different mirbase versions has been extensively analyzed, and the naming history of each mirna has been extracted. this enables the identification of mirna publications regardless of possible nomenclature changes. user interaction has been further refined allowing users to save results that they wish to analyze further. a connection to the ucsc genome browser is now provided, enabling users to easily preview predicted binding sites in comparison to a wide array of genomic tracks, such as single nucleotide polymorphisms. the web server is publicly accessible in www.microrna.gr/microt-v4.
acute promyelocytic leukemia (apml) is a subset of aml with characteristic clinical, morphological and genetic features. its incidence gradually increases, reaching a plateau during early adulthood, remaining constant until it diminishes after 60 years of age. the identification of specific chromosomal abnormality plays an important role in determining therapy and prognosis in certain subtypes of aml. data from the pediatric oncology group have shown that inv(16) / t(16;16), t(8;21), and normal karyotypes are associated with favorable prognostic outcome, whereas poorer outcome was observed in t(15;17) (without all- trans retinoic acid (atra) treatment), 11q23, and other abnormalities., several specific recurrent chromosome aberrations have been described in aml, both unbalanced and balanced rearrangements. balanced chromosome rearrangements are detected in approximately 2530% of adults with de novo aml and have attracted a great deal of attention not only because their molecular dissection has led to identification of genes involved in leukemogenesis but also because specific translocations and inversions are associated with clinical features and treatment outcome of patients harboring them.[46 ] in this article, we discuss the case study of a two - year - old child with unbalanced chromosome aberrations involving chromosome 17 with a cytogenetically normal chromosome 15. patient had a history of fever, general weakness and bleeding from the gums. on examination, his general condition was poor, with cervical and axillary lymph nodes. on systemic examination, he had hepatomegaly 23 cm, and splenomegaly 12 cm. at presentation, hemogram was hb 69 g / l, total count of 52 10/l and platelets 20 10/l. differential count showed neoplastic promyelocytes 80%, neutrophils 12%, lymphocytes 12%, bone marrow aspiration showed hyper cellular marrow with sheets of neoplastic promyelocytes (70 %). (a) peripheral smear : auer rods positive, (b) mpo positive cytogenetic study was carried out on cells from bone marrow aspiration. short - term culture of 24 and 48 h were set up using rpmi 1640 medium supplemented with 20% fetal bovine serum. after 1618 h, 50 l of colcemid at a final concentration of 10 g / ml was added for 30 min followed by hypotonic treatment, fixation in carnoy 's fixative. fifteen metaphases were analyzed which consistently showed 46xy, -17,+der(17)(17qter - cen-17q21 :) karyotype [figure 2 ]. short - term culture of 24 and 48 h were set up using rpmi 1640 medium supplemented with 20% fetal bovine serum. after 1618 h, 50 l of colcemid at a final concentration of 10 g / ml was added for 30 min followed by hypotonic treatment, fixation in carnoy 's fixative. fifteen metaphases were analyzed which consistently showed 46xy, -17,+der(17)(17qter - cen-17q21 :) karyotype [figure 2 ]. short - term culture of 24 and 48 h were set up using rpmi 1640 medium supplemented with 20% fetal bovine serum. after 1618 h, 50 l of colcemid at a final concentration of 10 g / ml was added for 30 min followed by hypotonic treatment, fixation in carnoy 's fixative. fifteen metaphases were analyzed which consistently showed 46xy, -17,+der(17)(17qter - cen-17q21 :) karyotype [figure 2 ]. to our knowledge, this is a rare cytogenetic abnormality in a child, involving only chromosome 17. the first report of aml - m3 with normal chromosome 15 with -17, ins(17 ; ?) (q11q21 ; ?) was by baranger. the present case demonstrated normal appearance of chromosome 15s, while one of the 17 homologues appeared to be an iso (17q), but upon detailed examination showed break on one of the arms at q21 band. the presence of 15q+ and i(17q-) is one of the most frequent abnormalities reported besides the standard translocation, but with an i(17q-) and two normal chromosome 15 is extremely rare. routinely cytogenetics, fluorescence in situ hybridization (fish), and polymerase chain reaction (pcr) analysis are employed for the diagnosis and precise localization of the fusion gene. but in our case, fish could not be performed and even before the cytogenetic results were available the patient died due to disseminated intravascular coagulation. hence, further molecular studies to establish the type of gene fusion transcript which could have had a prognostic value could not be ascertained. a small proportion of apml patients do not have t(15;17)/ pml - rara but do have other chromosomal aberrations and gene fusion, all these rearrangements and t(15;17) are very strongly correlated with characteristic marrow morphology in which abnormal promyelocytes predominate (fab- m3). the present who classification recognizes two main morphological subtypes of apml that includes a more frequent hyper granular variant form featured by abnormal dysplastic promyelocytes with abundant cytoplasmic granules and auer rods (faggots) and a less frequent micro granular variant of apml characterized by leukemic blast, bilobed nuclei with dusty and minute cytoplasmic granules. the evolution of aml involves leukemogenic events that occur in the stem cell (stem cell origin model) and favors self renewal while disrupting normal hematopoietic cell lineage development.[810 ]	acute myeloid leukemia (aml - m3) is associated with the translocation t(15;17)(q22;q12 - 21) which disrupts the retinoic acid receptor alpha (rara) gene on chromosome 17 and the pml gene on chromosome 15. we report a two - year - old patient with aml - m3 without the usual translocation t(15;17). cytogenetic studies demonstrated normal appearance of chromosome 15 while the abnormal 17 homologue was apparently a derivative 17, der(17)(17qter - cen - q21 :), the rearrangement distinctly shows deletion at 17q21 band and the morphology corresponding to an iso chromosome i(17q-). this case report is a rare cytogenetic presentation of acute promyelocytic leukemia (apml).
a 28-year - old woman, with primary infertility of three years was referred for laparoscopic myomectomy with a history of severe dysmenorrhea and a diagnosis of posterolateral wall myoma. on pelvic examination, a palpable myoma was noted on the posterior uterine surface, and the uterus was felt to be approximately 10 gestational weeks in size. a well - circumscribed mass measuring 5.16 6.0 4.25 cms was seen in the posterolateral fundus and was found to be impinging on the endometrial stripe. the patient was given a choice between conventional laparoscopic myomectomy and the single incision approach. a 2 cm vertical incision was made at the base of the umbilicus and peritoneal access was gained., norwalk, ct), [figure 1 ] was inserted through the incision. the device, made from an elastic polymer, was slightly hourglass shaped and could be deployed through 2 cm fascial incision. it contained four openings : one for insufflation via a right - angled tube and three that could accommodate trocars 5 to 12 mm in size. the compressibility of the elastic polymer allowed for the access ports to expand and form fit the space in which they resided, and the ports also passed through the working channels. the sils tm port has the capacity of up to three laparoscopic instruments of 5 to 12 mm after careful survey of the abdomen and pelvis, dilute vasopressin, 20 u, in 100 ml of saline solution, was injected subserosally over the posterior myoma. once the correct plane was entered, the myoma was dissected out of the uterus using a 5 mm myoma cork screw and blunt scissors. using mostly blunt dissection, the enucleation of the myoma was done [figure 2 and 3 ]. hemostasis was ensured and a piece of adhesion barrier (gynecare interceed ; ethicon inc., west somerville, new jersey) was then cut in half, introduced into the abdomen through one of the 5 mm trocars, and placed over the hysterotomy incision. the myoma was then grasped with a 12 mm claw forceps and electric morcellation was done using rotocut g1 morcellator morcellation (rotacut) the advantages obtained by electronic morcellation over manual morcellation from umbilical incision site are : reduction in operative timedecreased risk of hernia formation due to absence of tearing or stretching of fascialower risk of injury to the surrounding tissues. reduction in operative time decreased risk of hernia formation due to absence of tearing or stretching of fascia lower risk of injury to the surrounding tissues. as laparoscopic myomectomies are performed routinely in our center, rotocut is preferred over gynecare morcellex for its cost effectiveness and speed. it is reusable and can be used indefinitely with periodic replacement of the cutting blade. the fascia was closed with a running 0 polyglactin 910 (vicryl, somerville, nj) suture. the skin was then approximated with a series of interrupted 3/0 monocryl sutures (ethicon inc.). ten milliliters of 0.5% bupivacaine hydrochloride (sensorcaine) were injected into the incision site, and dermabond adhesive (ethicon inc.) was applied. the total procedure time (time from first incision to end of procedure, d and c) single - incision surgery has been reported to offer patients improved cosmetic outcomes as compared to multiport laparoscopic surgery, and possibly less postoperative pain, although these potential benefits have yet to be demonstrated in a well - designed prospective trial. a number of advantages have been proposed including cosmesis, less incisional pain, less blood loss, and the ability to convert to standard multiport laparoscopic surgery. an additional morcellation port is avoided as specimen retrieval / morcellation can be done through the umbilical incision. the primary limitations of sils are the restricted degrees of freedom of movement, lack of triangulation, the number of ports that that can be used, and the proximity of the instruments to each other during the operation all of which increase the complexity and technical challenges of the operation. many of these difficulties may be related to the technique of port placement and utilization during single incision laparoscopic surgery. a number of methods have been described for port access to perform sils, including multiple fascial punctures through one skin incision and use of novel port access devices. to further overcome the technical challenges of sils, different instruments that provide angulations and small profile trocars, endostitch, are being developed. the barbed suture greatly facilitates myometrial closure because there is no need to tie knots and there is no backsliding of the suture, which enables continuous wound closure with even distribution of tensile strength throughout the repair. these benefits of barbed suture are especially valuable in single - incision surgery, because intracorporeal knot tying can be more challenging here than in the multiport approach. currently, careful case selection is paramount, so that these procedures can be explored safely, with a low threshold to convert to standard laparoscopy, as indicated, for safety and quality of care. the use of novel port access devices, articulated instruments, and endostitch self - retaining sutures, makes the procedure easier, with a potential for saving time.	single port laparoscopic surgery (spls), also called sils is the natural extension of multi - incisional laparoscopic surgery, in the quest for reduction of traumatic insult and residual scarring to the patient. today with the evolution of newer instruments, bidirectional self - retaining sutures, and surgical experience we are able to perform many surgeries in gynecology.
we describe the restructuring that is taking place in primary health care (phc) in portugal and discuss how the new emerging organization and work practices reflect in care transitions and intra- and inter - organizational integration. phc represents an essential support for the restructuring that is taking place in health care in portugal. we conducted a case study on a health centre located in the centre region of portugal. health centres are reorganizing toward a new model characterized by an organizational structure based on functional units : family health unit (fhu), community care unit (ccu), and personalized healthcare unit (phu), which will be in place in all of them plus transversal units, common to a group of these arrangements. the overarch management body is the aces (agrupamento de centros de sade), that reports to a health regional administration (hra). in portugal, there are five hra. an aces may coordinate a number of fhu, ccu and phu but has only one public health unit and one shared resources care unit. units are based on multidisciplinary teamwork, having : specific missions, although inter - cooperative and complementary, organized in a network ; administrative autonomy ; proper instruments of organizational management ; well - defined leadership and clinical governance systems and mechanisms of representation and participation of the community and citizens. it is too soon to assess the implications that the reorganization of phc might have in the integration of care in portugal.	introductionwe describe the restructuring that is taking place in primary health care (phc) in portugal and discuss how the new emerging organization and work practices reflect in care transitions and intra- and inter - organizational integration.theory and methodsphc represents an essential support for the restructuring that is taking place in health care in portugal. we conducted a case study on a health centre located in the centre region of portugal. we identified and analyzed internal units and care transitions and conducted interviews.resultshealth centres are reorganizing toward a new model characterized by an organizational structure based on functional units : family health unit (fhu), community care unit (ccu), and personalized healthcare unit (phu), which will be in place in all of them plus transversal units, common to a group of these arrangements. the overarch management body is the aces (agrupamento de centros de sade), that reports to a health regional administration (hra). in portugal, there are five hra. an aces may coordinate a number of fhu, ccu and phu but has only one public health unit and one shared resources care unit. units are based on multidisciplinary teamwork, having : specific missions, although inter - cooperative and complementary, organized in a network ; administrative autonomy ; proper instruments of organizational management ; well - defined leadership and clinical governance systems and mechanisms of representation and participation of the community and citizens.conclusions and discussionit is too soon to assess the implications that the reorganization of phc might have in the integration of care in portugal.
inflammatory bowel disease (ibd) is a complex disease comprised of crohn s disease or crohn disease (cd) and ulcerative colitis (uc), two conditions that involve unpredictable and destructive inflammatory processes resulting in weight loss, diarrhea, abdominal pain, and fever.13 uc is restricted to the colon, whereas cd is characterized by skip lesions and can involve any site in the gastrointestinal tract.4 symptoms include abdominal pain, ulcers, bloody or nonbloody diarrhea, cramping, weight loss, anorexia, and fatigue. affected children can also exhibit stunted growth and delayed puberty.5 pathological features include ulceration, abscesses, inflammatory infiltrates, edema, mucin depletion, and loss of the normal crypt architecture. ibd leads to fistulas, perforations, infection, abscesses, and increases the risk of intestinal dysplasia and colon cancer in proportion to its duration.6,7 ibd is caused by dysregulation of the immune mechanisms that maintain homeostasis between the intestinal epithelium and commensal and pathogenic gut flora.3,811 genetic analyses have implicated the human genes atg16l, nod2/card15, ibd5, ctla4, tnfsf15, jak2, stat3, il23r, and ormdl3 as risk factors in ibd.1219 these clues, combined with basic research, have revealed that antimicrobial peptides, autophagy, endoplasmic reticulum stress, innate and adaptive immune cell function, t - helper (th)17 cells, regulatory t - cells, and cytokines (tumor necrosis factor [tnf]-, interleukin [il]-17, il-23/il-12, il-22, and il-6) are contributing factors in ibd.2023 these mediators initiate signaling pathways that activate key inflammatory transcription factors including nuclear factor kappa b (nfb) and signal transducer and activator of transcription (stat)3, which integrate and amplify signals from a wide range of intrinsic and environmental stimuli.2426 many cell compartments of the gut including enterocytes, paneth cells, t - cells, mature and immature myeloid cells, and vascular cells contribute to the regulation of nfb, stat3, and the inflammatory milieu.27,28 elucidating the complex interactions between intestinal cells, secreted proteins, and transcription factors, their modulation by factors in the gut mucosa and its environment, and how these interactions are disrupted in ibd are the necessary first steps to identifying new targets and curing ibd. targeted therapy and dietary chemoprevention strategies hold the promise of reducing the toxicities and risks associated with global immunosuppressive regimens that are currently being employed to treat ibd. sphingosine-1-phosphate (s1p) is a signaling lipid found in the circulation and in most tissues.29,30 s1p is derived from the recycling of endogenous human sphingolipids and the metabolism of sphingolipids found in dietary animal products that, like human tissues, contain sphingolipids, which are built upon a sphingosine structural backbone.29 s1p has many functions in angiogenesis, development, innate and adaptive immunity, and is a regulator of lymphocyte trafficking.31 a majority of s1p s biological functions have been linked to its ability to activate a family of five g protein - coupled receptors, s1p receptors 15 (s1pr15).29 however, s1p exerts some actions that have not yet been definitively or completely attributed to s1prs. for example, s1p serves as a major activator of the il-6/stat3 pathway implicated in the pathophysiology and genetic basis of ibd, as well as the pathogenesis of colon cancer.24,3238 in fact, s1p production appears to be oncogenic in colon cancer.39,40 s1p is also the cofactor for the tnf receptor associated factor 2 e3 ubiquitin ligase required for activation of nfb downstream of tnf- and nucleotide - binding oligomerization domain - containing protein 2.16,41 nuclear actions of s1p have also not been linked to s1pr functions.42 s1p is generated from sphingosine through the actions of sphingosine kinase (sphk) enzymes, as shown in figure 1. there are two isoforms of sk : the ubiquitously expressed major sk, sphk1 ; and the more tissue - restricted isoform, sphk2. s1p can be dephosphorylated by specific and nonspecific lipid phosphatases.43 however, the irreversible degradation of s1p to ethanolamine phosphate and hexadecenal is catalyzed by the conserved endoplasmic reticulum enzyme, sphingosine phosphate lyase (spl), which is expressed in differentiated enterocytes of the small and large intestine, paneth cells, and inflammatory cells44,45 (saba, unpublished data, 2014). spl is downregulated in colon cancer, leading to s1p accumulation in neoplastic intestinal tissues, thereby implicating spl in colon carcinogenesis.46,47 sphingolipids are implicated in the regulation of immune functions and key inflammatory pathways involving stat3 and nfb.32,48 further, there is high expression of the genes involved in sphingolipid metabolism in the small and large intestine, where they function in the metabolism of dietary sphingolipids.49 based on these findings, there has been interest in exploring the possible role of s1p signaling in ibd. the relevance of sphingolipids in the pathophysiology of ibd is heightened by the recent finding that polymorphisms of orosomucoid (orm)1-like 3, a homologue of the sphingolipid regulatory protein orm1, are genetically linked to ibd.19,50 this review will summarize the scientific evidence implicating s1p signaling as a mediator and potential target in ibd. in 2004 and 2005, several publications from one laboratory5153 demonstrated that s1p signaling through s1pr1 and metabolism by spl control lymphocyte egress from the thymus into the circulatory system. these studies showed that the pharmacological inhibition of spl (which disrupts the s1p gradient between the blood and tissues) or treatment with an s1pr1 modulator (which induces downregulation and attenuates signaling through this receptor) induced lymphopenia in mice. the role of s1p signaling and metabolism in the regulation of lymphocyte trafficking has been further confirmed through genetic approaches.54,55 most exciting was the fact that targeting the s1p signaling pathway blocked lymphocyte trafficking without necessarily reducing global immune functions as other immunosuppressive agents do. the strategy of targeting s1pr1 to induce lymphopenia was later shown to be therapeutically effective in the treatment of the human autoimmune disorder, multiple sclerosis.5659 other lines of evidence demonstrate that s1p signaling is linked to inflammation, autoimmune disease, and cancer.32 in 2010, the research group of lee demonstrated that the stat3 signaling pathway and the s1p / s1pr1 pathway coactivate one another in an inflammatory loop that promotes carcinogenesis. in an atherosclerosis mouse model, s1pr2 activation increased nfb activity in macrophages, one of the cell types implicated in the development of atherosclerosis.60 in another study, s1pr1 was found to directly activate the stat3 pathway via il-6 in a mouse model of autoimmune encephalomyelitis.61 these studies implicate the s1p axis in the pathophysiology of autoimmune disease and highlight the need to explore s1p targeting approaches in other autoimmune and inflammatory conditions. the results from preclinical studies employing various mouse models of colitis suggest that targeting s1p signaling and other sphingolipids may also be useful in the treatment of ibd.6265 fty720, krp-203, and w-061 are s1pr agonist / functional antagonists that have a high affinity for s1pr1, causing its internalization and downmodulation, as shown in figure 2a. these agents have been shown to reduce murine models of colitis produced by il-10 deficiency, dextran sodium sulfate (dss) treatment, and adoptive t - cell transfer.6669 in contrast to cd, which is characterized by a th1 cytokine profile, uc is defined by a th2 cytokine profile.70 th2 cytokines are necessary for the differentiation of cluster of differentiation (cd)4 + t - cells into natural killer t (nkt) cells. nkt cells produce il-13 following activation by glycolipid antigens that are found on the bacteria of the gut or in epithelial cells infected by bacteria.71 the lamina propria of uc patients is characterized by high expression of il-13 and il-13-producing cd4 + t - cells.72 nkt cells have been shown in vitro to cause cytotoxicity via il-13 and to negatively affect the epithelial barrier function of a monolayer of ht29.72 in the oxazolone mouse model of uc, an ex vivo culture of cd4 + t - cells isolated from fty720-treated mice showed reduced expression of proinflammatory cytokines il-13, il-5, and il-4, produced by th2 cells.73 this suggests that fty720 may have the potential to correct fundamental immune imbalances in the uc microenvironment. in a th1-driven 2,4,6-trinitrobenzenesulfonic acid (tnbs) mouse model of colitis, fty720 also showed efficacy in the reduction of colitis.74 treg cells are thought to play a key role in ibd by preventing the proliferation and activation of t lymphocytes. in this study, fty720 was found to directly affect the functional activity of cd4+cd25 + regulatory t - cells. administration of fty720 to tnbs - treated mice resulted in the decreased expression of th1 cytokines, namely il-12p70 and tnf-, and an upregulation of the immunosuppresive treg cytokines il-10 and transforming growth factor-. this was accompanied by an increase in the expression of cytotoxic t - lymphocyte antigen 4 and foxp3, both of which are markers of treg cells. the ability of fty720 to attenuate colitis in both th1- and th2-driven models of colitis makes it an attractive candidate for the treatment of ibd patients. in addition to the effects on colitis afforded by agents targeting s1prs, it was observed that mice lacking sphk1 were less susceptible than littermate controls to colitis induced by dss.75 this suggests that sphk1 could be targeted to reduce s1p signaling (figure 2b). compared to control mice, sphk1 null mice showed a similar expression of tnf-, but decreased expression of cyclooxygenase (cox)-2 and no systemic inflammatory response. the evidence suggests that targeting sphk1 with a specific inhibitor could attenuate inflammatory responses in ibd patients. in support of this notion, two inhibitors of sk, abc747080 and abc294640, prevented tnf- activation of nfb, prostaglandin e2 production, vascular cell adhesion molecule and intercellular adhesion molecule expression, and reduced clinical and histological signs of dss - induced colitis in c57bl/6 mice.76 the effects of the s1p axis on adaptive immunity are shown in figure 3. spl is highly expressed throughout the intestine, especially in the differentiated epithelial cells of the duodenum and jejunum and, to a lesser degree, in the ileum and colon.77,78 in a recent study,79 an inverse correlation between the colonic concentrations of s1p and spl s cofactor, pyridoxal-5-phosphate was found. of note, plasma pyridoxal-5-phosphate concentration was inversely linked with the concentration of inflammatory markers.79 these results suggest that spl expression, as well as its cofactor availability, may be important in preventing or reducing inflammation. patients suffering from ibd are at a higher risk of developing colon cancer the longer they suffer from unresolved inflammation.2 interestingly, we found that spl expression and activity were downregulated in adenomatous lesions of apcmin/+ mice.46 moreover, human colon cancer tissues expressed less spl than the normal adjacent tissue. it was demonstrated that spl expression is required for p38- and p53-induced cell death via caspase 2 activation.46 the results of this study raise the possibility that changes in spl expression or enzyme activity could contribute to the initiation of intestinal tumorigenesis. liang have provided new evidence supporting a role for sphk2 in the pathogenesis of colitis and colitis - associated cancer (cac). in their study, the azoxymethane (aom)/dss mouse model of cac was implemented in both wild type and sphk2 knockout (ko) mice. they found that the tumor number, size, and load were higher in sphk2 ko mice compared to wild type mice.48 the sphk2 ko mice also exhibited elevated disease activity, suffering from severe diarrhea, as well as blood and weight loss. in addition, the ko mice exhibited shorter colons (indicating inflammation - induced stricture and fibrosis), extensive colon architectural damage, and immune cell infiltration.48 in performing biochemical analyses of the two groups, the authors made a surprising discovery. they found increased circulating and colonic s1p levels in sphk2 ko mice compared to controls. moreover, sphk1 protein and messenger ribonucleic acid levels were higher in the colons of sphk2 ko mice.48 the authors suggested that the upregulation of sphk1 could be the result of reduced nuclear sphk2. importantly, one of the transcriptional regulators of sphk1 expression, c - jun, is regulated by histone acetylation.42,80,81 therefore, in the colons of sphk2 ko mice, the authors suggest that increased histone deacetylase activity results in increased activation of c - jun, thereby driving increased sphk1 expression.48 s1p activates nfb, a transcription factor involved in the expression of many proinflammatory cytokines that contribute to the pathogenesis of ibd and cac.41,82,83 il-6, which is a nfb target gene, can activate stat3, a regulator of s1pr1 expression.32,84 accordingly, nfb activation, il-6 expression and s1pr1 expression were all increased in the colons of sphk2 ko mice following dss treatment to a greater extent than in controls.48 to determine the cell type responsible for il-6 secretion, the authors generated bone marrow chimeras by transferring bone marrow cells of either wild type or sphk2 ko mice to an irradiated recipient. sphk2 disruption in bone marrow cells alone was sufficient to recapitulate the sphk2 global ko mouse phenotype following dss treatment.48 flow cytometry analysis identified macrophages and dendritic cells as the major producers of il-6.48 however, intestinal epithelial cells were at least in part responsible for the increased colonic expression of il-6 in global sphk2 ko mice, as shown by immunohistochemistry.48 the authors proposed a model involving a feed - forward amplification loop in which the sk / s1p / s1pr1 axis leads to the activation of nfb and persistent stat3 activation in cac, as shown in figure 4.48 to disrupt the feed - forward cycle, the authors administered fty720, an s1pr1 functional antagonist that can also inhibit sphk1 by causing its proteasomal degradation (figure 2b).8587 administration of fty720 in sphk2 global ko mice treated with dss reduced disease activity, sphk1, il-6, and s1pr1 expression, and nfb and stat3 activity.48 fty720 could prevent cac in sphk2 ko mice if administered at the beginning of the aom / dss regimen, but not when it was administered at a later time point. this was true even though fty720 administration could still reduce stat3 activation and s1pr1 expression.48 the authors concluded that fty720 should be explored as a drug to treat cac in ibd patients, since it reduces both the expression of s1pr1 and sphk1, thereby disrupting the s1p / sphk1/s1pr1 signaling loop. each day, we consume between 0.30.4 g of sphingolipids though our diet.88 in the intestinal tract, these sphingolipids are digested to form sphingoid bases and ceramides.49 in the past three decades, the potential of sphingolipids as dietary bioactive molecules has been studied intensively in different mouse models of ibd and colon cancer. 1,2 dimethylhydrazine (dmh) is a carcinogenic compound used in mouse and rat models of cac.89,90 dmh promotes colon tumorigenesis by causing oxidative stress and inflammation.91 one report of a study employing the dmh mouse model found that dietary sphingomyelin can reduce the number of premalignant lesions and tumors by 20%.92 in another study, the plant sphingolipid soy glucosylceramide added to the diet of apcmin/+ or dmh - treated mice decreased the number of adenomas and aberrant crypt foci arising in these two respective models of colon cancer.93 the authors confirmed that the expression levels of two transcription factors involved in tumorigenesis, hypoxia - inducible factor 1 and transcription factor 4, were decreased in response to sphingolipid treatment.93 mazzei explored the effects of dietary sphingomyelin in the aom / dss mouse model of cac. they found that adding 1 g / kg of sphingomyelin in the mouse diet decreased both the disease activity index and the tumor number.94 they found that sphingomyelin attenuated the expression of proinflammatory cytokines (interferon-, il-17, and il-23), but induced antiinflammatory cytokines and receptors implicated in th2 and treg cell differentiation (il-4, il-3, il-13ra2, and il-10rb).94 in contrast, in an acute dss colitis model, gavage of 4 mg or 8 mg of sphingomyelin / day exacerbated the disease activity by causing increased apoptosis of the intestinal epithelial cells.95 this finding was associated with an increased activity of cathepsin d that mediated caspase-3- and caspase-9-induced apoptosis.95 using the il-10 ko mouse model of spontaneous colitis, the authors showed that sphingomyelin aggravated mucosal inflammation.95 these findings using a variety of mouse models, different sphingomyelin doses, and measuring different disease - related endpoints raise the possibility that dietary sphingolipids may have untoward effects depending on the state of the target tissue, and possibly the source and structure of the sphingolipid. they also suggest that there may be a tight range of sphingomyelin concentrations that will afford therapeutic benefit in cac. they are found in soy products and other natural sources.96 sphingadienes containing double bonds at c4,5 and c8,9 were shown to induce apoptosis in hct116, dld1, and sw480 colon cancer cell lines and to decrease tumor development in an apcmin/+ mouse model of colon cancer.96 sphingadienes act, at least in part, by blocking the translocation of akt from the cytoplasm to the membrane, thereby inactivating akt. this effect of sphingadienes leads to the induction of apoptosis and autophagy, processes that are repressed by akt.96 sphingadiene treatment also inactivated gsk-3 and, as a consequence, downregulated growth - activating wnt signaling.97 sphingadienes are being explored for their potential efficacy as antiinflammatory agents in ibd and in their ability to prevent the development of cac (degagne, unpublished data, 2014).29 importantly, unlike mammalian sphingolipids, sphingadienes can not be metabolized to s1p. further, since sphingadienes are not produced in appreciable amounts in humans, their metabolism by the enzymes present in human colonic epithelial cells is limited, their uptake into the bloodstream is minimal, and their action is local, making them appealing candidates for the long - term chemoprevention of cac. the studies described above highlight the possibility that strategies involving s1pr1 downregulation or inhibition that have proven useful in other autoimmune diseases may afford promise in the context of ibd. however, chemically targeting s1prs may be complicated by their diverse functions. for example, s1pr1 activity could prove critical in maintaining vascular integrity in the colonic mucosa. this concern is illustrated by results from preclinical studies testing s1pr1 disruption in mice suffering from experimental colitis.98 however, treatment of c57bl6/j mice with fty720 did not cause more bleeding than the control group in an experimental colitis model.98 of additional concern, administration of fty720 to mice in an experimental colitis model impaired mucosal immunity such that the mice were then unable to eliminate an enteric bacterial infection.99 fty720 treatment was associated with a shift in the immune cell populations present in the colon during the infection, decreasing the number of t and b lymphocytes and macrophages, and resulting in the decreased expression of th1 cytokines (il-1, inducible nitric oxide synthase [inos ], il-17a, il-6, and tnf-). even when the treatment was discontinued prior to infection, the mice were still unable to clear the bacterial infection.99 these findings suggest that the safety of strategies that modulate s1pr signaling in ibd patients must be investigated further. specifically, it will be critical to understand how s1pr modulation affects the innate immune response to a wide range of bacteria and viruses to which ibd patients are particularly susceptible. the recent article by liang has established the importance of sphk2 in inflammatory signaling through stat3. interestingly, others have shown that cd4 + t - cell sphk2 is required for suppressing il-2 responses that aggravate ibd via a stat5-dependent mechanism.100 sphk2 null mouse cd4 + t - cells have a hyperactive phenotype, increased proliferation, and cytokine secretions in response to il-2 signaling. moreover, these sphk2 null mouse cd4 + t - cells were not responsive to treg cell - mediated suppression. this study raises concerns regarding the use of sk inhibitors in treating ibd patients, as they could have adverse effects on immune cells by directly increasing cytokine secretions through their effects on cd4 + t - cells and also by counteracting the immunomodulatory role of treg cells. thus, the s1p signaling pathway may be affecting multiple aspects of the innate and adaptive immune response relevant to ibd. is the decreased expression of sphk2 a characteristic of or a precursor state to the development of ibd or colon cancer ? what are the key mechanisms by which s1p signaling (and downstream nfb and stat3 inflammatory nodes) promotes the switch from a state of inflammation to a preneoplastic state and, ultimately, to the development of cac ? at this time, the exact role of s1p in ibd remains incompletely understood. further, the potential for s1p to be used as a biomarker of disease activity has not been explored. in that regard, there is sparse information regarding the lipid metabolic profiles of ibd colons compared to controls. how lipid metabolomics, s1p degradation, and the enzymes of sphingolipid metabolism that are enriched in the gut epithelium may influence gut immunity and ibd still remain to be determined. the mechanisms that underlie the pathophysiology of ibd are complex and involve numerous signal transduction pathways that regulate innate and adaptive immunity in the gut. of particular interest are a triumvirate of interrelated pathways centered on the sphingolipid metabolite s1p, as well as on the nfb and stat3 inflammatory signaling pathways with which it interacts. s1p functions as a critical regulator of lymphocyte trafficking and a cofactor in inflammatory signaling.31 further, many s1p - related targets, including s1prs and the enzymes involved in s1p biosynthesis or degradation, are located in the epithelium of the small and large intestine wherein ibd arises.49 as such, these targets are of interest in the treatment of ibd and in the prevention of its progression to cac. in addition, dietary sphingolipids may represent another strategy for reducing colitis and preventing colon carcinogenesis. while some safety concerns remain, the preclinical evidence supports further investigation of these strategies in the context of ibd.	inflammatory bowel disease (ibd) is a complex disease that involves unpredictable and destructive inflammation in the gastrointestinal tract resulting in gastrointestinal symptoms, infection, and tissue destruction, and which can be associated with an increased risk of colon cancer. the underlying cause of ibd involves disruption of the innate and adaptive immune mechanisms that maintain homeostasis between the gut mucosa and its environment. elucidating how the homeostatic mechanisms controlling gut mucosal immunity and inflammation are disrupted in ibd represents the first steps to identifying novel therapeutic targets. sphingosine-1-phosphate (s1p) is a bioactive sphingolipid that is enriched in the blood and lymph, and functions in innate and adaptive immunity. s1p signaling regulates inflammation via its impact on the trafficking, differentiation, and effector functions of bone marrow - derived immune cells. s1p also activates nuclear factor kappa b and signal transducer and activator of transcription 3 inflammatory pathways. s1p is generated by the ubiquitously expressed lipid kinase, sphingosine kinase (sphk)1 and its tissue - restricted homolog, sphk2. s1p is irreversibly degraded by s1p lyase, which is highly expressed in enterocytes. recent studies targeting s1p metabolism and signaling have shown promise in preclinical models of ibd and have shed light on the mechanisms by which s1p signaling impacts ibd. the evidence suggests that targeting s1p signaling and metabolism may represent a novel strategy in treating ibd and it may reduce colon cancer risk by interrupting the progression from inflammation to carcinogenesis.
it is well - recognized that in chronic hemodialysis patients the rate of mortality is higher than that of general population. the cause of discrepancy is probably multifactorial, but malnutrition, anemia, hypoalbuminemia, reduced muscle mass and increased atherosclerosis are common in dialysis patients [1 - 3 ]. therapies designed to improve the nutritional status, control of anemia and atherosclerosis of hemodialysis patients might therefore be expected to improve outcome. anabolic agents, such as human growth hormone (hugh) can improve nitrogen balance, reduces urea generation and protein catabolic rate in long term hemodialysis patients. however hugh is expensive, can exacerbate hyperglycemia and may have a limited potential role as a long - term treatment for anabolism. the anemia of renal failure responses, directly to treatment with erythropoietin. because of the relatively high cost of recombinant human erythropoietin, the use of nandrolone decanoate (nd) a non-17alpha - alkylated androgen has been suggested instead of the above mentioned hormone. many studies have shown that nd not only increased rbc production, but also prolonged rbc survival. in spite that the drug is associated with high incidence of adverse effects including acne, virilization, abnormal liver function, discomfort from intramuscular injection, it can increase weight, triceps skinfold thickness, nitrogen balance and bone mineral density. moreover a reduction in the level of atherogenic lipoprotein (a) [lp(a) ] particles following nd administration to hemodialysis patients has been described. lp(a) is an important independent risk factor for atherosclerotic cardiovascular disease in general population as well as in hemodialysis patients [8 - 10 ]. the lipoprotein contributes at least partially to the increased cardiovascular risk seen in patients with renal failure. therefore it is clinically important to reduce high serum lp(a) by intervention [12 - 14 ]. lp(a) is a low - density lipoprotein particle in which apolipoprotein b-100 (apo b-100) linked by a single interchain disulfide bridge to a unique glycoprotein apolipoprotein(a) [apo(a) ]. in contrast to apo b-100, which remain relatively constant in molecular weight, apo(a), under genetic control, ranges between 300 and 800 kd [15 - 17 ]. the size of the apo(a) phenotypes f, b, s1, s2, s3 and s4 were inversely associated to plasma concentrations of lp(a) and family studies showed that a series of autosomal alleles at one locus are involved in determining both apo(a) size and lp(a) concentration. it is now demonstrated that plasma levels of lp(a) are controlled by synthesis rather than by catabolism. the purpose of this study was to analyze the effect of nd on nutritional status, hematological parameters, lipid profiles and serum levels of lp(a) in terms of differences in apo(a) isoforms in patients on hemodialysis. malnutrition, active hepatitis and benign prostatic hypertrophy (in males) were among the exclusion criteria. six patients were lost to follow - up (3 died from cardiac arrest, 2 received kidney transplant and 1 for abnormal liver function). sixty four patients (47 male and 17 female), mean age of 63 12 years completed the study period. the etiology of end - stage renal disease was diabetic nephropathy (n = 10), chronic glomerulonephritis (n = 17), polycystic disease (n = 5), urological problems (n = 5), chronic interstitial nephropathy (n = 4) and unknown (n = 23). all the patients underwent hemodialysis (4 hours) two to three times per week using cellulosic membrane. the dialysis schedule was not modified during period of the study. after taking of informed consent from the patients, nd 100 mg / i.m. fasting venous blood samples were collected before the start of nd treatment, at 2, 4 months during the treatment and 2 months after withdrawing of treatment. hemoglobin (hb) and hematocrit (hct) were measured by cell counter, and serum levels of total cholesterol (c), triglyceride (tg), high density lipoprotein cholesterol (hdl - c), albumin (alb) and creatinine (cr) in the collected samples were determined by using commercial reagents (randox) in an automated chemical analyzer (cobas mira). the serum levels of lp(a) were assayed by commercially available immunoturbidimetric kit using the same analyzer. the apo(a) phenotypes were determined by sodium dodecyl sulfate - polyacrylamide / agarose gel electrophoresis and immunoblotting with monoclonal apo(a) antibodies. apo(a) isoform sizes were determined by comparison of their sizes with an apo(a) standard (immuno - ag) electrophoresed on each gel which contained 5 different isoforms. low molecular weight (lmw) apo(a) phenotypes included all subjects who had at least one of the isoforms f, b, s1 or s2, whereas the high molecular weight (hmw) apo(a) phenotypes included all subjects with only s3 or s4 isoforms or with null type. paired t - test and wilcoxon signed ranks test as appropriate were used to assess significancy of differences between base and 2 months after withdrawing treatment. comparisons analysis between base, two and post of treatment values were performed using multiple comparison in one - way anova and friedman tests. all correlation were evaluated by liner regression and the statistical significance was set at p 0.1 and p > 0.2 respectively). the correlation between the baseline lp(a) levels and the degree of decreased lp(a) after 4 months of nd treatment [delta lp(a) ] in patients with high serum lp(a) concentration according to apo(a) molecular weight were analyzed in the patients with high serum lp(a) concentration. interestingly significant correlation in both high lp(a) with hmw phenotypes (r = 0.832, p < 0.01) (fig. 1) and high lp(a) with lmw phenotypes (r = 0.713, p < 0.01) patients groups were noticed (fig. correlation between basal serum lp(a)concentration and decrease in serum lp(a) after 4 months of nd treatment [delta lp(a) ] in patients with high serum lp(a) concentration in hmw molecular weight groups. delta lp(a) = lp(a) [after 4 months of nd treatment]-lp(a) [basal ] (r = 0.832 p < 0.01) correlation between basal serum lp(a)concentration and decrease in serum lp(a) after 4 months of nd treatment [delta lp(a) ] in patients with high serum lp(a) concentration in lmw molecular weight groups. delta lp(a) = lp(a) [after 4 months of nd treatment]-lp(a) [basal ] (r = 0.713 p < 0.01) changes of nutritional, hematological and lipid profile before the start of nd treatment, at, 2, 4 months during the treatment and 2 months after withdrawing treatment in 64 hemodialysis patients., comparison analysis between base, two and post of treatment values were performed using multiple comparisons by one - way anova., paired t - test was used to assess significancy of differences between base and 2 months after withdrawing treatment., comparison analysis between base, two and post of treatment values were performed using friedman test., wilcoxon signed ranks test was used to assess significancy of differences between the base and 2 months after withdrawing of treatment. effect of nd treatment on serum lp(a) according to basal lp(a) concentration and molecular weight of associated apo(a) isoforms. lp(a) levels are unaffected by lipid lowering dietary treatment, and are even increased by dietary trans fatty acids. furthermore, the lp(a) serum level is unaffected by most lipid lowering drugs [23,30 - 32 ]. neither treatment with hmg - coa reductase inhibitors, nor fibrate drugs have resulted in significant lowering of lp(a) levels. in the present study the effect of nd therapy on nutritional status, hematological parameters and lipid profiles with emphasis on lp(a) were evaluated in 64 stable hemodialysis patients. it has been reported that low levels of alb and cr were associated with a significantly greater risk of hemodialysis patients ' mortality. therefore therapeutic agents which increase alb and cr levels may decrease mortality in these patients. many studies have shown that treatment with nd resulted in significant increase in serum alb and cr levels. we also observed that treatment with nd increases the concentrations of the alb and cr and after the androgen withdrawal, levels of the parameters maintained at levels higher than the basal values at before of treatment. the findings suggest that nd treatment has beneficial effects on nutritional status in hemodialysis patients. it has been suggested that administration of androgen also has beneficial effect on erythropoiesis in patients on regular hemodialysis [2,5 - 7,34,35 ]. hb and hct values raised significantly with respect to basal values during the study although dose of the recombinant human erythropoietin were not changed during the therapy with nd. moreover after discontinuing use of the nd, levels of both parameters decreased slightly, but their levels still significantly were higher than basal values. our results suggest that nd can be used successfully in the treatment of anemia in hemodialysis patients. many studies concluded that androgen therapy is associated with high incidence of adverse effect in lipid profiles while others have shown that nd has no marked effect on the lipid profile. in this study evaluating the effect of the drug on lipid profiles showed significantly increase in c and marked decrease in hdl - c levels, without significant changes in tg level. although after withdrawal nd therapy the levels of c decreased and that of hdl - c increased, but the levels of both parameters were still significantly different from those of the basal values. the data confirm the finding of the others that, treatment with nd is associated with adverse effect on serum lipid profiles. we believe that the accompanying of lipid lowering agents during nd administration in hemodialysis patients maybe clinically useful and deserves another study. in our investigation significant reduction in the levels of lp(a) in hemodialysis patients after 4 months treatment with nd was observed. lp(a) is largely genetically determined by the rate of production of apo(a) and the protein levels mostly controlled by both synthesis and secretion [36 - 40 ]. the synthesis of apo(a) depends on the expression of apo(a) mrna in hepatocytes, which is regulated by transcriptional activity of the apo(a) gene. the secretion of apo(a) by hepatocytes in turns is affected by the molecular weight of the apo(a) isoforms. for comparing the transcriptional activity of the apo(a) gene in term of apo(a) molecular weight, all patients were classified into the following four groups : 1-high lp(a) concentration with hmw isoforms. because groups with high lp(a) concentration have higher transcriptional activity than groups with low lp(a) levels in same molecular weight isoforms, it is conceivable that, group 1 has a higher transcriptional activity than group 3, and group 2 has higher transcriptional activity than group 4. results have shown that nd lowered serum lp(a) concentration more effectively in patients with high lp(a) in both hmw and lmw isoforms (group 1, 2), and the decreased values for serum lp(a) are correlated with the pre - treatment (basal levels) of serum lp(a) in both groups. because it was established that, the secretory efficiencies of apo(a) in the same molecular weight isoforms are likely to be similar, a different in lp(a) reduction in the same molecular weight isoforms (group 1, 3 and group 2, 4) would represent a different in transcriptional activity of apo(a) gene. therefore it may be suggested that, nd lowers serum lp(a) concentration preferentially in patients with high serum lp(a) levels by effect on apo(a) gene transcription rather than effect on secretory efficiency of apo(a) in hemodialysis patients. in conclusion, nd administration has beneficial effects on nutritional status and treatment of anemia in hemodialysis patients. in spite of the fact that the drug has adverse effect on the lipid profiles, it reduces the level of lp(a) mostly in patients with high lp(a) levels irrespective of apo(a) isoform size.	malnutrition, anemia and increased atherosclerosis are the main causes of mortality in hemodialysis patients. therapies designed to improve the disorders might therefore be expected to improve outcome. the effects of nandrolone decanoate (nd), in 64 stable hemodialysis patients, were studied with respect to the following parameters : nutritional status, hematological indexes, lipid profiles including serum levels of lipoprotein(a) [lp(a) ] in terms of differences in apolipoprotein(a) [apo(a) ]. the patients were treated with nd at dose of 100 mg / i.m./week for 4 months. after 2 and 4 months of treatment the elevations in the serum levels of albumin (p 30 mg / dl) than those of with low lp(a) (< 30 mg / dl), irrespective of apo(a) molecular weight. it may be suggested that, nd has beneficial effect on nutritional status and treatment of anemia in hemodialysis patients. in spite the adverse effect of nd on lipid profile, it decreases lp(a) mostly in patients with high serum lp(a) preferently by the effect on apo(a) gene transcriptional activity.
the goal of endodontics is to prevent or treat apical periodontitis and most of the endodontic failures occur as the result of leakage of irritants through improperly sealed root end fillings into periradicular tissues.[25 ] an ideal orthograde or retrograde root canal filling material should seal the pathways of communication between root canal system and its surrounding tissues. the materials used in root canal therapy, particularly root end filling, are frequently in direct contact with soft and hard periodontal tissues ; therefore, a root filling material is necessary to be highly biocompatible and nontoxic. in 1990s, mineral trioxide aggregate (mta), a new root - ending endodontic material, was developed at the university of loma linda. mta is now used extensively in endodontics for pulp capping, pulpotomy, repair of root perforations, root end filling, root canal filling, and apical barrier formation in teeth with necrotic pulps and open apices. despite all the benefits listed for mta, it has also some disadvantages. the main drawbacks of mta include the potential for tooth discoloration, presence of some toxic elements in the material composition, high cost, long setting time, difficult handling, and difficulty in its removal after setting. efforts have been made to overcome these shortcomings. introducing new substitutes for mta should await comprehensive investigations, and new formulations have to be tested in vitro as well as in vivo before applying in human beings. recently, investigators of torabinejad dental research center at isfahan university of medical sciences (iums), isfahan, iran, have formulated new cements to be used in endodontics. these materials include calcium aluminate -aluminate cement (caac), calcium aluminate -aluminate plus cement (caac plus), and a mixture of 1 to 1 wollastonite and caac cement (wolca). caac contains calcium aluminates (60 - 70% ca, 10 - 15% ca2, and 0 - 5% c12a7) and alpha aluminate (-al2o3 5 - 15%). caac plus is a mixture of caac and 5% by weight sodium hexametaphosphate (na - hmp) to improve physical properties of caac. wollastonite is a naturally occurring calcium silicate (casio3) with a theoretical composition of 48.3% cao and 51.7% sio2. although there is an extensive knowledge on the biocompatibility of mta,[1517 ] the biological properties of these new cements (caac, caac plus, and wolca cement) have not been evaluated. thus, the purpose of this study was to compare the biocompatibility of these cements with each other and mta in subcutaneous connective tissue of rats. the protocol of this study was approved ethically by research council of the iums. in this experimental animal study, 27 healthy male wistar rats weighing 250 to 300 g were used. rats were anesthetized with an intramuscular injection of ketamine (60 mg / kg, alfasan, woerden - holland), acepromazine (2.5 mg / kg, alfasan, woerden - holland), and atropine (0.04 mg / kg, alfasan, woerden - holland). the dorsal skin was shaved and disinfected with povidone - iodine solution (10%) (daroupakhsh, tehran, iran). 15 scalpel blade and pockets were prepared in one direction by undermining the incisions longitudinally by blunt dissection for 20 mm. sterile polyethylene tubes (1.5 mm of inner diameter and 7 mm in length) were filled with mta (dentsply tulsa dental, tulsa, ok) caac, caac plus, and wolca cements which have been prepared according to manufacturer 's instructions, under aseptic conditions ; each tube were implanted in each subcutaneous pocket of rats. wounds were sutured for 7 days. at the end of experimental periods of 7, 14, and 30 days after histological processing, tissue samples were serially sectioned longitudinally to a thickness of 4 m and stained with hematoxylin and eosin. sections were evaluated under a light microscope (olympus ch30 rf200, olympus optical co., ltd. japan) equipped with a digital camera (sony exwavehad, tokyo, japan) using 10 and 40 objective lenses by two independent examiners in a blind manner. the occurrence of inflammatory response were scored according to previously established scores 0 (no reaction) for absence of inflammatory cells ; 1 + (mild reaction) for presence of mild chronic inflammatory infiltrate, or few eosinophilic or giant cells ; 2 + (moderate reaction) for presence of moderate chronic inflammatory infiltrate, or some eosinophilic or giant cells, or 3 + (severe reaction) for presence of an intense chronic inflammatory infiltrate, large number of eosinophilic or giant cells. differences between the inflammatory responses of sites were statistically analyzed using friedman test while wilcoxon test was used to compare individual pairs of groups. differences between the three sets of data were statistically analyzed by kruskal - wallis and mann - whitney tests. statistically significant differences were found between sites on 7, 14, and 30 days following implantation (p=0.018, p0.05). there were no statistically significant differences between mean inflammatory scores of matched sites in different experimental periods, except caac plus group, in which inflammation increased with time (p=0.024). photomicrographs of different inflammatory reactions are presented in figures 1 to 4. the 30-day mta specimen with grade + inflammation (400 mag) mild inflammatory cells (ic) are infiltrated in surrounding connective tissue (ct) the 30-day wolca specimen with grade + + inflammation (400 mag) moderate infiltration of inflammatory cells (ic) can be seen in connective tissue (ct) and around the capillaries (v) the 30-day caac plus specimen with grade + + + inflammation (400 mag) severe inflammatory cells (ic) are infiltrated in connective tissue the 30-day negative control specimen with grade 0 inflammation (100 mag) inflammatory cells around the connective tissues (ct) and muscles (m) are absent the term biocompatibility is often described as the ability of a material to perform with an appropriate host response in a specific application. because of continuous introduction of new dental materials, evaluation of their biologic potential is necessary. according to iso-6876 and 10993 - 5 standards, tissue implantation of different materials in the body of laboratory animals has been proposed. although data from laboratory animals could not be extended to human beings, it is considered as a valuable method to evaluate their biological properties. rat subcutaneous implantation studies are acceptable experimental models for this assessment and the inert nature of polyethylene tubes makes them suitable for implantation studies, so implantation of polyethylene or silicon tubes filled with endodontic materials into subcutaneous connective tissue of rats simulates in situ conditions of the materials. numerous studies have used mta to seal the natural, pathological, and iatrogenic communications between root canal system and periapical tissues. biocompatibility of mta has been reported in many in vitro and in vivo studies.[510151626 ] also, biocompatibility of new materials should be evaluated to ensure that a new material does not cause irritation, unwanted reactions, or tissue necrosis compared with control groups. the severity of inflammation against caac plus cement was higher than other test materials at all periods and the inflammatory response against this material increased with time. at 7 days after implantation of materials, no statistically significant difference was observed between experimental groups, and at 14 and 30 days, severity of inflammation against caac plus cement was significantly more than other groups. in this study, empty polyethylene tubes as negative control group revealed no inflammation to mild inflammatory response, which is similar to previous studies. initial inflammatory response to empty tubes is probably the result of surgical process of tube implantation. at 7 days, the mta group displayed a mild - to - moderate inflammatory response which was reduced to a mild reaction after 30 days. caac and wolca cement groups also revealed a similar response to mta which was not significantly different. according to these results, caac is a biocompatible material which is consistent to previous in vitro studies on biocompatibility of similar calcium aluminate cements. wolca cement is a mixture of caac and wollastonite in a ratio of 1 : 1. wollastonite is a naturally occurring calcium silicate, the composition of which is similar to mta. it seems that the new material made from mixing two biocompatible cements (caac and calcium silicate) is still biocompatible. weak inter - particle bonds in slurry of flocculated particles make the slurry more viscous than slurry of dispersed particles. in other words, if slurry contains highly dispersed particles, it will have a low viscosity. since the internal structure of dispersed slurry approaches that of liquid and dispersed particles could be packed more firmly than flocculated particles if particles in slurry are not sufficiently dispersed, the particle charge can be increased by adding a polyelectrolyte such as na - hmp. caac plus contains 5% by weight na - hmp as a dispersant to get these advantages, but the severity of tissue inflammation against caac plus increased by the time. evaluated the effects of adding na - hmp on basic properties of calcium phosphate cement and mentioned that although na - hmp made this cement more stable and improved its injectability properties, it weakened other basic properties of this cement - like compressive strength and increased its setting time. according to these findings, it seems that severe inflammatory response to caac plus cement can be due to its prolonged setting time. this is consistent with other investigations which have shown that delay in curing reaction of a substance causes an acute inflammatory response. previously, the ability of na - hmp to change the electrical charge of the materials has been demonstrated which alters the proteins and cells absorbed to the material 's surface. this issue can also explain the difference between the biocompatibility of this cement and caac. according to the results of the current study, biocompatibility of caac and wolca cements were comparable with that of mta, but caac plus showed higher inflammatory response than mta and is not biocompatible.	background : introducing new endodontic cements should await comprehensive investigations and new formulations have to be tested in vivo before applying in human beings. so, the purpose of this study was to compare the biocompatibility of new endodontic cements, calcium aluminate -aluminate cement (caac), calcium aluminate -aluminate plus cement (caac plus), and a mixture of wollastonite and caac cement (wolca) and mineral trioxide aggregate (mta), in subcutaneous connective tissue of rats.materials and methods : twenty - seven wistar rats were divided into three groups of 7, 14, and 30 experimental days. sterile polyethylene tubes were filled with mta, caac, caac plus, and wolca cement and implanted subcutaneously. empty tubes were implanted as negative control. after the experimental periods, animals were sacrificed by anesthetic overdosing. the occurrence of inflammatory responses was scored according to the previously established scores. data were statistically analyzed using friedman, wilcoxon, kruskal - wallis, and mann - whitney tests. the level of significance was 5% (p<0.05).results : there was a statistically significant difference between experimental and negative control sites in each group (p<0.05). caac plus showed the highest mean scores of inflammation, compared with mta, caac, and wolca cement sits at the end of all periods (p<0.05). there were no statistically significant differences between inflammatory scores of each site in different experimental groups, except caac plus sites, in which inflammation increased significantly with time (p<0.05).conclusion : according to the results of the current study, biocompatibility of caac and wolca cement were comparable with that of mta, but caac plus induced an inflammatory response higher than mta, therefore is not biocompatible.
supplementary material is available for this article at 10.1007/s13659 - 012 - 0038 - 8 and is accessible for authorized users.	three new triterpenoids, 3-hydroxy - urs-30-p - z - hydroxycinnamoyl-12-en-28-oic - acid (1), 3-hydroxy - olean-30-p - ehydroxycinnamoyl-12-en-28-oic - acid (2) and 3,6-dihydroxy - urs-14-en-12-one (3), together with seven known triterpenoids, were isolated from the roots of rubia schumanniana. their structures were established by means of spectroscopic analysis. all compounds were evaluated for cytotoxic activity, and compounds 26 showed cytotoxicity with the ic50 values of 10.7518.87 g / ml. electronic supplementary materialsupplementary material is available for this article at 10.1007/s13659 - 012 - 0038 - 8 and is accessible for authorized users.
epidermolysis bullosa (eb) consists of a heterogeneous group of rare autosomal dominant or recessive disorders, characterized by epithelial fragility. patients with recessive dystrophic epidermolysis bullosa (rdeb) often develop chronic constipation caused by painful defecation from blistering and tearing due to skin fragility, as well as by poor motility from opioid analgesia, which is frequently used in those with severe eb. as a result of straining, anal fissuring may further develop. due to these painful fissures and ongoing constipation, chronic anal fissures and anal sphincter spasm may result. an anal fissure is a tear in the anoderm (squamous epithelium) distal to the dentate line. by definition, an acute anal fissure heals within 6 weeks with conservative local management, whereas a chronic anal fissure fails conservative management, requiring more aggressive measures. this case demonstrates the successful use of botulinum toxin type a (btx - a) to relieve anal sphincter spasms and improve chronic anal fissures from eb. a 20-year - old female with generalized, severe rdeb gradually developed chronic anal fissures, resulting in severe anal sphincter spasms and severe, disabling pain. her quality of life (qol) was greatly diminished by this symptom (qoleb pilot score of 42/75 ; frew., 2009). she had inherited two heterozygous deletion mutations in the col7a1 gene, c[4918del ] in exon 52 and [7634del ] in exon 102, predicting two frameshift mutations, p. [gly1640fs ] and [gly2545fs ], leading to presumed downstream premature termination codons (venugopal., 2013). blistering was extensive from early infancy, causing cutaneous scarring and mitten deformities of hands and feet. she developed known complications of eb, including squamous cell carcinoma, hepatic amyloidosis (chaptini., 2015), premature and extensive dental caries, esophageal strictures, osteoporosis, hypogonadotrophic hypogonadism, and bilateral inferior exposure keratopathy. previous procedures included surgeries to correct hand contractures, right lower - lid ectropion repair, and regular iron and blood transfusions for anemia. medications included long - standing phenytoin 50 mg three times daily to aid wound healing, pantoprazole, cholecalciferol, estradiol / norethisterone, pregabalin, tramadol, oxycodone, and lactulose. g / l), hyponatremia (132 mmol / l), hypoalbuminemia (21 g / l), creatinine 48 mol / l, and liver function tests (lfts) demonstrating a cholestatic picture. anal manometry showed resting pressure 64 cmh2o (range : 54 - 104), reduced squeeze pressure 147 cmh2o (range : 179 - 317), and sphincter length 3.5 cm (range : 2.5 - 5). conservative measures for her anal fissures were trialed without success, including laxatives and a high - fiber diet. glyceryl trinitrate 0.2% ointment and zinc barrier were also unsuccessful. when she was referred in november 2008 for participation in a cell therapy clinical trial in sydney in 2009 (venugopal., 2013), we were asked about how to manage the fissuring. after topical tacrolimus failed to relieve any inflammation, the suggestion was made to consider intramuscular btx - a as a therapeutic intervention. over a period of 2 years starting from age 19 years, she underwent five btx - a injections into the internal anal sphincter (one injection approximately every 5 months), performed by a colorectal surgeon. in each case, a general anesthetic was used, and 50 units of btx - a in 2.5 ml of normal saline were injected into the internal anal sphincter in all four quadrants. the patient experienced a 50% subjective improvement in her pain and spasms for at least 1 month after each injection, followed by residual improvement for many months. four years after the cessation of injections, the patient s symptoms were still improved from baseline. chronic anal fissures often complicate rdeb, as these patients develop constipation early on in life because of anal discomfort and medications. once the tear in the anus mucosa occurs, it begins a cycle leading to repeated injury. the exposed internal sphincter muscle underneath the tear goes into spasm. as well as causing severe pain, the spasm pulls the edges of the fissure apart, impairing wound healing. the spasm also causes further tearing of the mucosa with each following bowel motion. in this case, btx - a injections successfully relieved the anal sphincter spasm and pain secondary to recurrent anal fissures by interrupting the cycle of repeated injury. botulinum toxin, produced by clostridium botulinum, blocks cholinergic nerve terminals and inhibits release of acetylcholine from nerve endings. it has been used to successfully treat certain spastic disorders of skeletal muscle, such as blepharospasm and torticollis, as well as wrinkles and spastic contractures in cerebral palsy. although there have been no documented cases of its use in eb for anal sphincter spasm or fissuring, it is commonly used to treat idiopathic anal sphincter spasm, typically given as injections around the anal canal (10 to 100 units total). it has also been reported to improve healing in patients with chronic anal fissures (maria., 1998b). despite variable doses and locations of the injection,, 2004, brisinda., 2007, jost and schrank, 1999, maria., 1998a, mnguez., 1999) the healing rate appears to be related to the dose and probably to the number of puncture sites (maria., 1998a, mnguez., 1999). in a randomized controlled trial, where 100 patients were treated with either btx into the internal anal sphincter or 0.2% nitroglycerin ointment for 8 weeks, after 2 months the fissures were healed in 46 (92%) of 50 patients in the btx group and in 35 (70%) of 50 in the nitroglycerin group (p = 0.009). three patients in the btx group reported adverse effects (p < 0.001) from mild incontinence to flatus that lasted 3 weeks after treatment but disappeared spontaneously (brisinda., 2007). in another randomized controlled trial, 50 patients with chronic anal fissures received injections of 50 units of botox formulation (group i), and 50 patients received injections of 150 units of dysport toxin (group ii ; brisinda., 2004). after 2 months, 46 patients in group i and 47 patients in group ii had a healed scar. in group i patients, the mean resting anal pressure was 41.8% lower and the maximum voluntary squeeze pressure was 20.2% lower than the baseline value. in group ii patients, the resting anal pressure and maximum voluntary squeeze pressure were 60.0 12.0 mmhg and 71.0 30.0 mmhg, respectively. there were no relapses in the 21 months of follow - up (brisinda., 2004). similarly, in a study where 30 patients with chronic anal fissures were randomized to intra - sphincteric btx - a or saline injection, after 2 months significantly more patients who had received btx - a had healed (73% versus 13% ; maria., 1998a). this was supported by another study where repeated btx - a therapy was used in 50 patients who failed initial (insufficient) btx - a treatment. nineteen of 20 patients (95%) treated with five units of btx - a were pain - free within 1 week, and healing was observed in 70% by 3 months. twenty - two of 30 patients (73%) treated with 10 units of btx - a were pain - free within 1 week, and 19 (63%) demonstrated healing after 3 months (jost and schrank, 1999). botulinum toxin injections have been used twice before in patients with eb in the literature for reasons other than anal sphincter spasm. btx - a has been used to treat palmar and plantar hyperhidrosis, which was found to be effective and safe (schnider., 1997, vadoud - seyedi, 2004). as hyperhidrosis contributes to frictional trauma and the development of blisters in eb simplex (ebs), it was thought that btx - a may have a therapeutic role in this condition. hence, btx - a was used in a double - blinded - study of a 43-year - old woman with ebs, localized type, to treat blisters and erosions on the plantar aspect of her feet (abitbol and zhou, 2009). the foot treated with btx - a had decreased pain and perspiration and a 64% decrease in blister formation after 3 months compared to the control foot, which received normal saline injections (abitbol and zhou, 2009). furthermore, a retrospective study of 14 patients (6 with ebs) with foot blisters who had received botulinum toxin therapy showed that 13 (93%) patients reported reduced blistering and pain, with a mean effect of 3 months (swartling., 2010). the mechanism of action of btx - a in the above cases is via blocking the autonomic cholinergic junctions of the post - ganglionic sympathetic fibers to the sweat glands, thus preventing hyperhidrosis. the mechanism of action in improving chronic anal fissures and spasm, as depicted in this case, is likely mainly due to cholinergic nerve terminal blockade, preventing spasm, as well as blockade of the post - ganglionic sympathetic fibers, preventing sweating, and subsequent fissuring. the main adverse effects after local injection of btx - a are weakness in the muscles adjacent to the treatment sites and, in the perianal area, fecal incontinence. beneficial effects have been shown to last from 4 months to 1 year (swartling., 2010). the long - term outcome of btx - a treatment has not been well described in the literature ; recurrence may be common. the report with the longest follow - up (42 months) showed that in 57 patients who had healed anal fissures from btx 6 months after injection, a recurrent fissure was observed in 22 patients (42% ; minguez., 2002). however, in the case described, long - term benefits remained after 4 years, indicating that this is a useful adjunctive treatment for rare patients with these disabling symptoms. this case demonstrates the successful treatment of chronic anal fissures and anal sphincter spasm with botulinum toxin a injections, with nil adverse effects. botulinum toxin a injections should be considered as a treatment for anal sphincter spasm secondary to recurrent anal fissures in eb when conservative measures fail.	we report a 20-year - old female with generalized, severe, recessive dystrophic epidermolysis bullosa who developed secondary chronic anal fissures. this resulted in anal sphincter spasm and severe, disabling pain. she was treated with five botulinum toxin a injections into the internal anal sphincter over a period of 2 years and gained marked improvement in her symptoms. this case demonstrates the successful use of botulinum toxin a injections to relieve anal sphincter spasm and fissuring, with long - term improvement.
the setting was kaiser permanente northern california (kpnc), an integrated health care delivery system that provides medical care for approximately one - third of the underlying population in the san francisco bay area. the source population consisted of women kpnc members who completed a voluntary multiphasic health checkup (mhc) at the kaiser permanente oakland medical center between 1984 and 1995. kpnc members at this facility were invited to complete a comprehensive health check - up upon enrollment. the mhc consisted of a clinic visit for the completion of questionnaires and clinical measurements, including blood pressure, weight, and serum glucose and cholesterol (measured in serum obtained from a random blood draw). the goal of the mhc was to provide health maintenance through early diagnosis (15). bmi was calculated as weight in kilograms divided by the square of height in meters ; height was measured using a stadiometer and weight using a balance beam scale. information on age, sex, race / ethnicity, education level, cigarette smoking, family history of diabetes, medical history, alcohol consumption, coffee consumption, and use of medications and hours since last food ingestion was collected using self - administered questionnaires (15). serum glucose was measured on serum obtained from a random blood draw using the hexokinase method, and total cholesterol was assessed using a kodak ektachem chemistry analyzer by the regional laboratory of kpnc at the time of the mhc exam. this laboratory participates in the college of american pathologists ' accreditation and monitoring program. among women 1545 years of age who participated in the mhc from 1985 to 1996 (n = 27,743 with clinical and questionnaire data, as well as an extra serum sample), we identified 4,098 women who subsequently delivered an infant by 2010 by searching the kpnc hospitalization database and the pregnancy glucose tolerance and gdm registry (16), an active surveillance registry that annually identifies all pregnancies resulting in a live birth or stillbirth among kpnc members. it also captures the results of all screening and diagnostic tests for gdm from kpnc s electronic laboratory database (data available since 1994). this is a nested case - control study within a cohort of 4,098 women who took part in an mhc exam, had an extra tube of serum stored for future use, and had a subsequent pregnancy on average, 6 years after the mhc exam. all cohort members who went on to develop gdm were included as case subjects ; two control subjects were selected for each case from among women not meeting the gdm case definition. we identified 267 women with gdm according to the kpnc electronic databases : case subjects had either 1) glucose values obtained during a standard 100-g, 3-h oral glucose tolerance test that met the american college of obstetricians and gynecologists plasma glucose thresholds for gdm (18) in the laboratory database (n = 228) or 2) a hospital discharge diagnosis of gdm in the electronic hospital discharge database for pregnancies occurring before the electronic laboratory data were available (prior to 1994 ; n = 39). standardized medical chart review was conducted by trained abstractors to confirm that these 267 women had a 100-g, 3-h oral glucose tolerance test meeting the american college of obstetricians and gynecologists criteria (18) for gdm (plasma glucose thresholds : fasting 5.3 mmol [95 mg / dl ], 1 h 10.0 mmol / l [180 mg / dl ], 2 h 8.6 mmol / l [155 mg / dl ], and 3 h 7.8 mmol / l [140 mg / dl ]). case subjects were excluded if at the time of the mhc exam they had a random glucose > 200 mg / dl (n = 6), no indication of gdm during the index pregnancy (n = 4) or impaired glucose tolerance with insufficient follow - up testing (n = 1), leaving a total of 256 confirmed cases of gdm. from among those women without an indication of gdm, control subjects were randomly selected ; two control subjects were individually matched to each case on year of mhc serum collection date (3 months), age at mhc serum collection (2 years), number of intervening pregnancies (0, 1, 2), and age at delivery of the index pregnancy (2 years). we matched for the year of serum collection to account for any potential degradation in the quality of the serum over time, thereby assuring the sample storage time was approximately the same for case and control subjects. since gdm is more common in older women, we matched on age at serum collection and age at delivery. we matched on number of pregnancies to account for any differences in pregnancies between the initial exam and the index pregnancy. control subjects were excluded from the analysis if they had glucose values diagnostic of gdm found during medical chart abstraction (n = 5), had an abnormal screening glucose but no follow - up diagnostic glucose test (n = 5), or had one abnormal glucose value on the diagnostic glucose test (n = 5), suggestive of mild gdm. of serum samples were thawed, aliquoted, and transported in batches on dry ice to the laboratory of p.j.h. at the university of california, davis, for analysis. serum adiponectin was measured with a commercially available radioimmunoassay (millipore [formerly linco research ]) using i - labeled murine adiponectin and a multispecies anti - adiponectin antibody. the assay has a sensitivity of 1 ng / ml and a linearity of 200 ng / ml. the intra- and interassay coefficients of variation are 200 mg / dl (n = 6), no indication of gdm during the index pregnancy (n = 4) or impaired glucose tolerance with insufficient follow - up testing (n = 1), leaving a total of 256 confirmed cases of gdm. from among those women without an indication of gdm, control subjects were randomly selected ; two control subjects were individually matched to each case on year of mhc serum collection date (3 months), age at mhc serum collection (2 years), number of intervening pregnancies (0, 1, 2), and age at delivery of the index pregnancy (2 years). we matched for the year of serum collection to account for any potential degradation in the quality of the serum over time, thereby assuring the sample storage time was approximately the same for case and control subjects. since gdm is more common in older women, we matched on age at serum collection and age at delivery. we matched on number of pregnancies to account for any differences in pregnancies between the initial exam and the index pregnancy. control subjects were excluded from the analysis if they had glucose values diagnostic of gdm found during medical chart abstraction (n = 5), had an abnormal screening glucose but no follow - up diagnostic glucose test (n = 5), or had one abnormal glucose value on the diagnostic glucose test (n = 5), suggestive of mild gdm. serum samples were thawed, aliquoted, and transported in batches on dry ice to the laboratory of p.j.h. at the university of california, davis, for analysis. serum adiponectin was measured with a commercially available radioimmunoassay (millipore [formerly linco research ]) using i - labeled murine adiponectin and a multispecies anti - adiponectin antibody. the assay has a sensitivity of 1 ng / ml and a linearity of 200 ng / ml. the intra- and interassay coefficients of variation are 6.2 years (the median time since exam), 5.0 (95% ci 2.012.0), compared with when it had been 6 h found similar adjusted ors associated with being in the lower two quartiles of adiponectin and gdm risk (quartile 2, 3.4 [95% ci 1.67.1 ], and quartile 1, 4.3 [95% ci 2.09.4 ]), compared with quartile 4. among this subset, we further adjusted for homeostasis model assessment of insulin resistance (homa - ir) and found that the ors associated with being in the lower two quartiles of adiponectin were slightly attenuated but remained significant (quartile 2, 3.2 [95% ci 1.56.9 ], and quartile 1, 3.7 [95% ci 1.78.1 ] compared with quartile 4). finally, we examined the association between adiponectin and gdm among a subset of women without the strongest risk factors for gdm : women who were normal weight (bmi < 25.0 kg / m) and had no family history of gdm (n = 55 case and n = 224 control subjects). among this subset of low - risk women, the or associated with continuous adiponectin was 0.70 (95% ci 0.560.88) for hmw and 0.84 (95% ci 0.760.92) for total adiponectin after adjustment for matching variables, bmi (continuous), parity, and race. in this nested case - control study, we found that lower adiponectin concentrations measured, on average, 6 years before pregnancy were associated with a 5.0-fold increased risk of developing gdm. we found similar associations between total and hmw adiponectin and gdm even when the measurement occurred 6 years before pregnancy, confirming the robustness of the association. of note, these relationships were independent of known risk factors for gdm, including bmi, age, and race / ethnicity, as well as markers of insulin resistance (specifically, glucose and insulin) that have been associated with adiponectin (7) concentrations and the development of reduced glucose tolerance in both pregnant and nonpregnant populations (21,22). our findings are among the first to suggest that low circulating adiponectin concentrations may predict gdm years prior to pregnancy and extend existing knowledge pertaining to pregravid risk factors for gdm. we found that the association between prepregnancy adiponectin and gdm risk remained a significant risk factor for gdm among the subset of women who were normal weight and had no family history of gdm : two strong risk factors for gdm. this finding is of clinical relevance because it suggests that adiponectin may help identify a group of high - risk women who may otherwise not be identified as being at high risk of developing gdm. our findings are consistent with previous studies of adiponectin and type 2 diabetes. a systematic review and meta - analysis of prospective studies examining adiponectin and incident type 2 diabetes found that higher adiponectin levels were associated with a 30% lower risk of type 2 diabetes (relative risk [rr ] 0.72 [95% ci 0.670.78 ]) per 1 log g / ml increment in adiponectin levels, consistent with a dose - response relationship (11). less is known about the role of adiponectin in gdm risk. a couple of studies assessing the prospective association between adiponectin levels in the first trimester of pregnancy and the risk of gdm found that women with gdm had lower levels of adiponectin compared with women who did not develop gdm (2325), which is consistent with the current study. other previous studies (26) with a small sample size examined adiponectin levels during the third trimester and gdm (24,27). however, since both total adiponectin (12) and hmw adiponectin (13) have been shown to decrease significantly in normal pregnancies, the previous studies were not able to assess whether the association between adiponectin and increased risk of gdm was related only to the physiologic changes that accompany normal pregnancy. pregnancy - induced changes such as rapid increases in body weight and fat, insulin resistance, inflammation, and lipids are related to both lower adiponectin and reduced glucose tolerance (7). the findings of our prospective study suggest that altered adiponectin levels in women with normal glucose metabolism years before pregnancy may lead to decreased glucose tolerance during pregnancy, such as gdm. the underlying etiology of gdm is believed to be diminished insulin secretion prepregnancy coupled with pregnancy - induced insulin resistance (5). adiponectin has been shown to promote -cell function and survival and decrease hepatic glucose output (thereby lowering systemic glucose levels) (28). therefore, low adiponectin levels may lead to both reduced insulin secretion and increased insulin resistance. in human studies, adiponectin has been shown to be inversely related to visceral adiposity (29) and liver fat accumulation (30) and positively correlated with truncal fat, all of which have been shown to be associated with insulin resistance and diabetes risk independent of bmi (28). we found no evidence that weight gain either before pregnancy affected the association between adiponectin and gdm regardless of baseline bmi. however, adiponectin levels have been shown to increase after significant weight loss either by caloric restriction or from weight loss surgery (gastric bypass) (28), and medications that increase the number of small adipocytes, such as thiazolidinediones, also increase adiponectin production (31). while this suggests that adiponectin can be modified, more information is needed to determine strategies for increasing circulating adiponectin concentrations to better inform possible prevention strategies for both gdm and type 2 diabetes. strengths of this study include our ability to exclude women with glucose values indicative of recognized, pregestational diabetes. we had the unique ability to look at adiponectin levels measured several years before pregnancy on a large number of gdm case and matched control subjects. we were able to control for markers of insulin resistance (homa - ir) among a subset, and our findings remained when adjusted for potential mediators. the study was limited by the lack of data on more informative measures of adiposity in addition to bmi, such as waist circumference or percent body fat, and we therefore were not able to assess whether the association between adiponectin and gdm was possibly mediated by increased visceral fat. we also lacked information on diet and physical activity changes that may have occurred from the baseline exam to the subsequent pregnancy ; therefore, we were unable to assess the impact of lifestyle changes on gdm risk in this study. we only had a single measurement of adiponectin, which may be subject to variation ; such misclassification would be nondifferential and bias our results toward the null hypothesis. finally, our samples were nonfasting ; however, the majority of studies have found either no or only a minor effect of feeding / fasting on circulating adiponectin concentrations (7), and our findings were similar in the subanalysis restricted to women who fasted for 6 h. in summary, after adjusting for potential confounding factors and clinical factors known to be related to insulin resistance, we found that low adiponectin concentrations, measured on average 6 years prior to pregnancy, were associated with a fivefold increased risk of gdm. circulating concentrations of total and hmw adiponectin represent potentially useful new biomarkers regarding who is at risk for gdm beyond the currently established clinical and demographic risk factors. future studies designed to be able to assess the sensitivity and specificity of adiponectin in predicting gdm will be valuable to help further clarify the clinical utility of these biomarkers.	objectiveto examine whether circulating total and high molecular weight (hmw) adiponectin concentrations, measured before pregnancy, are associated with subsequent risk of gestational diabetes mellitus (gdm).research design and methodsthis was a nested case - control study among women who participated in the kaiser permanente northern california multiphasic health check - up exam (19841996) with a serum sample obtained and who had a subsequent pregnancy (19842009). eligible women were free of recognized diabetes. case subjects were the 256 women who developed gdm. two control subjects were selected for each case and matched for year of blood draw, age at exam, age at pregnancy, and number of intervening pregnancies.resultscompared with the highest quartile of adiponectin, the risk of gdm increased with decreasing quartile (odds ratio [or ] 1.5 [95% ci 0.72.9 ], 3.7 [1.97.2 ], and 5.2 [2.610.1 ] ; ptrend < 0.001) after adjustment for family history of diabetes, bmi, parity, race / ethnicity, cigarette smoking, and glucose and insulin concentrations. similar estimates were observed for hmw (ptrend < 0.001). the combined effects of having total adiponectin levels below the median (< 10.29 mg / ml) and being overweight or obese (bmi 25.0 kg / m2) were associated with a sevenfold increased risk of gdm compared with normal - weight women with adiponectin levels above the median (or 6.7 [95% ci 3.612.5]).conclusionsprepregnancy low adiponectin concentrations, a marker of decreased insulin sensitivity and altered adipocyte endocrine function, is associated with reduced glucose tolerance during pregnancy and may identify women at high risk for gdm to target for early intervention.
several cell lines were isolated during independent experiments from dissected annulus fibrosus (af) tissue of mature bovine intervertebral discs (ivd) via a reproducible non - enzyme driven protocol. the cell lines were frozen at low passage number and they recovered well after freeze - thawing (see fig. preliminary characterization of the af cells was carried out with bovine specific rna probes derived from bovine genomic dna using plate rna in situ hybridisation (pish) for col1a1 and col2a1 expression, two structural proteins found in the mature ivd. less type - ii collagen fibers were described for the outer af in rabbit correlating with a common notion that type ii collagen is higher in the np than the af,. the dissected outer af of mature bovine ivds was the source for our af cell lines and we did not detect col2a1 expression by either rna in situ hybridization (sish),,, on sections of the outer af tissue or by pish on the cells derived from the outer af, while col2a1 expression was very prominent in cells of the np as shown by sish on the same section (fig. the discrepancy between our findings and that of increased col2a1 expression in the bovine af over the np reported by minoque. might reflect differences in defining the af. we see col1a1 expression in af and np cells by sish in vivo and by pish in vitro (fig. skinned bovine tails were collected fresh from local abattoirs, remained chilled and were processed within 2 h. tail pieces were immersed in 10% povidone - iodine solution, rinsed with tap water, followed by immersion in 70% etoh prior to removing all fat and muscle tissue. ivds were dissected away from adjacent vertebrae endplates, briefly dipped in 70% etoh and rinsed with 1 pbs/10% gentamicin prior to separating the outer af from the remaining ivd tissue. outer af tissue was cut into smaller pieces using sterile procedures and placed in uncoated as well as 0.1% gelatin coated 35 mm culture dishes (falcon). sterile filtered fbs - hi with 10% gentamicin and 5 g / ml amphotericin b (all gibco) was added prior to the incubation at 37c, 5% co2 and atmospheric o2. after 24 h the fbs mix was diluted 1:1 with standard dmem based growth medium containing 1 dmem with 4.5 g / l glucose, 1 pyruvate, 1 glutamax, 1 nonessential amino acids, 10% v / v hi - fbs, 0.48% v / v gentamicin (all gibco), 0.12 mm beta - mercapthoethanol (sigma) and additional 5 g / ml amphotericin b and the tissue. following 48 h of incubation cells had attached to the bottom of the wells and were expanded in fresh standard dmem based growth medium (see above). cell lines derived in such manner from af tissue could be passaged with 0.05% trypsin / edta (gibco) at 1:10 dilutions for more than 10 passages without slowing down in population growth or dramatic changes in morphology (fig. early and late passages were subjected to plate rna in situ hybridization (pish) for preliminary gene expression analysis (fig. during embryogenesis, the af part of the ivd is believed to be of sclerotomal origin,. cultured cells derived from the outer af of mature bovine caudal ivds with our procedure were assayed for the expression of two major collagen genes col1a1 and col2a1. the observed in vitro expression of these two genes mirrored the in vivo expression in cells of the mature the af : presence of col1a1 expression and absence of col2a1 expression in cells of the outer af (fig.	the adult bovine (bos taurus) intervertebral disc is primarily comprised of two major tissue types : the outer annulus fibrosus (af) and the central nucleus pulposus (np). we isolated several primary cell lineages of passage (p) 0 cells from the af tissue omitting typically used enzymatic tissue digestion protocols. the cells grow past p10 without signs of senescence in dmem + 10% fcs on 0.1% gelatin coated / uncoated surfaces of standard cell culture plates and survive freeze - thawing. preliminary analysis of the af derived cells for expression of the two structural genes col1a1 and col2a1 was performed by pish recapitulating the expression observed in vivo.
in 2006, epstein. reported that hypomagnesaemic hypoparathyroidism could be caused by long - term use of a proton - pump inhibitor (ppi), omeprazole 1. thereafter, case reports accumulated, in which ppis were shown to be associated with hypomagnesaemia 2 - 11, and in 2011, the us food and drug administration (fda) published a safety announcement that long - term use of ppis can lead to hypomagnesaemia 12. although recognized as a rare side effect of ppis, hypomagnesaemia is a serious condition that can be complicated by life - threatening arrhythmias and neurologic manifestations 10, 11. exactly how ppis could cause hypomagnesaemia has not been clarified, and controlled studies are required to delineate the mechanisms 13. symptoms include tetany, seizures, muscle cramps, vomiting, nausea, and diarrhea, but these are not always found in patients with hypomagnesaemia 10, 11. most reports on ppi - induced hypomagnesaemia concern omeprazole or esomeprazole, the s - isomer of omeprazole, but the recurrence after substitution by other ppis suggests that this is a class effect commonly found for ppis. the present study was performed to assess omeprazole and esomeprazole in terms of susceptibility to hypomagnesaemia, and to this end, more than a million case reports on adverse drug events submitted to the fda database were reviewed. input data for this study were taken from the public release of the data in the fda 's adverse event reporting system (aers), which covers the period from the first quarter of 2004 through the end of 2009. this database relies on spontaneous reports of adverse drug events by health professionals, consumers, and manufacturers. the data structure of aers is in compliance with international safety reporting guidance ich e2b issued by the international conference on harmonisation, consisting of 7 data sets : patient demographic and administrative information (demo), drug / biologic information (drug), adverse drug events (reac), patient outcomes (outc), report sources (rpsr), drug therapy start and end dates (ther), and indications for use / diagnosis (indi). the adverse drug events in reac are coded using preferred terms (pts) in the medical dictionary for regulatory activities (meddra) terminology. prior to analysis, all drug names were unified into generic names by a text - mining approach, because aers permits the registering of arbitrary drug names, including trade names and abbreviations. spelling errors were detected by a spell checker software, gnu aspell, and carefully confirmed by working pharmacists. foods, beverages, treatments (e.g. x - ray radiation), and unspecified names (e.g. beta - blockers) were omitted for this study, and the total number of omissions was 164,384. finally, duplicated reports were deleted according to the fda 's recommendation of adopting the most recent case number, resulting in a reduction in the number of reports from 2,231,029 to 1,644,220. a total of 22,017,956 co - occurrences were found in 1,644,220 reports, where a co - occurrence was a pair of a drug and an adverse drug event. in pharmacovigilance analyses, data mining algorithms have been developed to identify an association between a drug and an adverse drug event or a drug - associated adverse drug event as a signal that is reported more frequently than expected by estimating expected reporting frequencies on the basis of information on all drugs and all adverse drug events in a database 14 - 20. for example, the proportional reporting ratio (prr) 14, the reporting odds ratio (ror) 15, the information component (ic) 16, and the empirical bayes geometric mean (ebgm) 17 are widely used. indeed, the prr is currently used by the uk medicines and healthcare products regulatory agency (mhra), the ror by the netherlands pharmacovigilance centre, the ic by the world health organization (who), and the ebgm by the fda. all of these algorithms extract decision rules for signal detection and/or calculate scores to measure an association between a drug and an adverse drug event from a two - by - two frequency table of counts that involve the presence or absence of a particular drug and a particular adverse drug event occurring in case reports. these algorithms, however, differ from one another in that the prr and ror are frequentist (non - bayesian) ones, whereas the ic and ebgm are bayesian ones. in this section, only the scoring thresholds used in the present study are given, and the reader is referred to review articles for more extensive details of each statistical test 18 - 20. in this section, we define how the association between a drug and an adverse drug event is classified as a signal, when using each statistical test. using the prr, a signal is detected if the count of co - occurrences is 3 or more, and the prr is 2 or more with an associated value of 4 or more 14. for the ror, a signal is detected if the lower bound of the 95% two - sided confidence interval of ror exceeds 1 15. signal detection using the ic is done using the ic025 metric, a criterion indicating the lower bound of the 95% two - sided confidence interval of the ic, and a signal is detected if the ic025 value exceeds 0 16. finally, the eb05 metric, a lower one - sided 95% confidence limit of ebgm 17, is used and a signal is detected when eb05 is greater than or equal to the threshold value 2. in this study, the adverse drug events coded by pt numbers were listed as omeprazole- and esomeprazole - associated, when at least 1 of 4 indices met the criteria indicated above, and subsequently hypomagnesaemia was identified by the pt code number 10021027. input data for this study were taken from the public release of the data in the fda 's adverse event reporting system (aers), which covers the period from the first quarter of 2004 through the end of 2009. this database relies on spontaneous reports of adverse drug events by health professionals, consumers, and manufacturers. the data structure of aers is in compliance with international safety reporting guidance ich e2b issued by the international conference on harmonisation, consisting of 7 data sets : patient demographic and administrative information (demo), drug / biologic information (drug), adverse drug events (reac), patient outcomes (outc), report sources (rpsr), drug therapy start and end dates (ther), and indications for use / diagnosis (indi). the adverse drug events in reac are coded using preferred terms (pts) in the medical dictionary for regulatory activities (meddra) terminology. prior to analysis, all drug names were unified into generic names by a text - mining approach, because aers permits the registering of arbitrary drug names, including trade names and abbreviations. spelling errors were detected by a spell checker software, gnu aspell, and carefully confirmed by working pharmacists. foods, beverages, treatments (e.g. x - ray radiation), and unspecified names (e.g. beta - blockers) were omitted for this study, and the total number of omissions was 164,384. finally, duplicated reports were deleted according to the fda 's recommendation of adopting the most recent case number, resulting in a reduction in the number of reports from 2,231,029 to 1,644,220. a total of 22,017,956 co - occurrences were found in 1,644,220 reports, where a co - occurrence was a pair of a drug and an adverse drug event. in pharmacovigilance analyses, data mining algorithms have been developed to identify an association between a drug and an adverse drug event or a drug - associated adverse drug event as a signal that is reported more frequently than expected by estimating expected reporting frequencies on the basis of information on all drugs and all adverse drug events in a database 14 - 20. for example, the proportional reporting ratio (prr) 14, the reporting odds ratio (ror) 15, the information component (ic) 16, and the empirical bayes geometric mean (ebgm) 17 are widely used. indeed, the prr is currently used by the uk medicines and healthcare products regulatory agency (mhra), the ror by the netherlands pharmacovigilance centre, the ic by the world health organization (who), and the ebgm by the fda. all of these algorithms extract decision rules for signal detection and/or calculate scores to measure an association between a drug and an adverse drug event from a two - by - two frequency table of counts that involve the presence or absence of a particular drug and a particular adverse drug event occurring in case reports. these algorithms, however, differ from one another in that the prr and ror are frequentist (non - bayesian) ones, whereas the ic and ebgm are bayesian ones. in this section, only the scoring thresholds used in the present study are given, and the reader is referred to review articles for more extensive details of each statistical test 18 - 20. in this section, we define how the association between a drug and an adverse drug event is classified as a signal, when using each statistical test. using the prr, a signal is detected if the count of co - occurrences is 3 or more, and the prr is 2 or more with an associated value of 4 or more 14. for the ror, a signal is detected if the lower bound of the 95% two - sided confidence interval of ror exceeds 1 15. signal detection using the ic is done using the ic025 metric, a criterion indicating the lower bound of the 95% two - sided confidence interval of the ic, and a signal is detected if the ic025 value exceeds 0 16. finally, the eb05 metric, a lower one - sided 95% confidence limit of ebgm 17, is used and a signal is detected when eb05 is greater than or equal to the threshold value 2. in this study, the adverse drug events coded by pt numbers were listed as omeprazole- and esomeprazole - associated, when at least 1 of 4 indices met the criteria indicated above, and subsequently hypomagnesaemia was identified by the pt code number 10021027. the total number of co - occurrences with omeprazole and esomeprazole was 178,766 and 121,506, representing 0.812% and 0.552% of all co - occurrences in the database, respectively. in total, 818 and 743 adverse drug events were listed as omeprazole- and esomeprazole - associated with 55,904 and 48,481 co - occurrences, respectively. hypomagnesaemia ranked 85 among 818 omeprazole - associated adverse drug events, and 135 among 743 for esomeprazole. the statistical data on omeprazole- and esomeprazole - associated hypomagnesaemia are listed in table 1. an association with hypomagnesaemia was suggested for both ppis, but the association was more noteworthy for omeprazole. magnesium is an essential factor implicated in many biochemical and physiological processes, and its homeostasis is sophisticatedly regulated by intestinal absorption, renal excretion and other systems in the body 10, 11. hypomagnesaemia or hypermagnesaemia may arise from various types of disorders 10, 11. in 2006, in which a ppi, omeprazole, was shown to be associated with hypomagnesaemia 1. to date, about 10 case reports have been published with respect to ppi - associated hypomagnesaemia 2 - 9, and their findings can be summarized as ; 1) ppi long - term use was observed in patients with hypomagnesaemia, 2) symptoms did not occur until plasma concentrations were less than 0.5 mmol / l, 3) mechanisms by which the hypomagnesaemia occurred under ppi therapy remain unclear, 4) hypokalaemia often accompanied the hypomagnesaemia, 5) hypocalcaemia also frequently developed via impairment of parathyroid hormone secretion, 6) oral or parenteral supplement of magnesium was effective for temporary relief from symptoms, but unable to correct the plasma concentration of magnesium, and 7) withdrawal of ppi allowed to resolve the hypomagnesaemia 10, 11. hypomagnesaemia is understood to be a rare side effect of ppis, but epstein. hypomagnesaemia might be underdiagnosed, in part, due to the relatively low frequency of magnesium measurements in routine clinical analysis. if hypomagnesaemia is found in ppi users, it might be attributed to co - administered diuretics or other nephrotoxic drugs. it is important to perform clinical studies to clarify the true prevalence and risk factors, and to clarify the mechanisms by which hypomagnesaemia develops. to date, most case reports on ppi - associated hypomagnesaemia concern omeprazole or esomeprazole, but hypomagnesaemia is understood to be common for ppis. broeren. showed that hypomagnesaemia was resolved after the replacement of omeprazole with a h2-blocker, ranitidine, but the re - replacement of ranitidine with pantoprazole resulted in recurrence 5. they also documented another case in which the replacement of omeprazole with rabeprazole resulted in a further decrease in serum levels of magnesium 8. in this study, using 1,644,220 reports from 2004 to 2009, it was suggested that hypomagnesaemia was associated with omeprazole and esomeprazole, and was more noteworthy for omeprazole, suggesting the usefulness of the aers database and official pharmacovigilance tools. although pantoprazole, lansoprazole and rabeprazole were also analyzed, the numbers of co - occurrences were not large enough to detect signals. the first clinical report on ppi - associated hypomagnesaemia appeared in late 2006, which was on omeprazole and esomeprazole, and the ppi - associated hypomagnesaemia entered clinical consciousness slowly. the aers data used in this study were those from 2004 to 2009, and the latest data should be used to assess the associations with pantoprazole, lansoprazole and rabeprazole. the aers database is considered a valuable tool ; however, some limitations inherent to spontaneous reporting have been pointed out 18. first, the data occasionally contain misspelling and miswords, although the structure of aers is in compliance with the international safety reporting guidance. second, the system was started more than 10 years ago, and reporting patterns have changed over time. third, the adverse events are coded using hierarchical terms of pts of meddra, and changes in terminology over time also might affect the quality of the database. to overcome problems with data quality, we manually corrected mistakes in the data entities and deleted duplicates according to fda 's recommended method, resulting in the development of a novel system to analyze an association between a drug and an adverse drug event. previously, this system has been used to assess adverse drug events accompanying the use of platinum agents 21. the data obtained was consistent with clinical observations, suggesting the usefulness of the system 21. additionally, this system was used to evaluate susceptibility to hypersensitivity reactions for 14 anticancer agents, and it was found that the number of co - occurrences was an important factor in signal detection 22, 23. very recently, this system was applied to the evaluation of adverse drug events induced by statins 24, capecitabine 25 and tigecycline 26, and again the reproducibility of clinical observations was suggested, providing that the number of co - occurrences was large enough to detect a signal. it should be noted that there is no credible counterfactual means, e.g., a randomized control group, to identify an association between a drug and an adverse drug event as a signal, and therefore disease - oriented adverse events can be extracted as signals. for example, hypomagnesaemia was extracted as an omeprazole - associated adverse drug event, but might be common in patients with acid peptic disorders irrespective of the administration of ppis. generally, the results obtained using this system can be biased by unmeasured confounding factors, and flawed by incomplete data ; however, a comparison among ppis possibly offsets them, resulting in a rank - order of association according to the statistical metrics. in conclusion, the data obtained in this study do not provide sufficient evidence to recommend systematic monitoring of magnesium levels in plasma, but chronic exposure to a ppi can lead to severe hypomagnesaemia.	objective : case reports showing that proton - pump inhibitors (ppis), omeprazole and esomeprazole, can cause hypomagnesaemia have been accumulating since 2006. in this study, the reports submitted to the adverse event reporting system (aers) of the us food and drug administration (fda) were evaluated to assess omeprazole and esomeprazole in terms of susceptibility to hypomagnesaemia.methods : after a revision of arbitrary drug names and the deletion of duplicated submissions, the reports involving omeprazole and esomeprazole were analyzed. standardized official pharmacovigilance tools were used for the quantitative detection of a signal, i.e., an association between a drug and an adverse drug event, including the proportional reporting ratio, the reporting odds ratio, the information component given by a bayesian confidence propagation neural network, and the empirical bayes geometric mean.results : a total of 22,017,956 co - occurrences were found in 1,644,220 reports from 2004 to 2009, where a co - occurrence was a pair of a drug and an adverse drug event. in total, 818 and 743 adverse drug events were listed as omeprazole- and esomeprazole - associated, with hypomagnesaemia ranking 85th and 135th, respectively. although both ppis were associated with hypomagnesaemia, the statistical metrics suggested that the association was more noteworthy for omeprazole.conclusion : the data obtained in this study do not provide sufficient evidence to recommend systematic monitoring of magnesium levels in plasma, but chronic exposure to a ppi can lead to severe hypomagnesaemia.
grading of breast cancer relies largely on the microscopic examination of tissue slides stained with hematoxylin and eosin (h & e). this is a subjective process by its very nature, consequently leading to inter- and even intraobserver variability, potentially affecting the predicted patient prognosis and also the treatment modalities offered. the variability in breast cancer grading may, at least, in part, be responsible for the variability in rates of chemotherapy use between institutions. segmentation of areas containing tumor cells in standard h & e histopathology images of the breast (and several other tissues) is a key task for computer - assisted assessment and grading of histopathology slides. good segmentation of the tumor regions can not only highlight the slide areas consisting of tumor cells, but it is also vital for the automated scoring of immunohistochemical (ihc)-stained slides, to restrict the scoring or analysis to areas containing only tumor cells and avoid potentially misleading results from the analysis of stromal regions. furthermore, detection of mitotic cells is critical for calculating key measures such as the mitotic index ; a key criteria for grading several types of cancers, including breast cancer. we are aware of the existing technologies that are capable of detecting mitotic cells on slides stained with ihc stains (e.g., ki67, phh3, etc.). however, as we show in this article, tumor segmentation can allow detection and quantification of mitotic cells from the standard h & e slides with a high degree of accuracy, without the need for special stains, in turn making the whole process more cost - effective. although some algorithms for segmentation of tumor nuclei, quantitative evaluation of nuclear pleomorphism, detection and grading of lymphocytic infiltration in histology images, and automated malignancy detection have been reported in literature, tumor segmentation in breast histology images has not received much attention.[25 ] wang,., has proposed a supervised tumor segmentation approach for tissue microarray spots that exploits tissue architectural and textural features in the markov random field (mrf) based bayesian estimation framework. however, supervised segmentation of breast cancer histology images containing a highly complex texture often raises questions with regard to an algorithm 's ability to avoid overfitting, the issue of training overhead. feature - based segmentation approaches often use a filter bank to represent a pixel as a point in a high - dimensional feature space, posing the so - called curse of the dimensionality problem. a dimensionality reduction (dr) technique, giving a low - dimensional representation and preserving relative distances between features from the original feature space, is desirable to solve this problem. along these lines, viswanath., proposed an ensemble embedding framework and applied it to image segmentation and classification. the idea is to generate an ensemble of low dimensional embeddings (using a variety of dr methods, such as graph embedding), evaluate embedding strength to select the most suitable embeddings, and finally generate consensus embedding by exploiting the variance among the ensemble. however, a major limitation of this framework, in the context of histopathology image analysis, is its high storage and computational complexity, mainly due to the very high - dimensional affinity matrix required for graph embeddings. random projections (rps) have recently emerged as a computationally simple and efficient low - dimensional subspace representation, with a minor drawback : multiple rps may produce substantially different projections because of the very nature of the random matrices. although this may not be a big issue in certain applications (like multimedia compression etc.), it can not be ignored in applications like segmentation in low - dimensional feature space. khan., proposed an ensemble of multiple rps (which they termed ranpec, short for random projections with ensemble clustering) followed by majority voting, to address the issue of variability among multiple rps. they further showed that ensemble clustering of random projections onto merely five dimensions achieves higher segmentation accuracy than a well - known supervised dr method on breast histology images. in this article, we propose a fast and totally unsupervised tumor segmentation framework based on dividing the stromal tissue into two types : hypocellular stroma (hypocs) and hypercellular stroma (hypercs). the proposed algorithm employs the magnitude spectrum in the gabor frequency domain to segment the hypocs regions and the phase spectrum in the gabor frequency domain to segment the hypercs regions. the algorithm has been evaluated on 35 h & e stained breast histology images belonging to five different tissue slides. instead of evaluating the system using object - based criteria, we have incorporated a much stricter pixel - based quantitative evaluation criterion. the experimental results show that the proposed system achieves an f1-score of 0.89 (with respect to the gt markings) for pixel - based segmentation, in h & e images. the main contributions of this article are as follows : (a) we show that the magnitude and phase spectra of the frequency domain are effective in representing the complementary features of the hypocs regions and hypercs regions, respectively ; (b) we present a fast, unsupervised, and data - independent algorithm for pixel level classification of tumor versus stromal regions (by integrating the hypocs and hypercs segmentations) in breast histology images, and (c) we show that segmentation of the stromal regions in breast histology images plays a critical role in mitosis detection, leading to a more accurate calculation of the mitotic index : one of the three criteria used in the so - called nottingham breast cancer grading system. the remainder of this article is organized as follows. section 2 outlines details of the segmentation algorithm, in particular how the segmentation of hypocs and hypercs is performed in a low dimensional feature space. the article concludes with a summary of our results and some directions for future studies. the dataset consists of 35 hpf (high power field) images taken from five different breast cancer biopsy slides, stained with h & e, and scanned at 40 magnification, using an aperio scanscope slide scanner. each hpf has a digital resolution of 2084 2084 pixels. on the basis of the tissue morphology, the breast histology image contents can be divided into four regions [figure 1 ] : tumor, hypocellular stroma (hypocs), hypercellular stroma (hypercs), and tissue fat and/or retractions / artifacts (background). the background is removed during the pre - processing stage, on the basis of color thresholding, while the hypocs and hypercs regions are segmented by calculating features using the magnitude and phase spectra, respectively, in the frequency domain and performing a ranpec segmentation on these features. the algorithm pipeline can be subdivided into three stages : (1) pre - processing, to normalize the staining artifacts and remove the tissue fat, artifacts, and background ; (2) segmentation of hypocs and hypercs regions ; (3) post - processing to combine the result of background removal in (1) and segmentation in (2). a sample h & e - stained breast cancer histology image : (a) original image, and (b) overlaid image, with four types of contents shown in different colors. the tumor areas are shown in red, hypocs in purple, and hypercs in green. note the difference in morphology of the hypo- and hypercellular stromal regions overview of the proposed algorithm : hymap hybrid magnitude - phase (hymap) - based tumor segmentation stain color constancy is one of biggest challenges of the digitized images of h & e stained tissue slides. several factors such as thickness of the tissue section, dye concentration, stain timings, and stain reactivity, result in variable stain color intensity and contrast. we have evaluated various stain normalization methods, but have found the magee., method to be the most effective in terms of dealing with tissues containing large amount of retractions / staining artifacts. first the stain - normalized (color) tissue image is transformed from the rgb space into the ycbcr space. the luminance channel is then thresholded using an empirically determined, fixed, global threshold. the rough binary mask resulting from this thresholding is finally refined via morphological operations, in order to fill up the small gaps. finally, the stain - normalized and background - free image is converted into the cie 's lab color space and anisotropic diffusion is applied to its b channel, to remove the inherent camera noise while preserving the edges. a traditional approach to texture segmentation is inspired by the multi - channel filtering theory. the idea is to characterize an image by a bank of filters, to generate a set of features that are capable of discriminating texture patterns belonging to different categories. a two - dimensional gabor function consists of a sinusoidal plane wave of a certain frequency and orientation, modulated by a two - dimensional gaussian. a gabor filter in the spatial domain is given by the following equation : g,(x, y) = g(x, y)exp(j2(xcos + ysin)) (1) where g(x, y) is a gaussian kernel with a bandwidth of. the parameters and represent frequency and orientation of the 2d gabor filter, where varies between 0 and in regular intervals, f, and f denotes a set of possible frequencies, and is defined as follows. fl(i) < 0.25 (2) fh(i) = 0.25 + 2 / nc \| 0.25 < fh(i) < 0.5 (3) where i = 0,1,,log2(ncol/8), ncol is the width of the image in terms of the nearest power of 2. we then define the set f of possible frequencies as follows, for an image with 512 columns, for example, a otal of 84 gabor filters can be used (six orientations and 14 frequencies). the hypocellular stromal features are then computed by convolving the gabor filters g,() with norm (obtained from step 3 of algorithm 1), and computing local energy on the results of the convolution. phase information could be used as an important cue in modeling the textural properties of a region. murtaza., used local frequency estimates in the gabor domain over a range of scales and orientations to yield a signature, which was shown to efficiently characterize the texture of a village in satellite images. we chose the phase spectrum to represent the attributes of the hypercs regions in a breast histology image, due to the recently established efficacy of the phase in textures exhibiting randomness. let vi(x, y) denote the i gabor channel for the stain normalized and smoothened version of an input image i(x, y), where i =, 1,2,,ng, ng = n \|f\| and n denotes the number of orientations. we can represent it as follows, vi(x, y) = \|vi(x, y)\|exp(ji(x, y)) (5) where \|\| denotes the magnitude operator and i(x, y) denotes the local phase. the gradient of the local phase and its magnitude can then be computed as below, the phase gradient features are computed using (7) for each of the gabor filter responses, over a window of size n ranpec is a fast, unsupervised, and data - independent framework for dimensionality reduction and clustering of high - dimensional data points. the main idea of ranpec is to project high - dimensional feature vectors onto a relatively small number of orthogonal random vectors belonging to a unit ball and perform ensemble clustering in the reduced - dimensional feature space. by getting an ensemble of projections for each feature vector and then picking a cluster for a pixel by the majority voting selection criterion, ensures stability of the results among different runs. experimental results in suggest that promising classification accuracy can be achieved by random projections when using fast matrix operations in an unsupervised manner. the dataset consists of 35 hpf (high power field) images taken from five different breast cancer biopsy slides, stained with h & e, and scanned at 40 magnification, using an aperio scanscope slide scanner. each hpf has a digital resolution of 2084 2084 pixels. on the basis of the tissue morphology, the breast histology image contents can be divided into four regions [figure 1 ] : tumor, hypocellular stroma (hypocs), hypercellular stroma (hypercs), and tissue fat and/or retractions / artifacts (background). the background is removed during the pre - processing stage, on the basis of color thresholding, while the hypocs and hypercs regions are segmented by calculating features using the magnitude and phase spectra, respectively, in the frequency domain and performing a ranpec segmentation on these features. the algorithm pipeline can be subdivided into three stages : (1) pre - processing, to normalize the staining artifacts and remove the tissue fat, artifacts, and background ; (2) segmentation of hypocs and hypercs regions ; (3) post - processing to combine the result of background removal in (1) and segmentation in (2). a block diagram of the proposed tumor segmentation framework is shown in figure 2. algorithm 1 outlines the algorithmic details of the pipeline. a sample h & e - stained breast cancer histology image : (a) original image, and (b) overlaid image, with four types of contents shown in different colors. the tumor areas are shown in red, hypocs in purple, and hypercs in green. note the difference in morphology of the hypo- and hypercellular stromal regions overview of the proposed algorithm : hymap hybrid magnitude - phase (hymap) - based tumor segmentation stain color constancy is one of biggest challenges of the digitized images of h & e stained tissue slides. several factors such as thickness of the tissue section, dye concentration, stain timings, and stain reactivity, result in variable stain color intensity and contrast. we have evaluated various stain normalization methods, but have found the magee., method to be the most effective in terms of dealing with tissues containing large amount of retractions / staining artifacts. first the stain - normalized (color) tissue image is transformed from the rgb space into the ycbcr space. the luminance channel is then thresholded using an empirically determined, fixed, global threshold. the rough binary mask resulting from this thresholding is finally refined via morphological operations, in order to fill up the small gaps. finally, the stain - normalized and background - free image is converted into the cie 's lab color space and anisotropic diffusion is applied to its b channel, to remove the inherent camera noise while preserving the edges. a traditional approach to texture segmentation is inspired by the multi - channel filtering theory. the idea is to characterize an image by a bank of filters, to generate a set of features that are capable of discriminating texture patterns belonging to different categories. a two - dimensional gabor function consists of a sinusoidal plane wave of a certain frequency and orientation, modulated by a two - dimensional gaussian. a gabor filter in the spatial domain is given by the following equation : g,(x, y) = g(x, y)exp(j2(xcos + ysin)) (1) where g(x, y) is a gaussian kernel with a bandwidth of. the parameters and represent frequency and orientation of the 2d gabor filter, where varies between 0 and in regular intervals, f, and f denotes a set of possible frequencies, and is defined as follows. fl(i) < 0.25 (2) fh(i) = 0.25 + 2 / nc \| 0.25 < fh(i) < 0.5 (3) where i = 0,1,,log2(ncol/8), ncol is the width of the image in terms of the nearest power of 2. we then define the set f of possible frequencies as follows, for an image with 512 columns, for example, a otal of 84 gabor filters can be used (six orientations and 14 frequencies). the hypocellular stromal features are then computed by convolving the gabor filters g,() with norm (obtained from step 3 of algorithm 1), and computing local energy on the results of the convolution. phase information could be used as an important cue in modeling the textural properties of a region., used local frequency estimates in the gabor domain over a range of scales and orientations to yield a signature, which was shown to efficiently characterize the texture of a village in satellite images. we chose the phase spectrum to represent the attributes of the hypercs regions in a breast histology image, due to the recently established efficacy of the phase in textures exhibiting randomness. let vi(x, y) denote the i gabor channel for the stain normalized and smoothened version of an input image i(x, y), where i =, 1,2,,ng, ng = n \|f\| and n denotes the number of orientations. we can represent it as follows, vi(x, y) = \|vi(x, y)\|exp(ji(x, y)) (5) where \|\| denotes the magnitude operator and i(x, y) denotes the local phase. the gradient of the local phase and its magnitude can then be computed as below, the phase gradient features are computed using (7) for each of the gabor filter responses, over a window of size n ranpec is a fast, unsupervised, and data - independent framework for dimensionality reduction and clustering of high - dimensional data points. the main idea of ranpec is to project high - dimensional feature vectors onto a relatively small number of orthogonal random vectors belonging to a unit ball and perform ensemble clustering in the reduced - dimensional feature space. by getting an ensemble of projections for each feature vector and then picking a cluster for a pixel by the majority voting selection criterion, experimental results in suggest that promising classification accuracy can be achieved by random projections when using fast matrix operations in an unsupervised manner. the dataset consists of 35 hpf (high power field) images taken from five different breast cancer biopsy slides, stained with h & e, and scanned at 40 magnification, using an aperio scanscope slide scanner. on the basis of the tissue morphology, the breast histology image contents can be divided into four regions [figure 1 ] : tumor, hypocellular stroma (hypocs), hypercellular stroma (hypercs), and tissue fat and/or retractions / artifacts (background). the background is removed during the pre - processing stage, on the basis of color thresholding, while the hypocs and hypercs regions are segmented by calculating features using the magnitude and phase spectra, respectively, in the frequency domain and performing a ranpec segmentation on these features. the algorithm pipeline can be subdivided into three stages : (1) pre - processing, to normalize the staining artifacts and remove the tissue fat, artifacts, and background ; (2) segmentation of hypocs and hypercs regions ; (3) post - processing to combine the result of background removal in (1) and segmentation in (2). a block diagram of the proposed tumor segmentation framework is shown in figure 2. algorithm 1 outlines the algorithmic details of the pipeline. a sample h & e - stained breast cancer histology image : (a) original image, and (b) overlaid image, with four types of contents shown in different colors. the tumor areas are shown in red, hypocs in purple, and hypercs in green. note the difference in morphology of the hypo- and hypercellular stromal regions overview of the proposed algorithm : hymap hybrid magnitude - phase (hymap) - based tumor segmentation stain color constancy is one of biggest challenges of the digitized images of h & e stained tissue slides. several factors such as thickness of the tissue section, dye concentration, stain timings, and stain reactivity, result in variable stain color intensity and contrast. we have evaluated various stain normalization methods, but have found the magee., method to be the most effective in terms of dealing with tissues containing large amount of retractions / staining artifacts. first the stain - normalized (color) tissue image is transformed from the rgb space into the ycbcr space. the luminance channel is then thresholded using an empirically determined, fixed, global threshold. the rough binary mask resulting from this thresholding is finally refined via morphological operations, in order to fill up the small gaps. finally, the stain - normalized and background - free image is converted into the cie 's lab color space and anisotropic diffusion is applied to its b channel, to remove the inherent camera noise while preserving the edges. a traditional approach to texture segmentation is inspired by the multi - channel filtering theory. the idea is to characterize an image by a bank of filters, to generate a set of features that are capable of discriminating texture patterns belonging to different categories. a two - dimensional gabor function consists of a sinusoidal plane wave of a certain frequency and orientation, modulated by a two - dimensional gaussian. a gabor filter in the spatial domain is given by the following equation : g,(x, y) = g(x, y)exp(j2(xcos + ysin)) (1) where g(x, y) is a gaussian kernel with a bandwidth of. the parameters and represent frequency and orientation of the 2d gabor filter, where varies between 0 and in regular intervals, f, and f denotes a set of possible frequencies, and is defined as follows. fl(i) < 0.25 (2) fh(i) = 0.25 + 2 / nc \| 0.25 < fh(i) < 0.5 (3) where i = 0,1,,log2(ncol/8), ncol is the width of the image in terms of the nearest power of 2. we then define the set f of possible frequencies as follows, for an image with 512 columns, for example, a otal of 84 gabor filters can be used (six orientations and 14 frequencies). the hypocellular stromal features are then computed by convolving the gabor filters g,() with norm (obtained from step 3 of algorithm 1), and computing local energy on the results of the convolution. phase information could be used as an important cue in modeling the textural properties of a region. murtaza., used local frequency estimates in the gabor domain over a range of scales and orientations to yield a signature, which was shown to efficiently characterize the texture of a village in satellite images. we chose the phase spectrum to represent the attributes of the hypercs regions in a breast histology image, due to the recently established efficacy of the phase in textures exhibiting randomness. let vi(x, y) denote the i gabor channel for the stain normalized and smoothened version of an input image i(x, y), where i =, 1,2,,ng, ng = n \|f\| and n denotes the number of orientations. we can represent it as follows, vi(x, y) = \|vi(x, y)\|exp(ji(x, y)) (5) where \|\| denotes the magnitude operator and i(x, y) denotes the local phase. the gradient of the local phase and its magnitude can then be computed as below, the phase gradient features are computed using (7) for each of the gabor filter responses, over a window of size n ranpec is a fast, unsupervised, and data - independent framework for dimensionality reduction and clustering of high - dimensional data points. the main idea of ranpec is to project high - dimensional feature vectors onto a relatively small number of orthogonal random vectors belonging to a unit ball and perform ensemble clustering in the reduced - dimensional feature space. by getting an ensemble of projections for each feature vector and then picking a cluster for a pixel by the majority voting selection criterion, ensures stability of the results among different runs. experimental results in suggest that promising classification accuracy can be achieved by random projections when using fast matrix operations in an unsupervised manner. we generate all experimental results on three criteria : (1) considering the first pathologist 's markings (p-1) as the ground truth (gt) ; (2) considering the second pathologist 's markings (p-2) as gt ; (3) fusing p-1 and p-2 using the logical or rule (i.e., a pixel is considered to be tumorous if any one of the two pathologists marked the pixel as tumorous), and considering the fused image as gt. some of the hpf images contain large tumor regions with small islands of stroma here and there ; however, a majority of hpf images contain a fair share of hypo- and hypercellular stroma (approximately 33%, on an average). the average degree of disagreement between the two pathologists on gt images is 11.55 5.37%. all 35 images in the dataset are pre - processed in a similar manner, with stain normalization carried out as described in section 2.2, and the background removal performed to remove fat and other artifacts caused by staining and fixation. to segment hypocs, a total of 84 gabor textural features (14 scales and six orientations) are calculated for each pixel of the input image i. in order to generate hypercs, the pg features are calculated on 10 orientations and three scales. the ranpec segmentation framework is applied on both gabor and pg features, independently, yielding hypocs and hypercs segmentations, respectively. the ranpec framework requires two parameters : r the dimensionality of lower dimensional space, and nc the number of runs of the ensemble. as recommended in, we used r = 5 and nc = 20 in our experiments. we have compared our proposed algorithm (hymap) with ranpec using the same experimental setup as suggested in. the algorithms considered here are evaluated on three pixel - wise accuracy measures : precision, recall, and f1-score. the f1-score is a measure that combines precision and recall in a statistically more meaningful way. let tp denote the number of true positives, fp the number of false positives, tn the number of true negatives, and fn the number of false negatives ; the precision is defined as tp/(tp + fp), recall is defined as tp/(tp + fn), and the f1-score is defined as 2 (precision recall)/(precision + recall). figure 3 provides an illustration of the efficiency of hypocs segmentation [figure 3b ] and hypercs segmentation [figure 3c ] in capturing the complementary stromal subtypes. figure 4 provides an illustration of the proposed tumor segmentation algorithm on two different hpf images. the segmentation results obtained by combining hypocs and hypercs yield high f1-scores of 0.86 and 0.89, with respect to the fused gt. considering the degree of disagreement between the two pathologists (i.e., 11.5 5.37%), table 1 shows the segmentation accuracies (in terms of precision, recall, and f1- score) of the unreduced and reduced feature spaces resulting from automated tumor segmentation. note that the f1-scores obtained from hymap (0.88, 0.89, and 0.89) are higher when compared with those from the unreduced feature space (0.87, 0.88, and 0.88) and ranpec (0.85, 0.85, and 0.85). table 1 also reveals that the reduced textural feature space achieves f1-scores of 0.88, 0.89, and 0.89, suggesting, in turn, that dr removes the redundant features and preserves the distances between high dimensional feature spaces, thereby improving segmentation accuracy. illustration of complementary segmentations obtained by hypo- and hypercellular stroma segmentation : (a) original images ; (b) results of hypocs shown in slightly darker contrast, outlined in green color ; (c) results of hypercs shown in slightly darker contrast, outlined in green color visual results of tumor segmentation in two sample images : first row : original images with fused ground truth (gt), marked non - tumor areas shown in a slightly darker contrast with blue boundaries ; second row : results of combining hypocs and hypercs using the proposed framework (f1-score = 0.86 and 0.89, respectively) ; third row : visual illustration of segmentation accuracy, green = tp, red = tn, yellow = fn, and blue = fp quantitative results of tumor segmentation accuracy indicators (precision, recall, and f1-score) for 35 bc histopathology images using three feature spaces (1) unreduced (n = 114), (2) ranpec (n = 10, as in), and (3) hymap (n = 10), where n denotes the dimensionality of the feature space further to the accuracy of segmentation, we present an application of tumor segmentation to mitotic cells (mcs) detection in tumor areas. mc detection is critical for calculating key measures such as the mitotic index : one of the three criteria used in the nottingham grading system to grade breast cancer histology slides. khan., proposed a statistical approach that models the pixel intensities in mitotic and non - mitotic regions by a mixture of the gamma - gaussian mixture model. figure 5 visually illustrates how tumor segmentation can improve mitotic cell detection accuracy in breast histology images. using the algorithm reported in, figure 5a shows the results of mc detection without tumor segmentation and figure 5b shows the results of mc detection with tumor segmentation. figure 5c and figure 5d note that the number of false positives increase significantly (from 4 to 82) when tumor segmentation is not performed for mitotic cells detection. visual results of mitotic cell (mc) detection in a sample image : (a) results of mc detection without tumor segmentation (tp = 17, fp = 82) using. all the true positive mcs are shown in yellow color, while all the false positives are shown in green color. (b) results of mc detection with tumor segmentation (tp = 17, fp = 4). all the true positive mcs are shown in yellow color, while all the false positives are shown in green color. (c) zoomed - in version of a portion of (a) for better visibility. (d) zoomed - in version of a portion of (b) for better visibility in this article, we presented an algorithm for segmentation of tumor areas in breast histology images based on segmentation of the image into hypocellular stroma and hypercellular stroma regions, using the magnitude and phase spectra in the gabor domain. the complementary nature of the segmentation of two stromal subtypes was shown, resulting in high segmentation accuracy for the tumor areas. it was further demonstrated that the specificity of mitotic cell detection can be significantly enhanced when detection is restricted to the tumor areas. we anticipate further applications of our method to accurate, tumor - localized quantification / scoring of ihc stained slides and its validation on large - scale datasets.	background : segmentation of areas containing tumor cells in standard h&e histopathology images of breast (and several other tissues) is a key task for computer - assisted assessment and grading of histopathology slides. good segmentation of tumor regions is also vital for automated scoring of immunohistochemical stained slides to restrict the scoring or analysis to areas containing tumor cells only and avoid potentially misleading results from analysis of stromal regions. furthermore, detection of mitotic cells is critical for calculating key measures such as mitotic index ; a key criteria for grading several types of cancers including breast cancer. we show that tumor segmentation can allow detection and quantification of mitotic cells from the standard h&e slides with a high degree of accuracy without need for special stains, in turn making the whole process more cost-effective.method:based on the tissue morphology, breast histology image contents can be divided into four regions : tumor, hypocellular stroma (hypocs), hypercellular stroma (hypercs), and tissue fat (background). background is removed during the preprocessing stage on the basis of color thresholding, while hypocs and hypercs regions are segmented by calculating features using magnitude and phase spectra in the frequency domain, respectively, and performing unsupervised segmentation on these features.results:all images in the database were hand segmented by two expert pathologists. the algorithms considered here are evaluated on three pixel - wise accuracy measures : precision, recall, and f1-score. the segmentation results obtained by combining hypocs and hypercs yield high f1-score of 0.86 and 0.89 with re - spect to the ground truth.conclusions:in this paper, we show that segmentation of breast histopathology image into hypocellular stroma and hypercellular stroma can be achieved using magnitude and phase spectra in the frequency domain. the segmentation leads to demarcation of tumor margins leading to improved accuracy of mitotic cell detection.
alzheimer disease (ad) is a complicated neurodegenerative disease common in elderly people and it has become a major threat to the growing elderly population due to the lack of effective therapies. several strategies, including the prevention of amyloid buildup and the promotion of amyloid removal, have been tested in a range of clinical trials. however, none of these strategies showed significant efficacy [35 ]. more attention has been paid to those factors that affect the disease in its early stage and it is estimated that genetic risk factors contribute to approximately over 70% of the incidence of ad. as a result of this, the identification of genetic risk factors enables us to understand the disease mechanism in a sensible way. until now, only apolipoprotein e 4 (apoe4) allele has been confirmed to be associated with increased risk of ad development. the apoe4 gene is located in chromosome 19, which is one of several prominent chromosomes associated with the development of ad. recently, other genes in chromosome 19 associated with altered risks of ad, including abca7 (atp - binding cassette, sub - family a, member 7), cd33 (siglec-3), and tomm40 (translocase of the outer mitochondrial membrane 40) [911 ], have been identified by several large - scale genome - wide association studies focusing on disease - associated single - nucleotide polymorphisms (snps). the important role of abca7rs3764650 polymorphism (location : 19p13) in ad pathogenesis has been investigated by several studies that suggested different associations between gene snp and ad [1215 ]. apart from that, the cd33 (location : 19q13) gene was observed to be associated with various immune functions, including cell adhesion, anti - inflammatory signaling, and endocytosis. the protective allele a of rs3865444 in this gene has been revealed to be associated with a reduced risk of ad in genome - wide studies. furthermore, the tomm40 gene is also located in the 19q13 region, which is approximately 15 kb closer to apoe. therefore, the tomm40 gene should be investigated carefully due to the linkage disequilibrium with the e4 allele. relevant research also indicated that a kind of a mitochondrial protein is coded by the tomm40 gene. the genetic variants of rs157580 and rs2075650 in tomm40 have been revealed to be associated with altered risk of ad in independent genome - wide studies since 2008. considering these inconsistent associations between polymorphisms in the above genes and the development of ad, it is critical to carry out a meta - analysis in order to produce a consistent result. additionally, differences in sample sizes and heterogeneous populations may be considered as potential sources of heterogeneity among individual studies. therefore, we performed the present meta - analysis with increased statistical power using a well - established method to assess the association between genetic mutation and disease through incorporating all available published data. this meta - analysis on all eligible related studies was performed to evaluate the association between polymorphism of abca7 rs3764650 or cd33 rs3865444 or tomm40 rs157580/rs2075650 and the susceptibility to ad. all of the potential eligible studies were screened based on the electronic databases pubmed, embase, medline, and china national knowledge internet (cnki) up to 1 december 2014 using advanced searching strategies. single - nucleotide polymorphism, abca7, cd33 and tomm40, were used to search for relevant articles. systematic searching was performed using the combination of alzheimer disease, single - nucleotide polymorphism (snp) and each one of abca7, the following 4 criteria were used to determine the inclusion of studies : (1) studies must assess the association between polymorphism of abca7rs3764650 or cd33rs3865444 or tomm40rs157580/rs2075650 and the susceptibility to ad ; (2) case - control studies must be based on humans ; (3) sufficient information should be accessible (e.g., the study sample size for each research group, allele or genotype frequencies, effect sizes, and other useful information) ; (4) the diagnose of ad should meet the clinical criteria set by the world health organization. a predesigned data collection form was used by 2 independent reviewers to collect the following data : first author name, year of publication, country of research, ethnicity of study population, type of ad, mean age of the case and control groups, sex ratio in the case and control groups, and frequency distributions of allele and numbers of different genotypes in the case and control groups. if several subpopulations were presented in the original articles, then they were considered as separate studies in this meta - analysis. finally, relevant studies were selected and key data were collected by 2 independent reviewers. the associations between abca7 rs3764650 or cd33 rs3865444 or tomm40 rs157580 or rs2075650 polymorphisms and the susceptibility to ad were quantified using odds ratios (ors) with 95% confidence intervals (cis). the final ors and 95% cis for each snp were pooled from individual study ors and 95% cis. pooled ors and 95% cis were estimated by allelic models. for each snp, statistical heterogeneity among individual studies was inferred by q test and i statistic ; these 2 heterogeneity tests were used to calculate the variability among individual studies and the combined i metric was derived to assess the percentage of variation. meta - analysis was first performed with the fixed - effects model. if the p value of the q test was greater than 0.05 and the i statistic result was less than 50% (ph > 0.05 and i 0.05 and i < 50%), then there was no significant heterogeneity among individual studies and the fixed - effects model was suitable for analysis. on the other hand, if the p value of q test was less than or equal to 0.05 or if the i statistic result was greater than or equal to 50% (ph 0.05 or i 50%), then significant heterogeneity was presented in these studies and the a random - effects model was appropriate for meta - analysis. furthermore, subgroup analyses were performed by ethnic groups (caucasian and asian) to explore the effects of ethnicity on the association between gene polymorphisms and the susceptibility to ad. publication bias was indicated by the funnel plot and plot asymmetry was confirmed by the rank correlation test. if the p value of the rank correlation test was greater than 0.05, then there is no significant evidence of publication bias, and a symmetrical inverted funnel was approximately presented in the plot ; otherwise, there was significant publication bias. a 2-sided p value of 0.05 was selected as the significance level and all statistical analyses were performed using r software (version 3.1.2, copyright(c) 2014 the r foundation for statistical computing). a total of 175 articles were initially identified based on the predefined searching strategies, including 71 articles for abca7, 60 articles for tomm40, and 44 articles for cd33. then 33 of the 175 articles were excluded because of duplication and 142 articles were screened manually for potential available information. after that, 119 of 142 articles were further excluded for several reasons. for example, some articles did not include essential information to evaluate the effect size and others did not have full - text available for review. studies performed by hollingworth, naj, harold, and lambert were considered as independent studies because they included different ad subgroup analyses by different countries. similarly, studies by naj and carrasquillo were also treated as several independent studies because they were 2-stage studies including discovery and replication. a total of 19 739 cases and 39 746 controls were included for abca7 polymorphism studies. studies for cd33 comprised 49 414 cases and 73 933 controls, whereas studies for tomm40 comprised 11 490 cases and 16 094 controls. the study populations and structures varied among all studies and only a few studies stated that the study populations were matched by race and environmental factors. the results of meta - analysis for abca7 rs3764605 polymorphism are presented in figure 1. the pooled or was 1.20 (95% ci : 1.141.26, p value < 0.001), which suggested that abca7 rs3764605 allele g was significantly associated with an increased risk of ad. as indicated by figure 1, only 6 of 17 the studies showed significant association between abca7 rs3764605 polymorphism and ad. similarly, meta - analysis of cd33 polymorphism rs3865444 is displayed in figure 2, which revealed that the allele a of rs3865444 was a protective factor for ad onsets (or=0.94, 95% ci : 0.900.98, p value=0.003). furthermore, evidence indicated that the 2 polymorphisms of tomm40 had opposite associations (figure 3) ; genetic variant a of rs157580 was significantly associated with a reduced susceptibility to ad (or=0.62, 95% ci : 0.570.66, p value < 0.001), while allele a of rs2075650 polymorphism was a risk factor for ad (or=2.87, 95% ci : 2.463.34, p value < 0.001). table 1 shows that all polymorphisms except rs3764650 in abca7 (ph=0.362, i=20.42%) had significant heterogeneity among individual studies. therefore, subgroup analyses by ethnicity (asian and caucasian) indicated significantly different ors and 95% cis for polymorphisms of rs157580, rs2075650, and rs3865444. for instance, cd33 rs3865444 and tomm40 rs2075650 polymorphisms in the asian group did not have significant association with the susceptibility to ad (or=0.86, 95% ci=0.581.27 ; or=2.45, 95% ci=0.454.45). funnel plots were constructed to assess publication bias for each genetic variants group (figure 4a : abca7rs3764650, figure 4b : cd33rs3865444, figure 4c : tomm40rs2075650, figure 4d : tomm40rs157580). p value of the rank correlation test for each polymorphism was greater than 0.05 (table 1 : abca7rs3764650, p value=0.433 ; cd33rs3865444, p value=0.794 ; tomm40-rs157580, p value=0.945 ; tomm40rs2075650, p value=0.233), which suggested that there was no significant publication bias in each polymorphism study. the sensitivity analyses results revealed that no individual study significantly affected the overall value of ors and 95% cis. a total of 175 articles were initially identified based on the predefined searching strategies, including 71 articles for abca7, 60 articles for tomm40, and 44 articles for cd33. then 33 of the 175 articles were excluded because of duplication and 142 articles were screened manually for potential available information. after that, 119 of 142 articles were further excluded for several reasons. for example, some articles did not include essential information to evaluate the effect size and others did not have full - text available for review. studies performed by hollingworth, naj, harold, and lambert were considered as independent studies because they included different ad subgroup analyses by different countries. similarly, studies by naj and carrasquillo were also treated as several independent studies because they were 2-stage studies including discovery and replication. a total of 19 739 cases and 39 746 controls were included for abca7 polymorphism studies. studies for cd33 comprised 49 414 cases and 73 933 controls, whereas studies for tomm40 comprised 11 490 cases and 16 094 controls. the study populations and structures varied among all studies and only a few studies stated that the study populations were matched by race and environmental factors. the results of meta - analysis for abca7 rs3764605 polymorphism are presented in figure 1. the pooled or was 1.20 (95% ci : 1.141.26, p value < 0.001), which suggested that abca7 rs3764605 allele g was significantly associated with an increased risk of ad. as indicated by figure 1, only 6 of 17 the studies showed significant association between abca7 rs3764605 polymorphism and ad. similarly, meta - analysis of cd33 polymorphism rs3865444 is displayed in figure 2, which revealed that the allele a of rs3865444 was a protective factor for ad onsets (or=0.94, 95% ci : 0.900.98, p value=0.003). furthermore, evidence indicated that the 2 polymorphisms of tomm40 had opposite associations (figure 3) ; genetic variant a of rs157580 was significantly associated with a reduced susceptibility to ad (or=0.62, 95% ci : 0.570.66, p value < 0.001), while allele a of rs2075650 polymorphism was a risk factor for ad (or=2.87, 95% ci : 2.463.34, p value < 0.001). table 1 shows that all polymorphisms except rs3764650 in abca7 (ph=0.362, i=20.42%) had significant heterogeneity among individual studies. therefore, subgroup analyses by ethnicity (asian and caucasian) indicated significantly different ors and 95% cis for polymorphisms of rs157580, rs2075650, and rs3865444. for instance, cd33 rs3865444 and tomm40 rs2075650 polymorphisms in the asian group did not have significant association with the susceptibility to ad (or=0.86, 95% ci=0.581.27 ; or=2.45, 95% ci=0.454.45). funnel plots were constructed to assess publication bias for each genetic variants group (figure 4a : abca7rs3764650, figure 4b : cd33rs3865444, figure 4c : tomm40rs2075650, figure 4d : tomm40rs157580). p value of the rank correlation test for each polymorphism was greater than 0.05 (table 1 : abca7rs3764650, p value=0.433 ; cd33rs3865444, p value=0.794 ; tomm40-rs157580, p value=0.945 ; tomm40rs2075650, p value=0.233), which suggested that there was no significant publication bias in each polymorphism study. the sensitivity analyses results revealed that no individual study significantly affected the overall value of ors and 95% cis. in this meta - analysis, a systematic overview of case - control studies for assessing the association between genetic variants and the susceptibility to ad was performed. all the eligible databases were searched electronically and the potential articles were screened to be included in this meta - analysis. eventually, 25 articles investigating 4 snps of 3 ad candidate genes were included in this meta - analysis. the pooled results showed significant association between the 4 snps (rs3764650, rs157580, rs2075650, and rs3865444) and the susceptibility to ad. two snps (rs3764650 and rs2075650) were found to be significantly associated with increased risk of ad, whereas the other 2 snps (rs3865444 and rs157580) were significantly associated with a decreased risk of ad. in addition, subgroup analyses suggested that the association between snps and the susceptibility to ad was significantly different between the asian and caucasian groups for snp of rs157580, rs2075650, and rs3865444. abca7 is a new member of the a - subfamily, which is also an integral transmembrane adenosine triphosphates - binding cassette transporter. the biogenesis of high - density lipoprotein was mediated by its protein and combined with helical apolipoproteins and cellular lipid. moreover, cholinergic dysfunction, amyloid precursor protein processing, and a production of deposition, which are the neuropathologic landmarks of ad, are all regulated by local lipid homeostasis integrity. using reverse transcription - pcr analysis and northern blot, previous studies concluded that the abca7 gene is expressed in human brain, particularly on the ca1 hippocampal neurons [3941 ]. therefore, functions of the abca7 gene were considered to have important associations with the susceptibility to ad. furthermore, the results of our meta - analysis confirmed that rs3764650 allele g of abca7 was associated with a 20% increase in the risk of ad development. in addition, the evidence suggested that the association between snp (rs157580, rs2075650, and rs3865444) and the susceptibility to ad differed significantly between the asian and caucasian groups. unfortunately, there was no available data investigating how genetic variants in africans affect the susceptibility to ad, whereas another snp, rs115550680 in abca7, has been discovered to be significantly associated with ad in african - americans. based on haploview, polymorphism of rs115550680 is in linkage disequilibrium with rs3764650. among these 19 studies selected for our analysis, only 7 provided exact genotype numbers ; therefore, meta - analysis was carried out using the allelic model only. as a member of the sialic acid - binding immunoglobulin - like lectins (siglec) family previous research suggested that the main function of cd33 related to siglecs is immunological regulation, which is involved in the microglial cleansing process. moreover, griciuc. reported that the increased expressions of cd33 in microglial cells were observed in ad patients. however, inconsistent associations between rs3865444 polymorphism and the susceptibility to ad were suggested by several independent case - control studies. on the other hand, our meta - analysis results revealed that rs3865444 allele a contributed significantly to reduced risk of ad. however, the effect of rs3865444 allele a on the susceptibility to ad differed significantly between the asian and caucasian groups, as indicated by the subgroup analyses. although the rs3865444 risk allele (c) was found to have greater cell surface expression of cd33 in african - americans, the complete data were not available. among included studies on rs3865444, therefore, we strongly recommend that further studies should be carried out on africans and south asians to confirm the association between rs3865444 snp and the susceptibility to ad in other ethnic groups. tom40 is a subunit of the translocase of the outer membrane (tom), which is encoded by the tomm40 gene. tom40 is located in the mitochondria and is involved in transporting cytoplasmic peptides and proteins during mitochondrial biogenesis. the role of tom40 in regulating protein traffic across the outer mitochondrial membrane appears to be important in the development of ad, perhaps because of the unique bioenergetic requirements of neurons. two polymorphisms of tomm40 were first found to be significantly associated with ad in genome - wide studies. however, inconsistent associations were reported by basic case - control studies due to different study methods and target populations. to identify the genetic association between snps and susceptibility to ad, however, genotype data were only available in asians and caucasians and further studies need to be performed in other ethnic groups to assess the effect of ethnicity on the overall association between gene snp and the development of ad. to the best of our knowledge, this is the first study to investigate 4 snps of 3 different genes located on the same chromosome first, some articles without sufficient genotype information were excluded and this may have introduced selection bias and reduced the statistical power. second, the meta - analysis method is not able to cope with different study designs and population structures, which might contribute to the heterogeneity among individual studies. significant associations between the 4 snps (rs3764650, rs157580, rs2075650, and rs3865444) and susceptibility to ad were suggested by our study. meta - analysis based on 19 739 cases and 39 746 controls confirmed that abca7 rs3764650 allele g is a risk factor of ad in both caucasians and asians. furthermore, meta - analysis including 49 414 cases and 73 933 controls suggest that cd33 rs3865444 allele a is a protective factor of ad in caucasians but this association was not significant in asians. in addition, meta - analyses of 2 polymorphisms in tomm40 (rs157580 and rs2075650) gene yielded contrary results ; snp of rs157580 was significantly associated with a reduced risk of ad, whereas snp of rs2075650 was significantly associated with an increased risk of ad, but the association between the asian and caucasian groups was significantly different, as indicated by the heterogeneity test. therefore, we recommend that well - designed studies with larger sample sizes should be performed. genetic variants and other factors, such as individual biological characteristics and environmental factors, particularly in african and asian populations, should be investigated together to assess the interaction between different factors that may significantly affect development of ad.	backgroundalzheimer disease (ad) has become an epidemic within the growing elderly population and effective therapies of ad have not been discovered. genetic factors accounted for over 70% of the incidence of ad and the disease - related polymorphisms are located on chromosome 19, which is one of several prominent chromosomes related to the development of ad. many inconsistent associations between polymorphisms in abca7, cd33, and tomm40 genes and the susceptibility to ad have been suggested by several independent studies.material/methodsa comprehensive literature search for studies involving the association between gene polymorphisms and ad was performed, and we finally selected 3 genes (4 polymorphisms) for the meta - analysis : abca7 (rs3764650), cd33 (rs3865444), and tomm40 (rs157580, rs2075650).resultsa total of 25 articles investigating 3 genes (4 polymorphisms) were included in the meta - analysis. the pooled results of 4 polymorphisms were all significantly associated with the susceptibility to ad. the pooled effect of abca7 rs3764605 allele g was significantly associated with an increased the risk of ad (or=1.20, 95% ci : 1.141.26, p value < 0.001). similarly, our evidence suggested that allele a of tomm40 rs2075650 polymorphism was a risk factor for ad (or=2.87, 95% ci : 2.463.34, p value < 0.001). alleles a of cd33 rs3865444 and a of tomm40 rs157580 were both protective factors for ad onset (or=0.94, 95% ci : 0.900.98, p value=0.003 ; or=0.62, 95% ci : 0.570.66, p value < 0.001).conclusionsresults from the meta - analysis revealed that the pooled abca7 rs376465, cd33 rs3865444, tomm40 rs157580, and rs2075650 variants were significantly associated with the susceptibility to ad. however, the association differed significantly between asian and caucasian groups for snps of cd33 rs3865444, tomm40 rs157580, and rs2075650.
it consists of fusion between two adjacent vertebrae across the lamina and transverse processes.1 fusion depends on host and surgical factors including the selection of an appropriate graft. iliac crest autograft has long been considered the gold standard for bone graft procedures. however, its use is associated with significant disadvantages including insufficient availability, donor site pain, increased operative time, increased estimated blood loss, and longer hospital stay.2 many autologous or heterologous bone substitutes are used to reach fusion. currently, there are over 200 different commercial types of bone graft extenders, enhancers, and substitutes available from which to choose.3 the objective is to find an osteoinductive and osteoconductive substrate that is also safe and easy to apply. the efficacy of platelet - rich plasma (prp) as an osteoinductive material has been studied by several authors.4 5 6 preliminary results about using prp in posterolateral fusion have been encouraging.7 in this article, we analyze the effectiveness and practicality of using an heterologous cancellous bone substitute with autologous prp obtained directly in the operating theater. we evaluated 20 consecutive patients who underwent posterior decompression, vertebral fixation with pedicle screws, and posterolateral fusion from january 2012 to january 2013 at the department of neurological and neurosurgical sciences of sapienza university of rome. the inclusion criteria were degenerative or traumatic lumbosacral spine disease and normal platelet count (150 to 450 10/ml). the exclusion criteria were previous lumbosacral surgery, fixation involving dorsal vertebrae, neoplastic disease, blood dyscrasias, and abnormal platelet count. the following method was used : cancellous bone substitute (25 50 30 mm) soaked with autologous prp was implanted on the right hemifield and cancellous bone substitute (25 50 30 mm) soaked with saline solution on the left hemifield. tecnoss (torino, italy) sp - block soft bs7e (25 50 30 mm) was used as a bone substitute. if hydrated with saline solution for 5 to 10 minutes, it becomes flexible and malleable. autologous prp was obtained in the operating theater by taking peripheral venous blood from the patient. the regen system (regenlab, lausanne, switzerland) was used, consisting of an 8-ml patented test tube regen tht - sp and a regen centrifuge (h19-f regen centrigel) placed directly in the operating theater. the test tube containing 3 0.1 g of polyester gel and 1 0.05 ml of sodium citrate solution was centrifuged for 8 minutes at 3,100 rpm (1,500 g). this removes 95 to 100% of total red blood cells, with 80% of white blood cells and a platelet concentration between 2.5 and 4.5 10/l remaining. after centrifugation, the content was divided into red blood cells below the filter at the base of the tube and prp with buffy - coat superiorly. each tube contained 4.5 ml of prp, immediately available for application and enough to cover about two levels of arthrodesis unilaterally. the contact of the prp with the porosity of the cancellous bone substitute and the addition of 2 ml of calcium gluconate favored the activation of the coagulation cascade and platelet activation (fig. investigations into bone density were obtained by comparative analysis of region of interest (roi) on computed tomography (ct) scan images. a square area 0.4 cm in size (roi) is traced over the axial ct scan across the lamina and transverse processes. the electronic calculation of the mean pixel content (bidimensional unit of measurement) can be done in hounsfield units (hus). the numerical value obtained is the average of the densitometric values of the pixels contained in the area ; hu = 0 corresponds to water, 1,000 to air, and + 1,000 to cortical bone. (b) after centrifugation, red blood cells trapped below the filter of gel content at the base of the tube. the upper portion is divided in the platelet - rich plasma and in the upper buffy - coat ; (c) bone block ; (d) intraoperative picture. the 20 patients included 8 males and 12 females with mean age = 70 (standard deviation [sd ] = 10.9). the platelet count in the whole blood varied between 173 and 390 10/ml. a ct scan 6 and 12 months after surgery was performed in all patients (figs. 2 and 3). investigations into bone density were obtained by comparative analysis of roi (table 1). the data were analyzed with two repeated - measures variance analyses with value of density after 6 months and value of density after 12 months as factors (density on the right hemifield versus density on the left hemifield) using age, levels of arthrodesis, and platelet count (in the whole blood) as covariates. region of interest (roi) analysis on computed tomography scan 6 months after surgery. the main effect of value of density after 6 months was significant (f(1,19) = 5.522, p < 0.05) : the value of density on the right hemifield after 6 months was significantly different from the value of the density on the left hemifield (mean = 830.353 [sd 61.8 ] for the right hemifield versus mean = 632.890 [sd 59.5 ] for the left hemifield). the post hoc mean comparison showed that the value of density on the right hemifield was significantly greater than the value of density on the left hemifield after 6 months (= 197.463 ; f(1,19) = 43, p < 0.001). the interaction with age was marginally significant (f(1,19) = 3.700, p = 0.72) ; the interaction between the levels (f(1,19) = 2.070, p = 0.169) and the platelet count (f(1,19)= 0.64, p = 0.804) was not significant, which suggests no effects of these variables on results. these data demonstrate increased bone density using prp and heterologous cancellous block in the first 6 months after surgery (fig. the analysis of variance on the value of the density after 12 months showed that at this time no significant difference was observed in the two hemifields (f(1,19)= 1.100, p = 0.310). in fact, the mean of the value of the density on the right hemifield was 871.810 versus 837.011 of the left hemifield after 12 months (= 34.79 ; f(1,19) = 0.665, p = 0.426). also in this case, no interaction between the covariates of age (f(1,19) = 0.573, p = 0.460.), levels (f(1,19) = 1.407, p = 0.253), and platelet count (f(1,19) = 0.117, p posterolateral arthrodesis is a fusion between two adjacent vertebrae across the lamina and transverse processes.1 many autologous or heterologous bone substitutes are generally used as an implantation to reach fusion.3 the objective is to find an osteoconductive and osteoinductive substrate that is also safe and easy to apply. collagen binding to fibronectin promotes the anchorage of mesenchymal stem progenitors, on which it exerts its chemotactic action and induces differentiation into osteoblasts. it has a rigid and malleable consistency and maintains the original graft volume from 6 to 18 months from application. this is particularly important to allow the rapid development of the bony bridge between the transverse processes. this also reduce the risk of pseudarthrosis and nonunion present in arthrodesis performed with other substitutes.8 moreover, cancellous bone substitute can be used also in association with autologous bone graft (laminectomy bone chips) ; future investigations to analyze this option are necessary. in this study because posterior elements are intact in vertebral body fractures, the cohort of patients is homogeneous. fusion was assessed with computerized analysis of roi, a square area 0.4 cm in size traced over the axial ct scan across the lamina and transverse processes. it is important to emphasize that the numerical value obtained from this measurement is the average of the densitometric values of the pixels contained in the area studied. prp has an osteoinductive effect increasing the bone regeneration and production.9 10 this is due to the action of growth factors released by platelets upon activation. platelets are a potential source of multiple autologous growth factors, and proteins are involved in the physiologic processes of healing and tissue regeneration. the products rich in platelets have the goal to replace the blood clot with a preparation enriched in platelets that, once activated, secrete large amounts of proteins and growth factors (platelet - derived growth factor, epidermal growth factor, transforming growth factor-, vascular endothelial growth factor, insulin - like growth factor 1). they exert mitogenic action on osteoblasts and induce the differentiation of mesenchymal stem progenitors into osteoblasts.11 autologous prp can be obtained directly in the operating theater. prp has to be prepared to effectively separate the platelets from red blood cells, taking care not to damage the platelets. this method is important because the growth factors contained within the platelet -granules are activated at the same time that the granules themselves merge with membrane platelet.12 13 14 the properties of the prp in tissue repair have already been documented in numerous studies. therefore, the molecular mechanisms through which the growth factors stimulate repair are complex and depend on the different types of tissues and types of cells that receive the messages.15 prp is obtained by platelet sequestration and concentration. the exact relationship between the platelet count and the concentration of growth factors remains unclear. no correlation between bone density and whole blood platelet count was found in our series. several in vitro studies seem to point out that the content of platelet growth factors is quite variable among individuals and is not necessarily proportional to the platelet count.16 prp is an autologous product and is completely safe for the patient. moreover, it promotes leukocyte chemotaxis and has antibacterial effect and also increases tissue repair and remodeling.16 several studies demonstrated that iliac crest autograph is the gold standard for bone graft procedures including posterolateral fusion.2 3 as its use is associated with significant disadvantages (including insufficient availability, donor site pain, increased operative time, increased estimated blood loss, longer hospital stay), many surgeons prefer to use heterologous bone substitutes even if it is proved that their validity is lower. in this study the validity of our method has not been compared with autograph, but the technique can be improved in the future. prp used with cancellous bone substitute increases the speed of bone production in posterolateral arthrodesis joining osteoinductive and osteoconductive effect.	study design prospective cohort study. objectives to analyze the effectiveness and practicality of using cancellous bone substitute with platelet - rich plasma (prp) in posterolateral arthrodesis. methods twenty consecutive patients underwent posterolateral arthrodesis with implantation of cancellous bone substitute soaked with prp obtained directly in the operating theater on the right hemifield and cancellous bone substitute soaked with saline solution on the right. results computed tomography scans at 6 and 12 months after surgery were performed in all patients. bone density was investigated by comparative analysis of region of interest. the data were analyzed with repeated - measures variance analyses with value of density after 6 months and value of density after 12 months, using age, levels of arthrodesis, and platelet count as covariates. the data demonstrated increased bone density using prp and heterologous cancellous block resulting in an enhanced fusion rate during the first 6 months after surgery. conclusions prp used with cancellous bone substitute increases the rate of fusion and bone density joining osteoinductive and osteoconductive effect.
crimean - congo hemorrhagic fever virus belongs to the genus nairovirus in the bunyaviridae family, and is a human pathogen that can cause a severe, and often fatal, hemorrhagic fever. cchf virus is the most geographically widespread tick - borne virus of medical importance (ergonul 2006). the disease is endemic in large areas of sub - saharan africa and the middle and far east, as well as in eastern europe. a significant increase of cases in countries such as kosovo, albania, turkey, iran, and greece has recently been observed (alavi - naini. the cchfv genome has been isolated from at least 31 different tick species in the ixodidae (hard ticks) and argasidae (soft ticks). hyalomma spp ticks are considered the most important in the epidemiology of cchfv ; however, the virus has also been isolated from ticks of other genera (i.e., rhipicephalus, boophilus, dermacentor, haemaphysalis, and ixodes spp, (saijo. humans in high - risk occupations (e.g., slaughterhouse workers, shepherds, health care workers, and veterinarians) are prone to cchf infection (garcia. this study was performed to ascertain the prevalence of cchfv in ticks present on the one - humped camel (camelus dromedarius), and to estimate the prevalence of cchfv igg in the one - humped camel s sera in three provinces : khorassan - e - shomali, khorassane - razavi, and khorassan - e - jonoobi of iran. the study was conducted in three provinces : khorassan - e - shomali, khorassan - e - razavi, and khorassan - e - jonoobi located at 5517, 6115e and 3024, 3817n in northeastern and east of iran (fig. 1). khorassan - e - shomali is a mountainous region, with temperate, cold weather. khorassan - e - razavi is a semi - desert region that has mild weather. khorassan - e - jonoobi is a semi - desert region that experiences arid conditions. the average annual rainfall is approximately 300400 mm in the northern areas (khorassan - e - shomali) and 150 mm in the central and southern areas (khorassan - e - razavi and khorassan - e - jonoobi). there are approximately 25 million camels in the world, and nearly 150,000 one - humped camels are in iran, this is 0.6% of the world camel population, and 3.8% of the asian camel population (fao 2011). the majority of iran s camels are dromedaries, and they are scat - tered across the country s provinces. in the khorassan provinces the camel population is 37,400 (ministry of agriculture jihad, 2002), but in the authors experience at present the actual number is several times greater, the majority of these are in khorassan - e - shomali and khorassan - e - jonoobi. study area in khorassan - e - shomali, khorassan - e - razavi and khorassan - e - jonoobi are shown with asterisks amplification of the s segment of the cchfv genome using rt - pcr, in tick samples from the khorasan province. (pc : positive control, nc : negative control, s1 - 10 : samples, s1, s2, s3, s4, s5, s6, s7 and s9 are negative, s8 and s10 are positive) from may 2012 to january 2013, eleven cities and towns were randomly selected from three provinces : khorassan - e - shomali, khorassan - e - razavi and khorassan - e - jonoobi as clusters, and at least 14 one - humped camels were sampled from each cluster. from each animal, two or three ticks were collected and placed in separate sterile tubes ; the tubes were labeled with the date of collection, animal number, sex, age, and area. collected ticks were sent to the laboratory and identified under a stereo microscope using general identification keys (hoogstraal 1979, apanaskevich and filippova 2007). the samples were then pooled according to the area, sex and species of tick (each pool routinely contained 18 ticks, but occasionally the number was greater) and immediately sent to the arboviruses and viral hemorrhagic fevers laboratory (national reference laboratory), pasteur institute of iran, and stored at 70 c until analysis. for the serum assay, 20 ml of blood collected from the jugular vein of each camel then the sample was labeled with the date of collection, animal number, sex, age, and area. the samples were immediately sent to a laboratory and centrifuged at 5000 rpm for 10 min. the sera were then separated and transferred into holding tubes and sent to the arboviruses and viral hemorrhagic fevers laboratory and stored at 70 c until analysis. according to previous studies in animals, tick infectious was 10% and with assumption 5% accuracy, 95% confidence interval and design effect equal 1.5 we need at least 207 ticks. with assumption 20% seropositivity in the studied animals, 8% accuracy, 95% degree of confidence, and design effect equal 1.5, the sample size will be 144, each cluster has at least 14 sera. reverse transcriptase polymerase chain reaction : ticks were individually washed twice by pbs (pbs, ph 7.4) and crushed with a mortar and pestle in 200300l of pbs. the extracted viral rna was stored at 70 c until analysis. for the rt - pcr, a master mix was prepared as follows : 28 l of rnase free water, 10 l of buffer (5 conc), 2 l of dntp mixture, 2l of enzyme mixture containing reverse transcriptase and taq dna polymerase enzymes, 1l of primer f2 (5-tggacaccttcacaaactc-3), 1l of primer r3 (5-gacaattccctacacc-3), 1l of rnase inhibitor and 5l of extracted viral rna as template. the f2 and r3 primers amplify a 536 bp fragment inside the s - segment of the cchfv genome, 536 bp fragment is rt - pcr target. the thermal cycling program for the rt - pcr, included 30 min at 50 c for reverse transcription reaction (cdna synthesis), 15 min at 95 c for activation of hot star taq dna polymerase and inactivation of reverse transcriptase, followed by 35 cycles of 95 c for 30 sec, 50 c for 30 sec, 72 c for 45 sec, and a final extension at 72 c for 5 min. for gel - based rt - pcr product analysis, 5l of the pcr product was mixed with 1l loading buffer (6 conc). then, the mixture was load in agarose gel 1.5%, and visualized with ethidium bromide (chinikar., the elisa plates were coated overnight at 4 c with mouse hyperimmune ascitic fluid diluted at 1:1000 in 0.05% tween 20-pbs containing 5% skim milk as a saturating reagent. the native or the recombinant antigen (produced in our laboratory) diluted in pbstm (pbs containing 0.05% tween and 3% skim milk) was added to the plates and the plates were incubated for 1h at 37 c and extensively washed. serum samples diluted in pbstm were added, and the plates were incubated for 1h at 37 c. after washing, the peroxidase - labeled anti - human or animal immunoglobulin diluted in pbstm was added to each weel and the plates were incubated for 1 h at 37 c. the plates were then washed 3 times with pbs containing 0.5% tween (pbst). finally, hydrogen peroxide and tetramethylbenzidine (tmb) were added and the plates were incubated for 15 min at room temperature. the enzymatic reaction was stopped by the addition of sulphuric acid (4n) and the plates read by elisa reader (anathos 2020) at 450 and 620 nm. taken together, an igg - positive serum was considered as positive control and a negative serum taken as negative control in the igg elisa (garcia. descriptive statistics (i.e. prevalence and percentages) were used to summarize the quantitative variables. the study was conducted in three provinces : khorassan - e - shomali, khorassan - e - razavi, and khorassan - e - jonoobi located at 5517, 6115e and 3024, 3817n in northeastern and east of iran (fig. 1). khorassan - e - shomali is a mountainous region, with temperate, cold weather. khorassan - e - razavi is a semi - desert region that has mild weather. khorassan - e - jonoobi is a semi - desert region that experiences arid conditions. the average annual rainfall is approximately 300400 mm in the northern areas (khorassan - e - shomali) and 150 mm in the central and southern areas (khorassan - e - razavi and khorassan - e - jonoobi). there are approximately 25 million camels in the world, and nearly 150,000 one - humped camels are in iran, this is 0.6% of the world camel population, and 3.8% of the asian camel population (fao 2011). the majority of iran s camels are dromedaries, and they are scat - tered across the country s provinces. in the khorassan provinces the camel population is 37,400 (ministry of agriculture jihad, 2002), but in the authors experience at present the actual number is several times greater, the majority of these are in khorassan - e - shomali and khorassan - e - jonoobi. study area in khorassan - e - shomali, khorassan - e - razavi and khorassan - e - jonoobi are shown with asterisks amplification of the s segment of the cchfv genome using rt - pcr, in tick samples from the khorasan province. (pc : positive control, nc : negative control, s1 - 10 : samples, s1, s2, s3, s4, s5, s6, s7 and s9 are negative, s8 and s10 are positive) from may 2012 to january 2013, eleven cities and towns were randomly selected from three provinces : khorassan - e - shomali, khorassan - e - razavi and khorassan - e - jonoobi as clusters, and at least 14 one - humped camels were sampled from each cluster. from each animal, two or three ticks were collected and placed in separate sterile tubes ; the tubes were labeled with the date of collection, animal number, sex, age, and area. collected ticks were sent to the laboratory and identified under a stereo microscope using general identification keys (hoogstraal 1979, apanaskevich and filippova 2007). the samples were then pooled according to the area, sex and species of tick (each pool routinely contained 18 ticks, but occasionally the number was greater) and immediately sent to the arboviruses and viral hemorrhagic fevers laboratory (national reference laboratory), pasteur institute of iran, and stored at 70 c until analysis. for the serum assay, 20 ml of blood collected from the jugular vein of each camel then the sample was labeled with the date of collection, animal number, sex, age, and area. the samples were immediately sent to a laboratory and centrifuged at 5000 rpm for 10 min. the sera were then separated and transferred into holding tubes and sent to the arboviruses and viral hemorrhagic fevers laboratory and stored at 70 c until analysis. according to previous studies in animals, tick infectious was 10% and with assumption 5% accuracy, 95% confidence interval and design effect equal 1.5 we need at least 207 ticks. with assumption 20% seropositivity in the studied animals, 8% accuracy, 95% degree of confidence, and design effect equal 1.5, the sample size will be 144, each cluster has at least 14 sera. reverse transcriptase polymerase chain reaction : ticks were individually washed twice by pbs (pbs, ph 7.4) and crushed with a mortar and pestle in 200300l of pbs. the extracted viral rna was stored at 70 c until analysis. for the rt - pcr, a master mix was prepared as follows : 28 l of rnase free water, 10 l of buffer (5 conc), 2 l of dntp mixture, 2l of enzyme mixture containing reverse transcriptase and taq dna polymerase enzymes, 1l of primer f2 (5-tggacaccttcacaaactc-3), 1l of primer r3 (5-gacaattccctacacc-3), 1l of rnase inhibitor and 5l of extracted viral rna as template. the f2 and r3 primers amplify a 536 bp fragment inside the s - segment of the cchfv genome, 536 bp fragment is rt - pcr target. the thermal cycling program for the rt - pcr, included 30 min at 50 c for reverse transcription reaction (cdna synthesis), 15 min at 95 c for activation of hot star taq dna polymerase and inactivation of reverse transcriptase, followed by 35 cycles of 95 c for 30 sec, 50 c for 30 sec, 72 c for 45 sec, and a final extension at 72 c for 5 min. for gel - based rt - pcr product analysis, 5l of the pcr product then, the mixture was load in agarose gel 1.5%, and visualized with ethidium bromide (chinikar. for igg antibody detection, the elisa plates were coated overnight at 4 c with mouse hyperimmune ascitic fluid diluted at 1:1000 in 0.05% tween 20-pbs containing 5% skim milk as a saturating reagent. the native or the recombinant antigen (produced in our laboratory) diluted in pbstm (pbs containing 0.05% tween and 3% skim milk) was added to the plates and the plates were incubated for 1h at 37 c and extensively washed. serum samples diluted in pbstm were added, and the plates were incubated for 1h at 37 c. after washing, the peroxidase - labeled anti - human or animal immunoglobulin diluted in pbstm was added to each weel and the plates were incubated for 1 h at 37 c. the plates were then washed 3 times with pbs containing 0.5% tween (pbst). finally, hydrogen peroxide and tetramethylbenzidine (tmb) were added and the plates were incubated for 15 min at room temperature. the enzymatic reaction was stopped by the addition of sulphuric acid (4n) and the plates read by elisa reader (anathos 2020) at 450 and 620 nm. taken together, an igg - positive serum was considered as positive control and a negative serum taken as negative control in the igg elisa (garcia. descriptive statistics (i.e. prevalence and percentages) were used to summarize the quantitative variables. a total of 200 one - humped multi - purpose camels (rearing for milk, meat, riding and offspring) were examined. tick infestation was observed in 170 of them, and 480 ixodid ticks (133 females and 347 males) were collected from different regions in the khorassan - e - shomali, khorassan - e - razavi and khorassan - e - jonoobi (table 1). positivity rates, number, and sex of ticks collected from one - humped camels from khorassan - e - shomali, khorassan - e - razavi and khorassan - e - jonoobi in east and northeast iran in the current study, only four species of hyalomma genus were observed. population frequency of h. dromedarii (90.7%) was higher than others and h. asiaticum had the lowest frequency (0.4%). hyalomma marginatum comprised about 2.9% and h. anatolicum accounted for 6% of total collected species. h. dromedarii is the most dominant tick species of camel in the khorassan region and a one humped camel is a suitable host. hyalomma dromedarii, h. anatolicum and h. marginatum were collected from all three provinces but in contrast, h. asiaticum only was collected from khorassan - e - razavi s one - humped camels (table 2). the sex, species and cchf - positive rate of ticks infesting one - humped camels the cchfv genome was found in 49 (10.2%) of 480 ticks, and three (6%) of 50 pools. the cchfv genome was detected in two out of four tick species, and of these, 42 (85.7%) belonged to h. dromedarii and 7 (14.3%) belonged to h. anatolicum (table 2). the viral genome was detected in tick samples from three cities in khorassan - e - jonoobi. the positivity rates were as follows : boshroyeh, 25 out of 480 (51%), birjand, 17 out of 480 (34.7%), and nehbandan, 7 out of 480 (14.3%, table 1). all three provinces, and six out of eleven cities and towns, were igg - positive for cchfv. nine (5.3%) out of 170 camels were igg - positive, this means that the igg prevalence was 5.3%. the positivity rates for the provinces varied, and were as follows : boshroyeh, 12.5%, kanimani, 7.14 %, birjand, 8%, nehbandan, 16.67%, chehl dokhtaran, 7.14%, and sabzevar, 6.66%. the highest rate of igg - positive samples was found in nehbandan (16.67%, two out of 12 sera), khorassan - e - jonoobi. eight out of the nine positive samples were collected from female camels (table 3). igg antibody - positive sera collected from one - humped camels from khorassan - e - shomali, khorassan - e - razavi and khorassan - e - jonoobi in this study, h. dromedarii was the most dominant species of tick on one - humped camels, and this is in agreement with the results obtained by salimabadi (2010) in iran and lawal. crimean - congo hemorrhagic fever infection was detected in 49 (10.2%) of 480 tick samples and this is a higher percentage than that previously reported by salim - abadi (3.79%) in the yazd provinces of iran (2011). hyalomma ticks are the primary vectors for the transmission of cchfv throughout europe, asia, the middle east, and africa (ergonul 2006). although hyalomma ticks are considered the most important vector and reservoir for the cchfv, the virus has also been reported in other tick genera (tahmasebi. 2010). the cchfv genome was detected in two out of the four tick species that were collected (h. dromedarii, 85.7% and h. anatolicum, 14.3%). (2011), therefore, these results suggest that h. dromedarii and h. anatolicum act as vectors for cchfv in one - humped camels. hyalomma dromedarii is distributed throughout the world wherever camels are present, and h. anatolicum is widely distributed throughout iran. hyalomma anatolicum transmits at least five arboviruses, and is a significant vector of cchfv to humans (nabian and rahbari 2008). (2010) detected the cchfv genome in rhipicephalus bursa (in one of the three ticks that were sampled), but we failed to discover r. bursa in our study. in the present study, we only found the cchfv genome in one - humped camel ticks in khorassan - e - jonoobi (birjand, boshroyeh, and nehbandan). this province has an unusual geographical location, as it borders afghanistan to the east, the sistan - va - baluchistan province of iran in the south, and the khorassan - e - razavi province of iran in the north. since 2000, the disease has infected 23 out of 31 provinces in iran : sistanva - baluchistan (283 confirmed human cases), isfahan (44 confirmed cases), fars (26 confirmed cases), tehran (17 confirmed cases), and khorasan (12 confirmed cases) (chinikar. 2012). notably, sistan - va - baluchistan province (south of khorassan - e - jonoobi) has not just had the highest number of cchfv cases, but cchf infections have been observed in this area since 2000 (chinikar. 2012), as it shares a border with two cchf - endemic countries : pakistan and afghanistan (chinikar. 2010). the unusual location of khorassan - e - jonoobi, which is connected in the north, south, and east to heavily infected or endemic areas of cchf, may explain why it was the only cchfv - positive area found in the study. we could not ascertain why all the reverse transcriptase polymerase chain reaction - positive ticks were male, but this may have been due to the presence of more males than females on the animals, and also in our samples (347 males vs 133 females). during the last decade, an increasing number of human cchfv infections have been reported in various regions of iran (chinikar. igg - positive serum samples collected from sheep, goats, cows, and humans have been frequently reported in different parts of the country (saidi. we found cchfv igg antibodies in one - humped camel sera in all three provinces studied, and these results, as well as those from a previous study by chinikar. (2012), may indicate that cchf is endemic to these regions, or that it has spread from neighboring countries. in total, nine (5.3%) out of 170 camels were igg positive. (1975), who found that cchf samples were negative in all 157 camels that they had studied, from south and southeast iran. (2000) found that 17 (16%) out of 109 camels sampled in oman were tested positive for the cchf igg antibody. sistan - va - baluchistan has been the most cchfv - infected province in iran since 2000, because, as mentioned previously, it shares a border with two cchf - endemic countries, pakistan and afghanistan (izadi. khorasan, which is connected to heavily infected or endemic areas of cchf, shares a large border area with neighboring countries, shares common pasture with herds from cchf - endemic areas, and experiences the illegal importation of animals across the border, which may explain why it is a cchf - positive area. another important issue is the presence of disease reservoirs and vectors, such as hyalomma spp. the cchfv genome has been isolated from at least 31 different tick species in ixodidae (hard ticks) and argasidae (soft ticks) (saijo. 2002, tahmasebi. 2010). as champour (2013) noted, the main tick species that affect one - humped camels in this region are h. dromedarii, h. anatolicum, h. marginatum, and h. asiaticum. hyalomma spp ticks are considered the most important species in the epidemiology of cchfv in camels in this area (hoogstraal 1979). six out of the eleven cities and towns studied yielded one - humped camel serum that was igg - positive for cchf, and it has been shown that cchf is distributed across these three provinces. we could not ascertain why almost all of the positive - igg sera samples were collected from female camels (8/9), but this may have been due to the presence of more females than males in the samples (136 females vs 34 males), possibly because female camels remained in herds longer than did males at the time of sampling. our results reveal a lower prevalence of seropositivity in one - humped camels (5.3%) than in other domestic animals (30%) in iran (chinikar. 2009). however, due to the fact that camels remain in herds for longer than do other animals (champour. 2013), and because camel pasture is very widely geographically distributed, this small percentage has a significant effect on the epidemiology of cchf. the importance of camels in the epidemiology of cchf in russia and astrakhan oblast has been previously reported by kurbanov, berezin, and chumakov (steele 1994). because cchf is a serious threat to iran, imported animals, particularly one - humped camels that carry a large number of ticks, should be inspected and treated carefully. continued surveillance and strictly enforced importation and quarantine practices will be required to prevent human exposure and the on - going dispersal of infected ticks and livestock in these regions. the use of commercially available insect repellent, and the use of clothing impregnated with permethrin, can give some protection against tick bites (telmadarraiy. further surveys of human and animal populations, particularly of those animals imported from neighboring countries to these regions, are recommended, in order to provide a better understanding of the distribution and epidemiology of the virus in these provinces.	background : this comprehensive study was conducted on multi - purpose one - humped camel (camelus dromedarius) sera and ticks to assess the epidemiological aspects of the crimean - congo hemorrhagic fever virus (cchfv) in northeast iran.methods:from may 2012 to january 2013, eleven cities were randomly selected in the khorasan provinces of iran as clusters, and at least 14 one - humped camels were sampled from each area. reverse transcriptase polymerase chain reaction was used for the detection of the cchfv genome in ticks. sera were analyzed using specific enzyme - linked immunosorbent assay tests.results:four hundred and eighty ixodid ticks were collected, and the genome of the cchfv was detected in 49 (10.2%) out of 480 ticks. the cchfv genome was detected in two out of four tick species, and in tick samples from three cities in khorassan - e - jonoobi. all three provinces, and six out of eleven cities, were cchfv - specific igg - positive. in total, nine (5.3%) out of 170 one - humped camels were igg - positive. the highest rate of igg - positive samples was found in nehbandan (16.67%).conclusion : continued surveillance and strictly enforced importation and quarantine practices should be implemented to prevent human exposure and the on - going dispersal of infected ticks and livestock in these regions. it is recommended that acaricides be used to prevent cchf transmission to humans, and to reduce the tick population. in addition, care should be taken by abattoirs workers and people who work with one - humped camels.
to describe a case of choroidal neovascularization (cnv) following photorefractive keratectomy (prk) to correct myopia. we performed prk in both eyes of a 20-year - old girl to correct myopia. refractive error was 4.75 2.25 5 in the right eye and 5.00 1.25 180 in the left eye. metamorphopsia was noticed by the patient in the right eye one month after the surgical procedure. fluorescein angiography and optical coherence tomography (oct) were performed which were compatible with cnv. after three monthly intravitreal bevacizumab injections, sub - retinal hemorrhage and intraretinal fluid resolved, but subretinal scar remained without any visual acuity improvement. also, refractive surgeons should consider cnv development in cases with visual compliant or metamorphopsia following prk. there are some reports of cnv after laser in situ keratomileusis (lasik) surgery, but to our knowledge, only one case of cnv following prk has been reported. here prk was performed in both eyes of a 20-year - old girl to correct myopia. the procedure was performed at refractive surgery center of baqiyatallah hospital, tehran, iran. a preoperative examination including refraction and best corrected visual acuity (bcva) measurements, slit - lamp examination, intraocular pressure (iop) measurement, and fundoscopy with dilated pupil was performed by the surgeon and reported as normal. preoperative refractive error was 4.75 2.25 5 and 5.00 1.25 180 in the right and left eye, respectively. alcohol - assisted corneal epithelium debridement was done, and mitomycin c was used at the end of the procedure. one month after surgical procedure, metamorphopsia developed in the right eye, and visual acuity reduction happened 3 months later. the patient was referred to retina clinic of the baqiyatallah hospital, tehran, iran 4 months after prk. on fundus examination there was an area of subretinal hemorrhage in the para foveal area associated with macular edema of the right eye. fluorescein angiography showed para foveal leakage (fig. 1), and optical coherence tomography (oct) revealed a hyper - reflective sub - retinal material associated with intraretinal fluid and pigment epithelial detachment (fig. although the effect of intravitreal anti vascular endothelial growth factor (anti - vegfs) in the cases of cnv following excimer laser refractive surgery has not been completely understood, we used 1.25 mg intravitreal bevacizumab (avastin ; genetech inc, south san francisco, california, usa) for treatment. after 3 monthly intravitreal injections, sub - retinal hemorrhage resolved and intraretinal fluid improved, but sub - retinal scar developed (fig. 3a and b). one month after the 3rd intravitreal injection, the bcva was 20/400, and no visual acuity improvement was observed. high myopia has been reported as a major cause of cnv formation (62%) in young patients. in the present report neo reported 3 cases of unilateral cnv after lasik for high myopia. among their patients, the mean spherical equivalent was 11.42 d (range from 6.75 to 20.00 d). they used a combination of intravitreal ranibizumab and photodynamic therapy (pdt) with verteporfin for the treatment. the mean bcva was 0.44 logmar and 0.17 logmar at presentation and after treatment, respectively. saeed described a case of cnv after lasik for correction of low myopia. the refractive error in their patient was 2.75 d in both eyes, and cnv developed in one eye 3 months after surgery. although loewenstein described macular hemorrhage in three patients with high myopia (13.00 to 20 d) after prk, to date, there has only been one report of cnv after prk. ruiz - mareno and colleagues evaluated the incidence of cnv in 5963 eyes undergoing prk for the correction of myopia. cnv developed in one eye after correction of 12.00 d of myopia 26 months after prk. the same researchers reported one case of cnv after the same number of prk surgeries. progressive elongation of the axial length and degenerative changes of the choroid in highly myopic eyes may cause the linear breaks in bruch 's membrane which are called lacquer cracks. lacquer cracks may be related to development of cnv.9, 10 myopic changes and lacquer cracks were not observed in the fundus examination of our case. iop elevation induced during lasik surgery may result in posterior segment pathologies and may be a factor for cnv formation. iop elevation does not happen during prk, and this may be the cause of lower reports of cnv formation following prk compared with lasik. acoustic shock waves produced by the excimer laser are another factor proposed to play a role in the cnv formation after both prk and lasik. cnv is not common in a 20-year - old patient without obvious myopic chorioretinal changes and lacquer cracks. also, our patients visual symptoms developed only 1 month after prk surgery. therefore, prk may play a role in the development of cnv in our case. since idiopathic and myopic cnv can not be ruled out in our case, the cause - and - effect relationship between the surgical procedure and cnv formation is not established. in conclusion also, refractive surgeons should consider cnv development in cases with visual compliant or metamorphopsia following prk.	purposeto describe a case of choroidal neovascularization (cnv) following photorefractive keratectomy (prk) to correct myopia.methodswe performed prk in both eyes of a 20-year - old girl to correct myopia. refractive error was 4.75 2.25 5 in the right eye and 5.00 1.25 180 in the left eye.metamorphopsia was noticed by the patient in the right eye one month after the surgical procedure. the patient was referred 3 months later when visual loss happened.resultsfluorescein angiography and optical coherence tomography (oct) were performed which were compatible with cnv.after three monthly intravitreal bevacizumab injections, sub - retinal hemorrhage and intraretinal fluid resolved, but subretinal scar remained without any visual acuity improvement.conclusionsit seems that cnv may occur after prk in myopic eyes. also, refractive surgeons should consider cnv development in cases with visual compliant or metamorphopsia following prk.
a central question in neuroscience is how memories are formed and stored in the brain. studies in laboratory animals have demonstrated that learning occurs through activity - dependent synaptic strength modification. given the sequential nature of many of our memories, it may come as no surprise that the temporal order of neuronal activity is a key determinant of synaptic plasticity. the order of presynaptic and postsynaptic action potential firing within a millisecond time window leads to either strengthening or weakening of synapses [26 ]. the involvement of postsynaptic ionotropic n - methyl - d - aspartate receptors (nmdars) in synaptic plasticity and spike - timing - dependent plasticity (stdp) has been well established. coincident pre- and postsynaptic firing is detected by postsynaptic nmdars (post - nmdars) that take on the role of coincidence detectors due to their multiple requirements for activation, which include the binding of glutamate, a signal of presynaptic activity, and depolarisation, a signal of postsynaptic activity. the depolarisation of the receptor is necessary in order to remove the magnesium ion (mg) blocking the channel pore at more hyperpolarised potentials, and is thought to be delivered by back propagation of somatic action potentials. activated postnmdars then permit the influx of calcium (ca), which can set in motion intracellular ca - dependent mechanisms that lead to transient or lasting changes in synaptic strength via changes in postsynaptic -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (ampars) expression and phosphorylation. temporal rules for spike - timing - dependent plasticity (stdp) are not the same for every synapse ; a diversity exists depending on brain area, neuron type, and location along dendrites [1114 ]. in juvenile rodent hippocampus, the window for synaptic modification is restricted to about 40 ms [1417 ] and a sharp switch of the direction of synaptic change exists at the 0 millisecond timing interval. in neocortical pyramidal neurons, the shape of the temporal stdp window depends on the location of synapses along the apical dendrite. in layer (l) 5 pyramidal neurons, proximal and distal synapses exhibit a progressive distance - dependent shift in the timing requirements for the induction of long - term potentiation (ltp) and long - term depression (ltd) [18, 19 ]. the mechanisms underlying these differences in timing rules rely on postsynaptic ca dynamics induced by back propagating action potentials : synapses at proximal dendritic locations experience sharper dendritic ca dynamics than distal synapses due to broadening of the action potential at distal dendrites [10, 1821 ]. as a result of dendritic depolarisation, more ca enters the postsynaptic neuron through nmdars and voltage - gated ca channels (vgccs) [10, 18 ]. in recent years, it has become clear that other factors beyond ca influx through postnmdars control stdp and contribute to a diversity of timing rules at glutamatergic synapses [22, 23 ]. in particular presynaptic ionotropic receptors, such as nmdars and nicotinic acetylcholine receptors (nachrs), presynaptic ionotropic receptors located on presynaptic terminals are ideally suited to influence the efficacy of synaptic transmission by directly affecting neurotransmitter release [2426 ]. short- and long - term activity - dependent modulation of the efficacy of synapses is crucial for regulating the flow of information throughout the nervous system and has been implicated in many neural processes, including learning. for several of the presynaptically located ionotropic glutamate receptors ampars, kainate receptors (kars) and nmdars it has been reported that they not only regulate neurotransmitter release, but are also involved in short- and long - term modification of synapse strength. for instance, hippocampal ca3 mossy fibre synapses onto pyramidal neurons show frequency facilitation on a seconds time - scale that involves activation of presynaptic kainate autoreceptors. on a minutes time scale, these presynaptic kainate receptors participate in the induction of ltp. however, in these studies, the role of presynaptic kainate receptors in the temporal rules of spike - timing - dependent plasticity was not considered. there is an abundance of anatomical and physiological evidence for the existence of presynaptic nmdars (pre - nmdars) in the mammalian neocortex, and many noncortical areas including the striatum [31, 32 ], hippocampus [3335 ], and cerebellum [3638 ]. physiological evidence for the existence of prenmdars came from the observation that activation of nmdars could lead to changes in transmitter release. it is now clear that prenmdars are not only involved in modulating transmitter release, but also have a prominent role in synaptic plasticity [30, 40 ]. in fact, in several cortical areas, spike - timing - dependent synaptic depression (tltd) depends exclusively on prenmdars and not on postnmdars. the involvement of prenmdars in stdp was first shown at synapses between connected pairs of visual cortex l5 pyramidal neurons. at these synapses, a stimulation protocol where the postsynaptic neuron was brought to spike before the presynaptic neuron (post - before - pre) induced tltd that was sensitive to nmda antagonists. both cv - analysis and the reduction in short - term depression that accompanied tltd indicated a presynaptic expression mechanism. the authors reasoned that since pre- and postsynaptic activity was required for tltd induction, but expression was presynaptic, a retrograde messenger would be required. a prime candidate was endocannabinoids (ecb), which are known retrograde messengers, capable of modulating presynaptic neurotransmitter release through cb1 receptors (cb1r) located on presynaptic terminals (wilson and nicoll). tltd was indeed found to be dependent on ecb signaling, since it was blocked by the cb1 receptor antagonist am251. ecb release by neurons is typically triggered by an increase in intracellular ca concentration (dimarzo [43, 44 ]). indeed, postsynaptic ca chelation with intracellular bapta blocked the induction of tltd. presynaptic activity alone in presence of the cb1r agonist acea without postsynaptic spiking led to ecb - dependent ltd (cltd), suggesting the requirement of postsynaptic activity for tltd serves only to trigger the release of ecbs. surprisingly, cltd was still sensitive to bath applied nmdar antagonists, but since cltd was independent of postsynaptic activity, it is unlikely that the nmdars are located postsynaptically, because these would not be activated without postsynaptic depolarization. also, nmdar stimulation led to an increase in mepsc frequency, suggesting prenmdars were located presynaptically. based on these observations, the authors concluded that the most parsimonious explanation was that nmdars involved in tltd are located presynaptically. more reports on prenmdar - dependent tltd in visual cortex and somatosensory cortex soon followed. there, tltd was also shown to be sensitive to bath applied nmdar antagonists, but to be independent of postnmdars, since tltd persisted when postnmdars were blocked by loading postsynaptic neurons with the use - dependent nmdar blocker mk-801 [45, 46 ] or by hyperpolarizing the postsynaptic neuron at the time of the presynaptic spike. the non - postsynaptic nmdars were assumed to be located presynaptically from the observed effect of nmdar stimulation on the frequency of spontaneous excitatory postsynaptic currents (epscs) and the amplitude of evoked ampar - mediated epscs, or by immunohistochemistry. the definite proof that nmdars involved in tltd were indeed located on presynaptic neurons came from an elegant study in the rodent barrel cortex, where stdp plays a role in sensory whisker map formation. in l4 to l2/3 synapses, a pre - before - post induction protocol induced timing - dependent ltp (tltp), and the reverse (post - before - pre) induced timing - dependent ltd. rodriguez - moreno and paulsen demonstrated that postsynaptic mk-801 blocked tltp, but not tltd whereas presynaptic mk-801 blocked tltd, but not tltp. these results showed that tltp and tltd are dependent on different nmdars, namely postnmdars and prenmdars, respectively. it is important to note that most of the examples of tltd reported above are assumed to be mediated by nmdars located on, or at least close to, the synaptic terminals, because of the observed effects of nmdar stimulation on transmitter release [41, 45, 46 ]. the reasoning behind this is that the increase in intracellular ca following nmdar activation is spatially limited to micro- or nanodomains, so in order for nmdar activation to affect the ca sensitive transmitter release processes, these receptors must lie close to the synaptic terminal. the legitimacy of this assumption has been questioned, however, by the recent finding that subthreshold depolarization following activation of somatodendritic nmdars can affect axonal ca levels through recruitment of vgccs. moreover, a follow - up study failed to detect changes in axonal ca levels when directly applying nmda to axonal compartments of visual cortex l5 pyramidal neurons. these new insights call for some caution when interpreting nmdar - mediated effects on synaptic transmission. therefore, although it remains difficult to imagine how such somatodendritic nmdars on presynaptic neurons would be recruited by tltd induction paradigms used in the above studies, their involvement can not be excluded. to date, all forms of cortical prenmdar - dependent stdp reported in the literature involve tltd [23, 41, 45, 46, 51 ], so it is unknown whether these presynaptic receptors can also mediate tltp. however, duguid and smart reported an intermediate form of ltp of inhibition in basket and stellate cell synapses onto purkinje cells in the cerebellum ; pairing presynaptic spiking with postsynaptic depolarisation resulted in a short period (2 - 3 min) of depolarisation - induced suppression of inhibition (dsi), which was followed by a prolonged period (up to 15 minutes) of depolarisation - induced potentiation of inhibition (dpi). secondly, dpi relies on prenmdars since it is abolished by ap-5, but postsynaptic purkinje cells do not express nmdars at this age. in addition, nmdar subunits colocalised with gad65/67 and synaptophysin, strongly suggesting that nmdars are located at the presynaptic terminal. these results show that synaptic activity- and prenmdar - dependent plasticity can also be involved in potentiating synapses. having nmdars at presynaptic terminals involved in stdp raises questions on the nature of the underlying induction and expression mechanisms ; firstly, how do prenmdars become activated ? secondly, how does prenmdar activation lead to a lasting change in synaptic efficacy ? and thirdly, where is the change expressed ? in all the examples mentioned above, tltd was accompanied by changes in short - term plasticity. this most likely reflects changes in release probability, pointing to a presynaptic site of expression. it is not unlikely that it is the presynaptic influx of ca through activated prenmdars that triggers the lasting change in release probability. to date, the precise mechanisms by which such an nmdar - mediated ca influx can induce such changes have not been directly investigated, so the answer to the second question remains elusive. how are prenmdars activated ? as mentioned before, nmdars require both depolarisation and binding of glutamate to become activated. presynaptic action potential firing provides an obvious source of depolarisation to prenmdars, but the source of glutamate acting on these receptors is less obvious. a number of possible sources can be identified (figure 1). firstly, as other presynaptic receptors, prenmdars can be activated by neurotransmitter released from the same nerve terminals on which the receptors themselves are located, thereby acting as autoreceptors [24, 26, 39 ]. alternatively, glutamate may be released postsynaptically and act as a retrograde signal to activate prenmdars. finally, glutamate can derive from sources outside the synapse, such as spill - over from synapses in the vicinity or glutamate release from nearby astrocytic processes. at first glance, a role for prenmdars as auto - receptors on glutamatergic terminals may seem unlikely, because by the time glutamate released from the terminal on which the receptors are located has reached the prenmdars, the depolarisation causing its release may already have ended. however, the prenmdars on which tltd of mouse barrel cortex l4 to l2/3 synapses depends were shown to contain nr2c / d subunits, which are known to be less voltage - sensitive. therefore, they may be well - suited as prenmdars in this form of tltd, being able to activate when glutamate binds even without a strong depolarisation. but tltd does not always rely on less voltage - sensitive nmdars ; prenmdar - dependent tltd at rat l5 to l5 visual cortex pyramidal neuron synapses and mouse l2/3 horizontal connections in barrel cortex relied on nr2b subunit - containing nmdars, which tend to have a higher voltage dependency. since nmdars are heteromeric structures, it remains possible that other nmdar subunits coassemble with nr2b to make the receptor less voltage sensitive. if prenmdar - dependent tltd relies on nmdars with low voltage - sensitivity, glutamate binding with only a mild depolarisation could be sufficient for channel opening and prenmdars could function as auto - receptors after all. prenmdars could also be activated by postsynaptically released glutamate, which could ensure that nmdars are glutamate bound at the time of the presynaptic action potential. this was shown to be the case in dpi of interneuron to purkinje cell synapses. since these synapses are gabaergic, prenmdars will not act as auto - receptors. by pharmacologically blocking eaat - mediated glutamate reuptake, the hypothesis was tested that retrograde postsynaptic release of glutamate could activate prenmdars. thus, the authors concluded that postsynaptically released glutamate may be responsible for activating prenmdars in this form of plasticity. although dendritic glutamate release has been reported in cortical pyramidal neurons as well, it has thus far not been investigated whether prenmdar - dependent tltd also relies on retrograde glutamate signalling. spill - over from neighbouring glutamatergic synapses has been suggested before as a source of glutamate in other forms of prenmdar - dependent plasticity [56, 57 ]. however, in these studies neighbouring glutamatergic synapses were explicitly stimulated during plasticity induction. as a consequence, tltd at a specific synapse with prenmdars would then only occur if neighbouring glutamatergic synapses would be active. in recent years, it has become clear that glial cells are intimately involved in the active control of neuronal activity, synaptic transmission, and plasticity. this has led to the concept of the tripartite synapse [5860 ], where communication is not limited to the pre- and postsynaptic neuronal elements, but where there is also a bidirectional communication between neurons and the astrocytes ensheathing the synapse. the potential importance of such astrocyte - neuron communication for synaptic plasticity was demonstrated recently in a study showing that astrocytic release of the neuromodulator d - serine was required for ltp at schaffer collateral synapses onto ca1 pyramidal neurons, although this is not without dispute. it is not unthinkable that astrocytes fulfill a similar role in prenmdar - dependent tltd by releasing glutamate. in fact, astrocytes have been reported to have the necessary intracellular machinery at their disposal for regulated exocytosis of glutamate and such astrocyte - derived glutamate can readily activate prenmdars. interestingly, prenmdars have been observed in extrasynaptic regions of presynaptic terminals closely apposed to glutamate - containing synaptic - like microvesicles in astrocytic processes. astrocytes express cb1 receptors which upon stimulation can trigger increases in intracellular ca levels leading to glutamate release [64, 65 ]. therefore, postsynaptically released ecbs may deliver signals of postsynaptic activity to nearby astrocytic processes. indeed, postsynaptically released ecbs have been shown to potentiate synapses in hippocampus by inducing glutamate release from astrocytes which in turn activated presynaptic metabotropic glutamate receptors [65, 66 ]. since prenmdar - dependent tltd at rat l5 to l5 visual cortex synapses, rat l4 to l2/3 barrel cortex synapses, and mouse l2/3 to l2/3 barrel cortex synapses, depended on ecb signalling as well, ecb signalling may be a general mechanism in prenmdar - dependent plasticity, serving to elicit glutamate release from astrocytes. the sequence of events that would have to take place in the case of ecb- and prenmdar - dependent tltd would be as follows ; during post - before - pre activity the postsynaptic neuron spikes first, allowing an increase in postsynaptic intracellular ca levels, which induces postsynaptic ecb release. activation of astrocytic ecb receptors induces increases in intracellular ca levels of the astrocyte which leads to the release of glutamate that binds to prenmdars. the depolarisation associated with following presynaptic action potentials then activates prenmdars and the subsequent influx of ca triggers some as yet unknown intracellular mechanism that leads to a persistent reduction of glutamate release. this scenario has one obvious difficulty ; the fact that prenmdar - dependent tltd can be induced using pre - before - post pairing intervals of only a few milliseconds puts severe time constraints on all the steps necessary within such a model. this issue can potentially be resolved by considering the time - course of astrocytic ca signals, which typically take place on a seconds timescale [6770 ]. therefore, ecb - mediated ca signals in astrocytes induced by the first pairings in the plasticity induction protocol may ensure glutamate levels are elevated during subsequent pairings. definite proof of this sequence of events from postsynaptic ecb release to prenmdar activation by astrocytic glutamate release awaits experimental testing. recently, banerjee. reported that in mouse barrel cortex l4 to l2/3 synapses, prenmdar - dependent tltd was ecb independent. these results raise the question of what other signalling mechanisms could be at play here. one candidate molecule would be nitric oxide (no), which has been shown to play a role in prenmdar - dependent cerebellar ltd. in fact, no has been implicated in mediating the presynaptic component of tltp at the same barrel cortex l4 to l2/3 synapses in mice. no derived from the postsynaptic neuron where it was released in response to postsynaptic depolarisation. application of an no donor resulted in an increase in miniature epsc frequency, indicating a presynaptic action and suggesting that no is indeed employed as a retrograde messenger at these synapses. since no has also been shown capable of eliciting vesicular glutamate release by astrocytes, it is possible that prenmdar - dependent tltd in the mouse brain occurs through recruitment of astrocytes by no signalling. barrel cortex tltd of l4 to l2/3 synapses and tltd of visual cortex l5 to l5 synapses are two cases of prenmdar - dependent plasticity that share many similarities ; both require specifically timed pre- and postsynaptic activity, both are expressed presynaptically, and both require activation of both cb1rs and prenmdars. however, some differences seem to exist. most importantly, as pointed out by duguid and sjstrm, in the presence of cb1 agonists, cltd could be induced in barrel cortex l4 to l2/3 synapses by trains of presynaptic stimulations delivered at either high (30 hz) or low (0.1 hz) frequencies. this was not the case in l5 visual cortex neurons, where cltd was only induced at stimulation frequencies higher than 15 hz. the latter finding is intriguing, because tltd at this synapse can be induced at low (0.1 hz) post - before - pre pairing frequencies. this suggests that at lower stimulation frequencies, some additional mechanism is needed besides ecb signalling. possibly, as proposed by duguid and sjstrm, tltd at low stimulation frequencies relies on an additional retrograde signal from the postsynaptic cell. as yet, the nature of this additional messenger can only be guessed at, but perhaps investigating the involvement of no would be a good place to start. together, these results indicate that prenmdars often require the involvement of other signalling molecules or messenger systems to fulfill their role in plasticity. it is important to know what precisely leads to prenmdar activation during stdp induction, as it has computational consequences for the role of prenmdar - dependent tltd in information processing. prenmdars functioning as auto - receptors would mean they are detectors of specific intrinsic activities of the synapse. however, if prenmdars are activated by glutamate from neighbouring cells, prenmdar - dependent tltd would be not only a reflection of coinciding pre- and postsynaptic activity, but also of coinciding activity of neurons and possibly astrocytes in the surrounding network. acetylcholine (ach) is one of the major neurotransmitters in the brain involved in regulating neuronal network activity. the effects of ach are mediated by two types of receptors ; the metabotropic muscarinic receptors (machrs) and the ionotropic nachrs. nachrs are ion channels which open upon the binding of ach, permitting the influx of multiple ionic species, most notably sodium and calcium, resulting in membrane depolarisation. brain nachrs are composed of multiple subunits, either heteromeric combinations of (210) and (24) subunits or homopentamers consisting of 7 subunits. the precise subunit composition has a profound effect on the biophysical (ca permeability, kinetics) and pharmacological properties (affinity, desensitization) of the receptor [73, 74 ]. these receptors are present throughout the brain, and are often found at somatodendritic locations, where they influence the excitability of the cell. however, just as nmdars, nachrs can also be found at presynaptic terminals in several brain regions, where they directly modulate excitatory glutamatergic transmission [7581 ]. most of these presynaptic nachrs contain 7 subunits and are thereby highly ca permeable, ideally suited to modulate the release of synaptic vesicles. activation of presynaptic nachrs can induce synaptic plasticity. in the ventral tegmental area (vta) of the mesolimbic dopamine system, which is involved in reward processing, glutamatergic synapses on dopaminergic neurons can undergo ltp when presynaptic activation is paired with postsynaptic activation, similar to cortical glutamatergic synapses [78, 83 ]. stimulation of presynaptic nachrs on these synapses by nicotine also induced ltp when this activation coincided with postsynaptic activity. the amount of ltp that was induced correlated with the level of increase in excitatory synaptic transmission induced by nachr activation. these effects on synaptic transmission were insensitive to ttx, indicating that the nachrs involved are located on, or close to the presynaptic terminals. both changes in excitatory synaptic transmission and nicotine - induced ltp were mediated by 7 subunit - containing nachrs. nicotine - induced ltp of glutamatergic inputs to da neurons depended on nmdar activation, which required postsynaptic depolarisation to remove the mg blockade. it was recently shown that pre- as well as postsynaptic nachrs in the vta are involved in increasing glutamatergic synapse function, and initiating glutamatergic synaptic plasticity, which may be an important, early neuronal adaptation in nicotine reward and reinforcement. nachrs can also modulate the rules for stdp, from locations further upstream than the presynaptic terminal. in l5 pyramidal neurons of mouse medial prefrontal cortex (mpfc), pairing presynaptic and postsynaptic activity at 5 ms intervals induced a long - term strengthening of glutamatergic inputs. when nachrs were stimulated with nicotine, tltp was eliminated and a depression of the excitatory inputs was observed. this nicotinic modulation of plasticity was abolished by inhibitors of gaba type a (gabaa) receptors, indicating the effects of nicotine were due to its actions on presynaptic interneurons. different types of gabaergic interneurons found in the pfc l5 express nachrs on their somas that activate these neurons when nicotine is present. thereby, nachr stimulation enhanced gabaergic inputs to l5 pyramidal neuron dendrites, resulting in reduced ca entry during action potential back - propagation from the soma [22, 85 ]. increasing dendritic action potential propagation by burst - like stimulation of the pyramidal neuron in the presence of nicotine could restore postsynaptic ca to levels comparable to those seen in the absence of nicotine, and restored stdp as well, indicating that strong postsynaptic stimulation could overcome the nicotinic modulation. thus, activation of nachrs expressed by mpfc interneurons that inhibit dendrites can alter the rules for induction of stdp. in mouse hippocampus, timing - dependent plasticity can be modulated through a similar recruitment of inhibition by nachrs on presynaptic interneurons. nachr activity could bidirectionally modulate plasticity, and the sign of synaptic change was critically dependent on the timing and localisation of nachr activation. in ca1 pyramidal neurons, pairing high - frequency stimulation (hfs) of schaffer collaterals with postsynaptic depolarisations resulted in short - term potentiation (stp) of these synapses. with machrs blocked by atropine, a puff of ach in dendritic regions of the cell during plasticity induction boosted stp into ltp. if, however, the ach puff was aimed at a neighbouring connected interneuron, the same protocol could no longer induce stp. moreover, stimulating nachrs on nearby interneurons during a stronger plasticity induction protocol, capable of inducing ltp in control conditions, converted ltp into stp. this demonstrates that timing and localization of nachr activity in the hippocampus can determine whether ltp will occur or not. although the authors did not further investigate the mechanisms underlying the blockade of plasticity by interneuronal nachr activation, it is tempting to speculate that the resulting increase in inhibitory input reduces postsynaptic ca signals in ca1 pyramidal neurons in a similar manner as it does in l5 neurons of the mpfc. plasticity induction by hfs does not involve back propagating action potentials, but increased inhibition may reduce the activation of postsynaptic voltage - dependent channels such as nmdars and vgccs that would otherwise be activated and promote synaptic potentiation. synaptic plasticity in the hippocampus has been associated with memory formation and synaptic plasticity in the pfc has been directly associated with attention and working memory. activation of nachrs alters processes of synaptic plasticity in cortical and hippocampal neuronal networks. by altering ca dynamics during active dendritic signalling in apical dendrites alternatively, nachrs may provide neuronal networks with the option to locally modulate synaptic plasticity, allowing a particular neuron or a particular synapse to respond differently than the average of the surrounding circuitry. by anatomical evidence shows that in rodent and human neocortex cholinergic nerve terminals establish classical synapses with closely apposed presynaptic and postsynaptic structures [89, 90 ], but direct physiological evidence for functional cholinergic synaptic transmission in the neocortex is lacking. in hippocampus, fast synaptic currents mediated by cholinergic transmission and 7 subunit - containing nachrs have been observed in interneurons, but not pyramidal neurons. slow, tonic modes of ach release may act on neurons in a diffuse manner, although ach is rapidly broken down by the substantial levels of acetylcholinesterase in the neocortex. whether rapid phasic ach changes act directly or in a diffuse manner is not known. recently it was shown that in the interpeduncular nucleus high - frequency (2050 hz) stimulation of ach neurons eventually generates postsynaptic nachr - mediated responses via volume transmission [93, 94 ]. regardless, the findings above suggest that during fast or slow ach signalling the rules for stdp may be altered for shorter or longer time. synapses can express multiple presynaptic ionotropic receptors that affect synaptic function and different types of ionotropic receptors can interact at the presynaptic level. for instance, activation of presynaptic ionotropic purinergic p2x receptors potentiates glutamate release due to the activation of 7-containing nachrs coexisting on rat neocortex glutamatergic terminals. considering the involvement of prenmdars and presynaptic nachrs in stdp, it would be interesting to examine whether these two species of receptors may also be found at the same synaptic terminals and if so, whether a similar interplay between nachrs and nmdars may occur. direct evidence for coexpression of presynaptic nachrs and nmdars there, axonal 7 nachrs were found to modulate prenmdar expression, implicating presynaptic 7 nachr / nmdar interactions in synaptic development and plasticity. firstly, in rat striatum, corticostriatal glutamate projections contain presynaptic 7 subunit - containing nachrs that upon stimulation elicit glutamate release. through microdialysis studies it was shown that nmdars could enhance glutamate release as well in this area, which the authors suggested was due to activation of prenmdars on cortico - striatal nerve endings. secondly, in rat hippocampus, presynaptic 7 subunit - containing nachrs have been reported to exist on excitatory presynaptic terminals, where they increase spontaneous and evoked glutamate release. these could well be the same synapses as where transmitter release - modulating prenmdars have been reported on a number of occasions [3335 ]. finally, in the neocortex where the prenmdar - dependent forms of tltd described above were observed, presynaptic nachrs thus, several candidate synapses exist for co - expression of presynaptic nmdars and nachrs. co - expression of these presynaptic ionotropic receptors could have several distinct, though not mutually exclusive, consequences for stdp. firstly, since presynaptic nachrs promote ltp, but prenmdars control ltd, there is the potential for an exciting competition to take place between potentiation and depression mechanisms at the presynaptic terminal. it must be noted, however, that all examples given of presynaptic nachrs promoting ltp are non - cortical (hippocampus, vta) and ltd promoting prenmdars are cortical. the most notable similarity between nachrs and nmdars is that they are both permeable to ca. in fact, upon activation, 7 subunit - containing nachrs permit a ca influx that rivals that of nmdars. the important difference with nmdars is, however, that nachrs do not have the voltage - dependent mg block. so, activation of nachrs at resting membrane potentials directly leads to ca influx without the need for depolarisation. at depolarized potentials (> 0 mv), however, an mg - dependent inward rectification takes place at nachrs that restricts the flow of current to very low levels [82, 101 ]. in that sense, activity of nachrs and nmdars may complement each other, acting at more or less distinct ranges of membrane potentials. thirdly, a direct interaction by which the activity of one receptor affects the other may exist. if nmdars and nachrs are expressed at the same synaptic terminal, local intracellular mg levels may lead to direct interaction between nachrs and nmdars ; activation of nmdars can result in a substantial increase in the intracellular concentrations of free mg. this particularly affects 7 subunit - containing nachrs, which have stronger mg - dependent inward rectification than 2 subunit - containing nachrs. therefore, at depolarized potentials, the increased mg levels following nmdar activation can act to inhibit nachrs and limit further ca influx through 7 subunit - containing nachrs. this crosstalk may represent a means by which rapid rise in intracellular ca concentrations via activation of nmdars and nachrs can be tightly controlled, so that intracellular ca overloading is avoided. such control over ca signals may be very important for plasticity processes and indeed, a coregulation of postsynaptic intracellular ca levels by nmdars and 7-containing nachrs to control synaptic plasticity has been proposed. the inverse, nachrs affecting the activity of nmdars, is also possible, albeit indirectly via intracellular signalling pathways. it has been shown that 7-containing nachrs can activate calcineurin (pp2b), a ca - sensitive enzyme, that when activated can lead to a reduction of the nmdar - mediated current decay time. by controlling the activity of pp2b, nachrs can regulate nmdar transmission and synaptic plasticity [103, 105, 106 ]. also, ca signals initiated by somatic or postsynaptic nachrs have been found to specifically reduce the amplitude of postnmdar - mediated currents through a ca - calmodulin - dependent process. having two routes through different ionotropic receptors towards plasticity modulation could endow the synapse with the ability to have different learning rules for different modes of processing, for example, in the presence or absence of ach. activation of these presynaptic receptors provides synapses with flexibility in the temporal rules for synaptic strengthening and weakening. thereby, the presence or absence of specific neurotransmitters can create windows during which specific timing of neuronal activity will lead to synaptic changes or not. for instance, hebbian plasticity is enhanced by behavioral relevance and attention, particularly in adults. attentional gating of plasticity may be provided by neuromodulators such as ach released in cortex by basal forebrain inputs. in addition, in barrel cortex, whisker map plasticity in s1 and other areas requires ach, and pairing of whisker stimuli with ach application drives receptive field plasticity. this suggests that presynaptic ionotropic receptors may fundamentally gate or modify hebbian learning rules during appropriate behavioral contexts. it will be interesting to learn from future research whether other types of presynaptic ionotropic receptors besides nmdars and nachrs are involved in controlling and shaping the rules for stdp.	throughout life, activity - dependent changes in neuronal connection strength enable the brain to refine neural circuits and learn based on experience. in line with predictions made by hebb, synapse strength can be modified depending on the millisecond timing of action potential firing (stdp). the sign of synaptic plasticity depends on the spike order of presynaptic and postsynaptic neurons. ionotropic neurotransmitter receptors, such as nmda receptors and nicotinic acetylcholine receptors, are intimately involved in setting the rules for synaptic strengthening and weakening. in addition, timing rules for stdp within synapses are not fixed. they can be altered by activation of ionotropic receptors located at, or close to, synapses. here, we will highlight studies that uncovered how network actions control and modulate timing rules for stdp by activating presynaptic ionotropic receptors. furthermore, we will discuss how interaction between different types of ionotropic receptors may create timing windows during which particular timing rules lead to synaptic changes.
a 52-year - old thai woman presented with asymptomatic annular erythematous plaques on the forehead and both cheeks that persisted for 2 years. dermatologic examination showed few discrete annular erythematous plaques on her forehead and both cheeks, 15 cm in diameter. there were solar lentigines and telangiectasias on the malar area, nose, and forehead as shown in fig. 1 and fig the routine histopathology demonstrated nodular and interstitial inflammatory cell infiltrate of histiocytes intermingled with some lymphocytes in the dermis as shown in fig. elastic stain showed elastotic material phagoticized by multinucleated cells and marked decrease of elastic tissue in some foci of the affected dermis (fig. 4). according to the clinical and histopathological findings, the dermatologic diagnosis was actinic granuloma (ag). complete blood count, liver enzyme, fasting blood glucose, and glycated hemoglobin were within normal range. the patient 's dermatologic condition was treated with prednisolone 15 mg / day for 6 weeks with a good response currently, she has been treated with hydroxychloroquine (200 mg / day), topical 0.1% mometasone furoate cream, broad spectrum sunscreen, and sun avoidance with partial improvement. it was also termed annular elastolytic giant cell granuloma, atypical necrobiosis lipoidica of the face and scalp, miescher 's granuloma of the face, and granuloma multiforme [1, 2, 3, 4 ]. ultraviolet (uv) radiation, especially uva, and heat are recognized as causal factors, by changing the antigenicity of elastic fibers. the immune response mediated by helper t cells to degenerated elastic tissue the typical cutaneous lesion of ag is an initially smooth, elevated, nonscaly, erythematous papule which centrifugally extends to an annular plaque with central clearing. they are usually distributed on chronically sun - exposed areas such as the face, neck, upper back, forearms, and dorsum of the hands. apart from the skin, conjunctival involvement has been reported in a few cases [6, 7 ]. there are some reports on the association between ag and internal diseases such as hematologic and solid malignancy, monoclonal gammopathy, temporal arteritis, erythema nodosum, and x - linked dominant protoporphyria [8, 9, 10, 11 ]. diabetes mellitus has been found in about 3740% of patient with ag, and may be caused by injury of elastic fiber from hyperglycemic state. as for the renal condition, focal segmental glomerulosclerosis has been described in association with various granulomatous diseases (e.g., sarcoidosis, wegener 's granulomatosis, churg - strauss syndrome, and kimura 's disease) [13, 14, 15, 16 ]. these include granuloma annulare, erythema annulare centrifugum, annular lichen planus, secondary syphilis, necrobiosis lipoidica, tinea corporis, and tuberculoid leprosy. the best method to obtain a precise histopathology is an elliptical biopsy across the annular rim and stained with elastic van gieson to demonstrate the three zones of elastic tissue change. in the first zone, solar elastosis is identified in the surrounding unaffected skin. in the second zone, a granulomatous reaction consisting of histiocytes and foreign - body type multinucleated cells is seen, with engulfment of elastotic fibers, representing the annular rim. in the third zone, an absence of elastic tissue in the superficial dermis is found in the center of the plaque. due to aesthetic concern in our case topical corticosteroids, intralesional corticosteroids, systemic corticosteroids, topical calcineurin inhibitors, phototherapy and photochemotherapy (narrowband uvb, puva, re - puva) have been used with some benefit [18, 19, 20 ]. cyclosporine a, dapsone, pentoxifylline, isotretinoin, and acitretin have been reported to be effective in some cases [21, 22, 23 ]. there are a few case reports with positive results from antimalarial therapy (chloroquine and hydroxychloroquine). to prevent the development of new lesions, patients should be instructed to avoid sun exposure and regularly use sunscreen. our patient had good response to a short course of low - dose prednisolone. however, rapid relapse occurred after the treatment was discontinued. currently, she has been treated with hydroxychloroquine (200 mg / day), topical 0.1% mometasone furoate cream, broad spectrum sunscreen, and sun avoidance with partial improvement. to the best of our knowledge, the correlation between ag and focal segmental glomerulosclerosis is still unidentified, further investigation is needed to establish the relationship between these two conditions. the authors declare no conflicts of interest. there was no funding for this work.	actinic granuloma is an uncommon granulomatous disease, characterized by annular erythematous plaque with central clearing predominately located on sun - damaged skin. the pathogenesis is not well understood, ultraviolet radiation is recognized as precipitating factor. we report a case of a 52-year - old woman who presented with asymptomatic annular erythematous plaques on the forehead and both cheeks persisting for 2 years. the clinical presentation and histopathologic findings support the diagnosis of actinic granuloma. during that period of time, she also developed focal segmental glomerulosclerosis. the association between actinic granuloma and focal segmental glomerulosclerosis needs to be clarified by further studies.
diabetes mellitus is one of the most frequent metabolic disorders that cause various central and peripheral nervous system complications such as neuropathy (1, 2). current therapeutic approaches are unable to completely relieve the neuropathic complications such as loss of sensation, pain and motor weakness (2). incurable diabetic side effects are typically related to sustained high glucose concentrations ; consequently, chronic hyperglycaemia is considered to be a key pathogenic factor of diabetic neuropathy and tissue damage (3, 4). both basic and clinical studies suggest that the oxidative stress caused by hyperglycaemia plays an important role in the pathogenesis of neurotoxicity (2). oxidative stress is stimulated by glucose through a combination of free radical production and decreased free radical scavenging. hydrogen peroxide is produced by the action of superoxide dismutase on superoxide, and it is produced in the mitochondria by elevated oxidative metabolism of glucose (5). elevated cellular oxidative stress induced by hyperglycaemia stimulates several glucose metabolic pathways that are associated with neuropathy progression. these include protein kinase c (pkc) activation, nadph redox imbalances, sorbitol and fructose accumulation, activation of nuclear enzyme poly (adp - ribose) polymerase, increased hexosamine pathway activity and superoxide overproduction and reduced levels of crucial anti - oxidative enzymes (2, 6). apoptosis is suggested as a potential mechanism for high glucose - induced neural cell death by both in vitro and in vivo studies (7, 8). several forms of chemical and physiological inducers of oxidative stress can promote apoptotic cell death. for example, hydrogen peroxide can induce apoptosis in various cell types, and this effect can be inhibited by antioxidants (9). studies have shown remedial properties of natural products or their active components, and antioxidant therapy in the prevention and/or treatment of chronic diseases such as neurodegenerative and cardiovascular disorders (10). ziziphus jujuba is broadly distributed in iran, and the fruit of this plant has gained wide attention in folk herbal medicine for the treatment of a wide range of disorders. chemical analysis of the z. jujuba fruit revealed that it contains flavonoids (quercetin and kaempferol) and phloretin derivatives (11). in our pervious study, we demonstrated the anti - oxidant activity of z. jujube in the central nervous system and improvements on spatial memory deficits induced by ethanol toxicity in rats (12). in the present study, we evaluated the effect of the z. jujuba fruit (zjf) aqueous extract on glucose induced neurotoxicity, and the potential anti - apoptotic effect of this extract on high glucose - treated pheochromocytoma (pc12) cells as an in vitro model of diabetic neuropathy. pc12 cells, which are derived from catecholamine - secreting adrenal chromaffin tumour in rats, are appropriate model for the assessment of neuronal cell death. dulbecco s modified eagle s medium (dmem), fetal bovine serum (fbs), heat - inactivated horse serum (hs), penicillin streptomycin solution and trypsin edta were purchased from gibco brl (grand island, ny, usa). 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazolium bromide (mtt) and dichlorofluorescein diacetate (dcfh - da) were acquired from sigma (st. louis, mo, usa). primary monoclonal anti--actin and primary polyclonal anti - caspase 3 antibodies were purchased from cell signaling technology, inc. fresh ripened fruits of z. jujuba were obtained from local herbal shops of khoramabad, iran during the october to november, 2012. identification of the plant was certified at the botany department of lorestan university, lorestan, iran. seeds were separated from fruits and about seven hundred grams of the pulp material were ground into fine powder. it was centrifuged at 4c for 20 min at 4000 g, and the supernatant was collected, lyophilized and stored at -20c until use. the extract was weighed and dissolved in phosphate buffered saline (pbs) to give 10 mg / ml extract as stock aliquot. pc12 cells were cultured in high glucose dmem supplemented with 10% fetal bovine serum, 5% heat - inactivated horse serum, penicillin (100 u / ml) and streptomycin (100 g / ml) at 37 c in a humidified atmosphere (90%) containing 5% co2. differentiated pc12 cells were plated at the density of 5000 per well in a 96 micro plate well for the cell viability assay. control cells were grown in dmem with 25 mm glucose, and the other cells (high glucose - treated) grown in dmem with 100 mm glucose. for protein extraction, cells were grown in a 6 well plate and allowed to attach and grow for 24 hr. then the cells were incubated in medium containing 100 mm glucose and different concentration of zjf extract for 24 hr. this method is based on reduction of 2- (4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) to formazan. mtt (0.5 mg / ml) was added to the 96-well plates and the cells were incubated for 2 hr at 37c. after removing medium, the resulting formazan was solubilized in 100 l dimethyl sulfoxide (dmso) per well. the optical density was determined at 570 nm by an automatic microplate reader (eliza mat 2000, drg instruments, gmbh). generation of intracellular reactive oxygen species (ros) was assessed by the oxidative - sensitive fluorescent probe dcfh - da. after treatment of cells with high - glucose media and zjf extract, attached cells were washed twice with pbs. pc12 cells were loaded with 10 m dcfh - da in culture media and incubated at 37 c for 30 min. fluorescence was evaluated with a micro - plate reader (perkin elmer victor 2) at 485 nm for excitation and at 530 nm for emission. western blot analysis was used to detect caspase-3 activation in pc12 cells. in brief, cultured cells were collected by trypsin - edta (0.5%), followed by two washes with cold pbs and lysed in cold lysis buffer [10 mm tris hcl (ph 7.4), 1 mm edta, 0.1% sodium dodecyl sulphate (sds), 0.1% na deoxycholate, 1% np-40 ; 2 g each of the protease inhibitors aprotinin, leupeptin, and pepstatin a ; and 0.5 mol / l pmsf ] and incubated on ice for 30 min. the homogenate was centrifuged twice at 14000 rpm at 4c for 20 min. protein concentrations of each fraction were measured with the bradford method (bio - rad laboratories, muenchen, germany). the protein samples from each fraction were separated via 10% sodium dodecyl sulphate- poly acrylamide gel electrophoresis (sds / page), and subsequently transferred to pvdf membrane for western blotting. membranes were probed using rabbit monoclonal antibody to caspase-3 (cell signaling technology, usa, 1:1000 overnight at 4c), and subsequently exposed to secondary hrp - conjugated igg. antigen - antibody complexes were then visualized by ecl system and exposed to lumi - film chemiluminescent detection film (roch, germany). photographs were digitized and the band intensity was quantified using lab work analyzing software (uvp, uk). all results are presented as the meansd the difference in cell viability (mean mtt assay) between groups was determined by one - way anova, followed by the tukey test. the values of caspase 3, and beta - actin band density were expressed as tested cleaved caspase-3/beta - actin ratio for each sample. dulbecco s modified eagle s medium (dmem), fetal bovine serum (fbs), heat - inactivated horse serum (hs), penicillin streptomycin solution and trypsin edta were purchased from gibco brl (grand island, ny, usa). 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazolium bromide (mtt) and dichlorofluorescein diacetate (dcfh - da) were acquired from sigma (st. louis, mo, usa). primary monoclonal anti--actin and primary polyclonal anti - caspase 3 antibodies were purchased from cell signaling technology, inc. fresh ripened fruits of z. jujuba were obtained from local herbal shops of khoramabad, iran during the october to november, 2012. identification of the plant was certified at the botany department of lorestan university, lorestan, iran. seeds were separated from fruits and about seven hundred grams of the pulp material were ground into fine powder. it was centrifuged at 4c for 20 min at 4000 g, and the supernatant was collected, lyophilized and stored at -20c until use. the extract was weighed and dissolved in phosphate buffered saline (pbs) to give 10 mg / ml extract as stock aliquot. pc12 cells were cultured in high glucose dmem supplemented with 10% fetal bovine serum, 5% heat - inactivated horse serum, penicillin (100 u / ml) and streptomycin (100 g / ml) at 37 c in a humidified atmosphere (90%) containing 5% co2. differentiated pc12 cells were plated at the density of 5000 per well in a 96 micro plate well for the cell viability assay. control cells were grown in dmem with 25 mm glucose, and the other cells (high glucose - treated) grown in dmem with 100 mm glucose. for protein extraction, cells were grown in a 6 well plate and allowed to attach and grow for 24 hr. then the cells were incubated in medium containing 100 mm glucose and different concentration of zjf extract for 24 hr. this method is based on reduction of 2- (4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) to formazan. mtt (0.5 mg / ml) was added to the 96-well plates and the cells were incubated for 2 hr at 37c. after removing medium, the resulting formazan was solubilized in 100 l dimethyl sulfoxide (dmso) per well. the optical density was determined at 570 nm by an automatic microplate reader (eliza mat 2000, drg instruments, gmbh). generation of intracellular reactive oxygen species (ros) was assessed by the oxidative - sensitive fluorescent probe dcfh - da. after treatment of cells with high - glucose media and zjf extract, attached cells were washed twice with pbs. pc12 cells were loaded with 10 m dcfh - da in culture media and incubated at 37 c for 30 min. fluorescence was evaluated with a micro - plate reader (perkin elmer victor 2) at 485 nm for excitation and at 530 nm for emission. western blot analysis was used to detect caspase-3 activation in pc12 cells. in brief, cultured cells were collected by trypsin - edta (0.5%), followed by two washes with cold pbs and lysed in cold lysis buffer [10 mm tris hcl (ph 7.4), 1 mm edta, 0.1% sodium dodecyl sulphate (sds), 0.1% na deoxycholate, 1% np-40 ; 2 g each of the protease inhibitors aprotinin, leupeptin, and pepstatin a ; and 0.5 mol / l pmsf ] and incubated on ice for 30 min. protein concentrations of each fraction were measured with the bradford method (bio - rad laboratories, muenchen, germany). poly acrylamide gel electrophoresis (sds / page), and subsequently transferred to pvdf membrane for western blotting. membranes were probed using rabbit monoclonal antibody to caspase-3 (cell signaling technology, usa, 1:1000 overnight at 4c), and subsequently exposed to secondary hrp - conjugated igg. antigen - antibody complexes were then visualized by ecl system and exposed to lumi - film chemiluminescent detection film (roch, germany). photographs were digitized and the band intensity was quantified using lab work analyzing software (uvp, uk). all results are presented as the meansd the difference in cell viability (mean mtt assay) between groups was determined by one - way anova, followed by the tukey test. the values of caspase 3, and beta - actin band density were expressed as tested cleaved caspase-3/beta - actin ratio for each sample. since the optimal glucose concentration for pc12 cell cultures is 25 mm, we simulated in vitro hyperglycemia by increasing the medium glucose level at the concentrations of 50, 75, 100, 125 and 150 mm for 24 hr. pc12 cells exposed to increasing levels of glucose at 50, 75, 100, 125 and 150 mm exhibited toxicity, reaching a maximal effect in 100 mm glucose, which resulted in 49.612.82% of relative cell viability. the glucose concentration of 100 mm was selected for further study as representative of hyperglycemic conditions that can decrease the viability of pc12 cells for assessing the protective effects of the zjf extract (figure 1). in this study, 24 hr treatment of cultured pc12 cells with different doses of zjf extract did not show toxic effects (figure 2). as shown in figure 3, zjf extract in the dose of 300 g / ml significantly inhibited high glucose - induced toxicity in pc12 cells after 24 hr ; whereas, zjf extract could not prevent cell damage in other concentrations (figure 3). effect of glucose on pc12 cell viability. pc12 cells exposed to increasing levels of glucose. data are expressed as mean sd ; n = 5 to 6 wells for each group ; p<0.01 and p<0.001 compared to control cells effects of various doses of ziziphus jujuba fruit (zjf) extract for 24 hr on pc12 cells viability. data are expressed as mean sd ; n = 56 wells for each group effects of various doses of ziziphus jujuba fruit (zjf) extract on high glucose - treated pc12 cell viability. high - glucose medium reduced cell viability and zjf extract (300 g / ml) protected the pc12 cells against high - glucose - induced cell death. data are expressed as meansd ; n = 5 to 6 wells for each group ; p<0.01 and p<0.001 compared to high glucose - treated cells to examine the intracellular ros in the hyperglycemic condition, we used the dcfh - da fluorescent method. as shown in figure 4, the levels of dcf - da fluorescence in pc12 cells treated 24 hr with high glucose media were increased markedly compared to control cells. furthermore, treatment with 300 g / ml zjf extract significantly decreased the production of the reactive oxygen spices in hyperglycemic cells (figure 4). effects of ziziphus jujuba fruit (zjf) extract on glucose - induced ros production in pc12 cells. data are expressed as meansd ; n= 5 to 6 wells for each group ; p<0.001 compared to control cells we examined caspase-3 activation by western blot analysis to evaluate the potential activity of zjf on preventing apoptosis after glucose induced cytotoxicity in pc12 cells. the pc12 cells were categorized to several groups including control, high glucose media and high - glucose media plus different concentrations of zjf extract. as shown in figure 5, 24 hr incubation with glucose at 100 mm enhanced expression of procaspase-3 protein compared to the control and vehicle groups. caspase-3 activation was increased in glucose treated cells after 24 hr. furthermore, treatment of pc12 cells with 300 g / ml zjf significantly antagonized high glucose - induced up - regulation of cleaved caspase-3 (figure 5). the activation of caspase-3 protein in pc12 cells exposed to high glucose medium and high - glucose plus 300 g / ml of ziziphus jujuba fruit (zjf) extract for 24 hr. each value in the graph is the mean sd band intensity for each group. since the optimal glucose concentration for pc12 cell cultures is 25 mm, we simulated in vitro hyperglycemia by increasing the medium glucose level at the concentrations of 50, 75, 100, 125 and 150 mm for 24 hr. pc12 cells exposed to increasing levels of glucose at 50, 75, 100, 125 and 150 mm exhibited toxicity, reaching a maximal effect in 100 mm glucose, which resulted in 49.612.82% of relative cell viability. the glucose concentration of 100 mm was selected for further study as representative of hyperglycemic conditions that can decrease the viability of pc12 cells for assessing the protective effects of the zjf extract (figure 1). in this study, 24 hr treatment of cultured pc12 cells with different doses of zjf extract did not show toxic effects (figure 2). as shown in figure 3, zjf extract in the dose of 300 g / ml significantly inhibited high glucose - induced toxicity in pc12 cells after 24 hr ; whereas, zjf extract could not prevent cell damage in other concentrations (figure 3). effect of glucose on pc12 cell viability. pc12 cells exposed to increasing levels of glucose. data are expressed as mean sd ; n = 5 to 6 wells for each group ; p<0.01 and p<0.001 compared to control cells effects of various doses of ziziphus jujuba fruit (zjf) extract for 24 hr on pc12 cells viability. data are expressed as mean sd ; n = 56 wells for each group effects of various doses of ziziphus jujuba fruit (zjf) extract on high glucose - treated pc12 cell viability. high - glucose medium reduced cell viability and zjf extract (300 g / ml) protected the pc12 cells against high - glucose - induced cell death. data are expressed as meansd ; n = 5 to 6 wells for each group ; p<0.01 and p<0.001 compared to high glucose - treated cells to examine the intracellular ros in the hyperglycemic condition, we used the dcfh - da fluorescent method. as shown in figure 4, the levels of dcf - da fluorescence in pc12 cells treated 24 hr with high glucose media were increased markedly compared to control cells. furthermore, treatment with 300 g / ml zjf extract significantly decreased the production of the reactive oxygen spices in hyperglycemic cells (figure 4). effects of ziziphus jujuba fruit (zjf) extract on glucose - induced ros production in pc12 cells. data are expressed as meansd ; n= 5 to 6 wells for each group ; p<0.001 compared to control cells we examined caspase-3 activation by western blot analysis to evaluate the potential activity of zjf on preventing apoptosis after glucose induced cytotoxicity in pc12 cells. the pc12 cells were categorized to several groups including control, high glucose media and high - glucose media plus different concentrations of zjf extract. as shown in figure 5, 24 hr incubation with glucose at 100 mm enhanced expression of procaspase-3 protein compared to the control and vehicle groups. caspase-3 activation was increased in glucose treated cells after 24 hr. furthermore, treatment of pc12 cells with 300 g / ml zjf significantly antagonized high glucose - induced up - regulation of cleaved caspase-3 (figure 5). the activation of caspase-3 protein in pc12 cells exposed to high glucose medium and high - glucose plus 300 g / ml of ziziphus jujuba fruit (zjf) extract for 24 hr. each value in the graph is the mean sd band intensity for each group. beta - actin was used as an internal control. p < 0.01 compared to control cells it is well known that diabetes and hypergly - caemic conditions elevate the formation of reactive oxygen species (ros) and decrease the cellular antioxidant defence capacity (5). hyperglycaemia produces oxidative stress, which leads to the production of superoxide and hydroxyl radicals, which in turn have direct toxic effects on nerve tissue (13). also, caspase (such as caspase-3 and -9) activation reportedly increases in high glucose conditions (13 - 16). our data in the present study revealed that glucose - induced toxicity in pc12 cells is mediated through ros generation and apoptosis. furthermore, 300 g / ml zjf aqueous extract successfully decreased the high glucose - induced ros generation and suppressed the activation of caspase-3, an apoptosis biomarker. for example, hydrogen peroxide can induce apoptotic cell death in several cell types (9). several pathology situations can result from the oxidative stress - induced apoptotic signalling that follows ros elevation and/or antioxidant diminutions, the disruption of intracellular redox homeostasis and irreversible oxidative reforms of dna, lipids and proteins (18). furthermore, caspase-3 activation through oxidative stress is important in the promotion of diabetic neuropathy (6). in recent years, the demonstration of anti - oxidative and neuroprotective characters of natural herbal products has drawn attention. it is notable that several natural herbal products reportedly protect neuronal cells from death and apoptosis in several neurodegenerative diseases (19, 20). zizyphus species (rhamnaceae) are broadly used as herbal medicines in different parts of the world, particularly in asia, for the treatment of various acute and chronic diseases (21). the zjf has been defined as the fruit of life, as it is an excellent source of vital functional components such as flavonoids, polyphenols, polysaccharides and saponins that are responsible for different biological activities including antitumor activity, improvement of central nervous system complaint, regulation of immune function, pain relief and decrease of blood glucose and triglyceride (12, 22 - 26). previous studies have shown that caffeic acid, p - coumaric acid, ferrulic acid and p - hydroxybenzoic acid have the highest amount of phenolic components in ziziphus that are responsible for the considerable antioxidant activity (27, 28). as mentioned before, our results revealed that hyperglycaemia leads to a significant increase in caspase-3 activation (as a main biochemical factor of apoptosis), and the zjf extract had a suppressing effect on its activation and eventually prevented high glucose - induced apoptosis in pc12 (figure 5). in addition, several studies show that some natural extracts suppress high glucose - induced neuronal cell dysfunction complications (such as diabetic neuropathy) through apoptosis inhibition (14, 29, 30). among the various natural antioxidants in ziziphus, caffeic acid and ferrulic acid additionally, caffeic acid prevents protein tyrosine kinase, lipoxygenase and cyclooxygenase activities (32 - 34). some studies show that caffeic acid can block / reduce the activation of tumour necrosis factor - alpha (tnf-) (35). in addition, tnf- activates nuclear factor kappa b (nf-b) (36, 37), which exists in neurons (8, 34). therefore, caffeic acid (one of the main components of z. jujuba) can prevent apoptosis induced by hyperglycaemic conditions. normally, the generation of ros and other free radicals are controlled by several innate scavenger molecules, which are typically found in the cell and quench free radicals. antioxidant enzymes such as superoxide dismutase, glutathione peroxidase (gpx) and several non - enzymatic free radical scavengers are involved in the antioxidant defence mechanisms (38). our previous study demonstrated that the zjf extract increased gpx activity in the hippocampus of ethanol - treated rats (12). with regard to this, the z. jujuba extract may prevent hyperglycaemic toxicity by elevating the intracellular antioxidant system (such as gpx) and detoxify the generation of the high glucose - induced mass of free radical and the generation of apoptosis. this study suggests that the aqueous extract of the zjf protects pc12 cells against high glucose - induced toxicity. the mechanisms underlying these effects may be due, at least in part, to reduced ros generation and apoptosis.	objective(s):the neuroprotective effect of fruit aqueous extract of ziziphus jujuba lam on glucose - induced neurotoxicity in pc12 cells as an appropriate in vitro model of diabetic neuropathy was investigated.materials and methods : cell viability was determined by the mtt assay. cellular reactive oxygen species (ros) generation was measured by dcfh - da analysis. cleaved caspase-3, a biochemical parameter of cellular apoptosis, was measured by western blot analysis.results:our data showed that a 4-fold elevation in glucose levels within the medium significantly reduced cell viability, increased intracellular ros and caspase-3 activation in pc12 cells after 24 hr. incubation of the high glucose medium cells with 300-g / ml z. jujuba fruit (zjf) extract decreased the high glucose - induced cell toxicity and prevented caspase-3 activation and excited ros generation.conclusion:thus, we concluded that the aqueous extract of z. jujuba protects against hyperglycaemia - induced cellular toxicity. this could be associated with the prevention of ros generation and neural apoptosis. moreover, the results suggest that the zjf has a therapeutic potential to attenuate diabetes complications such as neuropathy.
after more than 30 years since its first clinical use, cisplatin is still one of the most widely used drugs in anticancer chemotherapy. the action mechanism of cisplatin has been explained in its essential aspects, relatively to its interaction with dna. nevertheless, some essential chemical processes, related to what happens before the cisplatin reaches the dna, generally considered its final target, are still to be identified. among these processes, the best known is the formation of aquospecies, the main reaction of activation of the drug [2, 3 ] which occurs in the cytoplasmic compartment by hydrolysis of the chloride ligands. however, many other nongenomic biomolecules could be potential targets for platinum [4, 5 ]. sulfur - rich biomolecules, including free amino acids (cysteine and methionine), oligopeptides (glutathione), and proteins represent good targets for a soft metal such as pt [69 ]. moreover, the need to improve the cisplatin clinical protocol drives much research into better understanding of its antitumor activity mechanism. on the other hand, in order to overcome acquired cellular resistance to cisplatin, much effort is currently devoted to the discovery of new pt anticancer drugs. in the last years several pt(ii) and pt(iv) complexes have been synthesised, but only a few compounds, such as carboplatin and oxaliplatin [1, 11 ], are actually used in clinical therapy. many studies [1214 ], carried out by this research group [15, 16 ], aimed to understand not only the nuclear, but also the cytoplasmic events taking place in cisplatin - treated cells and able to induce apoptosis. this research group has long been involved in both the synthesis and preliminary evaluation of biological activity of the new pt complexes and in the subsequent studies of intracellular signal transduction, triggered by these molecules and by cisplatin itself [5, 1719 ]. recently, this group has synthesized and studied new platinum(ii) complexes containing acetylacetonate (acac) in the metal coordination sphere : [ptcl(o, o-acac)(dmso) ] (1a) with only one oxygen - bonded chelate acac (o, o-acac), [pt(o, o-acac)(-acac)(dmso) ] (2a) containing both an o, o-acac and a -bonded acac (-acac) and their dimethylsulphide (dms) analogues (1b and 2b) having the same key structures (scheme 1), that have shown interesting biological activities [2023 ] and in vitro antimetastatic activity. these compounds not only are able to induce apoptosis in endometrial cancer cells (hela), with activity up to about 100 times higher than that of cisplatin, but also show high cytotoxicity in cisplatin - resistant breast cancer cell lines (mcf-7). the [pt(o, o-acac)(-acac)(dms) ] complex (2b) with two acetylacetonate ligands, one o, o-chelate and the other one sigma - linked by methine in gamma position, is the more active among the tested complexes. as well as their specific biological activity, these complexes showed an interesting and selective chemical reactivity with nucleophiles with different hsab (hard - soft acid - base) character. indeed, in the complexes [ptcl(o, o-acac)l ] (l = dmso, dms), containing two ligands with different hard / soft character on the same metal, selective substitution reaction in the presence of further ligands was observed. the more hard ligand replaces the harder one, and the more soft replaces the softer one. however, in these complexes the replacement of cl with hard ligands is kinetically and thermodynamically less favoured with respect to the substitution reaction of dms or dmso with soft - type ligands. when only a soft ligand is present, as in the case of the complexes [pt(o, o-acac)(-acac)l ] (l = dmso, dms), the reaction takes place only in the presence of soft nucleophile, otherwise there is no reaction. these results, together with the biological studies, indicate that for these complexes, characterized by low reactivity with hard nucleophiles and specific reactivity with soft ligands, the dna could not be the main target. in this we reported the h nmr investigations on reactivity of the new compounds with hard and soft biological nucleophiles, such as nucleobases and sulfur amino acids, confirming selective reaction with the latter. in this work the well - known salmonella - his reversion test (ames ' test, a standard reverse mutation assay on the mutagenic capability of the complexes) on two salmonella typhimurium strains, ta98 and ta100, was performed. the bacteria reversed mutation assay (ames test), which is normally used to evaluate the mutagenic properties of test substrates, can be also used to assess the ability of tested compounds to interact with dna. h nmr spectra were recorded on a bruker avance dpx 400, using cdcl3, and d2o as solvent. h and c chemical shifts in cdcl3 were referred to tms, by using the residual protic solvent peaks as internal references. h and c chemical shifts in d2o were referenced to tsp (2,2,3,3-d(4)-3-(trimethyl - silyl)-propionic acid sodium salt), (h) = 0 ppm, as an external reference. pt chemical shifts were referenced to na2[ptcl6 ] (d(pt) = 0 ppm) in d2o as an external reference. [ptcl2(dmso)2 ] and k[ptcl3(dmso) ] were prepared according to previously reported procedures. a solution of acetylacetone (0.097 g, 0.973 mmol) and koh (0.027 g, 0.487 mmol) in methanol (5 ml) was added dropwise to a solution of k[ptcl3(dmso) ] (0.204 g, 0.487 mmol) in water (10 ml) at room temperature with stirring. after few minutes a yellow precipitate separated from the solution. the reaction mixture was left stirring overnight, and the pale yellow precipitate of [ptcl(o, o-acac)(dmso) ] (1a) was then isolated by filtration and dried under vacuum (yield 0.149 g, 75%). calcd for c7h13clo3spt (407.79) : c 20.62, h 3.21 ; found c 20.73, h 3.28. alternatively, a solution containing acetylacetone (0.097 g, 0.966 mmol) and koh (0.027 g, 0.483 mmol) in water (5 ml) was added dropwise to a suspension of cis-[ptcl2(dmso)2 ] (0.204 g, 0.483 mmol) in water (10 ml) at room temperature with stirring. the suspension was left under stirring for one day, and the solid was then filtered and dried under vacuum (yield 0.027 g, 26%). h nmr in cdcl3 (298 k) : 2.06s [3h, ch3(o, o-acac) ], 2.02s [3h, ch3(o, o-acac) ], 5.56s [1h, ch(o, o-acac) ], 3.44s [6h, ch3(dmso), jh - pt 40 hz ]. c nmr in cdcl3 (298 k) : 26.3 [2c, ch3(o, o-acac) ], 102.3 [1c, ch(o, o-acac) ], 185.1 and 185.9 [2c, co(o, o-acac) ], 44.3 [2c, ch3(dmso) ]. a solution of acetylacetone (0.358 g, 3.576 mmol) and koh (0.114 g, 2.860 mmol) in methanol (5 ml) was added dropwise to a suspension of cis-[ptcl2(dmso)2 ] (0.302 g, 0.715 mmol) in methanol (20 ml) at room temperature with stirring. the reaction mixture slowly became a pale yellow solution. after one day, the solvent was evaporated under vacuum, and the yellow residue was extracted with chcl3 (10 ml). the chloroform solution was then filtered to remove kcl and k(acac), pentane (30 quadrangular pale yellow crystals of [pt(o, o-acac)(-acac)(dmso) ] (2a) which separated out from the solution were filtered, washed with pentane, and dried under vacuum (yield 0.168 g, 50%). calcd for c12h20o5spt (471.441) : c 30.57, h 4.28 ; found c 30.73, h 4.34. hnmr in cdcl3 (298 k) : 2.00s [3h, ch3(o, o-acac) ], 1.95s [3h, ch3(o, o-acac) ], 5.53s [1h, ch(o, o-acac) ], 2.29s [6h, ch3(-acac) ], 4.79s [1h, ch(-acac), jh - pt 120 hz ], 3.31s [6h, ch3(dmso), jh - pt 19 hz ]. c nmr in cdcl3 (298 k) : 27.50 and 27.3 [2c, ch3(o, o-acac) ], 102.2 [1c, ch(o, o-acac) ], 185.8 and 184.9 [2c, co(o, o-acac) ], 30.9 [2c, ch3(-acac) ], 42.0 [1c, ch (-acac) ], 208.5 [2c, co(-acac) ], 42.9 [2c, ch3(dmso) ]. pt nmr in cdcl3 (298 k) : 3198. to a chloroform (3 ml) solution of 1a or 2a (0.1 g, 0.24 mmol for 1a and 0.1 g, 0.21 mmol for 2a) a large dms excess (0.224 g, 3.6 mmol for 1a and 0.263 g, 4.24 mmol for 2a) was added. the resulting yellow solution was added to pentane (10 ml) and kept at 5c for one day up to the formation of yellow needles crystals for 1b and pale yellow crystals for 2b. finally, (yield 0.075 g, 0.191 mmol, 80% for 1b. anal. calcd for c7h13clo2pts (391.773) : c 21.46 ; h 3.34 ; found : c 21.27 ; h 3.20 ; yield 0.078 g, 0.171 mmol, 82% for 2b. calcd for c7h13clo2pts (455.428) : c 31.65 ; h 4.43 ; found : c 31.72 ; h 4.56). k) : 1.97s [3h, ch3(o, o-acac) ], 1.88s [3h, ch3(o, o-acac) ], 5.48s [1h, ch(o, o-acac) ], 2.33s [6h, ch3(dms), jh - pt 48 hz ]. hnmr in cdcl3 of 2b (298 k) : 1.89s [3h, ch3(o, o-acac) ], 1.95s [3h, ch3(o, o-acac) ], 5.47s [1h, ch(o, o-acac) ], 2.20s [6h, ch3(-acac) ], 4.88s [1h, ch(-acac), jh - pt 124 hz ], 2.29s [6h, ch3(dms), jh - pt 51 hz ]. c nmr in cdcl3 of 1b (298 k) : 26.1 and 26.5 [2c, ch3(o, o-acac) ], 101.7 [1c, ch(o, o-acac) ], 184.9 and 182.9 [2c, co(o, o-acac) ], 22.1 [2c, ch3(dms) ]. c nmr in cdcl3 of 2b (298 k) : 27.50 and 27.1 [2c, ch3(o, o-acac) ], 101.6 [1c, ch(o, o-acac) ], 183.7 and 184.7 [2c, co(o, o-acac) ], 30.9 [2c, ch3(-acac) ], 40.5 [1c, ch (-acac) ], 207.2 [2c, co(-acac) ], 22.1 [2c, ch3(dms) ]. pt nmr in cdcl3 of 1b (298 k) : 2096. pt nmr in cdcl3 of 2b (298 k) : 2905. a solution containing the platinum complex (approximately 2 10 mmol) and an excess of guo, 5-gmp, or l - methionine (1.6 10 mmol) dissolved in d2o (1 ml) was placed in an nmr tube and the reaction monitored by h nmr spectroscopy. for all complexes tested, the reaction with guo and 5-gmp was negligible after 24 h, whereas the l - methionine instantly reacted with the initial pt complexes. two strains of salmonella typhimurium, ta98 and ta100 (kindly supplied by department of experimental studies and applied medicine, hygiene section, university of brescia), histidine auxotroph mutants, deficient in the synthesis of histidine (his), amino acid necessary for bacterial growth were used according with the method proposed by ames.. these strains contain other mutations that greatly increase the ability to detect mutagens : the mutation causes partial loss of the lipopolysaccharide barrier coating the surface of the bacteria and increases permeability to large molecules such as benzo[a]pyrene that do not penetrate the normal cell wall ; the mutation uvrb is a deletion of a gene coding for dna excision repair system, resulting in a high sensitivity to uv rays ; the r - factor plasmid, pkm101, carries ampicillin resistance gene. the histidine auxotrophs will only grow in a medium containing sufficient histidine supplement. to revert to histidine production (prototrophy), or become his+, a reverse mutation must occur in the original his mutation site (found in one of the genes involving histidine biosynthesis). each sample was investigated with the plate incorporation test : 100 l / ml of complexes (1a, 2a, and 2b), at different dilutions with dmso (from 0.01 g / plate to 30 g / plate), were added to 2 ml top agar and to 100 l of culture of s. typhimurium growth at optimal concentrations (10 u.f.c / ml). three plates were incubated for each of the dilutions tested. a solvent control (dmso) furthermore, a positive control was carried out using cisplatin, known as compound at strong mutagenic activity [32, 33 ], testing the same dilutions of new compounds. the results were expressed as numbers of net revertants, calculated as difference between the number of revertants / plate of tested compounds and the number of spontaneous revertants enumerated on the control plates. moreover, we calculated the mutagenicity ratio (mr)the ratio of the number of salmonella typhimurium revertants grown in the presence of the tested complex to the number of spontaneously appeared revertants (on the negative control). the synthesis of [ptcl(o, o-acac)(dmso) ] (1a), containing a single chelate acac, was straightforward. due to its low solubility in the reaction medium, it precipitates as a pale yellow powder from the reaction mixture of k[ptcl3(dmso) ] with acetylacetone and koh in water. complex 2a was obtained by treating cis-[ptcl2(dmso)2 ] with acetylacetone and koh in meoh and was isolated from their respective reaction mixtures by an appropriate workup procedure reported in the experimental section (scheme 2). in both cases, in order to prevent metal reduction, a slight excess of acetylacetone was used with respect to the calculated stoichiometric amount of koh based on starting platinum complex. it should be noted that the use of the scarcely soluble cis-[ptcl2(dmso)2 ] as a starting platinum complex for the preparation of 1a in meoh or water gave unsatisfactory results. due to the low solubility of cis-[ptcl2(dmso)2 ], the reaction with acetylacetone and koh in meoh resulted in an excess of acac in solution, even when using a stoichiometric or substoichiometric amount of acac and always gave a mixture of 1a, 2a and unreacted starting material. on the other hand, the reaction of cis-[ptcl2(dmso)2 ] with acetylacetone and koh in water, in which both the starting platinum complex and [ptcl(o, o- acac)(dmso) ] are sparingly soluble, gave analytically pure 1a although a longer reaction time was required and a lower yield was obtained. in the presence of dimethylsulfide (dms), [ptcl(o, o-acac)(dmso) ] (1a) and [pt(o, o-acac)(-acac)(dmso) ] (2a) complexes selectively undergo substitution of the sulfur ligand to give the analogous dms complexes [ptcl(o, o-acac)(dms) ] (1b) and [pt(o, o-acac)(-acac)(dms) ] (2b). interestingly, the substitution reaction appears to be very selective not only for 1a, where dmso is the only expected exchangeable ligand, but also for 2a, where, in principle, the chloroligand was also able to undergo substitution with dms. therefore, the synthetic procedures of 1b-2b complexes reported in the experimental section were developed, taking advantage of the selective reactivity showed by 1a-2a compounds towards soft ligand such as dms. for the synthesis of 1b and 2b complexes, an excess of dms was added to 1a and 2a, in order to complete the substitution reaction (scheme 3). by h nmr the reactivity of water soluble complexes (1a, 2a, and 2b) with biological nucleophiles (nucleobases and amino acids) was investigated. the poor water solubility of [ptcl(o, o-acac)(dms) ] (1b) prevented further investigation on its reactivity and biological activities. the reactions with soft biological nucleophiles, such as l - methionine (l - met), rapidly gave the same selective substitution of dmso or dms, already seen for these complexes towards classical soft nucleophiles (dms, pph3, ethylene, carbon monoxide). indeed, also in the presence of l - methionine excess both 1a and 2a complexes gave selective substitution reaction of dmso affording, respectively, to the neutral species [ptcl(o, o-acac)(l - met) ] [pt(o, o-acac)(-acac)(l - met) ]. moreover, this substitution reaction not only was more selective but also was faster. in figure 1 the h nmr time monitoring of reaction of 1a with l - met was reported. adding l - met excess to a solution of 1a in d2o, after only 5 minutes (the time needed to record a h nmr spectra) the substitution of dmso ligand and the coordination of l - met were observed by the decreasing of the signal at 3.44 ppm, assigned to the coordinated dmso ligand. at the same time, the increasing of the singlet at 2.6 ppm, attributed to the free dmso ligand, and the appearance of new signals related to the o, o-acac carrier ligand were detected. after 10 minutes, almost the entire starting complex was reacted with the l - met. the substitution reaction of 1a and 2a with soft biological nucleophiles (l - met) was more selective, especially in the case of 1a, where another good leaving group such as cl is present in the coordination sphere. contrary, in the reactions with biological nitrogen ligands, such as purines (guo, 5-gmp), both 1a and 2a showed little reactivity even after several hours. in the h nmr spectra in d2o of 1a in the presence of 5-gmp (figure 1) no new signals of coordinated or free dmso ligand assignable to substitution reaction products were observed. the same selective reactivity in the substitution of the soft sulfur ligand was observed in the reaction of the water soluble [pt(o, o-acac)(-acac)(dms) ] (2b) with l - methionine, guo, and 5-gmp. also in this case a very fast substitution reaction was noted in the presence of l - met excess. in figure 2 by addition of l - met excess to a deuterated water solution of 2b, the decreasing of singlet at 2.29 ppm, attributed to coordinated dms ligand, and the increasing of new signal of o, o-acac and -acac, assigned to the [pt(o, o-acac)(-acac)(l - met) ] species, were identified after only 10 minutes. analogously to 1a and 2a complexes, in the h nmr time monitoring of the reaction of 2b with guo or 5-gmp (figure 2) any substitution reaction occurred. such behaviour is very peculiar and suggests a possible selectivity in the substitution reaction at the metal centre in these systems ruled by the hard - soft characteristics of the leaving and incoming ligands. this selectivity could be also operating when the substitution at the metal involves biological sulfur ligands such as thiols or thioethers attached to proteins. furthermore, these compounds not only induced apoptosis in endometrial cancer cells (hela), but also showed high cytotoxicity in cisplatin - resistant breast cancer cell lines, with activity up to about 100 times higher with respect to cisplatin and in vitro antimetastatic activity. among all complexes, [pt(o, o-acac)(-acac)(dms) ] (2b) differently from cisplatin, for which the activity appears to be associated to both cellular accumulation and dna linking, intracellular total platinum amount analysis indicated a scarce reactivity of new complexes with dna, the principal biological target of cisplatin [21, 22 ]. moreover recent in vivo studies aimed to evaluate the outcomes of perinatal treatment with chemotherapeutic agents on key cns developmental processes such as neural cells proliferation, migration, and differentiation demonstrated that the brain platinum content after [pt(o, o-acac)(-acac)(dms) ] treatment was notably higher (approximately 4-fold as much) than after cisplatin. however, compared with cisplatin, [pt(o, o-acac)(-acac)(dms) ] induces less severe changes on fundamental events of neuroarchitecture development. all these data suggested that the cytotoxicity mechanisms of the new complexes may not necessarily require interaction with dna and that their cytotoxicity is associated only with the intracellular accumulation. the ames ' test, carried out on the new complexes, confirmed these results. the mutagenic activities of new complexes (1a, 2a, and 2b) with respect to cisplatin on two salmonella typhimurium strains (ta98 and ta100) were reported in figure 3 and table 1. as expected a rising of revertants at increasing doses of cisplatin was observed, reaching the highest number of net revertants at the highest tested doses (30 g / plate) on both strains. interestingly, at the same tested doses and on both salmonella typhimurium strains the new complexes showed negligible mutagenic activity. indeed, also at the highest tested doses no net revertants colonies were observed, whereas only cisplatin exhibited the well - known [32, 33 ] dose - dependent increase in revertants. hence, we can assert that the biological activity of the new pt(ii) acac complexes is related to the reaction with nongenomic biological targets. we have reported new pt(ii) -diketonate complexes with an intriguing chemical reactivity and interesting biological activities. the new complexes coordinate, instead of the mono- and bidentate amine ligands of the classical cisplatin analogues, o, o-acetylacetonate (acac) chelate as carrier ligand and dms or dmso ligands. due to their ability to induce apoptosis in endometrial cancer cells (hela) and in cisplatin - resistant breast cancer cell lines (mcf-7) with different pathways with respect to cisplatin, further investigations were performed on the reactivity of novel compounds with biological targets and on the mutagenic capability. indeed, differently from cisplatin, for which the activity appears to be associated with its intracellular accumulation and formation of dna adducts, the cytotoxicity of the new compound is only related to the intracellular accumulation. these complexes, besides their specific biological activity, showed an interesting and selective chemical reactivity towards nucleophile with different hsab (hard - soft acid - base) character. the same selective reactivity has been studied towards biological nucleophiles, such as nucleobase and amino acids. the new complexes showed also in these cases negligible reactivity with nucleobases (guo and 5-gmp) and gave selective substitution of dmso or dms with soft biological nucleophiles, such as l - methionine, suggesting that the cellular targets could be amino acid residues in proteins and enzymes involved in the apoptotic induction. interestingly, in the mutagenic tests carried out in this work on two salmonella typhimurium strains (ta98 and ta100) the new complexes showed, also at the highest tested doses, insignificant mutagenic activity with respect to cisplatin, known for its strong mutagenic activity and then used as a positive control. all these data suggest that the cytotoxicity mechanisms of the new -diketonate complexes may not necessarily require interaction with dna and that their biological activity is connected to the reaction with nongenomic biological targets.	new platinum(ii) complexes [ptcl(o, o-acac)(l) ] (1) and [pt(o, o-acac)(-acac)(l) ] (2) (l = dmso, a ; dms, b) containing a single chelated (o, o-acac) (1), or one chelated and one -bonded (-acac) acetylacetonate (2) have been synthesized. the new pt(ii) complexes exhibited high in vitro cytotoxicity on cisplatin sensitive and resistant cell lines and showed negligible reactivity with nucleobases (guo and 5-gmp) but selective substitution of dmso / dms with soft biological nucleophiles, such as l - methionine. in order to assess the ability of the new complexes with respect to cisplatin to induce apoptosis by interaction with nongenomic targets, the ames ' test, a standard reverse mutation assay, was carried out on two salmonella typhimurium strains (ta98 and ta100). interestingly, the new complexes did not show the well - known mutagenic activity exhibited by cisplatin and are, therefore, able to activate apoptotic pathways without interacting with dna.
chromatin is the complex of dna and histone proteins that packages eukaryotic dna into chromosomes. the nucleosome is the repeating structural subunit of chromatin and consists of 147 bp of dna wrapped around a histone octamer core. translational positioning of nucleosomes along the dna sequence influences the accessibility of regulatory sequences to the transcriptional machinery and can thereby regulate gene expression levels (for review, see hughes and rando, 2014, radman - livaja and rando, 2010). the average nucleosome - positioning profile over all yeast genes consists of a nucleosome - depleted region (ndr) of 150 bp, with well positioned 1 and + 1 nucleosomes upstream and downstream of the ndr, respectively. the transcription start site (tss) is located in the + 1 nucleosome, there is a regularly spaced nucleosomal array over the first kb of the gene body, and nucleosome positions become fuzzier toward the middle and end of the coding sequence (brogaard., 2012, because ndrs are thought to facilitate transcriptional activation by enabling access of regulatory proteins to their binding sequences, ndr formation or loss can lead to gene activation or silencing, respectively. the distribution of nucleosomes along the genome depends in part on the underlying dna sequence, with promoter regions enriched in poly a tracts mostly excluding nucleosomes (kaplan., 2009, yuan., 2005). in addition to poly a tracts that passively disfavor nucleosome assembly, ndrs can also be formed through active nucleosome removal from promoter regions by remodelers, such as rsc (parnell., 2008), or nucleosome displacement by general transcription factors (tfs), such as abf1 and rap1 (yarragudi., 2004, whereas dna sequence composition contributes to nucleosome occupancy in yeast, it is the action of chromatin remodelers and the transcriptional machinery that establishes precise nucleosome positioning over genes (gkikopoulos., 2011, hughes., 2012,, 2015, pointner., 2012, weiner., 2010). indeed, in vitro assembly of nucleosomes onto purified yeast genomic dna results only in nucleosome depletion over poly a tracts but little evidence for nucleosome positioning, whereas addition of yeast extract to such reconstitutions yields a more - accurate recapitulation of nucleosome positioning patterns observed in vivo (zhang., 2011). purified atp - dependent remodelers, such as chd1 and swi / snf family members, can generate ndrs at promoters and regularly spaced nucleosomal arrays over gene bodies similar to those seen in vivo, even in the absence of transcription (lieleg., however, such in vitro nucleosome reconstitutions do not perfectly match nucleosome positions observed in vivo (hughes., 2012). in vivo, the process of transcription plays a key role in nucleosome positioning, due both to the direct effects of rna polymerase on nucleosomes and to the effects of remodelers that are recruited to target genes during transcriptional activation or elongation (bintu., 2011, radman - livaja., 2011, studitsky., 1997, weiner., 2010). contrary to the steady - state landscape of nucleosome positioning, chromatin structure dynamics over the cell cycle, during which chromosomes are subject to dramatic perturbations caused by replication and mitosis, are not well characterized. dna replication initiates the disassembly of maternal nucleosomes ahead of the replication fork and their reassembly in its wake on one or the other daughter chromatid (alabert and groth, 2012). as nucleosomes can influence transcription, depending on their precise locations, replication provides an opportunity for the cell either to re - establish the same nucleosome - positioning profiles or to rearrange the nucleosomal landscape and thereby maintain or change its gene expression program, respectively. the process of nucleosome re - positioning after the disruption caused by replication is not well understood. specifically, it is not known how and where nucleosomes re - position themselves on newly replicated dna and how long it takes them to reconstitute the canonical mid - log positioning pattern. this is related to the questions of when transcription resumes after the disruption caused by dna replication, whether both new gene copies are transcribed, and whether transcription re - activation is a cause or a consequence of nucleosome positioning maturation. in order to address these questions, we have developed a method for genome - wide mapping of nucleosome positions on recently replicated dna in budding yeast : nascent chromatin avidin pull - down (nchap). in this method, we isolate newly synthesized dna at varying times after a pulse of the nucleotide analog 5-ethynyl-2-deoxyuridine (edu), which, along with micrococcal nuclease (mnase) digestion, allows us to follow genome - wide nucleosome - positioning dynamics after the passage of the replication fork on both leading and lagging dna copies. we find that nucleosomes assume their mid - log positions with varying rates at different genomic loci. because we find that highly transcribed genes exhibit a rapid return to their canonical chromatin architecture, we hypothesized that transcription participates in the regular phasing of nucleosomes in the gene body minutes after the passage of the fork. consistent with this, treatment with the rna polymerase inhibitor thiolutin interfered with chromatin maturation over coding regions. experiments in deletion mutants reveal a role for chd1 and isw1a in nucleosome phasing relative to the tss and a role for hir in determining the linker length between nucleosomes. in contrast to transcription - dependent maturation of gene body chromatin, aspects of promoter packaging, such as the ndr midpoint and the position of the + 1 nucleosome, appear to be determined earlier, possibly in the absence of transcription elongation. together, our data illuminate the genomic landscape of chromatin maturation following replication, and our methodology enables future genetic interrogation of the mechanisms responsible for chromatin maturation. in order to map the positions of nucleosomes on recently replicated dna, we developed nchap, which combines edu labeling of nascent dna (sirbu., 2011, wirges., 2007) with mnase digestion of chromatin. edu is a thymidine analog that is incorporated during the synthesis of new dna, and mnase is an endonuclease that preferentially cleaves dna in the linker regions between nucleosomes. cells are arrested in g1 with factor and released into s phase in the presence of edu. aliquots are taken at regular intervals following release into s phase, and cells are fixed with formaldehyde. fixed cells are then treated with mnase, and nucleosome - protected dna is isolated after cross - link reversal. edu from purified 150-bp fragments is conjugated to biotin azide in a click reaction, and biotinylated dna fragments are isolated using streptavidin - coated magnetic beads. illumina paired end sequencing libraries are prepared (adapted from borodina., 2011) from the nascent dna attached to beads. in order to differentiate between the lagging and the leading strand copies, the edu - containing nascent strand is separated from its template using primer extension from one end of the adaptor - ligated fragment (figure 1a). upstream of efficient replication origins will originate from the lagging strand copy, whereas the complementary crick reads will be from the leading copy. several controls validate the ability of nchap to identify newly replicated dna in relatively unperturbed cells. first, flow cytometry profiling of dna content shows that s phase progression is not impaired in the presence of edu (figure s1a). second, we tested the ability of our protocol to specifically capture the single edu - bearing strand of dna, using in - vitro - generated fragments that have incorporated edu in only one strand (figure s2). this fragment was then subjected to the same procedure that we used to generate nchap libraries as outlined in figures 1a, s2a, and s2b. following streptavidin pull - down of these test libraries, qpcr with strand - specific primers showed that the fraction of fragments with the edu - containing strand in the expected orientation was 70%85%. finally, we also generated dna fragments in which only one strand has incorporated edu in vivo, taking advantage of the 53 resection and gap - filling steps that occur during the repair of a double - strand break at the matalpha1 locus (reviewed in haber, 2012), which we induced in the presence of edu (figure s2c). here, we observe a 10-fold enrichment of the edu - containing strand in the expected orientation. overall, our tests show that our strand - specific library construction protocol can efficiently isolate the nascent dna strand and thus differentiate between leading and lagging strand copies of the genome. we next applied nchap to cells released from g1 arrest into s phase for varying lengths of time. nchap data at early time points reveal strong peaks surrounding known origins of replication (autonomously replicating sequence [ars ] ; nieduszynski., 2007, 2002), validating the ability of our protocol to specifically map nucleosomes assembled on newly synthesized dna. the enrichment of nchap data (blue peaks) around origins is not due to mnase bias toward replicated regions, because read density distributions from mnase - digested input fractions (before biotinylation and streptavidin pull - down) exhibit the relatively even occupancy expected of the nucleosome landscape in yeast (pink peaks). average nucleosome profiles from the nascent chromatin fraction resemble classic average nucleosome profiles, indicating that edu incorporation does not interfere with mnase digestion or nucleosome assembly (figure s3a). together, these data validate the ability of nchap to accurately identify nucleosome - protected regions of recently replicated dna in a strand - specific manner. during cell - cycle arrest and release, asynchrony among individual cells in the timing of g1 release and entry into s phase results in heterogeneity in the location of the replication fork in any given cell, meaning that, even for early firing regions of the genome, nchap will capture dna that has been replicated from 1 to as many as 20 min prior. consequently, in order to characterize the dynamics of chromatin maturation following replication, we carried out nchap across a time course in which asynchronous yeast was subject to a brief pulse of edu followed by a thymidine chase for varying lengths of time. an asynchronous population by definition contains cells at all stages of the cell cycle, including 15%40% of cells that are in s phase and which, as a population, will have replication forks located at every location along the genome. as a result, a relatively short pulse of edu will label short stretches of replicating dna covering the entire genome over the whole cell population. the thymidine chase stops further incorporation of edu, and subsequent fixation at regular time intervals provides snapshots of simultaneous nucleosome positioning changes shortly after replication in all replicating cells. in order to capture times as close as possible to the moment immediately after the passage of the replication fork, we sought to identify the minimal duration of the edu pulse that provided appreciable incorporation into replicated dna. we used flow cytometry profiling of cells labeled with fluorescein (fam)-conjugated edu to monitor the kinetics of edu incorporation in asynchronous cells (figure s4). edu labeling is detectable within 15 min of its addition to the culture (figure s4a), and the majority of replicating cells have incorporated edu after 2530 min (figure s4). edu incorporation could be delayed and slowed by growing yeast at suboptimal temperatures to extend the length of the cell cycle (figure s4b). at 30c, an initial lag phase of 15 min is followed by a gradual increase in the numbers of cells that have incorporated edu, as well as an increase in edu - fam fluorescence intensity per cell as genome replication progresses and more edu is incorporated in each s phase cell (figure s4c). a slower increase in the average cellular edu - fam intensity over time at 25c and 37c compared to 30c is consistent with slower or stalled replication forks, delayed s phase entry, and/or slower edu uptake and processing (figure s4c). because the fraction of edu - positive cells in the asynchronous cell population increases gradually and cells need to be incubated with edu for 25, 40, or 50 min (if grown at 30c, 37c, or 25c, respectively) before all cells that were in s phase at the moment of edu addition become edu positive (figure s4b), we conclude that the rates of edu import and processing can vary widely among different cells in the population, possibly due to variable expression of the edu transporter (hent1) and thymidine kinase (tk) that were introduced into our yeast strain. all subsequent experiments were performed at 30c. to assess whether we could effectively halt edu incorporation using an excess of cold thymidine, we pulsed asynchronous yeast with edu for 20 or 5 min and then assayed edu - fam at varying times after thymidine addition (figure s5). both the levels of edu - fam across the population and the fraction of edu - fam - positive cells stay constant up to 25 min after the thymidine chase (figures s5b and s5c), and then as cells enter a new round of replication, the fraction of edu - positive cells decreases, as expected for a successful thymidine chase. moreover, the average edu - fam intensity per cell remains low throughout the chase, consistent with edu being incorporated into only a small fraction of the genome, as intended (figure s5e). we carried out two separate pulse - chase time course experiments : one with a 20-min and another with a 5-min edu pulse (figure s5). we calculated the pearson correlation between the nucleosome - positioning profile for each gene in the yeast genome (from 500 bp upstream of the tss to the stop codon ; xu., 2009) in our nchap data and the corresponding profile from a mid - log total chromatin standard (weiner., 2010) for each time point (figure 2a ; table s1). for each gene, we define its maturation index as the average correlation over the time course between the nucleosome profiles from nascent chromatin and the total chromatin standard, with individual genes exhibiting a wide range of maturation indices (figures s3b and s3c). importantly, data for total chromatin consistently exhibit higher correlations to the standard than do nascent chromatin data (figure 2a ; plot below the heatmap), demonstrating that the wide range of maturation indices in nascent chromatin is not an artifact of variability in mnase digestion across the time course. progressive changes in nucleosome positioning on nascent dna are also evident from whole - genome pairwise correlation analysis (figure s6a ; table s2). what distinguishes genes that rapidly adopt their mature chromatin state following replication ? sorting genes according to their maturation index from the 20-min edu pulse experiment, we find that genes that have the highest maturation index are also generally highly transcribed during active growth in rich medium (kim., 2010). this trend is shown via a 50-gene running window average of rna pol2 occupancy and is even more evident when genes are divided into quintiles (1,245 genes each) of maturation indices : the average rna pol2 occupancy in the highest quintile (5) is 10-fold higher than in the lowest quintile (1 ; figure 2a, top middle and right panels). this suggests that the process of transcription plays a role in re - establishing nucleosome positions over genes after chromatin disruptions caused by dna replication. box plot and pairwise t test analysis of correlations to the standard for quintiles 1 and 5 from two biological replicates of the 20-min edu pulse experiment show that the two quintiles are significantly different in all time points from both replicates (p value of t test 0.05) and that most of the variability between replicates comes from early time points in the first quintile (figures s6b and s6c). although data from the 5-min edu time course differ quantitatively from data from the 20-min edu time course (figure 2b), in both datasets, nascent chromatin increasingly matches the mid - log standard as the time course progresses (figures 2a, bottom, and s7), and this trend is also replicated in the second 20-min edu pulse experiment (figure s7). moreover, the correlation of maturation indices in each quintile (defined above) with average rna pol2 occupancy is also preserved in both time courses when focusing on robustly measured genes (figure 2b). to examine the process of chromatin maturation more closely, we compared the average tss - aligned nucleosome profiles from nascent chromatin to the same profiles obtained from mid - log yeast, averaging data according to quintiles of genes grouped by maturation index. visual inspection of these averaged profiles reveals that nucleosomes become better defined over the first kb of the gene body over time (figures 2c2e). this improvement in nucleosome phasing can be quantitated using a measure of peak to trough for nucleosomes a low peak - to - trough ratio can either be due to a low average nucleosome occupancy at that position across the cell population or fuzzy positioning (nucleosomes are not placed at the same distance from the tss in all genes and in all cells). the average peak - to - trough ratios for nucleosomes + 2 to + 7 on nascent chromatin after a 20-min edu pulse reach a plateau between the 4- and 8-min time points for rapidly maturing (fifth quintile) genes, whereas, in the first quintile, the ratios are stabilized only 10 min later (figure 2d, top). similar rates of average peak / trough increase were confirmed in a second biological replicate of the 20-min edu pulse experiment (figure s8). a quadratic fit to the curves from figure 2d (top) reveals that genes in the first and fifth quintiles reach their half - maximal peak / trough 20 and 7 min after the chase, respectively. consistent results were obtained using the data from the 5-min edu pulse (figure 2e), albeit with somewhat more - rapid apparent rates of nucleosome phasing over gene bodies (figures 2d, bottom, and 2e). as the genes that exhibit comparatively rapid phasing of nucleosomes over gene bodies (the fifth quintile) are relatively highly transcribed, we propose that the regular phasing of nucleosomes downstream of the tss is a consequence of transcription elongation. in contrast to the relatively slow chromatin maturation observed over gene bodies, the + 1 nucleosome and the midpoint of the ndr are already in place at the beginning of our time course for the majority of genes (figures 2c and 2e). thus, the positions of the + 1 nucleosome and the ndr are determined early after the passage of the replication fork. indeed, promoter chromatin architecture is established so rapidly that it is impossible to pinpoint its exact kinetics using our assay, as even for the 5-min edu pulse time course, there is a 10-min window between edu addition and the first recorded time point, during which time edu is incorporated into dna at different moments in different cells (figure s4). to directly test the involvement of transcription in nucleosome positioning maturation, we treated cells with the rna polymerase inhibitor thiolutin in a parallel experiment with the 5-min edu pulse time course shown in figure 2 (figure s9). chromatin maturation is greatly impaired upon treatment with the inhibitor (figures 3, s6, and s7), thus providing experimental support to our hypothesis that transcription elongation is involved in the reordering of nucleosomes on nascent dna. importantly, thiolutin specifically affects nascent chromatin maturation, as total chromatin fractions from thiolutin - treated and untreated cells are nearly indistinguishable (figures 3, s6, and s7). note that thiolutin has been added after the edu pulse to avoid negative effects of the inhibitor on replication and edu incorporation. as expected, the average peak / trough ratios in thiolutin - treated cells are lower than in non - treated cells. we can still detect slow nucleosome phasing maturation, with peak / trough ratios increasing at a similar rate in the first and fifth quintiles, even in the absence of transcription (figure 3c), suggesting that there is also a transcription - independent mechanism responsible for nucleosome reorganization. still, we can not exclude the possibility that some residual transcription occurs in thiolutin - treated cells and accounts for the observed slow chromatin maturation. in any case, we conclude that transcription plays a central role in establishing chromatin architecture over gene bodies. in order to better understand the mechanisms involved in nucleosome positioning maturation, we repeated the 5-min edu pulse - chase experiment in mutants with deletions of hir1, chd1, or ioc3 chd1 and isw1a are both atp - dependent chromatin remodelers that associate with the gene body during transcription and are involved in nucleosome array organization over coding sequences (gkikopoulos., 2011, radman - livaja., 2012, smolle., hir1 is a subunit of the hir nucleosome assembly complex, which participates in histone turnover and replication - independent nucleosome assembly (green., 2005, lopes da rosa., 2011, ray - gallet., 2002). nucleosome phasing is globally reduced in all three mutants (although to a somewhat lesser extent in hir1) compared to wild - type (wt) profiles (figure 4b, left). the level of disorganization of nucleosomal arrays in gene bodies is comparable between nascent and total chromatin profiles, suggesting that perturbations caused by dna replication persist after s phase in the absence of these chromatin remodelers. more striking, however, is the difference in linker lengths between nascent and total chromatin profiles (figure 4b, right). nucleosomes appear to be less densely packed shortly after replication, with an average linker length of 20 bp compared to 13 bp in total chromatin in wt, chd1, and ioc3 cells alike. in hir1 cells, total and nascent chromatin have the same 20-bp linker length, suggesting that hir activity tightens the spacing between nucleosomes after replication, consistent with a recent report on hir1 effects on nucleosome positioning in nascent chromatin (fennessy and owen - hughes, 2016). as detailed above, the strand separation step in our library - generation protocol enables us to distinguish between nucleosome - positioning profiles on the leading and lagging daughter chromatids. the leading or lagging copy annotation is assigned according to the position of a gene relative to its closest replication origin and whether its reads map to the watson or the crick strand watson reads upstream and downstream of an origin will be lagging and leading copies, respectively. however, due to varying efficiencies of yeast origins (every origin is not activated in every s phase cell ; yang., 2010), leading and lagging annotations can only be unambiguously assigned to genes located near efficient origins of replication, leaving us with a set of 1,064 genes (figures s10a and s10b ; experimental procedures). nucleosome profiles of lagging and leading copies of each gene in this set and from every time point were compared to the corresponding profiles from the final (22 min) time point in the 20-min edu pulse - chase experiment (figure 2). the resulting correlation profiles were ordered according to the average difference (for all three experiments) in maturation indices between the lagging and the leading strand copies of each gene (figure 5a ; table s3). we could detect comparable differences in nucleosome positioning maturation on leading and lagging copies in the three datasets at 433 genes out 1,064, with 200 genes showing significant differences between leading and lagging copies (figures 5b and s11), suggesting that nucleosome re - positioning can occur independently on the two daughter chromatids. there is no global effect of leading or lagging strand replication on nucleosome re - positioning because maturation indices are higher on the leading or the lagging copy in equal proportions. lagging and leading profiles from total chromatin also show asymmetry in their correlations, albeit to a lesser extent than the nascent profiles (figures 5a and 5e). this is not due to mnase sequence bias toward the watson or the crick strands of individual genes, as all genes were compared to the corresponding watson or crick profiles from the 22-min time point standard. it is more likely that the asymmetry we observe in total chromatin profiles comes from the substantial fraction of s phase cells, which in this experiment represents 40% of the population (figure s9b). the relatively slower - maturing copies in the top and bottom quartile of the heatmap in figure 5a are enriched for genes in which the newly synthesized strand also serves as the template for transcription (figure 5c). this observation is consistent with at least two hypotheses : (1) edu incorporation on the template strand potentially interferes with rna pol2 initiation or elongation, thus delaying transcription - coupled chromatin maturation (figures s10c and s10d) or (2) asymmetric recruitment of chromatin remodeling enzymes and/or tfs to one copy, resulting in the preferential transcription of that gene copy. for example, asymmetric transcription following replication could result from transcription preferentially occurring on the leading strand copy when the newly synthesized copy of a gene s promoter and the replication fork are oriented in the same direction, potentially as a result of the underassembly of chromatin on the lagging strand immediately behind the fork. several observations suggest that asymmetric chromatin maturation results from differential expression of the two gene copies after replication. first, differences between maturation indices of the leading and lagging copies are substantially reduced in the presence of thiolutin, suggesting that differences in chromatin maturation dynamics on the two copies of a gene may be due to differences in transcription rates of the two copies, i.e., when neither gene copy is transcribed, chromatin maturation is equally slow on either copy (figures 5d and s11). second, to test whether edu interferes with transcription when it is incorporated in the template strand, we compared steady - state mrna levels in mid and late s phase of edu - treated and untreated synchronized cells using gene expression microarrays in two independent biological replicates (figure 6). consistent with recent studies (voichek., 2016), we find that rna levels of 95% of cell - cycle - independent genes do not change from mid to late s phase, which could be a consequence of either a 2-fold decrease in transcription rates on both copies or the expression of only one gene copy. for this group of genes, edu also had no effect on rna levels (figures 6a and 6b), arguing against the hypothesis that asymmetric chromatin maturation is an artifact of edu effects on transcription. moreover, although a small group of 343 genes (whose expression was not buffered after s phase) exhibited edu - dependent inhibition of transcription (figures 6b and 6c), these genes are not enriched for genes that show differences in chromatin maturation between the leading and the lagging gene copies. the non - buffered gene set is enriched for ribosomal genes and genes involved in translation (figure 6d). we propose that, for the majority of genes, rna production is buffered after genome replication by the suppression of transcription in one of the two copies (probably the one with the nascent strand as the transcription template), which we detect as a difference in transcription - dependent chromatin maturation between the two gene copies (figure 5). future studies will focus on the mechanisms that regulate gene expression levels in the genome after replication, which should clarify whether the two gene copies are differentially transcribed following replication. chromatin features change throughout the cell cycle, with the biggest perturbations occurring during dna replication and mitosis. histone proteins on the maternal genome are removed from the dna ahead of the replication fork and are recycled on one or the other daughter chromatid, with newly synthesized histones restoring a full complement of nucleosomes to both daughter genomes. here, we describe a method for following nucleosome positioning dynamics on newly replicated dna, which we call nchap. nchap allows us to isolate nascent chromatin and follow in parallel changes in nucleosome positioning on the leading and lagging strand chromatids shortly after the passage of the replication fork. whereas other studies have concentrated on proteomic analysis of bulk nascent chromatin (alabert., 2014, 2011), we provide a genome - wide locus - specific timeline of nucleosome footprint changes after replication. an earlier study, which mapped okazaki fragments, reported an enrichment of okazaki fragment ends at the positions of nucleosome dyad axes (smith and whitehouse, 2012). however, it is not clear whether nucleosome assembly on the lagging strand precedes okazaki fragment ligation, as proposed. given that we find that nucleosome maturation is not significantly faster on leading or lagging strand genomes (figure 5), we conclude that any lagging strand - specific nucleosome deposition processes must occur rapidly relative to the 10-min time resolution achieved here and that the nucleosome positioning maturation process described here takes place after okazaki fragment maturation. we propose that gene expression programs are maintained from one cell generation to the next by the formation of ndrs on daughter chromatids very early after the passage of the replication fork (figure 7a). due to the heterogeneous rates of edu import in the population, we can only conclude that promoter maturation happens within 10 min after replication fork passage, and this process most likely occurs independently of transcriptional elongation, because transcription - dependent nucleosome phasing is detected later on in our time course. it may involve a dna sequence that repels nucleosomes (such as poly a tracts) and consequently favors the (re)binding of tfs, chromatin remodelers, and other components of the transcription machinery that were probably present at the locus before replication. or an initially bound nucleosome may be evicted through the action of a chromatin remodeler recruited to the site by a sequence - specific tf, or the bound tf itself may prevent nucleosome binding. these tfs / remodelers presumably then help establish the positions of the + 1 and 1 nucleosomes. a similar model was recently proposed for promoter architecture re - establishment after replication in drosophila cells (ramachandran and henikoff, 2016). in regions without nucleosome - repelling sequences or without sequences for available tfs, nucleosomes are deposited at regular intervals shortly after the passage of the replication fork. at this stage, nucleosomes are slightly delocalized (i.e., not exactly in the same position), both on the two nascent copies within the same cell and in different cells in the population, resulting in average nucleosome profiles with low peak - to - trough ratios. remodelers that accompany the elongating rna pol2 (chd1 or isw1a) reorder nucleosomes in its wake and position them at more - regular intervals. consequently, nucleosomes on genes with higher rna pol2 occupancy will be better phased over the whole population. concomitantly, hir reduces the spacing between nucleosomes from on average 20 bp (found in nascent chromatin) to 13 bp (measured in total chromatin). we nevertheless found a significant number of genes with low rna pol2 occupancy and high maturation indices, which, as suggested by our results with thiolutin - treated cells, is probably due to a transcription - independent chromatin maturation process. this could be a consequence of the action of transcription - independent chromatin remodelers, although it is not clear why these remodelers only act on a subset of poorly transcribed genes. it is also possible that, in the absence of transcription, nucleosomes are reorganized passively through statistical positioning within discrete domains located between boundaries akin to chromosomal interaction domains (cids) recently observed in yeast (hsieh., 2015). alternatively, a transient burst of transcription right after replication could also be responsible for high maturation indices on these genes. a detailed analysis of rna pol2 occupancy kinetics on nascent dna will be necessary to distinguish between these possibilities. because the nascent strand in the slower - maturing copy tends to be the transcription template strand, observed differences in maturation rates between leading and lagging gene copies could be a consequence of gene expression buffering. whereas rna pol2 progression may be impaired when it encounters edu in the template strand, transcription of both copies of the gene after replication in the absence of edu is only seen at 343 mostly highly transcribed genes (note that these are not the same genes that exhibit copy - specific maturation behaviors). the reason there is no detectable edu effect on steady - state rna levels in most genes could be a consequence of recently reported buffering effects of h3k56 acetylation by rtt109 (voichek., 2016) that potentially operate via the preferential expression of only one gene copy after replication. we propose that the suppressed copy would generally be the one with the nascent strand as the transcription template strand. consequently, differences in maturation rates between the two gene copies might be caused by asymmetric h3k56 acetylation and recruitment of chromatin - remodeling enzymes and/or tfs to one of the copies, which would cause differences in transcription rates and nucleosome positioning maturation rates. interestingly, when the lagging nascent strand is the transcription template, the replication fork and rna polymerase advance in the same direction. conversely, replication and transcription travel in opposite directions when the transcription template strand is the leading nascent strand. it is therefore possible that tfs bound to promoters ahead of the fork rebind preferentially to the leading or lagging copy after replication as a consequence of the differential rates of okazaki fragment ligation and fork speed. as illustrated in figure 7b, it is less likely that rna pol2 (on the yet un - replicated promoter) and the replication fork will collide when replication and transcription travel in the same direction. as the fork travels unhindered, okazaki fragment ligation may lag behind the fork and tfs bound to the promoter ahead of the fork are more likely to rebind to the leading copy, thus favoring transcription and consequently chromatin maturation of the leading copy as observed. on the other hand, when the two travel in opposite directions, a head - on collision of the fork and rna pol2 that is ahead of the fork is more likely. the fork then possibly stalls and slows down, and okazaki fragment ligation now happens almost simultaneously with synthesis, which allows tfs to bind to the leading or lagging copy of the promoter. it is, however, difficult to imagine why there would be a bias toward the lagging copy as our results predict. future studies will test whether only one gene copy is transcribed after replication and consequently find out which copy is suppressed. taken together, future studies should further illuminate the mechanisms responsible for rapid establishment of nucleosome positions and could potentially identify subtle consequences of slow maturation on genome function. detailed protocols are available in the supplemental experimental procedures. all experiments (except those in figure s2c) were done with the strain pv1 (mata ade2 - 1 trp1 - 1 can1 - 1000 leu2 - 3,112 his3 - 11,15 gal psi+ rad5 + ura3::ura3/gpd - tk(7x) aur1c::adh - hent1 bar1::kanr). the experiment in figure s2c was done with the cvy61ho strain (mata ade2 - 1 his3 - 11,15 trp1 - 1 leu2 - 3,112 can1 - 100 bar1::hisg trp1::brdu inc [brdu = hsv - tk + hent1 ] pjh132 [gal::ho ura3 ]). mutant strains from figure 4 are as follows : hir1 : strain ac5 (mata ade2 - 1 his3 - 11,15 leu2 - 3,112 ura3 - 1 trp+ can1 - 100 gal psi+ rad5 + ura3::gdp - tk(7x) aur1c::adh - hent1 hir1::nat bar1::kanr);chd1 : strain rz12 (mata ade2 - 1 his3 - 11,15 leu2 - 3,112 trp1 - 1 ura3 - 1 can1 - 100 gal psi+ rad5 + ura3::gdp - tk(7x) aur1c::adh - hent1 chd1::leu2) ; andioc3 : strain rz15 (mata ade2 - 1 his3 - 11,15 leu2 - 3,112 trp1 - 1 ura3 - 1 can1 - 100 gal psi+ rad5 + ura3::gdp - tk(7x) aur1c::adh - hent1 ioc3::kanr). hir1 : strain ac5 (mata ade2 - 1 his3 - 11,15 leu2 - 3,112 ura3 - 1 trp+ can1 - 100 gal psi+ rad5 + ura3::gdp - tk(7x) aur1c::adh - hent1 hir1::nat bar1::kanr) ; chd1 : strain rz12 (mata ade2 - 1 his3 - 11,15 leu2 - 3,112 trp1 - 1 ura3 - 1 can1 - 100 gal psi+ rad5 + ura3::gdp - tk(7x) aur1c::adh - hent1 chd1::leu2) ; and ioc3 : strain rz15 (mata ade2 - 1 his3 - 11,15 leu2 - 3,112 trp1 - 1 ura3 - 1 can1 - 100 gal psi+ rad5 + ura3::gdp - tk(7x) aur1c::adh - hent1 ioc3::kanr). for the synchronization experiment in figure 1, cells were arrested in g1 with factor and then transferred into preheated media containing 10 m edu and pronase. aliquots were taken and fixed with 1% formaldehyde right before release and then at regular intervals after release starting at 25 min from media change. in the edu - thymidine pulse - chase experiments, exponentially growing cells were transferred to media with 10 m edu preheated at 30c. thymidine was added after 5 or 20 min and incubated for another 5 or 10 min. cells were then pelleted and transferred into fresh media with thymidine (and thiolutin when indicated), and aliquots were taken at indicated time points and fixed as above. cells from frozen pellets were spheroplasted by bead beating in the bullet blender (next advance). spheroplasts were treated with mnase, which was adjusted to the cell density in each tube in order to obtain 80%90% mononucleosomal - sized fragments after 20 min at 37c. after cross - link reversal, dna was extracted with phenol- chloroform - iso amyl alcohol (pci), and 150-bp mono - nucleosomal sized fragments were subsequently purified from 2% agarose gels. purified 150-bp fragments were mixed with biotin azide in a cubr solution. after a 2-hr incubation at 37c, biotinylated dna was incubated with streptavidin - coated magnetic beads (blocked with salmon sperm dna). following ligation with illumina genome sequencing adaptors with in - line barcodes, dna was subjected to primer (illumina pe primer 2.0) extension with 2deoxyuridine 5-triphosphate (dutp) to separate the nascent strand from its complement. after degradation of the dutp - containing strand with user enzyme, the nascent dna strand was pcr amplified. paired - end sequencing was done on a hiseq 2000 (illumina ; cnag) or on a next seq sequencer (illumina) in o.j.r.s laboratory. measurements were made with facscalibur (bd biosciences ; fl-1 filter ; forward scattered light [fcs ] size cutoff : 70). genomic dna was isolated from g1-arrested flash - frozen cell pellets and sonicated with the bioruptor pico cup sonicator. cells were released into s phase in media with or without 10 m edu, as above. fifty - milliliter aliquots were flash frozen in liquid nitrogen 32 and 40 min after release for rna isolation. total rna was isolated from frozen pv1 cell pellets with trizol and treated with dnase i. rna was reverse transcribed using oligodt as primers. the resulting cdna was dye coupled with cy5 or cy3 n - hydroxysuccinimide (nhs) esters and purified as described previously (liu., 2005). the cy5- or cy3-labeled cdna was mixed with cy3- or cy5-labeled genomic dna, respectively, and hybridized to agilent 8x15k yeast gene expression arrays. images were scanned with the innoscan 710 microarray scanner (innopsys) and processed with the mapix software. data were normalized by dividing the cy5/cy3 ratio for each probe with the average cy5/cy3 ratio for the whole array. sequences were aligned to the s. cerevisiae genome using blast - like alignment tool (blat). we kept reads that had at least one uniquely aligned 100% match in the paired - end pair. read count distribution was normalized to one by dividing each base pair count with the genome - wide average base - pair count. genes that are replicated from efficient origins were filtered as follows : (1) efficient origins were defined as origins whose read density peak heights were 0.6 at the 25-min time point in the experiment from figure 1. (2) genes that were within the boundaries of the read density area around efficient origins at the 25-min time point were considered as being replicated from that particular origin in most cells. analysis was done using in - house perl and r scripts (available upon request). the analysis in figures s6, s7, and s11 was performed using perl (statistics::ttest) and r scripts as detailed in the supplemental experimental procedures and figures s6, s7, and s11. m.r.- l. developed nchap, designed the experiments, performed the experiments in figure s2, developed analysis tools, analyzed the data, and wrote the manuscript. o.j.r. initiated the development of nchap, advised on experimental design and data analysis, and wrote the manuscript.	summarychromatin is thought to carry epigenetic information from one generation to the next, although it is unclear how such information survives the disruptions of nucleosomal architecture occurring during genomic replication. here, we measure a key aspect of chromatin structure dynamics during replication how rapidly nucleosome positions are established on the newly replicated daughter genomes. by isolating newly synthesized dna marked with 5-ethynyl-2-deoxyuridine (edu), we characterize nucleosome positions on both daughter genomes of s. cerevisiae during chromatin maturation. we find that nucleosomes rapidly adopt their mid - log positions at highly transcribed genes, which is consistent with a role for transcription in positioning nucleosomes in vivo. additionally, experiments in hir1 mutants reveal a role for hir in nucleosome spacing. we also characterized nucleosome positions on the leading and lagging strands, uncovering differences in chromatin maturation dynamics at hundreds of genes. our data define the maturation dynamics of newly replicated chromatin and support a role for transcription in sculpting the chromatin template.
we report on a 63-year - old, average frame, caucasian male found to have clinical stage t1c adenocarcinoma of the prostate who, after discussion of all options, elected to undergo laparoscopic radical prostatectomy (larp). a routine preoperative course then followed, including a medical history and physical examination, normal chest x - ray, electrocardiogram, basic metabolic panel and complete blood count. the procedure was performed with an operative time of 276 min and was without incident. blood loss was approximately 200 ml, and the patient was hemodynamically stable throughout the entire procedure. after reversal of anesthesia and extubation, he was reported to be able to move all extremities upon arrival to the post anesthesia care unit (pacu). a few moments later he was unable to demonstrate any movement of all four extremities. the anesthesia personnel were asked to first exclude drug reaction or persistent neuromuscular blockade from muscle relaxants ; neurology and spinal surgery consultation followed within the hour, and his sensation and muscle tone was found to be intact. deep tendon reflexes were grade ii throughout all extremities a non - contrast computed tomography (ct) scan of the brain was negative for acute hemorrhage. magnetic resonance imaging (mri) of the brain, thoracic and cervical spine proved to be negative for spinal cord compression or injury, and a magnetic resonance arteriogram (mra) of the brain showed no aneurysm or other lesion. neurology and spinal surgery neurological findings were consistent with pure motor quadriplegia with a differential diagnosis including an anterior spinal cord infarct or ischemia not evidenced by the mri. spinal cord injury protocol was initiated including bolus dose steroids followed by continuous steroid infusion. at 12 and 24 h post - surgery the patient still demonstrated complete motor quadriplegia. with the diagnosis of spinal injury becoming less favored, a trial of hyperbaric oxygen therapy to treat a presumptive diagnosis of gas air embolism (gae) was requested. the first hyperbaric oxygen therapy was started approximately 26 h postoperatively and led to an immediate ability to move his upper extremities and some minimal movement of his lower extremities. twelve hours later the second hyperbaric oxygen treatment appeared to further increase his motor function. an aggressive physical therapy regimen followed and over the course of the next 14 days a slow but steady recovery was appreciated. he is now more than 8 weeks postoperative and is fully recovered and functional in the community. laparoscopic radical prostatectomy is rapidly becoming the procedure of choice for surgical treatment of clinically localized prostate cancer. current recommended guidelines for positioning were followed and include having the patient in steep trendelenberg position, the legs placed in stirrups, and the knees flexed and lowered. all pressure points are padded with foam pads to prevent injury and the neck remains aligned and supported in a neutral position. postoperatively our patient reported a remote history of minor hyperextension cervical spine injury that required no surgery and left him with no deficits. there was no evidence of congenital abnormality or injury on any of our imaging studies. reversible motor paralysis is not a known complication of larp or other laparoscopic surgery that could be found in the current literature. however, there have been reports of specific deficits including paralysis in the literature that were attributed to gae by other authors [35 ]. the lack of significant response to steroids in our case led us to doubt spinal cord injury as the explanation for these deficits. negative mri findings agreed with this conclusion, and a repeat mri was performed 1 week postoperatively that showed no delayed findings consistent with injury. our first discussion of a diagnosis of gae or anterior spinal cord ischemia was entertained early on postoperative day one. with no current diagnostic modality to confirm this suspicion, a trial of hyperbaric oxygen therapy was suggested and with excellent response. most of the literature on gae and treatment modalities with hyperbaric oxygen therapy is based on decompression sickness and scuba diving injuries [68 ].. demonstrated venous outflow obstruction as the cause of paralysis following induced decompression sickness in dogs. the dogs were treated with dextran 70 with various outcomes prior to euthanization and autopsy. there was significant white matter destruction showing extensive spongy degeneration and focal zones of demyelination with sparing of the gray matter. these findings were felt to be consistent with venous occlusion from gas embolism leading to the paralysis. gae has been shown to produce similar spinal cord syndromes in other reports and typically has responded well to hyperbaric oxygen therapy. the pneumoperitoneum used in larp could have resulted in venous entry of gas embolus eventually traveling to the inferior vena cava (ivc). upon removal of the steep trendelenberg position, the gae could travel to the more proximal vena cava and cause obstruction of the lumbar veins and subsequently venous congestion compromising the spinal cord. this might explain the delay between the end of the procedure and the onset of motor paralysis. although the diagnosis of gae with spinal cord involvement was suspected, there is no typical presentation or firm method of diagnosis available. with a significant index of suspicion for a gae and a rapid recovery with hyperbaric oxygen therapy although no immediate response was recognized, steroid administration could have been responsible for the eventual recovery as well. regardless of the accurate etiology of the paralysis, we want to stress here the importance of early (i.e. < 8 h) multidisciplinary evaluation and treatment. treatment including steroid administration to cover spinal cord injury and hyperbaric oxygen therapy if gae is a consideration could be critical in this rare event.	laparoscopic radical prostatectomy (larp) has been accepted as first line therapy for clinically localized prostate cancer. complications have been low and outcomes are comparable to that of open surgery with potential benefits including shorter hospital stay, less pain and quicker return to normal activity. unexplained paralysis following larp is a rare entity with no reported cases in the current literature. we report a case of complete motor paralysis following larp. an extensive multidisciplinary evaluation did not definitively establish a diagnosis. aggressive multimodality treatment led to a complete recovery. our understanding of this phenomena with the possible etiology and treatment is discussed.
the patient was a 10 year - old boy with a height of 128 cm and a weight of 36 kg who complained of pain in the right lower abdomen. he was diagnosed with appendicitis, and it was decided to do an appendectomy. he had been diagnosed with grade 3 mental retardation at age 4 although an exact diagnosis was not possible because a dna test was not done. however, the parents were planning for a second child and had a dna test done in england when the patient was 10 and confirmed an 8p23 deletion. according to the past history, there were no abnormalities that accompanied the genetic disorder such as asthma, and dna tests were done on the parents for a family history but all were normal. based on the physical exam, the patient exhibited the characteristic facial features of microcephaly with a broad nasal bridge, short neck, and high arched palate. according to the airway examination, the interincisional distance was approximately 3 cm and the thyromental distance was approximately 5 cm when the mouth was opened at its widest maximum. the mallampati airway classification was grade 3 and difficulty in intubation was predicted ; there were no limitations in neck movement and there were no loose teeth but they were irregular. in an echocardiography conducted at another hospital, the patient had no heart problems that can accompany 50% of the people with deletion 8p23 syndrome, and there were no indication of ebstein 's malformation and tof. no abnormalities were found in the blood tests or computed tomography (ct) done before surgery. the patient exhibited severe mental retardation and although simple communication was possible, he was not able to use language properly. the patient responded impulsively and showed destructive and aggressive behavior characteristic to this syndrome when moving to the operating bed such as pulling hair and kicking with his feet. blood pressure before anesthesia was 125/70 mmhg ; heart rate was 115 beats / min ; oxygen saturation was at 100%, and the ecg was normal. in the emergency room before coming up to the operating room, a 22 g intravenous access was prepared on the right upper extremity but no medication before anesthesia was administered. as intubation was predicted to be difficult, a video glidescope was additionally prepared as well as a mccoy laryngoscope. before proceeding with anesthesia, preoxygenation 50 g of fentanyl and 150 mg of pentothal sodium was iv injected and mask ventilation was started with 4.0 vol% sevoflurane and 100% oxygen. after checking that the mask ventilation was operating properly, 30 mg rocuronium bromide was administered for rapid sequence intubation while sellick 's maneuver was done due to the risk of respiratory aspiration. after a minute, proper muscle relaxation was verified and the glottis were exposed using a macintosh laryngoscope. grade 3 in cormack and lehane airway classification was confirmed, and although intubation of an endotracheal tube with 6.5 mm envelope was attempted, it was unsuccessful so the video glidescope was used to carefully intubate an endotracheal tube with inserted stylet. directly after intubation, blood pressure was 130/88 mmhg ; heart rate was 100 beats / min, and oxygen saturation was at 100%. mechanical ventilation was started with volume controlled ventilation with a 400 ml tidal volume and a respiratory rate of 15/min, while maintaining anesthesia with 50% oxygen / n2o and 1.0 - 2.0 vol% of sevoflurane. the operation lasted a total of 1 hour and 35 minutes and 200 ml of ringer 's lactate solution was supplied. there were no abnormalities during surgery and after the surgery had finished, 7.5 mg of pyridostigmine and 0.3 mg of glycopyrrolate was iv injected to contend with muscle relaxation. spontaneous respiration was induced to confirm stable respiration. after confirming that the tidal volume was more than 5 ml / kg, the patient responded to the command to open his eyes, which indicated that he was awake. then, extubation was done, followed by 100% oxygen through a mask while checking the recovery of consciousness and muscle motility. respiration was regular in the recovery room but the patient displayed excessive behavior and impulsive responses shown before the anesthesia so 4 mg of nalbupine was iv injected and he was stabilized. deletion 8p syndrome is a very rare congenital disease with characteristic cardiac malformation, mental retardation, craniofacial anomalies, malformations of the digestive system, and neural developmental abnormalities. it was first detailed in 1973, and deletion 8p23 was first detailed in 1988. large terminal deletions can be discovered through basic chromosome testing but the latest molecular biological methods such as fluorescence in situ testing (fish testing) or array comparative genomic hybridization (array cgh) need to be used to discover or confirm small interstitial deletions. the frequency of 8p23 deletion syndrome is equal regardless of gender and the age of the parents, and it appears as either a first occurrence in one family (de novo) or through familial translocation. there are cases where a parent has the 8p deletion but their children have no abnormalities, or parents have no deletion but the children develop problems. deletion 8p syndrome causes developmental delays in nearly all the organs in the body so it exhibits diverse clinical signs. the most important and common symptom is cardiac malformations and mild to moderate intellectual disabilities and mental retardation. also, due to microcephaly, the size of the brain is smaller which can lead to more problems, and there are common reports of behavioral abnormalities such as excessive behavior, impulsive responses, and attention deficit. destructive and aggressive behavior and abrupt changes in response are characteristic, and they express themselves to others through behaviors such as pulling hair, hitting, biting, and kicking with their feet. in boys, there could be cryptorchidism or hypospadia and they may need surgical correction. hypotonia or hypertonia or a combination of these can occur in newborns ; in ophthalmology, there could be strabismus, myopia, or hyperopia but can be corrected with ordinary glasses, and serrated teeth form can occur. it usually does not affect pregnancy but there is a high possibility of giving birth to small for gestational age (sga) or large for gestational age (lga). the points to be considered in general anesthesia of patients with deletion 8p syndrome is that intubation will be difficult because of facial features such as microcephaly, a high arched palate, cleft palate, high narrow forehead, broad nasal bridge, short neck, and a large tongue ; congenital diaphragmatic hernia occurs in 20 - 30% of people with this syndrome but can be corrected through surgery, and esophageal motility disorder which leads to frequent gastroesophageal reflux so there is a high risk of aspirating stomach content resulting in aspiration pneumonia when inducing anesthesia or during recovery. therefore, the degree of difficulty in securing the patient 's airway should be checked beforehand, and the operator should be well - acquainted with the asa difficult airway algorithm. cornelia de lange syndrome (cdls) caused by mutation of the nipbl (nipped - b like) gene located at chromosome 5p13 is very similar to deletion 8p syndrome and has difficulties in intubation due to characteristic facial features, poor esophageal motility, and congenital diaphragmatic hernia and is easily exposed to aspiration.. failed in achieving an orotracheal intubation of a 8 year - old cdls patient with a cleft palate and tof so they used a laryngeal mask and bronchofiberscope to intubate, and fernandez - garcia. also did not use muscle relaxants in the general anesthesia of a 11 year - old cdls patient considering the difficulties in intubation and maintaining airway but successfully conducted anesthesia by using a laryngeal mask. conducted rapid sequence intubation using pentothal sodium and succinylcholine on a 9 year - old cdls patient undergoing surgery due to external injuries keeping in mind the possibility of aspiration and enterocleisis from malrotatio intestini. also in our case, there were no limitations in neck movement but intubation was anticipated to be difficult according to mallampati airway classification so video glidescope was prepared as well as a mccoy laryngoscope. in the first attempt, it was confirmed as a grade 3 cormack and lehane airway classification so a rapid sequence intubation using the video glidescope and sellick 's maneuver was done. a grade 3 cormack and lehane airway classification signifies that only the epiglottis is visible through proper use of the laryngoscope, and grade 4 is when no anatomical structure relevant to the airway but only soft tissue is visible. pre - anesthetic evaluation of deletion 8p syndrome patients can be very difficult because severe mental retardation and uncooperative behavioral disorders can make accurate examination and tests difficult. evaluation of cardiac function is essential in particular because there is the possibility of cardiac malformations. accompanying cardiac malformation such as avsd, ps, and vsd, ebstein 's malformation, tof, and hypoplastic left heart syndrome (hlhs) can congenitally occur. contended that from patients with deletion 8p23 syndrome, 75% of those with a terminal deletion and 94% of those with an interstitial deletion had cardiac malformation. in our case, an echocardiography was done as a pre - anesthetic evaluation to confirm that there were no cardiac malformations and cardiac output was also normal. in addition, since excessive behavior, impulsive responses, lack of cooperation, and self - injurious behavior from mental retardation can accompany most patients with deletion 8p syndrome, general anesthesia is preferred rather than regional anesthesia and occurrence of agitation in the recovery room must also be carefully observed and prevented. extubation is done after awakening because intubation is commonly difficult in patients with deletion 8p syndrome ; complications such as laryngospasm and coughing can emerge, and the risk of aspiration from gastroesophageal reflux is high. however if there is no evidence of gastroesophageal reflux and risk of aspiration in the pre - anesthetic evaluation, and intubation was not difficult, then extubation before awakening can be considered to reduce the excessive behavior and impulsive responses of the patient. the patient in our case showed pain and agitation after post - awakening extubation, and was stabilized by iv injecting of nalbuphine. in conclusion, there have been no anesthetic cases in korea of patients with deletion 8p syndrome and when a patient shows several characteristics mentioned here, pre - anesthetic evaluation of the patient such as assessing the degree of intubation difficulty according to facial deformities, cardiac malformation, and mental retardation must be thoroughly conducted to select a suitable method of anesthesia.	a deletion 8p syndrome is a relatively uncommon congenital disease characterized by mental retardation associated with multiple malformation that make anesthetic management a challenge. anesthetic management of a patient with deletion 8p syndrome may pose a serious problem mainly from difficult tracheal intubation, aspiration complication and cardiac malformation. we experienced a case of 10 year - old boy with a deletion 8p syndrome who underwent appendectomy under the general anesthesia. intubation was performed by video glidescope after unsuccessful attempt with macintosh laryngoscope. a high arched palate, short neck, poor patient cooperation due to mental retardation and occasional autistic behaviour made airway management difficult. this case should alert anesthesiologists to the greater difficulties of managing patients with deletion 8p syndrome.
lyme disease (ld), caused by the spirochete bacteria of the genus borrelia, is the most common vector - borne infectious disease in north america. more than 38,000 new cases were reported in the us in 2009,1 but underreporting is estimated to be six- to twelvefold, making the true number likely over 200,000 cases per year.2 ld is caused by five species of spirochete bacteria of the genus borrelia. borrelia burgdorferi sensu stricto is the main cause of ld in north america, while borrelia afzelii, borrelia garinii, b. burgdorferi, borrelia spielmanii, and borrelia bavariensis are the cause of most cases of ld in europe.3 ld was named after two towns in connecticut lyme and old lyme where the disease was recognized as a separate entity with the investigation of a cluster of children who experienced uncommon arthritic symptoms preceded by a characteristic skin rash during 19751976. this rash, termed erythema migrans (em), had been linked in europe to the bite of ixodes ticks. b. burgdorferi was first identified in 1982.4 ld manifests itself as a multisystem inflammatory disease that affects the skin in its early, localized stage, and then spreads to the joints, nervous system, heart, and other organ systems in its later disseminated stage.5 in the us, ld is transmitted by the bite of vector ticks of the genus ixodes, primarily by the deer tick ixodes scapularis.6 ixodid ticks are responsible for transmitting the spirochetes from mammals to humans. ixodid ticks have a 2-year life span, in which they pass through three developmental stages larva, nymph, and adult feeding only once per stage.4 ixodid ticks acquire b. burgdorferi by ingesting a blood meal from an animal reservoir like the white - footed mouse (peromyscus leucopus).2,7,8 in more urban areas, the reservoir is more diverse due to host availability, and will include chipmunks, shrews, squirrels, and even birds and lizards.9 the tick larvae overwinter and emerge the following spring in the nymphal stage, which is the stage of the tick that is most likely to transmit the borrelia infection.10 nymphs are responsible for 90% of human disease transmission ; nymphal stage ixodid ticks are rarely noticed because of their small size (6 weeks after onset of illness, rise to higher concentrations than igm antibodies, and can persist for months or years.34 first - generation elisas for the detection of anti - borrelia antibodies lack specificity. the inclusion of a second, more specific, serological method (western blotting) is used to exclude false - positive elisa samples.35,36 in the elisa assay, wells of plastic microwell plates are sensitized by passive absorption with b. burgdorferi antigen. any antigen - specific antibody in the sample will bind to the antigen coating the well.34,37 peroxidase - conjugated goat antihuman igm / igg antibody is added to the wells, and the plate is incubated. the conjugate will react with the human igm / igg antibody immobilized in the solid phase in step one.34,37,38 the microwells containing immobilized peroxidase conjugate are incubated with peroxidase substrate solution.37,38 hydrolysis of the substrate by peroxidase produces a color change. the color intensity of the solution depends on the antibody concentration in the original sample. the color intensity of the solution is measured photometrically, and an optical density value is calculated. then, an immunoglobulin - status ratio (isr) is calculated for each specimen by dividing the specimen optical density value by the cutoff optical density value. a positive isr means that antibodies to b. burgdorferi are presumptively detected in the serum specimen.34,3740 in western blotting, an antigen mixture prepared from b. burgdorferi strain b31 is separated by sodium dodecyl sulfate if antibodies to b. burgdorferi are present in the serum specimen, they will bind to antigens on the nitrocellulose membrane.42 bands in which a reaction has occurred will be detected by the addition of an enzyme - labeled antihuman igg or antihuman igm reagent that allows for the visualization of bands on the nitrocellulose membrane.41,42 practically speaking, the test produces something similar to a bar code with several lines or bands. each band represents antibodies to a different component of the b. burgdorferi bacteria.43,44 these specific band patterns have been internationally recognized. it is recommended that igm western blot is considered positive if at least two bands are present out of a total of five. it is recommended that igg western blot is considered positive if at least five bands are present out of a total of ten.38,4345 there has been laboratory variation reported that results in the spread of the sensitivity of the elisa assay. the sensitivity of the elisa assay varies from 29%40% in patients with em during the acute phase to 28%78% during the convalescent phase.4648 early ld and em are treated with antibiotics on an outpatient basis. doxycycline (100 mg twice daily by mouth), amoxicillin (500 mg twice a day by mouth), or cefuroxime (250 mg twice a day by mouth) for 1014 days are equally efficacious.7,4,49 doxycycline is often the drug of choice, because it is also considered the treatment for the gram - negative bacterium anaplasma phagocytophilum, a potential tick - borne coinfection.29 macrolides like azithromycin, clarithromycin, and erythromycin should not be selected, due to possible antibiotic resistance that has been identified.50,51 approximately 10%20% of patients treated for ld with a recommended 2-week course of antibiotics will have lingering symptoms of fatigue, headache, musculoskeletal pain, and lethargy.11 thirty - four percent of a population - based, retrospective cohort study in massachusetts were found to have arthritis or recurrent arthralgia, neurocognitive impairment, and neuropathy or myelopathy, for a mean of 6 years following treatment for ld.52 sixty - two percent of a cohort of 215 consecutively treated ld patients in westchester county, new york were found to have arthralgia, arthritis, and cardiac or neurologic involvement for an average of 3.2 years after treatment.53 this complication is commonly known as post - treatment ld syndrome (ptlds).54 however, in the absence of additional tests to rule out the eradication of the initial infections, others would argue that the more appropriate diagnosis is chronic ld.55 coinfections are a troubling complication for patients with chronic ld. the reported prevalence of coinfections in the us ranges between 4% and 28%. in the us, coinfections can include anaplasma phagocytophilum, which causes human granulocytic anaplasmosis, and babesiosis.56 the risk for coinfections is increased, due to the ixodid tick being a vector for all three.5658 symptoms of a babesiosis infection include fatigue, malaise, weakness, fever (> 38c), myalgia, arthralgia, and anorexia.56 severe babesiosis infections may progress to acute respiratory distress syndrome, disseminated intravascular coagulation, congestive heart failure, renal failure, myocardial infarction, splenic infarcts or splenic rupture, and death.56 records of 139 patients with babesiosis between 1982 and 1993 were analyzed, and nine patients (6.5%) died, 35 (25.2%) were admitted to the intensive care unit, and 35 (25.2%) required hospitalization for more than 14 days.59 rashes in patients with babesiosis are often reflective of concurrent infection with ld.60 we report the case of a patient who presented to a local primary care treatment facility with flu - like symptoms, headache, an expanding em rash, and later serological studies supporting the diagnosis of ld. following an antibiotic regimen twice the length and double the recommended treatment dose outlined by the infectious diseases society of america (idsa) there is a need for diagnostic tests sensitive enough and specific enough for identifying ld in all stages of infection. some clinicians will find controversy in the diagnosis and treatment of patients presenting with signs and symptoms of ld but lacking any dermatological presentation of the em rash and presenting with negative serological tests. current tests may prevent clinicians from being able to diagnose patients accurately who may not have obvious symptoms like em. those with clinical presentations distorted by coinfections will also likely experience a delay in their treatment. clinicians who hesitate to treat patients who do not display all of the diagnostic criteria required by the idsa may see their patients continue to progress from a subclinical phase to a more advanced phase of ld.	a 50-year - old woman from pulaski, virginia, presented to a local clinic with headaches, fever, generalized joint pain, excessive thirst and fluid intake, and a progressing rash on her back. on physical examination, she had a large circular red rash on her back with a bulls - eye appearance, 16 18 cm in diameter. serologic tests confirmed a diagnosis of lyme disease. the patient could recall a walk through the woods 3 weeks prior, although she never noticed a tick on her body. following a prolonged course of antibiotics, this case report presents a patient with ongoing symptoms consistent with post - treatment lyme disease.
the papillary fibroelastoma (pfe) is a rare and benign primary cardiac tumor, and the most frequently found tumor occurring in the cardiac valves. pfe has been found most often on valve leaflets, chordae tendineae, and both ventricles. we describe an interesting case of pfe originating from the anterior leaflet of the mitral valve mimicking vegetation. a 52-year - old woman was readmitted to a regional hospital due to wound infection after a previous mastectomy due to breast cancer. she received antibiotics for 2 weeks to treat a suspicious vegetation apparent on transthoracic echocardiography (fig. because the size of the cardiac mass had not changed, the patient was referred to our hospital for further evaluation. figure 1:transthoracic echocardiography shows a mobile and spherical mass of 1.0 cm in size attached to anterior mitral leaflet. parasternal long - axis view (a) and an apical four - chamber view (b). transthoracic echocardiography shows a mobile and spherical mass of 1.0 cm in size attached to anterior mitral leaflet. parasternal long - axis view (a) and an apical four - chamber view (b). gross specimen of excised mass reveals a friable mass with frond - like surface. on physical examination, the laboratory findings revealed 15 700/mm leucytosis, platelet count 3 251 000/mm and erythrocyte sedimentation rate 53 mm / h. tee revealed a mass of > 1.0 cm in size, mobile, attached to anterior mitral leaflet on the atrial aspect, and round with a homogenously speckled surface (fig. the cardiac mass was removed by surgery in order to reduce the risk of embolism as well as to rule out the infective endocarditis. under standard cardiopulmonary bypass, the mass was removed using a shave excision technique. however, there was significant mitral regurgitation after mass excision, and following excision of the mitral leaflets and chordae, a mechanical valve was implanted with preservation of the subvalvular apparatus of the mitral valve. macroscopically, the excised lesion was composed of a soft beige tissue with micropapillary projections and lobulated surface of 1.2 0.5 cm, entirely processed (fig. after the surgical excision, the mass was fixed in formalin, paraffin embedded, sectioned at 3 m thick and stained conventionally with hematoxylin and eosin. the histology examination revealed a papillary lesion composed of numerous papillary fronds with an acellular fibro - hialinous stroma (fig. 2c). in the excised margin in close contact with the non - pathologic endocardial tissue, the lesion has an infiltrative - like appearance, but the excisional margin itself was free of neoplasia. a higher magnification demonstrates a myxoid papillary structure lined by endothelial cells that express endothelial cell markers (fig. the postoperative course was uneventful, and the patient was discharged in a satisfactory condition on the seventh day. low magnification demonstrates a papillary configuration of paucicellular fronds (a, b h - e 10). a higher magnification demonstrates a myxoid papillary structure lined by endothelial cells that express endothelial cell markers (c h - e 40, d cd 34 20). low magnification demonstrates a papillary configuration of paucicellular fronds (a, b h - e 10). a higher magnification demonstrates a myxoid papillary structure lined by endothelial cells that express endothelial cell markers (c h - e 40, d cd 34 20) different mechanisms such as prior damage to the endothelium, hamartomatous origin and organizing emboli to cause pfe. if trauma is mechanically induced, pfe can occur in proximity to the iatrogenic injury. the presence of fibrin and elastin fibers within the fronds supports the hypothesis that pfe may arise from organizing thrombi. one of the most discussed theories is the microthrombus theory which stresses the fact that these lesions are acquired and they originate as small thrombi that serve as a nidus for further ecrescents to minor endothelial site injury of the valves or to previous diseased valves. pfe is considered also a hamartomatous growth of endocardial tissue that may give rise to neurologic symptoms when located on the left side of the heart. pfe is similar to lambl 's excrescences, but the site may be anywhere on the endocardium, and the lesion is large enough to potentially cause symptoms by embolization of attached thrombi or prolapse into a coronary orifice [4, 5 ]. the other theory is that pfes are true tumoral lesions that predominantly affect heart valves. the differential diagnosis of pfe includes other cardiac tumors, thrombus, vegetation and lambl 's excrescences. in contrast to myxoma, there are no vascular structures and associated inflammation within the fronds. pfe has several characteristic findings that may help to differentiate it from thrombus or vegetation. similar to vegetations, pfe is usually found on cardiac valves ; however ; pfe is often a solitary mass, usually of small size (< 1 cm), usually at the mid - portion of valve leaflets and with a frond - like characteristic surface. however, pfe may lack some of these findings, whereas vegetations may represent many of these characteristics. for that reason, pfe can often be differentiated by clinical information, blood cultures and laboratory tests. in this case, there was evidence of wound infection, so the clinical information was quite misleading. although pfes are benign, they can cause serious complications such as thromboembolism, myocardial ischemia, stroke and sudden death. surgical excision of pfe is recommended for symptomatic patients and asymptomatic patients with a fragile mass nature and frond - like papillary tissues of the tumor itself [8, 9 ]. right - sided pfes are asymptomatic and rarely cause pulmonary embolism, whereas left - sided can cause life - threatening complications. acute myocardial infarction, caused by a tumor occluding the coronary ostium or by embolization, may be the presenting symptom. cerebral embolization, either of fibrin or of a tumor fragment, has also been reported ; this may present as a transient ischemic attack or visual disturbance. considering the patient 's age, tumor mobility and tumor size, we decided to remove the cardiac mass to prevent potential complications such as embolization. the treatment of choice for pfe is surgical excision, which is safe without causing significant morbidity or mortality. when valvular involvement is present, excision with valve repair or replacement is curative. this report has received the ethics committee approval. we confirm that we have obtained consent to publish such an information from the patient to report individual patient data.	the papillary fibroelastoma (pfe) is a rare and benign primary cardiac tumor, and the most frequently found tumor occurring in the cardiac valves. with the introduction of echocardiography, the diagnosis of these tumors in living patients has been reported sporadically. the pfes have been found most often on valve leaflets, chordae tendineae, and both ventricles. we describe an interesting case of the pfe originating from the anterior leaflet of the mitral valve mimicking vegetation. the patient underwent successful surgical removal of the pfe.
in a recent issue of the journal of experimental medicine, middleton. demonstrated that mice with homozygous null mutations in alox15, which encodes 12/15-lipoxygenase, develop mpd (6). 12/15-lo incorporates oxygen into unsaturated lipids to generate short - lived peroxides that are ultimately converted to 12(s)-hydroxyecosatetraenoic acid and related products, which have pleiotropic effects on cell signaling and the inflammatory response. no hematologic abnormalities have been noted in the mutant mice until now, however (even though alox15-null mice have been used for many years in atherosclerosis studies), possibly because of the decreased penetrance of the phenotype in mixed genetic backgrounds. reported that although young (68-wk old) alox15 mice (in a c57bl/6 background) were normal, they uniformly developed features of an mpd by 1012 wk of age, including increased levels of circulating myeloid cells, splenomegaly, and infiltration of spleen by what appear to be proliferating, apoptosis - resistant, immature myeloid progenitors (6). these observations in alox15 mice suggest that loss or down - regulation of 12/15-lo could contribute to cml. indeed, the authors went on to show that 12/15-lo was undetectable in a cml cell line and that ectopic expression of 12/15-lo in these cells markedly decreased their proliferation and survival, effects that were partially reversed by treatment with an inhibitor of 12/15-lo. an important feature of human cml is the progression from the chronic phase, where differentiation of myeloid progenitors to neutrophils is close to normal, to blast crisis (bc), a terminal phase resembling acute myeloid leukemia (aml), where there is profound impairment of hematopoietic differentiation. interestingly, a minority (15%) of ageing alox15 mice developed a condition similar to bc, with progressive splenomegaly and increased marrow, splenic, and circulating immature myeloid cells. however, it is not clear whether these mice meet defining criteria for aml, such as > 20% marrow myeloblasts (7). the cause of morbidity and death in mice with bc - like disease was thought to be severe anemia, based on a relative decrease in mature erythrocytes in the bone marrow, but it is not apparent whether this correlated with decreased blood hemoglobin or hematocrit, which are the standard criteria for anemia. treatment of splenocytes from these mice in vitro with imatinib did not impair their proliferation or survival, nor was there any increase in tyrosyl phosphorylation of the abl substrate crk, suggesting that dysregulated abl activity is not involved. but the authors did establish an intriguing functional connection between 12/15-lo, the protein kinase akt, and interferon consensus sequence binding protein (icsbp ; also known as irf-8) (fig. (a) interferon- (ifn-) induces icsbp transcription through stat1. increased icsbp mediates an antileukemic effect through an unknown mechanism. abl kinase represses icsbp transcription through an unknown mechanism, but also activates multiple signaling pathways, including ras - mapk (leading to induction of bcl-2 gene transcription), stat5 (leading to bcl - x gene transcription), pi3k (through a grb2gab2 interaction) leading to akt activation, and src family kinases (lyn and hck). the net effect of bcr abl activity is to promote bcl-2 and bcl - x expression and to inhibit icsbp transcription. (c) in contrast, 12/15-lo may either activate pten or inhibit pdk1, both regulators of akt, leading to increased phosphorylation and cytoplasmic localization of icsbp, an effect mediated in part through an unknown tyrosine kinase. this may increase survival in myeloid progenitors through relief of iscbp - mediated inhibition of bcl-2 and bcl - x. several lines of evidence have previously implicated icsbp, an interferon - stimulated transcriptional repressor, as a suppressor of normal and cml myelopoiesis. icsbp transcripts are low to absent in chronic phase cml (8), and iscbp - deficient mice develop an mpd - like syndrome (9), in which the myeloid progenitors are hypersensitive to myeloid growth factors including granulocyte / macrophage colony - stimulating factor and interleukin-3 (10). in mouse bone marrow, icsbp activation is decreased by bcr abl, whereas enforced coexpression of icsbp attenuates both normal and bcr abl granulopoiesis (11). direct repression targets of icsbp in myeloid cells, which may account for this activity, include the antiapototic genes bcl - x (12) and bcl-2 (13).. found that nuclear icsbp protein levels were reduced in splenocytes from alox15 mice with mpd (6). this decrease in nuclear icsbp correlated with increased activation of akt, enhanced tyrosyl phosphorylation of icsbp, and elevated expression of bcl-2. prevention of akt activation by treatment with a phosphatidylinositol 3-kinase (pi3k) inhibitor reduced icsbp tyrosyl phosphorylation and increased nuclear icsbp levels, coincident with reduced bcl-2 levels and increased apoptosis. the mechanism through which 12/15-lo deficiency activates pi3k was not defined, but it is possible that some 12/15-lo products affect pi3k regulators such as pten or pdk1 (fig. the findings further suggest that a tyrosine kinase is involved in regulating icsbp, but aside from excluding abl, the authors did not pursue the identity of this kinase. one problem in diagnosing mpd is that the malignant cells are virtually identical to normal maturing myeloerythroid cells, so that distinguishing mpd from reactive conditions is difficult (1). an undiagnosed generalized inflammatory state in older alox15 mice could explain the myeloproliferation, but 12/15-lo and its products are generally considered to be proinflammatory, arguing against this possibility. because alox15 mice are initially normal, 12/15-lo deficiency alone may be insufficient for development of mpd ; additional events might be required for progression to mpd and subsequently to the blastic phase. in the middleton. paper, the authors could not determine whether the mpd or blast phase are clonal processes (6), but future retroviral marking studies may resolve this. they were unable to adoptively transfer disease from alox15 mpd mice by transplantation of bone marrow and/or splenocytes to syngeneic, unirradiated recipients, which probably reflects the very poor engraftment of donor hematopoietic stem cells under these conditions (14). however, they were able to efficiently transfer hematopoietic disease (defined as modest splenic enlargement with disruption of architecture) when donors in the blast phase were used, although the extent of donor engraftment and whether recipients developed fatal aml were not documented. these results suggest that the alox15 donor cells capable of transferring disease (i.e., the leukemia - initiating or leukemic stem cells) differ between the mpd and blast phases of the disease, which is reminiscent of human cml (15). it would also be informative to test whether alox15 myeloid progenitors were hypersensitive to cytokines, as in icsbp deficiency (10). cytokine hypersensitivity would connect decreased icsbp function directly with the enhanced myelopoiesis in alox15 mice, but it would also suggest that the disease is more like jmml (2) than cml, where the cytokine response is normal (16). ultimately, whether alox15 mice represent an informative model of cml will require further study. there are some tantalizing clinical correlates, including decreased 12-lo activity in cml marrow cells (17) and frequent loss at chromosome 17p, where alox15 is located, in cml disease progression. however, some functional connection between bcr abl and 12/15-lo (for example, does bcr abl alter 12/15-lo activity or expression ?) is needed to support the proposed role of 12/15 lo in cml (fig. although both bcr abl activity and 12/15-lo deficiency activate akt in myeloid progenitors, the effects on icsbp in the two myeloproliferative syndromes are distinct, with bcr abl decreasing icsbp transcripts and protein expression, whereas 12/15-lo deficiency impairs nuclear localization of icsbp but not its overall expression. abnormalities of the 12/15-lo pathway should be sought in ph - negative (atypical) cml and in chronic neutrophilic leukemia, two cml - like mpds that lack bcr abl (1), and in myeloid blast crisis of cml. whether or not the analogy to cml holds up, the alox15 mice are certain to provide important new insights into normal and malignant myelopoiesis. icsbp intersects with another well - studied hematopoietic signaling system that has been implicated in cml oncogenesis, the jak - stat pathway. activation of the latent transcription factor, signal transducer and activation of transcription 5 (stat5), in bcr abl - expressing cell lines and primary leukemia cells was recognized a decade ago (18), but the role of stat5 in the pathogenesis of cml has been controversial. abl may activate stat5 through direct phosphorylation, or the activation could be indirect, via phosphorylation by jak2 (19) or by src family kinases (20), both of which are activated in bcr abl - expressing cells (fig. 1). in a mouse retroviral bone marrow transduction / transplantation model of cml, initial studies using donor mice with targeted mutations in stat5a and stat5b suggested that stat5 was not absolutely required for induction of cml - like leukemia by bcr however, it is now widely recognized that the stat5a / b mutations used in these studies were hypomorphic rather than true null alleles (22). the role of stat5 in cml has been readdressed in two recent papers. in the first paper, induction of murine cml - like mpd was attenuated in donor hematopoietic cells with a single null mutation in stat5a (23), indicating that this stat5 isoform has a nonredundant function in bcr the second paper used novel mice that have the entire stat5ab locus deleted (24). stat5ab mice die perinatally, but fetal liver hematopoietic progenitors from these mice were incapable of generating leukemia in recipient mice after retroviral transduction with bcr abl (25). together with a recent report that sirna against stat5 in human cml patient samples impairs ph myeloid colony formation (26), these studies suggest that stat5 signaling contributes to bcr the stat5ab experiments addressed principally b lymphoid transformation and leukemogenesis rather than cml - like mpd, and the extent that nonmalignant stat5ab hematopoietic stem cells can contribute to stable myeloerythropoiesis after transplantation has not been defined. lastly, the important transcriptional targets of stat5 in cml must be determined. in this regard, there is considerable evidence that bcl - x, a target for repression by icsbp (12), is transcriptionally activated by stat5 in cml cells (27) and may contribute to increased survival (fig. as mentioned in the previous section, bcr abl activates multiple src family kinases through a mechanism that does not involve direct phosphorylation, and src kinase inhibitors and dominant - negative mutants impair bcr abl transformation in cultured cells (28). however, bcr abl can efficiently induce cml - like mpd in marrow from mice lacking the three src kinases principally expressed in myeloid progenitor and stem cells (lyn, hck, and fgr), suggesting that these src kinases have no role in the pathogenesis of chronic phase cml (29). in contrast, induction of b cell acute lymphoblastic leukemia (b - all), which is also bcr abl dependent, was dramatically impaired in the absence of any two of these three src kinases, suggesting a partially redundant requirement for src kinases in the pathogenesis of ph b - all and the b lymphoid bc stage of cml (29). subsequently, these findings were supported by the demonstration that sirna knockdown of lyn in primary cml cells impaired leukemic cell viability and colony formation in cells from patients with ph lymphoid bc, but had less effect on myeloid bc cells (30). several pharmaceutical companies have developed drugs that inhibit the kinase activity of both bcr abl and src kinases, some of which are active against many but not all of the bcr abl imatinib - resistant mutants (31, 32). the studies with lyn / hck / fgr - deficient mice showed that those particular src kinases were not required for induction of cml - like disease by bcr abl (29), but involvement of the other six src family members could not be excluded. when administered to mice with bcr abl - induced cml - like mpd or b - all, the abl kinase inhibitor imatinib (gleevec) prolonged the survival of mice with either disease, whereas cgp76030, a small molecule inhibitor of src kinases that also inhibits bcr abl at higher concentrations (28), was effective alone and synergized with imatinib in mice with b - all, but had no effect in mice with cml - like leukemia (29). biochemical analysis of primary leukemia cells showed that, at the doses used, cgp76030 inhibited src family kinases but not bcr abl. a novel genetic strategy has been developed to verify the in vivo therapeutic target of dual abl / src kinase inhibitors in the ph leukemias. abl t315i mutant is resistant to both imatinib (33) and the dual abl / src inhibitor dasatinib (bms-354825) (31). abl t315i are relatively resistant to imatinib but susceptible to dasatinib in vitro and in vivo (5), implying that inhibition of bcr abl alone is insufficient for therapeutic responses in ph b - all. the results further suggest that pure src kinase inhibitors will have little therapeutic activity in chronic phase cml patients, but could be useful in the treatment of ph b - all and cml lymphoid blast crisis. this prediction has been born out clinically, as patients with chronic and accelerated phase cml and the t315i mutation do not respond to dasatinib (34).	chronic myeloid leukemia (cml), which is caused by the bcr abl fusion tyrosine kinase, is one of the most intensively studied human cancers. abl kinase inhibitors have been spectacularly successful in treating cml, but disease persistence and acquired drug resistance can prevent eradication and cure of the leukemia. the development of better therapies will depend on a full understanding of signaling pathways in cml, facilitated by model studies using mutant mice.
sushi and sashimi are traditional japanese food, though in recent years they attracted a large number of european consumers. it was formerly used for carps, that were caught, gutted, salted and preserved between two stones, and left to ferment for a period ranged from one to three years. sushi is prepared with cold cooked rice acidified with vinegar, and it is shaped into bite - sized pieces and topped with raw or cooked fish, or formed into a roll with fish, eggs or vegetables, and wrapped in seaweed (nori) (atanassova., 2008). the sashimi, instead, is characterised by the use of fish and shellfish, which are cut into thin slices and served with several sauces (e.g. wasabi, soy sauce or ponzu sauce), and accompanied with daikon roots (food and environmental hygiene department hksar, 2000). sushi and sashimi are ready - to - eat (rte) food and regarded as a potentially hazardous. frequently, indeed, foodborne diseases related to sushi e sashimi consumption have been reported (adams., 2004 ; food and environmental hygiene department hksar, 2000 ; masotti., 2004 ; millard and rockliff, 2003 ; nsw food authority, 2006)., staphylococcus aureus, vibrio parahaemolyticus, vibrio cholerae, listeria monocytogenes, bacillus cereus can occur in materials used for their preparation such as fish, seafood products, vegetables and rice. in the light of the significant increase of consumption of traditional japanese foods in italy and due to the attention of ec reg. (european commission, 2005), the aim of this study was to investigate the microbiological quality of sushi and sashimi sold in messina and catania (southern sicily, italy). the study was carried out on 38 samples of sushi and 12 of sashimi collected from restaurants, sushi bar and take - away outlets of messina and catania. in addition to rice and nori seaweed, they were made with smoked fish (salmon, tuna and swordfish), canned tuna in oil, surimi, fish eggs, cream cheese, cucumbers, carrots, avocado, sesame or poppy seeds. all samples, transported to the laboratory under refrigerated condition, were analysed for the following bacteriological determinations : aerobic mesophilic bacteria (amb) (iso 4833:2004 ; uni, 2004), psychrophilic bacteria (pb) (iso 17410:2001 ; iso, 2001), specific spoilage organisms (ssos) (lyngby iron agar at 25c for 5 days), enterobacteriaceae (iso 21528 - 2:2004 ; iso, 2004), pseudomonas spp. (pseudomonas agar base with cfc supplement at 30c for 48 h), quantitative and qualitative determination of vibrio spp. (thiosulphate citrate bile salt sucrose agar at 37c for 24 h), coagulase - positive staphylococci and micrococci (iso 6888 - 2:1999 ; iso, 1999), bacillus cereus (iso 7932:2005 ; uni, 2005a), listeria monocytogenes (iso 11290 - 1:2005 and iso 11290 - 2:2005 ; uni, 2005b, 2005c) and salmonella spp. colonies of enterobacteriaceae, isolated from tuna sashimi, were identified by api 20 e (biomrieux, marcy letoile, france). then, they were cultured on niven medium, in order to evaluate their istidino - decarboxylase activity. in sushi samples, the amb ranged from 5.00 to 8.18 log cfu / g, and pb from 4.70 to 7.13 log cfu / g (table 1). the highest percentage of samples for amb (44.74%) and pb (47.37%) were from 6.00 to 7.00 log cfu / g, while for enterobacteriaceae (44.74%) from 4.00 to 5.00 log cfu / g. specific spoilage organisms and pseudomonas spp. ranged from 3.49 to 7.72 log cfu / g and from 3.26 to 8.00 log cfu / g respectively. coagulase - positive staphylococci were isolated in 16 samples (42.11%) with values from 2.00 to 3.60 log cfu / g. bacillus cereus was detected in 3 samples (7.89%), with values between 1.70 and 4.00 log cfu / g and vibrio spp. were observed in 15 samples (39.47%) ranging from 1.70 to 3.70 log cfu / g. in all sushi samples, no salmonella spp. and the ph of the rice ranged from 4.6 to 6.6 (mean 5.6), while the ph of fish from 5.06 to 8.4 (mean 6.06). in sashimi samples, the amb, pb and ssos were higher than 7.00 log cfu / g (table 2). the enterobacteriaceae and pseudomonas spp. were from 6.00 to 8.00 log cfu / g. were observed in 6 samples (50%) (2.00 log cfu / g), while in all 12 samples (100%) no salmonella spp. and twenty - five colonies of enterobacteriaceae, isolated from tuna sashimi, were identified as citrobacter freundii, klebsiella oxytoxa, serratia liquefaciens, rahnella aquatilis and raoultella ornithinolytica. the ph of sashimi samples examined ranged from 5.86 to 8.4 (mean 6.98). the microbiological limits for rte foods such as sushi and sashimi are defined by ec reg. sushi and sashimi characterised by shelf - life less than five days are rte foods unable to support the growth of l. monocytogenes. for this reason, l. monocytogenes must be less than 100 cfu / g during the shelf - life of these products. all samples examined in this study were in accordance with the microbiological criteria of ec reg. (2000), hong kong (food and environmental hygiene department hksar, 2000, 2007) and food standards australia (food standards australia new zealand, 2001 ; anzfa, 2001), 6 sushi samples (21.05%) examined were considered unsatisfactory for amb criteria (table 3). thirty - one samples (81.58%) were unsatisfactory for enterobacteriaceae and 6 (15.79%) for coagulase - positive staphylococci. seventeen samples resulted borderline for amb (44.74%), 6 for enterobacteriaceae (15.79%), 3 for b. cereus (7.90%) and 11 for coagulase - positive staphylococci (28.95%), as showed in table 3. high bacterial charges as well as potentially pathogen microorganisms were observed in sushi and sashimi examined as previously reported by several authors (adams., 1994 ;, 2008 ; barralet., 2004 ; millard and rockliff, 2003). in this (2008) described average amb values of 6.3 log cfu / g in fresh sushi samples. l. monocytogenes were found in 1.6 and 1.2% of samples respectively, while staphylococcus aureus showed a charge of 2.2 to 4.7 log cfu / g (atanassova., 2008). the microbiological status of sushi and sashimi reflects the microbiology of materials used for their preparation. is indeed related to the fish and shellfish products used (giuffrida and panebianco, 2008), while b. cereus has been reported in plant foods (especially rice) (eglezos., 2010). l. monocytogenes can occur in vegetables and dairy products (cheese), while the finding of s. aureus is an evidence of human contact during the preparation of food (nogara., 2004). this study confirms that the production of sushi and sashimi of good quality obviously depends on the choice of raw materials, as also reported by atanassova. a good rice acidification (ph of the rice must be less than 4.6) and the maintaining of cold chain during preparation and storage are also essential to obtain products of good microbiological status. finally, a proper training of personnel who manipulates this easily perishable food is desirable. further bio - molecular investigations will be necessary to confirm the istidino - decarboxylase activity of enterobacteriaceae strains isolated from tuna sashimi as suggested by mancusi. the presence of hista - mine - producing bacteria on sashimi could also represent a potential toxicological hazard.	sushi and sashimi are traditional japanese food, mostly consisting of raw seafood alone or in combination with rice. eating sushi and sashimi has become popular in many countries even outside japan. this food is not free from health risks such as ingestion of pathogenic bacteria or parasite. the aim of this study was to investigate on hygienic - sanitary quality of sushi and sashimi sold in the cities of messina and catania, southern italy. fifty samples (38 sushi and 12 sashimi) were analysed to determinate the aerobic mesophilic bacteria (amb), psycrophilic bacteria (pb), enterobacteriaceae, specific spoilage organisms (ssos), pseudomonas spp., coagulase - positive staphylococci, micrococci, vibrio spp., bacillus cereus, salmonella spp. and listeria monocytogenes. in sushi, amb ranged from 5.00 to 8.18 log cfu / g, pb from 4.70 to 7.13 log cfu / g, enterobacteriaceae from 1.41 to 6.67 log cfu / g, while ssos and pseudomonas spp. from 3.49 to 7.72 and from 3.36 to 8.00 log cfu / g, respectively. micrococci ranged from 3.53 to 5.03 log cfu / g and coagulase positive staphylococci were found in 16 samples (2.00 to 3.60 log cfu / g). bacillus cereus was found in 3 samples (1.70 to 4.00 log cfu / g), while vibrio spp. was found in 15 of the sushi samples (1.70 to 3.70 log cfu / g). in sashimi, the amb, pb and ssos values were higher than 7.00 log cfu / g, pseudomonas spp. and enterobacteriaceae were from 6.00 to 8.00 log cfu / g, while vibrio spp. were found in six samples with means of 2.00 log cfu / g. no salmonella spp. and listeria monocytogenes were detected in all sushi and sashimi samples.
breast cancer is the most common malignancy and the second most common cause of cancer - related mortality in women. successful strategies for screening, early diagnosis, prognosis, and risk stratification are needed to decrease mortality and increase the probability of curing the disease. currently, mammography is the gold standard of breast cancer screening and remains the only screening test proven to reduce mortality. indeed, mammographic sensitivity decreases significantly as breast density increases, with sensitivity reported to be as low as 45% in women with extremely dense breasts. conversely, mammography can also lead to overdiagnosis (i.e., detection of tumors that might not need intervention) and can lead to unnecessary treatment of some patients. therefore, considerable efforts have been undertaken to produce an effective screening method for early - stage breast cancer. both full - field digital mammography and computer - aided detection programs have been proposed, but results from these methods remain controversial. the ability of magnetic resonance imaging (mri) to detect the presence and extent of small tumors seems to exceed that of both mammography and ultrasound, with low specificity. however, additional investigations are still required to confirm this finding. finally, improving early - stage breast cancer screening for early detection to be an effective and practical approach, screening tests must satisfy four basic requirements. first, screening tests should be able to distinguish healthy individuals from cancer cases with a high degree of accuracy, sensitivity, and specificity (namely, low false - negative and false - positive rates). second, detection should be possible before the disease progresses to an advanced stage, or even prior to the first manifestation of clinical signs, when it is still curable. third, the test should ideally allow discrimination between lesions that are aggressive and require treatment and those that ultimately will do no harm, thus reducing the problem of overdiagnosis. therefore, fda - approved protein tumor markers currently used in clinical practice, such as circulating tumor cell (ctc) proteins, estrogen receptor (er), progesterone receptor (pr), her-2/neu, ca 15 - 3, and ca27.29, are not approved for screening or early diagnosis. rather, er and pr assays are recommended for prognosis and determining response to therapy, while her-2/neu is used to assess appropriate therapeutic options. moreover, er, pr, and her-2/neu measurements are based on immunohistochemistry, a method that requires invasive intervention such as biopsy to obtain samples. the level of ctcs is reported to be associated with cancer progression and survival. finally, ca15 - 3 and ca27.29 serum biomarkers are only recommended for monitoring disease state and response to therapy. development of a sensitive, specific, and reproducible assay to identify biomarkers that can accurately determine the onset of breast cancer, particularly noninvasive tumors, is an attractive goal. this assay should be applicable for routine clinical use, require minimal time, and present little risk for the patient (e.g., venipuncture). based on these criteria, autoantibodies are present in sera before tumor - associated antigens (taas) can be detected. autoantibodies also correspond to efficient biological amplification of taas and are secreted into serum prior to first clinical signs. moreover, antibodies are highly stable in serum samples and, unlike other polypeptides, are not subject to proteolysis, simplifying sample handling. they have a long lifetime in blood (t1/2 between 7 to 30 days, depending on the subclass of immunoglobulin) and may persist as long as a corresponding autoantigen that elicited the original specific humoral response. finally, antibodies are biochemically well - characterized and many reagents and techniques are available for their detection, simplifying assay development. in 1955, baldwin was the first to demonstrate that the immune system could react to a developing tumor. he showed that tumor extracts could cause considerable tissue destruction when injected into growing tumors in rats. tumors in these rats regressed, and the animals remained free of recurrent tumor growth. furthermore, injected rats were found to be immune to subsequent implants of the same tumor. these results suggested that the development of tumor immunity depends upon the presence of immunogenetic differences in the tumor - host relationship. also in 1955, graham j. b. and r. m. graham screened autoantibodies in the sera of 48 patients with gynecological cancers including cervical, ovarian, and uterine lesions using the complement - fixation technique. twelve of these samples demonstrated significant autoantibody titers, suggesting for the first time that autoantibodies could be used as a diagnostic tool for cancer. in 1970, taylor and odili identified the first neoantigen, which was highly similar to the t antigen of oncogenic dna virus, eliciting a specific humoral response in breast cancer. in the following years, using immunofluorescence approaches priori. confirmed the presence of autoantibodies in random sera from breast cancer patients. in 1975, wasserman. demonstrated that the incidence of autoantibodies at diagnosis of breast carcinoma was higher in patients who developed local recurrences or distal metastases within 2 years than in patients free from recurrence. although these results have not been be confirmed, this study was the first to use autoantibodies as prognostic biomarkers. considerable efforts have been made to identify autoantibodies and their antigen counterparts to detect and/or monitor cancer progression. over the past 10 years, several technical approaches have been developed (figure 1), and many studies have demonstrated the potential use of autoantibodies for early breast cancer detection. these molecules included p53, muc-1, heat shock proteins (hsp-27, hsp-60, and hsp-90), her2/neu / c - erg b2, gipc-1, c - myc, c - myb, cancer - testis antigens (ny - eso-1), brca1, brca2, endostatin, lipophilin b, cyclin b1, cyclin d1, fibulin, insulin - like growth factor binding protein 2 (igfbp-2), topoisomerase ii alpha (topo2), and cathepsin d (for review see [9, 15 ]). recently, an original study aimed to address the temporal relationship between breast cancer development and serum antibody responses against two previously identified taas (p53 and her-2/neu) with sera collected prior to diagnosis, at diagnosis, and during treatment. at the time of treatment, p53 and her-2/neu autoantibodies were significantly increased in the sera collected from patients with breast cancer. interestingly, comparison of antibody responses in prediagnostic samples and controls demonstrated that her2/neu and p53 antibodies can be detected in sera collected, on average, more than 150 days prior to diagnosis. although sample sizes were relatively small (33 cases and 45 controls), and although the percentage of patients producing autoantibodies against her2 and p53 in prediagnostic samples was also low (15% and 6%, resp.), these results confirm the potential usefulness of these markers as indicators of the early stages of carcinogenesis. in the past two years sun. provided the first evidence for the presence of circulating sox2 antibodies in breast cancer. the authors determined the expression levels of sox2 antibodies in sera from 282 breast cancer patients, 78 benign breast disease patients, and 194 healthy women, using indirect enzyme - linked immunosorbent assay (elisa). the results showed that sox2 antibodies were more prevalent in patients with breast cancer (18.4%) than in healthy women (2.6%, p < 0.0001) and patients with benign breast disease (6.4%, p = 0.011). based on the concentration of circulating sox2 antibodies, the investigators were able to discriminate between breast cancer patients and healthy controls (p < 0.001) and between breast cancer patients and those with benign breast disease (p < 0.001). furthermore, in breast cancer patients the prevalence of sox2 antibodies was associated with higher tumor grade and positive nodal status. liu. also identified a specific humoral response against the p90/cip2a antigen. in 256 sera samples (168 from breast cancer patients and 88 from normal individuals), the authors showed that p90/cip2a elicited higher autoantibody production in breast cancer (19.1%) than in normal volunteers (2.3%). these results were supported by the higher frequency of p90/cip2a expression in breast cancer tissues than in adjacent normal tissues. ye. have assessed the levels of cd25 and foxp3 autoantibodies levels, previously identified in lung and esophageal cancer [20, 21 ], in 152 breast cancer patients and 112 healthy individuals. however, patients with stage i primary breast cancer exhibited higher expression of cd25 autoantibodies than healthy controls. in addition, heo. observed by elisa that a mimotope for circulating anti - cytokeratin 8/18 autoantibody discriminated breast cancer patients from normal subjects with a sensitivity and a specificity of 50% and 82.61%, respectively. usually, only 1030% of cancer patients elicited a specific humoral response against a single taa. the reason for this low sensitivity could lie in the heterogeneous nature of breast cancer, whereby different proteins are aberrantly processed or regulated in patients with the same type of cancer. therefore, several studies have evaluated the usefulness of detecting various autoantibodies as a panel to increase the accuracy of a potential diagnostic test (table 1). were the first to assess the frequency of seven autoantibodies (p53, c - myc, her2, ny - eso-1, brca1, brca2, and muc1) in a ductal carcinoma in situ (dcis) population of 40 patients. interestingly, reproducibly elevated serum levels of autoantibodies were seen in at least one of the six antigens in 64% of primary breast cancer patients and 45% of patients with dcis, at a specificity of 85%. reported a multimarker signature combining hsp60, muc1, fkbp52, ppia, and prdx2 that reached sensitivity, specificity, and accuracy of 72.2, 72.6, and 72.4%, respectively, in dcis compared with healthy individuals. recently, the same group identified a panel of five new autoantibodies from 80 subjects (20 patients with early - stage dcis or primary breast cancer, 20 women with benign breast lesions, 20 healthy controls, and 20 women with autoimmune disease). the expression levels of these five markers were validated by elisa on a second set of sera (182 patients : 59 patients with primary breast cancer, 55 patients with dcis, and 68 healthy controls). the signature significantly discriminated early - stage cancer from healthy individuals (auc = 0.81 ; 95% ci : 0.740.86). interestingly, this value was high in both node - negative early - stage primary breast cancer patients (auc = 0.81 ; 95% ci : 0.720.88) as well as in dcis patients (auc = 0.85 ; 95% ci : 0.760.95). using microarray, ye. assessed imp1, p62, koc, p53, cmyc, survivin, p16, cyclin b1, cyclin d1, and cdk2 autoantibody levels in 122 patients. the antibody frequency to the individual taas in breast cancer was variable and ranged between 7.3% and 22.0%. however, with the successive addition of taas to a total of eight antigens, there was a stepwise increase in positive antibody reactions, reaching a sensitivity of 61.0% and a specificity of 89.0% in breast cancer. the positive and negative likelihood ratios were 5.545 and 0.438, respectively, which showed that the clinical diagnostic value of a parallel assay of eight taas was high. moreover, the positive predictive value (ppv) was 73.5% and the negative predictive value (npv) was 82.0%. using a t7 breast cancer complementary deoxyribonucleic acid phage library for tumor - associated antigens, dong. autoantibodies have been evaluated by elisa in 150 breast cancer, 150 normal, and 40 non - breast - cancer serum samples. autoantibodies were significantly higher in breast cancer patients relative to controls (p < 0.01), with an auc of 0.73 and 0.69 for hnrnpf and fth1 autoantibodies, respectively. even when the two biomarkers were combined, the specificity remained low (72.0%), while the sensitivity increased to 91.0%. however, when both of these autoantibody biomarkers combined with serum ca 15 - 3 values, the auc increased to 0.93, with 89.3% sensitivity and 93.8% specificity. mang. described significant and consistent differences in the level of autoantibodies targeting specific antigens in a population of 20 patients with dcis and 20 with early - stage breast cancer. in this protein microarray experimental study, a set of five autoantibody targets (rbp - jk, hmgn1, psrc1, cirbp, and echdc1) with the highest differential signal intensities was used to establish an autoantibody signature of the transition from dcis to early - stage cancer. the performance of this humoral signature was then assessed in an independent set of 120 newly diagnosed patients using elisa. the results showed that this signature could significantly discriminate dcis from invasive breast cancer (auc = 0.794 ; 95% ci : 0.6740.877). moreover, this panel could clearly distinguish low - grade dcis from high - grade dcis (auc = 0.749 ; 95% ci : 0.5810.866). interestingly, the autoantibody signature could significantly divide the dcis patients into groups with either poor prognosis or good prognosis (p = 0.01). taken together, these results suggested that examining the humoral response to preinvasive lesions could identify potential markers that accurately detect dcis patients at high risk for subsequent local recurrence. although several studies present hopeful preliminary results, there is a need to validate autoantibody signatures on a large prospective population. indeed, for biomarkers reach to the clinic, their original performance must be independently reproduced in subsequent validation studies. however, most of the studies cited above are limited by the size of the validation sample set. as an example in a breast cancer population, when muc1 autoantibody was assessed in prediagnostic sera from over 2000 women, distributed across one discovery set (273 cases versus 273 controls) and two validation sets (426 cases versus 426 controls and 303 cases versus 606 controls), validation is usually performed by elisa, an assay that is rapid and simple to carry out and can handle a large number of samples in parallel. however, multiplex analysis remains difficult because elisa processing is usually time consuming and expensive. the lumina immunobead platform (labmap, flowmetrix) uses digital signal processing capable of classifying polystyrene beads (microspheres) dyed with distinct proportions of red and near - infrared fluorophores., up to one hundred different detection reactions can be carried out simultaneously on the various bead populations in very small sample volumes. this technology has already been utilized in non - small - cell lung cancer autoantibodies detection [38, 39 ]. in 2009, kim. used the bead array platform for discovering signatures specific to primary nonmetastatic breast cancer and differentiating these patients from normal subjects using sensitive combinatorial classifiers. in his work, an antibody bead array of 35 markers was constructed, and an initial study population consisting of 98 breast cancer patients and 96 normal subjects was analyzed. multivariate classification algorithms were then used to find discriminating biomarkers, which were validated with another independent population of 90 breast cancer subjects and 79 healthy controls. serum concentrations of three autoantibodies (against epidermal growth factor, soluble cd40-ligand, and proapolipoprotein a1) were increased in breast cancer patients, whereas five autoantibodies (against high - molecular - weight - kininogen, apolipoprotein a1, soluble vascular cell adhesion molecule-1, plasminogen activator inhibitor-1, vitamin - d binding protein, and vitronectin) were decreased. the classifier was able to discriminate breast cancer patients from the normal population with high accuracy (87.6% to 91.8% according to classification method). this technique was developed for high throughput and multiparametric assays that allow for the identification of multiple tumor markers. combining various taas onto microstructured microarray under optimized conditions (spotting ph buffer, surface chemistry, and blocking procedure) could improve sensitivity and specificity of anti - taa autoantibody detection. a recent paper showed the utility of protein microarray for autoantibody detection in sera of breast cancer patients. the authors investigated both surface chemistry and protein immobilization conditions to improve sensitivity of the detection of tumor autoantibodies on these microarrays. ten proteins (cea, p53, her2, ny - eso-1, hsp60, hsp70, mycl1, chek2, hnrnpk, and nme1) were immobilized onto microstructured glass slides functionalized with six different surface chemistries to detect autoantibodies in sera of breast cancer patients. the authors demonstrated that there is not a unique surface chemistry suitable for all proteins and that immobilization parameters must be optimized for each protein. thus, to validate the best surfaces for protein immobilization and biological activity, sera from 29 breast cancer patients and 28 healthy donors were tested on taa microarrays. through a combination of five taas (hsp60, p53, her2-fc, ny - eso-1, and hsp70) immobilized on an optimized surface chemistry, 82.7% of breast cancer patients were specifically detected. the potential cost and time savings that could be realized by using these technologies relative to other methods provide a strong impetus for their routine use in both research and clinical settings. nevertheless, as with all clinical laboratory tests, questions of reproducibility, precision, and accuracy must be addressed to validate these assays. with the inherent heterogeneity of breast tumors and our limited understanding of the humoral immune response to cancer, there are some obstacles to autoantibody identification and their routine clinical use for early breast cancer detection recent published reports indicate an encouraging future for the implementation of sensitive and specific tests. it is conceivable that a humoral signature based on the detection of specific autoantibodies can be applied to the detection of cancer as well as to the tracking of disease progression and response to therapy. the most significant hope may be the use of such a signature for detection of cancers to which patients are predisposed by monitoring autoantibody profiles before the first clinical manifestation of symptoms. finally, investigators should pursue a transition from the current system of retrospective studies to prospective analyses of patients ' autoantibody responses and an assessment of this method 's efficacy in clinical settings.	the widespread use of screening mammography has resulted in increased detection of early - stage breast disease, particularly for in situ carcinoma and early - stage breast cancer. however, the majority of women with abnormalities noted on screening mammograms are not diagnosed with cancer because of several factors, including radiologist assessment, patient age, breast density, malpractice concerns, and quality control procedures. although magnetic resonance imaging is a highly sensitive detection tool that has become standard for women at very high risk of developing breast cancer, it lacks sufficient specificity and costeffectiveness for use as a general screening tool. therefore, there is an important need to improve screening and diagnosis of early - invasive and noninvasive tumors, that is, in situ carcinoma. the great potential for molecular tools to improve breast cancer outcomes based on early diagnosis has driven the search for diagnostic biomarkers. identification of tumor - specific markers capable of eliciting an immune response in the early stages of tumor development seems to provide an effective approach for early diagnosis. the aim of this review is to describe several autoantibodies identified during breast cancer diagnosis. we will focus on these molecules highlighted in the past two years and discuss the potential future use of autoantibodies as biomarkers of early - stage breast cancer.
all procedures for the animal experiment were reviewed and approved by the animal care and use committee of the national institute for physiological sciences, okazaki, japan. specific pathogen - free rats were purchased from charles river laboratories japan (kanagawa, japan). all rats were housed in an environmentally controlled room with a 12-h dark/12-h light cycle at a temperature of 23 2 c and humidity of 55 5% and were given free access to a laboratory diet (ce-2 ; clea japan, tokyo, japan) and filtered water. black coat color was used to identify wdb (a rat strain blk which we described in previous papers [14, 20, 21 ] has been renamed wdb / nips and registered to rat genome database, http://rgd.mcw.edu/wg/home, with the rgd i d 7411634). a homozygous cag / venus transgenic rat strain, generated with the crlj : wi background, was named cag / venus tg (wi). a knock - in rat strain, generated with es cells originating from the wdb strain, was named scale bars : 100 m.) were harvested and freed from their zonae pellucidae. 2b) were cultured for 7 days in (1) 2i medium consisting of 1 m mek inhibitor (pd0325901 ; stemgent, cambridge, ma, usa) and 3 m gsk3 inhibitor (chir99021 ; axon medchem bv, groningen, netherlands) in n2b27 medium, (2) 2i medium with 1,000 u / ml rat lif (esgro ; millipore, bedford, ma, usa), (3) 2i medium with 10 m forskolin (sigma - aldrich, st. louis, mo, usa), and (4) 2i medium with 1,000 u / ml rat lif and 10 m forskolin on a feeder layer of mitomycin c - treated mouse embryonic fibroblasts. 2c) were disaggregated and transferred to new culture vessels containing the same culture medium (passage-1 ; p1). these tentative es cell lines (fig. 2d) were maintained by medium exchange every other day and trypsinization / expansion every three days (p2 plus). gender of the es cell lines was determined by pcr amplification of the rat y chromosome - specific sry gene using a redextract - n - amp kit (sigma - aldrich). a 104-bp fragment of the rat sry gene was amplified by 1 cycle for 3 min at 94 c ; 38 cycles for 30 sec at 94 c, 30 sec at 55 c and 30 sec at 72 c ; and 1 cycle for 10 min at 72 c with a primer pair of sry - f (5-cat cga agg gtt aaa gtg cca-3) and sry - r (5-ata gtg tgt agg ttg ttg tcc-3). the expression of stem cell marker genes (oct-4, rnanog, fgf-4 and rex-1), a trophectoderm - specific marker gene (cdx2) and reference genes (gata6 and -actin) was examined by rt - pcr analysis. the primer sets used were the same as those described in our recent publication. briefly, total rna was extracted from each sample using a rneasy mini kit (qiagen, germantown, md, usa). the cdna was prepared using a superscript iii first - strand synthesis system (invitrogen, carlsbad, ca, usa) and amplified with takara la taq (takara bio, shiga, japan) by 30 cycles at 95 c for 30 sec, at 55 c (or 60 c in case of rnanog and gata6) for 30 sec and at 72 c for 60 sec. the potential of each es cell line for germline transmission was examined by the conventional approach using g1 generation offspring via chimeric rats. chimeric rats were prepared by microinjection of 1015 cells from each es cell line at p5 - 19 into the blastocoel cavity of wi e4.5 blastocysts, as described previously. when the albino wi - derived es cells (without fluorescent markers) were used, the host blastocysts were from the wdb female rats. all chimeric male rats that could develop to adulthood were used for the progeny test. contribution of the es cells in the resultant offspring was confirmed by their coat color. when germline - competent g1 offspring were not obtained, the progeny test was terminated with the negative data from at least 5 chimeric rats per es cell line and 4 litters per chimeric rat. data regarding the outgrowth rate and the establishment efficiency at p5 were analyzed by fisher s exact probability test using js - star 2012, a program available online (http://www.physics.csbsju.edu/stats/index.html). percentage data regarding the full - term development of es cell - injected blastocysts, chimeric rat production and germline - competent chimeras were arcsine transformed and analyzed by fisher s least significant difference test after one - way anova. all procedures for the animal experiment were reviewed and approved by the animal care and use committee of the national institute for physiological sciences, okazaki, japan. specific pathogen - free rats were purchased from charles river laboratories japan (kanagawa, japan). all rats were housed in an environmentally controlled room with a 12-h dark/12-h light cycle at a temperature of 23 2 c and humidity of 55 5% and were given free access to a laboratory diet (ce-2 ; clea japan, tokyo, japan) and filtered water. black coat color was used to identify wdb (a rat strain blk which we described in previous papers [14, 20, 21 ] has been renamed wdb / nips and registered to rat genome database, http://rgd.mcw.edu/wg/home, with the rgd i d 7411634). a homozygous cag / venus transgenic rat strain, generated with the crlj : wi background, was named cag / venus tg (wi). a knock - in rat strain, generated with es cells originating from the wdb strain, was named scale bars : 100 m.) were harvested and freed from their zonae pellucidae. 2b) were cultured for 7 days in (1) 2i medium consisting of 1 m mek inhibitor (pd0325901 ; stemgent, cambridge, ma, usa) and 3 m gsk3 inhibitor (chir99021 ; axon medchem bv, groningen, netherlands) in n2b27 medium, (2) 2i medium with 1,000 u / ml rat lif (esgro ; millipore, bedford, ma, usa), (3) 2i medium with 10 m forskolin (sigma - aldrich, st. louis, mo, usa), and (4) 2i medium with 1,000 u / ml rat lif and 10 m forskolin on a feeder layer of mitomycin c - treated mouse embryonic fibroblasts. 2c) were disaggregated and transferred to new culture vessels containing the same culture medium (passage-1 ; p1). these tentative es cell lines (fig. 2d) were maintained by medium exchange every other day and trypsinization / expansion every three days (p2 plus). gender of the es cell lines was determined by pcr amplification of the rat y chromosome - specific sry gene using a redextract - n - amp kit (sigma - aldrich). a 104-bp fragment of the rat sry gene was amplified by 1 cycle for 3 min at 94 c ; 38 cycles for 30 sec at 94 c, 30 sec at 55 c and 30 sec at 72 c ; and 1 cycle for 10 min at 72 c with a primer pair of sry - f (5-cat cga agg gtt aaa gtg cca-3) and sry - r (5-ata gtg tgt agg ttg ttg tcc-3). the expression of stem cell marker genes (oct-4, rnanog, fgf-4 and rex-1), a trophectoderm - specific marker gene (cdx2) and reference genes (gata6 and -actin) was examined by rt - pcr analysis. the primer sets used were the same as those described in our recent publication. briefly, total rna was extracted from each sample using a rneasy mini kit (qiagen, germantown, md, usa). the cdna was prepared using a superscript iii first - strand synthesis system (invitrogen, carlsbad, ca, usa) and amplified with takara la taq (takara bio, shiga, japan) by 30 cycles at 95 c for 30 sec, at 55 c (or 60 c in case of rnanog and gata6) for 30 sec and at 72 c for 60 sec. the potential of each es cell line for germline transmission was examined by the conventional approach using g1 generation offspring via chimeric rats. chimeric rats were prepared by microinjection of 1015 cells from each es cell line at p5 - 19 into the blastocoel cavity of wi e4.5 blastocysts, as described previously. when the albino wi - derived es cells (without fluorescent markers) were used, the host blastocysts were from the wdb female rats. all chimeric male rats that could develop to adulthood were used for the progeny test. contribution of the es cells in the resultant offspring was confirmed by their coat color. when germline - competent g1 offspring were not obtained, the progeny test was terminated with the negative data from at least 5 chimeric rats per es cell line and 4 litters per chimeric rat. data regarding the outgrowth rate and the establishment efficiency at p5 were analyzed by fisher s exact probability test using js - star 2012, a program available online (http://www.physics.csbsju.edu/stats/index.html). percentage data regarding the full - term development of es cell - injected blastocysts, chimeric rat production and germline - competent chimeras were arcsine transformed and analyzed by fisher s least significant difference test after one - way anova.	abstract this study was designed to investigate whether supplementation of 2i medium with leukemia inhibitory factor (lif) and/or forskolin would support establishment of germline - competent rat embryonic stem (es) cell lines. due to the higher likelihood of outgrowth rates, supplementation of forskolin with or without lif contributed to the higher establishment efficiency of es cell lines in the wdb strain. germline transmission competency of the chimeric rats was not influenced by the profile of es cell lines until their establishment. when the lif / forskolin - supplemented 2i medium was used, the rat strain used as the blastocyst donor, such as the wi strain, was a possible factor negatively influencing the establishment efficiency of es cell lines. once es cell lines were established, all lines were found to be germline - competent by a progeny test in chimeric rats. in conclusion, both lif and forskolin are not essential but can play a beneficial role in the establishment of genuine rat es cell lines.
malaria is a serious disease caused by the plasmodium parasite and transmitted through the bite of the anopheles mosquito. it is estimated that there were about 214 million cases of malaria in 2015, resulting in approximately 440,000 deaths, most of which originating from sub - saharan africa and in children under 5 years of age. malaria is characterized by signs and symptoms such as severe anemia, fever, vomiting, and fatigue. during the symptomatic phase of malaria several clinical complications these complications are anemia, cerebral malaria, placental malaria, and acute lung injury / acute respiratory distress syndrome (ali / ards). the ali / ards has been diagnosed in patients suffering from malaria caused by all the species that cause disease in humans, including p. knowlesi ; however it is more common in p. falciparum and p. vivax malaria. ali / ards is characterized by high morbidity and, although more common in adults, also affects children and pregnant woman. the most common manifestations of this syndrome are noncardiogenic pulmonary edema, increased phagocytic activity, dyspnea, reduction in the capacity of gas exchange, and increased levels of inflammatory mediators. ali / ards is most commonly caused by bacteria, sepsis, viral pneumonia, gastric aspiration, severe trauma, adverse drug reactions, and fungal or parasitic infections of the lung. the mechanisms that are critical in the initial and later stages of ali / ards are not well defined. however, it is known that the presence of intravascular fluid in the lungs, due to increased permeability of the alveolar capillary membrane, is the key pathophysiological mechanism of ali / ards. multiple factors are possibly involved in the increased vascular permeability, such as endothelium injury, increased levels of proinflammatory cytokines such as tnf- (tumor necrosis factor alpha), interleukin 1 (il-1), or il-6 and il-8, and endovascular occlusion associated with the accumulation of erythrocytes with reduced deformability, leukocytes, and platelets [6, 9 ]. different mouse models have been developed for the study of ma - ali / ards, showing similar aspects to human ali / ards [1013 ]. dba/2 strain mice develop ali / ards when infected with the parasite plasmodium berghei anka (pba). in this model, an average of 50% of the mice that die between days 7 and 12 after infection have dyspnea, hypoxemia, and reduced respiratory rate. postmortem studies revealed that these mice had pleural effusion, containing cells such as neutrophils, lymphocytes, monocytes, and macrophages [10, 14 ]. furthermore, the vascular endothelial growth factor (vegf) has been identified as critical in increased pulmonary vascular permeability, a hallmark of ali / ards [10, 15 ]. the protective role of ho-1 enzyme and carbon monoxide (co) in experimental severe malaria episodes has been demonstrated in animal models [10, 15, 16 ]. additionally, ho-1 was found to be increased in peripheral blood leukocytes, plasma, tissue macrophages, and monocytes of humans with severe malaria [17, 18 ]. ho-1 is an enzyme encoded by hmox-1 gene and is considered protective due to its anti - inflammatory, antiapoptotic, and antiproliferative actions in different cell types, including endothelial cells. this enzyme participates in the free heme degradation, generating equimolar amounts of co, iron, and biliverdin and plays a protective role in modulating tissue response to injury in various organs, including the lung [2022 ]. the malaria infection leads to the release of reactive oxygen species and free heme, harmful to the endothelial cells of the host. ho-1 catabolizes free heme into iron, biliverdin, and co, which are less toxic to the cells. therefore, some studies have shown that inducers of ho-1 such as hemin and cobalt protoporphyrin ix (coppix) protected mice infected with malaria, or suffering from other diseases such as polymicrobial sepsis, from developing ali / ards [16, 24, 25 ]. additionally, it was observed in previous publications that ho-1 inducers are protective against experimental cerebral malaria and that treatment of dba/2 mice with a co - releasing molecule is protective against ma - ali / ards. meanwhile, in this study, it was observed, for the first time that ho-1 expression is increased in pba infected dba/2 mice lungs which develop ali / ards and that the induction of ho-1 protects these mice from developing ali / ards. six- to ten - week - old male dba/2 mice were bred under pathogen - free conditions in isogenic mouse facilities, at the biomedical sciences institute of the university of so paulo. in all experiments, mice had ad libitum access to water and food (nuvilab cr-1, quintia s / a, so paulo, brazil). mice were intraperitoneally infected with 1 10 plasmodium berghei anka (clone 1.49l) infected red blood cells (irbcs), as previously described. all experiments were performed in accordance with the ethical guidelines for experiments with mice, and the protocols were approved by the animal health committee of the biomedical sciences institute of the university of so paulo (ceua number 146, page 136, book 2). the guidelines for animal use and care were based on the standards established by the brazilian college of animal experimentation (cobea). the mice were euthanized with an anesthetic overdose of ketamine (150 mg / kg) (vetbrands, brazil) and xylazine (15 mg / kg) (syntec, brazil), and consciousness was checked by testing the pedal reflex, heartbeats, and breathing movements. necropsy was performed in mice dying naturally from malaria or mice euthanized on the 20th day after infection (dai) to complete the experiment and to avoid animal suffering. the lungs were collected and fixed in buffered 10% formalin for 24 hours and 70% alcohol for 2448 hours and then embedded in paraffin, sectioned at 5 m onto slides, and stained with hematoxylin - eosin (he) according to standard protocol. histopathological analyses were performed under a axio imager m2 (zeiss) microscope using the axio cam hrc (zeiss) and the software axio vision, version 4.9.1.0. in order to determine the alveolar area percentage, 20 pictures per lung he section were taken and the software gimp, version 2.8.16 (https://www.gimp.org/) was used to determine the alveolar area in pixels of each picture. the percentage of alveolar area relatively to total area of the pictures were determined in ali / ards versus hp and in hemin treated versus nontreated (saline) mice. respiratory patterns (respiratory frequency (rf) and enhanced pause (penh)) were monitored on the 7th dai by placing the mice in the plethysmography chamber (wbp, buxco electronics, wilmington, north carolina, usa) for 10 minutes (basal level) as described before. the data were collected using biosystems xa software and included the rf (breaths / minute) and variables to calculate the penh, a theoretical variable that correlates with both pulmonary resistance and intrapleural pressure. the penh is calculated by the following equation : (1)penh = peak expiratory heightpeak inspiratory heightexpiratory timerelaxation time1. identification of ali / ards in mice before death was done as described before. in short, we used two groups of pba infected mice (1012 mice per group) : the survival group and the euthanized group in which the mice were euthanized on the 7th dai. in the survival group, in any mouse that died between the 7th and 12th dai showing pleural effusion or red and congested lungs at necropsy, the cause of death was attributed to ali / ards. in contrast, in mice without pleural effusion that died after 13th dai with pale lungs and high levels of parasitemia, the cause of death was attributed to hyperparasitemia (hp) and, consequently, anemia. in the euthanized group, mice were classified as having been likely to die of ali / ards or hp, by comparing their respiratory patterns and parasitemia levels with the survival group, in which the causa mortis was known, as previously published. to perform the immunohistochemistry of ho-1, slides containing the tissue sections were placed in an incubator at 60c for 20 minutes. then they were incubated in xylene twice for 15 min at 60c and afterwards passed in absolute ethanol, in 95% alcohol, in 70% alcohol, in distilled water, and finally in 1x pbs ph 7.2 to 7.4. for the antigen retrieval, the slides were incubated in sodium citrate buffer ph 6 for 45 minutes at 95c. endogenous peroxidase was blocked with 3% hydrogen peroxide for 15 minutes, twice, at room temperature and protected from light. the tissue was probed with rabbit polyclonal to ho-1 (1 : 1000) antibody (abcam, ab13243) overnight at 4c and then the kit reveal mouse / rabbit (spring - code spd-015) was used in accordance with the manufacturer 's instructions. the quantification of ho-1 in lung tissue was done by calculating the marked area in the entire tissue section. the calculation was done in imagej (version 1.50b) software (https://imagej.nih.gov/ij/), using the plugin ihc toolbox (http://rsb.info.nih.gov/ij/plugins/ihc-toolbox/index.html). fresh frozen mice lung tissues were sonicated and homogenized in ice using the radio - immunoprecipitation assay (ripa) buffer composed of 50 mm tris - hcl ph 8.0, 150 mm nacl, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate (sds), 1 mm sodium orthovanadate, 1 mm naf, and a protease inhibitor tablet. the samples were analyzed for protein content using a bradford protein assay (biorad) according to manufacturer 's instructions. each sample was quantified, and then 9 g of protein was loaded onto a 12% tris - glycine sds - polyacrylamide gel, according to the manufacturer 's protocol (biorad). the gel was transferred to a pvdf membrane by electrophoresis at 30 v for 16 h. the membrane was blocked in pbs with 0.1% tween 20 (pbs - t) and 10% nonfat milk at room temperature for 2 h. all antibodies were diluted in the same buffer (pbs - t) with 1% nonfat milk. the membrane was then probed with rabbit polyclonal ho-1 antibody (abcam, ab13243, 1 : 20,000) and then incubated for 1 h at room temperature. after incubation, the membrane was washed five times with pbs - t and incubated with horseradish peroxidase conjugated goat anti - rabbit igg (millipore, ap307p, 1 : 20,000) for 1 h at room temperature. after washing five times with pbs - t, an ecl system (clarity western ecl blotting substrate, biorad) was used for detection of the proteins in a chemidoc xrs+ system (biorad). the ho-1 expression was calculated by densitometry, in the software imagej (version 1.50b) of the ho-1 bands in the immunoblot relative to the housekeeping protein -actin (rabbit monoclonal to -actin, novus biologicals, nb600 - 501, 1 : 500,000). on the 7th dai elisas kits were used to quantify ho-1 levels in serum and macerated lung tissue (enzo lifesciences, adi-960 - 071) and bilirubin levels (an indirect measure of ho-1 activity) (biomatik, eku08395) according to the manufacturer 's instructions. the ho-1 level of the ali / ards and hp mice was expressed as fold increase in relation to noninfected mice. extraction of total rna from lungs of mice was performed using rneasy mini kit (qiagen), according to the manufacturer 's instructions. one microgram of total rna was reverse - transcribed to single - strand cdna using the first - strand cdna synthesis kit (roche) amv reverse transcriptase protocol (roche applied science). ho-1 transcripts in the cdna obtained from the reverse transcriptase reaction were quantified by real - time quantitative fluorogenic pcr performed in the 7500 fast instrument (applied biosystems). sybr green pcr master mix (applied biosystems) was used to quantify gene expression according to the manufacturer 's instructions. the gene expression was normalized by the housekeeping gene hypoxanthine guanine phosphoribosyltransferase (hprt) and using the relative quantification method 2 as described before. the primers used were as follows : ho-1 : 5-tctcagggggtcaggtc-3 (forward) and 5-ggagcggtgtctgggatg-3 (reverse) ; ifn- : 5-cacactgcatcttggctttg-3 (forward) and 5-tctggctctgcaggattttc-3 ; hprt : 5-tgctcgagatgtgatgaagg-3 (forward) and 5-tcccctgttgactggtcatt-3 (reverse). hemin (sigma - aldrich) was diluted in 0.2 m naoh to a final concentration of 5 mm and ph 7.4. hemin was administered in two doses : the first was two days before the infection and another at 4th dai in single doses of 17 mg / kg or with saline solution (control group), intraperitoneally. to measure parasitemias and survival rate of hemin treated and ho-1 induction by hemin was also used to measure lung vascular permeability and vegf and cytokine serum levels. to investigate lung vascular permeability on the 7th dai, the hemin treated or saline treated infected mice and noninfected mice were injected intravenously with 0.2 ml of 1% evans blue (sigma). the mice were euthanized 45 minutes later, and the lungs were weighed and placed in 2 ml of formamide (merck) for 48 hours at 37c. the amount of evans blue staining per gram of lung tissue was calculated from a standard curve. the lung permeability was expressed as fold increase in relation to the noninfected mice. on the 7th dai, hemin treated or saline treated infected mice and noninfected mice were anesthetized, and their serum was collected by cardiac puncture. an elisa kit (r&d systems) was used to quantify vegf levels in the serum according to the manufacturer 's instructions. the vegf level was expressed as fold increase in relation to that of the noninfected mice. the cytokine levels were measured in serum from the hemin treated and saline treated dba/2 mice. mice inflammation cba cytokine kit (cytometric bead array, becton - dickinson) was used to measure interferon, ifn-, tumor necrosis factor, tnf-, and il- and il-10 levels. this kit was used for dosing cytokines from lung tissue lysates using flow cytometer (bd facs calibur system, becton - dickinson) and using the software cell quest pro, version 5.2. the individual standard curve range for a given cytokine was determined according to manufacturer instructions. the primary microvascular lung endothelial cells (pmlec) were obtained from dba/2 mice, according to what was described before. after euthanasia, the animal 's body was disinfected with iodine alcohol. then all blood was taken from the mice by cutting the carotid artery. in a laminar flow chamber, the lung tissue was cut into fragments of approximately 1 mm which were distributed among 6-well polystyrene plates. after being supplemented, dmem culture medium (20% serum fetal bovine (fbs) and antibiotics) was added to each well. the plate was then incubated at 37c and 5% co2 for 72 h. after this period, the tissue fragments were removed and 50% of the medium was replaced. after 7 days of incubation, the cells were removed with trypsin 0.25% (edta, gibco) for 15 min and replaced in a culture flask of 75 cm. the trypsinization procedure was repeated every 5 to 7 days. finally, the cells were cultured for 15 to 20 days (3rd and 4th passage) until being used in the following trials. the isolated pmlec were characterized by immunofluorescence with the antibodies anti - vwf, anti - vcam, anti - ace, anti - cd62e, anti - enos, anti - cd31, and anti - ve - cadherin. to analyze the area of opening of interendothelial junctions (oij), that, the lung endothelial cells were plated in 24 well plates (7 10 cells / well), adhered to gelatin on glass coverslips, and maintained at 37c and 5% co2. the cells were stimulated with pba lysate for 3 h, after incubation with hemin (5, 10, and 20 m during 24 h), or solely with dmem culture medium, supplemented with 20% fbs, in triplicate. subsequently, the cells were fixed with 3.7% formaldehyde, permeabilized with acetone at 20c, and blocked with bovine serum albumin solution (1% bsa). each slide, with fully confluent cells, was chosen randomly and ten to twenty pictures were taken and scanned in a zig - zag way, from top to bottom. the images were acquired in the fluorescence axio imager m2 (zeiss) microscope using the axio cam hrc (zeiss) and the software axio vision, version 4.9.1.0. the total area of oij was measured in each picture using the software gimp, version 2.8.16 (https://www.gimp.org/). the increased lung vascular permeability was analyzed in pmlec plated on inserts of permeable membranes with pores of 0.4 m (transwell corning), pretreated with gelatin and coupled in 24-well polystyrene plates at a concentration of 2.2 10 cells per insert and maintained in dmem culture at 37c. after 96 hours, until the cells reach confluency, the extract was applied for 3 h after incubation with hemin (20 m during 24 h) or solely with 20% fbs supplemented dmem culture medium. subsequently, the culture medium was replaced by hank 's balanced salt solution and in the upper compartment of each insert (in contact with the cells) ; 200 l of evans blue was incubated at a concentration of 2 mg / ml at 37c. after 30 min, the liquid from the lower compartment was collected and analyzed in a spectrophotometer at a wavelength of 650 nm (nanodrop 2000, thermo scientific). finally, the concentration of evans blue in each sample was determined from a standard curve with previously known concentrations of evans blue (0.2 mg / ml to 0.0031 mg / ml). the data were analyzed by d'agostino - pearson normality test. whitney test and kruskal wallis test with dunn 's multiple comparisons test. the unpaired t - test and one - way anova with bonferroni multiple comparison test were used for parametric variables. statistical analyses were performed in graphpad prism version 5.0 (http://www.graphpad.com/scientific-software/prism/), including assessments of sensitivity and specificity. to establish cut - off from data, roc curves were generated using the results of the control group in medcalc version 8.2.1.0 (https://www.medcalc.org/). the development of ali / ards in the dba/2 mouse model occurred between the 7th and the 12th dai (figures s1a and s1b in supplementary material, available online at http://dx.doi.org/10.1155/2016/4158698), during which the mice have died presenting at necropsy reddish lungs, pleural effusion, and histologic changes such as congestion, alveolar edema and hemorrhage, inflammatory infiltrate, and damage to the alveolar wall (figure s1c). mice that survived after the 12th dai died on the 20th dai or were euthanized at the same day, showing grayish tone lungs, splenomegaly, and no pleural effusion, interstitial chronic pneumonia, and malarial pigment in the lung tissue (figure s1c). these two phenotypes were described in detail previously by our group [10, 14 ]. noninfected mice showed light pink lungs, and no liquid inside of the thoracic cavity was detected (figure s1c). these changes in lung histology were consistent with the percentage of alveolar area ; they were significantly lower in ali / ards than in hp mice (figure s1d). by using the predictive model to identify ali / ards or hp in mice before death, the expression of ho-1 in the development of ali / ards was determined in the serum and lungs of infected dba/2 mice and compared to noninfected mice. in order to observe the protein expression of ho-1, immunohistochemistry sections of lung tissue of dba/2 mice the expression of ho-1 was higher in lungs of ali / ards - developing mice than in hp - developing mice and noninfected mice. quantification of immunohistochemical stain of ho-1 in the lung was performed by image analysis (figure 1(a)). ho-1 protein levels (figure 1(b)) and ho-1 mrna expression (figure 1(c)), measured by western blot and qrt - pcr, respectively, in the mice lungs, were higher in ali / ards - developing mice compared to hp - developing mice on the 7th dai. also, ho-1 levels in the lung tissue cell lysate and in the sera were higher in ali / ards mice than in hp mice, as determined by elisa (figures 1(d) and 1(e)). in addition, we checked the activity of ho-1 measuring bilirubin in pba infected and noninfected mice by elisa, on the 7th dai. the levels of bilirubin were significantly higher in ali / ards - developing mice than in noninfected mice (figure 1(f)). however, the levels of bilirubin were not significantly different between ali / ards and hp mice. mice were treated with 17 mg / kg of hemin, an inductor of ho-1, on the second day before pba infection and on 4th dai. the efficiency of induction of ho-1 protocol was checked and confirmed, showing that hemin treatment increased mrna and protein levels of ho-1 in the lungs and in the serum of noninfected animals (figures s2a and s2b, resp.), which confirms it as an inducer of ho-1. the hemin treatment increased the survival with most of the mice alive after the 12th dai without developing ali / ards (figure 2(a)). additionally, the parasitemia was significantly different between the 5th and the 7th dai (figure 2(b)). mice treated with saline (control) and sacrificed on 7th dai presented necropsy reddish lungs, pleural effusion, congestion, alveolar edema and hemorrhage, inflammatory infiltrate, and damage to the alveolar wall (figure 2(c)), similarly to what was observed in mice that developed ali / ards (figure s1) [10, 14 ]. on the other hand, hemin treated mice showed a phenotype similar to noninfected mice : light pink lungs, and no liquid inside of the thoracic cavity was detected (figure 2(c)). as it was observed in ali / ards versus hp mice, saline treated mice also had significantly less percentage of alveolar area than hemin treated mice (figure 2(d)). regarding the respiratory parameters, it was found that hemin treatment led to a significant amelioration, characterized by a decrease in enhanced pause (penh) (figure 3(a)) and an increase in the respiratory frequency at 7th dai (figure 3(b)). also, treatment with hemin led to a significant reduction of ifn- mrna in lung and serum protein levels, il-10 in serum, mcp-1 protein in lung tissue lysates, and serum and tnf in serum (figures 3(c)3(h)). the treatment of pba infected mice with hemin led to a decrease of vegf serum levels (figure 4(a)), a potent inducer of ali / ards in pba infected dba/2. also, hemin treated mice showed protection of the alveolar capillary barrier, which can be seen by a reduction in lung vascular permeability after evans blue administration and quantification (figure 4(b)). in addition, we observed the same effect in pmlec when they were hemin treated after being stimulated with pba lysate for 3 hours, in a transwell plate (figure 4(c)). to understand a possible mechanism of ho-1 action in reducing pulmonary vascular permeability, pmlec were treated with hemin in 5, 10, and 20 m, before pba stimulus. the hemin action showed a significant reduction in the oij that are formed between the endothelial cells, when compared to nontreated cells (figure 4(d)). the dba/2 developed ali / ards between the 7th and 12th dai, with an incidence of 50% (average) when infected with pba [10, 14 ]. our results reinforce this finding, showing that the animals that began to die after the 7th dai until the 12th dai showed typical signs of ali / ards, such as pleural effusion, alveolar edema and hemorrhage, alveolar wall damage, inflammatory infiltrates, and lower percentage of alveolar area. the parasitemia reaches about 20% when mice start to develop ali / ards (7th dai) and this value has a slight decrease from the 12th dai onwards. interestingly, human malaria - associated ali / ards often occurs in patients who have already begun the antimalarial treatment, which also leads to a decrease in parasitemia. ho-1 showed a protective role in experimental episodes of severe malaria, including cerebral malaria [15, 16, 31 ]. moreover, treatment with ho-1 inhibitors such as zinc protoporphyrin ix (znppix) or tin protoporphyrin ix (snppix) led to an enhancement of ali / ards signals in the case of sepsis but had no effect in the cases of hyperoxia and experimental cerebral malaria [16, 24, 32 ]. however, our data showed that ali / ards - developing mice had increased levels of mrna and ho-1 protein, when compared with hp - developing mice and noninfected mice. additionally, the levels of bilirubin, which represent an indirect measure of ho-1 activity, were higher in ali / ards mice than in noninfected mice. although it is not known whether this increase in ho-1 levels and ho-1 activity constitutes an effort to revert the ali / ards phenotype in the infected mice, it was showed in previous publications that the treatment of p. berghei anka infected dba/2 mice with co (a product of ho-1) and with a co - releasing molecule protected them against ali / ards. therefore, in order to clarify whether ho-1 is protective in this model, its induction was performed by hemin. the treatment with the inducer of ho-1 (hemin) had beneficial effects in pba infected dba/2 mice and in pmlec in contact with pba lysate. in vivo, this treatment led to an improvement in the survival rate and in lung histology, with the absence of lung edema, higher alveolar area percentage, and the absence of pleural effusion at necropsy. it is an interesting effect, since hemin was observed to lyse malaria parasites in culture. additionally hemin treated mice infected with plasmodium chabaudi adami also have a reduction in parasitemia. additionally, hemin induces ho-1, which reduces the oxidative stress in the host, which may increase its capacity of parasite clearance. hemin also led to an improvement of respiratory parameters, with a decrease of enhanced pause (penh) and increase of respiratory frequency reinforcing the mitigation of clinical signals of ali / ards, alongside an increase in survival rate. these respiratory parameters were previously used in a predictive model of malaria - associated ali / ards in dba/2 mice. additionally, there is evidence of penh and respiratory frequency being altered in a model of lung injury by so2 exposure in rats. in addition to improvements in survival, lung histology, penh, and respiratory frequency, the treatment with hemin led to a significant decrease in the levels of the proinflammatory cytokines ifn-, mcp-1, and tnf-, which is in agreement with the survival increase and with improvement in lung parameters, as it was shown that tnf- induced pulmonary vascular endothelial injury in an animal model. additionally, lung neutrophil accumulation and lung leak were abrogated in tnf- knockout mice that were subjected to hemorrhagic shock. ifn- was also considered to be a key contributor to ali / ards in a hyperoxia mice model, where it was shown that this cytokine induced increases in lung alveolar permeability and neutrophil migration into lung air spaces. increased levels of mcp-1, chemokine involved in recruiting of monocytes, neutrophils, and lymphocytes, were found in pulmonary alveolar macrophages isolated from a rat model of immune complex - mediated acute inflammatory lung injury. this shows that hemin reduced the levels of inflammatory cytokines that are important in ali / ards, which corroborates with previous results showing that ho-1 induction by hemin has anti - inflammatory effects in models of sepsis and lps induced ali / ards in mice [25, 42 ]. as an anti - inflammatory cytokine, which was increased in hemin treated animals, which were protected against endotoxic shock and whose effect in ali / ards was shown to be protective, il-10 was not expected to be decreased in hemin treated animals in our data. however, this cytokine was observed to be increased in mice that developed ali / ards in our model before (unpublished data). the treatment with hemin resulted in a reduction in serum levels of vegf, a factor that promoted the development of ali / ards in this model, in a previous study. furthermore, siner and colleagues demonstrated that vegf is a potent inducer of ho-1 enzyme and that the induction of this enzyme reduced the acute lung injury in mice caused by hyperoxia. the same could be happening in our model of ma - ali / ards, because the vegf levels are increased on the 7th dai, coinciding with an increase of ho-1 levels in serum and lungs. on the other hand, it was shown that treatment of pba infected mice with vegf prevented them from developing experimental cerebral malaria. however, the contribution of vegf to the increase of ho-1 might not occur soon enough to revert the ma - ali / ards phenotype in our model. in this model, vegf caused a deleterious effect, increasing the lung vascular permeability. additionally, it was shown that alf492, a co - releasing molecule, protected pba infected dba/2 from ali and reduced vegf levels of treated mice. this corroborates our results that show a reduction in vegf levels on hemin treated mice. however hemin treatment was also shown to have deleterious effects in a rat model of neuroinflammation, where it was responsible for increased levels of reactive oxygen species, brain tissue loss, microglial activation, and neuronal death. in addition, improved pulmonary vascular permeability in hemin treated infected mice on the 7th dai was observed. this corroborates previous studies that showed a reduction in the breakdown of alveolar capillary barriers in models of ali / ards induced by lps. additionally, in experimental cerebral malaria, the induction of ho-1 reduced the permeability of the blood brain barrier in pba infected mice. also, in vitro, there was a reduction of the permeability of pmlec stimulated with pba and treated with hemin reinforcing the importance of ho-1 in this model. finally, pmlec stimulated with pba lysate and treated with different hemin concentrations led to a decrease in the opening of interendothelial junctions, indicating that hemin acts at the cellular level, protecting from the deleterious effect of the parasite in endothelial cells and consequently reducing the lung vascular permeability. this is in accordance with a previous publication where the presence of oij after acute lung injury initiation in an animal model was responsible for an increase in lung vascular permeability. this data supports the hypothesis that the increase in ho-1 levels after the 7th dai is a late effort to reverse the ali / ards phenotype. inducing ho-1 early in pba infection has a protective effect against ali / ards, making this enzyme a target for the prevention of ma - ali / ards.	malaria is a serious disease, caused by the parasite of the genus plasmodium, which was responsible for 440,000 deaths in 2015. acute lung injury / acute respiratory distress syndrome (ali / ards) is one of the main clinical complications in severe malaria. the murine model dba/2 reproduces the clinical signs of ali / ards in humans, when infected with plasmodium berghei anka. high levels of ho-1 were reported in cases of severe malaria. our data indicated that the ho-1 mrna and protein expression are increased in mice that develop malaria - associated ali / ards (ma - ali / ards). additionally, the hemin, a ho-1 inducing drug, prevented mice from developing ma - ali / ards when administered prior to the development of ma - ali / ards in this model. also, hemin treatment showed an amelioration of respiratory parameters in mice, high vegf levels in the sera, and a decrease in vascular permeability in the lung, which are signs of ali / ards. therefore, the induction of ho-1 before the development of ma - ali / ards could be protective. however, the increased expression of ho-1 on the onset of ma - ali / ards development may represent an effort to revert the phenotype of this syndrome by the host. we therefore confirm that ho-1 inducing drugs could be used for prevention of ma - ali / ards in humans.
rupture of intervertebral disc material into the intradural space is a rare event in lumbar disc disease but must be considered in the differential diagnosis of mass lesion causing nerve root or cauda equina syndromes. the pathogenesis of lumbar intradural disc herniation is most likely related to dense adhesion between the ventral dura mater and the posterior longitudinal ligament. intradural disc herniations are usually seen at l4-l5 and l3-l4, but have also been reported at other levels. there are approximately nine reports in the english literature of intraradicular disc herniation at l5-s1. however, intradural disc herniation at l5-s1 is quite rare. this report presents a case of intradural disc herniation at l5-s1 mimicking an intradural extramedullary spinal tumor. a 61-yr - old man was admitted to hospital having experienced pain in the lower back and both lower legs for 4 months and a sudden exacerbation of the symptoms for 3 days before admission. neurological examination revealed weakness of the extensor hallucis longus and decreased ankle reflex in both lower extremities. a sensory deficit over the saddle area was observed, but bladder and bowel function were normal. magnetic resonance imaging (mri) showed a mass - like lesion at the level of l5-s1 space (fig. 1). gadolinium - enhanced mri demonstrated a large disc herniation at the l5-s1 level with an intradural component (fig. subsequent durotomy demonstrated a 12 cm mass occupying the spinal canal with peripheral displacement of the nerve roots (fig. careful excision of the disc fragment revealed a 0.40.4 cm defect in the anterior thecal sac, which was firmly adherent. the patient 's postoperative period was uneventful and he gained full recovery in 3 months. intradural disc herniations comprise 0.27% of all herniated intradural disc herniation at l5-s1 discs (1). lumbar intradural disc rupture must be considered in the differential diagnosis of mass lesions causing nerve root or cauda equina syndromes. approximately 123 cases of intradural disc herniations have been reported since 1942 (2). the majority of them occurred at the l4-l5 levels and only 12 cases occurred at l5-s1. type b : herniation of a disc into the dural sheath in the preganglionic region of the nerve root. this terminology is confusing and actually indicates a special type of disc herniation through the dural sheath of the nerve root but not within the epineurium. therefore, describing type b as an intraradicular disc herniation is more specific and certain. type b intradural disc herniations are much less frequent. of the 12 cases of intradural disc herniation were reported at l5-s1, 9 belonged to type b intradural disc herniation and the other 3 could not be classified due to the absence of any comment of their type. ten cases of type b intradural disc herniation have been reported, and all were in the lumbar region : 9 in the s1 nerve root and one in the l5 nerve root (3 - 6). there are approximately nine reports in the english literature of intraradicular disc herniation at l5-s1. this report presents a case of type a intradural disc herniation at l5-s1 mimicking an intradural extramedullary spinal tumor. the exact mechanism of the dural tear by a herniated disc is not known clearly. an anatomical investigation revealed dense non - separable adhesions of the ventral dura to the posterior longitudinal ligament at the l4-l5 level in eight of 40 cadavers (7). it was suggested that adhesions formed congenitally or caused by trauma, surgery, inflammation, osteophytes or disc protrusion fixed the dural sac. a study of fresh adult cadavers by spencer. (8) demonstrated the existence of dural ligaments fixing the dura and nerve roots at their exit from the main dural sac to the posterior longitudinal ligament and vertebral body periosteum proximal to the intervertebral disc. distal fixation generally occurs at the intervertebral foramen where the epineural sheath of the spinal nerve is attached. these ligaments in certain cases also cause increased nerve root fixation, thereby allowing penetration of a ruptured disc to the s1 root. the reason why type a herniations occur at the l4-l5 intervertebral disc space and type b at l5-s1 is also yet be elucidated. the postulated mechanism is adhesions in both type a and type b. although intraradicular disc herniation are frequently seen at the l5-s1, type a intradural disc herniation could occur at the l5-s1, if adhesions between the ventral dura and the posterior longitudinal ligament are formed congenitally or caused by trauma, surgery, inflammation at the l5-s1 level. it is usually not difficult with current mri techniques to differentiate lumbar disc herniation from other conditions (2). contrast - enhanced mri scans are useful to differentiate a herniated disc from a disc space infection or tumor. a herniated disc fragment will rarely be enhanced centrally, which is attributed to vascular granulation tissue infiltrating the fragment (2). in our case, the lesion had intermediate signal intensity on t1-weighted and low signal intensity on t2-weighted mri scans and that led us to suspect an intradural extramedullary tumor lesion. however on contrast mri scans there was peripheral enhancement of the lesion, which is typical for a disc fragment (9). while treating lumbar disc disease, the possibility of an intradural disc herniation should be kept in mind for the success of the discectomy and the management of failed back syndrome. we herein present a case of intradural disc herniation at l5-s1 mimicking an intradural extramedullary spinal tumor that demonstrates the role and the importance of contrast mri in the diagnosis of intradural disc herniation.	intradural lumbar disc herniation is a rare pathological entity. the pathogenesis of intradural lumbar disc herniation is not known clearly. intradural disc herniations usually occurred at the l4-l5 levels but have also been reported at other levels. however, intradural disc herniation at l5-s1 is quite rare. there are approximately nine reports in the english literature of intraradicular disc herniation at l5-s1. we described a 61-yr - old man with suspected intradural mass at the level of l5-s1 space. the patient presented with pain in the lower back and both lower legs for 4 months and a sudden exacerbation of the symptoms for 3 days. gadolinium - enhanced magnetic resonance imaging (mri) demonstrated a large disc herniation at the l5-s1 level with an intradural component. l5 and s1 laminectomy was performed, and dura was swollen and immobile. subsequent durotomy was performed and an intradural disc fragment was removed. the patient had full recovery in 3 months. intradural lumbar disc herniation must be considered in the differential diagnosis of mass lesions in the spinal canal. contrast - enhanced mri scans are useful to differentiate a herniated disc from a disc space infection or tumor.
the success of root canal therapy depends on the method and the quality of instrumentation, irrigation, disinfection, and three - dimensional obturation of the root canal. endodontic instrumentation using either manual or mechanized techniques produces a smear layer and plugs of organic and inorganic particles of calcified tissue. the smear layer contains additional organic elements such as pulp tissue debris, odontoblastic processes, microorganisms, and blood cells in the dentinal tubules. a smear layer can create a space between the inner wall of the root canal and the obturating materials, thus preventing the complete locking and adherence of the root canal filling materials into the dentinal tubules. it also contains bacteria and bacterial by - products and thus must be completely removed from the root canal system. in addition, removal of the smear layer can allow intracanal medicaments to penetrate the dentinal tubules for better disinfection. among the different root canal irrigants for removal of smear layer, sodium hypochlorite, (naocl), and ethylene diamine tetra - acetic acid (edta) solution mtad, containing 3% doxycycline, 4.25% citric acid, and detergent (tween 80), has been shown to be clinically effective in removing the smear layer when used along with naocl. however, few reports suggest that the microhardness of dentin decreases when they are exposed to naocl or edta, or mtad. it has been indicated that microhardness determination can provide indirect evidence of mineral loss or gain in the dental hard tissues. it has been reported that these kinds of chemical irrigants caused alterations in the chemical structure of human dentin and changed the ca : p ratio of the dentin surface. any change in the ca : p may change the permeability and solubility characteristics of dentin which may also affect the quality of its adhesion to root canal sealers. moreover, edta and citric acid (component of mtad) strongly reacts with sodium hypochlorite, thus rendering the latter agent ineffective. therefore it is necessary to find a new, safe, and effective irrigating regimen during root canal preparation. a soft chelating irrigation protocol with etidronic acid, also known as 1-hydroxyethylidene-1, 1-bisphosphonate (hebp) or etidronate, has been proposed as a potential alternative to edta or citric acid because of its chelating ability.[1113 ] it shows no short - term reactivity with sodium hypochlorite. however the effect of hebp on microhardness of root dentin has not been evaluated so far in the literature. therefore, the purpose of this study was to evaluate the effect of 18% hebp solutions as a final rinse on the microhardness of human root canal dentin in comparison with 17% edta and mtad using vicker 's microhardness test. forty human single - rooted mandibular premolars, recently extracted for orthodontic reasons, were used for this study. the teeth were decoronated at the cementoenamel junction sectioned longitudinally starting from cervical with a low speed diamond saw (isomet, buehler ltd., the separated buccal and lingual root segments were horizontally mounted in autopolymerising acrylic resin so that their dentin was exposed. the dentin surfaces of the mounted specimens were grounded smooth with a series of increasingly fine emery papers (shor international corporation, mt. vernon, ny, usa) under distilled water to remove any surface scratches, and finally polished with 0.1 m alumina suspension (ultra - sol r, eminess technologies inc., all the groups were treated with different irrigants as per the following protocol : group i : the specimens were treated with distilled water (control).group ii : the specimens were treated with 1.3% naocl (prime dental products ltd., mumbai, india) for 20 minutes followed by 17% edta (dent wash prime dental products ltd., mumbai, india) for 1 minute.group iii : the specimens were treated with 1.3% naocl for 20 minutes followed by mtad (dentsply tulsa dental, tulsa, ok, usa) for 5 minutes.group iv : the specimens were treated with 1.3% naocl for 20 minutes followed by 18% hebp for 5 minutes. group ii : the specimens were treated with 1.3% naocl (prime dental products ltd., mumbai, india) for 20 minutes followed by 17% edta (dent wash prime dental products ltd., mumbai, india) for 1 minute. group iii : the specimens were treated with 1.3% naocl for 20 minutes followed by mtad (dentsply tulsa dental, tulsa, ok, usa) for 5 minutes. group iv : the specimens were treated with 1.3% naocl for 20 minutes followed by 18% hebp for 5 minutes. hebp solutions were freshly prepared by mixing the hebp powder (zschimmer and schwarz mohsdorf gmbh and co., kg, burgstadt, germany) with bi - distilled water to w / v concentrations of 18%. the surface hardness of the root dentin was determined in each specimen with a vicker 's hardness tester (matsuzawa mmt-7, matsuzawa seiki co., ltd., the indentations were made with a vicker 's diamond indenter at a minimum of three widely separated locations. the locations were chosen in apical, middle, and cervical region of the root canal wall. the indentations were made on the cut surface of each specimen using 300 g load and a dwell time of 20 seconds. the microhardness data were tabulated and statistically analyzed by one - way anova and the inter group comparison of means was conducted using a post hoc tukey 's multiple comparison test. forty human single - rooted mandibular premolars, recently extracted for orthodontic reasons, were used for this study. the teeth were decoronated at the cementoenamel junction sectioned longitudinally starting from cervical with a low speed diamond saw (isomet, buehler ltd., the separated buccal and lingual root segments were horizontally mounted in autopolymerising acrylic resin so that their dentin was exposed. the dentin surfaces of the mounted specimens were grounded smooth with a series of increasingly fine emery papers (shor international corporation, mt. vernon, ny, usa) under distilled water to remove any surface scratches, and finally polished with 0.1 m alumina suspension (ultra - sol r, eminess technologies inc., all the groups were treated with different irrigants as per the following protocol : group i : the specimens were treated with distilled water (control).group ii : the specimens were treated with 1.3% naocl (prime dental products ltd., mumbai, india) for 20 minutes followed by 17% edta (dent wash prime dental products ltd., mumbai, india) for 1 minute.group iii : the specimens were treated with 1.3% naocl for 20 minutes followed by mtad (dentsply tulsa dental, tulsa, ok, usa) for 5 minutes.group iv : the specimens were treated with 1.3% naocl for 20 minutes followed by 18% hebp for 5 minutes. group ii : the specimens were treated with 1.3% naocl (prime dental products ltd., mumbai, india) for 20 minutes followed by 17% edta (dent wash prime dental products ltd., mumbai, india) for 1 minute. group iii : the specimens were treated with 1.3% naocl for 20 minutes followed by mtad (dentsply tulsa dental, tulsa, ok, usa) for 5 minutes. group iv : the specimens were treated with 1.3% naocl for 20 minutes followed by 18% hebp for 5 minutes. hebp solutions were freshly prepared by mixing the hebp powder (zschimmer and schwarz mohsdorf gmbh and co., kg, burgstadt, germany) with bi - distilled water to w / v concentrations of 18%. the surface hardness of the root dentin was determined in each specimen with a vicker 's hardness tester (matsuzawa mmt-7, matsuzawa seiki co., ltd., the indentations were made with a vicker 's diamond indenter at a minimum of three widely separated locations. the locations were chosen in apical, middle, and cervical region of the root canal wall. the indentations were made on the cut surface of each specimen using 300 g load and a dwell time of 20 seconds. the microhardness data were tabulated and statistically analyzed by one - way anova and the inter group comparison of means was conducted using a post hoc tukey 's multiple comparison test. the mean and standard deviation values of the root dentin microhardness data for the control groups and 17% edta, mtad, and 18% hebp groups are listed in table 1. the microhardness values of control (group i), edta treated (group ii), mtad treated (group iii), and hebp treated (group iv) groups were 66.65 1.04, 51.63 0.86, 42.85 0.99, and 53.74 1.18, respectively. intergroup comparison showed that the reduction in microhardness was statistically significant among the experimental groups as well as between the experimental and control group (p<0.001). current concepts of chemo - mechanical preparation imply that chemicals should be applied on instrumented root canal surfaces in order to remove the smear layer following mechanical preparation. it has been proved that different irrigant activation protocols enhance the action of chelating agents. but the objective of our investigation was only to assess the chemical action of chelating agent as final rinse on the microhardness of root dentin. the irrigating regimens in all the experimental groups were standardized to 20 minutes of 1.3% naocl, which represents the working solution, followed by a final rinse 17% edta for 1 minute in group ii mtad for 5 minutes in group iii and 18% hebp for 5 minutes in group iv. the changes in the apatite / collagen ratio and flexural strength of mineralized dentin were also found to be insignificant with 1.3% naocl for 20 minutes. the process of the smear layer removal is usually more efficient in the coronal and middle third of the canals in the absence of surfactant. a relatively small canal diameter in the apical one - third exposes the dentin to a lesser volume of irrigants and hence compromising the efficiency of smear layer removal. therefore tooth specimens were split longitudinally to ensure complete distribution of irrigants throughout the root canal system. the ability of vicker 's microhardness test to detect surface changes after chemical treatment with edta and citric acid solutions for smear layer removal has been demonstrated. as microhardness of dentin may vary considerably within the same tooth, three indentations were made in the cervical, middle, and apical root canal wall and the means for each sample were calculated. in our study, the microhardness of the root dentin specimens following exposure with chelating agents reduced drastically when compared with the nontreated specimens (control group). the microhardness of root dentin following mtad as a final rinse was significantly less when compared to that of edta. this difference could be attributed to the increased depth of demineralization of mtad (812 m) in relation to edta (24 m). it has also been showed that mtad and 5% citric acid had higher demineralization kinetics compared to 17% edta. this could be attributed to the larger intertubular dentin area available for hybridization when a soft chelating irrigation protocol using hebp is applied. it has been suggested that the total available area of intertubular dentin is an important aspect in the hybridization process. the major retention is provided by micromechanical interactions of the bonding agent with the collagen matrix and the underlying mineralized zone in the intertubular dentin. accordingly, hebp increased the bond strength of resin - based sealers to root canal dentin compared with edta and mtad. moreover, there is evidence that partial depletion of surface ca may significantly alter the bond strength of some adhesive materials. the bonding quality of resin - based obturation material to root dentin has been evaluated following a soft chelating irrigation protocol by performing micropush - out and shear bond strength assessment. however, these bond strength values are useful only in indirect understanding the influence of a different irrigation protocol on dentin demineralization. the important aspect pertaining to the use of a soft chelating solution is the depth of demineralization which decides the sealing ability. under the limitations of the study, hebp as a final rinse appears to be a promising irrigation protocol with less impact on the mineral content of root dentin. however, further studies are required on the depth of demineralization and the sealing ability of resin - based sealers with the radicular dentin to provide more information on the clinical performance of hebp.	context : evaluation of microhardness of root dentin provides indirect information on the change in mineral content of root dentin thereby providing useful information on the bonding quality of resin - based root canal sealers.aim:this study evaluated the effect of 17% edta, mtad, and 18% hebp solutions on the microhardness of human root canal dentin using the vickers microhardness test.materials and methods : forty human single - rooted teeth were decoronated at the cementoenamel junction and sectioned longitudinally into buccal and lingual segments. eighty specimens were divided into four groups (n=20). group i was treated with distilled water (control), groups ii, iii, and iv were treated with 1.3% naocl as a working solution for 20 minutes followed by 17% edta, mtad, and 18% hebp respectively. the surface hardness of the root dentin was determined in each specimen with a vicker 's hardness tester. the values were statistically analyzed using the one - way anova and post hoc tukey multiple comparison tests.results:there was a statistical significant difference among all the groups (one - way anova ; p<0.001). among the experimental groups, hebp showed the highest dentin microhardness (53.74 mpa, p<0.001). least microhardness was found with mtad (42.85 mpa, p<0.001).conclusions : hebp as a final rinse appears to be a promising irrigation protocol with less impact on the mineral content of root dentin.
edentulous patients can have substantial difficulties using their conventional complete dentures due to a lack of retention, support, and stability and the related compromise in chewing ability.1 the treatment options to manage completely edentulous patients are either a complete denture or an implant - supported prosthesis. a mandibular implant overdenture has been shown to improve masticatory function and patient satisfaction in complete denture patients who prefer an implant overdenture option.2 meanwhile, a mandibular implant overdenture has been reported to be simpler and more cost effective than an implant fixed prosthesis.3 a two - implant overdenture in the mandible opposing a maxillary complete denture has even been considered the first treatment choice for completely edentulous patients.4,5 to enhance retention and stability of denture, various overdenture attachments systems can be used for mandibular implant overdentures. the most popular attachment systems are bar, ball, magnet types, and a number of individual mechanical attachments similar in size and function to the ball type. generally, the selection of an attachment system has been dependent on the experience and preference of practitioners. few studies have compared different attachments in a manner useful for clinical decision - making. a few systematic review articles have reported the implant survival rate,6 prosthetic complications7 and patient satisfaction8 of a mandibular overdenture without comparing attachment systems. the review by trakas.9 compared attachment systems based on various implant survival, prosthetic maintenance and patient satisfaction outcomes. however, this review had a lack of explanation about how data was collected according to inclusion and exclusion criteria. therefore, a systematic review on implant overdenture attachment systems is required to focus on various published outcomes. this systematic review aimed to address treatment outcomes depending upon attachment systems for mandibular implant supported overdentures in terms of 1) implant survival rate, 2) prosthetic maintenance and complications, and 3) patient satisfaction. the pico format (population, intervention, comparisons, outcomes)10 was used to define a clinical question with clear inclusion criteria. the specific question and inclusion criteria were clinical studies involving completely edentulous participants (p) requiring mandibular implant overdentures opposing conventional maxillary complete dentures (i). the chosen studies were then further divided according to overdenture attachment systems (primarily bar, ball, or magnet attachments) that were used (c). survival rate of implants, prosthetic maintenance and complications, and patient 's satisfaction were the outcomes (o) evaluated. a systematic literature search was conducted using the combined mesh terms " mandibular prosthesis " or " denture, overlay " and " dental implants " or " dental prosthesis, implant supported " and " clinical study " or " comparative study " or " outcome assessment " or " epidemiologic studies " or " intervention studies " or " patient satisfaction " and limited by " human " and " english " in the data base, medical literature analysis and retrieval system online (medline). the aim was to identify all publications reporting on attachment systems for mandibular supported overdentures up to august 1, 2010. the electronic search by combined mesh term was further augmented by hand search through the following journals : clinical implant dentistry and related research, clinical oral implants research, implant dentistry, international journal of oral and maxillo - facial implants, international journal of oral and maxillo - facial surgery, international journal of periodontics & restorative dentistry, international journal of prosthetics, journal of clinical periodontology, journal of dental research, journal of oral implantology, journal of oral and maxillo - facial surgery, journal of oral rehabilitation, journal of periodontology, journal of prosthetics, journal of prosthetic dentistry, and periodontology 2000. only rct, quasi - randomized and comparative clinical trial studies on mandibular implant overdentures (mio) until august, 2010 were selected if more than one type of overdenture attachment was reported. included studies also reported at least one of the sought outcomes (implant survival rate, prosthetic maintenance and complications, or patient 's satisfaction). to compare the studies between attachments on mio, all implants were conventionally loaded with delayed healing after extraction and before loading. finally, studies published in english were included the duration of follow - up period less than 1 year of function was excluded. both rigid types of overdenture applications, such as milled bar or combinations of attachment types, and cantilevered applications of attachments were excluded., two independent reviewers evaluated the selection of the articles according to the inclusion and exclusion criteria. extracted data were the sample size, patient age, observation period, type of implant, number of implant, type of attachment, treatment outcomes and the outcome of statistical analysis comparing any of the following quantifiable factors : 1) implant survival rate, 2) prosthetic maintenance and complications, 3) patient satisfaction. the implant survival rate denoted the raw percentage of implants still present at follow - up after initial placement of implants. prosthetic maintenance and complications denoted mechanical damage of the implant superstructures. among these, ' matrix or clip loosening ', ' detachment or loss of matrix ' and ' fracture of denture ' were included. prosthetic maintenance and complications were classified to what type and how often maintenance and complications relative to the attachment systems commonly occurred. patient satisfaction concerning chewing ability, phonetics, and social function were evaluated by questionnaire, visual analogue scale (vas), or in some cases by patient preference. data was insufficient to conduct a statistical meta - analysis on those factors, so data were descriptively analyzed. forty six publications were selected by independent screening of the titles and abstracts from the pubmed search. in addition, 3 publications were also included by hand search. based upon reading these 49 full text articles, a total of 24 studies were finally included (fig. 1). then the data on survival rate of implants, prosthetic maintenance and complications, and patient satisfaction were collected (table 2). fourteen studies reported the implant survival rate of implant supported overdentures. among them, four studies11 - 14 presented data on the implant survival rate according to attachment systems (table 3). the 3 year randomized controlled study by davis.13 showed a survival rate of 100% in the ball group and 91.7% in the magnet group. however, a follow - up publication of apparently the same study showed a slightly reduced survival rate 96.2% in the ball group and 91.7% in the magnet group.15 meanwhile, another the 3 year prospective study by davis.12 showed an implant survival rate of 95.8% in the bar, 100% in the ball, and 91.7% in the magnet attachment groups. according to a 19 month randomized controlled study by wismeijer.,14 an overall implant survival rate of 97.5% was found in the bar attachment groups (97.2% in single bar, 97.7% in triple bar) and 100% in the ball attachment group. ten other studies15 - 24 presented data of the implant survival rate without respect to the attachment groups ranging from 93.3% to 100%. thirteen studies presented data on the type of routine prosthetic maintenance and complications (table 3). most common prosthetic maintenance and complications events were replacement of an assay for magnet attachment, activation of a matrix or clip for ball or bar attachment.. showed denture base adjustment as the main complication for bar and ball attachment overdentures.25 fourteen studies compared the rate of prosthetic maintenance and complications according to attachment systems. according to naert.17,20,23 and davis.,12 the most frequent prosthetic maintenance and complications occurred for magnet attachments due to wear and corrosion. there was conflicting information among studies on the rate of prosthetic maintenance and complications for the ball and bar attachments. the study of walton,22 walton.26 and macentee.27 showed ball attachment had more prosthetic maintenance and complications. on the other hand, gotfredsen and holm21 showed that the bar attachments had more prosthetic maintenance and complications than ball attachments during the first year. kleis.24 found more maintenance and complications for locator type attachments than ball attachments. locators are an individual mechanical attachment roughly similar in size and function to a ball attachment. several studies11,13,15,21,28 showed the type and rate of prosthetic maintenance and complications in mandibular overdentures did not differ significantly according to the attachment system (table 3). most of these17,20,24,27 - 31 showed no significant differences in patient complaint, overall satisfaction according to vas and preference where available according to attachment systems. two studies11,32 mentioned that the patient satisfaction of magnet attachment was lower than that of other attachments. the studies by davis.11 showed the magnet group was less stable and chewing ability was less effective according to patients compared to the ball group. the 10-year randomized controlled study by naert.32 found that prosthesis stability and chewing comfort of mandibular overdentures were significantly lower in the magnet group than the bar and ball groups. however, davis.11 reported that both ball and magnet attachments improved patient satisfaction with chewing compared to complete dentures without implants. in addition naert.32 also reported that overall patient satisfaction with overdentures was higher than with complete dentures in bar, ball, and magnet attachment groups. this systematic review addressed implant survival rate, prosthetic maintenance and complications, and patient satisfaction of mandibular implant supported overdentures according to different attachment systems for edentulous patients. this included twenty rcts comparing different attachment systems and four prospective clinical trials which were of a lower level of evidence than rcts. based on the articles in which an observation period ranged from 1 to 10 years, the survival rates of the implants which supported the overdentures in the mandible, ranged from 91.7% to 100%, and the mean implant survival rate was over 98%, both of which supports the presumption that this treatment has a good prognosis in a long - term perspective. this high implant survival rate was coincident with the result of previous reports which showed an implant survival rate of more than 97.2% for mandibular fixed prosthesis and more than 97.1% for mandibular overdentures.33 four studies11 - 14 presenting data on implant survival according to attachment systems, did not specify censored data for a cumulative survival rate making it impossible to calculate an implant survival rate according to different attachment systems through meta - analysis. it has previously been reported that most prosthetic maintenance and complications occur during the first year of loading.27 in the present review pooled evidence was inconclusive in this regard. magnet attachments showed the most common prosthetic maintenance and complications due to wear and corrosion. corrosion of magnetic attachments occurs by breakdown of the encapsulating material and diffusion of moisture and ions through the seal.34 alnico alloys, which have been used in dentistry for many years as a magnet material, were especially easily corroded in saliva rapidly weakening their attractive force.35 however, recently rare - earth alloys, such as neodymium (ndfeb) and new laser - welding technique make it possible to produce a stronger and potentially more durable magnetic force.36,37 in spite of the improved performance of magnet attachments, well - organized long - term randomized controlled clinical trials have not been reported to date. the other most common maintenance requirements related to clip loosening in bar attachments and matrix loosening in ball attachments. however, there were conflicting data on whether bar or ball attachments required more maintenance. a vancouver group found that ball attachments (2.25 mm ball abutment with titanium alloy cap, nobel biocare) had more prosthetic maintenance and complications than bar attachments (round gold bar system, nobel biocare).22,26,27 the c - spring in the ti alloy cap which they used had a tendency to be loose or fractured due to excessive wear of the patrices to the springs encased within the matrix housings.38,39 on the other hands, gotfredsen and holm21 found that the bar attachment had more prosthetic maintenance and complications than the ball attachment (dalla bona spherical gold alloy male and female) at the first year. in addition it was generally more time consuming to replace clips or repair overdentures in the bar group compared to that in the ball group. new developed elliptical gold matrices of ball attachment had large wings to avoid detachment from denture base and made it possible to reduce complication and maintenance rate. some studies27,31 demonstrated that implant supported overdentures improved the participants ' overall satisfaction in comparison with the previous conventional complete dentures. in ten studies, it was mentioned that there were no significant differences in the preference of a specific attachment system in regard to aspects of pain, comfort, appearance, mastication, speech, stability and oral hygiene. two studies11,32 mentioned that patient satisfaction with magnet attachments were less than with bar and ball attachments. when comparing magnet with ball attachments, the magnet group also showed significantly less retention.11 although overall patients satisfaction was similar for the three attachment types, patients in the magnet group were less satisfied with denture stability and chewing ability.32 however, recently developed magnets with improved corrosion resistance and a stronger magnetic force, may still be a useful treatment option for edentulous patient with weak muscle disease such as parkinson 's disease patients, because they not only keep the denture stable, but also need less force to insert and remove the denture. the survival rate of implants appears likely primarily influenced by non - prosthetic factors, such as implant surface roughness, bone quantity and quality, smoking habits, and history of periodontitis.9 conversely a surgeon 's ability to place parallel implants may affects not only the implant survival rate but also, prosthetic maintenance and complications, and patient satisfaction. in one study, walton.40 found that a high complication rate with a ball attachment matrix could be due to the misalignment implants. the number of repairs was significantly higher when the implant analogues were inclined lingually more than 6.0 degrees or facially fewer than 6.5 degrees, which is usually the inclination of the lower incisor teeth.40 better consistency in the horizontal level, angulation, and distance from the midline of the two implants might bring fewer prosthetic complications and better patient satisfaction. the implant survival rate of mandibular implant overdentures seemed to be high regardless attachment systems.	purposethe aim of this systematic review was to address treatment outcome according to attachment systems for mandibular implant overdentures in terms of implant survival rate, prosthetic maintenance and complications, and patient satisfaction.materials and methodsa systematic literature search was conducted using pubmed and hand searching of relevant journals considering inclusion and exclusion criteria. clinical trial studies on mandibular implant overdentures until august, 2010 were selected if more than one type of overdenture attachment was reported. twenty four studies from 1098 studies were finally included and the data on implant survival rate, prosthetic maintenance and complications, patient satisfaction were analyzed relative to attachment systems.resultsfour studies presented implant survival rates (95.8 - 97.5% for bar, 96.2 - 100% for ball, 91.7% for magnet) according to attachment system. ten other studies presented an implant survival rate ranging from 93.3% to 100% without respect to the attachment groups. common prosthetic maintenance and complications were replacement of an assay for magnet attachments, and activation of a matrix or clip for ball or bar attachments. prosthetic maintenance and complications most commonly occurred in the magnet groups. conflicting findings were found on the rate of prosthetic maintenance and complications comparing ball and bar attachments. most studies showed no significant differences in patient satisfaction depending upon attachment systems.conclusionthe implant survival rate of mandibular overdentures seemed to be high regardless attachment systems. the prosthetic maintenance and complications may be influenced by attachment systems. however patient satisfaction may be independent of the attachment system.
asian - lineage highly pathogenic avian influenza (hpai) h5n1 virus has infected poultry in many countries of the world and is thought to be highly endemic in certain delta areas due to frequent viral incursions from large populations of migrating birds [1, 2 ]. as of 2011, the food and animal organization (fao) considered hpai h5n1 to be enzootic among aquatic birds in bangladesh, china, egypt, india, indonesia, and vietnam. the latest report from the world organization for animal health revealed outbreaks of hpai h5n1 that have occurred in bangladesh, bhutan, cambodia, china, india, korea, nepal, and vietnam in 2013 and in china, nepal, and vietnam during early 2014. large populations of aquatic migratory birds frequent delta areas in romania and evidence to date strongly suggests that they have introduced hpai strains into romania 's poultry. the first romanian hpai h5n1 incursions were detected in backyard poultry farms in october 2005 [5, 6 ]. the virus rapidly spread throughout the country until aggressive control measures were taken ; the epizootic halted in july 2006. laboratory studies demonstrated that the epidemic virus was similar to h5n1 viruses previously detected in southeast asia and turkey and that migrating birds were the likely source. a second hpai h5n1 outbreak took place in november 2007 and was contained in murighiol, tulcea county, romania. a third hpai h5n1 detection occurred among poultry in march 2010 in six backyard poultry farms in letea and in one backyard poultry farm in plauru, tulcea county. these two villages are also located in the danube delta. as wild bird die - offs occurred in the danube delta before, or in concert with these outbreaks, the authors of this paper reasoned that surveillance among migrating birds in the danube delta might serve as an early warning system for future avian influenza virus (aiv) incursions. as hpai viruses are thought to originate from low pathogenic avian influenza (lpai) viruses, it is more strategic to conduct surveillance for lpai viruses, particularly among wild birds before viruses enter domestic flocks. as opposed to hpai viruses, where signs of disease occurrence are often more visible due to high mortality rates, lpai often fails in causing overt signs of disease ; thus, surveillance can be challenging particularly among wild birds. traditional wild bird lpai surveillance systems often involve substantial costs for labor, trapping equipment, transportation, and laboratory studies. also, a number of factors can bias results ; for example, certain birds are easier to trap than others and seasonality can influence the composition of the wild bird population ; hence, trapping during certain time periods can influence the overall sample diversity. furthermore, trapping of wild birds can inflict injury. in contrast, monitoring domestic geese and ducks that mix with wild birds in a sentinel system has numerous advantages. surveillance costs are less as these geese and ducks are easier to sample and sampling can be recurrent so as to identify new lpai introductions over time. detections from such domestic geese and duck sentinel systems help to identify viruses that are prone to move into domestic birds before they become enzootic. detections of lpai in the aquatic environment can help public health officials initiate interventions to prevent the spread of lpai to poultry farms. with these advantages in mind, we established sentinel domestic geese and duck surveillance at geographically diverse sites within the danube delta. the danube delta is the second largest river delta in europe, after the volga delta. the total delta area is approximately 4,125 km of which approximately 85% is situated in tulcea county, romania. in 2008, we initiated active sentinel domestic geese (anser cygnoides) and duck (anas platyrhynchos) surveillance in the danube delta ; surveillance was first initiated in one location, cot candura, and then expanded to seven sites by august 2009 (figure 1). in an effort to use representative sites for sentinel bird surveillance, researchers selected sites that were geographically diverse, while also taking into account the geographic density of wild bird populations in different areas of the danube delta. specific locations near wetlands or lakes were targeted where previous aiv had been isolated from dead waterfowl or where primary reservoir species frequently gather. in the surveillance sites, shelters and open fenced enclosures (figures 2 and 3) were constructed to hold 20 domestic birds. the surveillance sites were located at (1) saon (n45 13 0, e28 33 0), (2) ceamurlia de jos (n440449, e2804326), (3) periprava (n4502105, e2903744) replaced in 2011 by letea (n4501005, e2900049), (4) cot candura (n45014 52, e2900444), (5) caraorman (n4500428, e2902455), (6) murighiol (n4500051, e2900737), and (7) enisala (n4405255, e2804859) (figure 1). surveillance was initiated in 2008 and is still in progress at the time of this report (february 2014). each year in early september, 140 young, free ranging domestic waterfowl were purchased and 20 were placed at each surveillance site (~15 geese and 5 ducks). sentinel geese and duck placement during this time period coincides with wild bird migration into the danube delta. both geese and ducks were used to increase the probability of contact with migrating wild geese and duck species. geese were chosen since they tend to lead the flock back to the pens in the evening, after a day spent on the lakes. the numbers of males to females (1 : 4) were chosen in order to avoid fighting among males. beginning in the third year of the surveillance, sentinel birds were banded such that the identity and health of each bird could be tracked over the influenza season. prior to bird banding, in order to ensure the naivety of the flock, any flock that had a positive swab sample was removed and replaced with verified, healthy birds. this was accomplished via testing birds for influenza a virus using real - time rt - pcr (rrt - pcr) and serology to ensure that they were nave to influenza a viruses prior to release at sentinel sites. the sentinel birds were free to move around in local waters and to mix with wild waterfowl. each evening the sentinel birds were encouraged with food to return to the protective enclosure. nearly every month, cloacal and tracheal swabs were collected by a veterinary team, which traveled to the sentinel sites via boat or car. the veterinary team also collected blood samples from a random selection of half of the sentinel birds in order to determine their serologic status. birds that were lost, seropositive, or rrt - pcr positive for influenza were replaced. the birds were tended by local villagers / fishermen who agreed to assist in the surveillance study. actions were taken to ensure animal health and well - being throughout the study via enlisting licensed public health veterinarians from the tulcea county health department to collect sera and swabs from the sentinel birds. additionally, the study was approved by the institutional animal care and use committee (iacuc) at the university of florida. tests were conducted in accordance with the diagnostic manual for avian influenza of the european union and the oie manual of diagnostic tests and vaccines for terrestrial animals (world organization for animal health, 2011). rna was extracted from swabs using viral rna kits (qiagen, alameda, ca, usa) and analyzed by rrt - pcr targeting the influenza a virus matrix gene. all positive samples were sent to the national reference laboratory for avian influenza, bucharest, romania, followed by the weybridge eu / oie / fao reference laboratory for further investigation (virus isolation, sequencing, and genotyping). positive samples were also sent to the global pathogens laboratory at the university of florida for molecular study. the detection of new aiv strains was quickly communicated to the local and central veterinary authority. from september 2009 through march 2013, serological samples were obtained from a random selection of half of the sentinel birds at each location. from september 2009 through september 2011, serological samples were characterized using an elisa blocking kit for aiv type a antibody detection (pourquier institute, now part of idexx laboratories, inc., westbrook, me, usa). all serological testing was conducted in the tulcea county sanitary veterinary and food safety laboratory. h5n2, h7n3, and h9n2 avian influenza antigens (istituto zooprofilattico sperimentale delle venezie, legnaro, italy) were used in the hi assays. the study used these antigens for screening since h5, h7, and h9 influenza strains have caused aiv outbreaks in humans and in birds. cloacal and tracheal swabs collected from each sentinel bird (5,520 each) and sera samples collected from a subset of sentinel birds (2,760) between september 2008 and april 2013 and september 2009 and april 2013, respectively, were screened for evidence of aiv infection. from 2009 through 2013, 47 swabs from 47 birds at 3 distinct locations screened positive for influenza a virus on five different occasions (table 1). h5n3 virus was present in 14 ducks and 20 geese, although no clinical signs were observed in sentinel or wild birds from the sentinel sites. the remaining 13 influenza a rrt - pcr positive samples from enisala and saon were confirmed by the romanian national reference laboratory and the global pathogens laboratory at the university of florida, respectively ; however, no virus was isolated or sequenced from the remaining 13 samples due to low viral load. h5n3, detected in 2009 at cot candura, was the first lpai virus ever isolated in romania. each new aiv strain detected was quickly communicated to both the local and national veterinary authorities. in the subset of previously immunologically nave sentinel birds, serological assays revealed elevated hi titers against h5, h7, and h9 viruses (table 2). we found elevated hi antibodies against the h5n2 antigen within 412 weeks after the first molecular detections of h5n3 virus. approximately 8 weeks after the hi elevations, antibody titers waned to levels < 1 : 16. national aiv surveillance programs within the european union vary in terms of the resources and the types of surveillance methods used. four main surveillance strategies are employed : (1) active surveillance involving testing of live caught, wild birds, (2) active surveillance of hunted birds, (3) active surveillance (periodic sampling) using sentinel, domestic birds kept in high - risk areas, and (4) passive surveillance and laboratory study of dead, wild birds or poultry when unusual mortality is detected. active surveillance involving wild caught birds proves suitable for detecting both hpai and lpai viruses in high - risk areas, while active surveillance of hunted birds has frequently been successful in detecting lpai viruses. use of the hunted bird approach however does not permit sampling for the entire respiratory season. active surveillance of sentinel domestic birds is used infrequently but has been useful in both hpai and lpai virus detections. romania relies upon the passive surveillance strategy among domestic and wild birds and hence, aiv surveillance is sparse and dependent upon the discovery and study of sick or dead wild birds. for comparison purposes, the romanian passive system of surveillance did not uncover any evidence of lpai viruses during the study period. when analyzing financial costs, use of methods 1 and 2 add additional financial constraints due to the requirement of additional traps and net cannons, prolonged time spent in the field, as well as the need for additional boats and gas. these incurred costs would supplement those already incurred by the sentinel surveillance method. among the sentinel geese and ducks included in the current surveillance study, we detected several lpai h5n3 viruses and found serological evidence that lpai h7 and h9 virus strains were also circulating (table 2). the detection of elevated and rapidly reduced serologic titers against lpai virus suggests that these infections may be subclinical, transient, and missed by periodic tracheal and cloacal swabs followed by molecular assays alone. detection of lpai in sentinel bird populations serves as an important precursor for preventing hpai outbreaks. prior to hpai outbreaks, circulating aivs undergo mutations and can transform into lpai, oftentimes without causing overt mortality in flocks until it mutates into a hpai. the public health response to both lpai and hpai remains the same : this includes culling infected birds. in lpai situations, this prevents the spread of lpai and contact with existing viruses in animals, which could result in mutation and hence, potential emergence of hpai. thus, surveillance for lpai in bird populations remains a key component in helping to prevent hpai outbreaks. during the study period, we posit that the sentinel surveillance did not detect h5n1 incursions since the distance between the closest sentinel site and the outbreak area was 15 km. in 2011, in order to enhance study detection, the periprava site was discontinued and a new sentinel site in letea was established. we propose that domestic geese and ducks can be used as an effective aiv sentinel surveillance system, especially in delta areas where the domestic birds can freely mix with large populations of migrating birds. using this method, we were able to detect the presence of h5n3 via molecular surveillance as well as find serological evidence of circulating h7 and h9 viruses. additionally, for our sentinel bird surveillance, we estimated that costs were less expensive than using other aiv surveillance methods such as trapping, hunting, or collecting wild bird carcasses. use of sentinel bird surveillance may also provide a more representative example of circulating viruses due to the species biases that accompanies hunting or sampling from deceased birds. thus, domestic bird surveillance is less invasive, less expensive, and more effective than using other surveillance strategies.	highly pathogenic avian influenza (hpai) h5n1 virus incursions from migrating birds have occurred multiple times in romania since 2005. beginning in september 2008 through april 2013, seasonal sentinel surveillance for avian influenza a viruses (aivs) using domestic geese (anser cygnoides) and ducks (anas platyrhynchos) in the danube delta was established by placing 15 geese and 5 ducks at seven sites. tracheal and cloacal swabs, and sera collections (starting in 2009) were taken monthly. we studied a total of 580 domestic birds and collected 5,520 cloacal and tracheal swabs from each and 2,760 sera samples. all swabs were studied with real - time reverse transcription polymerase chain reaction (rrt - pcr) for evidence of aiv. serological samples were studied with hemagglutination inhibition assays against avian h5, h7, and h9 influenza viruses. from 2009 to 2013, 47 swab specimens from cot candura, enisala, and saon screened positive for aiv ; further subtyping demonstrated that 14 ducks and 20 geese had cloacal evidence of h5n3 carriage. correspondingly, 4 to 12 weeks after these molecular detections, sentinel bird sera revealed elevated hi titers against h5 virus antigens. we posit that domestic bird surveillance is an effective method to conduct aiv surveillance among migrating birds in delta areas.
the splenium is a name of the posterior part of the corpus callosum (cc). in greek this word means a bandage strip tied around an injury or a damaged part of someone 's body. although the association of the name with the respective structure is not immediately clear from the most common sagittal images of the brain (figure 1(a)), which create an illusion of the cc as a structure that can only be artificially partitioned, the basal view of the splenium from gray 's atlas (figure 1(b)) completely justifies its name and shows that the splenium fibers connect occipital and parietal cortices, as well as inferior and medial temporal regions (including the posterior cingulate). according to anatomical tracing studies, the fiber composition of the splenium is heterogeneous : its anterior part includes thin late - myelinating fibers from parietal and medial temporal association areas, while the posterior part contains thick early - myelinating fibers linking primary / secondary visual areas [15 ]. most of the splenium fibers are reciprocal and connect the hemispheres homotopically, while some fibers are heterotopic, although homoareal, and others link different cortical areas [69 ]. the splenium connections are unevenly distributed across cortical areas both in humans and in nonhuman primates [7, 10, 11 ]. they are relatively dense and widely distributed in the extrastriate cortices, whereas in the striate cortex, callosal fibers are located in a narrow strip along the v1/v2 border representing the vertical meridian of the visual field. these basic aspects of the splenium organization are supplemented by recent neuroimaging findings. in vivo tracing diffusion tensor imaging (dti)studies describe a more detailed spatial organization of fibers within the human splenium [3, 5, 12 ]. according to these reports, the middle part of the splenium carries fibers connecting dorsal visual and association parietal areas, the superior - posterior part contains fibers linking posterior cingulate and retrosplenial cortices, and the inferior - anterior portion incorporates fibers from ventral visual areas. the neuroimaging data also question some features of splenial connectivity that had been established in animal and postmortem human studies. these include the notion that the primary visual cortex is mostly devoid of callosal connections : significant interindividual variability of connections between the striate cortices (with one - third of participants exhibiting direct interhemispheric projections in this area) has been found by putnam and colleagues. another example is the assumed symmetry of callosal connections : greater interhemispheric connectivity from the right hemisphere to the left one has been found in the extrastriate cortices. the diverse structural properties of the splenial fibers across brain areas suggest that they are involved in a variety of functions, while their considerable variation between subjects implies a contribution of the splenium to plastic changes in the course of human development. considering that the splenium is well defined anatomically and is easily accessible in animal models and in noninvasive human neuroimaging, this structure is of significant interest for basic neuroscience and clinical applications. this paper addresses the structural and functional development of the splenium based on the recent literature with an emphasis on the heterogeneity of its functions and mechanisms at different levels of the visual hierarchy. the development of the human cc was studied using both postmortem and in vivo mri - based techniques. a direct comparison of these methods in showed that, at least in terms of the cc area and shape, they provide consistent information. moreover, the two methods are complementary : while postmortem material allows a more precise identification of the cc borders, the in vivo imaging techniques are easily compatible with (neuro)psychological characteristics and permit a longitudinal study design, thus providing an inestimable advantage for the research into human development. the developing splenial fibers travel together with the hippocampal commissure, whereas the frontal fibers of the cc cross the midline separately from the anterior and hippocampal commissures [14, 15 ]. this developmental pattern as well as the partial cc ageneses and regional malformations suggests that the splenium can be considered a neocortical component of the hippocampal commissure, which carries fibers connecting the hippocampi together with those linking the posterior parietal, medial temporal, and medial occipital cortices of the two hemispheres [4, 5, 16 ]. anatomical reports show that the prenatal development of the human cc is characterized by a posterior - to - anterior gradient, with the prominent splenium emerging only in the 18th or 19th week of gestation [14, 1719 ]. after birth, the slower growth of the splenium compared to the genu is replaced by the opposite trend, with higher growth rates of the splenium than those of the genu [18, 20, 21 ]. similar nonuniform postnatal growth of the cc compartments was demonstrated with mri in baboons. in particular, by postnatal week 32, their midsagittal splenium area achieves 55% of the average adult size, whereas the genu and the anterior midbody attain only about 50%. as can be extrapolated from the monkey data, the total number of callosal fibers continues to increase until birth. nevertheless, at the end of gestation and during the first months after birth, the sagittal area of the cc reduces both in monkeys and in humans [13, 18 ]. since this coincides with the time of massive axonal elimination, the latter is suggested to be the main cause of cc reduction [1, 13 ]. further postnatal changes in the callosal sagittal area are interpreted as an interplay between continuing myelination, pruning, and the redirection of fibers. structural mri - based studies report the prolonged growth of the total cc area and splenium (among other cc subdivisions) from birth adulthood in nonhuman primates, including chimpanzees, bonnet macaques, and capuchin monkeys. since the end of the 1990s, several laboratories have applied mesh - based computational mri techniques to the analysis of the sagittal callosal area in children and adolescents [2730 ]. in this method, aimed toward longitudinal research, four - dimensional quantitative maps of growth patterns are reconstructed by computing a three - dimensional elastic deformation field, which rearranges the shape of the cc in the earlier scan into the shape in the later scan. these groups reported greater increase in the splenium than in the anterior cc regions in children and adolescents aged 418 years [27, 28 ], 615 years, and 722 years. alternative imaging methods provide converging results. to assess the cc development in healthy children of 315 years, kim and collaborators used multiecho t2 relaxometry based on the longer t2 relaxation times of water molecules within the axon and extracellular space unbound to macromolecules. during development, the axonal diameters in the splenium grow in parallel with the reduction of their density [1, 2 ]. therefore, the continuing increase of axonal size should correlate with the increase of t2 relaxation times. the measurements in genu and splenium revealed that the relaxation times significantly correlate with age only in the splenium, suggesting its prominent growth in the late childhood and adolescence. dti studies, although inconsistent about the anterior - to - posterior gradient of cc maturation, nevertheless show that the splenium develops gradually through adolescence [32, 33 ]. recently, in a large computational mesh - modeling mri study of 190 children and adolescents aged 518 years, luders and coauthors confirmed that the callosal area increases with age and revealed the age-, sex-, and region - specific rates of growth. in particular, in a result qualitatively similar to previous neuroimaging studies (e.g.,), the younger children showed the most pronounced growth in the anterior cc, while the splenium began to overtake the anterior parts of the cc starting from the age of 9 - 10 years in girls and of 11 - 12 years in boys. a synthesis of the postmortem anatomical and in vivo mri data suggests that periods of accelerated growth of the genu alternate with periods when the splenium picks up speed. such shifts occur around birth time (the splenium speeds up compared to the genu), in early childhood (the genu begins to outrun the splenium), and in middle childhood (the splenium once more takes the lead in growth). the mechanisms behind these changes seem to be age - specific. in the context of the first postnatal spurt of splenium growth, the data of chalupa and colleagues from their tract tracing studies in rhesus monkeys are of interest [35, 36 ]. they showed that, in late fetal development, the elimination of cc axons in the visual areas is less pronounced than that in the sensorimotor cortex. if the lower proportion of axonal retraction in the posterior areas is also characteristic for humans, this phenomenon could explain the higher splenium growth in the early postnatal period. the last period, characterized by an anterior - to - posterior gradient of the cc development, is in humans likely related to the protracted myelination of the splenium. myelination starts at 3 - 4 months after birth and continues into adulthood [21, 37 ]. in adults only 16% of the cc fibers remain unmyelinated. to analyze the link between cc area and myelination, fornari and colleagues (2007) used magnetization transfer imaging (mti) in children of 7 to 13 years of age mti estimates the efficiency of magnetization exchange in biological tissues between a pool of free protons in intra- and extracellular water and a pool of protons bound to macromolecules (for review, see). as shown in an in vitro experiment, the contribution of the myelin sheets to the mt contrast is nine times larger than the contributions of intra / extracellular water. a postmortem study of the multiple sclerosis brain demonstrated highly significant correlations between morphometric and mti measures of myelin content. since the most important contributors to the magnetization transfer effect are the extent, concentration, and integrity of myelin membranes, mti permits an accurate evaluation of changes in myelination in children, aging people, and populations with myelination abnormalities [4244 ]. consistently with previously reviewed reports, in a group of healthy children, the most robust direct correlation between the mti index of myelination and a child 's age has been shown by fornari and colleagues for the area of the splenium. the size of the cc in animals and humans increases with learning or training [4648 ]. it is likely that nonmonotonic growth of the splenium probably reflects its plastic tuning to the heterochronically maturing visual functions in childhood and adolescence. more specifically, the accelerated growth of the splenium in the first postnatal weeks / months coincides with the fast development of sensitivity to orientation, direction of motion, and disparity. another period of relatively high growth rates that starts in middle childhood accompanies improvement of the functions associated with spatial integration (see sections 5 and 6). before proceeding any further, it should be noted that the tasks performed by the cc within the framework of interhemispheric integration as well as the physiological mechanisms implementing these tasks remain to be studied further. at a functional level specifically, excitation refers to the tendency of one site to activate the symmetric location in the other hemisphere, while inhibition refers to the opposite effect. since cortico - cortical long - distance connections are mainly excitatory, the interhemispherically induced suppression of a response necessarily includes local inhibitory interneurons. therefore, the interhemispheric effects resulting from a summation of multiple diversified events at a neuronal / synaptic level require a very cautious interpretation at a network level, especially in noninvasive human research. in a decades ' long debate about the excitatory, inhibitory, or mixed nature of interhemispheric effects of the cc, the excitatory function seemed to get the majority of support. to this end, in 2005 bloom and hynd wrote the following : the available research, no matter how limited, primarily supports the notion that the corpus callosum serves a predominantly excitatory function. recent research has revealed a more complicated picture, in which the cc functions and mechanisms not only change along its anterior - to - posterior axis depending on the cortical area of origin / destination, but also vary within a singular area. the application of sophisticated experimental methods by the group of innocenti substantially enriched our understanding of the repertoire of splenial functions [5153 ]. by combining local reversible thermal inactivation in one hemisphere with optical imaging of intrinsic signals or electrophysiological recordings in the other hemisphere, these authors showed that the splenium fibers connecting visual areas 17/18 of the ferret modulate the driving thalamocortical input by means of inhibitory effects at short latencies and of excitatory effects at longer latencies. the latencies of inhibitory effects are compatible with higher conduction velocities of thick early - myelinating fibers, whereas the excitation is apparently driven by thinner axons with lower conduction velocities. their interplay with axonal geometry can change the synchronization of stimulus - driven local field potential. considering that synchronization serves to recruit neuronal populations to common activity [54, 55 ], such effects of the splenium might not be limited to the area of their destination a narrow strip at the 17/18 border but affect the functionality of a significant part of the area (see section 4). not much is known about the splenium functions in the extrastriate areas. however, comparing the splenium connections between the striate cortex, where they are thick (heavily myelinated), sparse, and concentrated along the border, and extrastriate cortices, where interhemispheric connections are thin, dense, and widely distributed, it is difficult to escape the conclusion that the functions of splenium fibers vary across visual areas. the conventional assumption is that the functional role of the splenium in a particular extrastriate area is defined by its specialization. for instance, olavarria and abel (1996) reported that callosal cells are assembled in regular protrusions into v2 of the monkey. these protrusions are distributed along the v1/v2 border at the intervals corresponding to the arrangement of thick and thin stripes. given that the stripes are specific to the organization of the v2 and correspond to the functional streams engaged in the processing of orientation and direction [57, 58 ], this structural evidence suggests some area - specific functions of the splenium beside establishing continuity across the visual field. one such function is figure - ground segregation, which refers to the ability of the visual system to segment images of the external world into objects and background. to this end, a mechanism has been proposed for the isolation of a figure from the background through the detection of its borders [59, 60 ]. it relies on inhibition among neurons with neighboring receptive fields tuned to the same feature. as a result, within a homogenous region, similarly tuned neurons mutually inhibit their activity, whereas at borders, such neurons are less inhibited due to regional heterogeneity. the receptive fields that implement this border - detecting mechanism are characterized by center - surround antagonism, that is, they have a receptive field center that is excited by a particular image feature and surround that is inhibited by the same feature. desimone and colleagues (1993) found that, in v4 of the monkey, the classical receptive fields (excitatory centers) are mostly limited to the contralateral visual field, while their suppressive surround might extend into the ipsilateral visual field up to 16 from the vertical meridian. in these experiments, dissection of the cc abolished much of the inhibition from the ipsilateral part of the surround, demonstrating its involvement in the core mechanisms of figure - ground segregation implemented in the v4. callosal connections are structurally, functionally, and developmentally similar to long - range intrahemispheric corticocortical connections [11, 62 ]. with the exception of the cc agenesis, there are no pathologies in which they are specifically involved. nonetheless, since intrahemispheric mechanisms within a single level of the visual hierarchy are realized via lateral intracortical horizontal fibers and short - range association fibers (u - fibers), the number of which is orders of magnitude greater than that of splenial fibers executing the same functions interhemispherically, one may speculate that the cc should have some adaptations compensating for its limited number of connections, and, therefore, interhemispheric networks should differ from the respective intrahemispheric networks. finally, the functions of the splenium may encompass communication among different levels of hierarchy. the inactivation of higher - order visual areas weakens the suppressive surround of neurons in lower - order areas, suggesting a role for top - down connections in this mechanism. the heterotopic splenial fibers [6, 9 ], especially those between association and primary visual areas, could mediate such feedback. as stated in the previous section, the interhemispheric synchronization of network activity can be involved in a variety of functions. the impact of the splenium in synchronizing the electrical activity between the hemispheres is supported by animal models and noninvasive human studies [6668 ]. kiper and colleagues examined interhemispheric synchronization in ferrets, in which, like in other mammals, the splenium fibers located along the v1/v2 border selectively connect neurons with the receptive fields having similar orientation preferences and placed near the vertical meridian of the visual field. for this structure of connectivity, the binding - through - synchronization hypothesis predicts an increase of interhemispheric synchronization in response to the bilateral collinear stimuli near the vertical meridian compared to the noncollinear stimuli. indeed, by contrasting differently oriented and located bilateral gratings before and after the section of the cc, the authors have shown that interhemispheric synchronization of epidural eeg increases in response to the isooriented gratings near the vertical meridian compared to the orthogonally oriented gratings, whereas callosotomy abolishes the effects of stimulus configuration. the same set of stimuli used in a noninvasive human study induces similar changes of interhemispheric synchronization in surface eeg, whereas the reduction of interhemispheric synchronization in the absence of the splenium in humans was shown in acallosal and split - brain individuals [6971 ]. it is safe to assume that even less dramatic changes in interhemispheric connectivity that occur in human postnatal development, for example, myelination of the splenium fibers, would also affect the interhemispheric synchronization of neural networks. oscillations provide a temporal frame for neuronal firing by means of synchronization of pre- and postsynaptic potentials [54, 55 ]. in the context of this discussion, oscillations in the eeg alpha band are of special interest. first, the alpha rhythm is the most prominent oscillatory activity that can easily be recorded by means of noninvasive surface eeg within a wide range of ages. second, it is generated by visual cortical circuits interacting with thalamocortical loops [72, 73 ] and has a relatively narrow frequency range between 8 and 12 hz. third, the alpha rhythm is characterized by a protracted course of development in children [74, 75 ] comparable with that of the cc. in 7- to 12-month - old infants, the activity that can be recorded over the occipital - parietal cortex within the frequency range 59 hz has the properties of alpha rhythm and is considered its precursor. alpha peak frequency logarithmically increases with age, providing the best estimate of maturation among the eeg parameters [74, 77 ]. in parallel, the spatial organization of alpha rhythm develops. in a high - density eeg study, srinivasan showed that, at the peak alpha frequency, the 6- to 11-year - old children demonstrate lower long - range synchronization between the anterior and posterior laplacian eeg signals in comparison to the young adults. thus, the typical feature of adult eeg high coherence between distant eeg signals in the alpha band is still absent in middle childhood. farber and knyazeva demonstrated an immaturity of long - range interactions for the case of interhemispheric connections. they analyzed the development of the interhemispheric coherence of alpha rhythm in 320 healthy children and adolescents aged 217 years. interhemispheric synchronization rapidly increased with age in early childhood (between 2 and 7 years), whereas in middle childhood and adolescence the increase rate progressively slowed down. the striking similarity between the trajectories of the alpha frequency and synchronization development and that of the white matter maturation [80, 81 ] suggest that the processes are closely related. theoretically, the frequency of coupled oscillators depends on connection strength and time delays between them [82, 83 ]. to this end, combined eeg - dti studies have found that, in adults, individual alpha frequency is linked to the structural properties of corticocortical and thalamocortical connections [84, 85 ]. the strongest correlation between an individual alpha frequency and fractional anisotropy, which reflects the joint contribution of fiber density and myelination, was found for the splenium. to summarize, although studies directly analyzing links between interhemispheric alpha synchronization and structural maturation of the splenium remain to be performed, the development of alpha rhythm in children seems to be closely linked to the maturation of the cc. moreover, the increase of interhemispheric alpha synchronization with age implies that the long - range interhemispheric interactions become an increasingly important regulator of visual functions. on the other hand, the relatively low level of functional cooperation between the hemispheres in the immature brain suggests the predominance of local intrahemispheric mechanisms underlying vision in young children. the extended structural and functional maturation of the splenium inspires me to consider the perceptual functions with protracted developmental trajectories, although it is not clear a priori whether such a gradual development depends on the inter- or the intrahemispheric mechanisms. most visual functions achieve adult levels within the first few months (e.g., contrast, motion, and orientation sensitivity) or the first few years (grating acuity and binocularity) of postnatal life. si refers to the processes that assemble local information across the visual field to implement a global representation of spatially extended objects in the brain. behavioral experiments consistently show that the basic mechanisms of spatio - temporal integration are available in the first months or even weeks of human life. infants treat the coherently moving parts of a display as belonging to the same object, differentiate upright from inverted biomotion displays, and integrate component motions into coherent pattern motion over large regions of space. yet the development of perceptual organization abilities takes a long trajectory through childhood and adolescence. thus, sensitivity to global form in glass patterns is adult - like only at 9 years of age. in a contour - detection task experiments with complex visual displays like hierarchical shapes and compound letters reveal that even in adolescence visual perception is biased toward representing local elements [92, 93 ]. furthermore, the organization principles, working in early life, improve with age and so does the ability to use collinearity for the integration of spatially distant line segments, which increases at least until 10 years of age. the neural basis of this protracted course of functional maturation is discussed in the following sections. in adults, cognitive performance correlates with the size of the callosal area and cognitive impairment with the demyelination of the splenium. apparently, myelination facilitates interhemispheric interaction by enhancing the coordination of interhemispheric input, which leads to a more efficient recruitment of the target neural population to common activity [98, 99 ]. if this is the case for the developing splenium in children, a correlation between its myelination and the activation of respective networks is to be expected. to test this in, we used a simple interhemispheric paradigm that requires only passive viewing of visual stimuli, verified earlier by us [67, 68 ] and by others in animal and human experiments. being minimally demanding, this task is applicable to groups of various age and health across the lifespan. specifically, subjects fixated on large high - contrast bilateral gratings including horizontal collinear coherently drifting gratings (stimulus cg) and noncollinear orthogonally oriented and drifting gratings (stimulus ng). of the two stimuli, only cg is fusible into a single image, while the ng is expected to induce a segmentation of the image between the right and left visual fields. functional magnetic resonance imaging (fmri) ng manifests highly reproducible activations (figure 2(a)) in the ventral - stream v3v / v4 areas [38, 98100 ]. in adults, these activations correlate with interhemispheric eeg synchronization [98, 99 ] and, therefore, can be considered a neural substrate of interhemispheric integration. first, we investigated whether the activation of these integration - specific areas is affected by splenium maturation. children of 713 years of age were scanned while they viewed the gratings. by implementing fmri and mti protocols in the same scanning session, we could estimate both functional and structural aspects of interhemispheric interaction. the activation associated with the cg > ng contrast was limited in children to the v3v part of the adults ' activation (figure 2(a)). this modulation of bold signal manifested by the networks presumably involved in the interhemispheric integration was correlated with the myelination of the splenial system of fibers. apparently, by changing the speed of transmission and the effective geometry of the cc fibers, myelination allows well - synchronized input to the opposite hemisphere, resulting in enhanced activation [9799 ]. in order to test other aspects of the development of interhemispheric interaction via the posterior callosal connections, we reanalyzed the fmri time series from this experiment with dynamic causal modeling (dcm), a method for evaluating effective connectivity, that is, the influence that one local neural system (source) exerts on another (target) [101, 102 ]. dcm differentiates positive coupling (excitation) that results in correlated increased activity between source and target regions from negative coupling (inhibition) that leads to a relative decrease in the target activation compared to the source. although the term inhibition is conventionally used in the dcm literature, its true meaning in this context is the suppression of activation response due to a variety of processes at a cellular level, including inhibition per se. the visual interhemispheric integration task described in the previous section is wellsuited for modeling effective connectivity since its neural substrate is a relatively restricted network, the nodes of which can be clearly identified, and the effects of the stimuli can be described in terms of factorial design. the latter allows one to model main factors as driving context - independent effects (in this case, stimulation with any grating stimulus compared to gray - screen (background)) and interactions, resulting from experimental manipulations, as modulatory (context - dependent) effects (here it is the effect of interhemispheric integration in response to cg compared to any grating stimulus). specifically, dcm allows an analysis of such an interaction in terms of modulatory connections, that is, by defining their architecture and the character of effect. we used two pairs of interhemispherically symmetric regions for the model : one pair in the primary visual cortex, where the driving input arrived, and another pair in the extrastriate visual cortex, where the response varied depending on the stimulus (figure 2(b)). the nodes were limited to the 4 mm radius spheres centered on the local maxima within these predefined territories. according to the probabilistic cytoarchitectonic atlas, one pair of nodes in each hemisphere occupied the territory on both sides of the v1/v2 border, while another one was located at the v3v / v4 border (figure 2(b)). in this model, the driving signals induced by visual stimulation arrive in the left and right primary visual cortices (v1l and v1r nodes of the model) and spread within the model between the v1l, v1r, v3l, and v3r nodes by means of reciprocal intrahemispheric, interhemispheric, homotopic, and heterotopic connections. on the assumption that each of these intrinsic connections can be modulated, the structure of modulatory connections reproduced the architecture of intrinsic connections. we used this model for comparison of children (the same group of 713 years as in) and adults that viewed the same gratings. the intrinsic (driving) effective connections (all excitatory) between the visual areas were significant in both groups and did not differ between children and adults, in keeping with a large body of evidence that basic visual networks integrated via long - distance reciprocal pathways are established early in the course of development. the modulation induced by the cg stimulus was conveyed by lateral and feedback connections, all of which were inhibitory. the strongest modulation manifested by strengthened mutual suppression was found between the primary visual areas in both subjects ' groups. a recent noninvasive human study provided converging evidence of transsplenial inhibition of neural responses. in these experiments of bocci and colleagues, the splenium input was manipulated with transcranial magnetic stimulation (tms), the effects of which were assessed with visual evoked potentials (veps) in response to the whole - field horizontal gratings. similar to the bilateral collinear gratings (stimulus cg) used by fornari with colleagues and knyazeva with colleagues [98, 99 ], this stimulus was interhemispherically identical. the unilateral tms of v1 increased the amplitudes of vep components generated in the striate and extrastriate areas of the contralateral hemisphere in response to the stimuli of medium - to - high contrast. considering that tms imposes inhibitory effect, that is, excludes callosal input, the increase of vep can be attributed to disinhibition. both our dcm results and the reviewed human findings are remarkably similar to the evidence from the already cited experimental study, in which the local cooling of area 17/18 in one hemisphere of the ferret reversibly eliminated callosal input to the symmetric area in the intact hemisphere. the effect of this manipulation consisted largely in the decrease of local field potential (lfp) in response to whole - field orthogonally oriented gratings and in the increase of lfp to isooriented gratings. a plausible interpretation encompassing all these findings is that orthogonally oriented gratings essentially represent two different stimuli, which activate the networks with different orientation and/or direction preferences through the thalamocortical and callosal pathways, while isooriented gratings activate the neurons similarly tuned in both hemispheres, thus extending their network over the two hemispheres. as a result, the orthogonally oriented gratings induce segmentation, while collinear gratings bring on integration between the visual hemifields. the basis of integration for large high - contrast gratings at the v1 level is no change in stimulus properties, that is, no borders. such stimuli are known to induce especially strong surround suppression, leading to a sparse population response [105107 ]. if this account holds true, the net result of converging thalamocortical and callosal inputs induced by a strong visual stimulus extending into both hemifields would be a suppression of the v1 response. therefore, the splenium can be involved in the adaptive process of neuronal response sparsification through suppressive mechanisms activated by redundant visual information. in a natural vision, when the entire retina is simultaneously stimulated, such a mechanism is essential for the efficient processing of moving images [105, 107 ]. moreover, it is likely that inhibition is more important for the processing of visual information in an awake animal than anesthetized animal models suggest [97, 108 ]. in addition to the lateral effective connections between the primary visual areas, effective feedback connections from the extrastriate v3v / v4 nodes convey inhibitory modulation induced by the isooriented stimulus in both groups (figure 2(b)). this is consistent with animal models, where the large spatial extent of surround suppression together with its short latent period suggests the involvement of feedback signals from the extrastriate cortex transmitted by fast myelinated fibers [65, 109, 110 ]. in our dcm model, since there are no assumptions about the number of synapses implementing a connection in dcm, it remains to be demonstrated whether the heterotopic callosal connections shown in animals and humans [6, 9 ] are implicated. the experiments of ban and colleagues (2006) suggest such a possibility. they have found that the bold response to the arcs presented symmetrically in the lower visual field quadrants is significantly lower compared to the response to the same arcs located asymmetrically (diagonally). in the absence of direct interhemispheric v1 connections between the low and high visual quadrants, this change of v1 activation is likely due to the top - down influences from the extrastriate areas. as demonstrated by lassonde and colleagues, children younger than 10 years of age show remarkably small deficits after callosotomy [112, 113 ]. although visual functions largely escaped examination, the set of various tasks including intermanual comparisons and naming of shapes and objects, as well as localization of touch, leave few doubts about close - to - normal performance even at their first neuropsychological assessment after the surgery and about the remarkably fast compensation of residual deficits. in contrast, children older than 10 years of age and adolescents show a full - blown split - brain syndrome. similar to adult split - brain patients, these children demonstrate a breakdown in interhemispheric communication, including the loss of intermanual transfer and integration of tactile information and difficulty naming objects held in the nondominant hand. cumulatively, the data of lassonde and colleagues suggest that some functions of the immature cc can be shared with alternative pathways, thus accounting for minimal postoperative deficits in young children. however, continuing development leads to the cortex rewiring through elimination of overproduced connections. ptito and lepore obtained direct evidence in favor of this view in experiments on cats with the posterior cc sectioned either before this structure reached maturity or after its maturation. to disconnect each eye from the contralateral hemisphere, all these animals had the optic chiasma sectioned in adulthood and then were monocularly trained on a visual discrimination task. only cats with early callosal transsection showed a capacity for the interhemispheric transfer of pattern discriminations. thus, in parallel with cc maturation, other connections become inaccessible, limiting plastic postoperative changes with age. yet the majority of functions are probably not strongly reorganized in the ontogenesis but gradually improve with cc development. our dcm - based findings shed new light on the nature of callosal functions with a protracted course of development. specifically, in contrast to excitatory connections that show no signs of changes between children and adults, interhemispheric modulatory connections (both lateral and descending) strengthen with age (figure 2(b)). the increase of interhemispheric suppression in the primary visual cortex of adults compared to that in children was the strongest effect observed. interestingly, although the strength of inhibitory connections correlated with age, it did not correlate with the mti indices of splenium myelination. this is in line with previously reviewed experimental evidence for the involvement of fast, that is, thick and early - myelinating, fibers in interhemispheric inhibitory effects. alternatively, since the cc neurons are generally excitatory but may target local inhibitory neurons, interhemispheric inhibition can be implemented via polysynaptic pathways with long - distance excitatory and local inhibitory components. then the correlation with age in the absence of a correlation with myelination apparently reflects the development of local connections. indeed, the local gabaergic mechanisms of the primary visual cortex analyzed postmortem manifest the extended development, which continues well into the second and third decades of life. it should be noted that from an ontogenetic perspective, the prolonged formation of transsplenial modulation between the primary visual areas challenges the conventional view that posits the prior maturation of the early visual cortex as a precondition for the later development of higher - order ventral stream regions. the modulatory effects transmitted in our model via interhemispheric top - down effective connections are also weaker in children than those in adults. considering the ages of the children in this group (713 years), the dcm evidence points to the slow formation of feedback connections, which might be a part of the neural network that enables collinearity detection. the available data on their structural maturation are limited to the connections between v2 and v1 [117, 118 ]. according to these postmortem anatomical studies, the upper layers of v1, which receive the feedback and callosal connections, seem to be immature at 5 years of age. the reviewed literature together with structural and functional mri, eeg, and dcm evidence obtained by the author 's group points to a slow structural development of the splenium in human ontogenesis and to a gradual formation of transsplenial effective connections conveying inhibitory influences. an important outcome of the protracted maturation of the mechanisms with splenial involvement is a greater efficiency of neuronal networks. reshaping interactions between interhemispherically distributed networks under various perceptual contexts allows sparse responses to superfluous information from the visual environment. another aspect of these processes is a reduction of well - known metabolic and structural redundancy in children 's brains [23, 119 ].	the splenium of the corpus callosum connects the posterior cortices with fibers varying in size from thin late - myelinating axons in the anterior part, predominantly connecting parietal and temporal areas, to thick early - myelinating fibers in the posterior part, linking primary and secondary visual areas. in the adult human brain, the function of the splenium in a given area is defined by the specialization of the area and implemented via excitation and/or suppression of the contralateral homotopic and heterotopic areas at the same or different level of visual hierarchy. these mechanisms are facilitated by interhemispheric synchronization of oscillatory activity, also supported by the splenium. in postnatal ontogenesis, structural mri reveals a protracted formation of the splenium during the first two decades of human life. in doing so, the slow myelination of the splenium correlates with the formation of interhemispheric excitatory influences in the extrastriate areas and the eeg synchronization, while the gradual increase of inhibitory effects in the striate cortex is linked to the local inhibitory circuitry. reshaping interactions between interhemispherically distributed networks under various perceptual contexts allows sparsification of responses to superfluous information from the visual environment, leading to a reduction of metabolic and structural redundancy in a child 's brain.
because a cervical pedicle screw has more rigid fixation than other posterior fixation techniques, including posterior wiring, lateral mass screw, or facet screw fixation. cervical pedicle screw not only allow for shorter instrumentation with sagittal correction11 - 13,19,31,34), but also are valuable for simultaneous posterior decompression and reconstruction1 - 3). then, the cervical pedicle screw insertion has become popular in a wide range of cervical spine related disorder (traumatic, degenerative, inflammatory, and neoplastic conditions). however, cervical pedicle screw insertion is technically demanding because of the anatomical variations in cervical pedicle size, lack of anatomical landmarks, small pedicle diameter, and the large convergence angle of cervical pedicles14,22). the potential risk of neurovascular injury remain the issue preventing wide acceptance17,24). therefore, accurate and safe cervical pedicle screw insertion techniques are necessary. following the first description of cervical pedicle screw insertion by abumi.1), different surgical techniques have been developed and evaluated, including the techniques relying on anatomical landmarks for cervical pedicle screw insertion, techniques with direct exposure of the pedicle by laminoforaminotomy to palpate the medial and superior pedicle walls, by the funnel technique, and the computer - assisted navigation system5 - 8,15,16,18,21,23,25,33,37). the accuracy of cervical pedicle screw insertion varied significantly in literature, ranging from 16.8 to 97%16,21,23,29,32). the navigation system has been shown to improve screw accuracy significantly, but has limited application due to its high cost and lengthy registration procedure. furthermore, surgical skills and experience are still needed and the surgeon should although a number of studies have evaluated the morphometry of cervical pedicles to support accurate placement of pedicle screws, the results are inconclusive14,27). therefore, several techniques and computer - assisted navigation systems for cervical pedicle screw insertion have been advocated. the objective of this study was to present the cervical pedicle screw insertion technique with direct exposure of the pedicle by laminoforaminotomy, and evaluate the accuracy of pedicle screw placement and validity of pedicle screw fixation in patients. but, the authors did not evaluate the accuracy and safety of pedicle screw placement in cadavers before applying this technique to patients. a total of 12 consecutive cases in which 104 cervical pedicle screws had been inserted from c3 to c7 were reviewed for this study. the ratio of men to women was 3 : 1 and the mean age was 54 years (range, 31 - 75 y). preoperative diagnosis was myelopathy due to cervical spondylosis in 6 cases, ossification of the posterior longitudinal ligament in 3 cases, traumatic lesion in 3 cases. our institutional ethics committee has approved the human protocol for this investigation that all investigations were conducted in conformity with ethical principles of research, and that informed consent was obtained. to evaluate the anomaly and dominant side of vertebral artery, we checked vascular - enhanced computed tomography (ct) scans in all patients. on preoperative ct scans, the convergence angle and the minimal diameter of the pedicle on the axial plane were measured for each cervical vertebra. the length of the screw was decided to reach the anterior one - third of the vertebral body on the preoperative ct images. those with a pedicle diameter of less than 3.5 mm or a pedicle convergence angle of over 60 degrees were excluded from cervical pedicle screw insertion. the authors considered every pedicle to be built as a funnel with a wide posterior base, which narrows towards the isthmus. a distinct characteristic of the cervical pedicles is that the lateral pedicle wall is always the thinnest cortex14,27). therefore, the medial pedicle cortex was used as a safe guide into the pedicle isthmus, then through it and then into the vertebral body. the arch of the medial pedicle wall, under which advancement into the pedicle proceeded, was always identified. patients were placed in the prone position with the head fixed using a mayfield clamp. a standard midline incision was made and paravertebral muscles were dissected and retracted laterally to expose facet joints. under the lateral fluoroscopic guide and the basis of the lateral mass depth, and the convergence angle of the pedicle obtained from a preoperative computed tomography (ct) scans, 3 mm cutting burr was used to remove the outer cortex of the lateral mass over the pedicle entrance in the slightly lateral to the center of the facet, close to posterior margin of superior articular surface, the appropriate convergence angle to visualize the entrance of the pedicle (fig. the pedicle screw insertion point is indicated by a cancellous bone at the pedicle entrance, which is commonly observed to be reddish because of a bloody cancellous bone. a laminoforaminotomy was performed to provide visual and tactile cues regarding the orientation of the pedicle medial and superior walls. the ligamentum flavum at each level was gently dissected free from the inferior aspect of the superior laminar arch and from the superior aspect of the inferior laminar arch using a small curved curette. thereafter, the inferior aspect of the superior lamina and the superior aspect of the inferior lamina were removed in varying amounts using a 3 mm cutting burr followed by 1 mm and 2 mm kerrison punches (fig. the ball - tip hook then could be used to identify the medial and superior walls of the pedicle before the insertion of each screw (fig. the direction in the sagittal plane of the pedicle probe and screw was intraoperatively determined using lateral fluoroscopic imaging. the pedicle was probed as close to the medial wall as possible by gentle manual pressure using a 15-degree, 2 mm - diameter curved gear shift probe (fig. after probing about 2 cm depth, lack of pedicle perforation was confirmed using a ball - tip probe. if perforation was detected within the pedicle, the trajectory was changed or the segment was skipped. then sequential drilling, tapping, and screwing were performed. for preventing iatrogenic neural tissue injury during rod connection procedure, rods were connected to the screws by appropriately sized slotted connectors before posterior decompression. using preoperative and postoperative vascular enhanced ct axial images, the diameter and convergence angle of the each pedicle and the screws were measured and the difference between pedicles and screws in diameter and convergence angle were analyzed (fig. the degree of perforation was classified as grade 0 if the screw was located within the pedicle ; grade 1, if perforation was made by the screw by less than 25% of the screw diameter ; grade 2, if perforation was made by 25% to 50% of the screw diameter ; and grade 3, if perforation was made by over 50% of the screw diameter (fig. 4). grade 0 and 1 were classified as the correct position and the others, as the incorrect position (fig. the direction of pedicle perforation was assessed ; medial, lateral, cranial, and caudal. the intraobserver and interobserver agreement rate and k values were obtained to check errors between 2 observers in grading of the pedicle perforation. operative time, intraoperative blood loss, and pedicle screw - related complications such as vertebral artery injury, nerve root injury, or irritation sign were analyzed. to evaluate the anomaly and dominant side of vertebral artery, we checked vascular - enhanced computed tomography (ct) scans in all patients. on preoperative ct scans, the convergence angle and the minimal diameter of the pedicle on the axial plane were measured for each cervical vertebra. the length of the screw was decided to reach the anterior one - third of the vertebral body on the preoperative ct images. those with a pedicle diameter of less than 3.5 mm or a pedicle convergence angle of over 60 degrees were excluded from cervical pedicle screw insertion. the authors considered every pedicle to be built as a funnel with a wide posterior base, which narrows towards the isthmus. a distinct characteristic of the cervical pedicles is that the lateral pedicle wall is always the thinnest cortex14,27). therefore, the medial pedicle cortex was used as a safe guide into the pedicle isthmus, then through it and then into the vertebral body. the arch of the medial pedicle wall, under which advancement into the pedicle proceeded, was always identified. patients were placed in the prone position with the head fixed using a mayfield clamp. a standard midline incision was made and paravertebral muscles were dissected and retracted laterally to expose facet joints. under the lateral fluoroscopic guide and the basis of the lateral mass depth, and the convergence angle of the pedicle obtained from a preoperative computed tomography (ct) scans, 3 mm cutting burr was used to remove the outer cortex of the lateral mass over the pedicle entrance in the slightly lateral to the center of the facet, close to posterior margin of superior articular surface, the appropriate convergence angle to visualize the entrance of the pedicle (fig. the pedicle screw insertion point is indicated by a cancellous bone at the pedicle entrance, which is commonly observed to be reddish because of a bloody cancellous bone. a laminoforaminotomy was performed to provide visual and tactile cues regarding the orientation of the pedicle medial and superior walls. the ligamentum flavum at each level was gently dissected free from the inferior aspect of the superior laminar arch and from the superior aspect of the inferior laminar arch using a small curved curette. thereafter, the inferior aspect of the superior lamina and the superior aspect of the inferior lamina were removed in varying amounts using a 3 mm cutting burr followed by 1 mm and 2 mm kerrison punches (fig. the ball - tip hook then could be used to identify the medial and superior walls of the pedicle before the insertion of each screw (fig. the pedicle was probed as close to the medial wall as possible by gentle manual pressure using a 15-degree, 2 mm - diameter curved gear shift probe (fig after probing about 2 cm depth, lack of pedicle perforation was confirmed using a ball - tip probe. if perforation was detected within the pedicle, the trajectory was changed or the segment was skipped. then sequential drilling, tapping, and screwing were performed. for preventing iatrogenic neural tissue injury during rod connection procedure, rods were connected to the screws by appropriately sized slotted connectors before posterior decompression. local bone harvested from the decompression site was grafted in the lateral mass bone defect. patients were placed in the prone position with the head fixed using a mayfield clamp. a standard midline incision was made and paravertebral muscles were dissected and retracted laterally to expose facet joints. under the lateral fluoroscopic guide and the basis of the lateral mass depth, and the convergence angle of the pedicle obtained from a preoperative computed tomography (ct) scans, 3 mm cutting burr was used to remove the outer cortex of the lateral mass over the pedicle entrance in the slightly lateral to the center of the facet, close to posterior margin of superior articular surface, the appropriate convergence angle to visualize the entrance of the pedicle (fig. the pedicle screw insertion point is indicated by a cancellous bone at the pedicle entrance, which is commonly observed to be reddish because of a bloody cancellous bone. a laminoforaminotomy was performed to provide visual and tactile cues regarding the orientation of the pedicle medial and superior walls. the ligamentum flavum at each level was gently dissected free from the inferior aspect of the superior laminar arch and from the superior aspect of the inferior laminar arch using a small curved curette. thereafter, the inferior aspect of the superior lamina and the superior aspect of the inferior lamina were removed in varying amounts using a 3 mm cutting burr followed by 1 mm and 2 mm kerrison punches (fig. the ball - tip hook then could be used to identify the medial and superior walls of the pedicle before the insertion of each screw (fig. the direction in the sagittal plane of the pedicle probe and screw was intraoperatively determined using lateral fluoroscopic imaging. the pedicle was probed as close to the medial wall as possible by gentle manual pressure using a 15-degree, 2 mm - diameter curved gear shift probe (fig. after probing about 2 cm depth, lack of pedicle perforation was confirmed using a ball - tip probe. if perforation was detected within the pedicle, the trajectory was changed or the segment was skipped. then sequential drilling, tapping, and screwing were performed. for preventing iatrogenic neural tissue injury during rod connection procedure, rods were connected to the screws by appropriately sized slotted connectors before posterior decompression. using preoperative and postoperative vascular enhanced ct axial images, the diameter and convergence angle of the each pedicle and the screws were measured and the difference between pedicles and screws in diameter and convergence angle were analyzed (fig. the degree of perforation was classified as grade 0 if the screw was located within the pedicle ; grade 1, if perforation was made by the screw by less than 25% of the screw diameter ; grade 2, if perforation was made by 25% to 50% of the screw diameter ; and grade 3, if perforation was made by over 50% of the screw diameter (fig. 4). grade 0 and 1 were classified as the correct position and the others, as the incorrect position (fig. the direction of pedicle perforation was assessed ; medial, lateral, cranial, and caudal. the intraobserver and interobserver agreement rate and k values were obtained to check errors between 2 observers in grading of the pedicle perforation. operative time, intraoperative blood loss, and pedicle screw - related complications such as vertebral artery injury, nerve root injury, or irritation sign were analyzed. the interobserver agreement rate was 87% for the grade of pedicle perforation (mean k=0.65), and intraobserver agreement rate was 93% (mean k=0.77). the number of screws inserted was 16 at c3, 18 at c4, 24 at c5, 24 at c6, and 22 at c7 (table 1). laminectomy with fusion was performed in 8 cases, anterior cervical discectomy and fusion in 1, cervicothoracic fusion in 3. the mean axial diameter of cervical pedicles was 5.01.0 mm and the mean convergence angle was 41.25.1 degrees. all the screws had a diameter of 4 mm and the mean convergence angle of the screws inserted was 36.64.2 degrees. the mean difference between the preoperative convergence angle of the pedicles and the convergence angle of the inserted screws was 4.574.3 degrees. perforation of cervical pedicles occurred in 29 screws (27.9%) : grade 1 in 20 (19.2%), grade 2 in 6 (5.8%), and grade 3 in 3 (2.9%) (table 2). overall, the correct position (perforation of grade 0, 1) was found in 95 screws (91.3%) and the incorrect position (perforation of grade 2, 3) in 9 screws (8.7%). the mean difference between the convergence angle of pedicles and that of screws was 3.672.7 degrees in the correct position group and 10.043.4 degrees in the incorrect position group. incorrect position occurred in 1 screw (6.2%) at c3, 2 (11.1%) at c4, 3 (12.5 %) at c5, 2 (8.3%) at c6, and 1 (4.5%) at c7 (table 1). the direction of perforation was lateral in 21 (72.4%), followed by medial in 6 (20.6%), and caudal in 2(6.8%) screw. postoperative vascular - enhanced ct scan confirmed that the blood flow of the vertebral artery maintained in all cases. biomechanical studies have reported that cervical pedicle screws provide greater stability than other posterior cervical fixation procedures12,13,30). however, cervical pedicle screw insertion is more technically demanding than in thoracic or lumbar vertebrae because of the smaller pedicle dimension, the individual variations in pedicle anatomy, and the potential risk of injury to neurovascular structures4,14,24,26,28,35). the cervical pedicle screw insertion technique was first described by abumi.1). since abumi technique of cervical pedicle screw insertion, several reports have discussed various methods to insert cervical pedicle screw in easier and safer techniques. however, the methods for precisely and reproducibly determining the ideal entry points and trajectories are yet to be defined. recently, an anatomical study of subaxial cervical pedicles and lateral masses using ct scans of adult volunteers provided entry points and trajectories for subaxial cervical pedicle screw insertion. zheng.38) reported a high success rate using subaxial cervical pedicle screw insertion, with an overall accuracy of 83.3%, including a non - critical perforation of 13.3% and a critical perforation of 3.3%. this success can be achieved using the recently proposed guidelines and the oblique view obtained through fluoroscopy. however, landmarks to the cervical pedicle entrance alone are insufficient for achieving accurate cervical pedicle screw insertion because of the variation in pedicle anatomy15,22). then, several insertion techniques using direct pedicle exposure have been advocated in clinical or cadaver studies14,16,22,23). abumi.1) described a technique in which the cortex at the insertion point is penetrated using a high speed burr, resulting in direct observation of the pedicle entrance. they also reported a pedicle - perforation rate of 6.7% in another clinical report4). karaikovic.16) used the funnel technique, in which the entrance into the pedicle and vertebral body was identified by removing the outer cortex. they used the medial cervical pedicle cortex as a guide into the vertebral body through the pedicle isthmus. this study reported a screw perforation rate of 16.8%, including non - critical perforations of 9.7% and critical perforations of 7.1%, using the funnel technique. miller.23) used partial laminectomy and a tapping technique in which the entrance point for screw insertion and angulations for screw placement were guided by direct determination of the superior, medial, and inferior borders of the pedicle through the laminar window opening. ludwig.21) inserted a cervical pedicle screw insertion after a laminoforaminotomy, which provides visual and tactile cues regarding orientation of the medial, and superior pedicle walls. yoshimoto.36) observed incomplete perforation in 7.3% and complete perforation in 3.7%. in 100 cases of cervical pedicle screw insertion using an oblique view in the year 2006, yukawa.37) reported incomplete perforation in 10.3% and complete perforation in 4%. in 2005, neo.24) mainly applied the abumi technique to 18 cases and found pedicle perforation in 25%, a rate higher than previous reports. on the other hand, several authors have reported cervical pedicle screw insertion techniques using computer - assisted navigation systems and computer - assisted navigation systems have lower pedicle screw perforation than free - hand techniques (direct pedicle exposure techniques)9,18,32). kotani.18) reported that the screw misplacement rate was significantly lower in a computer - assisted group (1.2%) than in a free - hand group (6.7%). increasing use of three - dimensional fluoroscopy - based computer - assisted navigation systems has recently been reported9,10). three - dimensional fluoroscopy is superior to conventional ct - based image guidance because anatomical registration is not required and real - time updates of the spine position can be obtained intraoperatively. ito.10) reported that the rate of pedicle perforations was 2.8% in the absence of clinically significant perforation when a three - dimensional fluoroscopy - based navigation system was used. ishikawa.9) reported that the prevalence of pedicle perforation was 18.7% in a three - dimensional fluoroscopy - based navigation group and 27% in a conventional free - hand group. although computer - assisted navigation systems can improve the accuracy of cervical pedicle screw insertion, there are also some disadvantages, such as the requirement of very expensive system costs. these results can not be compared directly because the criteria for assessing development and degree of pedicle perforation vary. we classified perforation of less than 1 mm as the correct position and perforation of over 1 mm, as the incorrect position. this result seems higher perforation rate than some of previous studies, because we included pedicle perforation of less than 1 mm at classification. the perforation rate could vary according to each grading system, which has not standardized yet. the difference lies in the laminoforaminotomy performed to provide visual and tactile cues regarding the orientation of the pedicle medial and superior walls. in addition, the use of a 15-degree, 2 mm - diameter curved gear shift probe is expected to reduce the risk of lateral perforation by guiding the trajectory along the strong medial cortex of the pedicle, which is about two times thicker than lateral cortex. the method used in this study requires removal of the bone in the lateral mass, similar to the abumi and funnel techniques. however, kowalski.20) reported no significant difference in the biomechanical pullout strength of cervical pedicle screw when the lateral mass cancellous bone is removed. the cervical pedicle screw insertion technique with direct exposure of the pedicle by laminoforaminotomy did not require special preoperative preparation or an intraoperative environment and showed that good screw position placement and strong screw fixation stability. we performed cervical pedicle screw insertion using the technique with direct exposure of the pedicle by laminoforaminotomy and with 91.3% correct position without clinical complications. this technique could be considered relatively safe and easy method to insert cervical pedicle screw.	objectiveto present the accuracy and safety of cervical pedicle screw insertion using the technique with direct exposure of the pedicle by laminoforaminotomy.methodswe retrospectively reviewed 12 consecutive patients. a total of 104 subaxial cervical pedicle screws in 12 patients had been inserted. we also assessed the clinical and radiological outcomes and analyzed the direction and grade of pedicle perforation (grade 0 : no perforation, 1 : 50% of screw diameter) on the postoperative vascular - enhanced computed tomography scans. grade 2 and 3 were considered as incorrect position.resultsthe correct position was found in 95 screws (91.3%) ; grade 0 - 75 screws, grade 1 - 20 screws and the incorrect position in 9 screws (8.7%) ; grade 2 - 6 screws, grade 3 - 3 screws. there was no neurovascular complication related with cervical pedicle screw insertion.conclusionthis technique (technique with direct exposure of the pedicle by laminoforaminotomy) could be considered relatively safe and easy method to insert cervical pedicle screw.
anxiety disorders are currently the most common neuropsychiatric disorders in the usa and europe (andlin - sobocki., 2005 ; kessler., 2005) and represent one of the major health problems in the western world (who, 2004). in the aetiology of an anxiety disorder, the simultaneous occurrence of various intrinsic and extrinsic factors, including those relating to genes and environment, seems to be important. among these, there is increasing evidence of a role of poor nutrition (popkin, 2006) in the development of some psychiatric diseases (coppen and bolander - gouaille, 2005 ; kroll, 2007), despite an apparent over - nutrition in our industrialised and globalised societies. altered values of diverse micronutrients, including electrolytes, minerals and vitamins, can be linked to symptoms in psychiatric patients (armstrong., 2007 ; thys - jacobs, 2000 ; zender and olshansky, 2009). in this respect, changes in magnesium (mg) homeostasis have been shown to contribute to affective disorders although some inconsistencies exist between the studies (for review see murck, 2002). furthermore, a causal relationship between mg and anxiety is suggested in mice with low plasma mg levels which display increased anxiety- and/or depression - related behaviour, irrespective of whether these depleted levels are natural or experimentally induced (laarakker., 2011 ; singewald., 2004), although in humans equivalent evidence is scarce (jacka., 2009 ; seelig, 1994). in further support of such a causal relationship, mg supplementation reduces the anxiety - related behaviour of mice (see for example poleszak., 2004). stimulated by these findings, it has been recently suggested that hypomagnesaemia is a possible physiological indicator of anxiety (laarakker., 2011). in the search for neurobiological mechanisms underlying abnormal anxiety, it may be that mg is an interesting player as it is one of the essential ions in the brain affecting many intracellular and interneuronal processes. for example, mg has been shown to modulate both glutamatergic neurotransmission (via a voltage - dependent block of nmda receptors) (haddad, 2005) and gabaergic neurotransmission, and to affect numerous transduction pathways, including that of proteinkinase c (for review see murck, 2002). in addition, mg has been shown to control the activity of the hypothalamic - pituitary adrenocortical (hpa) axis (murck and steiger, 1998), which is considered to be the main stress response system (for review see e.g. young., 2008). in the course of hpa axis activation by the corticotropin releasing hormone (crh), synthesised in the paraventricular hypothalamic nucleus (pvn), adrenal corticotropin hormone (acth) is released from the anterior pituitary into the blood stream which, in turn, stimulates the secretion of corticosterone. hpa axis activation via crh is involved in the generation of many different autonomic, hormonal and behavioural changes during stress and elicits anxiety symptoms (for review see lowry and moore, 2006 ; young., it has been suggested that dysregulations in the crh - hpa axis system may contribute to pathological anxiety (for review see charney and drevets, 2008 ; millan, 2003 ; young., 2008). on the basis of these data, it may be hypothesised that an altered hpa axis function underlies the enhanced anxiety of mg deficient mice that is elicited by dietary mg restriction (when mice are limited to 10% of their daily requirement) which results in a 45% reduction of plasma mg levels (singewald., 2004). to address this possibility, we initially explored the robustness and validity of the model by inducing hypomagnesaemia according to our previous protocol for c57bl/6j mice (singewald., 2004) in two additional mouse strains with divergent levels of inborn anxiety (c57bl/6n and balb / c mice) (for review see e.g. belzung and griebel, 2001 ; sartori., 2011), and then tested the effects of a clinically established benzodiazepine and the two antidepressant drugs, paroxetine and desipramine, with established clinical efficacy in patients with anxiety disorder (for review see plag., 2009) and/or with demonstrated sensitivity in mg deficient mice to reduce depression - like behaviour (singewald. the emotional behaviour of mg deficient mice was assessed in a battery of anxiety tests including the open field test, the light / dark test, the stress - induced hypothermia test, and the hyponeophagia test, each of which are thought to mimic different aspects of human anxiety and which differ in their sensitivity to detecting the anxiolytic properties of drugs (for review see crawley, 2000 ; cryan and holmes, 2005 ; dulawa and hen, 2005 ; vinkers. we then quantified markers of hpa axis function including crh gene expression and plasma acth levels in experimentally naive or unstressed mg deficient mice. finally, functional relevance of potentially altered transcriptional processes in the hpa system was investigated in mg deficient mice using mapping of the immediate - early gene c - fos as a marker of neuronal activation in response to an anxiety - provoking situation. rather than addressing every aim in each strain, we allocated specific experiments to either the c57bl/6n or the balb / c strain (see methods). thus, using a reduced number of animals following the principles of animal welfare, this experimental design allowed revealing most information complementary to the one gained in c57bl/6n mice (e.g. crh expression in the pvn) by adding further aspects (e.g. the influence of inborn anxiety levels by using the balb / c strain). male c57bl/6n and balb / c mice were purchased from charles river (germany). in all experiments adult animals (8 weeks old at the onset of the experiment) were used. they were group - housed (up to 10 animals per cage) side - by - side in a temperature- (22 2 c) and humidity- (5060%) controlled animal care facility unit under a 12 h light / dark cycle (lights on at 07:00 h), and provided with pelleted food and water ad libitum. all experimental procedures were designed to minimise animal suffering as well as the number of animals used and were approved by the national ethical committee on animal care and use (bundesministerium fr wissenschaft und forschung) in compliance with international laws and policies. mice assigned to mg deficient groups were allowed to freely access a 0.005% mg containing diet (ssniff spezialditen, germany), which provided about 10% of the daily mg requirement (kantak, 1988) as previously described (singewald., 2004). control mice were fed a normal, 0.2% mg containing diet (ssniff spezialditen, germany) which offers four times more than the minimum mg requirement (kantak, 1988), and hereafter are always referred to as controls. in mg deficient mice chronic treatment with either desipramine or paroxetine was given via the drinking water. a daily drug intake of approximately 30 mg / kg desipramine (sigma aldrich) and 5 mg / kg paroxetine (kindly provided by glaxosmithkline, uk), respectively, was achieved by adapting the concentrations of the drugs in the drinking solutions according to mean drinking volume and bodyweight per cage. however, the individual dose obtained may have varied due to variations in the mice in terms of water consumption and bodyweight. mice were kept under the assigned experimental condition (diet / drug treatment) for at least three weeks until the completion of experiments. diazepam (1 mg / kg, dissolved in purified water ; sigma, germany) or vehicle were administered i.p. to mice in their home cages. mice were left undisturbed in their home cages for three weeks from the start of diet and/or chronic drug treatment until the onset of behavioural experiments. after animals had been habituated to the testing room for at least 24 h, behavioural experiments were carried out between 09:00 h and 14:00 h. testing chambers were carefully cleaned with tap water and a damp towel after each animal had been tested. in order to decrease the number of animals used, the behavioural experiments, with at least two days of rest between each, were conducted in the same animals starting with the stress - induced hyperthermia test (lecci., 1990), the open field test (broadhurst, 1961 ; defries., 1974), the light / dark test (crawley, 1981), and the hyponeophagia test (bodnoff., 1988) according to protocols used previously in our lab (busquet., 2009 ; mice were housed singly for at least 24 h prior to testing. with the mouse in its homecage, a glycerol lubricated thermistor probe connected to a digital thermometer (dm 852, ellab, denmark) was inserted into its rectum and left there until the temperature was stable for 20 s. the accuracy of the thermometer was 0.1 c. rectal temperature was measured in each mouse at t1, and then at t2 10 min later. as the intubation of the rectal probe for assessing the core temperature has been shown to sufficiently initiate a stress response in mice (bouwknecht., 2007), the difference between temperatures t2 and t1 was taken as evidence of stress - induced hyperthermia. at the end of the stress - induced hyperthermia test, animals were group housed again for the following tests. the open field consisted of a plastic box (41 41 41 cm) equipped with an automated activity monitoring system (truscan, coulbourn instruments, usa). the area of the open field, illuminated with 150 lux, was divided into a 28 28 cm central zone and a surrounding border zone. mice were placed individually into the periphery of the open field and allowed to explore it for 10 min. the following anxiety - related parameters were recorded : entries into the central zone, time spent in it, number of rearings, and the overall distance travelled. the light / dark testing arena (41 41 41 cm ; truscan, coulbourn instruments, usa) was divided into two halves, comprising a white, aversive compartment illuminated with 400 lux at floor level and a dark, safe compartment covered by a black top illuminated with 10 lux. the compartments were connected by a small opening (7 7 cm) located in the centre of the partition at floor level. animals were individually placed into the dark compartment facing away from the opening and allowed to freely explore the apparatus for 10 min. during the 10 min testing period the behaviour displayed by each mouse was automatically registered and the following anxiety - related parameters quantified : the latency to the first entry into the lit compartment, number of entries into the lit compartment, time spent in the lit compartment, number of rearings and overall distance travelled by the mice. this paradigm was carried out in the open field arena (see above) with lighting set at 40 lux. animals were placed into a corner of the arena and the latency they took to start consuming oatflakes placed in the centre of the testing arena was registered, as well as the distance travelled. in a separate cohort of the c57bl/6n strain, mice were fed either the control diet, the mg restricted diet, or the mg restricted diet and concomitant chronic desipramine treatment as described above and were left undisturbed in their home cages for three weeks. these experimentally naive mice were then sacrificed by carbon dioxide inhalation in a saturated chamber in accordance with established welfare guidelines (hackbarth., 2000) and their brains were quickly removed. consecutive, frozen sections (12 m) at the level of the pvn, central amygdala and barrington s nucleus were prepared on slides (paxinos and franklin, 2001) and further processed for in situ hybridisation as previously described (keck., 2005 ; sartori., briefly, a specific, [s]-labelled oligonucleotide probe (microsynth, balgach, switzerland) directed against bases 64111 of the rat prepro - crh clone (jingami., 1985) showing high homology with the murine gene (accession number nm_205769.1) was hybridised to brain sections at 35 c overnight. subsequently, slides were washed in 0.5 saline sodium citrate three times at 55 c followed by two washes at room temperature. dry slides were exposed to biomax mr film (kodak, usa) for 714 days. quantitative analysis was performed on digitised autoradiograms using image analysis software (analysis, soft imaging systems, germany). in two sections per animal, mean grey values from regions of interest were determined and converted into optical densities, and then further into nci / g tissue equivalent using autoradiographic [c]-microscales (amersham biosciences, uk) corrected for [s ]. at least five days after the last behavioural experiment, c57bl/6n mice assigned to the control diet, the mg deficient diet, or the mg deficient diet and concomitant chronic treatment with desipramine were euthanised using carbon dioxide in a saturated chamber (hackbarth., 2000). trunc blood was collected from the sacrificed animals in edta - coated vials (greiner bio - one gmbh, austria) and centrifuged (3000 rpm) for 10 min at 4 c. plasma acth and corticosterone concentrations were determined using commercially available kits (mp biomedicals, usa), with an intra- and inter - assay variability of less than 10% and a lower detection limit of 6 pg / ml and 8 ng / ml, respectively. 30 min after i.p. injection of either diazepam (1 mg / kg) or vehicle, balb / c mice were subjected to the open arm exposure test as previously described (muigg., 2009). during the 10-min testing period in which the animals were allowed to freely explore the open arm of an elevated plus maze, entries into and time spent in the proximal and distal compartments of the open arm as well as the total distance travelled were automatically quantified by the videomot tracking system (tse systems, germany). two hours later mice were deeply anaesthetised (200 mg / kg sodium pentobarbital, i.p.) and transcardially perfused with saline followed by paraformaldehyde solution (4% in 0.1 m phosphate buffered saline, ph 7.4), and their brains were removed. coronal brain sections (100 m) at the level of the pvn were processed for c - fos immunocytochemistry using a polyclonal rabbit anti - c - fos primary antibody (1:20,000 ; santa cruz biotechnology, usa), a biotinylated goat anti - rabbit secondary antibody (1:200 ; vector laboratories, usa) and an avidin - biotin - horseradish peroxidase procedure (vectastain abc kit, vector laboratories, usa) with 3,3-diaminobenzidine (sigma, germany) as chromogen according to previous protocols (muigg., 2009 the pvn cells containing a nuclear brown - black reaction product were considered to be c - fos - positive cells and their number was counted bilaterally in a representative tissue area of 0.01 mm with the help of a light microscope (olympus bx-40) equipped with an ocular grid. variations in n - numbers between tests and/or assays are explained by the combination of at least two separate sets of experiments using different n - numbers and the early withdrawal of some animals for other experiments, in part reported elsewhere (e.g. whittle., 2011). first, all experimental groups were tested for statistically significant outliers using the grubb s test. data were then tested for homogeneity of variances using levene s test. if a parametric distribution was revealed, the data were further analysed using either a one- or two - way anova followed by fisher s lsd test when allowed. whitney u tests. statistical significance was set at p < 0.05 while non - significant p values were reported as n.s. at the beginning of the experiments, c57bl/6n mice assigned to control diet, mg deficient diet or mg deficient diet chronically treated with either desipramine or paroxetine did not differ in bodyweight (mean bodyweight of 22.5 0.1 g). after three weeks under the experimental conditions (diet / drug), bodyweight gain differed between experimental groups (f1,22 = 31.520, p < 0.001). within this time period mg deficient animals (+ 5.1 1.1%) put on more bodyweight than control animals (+ 2.4 0.8% ; p < 0.05). compared with the mg deficient group, the gain in bodyweight was increased in mg deficient mice chronically treated with paroxetine (+ 10.9 1.0% ; p < 0.001) while the mg deficient mice chronically treated with desipramine even lost weight (2.9 1.1% ; p < 0.001). similar to c57bl/6n mice, bodyweights did not differ between control mice and mg deficient mice (mean bodyweight of 20.0 0.1 g) of the balb / c strain at the beginning of the experiments. three weeks of feeding a mg restricted diet caused a reduced increase in bodyweight in balb / c mice (control : + 11.8 1.3% : mg deficient : + 5.8 1.5 g ; p < 0.01). using the stress - induced hyperthermia paradigm (for review see vinkers., 2008), anxiety - related behaviour of c57bl/6n mice assigned to either the control diet, the mg deficient, or the mg deficient diet and parallel chronic paroxetine treatment was tested (table 1). basal body temperature of control mice was low reaching 35.8 0.2 c, which indicates that animals were not pre - stressed under the given experimental conditions. there was a significant difference between mice in the body temperature t1 under unstressed conditions (f1,22 = 6.400, p < 0.01). subsequent post - hoc analysis designated increased basal body temperature in mg deficient animals compared with controls and paroxetine - treated mg deficient mice. the mild stress of measuring t1 caused a rise in body temperature (t2) which by this stage no longer differed between the three experimental groups. the stress - induced rise in body temperature was smaller in mg deficient mice than it was in either control or paroxetine - treated mg deficient mice (f1,21 = 4.132, p < 0.05). animals of the c57bl/6n strain were then tested in the open field test and light / dark test which are based on an exploration - avoidance conflict (for review see e.g. cryan and holmes, 2005). in the open field test (table 1), experimental groups differed significantly in terms of number of entries into the centre of the testing arena (f2,27 = 5.021, p < 0.05), time spent there (f2,26 = 5.284, p < 0.05 ; fig. 1) as well as number of rearings (f2,27 = 6.381, p < 0.01). specifically, compared with mice fed the control diet, these measures were lower in mg deficient and paroxetine - treated mg deficient animals. in the light / dark test (table 1 ; fig. 1), we observed a significant difference in the latency to enter the brightly lit, aversive compartment of the light / dark chamber (f2,27 = 7.122, p < 0.01) as this latency was increased in both groups fed a mg restricted diet (fig. 1). furthermore, there was a significant group effect in terms of the number of entries into (f2,27 = 3.430, p < 0.05) and time spent (f2,27 = 5.045, p < 0.05) in the lit compartment of the light / dark test chamber, and in the rearing numbers (f2,27 = 7.361, p < 0.01). paroxetine - treated mg deficient mice displayed reduced values in all three parameters compared with both control and mg deficient groups (table 1). next, we subjected all c57bl/6n mice to the hyponeophagia paradigm, one of the few tests which are sensitive to the anxiolytic effects of chronic antidepressant treatment (bodnoff., 1989 ; dulawa and hen, 2005 ; gordon and hen, 2004). there was a significant group effect in the latency to eat (f3,58 = 11.828, p < 0.001) the preferred food placed into the centre of the testing arena (fig. 2). mg deficiency caused an increase in the latency to eat. in mg deficient mice chronic desipramine treatment reduced the latency to eat compared with untreated mice while long - term treatment with paroxetine did not affect this behavioural parameter (fig. 2). mg deficiency and long - term antidepressant treatments did not alter general locomotor activity as indicated by the distances travelled in the open field test (n.s. ; table 1), the light / dark test (n.s. ; table 1), and the hyponeophagia test (n.s. ; fig. 2). all together these findings suggest that chronic mg deficiency was anxiogenic and that chronic desipramine, but not paroxetine treatment was effective in reducing anxiety in this model. in c57bl/6n mice prepro - crh mrna expression, as visualised by in situ hybridisation, was high in the pvn and moderate in the central amygdala and barrington s nucleus (table 2), which is in good accordance with previous studies (e.g. keegan., 1994). in the pvn a significant group effect was observed (f2,29 = 12.486, p < 0.001) as the abundance of prepro - crh mrna was increased in mg deficient mice compared with mice fed the control diet (p < 0.001), and chronic treatment with desipramine normalised this effect in mg deficient mice (p < 0.001) (table 2, fig. 3). in contrast to the pvn, prepro - crh mrna expression did not differ between experimental groups in the central amygdala (n.s.) or in the barrington s nucleus (n.s.) (table 2). under unstressed conditions plasma acth levels (h2,46 = 7.466, p < 0.05), but not corticosterone levels (n.s.), differed between experimental groups of the c57bl/6n strain (table 3). mg deficient animals showed increased plasma acth levels compared with controls fed a normal mg containing diet (p < 0.01). chronic treatment with desipramine decreased the elevated acth plasma levels of mg deficient mice, which no longer differed in this respect from control mice (n.s.). in an attempt to gain additional evidence for a possible functional relevance of increased pvn prepro - crh mrna expression in the mg deficiency - induced enhanced anxiety - related behaviour, c - fos expression was used to map neuronal activation in the pvn in response to the mild emotional challenge of open arm exposure. as with the c57bl/6n strain, mg deficiency increased anxiety - related behaviour in balb / c mice which was reversed by diazepam treatment as indicated by significant dietary and drug effects in the measures percentage of entries into the anxiogenic distal part of the open arm (diet : f1,35 = 12.615, p < 0.01 ; drug : f1,35 = 15.359, p < 0.001, table 4, fig. 0.05 ; drug : f1,36 = 33.519, p < 0.001 ; table 4). furthermore, diazepam significantly affected the percentage of time spent in the distal compartment (f1,35 = 10.352, p < 0.01) (table 4). exposure to the open arm of an elevated plus maze induced c - fos expression in the pvn (fig. two - way anova revealed a significant diet drug interaction in terms of the number of c - fos - positive cells in the magnocellular portion of the pvn (f1,36 = 20.8301, p < 0.001) while there was a dietary effect in its parvocellular portion (f1,36 = 4.187, p < 0.05 ; data not shown). specifically, mg deficiency increased emotional challenge - induced c - fos expression and diazepam normalised it in balb / c mice (fig. in the present study we have shown that dietary mg restriction reproducibly enhanced anxiety - related behaviour in mice and that this effect was robust in terms of different strains and paradigms used. mg deficiency was associated with an increased transcription of prepro - crh in the pvn, the main output region of the hpa axis, and elevated plasma acth levels pointed to an up - regulated stress system. indeed, in mg deficient mice expression of the immediate - early gene c - fos was increased in the pvn in response to a mildly anxiety - provoking situation indicating functional over - reactivity of this brain area. in parallel with a reversal of the behavioural changes brought about by mg deficiency, the observed abnormalities in the hpa axis system these data support a relationship between low mg levels and both anxiety - related behaviour and a modulated stress axis. in the present study chronic feeding of c57bl/6n mice with a low mg containing diet enhanced anxiety - like behaviour in the open field and light / dark test compared with controls. these findings are in line with our previous results using the c57bl/6j substrain (singewald., 2004) which were independently confirmed by muroyama. in addition, we included another anxiety test, the hyponeophagia test, which refers to the inhibition of feeding in rodents upon exposure to novelty and which is one of the limited number of tests available in terms of its sensitivity to the anxiolytic effects of chronic, but not acute antidepressant treatment (for review see dulawa and hen, 2005). in this test the measures of anxiety - related behaviour were also enhanced in mg deficient mice compared with mice fed the control diet. in contrast to our previous study (singewald., 2004), bodyweight gain was enhanced in mg deficient compared with control mice excluding the argument that reduced appetite may have contributed to the enhanced latency to feed. this, however, seems unlikely since the enhanced latency to feed was fully reversed by desipramine which is reported to exert anorexic effects or no change in bodyweight gain, rather than orexigenic effects at the beginning of treatments (gobshtis., 2007 ; sartori., 2004 ; yalcin., 2005). in the open field test, light / dark test and hyponeophagia test there was a trend towards a reduced anxiety - induced locomotion in mg deficient mice compared to mice fed the control diet. however, it is unlikely that the enhanced anxiety - related behaviour induced by mg deficiency is influenced by unspecific effects on locomotion or due to a general motor impairment as mg deficient and control groups do not differ in locomotor activity in their home cages as well as in the rotarod test (singewald., 2004). nevertheless, we examined the anxiogenic effects of mg deficiency in a test that is entirely independent of locomotion, the stress - induced hyperthermia test. we found that even under unstressed conditions, body temperature was increased in mg deficient mice compared with control mice which seemed to limit their hyperthermic response to exposure to the mild stress. the increased basal body temperature may therefore point towards a kind of chronically - stressed state induced by mg deficiency which caused a new set - point for body temperature (keeney., indeed, elevated basal body temperature is reported in chronically - stressed animals (hayashida., 2010 ; keeney., 2001) as well as in mutant mice (guilloux., 2011) in further support of this idea, the hpa - stress axis seems to be up - regulated in mg deficient compared with control mice (see below). during the open arm exposure test mg deficiency also increased anxiety - related behaviour in balb / c mice, a mouse strain with emotionality reported to be different to or even opposite that of c57bl/6 mice (griebel., 2000). this effect coincided with an attenuated distance travelled which is thought to be a sign of increased neophobia reflecting trait anxiety, observed particularly in balb / c mice (for review see belzung and griebel, 2001). thus, it seems that dietary mg deficiency further increased the innate anxiety of this strain. in addition to experimentally reducing plasma mg levels, natural low mg levels across mouse strains have also been associated with enhanced anxiety - like behaviour. for example, a / j mice characterised by enhanced anxiety - related behaviour compared with c57bl/6 mice have lower plasma mg levels (laarakker., 2011). we next tested whether it was possible to attenuate the increased anxiety - related behaviour of mg deficient mice by the application of clinically effective pharmacotherapies., 2004 ; bentz., 2010 ; soravia., 2006 ; for review see millan, 2003) reversed the enhanced anxiety - related behaviour of mg deficient balb / c mice, while it was ineffective in control mice suggesting efficacy of diazepam particularly in subjects with high emotionality. likewise, specificity of action of diazepam is described in the hab mouse model (kromer., 2005) and various psychopathological rat models of enhanced anxiety including the hab rats (liebsch., 1998). in addition to the classical anxiolytic drug diazepam, chronic desipramine treatment also reduced anxiety - related behaviour of mg deficient c57bl/6n mice in the hyponeophagia test (the present study), while it was not effective in altering anxiety - like behaviour in the open field test and light / dark test (singewald., 2004) whose limited sensitivity to the anxiolytic effects of chronic antidepressant treatments is well known (for review see belzung, 2001). anxiolytic effects of chronic desipramine treatment have also been reported in zinc - deficient mice displaying signs of enhanced anxiety - related behaviour (whittle., 2009) as well as in rodents with normal levels of anxiety (bodnoff., 1989, 1988 ; hyponeophagia - based models in mice and rats predict the anxiolytic effects of antidepressants in a manner that is consistent with the time - course of their effects in humans (for review see dulawa and hen, 2005). in the clinical setting imipramine, the prodrug of desipramine, is used in the treatment of anxiety disorders that include generalised anxiety disorder, panic disorder and post - traumatic stress disorder (for review see plag., 2009). the findings of anxiolytic effects resulting from diazepam and chronic desipramine treatment in mg deficient mice further underlines the validity of the mg deficiency model of enhanced anxiety - related behaviour in mice. in contrast to desipramine, chronic treatment with paroxetine did not affect anxiety - related behaviour in the hyponeophagia test. this finding is not due to possible underdosing as a dose of 5 mg / kg paroxetine has been recently shown to be effective in reducing depression - like behaviour in mg deficient mice (whittle., 2011). although, to our knowledge, paroxetine has not so far been tested in the hyponeophagia test, another ssri, fluoxetine, has proven to exert anxiolysis following chronic, but not acute, application in rodents (bodnoff., 1989 ; dulawa., 2004 ; 2003), suggesting that mg deficient mice are insensitive to the anxiolytic effects of chronic ssri (paroxetine) treatment. this finding is of potential research interest, as mg deficient mice may model a considerable proportion of patients with an anxiety disorder that does not respond to ssri treatment (liebowitz, 1997 ; for review see zamorski and albucher, 2002). in an attempt to gain insight into neurobiological mechanism(s) underlying the enhanced anxiety - related behaviour of mg deficient mice, we followed a hypothesis - driven approach rather than using an unbiased technique (see also german - fattal., 2008) that was previously used in our lab, where brain protein changes were identified that were correlated with the altered depression - related behaviour of mg deficient mice (whittle., 2011). mg modulates various neurobiological mechanisms, including neurotransmitter systems and the hpa axis (for review see murck, 2002). the hpa axis is an interesting substrate as it is known to play a role in stress processing and in normal and pathological anxiety (for review see charney and drevets, 2008 ; millan, 2003 ; young., specifically, as mg reduces hpa axis activity (held., 2002 ; murck and steiger, 1998), a disinhibition of this system during mg deficiency may be postulated. in order to address this hypothesis, we investigated markers of hpa axis activity, including prepro - crh transcription and acth plasma levels, in experimentally naive or unstressed control mice, mg deficient mice and mg deficient mice chronically treated with desipramine, the drug that was shown to be behaviourally active. the abundance of prepro - crh mrna was enhanced in the pvn, the main output region of the hpa axis, of mg deficient mice compared with control mice. in addition, acth plasma levels were elevated in mg deficient mice, indicating that the increase in prepro - crh transcription translated into increased acth release from the pituitary, while corticosterone plasma levels did not differ between experimental groups. a similar divergence between baseline plasma acth and corticosterone levels has been previously reported in low aggressive mice and may be explained by altered adrenocortical sensitivity to acth (veenema., 2003). in mg deficient mice the findings of elevated abundance of prepro - crh mrna and acth release point towards an up - regulated set - point of the hpa axis during mg deficiency which is also observed in some, but not all patients with an anxiety disorder (for review see charney and drevets, 2008 ; young., 2008), and this is therefore suggested to contribute to the enhanced anxiety - related behaviour of mg deficient mice. in further support of this, we observed increased neuronal activity in the pvn of mg deficient balb / c mice in response to the open arm of an elevated plus maze which, though considered as being mildly anxiogenic, is able to cause the release of stress hormones and to induce neuronal activation in rodents compared with an unstressed condition (muigg., 2009 ; nguyen., 2009 ; salome., interestingly, while stress - induced neuronal activation has been shown to be blunted in cortical areas of balb / c mice compared with c57bl/6 mice, pvn activation is similar between the two strains (omahony., 2010) supporting the use of balb / c mice complementary to c57bl/6n mice in the present study. the finding of an over - reactive pvn in mg deficient animals, thus, demonstrates functional relevance of the pvn in mediating hyper - anxiety. this over - reactivity of the pvn may be triggered by the enhanced transcription rate of prepro - crh. like mg deficient mice, hab mice and rats with high trait anxiety (kromer. also show increased c - fos expression in the pvn compared with their low anxiety (lab) counterparts in response to open arm exposure (muigg. crh levels have been shown to be up - regulated by a number of different stressors (for review see e.g. holsboer, 2000), including neonatal maternal separation, a proposed animal model of anxiety and depression (for review see sartori., 2011), and in rats with high trait anxiety (bosch., 2006). in line with the present findings, crh concentrations in the cerebrospinal fluid are elevated in chronic, combat - related post - traumatic stress disorder (baker., 1999 ; however, such reported abnormalities of the hpa axis are inconsistent between studies as, for example, basal plasma or urine cortisol concentrations have been shown to be increased, unaltered or decreased in patients with anxiety disorder (for review see charney and drevets, 2008 ; young., 2008). in mg deficient mice, application of the benzodiazepine diazepam reduced the hyper - reactivity of the pvn in response to a mild emotional challenge. the ability of benzodiazepines to decrease stress - induced neuronal activation in the pvn has been previously shown in mice (imaki., 1995) and rats (beck and fibiger, 1995 ; de medeiros., 2005). furthermore, chronic desipramine treatment via the drinking water normalised crh overexpression in the pvn and elevated acth plasma levels. tricyclic antidepressants have been shown to attenuate hpa axis activity which is thought to contribute to their anxiolytic and antidepressant actions (for review see holsboer, 2000). both findings further support the idea of a critical involvement of the hpa axis, and in particular of the pvn, in terms of contributing to the anxiogenic effects of mg deficiency. however, it should be noted that due to an existing comorbid link between anxiety and depression, it is not possible to make a clear distinction between mechanisms underlying anxiety alone. indeed, mg deficient mice are also characterised by a pro - depressive phenotype which can be reversed by chronic antidepressant treatments including desipramine and paroxetine (muroyama., 2009 ; singewald., 2004 ; thus, it may be that the up - regulated hpa axis in mg deficient mice also is of relevance to the enhanced depression - like behaviour. in humans too, depression has been shown to be associated with an abnormally elevated hpa axis which is restored after clinical improvement (for review see e.g. holsboer, 2000). taken together, the present findings demonstrate the robustness and validity of the mg deficiency model as model of enhanced anxiety - related behaviour and further supports emerging evidence in humans that reduced mg levels are associated with different facets of anxiety behaviour (jacka., 2009 ; seelig, 1994). hence, an inverse relationship between mg and anxiety is suggested by these data. in terms of the induced anxiogenesis, the mg deficiency model appears to be reproducible across mouse strains, regardless of the level of inborn anxiety displayed. finally, it is suggested that dysregulations in the hpa axis may contribute to the hyper - emotionality induced by dietary induced hypomagnesaemia.	preclinical and some clinical studies suggest a relationship between perturbation in magnesium (mg2 +) homeostasis and pathological anxiety, although the underlying mechanisms remain largely unknown. since there is evidence that mg2 + modulates the hypothalamic - pituitary adrenal (hpa) axis, we tested whether enhanced anxiety - like behaviour can be reliably elicited by dietary mg2 + deficiency and whether mg2 + deficiency is associated with altered hpa axis function. compared with controls, mg2 + deficient mice did indeed display enhanced anxiety - related behaviour in a battery of established anxiety tests. the enhanced anxiety - related behaviour of mg2 + deficient mice was sensitive to chronic desipramine treatment in the hyponeophagia test and to acute diazepam treatment in the open arm exposure test. mg2 + deficiency caused an increase in the transcription of the corticotropin releasing hormone in the paraventricular hypothalamic nucleus (pvn), and elevated acth plasma levels, pointing to an enhanced set - point of the hpa axis. chronic treatment with desipramine reversed the identified abnormalities of the stress axis. functional mapping of neuronal activity using c - fos revealed hyper - excitability in the pvn of anxious mg2 + deficient mice and its normalisation through diazepam treatment. overall, the present findings demonstrate the robustness and validity of the mg2 + deficiency model as a mouse model of enhanced anxiety, showing sensitivity to treatment with anxiolytics and antidepressants. it is further suggested that dysregulations in the hpa axis may contribute to the hyper - emotionality in response to dietary induced hypomagnesaemia.this article is part of a special issue entitled anxiety and depression.
the widespread use of complementary and alternative medicines (cam) among patients of biomedical practitioners has been widely documented (13). it might be implicitly accepted (which may explain why it is less commonly acknowledged) that patients of cam practitioners are almost always patients of conventional physicians, as well (4). however, coordination of care rarely occurs when the providers are from two distinct traditions, i.e. conventional biomedicine and traditional chinese medicine (tcm). nomenclature for referring to medical disciplines is a subject in its own right (5). integrative education is, in this example, the process of training conventional biomedical and tcm practitioners in each tradition such that patient care may be effectively coordinated. when practitioners from these distinct and prevalent medical systems are familiar with the diagnosis and treatment plan of the other, the patient can only benefit. a bilateral educational model ensures that students in each tradition are taught by experts from each tradition. it should be obvious that the process of integrative education, e.g. placing practitioners from each tradition in the clinics and the classrooms, is not balanced. this imbalance is evident in discussion and implementation of how to integrate academically. stating the obvious in the case of integrative medicine is important, especially when the two disciplines differ so fundamentally in their approach to patient care. educational models in the united states that promote the integration of tcm and conventional medicine are far from standard and decidedly one - sided. the conventional medical model places a very high priority on understanding the biological mechanisms of action that underlie acupuncture as a prerequisite of practice. it follows logically that tcm practitioners should know more about the biomedical approach. in our pain management professional acupuncture and oriental medicine doctorate program there is a strong belief that learning more orthopedics only strengthens the ability of the tcm practitioner to practice more effectively, especially when integrating with conventional medical providers. however, if understanding the mechanism of action for serotonin uptake inhibitors is not necessary for diagnosing and treating depression in conventional medical practice it certainly should not be in tcm. the process of integration is more than teaching the biomedical approach to tcm students or surveying tcm for medical students. surveys of integrative education tend to focus on conventional medical schools, suggesting tcm schools are only involved at the point of service delivery. a recent survey of nine leading academic medical centers in canada revealed a variety of models for exposing medical students to cam. a common model included recruiting cross - trained cam providers to lecture or otherwise interact on campus. one school employed an elective exchange model wherein medical students met with tcm students to discuss approaches to patient care (6). a survey of 19 us osteopathic medical schools showed that all but one included cam instruction. teaching, which originated across different clinical departments, did not exceed 20 h and typically occurred in the first two years. surprisingly, 18 of the 25 identified instructors were reported as cam providers (7). a cam instruction survey within us medical schools seventy - five schools (64% of 117) reported offering cam instruction, mostly as electives (84 of 123 courses, 68%), for the most part through family medicine and medicine / internal medicine departments (52 or 42%). education that leads to integration will be successful when more instructional models strive to represent how each tradition approaches the patient. integrative education that balances both perspectives on patient care must take place and should no longer be challenged as a training goal. nevertheless, harsh challenges can be found in the academic literature. a recent editorial in a croatian medical journal stated that scientific proof of cam effectiveness based on mechanisms of action are not to be found concluding that cam is a a letter to the editor (11) responded to an article suggesting that increased frequency of cam use by patients justifies inclusion of cam instruction in medical school curricula (12). the writer concluded that to do so would drop the standards for medical curriculum to below those for medical practice effectively dumbing down medical education. while integrative education may remain somewhat controversial within us academic medical centers, many conventional medicine educators and students recognize that, in the least, physicians must be able to communicate with their patients about the cam treatments patients seek out on their own (1315). the challenges academic medicine faces developing an integrative curriculum typically focus on introducing cam practices as factoids instead of complicated systems of knowledge. the cam practices commonly featured are herbal medicine, acupuncture, homeopathy, complementary nutrition, mind body therapies and massage (13,16,17). instruction in cam methods that might lead to change in physician practice is rare. generally, the emphasis is on the importance of improving physician patient relationship and enriching the [medical provider ] both professionally and personally (13). it is plain that tcm practitioners must know how to interact with conventional providers and the medical system in general. this simple recognition is another case of stating the obvious when it comes to integration models. we found only one reference that described what we would consider a bilateral model, e.g. recognizing the importance of educating cam practitioners to interact with conventional physicians, the public, and policy makers (18). we argue the first step in establishing integrative education models is not a matter of content but one of acculturation versus assimilation. preservation of tcm values and knowledge as tcm is integrated with conventional medicine is preferred to changing tcm so that it more closely resembles or even becomes a subset of conventional medicine. in assimilation, the values and knowledge of tcm are subordinated to those of conventional medicine, the outcome of which leaves the techniques without a theoretical framework (figs 1 and 2). the potential benefit of a medical model that integrates eastern and western medicines is documented in a 2004 article (19). the language is worth noting for its optimism and respect in a balanced discussion of how tcm and biomedicine converge to reveal new understanding and treatment approaches for functional somatic syndromes : the convergence of these biomedical models with the ancient healing tradition of tcm may provide novel perspectives in understanding these challenging and elusive disorders. sensitivity to the dominance of conventional medicine is certainly acute among tcm providers. the primacy of conventional medicine, the secondary role of other traditional medicines and the barriers this simple dyad presents to providers and patients is evident, it has been argued, in the use of terms like alternative and complementary (5). biomedical reductionist versus chinese wholistic philosophies have been scrutinized and questioned with tremendous implications for mutual comprehension and east west integration (20). the atomistic approach that has resulted in the identification of bacteria and viruses leading to effective treatment methods has no complement in the tcm approach to health where the person and the disease are inseparable and person - focused treatment logically overcomes disease by restoring individual balance. the difference is akin to the shift in modern physics, whereby quantum, chaos and complexity theories have overtaken atomistic linear models of 17th and 18th century physics for most phenomena (21). an approach that dominated scientific thought for 500 years has been supplanted because it no longer provides direction for understanding phenomena empirically observed but scientifically unexplainable. this simplified example illustrates the open - mindedness that must accompany integration of tcm and conventional biomedicine. to paraphrase pritzker (21) the intuitive practices that are highly valued in tcm this judgment may be inappropriate given that the familiar western concepts of quantification, objectivity and scientific rigor are without complement when compared with the eastern canons of intuition, tendency and dynamism in comprehending health. it seems intuitively true that tcm is intrinsically more integrative, providing a more effective model for integrating eastern and western medical cultures. conventional medicine is based on the process of gathering evidence to eliminate competing diagnoses in order to arrive at the specific correct diagnosis. tcm is based on gathering information leading to recognition of a familiar pattern for which a treatment plan that addresses the entire person is recommended. conventional medical hegemony in terms of knowledge and values must be recognized and suspended when attempting to understand tcm. qi is a fundamental chinese concept with at least 2000 years of history in chinese medicine. it is a word used by billions of chinese people everyday, yet, for the great majority of biomedical scientists, it is something unproven, even fantastic. it might be better for conventional medicine to approach tcm in the same manner as the therapeutic effects of prayer or positive guided imagery. suspension of disbelief, a foundational concept in cultural anthropology, must be the first skill applied by medical students when learning the principles of tcm. likewise, conventional medical instruction for tcm students must be delivered by western medical instructors. invaluable, forcing thinking outside the box. among the merits are the opportunity to look at conventional medicine from a different perspective the development of critical appraisal, and the acquisition of vital information about the practice of cam (22). in a bilateral educational model, knowledge and values flow in both directions, and are informed by each system 's theoretical framework. tcm content is taught by tcm experts and biomedical content is taught by biomedical experts (fig. the consortium of academic health centers for integrative medicine implementation guide for curriculum in integrative medicine (23) lists 11 modules totaling 100 h. while the guide is truly commendable and represents a step towards integration it is fundamentally assimilative. only three of the modules call for a cam provider paired with academic medical faculty. these include a 4 week long evidence - based integrative medicine course. introduction to herbal medicine two recent physician surveys (24,25) found deficits in knowledge and substantial room for improvement in knowledge, attitudes and clinical practice regarding herbs. at least one study (26) reported that 78% of cam courses taught in medical schools were taught by cam practitioners or prescribers of cam therapies. if it is the case that the preponderance of cam practitioners or prescribers of cam therapies teaching medical school cam courses are mds, then it is likely that cam knowledge and values are being lost in translation. the approach we have taken in our acupuncture and oriental medicine doctorate program is to pair conventional tcm with conventional medicine teachers. we believe, over time, synergy will yield something greater than the sum of the individual systems : teachers of integrative medicine informed by and informing both traditions.	unstated and unacknowledged bias has a profound impact on the nature and implementation of integrative education models. integrative education is the process of training conventional biomedical and traditional chinese medicine practitioners in each tradition such that patient care may be effectively coordinated. a bilateral education model ensures that students in each tradition are cross - taught by experts from the other tradition, imparting knowledge and values in unison. acculturation is foundational to bilateral integrative medical education and practice. principles are discussed for an open - minded bilateral educational model that can result in a new generation of integrative medicine teachers.
angle classified malocclusion in the sagittal plane based on the dental relationship and ignoring the skeletal relation. the disto - occlusion is categorized into division 1 and division 2 based on the spatial orientation of upper anterior teeth. apart from these basic features, there were no characteristic features pertaining to class ii div 2 in the literature. the class ii div 2 malocclusion is rare and procuring the study sample is always a difficult task. even though angle gave the classification of malocclusion in 1890s, there is still lack of clarity regarding the classical features of class ii div 2 malocclusion. moorrees., buschang., and walkow and peck analyzed the study models of class ii div 1 and div 2 and summarized that class ii div 2 malocclusion exhibited decreased intercanine width. assessed the cephalometric parameters between these two groups and found that mandibular retrognathism was a similar feature in both the groups. in the current scenario, an orthodontist should be abreast with the classical appearance of a malocclusion, as this may help the professional to choose the best treatment possible for the patient. the aim of this study is to differentiate the cephalometric and transverse arch dimensions between angle 's class ii div 1 and div 2 malocclusions, in order to understand the diagnostic features of class ii div 2 malocclusion. the diagnostic study casts and the lateral cephalometric radiographs required for the study were obtained from the hospital records of drs sudha and nageswara rao siddhartha institute of dental sciences. a total of 612 pre - treatment records were obtained, with age ranging from 14 to 25 years ; of these, 317 were class ii div 1 and 295 were class ii div 2 malocclusions. the inclusion criteria were angle 's class ii molar relationship on both the sides, with all the permanent teeth erupted, and an increased horizontal and vertical overlapping greater than 5 mm and 4 mm, respectively, for class ii div 1 malocclusion and overjet of 3 mm and 100% overbite for class ii div 2 malocclusion. the transverse arch width dimensions were measured by using digital vernier calipers (parameters used are explained in figures). an appropriate statistical test was used to assess the cephalometric variables and transverse arch dimensions between the study groups. intragroup evaluation was done at first to rule out sexual difference within the study groups. after the final verification that the sex did not have a variable difference, males and females in either of the study group were combined to evaluate the cephalometric and transverse arch dimensions. the vertical linear variables and dental variables on the cephalometric radiograph [figures 14 and table 1 ] revealed a notable variation (jarabak ratio, lower facial third and down 's mandibular plane angle). skeletal sagittal parameters skeletal vertical parameters angular skeletal vertical parameters linear dental and soft tissue parameters mean comparison of cephalometric parameters between class ii div 1 and class ii div 2 groups the maxillary transverse arch dimensions [figure 5 and table 2 ] were comparatively more in angle 's class ii div 2 group of malocclusion. a notable difference was not found with respect to the mandibular arch width parameters [figure 6 and table 2 ]. (a) maxillary intercanine width (b) basal arch width at maxillary canines (c) maxillary intermolar width (d) basal arch width at maxillary molars mean comparison of arch width parameters between class ii div 1 and class ii div 2 groups mandibular arch width parameters. (a) mandibular intercanine width (b) basal arch width at mandibular canines (c) mandibular intermolar width (d) basal arch width at mandibular canines in 1950s, studies were conducted in the department of orthodontics, university of illinois to evaluate the dental features and skeletal arrangement among various malocclusions. vallera and nelson reported that analyzing cephalometric radiograph helps the orthodontist to arrive at treatment planning. reported that the transverse arch dimensions and the apical bases, too, have a diagnostic potential. hence, we have evaluated both cephalometric and transverse arch dimensions in the present study. the sna, snb, and anb angles were measured in both the groups because of their importance in orthodontic diagnosis. to analyze the position and spatial orientation of bony bases, certain important parameters from mcnamara analysis and schwartz analysis the dental parameters included in the study were orientation of upper incisor with sella - nasion plane (ui - sn plane) and lower incisor to mandibular plane (go - me). facial angle, skeletal convexity, h - line angle, and lower lip to ricketts e - line were included as a part of the study. both the groups were shown to have class ii skeletal bases with a mild retrognathic mandible. these results were in accordance with pancherz. and isik. and contrasted with rosenblum, demisch. the skeletal vertical parameters [figures 2 and 3 ] showed a clear hypodivergent growth pattern with decreased lower facial thirds in class ii div 2 group of malocclusion [table 1 ]. the maxillary anterior teeth were absolutely retroclined in the class ii div 2 group, as per angle 's abbreviation of class ii div 2 malocclusion. lower anteriors were near normal without much difference between both the groups [table 1 ]. there was no significant difference between the two study groups with respect to the soft tissue measurements except for the linear measurement of lower lip to ricketts e - line. the lower lip was slightly behind the ricketts e - line in class ii div 2 group. this might be the reason for prominent chin, deep mentolabial sulcus, and poor retention (excessive pressure exerted by lower lip on the upper anteriors). the maxillary arch width parameters [figure 5 and table 2 ] were increased with a statistically significant difference in class ii div 2 group of malocclusion. these results educate the orthodontist to choose for non - extraction mode of therapy, unless the patient 's soft tissue integument demands for extraction. as there is normal or increased maxillary arch width and narrowing of mandibular arch width, there is more probability for the occurrence of malalignment in the lower arch in class ii div 2 group. the main setback of the study is that we have focused only on the lateral cephalograms and study models, not considering the clinical examination. the study had focused only on the local population and, therefore, some results may be contradicting with the world 's averages. the sna, snb, and anb angles were measured in both the groups because of their importance in orthodontic diagnosis. to analyze the position and spatial orientation of bony bases, certain important parameters from mcnamara analysis and schwartz analysis the dental parameters included in the study were orientation of upper incisor with sella - nasion plane (ui - sn plane) and lower incisor to mandibular plane (go - me). facial angle, skeletal convexity, h - line angle, and lower lip to ricketts e - line were included as a part of the study. both the groups were shown to have class ii skeletal bases with a mild retrognathic mandible. these results were in accordance with pancherz. and isik. and contrasted with rosenblum, demisch., and peck the skeletal vertical parameters [figures 2 and 3 ] showed a clear hypodivergent growth pattern with decreased lower facial thirds in class ii div 2 group of malocclusion [table 1 ]. this is in accordance with houston, bjork and skeiller, pancherz., karlsen, and peck. the anticlockwise rotation in class ii div 2 malocclusion may be because of lack of incisor support. the maxillary anterior teeth were absolutely retroclined in the class ii div 2 group, as per angle 's abbreviation of class ii div 2 malocclusion. lower anteriors were near normal without much difference between both the groups [table 1 ]. there was no significant difference between the two study groups with respect to the soft tissue measurements except for the linear measurement of lower lip to ricketts e - line. the lower lip was slightly behind the ricketts e - line in class ii div 2 group. this might be the reason for prominent chin, deep mentolabial sulcus, and poor retention (excessive pressure exerted by lower lip on the upper anteriors). the maxillary arch width parameters [figure 5 and table 2 ] were increased with a statistically significant difference in class ii div 2 group of malocclusion. the results of the present study were similar to buschang., staley., and sayin and turkkahraman. these results educate the orthodontist to choose for non - extraction mode of therapy, unless the patient 's soft tissue integument demands for extraction. as there is normal or increased maxillary arch width and narrowing of mandibular arch width, there is more probability for the occurrence of malalignment in the lower arch in class ii div 2 group. nowadays the treatment plan is mainly based on the soft tissue integument of the patient. the main setback of the study is that we have focused only on the lateral cephalograms and study models, not considering the clinical examination. the study had focused only on the local population and, therefore, some results may be contradicting with the world 's averages. the classical features of angle 's class ii div 2 group of malocclusion were as follows : orthognathic maxilla and a mild retrognathic mandiblemarked horizontal growth pattern with forwardly rotated mandibular baseskeletal deep biteretroclined upper incisors with near - normal lower anteriorslower lip placed slightly behind e - line with prominent chinincreased transverse maxillary values (intercanine and intermolar widths ; basal arch width at canines and molars)restricted mandibular arch width, hence, increased chances for crowding in lower arch. orthognathic maxilla and a mild retrognathic mandible marked horizontal growth pattern with forwardly rotated mandibular base retroclined upper incisors with near - normal lower anteriors lower lip placed slightly behind e - line with prominent chin increased transverse maxillary values (intercanine and intermolar widths ; basal arch width at canines and molars) restricted mandibular arch width, hence, increased chances for crowding in lower arch.	statement of problem : a thorough knowledge of the salient features of malocclusion makes the practitioner to come to a proper diagnosis and to formulate proper mechanotherapy. it also helps to predict the prognosis, prior to the onset of treatment process. among the various malocclusions, class ii div 2 occurs the least often. the literature review does not clearly describe the classical skeletal and dental features of angle 's class ii div 2 malocclusion.purpose of study : the aim of this study is to describe the unique features of angle 's class ii division 2 malocclusion.materials and methods : a total of 612 pre - treatment records (study models and cephalograms), with age ranging from 14 to 25 years, were obtained from the hospital records of drs sudha and nageswar rao siddhartha institute of dental sciences. among these samples, 317 were class ii div 1 and 295 were class ii div 2. the lateral cephalograms were analyzed by using kodak software and the arch width analysis was calculated by using digital vernier calipers.results:student's t test was used for the study. on the cephalograms, the vertical skeletal measurements and few of the dental variables showed a significant difference. on the plaster models, the maxillary transverse measurements revealed a notable discrimination between the groups.conclusion:angle's class ii div 2 malocclusion has a marked horizontal growth pattern with decreased lower facial thirds, palatally inclined upper anteriors, and remarkably increased transverse maxillary arch dimensions.
however, the majority of hematospermia is related to infection in prostate and seminal vesicle. symptom relief can be achieved for some of the patients after a course of anti - inflammatory therapy. however, symptoms in a small portion of the patients can not be improved after medication and the hematospermia persists [1, 2 ]. from august 2009 to september 2013, we evaluated and treated 20 patients with recurrent hematospermia using the pediatric ureteroscopy with satisfactory outcome. first of all, all the following work was conducted in accordance with the declaration of helsinki (1964). the study was conducted with the understanding and the consent of the patients as well as approval from the ethical committee of the affiliated ganzhou city people 's hospital of nanchang university, china. the study consists of 20 patients with age ranging from 25 to 48 years and a mean age of 36 years. the duration of the hematospermia ranges from 6 to 48 months with a mean duration of 18 months. the color in patients with longer interval of ejaculation was dark reddish or coffee colored. these include cbc, urinalysis, coagulation function index, and prostate fluid routine examination and culture. per the prostate routine exam, there were 8 cases with elevated wbc count accompanied by decreased lecithin corpuscle. per the prostate fluid culture, there were 2 cases infected with e. coli, 1 case with staphylococcus aureus, and one case with proteus syndrome. transrectal color doppler revealed one case with seminal vesicle cyst, 6 cases with inflamed prostate accompanied by calcification, and 5 cases with unilateral or bilateral seminal vesicle stones. all patients underwent the seminal vesiculoscopy using 67.5f pediatric ureteroscope under epidural anesthesia in lithotomy position. pancystoscopy was performed initially to investigate prostate, bladder, and bilateral ureteral orifices before withdrawing the ureteroscope to the position of verumontanum. under direct visualization, we initially located the opening of the verumontanum. with the guidance of an epidural catheter and mild flushing with saline, we then entered prostatic utricle for investigation. after careful examination of the prostate utricle, we withdrew the ureteroscope to the opening of the prostate utricle and looked for openings of bilateral ejaculatory ducts outside at 5 and 7 o'clock positions, under continuous saline flushing. seminal vesicle was then entered under the guidance of the epidural catheter. for patients with unclear ejaculatory duct openings, we used transurethral plasmakinetic resectoscope to resect the verumontanum to expose the openings. under the direct vision of ureteroscope, we examined carefully if there were congestion or edema on seminal vesicle mucosa, active bleeding or old blood clot, stone, anatomical abnormalities such as cyst, and new growth. for those small stones, sand - like stones, and old blood clots, we manually removed them using alligator forceps or flushed them out under pressure produced by bolus injection. for those larger stones inside the seminal vesicle, we cleared them by using holmium laser (power of 2 joules, frequency 15 hz) to powderize them followed by saline flushing. for seminal vesicle cysts postoperatively, every patient was placed a foley catheter and was treated with intravenous antibiotic for 35 days. the operative time ranged from 25 to 90 minutes with a mean duration of 35 minutes. eighteen of the 20 cases underwent exam, saline flushing, and antibiotic irrigation bilaterally while for the other 2 cases the exam and treatment were performed unilaterally. in 5 patients there were seminal vesicle stones (left side, 3 cases ; right side, 2 cases, figure 1) which were flushed out after being powderized by holmium laser. in two patients stones were found in prostate utricle (figure 2), which were extracted manually using alligator forceps. eleven cases were found to have edema and congestion in the inner wall of seminal vesicle cavities and a trace of old blood clots, consistent with seminal vesicle wall inflammatory hemorrhage (figure 4). we followed the cases for 6 to 12 months and found that that there were rare complications. no postoperative epididymitis, retrograde ejaculation, urinary incontinence, and urethral stricture were found in this study. there was one case noticed to have recurrent hematospermia 6 months after surgery with transrectal color doppler ultrasound revealing a 1.20.8 cm left sided para - seminal vesicle cyst, which was improved after a course of anti - inflammatory and physical therapy., the etiology can be divided into 3 types including organic, functional, and exigent. the most common circumstance for organic cause is seminal vesicle and prostate inflammation and infection. other circumstances are ejaculatory duct obstruction, seminal vesicle and prostate stone or cyst, seminal vesicle tuberculosis, and ejaculatory duct injury including iatrogenic factors such as transrectal prostate biopsy [39 ]. in addition the common circumstances for the functional cause are excessive masturbation, excessive sexual intercourse, or too long abstinence time. the exigent cause of the hematospermia could result from minor damage to the ejaculatory duct. seminal vesicle is the organ to store sperm with abundant vascular layer. when there is inflammatory reaction in the seminal vesicle, the mucosa became congested and edematous and the hematospermia occurs, which often leads to severe stress and fear to the patient. in most patients, the hematospermia may resolve spontaneously in a few weeks or after a course of sensitive antibiotic. however, a small portion of the patients may have recurrent hematospermia resistant to routine therapy, which poses a big challenge for clinical diagnosis and treatment [3, 4 ]. according to the literature data retrieval, conventional treatment of hematospermia includes systemic administration and local treatment, the latter including prostate massage, hot water bath, physical therapy to improve local blood circulation and promote inflammation absorption and elimination. due to the special anatomical and physiological characteristics of seminal vesicle and prostate, the treatment efficacy is often poor or not to work for the persistent hematospermia. therefore, we used the 67.5f ureteroscope to investigate the seminal vesicle for those patients with persistent hematospermia, achieving good outcome in both finding out the etiology and delivering therapy. there are different methods to enter a scope for vesiculoscopy for those patients with unclear ejaculatory duct openings. some people performed blind insertion through prostate utricle using the scope body forcefully to create a hole, while others used transurethral plasmakinetic resection of verumontanum to expose the ejaculatory duct to enter the scope under direct visualization. we performed one of our 20 cases using the aforementioned blind insertion, which is the case in which recurrent hematospermia was found 6 months after the surgery with a color doppler ultrasound revealing the left sided para - seminal vesicle cyst. we think this could be caused by seminal vasculitis resulting from blind insertion injury to the ejaculatory duct and incomplete absorption of the intraoperative saline extravasations. but because of the small number of cases and without the blind comparative study, the exact cause of the recurrence needs further investigation. in our study, we found that the most important causes of hematospermia are inflammation, stenosis, and cyst. the infection, hemorrhage, and impeded drainage from narrowed ejaculatory duct opening may contribute to the formation of stones, posing a vicious cycle because the stone will further aggravate the stenosis leading to infection and stone formation. for those cases with ejaculatory ducts stenosis with bad exposure of ejaculatory duct opening, we used the transurethral plasmakinetic resection of verumontanum to expose spacious ejaculatory duct openings, not only removed the ejaculatory duct stenosis, but also dredged the ejaculatory duct. (1) the ejaculatory duct openings are most commonly located at 5 and 7 o'clock positions outside the prostate utricle, which allow the 67.5f pediatric ureteroscopy to enter under direct visualization in most circumstances. however, it is very important to use epidural catheter as a guide instead of ureteral catheter to avoid edematous effect on the openings and bleeding which will make it difficult to find the openings afterwards. compared to the ureteral catheter, the epidural catheter is smaller and smoother with better flexibility. infusing saline through the epidural catheter can facilitate to extend ejaculatory duct openings by maintaining a pressure. we prefer a pressure at 200 mmhg and a speed at 0.2 l / min. (3) we used transurethral plasmakinetic resection of verumontanum instead of traditional vaporization resection for those patients with unclear ejaculatory duct openings, mainly because plasmakinetic resection does not produce coke attachments, reducing the likelihood of postoperative scar stenosis. (4) for patients with intractable hematospermia, we recommend prostate fluid culture and drug susceptibility before the vesiculoscopy and intraoperative irrigation of the seminal vesicle cavities using sensitive antibiotic plus saline. in summary, we think seminal vesiculoscopy using a pediatric ureteroscope develops a new way to treat refractory hematospermia and plays an important role in promoting the understanding of the cause and treatment of hematospermia. combining literature reports with our preliminary practical experience, the authors think that the pediatric ureteroscopic examination of seminal vesicle has the advantages of being simple operation, of less trauma, safe, and effective and can be widely used as one of effective methods of diagnosis and treatment of intractable hematospermia. but because of the small number of cases we carried out in the study and the lack of multicenter clinical study of large samples, especially the possible negative thermal effect of holmium laser lithotripsy on mucosa of seminal vesicle by using power of 2 joules and frequency of 15 hz, the overall and long - term efficacy still needs further observation. also, we understand that, in case of midline seminal vesicle cyst, we may be able to find the thin lining of mucosa to puncture and introduce the scope to seminal vesicle easily without a need of transurethral resection of verumontanum.	to describe a novel technique of transurethral seminal vesiculoscopy using a pediatric ureteroscope in the diagnosis and management of persistent hematospermia, a retrospective study was carried out for 20 patients with recurrent hematospermia whom we evaluated and treated using a 67.5f (6f front end and 7.5f rear end) pediatric ureteroscope from august 2009 to september 2013. for the 20 patients, the age ranges from 25 to 48 years with a mean age of 36 years. the duration of the hematospermia ranges from 6 to 48 months with a mean duration of 18 months. transurethral seminal vesiculoscopy was successfully performed in the 20 cases and the mean operative time was 35 min (ranges from 25 to 90 min). among the 20 patients, 11 patients were found to have seminal vesiculitis, five were with seminal vesicle stone, one was with prostatic utricle stone, one was with prostate cyst, and one was with ejaculatory duct obstruction. the mean follow - up period was 7 months (ranged from 6 to 12 months). hematospermia in 19 cases disappeared after the surgery and only in one patient the hematospermia recurred 6 months after the surgery. the cure rate was 95%. this study indicated that transurethral seminal vesiculoscopy could be performed easily using a semirigid pediatric ureteroscope with few complications and is an effective therapeutic approach for persistent hematospermia.
	we explore the limits of modifying metal work functions with large molecular dipoles by systematically increasing the dipole moment of archetype donor acceptor molecules in self - assembled monolayers on gold. contrary to intuition, we find that enhancing the dipoles leads to a reduction of the adsorption - induced change of the work function. using atomistic simulations, we show that large dipoles imply electronic localization and level shifts that drive the interface into a thermodynamically unstable situation and trigger compensating charge reorganizations working against the molecular dipoles. under certain circumstances, these are even found to overcompensate the effect that increasing the dipoles has for the work function.
rupture of an atherosclerotic plaque is the primary underlying cause of most acute coronary events and strokes [1, 2 ]. a vulnerable plaque (vp) is considered to be prone to rupture. it is characterized as a large pool of lipids with an overlying thin fibrous cap that is heavily infiltrated by macrophages [3, 4 ]. besides plaque components, morphological characteristics such as lesion eccentricity compared with concentric plaques, eccentric plaques are usually associated with not only cerebrovascular and cardiovascular symptoms, but also the progression of atherosclerotic disease [57 ]. to predict vp rupture, methods such as intravascular ultrasound (ivus) and magnetic resonance imaging (mri) have been used to identify directly vp features in vivo in some experimental studies [8, 9 ]. however, the lower resolution of these modalities has restricted their application for evaluation of the microstructural features of atherosclerotic plaques. optical coherence tomography (oct) is a novel intravascular imaging modality that uses near - infrared light with high image resolution (10 m). oct allows for unparalleled imaging of the plaque composition, such as fibrous - cap thickness (fct) and lipid content. it has been considered to be one of the most promising tools to detect the key features of plaque rupture and to assess the vascular response to stenting [11, 12 ]. the further study of the development of vps and intra - arterial treatment of stabilizing plaques is important, and an appropriate animal model is required. the aim of the present study was to establish an animal model with morphological and structural characteristics similar to those seen in human vps. we also wished to evaluate the reliability of oct technology for repeated observation of lesion features in vivo. the study protocol was approved by the ethics committee of the second affiliated hospital of harbin medical university (harbin, china). sixteen adult male new zealand white rabbits (3.0 kg) were housed continuously at the animal care facilities of the second affiliated hospital of harbin medical university. sixteen rabbits were fed a 1% high - cholesterol diet (hcd) for 2 weeks before and 12 weeks after endothelium denudation. they were divided into two groups according to the pattern of vessel injury. in group a (n = 8), eccentric endothelial denudation was induced by an inflexible modified metal needle which had a blunt and rounded front head (diameter, 0.0285 inch) ; a partial and longitudinal injury to the vascular wall was generated (figure 1). for rabbits in group b (n = 8), they were used as controls to compare the normal histological and oct appearance of the vascular wall to that of diseased rabbits. after 2 weeks of the 1% hcd diet, rabbits were anesthetized with ketamine (5 mg / kg), xylazine (5 mg / kg), and acepromazine (0.75 mg / kg) given via the intramuscular route. rabbits were heparinized and a median incision in the neck region made under local anesthesia induced by 1% lidocaine hydrochloride. after the right common carotid artery was exposed surgically, a 1 - 2 mm arteriotomy was made at the external carotid artery. oct was obtained using a time - domain oct system (light lab imaging, westford, ma, usa) with an image wire (crossing profile, 0.014 inches ; light lab imaging) at a pullback speed of 3 mm / s during intermittent flushing with 0.9% (physiological) saline through the guiding catheter to displace blood transiently. after withdrawing the oct image wire, a 3-f fogarty catheter was introduced through the same cut - down in the rabbits of group b. the balloon was then inflated with 0.3 ml saline, and the catheter gently retracted to the external carotid artery as described previously. in group - a rabbits, the modified metal needle replaced the balloon and was used to make a partial and longitudinal endothelial denudation. vessel injury was completed thrice in each rabbit of groups a and b. the balloon or needle was then removed. one hour later, the oct image wire was introduced through the same cut - down again. with the saline flush, serial oct images were obtained. after pulling out the oct image wire, postoperative therapy was aspirin (100 mg, p.o.) and ampicillin (250 mg, i.m.) for the next 3 days. twelve weeks later, oct was repeated immediately on the same carotid artery before killing. oct was undertaken thrice before injury as well as 1 h and 12 weeks after injury. the procedures detailed above were undertaken in the right common carotid artery of each rabbit (figure 1). each carotid artery was cut into three segments (proximal, middle, distal). the proximal end of each segment was marked with a suture ligature on its anterior side to avoid confusion. samples were embedded in paraffin and cut into serial transverse sections of 5 m for histopathological and immunohistochemical analyses. hematoxylin and eosin (h&e) and masson 's trichrome staining were carried out to show structural changes in vascular walls and to detail the plaque components. if atherosclerotic lesions were identified by morphological analyses, additional immunohistochemical staining of macrophages and smooth muscle cells (smcs) was carried out as described by tian.. staining with picrosirius red was also done to identify the type of collagen fibers within the fibrous caps of plaques under polarized light (type i, orange ; type iii, green). plasma levels of total cholesterol (tc) and triglycerides (tg) were detected using standard enzymatic techniques. levels of high - density lipoprotein - cholesterol (hdl - c) and low - density lipoprotein - cholesterol (ldl - c) were also measured. normal oct and the corresponding histology cross - sections of carotid arteries were acquired from uninjured and normal - diet rabbits. acute vessel injury was evaluated by oct after balloon and needle injury. according to previous histology results, mild injury was defined as endothelial denudation with or without punctated breaks in the internal elastic lamina (iel) and without medial laceration. severe injury was defined as disruption of the media and even large lacerations of media extending through the adventitia. plaque type as well as the length of the plaque and lipid core were determined by oct as described previously [10, 15, 17 ]. fct was measured at five locations of the fibrous cap on each cross - sectional image and the average value calculated. measurements of the mean fct, the mean area of the lumen, and the mean value of the arc of the lipid core were calculated at 1 cross - sectional image per 1 mm interval of lesion. the percentage of luminal stenosis was calculated by comparing the mean lumen area of the lesion to that of a reference site using the following formula : (1)% stenosis=[1(mean lumen arealesionlumen areareference)100 ]. within 10 mm of the distal and proximal end of the plaque the reference lumen area was measured as the mean value between the distal and proximal reference lumen area. finally, the presence of thrombus and plaque fissure was also noted. oct images were analyzed by two independent observers who were blinded to the procedural characteristics. the right carotid artery bifurcation and its big branch were used as the internal anatomic markers for localization to match histological and oct cross - sectional images as well as to match oct images at different time points. atherosclerotic plaques identified at histology were classified according to criteria set by the american heart association (aha). the area and thickness of cross - sectional media and plaques were derived from the inner sides of the outlines of the lumen and the adventitia. the eccentricity index (ei) was calculated as the ratio between the minimal and maximal thickness of media and plaque (mpt) at the cross - sectional image of maximal plaque formation. the plaque was defined as an eccentric plaque if the ei was 0.5 and as a concentric plaque if < 0.5. furthermore, the area percentage of macrophages foam cells and smcs was quantified (at 100 magnification) within digitized immunohistochemically stained plaques. the measured region of interest included the maximum amount of macrophages and an overlying fibrous cap. all slides were analyzed by computerized planimetry using image - pro plus 4.5 (media cybernetics, silver spring, md, usa). the student 's t - test was done to evaluate the level of significance for a given measurement between the two groups. the intraclass correlation coefficient (icc) was calculated to evaluate the agreement between oct and histological findings. statistical analyses were done using sas ver9.1.3 (sas institute, cary, nc, usa). of 4 rabbits that did not complete the experiment protocol, 2 from group a died prematurely from anesthesia accidents, and 2 from group b had chronic total occlusion (cto) in the distal right carotid artery identified by histological analyses. cto lesions comprised mainly macrophage foam cells and extracellular lipids (figure 3(f)). the baseline levels of tc, tg, hdl - c, and ldl - c were 2.46 1.51, 1.17 0.37, 0.87 0.33, and 0.66 0.45 mmol / l, respectively. the levels of tc, tg, hdl - c, and ldl - c increased to 37.86 1.93, 16.71 4.46, 10.53 3.04, and 28.08 5.26 however, a significant difference was not observed in any of these measured plasma lipid levels between the rabbits in group a and group b (supplementary table, available at doi:10.1155/2012/469726). mild injury and severe injury were found in both injury groups (figure 2). further statistical comparisons between the two injury patterns were not carried out because an accurate number of total injured cross - sections was not available. tissue sections from control rabbits showed layering of normal carotid arteries (figures 3(a1)3(a4)). the media contained abundant collagen (figure 3(a2)), smcs (figure 3(a3)), and few macrophages (figure 3(a4)) and they showed high backscattering intensity on oct images (figure 3(a1)). the adventitia (which consists primarily of loose connective tissue) exhibited the low backscattering intensity. the intima could not be detected because its thickness was < 10 m. in the experimental groups, the oct appearance of one lesion (figure 3(b1)) showed highly reflective subintimal areas. these were documented as type - iii lesions and comprised smcs (figure 3(b3)) and small extracellular accumulations of lipids (figure 3(b4)) at histology. a total of 23 lesions in 12 carotid arteries with a fibrous cap and large extracellular lipid cores were documented as advanced plaques (type - iv lesion) according to criteria noted previously. oct images of type - iv lesions (figure 3(c1)) were characterized by signal - poor regions (lipid pools, figure 3(c3)) with an overlying thin fibrous cap (figure 3(c3)) which consisted mainly of collagen fibers (type i, orange ; type iii, green ; figure 3(c4)). the oct appearance of type - v plaques was similar to that of type - iv lesions but with a thickened fibrous cap (figure 3(d4)) overlaying a lipid core. the oct and corresponding histological images of type - vi plaques (figures 3(e1)3(e4)) showed a visible adherent white thrombus attached to a lipid - rich fibrous plaque. additionally, neovessels, cholesterol crystals, and intraplaque hemorrhage were observed by histopathological examination in the atherosclerotic lesions of the experimental groups (figures 3(g)3(i)). cholesterol crystals could not be detected by oct because of the barrier of lipids and thick caps. oct was used to correctly diagnose 255/261 (97.7%) as advanced lesions and misdiagnosed 6/261 (2.3%) type - iii cross - sections as type - iv lesions. the oct features of advanced lesions in group a (n = 12) and group b (n = 11) were collected and compared in vivo (table 1). significant differences between group a and group b were not observed with respect to the average and minimum fct, lipid - core length, plaque length, average lumen area, and the average stenosis ratio. atherosclerotic plaques with maximum formation were selected from each right carotid artery of rabbits in the two groups (figure 4). twelve plaques were selected and the microstructures between the two groups were compared (table 2). although the two injury patterns could initiate eccentric lipid - rich plaques, those from the needle - injury group exhibited a higher ei, thinner fct, and greater infiltration of inflammatory cells. plaques from the balloon - injury group had greater lipid content in the mean plaque area and mean plaque thickness (mean, minimum, and maximum), but there was no significant difference in minimum fct and lumen area. thirteen representative oct and corresponding histology cross - sections were selected from 23 advanced lesions to ascertain the agreement between oct and histopathology. compared with histopathology, oct measurement of the mean plaque area showed an acceptable correlation, whereas measurements of fct and the lipid arc by oct were extremely accurate (table 3). because of the shrinkage of the lumen of tissue sections, oct measurements of lumen area were significantly different compared with the corresponding histological examination (table 3). in addition, mean plaque thickness and the ei could not be detected by oct due to its limited penetrating power of lipid content. in the present study, we modified the pattern of denudation of endothelial cells and developed a more common model of eccentric vps that reproduced the features of human vps. in the development of atherosclerotic diseases, balloon injury and hyperlipidemia are thought to cause injury to endothelial cells and result in further atherosclerotic lesions [20, 21 ]. a combination of mechanical damage and lipid toxicity has been shown to accelerate atherogenesis and generate types of atherosclerotic plaques within 812 weeks in rabbits [14, 15, 22 ]. balloon injury might cause concentric denudation of endothelial cells lining the luminal site of vessel walls. after endothelial injury, a series of pathologic processes lead to the development of atherosclerotic plaques at injury sites. our results showed that balloon injury could generate eccentric plaques, but their ei was low and the morphology of plaques was concentric (or approx. hence, we chose a modified needle which had a blunt and rounded front head to cause partial and longitudinal injury to vascular walls. after 12 weeks of an hcd, histology results and oct showed that the needle - injury group could generate more eccentric plaques which had a similar morphology to that of human vps compared with the balloon - injury group. in humans, a thin - capped fibroatheroma (tcfa) is defined as a plaque with a thin fibrous cap (thinnest part, 65 m) overlying a large lipid pool (2 quadrants). this critical threshold of 65 m has been derived from the fragments of atherosclerotic plaques observed in postmortem examinations. the thinnest fct derived from the fragments of plaques in the needle - injury group (48.20 25.08 m) was close to the values noted in humans (65 m) and rabbits (57 15 m). in addition, the oct - detected average fct in the two groups was close to the critical fct of rupture - prone coronary plaques in humans (< 80 m for the thinnest fct and < 188 m for the most representative fct). furthermore, those plaques had a large lipid arc of 2 quadrants. hence, the plaques of the needle - injury group were equipped with the representative features of human rupture - prone plaques. in all 12 rabbits, we observed advanced atheromatous lesions by oct and histological examination. human autopsy studies have shown that advanced lesions (types iv and va) are especially prone to disruption. those tears may occur more frequently in regions of lesions with greater infiltration of macrophage foam cells. indeed, local inflammation can induce the expression of collagenase and inhibit the expression of proteolytic inhibitors, thereby rendering the fibrous cap weak and susceptible to rupture [23, 27 ]. by contrast, vascular smcs may be important in maintaining plaque stability through the formation of a firm fibrous cap. in the present study, however, plaques in the needle - injury group had more macrophage foam cells compared with those of the balloon - injury group. in addition, the neovascularization of plaques and intraplaque hemorrhage also increase the vulnerability of plaques [28, 29 ]. in the present study, hence, erythrocytes might intrude from the lumen into the plaque, resulting in intraplaque hemorrhage. the needle - injury group could generate more eccentric, lipid - rich plaques whose caps were thin, fibrous and infiltrated with macrophages. the present study showed that oct was a useful tool to identify the degree of injury immediately after endothelial denudation and for evaluation of the prognosis. previous histological examination indicated that severe injury to the vascular wall resulted in a larger neointimal lesion compared with a mild degree of injury. it is possible that removal of the medial barrier facilitated the intimal migration of adventitial cells. previous results were identified according to the histological examination or autopsy and could not be used to detect immediate vascular injury in situ of the same vessel due to the limitation of intravascular imaging facilities. oct could be used to correctly detect advanced lesions and their microstructural features in rabbits. for advanced plaques (type iv) in humans, intracoronary oct has been reported to accurately identify lipid pools, acute thrombus, and fct. in the present study, oct provided clear imaging and quantitative analyses of lipid pools and fct in vivo in rabbits. the correlation observed between histological and oct measurements may allow evaluation of the effects on fct and the lipid arc in plaques under different drug treatments without killing the animals. in the present study, most rabbits underwent final oct at the distal region of the common carotid artery (arrow (2) of figure 1). after ligating the distal end of the common carotid artery, the big - branch vessel could guarantee unobstructed blood flow. hence, it provided the possibility of further imaging which could be undertaken at the site of the big - branch vessel (arrow (3) of figure 1). the unique anatomic structure of the carotid artery in rabbits permits serial in vivo monitoring of the evolving characteristics of the same lesion over time. our previous animal study demonstrated the feasibility of oct to evaluate the same artery twice in vivo. utilizing this rabbit model, oct could be used to not only accurately evaluate vp features, but also to observe and measure the same plaques more than once. this model could guide the design of invasive imaging approaches for the detection and treatment of vps. one limitation of this rabbit model was the absence of normal human - like layering of the intima and media in the coronary vessel wall in oct images for normal rabbits. this could be because the carotid artery of rabbits is elastic whereas that of humans is muscular. besides, oct shows poor penetration of tissue (1 - 2 mm) and high attenuation in lipid plaques. the binding between the media and adventitia was also unclear in oct images in this model. oct could not be used to detect definitively the thickness of the media and neointima. we could not recommend measurement of the thickness of the media and neointima by oct in this rabbit model. one way to overcome this limitation of penetration is to combine oct with other modalities capable of imaging through blood. ivus can be used to detect binding between the media and adventitia, and has been shown to provide additional information that may be related to plaque vulnerability. another limitation was that the method of triggering plaque rupture was not done before killing. therefore, further studies are needed to evaluate the appearance between ruptured and nonruptured plaques in vivo in oct images. we developed an animal model of vps with more eccentric plaques and a similar morphology to that seen in human vps. oct is a useful tool to detect the degree of acute injury as well as advanced lesions and their microstructural features in vivo. a combination of oct and this modified model of vps could be an important research tool for the understanding and treatment of vps in acute coronary syndromes.	we aimed to establish a rabbit model of vulnerable plaques (vps) with the morphology and component characteristics of human vps and to evaluate the microstructural features of vps in vivo using intravascular optical coherence tomography (oct). twelve rabbits underwent endothelial denudation of the carotid artery and consumed a 1% high - cholesterol diet (hcd). they were equally divided into two groups : group a (modified needle injury) and group b (balloon injury). oct was undertaken thrice before injury as well as 1 h and 12 weeks after injury. the degree of acute artery injury after endothelial denudation was detected by oct. twelve weeks after injury, oct showed that both groups generated vps which had thin fibrous caps and a large lipid core, whereas plaques in group a had smaller lipid arcs (p < 0.0001). histological findings demonstrated that a larger eccentricity index (ei) (p < 0.05) and greater infiltration of macrophages (p < 0.05) in group a than in group b. qualitative and morphometric analyses of plaques showed a significant correlation between histological and oct measurements. a combination of modified endothelial denudation and an hcd in rabbits produced more eccentric lesions similar to those seen in humans. these data suggest that oct could be a useful tool for evaluation of the degree of injury and vps in vivo.
endogenous peptides, protein, and oligonucleotides are among the main drugs which attract much attention because of their great potentials in treating chronic diseases. however, the extreme in vivo environment of human body has always limited the therapeutic applications of these substances [2, 3 ]. polymeric nanoparticles have attracted much attention as delivery systems due to their ability in overcoming the physiological barriers and protecting and targeting the loaded substances to specific cells [4, 5 ]. naturally occurring polymers such as chitosan (cs) cs is a biodegradable polysaccharide, and it is derived from deacetylation of chitin. apart from its biocompatibility, the low toxicity, hemostatic, and bacteriostatic properties also contribute to its various applications in pharmaceutical field [911 ]. several anions have been investigated to crosslink cs like sodium sulphate and dextran sulphate (ds). ds is able to modify protein and sirna entrapment efficiency (ee) without the use of hardening agents and control the rate of drug release due to its high charge density. besides ds is a cheap material, it produces mechanically more stable nanoparticles compared to the pentasodium tripolyphosphate (tpp) [16, 17 ]. several studies had reported the unique features of chitosan nanoparticles (cs nps) using ds. however, the modulation of preparative parameters on their physical characteristics is still not fully investigated, for example, the influence of ds steric hindrance on the electrostatic attraction between cs and bsa. furthermore, the determinant of a successful drug delivery system is dependent on its physical characteristics and stability. therefore, the objectives of present study were to modulate preparative parameters to obtain nanosized particles of cs nps and to determine their colloidal stability at different storage temperatures and in various suspending mediums. low molecular weight chitosan (70 kda with the degree of deacetylation 75%85%), acetic acid glacial, phosphate buffered saline (pbs), bovine serum albumin (bsa, 46 kda), and bradford reagent was purchased from sigma - aldrich inc., usa. double - stranded sirna (sense : 5-gauuauguccgguuauguauu-3, antisense : 3-uacauaaccggacauaaucuu-5) was purchased from thermoscientific dharmacon, usa. protein ladder (high range), laemmli sample buffer, 10x tris / glycine / sodium dodecyl sulfate buffer, ammonium persulfate, tetramethylenediamine (temed), 2% bis solution, and 40% acrylamide solution were purchased from bio - rad, usa. tris - hcl buffer was obtained from invitrogen, usa. all other chemicals used were of analytical grade. cs and ds solution were dissolved in 1% v / v acetic acid and distilled water, respectively. ph of cs solution was adjusted to ph 4 by adding 1 m naoh or 1 m hcl. ds solution (0.05%, 0.1%, 0.15%, 0.2%, and 0.25% w / v) was added dropwise into cs solution (0.1% w / v) under magnetic stirring (wisestir digital multipoint magnetic stirrer ms - mp8, daihan scientific, korea) at 250 rpm for 15 min to form nanoparticles. the resultant nanoparticles were washed and harvested by ultracentrifugation (optima l-100 xp ultracentrifuge with a rotor nv 70.1, beckman - coulter, usa) twice at 12 500 rpm for 15 min at 10c. for bsa association into cs nps, bsa was dissolved in cs solution (0.1% w / v, ph 4) to produce a final concentration of 1 mg / ml. for sirna association into cs nps, 3 l of sirna (15 g/l) in deionized water was added to ds solution (0.05%, 0.1%, 0.15%, 0.2%, and 0.25% w / v) before adding this dropwise to cs solution (0.1% w / v). electrophoretic mobility measurements (e) of cs nps were performed with a zetasizer nano zs (malvern instruments, uk) and e was measured against waiting time. particle size, surface charge, and polydispersity index (pdi) of freshly prepared cs nps were measured using a zetasizer nano zs (malvern instruments, uk), based on the photon correlation spectroscopy (pcs) techniques. morphological characterization of unloaded cs nps, bsa / sirna loaded cs nps (ds : cs weight ratio of 0.5 : 1, 1 : 1) was carried out by using transmission electron microscopy (tem), tecnai spirit, fei, eindhoven (the netherlands). bsa / sirna loaded cs nps were separated from the solution by ultracentrifugation (optima l-100 xp ultracentrifuge with a rotor nv 70.1, beckman - coulter, usa) at 14000 rpm for 30 min. bsa content in the supernatant was analyzed by a uv - vis spectrophotometer at 595 nm (u.v-1601 ; shimadzu, japan) using the bradford protein assay as per manufacturer instruction. sirna content in the supernatant was analyzed by a uv - vis spectrophotometer at 260 nm. the bsa / sirna entrapment efficiency (ee) was calculated using the following equation:(1)ee(%)=(total amount of bsa / sirna added)(free amount of bsa / sirna)(total amount of bsa / sirna added) 100. freshly prepared cs nps (made from 0.05% and 0.1% w / v of ds and cs solution, resp.) were centrifuged at 12 500 rpm for 15 min prior to storing. after ultracentrifugation, the obtained pellets were resuspended in either distilled water (measured ph of 6.6) or pbs ph 7.4. the particle size and surface charge were measured at predetermined storage time durations (0, 1, 2, 3, 5, 8, and 14 days), and at either ambient temperature or 4c. the release of bsa / sirna was determined from cs nps with the highest ee (ds : cs ratio 1 : 1, ee = 98% 0.2 and 95 4, resp.). bsa / sirna loaded cs nps were suspended in tris - hcl buffer solution (ph 7.4, 4 ml) and placed on a magnetic stirrer with a stirring speed of 100 rpm at 37c (ms mp8 wise stir wertheim, germany) for 48 h at 37c. at predetermined time intervals (0, 0.5, 1, 2, 4, 6, 12, 20, 24, and 48 h), samples were centrifuged at 14 000 rpm for 30 min at 10c. then, the supernatant was decanted and replaced with an equivalent volume of fresh buffer solution. the amount of released bsa / sirna in the supernatant was analyzed by a uv - vis spectrophotometer (u.v-1601 ; shimadzu, japan) at a wavelength of 280 and 260 nm, respectively. the integrity of bsa released from cs nps was determined by sds - page (12% resolving and 10% stacking gel) using mini - protein system (bio - rad, usa). bsa samples were mixed with laemmli sample buffer in 1 : 1 ratio and heated at 95c for 5 min. samples (15 l) were loaded into the wells and the gel was run using a mini - protein system tetra cell at a constant voltage of 150 v for 90 min with a running buffer containing 25 mm tris, 192 mm glycine, and 0.1% sds at ph 8.3. the sample bands were stained for 40 min with 0.1% coomassie blue solution containing 40% acetic acid and 10% methanol, followed by staining overnight with a solution of 40% acetic acid and 10% methanol. all the data were presented as mean standard deviation (sd). statistical analysis (anova test and tukey 's posthoc analysis) was performed by using the statistical package for the social (spss) programme version 15. figure 1(a) demonstrates the results of electrical mobility (e) against waiting time. from the graph, it could be observed that the e remained plateau and constant after 30 min. this demonstrates that the formation of stable electrical double layer (e.d.l.) was not instantaneous but required some moments. the effects between cs concentration and ds final concentrations on the size of cs nps are presented in figure 1(b). it was observed that most of the cs nps with the size of less than 500 nm were obtained at a low cs concentration (0.1% w / v). an increasing trend in particle size could be observed with increasing the ds concentration from 0.05 to 0.25% w / v. in general, ds concentration of 0.05% w / v (low concentration) produced nanoparticles with particle size less than 500 nm. contrary to that, large nanoparticles (> 1000 nm) were obtained when concentration of both polymers was increased to 0.25% or above. based on the results, ds concentrations from 0.05 to 0.20% w / v were selected for the following studies. furthermore, an increase in the ds : cs weight ratio (higher density of negative charges from ds present in the system) led to an increase in particle size but a decrease in particle surface charge (table 1 (above)). as the cs weight exceeded the mass of ds, a positive value of + 56.2 1.5 mv was obtained. however, particle surface charge decreased to 34.7 4.34 mv when more negatively charged ds was added. it was continuously decreasing when the ds : cs weight ratio had reached to 2.5 : 1. this was expected to be due to an excess of ds molecules accumulated on the surface of nanoparticles. table 1 (below) shows that ds 0.2% w / v possessed the largest particle size after being loaded with bsa. particle size for ds at concentration of 0.1 and 0.15% w / v was also larger than the empty ones (p 90%) for all ds : cs ratios. storage temperature and suspending medium were found to be the factors that could influence the stability of cs nps. cs nps were labile and tend to destabilize at ambient temperature but withhold this labile behavior when cool environment (24c) was provided. in addition, cs nps had better stability in distilled water than in pbs which might be due to hydrogen bonds that formed between water molecules and ionizable groups of cs nps.	chitosan nanoparticles (cs nps) exhibit good physicochemical properties as drug delivery systems. the aim of this study is to determine the modulation of preparative parameters on the physical characteristics and colloidal stability of cs nps. cs nps were fabricated by ionic interaction with dextran sulphate (ds) prior to determination of their storage stability. the smallest cs nps of 353 23 nm with a surface charge of + 56.2 1.5 mv were produced when cs and ds were mixed at ph 4 and with a ds : cs mass ratio of 0.5 : 1. an entrapment efficiency of 98% was achieved when bsa / sirna was loaded into the nanoparticles. the results also showed that particle size and surface charge of cs nps were slightly changed up to 2 weeks when stored at 4c. greater particle size and surface charge were obtained with increasing the concentration of ds. in conclusion, nps were sufficiently stable when kept at 4c and able to carry and protect protein.
the first to report about microbial butanol production was pasteur in 1862 (pasteur 1862). pasteur named the culture vibrion butyrique most probably though it was a mixed bacterial culture comprising at least one clostridium strain (drre 2007). while scientific interest was documented by some further reports, commercial interest was only triggered in the beginning of the 20th century by the need for synthetic rubber production (jones and woods 1986). at this time he isolated a new bacterial culture, readily fermenting starchy material into acetone and butanol. starting from there, an industrial solvent production process was developed, based on available plants for ethanol fermentation. interestingly, weizmann was a chemist by training and he set about training himself to become a microbiologist as necessary. this underlines the close connection of industrial microbiology with chemistry, which tends to get a bit lost with the advent of genetic engineering and changing study courses. nevertheless, renewed close collaboration of these two sciences is highly relevant now more than in the past, as exemplary outlined by the thoughts of dusselier, mascal and sels (2014), who provided a chemist 's view of the biorefinery. in 1915, a patent was issued claiming acetone butanol fermentation with c. acetobutylicum, and industrial production on large scale commenced in 1916. the outbreak of wwi led to a high demand of acetone for smokeless powder production, shifting the interest from butanol as a product to acetone. during the war, the requirement for acetone resulted in the accumulation of butanol as an unwanted by - product of the fermentation which was stored. after the war, butanol again became a commercially important chemical this time for the production of quick - drying lacquers, for the rapidly growing automobile industry. starch was originally the carbon source on which the fermentation was based. however, molasses became cheaply available in large quantities and became the main carbon substrate for solvent production. this became possible only because new bacterial strains, readily fermenting sugars, had been isolated by then. during wwii, the importance of microbial processes for acetone and butanol production declined rapidly after the war. one reason was that petrochemistry was gaining scale and importance, and the production of solvents from petroleum became very cheap. another reason was that molasses became increasingly sought after for the feed of cattle, leading to a significant price increase of the substrate. today, 50 years later, the weizmann process and modern derivatives of it are gaining interest and importance once more. this time, the driver is the need for sustainability the desire to shift back from petroleum to readily renewable resources, decreasing the carbon footprint and avoiding toxic chemicals as far as possible. figure 1 depicts very generally the concept of microbial chemical production in biorefineries. a renewable resource, which can be biomass-, or waste - derived is converted into a substrate stream amenable to microbial conversion. importantly, the product must be purified before reaching the market. while clearly the bioprocess is central, and very often of most interest to researchers, the price of the chemical or fuel produced, which decides over failure or success of the approach, is dictated mainly by substrate and purification costs (porro. this thematic issue will work along those lines, highlighting efforts not only on the microbiology itself, but on all steps of the process chain. a renewable resource is converted into a substrate stream, which can be microbially converted into a base chemical. the chemical has to be purified before it can be upgraded to more advanced products. a general overview about past achievements and pending challenges of (acetone-) butanol fermentation is given by moon. they set the process into its historic and economic contexts in relation to the newest developments. the current frontiers of the process, with a particular focus on cell and metabolic engineering, are given by chen and liao (2016). as outlined before, substrate and purification costs are decisive for the commercial success of any chemical (and concomitantly microbial) process. while starch was historically the substrate of choice (and is still a major substrate for bioethanol production outside the countries which can grow sugar cane), a shift to molasses as a cheap waste product kept the profitability of the process high. with the opening of additional markets for molasses, its price increased, significantly contributing to the death of microbiological acetone and butanol fermentation. nowadays, lignocellulose is seen as a promising carbon source for chemicals (hasunuma. it is abundant and not in direct competition with food production and a lot of effort is therefore being invested into the development of its use. a relevant example for biobutanol production is the proposition of simultaneous saccharification and fermentation of pretreated corn stover (dong. an innovative co - culture system of recombinant escherichia coli for the bioconversion of cellulose hydrolysate has been suggested by saini (2016). 2016), give a general overview about the use of corn stover as carbon source with a focus on economical aspects. finally, the use of gaseous substrates for the fermentation of solvents is a very different approach (drre 2016), which can be based on various sources for carbon - rich gas, opening ways to valorize industrial off gases or gasified carbon - rich waste streams. a major focus of this thematic issue will therefore be to present different views and approaches for the microbial process itself, from its history to the high end of microbial cell design. the history and importance of this process have led to a range of studies and reports, which can be used as a model for developing other microbial production processes. this thematic issue aims therefore to show the reader how modern technologies and new discoveries in all fields of microbiology can be employed for industrial needs. they are spore forming bacteria what has medical relevance for some of the species and which has technical relevance in context of solvent fermentation. (2016) used flow cytometry to evaluate population heterogeneity with particular focus on spore numbers and quality during a solventogenic fermentation process. spore formation constitutes a special challenge for the provision of the inoculum for large - scale microbial processes. sandoval - espinola, chinn and bruno - barcena (2015) report about an optimized strategy for inoculum preparation. the influence of calcium and zinc ions on fermentation performance has been characterized by wu. however, the tolerance of the production organisms to high solvent concentrations remains a major challenge. high concentrations are needed to decrease purification costs, but are detrimental to the survival and productivity of the cultures. peabody and kao (2016) give an overview of the recent progress in increasing microbial solvent tolerance. it is a complex property of living cells, which is only partially understood and rational engineering approaches have therefore been difficult. an interesting approach by jones, venkataramanan and papoutsakis (2016) shows that an increase of butanol tolerance is achieved by overexpression of two stress - responsive small, non - coding rnas. discovery of these new signalling molecules has brought a completely new level of cellular organisation to light, with implications for process understanding and bioengineering. this indicates once more that the common separation between fundamental and applied research is in most cases not useful. fundamental research is not only a prerequisite for the development of applications, but engineering cell factories can lead to many fundamental discoveries. essalem and mitchell (2016) describe the characterization of a clostridial glucose - mannose phosphotransferase system, shedding light on sugar uptake and guiding further metabolic engineering approaches which bring us to the next field of interest. new methods for mutagenesis are being developed, such as an inducible transposon system for efficient random mutagenesis furthermore, genetic engineering and thereby rational strain design are gaining importance also for abe fermentation. the understanding of the metabolism of the cells is the basis for rational strain design. metabolic modelling helps to identify genetic targets, which might very well be non - intuitive. dash, ng and maranas (2016) give a concise overview about current developments and applications in the metabolic modelling of clostridia. liao, seo and lu (2016) shed some light on approaches for systems - level modelling of the entire fermentation process. while abe processes relied historically on clostridia as productions hosts, other microorganisms are now attracting attention the exploitation of biodiversity becomes important. branduardi and porro (2016) present a general outline of the requirements and provisions needed for a viable cell factory for butanol production. they conclude that yeasts might very well be alternative production hosts, thereby agreeing with kuroda and ueda (2016) who summarize recent engineering approaches using baker 's yeast for advanced biobutanol production. escherichia coli has also been suggested as an alternative production host (saini. another aspect in this context is that in nature many metabolic tasks are split over different species. digestion of cellulose and hemicellulose and conversion of the carbon into volatile organic acids (which are the actual nutritional basis of the ruminant) are achieved by a complex community of microorganisms carrying out different biochemical reactions (sauer, marx and mattanovich 2012). such concepts are in their infancy in the field of industrial microbiology, but are now being considered for microbial butanol production, as shown by saini. friedl (2016) summarizes various aspects of possible downstream processing strategies. in situ recovery of the solvents intelligent bioreactor design can increase productivity from the downstream side of the process by influencing the microbe (li. clostridial fermentations proceed via two very distinct stages an acidogenic phase, where organic acids such as butyric acid are the main products, followed by the solventogenic phase, when the acids are converted to the corresponding alcohols. historically, the acids have been of minor industrial importance, while the alcohols were sought after. however, the acids can be converted in many ways to alcohols or other products of interest as outlined by sjblom. the message here is to keep the eyes open to find creative solutions to pending problems based on nature 's capabilities. about 100 years after the first industrial exploitation of the clostridial acetone butanol fermentation this process is still of high interest, commercially as well as scientifically. it still poses many questions, holds a lot of secrets, and many aspects have yet to be understood. future generations of scientists will have enough to do to understand and optimize the underlying metabolic processes. at the same time, it is one of the oldest and best studied microbial chemical production processes, so we can learn a lot from its development. the centennial year is a good point to stop for a moment, look at this development and think about what it has to teach us for many other endeavours. this is of special relevance now, as our societies strive to free ourselves from our dependence on petroleum. i hope that this thematic issue of fems microbiology letters contributes to this thinking, and the development of new ideas based on past experience. the author 's research on microbial chemical production is being financed by the christian doppler research society, by the austrian research promotion agency ffg and by the federal ministry of science, research and economy (bmwfw), the federal ministry of traffic, innovation and technology (bmvit), the styrian business promotion agency sfg, the standortagentur tirol, the government of lower austria and zit technology agency of the city of vienna through the comet - funding program managed by the austrian research promotion agency ffg.	microbial production of acetone and butanol was one of the first large - scale industrial fermentation processes of global importance. during the first part of the 20th century, it was indeed the second largest fermentation process, superseded in importance only by the ethanol fermentation. after a rapid decline after the 1950s, acetone - butanol - ethanol (abe) fermentation has recently gained renewed interest in the context of biorefinery approaches for the production of fuels and chemicals from renewable resources. the availability of new methods and knowledge opens many new doors for industrial microbiology, and a comprehensive view on this process is worthwhile due to the new interest. this thematic issue of fems microbiology letters, dedicated to the 100th anniversary of the first industrial exploitation of chaim weizmann 's abe fermentation process, covers the main aspects of old and new developments, thereby outlining a model development in biotechnology. all major aspects of industrial microbiology are exemplified by this single process. this includes new technologies, such as the latest developments in metabolic engineering, the exploitation of biodiversity and discoveries of new regulatory systems such as for microbial stress tolerance, as well as technological aspects, such as bio- and down - stream processing.
glucose meters have been used in the hospital setting for decades. traditionally glucose meters were used in the hospital to dose subcutaneous insulin for patients with diabetes when they were hospitalized. as even well - controlled diabetic patients will have their insulin needs, diet and caloric requirements change during periods of acute illness ; glucose must be measured frequently (four or more times per day) before meals and/or insulin dosing in the hospital. although most hospital laboratories offer a measurement of serum or plasma glucose, hospitals and healthcare systems find it both convenient and efficient to measure capillary whole blood glucose at the bedside in order to expedite insulin dosing. this can help insure that glucose values are taken before (rather than after) meals are consumed, as it is the pre - prandial blood sugar value that is most often used to dose insulin. in 2001 van den berghe and colleagues changed the landscape of glucose control in the hospital by studying the impact of tight glycemic control (maintaining blood glucose between 80 - 110 mg / dl) among critically ill patients (both diabetic and non - diabetic) after cardiovascular surgery. van den berghe s original study sought to determine whether closely controlling glucose levels in patients in a surgical intensive care unit (icu) would improve patient outcome. in the study 1500 patients were divided into two groups : one control group that received what was conventional treatment of hyperglycemia in the icu at that time (subcutaneous or intravenous insulin to keep glucose levels less than 200 mg / dl), and an experimental group that received intravenous insulin to keep blood glucose at relatively normal levels of 80 - 110 mg / dl the experimental group that received intravenous insulin to keep blood glucose relatively normal had much better health outcomes than the control group (mortality decreased 34%, renal failure 41%, bloodstream infections 46%). the outcomes were startling to critical care experts, and almost overnight changed the standard of care in critical care medicine from a relaxed attitude towards hyperglycemia in the icu to vigilant glucose monitoring and insulin treatment to maintain normal or near - normal blood glucose levels. subsequent studies found that depending upon the patient population (medical vs. surgical icu), icu nutrition practices, and protocols to dose insulin and monitor glucose ; intensive glycemic control was of either benefit in only some icu patients or not beneficial at all. finally, in 2011 a multi - center trial called nice - sugar was performed to determine what level of glycemic control was optimal in the icu setting. unlike the preliminary studies done by dr. van den berghe, nice - sugar did not compare conventional treatment to more rigorous management of glycemic control ; as by that time some active management of glucose levels in the icu was standard of care. rather, nice - sugar compared two different glucose management strategies one aimed at controlling glucose levels among critically ill patients to near - normal levels (similar to the van den berghe strategy) and one that aimed for slightly higher (140 - 180 mg / dl) glucose levels. nice - sugar, performed in over 40 medical centers, found that patients assigned to the higher (160 mg / dl), time within intended target glucose range, and glycemic variability were all detrimentally affected when precision increased beyond 5 - 10% cv. these studies differed in the type of insulin dosing modeled (subcutaneous vs. intravenous), glucose target ranges assumed, and frequency of glucose monitoring. however both raise concerns about the use of glucose meters to manage patients on intravenous insulin in the icu. both studies suggest a threshold effect of either glucose meter total erroror imprecision ; with a suggested minimum total error of 10 - 15% and imprecision of 160 mg / dl), time within intended target glucose range, and glycemic variability were all detrimentally affected when precision increased beyond 5 - 10% cv. these studies differed in the type of insulin dosing modeled (subcutaneous vs. intravenous), glucose target ranges assumed, and frequency of glucose monitoring. however both raise concerns about the use of glucose meters to manage patients on intravenous insulin in the icu. both studies suggest a threshold effect of either glucose meter total erroror imprecision ; with a suggested minimum total error of 10 - 15% and imprecision of < 5%. because a number of previous studies demonstrated total error greater than 10 - 15% when glucose meters are used on icu patients, this has fueled concern about their use in this context. while studies of glucose meter use among critically ill patients have demonstrated both systematic differences (generally positive bias) and variability between glucose meter and laboratory glucose values, a few studies have concluded that the use of glucose meters during glycemic control may be appropriate. one study used parke s error grid analysis to assess the clinical impact of glucose meter errors when arterial, venous or capillary samples were used to dose glucose meters. these authors concluded that glucose meters may be appropriate for use in glycemic control protocols when arterial or venous (but not capillary) samples are used. however it is not clear whether use of the parke s error grid is appropriate for assessing the clinical impact of glucose meter errors in the context of intravenous insulin therapy during icu glycemic control protocols. another study also examined differences between glucose meter and laboratory glucose when either arterial, venous or capillary samples from critically ill patients were used. this study examined the number and magnitude of insulin dosing errors when glucose meter (compared to laboratory glucose) results were used to make insulin dosing decisions using the institutional glycemic control protocol (target glucose 80 - 110 mg / dl). this study found that errors in the measurement of both venous catheter and capillary glucose resulted in more frequent large (2 or more insulin dosing categories) dosing errors ; whereas use of arterial catheter whole blood on the glucose meter resulted in predominantly one category dosing errors. finally one study used consensus error grid and bland altman analysis to study whole blood glucose accuracy using several different devices ; and found that by limiting sample type to arterial blood that some glucose meters were accurate enough to be used during glycemic control in assessing the appropriateness of glucose meter use in the icu, choice of sample type is an essential consideration. a number of studies have demonstrated that capillary glucose can be highly inaccurate in patients in shock, or patients with edema or poor tissue perfusion several studies have also demonstrated systematic overestimation of glucose values when venous catheters are used to obtain venous whole blood for analysis on some glucose meter technologies. arterial whole blood is very likely the best sample choice for monitoring whole blood glucose in critically ill patients. in considering the evidence for and against use of glucose meters in the icu, one should pay special attention to sample source as a potential cause for poor glucose meter performance. other investigators have studied whether other factors may be more important than glucose monitor accuracy in determining the effectiveness of a glycemic control protocol. one study compared use of a standardized insulin infusion protocol to physician - directed intravenous insulin dosing in a mixed medical / surgical icu. use of the standardized infusion protocol reduced the rate of hypoglycemia from 16% to 4%, and also reduced the frequency of dextrose rescue. patients using the standardized protocol reached target glucose faster and maintained blood glucose in the target range (81 - 110 mg / dl) longer. glucose in this study was monitored using capillary samples on a glucose meter, perhaps the least desirable sample for critically ill patients. even with this limitation, the study demonstrated that execution of a standardized infusion protocol can improve at least short - term outcomes (hypoglycemia, time in therapeutic range). another study demonstrated that by using an insulin infusion protocol that focused on velocity of glucose change (rather than absolute glucose levels), glucose meters could be used to maintain blood glucose in the range of 100 - 139 mg / dl with very little (0.3% of all glucose values < 60 mg / dl) hypoglycemia. another investigator has described a collaborative approach to establishing both glucose target ranges and insulin infusion algorithms based upon practice and nursing leader opinions about what could be safely accomplished. using this approach they implemented an initial glycemic control protocol to keep glucose levels among critically ill patients below 140 mg / dl they used hourly capillary glucose meter and/or laboratory serum / plasma glucose for all patients on intravenous insulin and observed a rate of severe hypoglycemia (glucose < 40 mg / dl) of 0.38%. when staff in the icu was comfortable with the under 140 protocol, the target glucose range was decreased to 80 - 125 mg / dl with only a modest increase in severe hypoglycemia (0.92%). the authors concluded that by taking an incremental approach to glycemic control, starting with a higher target range and lowering the range only after staff demonstrated they could reliably execute the protocol, safe and effective glycemic control was possible using glucose meters for some monitoring. a more common approach to improving outcomes during glycemic control is to use information technology solutions to computerize insulin doses based upon trended (rather than individual) glucose values. this approach mitigates the risk of hypoglycemia from a single aberrant glucose meter value. using this approach one study demonstrated that rates of severe hypoglycemia were 4.25% when mostly capillary whole blood glucose meter values were used to dose insulin among 4588 critically ill patients managed on a glycemic control protocol with an 81 - 110 mg / dl target range. these authors went on to investigate causes of hypoglycemia among all incidents where glucose fell below 40 mg / dl the authors found that ~ 70% of hypoglycemic episodes could be attributed to delay in obtaining glucose measurement ; suggesting that human error (rather than measurement error) is responsible for the most insulin - induced hypoglycemia during traditional tight glycemic control protocols. the same authors compared the computerized infusion protocol to a paper - based protocol and found that using a computerized protocol improved the time in therapeutic range, mean blood glucose level, and percent of blood glucose measurements below 70 mg / dl.. finally a study over a one month period in three intensive care units at one institution found that using arterial whole blood to dose glucose meters, and relying upon consistent hourly glucose measurements performed by laboratory (rather than nursing) staff, rates of severe hypoglycemia were 1.4% despite a relatively low glucose target range of 80 - 130 mg / dl in addition, 86% of severe hypoglycemic episodes observed were due to protocol violations (missed hourly glucose measurements or failure to change insulin infusion rate according to protocol instructions). when the glucose target range was changed to 110 - 150 mg / dl (with no change in glucose meter used or measurement frequency), no episodes of hypoglycemia were observed in 211 patients over one month. a larger study (three months, 1503 patients) within the same icu units found a rate of severe hypoglycemia of 0.25 %. collectively these studies highlight several key points that must be considered before determining the appropriateness of glucose meters for managing glycemic control in the icu. the choice of sample type (arterial whole blood preferred) may be as or more important than the type of glucose monitor used for whole blood glucose measurement. glucose meters have been used in effective glycemic control protocols demonstrating both low rates of severe hypoglycemia and reliable glycemic control in the icu. elements of effective protocols are computerized (rather than paper - based) insulin dosing algorithms, collaboration and teamwork to determine the appropriate glucose target for a given hospital or icu population, and use of frequent (often hourly) arterial whole blood sampling for all patients on intravenous insulin. while many studies demonstrating poor performance of glucose meters in critically ill patients used older glucose meter technologies, newer technologies with improved accuracy have recently become available. some recent studies have demonstrated that newer glucose meter technologies can meet even the more stringent clsi poct12-a3 accuracy guidelines (12.5% for values above 100 mg / dl) when used in the intensive care unit. meters that meet more stringent accuracy guidelines such as poct12-a3 would be performing within the 10 - 15% total error allowance predicted to minimize large insulin dosing errors in the context of icu glycemic control. with the improved performance of newer glucose meters, one might think that the issue of glucose meter accuracy in the icu was close to resolution. to add fuel to the ongoing controversy about glucose meter use in the icu, the food and drug administration (fda) released draft guidelines suggesting that improved accuracy was necessary for any future glucose monitors intended for hospital use. while the guidelines are still in draft form at the time of this review, fda draft guidance criteria suggested that 99% of glucose meter values should be within 10% of the reference or true glucose value. there is concern among some that tightening accuracy criteria to this level could impede the development of new meters and monitors, without improving the quality of care delivered in the icu during glycemic control. amidst this cloud of confusion and controversy surrounding glucose meter use in the icu, what is the point of care program to do ? first and foremost, consider the entire glycemic control protocol in use within your institution, and the role that glucose meters play in the overall scheme of glycemic control. eliminating the use of glucose meters in support of intravenous insulin protocols, without first considering alternatives and implications, would almost certainly have an adverse effect on patient care. understand the effectiveness of the glycemic control protocol (rates of hypo and hyperglycemia, time within intended glucose range) as implemented, and the systematic issues that may be leading to adverse outcomes such as hypoglycemia. if the major issues are remembering to obtain glucose values in a timely manner to facilitate insulin dosing decisions, or communicating glucose results to providers in a timely manner, then changing glucose measurement devices (especially away from the bedside) would not be expected to improve outcome. if spurious glucose results have been observed in some icu patients, determine whether common interferences (low hematocrit, some medications) in the icu environment may be affecting the glucose meter technology in use. if user errors such as incorrect strip codes or under - dosing of strips are suspected ; consider switching to a glucose meter technology that reduces the likelihood of these errors and examining training and competency systems. hospitals and point of care programs should also consider the sample type (capillary, arterial or venous whole blood) routinely used for bedside glucose measurements, before making a decision to switch technologies or glucose measurement devices. if capillary sampling is being used as the predominant sample type, switching to arterial whole blood may improve measurement accuracy without requiring large changes in workflow or testing processes. finally, consider evaluating the accuracy of the device being used by comparing whole blood glucose meter values to laboratory serum or plasma glucose obtained from icu patients. if the vast majority of glucose meter values are not within 15% of lab glucose values, then it is likely that more accurate glucose measurements are both possible and desirable. simulation models have provided the best evidence available to relate glucose meter accuracy to insulin dosing errors during glycemic control in the icu. however they do not provide a way to measure the impact of glucose meter error on patient outcome. studies directly relating glucose monitor accuracy to glycemic control outcome (mortality, infections, transfusions, etc) or effectiveness (hypoglycemia, hyperglycemia, time in therapeutic range) are needed to understand the level of glucose meter accuracy required for management of critically ill patients on intravenous insulin therapy.	glucose meters are a fast and convenient way to measure circulating blood glucose. like many technologies in healthcare, the use of glucose meters within the hospital has evolved significantly over the last few decades. this change has been driven predominantly by changes in the approach to glycemic control for critically ill patients. both glycemic control in the intensive care unit (icu), and use of glucose meters to manage insulin dosing during glycemic control, are likely to remain controversial topics in the years to come. this review will elaborate on the evidence for and against use of glucose meters in the icu to monitor glucose concentrations during glycemic control, and provide some tips for point of care programs on how to evaluate glucose monitors for this purpose.
kidney transplantation is the treatment of choice for end - stage renal disease.1 a total of 292,427 kidney transplants were performed in the us by the end of 2009.2 the use of better donor recipient selection algorithms, novel surgical techniques, and improved post - transplant care have all led to a significant improvement in short - term graft survival, which currently approaches 90% in the first year after transplant.3 however, the most significant impact in this improvement is the introduction of more potent immunosuppressive therapy. the goal of immunosuppressive therapy in renal transplantation is to minimize acute and chronic rejection while, at the same time, balancing these beneficial effects with their adverse effects, which include the development of increased cardiovascular risk factors, infections, and malignancies. current immunosuppression strategies are primarily based on an induction regimen using a monoclonal or polyclonal antibody, followed by maintenance immunosuppression consisting of calcineurin inhibitors (cyclosporine or tacrolimus), an antiproliferative agent (mycophenolate mofetil), and low - dose corticosteroids (prednisone).4 unfortunately, improvement in short - term graft survival has not been reflected in improved long - term outcomes.5 five - year graft survival continues to lag behind and is currently estimated to be approximately 72%.6 the primary causes of late allograft loss include chronic allograft dysfunction and death with a functioning graft.7 chronic allograft dysfunction can result from multiple causes, including chronic immune - mediated injury, interstitial fibrosis, and tubular atrophy, as well as the chronic toxic effect of calcineurin inhibitors.8 histologic data from protocol renal allograft biopsies demonstrated the presence of calcineurin inhibitor - induced nephrotoxicity in 50% of renal transplant recipients at two years and 100% at 10 years after transplant.8 long - term mortality in the adult renal transplant recipient is also estimated to be nearly four times that of the general population.9 a large proportion of this decreased patient survival is secondary to an increased burden of cardiovascular disease and infections in renal transplant recipients.10 calcineurin inhibitors have been associated with the development and worsening of hypertension and hyperlipidemia, as well as diabetes.11 various calcineurin inhibitor minimization and withdrawal strategies have been attempted, with mixed results.12,13 the use of mtor (mammalian target of rapamycin) inhibitors (sirolimus and everolimus) for calcineurin inhibitor minimization / withdrawal has been hampered by an adverse side effect profile.14 therefore, calcineurin inhibitors currently remain the cornerstone of maintenance immunosuppression in renal transplant recipients. by the end of 2007, close to 99% of renal transplant recipients were discharged on calcineurin inhibitors.15 the current trend in drug development is focused on preservation of long - term function and minimization of the adverse reactions of immunosuppressive drugs. multiple small molecules and biologic agents are currently being studied.16,17 t cell costimulation blockade is a promising approach and is being intensively investigated since the development of belatacept. in this review we discuss the mechanism of action, preclinical and clinical data, and the side effect profile of belatacept. the ability of t cells to recognize nonself antigens is critical for an effective immune response.18 antigen - presenting cells (dendritic cells, macrophages, and b cells) are specialized cells capable of activating t cells. to trigger an adequate alloimmune response, first, nonself or alloantigens are displayed by major histocompatibility complex (mhc) molecules on the surface of antigen - presenting cells (see figure 1). allopeptide complex engages a specific t cell receptor, leading to initiation of the signaling process from the cd3 complex. calcineurin pathway, which activates the nuclear factor of activated t cell. to amplify the t cell response further, signal 2 or costimulation must be transmitted (see figure 1). this signal is mediated by the interaction of cd80 (b7.1) and cd86 (b7.2) on antigen - presenting cells with specific t cell receptors (cd28 and its homolog, cytotoxic t lymphocyte - associated antigen 4 [ctla4 ]). cd28, a disulfide - linked homodimeric transmembrane member of the immunoglobulin superfamily, is constitutively expressed on all nave cd4 and cd8 t cells, although some mature t cells, especially memory cd8 t cells, are cd28-negative.19 in contrast with cd28, ctla4 delivers signals that attenuate t cell proliferation. ligation of cd28 by cd80/86 is required for clonal expansion of nave cd4 t cells. because ctla4 has a higher affinity than cd28 for the cd80/86 ligand, it binds to most of the available molecules, effectively shutting down further t cell proliferation. thus, costimulatory molecules can provide positive or negative signals to t cells. for complete t cell activation and differentiation signal, t cells will either undergo apoptosis or develop donor - specific anergy.20 activation of signals 1 and 2 leads to expression of cytokines, especially interleukin-2. these cytokines activate the mtor via the janus kinase 3 and phosphoinositide-3 kinase signal transduction pathways, leading to further propagation of the lymphocyte cell cycle. ctla4-ig (abatacept) was the first molecule to be developed by fusion of the extra - cellular domain of ctla4 with the constant region fragment (fc) of human igg1 to increase its serum half- life.21 given the higher affinity of ctla4 for cd80/86, ctla4-ig should theoretically block antigen - presenting cell stimulation of t cells through cd28, thereby terminating the immune response (see figure 1). however, the fc region can independently bind to multiple receptors that modulate immune responses, including antibody - dependent cellular cytotoxicity and complement - dependent cytotoxicity. thus, in abatacept, a series of directed cysteine to serine mutations were introduced in the hinge region to reduce this fc- mediated binding.21 although abatacept proved to be highly efficacious for autoimmune t cell - mediated autoimmune disorders, such as rheumatoid arthritis and psoriasis, it was found to be an inadequate maintenance immunosuppressive agent in nonhuman primate models of transplantation.2224 studies into potential reasons for this disconnect found that although ctla4 binds with a much higher avidity to cd80 and cd86 than does cd28, ctla4-ig was significantly less potent at inhibiting cd86-dependent as opposed to cd80-dependent costimulation.25 thus, it was reasoned that a ctla4-ig protein with a higher avidity for cd86 could be developed. using a rational mutagenesis and screening strategy, a daughter molecule, lea29y (belatacept, bristol - myers squibb, new york, ny, usa), with two amino acid substitutions (l104->e and a29->y), was developed.26 belatacept was found to bind four times more avidly to cd86 and two times more avidly to cd80 than the parent compound, abatacept. this improved binding results in an approximately 10-fold more potent inhibition of t cell activation.26 flow cytometric studies in renal transplant patients have demonstrated that belatacept saturates both cd80 and cd86 receptors in whole blood and dendritic cell cultures, although the belatacept concentrations required for cd86 receptor saturation were approximately 10-fold higher than those required for cd80 saturation.27 in a study published only in abstract form, davis reported that, like abatacept, belatacept did not mediate antibody - dependent cellular cytotoxicity or complement - dependent cytotoxicity of target b cells through its fc domain. these findings suggest that the immunomodulatory activity is mediated predominantly through inhibition of cd28 signaling.28 in humans, cd4 + cd25 + regulatory t cells (tregs) constitute 5%15% of peripheral cd4 + t cells, and are surmised to have an important suppressive role in the pathologic immune responses after transplantation.29 the fork head transcription factor, foxp3, is essential for the development of tregs. interleukin-2 and cd28 costimulation has been shown to be an essential survival factor for tregs.30 thus, drugs that target these pathways could have a potentially deleterious effect on treg survival.31 reassuringly, data from a phase ii belatacept renal transplant study showed that costimulation blockade did not interfere with treg homeostasis. the authors presented several hypotheses as to why treg homeostasis was not altered by belatacept, including the possibility that human tregs might not be as sensitive to cd28 costimulation blockade as mouse tregs ; that allotransplantation could result in the development of cd28-independent adaptive tregs ; that other costimulatory molecules, like cd2, could function as a substitute for the cd28 pathway ; and, finally, that long - term intermittent dosing of belatacept could have a subsaturating effect on cd86, allowing tregs to receive sufficient cd28 signaling in order to maintain their survival. a significant increase in intragraft foxp3 + the authors surmised that this may lead to better resolution of graft rejection episodes and potentially promote tolerance.32 the number of patients was too small, however, to evaluate the impact of this finding on overall graft survival. various in vitro and in vivo studies have examined the efficacy of combined cd80/86 blockade. vierboom showed that a combination of anti - cd80 and anti - cd86 monoclonal antibodies resulted in a complete abrogation of the primary alloimmune response among peripheral mononuclear blood cells obtained from rhesus monkeys.33 in an animal model, kirk demonstrated that administration of anti - cd80 and anti - cd86 monoclonal antibodies resulted in a delayed onset of acute allograft rejection without global t cell or b cell depletion. however, although treatment with monoclonal antibodies alone (without other immunosuppressive drugs) was sufficient to maintain graft survival, rejection occurred as soon as the treatment ceased, suggesting that the therapy was not tolerogenic.34 montgomery showed a greater rejection - free survival rate but a lack of durable tolerance with combination anti - cd80, anti - cd86, and anti - cd154 monoclonal antibodies.35 in a study on human and pig peripheral mononuclear blood cells, emamaullee showed that both belatacept and basiliximab, either as monotherapy or as combination therapy, potently inhibited allogeneic immune responses.36 table 1 lists the results of various animal studies examining the use of belatacept in solid organ transplantation. in a nonhuman primate model, larsen s landmark study demonstrated that belatacept monotherapy was inferior to combination therapy with belatacept and conventional immunosuppressive drugs in preventing allograft rejection.26 importantly, belatacept did prevent development of donor - specific antibodies, which is a major contributor to chronic allograft loss in clinical settings.37 table 2 lists the ongoing and completed clinical trials investigating the use of belatacept in renal transplantation. the first clinical trial on the use of belatacept in renal transplantation was a phase ii noninferiority trial comparing the efficacy of belatacept with cyclosporine for prevention of acute rejection at six months post - transplant.38 in a partially blinded, parallel group, multicenter study, the belatacept study group randomized 218 renal transplant recipients to receive a more intensive (11 infusions of 10 mg / kg over the first six months, followed by 5 mg / kg every 48 weeks) or less intensive (five infusions of 10 mg / kg over the first three months, followed by 5 mg / kg every 48 weeks) belatacept regimen or cyclosporine. all patients also received mycophenolate mofetil and corticosteroids as maintenance immunosuppression and induction with basiliximab. at six months, the incidence of acute rejection was similar in all three groups, being 7%, 6%, and 8% in the more intensive belatacept, less intensive belatacept, and cyclosporine groups, respectively. the grades of acute rejection were also similar, although the less intensive belatacept group experienced a higher incidence of subclinical rejection and treated episodes of subclinical rejection (20% and 15%) compared with the more intensive belatacept (9% and 8%) and cyclosporine (11% and 7%) groups. most importantly, glomerular filtration rate, as measured by iohexol clearance, was significantly higher in the belatacept groups compared with the cyclosporine arm. consistent with this finding, protocol biopsies demonstrated a 15%24% reduction in the incidence of chronic allograft nephropathy in the belatacept groups. the belatacept groups had a statistically significant lower risk of developing diabetes and need for treatment of hyperlipidemia, and a lower incidence of hypertension. in a recently presented phase ii study, 89 epstein barr virus (ebv) seropositive adult renal transplant recipients were randomized 1:1:1 to receive belatacept + mycophenolate mofetil (n = 33), belatacept + sirolimus (n = 26), or tacrolimus + mycophenolate mofetil (n = 30). although the overall glomerular filtration rate was better in the belatacept - treated groups, acute rejection rates were higher in the belatacept + mycophenolate mofetil arm. at the end of 12 months, 2/33 patients in the belatacept + mycophenolate mofetil group and 2/26 patients in the belatacept + sirolimus group had lost their allograft compared with none in the tacrolimus + mycophenolate mofetil group. the authors concluded that use of belatacept in renal transplant recipients may enable calcineurin inhibitor and corticosteroid avoidance, with acceptable rates of acute rejection and improved glomerular filtration rate, although graft loss remains a concern.39 benefit (belatacept evaluation of nephroprotection and efficacy as first line immunosuppression trial) is a three- year, phase iii clinical trial that randomized patients to three groups, ie, cyclosporine (n = 231), less intensive belatacept (n = 231), and more intensive belatacept (n = 225). based upon the results published so far, patient and graft survival are similar across the three groups at both one year (96% more intensive belatacept ; 96% less intensive belatacept ; and 93% cyclosporine) and two years (94% more intensive belatacept ; 95% less intensive belatacept ; and 91% cyclosporine) post - transplant. 40,41 at the end of one year, although the incidence of acute rejection was greater for more intensive (22%) and less intensive (17%) belatacept compared with cyclosporine (7%), no apparent impact on graft survival was demonstrated. banff iib acute rejection occurred more frequently in belatacept - treated (less intensive 5% ; more intensive 10%) compared with cyclosporine - treated patients (1%). at the end of two years, glomerular filtration rate continued to be significantly higher (1517 ml / min) in the belatacept - treated patients. belatacept - treated patients also had sustained benefits in their cardiovascular and metabolic risk profile. benefit - ext (belatacept evaluation of nephroprotection and efficacy as first - line immunosuppression trial- extended criteria donors) is a three - year, randomized phase iii study in renal transplant recipients receiving an extended criteria donor kidney allograft (n = 543).42 the rationale for this trial was that because extended criteria donor recipients have a heightened risk of allograft loss, they might derive a significant benefit from the non - nephrotoxic belatacept. at the end of the first year, renal function was statistically superior for more intensive belatacept versus cyclosporine (52 ml / min more intensive belatacept ; 45 ml / min cyclosporine) but not for less intensive belatacept (50 ml / min) versus cyclosporine. surprisingly, the incidence of chronic allograft nephropathy was similar (45% more intensive belatacept ; 46% less intensive belatacept ; 52% cyclosporine) across the three groups. the recently reported two - year results echo the findings of the first year of the study, although only 64% of the originally enrolled subjects completed the study.43 in the abstract, the authors conclude that the more intensive belatacept regimen does not confer any additional efficacy over the less intensive regimen. based upon current data, it seems likely that benefit - ext might not meet its primary endpoint of better graft survival with belatacept therapy at the end of the three - year study period. encouragingly, though, diabetes and cardiovascular risk factors were lower in the belatacept - treated patients. based upon an interim report, another phase ii study is being conducted in stable renal transplant recipients maintained on calcineurin inhibitor - based regimens. patients 636 months post - transplantation (n = 173) with glomerular filtration rates of 3575 ml / min were randomized to either belatacept 5 mg / kg (less intensive) or continued treatment with a calcineurin inhibitor. seven percent of the belatacepttreated patients had acute rejection compared with none in the calcineurin inhibitor group. although patient and graft survival remained similar in both groups, glomerular filtration rate improved significantly in the belatacept group at the end of 12 months.44 a systematic review of randomized controlled trials was recently presented at the 2010 international congress of the transplantation society meeting.45 based upon the report, patients treated with belatacept had a 69% lower chance of dying compared with those treated with tacrolimus. the odds ratios (ors) of graft loss with belatacept were not statistically different relative to cyclosporine (or = 0.70, 95% credible interval 0.321.50) or tacrolimus (or = 0.82, credible interval 0.351.84), although acute rejection odds were significantly higher (or = 2.61, credible interval 1.294.91) relative to tacrolimus. the odds of development of new - onset diabetes after transplantation were significantly lower (or = 0.19, credible interval 0.080.42) with belatacept compared with both calcineurin inhibitors. multiple other trials, including the use of belatacept along with various agents like sirolimus and alemtuzumab, are currently in progress.46 because tacrolimus has largely replaced cyclosporine as the calcineurin inhibitor of choice, trials comparing tacrolimus with belatacept would be crucial in confirming the results presented above. ashman reported successful use of belatacept as maintenance immunosuppression in a young kidney transplant patient who developed de novo thrombotic microangiopathy serially in association with cyclosporine, tacrolimus, and sirolimus.47 a compassionate use study to make belatacept available for renal transplant recipients who are intolerant to or having contraindications to calcineurin inhibitors and/or mtor inhibitors is currently enrolling patients. in human studies, belatacept appears to have predictable pharmacokinetics. no definitive relationship, however, has been established between belatacept serum concentration and the risk of acute rejection. a pooled analysis of 1425 intent - to- treat patients (more intensive belatacept, n = 477 ; less intensive belatacept, n = 472 ; cyclosporine, n = 476) with a median follow - up of 2.4 years demonstrated that the incidences of serious adverse events were comparable in all three groups.48 the overall incidence of malignancies was slightly higher in the more intensive belatacept group (10% more intensive belatacept ; 6% less intensive belatacept ; 7% cyclosporine). a total of 15 cases of post - transplant lymphoproliferative disease, including eight cases involving the central nervous system, were reported. of these posttransplant lymphoproliferative disease cases, eight occurred on more intensive belatacept, five on less intensive belatacept, and two on cyclosporine. six of eight central nervous system post - transplant lymphoproliferative disease cases were reported from the more intensive belatacept group and in two of eight from the less intensive belatacept group. ebv seronegative status was found to be the strongest risk factor. although rates of polyomavirus infections were similar, herpes virus infections were higher in the belatacept groups. the incidence of death and serious infections was lowest in the less intensive belatacept group. vincenti recently published the five - year safety data of their initial phase ii study.49 belatacept - treated patients did not have a higher frequency of serious infections or post - transplant lymphoproliferative disease compared with cyclosporine. serious cardiac disorders occurred more frequently with cyclosporine (2% for belatacept versus 12% for cyclosporine). haidinger reported a renal transplant recipient on belatacept who developed fatal pneumocystis jiroveci pneumonia four years post - transplant.50 cytomegalovirus infection preceded the pneumonia, illustrating that excessive immunosuppression can result, even with a lymphocyte - specific regimen. various in vitro and in vivo studies have examined the efficacy of combined cd80/86 blockade. vierboom showed that a combination of anti - cd80 and anti - cd86 monoclonal antibodies resulted in a complete abrogation of the primary alloimmune response among peripheral mononuclear blood cells obtained from rhesus monkeys.33 in an animal model, kirk demonstrated that administration of anti - cd80 and anti - cd86 monoclonal antibodies resulted in a delayed onset of acute allograft rejection without global t cell or b cell depletion. however, although treatment with monoclonal antibodies alone (without other immunosuppressive drugs) was sufficient to maintain graft survival, rejection occurred as soon as the treatment ceased, suggesting that the therapy was not tolerogenic.34 montgomery showed a greater rejection - free survival rate but a lack of durable tolerance with combination anti - cd80, anti - cd86, and anti - cd154 monoclonal antibodies.35 in a study on human and pig peripheral mononuclear blood cells, emamaullee showed that both belatacept and basiliximab, either as monotherapy or as combination therapy, potently inhibited allogeneic immune responses.36 table 1 lists the results of various animal studies examining the use of belatacept in solid organ transplantation. in a nonhuman primate model, larsen s landmark study demonstrated that belatacept monotherapy was inferior to combination therapy with belatacept and conventional immunosuppressive drugs in preventing allograft rejection.26 importantly, belatacept did prevent development of donor - specific antibodies, which is a major contributor to chronic allograft loss in clinical settings.37 table 2 lists the ongoing and completed clinical trials investigating the use of belatacept in renal transplantation. the first clinical trial on the use of belatacept in renal transplantation was a phase ii noninferiority trial comparing the efficacy of belatacept with cyclosporine for prevention of acute rejection at six months post - transplant.38 in a partially blinded, parallel group, multicenter study, the belatacept study group randomized 218 renal transplant recipients to receive a more intensive (11 infusions of 10 mg / kg over the first six months, followed by 5 mg / kg every 48 weeks) or less intensive (five infusions of 10 mg / kg over the first three months, followed by 5 mg / kg every 48 weeks) belatacept regimen or cyclosporine. all patients also received mycophenolate mofetil and corticosteroids as maintenance immunosuppression and induction with basiliximab. at six months, the incidence of acute rejection was similar in all three groups, being 7%, 6%, and 8% in the more intensive belatacept, less intensive belatacept, and cyclosporine groups, respectively. the grades of acute rejection were also similar, although the less intensive belatacept group experienced a higher incidence of subclinical rejection and treated episodes of subclinical rejection (20% and 15%) compared with the more intensive belatacept (9% and 8%) and cyclosporine (11% and 7%) groups. most importantly, glomerular filtration rate, as measured by iohexol clearance, was significantly higher in the belatacept groups compared with the cyclosporine arm. consistent with this finding, protocol biopsies demonstrated a 15%24% reduction in the incidence of chronic allograft nephropathy in the belatacept groups. the belatacept groups had a statistically significant lower risk of developing diabetes and need for treatment of hyperlipidemia, and a lower incidence of hypertension. in a recently presented phase ii study, 89 epstein barr virus (ebv) seropositive adult renal transplant recipients were randomized 1:1:1 to receive belatacept + mycophenolate mofetil (n = 33), belatacept + sirolimus (n = 26), or tacrolimus + mycophenolate mofetil (n = 30). although the overall glomerular filtration rate was better in the belatacept - treated groups, acute rejection rates were higher in the belatacept + mycophenolate mofetil arm. at the end of 12 months, 2/33 patients in the belatacept + mycophenolate mofetil group and 2/26 patients in the belatacept + sirolimus group had lost their allograft compared with none in the tacrolimus + mycophenolate mofetil group. the authors concluded that use of belatacept in renal transplant recipients may enable calcineurin inhibitor and corticosteroid avoidance, with acceptable rates of acute rejection and improved glomerular filtration rate, although graft loss remains a concern.39 benefit (belatacept evaluation of nephroprotection and efficacy as first line immunosuppression trial) is a three- year, phase iii clinical trial that randomized patients to three groups, ie, cyclosporine (n = 231), less intensive belatacept (n = 231), and more intensive belatacept (n = 225). patients received induction with basiliximab and were maintained on mycophenolate mofetil and corticosteroids. based upon the results published so far, patient and graft survival are similar across the three groups at both one year (96% more intensive belatacept ; 96% less intensive belatacept ; and 93% cyclosporine) and two years (94% more intensive belatacept ; 95% less intensive belatacept ; and 91% cyclosporine) post - transplant. 40,41 at the end of one year, although the incidence of acute rejection was greater for more intensive (22%) and less intensive (17%) belatacept compared with cyclosporine (7%), no apparent impact on graft survival was demonstrated. banff iib acute rejection occurred more frequently in belatacept - treated (less intensive 5% ; more intensive 10%) compared with cyclosporine - treated patients (1%). at the end of two years, glomerular filtration rate continued to be significantly higher (1517 ml / min) in the belatacept - treated patients. belatacept - treated patients also had sustained benefits in their cardiovascular and metabolic risk profile. benefit - ext (belatacept evaluation of nephroprotection and efficacy as first - line immunosuppression trial- extended criteria donors) is a three - year, randomized phase iii study in renal transplant recipients receiving an extended criteria donor kidney allograft (n = 543).42 the rationale for this trial was that because extended criteria donor recipients have a heightened risk of allograft loss, they might derive a significant benefit from the non - nephrotoxic belatacept. at the end of the first year, patient and graft survival was similar across the three groups. renal function was statistically superior for more intensive belatacept versus cyclosporine (52 ml / min more intensive belatacept ; 45 ml / min cyclosporine) but not for less intensive belatacept (50 ml / min) versus cyclosporine. surprisingly, the incidence of chronic allograft nephropathy was similar (45% more intensive belatacept ; 46% less intensive belatacept ; 52% cyclosporine) across the three groups. the recently reported two - year results echo the findings of the first year of the study, although only 64% of the originally enrolled subjects completed the study.43 in the abstract, the authors conclude that the more intensive belatacept regimen does not confer any additional efficacy over the less intensive regimen. based upon current data, it seems likely that benefit - ext might not meet its primary endpoint of better graft survival with belatacept therapy at the end of the three - year study period. encouragingly, though, diabetes and cardiovascular risk factors were lower in the belatacept - treated patients. based upon an interim report, another phase ii study is being conducted in stable renal transplant recipients maintained on calcineurin inhibitor - based regimens. patients 636 months post - transplantation (n = 173) with glomerular filtration rates of 3575 ml / min were randomized to either belatacept 5 mg / kg (less intensive) or continued treatment with a calcineurin inhibitor. seven percent of the belatacepttreated patients had acute rejection compared with none in the calcineurin inhibitor group. although patient and graft survival remained similar in both groups, glomerular filtration rate improved significantly in the belatacept group at the end of 12 months.44 a systematic review of randomized controlled trials was recently presented at the 2010 international congress of the transplantation society meeting.45 based upon the report, patients treated with belatacept had a 69% lower chance of dying compared with those treated with tacrolimus. the odds ratios (ors) of graft loss with belatacept were not statistically different relative to cyclosporine (or = 0.70, 95% credible interval 0.321.50) or tacrolimus (or = 0.82, credible interval 0.351.84), although acute rejection odds were significantly higher (or = 2.61, credible interval 1.294.91) relative to tacrolimus. the odds of development of new - onset diabetes after transplantation were significantly lower (or = 0.19, credible interval 0.080.42) with belatacept compared with both calcineurin inhibitors. multiple other trials, including the use of belatacept along with various agents like sirolimus and alemtuzumab, are currently in progress.46 because tacrolimus has largely replaced cyclosporine as the calcineurin inhibitor of choice, trials comparing tacrolimus with belatacept would be crucial in confirming the results presented above. ashman reported successful use of belatacept as maintenance immunosuppression in a young kidney transplant patient who developed de novo thrombotic microangiopathy serially in association with cyclosporine, tacrolimus, and sirolimus.47 a compassionate use study to make belatacept available for renal transplant recipients who are intolerant to or having contraindications to calcineurin inhibitors and/or mtor inhibitors is currently enrolling patients. no definitive relationship, however, has been established between belatacept serum concentration and the risk of acute rejection. a pooled analysis of 1425 intent - to- treat patients (more intensive belatacept, n = 477 ; less intensive belatacept, n = 472 ; cyclosporine, n = 476) with a median follow - up of 2.4 years demonstrated that the incidences of serious adverse events were comparable in all three groups.48 the overall incidence of malignancies was slightly higher in the more intensive belatacept group (10% more intensive belatacept ; 6% less intensive belatacept ; 7% cyclosporine). a total of 15 cases of post - transplant lymphoproliferative disease, including eight cases involving the central nervous system, were reported. of these posttransplant lymphoproliferative disease cases, eight occurred on more intensive belatacept, five on less intensive belatacept, and two on cyclosporine. six of eight central nervous system post - transplant lymphoproliferative disease cases were reported from the more intensive belatacept group and in two of eight from the less intensive belatacept group. ebv seronegative status was found to be the strongest risk factor. although rates of polyomavirus infections were similar, herpes virus infections were higher in the belatacept groups. the incidence of death and serious infections was lowest in the less intensive belatacept group. vincenti recently published the five - year safety data of their initial phase ii study.49 belatacept - treated patients did not have a higher frequency of serious infections or post - transplant lymphoproliferative disease compared with cyclosporine. serious cardiac disorders occurred more frequently with cyclosporine (2% for belatacept versus 12% for cyclosporine). haidinger reported a renal transplant recipient on belatacept who developed fatal pneumocystis jiroveci pneumonia four years post - transplant.50 cytomegalovirus infection preceded the pneumonia, illustrating that excessive immunosuppression can result, even with a lymphocyte - specific regimen. belatacept, a selective t cell costimulation blocker, is a promising new therapy for maintenance immunosuppression among renal transplant recipients. it was originally anticipated that costimulation blockade would be successful in achieving immunologic allograft tolerance, but based upon current data this does not appear to be the case. thus, the new paradigm revolves around the use of belatacept (among other molecules) for avoidance of calcineurin inhibitor nephrotoxicity and minimization of long - term cardiovascular and metabolic side effects. although scheduled monthly infusions might improve compliance among a certain patient subset, eg, children, most mobile patients and those in remote locations could find themselves unable to adhere to such therapy. furthermore, because the drug has a long half - life of 810 days, it might be difficult to dose patients battling with life - threatening infections appropriately. owing to concerns about an increased post - transplant lymphoproliferative disease risk in ebv seronegative patients, current belatacept trial protocols have now been modified to enroll ebv seropositive patients only. unfortunately, this exclusion will complicate the introduction of this drug for young patients, who might derive the maximum long - term benefit from non - nephrotoxic regimens. on a positive note, animal and human studies have demonstrated that the use of belatacept can lead to better renal function, along with a lower incidence of diabetes and cardiovascular risk factors. although acute rejection seems to be more frequent with belatacept, so far there are no data to suggest that long - term renal allograft survival is shortened. of course, the observed benefits in renal function as measured by calculated glomerular filtration rate will need to be confirmed using hard endpoints, including patient and allograft survival.	the last several decades have witnessed a substantial decrease in the incidence of acute allograft rejection following kidney transplantation, although commensurate improvements in long - term graft function have not been realized. as a result, the primary focus of new immunosuppressive drug development has expanded to include ease of use and improved side effect profile, including reduced nephrotoxicity, in addition to the more traditional goal of improved short - term outcomes. a number of novel drugs are currently under investigation in phase i, ii, or iii clinical trials, primarily to replace the nephrotoxic but highly effective calcineurin inhibitors. belatacept is a humanized antibody that inhibits t cell costimulation and has shown encouraging results in multiple phase ii and iii trials. this article reviews the mechanism of action of belatacept, as well as published and preliminary results of the phase i iii clinical trials involving this novel immunosuppressive agent.
a 59-year - old asymptomatic diabetic male was referred to our retinal service with presumed diagnosis of asteroid hyalosis. the patient had undergone laser treatment and intravitreal preserved triamcinolone acetonide injection for clinically significant diabetic macular edema in the right eye 4 years previously. the right eye showed focal laser scars in the macula and several shiny refractile crystalline deposits [fig. 1 ]. optical coherence tomography localized these deposits to the posterior hyaloid [fig. 2 ]. these deposits were isofluorescent and not associated with specific angiographic abnormalities [fig. 3 ]. the patient has been followed for two additional years without any change in the visual acuity or status of the macula. although a posterior vitreous separation has not developed, the crystals have been observed to migrate slowly in distribution. a fundus photograph of the right eye demonstrating small pre - retinal crystals concentrated in the macula, although peripheral crystals are noted (arrow) para - foveal optical coherence tomography scan of the right eye demonstrating triamcinolone crystal deposits on the posterior hyaloid surface (arrow) fluorescein angiogram of the right eye revealing several micro - aneurysms and focal laser scars, but no crystals, which are isofluorescent triamcinolone has been shown to aggregate into crystalline structures that may resist the washout process and accumulate over the macula. a recent study has identified intra - vitreal triamcinolone acetonide injections, both preserved and preservative - free, as a cause of crystalline retinal deposits. our case report also identifies triamcinolone crystal deposition, all on the posterior hyaloidal surface. with follow - up now extending greater than 6 years, no functional toxic effects have been noted in our patient. whether the crystals retain any biological activity is unclear. in our case and the previous series no adverse visual effect or we suggest the term drug - induced benign hyaloidopathy and recommend this to be included as a differential diagnosis of crystalline maculopathy and asteroid hyalosis.	we report a case of unusually long persistence of triamcinolone crystals after intra - vitreal injection. crystals were noted on fundus examination predominantly confined to the posterior pole. optical coherence tomography localized the crystals to the posterior hyaloidal surface. over 6 years of follow - up the patient has retained good visual acuity and no observable changes in the retina. as the condition clinically resembles both crystalline maculopathy and asteroid hyalosis, we suggest the term drug - induced benign crystalline hyaloidopathy.
solid pseudopapillary neoplasm (spn) is an indolent epithelial tumor with a low malignant potential that occurs predominantly in females with a mean age of 28 years. the clear cell variant of spn is even more unusual with only six papers reported in the english literature. we present the cytologic features of clear cell variant of spn with the broad differential diagnosis and a review of past studies. ultrasonogram showed a solid / cystic mass, 2.6 cm in diameter, in the tail of the pancreas. percutaneous ultrasound - guided fine needle aspiration biopsy (fnab) was performed using a 22-gauge needle. the excess material was fixed in an alcohol - formalin solution for cell block preparation and paraffin sections were stained by hematoxylin and eosin. the material was highly cellular and consisted of a monotonous population of loosely cohesive small to medium - sized, round to cuboidal epithelioid cells exhibiting high nuclear / cytoplasmic ratio, finely granular salt and pepper chromatin and nuclear grooves. pseudopapillary structures with arborizing capillaries and metachromic core on diff - quik - stained smears were lined by epithelioid cells. acinar and rosette - like formations, as well as single neoplastic cells were observed [figure 1 ]. many tumor cells exhibited single or multiple clear cytoplasmic vacuoles, displacing the nucleus [figures 2 and 3 ]. the vacuoles were well - defined and variable in size, producing coin - shaped empty spaces in the cytoplasm [figures 4 and 5 ]. coin - shaped vacuoles were mimicking signet - ring appearance on the cell - block [figures 6 and 7 ]. periodic acid - schiff (pas) and mucicarmine stains were negative on the cell block sections. typical complex - branching pseudopapillary structures surrounded with single and/or loosely cohesive neoplastic cells (pap, smear) the clear vacuoles were often single but numerous clear vacuoles were also observed both in neoplastic groups and single, discohesive, plasmocytoid neoplastic cells (diff - quik, smears) small groups of cells with clear cell vacuoles in variable sized (diff - quik, smear) intracytoplasmic vacuoles are well defined like coin shaped (diff - quik, smears) vacuoles can be seen often and easily in diff - quik stained smears (diff - quik, smears) the vacuoles can be seen foamy to clear on cell block preparation (h and e, cell block) the vacuoles can mimick signet - ring appearance on cell block preparation (h and e, cell block) tumor cells showed strong nuclear staining for -catenin and cytoplasmic staining for vimentin and cd10 [figures 810 ]. neoplastic cells shows nuclear immuno reactivity with beta - catenin (cell block) strong cytoplasmic immuno reactivity with vimentin antibody (cell block) cd10 also showed strong cytoplasmic immuno reactivity similar to vimentin (cell block) spn is a rare tumor, which accounts for 1 - 3% of all non - endocrine tumors of the pancreas. this tumor tends to occur in young adult females and is generally localized in the body and tail of the pancreas. many theories have been hypothesized for the origin of the tumor including small duct epithelium, acinar cells, endocrine cells, totipotential stem cells or primitive cells capable of both exocrine and endocrine differentiation along with genital ridge - related cells that being incorporated into the pancreas during early embryogenesis. massive necrosis results in cyst formation in these tumors, while viable tumor cells loosely adhere to the blood vessels, giving the appearance of pseudopapillary formation. differential diagnosis of spn includes other solid, cystic and low grade malignant pancreatic neoplasms such as mucinous tumors, serous cystadenoma, neuroendocrine tumors, acinar cell carcinoma (acc) and pancreatoblastoma (pb). the combination of cytomorphologic features with pseudopapillary formation, myxohyaline change in vessels and cell degeneration produces various patterns such as acinar, trabecular, pseudorosette and single discohesive cells. immunocytochemistry is a highly useful tool in the differential diagnosis of this tumor. in the present case, striking intracytoplasmic vacuoles clear cell dominated pattern spn was first described in 2006 by albores - saavedra. they demonstrated three cases of spn with classic clinical features ranging in age from 26 to 32 years, in which the tumor cells had cytoplasmic vacuoles. ultrastructurally, the cytoplasm contained a moderate amount of smooth and rough endoplasmic reticulum but no glycogen particles, lipid, mucin or neurosecretory granules. the authors stated that the vacuoles were formed by dilatation of the smooth endoplasmic reticulum and mitochondria. he acclaimed the need to be attentive particularly for endocrine tumors of the pancreas with clear cell morphology while diagnozing. the latter group of patients, which tend to be von hipple lindau syndrome, also the clear cell cytoplasm in these tumors due to the presence of lipid droplets. jhala., were first to report the cytologic features of the clear cell variant of spn in 2007, in a subsequent paper jhala. they concluded that the presence of large clear vacuoles was more in favor of spn than pen. hav., and tanino., reported one case each of this entity. in a recent report, zhao., emphasized the diagnostic challenges in the fnab of this variant. beside the classic mimics of spn mentioned above, clear cell variant presents even broader differential diagnosis including primary and metastatic clear cell adenocarcinomas, metastatic renal cell carcinoma, oncocytoma, adrenal cortical tumors, ectopic adrenal tissue in the pancreas and metastatic balloon cell melanoma. vacuoles can appear foamy in the papanicolaou - stained smears and cell block sections, in contrast to the air - dried, diff -quik stained smears which show coin - shaped clear vacuoles. the foamy appearance can simulate oncocytoma or oncocytic tumors such as the conventional and chromophobe renal cell carcinoma, oncocytic adrenocortical neoplasms and extra adrenal oncocytic paragangliomas. clear cell spn can be distinguished from its mimics by careful attention to the clinical history, cytomorphologic and architectural features and the histological and immunocytochemical characteristics. there may be still potential errors due to various presentations which result from the delphic origin of spn. immunocytochemistry can be the key in these scenarios, being cytokeratin negative and vimentin positive. intense nuclear staining for -catenin and focal positivity for alpha-1-antitripsin in pas positive globules are highly useful. there are many additional stains in the literature such as cd10, progesterone receptors, cyclin d1, galectin3 etc., neuroendocrine markers ; particularly nse, cd56 and synaptophysin can may be expressed by neoplastic cells of spn, but chromogranin a is uniformly negative, which along with the lack of epithelial markers help to differentiate this tumor from pen. the combination of negative staining for cytokeratins and strong nuclear staining for -catenin is also helpful in distinguishing this tumor from acc and pb, which are well - known mimics of spn. the most helpful immunocytochemical stains in distinguishing clear cell spn from other pancreatic neoplasms and extra - pancreatic clear cell tumors are demonstrated in table 1. immunophenotypic profile of classic type and clear cell variant of spn including its common mimickers to the best of our knowledge, only two previous papers have reported the fnab features of clear cell spn. we conclude that the clear cell variant of spn can be diagnosed cytologically by careful attention to the cytomorphological criteria, combined with immunocytochemical stains. all authors of this article declare that they qualify for authorship as defined by icmje http://www.icmje.org/#author. each author has participated sufficiently in the study and takes public responsibility for appropriate portions of the content of this article. as this is case report without identifiers, our institution does not require approval from institutional review board (irb) (or its equivalent). to ensure the integrity and highest quality of cytojournal publications, the review process of this manuscript was conducted under a double blind model (authors are blinded for reviewers and vice versa) through automatic online system.	solid pseudopapillary neoplasm (spn) of the pancreas is a rare tumor of uncertain malignant potential, predominantly affecting young adult females. we report a case of clear cell variant of spn, which was diagnosed by fine needle aspiration biopsy. the aspirate was highly cellular and exhibited delicate branching papillary structures with central capillaries covered with several layers of plasmacytoid tumor cells. acinar and rosette - like formations, as well as single neoplastic cells were also observed. an unusual cytologic feature was the presence of large, clear cytoplasmic vacuoles. the diagnosis of spn was confirmed by characteristic immunocytochemical staining pattern including nuclear staining for -catenin, cytoplasmic staining for vimentin and lack of reactivity for cytokeratin.
ankylosing diseases of the spine include two major entities : ankylosing spondylitis (as) and diffuse idiopathic skeletal hyperostosis (dish). these diseases have unique pathophysiologies but similar clinical presentations later in the disease course.1 as is a seronegative spondyloarthropathy, an inflammatory condition with negative laboratory markers common to autoimmune conditions (e.g., anti - nuclear antibody, serum rheumatoid factor). however, there is a genetic predisposition, with 90% of individuals with as being positive for the hla - b27 allele.2 the disease causes bony replacement of ligaments, fusion of joints of the axial spine including the intervertebral disks and facet joints, and subsequent osteoporosis. as generally starts at the sacroiliac (si) joints and progresses in a rostral direction. dish, on the other hand, is a noninflammatory condition without a clear pathognomonic mechanism or genetic predisposition, but it is correlated with advanced age, diabetes mellitus, hypertension, and obesity.3 dish also leads to the progressive ossification of longitudinal ligaments and most commonly affects the thoracic spine, followed by the lumbar and cervical segments. the prevalence of as is 0.1 to 1.4% in the general population and as high as 5 to 6% in hla - b27 (+) individuals.4 5 the prevalence of dish varies considerably based on age and the population studied. for example, a finnish study concluded that the prevalence in the general population above age 40 was 3.8% for men and 2.6% in women, and in those over 70 years, the prevalence was 10.1 and 6.8%, respectively. in an american study on patients over 50 years, the prevalence was found to be 25% for men and 15% for women.6 the diagnosis of as is based on the modified new york criteria, which include radiographic and clinical criteria.5 as generally begins in the second or third decade, but because the disease is progressive, it may not become clinically apparent until later in life. the diagnosis of dish is based strictly on radiographs, with anterolateral bridging ossifications seen on at least four contiguous vertebrae without involvement of the si or zygapophysial joints.7 the restricted motion and overall kyphotic spinal alignment of patients with as and dish increases the risk of hyperextension injuries from seemingly trivial trauma, such as falls from standing.8 in fact, patients with as have four times the rate of spinal fractures compared with the general public, and although the fracture risk for patients with dish has not been reliably quantified, it is hypothesized that the same mechanisms are at play.9 10 previous studies have shown that these fractures most commonly occur in the cervical spine, followed by thoracic and lumbar segments.8 11 because the spinal column becomes progressively fused, the fracture pattern is often similar to that of a long bone fracture, with lever arms above and below the fracture.11 the substantial lever arms can be quite unstable and place the patient at high risk for a neurologic injury, particularly if the cervical segment is involved. in addition, there is a high incidence of missed fractures and epidural hematomas in these patients, leading to higher rates of spinal cord injury. treatment for ankylosed - spine fractures may be operative or nonoperative, depending on patient age, medical comorbidities, fracture pattern, and neurologic status. based on larger patient series, the average age of patients with an ankylosed spine - related fracture ranges from 59 to 72 years, and they tend to have increased medical comorbidities compared with the overall patient population with thoracolumbar spinal trauma.8 11 12 conservative treatment generally consists of bracing and bed rest. however, previous studies have demonstrated a trend for higher complication rates in nonoperative patients, and one series showed a mortality rate of 51% in the nonoperative group.11 it should be noted that most patients who receive nonoperative treatment do so because of high surgical risk, and most morbidity is due to the development of pulmonary complications.11 13 14 due to the torque associated with the lever arms, in conjunction with poor, osteoporotic bone quality, surgical treatment often requires multiple points of pedicle screw fixation above and below the affected level to appropriately stabilize the spinal column and heal the fracture. most series on surgery for ankylosed - spine fractures were based on traditional open procedures with pedicle screw placement and creation of a fusion bed. although percutaneous pedicle screw placement has been criticized for a lack of data regarding rates of pseudarthrosis and hardware failure, a recently developed algorithm for the treatment of traumatic thoracolumbar injuries proposed reserving the use of percutaneous nonfusion techniques to patients with as / dish.15 given the high risk profile of most patients with as / dish, our approach has likewise been to treat these patients with minimally invasive (mis) stabilization procedures to reduce blood loss, physiologic stress, and perioperative morbidity. we hypothesize that mis treatment in this patient population leads to favorable disability profiles and health - related quality - of - life (hrqol) measures. we retrospectively identified consecutive patients with hyperextension thoracolumbar injuries admitted to a single level i trauma center between june 2009 and june 2014. all patients were diagnosed with either as or dish and an acute spine fracture based on computed tomography (ct) scans, and magnetic resonance imaging scans were obtained if there was any question to the diagnosis of fracture or concern for involvement of the neural structures (fig. data were collected by an institutional review board (irb)-approved protocol at the university of pennsylvania. in addition to data from the hospitalization, outpatient follow - up data including radiographs were collected at the latest point of follow - up available. patients were then contacted and administered the oswestry disability index (odi) and euroqol-5d (eq-5d) surveys via mail to assess postoperative disability and hrqol, respectively. preoperative computed tomography and magnetic resonance imaging scans demonstrating acute vertebral fractures (arrows) for patients 5 (a), 6 (b), 11 (c), and 10 (d). all surgeries were performed under general anesthesia by the senior author. because of the general kyphotic spinal alignment, all patients were positioned on a radiolucent wilson frame on the flat jackson operating table rather than in the prone modular jackson table (mizuho osi, union city, california, united states), which increases thoracolumbar extension. a midline skin incision was made, and the inferiormost spinous process was exposed to anchor the fiducial array for the stealthstation navigation system (medtronic, minneapolis, minnesota, united states). an o - arm intraoperative ct scan the midline skin incision was extended as needed, and the entry points were planned using the navigation probe. small, paraspinal fascial incisions were made, and the tip of a navigated drill guide was used to bluntly dissect down to bone. using the navigated drill guide, pilot holes were made, followed by a navigated tap and navigated screwdriver to place the pedicle screws with screw extensions (viper mis spine system, depuy - synthes, raynham, massachusetts, united states). rods were subsequently cut to size, contoured, and passed under the muscle and fascia through the screw heads and screw extensions. the rods were secured to the screws with locking caps and the screw extensions removed. patients 1 and 2 received orthotic braces to be worn for 6 weeks postoperatively, although no subsequent patient was assigned a brace. all surgeries were performed under general anesthesia by the senior author. because of the general kyphotic spinal alignment, all patients were positioned on a radiolucent wilson frame on the flat jackson operating table rather than in the prone modular jackson table (mizuho osi, union city, california, united states), which increases thoracolumbar extension. a midline skin incision was made, and the inferiormost spinous process was exposed to anchor the fiducial array for the stealthstation navigation system (medtronic, minneapolis, minnesota, united states). an o - arm intraoperative ct scan the midline skin incision was extended as needed, and the entry points were planned using the navigation probe. small, paraspinal fascial incisions were made, and the tip of a navigated drill guide was used to bluntly dissect down to bone. using the navigated drill guide, pilot holes were made, followed by a navigated tap and navigated screwdriver to place the pedicle screws with screw extensions (viper mis spine system, depuy - synthes, raynham, massachusetts, united states). rods were subsequently cut to size, contoured, and passed under the muscle and fascia through the screw heads and screw extensions. the rods were secured to the screws with locking caps and the screw extensions removed. patients 1 and 2 received orthotic braces to be worn for 6 weeks postoperatively, although no subsequent patient was assigned a brace. there were 11 patients in the study, 9 with as and 2 with dish. patient 6, whose fracture occurred subsequent to positioning for a brain tumor operation, was admitted approximately 2 months postoperatively for a cardiopulmonary event and died in the hospital. patient 5 returned to living in a nursing home due to advanced dementia and thus could not complete the functional outcomes surveys. patients 4 and 10 declined to participate in the outcomes surveys, but both live independently and report doing functionally well. all other patients returned to living independently. details on patient demographics, comorbidities, and surgical metrics are presented in table 1. the average follow - up time was 28 months (range : 5 to 58). the average age at surgery was 77 (range : 52 to 88), and 45% were male (5/11). all patients had an american society of anesthesiologists (asa) grade of iii, representing severe systemic disease. all fractures occurred in the thoracic, thoracolumbar junction, or lumbar spine, with an average of seven segments included in the operative construct. of the 11 patients, 5 (45%) suffered a low impact injury, with the most common mechanism being a fall from standing. all patients had an american spinal injury association (asia) impairment scale of asia - e (neurologically intact), except for patient 3, who was asia - d due to weakness associated with concomitant jumped cervical facets. average body mass index (bmi) was 34 (range : 20.4 to 44.5), with 73% (8/11) being obese (bmi 30). average operative time was 227 minutes (range : 79 to 449), and the average blood loss was 251 ml (range : 25 to 900 ml). the average postoperative length of stay was 14.4 days (range : 4 to 60 days), and 36% of patients (4/11) required wound revision for superficial breakdown, of which two (patients 7 and 11) grew organisms from surgical cultures. one patient (patient 3) had her iliac wing screws removed approximately 2 years after the initial surgery due to a bothersome prominence. abbreviations : as, ankylosing spondylitis ; asa, american society of anesthesiologists ; bmi, body mass index ; dish, diffuse idiopathic skeletal hyperostosis ; ebl, estimated blood loss ; los, length of stay ; mvc, motor vehicle collision ; n, no ; n / a, not available ; op, operation ; or, operating room ; ped, pedestrian ; y, yes. the average odi was 21.5% (range : 0 to 34%), corresponding to low to moderate disability (defined as 21 to 40%). the average eq-5d utility score was 0.77 (range : 0.60 to 1). the range of eq-5d utility scores in the u.s. population ranges from 0.109 to 1, where 1 represents perfect health, 0 represents death, and negative numbers are deemed to be a state worse than death per the sample cohort. we only report eq-5d values for patients who were alive with self - reported eq-5d surveys. abbreviations : asa, american society of anesthesiologists ; bmi, body mass index ; eq-5d, euroqol-5d ; los, length of stay ; odi, oswestry disability index. spine fractures in patients with ankylosing spinal disorders are often devastating events because most of the patients tend to be older with multiple medical comorbidities. in our series, the average combined patient age of 77 years was in line with average ages published in the literature, which range from 68 to 81.5 in combined as and dish cohorts.8 11 12 13 14 16 17 moreover, all patients in our series had at least one major medical comorbidity and an asa grade of iii, representing severe systemic disease, including reversible conditions such as multiorgan injury from polytrauma. the acquired global kyphosis in this patient population frequently causes decreased pulmonary compliance, as well. these factors cumulatively place patients at a higher operative risk than typical patients with spinal trauma. the rates of neurologic injury at presentation in ankylosed spine fractures tend to be high. one series found 58% of all patients presented with neurologic deficits, and another larger meta - analysis found that 67% of patients with as and 40% with dish presented with deficits.8 11 in particular, a finnish study found patients with as have greater than 11 times the risk of a spinal cord injury compared with the general population.18 additionally, these patients have high rates of delayed diagnosis and thus an increased risk of secondary injury. for example, in one series of 20 patients with as, the initial plain films were negative in 60% (12/20).19 caron quantified the danger of missed diagnoses ; 81% of patients with a delayed diagnosis experienced neurologic deterioration during the delay.11 it should be noted that these large series include cervical spine fractures, which not only are the most common fractured segment in ankylosing spinal diseases, but also carry the highest rates of neurologic deficits. in our series, all but one patient was neurologically intact. patient 3 had concomitant jumped cervical facets resulting in a spinal cord injury attributable to the cervical spinal cord. there was one delayed diagnosis : patient 6 was originally admitted for meningioma surgery and the fracture likely occurred during operative positioning. he had persistent back pain that led to neuroimaging several days later, identifying a spine fracture, but the patient did not have deficits attributable to the fracture. given the poor functional status and high operative risk of most patients with as and dish, most series on ankylosed spine fractures demonstrate high rates of nonoperative treatment, ranging from 33 to 46% among the combined cohorts.8 11 14 although we could not find any series that demonstrated a statistically significant difference in morbidity and mortality between operative and nonoperative cohorts, in general most major complications and deaths among nonoperative patients were related to the development of a pulmonary complication, presumably due to a combination of prolonged stasis, decreased pulmonary compliance from a kyphotic habitus, and bracing. when the operative risk is acceptable, we believe that early definitive surgical treatment of the fracture allows patients to mobilize earlier, preventing some of those adverse events from occurring. although a variety of surgical approaches have been used to treat these fractures, including anterior, posterior, and combined approaches, the most common approach based on the published series appears to be posterior instrumentation three levels above and three levels below the fracture. percutaneous instrumentation has become increasingly popular in the treatment of thoracolumbar trauma,20 21 22 23 24 25 and two recent series have been published on the specific use of mis in the treatment of spine fractures in ankylosing spinal disorders.16 17 however, the two series took divergent views on the number of levels to include in the construct ; one group sought to instrument the fewest number of levels (average of 1.8 levels), and the other primarily instrumented three levels above and below the fracture. patient 4 's sagittal computed tomography scan demonstrating an unstable t7 fracture with listhesis (a). postoperative anteroposterior (b) and lateral (c) x - rays of the operative construct demonstrating reduction of the fracture. positioning patients with ankylosed spines can be very precarious, as demonstrated by patient 6, who suffered an iatrogenic fracture. because these patients develop a global kyphosis with limited mobility, placing them in a prone position that increases lordosis may exacerbate their fracture, cause a new fracture, or cause neurologic injury due to the unstable lever arms flanking the fracture. although other series on percutaneous instrumentation for ankylosed spine fractures have used fluoroscopy as the primary imaging modality, we used ct guidance for the screw placement and intraoperative ct to confirm the appropriate screw position prior to completing the surgery. as yeoh have described, the fused joints and osteoporotic bone quality in ankylosing spinal disorders make x - rays very difficult to interpret.17 ct overcomes this challenge with increased detail that facilitates more accurate screw trajectories. additionally, the poor bone quality increases the risk of the screw loosening and hardware migration. ensuring good screw rod interface can help minimize the risk of hardware failure, thus it is critical to prebend the rods to the exact contour of the screw extensions about the skin and avoid using persuader - type devices to fit and lock the rods in place. we identified one large study specific to as / dish fractures that reported mean operative times of 264 minutes for patients with as, 203 minutes for patients with dish, and 220 minutes for control patients, along with mean blood loss of 960 ml, 788 ml, and 613 ml for each respective cohort.14 that study included fractures throughout all spinal segments and included a combination of long - segment and short - segment fusions, as well as a small number of mis fixations. although our mean operative time is similar, the mean estimated blood loss in our series was considerably less. given the small number of cases in this series, we believe that operative times will continue to decline as we move further along the learning curve. although none of the patients in our series required late revision of the instrumentation, 4/11 patients did require reoperation for wound breakdown. patients 7, 10, and 11 were all morbidly obese (bmi 37.7, 44.5, 43.1, respectively) with active diabetes. patient 3 developed a postoperative urinary tract infection and had the longest operative time of the series (449 minutes). it should also be noted that only patients 7 and 11 had grown organisms from cultures obtained during the operation (methicillin - resistant staphylococcus aureus and methicillin - susceptible s. aureus, respectively), although all four patients were treated with antibiotics. although none of the infections were deep to the fascia or required removal of instrumentation, all patients required hospitalization and a second surgical procedure. we had converted to a single midline skin incision as opposed to bilateral, paramedian incisions out of concern for devitalizing the skin in the midline between the parallel incisions. however, this method also increases the amount of the suprafascial dead space. in the obese patients, we placed a temporary subcutaneous drain to reduce the dead space and prevent seroma formation, but we will consider utilizing smaller parallel incisions if there are no pre - existing skin issues. outcomes in spine surgery have largely been reported as assessment of radiologic parameters (e.g., healed fracture, kyphotic angulation, etc.) or neurologic status (e.g., asia scale), although increasing attention has been given to patient - reported outcome measures in the field. in this series, we tracked the patient - reported health outcome measures using one disease - specific survey and one hrqol survey, the odi and eq-5d, respectively. to our knowledge, one other series reported odi results, and we are the first to report eq-5d values. yeoh performed mis long - segment stabilization on 10 patients with as and dish, and they reported an average postoperative odi of 16%, compared with a value of 21.5% in our series.17 our average eq-5d utility of 0.77 suggests that the patients have a reasonably high quality of life, although the precise improvement could not be determined due to a lack of preoperative values. because of the underlying diagnoses of as or dish, in addition to old age, these patients will naturally have a certain baseline level of disability and diminished hrqol the sport trial reported similar 1- and 2-year postoperative utility values in patients undergoing surgery for lumbar stenosis and degenerative spondylolisthesis.26 the results from our series suggest that patients undergoing percutaneous dorsal instrumentation for ankylosed spine fractures tend to have limited disability and high levels of health utility, despite old age, obesity, and multiple medical comorbidities in the average patient. for our patients, we only obtain imaging through 3 months postoperatively unless the patient has specific signs or symptoms to suggest neurologic deterioration or a hardware malfunction, as determined by irb restrictions. this approach precludes the ability to assess the long - term radiographic outcomes, such as fracture healing and deformity correction. although we have an average follow - up time of 28 months, hardware - related complications can be encountered several years after the surgery, thus the duration of postoperative imaging is determined by the discretion of the treating surgeon. limitations in this study include its retrospective nature, small number of patients, and lack of preoperative patient - reported outcome measures. moving forward unfortunately, because most of these patients present with acute trauma, obtaining preoperative patient - reported outcome measures will be exceedingly difficult, if not impossible, to reliably obtain. as and dish are two ankylosing spinal disorders that significantly increase the risk of spinal fractures. given the old age and multiple medical comorbidities associated with the average patient, surgery in this patient population is high risk, with high rates of postoperative morbidity and mortality. mis techniques in spine surgery are becoming increasingly common, both in routine practice and in trauma settings. based on our results, mis stabilization, when used on patients with good preoperative neurologic status, can be successfully used to manage spinal fractures in the patients with as and dish and can preserve a good postoperative quality - of - life with limited disability.	study design retrospective case series. objective ankylosing spondylitis (as) and diffuse idiopathic skeletal hyperostosis (dish) are two related diseases that significantly increase the risk of unstable spinal fractures from seemingly trivial trauma. given the older age and higher surgical risk profile of most of these patients, minimally invasive (mis) approaches to the treatment of such fractures may reduce operative risk and physiologic stress. methods eleven consecutive patients with hyperextension thoracolumbar injuries and a diagnosis of as or dish admitted to a single level i trauma center between june 2009 and june 2014 were retrospectively reviewed. all patients were treated with mis stabilization. in addition, the patients were administered the oswestry disability index and euroqol-5d surveys to evaluate patient - reported outcomes regarding disability and health - related quality of life, respectively. results of the 11 patients, 10 were alive at the time of review. the mean follow - up time was 28 months. the average age was 77 years old with a mean body mass index of 34. all patients had severe systemic disease, american society of anesthesiologists grade iii, with multiple medical comorbidities. seven segments on average were included in the operative construct. there were no instrumentation failures or nonunions requiring revision surgery. the average postoperative oswestry disability index was 21.5% (range : 0 to 34%), corresponding to low to moderate disability, and the average euroqol-5d utility score was 0.77 (range : 0.60 to 1), a similar average postoperative utility value to those published in the literature on elective surgery for degenerative lumbar conditions. conclusions mis stabilization, when used on patients with good preoperative neurologic status, can successfully manage spinal fractures in patients with as and dish and preserve a favorable postoperative quality of life with limited disability.
elastosis perforans serpiginosa (eps), characterized by transepidermal elimination of fragmented elastic fibres, clinically presents as hyperkeratotic papules. it 's rare association with pseudoxanthoma elasticum (pxe) has been documented in the literature. we report a case of pxe with associated lesions that were histopathologically compatible with eps. pxe is due to the mutations in the mrp6/abcc6 gene, which is a member of the atp - binding cassette (abc) family and acts as a transmembrane transporter primarily in the liver and the kidney, primary defect of which results in the accumulation of certain metabolic compounds with secondary calcification of elastic fibers. we also highlight the importance of the clinical finding of an exaggerated mental crease, which has been shown to be a sensitive and highly specific finding in patients under the age of 30 years with pxe. a 22-year - old female, third of four siblings born of first - degree consanguinity presented with a three - year history of excess folds of the skin on the sides of the neck and a six months history of asymptomatic eruption on the neck. there was no history of intermittent claudication, chest pain, seizures, or melena. physical examination revealed numerous small yellow brown non follicular papules about 12 mm in size arranged in a linear fashion on the sides of the neck. skin of the neck was soft, lax and was hanging in folds on the sides of neck and in both the axillae [figures 1 and 2 ]. a few brown to black distinctive hyperkeratotic nonfollicular papules about 5 mm in diameter arranged in a serpiginous fashion with a central plug, revealing a crateriform pit on dislodgment were seen on the sides of the neck [figure 3 ]. electrocardiogram (ecg), 2d echo, and radiography of the chest were normal. the ophthalmologic examination revealed the presence of angioid streaks in the fundus, which were clinically asymptomatic [figure 5 ]. lax skin demonstrated on the sides of neck lax skin hanging in folds in both the axillae hyperkeratotic nonfollicular papules and atrophic scars arranged in a serpiginous seen on the sides of the neck a single prominent mental crease angioid streaks in the fundus demonstrated on ophthalmoscopy histopathology of the biopsy specimen from a hyperkeratotic papule revealed hyperplastic epidermis. there were numerous fragmented, stringy, and curled elastic fibers giving the appearance of revealed wool in the upper- and mid - reticular dermis [figure 6 ]. special staining with von kossa stain revealed calcification of the elastic fibers in the reticular dermis and in the disrupted follicular infundibular wall. with the above clinical and histopathological evidence, we diagnosed the case as pxe with associated eps. histopathology revealing numerous fragmented, stringy, and curled elastic fibers giving the appearance of raveled wool in the upper- and mid - reticular dermis [h and e, 10 ] higher magnification revealing disrupted follicle surrounded by foreign body type granulomatous inflammation with several foreign body giant cells and neutrophils with small fragments of elastic fibers in the infundibular wall [h and e, 40 ] pxe (mim 264800), also known as systematized elastorrhexis or gronblad strandberg syndrome is an autosomal recessive disease of the connective tissue, which primarily affects the dermis, retina, and the cardiovascular system. the prevalence is currently estimated to be 1 in 25,000 - 70,000 live births. the diagnosis of pxe is most often made late in the second or third decade of life. genetic studies have identified mutations in the mrp6/abcc6 gene on chromosome 16p13.1, which is a member of the abc family and acts as a transmembrane transporter primarily in the liver and the kidney, primary defect of which results in the accumulation of certain metabolic compounds with secondary calcification of elastic fibers. characteristic skin lesions include yellow papules in a linear or reticulate pattern with soft, lax, wrinkled skin hanging in folds by the sides of the neck, below the clavicles, the axillae, abdomen, groins, perineum, and thighs described as eye involvement results from breaks in bruch 's membrane at the posterior aspect of the retina (angioid streaks), which is symptomless but may be complicated by retinal neovessels, recurrent hemorrhage, disciform scarring, and eventual loss of central vision. most of the cardiovascular manifestations are related to early arteriosclerosis as a consequence of degenerated elastic laminae, which manifest as disappearance of peripheral pulses, intermittent claudication, angina pectoris, rarely myocardial infarction, cerebral stroke, and visceral hemorrhage. early diagnosis is important if the ocular and cardiovascular complications are to be prevented. to facilitate and unify the clinical diagnosis for pxe, three major diagnostic criteria (characteristic yellow skin lesions in flexural sites, elastic fiber calcification in lesional skin, and ocular disease) and two minor criteria (histopathologic features in nonlesional skin and family history of pxe in a first - degree relative) have been adopted. the presence of exaggerated mental crease has been shown to be asensitive and highly specific finding in patients under the age of 30 years with pxe. it may be the result of deterioration of elastic tissue in the lip and chin, or blood vessels supplying the jaw. the clinical finding of a horizontal chin crease in the presence of central loss of vision, arterial occlusive disease, or acute gastrointestinal hemorrhage should prompt the physicians to examine their patients for other features of pxe. a rare association of pxe with eps such as lesions characterized by spontaneous perforating lesions with transepidermal elimination of fragmented elastic fibers, clinically presenting as hyperkeratotic papules has been reported. eps is classified into three types : (1) idiopathic ; (2) reactive, associated in 25% of cases with connective tissue diseases ; (3) induced by d - penicillamine. confusion exists whether these perforating lesions should be considered as eps associated with pxe or simply as perforating pxe. the first group includes patients previously diagnosed with generalized hereditary pxe, who in the course of their disease progression develop clinical lesions that are histopathologically indistinguishable from eps. the second group is characterized by transepidermal elimination of altered elastic fibers occurring in a localized, acquired form of pxe usually in the periumbilical region, who do not develop other ocular, vascular, and visceral manifestations of hereditary pxe. the term periumbilical perforating pxe was first proposed by hicks., in 1979, for the second entity. localized acquired cutaneous pseudoxanthoma elasticum, as they believed that the process was acquired and was lacking differentiating the above conditions is important as the prognosis differs and a close follow up is needed in the hereditary form of pxe to rule out the systemic involvement. we categorize our patient into the hereditary form with systemic involvement such as angioid streaks and having associated eps lesions. we also highlight the importance of prominent mental crease, which when identified in an individual less than 30 years should prompt a search for pxe, which is often diagnosed as in our patient.	elastosis perforans serpiginosa (eps), characterized by transepidermal elimination of fragmented elastic fibers, clinically presents as hyperkeratotic papules. eps is classified into three types : (1) idiopathic ; (2) reactive, with associated connective tissue diseases such as pseudoxanthoma elasticum (pxe), ehlers danlos syndrome, cutis laxa, marfan syndrome, osteogenesis imperfecta, down 's syndrome ; (3) the one that is induced by d - penicillamine. a rare association of eps with pxe, which is primarily a defect of transmembrane transporter protein with accumulation of certain metabolic compounds and secondary calcification of elastic fibers has been documented in the literature. we report a case of pxe with associated lesions that were histopathologically compatible with eps.
traumatic brain injury is a major cause of sustained morbidity and disability both in the military and civilian populations (tarapore., 2013). in the case of mild traumatic brain injury (mtbi), an initial brief change in mental state or consciousness is typically followed by postconcussion symptoms (cassidy., 2004), such as headaches, fatigue, and dizziness, which usually emerge on the day of injury and persist for at least the first few days thereafter (boccaletti., 2006). in most patients cognition recovers and symptoms resolve within 3 months ; however, up to 25% of patients (sigurdardottir., 2009) suffer residual symptoms, long - term impairment, and sometimes disability (levin, 2009). in a large percentage of patients mtbi is difficult to diagnose because of the absence of apparent external injuries and obvious focal brain lesions in conventional computed tomography (ct) or magnetic resonance imaging (mri) scans (tarapore., 2013), although mtbi - related morphological changes at a microscopic level have been recently reported using mri (pasternak., 2014 ; a common practice is to classify tbi as mild, moderate, or severe based on the level of consciousness assessed using the well - known glasgow coma scale (gcs, teasdale and jennet, 1974). some studies have attempted to classify tbi and predict long - term outcome based on ct findings (zhu., 2009) or the amount of exosomes released in the peripheral circulation due to injury (taylor and taylor, 2014). one mechanism by which tbi is thought to affect cognition and behavior is through changes in functional connectivity (castellanos., 2010 ; martino., 2011). functional brain connectivity refers to temporally correlated activity in spatially distinct brain regions and is believed to mediate neural communication among these regions (aertsen., 1989 ; salvador., 2005 several imaging studies have demonstrated the existence of a network of functionally connected brain regions that support a default mode of brain function (guskiewicz., 2005). changes in brain activity caused by focal lesions result in abnormal interactions among local and remote brain regions that are functionally connected (quigley., 2001 ; van cappellen van walsum., 2003), and these changes are reflected on the configuration of the default brain network. thus, functional connectivity analysis offers a unique opportunity to quantify changes in neurophysiological processes that correlate well with clinical symptoms (huang. 2009, huang. 2012 ; lewine., 2007). the literature shows several examples of pathological alterations, including increase (van dellen., 2012) and decrease (sharp 2010, rubinov, and bullmore 2013), which provide evidence that a balance in the synchronization level in healthy controls is required for optimal brain function. several brain connectivity studies in clinical populations focus on the description of topological differences with control group employing various synchronization measures, network metrics and statistics (sporns, tononi, and ktter 2005, guggisberg. stam., 2007 ; bassett., 2009 ; bullmore and sporns, 2009 ; castellano., 2010 ; dimitriadis., 2013b ; bigler, 2013). in the area of brain injury in particular, during the last two decades there has been a growing effort to characterize the structural and functional effects of mtbi using the full range of neuroimaging methods (see the excellent review by eierud., 2014), and various biomarkers have been proposed based on functional mri (fmri) and diffusion tensor imaging (dti) (kou., 2010 ; hunter., 2012, meg plays a special role because it measures neuromagnetic fields resulting from primary neuronal currents that are not subject to the distortion induced by the variable conductance of brain tissues (tarapore., 2013). 2011, qian. 2013) because they require no training or experience with cognitive tasks, and they impose minimal demands on a patient, which is especially important after brain injury. thus, resting state meg is particularly suitable for detecting changes in functional connectivity networks in patients with mtbi compared to matched controls. one of the very first studies using resting - state meg as a possible neurophysiological biomarker for mtbi provided clear evidence that analysis of functional connectivity patterns could be a valuable tool for early detection of mtbi (zouridakis., 2012). other studies showed abnormal slowing in brain areas affected by tbi (huang., 2014) and reduced overall functional connectivity in tbi patients compared to controls (tarapore., 2013). in particular, using resting - state meg and low - frequency source imaging, huang. (2012) showed 87% and 100% accuracy in detecting abnormalities in mild and moderate tbi, respectively. furthermore, lewine. (1999) used mri and meg to examine postconcussive symptomatology and were able to discriminate between healthy adults and individuals with resolved mtbi. another meg study attempting to characterize system complexity and potential neural network damage found reduced complexity in multiple brain areas in mtbi subjects relative to healthy controls (luo., 2013). other studies further demonstrated that decreased connectivity in resting - state meg may persist for years after mtbi, even in mild tbi cases (castellanos., 2011), but the abnormally reduced connectivity might improve over time across serial meg scans, suggesting neuroplasticity during recovery from tbi (tarapore., 2013). continuing our earlier attempt (zouridakis., 2012) to understand how mtbi affects communication in the human brain network, the present study extends our previous work in several ways : first, we employ a much larger sample of patients and controls, 31 mtbi patients and 50 normal controls, compared to only 10 and 10, respectively ; second, we use the phase locking value estimator and graph theory to compute functional connectivity graphs (fcgs), whereas in the earlier study we used granger causality as the estimator ; and third, we follow a tensor - based instead of vector - based approach for subject classification. here we consider fcgs as second - order tensors, which, after dimensionality reduction, are used for subject classification, whereas the earlier study considered the granger connectivity matrices as one - dimensional vectors and used them for subject classification ; and fourth, we performed the analysis in several frequency bands, instead of only one, based on recent studies that found differences in fcgs across frequency bands (illebrand., 2012 ; engel., 2013). the remainder of this paper is structured as follows : section 2 describes the data and the detailed analysis procedure, section 3 presents the classification results, section 4 is devoted to discussing our findings, and section 5 summarizes some concluding remarks. the overall methodology employed in this study is outlined in fig. 1 and includes the following steps : data collection, preprocessing, wavelet decomposition, computation of functional connectivity graphs (fcgs), selection of significant links, feature extraction, optimization of the number of features, designing a classifier, evaluating its performance, and assessing the topological properties of the fcgs. the present project is part of a larger study of mtbi, supported by the department of defense (dod). the definition of mtbi used in this study followed the guidelines of dod (assistant secretary, 2007) and the american congress of rehabilitation medicine (kay t., 1993). this work was approved by the institutional review boards (irbs) at the participating institutions and the human research protection official 's (hrpo) review of research protocols for dod. all procedures were compliant with the health insurance portability and accountability act (hipaa). subjects included in this analysis included a group of mtbi subjects from the dod project and a group of age - matched normal controls drawn from a database that was being assembled as a normative data repository at uthsc - houston. the mtbi group consisted of 31 right - handed mtbi participants (29.33 9.2 years of age). subjects were recruited from the emergency departments (eds) of two level 1 trauma centers and one level iii community hospital in a large ethnically diverse southwestern metropolitan area. subjects were recruited by healthcare professionals (rn, md, emt - p) who had clinical experience with brain injury patients, knowledge of research, and excellent interpersonal and problem - solving skills. screening occurred through review of data in the eds ' electronic healthcare system (ehs), consultation with ed staff, and subject interviews. special permission was obtained from the institutional irbs to administer the galveston orientation and amnesia test (goat) (levin., 2008) prior to obtaining informed consent to identify cognitive impairment that would preclude provision of informed consent. all subjects showed goat scores of 75 or greater and so have provided informed consent. the inclusion criteria for all subjects in the dod project included age 1850 years, injury occurring within the preceding 24 h, and no requirement for hospitalization for the injury for which the participant was enrolled. for mtbi subjects, the inclusion criteria also required the presence of a head injury (documented in medical records and/or verified by witnesses), glasgow coma scale (gcs) (teasdale and jennett, 1974) score 1315, loss of consciousness 3 for any body part, history of significant pre - existing disease (e.g., psychotic disorder, bipolar disorder, post - traumatic stress disorder (ptsd) diagnosed by a psychiatrist or psychologist, past treatment for alcohol dependence or substance abuse), blood alcohol level > 80 mg / dl at the time of consent, documentation of intoxication, left - handedness, and contraindications for mri (including claustrophobia and pregnancy). the demographics of mtbi subjects and the location of injury are given in table 1. the control group (group n) consisted of 55 right - handed age - matched normal subjects (29.25 9.1 years of age). previous hospitalization for mtbi, history of neurologic disorder, schizophrenia, bipolar disorder, substance abuse, and extensive dental work and implants incompatible with meg were also the exclusion criteria for the control subjects. the research protocol for the control group also received irb approval, and all participants provided written informed consent. subjects were asked to lie down and remain as still as possible during the recording procedure with eyes closed. data collection in this large multicenter study involved two different meg scanners, a 248-channel whole - head magnes wh3600 system (4d neuroimaging inc., san diego, ca) featuring axial gradiometer sensors, and a 306-channel elekta neuromag system (elekta ab, stockholm, sweden) featuring 204 planar gradiometers and 102 magnetometers. to make the recordings from various projects of the larger study comparable, all data collected with axial gradiometers were transformed to planar gradiometer field approximations using the sincos method implemented in fieldtrip (oostenveld., 2011). planar gradiometers have maximum sensitivity to superficial cortical sources directly under them (hmlinen, 1995), which makes them less sensitive to artifacts and distant disturbances. natick, ma, usa) and fieldtrip (oostenveld., 2011). axial gradiometer data were originally collected at a sampling rate of 1017.25 hz and bandpassed between 0.1200 hz using hardware filters, whereas the planar gradiometer data were collected at 1000 hz and bandpass filtered via software between 0.1200 hz. artifact reduction was performed based on independent component analysis (ica) as implemented in the eeglab toolbox (delorme and makeig, 2004). the extended informax algorithm (delorme., 2007a ; onton., 2006 ; romero., 2008) was used to remove components associated with eye, muscle, and cardiac artifactual activity (dimitriadis. in addition to visually inspecting the time course and topographical layout of all ics, we estimated their kurtosis and skewness values. components reflecting cardiac activity were recognized from their typical rhythmic pattern in the time domain and their widespread topography. ics related to muscle activity were identified based on statistical measures (kurtosis higher than a predefined threshold, kurtthr = 12, determined empirically), spectral characteristics (increased energy in the frequency range between 2060 hz), and topographies encompassing temporal brain areas (delorme., 2007b ; dimitriadis. artifactual ics were zeroed, whereas the remaining ics were used to generate artifact - free signals that were back - projected to the original meg sensor space via the inverse ica transformation. occasionally, activity from a bad sensor was replaced with the interpolated activity of its immediate neighbors after applying ica to avoid introducing dependencies in the recordings. this interpolation procedure was necessary for five control and two mtbi subjects for at most four meg sensors. meg signal oscillations in specific frequency bands were studied using wavelet transform analysis (mallat, 2008). after preprocessing, all data were further lowpass filtered and decimated by a factor of six, giving an effective sampling rate of 166 hz, which constrained the frequency bands of the wavelet transform to roughly conform to the classical frequency bands (assett., 2009). decimation was used to reduce the processing time, while the new sampling rate was higher than twice the minimum rate required by the nyquist theorem to avoid distortion of the meg signals in the effective range of frequencies of interest (160 hz). each frequency band was isolated by applying the maximum overlap discrete wavelet transform to each time series (daubechies, 1992) using the daubechies 5 wavelet in line with previous work (assett. thus, wavelet scale 1 (3060 hz) roughly corresponded to the classical low- band, scale 2 (1530 hz) to, scale 3 (815 hz) to, scale 4 (48 hz) to, and scale 5 (14 hz) to. we constructed fcg by quantifying the coupling between pairs of sensors using the phase locking value (plv) as an estimator (lachaux., 1999), separately for each frequency band. plv is defined as(1)plv=1nt=1nej(t)where (t) is the difference in phase between the two time series and n is the number of samples. if the phase difference varies by a little, plv is close to one ; otherwise it is close to zero. plv can assess phase relationships between two signals but it can not detect any causal relationships (lags or delays) between the signals. to construct intra - frequency functional connectivity matrices, we calculated the plv between the time series in each frequency band for all possible pairs of sensors to create the weights matrix fcg. through this process, we created 5 such connectivity matrices (,,,, and) per subject. for each subject, the aforementioned procedures yielded a fully connected, weighted, symmetric fcg representing the mutual influences among all cortical regions. the maximum number of possible undirected connections in a network with k nodes is nmax = k(k 1) / 2. when k = 248, the corresponding nmax = 30,628, and the fcg is extremely dense. therefore, it must be filtered so that the pattern of the most significant connections can emerge. we performed two kinds of filtering based on statistical analysis and graph theory principles. for statistical filtering a surrogate data method (lachaux., 1999) with multiple realizations was used to select the most significant fcg values at the 99% confidence level. generating 200 surrogates from the original time series was enough to reveal significant interactions (lachaux., 1999). surrogates were obtained by randomizing the phase of the original signals while keeping their spectral profile intact to remove all temporal correlations (theiler, 1992). after computing the surrogate plvs, we assigned a p - value to each connection in the fcg matrix by estimating the proportion of surrogate plv values that were higher than the observed values (theiler., 1992). to correct the effects of multiple comparisons, p - values were adjusted using the false discovery rate (fdr) method (benjamini and hochberg, 1995). a threshold of significance q was set such that the expected fraction of false positives was restricted to q 0.01 (dimitriadis., 2012 ; ioannides., 2012). graph theory - based analysis (bullmore and sporns, 2009 ; bassett and bullmore, 2009 ; he and evans, 2010 ; stam, 2010) was used to capture the structure of the neural system under investigation and the relationship between separation and integration of neural populations. small - word structures are characterized by a dense network of local connections and a limited number of long - range connections that provide efficient communication between distant nodes. efficiency in information transmission between nodes is measured as the inverse of the shortest distance between the nodes, while the average of all pair - wise efficiencies represents the global efficiency of the graph. the function cost relates to the energy expenditure needed for a network to maintain its efficiency, and it is given by the ratio of existing connections divided by the total number of possible pairwise connections in a network. global cost efficiency is defined as the global efficiency e at a given cost c minus the cost (e c), which typically has a positive maximum value at some cost cmax, for an economical small - world network. importantly, this metric of network topology is independent of arbitrary, investigator - specified thresholds. instead, the cost efficiency curve is estimated over a wide range of thresholds, and the behavior of the curve is summarized by its maximum value, which occurs at a data - driven connection density or cost c (bassett., 2009)1 the physical distance among the meg sensors was used to separate the local from the long - range connections. for each sensor, we computed its neighbors of order one, two, three, and four, i.e., neighbors in all directions that were one, two, three, and four steps away, based on the topographical layout of the meg sensors (zouridakis., 2012). to optimize the performance and generalizability of our methodology (hastie., 2003), we implemented a true external cross - validation procedure by splitting the data into two subsets : a training set that included 80% of the subjects (44 n and 25 mtbi) and was used for designing the classifier and a test set that included the remaining 20% of the subjects (11 n and 6 mtbi) and was used for assessing the classifier performance. as a metric, we used the correct classification rate, which was defined as the proportion of subjects in the test set for which the correct label was predicted. the entire cross - validation procedure was repeated 100 times to avoid any adaptation effects, and the average value of the correct classification rate was used to compute the overall accuracy of the procedure. the optimum number of feature to represent the data was determined using only the training dataset in a five - fold cross - validation procedure as described in fig. test subjects were removed from the analysis at the very beginning of the procedure, prior to feature selection and five - fold cross validation. most of the previous studies in this area represent fcgs as vectors in a high - dimensional space (shen., 2010 ; pollolini., 2010 ; richiardi., the main limitation of this approach (and the related feature extraction algorithms) is that it overlooks the inherent form of fcgs. each fcg has a natural tabular representation and hence it can be thought of as a second - order tensor. the relationship between the row and column vectors of the associated matrix might be important for deriving a suitable low - dimensional representation (projection), especially when the number of available connectivity patterns is small. to overcome the limitations of previous approaches, in this study we treated fcgs as tensors and resorted to tensor subspace analysis (tsa) (he and cai, 2005) as the most appropriate feature extraction algorithm. in our formulation, the tensor form was given by (subjects sensors sensors) (dimitriadis. fcgs from individual subjects were represented based on a recent methodology that blends ideas from multi - linear algebra and manifold data learning (he and cai, 2005). briefly, given a set of fcgs sampled from the space of functional connectivity patterns, we approximated the underlying manifold by first building an adjacency graph that captured the proximity relationships among the connectivity patterns, and then derived a tensor subspace that faithfully represented these relationships. mathematically, an fcg xn1n2 of size n1 n2, where n1 and n2 denote the dimensions of a second order tensor (in our case, n1 and n2 are equal to the number of meg sensors), can be thought of as a second order tensor in the tensor space. then, the generic problem of linear dimensionality reduction in the second order space is expressed as follows : given a set of m tensors x1,...,xmn1n2, find two transformation matrices u and v of size (n1l1) and (n2l2), respectively, that map these m tensors to a new set of tensors y1,...,yml1 l2, where yi = uxiv, l1 < n1, and l2 < n2, such that each new tensor yi represents uniquely a tensor xi in the original set. the method is of particular interest in the special case when x1, x2,, xm m, where m is a nonlinear sub - manifold embedded in n1n2. traditional linear dimensionality reduction algorithms, such as principal component analysis and linear discriminant analysis, find first - order mappings from to with l < n, but tsa finds a second - order mapping from n1 n2 to l1 l2 with l1 < n1 and domain of fcgs most probably forms a nonlinear sub - manifold embedded in the ambient space of second order tensors (lu.,, we attempted to find a linear subspace approximation to the sub - manifold in the sense of local isometry using a technique that is the tensorial counterpart of the locality preserving projection (he., 2005). given a set of m tensors { xi}i = 1:m, where each tensor is the tabular version of a single fcg having the group membership as class label (n or mtbi), tsa starts by building an (m m) weight matrix s that represents the nearest neighbor graph g among the tensors. the value of m is given by the total number of subjects in the training dataset. xj\|\|2/r) condition 1 0 otherwise}where the exponential is known as the heat kernel and is employed with the frobenius norm (golub and van loan, 1996), r is a control parameter usually referred to as radius of influence, and condition 1 states that tensors xi and xj share the same class label and each of them is among the k - nearest neighbors of the other, while the indices i and j vary across the m tensors (m equals the number of subjects in the training dataset). then tsa seeks two transformation matrices u and v which, when applied to each tensor, result in a mapping that preserves the neighborhood relationship encoded in graph g. mathematically, this is formulated in the following optimization problem,(3)minu, vijutxivutxjv2sij. denoting with d a diagonal matrix with elements dii=jsij, the above problem is reformulated as two coupled problems of eigenvector analysis (he., 2005):(4)(du su)v= duvdu=idiixituutxi, su=ijsijxituutxj (5)(dv sv)u= dvudv=idiixivvtxit sv=ijsijxivvtxjt. the optimal matrices u and v can be obtained by iteratively computing the generalized eigenvectors of eq. (4) and eq. (5) after initializing u with the identity matrix. tsa detects the intrinsic local geometric structure of the tensor space by learning the topology of a new tensor subspace of lower dimension. the objective function in eq. (3) incurs a heavy penalty if neighboring points xi and xj are mapped far apart. (3) ensures that when the original tensors xi and xj are close, the new tensors yi = u xiv and yj = u xjv will be close as well. the dimensionality of the reduced tensors yi, i.e., the number of eigenvectors used for mapping yi = u xi v, was optimized using a five - fold cross - validation procedure that achieved maximum separation between the n and mtbi groups. specifically, we randomly partitioned the training dataset into five equal sized subsets and used four subsets for building a k - nearest neighbor (k - nn) classifier (altman, 1992) and one subset for testing its performance. the number of neighbors k and the heat parameter were set in a similar way. our methodology employed the so - called extreme learning machine (elm) classifier (huang., 2006), an emerging learning technique that provides efficient unified solutions to generalized feed - forward networks, including single- and multi - layer neural networks. we chose this relatively new classification procedure because of its computational elegance and fast - learning capabilities, which lead to improved performance compared to other learning algorithms, like back propagation neural networks, radial basis function networks, and support vector machines (kim., 2009). the proposed scheme combined tensor subspace analysis with extreme learning machine classification and it was thus denoted as tsa+elm2. for comparison purposes, we used two additional popular classifiers as a baseline : the k - nn algorithm with the frobenius norm (horn and mathias, 1990) as a measure of similarity, and a support vector machine (svm) with a linear kernel (cortes and vapnik, 1995). furthermore, to demonstrate the superiority of the proposed tensorial approach the fcg tensors were first vectorized, i.e., converted to high dimensional vectors by traversing the corresponding matrices in a systematic way, and then their dimensionality was reduced using linear discriminant analysis (lda ; fisher, 1936) for feature extraction. the dimensions kept by lda were selected to optimize classification performance of the two groups. therefore, our analysis employed various combinations of approach (tsa, lda, and vectorial) and classifier (elm, k - nn, and svms). after selecting a classifier / approach combination, and optimizing it using the training data, the classifier was validated using the test data as follows : each fcg in the test set was compared against fcgs of known group membership (normal or mtbi) developed from the training dataset. the fcg was finally assigned to the group with which it had the highest similarity. following the significant link selection procedure described previously, the resulting fcgs were described using the well - known topological metrics of global and local efficiency defined for weighted graphs. the global efficiency ge of a network is given by(6)ge=1nin1n1jn, ji(dij)1where n represents the total number of nodes in the network and dij is the shortest path length between nodes i and j. ge is the inverse of the harmonic mean of paths between nodes and reflects the overall efficiency of parallel information transfer in the network (achard and bullmore, 2007 ; latora and marchiori, 2001). on the other hand, local efficiency le is a measure of fault tolerance in the network and is defined as(7)le=1n innodal_lei = 1ninj, hgi, j, hi(djh)1ki(ki1)where ki corresponds to the functional neighbors of the ith node, n represents the total number of nodes in the network, and dij is the shortest path length between nodes i and j. le indicates how well subgraphs exchange information when a particular node is eliminated (achard and bullmore, 2007). to identify cost - efficient functional networks, the same link selection procedure was applied to the average fcgs, independently for each frequency band and group. statistical analyses were performed using the wilcoxon rank sum test (p < 0.001). table 2 summarizes the classification performance obtained for various combinations of approach (vectorial, lda, and tsa) and classifier (k - nn, elm, and svms). for tsa, the parameters used were as follows, weight mode : eat kernel, neighbor mode : 10, supervised learning, and number of dimensions : 6 (dimitriadis. 2013a, dimitriadis. 2015). for the k - nn algorithm we used majority voting. for each of the three classifiers (k - nn, elm, and linear svm), the corresponding dimensions kept by lda in each frequency band were as follows, : (5, 6, 5), : (6, 6, 5), : (6 - 6 - 5), : (7 - 6 - 7), and : (6 - 6 - 5). as it can be seen in table 2, the proposed classification scheme of tsa+elm provided clear separation of the two groups, in all frequency bands, with high sensitivity and specificity. in particular, in the band normal controls and mtbi patients could be separated with 100% classification accuracy. the combination of lda + k - nn showed reduced classification accuracy (approximately 80% across all frequency bands) compared to tsa + knn (table 2a). on the other hand, the elm classifier showed improved performance compared to k - nn (table 2b) in all cases. using the svm classifier with tsa resulted in decreased performance compared to elm classifier (table 2c). however, the use of the svm classifier improved performance for both the lda and vectorial approaches compared to the elm classifier (table 2c). to demonstrate visually the ability of the proposed tsa+elm scheme to absolutely separate mtbi patients from normal controls in the frequency band, we computed a three - dimensional representation for the fcgs, each of which was originally described by a vector of 36 dimensions (in tsa each dimension of the second order tensor was 6, thus the total number of dimensions needed for the vector representation was 6 6 = 36). we estimated the pair - wise similarity between all possible pairs of vectors a and b across all subjects using cosine similarity (cos), which was given by(8)cosab = abab=i=1naibii=1nai2i=1nbi2 cos(a, b) measures the cosine of the angle between vectors a and b, and thus it is a judgment on their orientation, not on their magnitude. the final cos estimates were tabulated in a matrix with dimensions 86 86 (since 86 is the total number of subjects in the two groups). by employing multidimensional scaling (borg and groenen, 2003), a well - known dimensionality reduction technique, we were able to project the original multidimensional matrix onto a set of three - dimensional points. finally, we estimated the convex hull (borst., 1999) of the 3d points corresponding to each group. 2, which shows complete separation of the two groups and also illustrates their variance. 3 illustrates the average functional connectivity patterns for each frequency band and group after applying the cost efficiency thresholding scheme. as it can be seen in fig. 3, fig. 4, the functional connectivity profile of the control group exhibited a consistent network with stronger local connections (level 1 and 2 neighbors) whereas the mtbi group showed a variable network with stronger distant connections (level 3 and 4 neighbors). across most frequencies, with the exception of the band, in the n group more than 80% of the total number of connections was distributed locally (level 1 and 2 neighbors), whereas in the mtbi group, more than 60% of the connections were shared between distant sensors (level 3 and 4 neighbors). additionally, from to, the n group exhibited significantly stronger connections locally, whereas the mean strength of distant connections was significantly higher in the mtbi group (see fig. 5 shows, when long - range connections were compared across subjects, the n group exhibited mostly central - to - central or central - to - peripheral connections, especially in the to frequency bands, whereas the mtbi group showed connections involving primarily peripheral locations, practically bypassing fronto - central and centro - parietal brain regions. in all frequency bands, global efficiency (ge) values were in general higher in normal controls compared to mtbi patients, but without reaching significance (p = 0.61, p = 0.57, p = 0.61, p = 0.83, and p = 0.76). in contrast, local efficiency (le) values were significantly higher (p < 0.001) in the n group compared to the mtbi in all frequency bands, except for (fig. 6). in this study we used plv estimation and graph theory to investigate topological differences in the functional connectivity networks of mtbi patients and normal controls computed from resting - state meg. configuration, i.e., a dense network of local connections and a limited number of long - range connections. level 1 and 2 neighbors accounted for more than 80% of the total number of connections and, on average, they were significantly stronger compared to the ones seen in mtbi subjects. in contrast, mtbi subjects showed sparse arrangements of weak local connections and a large number of stronger distant connections. the average strength was significantly higher compared to the control group (fig. 4, fig. 5) however, these long - range connections in the mtbi group involved primarily peripheral locations, whereas in the controls, long - range links connected frontal - to - central or central - to - peripheral locations. these findings echo the results of our earlier study (zouridakis., 2012). marked differences between the two groups were seen in all frequency bands, except for, where the connection strength and sensor location were not significantly different between the two groups, at any neighborhood level. as an extension of our earlier work, in the present study we proposed a novel approach that features two improvements : estimation of functional connectivity graphs based on the plv estimator and a multivariate tensorial treatment of these graphs. as table 2 shows, the elm classifier provided 100% classification accuracy in the frequency band and more than 96% accuracy in the rest of the frequency bands studied. biomarker to characterize mtbi, instead of the typical univariate estimates, like relative power. this can be clearly seen in table s1 in the supplementary material, where we provide a detailed analysis based on relative power. our findings of frequency - specific differences between the two groups match current research on spontaneous activity. a number of studies used meg source localization (de pasquale., 2010 ; brookes., 2012 ; hipp, engel, and siegel 2011, hipp. 2012 ; hillebrand., 2012 ; bardouille and boe, 2012 ; marzetti., 2013) to explore two different modes of intrinsic coupling, namely phase coupling between band - limited oscillations and coupling between aperiodic fluctuations of signal envelopes (hipp., 2012). there is now evidence that the former is more sensitive to functional changes due to injury even in the absence of structural damage, as in the case of mtbi, while the latter can quantify functional abnormalities when structural damage predominates (ipp., 2012). these studies used various connectivity estimators, including power correlations (de pasquale., 2010 ; brookes, woolrich, and barnes 2012, brookes, woolrich, and barnes 2012 ; hipp., 2012), phase coherence (hipp., 2011 ; bardouille and boe, 2012), phase lag index (hillebrand., 2012), and imaginary coherence (marzetti., 2013). thus, our findings of band differences between the two groups using the plv estimator are in line with the current literature. in terms of topology, even though our analysis was performed at the sensor level, our findings follow closely the results of studies that explored frequency - dependent networks at the source level (hillebrand., 2012). in general, the brain areas that are part of the default - mode network showed a dense network of connections extended over the entire brain for low frequencies and a more anatomically constrained network for higher frequencies (buzsaki, g. and draguhn, 2004 ; fox and raichle, 2007 ; florin and baillet, 2015). we found that, in the range of high frequencies, both the number (80% of total connections) and strength of local connections were higher in the control group, whereas in the band there were fewer (only 20%) and weaker connections. additionally, we showed local topological differences with higher local efficiency for controls compared to mtbi subjects in the band, which resulted in 96.48% classification accuracy and partially matched previous findings of abnormal brain activity in mtbi patients in the band (huang. 2012). nevertheless, despite the very interesting results obtained, the present study has some limitations. for instance, we estimated fcgs at the sensor level, although some studies have applied source reconstruction methods to identify activation patterns at the source level (huang. 2009, huang. 2012 ; hillebrand., 2012). even though we can not interpret the activation patterns in terms of intracranial cortical locations, we are still able to describe statistically significant differences in the connectivity patterns of mtbi and control subjects using surface brain regions. another potential criticism of the present study could be attributed to volume conduction effects, i.e., the transmission of electromagnetic fields from a primary intracranial source through biological tissue towards the measurement sensors resulting in distorted recordings. although these effects can be reduced using connectivity estimators that are robust to volume conduction, such as the phase lag index (hillebrand., 2012) and imaginary coherence (marzetti., 2013), or through orthogonalization preprocessing (hipp., 2012), we decided not to employ any correction, since the absence of correlation between power and connectivity profiles in all frequency bands (see the supplementary material) was a strong argument that a common source did not alter the functional connectivity patterns ; otherwise, a common source would have changed also the signal power. furthermore, any possible volume conduction effects would be common to both groups, but we were looking only for differences between the two groups. finally, some details on subject demographics are incomplete, as table 1 shows, making it difficult to establish correlations between lesion locations and connectivity patterns for every participant in the study. the present study has focused on the development of noninvasive biomarkers that can be used to uniquely characterize mtbi patients and normal controls. it follows a general trend in current research in the area of neuroinformatics, where the big challenge for the next decade is the development and deployment of a large database with pre - computed brain connectivity profiles and derived network metrics (e.g., a connectome database) in both healthy and mtbi subjects (sporns, 2012). this would allow recording resting - state meg in the laboratory, estimating the related connectivity maps in both sensor and source space, querying the connectome database, and visualizing the results in a laboratory pc (akil. understanding brain function and visualizing the human connectome are slowly becoming a reality (human brain project, europe and usa)3. in the near future, computational approaches applied to connectomics will undergo rapid expansion, especially with shared neuroimaging resources, and complex network analysis and modeling (jirsa., 2010 ; sporns, 2012), and may be helpful in designing optimal recovery strategies following traumatic brain injury (irimia. the proposed methodology can estimate patterns of brain activation in the form of connectivity networks that are highly consistent and can uniquely characterize subject groups using resting state meg, a noninvasive modality with superior temporal resolution over other neuroimaging techniques. furthermore, resting state recordings are easy to obtain, as they do not require engagement from the subject, which makes such tests suitable for all sorts of patient types and age groups, including children. these encouraging findings support the hypothesis that functional connectivity patterns may be used as general biomarkers for predicting a subject 's brain state (dimitriadis. 2013a, dimitriadis. 2015), for detecting various brain diseases (dimitriadis, 2015), and for developing image - based tools that can provide more accurate diagnosis, help guide treatment, and monitor the effectiveness of intervention in mtbi patients.	mild traumatic brain injury (mtbi) may affect normal cognition and behavior by disrupting the functional connectivity networks that mediate efficient communication among brain regions. in this study, we analyzed brain connectivity profiles from resting state magnetoencephalographic (meg) recordings obtained from 31 mtbi patients and 55 normal controls. we used phase - locking value estimates to compute functional connectivity graphs to quantify frequency - specific couplings between sensors at various frequency bands. overall, normal controls showed a dense network of strong local connections and a limited number of long - range connections that accounted for approximately 20% of all connections, whereas mtbi patients showed networks characterized by weak local connections and strong long - range connections that accounted for more than 60% of all connections. comparison of the two distinct general patterns at different frequencies using a tensor representation for the connectivity graphs and tensor subspace analysis for optimal feature extraction showed that mtbi patients could be separated from normal controls with 100% classification accuracy in the alpha band. these encouraging findings support the hypothesis that meg - based functional connectivity patterns may be used as biomarkers that can provide more accurate diagnoses, help guide treatment, and monitor effectiveness of intervention in mtbi.
lymphedema (le) is a common and debilitating condition in breast cancer survivors characterized by regional swelling, typically in one or both arms. it is characterized by an accumulation of protein - rich fluids caused by an interruption in the axillary lymphatic drainage from the arm. due to the increased survival of breast cancer patients, le has emerged as an important long - term morbidity, resulting in functional, cosmetic, and psychological problems, all of which may significantly impact quality of life. despite the frequency of this condition, there are no uniform criteria for defining le. symptom duration is generally not stated as part of the definition of le, with a diagnosis of le made primarily on the presence of swelling alone. after the initial onset of symptoms, many patients experience a spontaneous resolution of swelling without progressing to a persistent condition. this transient swelling is typically of less than six - month duration. at the time of initial swelling, determining whether a patient has either transient lymphedema (tle) or persistent lymphedema (ple) is important, particularly among ple patients who must learn to cope with this life - long complication through the use of proactive education and management. in contrast, tle patients, whose symptoms can be expected to resolve naturally, may benefit by avoiding unnecessary treatment. however, the incidence, time course of tle, and risk factors associated with progression to ple from initial swelling remain poorly understood. in this study, we analyzed the incidence and risk factors for development of tle and ple after initial swelling and estimated the ratio of tle and ple based on the presence of known treatment - related factors. the study population consisted of 1,255 consecutive breast cancer patients who underwent curative breast surgery with axillary lymph node dissection (alnd) at the national cancer center, korea between may 2004 and april 2009. those with synchronous or metachronous contralateral breast cancer (n=7) and those with a follow - up period of 30% were scored as grades 2 or 3, respectively. assessments were made beginning at least 6 months postoperatively and regularly every 6 months until the last follow - up. le events were defined as any case of arm swelling, including both objectively and subjectively defined incidents. tle was defined as a single episode of arm swelling that resolved spontaneously at the next follow - up. ple was defined as arm swelling that persisted over two consecutive follow - up examinations. the rates of le were calculated using the kaplan - meier method, and the development of le was measured from the date of surgery. univariate cox proportional hazards models were used for evaluation of risk factors associated with the development of le. the factors included age (10), and regimen of chemotherapy (not performed or chemotherapy without taxane vs. chemotherapy with taxane). variables shown to be significant (p 30% were scored as grades 2 or 3, respectively. assessments were made beginning at least 6 months postoperatively and regularly every 6 months until the last follow - up. le events were defined as any case of arm swelling, including both objectively and subjectively defined incidents. tle was defined as a single episode of arm swelling that resolved spontaneously at the next follow - up. ple was defined as arm swelling that persisted over two consecutive follow - up examinations. the rates of le were calculated using the kaplan - meier method, and the development of le was measured from the date of surgery. univariate cox proportional hazards models were used for evaluation of risk factors associated with the development of le. the factors included age (10), and regimen of chemotherapy (not performed or chemotherapy without taxane vs. chemotherapy with taxane). variables shown to be significant (p 10), chemotherapy regimen (with taxane), and rt (breast with scrt) ; higher rates of ple development showed correlation with advanced stage at diagnosis (iii), type of surgery (mrm), n - aln (> 10), chemotherapy regimen (with taxane), and rt (breast with scrt). patients with more advanced stages of disease showed higher rates of le (12.4% in stage iii vs. 10.6% in stage i or ii for tle, 41.7% vs. 14.2% for ple ; p 10 for tle were 9% and 13%, respectively (p=0.001), compared with 13.7% and 31.3%, respectively, for ple (p 10), chemotherapy regimen (with taxane), and rt (breast with scrt) ; higher rates of ple development showed correlation with advanced stage at diagnosis (iii), type of surgery (mrm), n - aln (> 10), chemotherapy regimen (with taxane), and rt (breast with scrt). patients with more advanced stages of disease showed higher rates of le (12.4% in stage iii vs. 10.6% in stage i or ii for tle, 41.7% vs. 14.2% for ple ; p 10 for tle were 9% and 13%, respectively (p=0.001), compared with 13.7% and 31.3%, respectively, for ple (p 10 was identified as a risk factor for both ple and tle by univariate analysis. scrt was also a strong contributing factor, associated with a 2- to 4.5-fold increase in the risk of le. similarly, coen. reported a significant increase in the risk of le from 1.8% to 8.9% over a 10-year follow - up period in patients with bcs following the addition of scrt. in this study, scrt was identified as an independent risk factor for development of not only ple but also tle in a multivariate analysis. because le is progressive, and early diagnosis could lead to more effective treatment, diagnosis of le at the earliest possible stage is important. at first presentation of arm swelling, understanding of risk factors associated with ple and predicting whether this condition is expected to be self - limiting or persistent may enable clinicians to mitigate ple incidence and severity by providing proactive education and management. this study showed that the tendency of tle and ple after initial grade 1 swelling could be estimated using a combination of three treatment - related risk factors : n - aln, scrt, and taxane chemotherapy. in patients with no risk factor, ~90% of swelling events were limited in scope, and had completely disappeared within 6 months. however, in all other patients, the likelihood of edema persisting or progressing to a more serious form increased according to the number of risk factors. the approximate ratio of tle vs. ple was 1:1 in patients with one risk factor, 1:2 in patients with two factors, and 1:3 in those with three risk factors. as mentioned above, 95 of 119 grade 1 patients (80%) who had both subjective symptoms and an objective arm circumference difference progressed to ple, implying that patients who had at least one risk factor or both subjective and objective symptoms of le may require more careful follow - up. patient - related factors such as obesity, hand use, injury, and infection could be modified to reduce the risk for these patients. in a study of 109 patients with mild le at le diagnosis by bar ad., 52 (48%) progressed to more severe stages at some point during the first 5 years of follow - up. in our study, 49 of 351 patients (14%) initially diagnosed as grade 1 eventually progressed to grade 2, with one patient progressing to grade 3. limitations of our study include the lack of preoperative baseline information on arm circumference or the ability to interview patients about arm swelling. in a meta - analysis by disipio., even in the absence of pretreatment le status, prevalence was thought to be a reasonable estimate of incidence because the proportion of female patients with le before surgery for breast cancer has been reported to be very low. the subjective patient s perception of arm edema was dependent on patient recall or swelling sense at the time of the outpatient clinic visit. for objective measurements, only the upper arm circumference was measured ; hence, localized forearm or hand edema would not have been detected, although subjective assessments may have been sufficient to overcome this limitation. in addition, we used a percentage difference rather than an actual difference in arm circumference for consideration of different bmi among patients and body weight changes overtime in each patient. the other limitation is that our study did not include data regarding treatment of le. however, in our hospital, education on the modification of patient - related factors such as reducing excessive arm use, avoiding arm infection of injury, and weight control was mandatorily provided to all breast cancer patients regardless of swelling episodes. in addition, most patients with objective le at the first swelling episodes were managed using a compression stocking or bandage. in conclusion, one - third of initial swelling events were transient in nature, with the remaining two - thirds progressing to a more persistent form of le. among patients with tle, chemotherapy with taxane and rt covering the breast and supraclavicular region were identified as independent risk factors. the approximate incidence ratios of tle and ple in patients with initial grade 1 edema were estimated according to treatment - related factors, which could facilitate prediction and treatment of this life - long, undesired complication.	purposethe purpose of this study is to identify risk factors for transient lymphedema (tle) and persistent lymphedema (ple) following treatment for breast cancer.materials and methodsa total of 1,073 patients who underwent curative breast surgery were analyzed. tle was defined as one episode of arm swelling that had resolved spontaneously by the next follow - up ; arm swelling that persisted over two consecutive examinations was considered ple.resultsat a median follow - up period of 5.1 years, 370 cases of lymphedema were reported, including 120 tle (11.2%) and 250 ple (23.3%). initial grade 1 swelling was observed in 351 patients, of which 120 were limited to tle (34%), while the other 231 progressed to ple (66%). all initial swelling observed in tle patients was classified as grade 1. in multivariate analysis, chemotherapy with taxane and supraclavicular radiation therapy (scrt) were associated with development of tle, whereas scrt, stage iii cancer and chemotherapy with taxane were identified as risk factors for ple (p < 0.05). the estimated incidence of tle among initial grade 1 patients was calculated using up to three treatment - related risk factors (number of dissected axillary lymph nodes, scrt, and taxane chemotherapy). the approximate ratios of tle and ple based on the number of risk factors were 7:1 (no factor), 1:1 (one factor), 1:2 (two factors), and 1:3 (three factors).conclusionone - third of initial swelling events were transient, whereas the other two - thirds of patients experienced ple. estimation of tle and ple based on known treatment factors could facilitate prediction of this life - long complication.
the goal of the cervical surgery is to relieve the compression on the nerves and/or spinal cord, and anterior or posterior cervical approach is used to access the cervical spine depend on the type of surgery required6). the anterior approach can allow the surgeon access to almost the entire cervical spine, and often helps maintain the normal curvature of the spine4). the textbook of korean spinal neurosurgery society provide that general external landmarks : the hyoid bone corresponds to c3 ; the thyroid cartilage corresponds to c4 - 5 ; and the cricothyroid membrane identifies the c6 vertebral level3). cervical spine level estimation according to the external landmarks is useful, quick and less exposable to radiation, but, sometimes cervical level confusion to the target cervical level could be happen. herein, the authors reviewed the corresponding between the neck external landmarks and cervical levels. between january and december 2013, the authors retrospectively reviewed cervical lateral radiographs which were taken in single university hospital by various cervical problems. all radiographs and the patient 's information such as sex, age, and heights were collected using the retrograde medical chart review. the neck external landmarks were defined as mandible angle, hyoid, thyroid, and cricothyroid in radiographs : mandible angle as the posterior border at the junction of the lower border of the ramus of the mandible ; hyoid as the superior surface of anterior margin of hyoid ; thyroid as the inferior margin of laryngeal incisures ; and crocithyroid as the lower border of the laryngeal prominence which connected to median cricothyroid ligament. these landmarks and horizontally lined to cervical spine were corresponded in radiographs, and each reference level were measured as cervical vertebral body level or cervical disc level (fig. 1). for example, if the horizontal line were corresponded to c3 vertebral level, it was measured as " c3 ", and if the line were corresponded to the disc level between c5 and c6, it was measured as " c5.5 ". the authors used the imaging programs for horizontal line drawing between landmarks and cervical level. the results are expressed as the meanstandard deviation. paired student t - test was used to assess the statistical differences using sas software for windows (sas institute inc., the average reference levels of cervical landmarks were c2.13 with mandible angle, c3.54 with hyoid, c5.12 with thyroid, and c6.01 with cricothyroid (table 1, fig. 2 and 3). the reference levels of cervical landmarks were differently observed by sex, age, and height accordingly mandible from disc level of c1 and c2 to c3, hyoid from disc level of c2 and c3 to c5, thyroid from disc level of c3 and c4 to c7, and cricothyroid from c4 to disc level of c7 and t1. the reference levels were statistically differently observed according to the sex, age, and height (p 180 cm) showed more narrowed cervical reference level of mandible, hyoid, thyroid, and cricothyroid compared to others (180 cm) showed more narrowed cervical reference level of mandible, hyoid, thyroid, and cricohyoid compared to others (< 180 cm). the age also contributed the range of cervical reference level, and the range were slightly narrowed accordingly the ages of subjects (for example, the cricohyoid reference level was narrower from c4-c7.5 in 1st decades to c5.5-c7 in 7 decades. the mandible was comparatively observed in constant level (c2 - 3), but, the hyoid, thyroid, and cricothyroid showed relatively wider variation of reference cervical level according to the sexes with statistical differences (p<0.05). surface landmarks did not correspond to exact levels of the cervical spine, and only provide general reference points.	objectivea general orientation along the cervical spine could be estimated by external landmarks, and it was useful, quick and less exposable to radiation, but, sometimes it gave reference confusion of target cervical level. the authors reviewed the corresponding between the neck external landmarks and cervical levels.methodstotally 1,031 cervical lateral radiographs of different patients were reviewed in single university hospital. its compositions were 534 of males and 497 females ; 86 of second decades (10 - 19 years - old), 169 of third decades, 159 of fourth decades, 209 of fifth decades, 275 of sixth decades, and 133 of more than seventh decades (> 60 years - old). reference external landmarks (mandible, hyoid bone, thyroid cartilage, and cricothyroid membrane) with compounding factors were reviewed.resultsthe reference levels of cervical landmarks were c2.13 with mandible angle, c3.54 with hyoid bone, c5.12 with thyroid cartilage, and c6.01 with cricothyroid membrane. the reference levels of cervical landmarks were differently observed by sex, age, and somatometric measurement (height) accordingly mandible angle from c1 to c3, hyoid bone from disc level of c2 and c3 to c5, thyroid cartilage from disc level of c3 and c4 to c7, and cricothyroid membrane from c4 to disc level of c7 and t1.conclusionsurface landmarks only provide general reference points, but not correspond to exact levels of the cervical spine. intraoperative fluoroscopy ensures a more precise placement to the targeted cervical level.
in this century, new changes have resulted in rapid development of service - providing sectors, especially in the health sector (1). services provided in different parts of society are considered as the heart of value - creation in the economy (2). today, quality of services, especially in institutes with high volume of customers, including financial and care services, has found a great importance in such a way that it could be considered as an essential strategy to achieve the intended results in a competitive market (3). quality has been converted into a big challenge to meet expectations of service receivers and their satisfaction (4). the problem of lack of quality in providing services occurs in organizations that do not focus on identifying and meeting customers needs and demands. lack of direct relation of organizations with customers finally will result in improper performance and lack of accountability to customers expectations (5). evaluating the quality of health services is a prominent scale to measure the amount of realization of health and medical organizations (6). it should be noted that customers satisfaction is obtained when organizations that provide health services, such as hospitals, medical centers, and clinics, pay attention to customers expectations as well as their needs and implement required plans to meet customers expectations (7). in today s competitive market, promotions are transferred to the society directly by patients or their families, and this issue is very important for medical organizations providing positive experiences for patients and their families and positive perceptions of patients for services received are very important for service - providing organizations (8). the quality of health services generally is recognized as satisfaction of the patients who are referred to medical centers, which have a direct effect on quality and the customers satisfaction. therefore, most medical organizations seek to promote the quality of services to ensure that customers are satisfied with the services they receive (9). therefore, providing health care according to patients preferences, expectations, and demands could promote the quality of hospital services (10, 11). expectations are one of the main determinants of customer evaluation of the quality of services, and the main step in providing quality services is accurate identification of customers expectations. in fact, responding to patients expectations is considered as one of the current challenges of the health system, and, in spite of its importance, the identification of patients needs and demands has been neglected (12). customers compare their observations of the process of providing services with their expectations of those services, and they judge the quality of the services they receive based on the difference between their expectations and what they actually received. therefore, a quality control strategy is required to reduce these gaps (13). the servequal model is one of the most prominent tools to measure customers expectations and perceptions. it is a diagnostic method for introducing weak and strong points of the quality of organizations services (14). it measures the following five main dimensions of the quality of services : tangible aspects of service : physical facilities, equipment, appearance of personnel reliability of service provider : performing committed function accurately and reliably responsiveness of service provider : tendency toward helping and responding to customers needs assurance provided by service provider : ability of personnel to induce trust and reliability empathy of service provider with customers : personal attention to customers (15). this model has been used in many studies. in their study, safi. concluded that quality gaps existed in terms of all five dimensions of the quality of health services in the medical centers north of tehran. the lowest gap, i.e., 0.68, was in empathy, and the highest gap, i.e., 0.88, was related to physical aspect of services (16). in the study by alidoosti on the rural society of shahr - e - kord, the results suggested that health services provided in health providers of rural area are far from favorable, and 37% of the people in the study were weakly satisfied with the services provided (17). in a study of private hospitals in malaysia, the findings showed that there was a significant gap between customers perceptions and expectations of provided services that required basic interventions in these hospitals to reduce the gap of service quality (18). in the study by padma. in the hospitals of india, patients were regarded as main concern of health service providers and hospitals paid special attention to patients perceptions to promote the quality of services (19). various studies have been conducted all around the world on the quality of health services. in studies conducted by purcrea (20), john (21), chakravarty (22), zarei (23), nekoei - moghadam (24), li (25), abedi (26), xu (27), and handayani (28), it was specified that there is a negative gap between perceptions and expectations of customers concerning the quality of provided services in health care providing units. it was believed that the need to promote quality of services and paying more attention to individuals in the society were the critical factors for the organizations. garrard and lin, in their studies, evaluated servequal as a proper instrument to measure quality of services and stated that this instrument should be used to identify the gap between perceptions and expectations (14, 29). a study conducted in qazvin province, iran, showed that there was a gap in the quality of health services in this province. however, in this study, the gap distribution was not clear between service providers (30). with regard to the gap in quality of services in qazvin province (30) and the lack of studies conducted to measure the quality of health services in qazvin city, the topic of this study in comprehensive health plans of qazvin province and unified consensus of health exports of qazvin to conduct a study on determination of the study of health services among providers was the main reason to conduct this study. in addition, assuring health services is very important for the health system and people, since they are very critical. evaluating the quality of these services through people s perceptions and expectations therefore, the aim of this study was to investigate the quality of health - providing services according to residents of qazvin province in 2014. this descriptive study was conducted with residents of qazvin province, iran, in 2014. a samples size of 400 subjects was selected using the sample size formula and considering the maximum possibility of higher expectations of people compared to their perceptions, p = 0.50 and error coefficient of 5%. by considering cluster effect 2,, 1,002 subjects were studied for more accuracy. due to the rural population of 25% relative to urban population in qazvin, 253 rural and 749 urban samples were selected. cluster sampling was used and samples were selected randomly from the urban population of qazvin province. the counties of qazvin, takestan, abyek, booyinzahra, avaj, and alborz were investigated. the samples selected from qazvin, abyek, avaj, alborz, boein zahra, and takestan totaled 403, 51, 38, 121, 44, and 92, respectively. the difference between the numbers of urban and rural samples was because of their population ratio with the cities. in addition, among the rural population of abyeh, avaj, alborz, boein zahra, takestan and qazvin villages, 24, 16, 21, 56, 44 and 82 samples were investigated respectively. this questionnaire included two sections : background variables (education, age, place of receiving services, waiting time to receive services) and main questions on five dimensions of service quality (physical, reliability, responsiveness, assurance, and empathy) in perceptions and expectations parts. there were four questions each in the physical, reliability, and responsiveness fields and five questions in the assurance and empathy fields. quality of services was calculated using a 5-point likert scale, ranging from strongly agree to strongly disagree. to determine the quality gap, scoring of customers to present situation (perceptions) of quality of health services provided was compared with their scoring to desirable status (expectations) of quality of health services. if the resulting score were positive, it showed that services provided were higher than peoples expectations and in the case of being negative, it stated that health services provided did not meet peoples expectations, and a quality gap exists. zero score shows the lack of a quality gap, which demonstrates that health services provided are in line with peoples expectations. the place of receiving services was shown as an option in the questionnaire, with six answers, including hospital, clinic, medical center, doctors office, health providers of urban area, and health providers of rural area. in addition, the last place of referring to receive services was regarded as the option to be selected. the validity of the questionnaire was approved by zarei and nekoei - moghadam in iran (23, 24). research tools were selected based on cronbach s alpha score of 0.91 and test - retest method of 0.83 for reliability. data were collected by going to peoples houses in cities and villages and asking questions about the quality of units and health service providers in qazvin province. first, an explanatory workshop was held for interviewers, and its goal was to have experienced local forces of each area collect the data. the ethics committee of qazvin university of medical sciences approved this study with i d number of 8346. the importance of the study was explained to the participants by interviewers, and the participants were assured that the information they provided would be confidential. a consent form for participating in the study the mean age of the subjects was 32 9.9, and the average waiting time to receive services was 73 47 minutes. the frequency distribution of the education of the people in the study was 6% illiterate, 34% junior diploma 30% high school diploma, 10% associate diploma, 18% bachelor s degree, and 2% master s degree and higher. the lowest perceptions of the quality of health service providers were related to hospitals, with an average of 3.26, followed by doctors offices at 3.51. in addition, the highest perceptions of the quality of health service providing centers was related to health providers of urban area at 3.79 and medical centers at 3.70 (table 1). the least expectations of people concerning the quality of service providers were related to clinics at 4.49, and their highest expectations were related to hospitals at 4.68. respectively, the highest quality gap in health services was related to hospitals and doctors offices with the values of 1.42 and 1.01, respectively, and the lowest gap was related to health providers of urban area and health providers of rural area with the values of 0.80 and 0.88, respectively (table 1). the highest perception of 3.68 was related to junior diploma and the highest expectation of 4.69 was related to master s degree. furthermore, the lowest perception and expectations values of 3.30 and 4.48 were related to those who held associate s diplomas (table 2). there was a significant relationship between perceptions (p = 0.000), expectations (p = 0.01), and quality gap of health services (p = 0.000) with the health service provider. there was a significant relationship between people s perceptions and education (p = 0.000). however, no significant relationship was observed between quality gap of health services and education (p = 0.2) (table 3). study of quality gap in all health service providers and comparing them with each other was one of the main issues in this study that has never been found before in other studies and was considered as its strong point. in current study, the highest quality gap of 1.42 in health services was related to hospitals and the lowest quality gap of 0.80 was related to health service providers of health providers of urban area. in a study conducted by jenaabadi. in hospitals of zahedan, there was a significant difference in all dimensions of quality between average of perceptions and expectations score. quality gaps in tangibles, reliability, responsiveness, assurance, and empathy were 0.690, 0.685, 0.795, 0.755, and 0.840, respectively (31). in a study conducted of the hospitals of arak, the quality gap of health services was reported to be 1.20 (32), and that complied with the findings of this study. it appears that arak and qazvin are very similar in terms of results because both are industrial cities. in a study by mohammadi on a medical center in zanjan s study, the averages of people s perceptions and expectations for the quality of health services were 3.04 and 4.26, respectively, and the quality gap was 1.22 (33), which complied with the current study in terms of quality gap. the results of a study conducted in khorramabad showed that the quality gap of health services was 0.69 (34), which complied with our findings. the results of a study conducted by ghobadi on doctors offices of ardebil revealed a quality gap of 0.65 in health services (35). quality gap in doctors offices was higher in qazvin province than in ardebil, which requires reforms and fundamental policy making to reduce this gap. the results of a study conducted by noronesa in fasa revealed that doctors offices need fundamental reforms in terms of meeting patients expectations to promote quality of health services (36). in studies conducted by sahebzadeh in isfahan (37) and azizi in darab (38), customers satisfaction with health service providers was not favorable, and that requires policies and plans to promote present situation and move toward favorable status. in studies by butt in malaysia (39), rohini in india (40), bakar in turkey (41), and arasli in cyprus (42), there was a negative gap between patients perceptions and expectations in all dimensions of quality of services. in spite of different values of this gap in various studies, it is clear that there is a gap in providing health services all around the world, and fundamental interventions are required to reduce this gap in services. in a study by gorji (43) and mohammadnia (44), negative gaps were observed in quality of health services between customers perceptions and expectations. contrary to all studies conducted in iran, the research society of which was limited, in this study people were investigated in person in all areas of qazvin province by referring to people s houses, which is considered as one of the strong points of this study. in addition, in this study all health service providers, including hospitals, clinics, medical centers, health providers of urban area, doctors offices, and health providers of rural area were compared that have never considered before in other studies. studies by taner (45), schrder (46), and suki (47) estimated people s perceptions to be lower than their expectations in terms of quality of health services, which demonstrated a quality gap and lack of meeting expectations of people who were referred to receive health services. in a study conducted in peru, a relationship was found between people s satisfaction with health services and their education and type of services received (48). in a study conducted in indonesia, the results showed that trained staff and experienced physicians promote the quality of services and increase satisfaction (49). in a study conducted by al - borie in saudi arabia, a significant relationship was found between education and perceptions and satisfaction from the quality of health services (50) that complied with results of current study. of limitations of this study therefore, it was attempted to use local experts to collect data and investigate all regions as far as possible. the results of this study showed that there was a service quality gap in all health service providers ; however, it was higher in hospitals and doctors offices. since providing human and material resources of health system always involves problems, the practical importance of these findings lies in the fact that weak points could be emphasized more to reduce quality gap in services of hospitals and doctors offices. to reduce quality gap in services, it is suggested that authorities consider customers complaints and deal with them, make essential services available 24 hours a day, promote motivation of staff and physicians to pay more attention to customers, and the staff and physicians should consider the requests and reasonable needs of customers. conducting a complementary study on quality gap of services between state and private sectors in qazvin province with descriptive methodology could be an appropriate path for future studies in this field.	introductionquality is the center of attention in all service providing organizations that are effective in promoting satisfaction of patients who are referred to medical centers. the aim of this study was to investigate the quality of health service providers in a case study of qazvin, iran, in 2014.methodsthis descriptive study was conducted on 1,002 people who were residents of qazvin province (iran) in 2014. the people were selected randomly from the population of the study area. the main variables studied were education, perceptions, expectations, and gaps in service quality. the data collection tool was the standard servequal questionnaire. to determine the reliability of the research tool, we used cronbach s alpha coefficient and the test - retest method. statistical analyses were conducted using spss and the anova test.resultsthe mean age of people included in the study was 32 9.9 years, and the average waiting time to receive services was 73 47 minutes. hospitals and doctors offices had the highest quality gap of 1.420 0.82 and 1.01 0.75, respectively. the service quality gaps in medical centers, health providers of rural area, and health providers of urban area were 0.883 0.67, 0.882 0.83, and 0.804 0.62, respectively. there was a significant relationship between peoples perceptions and expectations concerning the quality of health services and their educational levels.conclusionthe higher gaps in quality in hospitals and in doctors offices require more attention. managers and policy makers should consider developing and implementing plans to reduce these gaps in quality and to promote better health services in these two sectors.
current use of laparoscopy in the evaluation and management of trauma patients has been a natural extension of this trend. although utilized for both blunt and penetrating injuries, laparoscopy has gained the most widespread acceptance as a useful tool in the management of patients with penetrating abdominal injuries. its ability to accurately determine anterior peritoneal penetration from stab and others have expanded its role beyond simply a screening tool for injury, to its current use in some centers as a diagnostic and therapeutic modality. the purpose of this study was to evaluate the role of laparoscopy in the trauma setting at our urban level ii trauma center. more specifically, we examined its use in the management of hemodynamically stable trauma patients with penetrating anterior abdominal injuries. we also sought to determine how our results compared with those of other centers regarding the widely accepted laparoscopic advantages of reduced morbidity, decreased rates of negative laparotomy, and shortened length of hospital stay (los). we conducted a retrospective chart review of 4541 trauma patients who were admitted to our level ii trauma center between july 1, 1999 and july 31, 2002. penetrating injuries accounted for 817 (18%) of these trauma admissions. there were 209 penetrating injuries isolated to the abdomen, of which 116 were gunshot wounds (gsw) and 93 were stab wounds (sw). the first group consisted of hemodynamically stable patients without evidence of abdominal peritoneal penetration based on physical examination, local wound exploration, or further confirmatory studies, including focused abdominal sonography for trauma (fast), computed axial tomography (ct), or diagnostic peritoneal lavage (dpl), or all of these. the additional confirmatory studies were not applied in a standard fashion to all patients, but rather selected based on clinical indication and attending physician preference. in addition, stab wounds with peritoneal penetration, but no peritoneal signs, also underwent a course of observation. the second group of patients was those with peritoneal signs or confirmed evidence of abdominal peritoneal penetration based on physical examination, local wound exploration, or further confirmatory studies. the third group of patients was taken to the operating room for laparoscopic evaluation due to questionable peritoneal penetration. criteria for laparoscopy was a hemodynamically stable patient with an injury trajectory suggestive of peritoneal penetration that could not reliably be ruled out based on physical examination or further confirmatory studies. no attempt was made to laparoscopically assess the extent of the intraabdominal injuries or make therapeutic interventions. laparotomy was negative if it was discovered after the abdomen was opened that no peritoneal penetration had occurred. a nontherapeutic laparotomy was one that confirmed peritoneal penetration with either no intraabdominal injuries or injuries so minor that they did not require surgical repair. the patients in these 2 groups were closed without therapeutic intervention and admitted to the floor for postoperative recovery. the laparoscopic findings were described as either positive or negative based on evidence of peritoneal penetration. figure 1 details the algorithm used for management of our patients with penetrating abdominal wounds. injury severity score (iss) and length of hospital stay (los) were calculated for each patient in the study. morbidity and mortality figures were also generated for each patient from our computer database by using standard methodology to define associated complications. penetrating abdominal injuries accounted for 209 of our trauma admissions, of which 116 were gsw and 93 were sw. gender breakdown of these patients was male, 171 (82%) and female, 38 (18%). thirty - three patients (gsw=9, sw=24) were observed in the emergency department based on their initial physical examination, radiologic studies, or additional confirmatory studies, or all of these. the majority of the gsw patients received immediate laparotomies due to the higher ballistic forces associated with these injuries. in general, gsw to the anterior abdomen required laparotomy unless the wound was tangential. table 1 provides the negative, nontherapeutic, and therapeutic outcomes in the formal laparotomy group with regards to the number and percentage of procedures and los for patients with gsw and sw. a review of the negative laparotomies revealed that possibly 8 of 10 gsw and all 14 stab wounds could have been done laparoscopically, based on the location of the entrance wound in the anterior abdominal wall. however, 2 of the negative gsw laparotomies assessed tangential wounds that would not have been well visualized by laparoscopy alone. all of these patients had anterior abdominal wounds, and 2 patients had flank wounds in addition to the anterior wounds. of these 22 patients, 7 had an additional confirmatory test prior to going to the operating room (table 2). eight sw were converted to an open procedure, and 7 had laparoscopy only (table 3). additional confirmatory test in patients managed with laparoscopy gsw = gunshot wound ; ct = computed tomography ; fast = focused abdominal sonography for trauma ; sw = stab wounds ; los = length of hospital stay. results of laparoscopy group results of laparotomies after conversion table 5 lists the number and percentage of gsw and sw patients who underwent laparotomy, laparoscopy, or were observed. mortality was 13% (gsw=20, sw=0) for the laparotomy group, and 0% (gsw=0, sw=0) for the laparoscopy group. morbidity was 27% (gsw=42, sw=7) in the laparotomy group and 14% (gsw=1, sw=2) in the laparoscopy group. these numbers reflect the increased severity of injury with the laparotomy group. however, we found no difference in morbidity or mortality when we compared data for the negative laparotomy group and the negative laparoscopy group. management of gunshot wounds versus stab wounds mean los was 7.9 days for the laparotomy patients and 3.0 days for the laparoscopy patients. this difference can be attributed to the higher severity of injury associated with the laparotomy group. however, a more useful comparison of los is that between the negative laparotomy group and the negative laparoscopy group, which revealed an los of 4.1 and 1.9, respectively. laparoscopic evaluation of the peritoneal cavity is not a new concept, having first been described by kelling over a century ago. the first reports demonstrating the utility of laparoscopic surgery in the evaluation of trauma patients were published soon thereafter in the 1920s. these early trauma studies investigated laparoscopy as a method to diagnose internal bleeding in patients with traumatic abdominal injuries. reports describing similar experiments were published sporadically throughout most of the last century. however, mainly due to technological limitations, it would be many years before laparoscopic surgical techniques would prove their practical utility and gain widespread acceptance. it is now evident that despite the lack of early enthusiasm in the surgical community for these new techniques, the laparoscopic pioneers correctly recognized the potential benefits of minimally invasive surgery in the trauma patient. recent technologic advances in optics and laparoscopic instrumentation have greatly broadened the applications for minimally invasive surgery. we now have a better understanding of the physiologic changes and complications associated with these procedures. increased training and utilization of minimally invasive procedures in all areas of surgery has come an increase in expertise among trauma surgeons. these techniques have evolved from what was once a novelty into an important part of every surgeon 's practice. for trauma patients, laparoscopy provides clear visualization of the peritoneal space and anterior abdominal wall, and unlike other diagnostic modalities, has the additional benefit of potential for therapeutic intervention. however, despite its increased use in trauma, the optimum role of laparoscopy in this setting has not yet been clearly defined. there continues to be variation among trauma centers on how best to optimize its application, taking full advantage of its benefits while overcoming its limitations. laparoscopy has been used as a screening, diagnostic, and therapeutic tool to evaluate both blunt and penetrating trauma at various centers. villavicencio and aucar authored an extensive review in 1997 in which they compared outcomes collected from 37 separate studies, involving over 1900 patients. a review of the data showed that it was most useful as a screening tool, missing only 1% of injuries and preventing 63% of patients from unnecessary laparotomies. the data were less encouraging when laparoscopy was used as a diagnostic tool, with missed injury rates reported between 41% and 77%. these numbers reflect a diagnostic accuracy that is unacceptable to most surgeons who have used the laparoscope less frequently in this manner. laparoscopy is infrequently used as a therapeutic tool ; however, several reports have demonstrated favorable results with laparoscopic repair of a variety of intraabdominal injuries, including the dia - phragm, liver, gallbladder, spleen and bowel. emergency department evaluation of the injured patient has evolved greatly over the years, based mainly on the technology of the time. trauma surgeons have had available a variety of diagnostics tools to assist with the management of their patients, including dpl, fast, and ct. the increased availability of laparoscopy now offers them even more flexibility during the workup of injured patients. certainly the evaluation of every trauma patient starts with the advanced trauma life support (atls) primary survey followed by a thorough physical examination. it is important to carefully inspect the patient 's wounds, because findings on the initial physical assessment usually determine the decision - making algorithm. local wound exploration is limited in its ability to determine specific intraabdominal injuries, but it can often determine peritoneal penetration and thereby avoid the need for further studies. in patients who are obtunded due to central nervous system dysfunction or intoxication as well as in those with distracting injuries to multiple body systems, the physical examination may be less reliable. wounds with tangential trajectories can also be difficult to accurately assess, necessitating additional diagnostic studies. diagnostic peritoneal lavage is simple, inexpensive, and can be quickly performed in the emergency department. however, dpl is limited by poor specificity, inability to accurately assess the retroperitoneum, and potential for missed hollow viscus or diaphragmatic injuries. it is also an invasive test that has some risk, albeit low, for procedural complications. although dpl has been considered the standard modality to assess traumatic intraabdominal injury for many years, its use is now declining in favor of more accurate, less invasive modalities, such as ultrasound (fast) and ct. we have significantly decreased the number of dpls performed at our institution in recent years as well, relegating its use to screening for late presenting potential hollow viscus injuries or rapid evaluation of blunt injuries if fast is unequivocal or unavailable. technological improvements have greatly enhanced the capabilities of ct and ultrasound, both of which now have an important role in trauma management. fast is now widely used in most trauma centers ; however, its major role is in blunt trauma. it has a low specificity for organ injury, but can effectively determine the presence of free fluid in the gravity dependent spaces of the peritoneal cavity. some centers have used positive fast examination findings as an indicator for laparotomy in penetrating trauma. improvements in ct speed and resolution have allowed for reliable evaluation of both the peritoneum and retroperitoneum. ct findings can often confirm or rule out peritoneal penetration based on a more clearly delineated wound trajectory or evidence of intraabdominal injury. unlike blunt injuries that can often be managed by observation and monitoring, ct confirmation of penetrating intraabdominal injury warrants laparotomy at our institution. the results of our study were consistent with those of other centers regarding the generally accepted laparoscopic advantages of decreased rates of negative laparotomy, shortened length of hospital stay, and quicker return to normal activity. the laparotomy patients had many more pulmonary complications including atelectasis, pneumonia, and respiratory failure. they also were found to have more wound complications including dehiscence, infection, and abscess formation. the decreased mobility of the laparotomy patients led to one deep vein thrombosis in this group. the results of our study did not show any difference in morbidity or mortality between the negative laparoscopy and the negative laparotomy groups. we found that a decrease occurred in los from 4.1 days in the negative laparotomy group to 1.9 days in the negative laparoscopy group. brandt looked at 21 trauma patients who underwent laparoscopic evaluation of both penetrating and blunt injuries, and found that emergency laparoscopy was 100 percent accurate in determining the need for laparotomy. a more recent retrospective review demonstrated a direct correlation between increased use of laparoscopy and decreased rates of negative laparotomy. our study revealed that laparoscopic evaluation spared many patients unnecessary laparotomies. after reviewing the operative reports of patients with negative laparotomies, it was determined that possibly 8 of 10 gsw and all 14 stab wounds could have been done laparoscopically. two of the 10 gsw patients had lateral or posterior injuries that required a more detailed evaluation of the retroperitoneum than would have been possible by laparoscopy alone. although we currently use the laparoscope solely as a screening tool for peritoneal penetration, the next logical progression for us is to conduct a more effective laparoscopic evaluation of specific organ structures in the trauma setting. this could potentially decrease or eliminate the conversions from laparoscopy to laparotomy that were nontherapeutic. this would entail a systematic laparoscopic evaluation of the entire peritoneal cavity, including running the small bowel, visualizing the diaphragm and solid organs, and accurately assessing for evidence of pelvic or retroperitoneal injury. the threshold for conversion would vary among surgeons based on laparoscopic expertise and confidence in the laparoscopic examination. data show that laparoscopy is a useful modality for evaluating and managing trauma patients with penetrating injuries. we have found that it is particularly helpful as a screening tool for anterior abdominal wall wounds and lower chest injuries to rule out peritoneal penetration. increased use of laparoscopy in select patients with penetrating abdominal trauma will decrease the rate of negative and nontherapeutic laparotomies, thus lowering morbidity, decreasing length of hospitalization, and provide for more efficient utilization of available resources. as technology and expertise among surgeons continues to improve,	background : minimally invasive surgery has become increasingly utilized in the trauma setting. when properly applied, it offers several advantages, including reduced morbidity, lower rates of negative laparotomy, and shortened length of hospital stay. the purpose of this study was to evaluate the role of laparoscopy in the management of trauma patients with penetrating abdominal injuries.methods:we conducted a 3-year retrospective chart review of 4541 trauma patients admitted to our urban level ii trauma center. penetrating abdominal injuries accounted for 209 of these admissions. patients were divided into 3 treatment groups based on the characteristics of their abdominal injuries. management was either observation, immediate laparotomy, or screening laparoscopy.results:thirty-three patients were observed in the emergency department based on their initial physical examination and radiologic studies. after emergency department evaluation, 154 patients underwent immediate laparotomy. in this group, 119 therapeutic laparotomies, 11 nontherapeutic laparotomies, and 24 negative laparotomies were performed. a review of the negative laparotomies revealed that possibly 8 of 10 gun shot wounds and all 14 stab wounds could have been done laparoscopically. twenty - two patients underwent laparoscopic evaluation, 9 of which were converted to open procedures.conclusion:minimally invasive surgical techniques are particularly helpful as a screening tool for anterior abdominal wall wounds and lower chest injuries to rule out peritoneal penetration. increased use of laparoscopy in select patients with penetrating abdominal trauma will decrease the rate of negative and nontherapeutic laparotomies, thus lowering morbidity and decreasing length of hospitalization. as technology and expertise among surgeons continues to improve, more therapeutic intervention may be done laparoscopically in the future.
thrombocytopenia absent radius (tar) is a rare syndrome associated with bilateral absence of radii, hypo - megakaryocytic thrombocytopenia, and presence of both thumbs.1 tar syndrome abnormalities include various systems, including skeletal, hematologic, and cardiac system abnormalities. this association of simultaneous involvement of various systems has been postulated due to the simultaneous development of the heart, radii, and megakaryocytes at 68 weeks gestation. the reported frequency of tar syndrome in medical literature has been expected to be around 0.42/100,000 population with unknown genetic inheritance pattern with few case reports showing both autosomal recessive and dominant pattern of inheritance. tar syndrome is the result of noncoding single nucleotide polymorphisms located in the 5utr region or the first intron of the gene rbm8a and also because of microdeletion of chromosome 1q21.1 region.2 this case report will help pediatricians to be aware of the associated problems with tar syndrome other than simply the functional disability associated with absent radius. we report an infant with tar syndrome with tetralogy of fallot which has not been reported in medical literature until now and this is the first case of its type. a 3-month old male infant who was the first child of a non - consanguineous couple and born at term with a birth weight of 2,480 grams was brought to the outpatient department with repeated episodes of bluish discoloration of lips on excessive crying. in the prenatal history the infant was delivered at 39 weeks by normal vaginal delivery with normal apgar score of 8/8/9 at 1, 5, and 10 minutes. the infant was exclusively breastfed and the couple did not consult any doctor for abnormalities before coming to our hospital. on physical examination the child was noted to be pink with 89%90% saturation on room air, bilateral absent radius, radial club hand and flexion against the palm (figures 1 and 2), and a grade 2/6 murmur in the left parasternal border, depressed. he was provisionally diagnosed to have tar syndrome on the basis of the physical findings. x - ray of upper limbs suggested absence of bilateral radius bone (figure 3). two - dimensional echocardiography revealed tetralogy of fallot physiology with ventricular septal defect (vsd), infundibular pulmonary stenosis with overriding of the aorta. complete blood count revealed thrombocytopenia and anemia with a platelet count of 53,000/mm and hemoglobin value of 9.2 gm / dl. genetic analysis showed interstitial microdeletion in 1q21.1 and a hypomorphic allele in rbm8a chromosomes, which confirmed our provisional diagnosis of tar syndrome. following admission the baby received three aliquots of single donor platelet transfusion and this increased the repeat platelet count to 142,000/mm. the infant underwent definitive repair of tetralogy of fallot (definitive repair included vsd closure and relief of rvot obstruction through a combination of infundibular muscle resection and pulmonary valvotomy). after the operation the baby was given post - operative care in the form of invasive ventilation for the following 48 hours and there was improvement in the neonate s saturation with a maximum of 96% and the infant was extubated successfully. the feeds were started on post - operative day 3 and gradually increased and breast feeding was started 7 days after the operation. the baby was discharged 2 weeks later and was to be followed up for the intermittent screening for thrombocytopenia and platelet transfusion for approximately 2 months. orthopedic / plastic surgeon consultation for the limb deformity was later planned, but the patient was later lost to follow - up. after enquiries it was discovered that the child had died, the grounds of which was unknown. tar is a rare syndrome associated with bilateral absence of radii, hypo - megakaryocytic thrombocytopenia, and presence of both thumbs.1 it was first reported in medical literature in 1959. the genetic inheritance pattern is uncertain with few documented reports mentioning autosomal recessive pattern, whereas others mention autosomal dominant pattern of inheritance. high - throughput sequencing studies suggest that tar syndrome is biallelic inherited with noncoding single nucleotide polymorphisms located in the 5utr region or the first intron of the gene rbm8a (coding for y14 protein, association to tar syndrome not yet cleared up) and a chromosome 1q21.1 region microdeletion.2 the pathophysiology of this term is mostly unknown, but theories have run from a common (unknown) injury in 68 weeks of gestation when the heart, limbs, and megakaryocytes develop simultaneously or due to the contiguous gene model which is founded on the premise that phenotypic findings are related when genes responsible for each defect are geographically associated in a chromosome. similarly, the pathophysiology for thrombocytopenia is largely unknown, but has been purported to be due to abnormal signal transduction to stimulator like thrombopoietin.3 thrombocytopenia begins in the neonatal period,4 50% of affected infants present with thrombocytopenia in the first week of life and approximately 90% develop thrombocytopenia by 4 months of age. other non - skeletal abnormalities commonly associated include gastroenteritis and cow s milk intolerance (47%), renal malformations (23%), cardiac defects like atrial septal defects, vsd, and very rarely tetralogy of fallot (15%), facial dysmorphism (53%), short stature (95%), macrocephaly (76%), and capillary hemangioma (24%).5 the predominant skeletal abnormality includes bilateral radial aplasia with presence of bilateral thumb. nevertheless, additional skeletal abnormalities are often observed, those including rarely more extensive upper limb malformations, phocomelia, and lower limb malformations in as many as 50% of the patients.5 tar syndrome is commonly associated with a combination of physical findings. tar syndrome is commonly labelled when there is a constellation of various physical findings : fanconi anemia is characterized by congenital anomalies, progressive bone marrow failure, and predisposition to cancers like myelodysplastic syndrome and acute myelogenous leukemia. the skeletal abnormalities include radial bone and thumb abnormalities, short stature, skin hyperpigmentation, and/or caf au lait macules. the presence of thumbs in our patient helped us to rule out fanconi anemia and genetic analysis confirmed our diagnosis.6roberts syndrome is inherited in autosomal recessive pattern and the affected patient has various problems which include severe growth retardation, cleft lip and palate, nose and ears anomalies, facial hemangioma, hypertelorism, microcephaly, oligodactyly or tetraphocomelia, renal malformations like polycystic and dysplastic kidneys, congenital heart defects, and enlarged male genitalia. the karyotype analysis shows typical railroad track appearance because of chromatic and premature centromere separation in metaphase spreads. the gene, which has been postulated in roberts syndrome as esco2 genetic, is located on 8p21.1 locus. oram syndrome (omim 142900) is associated with upper limb extremity abnormalities which include abnormality of radius, metacarpals, or carpal bones. our patient did not have any abnormality of metacarpal and carpal bones and genetic analysis showed interstitial microdeletion in 1q21.1 and a hypomorphic allele in rbm8a chromosomes which confirmed tar syndrome.8diamond - blackfan anemia / aase syndrome is characterized by erythroid hypoplasia, macrocytic anemia, and normal white blood cell and platelet counts. the affected patient usually has other congenital anomalies, such as triphalangeal, bifid, or subluxed thumbs, flattening of the thenar eminence, with a normal radius bone.. these patients usually have webbed neck, klippel - feil anomaly or sprengel deformity. the absence of radius and reduced platelet count helped us to differentiate diamond - blackfan anemia from tar syndrome and our suspicion was confirmed by genetic analysis.9 fanconi anemia is characterized by congenital anomalies, progressive bone marrow failure, and predisposition to cancers like myelodysplastic syndrome and acute myelogenous leukemia. the skeletal abnormalities include radial bone and thumb abnormalities, short stature, skin hyperpigmentation, and/or caf au lait macules. the presence of thumbs in our patient helped us to rule out fanconi anemia and genetic analysis confirmed our diagnosis.6 roberts syndrome is inherited in autosomal recessive pattern and the affected patient has various problems which include severe growth retardation, cleft lip and palate, nose and ears anomalies, facial hemangioma, hypertelorism, microcephaly, oligodactyly or tetraphocomelia, renal malformations like polycystic and dysplastic kidneys, congenital heart defects, and enlarged male genitalia. the karyotype analysis shows typical railroad track appearance because of chromatic and premature centromere separation in metaphase spreads. the gene, which has been postulated in roberts syndrome as esco2 genetic, is located on 8p21.1 locus. the genetic analysis of our patient ruled out robert syndrome in the index case.7 holt oram syndrome (omim 142900) is associated with upper limb extremity abnormalities which include abnormality of radius, metacarpals, or carpal bones. the only gene which has been known to cause holt oram syndrome, tbx5 gene, is located on chromosome 12 (12q24.1). our patient did not have any abnormality of metacarpal and carpal bones and genetic analysis showed interstitial microdeletion in 1q21.1 and a hypomorphic allele in rbm8a chromosomes which confirmed tar syndrome.8 diamond - blackfan anemia / aase syndrome is characterized by erythroid hypoplasia, macrocytic anemia, and normal white blood cell and platelet counts. the affected patient usually has other congenital anomalies, such as triphalangeal, bifid, or subluxed thumbs, flattening of the thenar eminence, with a normal radius bone. the absence of radius and reduced platelet count helped us to differentiate diamond - blackfan anemia from tar syndrome and our suspicion was confirmed by genetic analysis.9 in tar syndrome, significant laboratory findings include thrombocytopenia with normal platelet morphology in peripheral smear and hypo - megakaryocytic thrombocytopenia with small, basophilic, vacuolated megakaryocytes on bone marrow examination with normal or elevated plasma thrombopoietin levels.10 radial abnormality can be detected as early as 13 weeks by prenatal ultrasonography.11 affected infants have an increased risk of bleeding manifestations especially intracranial hemorrhage which is more common during the first 2 years of life. we suspect that cause of death in our index case could be because of intracranial hemorrhage as these infants are more prone to it. hence, these babies should follow general thrombocytopenic precautions like avoidance of trauma and avoidance of antiplatelet drugs. prophylactic platelet transfusions to raise the platelet count above 40,000/mm (extrapolated from thrombocytopenia with leukemia) is currently pursued in patients with high risk of clinically important hemorrhage. other recent treatment modalities include treatment with erythropoietin,12 il-6,13 and hematopoietic stem cell transplantation in refractory thrombocytopenic patients.14 splenectomy in adults has shown to improve thrombocytopenia. the risk of bleeding, thrombocytopenia is significant particularly in the first 2 years of life, subsequently platelet count increases with age. yet there is a reported increased risk of leukemia with tar syndrome.15 similar events have been described in the past, some of them have included additional findings. in a serial publication published by greenhalgh in 2002, all patients had documented thrombocytopenia and bilateral radial aplasia, 47% had lower limb anomalies, 47% intolerance to cow s milk, 23% renal anomalies, and 15% cardiac anomalies. congenital anomalies also included facial capillary hemangiomata, intracranial vascular malformation, sensorineural hearing loss, and scoliosis.5 menghsol reported a patient with tar syndrome who also had coarctation of the aorta and axial rotation of the kidney. other findings included adducted thumbs, radial aplasia, hypoplasia of the cerebellar vermis, and axial malrotation of the kidney.16 tar is a rare syndrome and is associated with bilateral absence of radii, hypo - megakaryocytic thrombocytopenia, and presence of both thumbs. tar syndrome abnormalities include various systems including skeletal, hematologic, and cardiac system abnormalities. babies with suspected tar syndrome should be assessed for other associated malformations of various systems like renal malformations, cardiac defects like atrial septal defects, vsd, and very rarely tetralogy of fallot, facial dysmorphism, short stature, macrocephaly, capillary hemangioma, and hematological malignancies in later life, requiring regular follow - up. antenatal diagnosis of tar syndrome is possible in the first trimester but that requires thorough antenatal checkup and regular follow - up. the risk of bleeding is highest during the first 2 years of life span after which the platelet count may improve. close surveillance for thrombocytopenia with regular follow - ups and platelet transfusion to raise platelet count above 40,000/mm are the mainstay of treatment. treatment of associated deformities requires a multidisciplinary approach that contributes to the successful management of such cases.	thrombocytopenia absent radius (tar) syndrome is a very rare and infrequently seen congenital disorder with an approximate frequency of 0.42/100,000 live births. it is associated with bilateral absence of radii, hypo - megakaryocytic thrombocytopenia, and presence of both thumbs. the other systems which are affected by tar syndrome include skeletal, hematologic, and cardiac systems. intracranial hemorrhages due to thrombocytopenia and cardiac disorders are a common association usually seen with this syndrome and are usual cause of death. we describe a 3-month - old infant who was diagnosed with tar syndrome on the basis of clinical features (thrombocytopenia and bilateral absent radius bone and confirmed by genetic analysis). the patient was diagnosed to have tetralogy of fallot, for which the infant was managed with definitive repair and thrombocytopenia was managed with platelet transfusion. infants with tar syndrome should be assessed for other associated malformations of various systems and followed up regularly and parents should be counseled for associated expected complications in these patients. we report an infant with tar syndrome with tetralogy of fallot, which has not been reported in medical literature until now and this is the first case of its type.
ten volunteers (eight men and two postmenopausal women) with type 2 diabetes were recruited for this study, which was approved by the university health research ethics board. participants met the following eligibility criteria : 1) between 30 and 65 years of age ; 2) not taking glucose - lowering medication or insulin ; 3) no changes in physical activity over the last 3 months and not planning on changing medication, physical activity, or diet over the course of the study ; and 4) hba1c 8%, resting blood pressure 140/90 mmhg, ldl cholesterol 3.5 mmol / l, and total : hdl cholesterol 5.0. the study used a factorial design and each participant was exposed to 4 conditions : 1) metformin and no exercise, 2) metformin and exercise, 3) placebo and no exercise, and 4) placebo and exercise. the order of the metformin versus placebo conditions was randomized by personnel not involved with the study, and allocation was concealed in sealed envelopes until participants completed the study. participants, study personnel, and investigators were blinded to the order of the placebo / metformin conditions. metformin or placebo was given for 28 days, immediately followed by the alternate condition for 28 days. on the last 2 days of each condition (days 27 and 28), participants returned to the exercise physiology laboratory for a nonexercise and exercise session, respectively. exercise was always performed on day 28 since the acute glucose - lowering effect of exercise may persist for at least 24 h (8). during the baseline, an exercise stress test with a 12-lead electrocardiogram was performed using a modified balke - ware treadmill protocol. each participant walked at a self - selected speed, determined as comfortable but fast, while the grade was increased by 2% each minute. this protocol was used to determine the peak oxygen uptake (vo2peak) and ventilatory threshold using the v - slope method. after the baseline visit, participants were given either metformin or placebo pills and were asked to maintain their routine physical activity and dietary habits. each participant consumed 500 mg of metformin with breakfast during the first week of the intervention followed by a 500-mg increase in each of the subsequent weeks until 1,000 mg were consumed with breakfast and supper during week 4 (total : 2,000 mg / day). on days 27 and 28 of the metformin and placebo conditions, participants arrived in the laboratory at 8:00 a.m. after a 12-h fast. fasting glucose was measured with a handheld glucose meter (one touch ultra ; lifescan, milpitas, ca), and then participants ate a standardized breakfast (549 kcal ; 56% carbohydrate, 30% fat, 14% protein) and took their assigned pills. at 10:00 a.m., an intravenous catheter was inserted into an antecubital vein kept patent with 0.9% sterile saline and the first blood sample was collected at 10:20 a.m. on day 27 of the metformin and placebo conditions, the participants remained at rest for the duration of the testing period. at 10:45 a.m. of day 28 during both conditions, participants performed a series of exercises that were selected to represent different intensities, modes, and energy systems. they began with 20 consecutive maximal leg extensions and flexions on a cybex ii isokinetic dynamometer (lumex). the isokinetic test was included since resistance exercise is recommended for people with diabetes (9) and since isokinetic testing helps avoid eccentric contractions under load, which might induce muscle damage and impact the subsequent treadmill tests. the angular velocity was set at 180 s (3.14 rads s), and measurements of peak torque, mean torque, and a fatigue index were calculated. after a 5-min rest period, each bout was separated by a 5-min rest period during which blood samples were drawn from the catheter. during the first exercise bout, this corresponded to the estimated average walking speed for individuals with type 2 diabetes in free - living conditions (10). the second bout also lasted 15 min and was completed at a speed and grade equivalent to an intensity below each participant s measured ventilatory threshold. the third bout was completed at an intensity above their ventilatory threshold and lasted 5 min. metabolic outcomes such as the volume of oxygen consumed (vo2) and the volume of carbon dioxide produced (vco2) during exercise were measured with a truemax metabolic measurement system (parvo medics, salt lake city, uttah). heart rate (hr) was measured using a polar heart rate monitor (polar electric, finland), and rate of perceived exertion was estimated with the borg scale. about 20 min after exercise (at 11:59 a.m.), another blood sample was taken immediately before the standardized meal (556 kcal ; 59% carbohydrate, 22% fat, 19% protein). participants remained in the laboratory, and blood samples were taken every 30 min for 2 h. each blood sample was first transferred into a 10-ml edta vacutainer tube. subsequently, 0.25 ml whole blood was transferred into 1.0 ml ice - cold 8% perchloric acid, and 2.0 ml was transferred into a tube with 67 l aprotinin. tubes were centrifuged and cooled before being moved to a 20c freezer until assays were completed. serum lactate, glucose, and nonesterified fatty acids (nefas) were determined enzymatically with spectrophotometric assays. glucagon and insulin were measured using commercially available radioimmunoassay (ria) kits (millipore, st. charles, mo and inter medico, markham, ontario, canada, respectively). plasma metformin concentrations were assessed in all plasma samples by a high performance liquid chromatographic. the assay was validated to a lower limit of quantitation of 7.8 ng / ml metformin based on 0.1 ml of human plasma (11). these were analyzed for calorie and macronutrient content using food processor sql (version 8.3.0 ; esha research, salem, or). habitual physical activity during each 4-week intervention was assessed using the godin leisure time questionnaire (12). finally, participants were asked to indicate their perception of which intervention (metformin or placebo) they had just completed on a 150-mm visual analog scales as well as symptoms such as nausea, headache, flatulence, abdominal discomfort, and indigestion. analyses were conducted using repeated - measures anova with treatment order added as a between - subject factor. to simplify the interpretation, the testing days were broken down into four periods : pre - exercise, exercise, postexercise, and postlunch. the number of within - factors and levels varied among these periods (e.g., postlunch was a 2 2 4 factorial anova to examine the effect of exercise, metformin, and time, respectively). insulin, glucagon, glucose, lactate, and nefas were log transformed before the statistical analyses to favor normality of residuals and homogeneity of variance. statistical tests were two - tailed, and p values of 0.05 were considered significant. ten volunteers (eight men and two postmenopausal women) with type 2 diabetes were recruited for this study, which was approved by the university health research ethics board. participants met the following eligibility criteria : 1) between 30 and 65 years of age ; 2) not taking glucose - lowering medication or insulin ; 3) no changes in physical activity over the last 3 months and not planning on changing medication, physical activity, or diet over the course of the study ; and 4) hba1c 8%, resting blood pressure 140/90 mmhg, ldl cholesterol 3.5 mmol / l, and total : hdl cholesterol 5.0. the study used a factorial design and each participant was exposed to 4 conditions : 1) metformin and no exercise, 2) metformin and exercise, 3) placebo and no exercise, and 4) placebo and exercise. the order of the metformin versus placebo conditions was randomized by personnel not involved with the study, and allocation was concealed in sealed envelopes until participants completed the study. participants, study personnel, and investigators were blinded to the order of the placebo / metformin conditions. metformin or placebo was given for 28 days, immediately followed by the alternate condition for 28 days. on the last 2 days of each condition (days 27 and 28), participants returned to the exercise physiology laboratory for a nonexercise and exercise session, respectively. exercise was always performed on day 28 since the acute glucose - lowering effect of exercise may persist for at least 24 h (8). during the baseline, an exercise stress test with a 12-lead electrocardiogram was performed using a modified balke - ware treadmill protocol. each participant walked at a self - selected speed, determined as comfortable but fast, while the grade was increased by 2% each minute. this protocol was used to determine the peak oxygen uptake (vo2peak) and ventilatory threshold using the v - slope method. after the baseline visit, participants were given either metformin or placebo pills and were asked to maintain their routine physical activity and dietary habits. each participant consumed 500 mg of metformin with breakfast during the first week of the intervention followed by a 500-mg increase in each of the subsequent weeks until 1,000 mg were consumed with breakfast and supper during week 4 (total : 2,000 mg / day). on days 27 and 28 of the metformin and placebo conditions, participants arrived in the laboratory at 8:00 a.m. after a 12-h fast. fasting glucose was measured with a handheld glucose meter (one touch ultra ; lifescan, milpitas, ca), and then participants ate a standardized breakfast (549 kcal ; 56% carbohydrate, 30% fat, 14% protein) and took their assigned pills. at 10:00 a.m., an intravenous catheter was inserted into an antecubital vein kept patent with 0.9% sterile saline and the first blood sample was collected at 10:20 a.m. on day 27 of the metformin and placebo conditions, the participants remained at rest for the duration of the testing period. at 10:45 a.m. of day 28 during both conditions, participants performed a series of exercises that were selected to represent different intensities, modes, and energy systems. they began with 20 consecutive maximal leg extensions and flexions on a cybex ii isokinetic dynamometer (lumex). the isokinetic test was included since resistance exercise is recommended for people with diabetes (9) and since isokinetic testing helps avoid eccentric contractions under load, which might induce muscle damage and impact the subsequent treadmill tests. the angular velocity was set at 180 s (3.14 rads s), and measurements of peak torque, mean torque, and a fatigue index were calculated. after a 5-min rest period, each bout was separated by a 5-min rest period during which blood samples were drawn from the catheter. during the first exercise bout, this corresponded to the estimated average walking speed for individuals with type 2 diabetes in free - living conditions (10). the second bout also lasted 15 min and was completed at a speed and grade equivalent to an intensity below each participant s measured ventilatory threshold. the third bout was completed at an intensity above their ventilatory threshold and lasted 5 min. metabolic outcomes such as the volume of oxygen consumed (vo2) and the volume of carbon dioxide produced (vco2) during exercise were measured with a truemax metabolic measurement system (parvo medics, salt lake city, uttah). heart rate (hr) was measured using a polar heart rate monitor (polar electric, finland), and rate of perceived exertion was estimated with the borg scale. about 20 min after exercise (at 11:59 a.m.), another blood sample was taken immediately before the standardized meal (556 kcal ; 59% carbohydrate, 22% fat, 19% protein). participants remained in the laboratory, and blood samples were taken every 30 min for 2 h. each blood sample was first transferred into a 10-ml edta vacutainer tube. subsequently, 0.25 ml whole blood was transferred into 1.0 ml ice - cold 8% perchloric acid, and 2.0 ml was transferred into a tube with 67 l aprotinin. tubes were centrifuged and cooled before being moved to a 20c freezer until assays were completed. serum lactate, glucose, and nonesterified fatty acids (nefas) were determined enzymatically with spectrophotometric assays. glucagon and insulin were measured using commercially available radioimmunoassay (ria) kits (millipore, st. charles, mo and inter medico, markham, ontario, canada, respectively). all assays were run in duplicate. plasma metformin concentrations were assessed in all plasma samples by a high performance liquid chromatographic. the assay was validated to a lower limit of quantitation of 7.8 ng / ml metformin based on 0.1 ml of human plasma (11). these were analyzed for calorie and macronutrient content using food processor sql (version 8.3.0 ; esha research, salem, or). habitual physical activity during each 4-week intervention was assessed using the godin leisure time questionnaire (12). finally, participants were asked to indicate their perception of which intervention (metformin or placebo) they had just completed on a 150-mm visual analog scales as well as symptoms such as nausea, headache, flatulence, abdominal discomfort, and indigestion. analyses were conducted using repeated - measures anova with treatment order added as a between - subject factor. to simplify the interpretation, the testing days were broken down into four periods : pre - exercise, exercise, postexercise, and postlunch. the number of within - factors and levels varied among these periods (e.g., postlunch was a 2 2 4 factorial anova to examine the effect of exercise, metformin, and time, respectively). insulin, glucagon, glucose, lactate, and nefas were log transformed before the statistical analyses to favor normality of residuals and homogeneity of variance. statistical tests were two - tailed, and p values of 0.05 were considered significant. baseline characteristics are presented in table 1. some reported mild to moderate gastrointestinal side effects during the metformin intervention ; but all participants except one (final metformin dosage, 1,500 mg / day) were able to tolerate the maximum dosage of 2,000 mg / day during the last week of the intervention. oxygen consumption (vo2), respiratory exchange ratio (rer), hr, and rating of perceived exertion (rpe) during exercise are shown in table 2. there was a main effect of exercise intensity (all p 0.01) and no interaction effect (all p 0.18) for vo2, rer, hr, and rpe. the first 15-min bout was performed at 33.9 5.4% of vo2peak, the second 15-min bout averaged 67.2 7.3% of vo2peak, and the third 5-min bout averaged 79.4 8.8% of vo2peak. vo2 was not affected by metformin (p = 0.60). however, mean rer was lower in the metformin condition (0.96 0.02 vs. 0.98 0.02 ; p = 0.03). mean hr was significantly higher in the metformin condition (124 9 vs. 118 8 beats per minute [bpm ] ; p = 0.009). the mean subjective ratings of perceived exertion during exercise were similar in the metformin and placebo conditions. however, participants reported a higher perceived exertion on their first exercise day regardless of whether they were on metformin or placebo. as well, when considering treatment order in the analyses, rpe was higher in the metformin condition (p = 0.03). effect of metformin on exercise - related outcomes data are reported as mean sd. analyses were adjusted for treatment order (i.e., metformin first vs. placebo first). plasma metformin concentrations were higher on the exercise day compared with the nonexercise day 25 min before exercise (1,897 352 vs. 1,594 363 ng / ml ; p = 0.02) and 20 min after exercise (2,230 335 vs. 1,893 323 ng / ml ; p = 0.01). plasma metformin concentrations showed a significant time by exercise interaction (p = 0.05) during the 2-h postmeal period, with metformin concentration becoming similar near the end of the 2-h period. fasting glucose was lower in the metformin condition compared with the placebo condition (6.4 0.6 vs. 7.2 0.6 mmol / l ; p = 0.02). 1, the mean glucose concentration continued to be lower throughout the day in the metformin conditions compared with placebo, with the difference becoming statistically significant after exercise. according to the sample taken immediately before lunch, exercise lowered plasma glucose concentration in the placebo condition but not in the metformin condition (1.1 2.0 vs. 0.1 1.1 mmol / l, respectively) ; however, the metformin exercise interaction was not significant (p = 0.17). the metformin exercise interaction reached statistical significance during the 2-h postlunch period (p = 0.05), suggesting that exercise caused an increased glycemic response in the metformin condition but not in the placebo condition. data are reported as mean sd (except for insulin : mean sem). analyses were adjusted for treatment order (i.e., metformin first vs. placebo first). na, not applicable ; mod, moderate ; vig, vigorous ; ex, exercise., metformin + no exercise ;, metformin + exercise ;, placebo + no exercise ;, placebo + exercise. effect of exercise ; effect of time ; effect of metformin ; exercise by metformin interaction ; \|\|exercise by time interaction ; metformin by time interaction. throughout the entire sessions, lactate concentrations were higher in the metformin condition compared with placebo (all p 0.05). lactate concentrations increased with increasing exercise intensity and remained elevated for 20 min after exercise (both p 0.52). there was no difference in the amount of physical activity completed during the metformin versus placebo conditions (godin leisure time questionnaire score 36 21 vs. 52 36 ; p = 0.13). participants did not report experiencing any difference in symptoms such as abdominal discomfort between conditions. participants rated a higher likelihood of taking metformin while they were in the metformin condition compared with placebo (89 40 mm vs. 62 47 mm on the 150-mm visual analog scale), but the difference did not reach statistical significance (p = 0.20). body mass was similar after the 28-day placebo condition versus after the 28-day metformin condition (86.7 19.0 vs. 86.7 18.9 kg ; p = 0.93). plasma metformin concentrations were higher on the exercise day compared with the nonexercise day 25 min before exercise (1,897 352 vs. 1,594 363 ng / ml ; p = 0.02) and 20 min after exercise (2,230 335 vs. 1,893 323 ng / ml ; p = 0.01). plasma metformin concentrations showed a significant time by exercise interaction (p = 0.05) during the 2-h postmeal period, with metformin concentration becoming similar near the end of the 2-h period. fasting glucose was lower in the metformin condition compared with the placebo condition (6.4 0.6 vs. 7.2 0.6 mmol / l ; p = 0.02). 1, the mean glucose concentration continued to be lower throughout the day in the metformin conditions compared with placebo, with the difference becoming statistically significant after exercise. according to the sample taken immediately before lunch, exercise lowered plasma glucose concentration in the placebo condition but not in the metformin condition (1.1 2.0 vs. 0.1 1.1 mmol / l, respectively) ; however, the metformin exercise interaction was not significant (p = 0.17). the metformin exercise interaction reached statistical significance during the 2-h postlunch period (p = 0.05), suggesting that exercise caused an increased glycemic response in the metformin condition but not in the placebo condition. data are reported as mean sd (except for insulin : mean sem). analyses were adjusted for treatment order (i.e., metformin first vs. placebo first). na, not applicable ; mod, moderate ; vig, vigorous ; ex, exercise., metformin + no exercise ;, metformin + exercise ;, placebo + no exercise ;, placebo + exercise. effect of exercise ; effect of time ; effect of metformin ; exercise by metformin interaction ; \|\|exercise by time interaction ; metformin by time interaction. throughout the entire sessions, lactate concentrations were higher in the metformin condition compared with placebo (all p 0.05). lactate concentrations increased with increasing exercise intensity and remained elevated for 20 min after exercise (both p 0.52). there was no difference in the amount of physical activity completed during the metformin versus placebo conditions (godin leisure time questionnaire score 36 21 vs. 52 36 ; p = 0.13). participants did not report experiencing any difference in symptoms such as abdominal discomfort between conditions. participants rated a higher likelihood of taking metformin while they were in the metformin condition compared with placebo (89 40 mm vs. 62 47 mm on the 150-mm visual analog scale), but the difference did not reach statistical significance (p = 0.20). body mass was similar after the 28-day placebo condition versus after the 28-day metformin condition (86.7 19.0 vs. 86.7 18.9 kg ; p = 0.93). in the 1960s and 1970s several studies had investigated the effect of metformin on exercise performance because of concerns over lactic acidosis (see reference 13 for a detailed review). although the combination of metformin and exercise was perceived as safe, interest in this area has reemerged in recent years (4,7,14,15). the current study is unique in that it focused on continuous exercise at several submaximal intensities that are relevant to activity patterns of people with type 2 diabetes and that we examined the interaction between exercise and metformin on the glycemic and hormonal responses to a subsequent meal. we found that metformin increased lipid oxidation as evidenced by a lower rer during all three submaximal intensities of exercise. according to nonprotein rer tables, this would correspond to an increased lipid oxidation from 16 to 26% of total energy expenditure when walking at 3.5 km / h. however, metformin increased submaximal hr and lactate concentrations, which are opposite to the direction of changes expected with regular exercise training. in the current study, interestingly, sharoff. (4) also found an increased hr of about 8 and 5 bpm during exercise at 65 and 85% of vo2peak, respectively ; however, in their study the increase in hr did not reach statistical significance. in our study, a higher rating of perceived exertion in the metformin condition was also observed, although participants were all able to complete the exercise bouts. taken together, this suggests that metformin has the potential to lower some patients selected exercise intensity since perceived exertion and hr are common feedback modalities and are frequently used to prescribe exercise intensities. although statistical significance was not reached, peak and mean torque for knee extension were lower in the metformin condition. lower mean torque may have been expected based on the reduced muscle atp concentrations observed by week 4 of metformin treatment in the study by musi. maximal metformin concentrations are typically reached 120240 min after a dose. in the current study, we observed greater plasma metformin concentrations 25 min before exercise and 20 min after exercise compared with samples taken at the same times on the rest day (150 and 225 min postdose). the reasons for these higher concentrations are unknown but may have been caused by the anticipatory and stress responses to exercise, which are known to increase hr and blood pressure while redistributing blood flow to tissues such as skeletal muscle (17). hence, the alteration in blood flow may have caused a transient decrease in the distribution of drug to certain tissues, including the liver. indeed, this may have contributed to the reduced hypoglycemic effect of metformin after exercise even though plasma concentrations were higher at some time points. a reduced renal blood flow could increase plasma concentrations of drugs such as metformin, which are primarily eliminated by the kidneys (18). this may have also contributed to some of the higher concentrations measured in the exercise group, although only a more complete assessment of plasma metformin concentrations and urinary recovery could answer this question. a limitation of the current study is that the three blood samples taken immediately after each aerobic exercise bout were not taken at the corresponding times on the nonexercise days. although some previous studies had suggested that the effects of exercise and metformin on insulin sensitivity (4) or on the risk of diabetes (3) are not additive, our results suggest that in some conditions the combination may in fact be less effective at lowering the glycemic response to a meal than metformin alone. the reasons for this are not clear, but may be related to the strong counterregulatory response when the two were combined. in our study, the glucagon concentrations peaked immediately before lunch and were highest in the combined metformin and exercise condition. in support of the notion that glucose production may have been increased by the higher glucagon concentrations, sharoff. (4) showed that hepatic glucose production was increased 2 h after exercise with metformin, unchanged by metformin alone, and decreased by exercise alone (4). (4) are that in the latter study participants were nondiabetic and 2 to 3 weeks of metformin use did not appear to alter insulin sensitivity or resting glucose concentration. nonetheless, taken together these studies provide interesting insight on glucose homeostasis after metformin and exercise. the lack of improvement in postmeal (lunch) plasma glucose concentrations on the exercise days should not discourage the use of exercise as a treatment modality. rather, this study emphasizes that it may be important to further consider the timing of exercise and meals to obtain optimal glycemic benefits. for example, others have shown that exercising in the fasting state (a condition that also leads to pronounced counterregulatory responses) was much less effective at lowering plasma glucose than was exercising after a meal (19,20). furthermore, it is important to remember that the exercise protocol in the current study ended with 5 min of exercise at an intensity above ventilatory threshold. (4), the exercise protocol ended with 10 min at 85% of vo2peak. high intensity exercise (i.e., above ventilatory threshold) in the postabsorptive state is known to cause an increase in counterregulatory hormones and glucose in type 2 diabetes (21). however, high intensity exercise performed 45 min after the beginning of breakfast led to a decreased glycemic response to a meal that was provided 2.5 h after exercise (22). it would be of interest to examine if interactions between metformin and exercise on glucose homeostasis would be as pronounced after lower intensity exercise. type 2 diabetes is characterized not only by insulin deficiency but also by hyperglucagonemia (23). we are aware of nonexercise studies that have suggested that metformin may increase glucagon concentrations, but the increases were not statistically significant (24,25). in the nonrandomized exercise studies by cunha. (14,15), glucagon concentrations were significantly higher in the participants with type 2 diabetes taking metformin compared with those taking glibenclamide or those with normal glucose tolerance. although speculative, the glucose - lowering benefits of metformin could be further enhanced by strategies that could help minimize the exercise - induced increased glucagon levels such as exercising after a meal. in conclusion, our study reports several novel findings regarding the concomitant use of metformin and exercise, specifically : 1) increased hr during exercise with metformin, 2) higher plasma metformin concentrations with exercise, and 3) nonadditive effects of metformin and exercise on the glycemic response to feeding. in our opinion, the magnitudes of these effects were small but have the potential to reduce the effectiveness of this therapeutic combination in diabetes treatment. additional research could help optimize the concurrent use of these important and widely prescribed treatment modalities for diabetes.	objectiveto determine the effect of metformin on the acute metabolic response to submaximal exercise, the effect of exercise on plasma metformin concentrations, and the interaction between metformin and exercise on the subsequent response to a standardized meal.research design and methodsten participants with type 2 diabetes were recruited for this randomized crossover study. metformin or placebo was given for 28 days, followed by the alternate condition for 28 days. on the last 2 days of each condition, participants were assessed during a nonexercise and a subsequent exercise day. exercise took place in the morning and involved a total of 35 min performed at three different submaximal intensities.resultsmetformin increased heart rate and plasma lactate during exercise (both p 0.01) but lowered respiratory exchange ratio (p = 0.03) without affecting total energy expenditure, which suggests increased fat oxidation. metformin plasma concentrations were greater at several, but not all, time points on the exercise day compared with the nonexercise day. the glycemic response to a standardized meal was reduced by metformin, but the reduction was attenuated when exercise was added (metformin exercise interaction, p = 0.05). glucagon levels were highest in the combined exercise and metformin condition.conclusionsthis study reveals several ways by which metformin and exercise therapies can affect each other. by increasing heart rate, metformin could lead to the prescription of lower exercise workloads. furthermore, under the tested conditions, exercise interfered with the glucose - lowering effect of metformin.
the human gastric pathogen helicobacter pylori (h. pylori) is a microaerophilic, spiral - shaped, gram - negative bacterium responsible for the majority of peptic ulcer diseases in humans. h. pylori has been shown to be the causative agent of type b gastritis and peptic ulcerations [2, 3 ]. infection with h. pylori increases the risks of developing gastric carcinoma and mucosa - associated lymphoid tissue (malt) lymphoma [4, 5 ]. continuously exposed to acidic ph during the process of colonization in the gastric mucus layer, h. pylori requires mechanisms to survive acid shocks and to enable growth in such acidic conditions. this bacterium expresses two nickel - containing enzymes : urease and hydrogenase, both of which is important for its colonization. the h. pylori urease consists of 12 urea and 12 ureb, and activation of this apoenzyme requires the insertion of 24 nickel ions. urease hydrolyzes urea into carbon dioxide and ammonia, thereby neutralizing the nearby environment [79 ]. [ni - fe ] hydrogen - uptake hydrogenase contains a heterobimetallic center in the large subunit with nickel coordinating to four cysteines, and permits respiratory - based energy production for the bacteria in the mucosa [10, 11 ]. therefore, h. pylori needs significant amounts of nickel to satisfy its requirement for the maturation of the nickel - containing enzymes. two kinds of high - affinity, nickel - specific uptake systems are found in h. pylori : atp - binding cassette (abc)-type transporter (abc abcd) and nickel - cobalt permease (nixa) [13, 14 ]. however, when excess nickel ions accumulate, they exhibit toxic effects and thus inhibit bacterial growth [1517 ]. h. pylori has developed a system to maintain nickel homeostasis, keeping a balance between the import, intracellular storage, and export of nickel ions. analysis of the h. pylori genome sequence has discovered several putative ion binders and membrane transporters involved in metal homeostasis. however, the related nickel - interacting proteins have not been fully identified so far. immobilized - metal affinity chromatography (imac) is a separation technique commonly used for fractionating proteins based on their different binding affinities of the surface - exposed amino acids towards immobilized metal ions [19, 20 ]. ni belongs to the group of intermediate metal ions, preferring coordination to nitrogen, oxygen, and sulfur, and especially favors binding to the proteins with two exposed vicinal histidines. elution of the target proteins is achieved by lowering the ph or by adding a competing reagent such as imidazole. two - dimensional gel electrophoresis (2-de) is a common choice for separating cellular proteins first by their isoelectric point (pi) and then by molecular weight (mw). however, low - abundance proteins may not be detected in 2-de due to its limited separation capacity. imac may compensate this, in some way, by specifically serving to enrich the low - abundance metal - binding proteins and to reduce protein complexity. in this study, proteomic technology was employed for the first time to isolate and identify candidate proteins involved in nickel transport, storage, and utilization in h. pylori, by integrating the powerful tools of ni - imac, 2-de, and matrix - assisted laser desorption time - of - flight mass spectrometry (maldi - tof ms). those proteins with surface active coordinating residues for binding nickel (and maybe other metals with similar coordinating features) the information obtained from the identification and functional analysis of these nickel - related proteins may improve our understanding of nickel homeostasis in bacteria. xia (department of medicine, the university of hong kong), and cultured in the basal medium, brucella broth (difco) with 5% (v / v) fetal bovine serum (fbs ; gibco / brl life technologies), for 72 hours with orbital shaking (100 rpm) at 37c in an anaerobic jar with a microaerobic gas - generating kit (oxoid). solutions used in this study were prepared with ultra - pure water (18.2 m ; millipore). h. pylori 11637 cells grown in 100 ml liquid media to mid - log phase were pelleted at 8000g for 5 minutes at 4c, and washed three times with 10 ml ice - cold phosphate buffered saline (pbs). cell pellets were resuspended in 10 ml of ice - cold buffer a (20 mm sodium phosphate buffer, 500 mm nacl, 10 mm imidazole, 1 mm pmsf, ph 7.4). bacteria were ruptured with sonication in the presence of 1% v / v triton x-100. proteins were recovered (10,000g, 30 minutes), and the supernatant was filtered through a 0.45 m cellulose acetate syringe filter (iwaki glass, japan) before being loaded onto a house - made, buffer a - equilibrated ni - nta agarose column (0.5 ml, qiagen). after washing with 10 column volumes of buffer a, proteins were eluted with buffer b (20 mm sodium phosphate buffer, 500 mm nacl, 500 mm imidazole, ph 7.4). the fractions were concentrated with centricon ym-3 (millipore), and buffer - exchanged into rehydration solutions (8 m urea, 4% chaps, 1 mm pmsf, 20 mm dtt, 2% pharmalyte ph 311) with plusone 2-d clean - up kit (amersham biosciences, buckinghamshire, uk). protein concentrations were determined by bca assay (bio - rad, calif, usa) using bsa as the standard. 2-de was carried out with an ipgphor ii isoelectric focusing (ief) unit and hoefer se 600 ruby electrophoresis unit (amersham biosciences) according to the method detailed elsewhere. briefly, 100 g of h. pylori protein samples were diluted in rehydration solutions containing traces of bromophenol blue. ief was carried out with precast 13-cm immobiline drystrip (ipg strips ; amersham biosciences) to generate a nonlinear ph gradient of 310. following ief, strips were immediately used for the second dimensional sds - page (10). proteins were visualized with silver stain, and protein molecular weight (mw) was calibrated with prestained sds - page marker (broad range ; bio - rad). to minimize gel to gel variations the silver - stained gels were scanned (image scanner ; amersham biosciences) and analyzed with imagemaster 2d elite software (amersham biosciences). the normalized intensity (ni) for each protein spot was calculated as the ratio of the spot intensity versus the sum of intensities of the spots present in the whole gel. protein spots were cut out from the silver - stained gels and enzymatically digested overnight with sequence grade porcine trypsin (promega) at 37c. the masses of the digested peptides were determined with a voyager - de str biospectrometry workstation (applied biosystems, calif, usa). protein identification was performed by searching the ncbinr protein database using protein prospector (http://prospector.ucsf.edu) (3), with pyroglutamic acid modification of n - terminal glutamine, oxidation of methionine, and protein n - terminal acetylation as permissible modifications. the criteria for searching were set at 50 ppm or better mass accuracy, at least four matching peptide masses, as well as theoretical mw and isoelectric point (pi) matching to the estimated values from gels. to investigate nickel binding proteins in h. pylori under physiologically relevant conditions, a relatively mild and nondenaturing lysis method was applied prior to nickel - imac (ni - imac) loading. our previous experiments have shown 1-d gels for the total extract of h. pylori 11637 cells and extract fractions eluted from a ni - imac column, displaying many protein bands for both cases. we have now further separated and identified nickel - interacting proteins in h. pylori using 2-de. the gel image of whole cell extracts of h. pylori 11637 (see figure 1(a)) has a protein distribution very similar to the standard proteome pattern of h. pylori 26695. more than 800 protein spots were separated, with molecular weights ranging from 6 to 200 kda and pi values from 3 to 10. figure 1(a) shows a representative gel image of ni - related proteins in h. pylori 11637. the ratio between the number of ni - binding proteins and total extracted proteins of whole cell lysates was 1:57, indicating that nickel specifically binds to a restricted number of proteins. qualitative comparison of 2-d gels between the ni - enriched eluates (see figure 1(a)) and the total extracts (see figure 1(b)) revealed remarkably different spot patterns. ni - binding protein spots were excised, trypsin - digested, and subjected to maldi - tof ms sequencing. mass spectra of tryptic peptides were used to identify proteins by matching to the spectra of the protein sequence databases, and the identification was further verified by comparing to the standard proteomic map of h. pylori. in total, twenty two proteins were identified as candidate ni - interacting proteins, as marked in figure 1(a) and summarized in table 1. some of the protein spots could not be annotated due to no substantial ms signals or inadequate peptide coverage for confident identification. interestingly, five of the proteins identified, including urea, hspb, fumarase, pfr, and hypothetical protein jhp0301, show more than one spot in the 2-de map, indicating the presence of posttranslational modifications. protein isoforms typically present themselves as a series of spots that differ slightly in their molecular masses and pi, that is, numerous closely spaced spots in 2-de profiles [2628 ]. five proteins (hspa, hspb, fumarase, tsaa, and napa) were also identified in the bi - interacting profile, indicating that there was some correlation between bi and ni interactions. the ni - interacting proteins can be classified into five general functional categories : cellular processes (hspa, hspb, tsaa, and napa), enzymes (urease, fumarase, guab, cad, ppase, and dmpi), membrane - associated proteins (om jhp1427 and hpaa), iron storage protein (pfr), and hypothetical proteins (hp0271, hp jhp0216, hp jhp0301, hp0721, hp0614, and hp jhp0118). nickel homeostasis is required for the establishment of h. pylori infection in animals, necessitating a balance in the nickel import, storage, and delivery for the synthesis and activity of nickel - dependent metalloenzymes. consequently, an analysis to identify the nickel - interacting proteins in h. pylori may be able to reveal candidate proteins for further characterization and validation to provide drug targets. in order to live in the acidic gastric environment, h. pylori continuously synthesize urease catalyzing the hydrolysis of urea to ammonia and carbamate to elevate ph. both subunits of urease were observed in the 2-de gel after ni - nta enrichment, suggesting each of them can bind nickel ions (see figure 1(a)). expression of active urease is essential for h. pylori infection in all animal models and for acid survival in vitro. urease activity is significantly enhanced in the presence of nickel, additional incorporation of ni into the apoenzyme is thus the major regulating event upon higher ni availability. heat shock proteins are a group of highly conserved, abundantly expressed proteins with protective advantage through their functions as molecular chaperones, assisting proper folding of a number of substrate proteins that are otherwise destined to aggregation [30, 31 ]. urease activity was increased four folds in e. coli when coexpressed with hspa, suggesting that hspa possibly plays an important role in the activation of urease, probably by means of its ni - binding domain in the c - terminus. hspa has two distinct ni - binding sites : a high - affinity site (kd 2.8 m) and a lower affinity site (kd 30 m). the h. pylori groel homologue hspb belongs to the hsp60 family, and has been shown to increase the risk of gastric carcinoma, possibly by directly inducing the hyperproliferation of gastric cells. hspb also was suggested to be responsible for the protection and regulation of urease activity. many identified ni - interacting proteins in h. pylori are involved in antioxidant, antitoxic, and antiinflammation machinery. pylori tsaa is a major component of the thioredoxin - dependent thiol - specific antioxidant (tsa) system that catalyzes the reduction of hydroperoxides and peroxynitrite. the cleavage of tsaa suppresses the protecting response of h. pylori cells against oxidative stress in two possible ways : directly by the way of nucleophilic attacking the peptide bond through metal - bound hydroxide ions, and/or indirectly by way of stimulating the activities of specific proteases. it was named because of its ability to mediate neutrophil adhesion to endothelial cells, and to bind to mucin and neutrophil glycosphingolipids. napa was identified as a 150 kda dna - binding dodecamer that protects cells from dna damage caused by free radicals under oxidative stress [39, 40 ]. napa was found to be positively regulated by iron, repressed by ferric uptake regulator (fur), and unaffected by copper, nickel, or zinc. an excess of iron is potentially harmful as it catalyzes the formation of toxic reactive oxygen species (ros) via fenton chemistry. ferritin protein pfr, the major iron storage protein of h. pylori, is also regulated by iron, nickel, zinc, and copper. the accumulation of this protein under iron - rich conditions allows h. pylori to maximize the iron storage capacity in response to an increase in iron availability. it is essential for the bacteria to adapt to low - iron conditions [4245 ]. obviously, ni - imac is also able to trap pfr with its cation binding affinity. immobilized - nickel affinity chromatography and proteomic analysis were integrated to separate and identify the ni - interacting proteins in h. pylori. the results suggest that ni - interacting proteins are mainly involved in cellular processes, oxidative stress, and metabolism of the bacteria. this study demonstrated that metalloproteomic technique can be utilized to efficiently identify metal - related proteins that may play crucial roles in metal homeostasis. these proteins may be potential targets for designing and constructing drugs to suppress the bacterial infection.	helicobacter pylori (h. pylori) is a widespread human pathogen causing peptic ulcers and chronic gastritis. maintaining nickel homeostasis is crucial for the establishment of h. pylori infection in humans. we used immobilized - nickel affinity chromatography to isolate ni - related proteins from h. pylori cell extracts. two - dimensional gel electrophoresis and mass spectrometry were employed to separate and identify twenty two ni - interacting proteins in h. pylori. these ni - interacting proteins can be classified into several general functional categories, including cellular processes (hspa, hspb, tsaa, and napa), enzymes (urease, fumarase, guab, cad, ppase, and dmpi), membrane - associated proteins (om jhp1427 and hpaa), iron storage protein (pfr), and hypothetical proteins (hp0271, hp jhp0216, hp jhp0301, hp0721, hp0614, and hp jhp0118). the implication of these proteins in nickel homeostasis is discussed.
the 2010 human influenza a (h1n1) virus pandemic seriously affected many countries, including kuwait. in children, respiratory involvement usually occurs with h1n1 ; extra pulmonary problems are not common.1 liver involvement is rare and needs early identification and treatment. a 9-year - old child was admitted with intermittent low - grade fever, cough, vomiting, and abdominal pain lasting for one week. he received oral antibiotics ; the fever subsided initially but reappeared after a few days, along with jaundice. he was previously healthy, with no past history of liver disease. on examination, he was alert and oriented, his temperature was 39c, he was icteric, appeared toxic, was sweating, had a respiratory rate of 30 breaths / minute, and had congested tonsils ; a respiratory system exam showed prolonged expiration with expiratory rhonchi. there were no signs of meningeal irritation, and the rest of his physical examination was unremarkable. investigations revealed a hemoglobin (hb) count of 14.5 g / dl, a total leukocyte count of 3.37 10, neutrophils 19%, lymphocytes 68%, a platelet count of 255 10. urinalysis showed mild urobilinogen and ketones, but a urine culture was sterile after 48 hours of incubation. mmol / l, alanine amino - transferase was 1763 u / l, aspartate amino - transferase was 1871 units / l, alkaline phosphatase was 246 units / l, and gamma glutamyl transferase was 107 units / l. g / l, serum ammonia count 74 mmol / l, and serum lactate 2.06 mmol / l. the patient had normal serum amylase and lipase levels, a negative cold agglutinin test, a normal ultrasound of the abdomen, and a negative chest x - ray. nasal and throat swabs for h1n1 were positive by a reverse transcription polymerase chain reaction (pcr) test. a hepatitis a, b, and c serological screen was negative, and his serum acetaminophen level was normal. furthermore, an additional work - up to rule out other causes of fulminant liver failure was performed, including negative blood tests for herpes simplex virus pcr, adenovirus pcr, epstein his immunoglobulins (igg, igm, and iga) were within normal limits ; he also had a negative antinuclear antibody (ana) < 1:40 titer and a negative anti - smooth muscle antibody and anti - liver kidney microsomal antibody (anti - lkm), ruling out the possibility of autoimmune hepatitis. finally, his serum amino acid and urine organic acids were unremarkable. he was treated according to centers for disease control and prevention (cdc) guidelines2,3 with tamiflu (oseltamivir ; genentech, san francisco, ca) for five days and other supportive measures, including fresh frozen plasma, iv - administered vitamin k, lactulose, and prophylactic intravenous antibiotics. severe infection is characterized by pneumonia, sepsis, septic shock, and multi - organ failure. extra - pulmonary involvement is rare in uncomplicated human infections.1 studies of mouse models suggest multiple organ localization, including the lungs, heart, thymus, liver, and spleen.4 snchez - torrent reported h1n1 encephalitis in a 3-month - old infant from spain.5 hepatic involvement is not frequent and accounts for less than 3% of all cases.1 carrillo - esper, in 2010, reported two adult h1n1 patients with hepatic involvement.1 el - shabrawi, in 2011, reported a 10-month - old child with acute myocarditis and fulminant hepatic failure associated with h1n1.6 the subject of the current case report had acute hepatic failure that presented as jaundice, elevated liver enzymes, and coagulopathy. most of the other causes of liver failure had been ruled out by relevant investigations. he responded well to antiviral and other supportive treatment, and showed full clinical and laboratory recovery. no viral replication is needed to produce hepatic damage, as there is evidence of hepatic oxidative stress and a decrease in antioxidant defenses even when the virus is isolated only from the lungs. this might be explained by the production of pro - inflammatory cytokines in the respiratory airway that leads to changes in hepatic metabolism and enzymatic activities.7,8 even though hepatic complications are rare in pediatric h1n1 cases, in reporting this case we would like to draw the attention of pediatric health care professionals to the importance of early recognition, focused investigations, diagnosis, and treatment of complicated human h1n1 infection.	liver involvement in pediatric influenza a (h1n1) infection is rare. focused clinical evaluation and laboratory tests can rule out or identify hepatic complications early on. here we report on a 9-year - old boy treated by the gastroenterology, hepatology, and nutrition unit of al - adan hospital s pediatric department. the patient, who was infected with h1n1 during the 2010 pandemic, showed symptoms of associated acute hepatic failure, was managed conservatively, and recovered completely following treatment. the author would like to draw the attention of pediatricians to the hepatic aspect of human h1n1 infection in order for them to recognize it early and treat it in a timely manner.
milk is the major source of income generated on dairy farms, and over the past several decades, milk production by dairy cows has increased markedly. however, this improvement comes at the cost of higher incidences of reproductive health problems and reduced fertility. large energy requirements at the onset of lactation in high - producing dairy cows results in a severe negative energy balance during the early lactation period, which may adversely impact postpartum health and fertility. as a result of milk production quotas, reductions in production costs can allow dairy farms to maintain profitability. for example, the costs associated with raising replacement heifers have been estimated to be approximately 20% of the overall dairy herd operating cost. moreover, involuntary culling is caused mainly by reproductive failure. thus, more attention is now being given to reproductive health management ; but correlations between cow parity, milk yield, nutritional status, periparturient health, and fertility require clarification in korean dairy herds. the objectives of this study were to determine the effect of parity on milk production, body condition change during early lactation, periparturient health, culling due to reproductive failure, and reproductive performance in korean dairy herds. all herds contained 50 or more cows and received regular reproductive health checkups every 2 to 4 weeks from veterinarians at the college of veterinary medicine, chungbuk national university. the cows were maintained in free - stall facilities and fed a total mixed ration, based on brewer 's grain, alfalfa hay, cotton seed, beet pulp, sweet sorghum, tall fescue, oat hay, and additives. data were collected from 1,290 calvings in eight dairy herds from october 2000 to may 2005. data were collected on milk yield, body condition score, cow parity, calving condition, disease occurrence, culling due to reproductive failure, and reproductive status. body condition was scored using standard procedures based on a scale of 1 to 5, as described by edmonson.. abnormal partus included ; dystocia (veterinary - assisted calving or pulling with extreme force), caesarean section, twins, or stillbirth. the postpartum reproductive and metabolic disorder definitions used in this study are similar to those used in previous studies. retained placenta was defined as the retention of the fetal membrane for > 24 h. metabolic disorders (abomasal displacement, milk fever, or ketosis) were diagnosed by clinical signs observed by a veterinarian and/or the farmer concerned within 4 weeks postpartum. abomasal displacement was diagnosed by a pinging sound upon abdominal auscultation by a veterinarian, and all cases were corrected by surgery. milk fever was diagnosed by the presence of the following clinical signs : weakness, cold skin, and a favorable response to calcium therapy. ketosis was defined as the presence of the following clinical signs ; anorexia, depression, and an odor of acetone in breath. endometritis was diagnosed at 4 weeks postpartum by examination by the corresponding author, by the presence of the following clinical signs ; cloudy discharge and an enlarged uterus observed by rectal examination with or without other clinical signs. ovarian cysts were diagnosed from 4 to 16 weeks postpartum by repeated ultrasonographic examinations at 2 to 4 week intervals (sonoace 600 with a 5.0 mhz linear - array transducer ; medison, korea). ultrasonographic evaluations were based on ovarian structures of larger than 25 mm internal diameter with a wall less than 3 mm thick (follicular cyst) or with a wall greater than 3 mm thick (luteal cyst) in the absence of a normal corpus luteum. cows diagnosed with ovarian follicular cysts beyond 8 weeks postpartum were treated with 100 g fertirelin acetate (gnrh ; conceral, korea) and cows diagnosed with luteal cysts were treated with 25 mg pgf2 (lutalyse ; pharmacia & upjohn, belgium). the cows with endometritis were treated with one intrauterine infusion of 1500 mg oxytetracycline hydrochloride solution (metrijet 15 ; intervet, u.k.) or 2% povidone - iodine solution (korea pharma, korea), and retreated if necessary. the voluntary waiting period from calving to first artificial insemination (ai) established for this study was 50 days. the conception to ai ratio were determined per rectum 60 to 70 days after ai by both ultrasonographic observation and manual palpation. reproductive performance data were collected for a minimum of 7 months postpartum or until pregnancy or culling. parity in these herds was categorized as 1, 2, 3, 4, or 5 or higher. body condition score changes versus parity from calving until months 5 of lactation were also compared by anova. the occurrences of periparturient disorders and culling rate among the various parities were evaluated using the chi - square test or fishers ' exact test. all herds contained 50 or more cows and received regular reproductive health checkups every 2 to 4 weeks from veterinarians at the college of veterinary medicine, chungbuk national university. the cows were maintained in free - stall facilities and fed a total mixed ration, based on brewer 's grain, alfalfa hay, cotton seed, beet pulp, sweet sorghum, tall fescue, oat hay, and additives. data were collected from 1,290 calvings in eight dairy herds from october 2000 to may 2005. data were collected on milk yield, body condition score, cow parity, calving condition, disease occurrence, culling due to reproductive failure, and reproductive status. body condition was scored using standard procedures based on a scale of 1 to 5, as described by edmonson.. abnormal partus included ; dystocia (veterinary - assisted calving or pulling with extreme force), caesarean section, twins, or stillbirth. the postpartum reproductive and metabolic disorder definitions used in this study are similar to those used in previous studies. retained placenta was defined as the retention of the fetal membrane for > 24 h. metabolic disorders (abomasal displacement, milk fever, or ketosis) were diagnosed by clinical signs observed by a veterinarian and/or the farmer concerned within 4 weeks postpartum. abomasal displacement was diagnosed by a pinging sound upon abdominal auscultation by a veterinarian, and all cases were corrected by surgery. milk fever was diagnosed by the presence of the following clinical signs : weakness, cold skin, and a favorable response to calcium therapy. ketosis was defined as the presence of the following clinical signs ; anorexia, depression, and an odor of acetone in breath. endometritis was diagnosed at 4 weeks postpartum by examination by the corresponding author, by the presence of the following clinical signs ; cloudy discharge and an enlarged uterus observed by rectal examination with or without other clinical signs. ovarian cysts were diagnosed from 4 to 16 weeks postpartum by repeated ultrasonographic examinations at 2 to 4 week intervals (sonoace 600 with a 5.0 mhz linear - array transducer ; medison, korea). ultrasonographic evaluations were based on ovarian structures of larger than 25 mm internal diameter with a wall less than 3 mm thick (follicular cyst) or with a wall greater than 3 mm thick (luteal cyst) in the absence of a normal corpus luteum. cows diagnosed with ovarian follicular cysts beyond 8 weeks postpartum were treated with 100 g fertirelin acetate (gnrh ; conceral, korea) and cows diagnosed with luteal cysts were treated with 25 mg pgf2 (lutalyse ; pharmacia & upjohn, belgium). the cows with endometritis were treated with one intrauterine infusion of 1500 mg oxytetracycline hydrochloride solution (metrijet 15 ; intervet, u.k.) or 2% povidone - iodine solution (korea pharma, korea), and retreated if necessary. the voluntary waiting period from calving to first artificial insemination (ai) established for this study was 50 days. the conception to ai ratio were determined per rectum 60 to 70 days after ai by both ultrasonographic observation and manual palpation. reproductive performance data were collected for a minimum of 7 months postpartum or until pregnancy or culling. parity in these herds was categorized as 1, 2, 3, 4, or 5 or higher. body condition score changes versus parity from calving until months 5 of lactation were also compared by anova. the occurrences of periparturient disorders and culling rate among the various parities were evaluated using the chi - square test or fishers ' exact test. during the study period, the average percentages of cows with parities of 1, 2, 3, 4, or 5 or higher were 31.1, 27.9, 20.5, 11.8, and 8.7%, respectively. mean 305 day milk yield increased (p 0.05, fig. 5). the data presented here demonstrate that advances in parity increase the risk of periparturient disorders and the incidence of culling due to reproductive failure in dairy herds. we suggest that severe loss and delayed body condition recovery due to increased milk yield during the early lactation period are responsible. in the present study, mean 305 day milk yield increased with parity, which is consistent with previous reports. body condition loss during early lactation, which reflects a negative energy balance, was found to be aggravated by parity, as has been reported by others. the inverse correlation between body condition score and increased parity during early lactation is presumed to be related to milk yield, which is also consistent with previous results, and demonstrates that body condition scores, which reflect the amount of fat mobilized during early lactation, decrease as milk yield increases. it has been reported that excessive lipid mobilization from adipose tissue, noted even in clinically normal cows during early lactation, may be linked to higher incidences of periparturient health disorders. our finding of increased risks of a retained placenta and metabolic disorder in association with parity have been reported by others. metabolic disorder is influenced by increased milk yield and with a severe loss and delayed recovery of body condition during early lactation, and these are clearly and positively correlated with parity. moreover, the correlation between an increased risk of endometritis and advancing parity, found in the present study, is in agreement with a previous study but contradicts another. this latter study found that the risk of endometritis is highest in first parity cows. on the other hand, an increased risk of endometritis may result from over - fattening and an increased rate of stillbirth in first parity cows. however, our data do not support this hypothesis, and we regard our finding of an increased risk of a retained placenta in high parity animals as an important finding. moreover, profound periparturient impairment of neutrophil function associated with advancing parity might increase susceptibility to endometritis. the increased risk of abnormal partus in cows with a parity of 4 was mainly caused by an increased occurrence of twins in this parity group, although the reason for this higher incidence was not clarified. in has been reported that the incidence of ovarian cysts is lower in cows with a parity of 1 than in other parities. an increased incidence of ovarian cysts in animals with a higher parity may be related to high milk yield, and a clear relationship between milk yield and the incidence of ovarian cysts has been reported on a number of occasions. taken together, these results suggest that increased milk yield and concurrent severe body condition loss during early lactation elicit a severe energy deficit, which may be related to the occurrence of postpartum reproductive (ovarian cysts) and metabolic diseases. the major reason for involuntary culling is reproductive failure. our finding that culling rates due to reproductive failure increase with advancing in parity is consistent with previous studies. moreover, postpartum reproductive diseases can affect fertility and cause delayed conception, which may indirectly lead to culling. likewise, in our study, the increased incidence of postpartum diseases associated with parity may have a reduced fertility and led to an increased need for culling. in the report of seegers., cows culled for reproductive disorders early in their lives (parity 1 or 2) were high - yielding cows that were presumed to have had a negative energy balance during the early lactation period, a condition that is exacerbated in young and/or high - producing cows. in fact, increased culling due to reproductive failure associated with parity increases might be attributed to a series of body stresses including high milk yield, concurrent exaggerated body condition loss, as well as to postpartum metabolic and reproductive diseases during the lactation period. the mean intervals from calving to first service and conception for cows with different parities were no different. however, the relationship between parity and fertility is difficult to determine because of the confounding effect of culling under farm conditions. older cows are reportedly less likely to conceive, although we did not find this. our correlation between fertility and parity may be explained by our observation that culling rates increased with parity increases. however, the reproductive performance of cows culled was not included in this study. in conclusion, this study shows that increases in parity increase milk yield, body condition loss during early lactation, and the risk of periparturient disorders and of culling due to reproductive failure in dairy herds.	the objectives of this study were to determine the effects of parity on milk production, body condition change, periparturient health, and culling in korean dairy herds. the data utilized included ; milk yield, body condition score, cow parity, calving condition, periparturient disorders, culling, and reproductive status, which were recorded from 1290 calvings in eight dairy herds. the mean milk yield in cows over 305 days increased with increasing parity (p < 0.01). cows with parities of 3, 4, and 5 or higher lost more body condition than those with a parity of 1 during month 1 of lactation (p < 0.01), and body condition recovery by cows with parities of 4 and 5 or higher was slower (p < 0.01) than recovery by cows with parities of 1, 2, or 3 until month 3 of lactation. the risk of retained placenta, metabolic disorder, and endometritis also increased with advancing parity (p < 0.05). moreover, the incidence of ovarian cysts was lower in cows with a parity of one than in cows with greater parities (p < 0.01). culling rate due to reproductive failure also increased with advancing parity (p < 0.01). these results suggest that parity increases milk yield, body condition loss during early lactation, the risk of periparturient disorders, and culling due to reproductive failure in dairy herds.
tuberculosis is the most common human immunodeficiency (hiv) virus - related opportunistic infection in india and caring for patients with both diseases is a major public health challenge. it is estimated that 60 - 70% of hiv - positive persons will develop tuberculosis in their lifetime. approximately, 50% of adult indian population is infected with mycobacterium tuberculosis, and the spread of hiv infection could lead to a potentially explosive increase in the number of cases of tuberculosis. about 1.8 million new cases of tuberculosis are occurring annually in india, whereas the pool of hiv - infected individual is quite large (~2.5 million). therefore, there is always a propensity for deadly synergic interactions between hiv and tuberculosis. hiv infection is the most important known risk factor that favors progression to active tb from latent infection by suppressing the immune response against tuberculosis. exogenous reinfection can also occur as hiv - infected individuals fail to contain new infections. the world health organization (who) reported in 2007 that the african region accounted for most hiv - positive tuberculosis cases (79%), followed by the southeast asia region (mainly india), which had 11% of total cases. although the prevalence of hiv infection among patients with tuberculosis ranges from 50% to 80% in many settings in sub- saharan africa, in other parts of the world it varies from 2% to 15%. studies from india have reported hiv seropositivity rates varying from 0.4 to 20.1%. on the other hand, approximately 50% of the hiv - infected people in india are coinfected with m. tuberculosis and approximately 200,000 of these coinfected persons will develop active tb each year in association with hiv infection. unlike other opportunistic infections, tuberculosis can occur at any stage of hiv disease, and its manifestations depend largely on the degree of immunosuppression. twenty - five to 65% of hiv - infected persons have been reported to have active tb of one organ or the other in developing countries. despite the burden of hiv, the epidemiology of tuberculosis in india is being primarily driven by the non - hiv tb - infected pool. effective implementation of ongoing revised national tb control programme is expected to markedly change the number of new tb cases occurring at any level of hiv prevalence in the country. the aim of the present study is to record the clinical, radiological profile of pulmonary and extrapulmonary tuberculosis (eptb) in hiv - seropositive persons. it was a prospective study, done over a period of 1 year (from 1st june, 2008 to 31 may, 2009) in the department of medicine at indira gandhi medical college (i.g.m.c), shimla. hospital, shimla and on follow - up at antiretroviral treatment (art) center were evaluated. hiv infection was diagnosed using three antigenically different rapid kits as per the national hiv testing policy (elisa / rapid / simple) and cd4 cell counts were determined by flow - cytometry technique using facs count machine with facscount reagents (becton dickinson, usa). art was started for eligible patients and was guided by baseline and 6-monthly cd4 counts in accordance with the national art guidelines. following investigations were done to establish the diagnosis of tuberculosis : ziehl - neelsen (z.n) of acid fast bacilli (afb) from given specimen or culture if indicated.histopathological demonstration of typical caseous granulomatous reaction.suggestive clinical profile and empirical response to antitubercular therapy (att).radiological features suggestive of tubercular lesions.pleural/ascitic fluid analysis showing evidence of lymphocytic exudative effusion and csf showing lymphocytic pleocytosis with hypoglycorrhachia (low csf glucose). ziehl - neelsen (z.n) of acid fast bacilli (afb) from given specimen or culture if indicated. pleural / ascitic fluid analysis showing evidence of lymphocytic exudative effusion and csf showing lymphocytic pleocytosis with hypoglycorrhachia (low csf glucose). hiv infection is the strongest of all known risk factors for the development of tuberculosis. of the 87 patients included in our study, males were affected more than females (65.51% males and 34.48% females). the most common affected age group in our study was 31 - 40 years and mean age of the patients was 34.94 year (range 22 - 56 years). unprotected heterosexual contact with professional sex workers was found as the most common mode of hiv transmission. fever, weight loss and cough were the commonest symptoms on presentation [table 1 ]. clinical features of neurological involvement were present in 22 (25.28%) patients with mean duration of 1 week prior to diagnosis. majority of patients with neurological complaints presented with headache and altered sensorium. beside tuberculosis, anemia was detected in 40 patients (45.97%) and esr was raised in 56 (64.36%) patients. showing common symptoms in 87 patients chest x - ray was done in all the 87 patients and pulmonary infiltrates was the commonest finding [table 2 ]. majority of patients with spleenomegaly had hypoechoic lesions in spleen. showing chest x - ray findings computed tomography scan (ct scan) of head was done in three patients and all of them had radiological features suggestive of meningitis. magnetic resonance imaging (mri) of brain done in four patients revealed infarcts and features of meningitis in two patients. two patients had evidence of cns tuberculoma and one had hydrocephalous along with cns tuberculoma. forty - three (49.42%) of them had cd4 cell count 100 cells/l and 71 (81.60%) had cd4 cell count 200 cells/l and 16 had cd4 count > 200 cells/l. mean cd4 cell count was 123 cells/l (in males 119 cells/l and in females 129 cells/l). pulmonary tuberculosis (ptb) majority of patients with pulmonary and eptb had cd4 count below 200/cumm (81.08% and 86.15% respectively). sputum smear - negative tuberculosis was diagnosed on the basis of clinical symptomatology, chest x - ray findings, raised esr and response to antitubercular chemotherapy. commonest form of eptb in our study was cns tuberculosis detected in 22 (33.84%) patients followed by abdominal tuberculosis in 17 (26.15%) patients. commonest form of eptb in female patients was abdominal tuberculosis and in male patients was cns tuberculosis. tubercular meningitis was the commonest manifestation of cns tuberculosis noticed in 20 (90.90%) patients. analysis of cerebrospinal fluid showed pleocytosis (mean white cell count 204/cmm with mean lymphocyte percentage of 65.61%), hypoglycorrhachia (mean glucose level, 33.14 mg / dl) and mean protein level of 187 mg / dl. all patients with meningitis were immediately started on att along with steroids after cryptococcal meningitis being ruled out by india ink staining. commonest form of abdominal tuberculosis was tubercular lymphadenopathy followed by spleenic tuberculosis and ascites, respectively. imaging studies by usg and ct scan abdomen, analysis of ascitic fluid and response to att were the criteria used for diagnosis of abdominal tuberculosis. ten out of 11 patients with hypoechoic lesions in spleen on usg had cd4 count below 200 cells/l. axillary lymphadenopathy was the commonest lymph node involvement present in 16 patients followed by cervical lymphadenopathy in 12 patients. fourteen patients had tubercular lymphadenopathy which was diagnosed on the basis on fine needle aspiration cytology (fnac) and lymph node biopsy. ziehl - neelson staining for afb was positive in eight out of 14 patients (57.14%) with lymph node tuberculosis. rest of the patients were diagnosed on the basis of granulomatous lymphadenitis and response to att. table 3 depicts the distribution of isolated pulmonary and eptb. all patients with disseminated tuberculosis had cd4 cell count less than 200/cumm. this observation is statistically significant (p - value < 0.042). showing distribution of pulmonary and extrapulmonary tuberculosis recently, hiv estimate for india has been revised to less than half ; however, most of this drop is not due to an actual decrease in hiv burden but due to availability of more reliable population - based data. tuberculosis is the most common opportunistic infection in hiv - positive persons and may develop at any stage of the disease. maximum patients in our study were in the age group of 31 - 40 years (68.96%) and majority (65.61%) were males. commonest finding noted in chest radiography was pulmonary infiltrates. other features are pleural effusion, cavity, military shadows and fibrotic changes. twenty - one (63.63%) patients had abnormality in upper zone and 12 patients (36.36%) had lower and mid - zone involvement. other studies also reported higher prevalence of eptb of 53 - 63% of total tuberculosis cases in hiv - infected patients, and was seen more frequently in severely immunocompromised hiv patients. overall 71 out of 80 (81.60%), seven patients with all form of tuberculosis had cd4 count below 200/cumm. this study showed that the prevalence of tuberculosis, both pulmonary and eptb, in hiv - infected patients is significantly higher with majority having cd4 cell count < 200/cmm. commonest form of eptb in our study group was cns tuberculosis diagnosed in 22 (33.84%) patients followed by abdominal tuberculosis in 17 (26.15%), peripheral tubercular lymphadenitis in 14 (21.53%) and pleural effusion in 12 (18.46%) patients. brig s k sharma in a study eptb in hiv - positive patients noted lymph node involvement as the most common eptb site followed by spleen. the difference in our study could be because of adequate inclusion of hospitalized immunocompromised patients. many of our patients were admitted with meningoencephalitis, pleural effusion, organomegaly and on investigations found to be immunocompromised. hence our study is more representative as it has both in hospital and community based data. fourteen patients in our study group had tubercular lymphadenitis which was diagnosed on basis on fnac and lymph node biopsy. zn staining for afb was positive in eight out of 14 (57.14%) patients with lymph node tuberculosis. the diagnostic role of fnac and afb smear examination has also been reported by artenstein and fine needle aspiration is more reliable in patients with hiv infection because of the higher mycobacterial burden, and should be the initial diagnostic procedure in these patients. tubercular coinfection is common in hiv infected and more so with falling cd4 cell level. the salient features of our study were higher prevalence of eptb in hiv - infected patients unlike many other indian studies. commonest eptb was cns tuberculosis unlike most other indian studies where either lymphnode or pleura were the commonest site of involvement. in hospital mortality in patients of cns tuberculosis was lower in our study perhaps due to high index of suspicion, prompt diagnosis and early institution of treatment. there is a definite role of fnac in diagnosis of tubercular lymphadenitis in hiv - positive patients. early diagnosis of tuberculosis and prompt institution of att reduces mortality and morbidity significantly. in resource poor areas, the diagnosis can be established with cytological / biochemical analysis of fluid, histopathological examination and zn staining of tissue coupled with radiological features and response to att. therefore, adequate knowledge of the manifestations of tuberculosis in hiv - infected patients is absolutely necessary for optimal management and to reduce mortality and morbidity.	background : hiv / aids pandemic is responsible for the resurgence of tb worldwide, resulting in increased morbidity and mortality. hiv and mycobacterium tuberculosis have a synergistic interaction ; each propagates progression of the other. coinfection with hiv infection leads to difficulties in both the diagnosis and treatment of tuberculosis, increase risk of death, treatment failure and relapse.objective:the aim of the present study is to study the clinical, radiological profile of pulmonary and extrapulmonary tuberculosis (eptb) in hiv - seropositive patients and their relationship to cd4 counts.materials and methods : it was a prospective study conducted over a period of 1 year in the department of medicine, indira gandhi medical college, shimla. we examined 87 hiv - infected patients with associated tuberculosis recruited from the department of medicine and antiretroviral center and were subjected to thorough clinical examination, x - ray chest, tuberculin testing and sputum examination for afb and necessary relevant investigations for eptb.results:most common affected age group was 31 - 40 years. eptb is the commonest form of tb in our study detected in 65 patients. commonest eptb was cns tuberculosis. disseminated tuberculosis was only found in patient with cd4 count less than 200/cmm. majority of lymph node tb was diagnosed by fine needle aspiration cytology (fnac) examination. all patients with afb - positive lymph node had cd4 count below 200/cum.conclusions : the results of this study provide information regarding the various forms of tb and their presentation in hiv - infected persons. early diagnosis of tuberculosis and prompt institution of antitubercular treatment (att) reduces mortality and morbidity significantly. in resource - poor areas, the diagnosis can be established with cytological / biochemical analysis of fluid, histopathological examination and zn staining of tissue coupled with radiological features and response to att. therefore, adequate knowledge of the manifestations of tuberculosis in hiv - infected patients is absolutely necessary for optimal management and to reduce mortality and morbidity.
total knee arthroplasty (tka) improves joint function in patients with painful joint disease and reduces pain12). the clinical outcomes of tka have improved over time, and the 10-year revision - free survival rate has been reported to range from 96% to 98%345). however, tka has been found to induce the most severe postoperative pain among all orthopedic surgical procedures, with severe pain during the acute phase thought to cause chronic pain67). postoperative methods for analgesia after tka include epidural anesthesia, local infiltration analgesia (lia), and femoral nerve block (fnb)8910). although epidural anesthesia has good analgesic effects, it limits the use of anticoagulants against lower limb deep venous thrombosis and can lead to adverse events, such as nausea and vomiting. although both have shown favorable postoperative analgesic effects, they have not been directly compared. moreover, the analgesic effect of these procedures may not be consistent. lia and fnb differ in their effects on these nerves because lia involves an injection into the entire knee joint, whereas fnb has analgesic effects only in the femoral nerve area. in this study, under the hypothesis that the analgesic effect of lia would be superior to that of fnb, we compared the analgesic effects of lia and the single - shot fnb after tka and attempted to identify factors associated with their analgesic effects. this study was approved by the kyoto prefectural university of medicine review board (erb - c-3 - 2). the subjects consisted of 66 patients (72 knees ; 15 knees in 14 males and 57 knees in 52 females) who underwent tka for osteoarthritis of the knee at our hospital between december 2012 and august 2014. patients were excluded if they had a primary constrained prosthesis, rheumatoid arthritis, osteonecrosis, valgus knee, or underwent surgery with epidural anesthesia. patients undergoing surgery on odd days received lia and those who underwent surgery on even days received fnb. lia, consisting of 5 mg of morphine, 150 mg of 0.75% ropivacaine, 0.3 mg of epinephrine, and 4 mg of betamethasone diluted in 50 ml of saline, was administered by the operator after completion of all femoral and tibial osteotomy steps, immediately before cement fixation of the implant. the lia solution was injected into the tissues around the joint, i.e., the posterior articular capsule of the knee joint, patellar retinaculum, subcutaneous tissue, and pes anserinus. using an electric stimulator, single - shot fnb, consisting of 150 mg of 0.75% ropivacaine, was administered by an anesthesiologist under echo guidance to the inguinal region above the femoral nerve upon completion of surgery. general anesthesia was induced by administration of propofol and fentanyl and maintained with sevoflurane and fentanyl. surgery was performed using the posterior stabilized (ps)-type model of nexgen lps - flex (zimmer, warsaw, in, usa) or scorpio nrg (stryker, mahwah, nj, usa) and the parapatellar approach. as a tourniquet has been reported to reduce early postoperative pain, a tourniquet was applied immediately before fixation of the prosthesis until completion of skin sutures11). a drainage tube was placed in the knee joint at surgery and removed two days later. on the day after surgery, early postoperative ambulation, range of motion training, and quadriceps muscle training were performed in response to pain. whenever possible, parallel bar walking was started on postoperative day 4. cane assisted walking was permitted about 2 weeks after surgery and stair climbing at 3 weeks after surgery. the rescue analgesic consisting of 25 or 50 mg of diclofenac, depending on pain severity, was administered as a suppository. pain was evaluated from the day of surgery to 5 days after surgery using a visual analogue scale (vas). the pain vas consisted of a line, 10 cm in length, on which patients indicated current pain from 0 (no pain) to 10 (worst possible pain). other parameters recorded included times (days) to 90 of knee joint flexion, parallel - bar walking, and t - cane walking ; range of motion (flexion and extension) of the knee joint at the time of discharge ; length of hospital stay ; and adverse events. as one study reported that almost all patients with severe pain had vas scores of > 54 mm, with a mean score of 75 mm12), patients with postoperative vas scores of < 54 for 5 days after surgery were designated as the low vas group, and those with vas scores of 54 at least once were designated as the high vas group. age, sex ratio, body mass index, preoperative vas, preoperative femorotibial angle (fta), preoperative range of motion of the knee joint (flexion and extension angles), and preoperative knee score were compared within each group (lia and fnb) between patients with vas scores of < 54 and those with vas scores of 5413). the fta was defined as the lateral angle between the femoral axis and the tibial axis. intraoperative factors compared included operation time, defined as the time from skin incision to the end of wound closure, and blood loss (intraoperative drainage), as measured with gauze and a suction tube. outcomes in the lia and fnb groups were compared using mann - whitney u tests for vas ; amount of diclofenac sodium used ; numbers of days to achieve 90 knee flexion, initiation of parallel - bar walking, and t - cane walking ; and range of motion (flexion and extension) of the knee joint at the time of discharge. patient characteristics, operation time, and blood loss were compared in the low and high vas groups using mann - whitney u - test. factors independently associated with high vas were determined by a stepwise multivariate logistic regression analysis. the available data were assessed by a power analysis with an alpha level of 0.05. all statistical analyses were performed using ibm spss ver. 21.0 (ibm co., armonk, ny, usa), with p<0.05 defined as statistically significant. measurements were expressed as meanstandard deviation. outcomes in the lia and fnb groups were compared using mann - whitney u tests for vas ; amount of diclofenac sodium used ; numbers of days to achieve 90 knee flexion, initiation of parallel - bar walking, and t - cane walking ; and range of motion (flexion and extension) of the knee joint at the time of discharge. patient characteristics, operation time, and blood loss were compared in the low and high vas groups using mann - whitney u - test. factors independently associated with high vas were determined by a stepwise multivariate logistic regression analysis. the available data were assessed by a power analysis with an alpha level of 0.05. all statistical analyses were performed using ibm spss ver. 21.0 (ibm co., armonk, ny, usa), with p<0.05 defined as statistically significant. pain vas scores were similar between the lia and fnb groups on days 05 after surgery (fig. the mean dosage of diclofenac per patient was significantly lower in the lia group than in the fnb group (3445 mg vs. 5645 mg ; p=0.048) (table 1). times to achieve 90 flexion of the knee joint (2.71.3 days vs. 2.31.2 days ; p=0.19), to initiate parallel - bar walking (4.11.3 days vs. 3.91.0 days ; p=0.57), to start t - cane walking (13.63.9 days vs. 13.94.0 days ; p=0.75), and to discharge (36.06.2 days vs. 34.66.9 days ; p=0.39) were similar between the lia group and fnb group. assessment of ranges of motion at discharge showed similar results between the two groups for extension (2.03.1 vs. 3.44.5 ; p=0.14) and flexion (124.611.3 vs. 124.87.4 ; p=0.95). adverse events in the lia group included nausea in five patients and itching and chest discomfort in one each. adverse events in the fnb group included nausea in three patients and numbness of the region innervated by the femoral nerve in two. no patient in either group experienced a serious adverse event, such as respiratory inhibition, infection, nerve palsy, or local anesthetic intoxication. the low vas group with a high analgesic effect included 37 knees : 25 treated with lia and 12 with fnb ; whereas the high vas group with a low analgesic effect included 35 knees : 19 treated with lia and 16 with fnb (p=0.24). the demographic and clinical characteristics of these two groups are summarized in tables 2 and 3. a comparison of the lia - treated patients with low and high vas scores showed significant differences in sex ratio and preoperative range of extension (table 4). preoperative vas score was similar in the lia group with low and high postoperative vas scores (47.131.1 vs. 46.130.1 ; p=0.93). of these lia - treated patients, two males and 23 females had low vas scores, and six males and 13 females had high vas scores (p=0.04). the preoperative ranges of extension in these two subgroups were 5.2 and 9.0, respectively (p=0.03), whereas their postoperative ranges of extension were 1.8 and 2.4, respectively (p=0.66). a comparison of the fnb - treated patients with low and high postoperative vas scores showed no significant differences in any demographic or clinical factors. the available data were assessed by a power analysis with the power ranging from 5.0% to 54.3% (table 5). the objective of this study was to compare the analgesic effects of lia and single - shot fnb after tka and to retrospectively examine factors influencing these analgesic effects. the dosage of diclofenac was significantly lower in the lia group than in the fnb group. male gender and preoperative limited range of extension were associated with high postoperative vas scores in the lia group, indicating that the postoperative analgesic effects of lia were smaller in patients with more severe flexion contracture before surgery and in male than female patients. tka is a superior reconstruction method for knee joint function, and long - term outcomes have improved and stabilized over time. evaluations of the postoperative outcomes of tka have included both long - term survival of the implant as well as patient satisfaction. patient satisfaction has been reported lower after tka than after total hip arthroplasty due to postoperative pain141516). several recent studies evaluating the analgesic effects of lia and fnb have reported conflicting results, with one finding that the analgesic effect of lia was superior and the other finding that fnb was superior1718). this study found equivalent pain vas scores in the two groups, whereas analgesic dosage was lower in the lia group than in the fnb group, suggesting that lia has a greater analgesic effect than fnb. the superior analgesic effect of lia may be due to the inaccessibility of fnb to the sciatic nerve area. when combined with sciatic nerve block, however, lia and fnb provided similar pain relief after tka19). these findings suggest that lia has analgesic effects on areas of both the femoral and sciatic nerves. severe preoperative pain in females and in young patients were found to predict pain after tka20), but intraoperative factors were not evaluated. we observed no significant differences in intraoperative factors between groups of patients with low and high vas scores. although analgesic effect was lower in male than in female patients, analgesia was not significantly associated with age or preoperative pain. in agreement with previous results, we found that pain after tka was more severe in female than in male patients21). preoperative limitation of extension was significantly more severe in patients with high than low preoperative vas scores who underwent lia. after surgery, however, extension angle improved to within 5 in both groups, with no significant difference between groups. these findings suggest that postoperative pain increases as severe preoperative flexion contracture improves after surgery, thus reducing the effect of lia. flexion contracture has been reported to be caused by contracture of the posterior cruciate ligament (pcl), posterior femoral osteophyte, posterior articular capsule, and musculotendinous tissue, such as the hamstring22). we used ps - type implants in all knees and resected the pcl during surgery. the posterior femoral osteophyte was resected when osteotomy was performed on the posterior femoral malleolus, followed by dissection of the posterior articular capsule. thus, it is unlikely that these treatments contributed to the difference between the two groups in postoperative analgesic effect. however, musculotendinous tissue posterior to the knee joint, such as the hamstring, was not treated surgically. in patients with severe preoperative flexion contracture, these tissues were likely stretched markedly after surgery, resulting in severe postoperative pain and reducing the analgesic effect. this study showed that the dosage of analgesics used after surgery was lower in the lia than in the fnb group and that it is necessary to increase analgesia after tka in male patients and in patients with knees showing flexion contracture. a cadaver study found that injection of 60 ml india ink infiltrated into posterior knee joint tissue23). modification of the analgesic method, such as an increase in the volume of posteriorly injected lia, may be necessary for knees with flexion contracture. primarily, it was underpowered to detect a statistically significant difference between the effects of lia and fnb. a comparison of the analgesic effects of lia and fnb after tka found that the dosage of concomitantly administered analgesics was lower in the lia group, suggesting that lia was superior to fnb as analgesia after tka. the postoperative analgesic effect of lia was lower in patients with more severe flexion contracture before surgery and in male than in female patients.	purposethis study compared the analgesic effects of local infiltration analgesia (lia) and femoral nerve block (fnb) after total knee arthroplasty (tka) and assessed factors associated with analgesia obtained by these two methods.materials and methodsstudy subjects included 66 patients (72 knees) who underwent tka for osteoarthritis of the knee. pain visual analogue scale (vas), the amount of analgesics used, number of days to achieve 90 of flexion of the knee joint, date of initiating parallel - bar walking, range of motion of the knee joint at discharge, and adverse events were investigated.resultsthe vas scores did not differ significantly between two groups, whereas the amount of analgesics used was significantly lower in the lia group. preoperative flexion contracture was significantly more severe in the lia group with high vas compared with low vas. no serious adverse event occurred in the lia or fnb group.conclusionsthe lower analgesic usage in the lia group than the fnb group indicates that the analgesic effect of lia was greater than that of singleshot fnb after tka. there were no serious complications in either group. the postoperative analgesic effect of lia was smaller in patients with severe than less severe preoperative flexion contracture.
after decades of neglect, the search for novel, effective antimalarials has been revived primarily due to emergence of drug resistance. widespread resistance has been reported not only for 8-aminoquinolines but recent reports indicate the appearance of increased parasite tolerance to artemisinin analogues, potentially predicating clinical resistance. the absence of highly efficacious malaria vaccines highlights the importance of developing small - molecule drugs. with support from the wellcome trust and medicines for malaria venture (mmv), the ngbs consortium (the novartis institute for tropical diseases (nitd), the genomics institute of the novartis research foundation (gnf), the biomedical primate research centre (bprc), and the swiss tropical and public health institute (stphi)) was formed and set up to discover new antimalarials which are effective against multidrug resistant parasite strains here we report our continued effort to optimize a series of imidazolopiperazine compounds in search of a preclinical candidate. as described previously, an extensive sar study on the three peripheral parts had yielded decent in vitro and in vivo antimalarial activities as well as favorable physiochemical and pharmacokinetic properties. our efforts toward the first - generation compounds led to candidates (like compound 1) with double - digit nanomolar potency and moderate oral exposure in mice. however, most potent compounds with an unsubstituted piperazine had poor oral exposure which resulted in inferior in vivo efficacy when administered orally, for example, compounds 28 and 31 (scheme 1) in the previous communication (figure 1). we were intrigued by the possibility of further refining the core structure of the molecule to further improve the properties. our hypothesis was further reinforced by chemical stability and mouse in vitro metabolic stability results which led to the identification of two biologically inactive metabolites (1a and 1b) arising from the oxidation of piperazine ring. our plan was to substitute three possible positions (positions 5, 6, and 8) to evaluate the corresponding effects on potency as well as a means to improve oral exposure. central piperazine ring oxidation is a major metabolic and chemical instability site in compound 1. on the basis of the designed molecules, it was clear that previous synthetic routes were not amenable for the desired compounds because we could not utilize the 3 + 2 cyclization. distinct syntheses were designed and executed to obtain substitution at different positions of a piperazine ring. the synthesis started with an alkylation reaction between cbz - protected glycine derivative 2 and bromide 3. adduct 4 was then refluxed with nh4oac in toluene to give imidazole 5 with removal of water using a dean stark apparatus. regioselective n - alkylation with ethyl 2-bromoacetate followed by a one - pot deprotection / cyclization sequence furnished the lactam core, which was reduced by borane to furnish amine 8. the quaternary -carbon did not pose a significant amount of steric hindrance for the hatu mediated amidation reaction with n - boc - glycine. amide 9 was obtained at room temperature in decent yield and further was brominated to provide intermediate 10. then a standard buchwald hartwig amination reaction with substituted aniline followed by deprotection finished the synthesis of compound 12 with the desired alkyl modification at position 8. reagents and conditions : (a) k2co3, dmf, rt, 84% ; (b) nh4oac, toluene, reflux, 88% (c) ethyl 2-bromoacetate, cs2co3, dmf, rt, 83% ; (d) pd / c, h2 (balloon), meoh, rt, 91% ; (e) bh3thf, thf, reflux, 95% ; (f) hatu, diea, ch2cl2, rt, 70% ; (g) br2, acoh, ch2cl2, rt, 100% ; (h) arnh2, pd2(dba)3, xantphos, cs2co3, dioxane, 150 c, 89% (for example 22, ar = 4f - ph and r1 = r2 = me) ; (i) tfa, ch2cl2, rt, 52% (for example 22, ar = 4f - ph and r1 = r2 = me). scheme 3 describes the synthesis of 6,6-dimethyl analogues. analogous chemistry to compound 12 was used for the first two steps to provide imidazole 5a. the compound was regioselectively n - alkylated with methallyl chloride to obtain the key intermediate for the piperazine cyclization step. the intramolecular hydroamination reaction proved to be difficult, and after extensive screening, treating the substrate 5a with a 5:1 mixture of acetic acid and methanesulfonic acid at 210 c produced the cyclized core with simultaneous loss of the carbamate protecting group. despite the harsh reaction condition, an acceptable yield of the product (39%) was obtained and the regiochemistry was confirmed by 2d nmr of the intermediate. the cyclized core was subjected to amide coupling with n - boc glycine to yield intermediate 13. the final steps in the sequence are analogous to scheme 2 to furnish analogue 14. the sequence involved six steps from intermediate 5a with an overall yield of 3.7%. the synthesis of the 5,5-dimethyl modified analogues diverged from their 8,8-dimethyl modified counterparts at intermediate 7a. n - pmb protection was necessary for lactam 7a before the nah - mediated bis - methylation at the 5-position (scheme 4). the bicyclic intermediate was then deprotected and amine was subjected to a hatu mediated amidation to obtain compound 7c. the final sequence involved bromination followed by palladium - catalyzed aniline amination and boc - deprotection to furnish 5,5-dimethyl modified compound 26. the sequence involved eight steps from intermediate 7a with an overall yield of 10.9%. reagents and conditions : (a) methallyl chloride, k2co3, ki, dmf, rt, 55% ; (b) acoh / msoh (6:1), 210 c, 39% ; (c) n - boc - glycine, hatu, diea, dmf, rt ; 57% ; (d) br2, acoh, ch2cl2, rt ; 55% ; (e) arnh2, pd2(dba)3, xantphos, cs2co3, dioxane, 150 c ; (f) tfa, ch2cl2, rt, 55% over 2 steps (for example 25, ar = 4f - ph). reagents and conditions : (a) pmbcl, koh, dmf, 0 c to rt, 63% ; (b) mei, nah, dmf, rt, 86% ; (c) bh3thf, thf, reflux, 100% ; (d) tfa, 70 c, quantitative ; (e) n - boc - glycine, hatu, tea, dmf, rt, 49% ; (f) br2, acoh, ch2cl2, 90% ; (g) arnh2, pd2(dba)3, xantphos, cs2co3, dioxane, 150 c ; (h) tfa, ch2cl2, rt, 46% over 2 steps for example 26 where ar = 4f - ph. we began exploring the sar on the piperazine ring by substituting at the 8-position first, and we decided to use 4-f - aniline and 3-f-4-cl aniline based on our previous work at the r6 position (table 1). adding a methyl group to the 8-position led to loss of potency by 10-fold (cmpd 17), but the compound was still equipotent to compound 15 (scheme 5). moreover, there was no profound effect on the potency by changing the absolute configuration of the methyl group. increasing the size of substituent to isopropyl group led to dramatic loss in potency (examples 19 and 20). a dimethyl group on the 8-postion not only led to more favorable compound in terms of completely blocking the benzylic carbon as indicated by our metabolite studies but also removing the stereogenic carbon. surprisingly, substitution of 3cl-4f aniline in the 8-disubstitution case provided a less potent compound when compared to other anilines (21 vs 22, 23, or 24). emboldened by the increase in the potency based on the substitution on the 8-position, we decided to study the effect of dime group on the ethylene carbons of the piperazine ring. on the basis of examples 25 and 26, it was abundantly clear that the dimethyl group on the 8-position had the most significant positive effect on potency. the substitution on the other positions of the piperazine ring only provided modest potencies when compared to 8-substitution, with activities in the double - digit nanomolar range. as the 5-substituted compounds and the 6-substituted compounds were less potent, we decided to focus our attention on the effect of the amide substituent of the piperazine nitrogen with the 8,8-dimethyl group constant. our previous efforts demonstrated that dimethyl glycine had slightly inferior potency but offered superior oral exposure. in the case of the substituted piperazines, installation of methylalanine amide provided equipotent compounds (15 vs 27, 22 vs 28, and 23 vs 29). we proceeded to further understand the overall effect of the dimethylpiperazine on the minimal pharmacophore. after studying the effects of various substitutions on the piperazine ring and obtaining a significant improvement in activity, we decided to reconfirm the minimal pharmacophore needed for antiplasmodial activity. on the basis of the earlier results, we were able to achieve antiplasmodial activity with ec50 in 160400 nm range for the core devoid of the amino acid substituent on the piperazine nitrogen (compound 31, table 2). modification of the piperazine ring into the corresponding lactam led to a 7-fold decrease in activity in the micromolar range (32). however, the antiplasmodial activity is regained by introduction of dimethyl substituent on the 8-position with a 7-fold improvement (33). the most optimal compound from this lactam subseries was a compound with 3cl-4f aniline substitution with an activity around 50100 nm (34). the dimethyl substitution on the reduced piperazine core led to 10-fold improvement in potency (31 vs 35) with compounds to 10 nm range. these results led us to conclude that the amino acid substitution had minimal to no effect on the potency of the compound with substitution on the 8-position of the imidazolopiperazine core and that the basicity of the piperazine nitrogen could be modulated without complete loss in antimalarial activity. in parallel to the sar studies to optimize the potency of the compounds series, we evaluated the in vitro adme and in vivo pharmacokinetic properties of key analogues. in general, this series of compounds had very good lipinski 's rule - of - five compliance and most of the analogues were highly soluble (> 175 m at ph 6.8), with the neutral lactams being least soluble among all the compounds. table 3 summarizes the in vitro adme profiles of the important compounds. typically, a range of values in the permeability assays was observed. in the caco-2 assays, the core piperazine compounds (both lactams as well as nonamide piperazine compounds) exhibited high apical to basal values (31, 32, 34, and 35). the permeability of the compounds was reduced once the side chain amino acid was installed, but metabolic stability is improved (28 vs 35). however, the % absorbed was lower in the pampa assay for compounds with poor permeability in the caco-2 assay. upon comparing the calculated log p values with pampa as well as caco-2 values unsurprisingly, log p of 4.5 or higher was needed for having excellent percentage absorbed values in pampa assay (see supporting information). removing the basicity of the piperazine ring by introduction of carbonyl group improved the mouse and human microsome stability of the compound, presumably by blocking the ethylene bridge (31 vs 32) but is not evident in rat microsomes. introduction of the dimethyl group on the 8-position of the piperazine ring does not improve overall metabolic stability of the compound (15 vs 28 or 31 vs 35) despite the metabolite identification results presented earlier. this may be due to the ethylene bridge in the piperazine ring representing a metabolic soft spot and enabling metabolic switching. in general, this scaffold exhibited poor rat metabolic stability when compared to other species. one interesting finding with the substituted piperazine analogues was the improvement of activity against the herg channel as determined in the automated patch clamp assay. compared to the unsubstituted piperazine analogue 27, we were able to decrease inhibition of the herg channel by at least 23-fold, with most analogues (such as 22) suggesting a lower potential for qt interval prolongation in compounds with a substituted piperazine. compounds series found to have favorable potency and in vitro adme properties were tested in snapshot pk, an abbreviated pk study shown to accurately bin compounds into low, moderate, or high oral exposure. compounds predicted to have reasonable oral exposure would be tested in mice using both po and iv routes of administration (table 4). the in vivo clearance generally was low to moderate and significantly improved when compared to the corresponding unsubstituted analogues (15, 22). this resulted in higher maximum concentration as well as improvement in auc values in the oral arms as well. lactam compound 34 exhibited highest oral auc than all other analogues, and it also had lower intrinsic clearance with moderate volume of distribution. to test whether red cell partitioning was occurring to explain the high volume of distribution for the basic compounds, we carried out an in vitro experiment in which compound 22 was incubated in nave rat whole blood as well as plasma for 30 min. after centrifuging the blood and quantifying the amount of compound in the red blood cells, we determined that the concentration of the compound was 3.47 times higher in whole blood than in plasma alone, indicating that the compound is either adhering to the rbc s or getting distributed inside the cells. po dosing ; dose (mg / kg), 20 ; species / strain, mouse, male balb / c ; formulation, 2.5 mg / ml in peg300/d5w, 3:1, solution ; vd = volume of distribution ; cl = clearance ; cmax = maximal concentration ; tmax = time at maximal concentration ; auc = area under the curve ; f = oral bioavailability. because we had examples of compounds with different modifications with acceptable bioavailability, we decided to carry on the efficacy experiment in mice. the in vivo antimalarial activities of the optimized compounds were evaluated using a plasmodium berghei mouse survival model. in this test, groups of three p. berghei infected mice were treated orally with compound one day after infection. the key readouts were the percentage of parasitemia reduction on day 3 postinfection compared to the untreated mice and the prolongation of survival of the infected mice. table 5 summarizes the in vivo efficacy test results of compound in comparison with standard antimalarials chloroquine (cq) and artesunate (as). it was observed that compounds 22, 24, and 29 exhibited > 99% parasitemia reduction at a low dose of 30 mg / kg with a modest survival of 15 days or higher. increasing the dose to 100 mg / kg or dosing the compound 3 30 mg / kg did not lead to increase in survival. currently, we are investigating the possibility of recrudescence of parasites or development of resistance for the compound in vivo to better explain the lack of curative activity in this model. we decided to determine the ed99 representative examples for the substituted and nonsubstituted piperazine cores and found the substituted piperazine core was 10-fold more efficacious (compound 22 ed99 = 2.2 mg / kg) than the unsubstituted core (compound 1 ed99 = 26.5 mg / kg). compounds 34 and 36 clearly proved inferior both in terms of parasitemia reduction as well as survival in mice, indicating the need for the amino acid side chain in providing better mouse efficacy. activity = average parasitemia reduction ; survival = average lifespan after infection (67 days for untreated control mice) ; compounds were formulated in 75% peg300/25% d5w (5% dextrose in water) ; cq, chloroquine ; as, artesunate. 10% ethanol, 30% peg400, 6% vitamin etpgs. on the basis of the efficacy results rat was chosen as the second animal species for oral exposure evaluation as it may serve as the in vivo preclinical toxicity species. taking into account the nonoptimal metabolic stability data for rats for this scaffold, we decided to study the oral exposure of the compound in rat for a period of 5 h in the oral arm only to get an estimation of the exposure (table 6). -methylalanine subclass of compounds like 29 did not exhibit any in vivo exposure and were not further pursued. in line with the better rat metabolic stability (er = 0.68), nonbasic lactam compound 34 demonstrated the best rat exposure despite lower mouse efficacy. po dosing ; dose (mg / kg), 20 ; species / strain, rat, wistar ; formulation, 2.5 mg / ml in peg300/d5w, 3:1, solution ; blq, below level of quantification ; na = could not be determined. as the oral exposure of compounds 22 was low in rats but demonstrated superior mouse efficacy, we decided to dose compound 22 at higher doses to make sure that sufficient multiples of exposure of the mouse efficacious dose could be achieved (table 7). we observe that when the dose was increased to 30 mg / kg from 10 mg / kg, there was overproportional 7-fold increase in auc as well as 2-fold increase in bioavailability. increasing the dose further to 100 mg / kg dose with suspension formulation led to 55-fold increase in auc with a 3-fold increase in oral bioavailability. the auc at the mouse efficacious dose (2 mg / kg) was 780 hnm, therefore a 45 exposure multiple is achieved at 100 mg / kg in the rat. the multiple of exposure provides a sufficient window to use the rat as a rodent toxicology species. it is unclear at this time if this overproportional exposure at higher doses is due to saturating metabolism or an efflux transporter, but given the partial efflux observed in caco-2, this may explain this multiple dose exposure results. compound 22 was subjected to a series of assays to evaluate the toxicity profile. this drug candidate was inactive on a panel of human cytochromes p450 (ic50s are greater than 6 m for cyp1a2, cyp2c19, cyp2c9, cyp2d6, and cyp3a4), and the observed cytotoxicity cc50 were all greater than 12 m on a panel of mammalian cell lines (293 t, ba / f3, cho, hep2, hela, huh7), which translates to an adequate selectivity index (si > 100). compound 20 was found negative in the mini - ames and micronucleus tests, which indicated a low mutagenic potential. finally, considering the efficacious plasma cmax in the mouse at 2 mg / kg is low (75 nm), the risk of cardiotoxicity is considered to be minimal where the herg channel inhibitory activity ic50 = 13.4 m, providing a greater than 170-fold window. imidazolopiperazine compounds had been proven to be effective against multidrug resistant parasite strains with minimum toxicity to host - cell lines. however, early lead compound such as compound 15 and 27 still suffer from moderate potencies, relatively poor rat pk, and potent herg inhibitory activity. directed by the identification of in vitro metabolites formed by compound 1, the piperazine core of 15 was systematically modified by a gem - dimethyl functionality at all three possible places. the 8,8-dimethyl analogues demonstrated excellent potency as well as improved oral exposure in the mouse. this also allowed us to use the glycine amide that reduced our herg risk when compared to 27. the first generation of compounds exhibited efficacy that was comparable to the known antimalarials in the p. berghei mouse model, however, compound 22 was more efficacious than chloroquine and artesunate especially when parasitemia reduction is compared in terms of ed99 values. although compound 22 demonstrated poor rat pk at low doses, with higher doses it was able to achieve the necessary exposure multiples calculated based on the plasma concentration found to be efficacious in the mouse model. compound 22 is thus currently under preclinical safety assessment in rats as a potential candidate for human trials. plasmodium falciparum cultures were grown in o rbcs using culturing media : rpmi 1640 media (no phenol red) with l - glutamine and containing 0.05 mg / ml gentamicin, 0.014 mg / ml hypoxanthine, 38.4 mm hepes, 0.20% sodium bicarbonate, 0.20% glucose, 3.4 nm naoh, 5% human serum, and 1.25% albumax. using traditional p. falciparum protocols published previously, 25 ml cultures were maintained in 75 cm flasks (fisher cat. 10 - 126 - 41) at 5% hematocrit and parasitemia ranging between 1% and 10%. once parasitemia reached 810%, cultures are diluted down to 1% or higher depending on needs. cultures were gassed for approximately 30 s to 1 min using a blood gas mixture to maintain a gas composition of 96% nitrogen, 3% carbon dioxide, and 1% oxygen and incubated at 37 c. on day 1 of the assay, the smear was fixed onto the slide by placing in methanol for 30 s, stained in 10% geimsa stain, and the percent of parasitized erythrocytes vs uninfected erythrocytes was determined by microscopy with a light microscope. p. falciparum strains to be used for screening were prepared with screening media (culturing media without human serum but supplemented with 0.5% albumax ii) and fresh erythrocytes. twenty l of screening media were dispensed via the microflo (biotek) into 384-well, black clear - bottom assay plates (clear gnf custom plates by griener bio - one). then 50 nl of compounds were transferred using a platemate plus (matrix) or the gnf systems pintool into the assay plates containing screening media to a final maximum concentration of 10 m in a 12 point dose response (1/2 log serial dilution). on the basis of the measured parasitemia levels, each parasite strain was diluted to yield 0.5% parasitemia, and 50% hematocrit blood (uninfected erythrocytes) was added to a concentration of 4.17%. this diluted culture was used in dispensing the remaining 60% of the assay volume (30 l) into the prewarmed assay plates via the microflo liquid dispenser. the final parasitemia was 0.3% and final hematocrit was 2.5% in a total volume of 50 l. assay plates were incubated at 37 c for 72 h in a gas chamber filled with low oxygen blood gas. after the 72 h incubation (equivalent to 12 cycles during the blood stage), a prepared mixture of the lysis buffer (5 mm edta, 1.6% triton x-100, 20 mm tris - hcl, 0.16% saponin) in water and sybr green detection (0.1%) reagent was dispensed at 10 l per well using the microflo. cultures were incubated for an additional 24 h at 25 c before measuring fluorescence intensity using the envision plate reader (perkin - elmer) with a 505 dichroic mirror. data were normalized based on maximum fluorescence signal values for dmso treated wells (no inhibition by compound) and the minimum fluorescence signal values for wells containing the highest concentration of inhibitor control compounds, for example, pyrimethamine at a final concentration of 10 m. data were analyzed on a plate - by - plate basis and compared to reference compounds that were always included on every plate, typically artemisinin, mefloquine, and pyrimethamine. ec50 values were obtained using a custom curve fitting model, and a standard logistic regression model was applied for curve fitting. the quality of the assay run was assessed by the performance of the reference compounds where the ec50 must be within 3-fold of the standard reference values for the assay plate to pass requisite data quality control needs. additionally, all compounds were typically assayed at a minimum in duplicate (independent assay plates) and ec50 values ideally must not vary by more than 2-fold between plates. the metabolic stability of drug candidates was determined in human, mouse, and rat liver microsomes using the compound depletion approach, quantified by lc / ms / ms. the assay measured the rate and extent of metabolism of chemical compounds by measuring the disappearance of the parent compound. the assay determined the compound s in vitro half - life (t1/2) and hepatic extraction ratios (er) and predicted metabolic clearance in human, rat, and mouse species. the compound concentration was determined by uv absorption measured with a spectramax190 microplate spectrophotometer (molecular devices corporation, sunnyvale, ca, usa) at absorption wavelengths between 260 and 290 nm. the caco-2 assay was carried out in a 96-well format, and compound concentrations in each chamber were measured by lc / ms. the assay offers apparent permeability data of test compounds from the apical to the basolateral chambers [papp (a b) ] and from the basolateral to the apical chambers [papp (b a) ]. the results can be used to predict the in vivo oral absorption (using papp (a b)) and the transport mechanism of the test compounds across gi membrane (ratio of papp (b a):papp (a b) and log p lipophilicity data). the assay utilizes electrophysiology measurement on the electric current passing through herg channel that is heterologously expressed in a stable cho cell line. channels were open by a herg - specific voltage protocol, and the compound effects were directly characterized by their block of the herg current. all compounds were tested by 4-point dose response curves, and ic50 values (between 0.2 and 30 m) were measured. in - life studies were carried out under protocols approved by the animal care and use committee (iacuc) of gnf. all compounds were formulated at a concentration of 2.5 mg / ml in 75% peg300 and 25% d5w (5% dextrose in distilled water) solution and were filtered using a 0.45 m syringe filter. the filtered solutions were dosed intravenously via the lateral tail vein at 5 mg / kg with a dosing volume of 2 ml / kg to male balb / c mice (n = 3 per group). the filtered solutions were also administered orally at 20 mg / kg (with a dosing volume of 8 ml / kg) to another group of mice (n = 3 per group). six blood samples of 50 l each were collected serially from each animal via retro orbital sinus up to 24 h after dosing. blood samples were collected into heparin microtainer and centrifuged and plasma separated and frozen until analysis. compounds were formulated at a concentration of 2.5 mg / ml in 75% peg300 and 25% d5w solution. in rat snapshot pk, two rats (wistar rats) were administrated orally at 10 mg / kg dose and four blood samples (100 l) were taken at 0.5, 1, 3, and 5 h post dosing. before the sample analysis, the plasma samples were pooled based on the sampling time. in the rat full pk studies, the formulations were filtered before the administration to rats. three rats were dosed intravenously (3 mg / kg), and three rats were administrated orally (10 mg / kg). six blood samples (100 l per sample) were collected serially from each rat within 24 h post dosing. plasma samples (20 l) were extracted with solution of acetonitrile : methanol (3:1) containing internal standard. the samples were vortexed and then centrifuged with an eppendorf centrifuge 5810r (eppendorf, hamburg, germany) at a setting of 4000 rpm for 5 min at 4 c. the supernatant was transferred to a clean 96-well analysis plate for lc / ms / ms analysis. an aliquot (10 l) was injected onto a phenomenex c18 guard column (4 mm 2 mm) followed by a zorbax sb - c8 analytical column (2.1 mm 30 mm, 3.5 m, agilent technologies inc., separation was carried out using a gradient elution method with a mobile phase consisting of 0.05% formic acid in water (solvent a) and 0.05% formic acid in acetonitrile (solvent b). the hplc system, consisting of agilent 1100 series binary pump (agilent technologies inc.), agilent 1100 series micro vacuum degasser (agilent technologies inc.), htc pal ctc analytics autosampler (leap technologies, carborro, nc), and vici two - position actuator (valco int., houston, tx, usa) was interfaced to a mds sciex api-4000 triple quadruple mass spectrometer (mds inc., mass spectral analyses were carried out using electrospray source in the positive ion mode and using multiple reaction monitoring (mrm) for quantification. mrm transition of compound 15 383.9/101.2, compound 22 412.2/312.1, compound 24 430.1/330.1, compound 27 412.2/327.2, compound 29 436.1/308.1, compound 34 430.0/346.1, and compound 36 355.0/272.3 was used with its optimized ms parameters, respectively. the mean value from the three animals at each time point was plotted against time to give plasma concentration time profile. pharmacokinetic parameters were determined using watson lims, version 7.2 (thermo electron corporation, pa, usa), by noncompartmental analysis. pharmacokinetic parameters for compounds were calculated by noncompartmental regression analysis using an in - house fitting program developed at gnf. the oral bioavailability (f) was calculated as the ratio between the area under the curve (auc) following oral administration and the auc following intravenous administration corrected for dose (f = aucpo doseiv / auciv dosepo). all in vivo efficacy studies were approved by the veterinary authorities of the canton basel - stadt. in vivo antimalarial activity was usually assessed for groups of three female nmri mice (2022 g) intravenously infected on day zero with 2 10 erythrocytes parasitized with p. berghei gfp anka malaria strain (pbgfpcon donation from a. p. waters and c. j. janse, leiden university). chloroquine and artesunate were formulated in 10% ethanol, 30% peg400, and 6% vitamin e tpgs. compounds were administered orally in a volume of 10 ml / kg either as a single dose (24 h post infection) or as three consecutive daily doses (24, 48, and 72 h post infection). with the single dose regimen, parasitemia was determined (72 h post infection) and for the triple dose regimen (96 h post infection) using standard flow cytometry techniques. activity was calculated as the difference between the mean percent parasitemia for the control and treated groups expressed as a percentage of the control group. the survival time in days was also recorded up to 30 days after infection. a compound was considered curative if the animal survived to day 30 after infection with no detectable parasites. unless otherwise noted, materials were obtained from commercial suppliers and were used without purification. removal of solvent under reduced pressure refers to distillation using bchi rotary evaporator attached to a vacuum pump (3 mmhg). products obtained as solids or high boiling oils were dried under vacuum (1 mmhg). purification of compounds by high pressure liquid chromatography was achieved using a waters 2487 series with ultra 120 5 m c18q column with a linear gradient from 10% solvent a (acetonitrile with 0.035% trifluoroacetic acid) in solvent b (water with 0.05% trifluoroacetic acid) to 90% a in 7.5 min, followed by 2.5 min elution with 90% a. h nmr spectra were recorded on bruker xwin - nmr (400 or 600 mhz). proton resonances are reported in parts per million (ppm) downfield from tetramethylsilane (tms). h nmr data are reported as multiplicity (s singlet, d doublet, t triplet, q quartet, quint quinted, sept septed, dd doublet of doublets, dt doublet of triplet, bs broad singlet), number of protons, and coupling constant in hertz. for spectra obtained in cdcl3, dmso - d6, and cd3od, the residual protons (7.27, 2.50, and 3.31 ppm, respectively) were used as the reference. analytical thin - layer chromatography (tlc) was performed on commercial silica plates (merck 60-f 254, 0.25 mm thickness) ; compounds were visualized by uv light (254 nm). 100c, isco) or using silica gel (merck kieselgel 60, 230400 mesh). the purity and quantitative analysis were determined with a waters zq 2000 lc / ms system, which employed an acquity uplc system (binary pump, column compartment, autosampler, and diode array uv detector). the eluent was split between the mass spectrometer and an antek chemiluminescent nitrogen detector (clnd). the mobile phases used were (a) 95% h2o/5% meoh / ipa (75/25, v / v) + 0.05% formic acid and (b) meoh / ipa (75/25, v / v) + 0.035% formic acid. a gradient hplc method with flow of 0.4 ml / min started at 2% b, with a hold of 1.0 min before a linear increase to 95% b at 3.50 min, with a 30 s hold. from 4.04.25 min, the column used was a thermo syncronis c18 2.1 mm 30 mm, 3 m. the mass spectrometer was operated in positive mode, with a spray voltage of 3.2 kv and cone voltage of 30 v. the source and desolvation temperatures were 130 and 400 c, respectively, with 600 l / h of nitrogen desolvation flow. all the final compounds reported in this communication had the purity of at least 95%. elemental analyses were carried out by midwest microlabs llc, indianapolis, in, usa. compound 1a was prepared from 1 by the following method : to a stirred solution of 1 (12 mg, 0.03 mmol) was added mno2 (52 mg, 0.60 mmol). the reaction mixture was stirred at room temperature (rt) for 2 h. lc / ms test showed that 1 was almost consumed and the desired product ([m + 1 ] = 428) was detected as one of the major peaks. the residue was subjected to a mass - triggered hplc purification to give 1a as well as 1b. h nmr for 1a (400 mhz, cd3od) 7.597.63 (m, 2h), 7.09 (t, j = 6.4 hz, 2h), 6.957.02 (m, 1h), 6.466.52 (m, 1h), 6.346.38 (m, 1h), 5.92 (s, 1h), 4.33 (dd, j = 4.4, 14 hz, 1h), 3.96 (dd, j = 4.8, 13.2 hz, 1h), 3.703.78 (m, 1h), 3.463.54 (m, 1h), 1.73 (s, 3h), 1.36 (s, 3h). cd3od) 7.757.78 (m, 2h), 6.957.01 (m, 3h), 6.446.50 (m, 1h), 6.326.36 (m, 1h), 4.19 (dd, j = 1.6, 6.8 hz, 2h), 4.00 (dd, j = 1.6, 6.8 hz, 2h), 1.47 (s, 6h). to a solution of 2-bromo-1-(4-fluorophenyl)ethanone (46.5 g, 214.29 mmol) in dmf (400 ml) was added 2-(benzyloxycarbonyl)-2-methylpropanoic acid (55.8 g, 236.4 mmol) and potassium carbonate (35.4 g, 256.5 mmol). the resulting solution was extracted with ethyl acetate (2 800 ml), and the combined organic layer was washed with water (2 800 ml) and then brine (1 800 ml). this resulted in 67 g (84%) of compound 4 as a white solid. to a solution of compound 4 (70 g, 187.7 mmol) in toluene (700 ml) was added nh4oac (144.5 g, 1.88 mol). the resulting solution was heated to reflux for 3 h in an oil bath. the resulting solution was extracted with ethyl acetate (2 500 ml), and the combined organic layer was washed with water (2 800 ml) and brine (1 800 ml). this resulted in 58 g (88%) of compound 5 as a white solid. h nmr (300 hz, dmso - d6) 11.91 (s, nh), 7.807.75 (m, 2h), 7.547.12 (m, 8h), 4.97 (m, 2h), 1.6 (s, 6h). to a solution of compound 5 (58 g, 164.3 mmol) in dmf (400 ml) was added cesium carbonate (134 g, 411.0 mmol). this was followed by the addition of ethyl 2-bromoacetate (33 g, 197.6 mmol) dropwise with stirring at rt in 30 min. the resulting solution was extracted with ethyl acetate (2 700 ml), and the combined organic layer was washed with water (2 800 ml) and brine (1 800 ml). the resulting mixture was dried concentrated under vacuum. this resulted in 60 g (83%) of compound 6 as a yellow solid. h nmr (300 hz, dmso - d6) 7.767.72 (m, 2h), 7.327.28 (m, 5h), 7.087.03 (m, 3h), 5.08 (m, 2h), 4.87 (m, 2h), 4.23 (q, j = 5.4 hz, 2h), 2.0 (s, 6h), 1.28 (t, j = 5.4 hz, 3h). to a solution of compound 6 (70 g, 159.45 mmol, 1.00 equiv) in methanol (800 ml) was added palladium on carbon (10 g). this resulted in 7 (38 g, 145.56 mmol, 91%) as a white solid. lc - ms : (es, m / z) [m + h ] calcd for c14h14fn3o, 260 ; found, 260. h nmr (300 hz, dmso - d6) 7.797.74 (m, 2h), 7.137.07 (m, 3h), 6.35 (s, 1h), 4.73 (s, 2h), 1.79 (s, 6h). to a stirred solution of compound 7 (5 g, 19.2 mmol, 1 equiv) was dissolved in 50 ml of thf, 1 m borane / thf complex (57 ml, 57 mmol, 3 equiv) was added slowly and reaction was refluxed overnight. the crude product of 8 (4.5 g, 18.3 mmol, 95%) was used in the next step. lc - ms : (es, m / z) [m + h ] calcd for c14h17fn3 246, found 246. h nmr (dmso, 300 hz) 7.757.70 (m, 2h), 7.4 (s, 1h), 7.14 (t, j = 9.0 hz, 2h), 3.9 (t, j = 5.4 hz, 2h), 2.51 (t, j = 5.4 hz, 2h), 1.41(s, 6h) ; nh proton not observed. to a stirred solution of compound 8 (2.9 g, 11.82 mmol, 1.1 equiv) and 2-(tert - butoxycarbonylamino)acetic acid (2.27 g, 13 mmol, 1.1 equiv) in 15 ml of dichloromethane were added diea (2.47 ml, 14.18 mmol, 1.2 equiv) and hatu (5.39 g, 14.18 mmol, 1.2 equiv). the reaction mixture was stirred at rt for 8 h. hplc / ms analysis showed that desired product compound 9 was the major product. the organic layer was washed with washed with water (1 30 ml), followed by saturated nahco3 (1 30 ml) and finally with brine (1 30 ml). the resulting oil was purified using column chromatography with hexanes / ethyl acetate (0100% linear gradient) used as eluant. the desired product compound 9 was obtained as oil (3.3 g, 8.27 mmol, 70%). h nmr (300 hz, dmso - d6,) 7.777.72 (m, 2h), 7.54 (s, 1h), 7.207.14 (m, 2h), 6.846.80 (m, 1h), 4.07 (s, 2h), 3.90 (d, j = 3.0 hz, 2h), 3.70 (s, 2h), 1.80 (s, 6h), 1.40 (s, 9h). lc - ms : (es, m / z) [m + h ] calcd for c21h28fn4o3, 403 ; found, 403. to a stirred solution of compound 9 (3.02 g, 7.51 mmol) in dichloromethane (30 ml) was added br2 (0.43 ml, 8.26 mmol) in acetic acid (3 ml). hplc / ms test showed that desired product (ii) was the only peak. the product was confirmed by 400 mhz proton nmr to be the title compound 10. the product was used in the next step without further purification and exhibited quantitative mass recovery. lc - ms : (es, m / z) [m + h ] calcd for c21h27brfn4o3, 482 ; found, 482. h nmr (meoh - d4, 400 hz) 7.847.81 (m, 2h), 7.14 (t, j = 8.8 hz, 2h), 4.094.01 (m, 4h), 3.81 (t, j = 4.8 hz, 2h), 1.89 (s, 6h), 1.46 (s, 9h). (s)-2-amino-1-(3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8-methyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 17 was prepared from compound 10 (r1 = me, r2 = h) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline followed by a tfa mediated deprotection in a protocol similar to compound 22. h nmr (meoh - d4, 400 mhz) 7.617.58 (m, 2h), 7.167.12 (m, 1h), 7.077.02 (m, 2h), 6.476.42 (m, 2h), 5.74 (m, 1h), 4.073.68 (m, 6h), 1.56 (d, j = 6.8 hz, 3h). (r)-2-amino-1-(3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8-methyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 18 was prepared from compound 10 (r1 = me, r2 = h) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline followed by a tfa mediated deprotection in a protocol similar to compound 22. (s)-2-amino-1-(3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8-isopropyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 19 was prepared from compound 10 (r1 = isopropyl, r2 = h) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline followed by a tfa mediated deprotection in a protocol similar to compound 22. h nmr (meoh - d4, 400 mhz) 7.617.58 (m, 2h), 7.267.20 (m, 3h), 6.456.38 (m, 2h), 5.5 (d, j = 7.9 hz, 1h), 4.113.75 (m, 6h), 2.362.31 (m, 1h), 1.15 (d, j = 6.7 hz, 3h), 0.95 (d, j = 6.7 hz, 3h). (r)-2-amino-1-(3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8-isopropyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 20 was prepared from compound 10 (r1 = isopropyl, r2 = h) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline followed by a tfa mediated deprotection in a protocol similar to compound 22. h nmr (meoh - d4, 400 mhz) 7.697.67 (m, 2h), 7.277.20 (m, 3h), 6.686.65 (dd, j = 2.5 hz, j = 11.0 hz, 1h), 6.606.55 (dd, j = 2.25 hz, j = 8.7 hz, 1h), 5.78 (d, j = 7.7 hz, 1h), 4.424.02 (m, 4h), 3.943.87 (m, 2h), 2.55 (m,1h), 1.24(d, j = 6.8 hz, 3h), 1.05 (d, j = 6.8 hz, 3h). m / z = 460.2 (m + 1). 2-amino-1-(3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 21 was prepared from compound 10 (r1 = me, r2 = me) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline followed by a tfa mediated deprotection in a protocol similar to compound 22. h nmr (meoh - d4, 400 mhz) 7.597.55 (m, 2h), 7.207.13 (m, 3h), 6.60 (dd, j = 11.2, 10.8 hz), 1h), 6.53 (dd, j = 2.4, 0.8 hz, 1h), 4.0 (m, 4h), 3.78 (m, 2h), 2.00 (s, 6h). elemental analysis (compound + 2.0 hcl + 2.0 h2o) : % c, 47.62 ; % h, 5.09 ; % n, 12.62 ; (calc). % c = 47.54/47.38 ; % n = 12.44/12.46 ; % h = 4.62/4.57 (experimental). 2-amino-1-(2-(4-fluorophenyl)-3-(4-fluorophenylamino)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : in a glass vial, cs2co3, 4f - aniline (0.462 g, 4.1 mmol, 2.0 equiv), pd2(dba)3 (0.095 g, 0.104 mmol, 0.05 equiv), xantphos (0.120 g, 0.208 mmol, 0.1 equiv), and dioxane were stirred for 5 min at rt. compound 10 (1 g, 2.08 mmol, 1.0 equiv) was added to the reaction mixture, after which the reaction mixture was degassed for 15 min and then stirred at 120 c under n2 for 8 h. hplc / ms test showed that the starting material compound 10 was consumed and desired product was formed predominantly along with some 9. the reaction mixture was concentrated and then purified by normal phase column chromatography (silica gel 80 g) using a gradient of 1000% to 0100% hexane : etoac. the organic layer was concentrated at reduced pressure to yield the boc derivative (950 mg, 89%) yield. lc - ms : (es, m / z) [m + h ] calcd for c27h32f2n5o3, 512 ; found, 512. the boc compound was treated with 20% tfa in ch2cl2 (50 ml) and was added to the mixture. after the completion of this reaction (monitored by lcms), the resulting mixture was concentrated under reduced pressure. the resulting residue was purified by reverse phase hplc to yield product as a tfa salt. lc - ms : (es, m / z) [m + h ] calcd for c22h23f2n5o, 412.2 ; found, 412.1. h nmr (meoh - d4, 400 hz) 7.617.57 (m, 2h), 6.94 (t, j = 8.8 hz, 2h), 6.81 (t, j = 8.8 hz, 2h), 6.47 (m, 2h), 3.72 (m, 2h), 3.58 (m, 2h), 3.42 (m, 2h), 1.85 (s, 6h). elemental analysis compound 22 with 0.65 equiv h2o : c, 62.44 ; n, 16.55 ; h, 5.79 (calculated). c = 62.54/62.44 ; n = 16.35/16.29 ; h = 5.52/5.61 (experimental). the regiochemistry of the dime groups was unambiguously assigned using 2-d nmr techniques (table 8). 2-amino-1-(2-(4-fluorophenyl)-8,8-dimethyl-3-(p - tolylamino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 23 was prepared from compound 10 (r1 = me, r2 = me) by a pd2(dba)3 mediated amination reaction with 4-methylaniline followed by a tfa mediated deprotection in a protocol similar to compound 22. h nmr (meoh - d4, 400 mhz) 7.797.69 (m, 2h), 7.21 (t, j = 8.4 hz, 2h), 7.04 (d, j = 8.0 hz, 2h), 6.76 (d, j = 8.2 hz, 2h), 4.11 (s, 2h), 3.88 (m, 4h), 2.22 (s, 3h), 2.12 (s, 6h). lc / ms major mass : 408.1 (m + h). 2-amino-1-(3-((3,4-difluorophenyl)amino)-2-(4-fluorophenyl)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 24 was prepared from compound 10 (r1= me, r2 = me) by a pd2(dba)3 mediated amination reaction with 3,4-difluoroaniline followed by a tfa mediated deprotection in a protocol similar to compound 22. h nmr (meoh - d4, 400 mhz) 7.737.69 (m, 2h), 7.21 (t, j = 8.8 hz, 2h), 7.067.03 (m, 1h), 6.766.65 (m, 1h), 6.536.51 (m, 1h), 4.084.06 (m, 4h), 3.863.83 (m, 2h), 2.07 (s, 6h). elemental analysis (compound + 2.9 hcl) : % c, 49.37 ; % h, 4.69 ; % n, 13.09 ; (calc). % c = 49.43/49.06 ; % n = 12.89/12.77 ; % h = 4.7/4.81 (experimental). 13 was prepared from compound 5a by the following way : to a stirred solution of compound 5a (1.1 g, 3.38 mmol, 1.00 equiv) in dmf (30 ml) was added 3-chloro-2-methylprop-1-ene (500 mg, 5.49 mmol, 1.50 equiv), potassium carbonate (560 mg, 4.06 mmol, 1.10 equiv), and potassium iodide (1.12 g, 6.75 mmol, 2.00 equiv) at rt. the mixture was washed with brine (3 10 ml), dried over sodium sulfate, and concentrated under vacuum. the residue was applied onto a silica gel column with petroleum ether / etoac (5:1) to give 0.7 g (55%) of compound 5a-1 as a light - yellow solid. m / z = 380 (m + 1). into a 30 ml sealed tube, was placed compound 5a-1 (2.0 g, 5.28 mmol, 1.00 equiv), acetic acid (12 ml), and methanesulfonic acid (2 ml). the reaction mixture was stirred for 12 h at 260 c (the temperature of the sand bath). the aqueous layer was washed with ethyl acetate (3 10 ml). the aqueous layer was extracted with ethyl acetate (3 10 ml). the combined organic layers were washed with brine (3 10 ml), dried over sodium sulfate, and concentrated under vacuum. the solid was collected by filtration and washed with 5 ml of n - hexane to give 500 mg (39%) of compound 5a-2 as a white solid. h nmr (400 mhz, cdcl3) 7.707.65 (m, 2h), 7.046.9 (m, 2h), 6.96 (s, 1h), 4.12 (s, 2h), 3.69 (s, 2h), 1.23 (s, 6h). to a stirred solution of compound 5a-2 (280 mg, 1.14 mmol, 1.0 equiv) in dmf (10 ml) was added 2-(tert - butoxycarbonyl)acetic acid (600 mg, 3.43 mmol, 3.0 equiv), hatu (1.3 g, 3.42 mmol, 3.0 equiv), and diea (880 mg, 6.82 mmol, 6.0 equiv) at rt. the organic layer was washed with brine (3 10 ml), dried over sodium sulfate, and concentrated under vacuum. the residue was applied onto a silica gel column with ch2cl2/meoh (10:1) to give 280 mg (57%) of compound 13 as a brown solid. h nmr (400 mhz, cdcl3) 7.727.68 (m, 2h), 7.23 (s, 1h), 7.137.07 (m, 2h), 5.46 (s, nh), 4.65 (s, 2h), 4.054.04 (m, 2h), 3.97 (s, 2h), 1.551.39 (m, 15h). the structure of 13 was verified by hmqc, cosy, hmbc, and roesy (table 9). 2-amino-1-(2-(4-fluorophenyl)-3-(4-fluorophenylamino)-6,6-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : to a stirred solution of compound 13 (386 g, 0.96 mmol) in 6 ml of dichloromethane was added br2 (55 l, 1.06 mmol) in acetic acid (2 ml). after neutralization, the residue was subjected to flash chromatography (40 g, 0100% ethyl acetate in hexane, 50 min, dry loading) purification to give 256 mg (55%) of the title compound 13a as a colorless oil. h nmr (400 mhz, cdcl3) 7.877.83 (m, 2h), 7.03 (t, j = 8.8 hz, 2h), 5.46 (s, 1h), 4.5 (s, 2h), 3.97 (d, j = 4.2 hz, 2h), 3.81 (s, 2h), 1.45 (s, 6h), 1.39 (s, 9h). example 25was obtained from 13a by a pd2(dba)3 mediated amination reaction with 4-fluoroaniline followed by a tfa mediated deprotection by analogy to compound 22 (55% yield over 2 steps). h nmr (400 mhz, dmso - d6) : 8.06 (m, 2h), 7.98 (m, 1h), 7.80 (m, 2h), 7.16 (m, 2h), 6.98 (m, 2h), 6.60 (m, 2h), 4.64 (m, 2h), 4.02 (m, 2h), 3.80 (m, 2h), 1.38 (s, 6h). 2-(4-fluorophenyl)-7-(4-methoxybenzyl)-5,5-dimethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine : compound 7b was prepared from compound 7a (r1 = r2 = h) by the following way : to a solution of compound 7a (231 mg, 1.0 mmol) in dmf (10 ml) were added koh (168 mg, 3.0 mmol) and pmbcl (405 l, 3.0 mmol) at 0 c. the reaction mixture was stirred at the same temperature for 2 h and at rt for 2 additional hours. the hplc fraction were evaporated under reduced pressure and neutralized by satd nahco3. the organic layer was concentrated under reduced pressure to give compound 7a-1 (221 mg, 63%) as a white solid. h nmr (400 mhz, cdcl3) 7.677.63 (m, 2h), 7.287.25 (m, 2h), 7.077.02 (m, 2h), 6.896.86 (m, 2h), 4.74 (s, 2h), 4.70 (m, 2h), 4.53 (s, 2h), 3.79 (s, 3h). to a solution of compound 7a-1 (253 mg, 0.72 mmol) in dmf (15 ml) were added 60% sodium hydride (87 mg, 0.085 mmol) and methyl iodide (0.45 ml, 7.2 mmol) at rt. the reaction mixture was stirred at rt for 2 h. the reaction mixture was quenched with methanol and directly subjected to mass - triggered hplc purification to give 7a-2 as a white solid after evaporation of acetonitrile water in vacuo. the tfa salt was subsequently neutralized by saturated sodium bicarbonate solution and extracted with dichloromethane to get the compound as a free base. the organic layer was dried over sodium sulfate and concentrated under reduced pressure to yield the desired compound 7a-2 (234 mg, 86%). the structure of 7a-2 was verified by 2-d nmr techniques (table 10). to a solution of compound 7a-2 (170 mg, 0.45 mmol) in thf (9 ml) was added 1.0 n bh3thf (2.70 ml, 2.70 mmol) at rt. the reaction mixture was stirred at reflux for 2 h. pd / c was added (gas generated). the reaction mixture was stirred for 1 h. solid was filtered off, and solvent was removed. the product was assumed to be of 100% yield and used in the next step without further purification. h nmr (400 mhz, cd3od) 8.1 (s, 1h), 7.747.70 (m, 2h), 7.34 (d, j = 8.6 hz, 2h), 7.27 (m, 2h), 6.94 (d, j = 8.6 hz, 2h), 3.91 (s, 2h), 3.80 (s, 3h), 3.79 (s, 2h), 2.87 (s, 2h), 1.61 (s, 6h). 7-(4-methoxybenzyl)-2-(4-fluorophenyl)-5,5-dimethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine (1.07 g, 2.93 mmol, 1.00 equiv) (7b) was added to trifluoroacetic acid (15 ml). the resulting solution was stirred for 2 h at 70 c in an oil bath. the ph value of the solution was adjusted to 8 with aqueous sodium bicarbonate. 7b-1 was extracted from the aqueous layer using 3 100 ml of ethyl acetate. this resulted in 7b-1 (718 mg, quantitative) as a yellow solid. to a solution of 7b-1 (750 mg, 3.06 mmol, 1.00 equiv) in n, n - dimethylformamide (30 ml) were added 2-(tert - butoxycarbonyl)acetic acid (850 mg, 4.86 mmol, 1.50 equiv), hatu (1.3 g, 3.42 mmol, 1.10 equiv), and triethylamine (650 mg, 6.44 mmol, 2.00 equiv). the resulting solution was stirred overnight at rt. the resulting mixture was washed with brine (3 100 ml), dried over anhydrous sodium sulfate, and concentrated under vacuum. the residue was applied onto a silica gel column with petroleum ether / ethyl acetate (4:1). this resulted in 600 mg (49%) of 7c as a pale - yellow solid. h nmr (300 mhz, cdcl3) : 7.75 (m, 2h), 7.22 (s, 1h), 7.137.07 (m, 2h), 4.994.87 (m, 2h), 4.12 (s, 2h), 3.91 3.65 (m, 2h), 1.57 (m, 6h), 1.43 (s, 9h). to a stirred solution of compound 7b-2 (583 g, 1.45 mmol) in dichloromethane (6 ml) was added br2 (82 l, 1.60 mmol) in acetic acid (2 ml). after neutralization, the residue was subjected to flash chromatography (40 g, 0100% ethyl acetate in hexane, 50 min, dry loading) purification to give 628 mg (90%) of the title compound 7c as a yellow solid. h nmr (400 mhz, cdcl3) 7817.78 (m, 2h), 7.07 (t, j = 8.6 hz, 2h), 5.28 (s, nh), 4.844.7 (m, 2h), 4.073.58 (m, 4h), 1.491.44 (m, 6h), 1.28 (s, 9h). 2-amino-1-(2-(4-fluorophenyl)-3-(4-fluorophenylamino)-5,5-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 26 was prepared from compound 7c by a pd2(dba)3 mediated amination reaction with 4-fluoroaniline followed by a tfa mediated deprotection in 46% yield over 2 steps. h nmr (400 mhz, cd3od) : 7.57 (m, 2h), 7.02 (m, 2h), 6.77 (m, 2h), 6.52 (m, 2h), 4.974.91 (m, 2h), 4.093.74 (m, 4h), 1.56 (s, 3h), 1.51 (s, 3h). 2-amino-1-(2-(4-fluorophenyl)-3-(4-fluorophenylamino)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)-2-methylpropan-1-one : compound 28 was prepared from compound 8 (r1 = r2 = me) in the following fashion using a slightly modified route to the one described in scheme 2. to a stirred solution of compound 8 (1.48 g, 6.04 mmol) and net3 (6.0 g, 59.4 mmol) in dichloromethane (20 ml) was added 2-bromo-2-methylpropanoyl bromide (14 g, 60.9 mmol) dropwise at rt. after being stirred for 3 h at rt, the reaction was then quenched by the addition of water (30 ml). the combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. this resulted in crude product as a dark solid, which was washed with etoac : petroleum ether (1:10) to remove the impurities to produce compound 8 - 1 as a gray solid. (2.0 g, 5.08 mmol, 1.00 equiv) in dmf (10 ml) was nan3 (1.0 g, 15.38 mmol, 3.00 equiv) at rt. the resulting mixture was washed with 3 20 ml of brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. the residue was applied onto a silica gel column with petroleum ether / etoac (5:1) to give compound 8 - 2 as a white solid. m / z 357 (m + h). to a stirred solution of compound 8 - 2 (1.2 g, 3.37 mmol, 1.00 equiv) in methanol (20 ml) was added pd / c (80 mg, 0.75 mmol, 0.20 equiv) at rt. m / z 331 (m + h). to a stirred solution of compound 8 - 3 (910 mg, 2.76 mmol, 1.00 equiv) in thf (50 ml) was added boc anhydride (3.2 g, 14.68 mmol, 5.00 equiv), followed by aqueous naoh (1n, 6 ml, 2.0 equiv) at rt. the organic layer was washed with 3 10 ml of brine, dried over na2so4, and concentrated under vacuum. the solid was collected by filtration and washed with n - hexane (5 ml) to give compound 8 - 4 as a white solid. h nmr (300 mhz, cdcl3) 7.757.71 (m, 2h), 7.097.02 (m, 3h), 4.84(s, nh), 4.094.0 (m, 4h), 1.98 (s, 6h), 1.55 (s, 6h), 1.45 (s, 9h). to a stirred solution of compound 8 - 4 (32 mg, 0.074 mmol) in dichloromethane (3 ml) was added br2 (4.2 l, 0.082 mmol) in acetic acid (1 ml). after neutralization, the residue was subjected to flash chromatography (4 g, 060% ethyl acetate in hexane, 16 min) purification to give the compound 8 - 5 as colorless oil, was collected by filtration, and washed with n - hexane (5 ml) to give compound 8 - 4 as a white solid. h nmr (400 mhz, cdcl3) 7.917.88 (m, 2h), 7.107.04 (m, 2h), 5.11(s, nh), 4.01 (m, 2h), 3.96 (s, 2h), 1.94 (s, 6h), 1.51 (s, 6h), 1.41 (s, 9h). compound 28 was prepared from compound 8 - 5 by a pd2(dba)3 mediated amination reaction with 4-fluoroaniline followed by a tfa mediated deprotection. h nmr (400 mhz, meoh - d4) 7.66 (dd, j = 9.2, 5.2 hz, 2h), 7.11 (t, j = 8.8 hz, 2h), 6.81 (t, j = 8.8 hz, 2h), 6.726.68 (m, 2h), 4.043.99 (m, 4h), 2.01 (s, 6h), 1.66 (s, 6h). 2-amino-1-(2-(4-fluorophenyl)-8,8-dimethyl-3-(p - tolylamino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)-2-methylpropan-1-one : compound 29 was prepared from 35 by the following way : to a stirred solution of 35 (21 mg, 0.06 mmol) and et3n (83 l, 0.60 mmol) in dry dichloromethane (6 ml) was added 2-bromo-2-methylpropanoyl bromide (71 l, 0.60 mmol). the reaction mixture was stirred at rt for 5 h. the reaction mixture was concentrated and subjected to mass - triggered lc / ms purification directly. the obtained solution was concentrated to give 22 mg (73%) of compound 35 - 1as yellow oil after neutralization. h nmr (400 mhz, meoh - d4) 7.717.66 (m, 2h), 7.237.19 (m, 2h), 7.02 (d, j = 8.4 hz, 2h), 6.66 (d, j = 8.4 hz, 2h), 4.31 (m, 2h), 4.09 (m, 2h), 2.21 (s, 3h), 2.06 (s, 6h), 2.00 (s, 6h). to a solution of compound 35 - 1 (22 mg, 0.044 mmol) in dmf (3 ml) was added nan3 (8.6 mg, 0.132 mmol) at rt. the reaction mixture was stirred at rt for 2 h. the reaction mixture was directly subjected to mass - triggered hplc purification directly. the obtained mecn / aqueous solution was combined and concentrated to give 15 mg (75%) of compound 35 - 2 as yellow oil after neutralization. h nmr (400 mhz, meoh - d4) 7.70 7.66 (m, 2h), 7.237.19 (m, 2h), 7.04 (d, j = 8.3 hz, 2h), 6.66 (d, j = 8.4 hz, 2h), 4.26 (m, 2h), 4.074.05 (m, 2h), 2.24 (s, 3h), 2.03 (s, 6h), 1.58 (s, 6h). to a solution of compound 35 - 2 (15 mg, 0.033 mmol) in meoh (3 ml) was added 10% pd / c (4 mg, 0.003 mmol) at rt. the reaction mixture was stirred at rt for 2 h. solid was filtered off, and solvent was removed. the reaction mixture was directly subjected to mass - triggered hplc purification to give 15 mg (100%) of the title compound yellow oil. h nmr (meoh - d4, 400 hz) 7.787.75 (m, 2h), 7.066.97 (m, 4h), 6.56 (d, j = 8.4 hz, 2h), 5.16 (s, nh), 4.33 (t, j = 4.8 hz, 2h), 3.74 (t, j = 4.8 hz, 2h), 2.29 (s, 3h), 1.95 (s, 6h), 1.45 (s, 6h). 2-amino-1-(3-(3,4-difluorophenylamino)-2-(4-fluorophenyl)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)-2-methylpropan-1-one : compound 30 was prepared from compound 8 - 5 by a pd2(dba)3 mediated amination reaction with 3,4-difluoroaniline followed by a tfa mediated deprotection.h nmr (400 mhz, meoh - d4) 7.637.60 (m, 2h), 7.14 (t, j = 8.4 hz, 2h), 7.01 (dd, j = 18.8, 8.8 hz, 1h), 6.686.63 (m, 1h), 6.5 (m, 1h), 4.03 (m, 2h), 4.01 (m, 2h), 2.01 (s, 6h), 1.66 (s, 6h). 2-(4-fluorophenyl)-n-(p - tolyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazin-3-amine : the details of the synthesis involving an ugi reaction between 4-fluorobenzaledehyde, 2-aminopyrazine, and 1-isocyano-4-methylbenzene has been reported in the previous communication.h nmr (400 mhz, meoh - d4) 7.73 (d, j = 6.8 hz, 2h), 7.006.99 (m, 4h), 6.49 (d, j = 8.0 hz, 2h), 4.01 (s, 2h), 3.72 (m, 2h), 3.15 (m, 2h), 2.19 (s, 3h). 2-(4-fluorophenyl)-3-(p - tolylamino)-7,8-dihydroimidazo[1,2-a]pyrazin-6(5h)-one : compound 32 was synthesized from compound 7 (r1 = r2 = h) pd2(dba)3 mediated coupling reaction with p - toluidine. h nmr (400 mhz, dmso - d6) 8.55 (s, 1h), 7.777.75 (m, 2h), 7.267.14 (m, 4h), 7.016.95 (m, 2h), 6.53 (m, 1h), 4.53 (m, 2h), 4.22 (m, 2h), 2.17 (s, 3h). 2-(4-fluorophenyl)-8,8-dimethyl-3-(p - tolylamino)-7,8-dihydroimidazo[1,2-a]pyrazin-6(5h)-one : compound 33a was prepared from 7 by the following way : to a stirred solution of compound 7 (390 mg, 1.51 mmol, 1.00 equiv) in dichloromethane (20 ml) was added nbs (0.28 g, 1.00 equiv). the solid was filtered out, and the mixture was washed with a saturated solution of na2s2o3 and dried over na2so4. the solids was purified by silica gel chromatography (petroleum ether / etoac = 1:2) to result in 432 mg (85%) of the title compound as a white solid. h nmr (300 mhz, dmso - d6) 8.76 (s, 1h), 7.947.89 (m, 2h), 7.317.25 (m, 2h), 4.57 (s, 2h), 1.56 (s, 6h). compound 33 was prepared from compound 33a by a pd2(dba)3 mediated amination reaction with p - toluidine. h nmr (meoh - d4, 400 hz) 7.627.58 (m, 2h), 7.05 (t, j = 8.4 hz, 2h), 6.92 (d, j = 8.4 hz, 2h), 6.52 (d, j = 8.4 hz, 2h), 4.42 (s, 2h), 2.12 (s, 3h), 1.71 (s, 6h). 3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8,8-dimethyl-7,8-dihydro imidazo[1,2-a]pyrazin-6(5h)-one : compound 34 was prepared from compound 7b by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline. h nmr (meoh - d4, 400 hz) 7.747.70 (m, 2h), 7.21 (t, j = 8.4 hz, 1h), 7.08 (m, 2h), 6.496.40 (m, 2h), 4.43 (s, 2h), 1.72 (s, 6h). 2-(4-fluorophenyl)-8,8-dimethyl - n - p - tolyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazin-3-amine : compound 35 was prepared from compound 35 - 1 (shown below and prepared by analogy to compound 9 and using cbz - glycine) by a pd2(dba)3 mediated amination reaction with p - toluidine followed by a 6n hcl mediated hydrolysis. h nmr (meoh - d4, 400 hz) 7.787.75 (m, 2h), 7.066.97 (m, 4h), 6.56 (d, j = 8.4 hz, 2h), 5.16 (s, nh), 4.33 (t, j = 4.8 hz, 2h), 3.74 (t, j = 4.8 hz, 2h), 2.29 (s, 3h), 1.95 (s, 6h). n,2-bis(4-fluorophenyl)-8,8-dimethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazin-3-amine : compound 36 was prepared from compound 35 - 1 by a pd2(dba)3 mediated amination reaction with 4-fluoroaniline followed by a 6n hcl mediated hydrolysis. h nmr (400 mhz, meoh - d4) 7.627.58 (m, 2h), 7.127.08 (m, 2h), 6.686.63 (m, 2h), 6.67 (m, 2h), 4.16 (m, 2h), 3.78 (m, 2h), 1.92 (s, 6h). unless otherwise noted, materials were obtained from commercial suppliers and were used without purification. removal of solvent under reduced pressure refers to distillation using bchi rotary evaporator attached to a vacuum pump (3 mmhg). products obtained as solids or high boiling oils were dried under vacuum (1 mmhg). purification of compounds by high pressure liquid chromatography was achieved using a waters 2487 series with ultra 120 5 m c18q column with a linear gradient from 10% solvent a (acetonitrile with 0.035% trifluoroacetic acid) in solvent b (water with 0.05% trifluoroacetic acid) to 90% a in 7.5 min, followed by 2.5 min elution with 90% a. h nmr spectra were recorded on bruker xwin - nmr (400 or 600 mhz). proton resonances are reported in parts per million (ppm) downfield from tetramethylsilane (tms). h nmr data are reported as multiplicity (s singlet, d doublet, t triplet, q quartet, quint quinted, sept septed, dd doublet of doublets, dt doublet of triplet, bs broad singlet), number of protons, and coupling constant in hertz. for spectra obtained in cdcl3, dmso - d6, and cd3od, the residual protons (7.27, 2.50, and 3.31 ppm, respectively) analytical thin - layer chromatography (tlc) was performed on commercial silica plates (merck 60-f 254, 0.25 mm thickness) ; compounds were visualized by uv light (254 nm). 100c, isco) or using silica gel (merck kieselgel 60, 230400 mesh). the purity and quantitative analysis were determined with a waters zq 2000 lc / ms system, which employed an acquity uplc system (binary pump, column compartment, autosampler, and diode array uv detector). the eluent was split between the mass spectrometer and an antek chemiluminescent nitrogen detector (clnd). the mobile phases used were (a) 95% h2o/5% meoh / ipa (75/25, v / v) + 0.05% formic acid and (b) meoh / ipa (75/25, v / v) + 0.035% formic acid. a gradient hplc method with flow of 0.4 ml / min started at 2% b, with a hold of 1.0 min before a linear increase to 95% b at 3.50 min, with a 30 s hold. from 4.04.25 min, the column used was a thermo syncronis c18 2.1 mm 30 mm, 3 m. the mass spectrometer was operated in positive mode, with a spray voltage of 3.2 kv and cone voltage of 30 v. the source and desolvation temperatures were 130 and 400 c, respectively, with 600 l / h of nitrogen desolvation flow. all the final compounds reported in this communication had the purity of at least 95%. elemental analyses were carried out by midwest microlabs llc, indianapolis, in, usa. compound 1a was prepared from 1 by the following method : to a stirred solution of 1 (12 mg, 0.03 mmol) was added mno2 (52 mg, 0.60 mmol). the reaction mixture was stirred at room temperature (rt) for 2 h. lc / ms test showed that 1 was almost consumed and the desired product ([m + 1 ] = 428) was detected as one of the major peaks. the residue was subjected to a mass - triggered hplc purification to give 1a as well as 1b. h nmr for 1a (400 mhz, cd3od) 7.597.63 (m, 2h), 7.09 (t, j = 6.4 hz, 2h), 6.957.02 (m, 1h), 6.466.52 (m, 1h), 6.346.38 (m, 1h), 5.92 (s, 1h), 4.33 (dd, j = 4.4, 14 hz, 1h), 3.96 (dd, j = 4.8, 13.2 hz, 1h), 3.703.78 (m, 1h), 3.463.54 (m, 1h), 1.73 (s, 3h), 1.36 (s, 3h). h nmr for 1b (400 mhz, cd3od) 7.757.78 (m, 2h), 6.957.01 (m, 3h), 6.446.50 (m, 1h), 6.326.36 (m, 1h), 4.19 (dd, j = 1.6, 6.8 hz, 2h), 4.00 (dd, j = 1.6, 6.8 hz, 2h), 1.47 (s, 6h). to a solution of 2-bromo-1-(4-fluorophenyl)ethanone (46.5 g, 214.29 mmol) in dmf (400 ml) was added 2-(benzyloxycarbonyl)-2-methylpropanoic acid (55.8 g, 236.4 mmol) and potassium carbonate (35.4 g, 256.5 mmol). the resulting solution was extracted with ethyl acetate (2 800 ml), and the combined organic layer was washed with water (2 800 ml) and then brine (1 800 ml). this resulted in 67 g (84%) of compound 4 as a white solid. to a solution of compound 4 (70 g, 187.7 mmol) in toluene (700 ml) was added nh4oac (144.5 g, 1.88 mol). the resulting solution was heated to reflux for 3 h in an oil bath. the resulting solution was extracted with ethyl acetate (2 500 ml), and the combined organic layer was washed with water (2 800 ml) and brine (1 800 ml). this resulted in 58 g (88%) of compound 5 as a white solid. h nmr (300 hz, dmso - d6) 11.91 (s, nh), 7.807.75 (m, 2h), 7.547.12 (m, 8h), 4.97 (m, 2h), 1.6 (s, 6h). to a solution of compound 5 (58 g, 164.3 mmol) in dmf (400 ml) was added cesium carbonate (134 g, 411.0 mmol). this was followed by the addition of ethyl 2-bromoacetate (33 g, 197.6 mmol) dropwise with stirring at rt in 30 min. the resulting solution was extracted with ethyl acetate (2 700 ml), and the combined organic layer was washed with water (2 800 ml) and brine (1 800 ml). the resulting mixture was dried concentrated under vacuum. this resulted in 60 g (83%) of compound 6 as a yellow solid. h nmr (300 hz, dmso - d6) 7.767.72 (m, 2h), 7.327.28 (m, 5h), 7.087.03 (m, 3h), 5.08 (m, 2h), 4.87 (m, 2h), 4.23 (q, j = 5.4 hz, 2h), 2.0 (s, 6h), 1.28 (t, j = 5.4 hz, 3h). to a solution of compound 6 (70 g, 159.45 mmol, 1.00 equiv) in methanol (800 ml) was added palladium on carbon (10 g). this resulted in 7 (38 g, 145.56 mmol, 91%) as a white solid. lc - ms : (es, m / z) [m + h ] calcd for c14h14fn3o, 260 ; found, 260. h nmr (300 hz, dmso - d6) 7.797.74 (m, 2h), 7.137.07 (m, 3h), 6.35 (s, 1h), 4.73 (s, 2h), 1.79 (s, 6h). to a stirred solution of compound 7 (5 g, 19.2 mmol, 1 equiv) was dissolved in 50 ml of thf, 1 m borane / thf complex (57 ml, 57 mmol, 3 equiv) was added slowly and reaction was refluxed overnight. the crude product of 8 (4.5 g, 18.3 mmol, 95%) was used in the next step. lc - ms : (es, m / z) [m + h ] calcd for c14h17fn3 246, found 246. h nmr (dmso, 300 hz) 7.757.70 (m, 2h), 7.4 (s, 1h), 7.14 (t, j = 9.0 hz, 2h), 3.9 (t, j = 5.4 hz, 2h), 2.51 (t, j = 5.4 hz, 2h), 1.41(s, 6h) ; nh proton not observed. to a stirred solution of compound 8 (2.9 g, 11.82 mmol, 1.1 equiv) and 2-(tert - butoxycarbonylamino)acetic acid (2.27 g, 13 mmol, 1.1 equiv) in 15 ml of dichloromethane were added diea (2.47 ml, 14.18 mmol, 1.2 equiv) and hatu (5.39 g, 14.18 mmol, 1.2 equiv). the reaction mixture was stirred at rt for 8 h. hplc / ms analysis showed that desired product compound 9 was the major product. the organic layer was washed with washed with water (1 30 ml), followed by saturated nahco3 (1 30 ml) and finally with brine (1 30 ml). the resulting oil was purified using column chromatography with hexanes / ethyl acetate (0100% linear gradient) used as eluant. the desired product compound 9 was obtained as oil (3.3 g, 8.27 mmol, 70%). h nmr (300 hz, dmso - d6,) 7.777.72 (m, 2h), 7.54 (s, 1h), 7.207.14 (m, 2h), 6.846.80 (m, 1h), 4.07 (s, 2h), 3.90 (d, j = 3.0 hz, 2h), 3.70 (s, 2h), 1.80 (s, 6h), 1.40 (s, 9h). lc - ms : (es, m / z) [m + h ] calcd for c21h28fn4o3, 403 ; found, 403. to a stirred solution of compound 9 (3.02 g, 7.51 mmol) in dichloromethane (30 ml) was added br2 (0.43 ml, 8.26 mmol) in acetic acid (3 ml). hplc / ms test showed that desired product (ii) was the only peak. the product was confirmed by 400 mhz proton nmr to be the title compound 10. the product was used in the next step without further purification and exhibited quantitative mass recovery. lc - ms : (es, m / z) [m + h ] calcd for c21h27brfn4o3, 482 ; found, 482. h nmr (meoh - d4, 400 hz) 7.847.81 (m, 2h), 7.14 (t, j = 8.8 hz, 2h), 4.094.01 (m, 4h), 3.81 (t, j = 4.8 hz, 2h), 1.89 (s, 6h), 1.46 (s, 9h). (s)-2-amino-1-(3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8-methyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 17 was prepared from compound 10 (r1 = me, r2 = h) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline followed by a tfa mediated deprotection in a protocol similar to compound 22. h nmr (meoh - d4, 400 mhz) 7.617.58 (m, 2h), 7.167.12 (m, 1h), 7.077.02 (m, 2h), 6.476.42 (m, 2h), 5.74 (m, 1h), 4.073.68 (m, 6h), 1.56 (d, j = 6.8 hz, 3h). r2 = h) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline followed by a tfa mediated deprotection in a protocol similar to compound 22. (s)-2-amino-1-(3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8-isopropyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 19 was prepared from compound 10 (r1 = isopropyl, r2 = h) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline followed by a tfa mediated deprotection in a protocol similar to compound 22. h nmr (meoh - d4, 400 mhz) 7.617.58 (m, 2h), 7.267.20 (m, 3h), 6.456.38 (m, 2h), 5.5 (d, j = 7.9 hz, 1h), 4.113.75 (m, 6h), 2.362.31 (m, 1h), 1.15 (d, j = 6.7 hz, 3h), 0.95 (d, j = 6.7 hz, 3h). (r)-2-amino-1-(3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8-isopropyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 20 was prepared from compound 10 (r1 = isopropyl, r2 = h) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline followed by a tfa mediated deprotection in a protocol similar to compound 22. h nmr (meoh - d4, 400 mhz) 7.697.67 (m, 2h), 7.277.20 (m, 3h), 6.686.65 (dd, j = 2.5 hz, j = 11.0 hz, 1h), 6.606.55 (dd, j = 2.25 hz, j = 8.7 hz, 1h), 5.78 (d, j = 7.7 hz, 1h), 4.424.02 (m, 4h), 3.943.87 (m, 2h), 2.55 (m,1h), 1.24(d, j = 6.8 hz, 3h), 1.05 (d, j = 6.8 hz, 3h). 2-amino-1-(3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 21 was prepared from compound 10 (r1 = me, r2 = me) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline followed by a tfa mediated deprotection in a protocol similar to compound 22. h nmr (meoh - d4, 400 mhz) 7.597.55 (m, 2h), 7.207.13 (m, 3h), 6.60 (dd, j = 11.2, 10.8 hz), 1h), 6.53 (dd, j = 2.4, 0.8 hz, 1h), 4.0 (m, 4h), 3.78 (m, 2h), 2.00 (s, 6h). elemental analysis (compound + 2.0 hcl + 2.0 h2o) : % c, 47.62 ; % h, 5.09 ; % n, 12.62 ; (calc). % c = 47.54/47.38 ; % n = 12.44/12.46 ; % h = 4.62/4.57 (experimental). 2-amino-1-(2-(4-fluorophenyl)-3-(4-fluorophenylamino)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : in a glass vial, cs2co3, 4f - aniline (0.462 g, 4.1 mmol, 2.0 equiv), pd2(dba)3 (0.095 g, 0.104 mmol, 0.05 equiv), xantphos (0.120 g, 0.208 mmol, 0.1 equiv), and dioxane were stirred for 5 min at rt. compound 10 (1 g, 2.08 mmol, 1.0 equiv) was added to the reaction mixture, after which the reaction mixture was degassed for 15 min and then stirred at 120 c under n2 for 8 h. hplc / ms test showed that the starting material compound 10 was consumed and desired product was formed predominantly along with some 9. the reaction mixture was concentrated and then purified by normal phase column chromatography (silica gel 80 g) using a gradient of 1000% to 0100% hexane : etoac. the organic layer was concentrated at reduced pressure to yield the boc derivative (950 mg, 89%) yield. lc - ms : (es, m / z) [m + h ] calcd for c27h32f2n5o3, 512 ; found, 512. the boc compound was treated with 20% tfa in ch2cl2 (50 ml) and was added to the mixture. after the completion of this reaction (monitored by lcms), the resulting mixture was concentrated under reduced pressure. the resulting residue was purified by reverse phase hplc to yield product as a tfa salt. lc - ms : (es, m / z) [m + h ] calcd for c22h23f2n5o, 412.2 ; found, 412.1. h nmr (meoh - d4, 400 hz) 7.617.57 (m, 2h), 6.94 (t, j = 8.8 hz, 2h), 6.81 (t, j = 8.8 hz, 2h), 6.47 (m, 2h), 3.72 (m, 2h), 3.58 (m, 2h), 3.42 (m, 2h), 1.85 (s, 6h). elemental analysis compound 22 with 0.65 equiv h2o : c, 62.44 ; n, 16.55 ; h, 5.79 (calculated). c = 62.54/62.44 ; n = 16.35/16.29 ; h = 5.52/5.61 (experimental). the regiochemistry of the dime groups was unambiguously assigned using 2-d nmr techniques (table 8). 2-amino-1-(2-(4-fluorophenyl)-8,8-dimethyl-3-(p - tolylamino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 23 was prepared from compound 10 (r1 = me, r2 = me) by a pd2(dba)3 mediated amination reaction with 4-methylaniline followed by a tfa mediated deprotection in a protocol similar to compound 22. h nmr (meoh - d4, 400 mhz) 7.797.69 (m, 2h), 7.21 (t, j = 8.4 hz, 2h), 7.04 (d, j = 8.0 hz, 2h), 6.76 (d, j = 8.2 hz, 2h), 4.11 (s, 2h), 3.88 (m, 4h), 2.22 (s, 3h), 2.12 (s, 6h). lc / ms major mass : 408.1 (m + h). 2-amino-1-(3-((3,4-difluorophenyl)amino)-2-(4-fluorophenyl)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 24 was prepared from compound 10 (r1= me, r2 = me) by a pd2(dba)3 mediated amination reaction with 3,4-difluoroaniline followed by a tfa mediated deprotection in a protocol similar to compound 22. h nmr (meoh - d4, 400 mhz) 7.737.69 (m, 2h), 7.21 (t, j = 8.8 hz, 2h), 7.067.03 (m, 1h), 6.766.65 (m, 1h), 6.536.51 (m, 1h), 4.084.06 (m, 4h), 3.863.83 (m, 2h), 2.07 (s, 6h). elemental analysis (compound + 2.9 hcl) : % c, 49.37 ; % h, 4.69 ; % n, 13.09 ; (calc). % c = 49.43/49.06 ; % n = 12.89/12.77 ; % h = 4.7/4.81 (experimental). 13 was prepared from compound 5a by the following way : to a stirred solution of compound 5a (1.1 g, 3.38 mmol, 1.00 equiv) in dmf (30 ml) was added 3-chloro-2-methylprop-1-ene (500 mg, 5.49 mmol, 1.50 equiv), potassium carbonate (560 mg, 4.06 mmol, 1.10 equiv), and potassium iodide (1.12 g, 6.75 mmol, 2.00 equiv) at rt. the mixture was washed with brine (3 10 ml), dried over sodium sulfate, and concentrated under vacuum. the residue was applied onto a silica gel column with petroleum ether / etoac (5:1) to give 0.7 g (55%) of compound 5a-1 as a light - yellow solid. m / z = 380 (m + 1). into a 30 ml sealed tube, was placed compound 5a-1 (2.0 g, 5.28 mmol, 1.00 equiv), acetic acid (12 ml), and methanesulfonic acid (2 ml). the reaction mixture was stirred for 12 h at 260 c (the temperature of the sand bath). the aqueous layer was washed with ethyl acetate (3 10 ml). the aqueous layer was extracted with ethyl acetate (3 10 ml). the combined organic layers were washed with brine (3 10 ml), dried over sodium sulfate, and concentrated under vacuum. the solid was collected by filtration and washed with 5 ml of n - hexane to give 500 mg (39%) of compound 5a-2 as a white solid. h nmr (400 mhz, cdcl3) 7.707.65 (m, 2h), 7.046.9 (m, 2h), 6.96 (s, 1h), 4.12 (s, 2h), 3.69 (s, 2h), 1.23 (s, 6h). to a stirred solution of compound 5a-2 (280 mg, 1.14 mmol, 1.0 equiv) in dmf (10 ml) was added 2-(tert - butoxycarbonyl)acetic acid (600 mg, 3.43 mmol, 3.0 equiv), hatu (1.3 g, 3.42 mmol, 3.0 equiv), and diea (880 mg, 6.82 mmol, 6.0 equiv) at rt. the organic layer was washed with brine (3 10 ml), dried over sodium sulfate, and concentrated under vacuum. the residue was applied onto a silica gel column with ch2cl2/meoh (10:1) to give 280 mg (57%) of compound 13 as a brown solid. h nmr (400 mhz, cdcl3) 7.727.68 (m, 2h), 7.23 (s, 1h), 7.137.07 (m, 2h), 5.46 (s, nh), 4.65 (s, 2h), 4.054.04 (m, 2h), 3.97 (s, 2h), 1.551.39 (m, 15h). the structure of 13 was verified by hmqc, cosy, hmbc, and roesy (table 9). 2-amino-1-(2-(4-fluorophenyl)-3-(4-fluorophenylamino)-6,6-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : to a stirred solution of compound 13 (386 g, 0.96 mmol) in 6 ml of dichloromethane was added br2 (55 l, 1.06 mmol) in acetic acid (2 ml). after neutralization, the residue was subjected to flash chromatography (40 g, 0100% ethyl acetate in hexane, 50 min, dry loading) purification to give 256 mg (55%) of the title compound 13a as a colorless oil. h nmr (400 mhz, cdcl3) 7.877.83 (m, 2h), 7.03 (t, j = 8.8 hz, 2h), 5.46 (s, 1h), 4.5 (s, 2h), 3.97 (d, j = 4.2 hz, 2h), 3.81 (s, 2h), 1.45 (s, 6h), 1.39 (s, 9h). example 25was obtained from 13a by a pd2(dba)3 mediated amination reaction with 4-fluoroaniline followed by a tfa mediated deprotection by analogy to compound 22 (55% yield over 2 steps). h nmr (400 mhz, dmso - d6) : 8.06 (m, 2h), 7.98 (m, 1h), 7.80 (m, 2h), 7.16 (m, 2h), 6.98 (m, 2h), 6.60 (m, 2h), 4.64 (m, 2h), 4.02 (m, 2h), 3.80 (m, 2h), 1.38 (s, 6h). 2-(4-fluorophenyl)-7-(4-methoxybenzyl)-5,5-dimethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine : compound 7b was prepared from compound 7a (r1 = r2 = h) by the following way : to a solution of compound 7a (231 mg, 1.0 mmol) in dmf (10 ml) were added koh (168 mg, 3.0 mmol) and pmbcl (405 l, 3.0 mmol) at 0 c. the reaction mixture was stirred at the same temperature for 2 h and at rt for 2 additional hours. the hplc fraction were evaporated under reduced pressure and neutralized by satd nahco3. the organic layer was concentrated under reduced pressure to give compound 7a-1 (221 mg, 63%) as a white solid. h nmr (400 mhz, cdcl3) 7.677.63 (m, 2h), 7.287.25 (m, 2h), 7.077.02 (m, 2h), 6.896.86 (m, 2h), 4.74 (s, 2h), 4.70 (m, 2h), 4.53 (s, 2h), 3.79 (s, 3h). to a solution of compound 7a-1 (253 mg, 0.72 mmol) in dmf (15 ml) were added 60% sodium hydride (87 mg, 0.085 mmol) and methyl iodide (0.45 ml, 7.2 mmol) at rt. the reaction mixture was stirred at rt for 2 h. the reaction mixture was quenched with methanol and directly subjected to mass - triggered hplc purification to give 7a-2 as a white solid after evaporation of acetonitrile water in vacuo. the tfa salt was subsequently neutralized by saturated sodium bicarbonate solution and extracted with dichloromethane to get the compound as a free base. the organic layer was dried over sodium sulfate and concentrated under reduced pressure to yield the desired compound 7a-2 (234 mg, 86%). the structure of 7a-2 was verified by 2-d nmr techniques (table 10). to a solution of compound 7a-2 (170 mg, 0.45 mmol) in thf (9 ml) was added 1.0 n bh3thf (2.70 ml, 2.70 mmol) at rt. the reaction mixture was stirred at reflux for 2 h. pd / c was added (gas generated). the reaction mixture was stirred for 1 h. solid was filtered off, and solvent was removed. the product was assumed to be of 100% yield and used in the next step without further purification. h nmr (400 mhz, cd3od) 8.1 (s, 1h), 7.747.70 (m, 2h), 7.34 (d, j = 8.6 hz, 2h), 7.27 (m, 2h), 6.94 (d, j = 8.6 hz, 2h), 3.91 (s, 2h), 3.80 (s, 3h), 3.79 (s, 2h), 2.87 (s, 2h), 1.61 (s, 6h). 7-(4-methoxybenzyl)-2-(4-fluorophenyl)-5,5-dimethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine (1.07 g, 2.93 mmol, 1.00 equiv) (7b) was added to trifluoroacetic acid (15 ml). the resulting solution was stirred for 2 h at 70 c in an oil bath. the ph value of the solution was adjusted to 8 with aqueous sodium bicarbonate. 7b-1 was extracted from the aqueous layer using 3 100 ml of ethyl acetate. this resulted in 7b-1 (718 mg, quantitative) as a yellow solid. to a solution of 7b-1 (750 mg, 3.06 mmol, 1.00 equiv) in n, n - dimethylformamide (30 ml) were added 2-(tert - butoxycarbonyl)acetic acid (850 mg, 4.86 mmol, 1.50 equiv), hatu (1.3 g, 3.42 mmol, 1.10 equiv), and triethylamine (650 mg, 6.44 mmol, 2.00 equiv). the resulting solution was stirred overnight at rt. the resulting mixture was washed with brine (3 100 ml), dried over anhydrous sodium sulfate, and concentrated under vacuum. the residue was applied onto a silica gel column with petroleum ether / ethyl acetate (4:1). this resulted in 600 mg (49%) of 7c as a pale - yellow solid. h nmr (300 mhz, cdcl3) : 7.75 (m, 2h), 7.22 (s, 1h), 7.137.07 (m, 2h), 4.994.87 (m, 2h), 4.12 (s, 2h), 3.91 3.65 (m, 2h), 1.57 (m, 6h), 1.43 (s, 9h). to a stirred solution of compound 7b-2 (583 g, 1.45 mmol) in dichloromethane (6 ml) was added br2 (82 l, 1.60 mmol) in acetic acid (2 ml). after neutralization, the residue was subjected to flash chromatography (40 g, 0100% ethyl acetate in hexane, 50 min, dry loading) purification to give 628 mg (90%) of the title compound 7c as a yellow solid. h nmr (400 mhz, cdcl3) 7817.78 (m, 2h), 7.07 (t, j = 8.6 hz, 2h), 5.28 (s, nh), 4.844.7 (m, 2h), 4.073.58 (m, 4h), 1.491.44 (m, 6h), 1.28 (s, 9h). 2-amino-1-(2-(4-fluorophenyl)-3-(4-fluorophenylamino)-5,5-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 26 was prepared from compound 7c by a pd2(dba)3 mediated amination reaction with 4-fluoroaniline followed by a tfa mediated deprotection in 46% yield over 2 steps. h nmr (400 mhz, cd3od) : 7.57 (m, 2h), 7.02 (m, 2h), 6.77 (m, 2h), 6.52 (m, 2h), 4.974.91 (m, 2h), 4.093.74 (m, 4h), 1.56 (s, 3h), 1.51 (s, 3h). 2-amino-1-(2-(4-fluorophenyl)-3-(4-fluorophenylamino)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)-2-methylpropan-1-one : compound 28 was prepared from compound 8 (r1 = r2 = me) in the following fashion using a slightly modified route to the one described in scheme 2. to a stirred solution of compound 8 (1.48 g, 6.04 mmol) and net3 (6.0 g, 59.4 mmol) in dichloromethane (20 ml) was added 2-bromo-2-methylpropanoyl bromide (14 g, 60.9 mmol) dropwise at rt. after being stirred for 3 h at rt, the reaction was then quenched by the addition of water (30 ml). the combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. this resulted in crude product as a dark solid, which was washed with etoac : petroleum ether (1:10) to remove the impurities to produce compound 8 - 1 as a gray solid. (2.0 g, 5.08 mmol, 1.00 equiv) in dmf (10 ml) was nan3 (1.0 g, 15.38 mmol, 3.00 equiv) at rt. the resulting mixture was washed with 3 20 ml of brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. the residue was applied onto a silica gel column with petroleum ether / etoac (5:1) to give compound 8 - 2 as a white solid. m / z 357 (m + h). to a stirred solution of compound 8 - 2 (1.2 g, 3.37 mmol, 1.00 equiv) in methanol (20 ml) was added pd / c (80 mg, 0.75 mmol, 0.20 equiv) at rt. m / z 331 (m + h). to a stirred solution of compound 8 - 3 (910 mg, 2.76 mmol, 1.00 equiv) in thf (50 ml) was added boc anhydride (3.2 g, 14.68 mmol, 5.00 equiv), followed by aqueous naoh (1n, 6 ml, 2.0 equiv) at rt. the organic layer was washed with 3 10 ml of brine, dried over na2so4, and concentrated under vacuum. the solid was collected by filtration and washed with n - hexane (5 ml) to give compound 8 - 4 as a white solid. h nmr (300 mhz, cdcl3) 7.757.71 (m, 2h), 7.097.02 (m, 3h), 4.84(s, nh), 4.094.0 (m, 4h), 1.98 (s, 6h), 1.55 (s, 6h), 1.45 (s, 9h). to a stirred solution of compound 8 - 4 (32 mg, 0.074 mmol) in dichloromethane (3 ml) was added br2 (4.2 l, 0.082 mmol) in acetic acid (1 ml). after neutralization, the residue was subjected to flash chromatography (4 g, 060% ethyl acetate in hexane, 16 min) purification to give the compound 8 - 5 as colorless oil, was collected by filtration, and washed with n - hexane (5 ml) to give compound 8 - 4 as a white solid. h nmr (400 mhz, cdcl3) 7.917.88 (m, 2h), 7.107.04 (m, 2h), 5.11(s, nh), 4.01 (m, 2h), 3.96 (s, 2h), 1.94 (s, 6h), 1.51 (s, 6h), 1.41 (s, 9h). compound 28 was prepared from compound 8 - 5 by a pd2(dba)3 mediated amination reaction with 4-fluoroaniline followed by a tfa mediated deprotection. h nmr (400 mhz, meoh - d4) 7.66 (dd, j = 9.2, 5.2 hz, 2h), 7.11 (t, j = 8.8 hz, 2h), 6.81 (t, j = 8.8 hz, 2h), 6.726.68 (m, 2h), 4.043.99 (m, 4h), 2.01 (s, 6h), 1.66 (s, 6h). 2-amino-1-(2-(4-fluorophenyl)-8,8-dimethyl-3-(p - tolylamino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)-2-methylpropan-1-one : compound 29 was prepared from 35 by the following way : to a stirred solution of 35 (21 mg, 0.06 mmol) and et3n (83 l, 0.60 mmol) in dry dichloromethane (6 ml) was added 2-bromo-2-methylpropanoyl bromide (71 l, 0.60 mmol). the reaction mixture was stirred at rt for 5 h. the reaction mixture was concentrated and subjected to mass - triggered lc / ms purification directly. the obtained solution was concentrated to give 22 mg (73%) of compound 35 - 1as yellow oil after neutralization. h nmr (400 mhz, meoh - d4) 7.717.66 (m, 2h), 7.237.19 (m, 2h), 7.02 (d, j = 8.4 hz, 2h), 6.66 (d, j = 8.4 hz, 2h), 4.31 (m, 2h), 4.09 (m, 2h), 2.21 (s, 3h), 2.06 (s, 6h), 2.00 (s, 6h). to a solution of compound 35 - 1 (22 mg, 0.044 mmol) in dmf (3 ml) was added nan3 (8.6 mg, 0.132 mmol) at rt. the reaction mixture was stirred at rt for 2 h. the reaction mixture was directly subjected to mass - triggered hplc purification directly. the obtained mecn / aqueous solution was combined and concentrated to give 15 mg (75%) of compound 35 - 2 as yellow oil after neutralization. h nmr (400 mhz, meoh - d4) 7.70 7.66 (m, 2h), 7.237.19 (m, 2h), 7.04 (d, j = 8.3 hz, 2h), 6.66 (d, j = 8.4 hz, 2h), 4.26 (m, 2h), 4.074.05 (m, 2h), 2.24 (s, 3h), 2.03 (s, 6h), 1.58 (s, 6h). to a solution of compound 35 - 2 (15 mg, 0.033 mmol) in meoh (3 ml) was added 10% pd / c (4 mg, 0.003 mmol) at rt. the reaction mixture was stirred at rt for 2 h. solid was filtered off, and solvent was removed. the reaction mixture was directly subjected to mass - triggered hplc purification to give 15 mg (100%) of the title compound yellow oil. h nmr (meoh - d4, 400 hz) 7.787.75 (m, 2h), 7.066.97 (m, 4h), 6.56 (d, j = 8.4 hz, 2h), 5.16 (s, nh), 4.33 (t, j = 4.8 hz, 2h), 3.74 (t, j = 4.8 hz, 2h), 2.29 (s, 3h), 1.95 (s, 6h), 1.45 (s, 6h). 2-amino-1-(3-(3,4-difluorophenylamino)-2-(4-fluorophenyl)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)-2-methylpropan-1-one : compound 30 was prepared from compound 8 - 5 by a pd2(dba)3 mediated amination reaction with 3,4-difluoroaniline followed by a tfa mediated deprotection.h nmr (400 mhz, meoh - d4) 7.637.60 (m, 2h), 7.14 (t, j = 8.4 hz, 2h), 7.01 (dd, j = 18.8, 8.8 hz, 1h), 6.686.63 (m, 1h), 6.5 (m, 1h), 4.03 (m, 2h), 4.01 (m, 2h), 2.01 (s, 6h), 1.66 (s, 6h). 2-(4-fluorophenyl)-n-(p - tolyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazin-3-amine : the details of the synthesis involving an ugi reaction between 4-fluorobenzaledehyde, 2-aminopyrazine, and 1-isocyano-4-methylbenzene has been reported in the previous communication.h nmr (400 mhz, meoh - d4) 7.73 (d, j = 6.8 hz, 2h), 7.006.99 (m, 4h), 6.49 (d, j = 8.0 hz, 2h), 4.01 (s, 2h), 3.72 (m, 2h), 3.15 (m, 2h), 2.19 (s, 3h). 2-(4-fluorophenyl)-3-(p - tolylamino)-7,8-dihydroimidazo[1,2-a]pyrazin-6(5h)-one : compound 32 was synthesized from compound 7 (r1 = r2 = h) pd2(dba)3 mediated coupling reaction with p - toluidine. h nmr (400 mhz, dmso - d6) 8.55 (s, 1h), 7.777.75 (m, 2h), 7.267.14 (m, 4h), 7.016.95 (m, 2h), 6.53 (m, 1h), 4.53 (m, 2h), 4.22 (m, 2h), 2.17 (s, 3h). 2-(4-fluorophenyl)-8,8-dimethyl-3-(p - tolylamino)-7,8-dihydroimidazo[1,2-a]pyrazin-6(5h)-one : compound 33a was prepared from 7 by the following way : to a stirred solution of compound 7 (390 mg, 1.51 mmol, 1.00 equiv) in dichloromethane (20 ml) was added nbs (0.28 g, 1.00 equiv). the solid was filtered out, and the mixture was washed with a saturated solution of na2s2o3 and dried over na2so4. the solids was purified by silica gel chromatography (petroleum ether / etoac = 1:2) to result in 432 mg (85%) of the title compound as a white solid. h nmr (300 mhz, dmso - d6) 8.76 (s, 1h), 7.947.89 (m, 2h), 7.317.25 (m, 2h), 4.57 (s, 2h), 1.56 (s, 6h). compound 33 was prepared from compound 33a by a pd2(dba)3 mediated amination reaction with p - toluidine. h nmr (meoh - d4, 400 hz) 7.627.58 (m, 2h), 7.05 (t, j = 8.4 hz, 2h), 6.92 (d, j = 8.4 hz, 2h), 6.52 (d, j = 8.4 hz, 2h), 4.42 (s, 2h), 2.12 (s, 3h), 1.71 (s, 6h). 3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8,8-dimethyl-7,8-dihydro imidazo[1,2-a]pyrazin-6(5h)-one : compound 34 was prepared from compound 7b by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline. h nmr (meoh - d4, 400 hz) 7.747.70 (m, 2h), 7.21 (t, j = 8.4 hz, 1h), 7.08 (m, 2h), 6.496.40 (m, 2h), 4.43 (s, 2h), 1.72 (s, 6h). 2-(4-fluorophenyl)-8,8-dimethyl - n - p - tolyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazin-3-amine : compound 35 was prepared from compound 35 - 1 (shown below and prepared by analogy to compound 9 and using cbz - glycine) by a pd2(dba)3 mediated amination reaction with p - toluidine followed by a 6n hcl mediated hydrolysis. h nmr (meoh - d4, 400 hz) 7.787.75 (m, 2h), 7.066.97 (m, 4h), 6.56 (d, j = 8.4 hz, 2h), 5.16 (s, nh), 4.33 (t, j = 4.8 hz, 2h), 3.74 (t, j = 4.8 hz, 2h), 2.29 (s, 3h), 1.95 (s, 6h). n,2-bis(4-fluorophenyl)-8,8-dimethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazin-3-amine : compound 36 was prepared from compound 35 - 1 by a pd2(dba)3 mediated amination reaction with 4-fluoroaniline followed by a 6n hcl mediated hydrolysis. h nmr (400 mhz, meoh - d4) 7.627.58 (m, 2h), 7.127.08 (m, 2h), 6.686.63 (m, 2h), 6.67 (m, 2h), 4.16 (m, 2h), 3.78 (m, 2h), 1.92 (s, 6h).	on the basis of the initial success of optimization of a novel series of imidazolopiperazines, a second generation of compounds involving changes in the core piperazine ring was synthesized to improve antimalarial properties. these changes were carried out to further improve the potency and metabolic stability of the compounds by leveraging the outcome of a set of in vitro metabolic identification studies. the optimized 8,8-dimethyl imidazolopiperazine analogues exhibited improved potency, in vitro metabolic stability profile and, as a result, enhanced oral exposure in vivo in mice. the optimized compounds were found to be more efficacious than the current antimalarials in a malaria mouse model. they exhibit moderate oral exposure in rat pharmacokinetic studies to achieve sufficient multiples of the oral exposure at the efficacious dose in toxicology studies.
chagas disease or american trypanosomiasis is caused by the parasite trypanosoma cruzi, which may produce acute and chronic manifestations. during the acute phase, most cases are indeterminate, but some patients may develop severe myocarditis or meningoencephalitis, which can be lethal. the chronic phase exhibits four major clinical variants : (i) the indeterminate form represents 5070% of cases and develops without evident clinical and pathological signs ; (ii) the cardiac form encompasses 1020% of cases and usually presents progressive congestive heart failure, various cardiac arrhythmias, thromboembolic events, and sudden death [1, 2 ] ; (iii) the digestive form (810% of cases) is characterized by clinical signs of megaesophagus, megacolon, or both ; (iv) the cardiodigestive or mixed form (8% of cases) comprises clinical and pathological signs of cardiac and digestive involvement. these clinical syndromes are considered to be a consequence of autonomic neuronal loss, microvascular derangements, and chronic organ inflammation directly dependent on parasite persistence or by immune system cells [3, 4 ]. in parallel with the chronic parasite infection, the immune response against parasite antigens key features of the immunity in the chagas disease include (i) predominance of partially activated t cd8 lymphocytes in cardiac inflammatory infiltrates, accompanied by high production of nitric oxide (no), il-12, monocyte chemoattractant protein-1 (mcp-1), and ifn- by infiltrating macrophages ; (ii) preponderant ifn- production by cytolytic natural killer (nk) cells in the acute phase of the disease to control tissue and systemic parasite burden ; and (iii) polyclonal b lymphocyte response [613 ]. cytokine patterns in chagas disease are characterized by t - helper (th)1 polarization in the acute phase, with predominance of ifn- and tnf production. during the chronic phase, a coexistence of the th1 (il-12, ifn-, and tnf) and th2 profiles (il-4 and il-10) may be observed, and the equilibrium between these profiles may be relevant to disease morbidity [3, 14, 15 ] ; that is, a th1 response aggravates and a th2 response produces a better outcome in murine trypanosomiasis and human chagas disease [1620 ]. besides cytokines, other immunomodulatory molecules such as pd-1 and ctla-4 are also involved in acute and chronic trypanosomiasis [2025 ]. in this context, immunomodulatory molecules may play an important role for disease evolution, controlling or exacerbating the immune response against t. cruzi itself or tissue modifications induced by parasites. among immunomodulatory molecules, the human leucocyte antigen- (hla-) g is a nonclassical histocompatibility class ib molecule, which has a well - recognized role in controlling several branches of the immune response, inhibiting t cell proliferation, nk, and cytotoxic t lymphocytes, inducing regulatory t cells and tolerizing dendritic cells. these effects are primarily due to the preferential interaction of the hla - g molecule with the ig - like transcript (ilt)2, ilt4, and killer cell immunoglobulin - like (kir2dl4) receptors, which induce inhibitory intracellular signals via tyrosine - based (itim) motifs. hla - g mrna has restricted expression in nonpathological conditions, for organs such as the placenta, thymus, heart, intestines, brain, and skin, and the constitutive expression of the hla - g molecule has been observed on placenta, thymus, cornea, pancreas, brain, erythroid cells, and blood cell surfaces [2934 ]. on the other hand, hla - g neoexpression has been observed in several situations, including tumors, viral infections, engrafted tissues, and autoimmune and inflammatory diseases. depending on the underlying condition, the expression may be advantageous when the blockade of the immune response is desirable, such as in autoimmune disorders and in allografting, whereas hla - g expression in tumors and chronic viral infections may be harmful [35, 36 ]. at the coding region, the hla - g gene presents limited polymorphisms compared to classical hla class i (hla - a / b / c) genes, exhibiting only 50 alleles (the international immunogenetics database (imgt), v3.16.0) and coding 16 different membrane - bound full length proteins. the regulatory regions also present several polymorphisms that coincide with or are close to binding sites for transcription factors (promoter region) or are targets for microrna binding (3 untranslated region (3utr)). at least 29 variation sites have been described at the promoter region and 16 variants have been identified at the hla - g 3utr. considering that the regulatory regions are involved on the magnitude of hla - g gene expression, the study of these regions is relevant. in the healthy brazilian population, at least eight hla - g 3utr variation sites have been described [37, 39 ], and some of them have been associated with plasma soluble hla - g (shla - g) levels. the hla - g murine functional homolog qa2, encoded by h2-q7/q9 gene, also presents restricted tissue expression, and qa2 mrna has been reported in thymus, liver, intestines, spleen, placenta, and brain. qa2 also modulates the immune response by interacting with as yet unidentified nk cell receptors [4145 ]. little information is available regarding hla - g and the qa2 murine functional homolog in acute and chronic parasitic diseases. considering that (i) no information is available regarding the role of these immunoregulatory molecules in t. cruzi infection ; (ii) chagas disease is a chronic disorder, in which several mechanisms of immunomodulation have been described in association with its pathogenesis ; (iii) clinical variants of chagas disease may depend on parasite and host genetic factors ; (iv) experimental trypanosomiasis may help in the understanding of the human disease counterpart, we designed a study encompassing the hla - g tissue expression and hla - g 3utr polymorphic site typing in patients presenting chagas disease, stratified according to major clinical variants. in addition, we evaluated the transcriptional level of the h2-q7/q9 (qa2), and other immunoregulatory h2-t23 (qa1), ctla-4, and pdcd1 (pd-1) genes in mouse (balb / c and c57bl/6) affected tissues during experimental acute and early chronic infection caused by the cl strain of t. cruzi and correlated the expression of these genes with other mediators of inflammation, including inf-, inducible nitric oxide synthase (nos-2), and il-10. the protocol of the study was approved by the local research ethics committee (protocol number 11237/2009) and written informed consent was obtained from all participants. a total of 177 chronic chagas disease patients exhibiting positive serology for t. cruzi antigens followed at the divisions of cardiology and gastroenterology of the department of medicine of the school of medicine of ribeiro preto, university of so paulo, brazil, and 155 healthy individuals from the same region of patients, exhibiting negative serology for t. cruzi infection, were studied. echocardiography, esophagus and colon contrast x - ray examinations, and esophagus electromanometry were performed to classify patients into cardiac (n = 52, exhibiting or not heart failure), digestive (n = 62, exhibiting megaesophagus, megacolon, or both), cardiodigestive or mixed (n = 24, presenting any combination of cardiac and digestive forms), and indeterminate (n = 39) variants. genomic dna was extracted from peripheral blood leucocytes using a standard salting - out procedure. genotyping of the variation sites at hla - g 3utr was performed by sequencing analyses as previously described. fifty - four specimens of tissues obtained from chagas disease deceased patients were analyzed, of which 30 were derived from heart, 13 from colon, and 11 from esophagus. in parallel, 20 tissue specimens (8 from heart, 6 from esophagus, and 6 from sigmoid colon) of deceased individuals without chagas disease and exhibiting no histopathological tissue alteration were analyzed. three specimens of trophoblast tissue (positive control for hla - g expression) were also analyzed. the immunohistochemical analysis was performed using the primary antibody mem / g9 (hla - g1 and shla - g5 specific mouse igg) (exbio, prague, czech republic), diluted 1 : 200, as previously described. briefly, after defining the range of brown color considered being positive, images were converted to 256 shades (8-bit) of gray. then, the grayscale images were converted into a binary (black and white) variable to define the cutoff point. the threshold was adjusted, and the brown areas became black portions in the binary image. immunostained areas were evaluated using the imagej software (national institutes of health, bethesda, md). male eight - week - old c57bl/6 and balb / c mice were intraperitoneously (ip) injected with bloodstream cl strain forms of t. cruzi, using three infected and three noninfected animals in two independent experiments. the mice were cared for according to the institutional guidelines on ethics in animal experiments and all protocols were approved by the local ethics committee on animal care and research (process number 172/2009). for the acute infection, mouse survival was verified daily for 30 days and the parasitemia was quantified microscopically by counting the parasites in 5 l of citrated blood obtained from the tail lateral vein from day 7 until day 29 after infection. parasite load was analyzed in the heart at day 24 after infection. for the early chronic infection, 10 bloodstream forms of t. cruzi were ip injected and mouse survival was verified daily for 60 days. parasite load was analyzed in the heart at day 60 after infection. gene expression was detected in the heart and esophagus at 24 and 60 days after infection (dpi), for acute and early chronic infections, respectively. although qa2, qa1, and pd-1 are the encoded molecules of the h2-q7/q9, h2-t23, and pdcd1 genes, respectively, to facilitate understanding we will refer in this text to qa2, qa1, and pd-1 expression as synonyms of their respective gene expressions. total rna was extracted from tissues using the trizol reagent (invitrogen, carlsbad, ca) and was reverse transcribed to obtain cdna with the superscript iii (invitrogen) transcriptase reverse. sybr green mix - based real - time quantitative pcr assays were performed using a steponeplus system (applied biosystems, foster city, ca). data were normalized according to the expression of the glyceraldehyde phosphate dehydrogenase (gadph) housekeeping gene and relative expression of each mrna was calculated with the 2 method. primer sequences included (i) h2-q7/q9-forward 5-atg gcg acc att gct gtt gt-3, h2-q7/q9-reverse 5-tcc aat gat ggc cac agc t-3 ; (ii) h2-t23-fwd 5-gca caa gtc aga ggc agt cg-3, h2-t23-rev 5-tgc agg tat gcc ctc tgt tg-3 ; (iii) ctla-4-fwd 5-acc tct gca agg tgg aac tca-3, ctla-4-rev 5-cca tgc cca caa agt atg gc-3 ; (iv) pd-1-fwd 5-ttc agg ttt acc aca agc tgg-3, pd-1-rev 5-tga caa tag gaa acc ggg aa-3 ; (v) inf--fwd 5-gca tct tgg ctt tgc agc t-3, inf--rev 5-cct ttt tcg cct tgc tgt tg-3 ; (vi) nos-2-fwd 5-cga aac gct tca ctt cca a-3, nos-2-rev 5-tga gcc tat att gct gtg gct-3 ; (vii) il-10-fwd 5-tgg aca aca tac tgc taa cc-3, il-10-rev 5-gga tca ttt ccg ata agg ct-3 ; (viii) gapdh - fwd 5-tgc agt ggc aaa gtg gag at-3, gadph - rev 5-cgt gag tgg agt cat act gga a-3. allele and genotype frequencies were estimated by direct counting, and adherences of phenotypical proportions to expectations under the hardy - weinberg equilibrium (hwe) were tested by the complete enumeration method using the genepop 3.4 software. linkage disequilibrium (ld) between variation sites at hla - g 3utr was evaluated by means of a likelihood ratio test of linkage disequilibrium implemented at the arlequin software. hla - g 3utr haplotypes of each individual were inferred for each group of patients (stratified according to the four clinical variants) and for the whole group of patients. two distinct computational methods that do not take any prior information into account were used for this purpose : (1) the em algorithm implemented with the pl - em software and (2) a coalescence - based method implemented in the phase v2 software. therefore, the haplotype pair of each subject was independently inferred by four approaches ; whenever these four approaches resulted in nonconsensual inference for a given subject, he was removed from all procedures that used haplotype data as input. the frequency of each allele, genotype, or haplotype was compared between patients and controls by the two - sided fisher exact test, with the aid of the graphpad instat 3.05 software, which was also used to estimate the odds ratio (or) and its 95% confidence interval (ci). for the hla - g tissue expression, gene expression, and parasitism load, statistical analyses were performed using student 's t - test or mann - whitney tests and the relationship between immunomodulatory and mediators of inflammation genes was performed using the spearman correlation, both with the aid of the graphpad prism 5 v5.0b software. to facilitate data presentation, hla - g 3utr variation sites were compared between (i) patients (considered as whole) with controls (healthy individuals) ; (ii) patients presenting clinically detectable disease (cdm) (cardiac (c) plus digestive (d) plus mixed (m) forms) with controls ; (iii) patients presenting cdm with indeterminate patients (indeterminate form i) ; and (iv) patients stratified according to the four clinical variants with each other and with controls. the following hla - g 3utr variation sites were observed in this study : 14-base pair insertion / deletion (i / d ; rs66554220) ; + 3001c / t (rs not available) ; + 3003t / c (rs115689421) ; + 3010c / g (rs116152775) ; + 3027a / c (rs115810666) ; + 3035c / t (rs115100128) ; + 3142g / c (rs115928989) ; + 3187a / g (rs114317070) ; and + 3196c / g (rs115045214). overall, these variation sites adhered to the hardy - weinberg equilibrium, except + 3010c / g in cardiac and whole group of patients and + 3142g / c in digestive and whole group of patients. table 1 shows the genotype and allele frequencies in patients with chronic chagas disease and controls. 14-base pair i / d and + 3001c / t : no significant differences were observed when the five types of comparisons were performed. the + 3001 t allele was observed in only one chagas disease patient presenting exclusively the digestive form. + 3003t / c : the + 3003cc genotype was not observed in chagasic patients. compared to controls, (i) the + 3003tc genotype frequency was decreased in the whole group of patients (p = 0.0131) and in the digestive form (p = 0.0462) ; (ii) the frequency of the + 3003c allele was decreased in the whole group of patients (p = 0.0101) and in patients exhibiting the digestive form (p = 0.0448) ; (iii) + 3003tt genotype frequency was overrepresented in the whole group (p = 0.0091) and in digestive (p = 0.0321) patients ; and (iv) + 3003 t allele frequency was increased in whole group (p = 0.0101) and in the digestive group (p = 0.0448) of patients. + 3010c / g : compared to controls, the + 3010cg genotype was underrepresented in the whole group of patients (p = 0.0322) and in those presenting the cardiac variant (p = 0.0098). + 3027a / c : a decreased frequency of the + 3027ac genotype was observed in the digestive form compared to control (p = 0.0069) and to patients with the indeterminate (p = 0.0026), cardiac (p = 0.0076), and mixed (p = 0.0050) forms. the frequency of the + 3027cc genotype was increased in patients exhibiting the digestive form compared to indeterminate (p = 0.0026), cardiac (p = 0.0076), mixed (p = 0.0050), and control (p = 0.0071) groups. the + 3027aa and + 3027ac genotypes were not observed in patients with the digestive form. as consequence, the digestive group exhibited a decreased frequency of the + 3027a allele compared to controls (p = 0.0046) and to patients with the indeterminate (p = 0.0029), cardiac (p = 0.0083), and mixed (p = 0.0055) forms. + 3035c / t : the + 3035cc genotype was overrepresented in patients with the digestive form compared to control (p = 0.0024) and to patients with cardiac (p = 0.0247) and mixed (p = 0.0404) forms. a decreased frequency of the + 3035ct genotype was observed in patients exhibiting the digestive form compared to mixed (p = 0.0404) and control (p = 0.0095) groups. the + 3035c allele was overrepresented in the group of patients with the digestive form compared to control (p = 0.0020), indeterminate (p = 0.0346), cardiac (p = 0.0118), and mixed (p = 0.0489) groups. + 3142g / c : the + 3142gg genotype frequency was significantly lower in digestive patients compared to indeterminate (p = 0.0449), cardiac (p = 0.0363), and mixed (p = 0.0346) forms. the + 3142gc genotype frequency was lower in cardiac patients compared to digestive (p = 0.0211) and control (p = 0.0061) groups. in addition, + 3142gc genotype frequency was decreased in the whole group of patients group compared to the control group (p = 0.0459). + 3187a / g : compared to controls, an increased frequency of the + 3187gg genotype was observed in the whole group (p = 0.0169) and in the group of patients with the cardiac form (p = 0.0459). + 3196c / g : patients exhibiting the mixed form presented a decreased frequency of the + 3196cc genotype (p = 0.0499) and an increased frequency of + 3196gc genotype (p = 0.0258). in addition, + 3196cg genotype was increased in the mixed group compared to the indeterminate variant (p = 0.0188). when cdm patients (excluding the indeterminate group) were compared to controls, the following results were observed : (i) the + 3003ct genotype frequency was decreased in patients (p = 0.0128) ; (ii) the frequency of the + 3003tt genotype was increased in patients (p = 0.0088) ; (iii) + 3003c allele frequency was decreased in patients (p = 0.0097) and + 3003 t allele presented the opposite association pattern (p = 0.0097) ; (iv) the + 3187gg genotype frequency was overrepresented in patients (p = 0.0282) ; (v) + 3196cc genotype frequency was decreased (p = 0.0259) and + 3196gc genotype frequency was increased (p = 0.0325) in patients. when cdm patients were compared to the indeterminate form, only the + 3196gc genotype was increased in cdm patients (p = 0.0440). on the other hand, when indeterminate patients were compared to healthy controls, no significant results were observed. table 2 shows the odds ratio and 95% confidence interval values obtained for all significant comparisons. to further understand how the ensemble of variation sites participate in chagas disease susceptibility and considering that these variation sites were in linkage disequilibrium (results not shown), we analyzed the frequency of the hla - g 3utr haplotypes observed for patients and controls, and the results of the statistical analyses are shown in tables 3(a) and 3(b). eleven out of 177 patients have been removed from all analyses that used haplotype data as input, since results for the four approaches of haplotype inference resulted in nonconsensual results. follow the nomenclature previously described by our group, which has been extensively used in the literature. compared to controls, (i) utr-4 was underrepresented in the whole group of patients (p = 0.0005), cdm (p = 0.0008), digestive (p = 0.0274), and cardiac (p = 0.0043) forms ; (ii) utr-13 was overrepresented in the indeterminate group (p = 0.0320) ; (iii) utr-14 was overrepresented in cardiac patients (p = 0.0135). a decreased frequency of the utr-7 haplotype was observed in the digestive group compared to indeterminate (p = 0.0056), mixed (p = 0.0053), and control (p = 0.0048) groups. in addition, utr-13 was also increased in patients presenting the indeterminate form when compared to cdm patients (p = 0.0415). hla - g molecule expression was evaluated in the major organs affected by the disease, including heart, colon, and esophagus. compared to non - chagasic tissues, hla - g expression was significantly decreased in chagasic heart (p = 0.0105 ; figures 1(a), 1(b), and 1(c)) and colon (p = 0.0485, figures 1(d), 1(e), and 1(f)). hla - g expression in individuals without chagas disease was primarily observed on cardiac muscle cells and no cellular infiltration was observed (figure 1(a)), whereas specimens from chagas patients exhibiting cardiomegaly showed lesser hla - g expression on cardiac muscle cells together with an infiltration of mononuclear cells (lymphocytes and plasma cells) exhibiting hla - g expression (figure 1(b)). the hla - g immunolabeling in esophagus of chagas patients with esophagomegaly was closely similar to that observed for individuals without chagas disease (figures 1(g), 1(h), and 1(i)). the transcriptional level of qa2 in acute and early chronic infections was studied in heart and esophagus specimens obtained from balb / c and c57bl/6 infected mice, using the t. cruzi cl strain. first, we characterized the acute and early chronic infections regarding the animal survival rate, tissue parasite load, and blood parasitism. survival rates showed no significant differences for balb / c and c57bl/6 mice for both acute (figure 2(a)) and early chronic (figure 2(b)) infections. during acute infection, the heart parasite load was 2-fold increased in c57bl/6 mice in relation to balb / c group (p < 0.05 ; figure 2(c)). no significant difference was observed between the two groups during the early chronic infection (figure 2(d)). in addition, when we analyzed the bloodstream forms in the acute infection, balb / c mice showed an increased parasitism level at days 21, 23, and 25 compared to c57bl/6 mice (p < 0.05 ; figure 2(e)). during acute infection, the heart and esophagus transcriptional levels of qa2 were significantly increased for both mouse strains, when compared to noninfected mice. compared to the control group, the heart expression of qa2 in balb / c and c57bl/6 was 28-fold and 25-fold increased, respectively (p < 0.05 ; figure 3(a)), and the esophagus expression of qa2 was 17-fold and 16-fold increased in balb / c and c57bl/6, respectively (p < 0.05 ; figure 3(b)). acutely infected balb / c mice showed increased transcriptional levels of qa2 in heart and esophagus, which were 7-fold and 14-fold higher than early chronically infected mice, respectively (p < 0.05 ; figures 3(a) and 3(b)). the transcriptional level of qa2 was 6-fold higher in heart of acutely infected c57bl/6 mice compared to early chronic infection (p < 0.05 ; figure 3(a)). no significant difference in terms of qa2 expression was observed between noninfected and early chronically infected mice in heart and esophagus of balb / c and c57bl/6 mice (figures 3(a) and 3(b)). to verify whether other known immunomodulatory genes were concomitantly modulated during the infection by cl strain of t. cruzi our results showed that all of them were significantly augmented in heart and esophagus of balb / c strain when compared to noninfected and early chronically infected mice (p < 0.05 ; figures 3(a) and 3(b)). c57bl/6 mice showed an increased transcriptional level of qa1 in heart and increased levels of ctla-4 in esophagus and pd-1 in both heart and esophagus, during the acute infection (p < 0.05), compared to controls and early chronically infected animals (figures 3(a) and 3(b)). moreover, during the acute infection, qa1 was 4-fold higher in esophagus of balb / c strain compared to c57bl/6 animals, and pd-1 expression was 2-fold higher in heart of c57bl/6 mice compared to balb / c strain (p < 0.05 ; figures 3(a) and 3(b)). no significant difference was observed between noninfected and early chronically infected mice in heart and esophagus specimens obtained from balb / c and c57bl/6 mice (figures 3(a) and 3(b)). considering that nonclassical histocompatibility genes may be influenced by the action of inflammatory mediators, we evaluated the transcriptional levels of inf-, nos-2, and il-10 genes in the heart to observe the relationship between these genes and the nonclassical genes. inf-, nos-2, and il-10 expression was increased in both balb / c and c57bl/6 strains during the acute infection compared to controls and early chronically infected animals (p < 0.05 ; figure 3(c)). the comparisons between strains showed that inf- expression was 4-fold higher in c57bl/6 than in balb / c (figure 3(c)). during the early chronic infection, the transcriptional levels of these genes were similar to noninfected mice for both balb / c and c57bl/6 strains (figure 3(c)). when we studied the relationship between these genes, we observed a positive correlation between qa2 and inf- (p = 0.0417 ; r = 1.0000) and qa2 and nos-2 (p = 0.0417 ; r = 1.0000) in heart specimens of both mouse strains during acute t. cruzi infection. the present study provides consistent data regarding the involvement of hla - g and its qa2 murine functional homolog in the t. cruzi infection. hla - g gene polymorphism and hla - g tissue expression have been evaluated in several diseases ; however, little is known about the role of these molecules in the human or experimental t. cruzi infections. the association of genetic markers with infectious disease has posed intriguing questions regarding the comparisons of gene frequencies between individuals with the disease with healthy individuals who had never been exposed to the specific infectious agent. due to the myriad of chronic clinical forms observed in chagas disease, part of these concerns may be circumvented by the possibility of comparing patients presenting well recognized clinical variants with patients who were infected by t. cruzi and have not developed clinically detected forms (indeterminate). when patients as a whole were compared to healthy individuals, some hla - g 3utr alleles / genotypes were overrepresented (+ 3003 t allele and + 3003tt and + 3187gg genotypes), while others were underrepresented (+ 3003c allele, and + 3003ct, + 3010gc, + 3042gc genotypes) in chagas disease patients. when symptomatic patients (cdm) were compared to healthy controls, similar results were obtained, indicating that these polymorphic sites were indeed associated with the clinically detected chagas disease. in contrast, when indeterminate patients were compared to controls, the frequency of the hla - g 3utr alleles / genotypes was closely similar. this result corroborates the role of the aforementioned hla - g 3utr alleles and genotypes in the clinically recognized chagas variants. the stratification of chagas patients into clinical variants showed that the + 3027cc and + 3035cc genotypes and the + 3027c and + 3035c alleles were strongly associated with the digestive form of chagas disease. in addition, the + 3142gc genotype was associated with a decreased risk to cardiac form development, when compared to the digestive form. these results indicate that genetic susceptibility to digestive or cardiac forms may be distinct. indeed, previous studies conducted by our group evaluating classical histocompatibility (hla - a, -b, -drb1 and dqb1) antigens / alleles and other immunoregulatory genes, such as ctla-4, also showed a differential pattern of susceptibility according to the chagas clinical variant. no specific posttranscription regulation mechanism has been reported concerning the + 3027a / c and + 3035c / t polymorphic sites ; however, in a previous in silico study, we reported several mirnas that may target the + 3027a / c and + 3035c / t polymorphic sites. when hla - g 3utr polymorphic sites were considered as haplotypes, decreased utr-4 and utr-7 frequencies were associated with the clinically detectable chagas disease and with the digestive form, respectively. on the other hand, noteworthy, particular posttranscription elements that are present in these utrs also exhibited a similar differential behavior when their frequencies were individually evaluated, further corroborating the role of hla - g 3utr sites in disease susceptibility. our group demonstrated that specific hla - g 3utr haplotypes are associated with differential soluble levels of hla - g (shla - g), being utr-1 associated with high shla - g and utr-5 and utr-7 with decreased shla - g levels. however, there are no studies associating hla - g 3utr haplotypes with tissue hla - g expression. considering that cytokines, chemokine, and immunomodulatory molecules play an important role in the pathogenesis of many diseases, including chagas disease, and gene polymorphisms may influence the expression of these molecules, different studies have reported the association between different clinical forms of chagas disease and polymorphisms at tnf [5761 ], il-1, il-10, il-4, inf-, tgf-1, il-12b, cxcl9, cxcl10, ccr5 [6870 ], ccl2, and ctla-4 genes. regarding hla - g expression on tissue cells, patients with the digestive form exhibited decreased expression in colon but not in esophagus specimens, contrasting with patients exhibiting the cardiac variant who presented a decreased expression on myocardium cells. many segments of the digestive tract and normal myocardium fibers may constitutively express hla - g ; however, the meaning of these findings has not been completely elucidated. the best characterized constitutive hla - g expression is in the placenta, where it may provide protection to the fetus against the cytotoxic cell immune response against paternal antigens. hla - g constitutive expression in gut and heart may in fact present a similar protective effect. indeed, increased plasma levels of shla - g and myocardium hla - g expression have been associated with decreased cellular acute rejection and better graft survival in patients submitted to heart transplantation [7375 ]. then, the loss of hla - g expression on myocardium cells may facilitate the action of infiltrating lymphomononuclear cells after tissue injury caused by t. cruzi. on the other hand, myocardium infiltrating lymphomononuclear cells since myocardium damage has been primarily attributed to fiber losses due to the action of immune cytotoxic cells, one may consider that hla - g lymphomononuclear cell expression may not be the only regulatory surface cell molecule. in addition, it is important to emphasize that all these findings were observed in heart specimens of deceased patients, and the temporal hla - g expression during the chronic phase has not been evaluated as yet. overall, the outcome of the experimental t. cruzi infection may depend on several factors, including (i) the inoculated strain, (ii) the amount of parasites, and (iii) the genetic background of the animal. several of these combinations have been used to induce acute or early chronic infection. balb / c mouse has been considered to be susceptible to acute t. cruzi infection induced by the y and tulahun strains, due to the predominance of a th2 immune response, characterized by high production of il-4 and il-10 [3, 76 ]. on the other hand, c57bl/6 mouse is considered to be resistant to acute t. cruzi infection induced by y and tulahun strains, since the mouse produces a th1 immune response with the production of inf-, il-12, tnf, and no [3, 14, 15 ]. in the present study, we used the less virulent t. cruzi cl strain that depending on the dose may produce severe acute or lead to the development of early chronic infection. in balb / c mice, the acute infection by cl strain induced higher heart parasitism and parasitemia with increased production of proinflammatory mediators. since balb / c is more susceptible to t. cruzi infection, chronic infection is difficult to be induced by more virulent strains. on the other hand, little is known about the acute and early chronic infection induced by cl strain in c57bl/6 mice. with the use of the cl strain we did not observe a differential susceptibility in balb / c and c57bl/6 mice in terms of mouse survival ; however, balb / c showed higher parasitemia, whereas c57bl/6 presented higher heart parasitism. therefore, the c57bl/6 controlled cl strain parasitemia but not heart tropism, and the inverse occurred with the balb / c mouse. similarly to hla - g, the qa2 molecule may also control the immune response by inhibiting nk cells. during acute t. cruzi infection, the transcriptional level of qa2 was increased in heart and esophagus in both balb / c and c57bl/6 mouse strains, and the increased expression was not related to resistance or susceptibility to the acute infection. although little attention has been devoted to the role of immunomodulatory nonclassical mhc molecules in experimental infection, qa2 expression may be induced to counterbalance the action of proinflammatory and other anti - inflammatory or immunomodulatory molecules. indeed, the transcriptional levels of the qa1, ctla-4, pdcd1, inf-, nos-2, and il-10 genes were also induced in the acute t. cruzi infection, as shown in this study. tissue damage observed in the affected tissues is due to the inflammatory process generated by an exacerbated immune response triggered against the parasite, and the high production of ctla-4, pd-1, and il-10 is related to control inflammatory process during t. cruzi infection [3, 14, 15, 23, 24, 78 ]. our data suggest that the augmentation of immunomodulatory gene expression during the acute phase, including qa2 gene, may be associated with the control of tissue damage caused by inflammatory process, cytolysis, and fibrosis. regarding the early chronic infection, the expression of immunomodulatory, proinflammatory, and anti - inflammatory genes analyzed in this study was closely similar to noninfected mice in both balb / c and c57bl/6 strains, and both animal groups were able to evolve into the early chronic infection, presenting only a little amount of t. cruzi in the heart. considering that mice were exposed to decreased number of the less virulent cl strain, it is reasonable to conclude that both balb / c and c57bl/6 mice had time and chance to mount an adequate immune response against the parasites. we present several lines of evidence pointing to the participation of the immunomodulatory molecules hla - g and its functional murine homolog qa2 on human and experimental t. cruzi infection. in terms of chagas disease susceptibility, hla - g 3utr alleles / genotypes / haplotypes exhibited differential frequencies in infected / diseased patients when compared to only infected patients and healthy controls. the observation of a decreased hla - g expression on cardiac muscle and colonic cells associated with the increased expression of hla - g on myocardium infiltrating lymphomononuclear cells may reflect a lack of protection of these tissues that constitutively express hla - g. in experimental infection, the mouse genetic background exerted only a mild influence on the overall response against the t. cruzi cl strain. the transcriptional levels of qa2, qa1, ctla-4, pdcd1, inf-, nos-2, and il-10 genes were induced only during the acute t. cruzi infection in balb / c and c57bl/6 mice, indicating a fine balance between pro- and anti - inflammatory genes.	genetic susceptibility factors, parasite strain, and an adequate modulation of the immune system seem to be crucial for disease progression after trypanosoma cruzi infection. hla - g and its murine functional homolog qa2 have well - recognized immunomodulatory properties. we evaluated the hla - g 3 untranslated region (3utr) polymorphic sites (associated with mrna stability and target for microrna binding) and hla - g tissue expression (heart, colon, and esophagus) in patients presenting chagas disease, stratified according to the major clinical variants. further, we investigated the transcriptional levels of qa2 and other pro- and anti - inflammatory genes in affected mouse tissues during t. cruzi experimental acute and early chronic infection induced by the cl strain. chagas disease patients exhibited differential hla - g 3utr susceptibility allele / genotype / haplotype patterns, according to the major clinical variant (digestive / cardiac / mixed / indeterminate). hla - g constitutive expression on cardiac muscle and colonic cells was decreased in chagasic tissues ; however, no difference was observed for chagasic and non - chagasic esophagus tissues. the transcriptional levels of qa2 and other anti and proinflammatory (ctla-4, pdcd1, il-10, inf-, and nos-2) genes were induced only during the acute t. cruzi infection in balb / c and c57bl/6 mice. we present several lines of evidence indicating the role of immunomodulatory genes and molecules in human and experimental t. cruzi infection.
	xavier university school of medicine admits students mainly from the united states and canada to the undergraduate medical program. a previous study conducted in june 2013 used the dundee ready educational environment measure to measure the educational environment and impact of different teaching and learning methods in the program. the present study aims to obtain information about students ' perceptions of changes in the educational environment, which underwent modifications in teaching and learning, in january 2014. information was collected about the participants ' semester of study, gender, nationality, and age. students ' perceptions of the educational environment were documented by noting their degree of agreement with a set of 50 statements grouped into five categories. average scores were compared among different groups. the mean total and category scores were compared to those of the 2013 study. sixty of the sixty - nine students (86.9%) who enrolled in the undergraduate medical program participated in the survey. the majority were male, aged 20 - 25 years, and of american nationality. the meansd total score was 151.3218.3. the mean scores for students ' perception in the survey categories were perception of teaching / learning (38.45), perception of teachers (33.90), academic self - perceptions (22.95), perception of atmosphere (36.32), and social self - perception (19.70). there were no significant differences in these scores among the different groups. all scores except those for academic self - perception were significantly higher in the present study compared to the previous one (p<0.05). the above results will be of particular interest to schools that plan to transition to an integrated curriculum.
qm / mm methods (for reviews, see, e.g., refs (17)) have been used extensively in studies of enzymatic reactions. in order to obtain reliable results, it is crucial to perform extensive sampling and also to use a reliable qm model for either calibrating a semiempirical model (e.g., the evb model) or actual determination of ab initio (ai) qm (ai)/mm free energy surfaces. in considering the reliability of such methods, one of the obvious issues is the size of the qm region. the size problem is frequently attributed to the treatment of the boundaries between the qm and mm regions, but this issue is not so serious if one uses the same models in the reference water reaction and in the enzyme active site. nevertheless, one would like to know the limitations imposed by the size of the qm region. here we noted that the great advances in computer power and the use of specialized computers (e.g., gpu) allows one to use very large qm regions and the results of the corresponding calculations can be used to imply that it is essential to use large qm regions to obtain reliable results. thus, it is important to conduct careful analysis of the size dependence of qm / mm calculations. the issue of the size dependence in qm clusters has been a subject of significant interest. similarly, the size dependence of the qm region in qm / mm models has been the subject of recent studies. some studies explored the size dependence of qm / mm calculations of spectroscopic properties. more relevant works (from the perspective of this paper) examined the energy difference in enzymatic reactions. however, as much as enzymatic reactions are concerned, the most important issue is the reliability of the calculated activation free energy, that should reflect an extensive averaging, that has not been performed in most of the previous studies. one exception is the study of ref (26), which evaluated the free energy of proton transfer (pt) in lysozyme for qm regions of different sizes. this work found that for a qm with a radius of less than 6 we can have errors of about 3 kcal / mol. here we note that pt reactions involve a rather small change in charge separation and one can expect a larger dependence on the size of the qm region in reactions that involve a larger change in charge distribution. thus, we focus in this work on the dependence of the calculated activation free energy on the size of the qm region in the reaction of the enzyme comt (catechol - o - methyl transferase) that catalyzes the transfer of a methyl group from sam (s - adenosyl - l - methionine) to a catecholate ion via an sn2 type reaction, as shown in scheme 1. the sam unit is truncated to the methyl group for clarification. the issue of the size of the qm region in comt was highlighted recently in a study which implied that the inclusion of large protein residues (up to 500 atoms) in the qm region is needed in order to obtain convergent results. while ref (11) only looked at the convergence of ground state properties including the donor acceptor distance, ref (12) examined the trend in activation energy, but this was done by evaluating the single point energy change upon moving to the transition state (rather than the activation free energy). nevertheless, such results may discourage further use of qm / mm calculations with medium qm size and thus require further systematic studies that will be performed in this work. in choosing comt as our benchmark it belongs to the general class of methyltransferases that plays an important role in human physiology and several diseases such as cancer, and genetic diseases. comt degrades catecholamines such as dopamine and has thereby attracted a major interest in its probable role in dopamine related pathologies, in particular parkinson s disease. v158 m comt polymorphism is speculated to be responsible for the violent behavior of schizophrenic patients. it is therefore imperative to study such enzymes and understand their function at the molecular level. it should also be noted that the comt catalyzed decomposition of dopamine follows a parallel pathway to the reaction catalyzed by mao b, which was recently studied using the evb method. hopefully, the present study can also be useful by shedding additional light on the results and in some cases limitations of previous qm / mm studies of comt. our study focuses on the dependence of the activation free energy of the reaction of comt on the size of the qm region. for very small sizes where, for example, the mg is treated classically, we find an obvious deviation from the converging results (although this problem can be fixed by semiempirical approaches with proper parametrization of the qm / mm interactions). for larger systems, our study used the semiempirical pm6 method (evaluated by the mopac2009 package) to treat the qm region, whereas the rest of the protein was treated classically, using the enzymix force field. the qm / mm interface between the qm and mm surfaces was implemented through the corresponding module of the molaris package. the initial coordinates for the qm / mm dynamics were taken from the crystal structure of the human, soluble form of comt (s - comt). the first step for each system involved a relaxation by slowly heating the protein from 30 to 300 k for at least 45 ps. the subsequent md simulations involved time steps of 1 fs at 300 k. the simulation was divided into 31 frames with each frame simulated for 10 ps. (o c), was constrained using a force constant of 100 kcal / mol and varied from 1.0 to 2.2. the pmf was obtained by combining the individual simulation windows, using the weighted histogram analysis method (wham) approach. further calculations for a few systems (i, ii, and iii) were done for a longer time, by dividing the simulation into 51 frames and running each window for 30 ps. this yielded similar results to the shorter runs, and therefore, for all other systems, we only carried out shorter runs. the boundary conditions for the simulations were introduced by immersing the protein in an 18 sphere of water molecules using the surface - constraint all - atom solvent (scaas) boundary conditions. the local reaction field method (lrf) which is similar to having no cutoff for electrostatic interactions is used to treat the long - range effects. the geometric center of the reacting system was taken as the center of the simulation sphere. the positions of all atoms beyond 20 from the center of the systems were fixed as found in the initial crystal structure, and their nonbonded interactions with the atoms within the simulation sphere were turned off. to determine the protonation state, we used our coarse grained (cg) model. subsequently, all residues that lie within 12 from the center of the sam and catecholate anion were explicitly ionized during the simulation. a large charge charge dielectric constant was used to treat the effect of the more distanced ionizable residues. three different starting configurations were generated for the simulations by taking structures from a long trajectory at a time difference of 45 ps. the resulting structures were used in the qm / mm simulation, and the final result was taken as the average of the three runs. obviously, more correct barriers should be obtained with high level qm methods, but the issue here is the size dependence of the barrier and not obtaining an accurate barrier. of course, we hope that the size dependence of the pm6 is related to the size dependence in more rigorous models. our study focused on the activation barrier for the methyl transfer step, which is depicted in scheme 1. the different residues considered as a part of the qm region are shown in figure 1. the active site consists of mg ion coordinated to the catecholate ion, two aspartate residues, asparagine residue, and a water molecule. the transition state with the mg ion and the ligands in the primary shell is shown in figure 1c for a representative case. (b) a schematic representation of the active site of comt enzyme with some important residues. (c) the transition state for the methyl transfer reaction between sam and catecholate ion for a representative case (distances are in). in order to explore the size dependence of the qm region the smallest region consisted of the substrates alone, while the largest region included the metal and seven residues, which were selected on the basis of the distance from the substrates. the first qm region investigated (system i) is described in figure 2. this system includes only the two substrates, sam and the catecholate ion, and consists of 26 atoms in the qm region including the linked atoms. the mg metal ion was treated classically as a point charge with a repulsive van der waals center. system i with only the substrates (catecholate and sam) as the qm region and system iii with the substrates, mg ion, and the surrounding ligands as the qm region. the qm part is shown as sticks and part of the mm region as wires (the rest of the protein is not shown for clarity). the next system considered (system ii) also included only the substrates in the qm region. however, the mg was treated using the dummy atom approach developed by qvist and warshel. this approach, which involves the inclusion of six dummy atoms around the metal center, as depicted in figure 3, was shown to yield improved solvation energies. md6 model complex used instead of mg in the classical simulation of system ii, where the ds represent the dummy atoms and the ls are the general ligands around mg. subsequently, the mg ion and the surrounding ligands (h2o, asp169, asp141, and asn170) were included in the qm region (system iii shown in figure 2). it should be noted that only a part of the residues as shown in figure 2 was included in the qm region. inclusion of the complete residue did not alter the result significantly, and hence, in all further calculations, we used chopped residues as the ligands coordinated to the mg ion. the change in the barrier upon moving to system iii reflects qm charge transfer effects, which are discussed below. next, we considered the effect of individual amino acid residues near the active site by including them separately in the qm region, while still retaining the residues in system iii (figure 4). the different residues considered are glu199 (system iv), lys144 (system v), tyr68 (system vi), trp143 (system vii), and met40 (system viii). lys144 and glu199 form h - bonds with the catecholate ion and result in a charged qm system. the free energy barriers obtained for systems iv and v were 16.4 and 16.5 kcal / mol, respectively. during the simulations, the hydroxyl proton at the catecholate shuttles between the o atom of the catecholate and the o atom of the glu residue. as will be discussed below, this corresponds to another reaction path and should be considered accordingly. the inclusion of tyr 68 (which is considered to be an important residue as its mutation decreases significantly the activity) yields a barrier of 15.8 kcal / mol. the inclusion of met40 and trp143 yields activation free energies of 15.9 and 17.1 kcal / mol, respectively. we also included in the qm region tyr147 (via) and w38 (viia), which are approximately 10 away from the center of the substrates. this yields barriers of 16.4 and 16.9 kcal / mol, which are similar to the barriers in other systems where the residues are much closer to the active site. the activation free energies in kcal / mol are provided in parentheses. after studying the effect of individual residues, we included both lys144 and glu199 (system ix) together with the mg metal and its ligands (figure 4). these two residues lie closest to the substrates forming weak h - bonding interactions and are therefore included in most of the systems described hereafter. further, we included three residues together keeping the mg and its surrounding ligands, lys144 and glu199, constant. in system xiii, we studied the effect of another residue, trp43, on the activation barrier. the different systems created (figure 5) are (a) lys144, glu199, trp143 (system x) ; (b) lys144, glu199, tyr68 (system xi) ; (c) met40, tyr68, trp143 (system xii) ; and (d) trp 143, trp43, tyr68 (system xiii). in the final and largest qm systems, we included four residues together, namely, lys144, glu199, trp143, and met40, resulting in system xiv and residues lys144, glu199, trp143, and tyr68 that generated system xv. systems that include in the qm region three residues along with the mg and its surrounding ligands. the activation free energies in kcal / mol are provided in parentheses. in total, 15 different qm systems have been considered and it has been found that the free energy barrier lies in the range 14.519.4 kcal / mol. the largest system including seven residues does not show much improvement over the system containing the mg and ligands alone. on the basis of our results, we propose that mg and its surrounding ligands (system iii) are sufficient to be included in the qm region. furthermore, we also carried out qm / mm studies for the y68a mutant, where the experimental free energy barrier for the mutant is 20.0 kcal / mol whereas that of the wild type is 18.4 kcal / mol. in this mutant case, we only investigated systems iii and i. it was found that, while the calculated free energy for wild type with system i is 22.5 kcal / mol, the corresponding barrier for y68a is 26.8 kcal / mol (leading to an increase of 4.3 kcal / mol in the barrier). in the case of system iii, the barriers for the wt and the mutant are 14.5 and 16.6 kcal / mol, respectively (leading to a 2.1 kcal / mol increase in barrier). it can be seen that the trend on increase in barrier upon mutation is reproduced by both systems (where system i gives a significant overestimate). the results of the above study are summarized in figures 6 and 7 as well as table 1. the analysis of the results can start with the obvious finding that cases that do not include the ion and its ligands in the qm systems (cases i and ii) present a major approximation, since the corresponding qm treatment does involve major charge transfer (ct). as we pointed out, the inclusion of the mg ion in the qm region requires inclusion of the ligands of this ion, and such a treatment can be very expensive when one uses a reasonable level of the qm treatment. fortunately, treating the mg ion classically and using the proper parametrization can give reasonable results. the pmfs obtained for different systems for the pm6/mm potential, which have been calculated using the wham procedure. all systems include the substrates : sam and catechol (shown in wires). another case, which requires special attention, is the inclusion of glu199 in the qm system (in addition to system iii) without including additional residues. in this case, we find a partial proton transfer (pt) from the catecholate to glu199. however, we actually have here a trivial case of another reaction mechanism (namely, a pt to glu199). of course, one should examine whether we have a real pt or an artifact of the use of the pm6 method. in fact, the problem disappears already when we add lys144 or additional residues to the qm region. we also find that adding lys144 alone to system iii also leads to a deviation in the reaction free energy. now again we have a residue that is involved in the reaction, where a pt from the catechol to the lys can be considered as an initial step of the reaction (see also below). here a partial pt for the back reaction can appear in small qm regions, but again adding more residues leads to a disappearance of the deviations. we like to note that, although lys144 is the most likely candidate to accept the proton from catechol leading to the formation of catecholate ion, it is still unclear whether the proton stays on lys144 or is transferred to some other residue or water molecules. the close proximity of this residue to mg (3.2) in the crystal structure results in this ambiguity, as it may lead to the low pka of these residues. in fact, the finding that the lys144ala mutant leads to a minor reduction in kcat indicates that the lys does not play a major role in the rate acceleration. thus, although our previous evb calculations gave a somewhat larger catalytic effect with neutral lys144, we kept this residue ionized here (in the cases when it was in the classical region). regardless of the actual protonation state of the lys residue during the nucleophilic attack, our analysis of the size dependence is still valid for the model system used. overall, we note that, while the convergence of the activation free energies is encouraging, the convergence for the reaction free energies is not as good as that of the activation free energies (table 1), for single residues that can be involved in side reactions like pt. nevertheless, once other qm residues were included with the problematic residues, we obtained converging results. this feature was found for glu199, lys144, and, for less clear reason, trp143, where in all cases adding more residues leads to the disappearance of this problem. qm / mm approaches provide a major tool for evaluating the energetics of chemical reactions in complex molecular systems. however, some workers can assume that the results depend drastically on the size of the qm system and thus presumably the qm / mm idea is inherently a major approximation. apparently, what is meant by approximation is not uniformly agreed upon. for example, some may assume that an accurate result can be obtained only when the protein and the solvent are represented by a high level ab initio approach. however, obtaining reliable results also requires a major sampling and satisfying the requirement of both a proper sampling and a reliable qm approach is an extremely challenging task that would be hard to accomplish in the near future. there are ways to use large qm systems, including our constraint dft (cdft) approach, or the use of a fast processor or specialized computers that allow running with large qm systems. however, the requirement of extensive sampling makes it crucial to exploit qm / mm approaches with a limited size of the qm region, while exploring systematically what are the limitations of such strategies. this work explored the approximations associated with the use of a relatively small size for the qm region, by a systematic analysis of this issue while modeling the free energy profile of the reaction of comt. as seen from figure 8, the convergence of the activation free energy is quite reasonable (except for models i and ii, as discussed below, the reasons for those deviations are obvious). convergence of the activation free energy as a function of the number of qm residues. the numbers in brackets are the number of qm atoms for the given system. the deviations between the results with different sizes of the qm region are more pronounced when we compare the calculated exothermicity (table 1). in analyzing the corresponding results first, we note that the cases that do not include the ion and its ligands in the qm systems present a major approximation, since the corresponding qm treatment does involve charge transfer (ct). thus, we do have here an obvious case where one should try to include the mg in the qm treatment. fortunately, the ct effect can be captured by changing the charge on the mg and the ligands and by proper refinement of the van der waals parameter without the use of computationally expensive methods. the ct can also be represented by including the mg in an evb treatment that can reproduce this effect. furthermore, even without including the mg in the evb region, we can easily reproduce the trend obtained with the mg in the qm region by a standard evb treatment that includes parametrization of the reaction exothermicity. as stated in section iii, the convergence for the reaction free energy is not as good as the activation free energies. this is in particular true for single residues (i.e., glu199 and lys144) that can be involved in side reactions such as pt reactions. however, once such residues are surrounded by other qm residues, we obtain converging results. such a behavior seems to reflect instability of the qm calculations, which do not follow a simple additive trend. this is reflected in the observation that the problem does not occur when the problematic residues are part of the mm system. it is also likely that the size dependence will be reduced if we use a polarizable force field. it should also be noted that our reaction is not a conventional sn2 reaction, as it involves two charged species that react to give a neutral product, thereby posing difficulties that arise in an sn1 reaction. thus, the solvation free energy of the reactive ionic species might result in a sensitivity of the reaction free energy. some workers tend to attribute the main source of the size dependence problem to the connection between the qm and mm link atom. however, a part of this problem is drastically reduced by using hybrid orbitals. furthermore, the problem is drastically reduced if one uses the same boundaries for reactions in water and reactions in enzyme as is done with the evb method. it is important to point out that even with some dependence on the qm size it is possible to explore quite reliably the catalytic and mutational effect. this issue has been demonstrated here with the wt to y68a mutations in two different qm descriptions. such a finding, for a mutant that is the subject of significant controversy, is clearly important in view of the possible presumption that one must use large qm regions in exploring mutational effects. reference (12) obtained about 3 kcal / mol deviation from the final result for about 10 residues but then suggested that the convergence requires at least 30 residues (where the deviation from the converging result is about 23 kcal / mol). ignoring the issue that the calculations are based on a single point energy evaluation, we like to address the implication that one needs to get around 2 kcal / mol convergence before exploring catalytic problems. first, obtaining 2 kcal / mol errors due to the size of the system still allows exploring enzyme catalysis, when the difference between the barrier in enzyme and in water is frequently on the order of 10 kcal / mol. of course, it is also allowed (as shown here) to explore mutational effects. second, no first principle approach can at present give errors of less than 2 kcal / mol. that is, changing the level of the quantum method can lead to several kcal / mol errors and adding a polarizable force field can change the results by a few kcal / mol. obviously not performing sampling can lead to enormous errors. furthermore, very large errors can also be obtained from evaluation of electrostatic contributions without proper relaxation of the protein. such calculations may produce an unphysical low dielectric that would not provide sufficient screening for charge charge interaction. apparently, even proper free energy calculations still involve several kcal / mol errors due to incomplete sampling, which, for example, can miss water penetration effects. the above - mentioned errors are inherent to current calculations, and once we are aware of such errors, we can better assess the effectiveness of different approximations. for example, the error can frequently be reduced by using a well - defined reference (e.g., the wt barrier in mutational studies and the reaction in water in exploring catalysis). as usual, the best way of exploring different approximations is to change the length of the run, the system size, and other factors and to explore the stability of the calculations as well as the relationship to the observed quantity. it might also be pointed out here that there are various implications that the catalytic effect deduced from calculations with limited qm size (mainly the conclusion that the catalysis is associated with electrostatic effects) might be questionable and can be changed with larger qm sizes. now, not only have evb calculations seemed to give extremely stable conclusions, but also equally importantly, other proposed catalytic factors (e.g., dynamical effects, quantum effects, and more) have never been reproduced by consistent calculations regardless of the size used. overall, the present study indicates that the size dependence is not as crucial as one may suspect. in particular, the size dependence is significantly smaller than the catalytic effect. of course, the residues that are in direct contact with the reacting atoms can be involved in a major charge transfer or even in another reaction path. however, the qm description of residues that are not in direct contact do not change the activation barriers of chemical transformation in a major way.	although qm / mm calculations are the primary current tool for modeling enzymatic reactions, the reliability of such calculations can be limited by the size of the qm region. thus, we examine in this work the dependence of qm / mm calculations on the size of the qm region, using the reaction of catechol - o - methyl transferase (comt) as a test case. our study focuses on the effect of adding residues to the qm region on the activation free energy, obtained with extensive qm / mm sampling. it is found that the sensitivity of the activation barrier to the size of the qm is rather limited, while the dependence of the reaction free energy is somewhat larger. of course, the results depend on the inclusion of the first solvation shell in the qm regions. for example, the inclusion of the mg2 + ion can change the activation barrier due to charge transfer effects. however, such effects can easily be included in semiempirical approaches by proper parametrization. overall, we establish that qm / mm calculations of activation barriers of enzymatic reactions are not highly sensitive to the size of the qm region, beyond the immediate region that describes the reacting atoms.
the cohort for this study consisted of 603 patients who underwent breast resection at sun yat - sen university cancer center for breast cancer between january 3, 1990 and january 6, 2005. the age at diagnosis ranged from 19 to 82 years (median, 56 years). all patients had axillary lymph node dissections without evidence of lymph node metastasis and all primary tumors were confined to the breast (5.0 cm). tumor size and lymph node number were obtained from the original macroscopic and microscopic descriptions of specimens. eighty - eight percent of the patients underwent modified radical mastectomies, and the remaining 12% underwent partial breast resections. a total of 27 surgeons performed the operations, and 18 pathologists were involved in pathologic diagnosis. the patients were stratified into two groups based on the number of tumor - free lymph nodes removed : group a, 10 or fewer lymph nodes, and group b, more than 10 lymph nodes. follow - up information obtained from sun yat - sen university cancer center for each patient began at the date of surgery and ended with either the date of death or the study termination date (february 1, 2014). patients were followed for 1283 months, with a median follow - up time of 126 months. death information was directly collected by the cancer center through active physician follow - up or direct communications with next of kin. demographic data by the number of lymph nodes removed were analyzed by chi - square test. cox proportional hazards regression was used to examine the independent association between the number of tumor - free lymph nodes removed and the risk of death from breast cancer. models included age (< 40, 4150, 5160, 6170, and 71 or more years), marital status (not married, married, or unknown), tumor size, grade, and adjuvant therapy. tumor size was recoded as 1 cm or less (ajcc stage t1a and b), 1.1 to 2.0 cm (stage t1c), and 2.1 to 5.0 cm (stage t2). tumor grade was classified as 1 (well differentiated), 2 (moderately differentiated), 3 (poorly differentiated), or 4 (anaplastic or undifferentiated). the cohort for this study consisted of 603 patients who underwent breast resection at sun yat - sen university cancer center for breast cancer between january 3, 1990 and january 6, 2005. the age at diagnosis ranged from 19 to 82 years (median, 56 years). all patients had axillary lymph node dissections without evidence of lymph node metastasis and all primary tumors were confined to the breast (5.0 cm). tumor size and lymph node number were obtained from the original macroscopic and microscopic descriptions of specimens. eighty - eight percent of the patients underwent modified radical mastectomies, and the remaining 12% underwent partial breast resections. a total of 27 surgeons performed the operations, and 18 pathologists were involved in pathologic diagnosis. the patients were stratified into two groups based on the number of tumor - free lymph nodes removed : group a, 10 or fewer lymph nodes, and group b, more than 10 lymph nodes. follow - up information obtained from sun yat - sen university cancer center for each patient began at the date of surgery and ended with either the date of death or the study termination date (february 1, 2014). patients were followed for 1283 months, with a median follow - up time of 126 months. death information was directly collected by the cancer center through active physician follow - up or direct communications with next of kin. demographic data by the number of lymph nodes removed were analyzed by chi - square test. cox proportional hazards regression was used to examine the independent association between the number of tumor - free lymph nodes removed and the risk of death from breast cancer. models included age (< 40, 4150, 5160, 6170, and 71 or more years), marital status (not married, married, or unknown), tumor size, grade, and adjuvant therapy. tumor size was recoded as 1 cm or less (ajcc stage t1a and b), 1.1 to 2.0 cm (stage t1c), and 2.1 to 5.0 cm (stage t2). tumor grade was classified as 1 (well differentiated), 2 (moderately differentiated), 3 (poorly differentiated), or 4 (anaplastic or undifferentiated). in all cases, the proportion of patients with 10 or fewer tumor - free lymph nodes removed was 60.7%, and the proportion with more than 10 tumor - free lymph nodes removed was 39.3%. the 5- and 10-year survival rates were higher for patients in group a, who had 10 or fewer tumor - free lymph nodes removed, than for patients in group b, who had more than 10 tumor - free lymph nodes removed (88.0% and 66.4% vs. 69.2% and 51.5%, respectively, p < 0.001 ; figure 1). in the multivariate cox proportional hazards models with all patients, the adjusted hazard ratios (hrs) for risk of death from breast cancer were higher for patients with larger tumors (p < 0.001), patients with tumors of higher grade (p < 0.01), and patients who did not undergo adjuvant therapy (p < 0.001). it was also elevated for patients who were younger at diagnosis but was lower for married patients versus unmarried patients (table 2). compared with patients with primary tumor 1 cm or less in diameter, patients with primary tumor 1.1 to 2.0 cm in diameter and patients with primary tumor 2.1 to 5.0 cm in diameter had higher adjusted hrs for risk of death from breast carcinoma of 1.539 [95% confidence interval (ci), 1.2681.901 ; p < 0.001 ] and 2.351 (95% ci, 1.7962.889 ; p the adjusted hrs for risk of death from breast cancer were elevated for high or unknown tumor grade versus low tumor grade (adjusted hr = 1.288 and p = 0.006 for grades 3 and 4 ; adjusted hr = 1.795 and p < 0.001 for unknown grade). compared with patients who did not undergo adjuvant therapy, patients undergoing adjuvant therapy had a lower adjusted hr for risk of death from breast cancer (adjusted hr, 0.352 ; 95% ci, 0.2960.433 ; p < 0.001). the adjusted hr for patients with 10 or fewer nodes removed was significantly reduced compared with that for patients with more than 10 nodes removed (adjusted hr, 0.579 ; 95% ci, 0.4920.687 ; p < 0.001). kaplan - meier analysis indicated differences in survival curves between group a and group b (p < 0.001). in this study, we found that patients who had more than 10 tumor - free lymph nodes removed had a higher risk of death from breast cancer compared with patients who had 10 or fewer tumor - free lymph nodes removed. we chose 10 as a critical value because the median number of lymph nodes removed in this study was 10, which was similar to the value in a previous study. hence, simple pursuit of a higher number of tumor - free lymph nodes removed may be of little use for improving survival rate. our finding of an association between the number of tumor - free lymph nodes removed and the risk of death from causes other than breast cancer suggests possible confounding with comorbidity. in other words, surgeons may not want to perform axillary dissection, or may perform less extensive dissection, on patients with comorbid conditions. in contrast to previous reports, this study also included analysis of adjuvant therapy, which was useful for lymph node negative patients, as a possible confounding factor ; a previous report suggested adjuvant therapy had prognostic significance in patients with lymph node negative breast cancer. thus, although camp. reported results similar to ours, they did not take into account of adjuvant therapy as a possible confounding factor. our results were also in contrast to those from other studies. sosa. studied 464 patients with lymph node negative invasive breast cancer that was 2 cm or smaller in diameter and found better survival among those with 10 or more (up to 33) lymph nodes removed compared with those with fewer than 10 lymph nodes removed. however, their analysis did not include tumors larger than 2 cm in diameter, and the number of deaths was too small for separate analysis of mortality from breast cancer versus other causes. similarly, studies by moorman. and polednak are limited by misclassifications (as indicated by reabstracting and recoding audits of stage and disease extent), the number of lymph nodes removed, and tumor size. other factors associated with the risk of death from breast cancer including age at diagnosis, marital status, tumor size, tumor grade, and adjuvant treatment were consistent with those reported in the literature,,. the observation that women with more tumor - free lymph nodes removed had a higher risk of death from metastatic breast cancer may be explained by the lack of host immune responses or defense by lymph nodes. under this hypothesis, other studies have focused on the prognostic benefit of host immune responses to tumor in particular, peritumoral lymphocytic infiltrates and alterations in the architecture of tumor - draining lymph nodes such as sinus histiocytosis. recent studies have begun to define the cytokines underlying lymph node genesis and hyperplasia. hyperplasia after lymph node genesis can occur as a downstream reaction to inflammation and/or tumor. in another study, 25% of the breast carcinomas examined expressed mrna for vascular endothelial growth factor - c (vegf - c), which specifically induces lymphangiogenesis,. however, further exploration is needed for the role of lymphangiogenic and lymph node development factors, especially in the context of tumor size and grade, as these factors may play a role in tumor metastasis for lymph node - negative breast cancer. a large proportion of patients underwent resection for breast cancer before 1998, when advances in adjuvant therapy were slim in china. notably, whether adjuvant therapy should be used after dissection in patients with lymph node - negative breast cancer remains to be determined. even if removal of more nodes did not, in itself, improve survival, which is a controversial issue, adjuvant therapy may be withheld from some node - negative patients, including a subgroup misclassified as node - negative. weir. demonstrated the recovery of a small number of negative lymph nodes at axillary dissection likely understages patients and leads to undertreatment, resulting in an increased regional relapse rate and poorer survival. the implication of a small number of nodes removed is that it reduces the prognostic value of the negative nodal status. for a variety of reasons, positive nodes have likely been missed in some of these patients, resulting in more regional relapses and poorer survival. thus, increases would occur in both the proportion of lymph node - positive cases with extensive nodal involvement (easily detected clinically or by removal of only a few nodes) and the proportion of false - negatives among lymph node - negative cases. the 5-year and 10-year survival rates of lymph node - negative patients would ostensibly decrease over time, but overall survival of all breast cancer patients would not decline unless either removal of nodes had a direct therapeutic effect or changes occurred in the use of adjuvant therapies that improve survival. another trend may be toward the use of a combination of pathologic and biologic tumor characteristics as predictors of axillary node status, at least for small tumors and especially for cases in which adjuvant therapy decisions do not rest fully on confirmation of nodal status.	recently, there has been controversy about the relationship between the number of lymph nodes removed and survival of patients diagnosed with lymph node - negative breast cancer. to assess this relationship, 603 cases of lymph node - negative breast cancer with a median of 126 months of follow - up data were studied. patients were stratified into two groups (group a, 10 or fewer tumor - free lymph nodes removed ; group b, more than 10 tumor - free lymph nodes removed). the number of tumor - free lymph nodes in ipsilateral axillary resections as well as 5 other disease parameters were analyzed for prognostic value. our results revealed that the risk of death from breast cancer was significantly associated with patient age, marital status, histologic grade, tumor size, and adjuvant therapy. the 5- and 10-year survival rates for patients with 10 or fewer tumor - free lymph nodes removed was 88.0% and 66.4%, respectively, compared with 69.2% and 51.1%, respectively, for patients with more than 10 tumor - free lymph nodes removed. for patients with 10 or fewer tumor - free lymph nodes removed, the adjusted hazard ratio (hr) for risk of death from breast cancer was 0.579 (95% confidence interval, 0.492 - 0.687, p < 0.001), independent of patient age, marital status, histologic grade, tumor size, and adjuvant therapy. our study suggests that the number of tumor - free lymph nodes removed is an independent predictor in cases of lymph node - negative breast cancer.
we present here a case of hereditary adamts 13 deficincy presenting as recurrent acute kidney injury. a 26-year - old male presented with history of decreased urine output, red urine for past 7 days following an episode of upper respiratory tract infection. there was no history of edema, hypertension, flank pain, fever, and jaundice. on examination, he was conscious and alert, with pulse rate of 86 beats per minutes, blood pressure of 122/76 mmhg with no postural drop. general physical examination was remarkable for pallor and petechial rash over lower limbs with no evidence of icterus, lymphadenopathy. there was no hepatosplenomegaly, chest was clear to auscultation, and heart sounds were normal. laboratory evaluation showed anemia (hb 7.2 g / dl) with reticulocytosis (7%) thrombocytopenia (platelet 52000 / mm), azotemia (blood urea nitrogen 56 mg / dl, creatinine 5.2 mg / dl). transaminases, protrombin time, and bilirubin were normal. bleeding time, clotting time, and activated partial thromboplastin time was normal. ldh was elevated to 1540 ; serum haptaglobin was depressed to t that causes the amino acid change r1219w). during the molecular screening of adamts13 gene, a homozygous variation of adamts13 gene has been also found in the intron 8 (987 + 69a > c), but without a pathogenetic significance. the results obtained suggest that our patient is affected by a congenital deficiency of adamts13, determined by a heterozygous mutation of adamts13 gene. patient showed a good response to plasma therapy in the form of normalization of platelet count (2.4 lac in last follow up), stabilization of serum creatinine to 1.4 mg / dl. von willebrand factor (vwf)-cleaving protease activity, or a disintegrin and metalloprotease, with thrombospondin-1-like domains (adamts-13) is a plasma metalloprotease that cleaves vwf multimers after excretion by endothelial cells. in its absence, large vwf multimers, congenital adamts 13 deficiency is classically associated with inherited ttp with characteristic neurologic features, this is the first report of it presenting as recurrent acute renal failure (without neurological features) and later followed by hypertension, proteinuria and chronic renal insufficiency. this is also the first report of successful use of regular plasma infusion in this setting. although liver is the primary site for adamts 13 synthesis, local synthesis of this factor has been demonstrated in kidney which may explain kidney specific manifestations of the disease in some cases. heterozygous mutation in exon 26 causing very low levels of adamts 13 was responsible for recurrent renal failure in our case. this mutation has been described as a cause of adamts 13 deficiency in patients with ttp. after repeated episodes of acute kidney injury our patient started developing chronic renal insufficiency as manifested by new onset hypertension, proteinuria and azotemia. curiously replenishing deficient adamts 13 by institution of regular monthly plasma infusions not only prevented further aki episodes but also led to control of proteinuria, hypertension and stabilization of renal function. if adamts 13 deficiency can cause chronic kidney disease as evident in our case and in one recent case report ; it will be interesting to explore the adamts 13 deficiency as a cause of chronic kidney disease where etiology is not obvious. to conclude, we have described a case of hereditary adamts 13 deficiency with only renal involvement at presentation (without neurological manifestations) in the form of recurrent aki- with hypertension, proteinuria and chronic renal insufficiency developing in long run.	we report here a case of 26-year - old male who presented with history of recurrent acute renal failure associated with microangiopathic hemolytic anemia and thrombocytopenia. adamts 13 deficiency due to mutation in the gene encoding for adamts 13 was identified as the cause. after eight episodes of acute kidney injury (aki), patient started developing hypertension, proteinuria, and renal insufficiency. treatment with regular monthly plasma infusions prevented further episodes of aki and stabilized the renal function. hypertension and proteinuria are controlled with angiotensin ii receptor blockers.
jatropha curcas is a tree of the euphorbiaceae family which has been used, due to the high oil content (4060%) of its seeds, as an alternative source of biodiesel. the residual seed cake is a low - value byproduct left after oil extraction which, however, has a high protein content. this seed cake, also, is highly toxic to a number of animal species due to the presence of different types of antinutritional components such as phytic acid, trypsin inhibitors, phenolic compounds, lectins (curcin), and saponins in high amounts [3, 4 ]. in addition to these, phorbol esters have been identified as one of the main compounds responsible for j. curcas toxicity. these compounds are referred to as tigliane diterpenes in which two hydroxyl groups are esterified to fatty acids and are well known for their tumor promoting activity. however, edible or nontoxic provenances have been reported to exist in mexico [3, 7 ] which contain negligible amounts of phorbol esters though the levels of the other antinutritional compounds are similar to those found in the toxic varieties. this would allow the seed meal from edible varieties to be processed and used as an economic source of protein for both humans and animals. plant protein isolates are very important in the food industry due to their high protein contents, which can reach 90%. the methods of preparation generally include the solubilization of proteins in basic solutions (ph 811) followed by precipitation using different techniques. acid precipitation at the isoelectric point, which usually ranges from ph 4.5 to 5.0, is the general method for plant protein isolation. nevertheless, there is a need to analyze and optimize these methods in order to have an optimum protein recovery from j. curcas seed cake that allows the minimization of the content of the antinutritional compounds mentioned before to improve the quality of the protein obtained. response surface methodology (rsm) and taguchi 's orthogonal array method are well - known optimization techniques which allow the building of an experimental design with the smallest number of experimental runs. rsm is a collection of statistical and mathematical techniques used for modeling, analyzing, and optimizing problems in science and engineering. there are many reports on the use of rsm for the optimization of biotechnology processes [13, 14 ], food processes, phenolic compound extraction, and protein precipitation methods. the taguchi method uses orthogonal arrays to study a large number of variables with a small number of experimental runs and provides information about all the parameters that affect the responses. this method has been used in several fields including the chemical, biotechnological, and food industries. the effectiveness and improvement of a sequential integration of the taguchi method and rsm (tm - rsm) has been previously demonstrated. the taguchi method is not able to find the real optimum value and the rsm even with the best experimental designs uses many runs when the initial number of variables is high. in the sequentially integrated approach, the taguchi method is used to screen and optimize the qualitative and discrete factors and the rsm uses these results in a new experimental design to model and optimize the quantitative and continuous variables with more accuracy to produce better solutions. the purpose of this work was then to optimize the protein isolation process from j. curcas l. seed cake by the isoelectric precipitation method via a sequentially integrated optimization approach (tm - rsm). the influence of four different factors (solubilization ph, extraction temperature, nacl addition, and precipitation ph) on the protein and antinutritional compounds content of the isolate will be analyzed. the edible j. curcas seeds used in this study were obtained from ripe fruits harvested in yautepec, morelos, mexico. the whole seeds (kernels plus shells) the seed cake obtained was milled using a kitchen blender (oster 10-speed blender, osterizer). the residual oil present in this flour was removed using hexane (boiling point 60c) in a soxhlet apparatus for 10 h. the defatted flour was dried and finally passed through a sieve (20-mesh screen). the crude protein (n 6.25) content of j. curcas seeds, seed cake, flour, and isolates was determined according to the standard methods of the association of official analytical chemists. the protein isolates were prepared with the different conditions indicated in the experimental designs following the general scheme of makkar.. the flour was suspended in distilled water (1 : 10) with or without nacl and the suspension was adjusted to alkaline ph with 1 n naoh and stirred at constant temperature for 1 h. the suspension was then centrifuged at 18000 g for 30 min at 15c. the supernatant was filtered and collected. the ph of this solution was adjusted with 1 n hcl for protein precipitation. the precipitated proteins were collected by centrifugation (18000 g, 30 min, 4c) and freeze - dried. total phenolics were extracted using acidified methanol and quantified by the folin - ciocalteu reagent and expressed as tannic acid equivalents 100 g. the phytic acid content of the samples was determined by the colorimetric procedure described by vaintraub and lapteva. suitable aliquots were diluted with distilled water to make 3 ml and then used for the assay. results were expressed as mg 100 g phytic acid, using as a standard phytic acid (sodium salt ; sigma, st. total saponin content was determined using the spectrophotometric method described by hiai.. the concentration of saponins was interpolated from a standard curve of different concentrations of diosgenin in 80% aqueous methanol and expressed as diosgenin equivalents 100 g. there are many operational variables which can affect the protein extraction during protein isolates preparation ; therefore, only the four more influential factors were chosen : solubilization ph, extraction temperature, nacl addition, and precipitation ph. based on the taguchi method, the l9-orthogonal array was constructed as shown in table 1. the protein and total phenolics content (%) of the isolates were considered as the response variables. the anova procedure was used to determine the percentage of contribution of each factor to the responses and to model the relationship between these factors and the selected response. the most important factors were selected to perform a second experimental design, a three - level box - behnken response surface design for the optimization of the protein extraction technique. all results from experimental designs were analyzed using the software design expert 7.0.3 (stat - ease, inc. first, in order to identify the significant influence of each factor tested on the response variables, an anova was performed with the results from the taguchi design shown in table 1. the influence of solubilization ph, extraction temperature, precipitation ph, and nacl concentration of the extraction solution on the protein and phenolics content of the isolates is shown in table 2. extraction temperature and precipitation ph were the parameters with the strongest influence on the protein content of the isolates. as reported, appropriate heat treatments might partially break down hydrogen and disulfide bonds, resulting in an improvement in protein dissolution rate and leading to obtain an isolate with higher protein content when temperature is increased. it has been reported that extraction in the presence of nacl has improved the extractability of proteins from some seeds [26, 27 ]. our results show that, unlike other seeds, the protein recovery decreased when nacl is added to the extraction solution at the range tested (0.3 and 0.6 mol l) by a salting - out effect on the jatropha proteins. however, the nacl addition in the extraction solution has no considerable impact on the final protein content of protein isolates (0.10% contribution). conversely, the total phenolics content was significantly and adversely affected by nacl concentration in the solution extraction and almost negligibly by the precipitation ph. temperature, precipitation ph, and nacl addition were then selected as independent variables and solubilisation ph was set constant to an 11 value in all experiments for the rsm. the three levels of these variables were the same as those used in the l9-orthogonal array performed previously. protein, total phenolics, phytate, and saponin content, of the isolates were taken as response variables in a box - behnken 3 experimental design. the responses obtained from each run of the experimental design were analyzed by multiple regression analysis using the design expert software (stat - ease, inc. the quality of the fit of the obtained model equations was expressed by the determination coefficient r and the significance of the models and their coefficients was evaluated by one - way anova and student 's t - tests, respectively. a quadratic equation was established on the basis of analysis of the box - behnken experimental data as follows : (1)protein content=+359.500930.41282t116.19415ph+7.69070nacl0.26063tnacl+6.79515e003t2 + 12.67662ph2. extraction temperature and precipitation ph were the parameters that affected more strongly the protein content of the isolates. as stated above, appropriate heat treatments might partially break down hydrogen and disulfide bonds, resulting in an improvement in protein dissolution rate which leads to a protein isolate with higher protein content when temperature is increased. on the other hand, tested the extraction ph of j. curcas proteins and found the lowest yields at a ph range of 4.0 to 4.3 ; these results suggest that isoelectric point of most jatropha proteins is very close or equal to 4 ; thus, a substantial amount of the protein solubilised at the alkaline ph is recovered when precipitation is performed at ph 4 (figure 1). some of the protein isolates were dark brown in color due to shell pigments that were solubilised and precipitated along with the proteins. the use of nacl increased the lightness of isolates in relation to those obtained by extraction with water. observed the same behavior when testing the extraction and isolation of proteins from defatted gevuina avellana seeds. the content of total phenolics ranged from 319 to 694 mg 100 g of protein isolate. this response was significantly affected by nacl concentration in the solution extraction and slightly by precipitation ph (64.86 and 9.88% of contribution, resp.). figure 2 shows the surface plots for the effect of both independent variables on the total phenolics content of protein isolates. it is observed that the content of these compounds decreased to a minimum point when the maximum concentration of nacl was added. the effect of extraction temperature per se can be considered negligible compared to the effect of the other factors. however, there is an important effect of the interactions between extraction temperature and precipitation ph as well extraction temperature and nacl concentration. the analysis of the desirability function showed multiple combinations of factors that allowed a minimization of the phytate content of protein isolates when extraction is performed at 60c and nacl is added (0.4 m) (figure 3). this effect may be due to activation of endogenous phytases and acid phosphatases by effect of temperature. these enzymes mediate the phytates release, allowing reactions between these and nacl present in the solution extraction to form their respective sodium salts.. carried out solubility measurements of dodecasodium phytate in pure water and in nacl solutions at different ionic strengths and found that the phytate solubility decreases drastically with an increase in ionic strength. so, the decrease of phytate content in the protein isolates carried out in nacl solution could be explained by a salting - out effect on the phytate salts formed during the solubilization which are discarded along with other nonsoluble compounds by centrifugation. alkaline solutions were used for protein extraction, and under these conditions, saponins are converted into sodium salts that are well soluble in water. by analyzing the anova results, the precipitation ph appeared to have a negligible effect ; however, it plays an important role since the acidification of the alkaline extract, in order to precipitate proteins, converts the saponins into water - insoluble forms which precipitate along with protein. the interaction of temperature and precipitation ph and the interaction of precipitation ph and nacl concentration have the strongest influence on the content of saponins of the protein isolates. the protein isolates obtained using pure water as extraction solution have the highest amount of saponins when the highest temperature is tested and the subsequent acid precipitation is carried out at ph 4 (figure 4). using the same conditions of temperature and extraction solution is possible to minimize the content of saponins just by performing the precipitation step at ph 5. in contrast, when nacl is added into the extraction solution at the maximum ionic strength (0.6 m), the saponin content in the protein isolates decreases drastically when the extraction is performed at 20c and precipitation at ph 4, and this decrease is directly proportional to the temperature extraction and the precipitation ph (figure 4(b)). the conditions for obtaining a j. curcas protein isolate from the seed cake depended strongly on the extraction temperature, nacl concentration, and precipitation ph and were determined by rsm. the optimal values obtained, in order to maximize the protein content and minimize, at the same time, the antinutritional factors content were solubilization at 20c into a solution of nacl 0.6 m and precipitation at ph 4. under these conditions, it was possible to obtain a protein isolate with 93.21% of proteins, 316.5 mg 100 g of total phenolics, 2891.84 mg 100 g of phytates, and 168 mg 100 g of saponins, differing just slightly from the predicted values by 0.30% (93.3%), 0.27% (317.36), 13.13% (3327.99), and 14.17% (146.76), respectively. the use of the sequentially integrated optimization allowed us to produce a j. curcas isolate with a higher protein content (93.21%) than previously reported. other authors reported protein contents of 90.1, 89, and 82% in their products [1, 2, 21 ]. in this work, a sequentially integrated approach was successfully applied to the optimization of conditions to prepare a jatropha curcas protein isolate. response surface methodology using a 3 box - behnken design was used as the optimization tool for attaining the conditions for a high - protein and low - antinutritional factors (phytic, saponin, and phenolic compounds) protein isolate from edible j. curcas seed cake. the estimated optimal conditions were an extraction temperature of 20c, a precipitation ph of 4, and an amount of nacl in the extraction solution of 0.6 m. by varying the conditions of preparation, isolates with different concentrations of protein and antinutritional factors can be obtained. the protein content of the isolate was higher than that of other reported protein concentrates or isolates.	jatropha curcas seed cake is a protein - rich byproduct of oil extraction which could be used to produce protein isolates. the purpose of this study was the optimization of the protein isolation process from the seed cake of an edible provenance of j. curcas by an alkaline extraction followed by isoelectric precipitation method via a sequentially integrated optimization approach. the influence of four different factors (solubilization ph, extraction temperature, nacl addition, and precipitation ph) on the protein and antinutritional compounds content of the isolate was evaluated. the estimated optimal conditions were an extraction temperature of 20c, a precipitation ph of 4, and an amount of nacl in the extraction solution of 0.6 m for a predicted protein content of 93.3%. under these conditions, it was possible to obtain experimentally a protein isolate with 93.21% of proteins, 316.5 mg 100 g1 of total phenolics, 2891.84 mg 100 g1 of phytates and 168 mg 100 g1 of saponins. the protein content of the this isolate was higher than the content reported by other authors.
the most prevalent osteoarthritis (oa) localization is the knee joint, and symptomatic knee oa (koa) affects 24% of the general population. low socioeconomic level, age, and obesity are directly correlated with the disease incidence. in 2010, the educational attainment of the brazilian population aged 10 years or older included 50.2% with no education or incomplete primary education and 7.9% with a college degree. low income, level of education, increased longevity, and obesity are major factors in an increased incidence of oa in brazil. international guidelines for the treatment of koa suggest that the basic principles consist of the need for a combination of pharmacological and nonpharmacological treatments with a core set of initial measures, including information access / education, weight loss if overweight, and an appropriate exercise program. there are several reports of minor effects of educational programs on pain, function, time spent in gyms, and weight loss. a positive outcome from a previous week - long educational program for patients with osteoporosis propelled the present proposal for 2 days of lectures and workshops about oa, reinforced by telephone calls, for patients with koa. this study evaluates a multiprofessional conservative treatment for patients with koa with 4 different educational program options with or without bimonthly telephone calls. this study was performed at the department of orthopedics and traumatology in a tertiary hospital in brazil. this study followed the guidelines of the consort statements for randomized controlled trials and nondrug treatments. the care providers were 7 orthopedic surgeons, 4 psychologists, 3 social workers, 1 nutritionist, 5 occupational therapists, 3 physical therapists, and 2 physical educators who were either volunteers or on staff at the orthopedic institute. patients had to meet the following criteria : an outpatient aged 45 years or older with koa according to the american college of rheumatology clinical and radiological definition who had received standard care for oa in the past 6 months ; no other rheumatologic disease ; knee pain rated above 30 mm on a numerical scale and necessitating drug treatment ; and able to understand and agree with the informed consent. the exclusion criteria included participating in another program with nutritional education, engaging in another clinical trial, or undergoing surgery during the study not related to koa that would prevent daily regular exercises. by november 2011, 306 patients were under routine care for koa, that is, followed by orthopedic surgeons and undergoing blood tests for metabolic syndrome (and referred to a general practitioner for clinical control) and calcium metabolism, x - rays, densitometry, and more specific images (ultrasound and magnetic resonance imaging [mri ]) according to symptoms. all patients were prescribed diacerhein. nonsteroidal anti - inflammatory drugs (nsaids) were used only occasionally for severe pain and for short periods of time. vitamin d3 and calcium supplements were prescribed according to blood levels and bone densitometry results. when present, osteoporosis was treated with alendronate. based on x - ray results, that is, classification of the severity by 3 orthopedic surgeons using the kellgren and lawrence (k&l) classification, orthotics, such as valgus or varus insoles, canes, walkers, and custom - made hand orthotics, were prescribed. of the 306 total patients, 228 met the inclusion criteria and were interested in participating in the study. the medical team chose the visual analogue scale (vas), western ontario and mcmaster universities osteoarthritis index (womac [brazilian version ]), lequesne index, and 36-item short - form health survey (sf-36) questionnaires for the assessment of pain, function, and quality of life at all time points. the social work team asked patients to report how many years of school they had completed. after baseline evaluations and questionnaires, participants were randomly allocated into 4 groups (1 - 4, according to the intervals of 1, 2, and 3 months between the 2 days of lectures, or no lectures in the case of group 4) and 2 subgroups (a and b, according to the use of bimonthly telephone calls or no telephone calls, respectively) of 28 or 29 participants. all groups received a booklet on oa and a video with all of the lectures. group 4 was required to watch the video once at the hospital and only then received the take - home material. patients in groups 1 to 3 were asked to come to the hospital on 2 specific saturdays according to the intervals of each group. the program, explained partially in our short - term results, comprised 2 days (from 7:00 to 17:00 hours, with meals included) of lectures and workshops on causes and treatment of oa. the first day comprised lectures by all teams of professionals and workshops with the physical and occupational therapists. each professional team had a lecture of 30 to 40 minutes (orthopedic surgeons, psychology, physical therapy, occupational therapy, physical fitness, and social workers) or up to 80 minutes (nutritionist). the physicians introduced the anatomy of a joint and the pathology of oa, its causes, irreversibility, and management. the psychology team explained personality characteristics from childhood to adulthood, called attention to the difference between having and being a disease and being the results of your choices and not your conditions or feelings, and worked on coping skills. the physical therapists and the physical educators called attention to the importance of a regular exercise program and the differences between physical labor and such a program. while the physical therapists showed how to improve activity to move from pain to a state of no pain, physical fitness instructors focused on a variety of options of exercises to improve strength, resistance, and flexibility. the occupational therapists introduced the importance of protecting joints in daily activities through optimum ergonomic performance as well as by alternating different levels of energy expenditure. the nutritionist explained the importance of a well - balanced diet (reduced quantity, colorful, whole grains, eating every 3 hours, and low - calorie meals). the social work team showed where and how patients could and should include habits of regular leisure, sports and social gathering, and tasks. during the 2 workshops, the physical therapy team taught the patients the exercise series presented in the booklet and in the dvd, which was to be conducted at least 3 times a week. the occupational therapy workshop showed patients how to protect their joints during daily activities in a model house (bathroom, kitchen, bedroom, and workshop, particularly for men). the second intervention program was the same length (from 7:00 to 17:00 hours) and started with the social workers. the psychology team led a group session where patients shared their experiences with the program. the occupational therapy team asked the patients to participate in the planning of a week s activities with a goal of avoiding unnecessary heavy - duty tasks and alternating different levels of energy expenditure. the nutritionist reviewed the slide sequence shown in the first class and answered questions for each specific topic related to a well - balanced diet. the medical team closed the program by quizzing patients on the definition, causes and management of oa, and recalling highlights of each team s presentations. both days of lectures had 30- to 60-minute intervals, at 9:30 (30 minutes), 13:00 (1 hour), and 16:00 (30 minutes), for meals composed of the diet suggested by the nutritionist. the printed material contained summaries of all classes from the first intervention day, including the actual exercises performed at the physical therapists and physical educators workshops as well as practical recommendations for daily living offered by the occupational therapists. the dvd had the 3 workshops (physical therapy, fitness, and occupational therapy) and all 7 explanatory lectures given in the first intervention, lasting a total of 143 minutes. all patients were asked watch the dvd and/or read the booklet no less than 3 times. subgroup a patients received telephone calls from the medical team 2 months after the lecture and every other month after that until the 1-year reassessment. patients were asked about pain, medications, diet, occupational therapy participation, and social and/or physical activity and frequency. patients were reminded repeatedly to watch the dvd and/or read the material ; to exercise at least 3 times a week (preferably daily) ; and to change their social, occupational, and dietary habits. patients answered the questionnaires, physical activity query, and, during the medical interview, the need to follow a healthy diet, exercise (if possible, continuously increase exercise load), and change occupational and leisure habits was reinforced. weight and height were measured at baseline and at 1 year after the educational program. the psychology team evaluated the patients with a coping scale adapted to the brazilian population at 1 year. the coping scale evaluated the patient s coping skills in terms of being focused on problems, their emotions and religion / fantasy thinking, and whether they were seeking social support. the lequesne, womac, vas, and sf-36 scores and level of education were assessed at each visit according to group and telephone calls and to psychological coping results obtained at 1 year. this is a pilot study to evaluate the best (time wise) intervention to add multiprofessional education to koa clinical treatment. groups 1, 2, and 3 had classroom instruction from all professionals as well as the audiovisual and written instructions that group 4 also received. when signing the informed consent, patients were aware that the groups would differ according to time between classes, lack of classes, and telephone calls. two secretaries scheduled appointments, classes, and material retrieval and plotted the questionnaires results in excel. the kruskal - wallis test was applied for comparing oa severity, whereas the likelihood ratio was applied for race. summary measures (quantitative characteristics) and analysis of variance (anova), followed by tukey s multiple comparisons, were used to compare groups. the results of the coping questionnaire domains were described according to group and telephone calls. the summary measures (mean, standard deviation, and 95% confidence interval [ci ]) of the questionnaires scores were described according to group and compared with anova and tukey s multiple comparisons. the study was approved by the ethics committee for the analysis of research projects (cappesq) under protocol number 0622/11. this study followed the guidelines of the consort statements for randomized controlled trials and nondrug treatments. the care providers were 7 orthopedic surgeons, 4 psychologists, 3 social workers, 1 nutritionist, 5 occupational therapists, 3 physical therapists, and 2 physical educators who were either volunteers or on staff at the orthopedic institute. patients had to meet the following criteria : an outpatient aged 45 years or older with koa according to the american college of rheumatology clinical and radiological definition who had received standard care for oa in the past 6 months ; no other rheumatologic disease ; knee pain rated above 30 mm on a numerical scale and necessitating drug treatment ; and able to understand and agree with the informed consent. the exclusion criteria included participating in another program with nutritional education, engaging in another clinical trial, or undergoing surgery during the study not related to koa that would prevent daily regular exercises. by november 2011, 306 patients were under routine care for koa, that is, followed by orthopedic surgeons and undergoing blood tests for metabolic syndrome (and referred to a general practitioner for clinical control) and calcium metabolism, x - rays, densitometry, and more specific images (ultrasound and magnetic resonance imaging [mri ]) according to symptoms. all patients were prescribed diacerhein. nonsteroidal anti - inflammatory drugs (nsaids) were used only occasionally for severe pain and for short periods of time. vitamin d3 and calcium supplements were prescribed according to blood levels and bone densitometry results. when present, osteoporosis was treated with alendronate. based on x - ray results, that is, classification of the severity by 3 orthopedic surgeons using the kellgren and lawrence (k&l) classification, orthotics, such as valgus or varus insoles, canes, walkers, and custom - made hand orthotics, were prescribed. patients with impaired mobility and pain were referred to physical therapy and acupuncture. of the 306 total patients, the medical team chose the visual analogue scale (vas), western ontario and mcmaster universities osteoarthritis index (womac [brazilian version ]), lequesne index, and 36-item short - form health survey (sf-36) questionnaires for the assessment of pain, function, and quality of life at all time points. patients the social work team asked patients to report how many years of school they had completed. after baseline evaluations and questionnaires, participants were randomly allocated into 4 groups (1 - 4, according to the intervals of 1, 2, and 3 months between the 2 days of lectures, or no lectures in the case of group 4) and 2 subgroups (a and b, according to the use of bimonthly telephone calls or no telephone calls, respectively) of 28 or 29 participants. all groups received a booklet on oa and a video with all of the lectures. group 4 was required to watch the video once at the hospital and only then received the take - home material. patients in groups 1 to 3 were asked to come to the hospital on 2 specific saturdays according to the intervals of each group. the program, explained partially in our short - term results, comprised 2 days (from 7:00 to 17:00 hours, with meals included) of lectures and workshops on causes and treatment of oa. the first day comprised lectures by all teams of professionals and workshops with the physical and occupational therapists. each professional team had a lecture of 30 to 40 minutes (orthopedic surgeons, psychology, physical therapy, occupational therapy, physical fitness, and social workers) or up to 80 minutes (nutritionist). the physicians introduced the anatomy of a joint and the pathology of oa, its causes, irreversibility, and management. the psychology team explained personality characteristics from childhood to adulthood, called attention to the difference between having and being a disease and being the results of your choices and not your conditions or feelings, and worked on coping skills. the physical therapists and the physical educators called attention to the importance of a regular exercise program and the differences between physical labor and such a program. while the physical therapists showed how to improve activity to move from pain to a state of no pain, physical fitness instructors focused on a variety of options of exercises to improve strength, resistance, and flexibility. the occupational therapists introduced the importance of protecting joints in daily activities through optimum ergonomic performance as well as by alternating different levels of energy expenditure. the nutritionist explained the importance of a well - balanced diet (reduced quantity, colorful, whole grains, eating every 3 hours, and low - calorie meals). the social work team showed where and how patients could and should include habits of regular leisure, sports and social gathering, and tasks. during the 2 workshops, the physical therapy team taught the patients the exercise series presented in the booklet and in the dvd, which was to be conducted at least 3 times a week. the occupational therapy workshop showed patients how to protect their joints during daily activities in a model house (bathroom, kitchen, bedroom, and workshop, particularly for men). the second intervention program was the same length (from 7:00 to 17:00 hours) and started with the social workers. the psychology team led a group session where patients shared their experiences with the program. the occupational therapy team asked the patients to participate in the planning of a week s activities with a goal of avoiding unnecessary heavy - duty tasks and alternating different levels of energy expenditure. the nutritionist reviewed the slide sequence shown in the first class and answered questions for each specific topic related to a well - balanced diet. the medical team closed the program by quizzing patients on the definition, causes and management of oa, and recalling highlights of each team s presentations. both days of lectures had 30- to 60-minute intervals, at 9:30 (30 minutes), 13:00 (1 hour), and 16:00 (30 minutes), for meals composed of the diet suggested by the nutritionist. the printed material contained summaries of all classes from the first intervention day, including the actual exercises performed at the physical therapists and physical educators workshops as well as practical recommendations for daily living offered by the occupational therapists. the dvd had the 3 workshops (physical therapy, fitness, and occupational therapy) and all 7 explanatory lectures given in the first intervention, lasting a total of 143 minutes. all patients were asked watch the dvd and/or read the booklet no less than 3 times. subgroup a patients received telephone calls from the medical team 2 months after the lecture and every other month after that until the 1-year reassessment. patients were asked about pain, medications, diet, occupational therapy participation, and social and/or physical activity and frequency. patients were reminded repeatedly to watch the dvd and/or read the material ; to exercise at least 3 times a week (preferably daily) ; and to change their social, occupational, and dietary habits. patients answered the questionnaires, physical activity query, and, during the medical interview, the need to follow a healthy diet, exercise (if possible, continuously increase exercise load), and change occupational and leisure habits was reinforced. weight and height were measured at baseline and at 1 year after the educational program. the psychology team evaluated the patients with a coping scale adapted to the brazilian population at 1 year. the coping scale evaluated the patient s coping skills in terms of being focused on problems, their emotions and religion / fantasy thinking, and whether they were seeking social support. the lequesne, womac, vas, and sf-36 scores and level of education were assessed at each visit according to group and telephone calls and to psychological coping results obtained at 1 year. this is a pilot study to evaluate the best (time wise) intervention to add multiprofessional education to koa clinical treatment. groups 1, 2, and 3 had classroom instruction from all professionals as well as the audiovisual and written instructions that group 4 also received. when signing the informed consent, patients were aware that the groups would differ according to time between classes, lack of classes, and telephone calls. two secretaries scheduled appointments, classes, and material retrieval and plotted the questionnaires results in excel. this is a pilot study to evaluate the best (time wise) intervention to add multiprofessional education to koa clinical treatment. the authors aimed to have 30 patients in each group. groups 1, 2, and 3 had classroom instruction from all professionals as well as the audiovisual and written instructions that group 4 also received. when signing the informed consent, patients were aware that the groups would differ according to time between classes, lack of classes, and telephone calls. two secretaries scheduled appointments, classes, and material retrieval and plotted the questionnaires results in excel. the kruskal - wallis test was applied for comparing oa severity, whereas the likelihood ratio was applied for race. summary measures (quantitative characteristics) and analysis of variance (anova), followed by tukey s multiple comparisons, were used to compare groups. the results of the coping questionnaire domains were described according to group and telephone calls. the summary measures (mean, standard deviation, and 95% confidence interval [ci ]) of the questionnaires scores were described according to group and compared with anova and tukey s multiple comparisons. the study was approved by the ethics committee for the analysis of research projects (cappesq) under protocol number 0622/11. a total of 306 patients were assessed for eligibility, and 246 met the inclusion criteria. four groups (2a, 2b, 3a, and 3b) were composed of 28 patients each ; the other 4 groups (1a, 1b, 4a, and 4b) had 29 patients each. sixteen patients missed classes (due to lost interest, weather conditions that prevented access to the hospital, or being unable to attend classes when they were scheduled). at this point, the subgroups ranged from 25 (1a and 2b) to 29 (1b and 4b) participants. at the 4-month reassessment, 1 patient had undergone total knee replacement and 2 patients had died (groups 1b and 4a). five patients (group 2a = 1 patient, groups 4a and 4b = 2 patients each) missed the evaluation and, when called, decided not to continue in the study (either because they were not interested or lived too far away to attend follow - ups). at the 1-year reassessment, 2 patients had died (one from group 1a and another from group 3b). one patient from group 1a and 2 from group 4b missed the 1-year reassessment (figure 1). twenty - seven patients were lost, of whom eight were from group 4, seven from group 1, and six from groups 2 and 3. all groups were homogeneous for nominal valued features, such as degree of koa, gender, race, or affected side or bilaterality (table 1). the groups were similar in age (p =.121) and level of education (.643). the average bmi of group 2 was 32.8 and was significantly different from group 3 (average 29.8, p =.026, mean difference 3.01 1.06, 95% ci 0.25 - 5.76). descriptions of personal and clinical characteristics of patients according to group and results of statistical tests. the number of patients who performed some physical activity was 25 at baseline (11 light ; 12 moderate, and 2 vigorous) increasing to 123 (74 light, 40 moderate, and 9 vigorous activity) at 1 year. in tables 2 and 3, the summary measures of the scores obtained for the womac, womac pain, lequesne index, vas, and mental (mcs) and physical (pcs) domains of the sf-36 quality of life are presented. the analysis of these results with respect to group, telephone calls, level of education, bmi, and time of reassessment showed that the womac, mcs, and pcs scores changed according to time of reassessment (p =.022, p =.012, and p =.038, respectively ; table 4). quality of life and functional improvements occurred primarily between baseline and the short - term reassessment at 4 months (pcs : 1.55 0.58, p =.025, 95% ci 2.97 to 0.14 ; mcs : 2.15 0.79, 95% ci 4.06 to 0.24, p =.021 ; womac (table 5) : p =.023, 95% ci 0.33 - 6.03, mean difference 3.18 1.18) and lost significant growth at 1 year, except for mcs, for which the improvement was maintained after 1 year (mean difference 2.34 0.93, 95% ci 4.59 to 0.09, p =.039). the womac pain, vas, and lequesne scores had no significant differences among the factors evaluated (p >.05, table 4). womac, womac pain, lequesne index scores according to group, telephone calls, and evaluation periods. abbreviations : sd, standard deviation ; womac, western ontario and mcmaster universities osteoarthritis index. visual analogue scale (vas) and sf-36 quality of life scores according to group, telephone calls, and evaluation periods. abbreviations : sd, standard deviation ; sf-36, 36-item short - form health survey ; mcs, mental component summary ; pcs, physical component summary. results of anova for comparison of scores between group, telephone calls, and times of reassessment. abbreviations : vas, visual analogue scale ; anova, analysis of variance ; mcs, mental component summary ; pcs, physical component summary ; womac, western ontario and mcmaster universities osteoarthritis index ; sf-36, 36-item short - form health survey. results of multiple comparisons between womac, physical and mental sf-36 quality of life score, and times of reassessment. abbreviations : mcs, mental component summary ; pcs, physical component summary ; womac, western ontario and mcmaster universities osteoarthritis index ; sf-36, 36-item short - form health survey. level of education had weak but significant correlations with lequesne scores at 1 year (r =.162, p =.023) and with pcs at baseline and at 1 year (r =.206, p =.004 and r =.158, p =.026, respectively). the vas, womac, womac pain, and mcs and changes in vas, womac, womac pain, pcs, and mcs did not correlate with education (p >.05). twenty - seven (of the 72) patients reduced more than 2 points in bmi. the remaining patients maintained (72 patients) or increased bmi (54 patients). we searched for correlations between bmi and all variables studied and found that baseline bmi correlated with bmi at 1 year (r =.967, p = 0). changes in bmi correlated with the 1-year results of the womac (r =.172, p =.016), womac pain (r =.193, p =.007), lequesne (r =.197, p =.006), and mcs (r =.160, p =.027) and with changes in womac (r =.220, p =.002), womac pain (r =.199, p =.006), and vas (r =.170, p =.018). the coping domains were similar between groups (p >.05) and were not influenced by telephone calls (p >.05). the mcs results were directly related to problem focusing (baseline, r =.273, p = 0 ; 4 months, r =.170, p =.024 ; and 1 year, r =.200, p =.007) and inversely related to emotion - focused scores (baseline, r =.306, p = 0 ; 4 months, r =.279, p = 0 ; and 1 year, r =.388, p = 0). similar weak but significant correlations were found between coping focused on religion / fantasy thinking and womac, womac pain, vas, lequesne, and pcs scores at all time points (baseline, 4 months, and 1 year). all but pcs were direct correlations. pain and quality - of - life changes were not correlated with coping domains. patients who scored higher in problem focusing showed a direct correlation with improvement in the lequesne score (r =.148, p =.049). all groups were similar in gender, race, age, affected side, and oa severity (table 1). the bmi ranged from 29.8 (group 3) to 32.8 (group 2). patients in group 3 were less obese than those in group 2 (p =.026). patients were allowed to participate regardless of inability to walk without aid or severe chronic diseases as long as they were already in treatment with recommendations from their specialists as to restriction of fluids, medications, and exercise routine. this could be a reason for the 4 deaths during the follow - up period. considering the variables studied, telephone calls did not significantly improve the results (tables 2 4). the effect size (es) of telephone calls in the improvement of pain was 0.12 (95% ci 0.0 to 0.24) and in the improvement of function was 0.07 (95% ci 0.00 to 0.15). calling 110 patients every other month, with a time expenditure of between 10 and 30 minutes per patient, was time consuming and not as effective as when patients came to the hospital for a consultation, where measures could be taken to relieve pain and where patients were able to exchange experiences with other participants in the program while waiting for their consultation. the womac, pcs, and mcs scores varied according to times of evaluation (p =.022, p =.038, and p =.012, respectively) regardless of group or telephone calls. in general, the short - term improvements in pain and function favoring groups who attended classes were not significant at 1 year ; from a cost perspective, this result favors the use of educational material only. education is described to have a very small effect on pain, with an es of 0.06 (95% ci 0.03 to 0.10) and, on function, with an es of 0.06 (95% ci 0.02 to 0.10) ; however, there can be no changing of habits, improving exercise or reducing bmi without teaching the importance, the benefits, and how and when to do it. patients are more receptive to health information when it is presented in terms of potential gain and uses examples that are of the same gender and race. years and grade of obesity, degree of oa, functional impairment, adversity coefficient, lack or presence of depression, and social oeconomic problems also influence results. patients who were essentially problem focused showed a weak relation to function improvement (lequesne, p =.05) at 1 year. objectively, from the program, almost 100 patients changed from null to some physical activity. at baseline, only 25 practiced regular physical activity (11 light, 12 moderate, and 2 vigorous) and at 1 year 123 were practicing some physical activity of which 74 light, 40 moderate, and 9 performed vigorous regular activity. these results were not shown as improved scores in womac, lequesne, or pcs of the sf-36. seventy - two patients lost weight, but only 27 more than 2 points in bmi affecting somewhat the correlation between bmi and pain / functional scores. in the search for factors affecting compliance to the program, we found weak correlations between coping domains and questionnaire results. we did not find relation between the patients level of education and pain and functional improvements. there was some indication that bmi reduction correlated with 1-year results of pain, function, and quality of life and with improvements in function (womac) and quality of life, as expected, mainly because, of the 198, only 27 reduced more than 2 points in bmi. therefore, instead of offering educational material only, it might be better to intervene more frequently, allowing patients to exchange experiences with other participants and reinforce correct diet and exercise to maintain or even improve on the results obtained at 4 months. the limitations of the study are (1) lack of control over the use of analgesics or other nonpharmacological measure. the educational program should be the core measure to all other types of oa treatment. although educational programs are known to have a small es, in our experience, teaching patients with osteoporosis led to a high adherence to physical activity, medications, and diet after the educational program. therefore, all patients were already instructed to pursue physical activity, acupuncture, physical therapy, and orthotics before the educational interventions. anti - inflammatory drugs were prescribed only for periods of no longer than a week, if they were prescribed at all. at the assessments, medications were seldom changed, but the actual consumption of medications was not evaluated in this study. (2) patients with k&l grade iv were included. despite being equally distributed across all groups, (3) the difference in bmi between groups 2 and 3 could be a potential bias ; however, both groups were very similar in bmi changes during the study and showed no significant differences in changes in pain, function, and quality of life. (4) we did not analyze changes in hours of physical activity, percentage of body fat, or quality of food ingested before and after the program. we had no experience with our population, their level of education, or their capacity to answer questionnaires, so we decided to perform this prospective randomized study as a pilot study to identify the best approach to improve pain, function, and quality of life in our patients with koa. they did have difficulties answering questionnaires, but the interval between classes did not influence the results at 4 or 12 months. despite studies indicating that telephone calls improve results in the treatment of koa and hip oa, in our experience, this was time consuming and not effective at the 1-year follow - up. in our short - term results, we observed a superiority of results in patients who attended classes compared to those who only received the educational material. at 1 year, we could observe increased frequency and intensity of physical activity not translated effectively in improved pain and functional subjective scores. roughly, 12% (27 of 228 initial patients) reduced more than 2 points in bmi and increased physical activity to levels of improved pain, function, and quality of life. coping skills and educational level of patients may affect adherence to treatment, and we may need to adapt the program improve patient s adherence. to adapt the program to improve patients adhrence. the effect of this educational program in function and quality of life of patients with koa is subtle. interval between classes (1, 2, or 3 months) is not an important issue.	introduction : knee osteoarthritis (koa) is the most prevalent form of osteoarthritis. low socioeconomic level, age, and obesity are directly correlated with the incidence of the disease. education, exercise, and diet are the core recommendations of all koa treatment guidelines.objective:to evaluate the impact of a multiprofessional educational program on patients with koa.methods:of a total of 198 participants, 150 patients with koa attended 2 days of lectures (at 1- to 3-month intervals) and received educational material on osteoarthritis, and a control group (48 patients) received educational materials only. body mass index (bmi), frequency, and intensity of physical activity, pain, function, and quality - of - life scores were assessed at baseline and at 4 and 12 months after the educational program. bimonthly telephone calls were made to half of the participants. correlations between bmi, level of education, coping skills, functional, and pain results was procured.results:the groups were similar in terms of race, gender, affected side, and osteoarthritis severity. the results were not affected by the telephone calls or the patients level of education. at baseline, 25 performed physical activity, whereas 123 performed at 1 year. seventy - two (36.36%) patients decreased bmi (45 by 1 point and 27 by more than 2 points). there were some weak correlations such as bmi reduction with pain and functional improvements and with coping results. significant improvements in function and quality of life were found at 4 months. quality of life remained improved at 1 year.conclusion:the effect of this educational program in function and quality of life of patients with koa is very subtle. interval between classes (1, 2, or 3 months) is not an important issue.
the most common facial bones fracture is the mandible fracture.1 mandibular fracture patterns depend on multiple factors, including direction and amount of force, presence of soft tissue bulk, and biomechanical characteristics of the mandible such as bone density and mass or anatomic structures creating weak areas.2,3 the hypothesis that the presence of mandibular third molars (m3) is associated with an increased risk on angle fractures. the presence of the m3 has been repetitively shown to be associated with higher relative risk for angle fracture. these studies demonstrated that whenever the m3 was present, the risk of angle fracture increased 2-to 3-fold when it was compared with absence at m3.46 an impacted tooth is one that fails erupt into the dental arch within the expected time. the unerupted term includes both impacted teeth and erupting teeth.7 the impacted or unerupted m3 could weaken the mandible because the tooth occupies more osseous space.8 however, it is not known if the other impacted or unerupted teeth weaken the mandible or not. there is not any study supports the hypothesis which is pointing out the relationship between the presences of mandibular impacted or unerupted teeth and an increasing risk on mandibular fractures. in this study a retrospective review was conducted of patients who presented with fractured mandibles at university of ondokuz may s, department of oral and maxillofacial surgery, between 1995 and 2003. one hundred one patients having mandibular fractures and forty - one patients having impacted or unerupted teeth were identified. to assess the predictor variable, panoramic radiographs were used (presence or absence of impacted or unerupted teeth) and conclusion variable (presence or absence of mandibular fracture). in addition, with the help of the patients hospital charts and panoramic radiographs, their age, gender, type of fracture, radiologic evaluation, and amount of impacted or unerupted teeth were assessed. the severity of tooth impaction was classified as either complete or partial bony. in order to show a causal relationship between impacted or unerupted teeth and mandibular fractures, group ii also consisted of mandibular fractures but they are not related to impacted or unerupted teeth. the database was analyzed with the use of the spss version 10.0. to assess the relationships between the presence of impacted or unerupted teeth and the risk of mandibular fractures, the patients are at the age of 2 to 45 years (mean age, 17.51 years). in the 26 patients, mandibular fractures are related to impacted or unerupted teeth and remaning 15 patients also have mandibular fracture which is not related to any impacted or unerupted teeth. the relationship between impacted or un - erupted teeth and mandibular fracture status is summarized in table 1. in group i, impacted or unerupted teeth contributed to 63.41% (26/41) of fractures. patients having mandibular fractures and impacted or unerupted teeth had nearly a 1,73-fold increase the risk of mandibular fractures comparing with patients not having impacted or unerupted teeth. the impacted or unerupted teeth significantly increased the risk of fractures in group i (x2=5,29, p=.0215). table 2 summarizes the degree of impactation as complete or partial bony. in this study, fall caused, 18 (44%) ; fight, 9 (22%) ; motor vehicle accidents, 3 (7%) ; sports, 3 (7%) ; and others, 8 (20%) of the mandibular fractures. thus, it is seen that, was the most common cause of the mandibular fractures, with the effect of 44%. as it was seen, the most common fracture between the impacted or unerupted teeth was impacted third molars (54%) (table 3). consistent with other similar studies, the results of this study confirmed an increased risk of mandibular fracture when the impacted or unerupted teeth were present. it is hypothesized that the impacted or unerupted teeth increase the risk of mandibular fractures by occupying osseous space and thereby, the angle region is weakened. it is not true that the risk of mandibular fracture incidence depends on only one factor because it depends on the vector and also the amount of force, the musculature of the face, the architecture of the mandible and the presence or absence of m3.9 the hypothesis that m3 level of impaction further increases the risk of angle fractures originated with the work of reitzik.8 the reasoning of this hypothesis is that when m3 occupies more osseous space, it weakens the mandible against the outside stresses. this compares the mandibular angle, when an impacted m3 is present, with a region of pathologic weakness similar to various conditions (i.e., presence of a tumour or cysts, periapical pathosis, hyperparathyroidism, paget s disease, osteoporosis, and other metabolic conditions).10 falls, motor vehicle accidents, fights, sports, and others cause to maxillofacial fractures commonly. in this study, falls were the most common cause of these fractures, comprising 44% of the etiology of the fractures. similar to other investigators,11 - 13 we found that patient s age has an important role on the risk of fracture. sixty - three of the patients were under the age of twenty years. in our study, only 10 women sustained fractures, whereas 31 men did. huelke reported that fractures occur more frequently in dentate than in edentulous regions of the mandible. similar to these investigators, we found that the impacted or unerupted teeth in the dentate regions of mandible weakens the mandibular bone. in this study, the most common mandibular fractures were seen as impacted or unerupted third molars teeth area. after that, the most common mandibular fracture was seen as impacted or unerupted canin teeth which have the longest root in the mandible. the first specific aim of this study was to measure the association between the presence of impacted or unerupted teeth and the risk of mandibular fractures. we noted the significant association between the impacted or unerupted teeth presence and the risk of mandibular fractures statistically (p=.0215). the results of this study confirmed that, if there are impacted or unerupted teeth, the risk of mandibular fractures will increase.	objectivesin this retrospective study, we measured the relationship between the presences of impacted or unerupted teeth in the mandible and mandibular fractures.methodsthe records and radiographs of 41 patients with mandibular fracture associated with impacted or unerupted teeth were examined. the presence of impacted or unerupted teeth were assessed for each patient and related to the occurrence of fractures of mandible.resultspatients with fracture in the impacted or unerupted teeth area present had a 1,73 times greater chance of an mandibular fracture than patients with no fracture in the impacted or unerupted teeth area. there was a statistically significant variation in the risk for a mandibular fracture depending on impacted or unerupted teeth presence (x2=5.29, p <.05).conclusionthe presence of an impacted or unerupted teeth significantly increases the likelihood of an mandibular fracture.
host defence peptides (hdps) are an evolutionarily ancient component of the innate immune system of most multicellular organisms.1 they exhibit a wide range of biological activities from direct killing of invading pathogens to modulation of immunity and other biological responses of the host. in this article, we will refer to the overlapping classes of peptides with these activities as antimicrobial and immunomodulatory, respectively. despite the enormous diversity in their sequences and structures, most hdps share the following features : positive charge, high content of hydrophobic residues, and amphipathic fold.2 the structural diversity of natural peptides provides an excellent starting point for the production of artificial peptides and derivatives with more potent and desirable biological activities, for clinical and commercial applications.3 two major families of naturally occurring hdps have been distinguished : defensins and cathelicidins (table 1). defensins are cationic amphipathic peptides with an average length of 30 residues and a triple - stranded antiparallel b - sheet structure, stabilised by three disulphide bonds.4 defensins are further subdivided into three subfamilies of,, defensins, based on the pattern of disulphidebonding (table 1). the diversity of mammalian host defence peptides in humans, but not in cattle or mice, -defensins are found in the secretory granules of neutrophils and other leukocytes.5, 6 in most mammals other than cattle, a set of -defensins is also produced by paneth cells in the crypts of the small intestine.7, 8 these -defensins, also known as cryptidins, are synthesised as inactive precursors and activated by a removal of an n - terminal segment catalysed by metalloprotease matrilysin (mmp7) in mice and by trypsin in humans.9, 10 after microbial stimulation, the concentration of -defensins within the crypts is estimated to reach 10 mg / ml, which is more than sufficient for strong microbicidal action.11 this, and the association of crohn 's disease with dysregulation in cryptidin production,12 highlights the importance of a - defensins in the maintenance of immune homeostasis in the gut. -defensins are expressed by most epithelial cells, and their expression is often stimulated by proinflammatory stimuli and infection. they are present in the mucosal secretions of respiratory, gastrointestinal, and urogenital tracts, and in inflamed skin.1316 -defensins are also expressed by human monocytes, macrophages, and dendritic cells, and are important components of the azurophilic granules of bovine neutrophils.4 the much rarer -defensins are cyclic molecules, produced in neutrophils and monocytes of rhesus macaques through ligation of two -defensin - like peptides.17 as a result of their cyclic structure, the (moderate) microbicidal activity of -defensins is resistant to salt concentration. cathelicidins are the other major family of natural hdps, although they are grouped by their mechanism of production rather than sequence similarity. all cathelicidins are synthesised as inactive precursors, comprising an n - terminal cathelin - like domain followed by the peptide region, and are proteolytically processed to release mature active hdps.18 cathelicidins vary in length, sequence, and structure, having extended, -helical or -hairpin folds ; however, some are short linear molecules (for example, indolicidin, 13 amino acids), and these are ideal starting points for the design of synthetic peptides with optimised biological activity. bovine and porcine immune systems produce a large variety of cathelicidins, including bactenecin, indolicidin, pr-39, protegrins, prophenins, and many others.18 in contrast, in humans there is only one known cathelicidin precursor protein hcap18, which is proteolytically processed to yield mature cathelicidin ll-37 19 and, in the skin, a series of additional proteolytic derivatives with altered activities.20 ll-37 lacks disulphide bonds and is weakly structured in solution, but adopts an a - helical conformation when interacting with lipid bilayers. mice also have only one cathelicidin precursor, which is processed to produce mature peptide cramp, with 67% sequence identity to ll-37.21 ll-37 is found in the secretory granules of neutrophils and other leukocytes,22 and is also produced by mucosal epithelia and keratinocytes.2325 the expression of ll-37 is modestly upregulated by proinflammatory stimuli, and is also induced by the hif-1 and the vitamin d receptor pathways.2628 many other hdps that do not belong to the defensin or cathelicidin families have also been characterised in humans and other mammals. some important examples include : histatins, histidine - rich cationic peptides with antifungal activity present in the saliva;29 dermcidin, an anionic peptide present in human sweat with modest salt - insensitive antimicrobial properties;30 cationic peptide lactoferricin derived from an iron - binding protein lactoferrin and found in mucosal secretions, milk, and in the granules of leukocytes.31 the evolutionarily widespread distribution and the extreme diversity of hdps highlight their prominent role in immune defences. historically, this has been attributed to their antimicrobial activity but in recent years potent immunomodulatory properties of hdps have been characterised and suggested to be an important part of their biological function.19, 32 an in - depth understanding of the physiological roles and mechanisms of action of hdps is crucial for the development of artificial variants with optimised activity for therapeutic applications. antimicrobial properties of hdps are linked to their amphipathic fold, which allows the peptides to interact with lipid bilayers of microorganisms,2 and kill either through membrane disruption or by translocating across the membrane and inhibiting cytosolic targets. owing to the relatively nonspecific nature of the interactions between hdps and the anionic lipids of microbial membranes, many peptides have broad antimicrobial properties, targeting gram - positive and -negative bacteria, fungi, protozoa, and some viruses.1, 33 furthermore, hdps are highly effective against multidrug - resistant bacterial strains. peptides can also interact with mammalian membranes and this is at least partly responsible for cytotoxicity of some peptides at high concentrations.34 however, distinct composition of mammalian membranes, with preferential localisation of anionic phospholipids into the inner leaflet, offers some protection. furthermore, membrane interactions and antimicrobial properties of most hdps are strongly dependent on the composition of the media, being inhibited by divalent cations and serum.19, 14, 34 thus, although hdps almost certainly function as powerful microbicidal agents in some physiological settings, such as the phagolysosomes of neutrophils, intestinal crypts, and sites of acute inflammation, under other conditions where salt concentrations are high (100 mm monovalent and 2 mm divalent cations) and peptide concentrations are modest, their immunomodulatory activities are almost certainly more physiologically relevant. a wide range of hdps from different species have been shown to act as chemoattractants for cells of innate and adaptive immunity. as an example, human cathelicidin ll-37 attracts neutrophils, monocytes, t cells, and mast cells, using formyl peptide receptor - like 1 (fprl1), and a distinct gi - coupled receptor.35, 36 optimal chemotactic activity of ll-37 is observed in a concentration range that can be reached in vivo under inflammatory conditions.35 furthermore, synthetic peptide idr-1, which is protective in mouse models of bacterial infections, similarly chemoattracts neutrophils, acting through receptor fprl1.37, 38 apart from direct chemotactic effects, hdps also elicit other complex responses in leukocytes and epithelial cells, altering gene expression and behaviour to facilitate and to modulate immune responses (figure 1). for example, when ll-37 was used to stimulate primary human monocytes and macrophage cell lines, microarrays demonstrated the induction of a wide range of chemokines, chemokine receptors, and other genes involved in cell adhesion, communication, and motility.40 in particular, induction of chemokines il8, gro-, mcp1, mip-1, and mip-3, but not proinflammatory cytokines, was consistently seen with a wide range of natural and synthetic peptides. ll-37 induces global alterations in gene expression in monocytes, signalling through p38, erk, pi3k, and nf - kb pathways, and promoting expression of chemokines and other genes involved in cell communication and motility.32, (39) ll-37 also acts as a direct chemoattractant for monocytes, neutrophils, t cells, and mast cells through the fprl1 receptor,35 and an unknown gi - coupled receptor.36 ll-37 is also strongly anti - endotoxic and inhibits the production of proinflammatory cytokines in monocytes in response to lps,40 and also the maturation of monocyte - derived dendritic cells by tlr ligands.41 in contrast, ll-37 pretreatment of monocytes modulates the process of dendritic cell differentiation, enhancing their function 42 and in plasmacytoid dendritic cells (pdcs) ll-37 promotes responses to tlr9 ligands.43 other activities of ll-37 include the promotion of antimicrobial functions of neutrophils,44 mast cell degranulation,45 and il-1 processing in lps - primed monocytes.46 ll-37 also affects neutrophil and epithelial cell apoptosis, 4749 in keratinocytes, ll-37 induces il8, and promotes migration and wound healing, and these activities depend on adam family metalloproteinase, egfr and fprl1.50, (51) in airway epithelial cells, ll-37 activates p38 and erk pathways and induces chemokine secretion, and this is reported to be mediated by receptors : fprl1,35, (52) adam family metalloproteinase and egfr,53 and through active peptide internalisation.52 ll-37 also promotes angiogenesis, through fprl1 receptor on vascular endothelium, 54 hdps also act as powerful modulators of cellular responses to inflammatory stimuli. thus, ll-37 can inhibit secretion of inflammatory cytokines in response to lipoploysaccharide,40 and offer some protection in experimental endotoxaemia in mice.32 direct interaction with lipoploysaccharide accounts for only a fraction of the anti - endotoxic activity of ll-37, and transcriptional profiling of human monocytes stimulated with lipoploysaccharide, with and without ll-37, established a complex pattern of modulation of the lipoploysaccharide response, with a profound inhibition of proinflammatory genes, but retention of expression of chemotactic mediators and negative regulators of tlr signalling.40 on the basis of these studies, we have proposed that at least in some physiological settings ll-37 and possibly other peptides act to promote localised immunity to infection, while preventing systemic hyperinflammatory response. in contrast to the well - established anti - endotoxic activity of some peptides, the effects of peptides on cellular responses to other proinflammatory stimuli are cell type and stimulus dependent. immature monocyte - derived dendritic cells generated in the presence of ll-37 have increased phagocytic activity and, after maturation, show enhanced capacity to induce th1-polarised immunity.42 in contrast, simultaneous treatment of immature monocyte - derived dendritic cells with ll-37 and tlr ligands inhibits dendritic cell maturation.41 in plasmacytoid dendritic cells, ll-37 strongly augments responses to host and bacterial dna, and this activity may contribute to the pathology of psoriasis.43 furthermore, murine -defensin 2 was reported to act as an endogenous tlr4 ligand, promoting dendritic cell maturation and th1 polarisation of immune response.55 the biological importance of hdps in mammalian immunity has been investigated in several mouse models. the role of cryptidins in the gut is highlighted by the following reports : mmp7-knockout mice lacking mature cryptidins and nod2-null mice with reduced cryptidin production are more susceptible to oral challenges with escherichia coli and listeria,10, 56 whereas transgenic mice with a paneth cellspecific expression of human cryptidin hd5 are more resistant to oral challenges with salmonella.57 the role of cathelicidins in the immune defences of epithelial surfaces is addressed by the studies of cramp - null mice, which develop necrotic skin infections when challenged with group a streptococcus,58 and are also more susceptible to infections of the urinary tract.59 dysregulation of hdp production is also implicated in the pathology of a number of human diseases. in the specific granule deficiency and morbus kostmann syndromes, deficiencies in neutrophil hdps (and other proteins of neutrophil granules) are associated with persistent bacterial infections.60, 61 reduced hdp production in the skin is also associated with human disorders, with reduced ll-37 and -defensin levels in atopic dermatitis being linked to frequent skin infections.62 in contrast, abnormally high levels and altered proteolytic processing of cathelicidins are implicated in skin inflammatory conditions of rosacea and psoriasis.43, 63 disruption of hdp production and function at mucosal surfaces is also implicated in human pathologies, in particular in crohn 's disease and cystic fibrosis. it was suggested that the bacterial colonisation of the airways of cystic fibrosis patients, is at least in part, the result of inactivation of antimicrobial peptides by the high salt contents of airway fluids in cystic fibrosis.64, 65 mutations in nod2, associated with crohn 's disease, result in impaired production of cryptidins and this was proposed to contribute to the chronic intestinal inflammation by disrupting the interactions between the immune system and the microflora.12, 56 in summary, hdps with their direct antimicrobial properties and diverse immunomodulatory activities play an important role in mammalian immunity to infections and immune homoeostasis. the potential for these activities for clinical and commercial purposes remains to be fully explored. with increasing bacterial resistance to conventional antibiotics,66 there is a growing interest in exploiting the biological activities of hdps to target multidrug - resistant pathogens.3 however, despite much progress in the laboratory, so far only a few hdp - derived compounds have progressed into the clinic, with most clinical trials focusing on topical rather than systemic applications (table 2). this is a listing of known antimicrobial and/or immunomodulatory agents in development and/or clinical trials within private companies. only agents currently in clinical trials or likely to enter the clinic soon are recorded. this information is based on a prior review,3 updated by us primarily from company press releases and public presentations. several peptides that went through clinical trials but were not approved3 or polymyxins and gramicidin s that are generic anti - infectives that have long been available are not described the traditional approach to developing hdp - based therapeutics has focused on the antimicrobial properties of natural peptides. various methodologies for identifying, selecting, or designing highly active artificial antimicrobial peptides have been developed. one approach is to screen extensive libraries of semirandom peptides for bactericidal properties.67, 68 alternatively, systematic substitutions in the amino - acid sequence of natural antimicrobial hdps can be used to improve their activity. for example, combining highthroughput peptide synthesis on cellulose sheets with automated screening for antimicrobial properties using a luciferase reporter system allowed the development of bactenecin derivatives with dramatically improved activity.69 furthermore, several computer - aided approaches to analyse structure function relationships in natural and artificial peptide libraries permitted the prediction of peptide activity and design of novel peptides with stronger antimicrobial properties.70, 71 one of the limitations that need to be overcome before peptides can be widely used in the clinic is the high cost of peptide production. costs can be reduced to some extent by focusing on linear peptides of minimal length, and truncated derivatives of bactenecin and indolicidin with lengths of 812 amino acids are good candidates.69 however, more complex structures can be effectively generated on a large scale using recombinant technology72 and should certainly be pursued. lantibiotics are bacterial peptides with antimicrobial properties produced on an industrial scale for use as food preservatives. they make up one class of such compounds.73 their potential therapeutic value certainly warrants further investigation. another approach to reducing the costs of peptide therapeutics is to improve peptide stability and pharmacokinetics, thus decreasing the required dose. currently, this is investigated with protease - resistant d - amino acid peptides and various peptidomimetics.3 concerns have been raised that a widespread use of hdps in the clinic would select for pathogens resistant to natural immune defences. indeed, many bacterial species already possess modestly effective resistance mechanisms, including peptide degradation, sequestration, efflux, and chemical modifications of cell walls and membranes to reduce hdp binding;74, 75 resistance to hdps can be selected in the laboratory.76 nevertheless, hdps are less prone to inducing resistance than conventional antibiotics because they often use several microbicidal mechanisms simultaneously, target ing many microbial systems with low affinity rather than having one specific target.75 also, because of the diverse mechanisms of peptide action, the use of synthetic peptides that do not occur in nature could partly alleviate the problem of resistance to natural hdps. finally, immunomodulatory peptides that act on the host rather than on the pathogen offer a unique opportunity to minimise the direct selective pressures for pathogen resistance. recently, such an immunomodulatory peptide, an innate defence regulator idr-1, was shown to protect mice against bacterial infections, including infections with multidrugresistant pathogens, and this provides an important proof of principle for the immunomodulatory approach.38 the peptide was shown to act as a neutrophil chemoattractant37 and furthermore to induce chemokine production and promote cell recruitment in vitro and in vivo ; these activities may account for some of its protective effects. importantly this peptide, as well as many natural hdps, exerts anti - inflammatory and anti - endotoxic effects at the same time as promoting local clearance of infection.38, 40 thus, unlike other treatments aimed at boosting the immune system, such peptides can offer protection without the risk of inducing dangerous hyperinflammatory states. in the immediate future, the field of immunomodulatory peptide therapeutics faces the challenges of developing highthroughput screens for immunomodulatory activity, and also of firmly defining which in vitro activities correlate with the protection against infection and other desirable biological outcomes in vivo. furthermore, other activities of hdps, such as their roles in wound healing and angiogenesis, require further investigation to assess their potential therapeutic value.54 over the course of evolution, nature has created an impressive arsenal of hdps with extreme diversity in structure and biological activity. these can serve as excellent templates for development of both antimicrobial and immunomodulatory compounds, often combining both activities in the same molecule. these compounds can be used in combination with conventional antibiotics, and also to target resistant pathogens where conventional antibiotics fail. some peptides may even have potential against diseases of unknown aetiology (emerging infectious diseases), as their spectrum of activity is often very broad and may involve stimulation of the immune response of the host rather than targeting the pathogen. importantly, immunomodulatory peptides that target the host immune system rather than the pathogen also offer an excellent opportunity to minimise the risks of pathogen resistance to these compounds. the current challenge is to develop new biologically active synthetic peptides or mimetics thereof with improved pharmacokinetics, low toxicity, and low manufacturing costs for both topical and systemic applications.	the rapidly increasing incidence of multidrug - resistant infections and the alarmingly low rate of discovery of conventional antibiotics create an urgent need for alternative strategies to treat bacterial infections. host defence peptides are short cationic molecules produced by the immune systems of most multicellular organisms ; they are a class of compounds being actively researched. in this review, we provide an overview of the antimicrobial and immunomodulatory activities of natural host defence peptides, and discuss strategies for creating artificial derivatives with improved biological and pharmacological properties, issues of microbial resistance, and challenges associated with their adaptation for clinical use.
studies in living animals are critical to oncology research, and many experimental models have been exploited for drug development and basic studies [1, 2 ]. fast - growing tumors can be generated in mice by orthotopic or ectopic implantation of tumor cell lines. however, models exhibiting spontaneous oncogenesis better mimic human disease therefore, oncogene - driven or chemically induced tumor models have come into use more recently [3, 4 ]. in both spontaneous and transplanted tumor models, the most common readouts are primary tumor growth and metastatic spread, but accurate measurement of these parameters is challenging. unlike necropsy, noninvasive imaging techniques could offer an ideal solution as they allow measurement of tumor burden in the whole body without the need to sacrifice the animal. this makes longitudinal studies possible, simultaneously reducing the number of animals required and producing more robust data. these technologies are also sensitive and accurate enough to detect microscopic nodules, whose importance in human disease prognosis is increasingly recognized [5, 6 ]. several imaging techniques have recently become available for small animals. these include 2-deoxy-2-[18f]fluoro - d - glucose positron emission tomography (fdg - pet), t2-weighted magnetic resonance imaging (t2w - mri), and optical imaging, encompassing bioluminescence imaging (bli) and fluorescence imaging (fli). both fdg - pet and t2w - mri are used clinically in humans, whereas optical imaging is specifically used for research and preclinical studies. while each method has its own advantages, a detailed side - by - side comparison of their use for tumor imaging has yet to be carried out. the purpose of the present study is to compare practicality and performance of these four imaging techniques in the context of mouse tumor studies. specifically, we assessed four different parameters, namely, practicality, performance for small tumor detection, performance for tumor burden measurement, and performance for tumor identification. the performance for tumor burden measurement was conducted specifically for optical methods, since they are well adapted for this purpose. conversely, the performance for tumor identification was compared only between mri and fdg - pet, since optical methods can not be applied for this purpose in current tumor models. the b16 transplanted tumor model is well - defined and offers a high level of flexibility. b16 cells can be modified to express the transgenes required for detection by optical imaging, followed by injection of these cells into the animal by different routes to produce either subcutaneous or pulmonary lesions. tumor onset is predictable, so nodules can be tracked from their microscopic stage, making this model ideal to assess the practicality and performance of each technique for small tumor detection and tumor burden measurement. in contrast to transplanted tumor models, retaad tumors may arise at any location in the skin (cutaneous melanoma tumors) and internal organs (visceral metastases). this model is, therefore, particularly suited for the assessment of the performance of imaging techniques in tumor identification, as it makes it possible to calculate the specificity, the sensitivity, and the positive predictive value for tumor detection. tumors in spontaneous models usually do not express reporter genes and are, therefore, not suited for optical imaging technologies. therefore, we have used this model to compare fdg - pet and t2w - mri. b16-f10-luc cells (xenogen, alameda, calif, usa) express firefly luciferase (sequence from pgl3, promega) under the control of the sv40 promoter. b16-f10-rfp cells express dsred2 under the control of the cmv promoter [11, 12 ]. all studies were approved by the institutional animal care and use committee of the biological resource center and of singhealth. two perpendicular diameters (d1 d2) of the tumor were then measured using caliper, and were used to calculate the tumor volume (v) (v = 4/3 d1 d2/8). prior to necropsy, the investigator was unaware of the results of the imaging scans, rendering the two analyses independent. animals injected intravenously with b16 cells were examined for their lungs and peritoneal cavity as described. retaad mice develop tumors spontaneously with widespread metastases, so they were subjected to more extensive necropsy. for each mouse, a necropsy diagram was filled to document the location, size, and morphology of nodules. four mice subcutaneously injected with b16 cells were used to determine the smallest detectable tumor. ten retaad mice were used to assess performance in tumor identification. after fasting overnight, mice were prewarmed to 37c, and approximately 5.5 mbq of fdg (0.6 mm) (department of nuclear medicine, singapore general hospital) was administered intraperitoneally. micro - pet imaging was performed using a r4 micropet scanner (concordes microsystems inc.) with a ring diameter of 26 cm, 7.8 cm axial field of view and an average intrinsic spatial resolution of 1.75 mm. under isoflurane anesthesia, mice were subjected to 15 minutes of acquisition. for image reconstruction, an energy window of 350700 kev and a coincidence timing window of 6ns were used. two - dimensional histograms by fourier rebinning and image reconstruction by filtered backprojection were used. the image data were corrected for nonuniformity of the scanner response, dead time count losses, and physical decay to the time of injection. no correction was applied for attenuation, scatter, or partial - volume averaging, as these parameters are not critical for mouse models. in the reconstructed images, tumors were identified as regions of high uptake in study animals that were absent from images of control mice. to allow quantitative image analysis, regions of interest (roi) were manually drawn over areas of high uptake. within these regions, counting rates were converted to standardized uptake values (suvs) using a system calibration factor derived from the imaging of a mouse - size water - equivalent phantom containing f. four mice subcutaneously injected with b16 cells were used to determine the smallest detectable tumor. data were acquired at the singapore bioimaging consortium on a 9.4 t mri scanner (varian, palo alto, calif, usa) using a transmit - receive volume rf coil. a multislice 2d fast spin echo with periodically rotated parallel lines with enhanced reconstruction (propeller) pulse sequence was used to give high image quality and robustness to motion. brain scans were conducted using the following parameters : repetition time (tr) = 4000 ms ; effective echo time (te) = 51 ms ; echo spacing (esp) = 6.4 ms ; echo train length (etl) = 16 ; field - of - view = 25.6 25.6 mm ; blade matrix = 256 16 ; number of blades = 32 ; reconstructed matrix = 256 256 ; slice thickness = 1 mm ; slice gap = 0.5 mm ; slices = 5 ; averages = 1 ; orientation = axial ; readout bandwidth = 208 khz and acquisition time = 2 min 16 s. for the abdomen, several changes were made to accommodate the shorter t2 so that, te = 20 ms ; esp = 5.0 ms ; etl = 8 ; blade matrix = 128 8 ; reconstructed matrix = 128 128 ; slice gap = 0.2 mm. the resulting data were used to reconstruct images as described. tumors were identified as highly contrasted masses or nodules that were present in study animals but absent from control mice. for all bli experiments, studies to determine the smallest detectable tumors, accuracy for tumor burden measurement, and tissue attenuation used four, four, and six mice, respectively, each subcutaneously injected with b16 cells. to further demonstrate the possibility to use bli for accurate followup of tumor growth (as shown in figure 5), 4 unshaved mice were injected subcutaneously with b16 cells and another 4 were injected intravenously. for other experiments, 1525 minutes before imaging, mice were injected intraperitoneally with 200 l of d - luciferin (15 mg / ml in pbs) as described and then anesthetized using isoflurane. for in vitro imaging, cells were plated in pbs in flat - bottomed 96 well plates before d - luciferin was added to a final concentration of 1.5 mg / ml. immediately after necropsy, some tumors were excised and imaged ex vivo on tissue culture plates. these plates were scanned for 5 to 40 seconds, whereas mice were scanned for 30 to 60 seconds using the ivis spectrum photon - counting device optical imaging system (xenogen, alameda, calif, usa). bioluminescence signal was reported as total light emission within the region of interest (photon / s). specific signal was calculated as the ratio of bioluminescent signal in the region of interest to the bioluminescent signal in a background region containing no cells or tumors. a signal was defined as positive when it was greater than the sum of the mean background signal plus 2 standard deviations of the background signal. for all fli experiments, studies to determine the smallest detectable tumors, accuracy for tumor burden measurement, and tissue attenuation used four, four, and six mice, respectively, each subcutaneously injected with b16 cells. for in vivo imaging, animals were anesthetized using isoflurane and some mice were shaved as indicated in the figure legends. for in vitro imaging, cells were plated in pbs in flat - bottomed 96-well plates. immediately after in vivo imaging, some tumors were excised at necropsy and imaged ex vivo on tissue culture plates. plates or mice were scanned for 0.1 to 1 seconds using the ivis spectrum photon - counting device optical imaging system (xenogen, alameda, ca) with filters for red fluorescence (excitation 535 nm, emission 600 nm) and background fluorescence (excitation 465 nm, emission 600 nm). regions of interest were drawn and quantified using living image software version 2.5. specific signal was reported as the ratio of the fluorescence signal in the region of interest to the fluorescence signal in a background region containing no cells or tumors. a signal was defined as positive when it was greater than the sum of the mean background signal plus 2 standard deviations of the background signal. bli and fli specific signals (signal - to - noise ratio) for single time point experiments were compared using the mann - whitney test. the specificity was defined as (number of sites where no tumor was found)/(total number of sites without tumor confirmed at necropsy), where the total number of possible sites for tumor growth in each retaad mouse was 14 (cheeks, neck, genitals, flanks, forelimbs, hind limbs, and peritoneum, each time on the left or right side). the positive predictive value was defined as (number of tumors detected by imaging and confirmed at necropsy)/(total number of tumors detected by imaging). the sensitivity for tumor identification was defined as follows : (number of tumors detected by imaging and confirmed at necropsy)/(total number of tumors observed at necropsy). three parameters were taken into account : animal preparation, time for analysis, and ease of access to the technology (table 1). all techniques required anesthesia of the animal by isoflurane inhalation, taking approximately 4 minutes per mouse. the additional tracer injection and preincubation time for fdg - pet resulted in at least 3-fold longer preparation time per animal compared to the other techniques. shaving requires 10 minutes per animal, and while there has been debate on whether it can be omitted for optical imaging, we found it to be dispensable for bli (see below). because devices differ, we selected widely used platforms for comparison : ivis spectrum (xenogen) for optical imaging, r4 micropet (concordes microsystem) for fdg - pet, and 9.4 t mri (varian) for t2w - mri. the main differences in practicality between technologies were highlighted by the ease of scale - up to larger groups of animals. for example, the ivis spectrum allows parallel imaging of up to 5 mice, taking as little as 2 minutes to complete all the scans. in contrast, both fdg - pet and t2w - mri can typically image only one mouse at a time (maximum 2 in some settings), taking at least 15 minutes per scan. overall, this meant that optical scanning of 10 mice could be completed in 1 - 2 hours, whereas fdg - pet or t2w - mri would take 1330 hours. each technique has low reagent and consumable costs of around five us dollars per scan per animal. fdg can usually be obtained as surplus material from nuclear medicine departments or comes at a low cost compared to other pet reagents. equipment for optical imaging is accessible in many research institutes and costs less than five hundred thousand us dollars, with the pet scanner costing around six hundred thousand us dollars. overall bli and fli are the most practical techniques and are particularly suitable for large studies requiring high throughput imaging. the b16 melanoma model allowed us to assess the smallest tumors that could be accurately detected by the various techniques. four mice per technique were injected subcutaneously with 10 b16 cells, resulting in tumor growth at the injection site. these tumors were clinically undetectable up to day 10 after injection, but were evident at necropsy. after day 10, tumors become macroscopic (2 mm diameter and above) and were measured in living animal with a caliper. bli and fdg - pet detected nonpalpable tumors (< 1 mm), whereas the smallest tumors detected by t2w - mri and fli were 1 mm and 2 mm diameter, respectively (figure 1). bli detected microscopic tumors as early as 1 day after subcutaneous injection (figure 1(c)) when the nodules were too small to be detected even at necropsy. while this means that their presence could not be confirmed either visually or histologically, these tumors were actively growing, increasing their bli signal and overall, bli and fdg - pet are applicable for in vivo detection of microscopic tumors, whereas t2w - mri and fli are only applicable to palpable tumors. both optical imaging techniques can be used to follow transplanted tumor growth. to carry out a detailed comparison, we selected two b16-luc and b16-rfp clones showing equivalent performance for in vitro imaging (figure 2(a)) with similar growth rates in vitro (not shown) and in vivo (figure 2(b)). at day 1, 2, 3 and 5 after injection, only bli detected nonpalpable tumors (figure 2(c)). at further time points, the tumor - specific signal detected by bli was significantly greater than that seen by fli for tumors of equivalent size (figure 2(c)). both techniques may, therefore, be used to follow small macroscopic tumors, but only bli provides data at the microscopic stage. we investigated the higher sensitivity observed for bli compared to fli when performed in vivo. attenuation occurs when tissues around the tumor absorb some of the imaging excitation and emission signal, autofluoresce, leading to a reduction in the signal to noise ratio. to test this hypothesis, we measured signal reduction by comparing ex vivo (figure 3(a)) and in vivo (figure 3(b)) signals after tumor excision. the signal measured in vivo on shaved mice was reduced 3-fold for bli and 14-fold for fli compared to the signal of tumors ex vivo after excision (figure 3(b)), confirming the hypothesis that fli is more prone to tissue attenuation. to address the contribution of mouse hair to further signal attenuation again, the fli signal (over than 400 fold reduced) was more prone to attenuation than the bli signal (70 fold reduction) (figure 3(c)). in summary, tissue and hair surrounding the tumor significantly reduced the ability of fli to detect small tumors in vivo. we compared tumor volumes estimated in vivo by bli and fli, to those calculated by caliper measurements and found a good correlation between these two techniques (figure 4). bli has demonstrated an ability to detect microscopic tumors and to estimate tumor volumes in vivo with good accuracy. to further explore the power of the technique we injected unshaved mice either subcutaneously or intravenously with b16-luc cells and imaged them repeatedly. as expected, bli detected subcutaneous tumors earlier than clinical examination (figure 5(a)). moreover, the bioluminescent signal follows a characteristic gompertzian curve as expected for tumor growth, therefore more accurately reflecting the biology of the tumor compared to caliper measurements. bli detected a signal following intravenous injection of b16-luc only after a few minutes, which likely reflects the initial trapping of the injected cells in the lung. by day 4 most of these cells were cleared and the bli signal dropped, only to increase again as tumor growth occurred in the lungs, peritoneal cavity and at the point of injection (figure 5(b)). bli can, therefore, be used in shaved or unshaved mice for quantitative followup of tumor growth at both cutaneous and internal sites. spontaneous or carcinogen - induced tumor models are increasingly used for cancer research. in these animals, a variable number of tumors arise in a range of locations over a less predictable time course. the need to sacrifice the animal for information is a disadvantage of such models, but to replace necropsy by in vivo imaging, three criteria must be met. firstly, the technique must correctly predict the absence of tumors at normal sites and for nontumor bearing mice (specificity). secondly, the technique must identify tumors accurately, with a low rate of false positives (high predictive value). thirdly, the technique must be sufficiently sensitive to detect all the tumors that necropsy currently does. we compared the specificity, predictive value and sensitivity of fdg - pet and t2w - mri in the retaad spontaneous melanoma model. during the course of disease, these mice develop tumors of various sizes in wide - ranging anatomical locations, making the model an ideal test for the performance of these techniques. some background is evident in the bladder, heart, and eye regions, but this was expected due to the excretion and circulation of the probe, and the presence of the harderian glands. the same effect was seen in control mice, and these regions were accordingly excluded from analysis. a total of 10 retaad mice and 4 control mice were independently analyzed by fdg - pet and necropsy. of the 28 tumors identified by fdg - pet, 24 were confirmed by necropsy or histology, making the positive predictive value of the fdg - pet 86%. the 4 tumors that were not confirmed at necropsy were embedded in the muscles of the back and the limbs, sites for which histological analysis could not be carried out. an additional ten tumors were found at necropsy but not by fdg - pet, most likely because of low metabolic activity. only 4 tumors were predicted by fdg - pet but could not be confirmed at necropsy or histology. taking into account 14 possible sites for tumor growth for each of 14 mice analyzed, the specificity of fdg - pet was 98%. figure 7 shows a typical t2w - mri scan of a tumor - bearing retaad mouse. a total of 6 retaad mice and 4 control mice were independently analyzed by t2w - mri and necropsy. twenty - two tumors were identified by t2w - mri, of which 21 were confirmed at necropsy, making the predictive value of t2w - mri 95%. five small tumors (4 out of 5 were < 1 mm diameter) were observed at necropsy but not detected by imaging. taking into account 14 possible sites for tumor growth for each of 10 mice analyzed, the specificity of t2w - mri was 99%. overall, both fdg - pet and t2w - mri allow precise 3d visualization of tumors with good specificity, sensitivity, and accuracy and are, therefore, highly recommended for any study aiming at identifying tumors. interestingly, fdg - pet shows slightly lower sensitivity for tumor detection than mri, probably due to the fact that some tumors lack the minimal metabolic activity required for detection. therefore, fdg - pet would be the preferred choice if the assessment of metabolic activity is desired ; otherwise, mri is recommended (table 2). to compare bli and fli in vivo, we used tumors originating from two different b16 cell lines expressing firefly luciferase and dsred2, respectively. the two prototypical reporter genes have been chosen among the most commonly used and most efficient markers at the time of writing. results obtained might change in the future when new reporters are developed. with current reporter genes, both bli and fli detected their respective cell lines equally well in vitro, but when the cells were injected into mice and allowed to form tumors, only bli was able to image microscopic nodules. we showed that this difference was due to the tissues surrounding the tumor during in vivo imaging, a phenomenon known as tissue attenuation. this is especially relevant for fli as the tissue can absorb and scatter fluorescent light at both the excitation and emission level. for bli these experiments used subcutaneous tumors, but for internal tumors the differences between the techniques could only be expected to be magnified due to the increased optical path through the tissues. despite fli being less sensitive than bli with the instrumentation we used, it has been used successfully for whole - body imaging in other studies, even with very low cell numbers. for example, labeled proteins have enabled fluorescent imaging of tumor cell mobility, invasion and angiogenesis (reviewed in). hirakawa. successfully used fli to monitor the dissemination of very small numbers of gfp - labeled skin tumor cells to the proximal lymph nodes of mice in vivo. importantly, fli is so far the only imaging technology to give single - cell resolution or even subcellular resolution in vivo [23, 24 ]. while each technique tested is state of the art, imaging technologies are constantly being improved. for example, optical techniques are being modified to permit three - dimensional reconstruction of the bioluminescent source and tumor localization. it is even becoming possible to combine imaging modalities using multiple fusion reporter genes within the same animal (see, e.g.,). table 3 summarizes the main features of the imaging methods, with their primary advantages and disadvantages. estimating the real cost of the different technologies is difficult, and largely depends on equipment availability. however, on the basis of equipment costs, operating expenses, and the level of training required, optical imaging is normally less costly than t2w - mri and fdg - pet. while the design of the current study did not involve the comparison of all techniques using a single tumor - bearing animal, the b16 model is reproducible enough to carry out a fair comparison. by using groups of at least 4 mice to perform statistical analyses, we were able to detect some major differences between the various imaging modalities investigated. further studies that include larger number of animals could be performed in order to detect even more subtle differences between these various imaging techniques. in fact, doing so requires generation of transgenic mice expressing the reporter gene in the cell lineage of interest, followed by either carcinogen treatment or intercrossing with an oncogene driven transgenic mouse line. for example, vooijs. expressed luciferase under a pituitary gland - specific promoter in a model of spontaneous pituitary cancer. similarly, lyons. constructed an oncogen - driven prostate cancer model with luciferase expression in the prostate. consistent with our findings, both authors successfully monitored tumor growth in vivo using bioluminescence. therefore, optical imaging is a valid strategy, but it is time consuming for spontaneous tumor models. in contrast to optical imaging techniques, t2w - mri and fdg - pet can be applied to any tumor - bearing mice, including spontaneous tumor models. using a b16 mouse tumor model, we showed that t2w - mri and fdg - pet scans allow early detection of tumors and exhibit good sensitivity and positive predictive value when compared to necropsy. results obtained with pet scanning are related to tumor metabolism and glucose uptake by the tumors ; hence, they could vary from one tumor cell line to the other. however, published data have shown that pet sensitivity is high in other tumor models (see, e.g. t2w - mri performed slightly better, presumably because its basis is anatomical rather than requiring tumor metabolism, as in the case of fdg - pet. indeed, in treated cancer patients, some responsive tumors simply lose their metabolic activity while the tumor mass is unchanged. this typically translates into a tumor mass anatomically identified by mri or ct scan but fdg - pet negative. in addition, mri and fdg - pet are less affected than optical imaging by attenuation due to the depth of the tumor, and both have the significant advantage of providing precise locations of even small nodules. as mri and fdg - pet are used in the clinical setting, their application in preclinical research may help translate basic findings into clinical studies. in this context, if whole - body metabolic imaging is required, fdg - pet is the best option for longitudinal followup of tumor burden and can be combined with ct which we did not address here. t2w - mri is better used for specific body sections or to monitor the development of a particular tumor over time, as it may provide contrast and anatomical information related to location, volume, vascularization, and invasion. for example, diffusion - weighted imaging was successfully used to detect glioma tumors in rats, and more recently used for whole - body imaging. overall, this study has highlighted how various imaging techniques can be best used in different types of tumor models or to assess particular readouts. bli in particular offers fast, sensitive whole - body tumor imaging, even detecting microscopic tumors. bli could replace traditional caliper measurements, as it is able and well suited to determine tumor burden in longitudinal studies. however, the main disadvantage of optical techniques is the requirement for tumor cells to express a reporter gene. this has so far largely limited the use of bli and fli to transplanted tumor models. however, with time and resources, spontaneous tumor models that also express reporter genes will become increasingly available. in the meantime, however, this means that nonoptical methods are preferable for tumor detection in spontaneous models. both available techniques have specific advantages and disadvantages ; t2w - mri accurately reflects tumor volume and morphology, but it is more time consuming, whereas fdg - pet uniquely measures metabolic activity. in summary, each technique represents a valuable tool to study tumor - bearing animals, but the careful selection of the most appropriate method will be critical to maximize the benefit of their use.	introduction and purpose. monitoring solid tumor growth and metastasis in small animals is important for cancer research. noninvasive techniques make longitudinal studies possible, require fewer animals, and have greater statistical power. such techniques include fdg positron emission tomography (fdg - pet), magnetic resonance imaging (mri), and optical imaging, comprising bioluminescence imaging (bli) and fluorescence imaging (fli). this study compared the performance and usability of these methods in the context of mouse tumor studies. methods. b16 tumor - bearing mice (n = 4 for each study) were used to compare practicality, performance for small tumor detection and tumor burden measurement. using retaad mice, which develop spontaneous melanomas, we examined the performance of mri (n = 6 mice) and fdg - pet (n = 10 mice) for tumor identification. results. overall, bli and fli were the most practical techniques tested. both bli and fdg - pet identified small nonpalpable tumors, whereas mri and fli only detected macroscopic, clinically evident tumors. fdg - pet and mri performed well in the identification of tumors in terms of specificity, sensitivity, and positive predictive value. conclusion. each of the four methods has different strengths that must be understood before selecting them for use.
ag nanoparticles are currently of considerable interest and the subject of a significant literature due to the potential uses of the tunable light absorption, scattering and local field enhancement in the uv - visible region. there are two types of bimetallic nanoparticles : alloy particles (particles with a homogeneous distribution of two kinds of metals) and core shell structure particles (particles with heterogeneous arrangement of two kinds of metals leading to core shell structure). the composition or shell thickness dependent surface plasmon resonance (spr) absorption spectra of both core shell type and alloy model au as we know, only one spr peak occurs at around 520 nm for pure au spherical nanoparticles. similarly, only one spr peak occurs at around 400 nm for pure ag spherical nanoparticles. in bimetallic au ag nanoparticles, the spr band depends on the composition and the distribution of the two metals. for alloy type particles, the two kinds of metals are homogeneously distributed over the whole volume on an atomic scale, so there is still only one spr peak located between those of pure au and ag nanoparticles. ag bimetallic nanoparticles may shift from 400 nm to 520 nm linearly by changing the molar fraction of au from 0 to 100%. for core shell structure particles, one of the two metals constitutes the core of the structure, and the other one the external shell, the spr become complex because of the interface between core and shell. a direct approach to determining shell structure by transmission electron microscopy (tem), because a boundary between ag and au elements can be distinguished by bright and dark contrast in the tem imaging. in the core shell bimetallic au ag nanoparticles, two spr bands can be observed. due to the two interacting metals at the interface, spr peak positions for both silver and gold shift. for example, when au nanoparticle was coated by an ag shell, a blue shift for the original pure gold peak was observed. however, with increasing the amount of silver, the ag shell thickness grows resulting in a red - shift of the spr maximum for the silver fraction. at last, the au ag core shell structure bimetallic colloids show only one spr at around 402 nm, which can be attributed to the spr of silver particles alone. the question arises for the origin and the shift fashion of the two spr bands. in the report of hodak. however, the mechanism and physical picture of these two kinds of spr, especially the mechanism of collective electron oscillation at the interface, have not been studied in great detail so far. in this paper, we propose to investigate the origin of the spr at the interface of au ag core shell structure bimetallic nanowire by calculating the wall thickness dependent absorption spectra. the mechanism based on oscillatory surface electrons under coulombic attraction has been used to illuminate the shift fashion of spr bands that were observed experimentally and calculated theoretically. we choose a long nanowire as our model, because gold nanowire will become a kind of widely used element of optoelectronic nano - devices. furthermore, noble metallic nanowires and nanotubes have already been synthesized experimentally [14 - 16 ]. in our studies, the bimetallic composite model consists of a metallic wire core of radius r1 coated by a metallic wall of thickness r2 1. the dielectric functions of the core, wall, and embedding medium are 1, 2 and 3, respectively. it is important to note that 1 and 2 have real and imaginary frequency - dependent components and can be expressed as (1)(2) where b1 and b2 are dielectric function of bulk metal of core and wall which are due to inter - band transition and varies with frequency. p1 and p2 denote the plasmon frequency of the bulk metal of core and wall, 1 and 2 are size limit relaxation time of metallic nanowire and nanowall, and is the frequency of electromagnetic wave. 1, 2, 3are the dielectric functions for the metallic core, metallic wall and embedding regions, respectively;r1denotes the core radius andr2denotes the wall radius in our analysis, the radius of bimetallic nanowire changes from 10 to 25 nm, which is much smaller than the light wavelength (300800 nm). so the quasi - static approximation can be employed in this calculation. in this theory, the spatial variation of the electromagnetic field therefore, the metallic particle is subjected to an almost uniform field and oscillates like a dipole with polarization proportional to the incident electric field. now we consider one bimetallic nanowire illuminated by the light of wavelength. this incident light travels in the forward y direction and the nanowire to be infinitely extended perpendicular to x y plane. when the external light - induced electric field,, travels in the forward y direction and polarized in x axis direction, the solution for the local electric field in the bimetallic nanowire could be derived from laplace s equation. when r = r2, = 0 (here is the included angle of incident field makes with the position vector), the corresponding electric field is(3) the corresponding polarizability is(4) finally, we can obtain the absorption cross section by using scattering theory, (5) although the length of the nanowire in this calculation is infinite, the absorption cross sections per unit length of the nanowire can be finite. so we refer to such an absorption cross section per unit length as simply an absorption cross section in this study. the calculated absorption spectra from transverse spr of ag au core shell structure nanowire are shown in fig., the core radius r1 is fixed at 10 nm, whereas the wall thickness r2 r1 changes from 0 to 15 nm. when au wall thickness is 0 nm, the only peak at 410 nm has been ascribed to the transverse spr of pure ag nanowire. with increasing the au wall thickness, the other peak takes place at around 530 nm and red shifts slightly. this peak at 530 nm resulted from the transverse spr of out surface of au wall, the red shift is ascribed to the increasing size of au wall. meanwhile, the spr peak at 410 nm red shifts obviously with increasing the au wall thickness. all these spectral characters are in agreement with the experimental results. calculated light absorption spectra of ag au core shell structure nanowires with varying wall thickness, the core radiusr1 = 10 nm in order to find the effect of wall thickness on the shift of spr peaks, we also plot the absorption cross section as a function of wavelength and gold wall thickness, as shown in fig it is obvious that the increasing au wall thickness leads to the longer wavelength peak from au wall get intense and red shift slightly, and leads to the shorter wavelength peak decrease in intensity and red shift nonlinearly. the similar decrease and red shift of the shorter wavelength peak has also been observed experimentally in ag core / au shell nanoparticles by steinbruck. and lee.. shell structure nanowire, the longer wavelength spr peak gets intense very slowly with increasing the wall thickness. therefore, as au formed a thin wall layer (< 4 nm) on the ag core wire, the surface plasmon absorption band showed only one peak, which is in agreement with the experimental result. however, the shorter wavelength spr peak of interface red shifts and gets weak rapidly with increasing the au wall thickness within a few nanometers. therefore, when the au wall is too thick, the shorter wavelength spr peak from interface becomes very weak. so the absorption spectrum has only one distinct peak from out surface at longer wavelength, which is in agreement with the experimental result. shell structure nanowires with different gold wall thickness;babsorption cross section versus wavelength in au ag core the core radius is 10 nm in order to investigate the origin of the shorter wavelength spr peak of gold coated silver nanowire, we also calculated the absorption spectra of silver coated gold nanowire (i.e. au ag core shell structure nanowire), as shown in fig. however, increasing the ag wall thickness leads to the longer wavelength peak blue shift distinctly and nonlinearly (the shift fashion is similar to that of shorter wavelength peak in ag au core shell structure nanowire), and leads to the shorter wavelength peak red shift slightly (the shift fashion is similar to that of longer wavelength peak in ag au core have also been observed experimentally in au core / ag shell bimetallic nanoparticles by pande.. 3a and b, we find the shorter wavelength peak at around 400 nm in au ag core shell structure nanowire and the longer wavelength peak at around 530 nm in ag both of them come from the spr of outer metallic wall. on the other hand, the longer wavelength peak shifting from 550 to 480 nm in au ag core shell nanowire and the shorter wavelength peak shifting from 410 to 450 nm in ag au core both of them come from the spr of interface between metallic core and wall. when the metallic wire has been coated with another kind of metal, a core shell structure comes into being and the surface of the wire has transformed into an interface between wire and wall. due to the interacting of the two metals at the interface, the corresponding spr peak shifts obviously with changing the ratio of wire and wall radius. however, the spr peaks resulting from ag ag interface always shift nonlinearly with varying wall thickness, which is different from the linear shift fashion of alloy ag this phenomenon may be illuminated by the mechanism of spr in the bimetallic core wall interface. as we know, the spr of noble metallic particles is intimately related to the delocalized conduction band electrons that only weakly interact with the lattice of cations. the coulombic attraction between the negative electrons and the positive metal cations takes place and serves as the restoring force for the oscillatory electrons. the frequency of spr is equal to the intrinsic frequency of oscillatory electrons in nanoparticles, which is controlled by the bound grade of the electrons. the tightly bound the electrons are, the higher the spr energy and the intrinsic frequency are ; on the contrary, the weakly bound the electrons are, the lower the spr energy and the longer resonance wavelength are. when ag wire is coated by an au wall, the electrons in ag wire may transfer into the au wall, and the oscillatory electron cloud will suffer an attractive force from net positive charges in au wall (au may has more valence electrons than ag. for instance, au has more positive charges than ag), as shown in fig. therefore, the bound grade has been decreased and the spr peak red shifts. on the contrary, when au wire is coated with an ag wall, the oscillatory electron cloud will suffer a repulsive force from net negative charges in ag wall. because spr is a surface effect and the amplitude of the oscillatory electron cloud is small and limited, so the extra coulombic attraction only arises within thin wall. the shift of spr will get weak and stop when the wall thickness exceeds the threshold value. in conclusion, there are two kinds of transverse spr in au ag bimetallic nanowire. one is resulted from the outer surface of wall metal and is similar to that of pure metallic particle, the other is resulted from the interface between the core and wall metals and is similar to that of alloy particle. quasi - static calculations show that, for ag - au core - shell structure nanowire, increasing the au wall leads to the interface spr red shifts obviously and decreases, whereas the outer surface spr red shifts slightly and increases. for au shell structure nanowire, increasing the ag wall leads to the interface spr blue shifts obviously and decreases, whereas the outer surface spr red shifts slightly and increases. the net charges and extra coulombic force in metallic wall affect the energy of spr and leads the nonlinear shifting. this work was supported by the national natural science foundation of china under grant no.	transverse surface plasmon resonances (spr) in au ag and ag au core shell structure nanowires have been investigated by means of quasi - static theory. there are two kinds of spr bands resulting from the outer surface of wall metal and the interface between core and wall metals, respectively. the spr corresponding to the interface, which is similar to that of alloy particle, decreases and shifts obviously with increasing the wall thickness. however, the spr corresponding to the outer surface, which is similar to that of pure metal particle, increases and shifts slightly with increasing the wall thickness. a mechanism based on oscillatory surface electrons under coulombic attraction is developed to illuminate the shift fashion of spr from bimetallic core shell interface. the net charges and extra coulombic force in metallic wall affect the spr energy and the shift fashion.
in the early 1960s, researchers began to describe an important nuclear structure in eukaryotic cells that differed from the already well - known dna / histone - based chromatin (1). this structure, referred to as the nuclear matrix (nm), can be separated from the rest of the nucleus by applying dnase i digestion followed by salt extraction (2). many functional aspects of the nm have been described ; these include dna replication (3), dna transcription (4) and dna repair (5,6). the existence of the nm as an independent sub nuclear structure is not a proven reality but a widely accepted hypothesis that has profoundly influenced the literature : pubmed alone retrieves over 3000 articles associated when queried with the terms nuclear matrix or nuclear scaffold. the nm might still be an artificial result of the preparation methods rather than a real in vivo structure (79). however, the main facts that argue in favor of the existence of this controversial part of the nucleus are its observation in non - eluted nuclei through electron spectroscopic imaging (10), the existence of protocols to isolate the nm at physiological salt concentrations through electroelution of chromatin (11), the fact that chromatin loops (s / mar - dna sequences) bind to a non - chromatin network and finally the description of functional units that stay in their original place even after removing chromatin and soluble proteins from the nucleus (12). two main structural elements form the nm (13) : the internal nuclear matrix (inm) and the nuclear shell (or nuclear lamina). the inm is an aggregate of proteins, mainly the intermediate filaments lamins, numa (13) and hnrnp proteins (13,14). several non - inm proteins can be separated along with the inm through more careful preparation protocols (15,16). the protein composition of nuclear matrices in different organisms and cell types was discovered mainly by 2d gel electrophoresis, a method that separates proteins based on their isoelectric points (first dimension) and molecular weight (second dimension). nuclear matrices, once separated from the chromatin and the soluble compartments of the nucleus, contain very different proteins in tumor than in non - tumor cells (17,18). in cancer research, these differences provide early indications for different types of tumors. collecting and analyzing data about nm proteins may help to understand the relationship between those proteins and cancer and to discover nm - associated proteins that have not been implicated with the nm. the vast majority of proteins that have actually been associated experimentally with the nm are not annotated in public databases. thus, we have built and are maintaining nmpdb, a database with proteins that are associated to the nuclear matrix. first, we downloaded over 3000 abstracts from pubmed that resulted from queries with the terms nuclear matrix / matrices and nuclear scaffold. then we wrote a simple perl script that color - highlighted three types of phrases in the text (through html tagging) : (i) nuclear matrix terms, (ii) uniprot protein names and (iii) verbs describing binding processes such as to bind, to associate or to interact (figure 1). each abstract was followed by html elements that enabled the quick interactive subclassification of each protein into one of the following classes : (i) part of the internal nuclear matrix (inm), (ii)tightly associated with the inm (asc), (iii) affinity toward the inm changes depending on protein modification, cell type and/or current stage of the cell cycle (mix) and (iv) part of the nuclear shell / nuclear lamina (nus). at this point, we also removed abstracts that contained the search words but did not promise to add information to our database. finally, we collected the names of the organisms and the cell types in which the interaction with the nm was observed. currently, nmpdb contains over 3000 links to pubmed articles corresponding to about 400 unique proteins ; for about 300 of these proteins we could verify the links to their sequences through either uniprot (19) or genbank (20). only 62 of all proteins had significant sequence similarity to any protein with known high - resolution information about the 3d structure as deposited in the pdb (21). only 101 of the 300 proteins were very different in their sequences [hssp values below 0 (22) ], and about half had rather high levels of sequence similarity to at least one other protein in our set (hssp value > 10). of the 400 proteins, 42, were classified as inm, 198 as asc and 130 as mix ; very few (currently 13) were classified as nus. most proteins (301) are mammalian (predominantly human, rat and mouse) ; 29 are viral proteins (e.g. hiv, papyloma / hpv, epstein such viral proteins are typically involved in the transcription of host dna, it is not surprising that they are an abundant part of the nuclear matrix in infected cells. other organisms prominent in nmpdb are gallus gallus (chicken, with 16 proteins), drosophila melanogaster (fruit fly, with 14 proteins), saccharomyces cerevisiae (yeast, with 13 proteins) and caenorhabditis elegans (worm, with 6 proteins). all entries in the database contain the following fields : (i) origin (organism and cell types), (ii) type of nuclear matrix interaction / involvement (inm, asc, mix or nus), (iii) molecular mass and known or calculated pi for locating the protein on a 2d gel and (iv) reference (pubmed ids of articles describing the interaction). for some entries we provide additional links to other databases, give the actual protein sequence and collect sequence - based predictions. although links to uniprot implicitly link nmpdb to a variety of other databases, we also provide explicit links to omim (23), swiss-2dpage (24) and s / mart db (25)which contains the dna sequences that the respective protein binds to. we provide the following information for all proteins for which we have sequences : (i) the structural domain - like organization according to chop (26,27), (ii) predictions of secondary structure, solvent accessibility and membrane helices through profphd [b. rost, manuscript submitted ; (28,29) ], (iii) coiled - coil regions through coils (30), (iv) disordered regions through norsp (31,32). where possible, entries are also cross - linked to pep, a database with predictions for entire proteomes (33) that also contains sequence alignments. for 53 sequences in the database, we found pecific information about which part of the sequence is responsible and necessary for nm binding. these regions, usually referred to as nuclear matrix targeting signals (nmts), are also deposited in nmpdb if available. nmpdb can be accessed from http://www.rostlab.org/db/nmpdb/a search - engine interface that allows the querying by different database fields and the linking of queries through and, or and and - not. the complete nmpdb database can be downloaded via ftp. the content of the database, the meaning of the fields and the search interface are described in separate help pages. nmpdb annotates many times more proteins as nuclear matrix - associated (400) than other public databases such as uniprot (80 nm proteins), the nuclear protein database (34) (27 nm proteins) or the s / mart - db (25) (80 nm proteins). we manually update nmpdb once a week at the moment and hope to maintain at least monthly updates for the years to come. first, we downloaded over 3000 abstracts from pubmed that resulted from queries with the terms nuclear matrix / matrices and nuclear scaffold. then we wrote a simple perl script that color - highlighted three types of phrases in the text (through html tagging) : (i) nuclear matrix terms, (ii) uniprot protein names and (iii) verbs describing binding processes such as to bind, to associate or to interact (figure 1). each abstract was followed by html elements that enabled the quick interactive subclassification of each protein into one of the following classes : (i) part of the internal nuclear matrix (inm), (ii)tightly associated with the inm (asc), (iii) affinity toward the inm changes depending on protein modification, cell type and/or current stage of the cell cycle (mix) and (iv) part of the nuclear shell / nuclear lamina (nus). at this point, we also removed abstracts that contained the search words but did not promise to add information to our database. finally, we collected the names of the organisms and the cell types in which the interaction with the nm was observed. currently, nmpdb contains over 3000 links to pubmed articles corresponding to about 400 unique proteins ; for about 300 of these proteins we could verify the links to their sequences through either uniprot (19) or genbank (20). only 62 of all proteins had significant sequence similarity to any protein with known high - resolution information about the 3d structure as deposited in the pdb (21). only 101 of the 300 proteins were very different in their sequences [hssp values below 0 (22) ], and about half had rather high levels of sequence similarity to at least one other protein in our set (hssp value > 10). of the 400 proteins, 42, were classified as inm, 198 as asc and 130 as mix ; very few (currently 13) were classified as nus. most proteins (301) are mammalian (predominantly human, rat and mouse) ; 29 are viral proteins (e.g. hiv, papyloma / hpv, epstein since such viral proteins are typically involved in the transcription of host dna, it is not surprising that they are an abundant part of the nuclear matrix in infected cells. other organisms prominent in nmpdb are gallus gallus (chicken, with 16 proteins), drosophila melanogaster (fruit fly, with 14 proteins), saccharomyces cerevisiae (yeast, with 13 proteins) and caenorhabditis elegans (worm, with 6 proteins). all entries in the database contain the following fields : (i) origin (organism and cell types), (ii) type of nuclear matrix interaction / involvement (inm, asc, mix or nus), (iii) molecular mass and known or calculated pi for locating the protein on a 2d gel and (iv) reference (pubmed ids of articles describing the interaction). for some entries we provide additional links to other databases, give the actual protein sequence and collect sequence - based predictions. although links to uniprot implicitly link nmpdb to a variety of other databases, we also provide explicit links to omim (23), swiss-2dpage (24) and s / mart db (25)which contains the dna sequences that the respective protein binds to. we provide the following information for all proteins for which we have sequences : (i) the structural domain - like organization according to chop (26,27), (ii) predictions of secondary structure, solvent accessibility and membrane helices through profphd [b. rost, manuscript submitted ; (28,29) ], (iii) coiled - coil regions through coils (30), (iv) disordered regions through norsp (31,32). where possible, entries are also cross - linked to pep, a database with predictions for entire proteomes (33) that also contains sequence alignments. for 53 sequences in the database, we found pecific information about which part of the sequence is responsible and necessary for nm binding. these regions, usually referred to as nuclear matrix targeting signals (nmts), are also deposited in nmpdb if available. nmpdb can be accessed from http://www.rostlab.org/db/nmpdb/a search - engine interface that allows the querying by different database fields and the linking of queries through and, or and and - not. the complete nmpdb database can be downloaded via ftp. the content of the database, the meaning of the fields and the search interface are described in separate help pages. nmpdb annotates many times more proteins as nuclear matrix - associated (400) than other public databases such as uniprot (80 nm proteins), the nuclear protein database (34) (27 nm proteins) or the s / mart - db (25) (80 nm proteins). we manually update nmpdb once a week at the moment and hope to maintain at least monthly updates for the years to come. thanks to amos bairoch (sib, geneva), rolf apweiler (ebi, hinxton), phil bourne (san diego university) and their crews for maintaining excellent databases and to all experimentalists who enabled this database by publishing their nuclear matrix related results. this work was supported by grants r01-gm63029 - 01 from the national institute of health (nih), r01-lm07329 - 01 from the national library of medicine (nlm) and dbi-0131168 from the national science foundation (nsf).	the nuclear matrix (nm) is a structure resulting from the aggregation of proteins and rna in the nucleus of eukaryotic cells ; it is the sticky bit that remains after aggressive dnase digestion and salt extraction protocols. owing to the important role of the nm in dna replication, dna transcription and rna splicing, the expression pattern of nm proteins has become an important early indicator for numerous cancers / tumors. recent descriptions of the nm structure distinguish between a network - like internal nuclear matrix (inm) and a nuclear shell that connects the inm to the inner and outer nuclear membranes. a cautious nm preparation protocol reveals a coat of proteins on top of the inm ; these proteins are usually referred to as the nuclear matrix - associated proteins. here, we describe a new database (nmpdb at http://www.rostlab.org/db/nmpdb/) that currently contains details of 398 nm proteins. we collected these data through a semi - automated analysis of over 3000 scientific articles in pubmed. we could match these 398 proteins to 302 protein sequences in uniprot or genbank. our nmpdb repository annotates these links along with the following annotations : organism, cell type, pubmed identifier, sequence - based predictions of structural and functional features and for some entries the explicit sequence segment that is responsible for localization (nuclear matrix targeting signal).
, they may be misdiagnosed to have bronchial asthma as they present with inspiratory stridor and/or expiratory wheezing. it is difficult to accurately predict the distensibility of the trachea even with an array of preoperative tests like flow volume loop, computerized tomography (ct), and bronchoscopy. a 45 year old female, with body mass index (bmi) 24 kg / cm, presented to our hospital with shortness of breath on lying supine for the past 3 months, more so when lying on right side, hence she preferred to sleep on the left side. she was treated elsewhere as bronchial asthma and referred to our center for evaluation of hemoptysis. on examination, she was found to have a swelling in the left lobe of the thyroid, compressing and pushing the trachea to the right. ct of the neck and thorax revealed an enlarged left lobe of thyroid measuring 3.4 3.3 3.4 cm with dense nodular calcification, causing short segment external compression and tracheal luminal narrowing. the stenosis extended from upper margins of c7 to t2 vertebrae, with a length of 3.1 cm and minimal diameter of 0.31 cm [figure 1 ]. fine needle aspiration cytology (fnac) of the swelling revealed colloid goiter and she was posted for a left hemithyroidectomy. three - dimensional computerized tomography and corresponding virtual bronchoscopic images of our patient room air arterial blood gas analysis showed partial pressure of arterial oxygen (pao2) of 80.5 mmhg, paco2 of 41 mmhg, ph of 7.41. preoperative fiberoptic bronchoscopy with a 4.8 mm bronchoscope revealed tracheal compression by extraluminal mass with a smooth margin 1.5 cm below the glottis. preoperative fiberoptic bronchoscopy revealed tracheal luminal narrowing of about 80%, which was 1.5 cm below the glottis when the patient was explained about the risks and options, she was not willing for any major resection or procedure. after written informed consent, patient was nebulized with 5 ml of 4% lignocaine and pre oxygenated with 100% o2 for 10 min through a nasopharyngeal airway. intravenously (iv), glycopyrrolate 0.2 mg, incremental doses of midazolam (2 mg) and fentanyl (100 g) were given. a 4.5 mm fiberoptic bronchoscope (fob) threaded through a 5 mm internal diameter (i d) microlaryngeal tracheal tube (mlt) was passed nasally. the fob was introduced close to the mass, and gently pushed past the obstruction. a little resistance was noted but the tube could be passed below the mass without undue force. the fob was withdrawn and trachea ventilated with a tidal volume of 450 ml, 8 breaths /min, and i : e = 1:3. the left lobe of the thyroid being stony hard and densely adherent to trachea could not be completely resected. as the patient was not willing for tracheal resection and reconstruction, an isthumectomy and release of fibrotic tissue around the trachea was done. before closure, the 5.0 mm (i d) mlt was replaced with a 6.0 mm i d mlt over an aintree intubating catheter. as this could be passed without any resistance, it was followed by the passage of a 7.0 mm i d endotracheal tube with little resistance. postoperatively, patient was electively ventilated, anticipating airway edema. under steroid cover, 24 h later, the patient was extubated over a tube exchanger, with standby tracheostomy. as the patient did not want any major definitive surgery ; she was referred to a regional cancer institute for further management. while anesthetizing patients with tracheal stenosis, despite having newer imaging modalities like multi - slice, three - dimensional -ct, virtual bronchoscopy to determine the size and anatomy of the airway, it is difficult to accurately predict the distensibility of the trachea with the available preoperative tests. preoperative assessments of airway size and collapsibility may not be perfect in predicting patency of the airway and ease of ventilation during general anesthesia. on one hand, it is important to maintain a patent airway till it is secured with an endotracheal tube. on the other, it is important not to over - estimate the airway narrowing as this will result in the patient being ventilated with an unnecessarily small endotracheal tube. pulmonary function tests are useful in the functional assessment of obstruction, but were contraindicated in our patient because of hemoptysis. the ability to compensate breathing through a narrow airway may be better during general anesthesia than during wakefulness, because behavioral influences such as anxiety are eliminated and oxygen consumption reduced. however, azizkhan., reported total collapse of the airway by mediastinal masses causing > 50% narrowing of the trachea. this helped, as we were able to pass the fob beyond the mass when it was not possible during the preoperative bronchoscopy. the presence of hypercapnia preoperatively, in the absence of respiratory pathology, strongly suggests the potential for failure of both spontaneous and mechanical ventilation during general anesthesia. critical fixed narrowing through which anesthetized patients can breathe spontaneously without increase in partial pressure of carbon dioxide in the blood (paco2) is 4.0 - 4.5 mm i d. as our patient was maintaining normocapnia, we assumed that her dynamic airway would be at least this wide. as the tracheal diameter varies with respiration, it is inaccurate to rely on static ct images in cases of external compression. toyoto., used a 5.5 mm i d reinforced endotracheal tube with an external diameter of 7.5 mm in their patient whose ct image showed the narrowest portion as being 2.6 mm. the serial passage of increasing sizes of endotracheal tubes was similar to dilatation of stenosis under direct vision. the isthumectomy and release of fibrotic bands also decreased the external compression of the trachea and increased the airway caliber post surgery and we were able to provide symptom relief. this also made the anterior surface of the trachea accessible for a low tracheostomy, which was not possible preoperatively. in conclusion, newer imaging modalities are just an adjunct in the assessment of the compromised airway in case of dynamic compression. our experience is with a single case and probably, only randomized control trials can answer the question whether newer imaging modalities really help airway assessment in dynamic tracheal compression or not. clinical assessment will, however, remain very important in deciding the plan of management.	anesthetic management of a patient with tracheal stenosis is challenging. though we have newer imaging modalities like multislice, three - dimensional computerized tomography, virtual bronchoscopy to determine the size and anatomy of the airway, it is difficult to accurately predict the distensibility of the trachea with the available preoperative tests. with our experience in this case, we believe that newer imaging modalities are just an adjunct in the assessment of the compromised airway in case of dynamic compression. clinical assessment is very important in deciding the plan of management.
total hip arthroplasty (tha) is one of the most commonly performed operations in the united states, with over 280,000 procedures reported annually [1, 28, 46 ]. the benefits of tha in terms of reduced pain and improved function and quality of life (qol) for patients with debilitating hip disease have been well documented in the literature. furthermore, tha is a highly cost - effective intervention when compared with nonoperative management in patients with advanced osteoarthritis (oa) of the hip [13, 20 ]. however, concerns regarding high rates of tha failure among young, active patients and a desire to preserve bone for future revision operations led to the development of hip resurfacing arthroplasty (hra), which was first introduced in the united states in the 1970s. hra differs from tha in that the femoral head is resurfaced rather than resected, thereby preserving femoral bone stock, which could theoretically decrease the morbidity and improve patient outcomes associated with future revision operations. however, early clinical experience with hra was unfavorable, as high failure rates (13% to 34% within an average of 18 months to 3 years) were reported due primarily to aseptic loosening [6, 19, 23 ]. thus, the procedure fell out of favor among orthopaedic surgeons in the late 1980s [6, 26 ]. with the introduction of large - diameter metal - on - metal (mom) bearings, which are associated with lower wear rates and less deformation than conventional metal - on - polyethylene bearings, hra has been reintroduced in the united states amid both controversy and enthusiasm. proponents of mom hra point to the potential benefits in terms of femoral bone preservation and therefore less morbidity and better functional outcomes associated with future revision surgeries [2, 39, 45, 47, 50 ]. opponents argue the increased risks of early failure due to femoral neck fracture and increased costs associated with mom hra implants overshadow the yet - to - be proven long - term benefits. furthermore, since the value of mom hra in terms of improved patient outcomes and ease of future revision surgery have not been conclusively demonstrated, many health plans have developed payment policies limiting the use of mom hra to specific patient populations. decision analysis offers a useful approach to compare mom hra to tha by comparing the expected lifetime costs and cumulative gains in quality of life associated with mom hra to the expected lifetime costs and cumulative gains in quality of life associated with tha based on known information regarding the costs, probabilities of clinical outcomes (including complications and revision surgeries), and quality of life associated with each treatment strategy. this approach is consistent with the emerging field of comparative effectiveness research, which has been defined as the conduct and synthesis of research comparing the benefits and harms of different interventions and strategies to prevent, diagnose, treat, and monitor health conditions in the real world setting. the aims of this study were (1) to evaluate the comparative clinical effectiveness, costs, and cost - effectiveness of mom hra compared with tha by patient age and gender for the treatment of patients with advanced oa of the hip ; (2) to identify which clinical and demographic factors and costs have the greatest influence on the incremental lifetime improvement in quality of life, costs, and cost - effectiveness of mom hra versus tha ; and (3) to quantify the uncertainty in the estimates of the comparative clinical and cost - effectiveness of mom hra versus tha. we used a markov decision model to evaluate the clinical and economic consequences of mom hra compared to tha. the population studied was men and women aged 50 years or older undergoing mom hra or tha for advanced oa of the hip. a 30-year time horizon was used to evaluate the incremental clinical effectiveness (in terms of quality adjusted life - years (qalys) gained) and cost - effectiveness (cost per qalys gained) of mom hra and tha. the incremental cost - effectiveness of mom hra versus tha was examined from a healthcare system perspective (focusing on health care costs and patient quality of life) using hospital and professional reimbursement to estimate costs and quality adjusted life years to estimate effectiveness. the decision tree (fig. 1a b) begins with the decision to choose either mom hra or tha for patients with advanced oa of the hip. the model used intervals (markov cycles) of 1-year duration. while in each state during each yearly interval, patients experience a quality of life (qol) and incur direct medical costs ; in addition, transitions associated with revision surgery (conversion from hra to tha, major total revision tha [revision of both the acetabular and femoral components ], major partial revision tha [revision of the acetabular component only ], or minor revision tha [exchange of the modular acetabular liner and femoral head only ]) are associated with a short - term transitional decrement in qol (or disutility) and an increase in direct medical costs associated with revision surgery. the probability of transition between states depends on the patients age, gender, and type of procedure (mom hra or tha). for mom hra, the health states are year of initial mom hra, post - hra, post - conversion from hra to tha, post - major total revision tha, post - major partial revision tha, post - minor revision tha, death due to any hra or tha surgery, and death due to other causes. thus, the mom hra cohort may experience an initial failure (requiring conversion from hra to tha) or a subsequent failure requiring revision after tha. for the tha cohort, the disease states are year of initial tha, post - tha, post - major total revision tha, post - major partial revision tha, post - minor revision tha, post - second major total revision tha, post - second major partial revision tha, post - second minor revision tha, death due to any tha surgery, and death due to other causes. decision analysis software (treeage pro 2008, williamstown, ma) was used to create a markov decision model.fig. b(a) a markov decision tree compares the clinical outcomes for mom hra and tha patients. mom tha and primary tha are represented as markov nodes (m). the branches are the markov states. conversion from hra to tha is analogous to first major revision in the primary tha alternative. (b) the detailed outcomes in the post - conversion from hra to tha branch are shown. (a) a markov decision tree compares the clinical outcomes for mom hra and tha patients. mom tha and primary tha are represented as markov nodes (m). conversion from hra to tha is analogous to first major revision in the primary tha alternative. (b) the detailed outcomes in the post - conversion from hra to tha branch are shown. information on implant survivorship was sought from large national or multicenter registries with implant survival data of sufficient duration to estimate annual age, gender, and procedure specific probability of implant survival and implant failure. additionally, because the probability of implant failure varies by year of followup, we sought data of sufficient duration (5 or more years after initial surgery). the australian orthopedic association (aoa) national joint replacement registry hip and knee arthroplasty annual report provides gender and decade of age stratified cumulative percent revision for 5 to 7 years of followup for 9956 patients who received hra for primary diagnosis of oa (excluding infection) and 109,972 patients who received primary conventional tha for a primary diagnosis of oa. the report also summarizes the type of revision (major total revision, major partial revision, and minor revision) and probability of subsequent revision for 2616 revision tha procedures. annual probability of revision of mom hra and tha was estimated from the summary gender- and age - stratified data in the aoa national joint replacement registry 2008 report by fitting a general failure time model (weibull distribution), which allowed for time varying hazard of failure for each gender and age stratum for 5 years of followup (the longest followup interval for which data was available for all strata). as indicated in the aoa registry report, the probability of failure in each stratum was highest in the first year of followup and declined thereafter, resulting in cumulative revision curves that increased with time but at lower rates after the initial year. the analyses of mom hra and tha are summarized separately for six gender and age strata to correspond with the available data on failure rates in the aoa registry report. for both genders, the analysis used a patient age of 50 years representing the age younger than 55 years stratum, a patient age of 60 years representing the ages 55 to 64 years stratum, and a patient age of 70 years representing the age 65 to 74 years stratum. the probabilities of perioperative mortality for mom hra, tha, and all revision thas were derived from the literature [15, 27, 3032, 37, 41, 44, 51, 52 ]. annual gender- and age - specific all - cause mortality rates were based on united states life tables. the effectiveness of each surgical procedure was based on the quality - adjusted life - years associated with each procedure. the qol values range from 0 (death) to 1 (perfect health) and reflect the average qol associated with that health state. the qol weights for patients with advanced oa of the hip and patients with successful primary tha were obtained from the literature [13, 20, 28, 29, 35 ]. the qol values for patients with successful mom hra, conversion from hra to tha, and revision tha were derived from literature comparing each of these health states to patients with primary tha [5, 25, 43 ]. perioperative morbidity and recovery were captured by applying a lower qol for a defined period of time after each surgical intervention (longer for revision than primary procedures). qol weights that are measured by methods that reflect patient preferences for a health state are described as utilities in the health economics literature. costs incorporated into the model included both hospital and professional fees for primary tha, revision tha, mom hra, and conversion from hra to tha. hospital costs were based on average medicare payments for diagnosis - related groups 544 (primary lower extremity arthroplasty procedures) and 545 (revision lower extremity arthroplasty procedures) for fiscal year 2008. similar to previously published cost - effectiveness analyses [40, 48 ], medicare reimbursement was chosen (even though the patient population being studied included men and women older than 50 years) since it more closely reflects the actual costs associated with hra and tha procedures, as opposed to private payer reimbursement, which is based on a negotiated rate, which often exceeds the true costs of the procedure. revision tha procedure costs were further delineated by procedure complexity, such as isolated femoral component revision, acetabular component revision, both component revision, or femoral head and liner exchange only, based on previously published data. device costs for primary tha, revision tha, and hra were obtained from published sources. costs associated with ambulatory visits and radiographs were also included in the analysis, based on average professional fees for evaluation and management services and both professional and technical fees for hip radiographs. the clinical course for patients who receive mom hra was compared to the clinical course for patients who receive tha by comparing the cumulative discounted total quality - adjusted years of life (qalys) and cumulative costs of mom hra with the cumulative discounted total qalys and cumulative costs of tha. the measures used in the comparison were the incremental qalys (a measure of effectiveness), the incremental costs, and the incremental cost - effectiveness ratio (icer), which is the ratio of the incremental costs to the increment effectiveness. in accordance with the recommendations of the panel on cost - effectiveness, we discounted all costs and utilities and report reference case estimates for a discount rate of 5%. the base case estimates for the probabilities, utilities, and costs were derived from the literature (table 1).table 1variables used in cost - effectiveness analysis and ranges for sensitivity analysesvariablevaluelow valuehigh valuecitationscosts hra$17,178$12,883$34,355[11, 38 ] primary tha$15,178$11,383$30,355[11, 38 ] hra conversion to tha$18,460$13,845$36,920[11, 38 ] major total revision$21,195$15,896$42,391[9, 38 ] major partial revision$18,155$13,616$36,311[9, 38 ] minor revision$16,367$16,275$32,735 incremental cost of hra implant$20000$2000 outpatient visit and radiography$129$97$257probability of clinical outcomes hra failure0.0045 00.0225[4, 5 ] primary tha failure0.0055 00.0084[4, 5 ] hra conversion tha failure0.0055 00.0084[4, 5 ] major total revision arthroplasty failure0.069500.07 major partial revision arthroplasty failure0.065000.065 minor revision arthroplasty failure0.097700.1 major total revision arthroplasty (proportion of all revisions)0.050.0250.1 major partial revision arthroplasty (proportion of all revisions)0.4950.250.75 minor revision arthroplasty (proportion of all revisions)0.4550.250.75 death, hra0.0060.0010.015[15, 27, 32, 41, 51 ] death, primary tha0.0060.0010.015[15, 27, 32, 41, 51 ] death, hra conversion to tha0.0120.0030.022[15, 27, 32, 41, 51 ] death, major total revision arthroplasty0.0120.0030.022[15, 27, 32, 41, 51, 52 ] death, major partial revision arthroplasty0.0120.0030.022[15, 27, 32, 41, 51, 52 ] death, minor revision arthroplasty0.0120.0030.022[15, 27, 32, 41, 51, 52 ] death, all - cause mortality0.006utility (quality of life) severe osteoarthritis before hra or tha0.50[8, 13, 20, 29 ] post - primary tha0.920.660.92[20, 29, 35 ] post - hra0.920.660.92 post - hra conversion to tha0.920.820.92 post - first major total revision arthroplasty0.840.580.90 post - first major partial revision arthroplasty0.840.580.90 post - first minor revision arthroplasty0.880.800.92 post - second major total revision arthroplasty0.760.500.82 post - second major partial revision arthroplasty0.760.500.82 post - second minor revision arthroplasty0.80.760.84 short - term morbidity major total or major partial revision arthroplasty0.200.200[32, 52 ] short - term morbidity minor revision arthroplasty0.100.100[32, 52]modeling variables discount rate0.0500.05 followup (years)30030 probabilities vary by age strata, gender, and year after surgery ; estimate shown for men younger than 55 years ; probabilities vary by year after surgery ; probabilities vary by age and gender ; estimate shown for men younger than 55 years ; hra = hip resurfacing arthroplasty. variables used in cost - effectiveness analysis and ranges for sensitivity analyses probabilities vary by age strata, gender, and year after surgery ; estimate shown for men younger than 55 years ; probabilities vary by year after surgery ; probabilities vary by age and gender ; estimate shown for men younger than 55 years ; hra = hip resurfacing arthroplasty. one - way sensitivity analyses were performed for each of the independent variables (table 1). in these analyses, each variable was varied from 50% to 200% of the point estimate (table 1), per decision analysis modeling convention, and the impact of each variable on the icer was calculated. one - way sensitivity analyses for selected variables (discount rate, difference in utility [qol ] after conversion from hra to tha compared to primary tha, and incremental cost of hra compared to tha) were calculated for each gender and age stratum. one - way sensitivity analyses were used to identify thresholds for selected independent variables where mom hra would be cost - saving compared to tha and thresholds where mom hra would be considered cost - effective based on an icer of $ 50,000 per qaly. two - way sensitivity analyses were performed to identify ranges for the incremental cost of hra compared to tha and difference in utility (qol) after conversion from hra to tha compared to primary tha where mom hra or tha was optimal based on net monetary benefits, using a willingness to pay threshold of $ 50,000 per qaly gained. a probabilistic sensitivity analysis (monte - carlo sensitivity analysis) was performed to evaluate the combined impact of the individual independent variables jointly on the incremental costs, incremental qalys gained, and the icers. in this analysis, each variable was represented as a probability distribution (table 2) and a random sample for each variable was drawn from its probability distribution and entered into the model. the incremental costs, incremental qalys gained, and the icers and their 95% confidence intervals were calculated from a monte carlo simulation using 10,000 samples for each gender and age stratum. an acceptability curve showing the proportion of samples for each gender and age stratum that were below a given willingness to pay threshold was calculated and graphed for a willingness to pay range of $ 0 to $ 100,000.table 2variables and distributions for probabilistic sensitivity analysisvariablevaluelow valuehigh valuedistributionmeansdcosts hra$17,178$12,883$34,355gamma17,178219161.4696279.4552 primary tha$15,178$11,383$30,355gamma15,178193661.4637246.9427 hra conversion to tha$18,460$13,845$36,920gamma18,460233562.5014295.3535 major total revision$21,195$15,896$42,391gamma21,195270461.4403344.9689 major partial revision$18,155$13,616$36,311gamma18,155231661.4491295.4479 minor revision$16,367$16,275$32,735gamma16,367208861.4437266.3740 incremental cost of hra implant$20000$2000gamma200025561.514832.5125 outpatient visit and radiography$129$97$257gamma1291665.00391.9845probability of clinical outcomes multiplier for hra failure10.501.5gamma10.11001 multiplier for primary tha failure10.51.5gamma10.11001 multiplier for hra conversion tha failure10.51.5gamma10.11001 multiplier for major total revision arthroplasty failure10.51.5gamma10.11001 multiplier for major partial revision arthroplasty failure10.51.5gamma10.11001 multiplier for minor revision arthroplasty failure10.51.5gamma10.11001 major total revision arthroplasty (proportion of all revisions)0.050.0250.10dirichlet (normalized beta)0.0551 major partial revision arthroplasty (proportion of all revisions)0.4950.250.75dirichlet (normalized beta)0.49549.51 minor revision arthroplasty (proportion of all revisions)0.4550.250.75dirichlet (normalized beta)0.45545.51 death, hra0.0060.0010.015beta0.0060.00255.7254948.5146 death, primary tha0.0060.0010.015beta0.0060.00255.7254948.5146 death, hra conversion to tha0.0120.0030.022beta0.0120.0055.6909468.5491 death, major total revision arthroplasty0.0120.0030.022beta0.0120.0055.6909468.5491 death, major partial revision arthroplasty0.0120.0030.022beta0.0120.0055.6909468.5491 death, minor revision arthroplasty0.0120.0030.022beta0.0120.0055.6909468.5491health state utility (quality of life) severe osteoarthritis before hra or tha0.500.320.85beta0.500.10250.02 post - primary tha0.920.660.92beta0.920.0442.323.68 post - hra0.920.660.92beta0.920.0442.323.68 post - hra conversion to tha0.920.820.92beta0.920.0442.323.68 post - first major total revision arthroplasty0.840.580.90beta0.840.0470.5613.44 post - first major partial revision arthroplasty0.840.580.90beta0.840.0470.5613.44 post - first minor revision arthroplasty0.880.800.92beta0.880.0458.087.92 post - second major total revision arthroplasty0.760.500.82beta0.760.0486.6427.36 post - second major partial revision arthroplasty0.760.500.82beta0.760.0486.6427.36 post - second minor revision arthroplasty0.80.760.84beta0.800.048020 short - term morbidity reduction major total or major partial revision arthroplasty0.2000.20beta0.200.0512.851.2 short - term morbidity reduction minor revision arthroplasty0.1000.10beta0.100.02514.4129.6 and are the two parameters of the gamma distribution or the beta distributions ; a multiplier randomly sampled from a gamma distribution was used to generate samples for the probability of failure because the underlying clinical probabilities for failure of tha and hra vary by age, gender, and interval since surgery and the clinical probabilities of major total revision, major partial revision, and minor revision arthroplasty failure vary by interval since surgery ; hra = hip resurfacing arthroplasty. variables and distributions for probabilistic sensitivity analysis and are the two parameters of the gamma distribution or the beta distributions ; a multiplier randomly sampled from a gamma distribution was used to generate samples for the probability of failure because the underlying clinical probabilities for failure of tha and hra vary by age, gender, and interval since surgery and the clinical probabilities of major total revision, major partial revision, and minor revision arthroplasty failure vary by interval since surgery ; hra = hip resurfacing arthroplasty. over a 30-year followup period, mom hra patients would experience modestly higher lifetime gains in quality adjusted life years and have moderately higher health care costs compared to patients who have primary tha, depending on their age and gender. the cost - effectiveness of mom hra compared to tha varies markedly by age and gender with preferable (lower) incremental cost - effectiveness ratios in men compared to women and in younger patients compared to older patients : the lowest icer was $ 28,614 for men aged 5565 and the highest was $ 2,483,435 for women age 6574an 87-fold difference (table 3). the icer was less than the $ 50,000 per qaly threshold for three of the age and gender strata studied : men less than age 55 ($ 48,882/qaly), men ages 5564 ($ 28,614/qaly), and women less than age 55 ($ 47,468/qaly) (table 3).table 3cost - effectiveness of hra compared to primary tha by gender and age stratastratastrategycostincremental costeffectiveness (qalys)incremental effectiveness (qalys)icer ($ /qalys)men < 55 yearstha$17,80812.299hra$19,495$168712.3340.03548,882men 5564 yearstha$17,88210.607hra$19,171$128910.6520.04528,614men 6574 yearstha$17,1848.138hra$19,009$18258.160.02283,699women < 55 yearstha$18,59112.866hra$21,047$245612.9170.05247,468women 5564 yearstha$17,87411.528hra$22,005$413111.5380.009435,800women 6574 yearstha$17,2319.208hra$20,956$37269.210.0022,483,435qaly = quality - adjusted life year ; icer = incremental cost - effectiveness ratio ; hra = hip resurfacing arthroplasty. cost - effectiveness of hra compared to primary tha by gender and age strata qaly = quality - adjusted life year ; icer = incremental cost - effectiveness ratio ; hra = hip resurfacing arthroplasty. the variables that had the most influence on the model results were the annual probability of mom hra and tha failure, the cost of mom hra and tha, operative mortality of mom hra and tha, and the qol after conversion from hra to tha (fig. 2). the one - way sensitivity analysis of the icer to the difference in qol after conversion from hra to tha compared to primary tha indicated that the icer for mom hra was very sensitive to the differences in qol after conversion from hra to tha for both men and women less than age 55, but not for men age 5564 (fig. mom hra would be cost - saving over the 30 year time horizon if the incremental cost of the hra implants compared to the primary tha implants was less than $ 313 for men aged less than age 55 years, less than $ 711 for men aged 55 to 64 years, and less than $ 175 for men aged 6574 years (fig. 4). the two - way sensitivity analysis indicated that the impact of the incremental cost of mom hra and the difference in qol on the cost - effectiveness of mom hra varied depending on age and gender. in general, over a wide range of values for the qol reduction after hra conversion and the incremental cost of hra conversion, mom hra was more favorable compared to tha for men than for women and for younger patients (age less than 55) compared to older patients (age 65 or older) (fig. 2one - way sensitivity analyses of icer to probabilities of clinical outcomes, costs, and qol are shown. the width of each bar indicates the range of the icer as each independent variable changes over its range. the upper value for the icer is over $ 7,627,147 at the upper value of the annual probability of hra failure (0.0225). the graph shows that the factors that have the greatest impact on the model results are the probability of hra failure, cost of hra and primary tha, probability of primary tha failure, probability of operative death from hra and primary tha, and quality of life after conversion of hra to tha.fig. 3a graph shows a one - way sensitivity analysis to difference in qol after conversion from hra to tha compared to primary tha by gender and age strata. the icer increased rapidly with small differences in the quality of life after conversion of hra to tha compared to primary tha for men age less than age 55, men age 55 to 64, and women less than age 55. men, age 55 to 64 had a more favorable (lower) icer with much smaller change in icer as the difference in quality of life after conversion from hra to tha increased.fig. 4the graph shows a one - way sensitivity analysis to incremental cost of hra compared to tha by gender and age strata. for both men and women, there is a linear relationship of the icer to the incremental costs of mom hra implants. mom hra would be cost saving (icer intercept = 0) if the incremental cost of mom hra were less than $ 313 for men less than age 55 years, less than $ 711 for men age 55 to 64 years, and less than $ 175 for men aged 65 to 74 years. for women in each age stratum, the costs of the mom hra treatment strategy are higher than the costs of the tha at every value of incremental cost of the mom hra implant compared to tha and there is no cost - saving threshold. in women less than age 55, the icer of mom hra is less sensitive to the incremental cost of the hra implants compared to tha, due to the higher probability of hra failure in women than in men.fig. dthese graphs show two - way sensitivity analyses of incremental cost of hra compared to primary tha and difference in qol after conversion from hra to tha compared to primary tha for (a) men younger than 55 years, (b) men aged 65 to 74 years, (c) women younger than 55 years, and (d) women aged 65 to 74 years. the graph area shows the combination of the incremental cost of hra and difference between qol after conversion from hra to tha and primary tha where mom hra (black) or primary tha (white) is optimal based on net monetary benefits analysis with a willingness to pay threshold of $ 50,000 per qaly. in general, over a wide range of values for the qol reduction after conversion from hra to tha and the incremental cost of hra conversion, mom hra was more favorable compared to tha for men than for women (fig. 5b versus 5d) and for younger patients (age less than 55) compared to older patients (age 65 or older) (fig.. one - way sensitivity analyses of icer to probabilities of clinical outcomes, costs, and qol are shown. the width of each bar indicates the range of the icer as each independent variable changes over its range. the upper value for the icer is over $ 7,627,147 at the upper value of the annual probability of hra failure (0.0225). the graph shows that the factors that have the greatest impact on the model results are the probability of hra failure, cost of hra and primary tha, probability of primary tha failure, probability of operative death from hra and primary tha, and quality of life after conversion of hra to tha. a graph shows a one - way sensitivity analysis to difference in qol after conversion from hra to tha compared to primary tha by gender and age strata. the icer increased rapidly with small differences in the quality of life after conversion of hra to tha compared to primary tha for men age less than age 55, men age 55 to 64, and women less than age 55. men, age 55 to 64 had a more favorable (lower) icer with much smaller change in icer as the difference in quality of life after conversion from hra to tha increased. the graph shows a one - way sensitivity analysis to incremental cost of hra compared to tha by gender and age strata. for both men and women, there is a linear relationship of the icer to the incremental costs of mom hra implants. mom hra would be cost saving (icer intercept = 0) if the incremental cost of mom hra were less than $ 313 for men less than age 55 years, less than $ 711 for men age 55 to 64 years, and less than $ 175 for men aged 65 to 74 years. for women in each age stratum, the costs of the mom hra treatment strategy are higher than the costs of the tha at every value of incremental cost of the mom hra implant compared to tha and there is no cost - saving threshold. in women less than age 55, the icer of mom hra is less sensitive to the incremental cost of the hra implants compared to tha, due to the higher probability of hra failure in women than in men. these graphs show two - way sensitivity analyses of incremental cost of hra compared to primary tha and difference in qol after conversion from hra to tha compared to primary tha for (a) men younger than 55 years, (b) men aged 65 to 74 years, (c) women younger than 55 years, and (d) women aged 65 to 74 years. the graph area shows the combination of the incremental cost of hra and difference between qol after conversion from hra to tha and primary tha where mom hra (black) or primary tha (white) is optimal based on net monetary benefits analysis with a willingness to pay threshold of $ 50,000 per qaly. in general, over a wide range of values for the qol reduction after conversion from hra to tha and the incremental cost of hra conversion, mom hra was more favorable compared to tha for men than for women (fig. 5b versus 5d) and for younger patients (age less than 55) compared to older patients (age 65 or older) (fig. the probabilistic sensitivity analysis demonstrated wide variation in the icers due to the overall simultaneous variation in the many underlying factors that may influence the clinical effectiveness and costs of mom hra and tha (table 3). the acceptability curves can be interpreted as the probability (or confidence) that the icer is less than a certain willingness to pay threshold. the probabilities that the icers are less than or equal to $ 100,000 per qaly were less than 75% for all strata (fig. 6), indicating that variation in costs of hra, failure rates of hra and tha, and quality of life difference after conversion of hra to tha have a large impact on the cost and clinical effectiveness of mom hra compared to tha. however, it should be noted that the impact is similar for each age and gender strata and unlikely to change the age and gender specific ranking of the incremental cost and clinical effectiveness of mom hra compared to tha.fig. 6an acceptability curve from the probabilistic sensitivity analysis shows the probability that icer is below a particular willingness to pay threshold based on the simulation using 10,000 samples for each gender and age stratum. the probability (confidence) that the icer was less than or equal to $ 100,000 per qaly gained was only 63% for men less than age 55, 75% for men the uncertainty illustrated by these acceptability curves indicates that variation in costs of hra, failure rates of hra and tha, and quality of life difference after conversion of hra to tha have a large impact on the comparative clinical and cost - effectiveness of mom hra. an acceptability curve from the probabilistic sensitivity analysis shows the probability that icer is below a particular willingness to pay threshold based on the simulation using 10,000 samples for each gender and age stratum. the probability (confidence) that the icer was less than or equal to $ 100,000 per qaly gained was only 63% for men less than age 55, 75% for men ages 5564, and 68% for women less than age 55. the uncertainty illustrated by these acceptability curves indicates that variation in costs of hra, failure rates of hra and tha, and quality of life difference after conversion of hra to tha have a large impact on the comparative clinical and cost - effectiveness of mom hra. despite the widely reported success of tha using conventional implants [7, 14, 16, 33, 42 ], new techniques and technologies are constantly being introduced into the marketplace, with the goal of improving clinical outcomes and reducing failure and reoperation rates. when evaluating any new technique or technology for use in clinical practice, it is important to consider the potential clinical benefits, risks, and economic costs associated with its use, preferably in comparison to the gold standard. mom hra offers potential advantages over conventional tha in terms of femoral bone preservation and ease of future revision surgery, especially in younger, more active patients who are more likely to require revision surgery. however, the benefits of mom hra compared to primary tha for patients with advanced oa of the hip have not been conclusively demonstrated in clinical trials or long - term observational cohort studies. we used decision analysis to compare the expected gains in quality of life, increase in costs, and cost - effectiveness of mom hra by age and gender, identify key factors that influence the cost and clinical effectiveness of mom hra compared to tha, and the uncertainty in these estimates. our decision analysis used data on 57 year outcomes of mom hra and tha by age and gender from the aoa national registry based on 109,972 tha patients and 9,956 mom - hra patients. national joint registry outcomes are more likely to represent clinical practice in the community and less subject to the selection bias and referral bias that might influence outcomes in studies from single centers or academic institutions. while our study provides novel information regarding the comparative effectiveness of mom hra and tha, the limitations of the methodology should be considered when interpreting the results. as is true with any decision analysis model, the validity and generalizability of the results for instance, although long - term implant survival is available for tha, only midterm survival is available for mom hra. furthermore, there are no direct estimates of qol following successful hra or conversion of hra to tha, so these values were derived from comparisons of qol and function in patients with hra and tha. while this introduces uncertainty into the model, sensitivity analysis was used to test the robustness of the model results and the conclusions. one of the advantages of decision analysis modeling is the ability to use sensitivity analysis to determine threshold values for critical input variables (e.g., age, risk of complications, cost) which influence the comparative effectiveness of each treatment option. moreover, by including a wide range of values for the model variables in our probabilistic sensitivity analysis, our study provides a more realistic estimate of the true uncertainty of the comparative effectiveness of mom hra compared to tha. our results indicate mom hra could be both clinically advantageous and cost - effective in appropriately selected men under the age of 65 years and women under the age of 55 years, when considering the initial and subsequent risks, costs, and benefits accrued over a 30-year period. previously evaluated the cost - effectiveness of mom hra compared to watchful waiting and tha in two groups of patients who were likely to outlive the lifespan of their prosthesis : patients younger than 65 years and those older than 65 years who participated in activities predicted to shorten the lifespan of their prosthesis. data were obtained from an extensive literature search, and costs were obtained from the british national health services price index. the investigators found that tha dominated mom hra throughout the 20-year followup period of the markov model, due to the higher cost of mom hra and also the higher revision rate resulting in lower quality adjusted life - years. mom hra became more cost - effective as the revision rate of tha increased or revision rate of mom hra procedures decreased. an annual revision rate for mom hra of 1.52% was used based on a 1996 study by mcminn.. this was compared to a revision rate of 1.36% for tha for active, young patients and 1.14% for older, less active patients. our study uses more recent data with lower age- and gender - specific failure rates for both mom hra and tha obtained from a large, national joint replacement registry and explores a larger number of potential factors that may influence the comparative effectiveness of mom hra compared to tha. our probabilistic sensitivity analysis quantifies the simultaneous impact of uncertainty in 35 independent variables (15 probabilities of clinical outcomes, 12 quality of life utilities, and eight cost estimates) on the incremental effectiveness, incremental costs, and icer of mom hra compared to tha. the acceptability curves provide an upper bound for the confidence that the icer is less than $ 100,000 per qaly gained. thus, we can only be 63% confident that the icer is less than $ 100,000 per qaly for men less than age 55, 75% confident for men age 5475, and 68% confident for women less than age 55. the limited information about the underlying parameters that could influence the comparative effectiveness of mom hra versus tha results in the wide variation in our estimate of the icer and emphasizes the need to include measurements of quality of life and resource use in future studies of the clinical outcomes of hra and tha. new surgical techniques and technologies are constantly being introduced into orthopaedic practice in the united states, many of which offer the promise of better clinical outcomes, often at a higher cost. in an era of limited healthcare resources, it is imperative to consider the comparative clinical and cost - effectiveness of new interventions and technologies vis - - vis the gold standard technique. this is especially important in the field of hip reconstructive surgery, where the gold standard treatment (tha) has been associated with excellent patient outcomes and long - term durability. given the higher costs associated with mom hra implants and the uncertainty that exists with respect to the downstream clinical risks and benefits associated with this new technology, the results of our study offer clinicians, patients, and policy makers the opportunity to consider the incremental risks, benefits, and costs that influence the comparative effectiveness of mom hra and tha.	backgroundmetal - on - metal hip resurfacing arthroplasty (mom hra) may offer potential advantages over total hip arthroplasty (tha) for certain patients with advanced osteoarthritis of the hip. however, the cost effectiveness of mom hra compared with tha is unclear.questions/purposesthe purpose of this study was to compare the clinical effectiveness and cost - effectiveness of mom hra to tha.methodsa markov decision model was constructed to compare the quality - adjusted life - years (qalys) and costs associated with hra versus tha from the healthcare system perspective over a 30-year time horizon. we performed sensitivity analyses to evaluate the impact of patient characteristics, clinical outcome probabilities, quality of life and costs on the discounted incremental costs, incremental clinical effectiveness, and the incremental cost - effectiveness ratio (icer) of hra compared to tha.resultsmom hra was associated with modest improvements in qalys at a small incremental cost, and had an icer less than $ 50,000 per qaly gained for men younger than 65 and for women younger than 55. mom hra and tha failure rates, device costs, and the difference in quality of life after conversion from hra to tha compared to primary tha had the largest impact on costs and quality of life.conclusionsmom hra could be clinically advantageous and cost - effective in younger men and women. further research on the comparative effectiveness of mom hra versus tha should include assessments of the quality of life and resource use in addition to the clinical outcomes associated with both procedures.level of evidencelevel i, economic and decision analysis. see guidelines for authors for a complete description of levels of evidence.
in 1984, it was first reported that h. pylori, a gram negative, spiral shaped, microaerophilic bacterium that colonizes the stomach and is involved in the pathogenesis of duodenal ulceration, gastric cancer, gastric ulceration, and active chronic gastritis [1, 2 ]. dyspepsia is a common term used for abdomen pain and complimented with other gastrointestinal symptoms. nonulcer dyspepsia is also known as functional dyspepsia which found to be associated with h. pylori infection [3, 4 ]. h. pylori infection was found to be associated with gastric cancer, peptic ulcer, duodenal ulcer, gastric carcinoma [58 ], and so forth. prolidase (ec 3.4.13.9), proline dipeptidase, degraded dipeptides with hydroxyproline or proline as c - terminal amino acid [9, 10 ]. its activity has been reported in leukocytes, erythrocytes, plasma, and the various organs such as brain, heart, kidney, uterus, thymus, and dermal fibroblasts. prolidase activity has been reported in various disorders, like osteoarthritis, chronic liver disease, and osteoporosis [1215 ]. it has been observed that increased oxidative stress associated with gastroduodenal mucosa inflammation in h. pylori infected individuals. it has been reported that h. pylori infection induced inflammation and can cause gastric atrophy and it is related to increased oxidative stress [18, 19 ]. serum prolidase activity and oxidative status in h. pylori infected individuals were studied by aslan. in 2007, among turkey population. the prevalence of h. pylori infection varies from country to country by geographic area, ethnicity, age, race, and socioeconomic status [21, 22 ] and consists of large diversity of strains [2227 ]. due to its different prevalence and diversity of strains, we aimed to observe oxidative stress and serum prolidase activity in patients with h. pylori infected nonulcer dyspepsia among indian subjects. with serum prolidase activity, we also estimated total antioxidant capacity (taoc), total oxidant status (tos), and oxidative stress index (osi) in the above cases. this study was done in the department of biochemistry, department of gastroenterology, and department of pathology, institute of medical sciences, banaras hindu university, varanasi, india. a total of 238 subjects were selected after informed consent. out of 238, 188 subjects were patients of nonulcer dyspepsia (nud) and 50 healthy individuals followed up examination. among nonulcer dyspeptic patients, 106 h. pylori positive and 82 h. pylori negative patients were observed. patients with h. pylori positive nud were considered as cases, while patients with h. pylori negative nud were considered as control-1 group. one was fixed with 10% formalin for histopathological examination in the department of pathology, and the other was treated with 10% urea solution containing phenol red indicator (0.2%) for the rapid urease test. the serum was separated from the cells by centrifugation at 3000 rpm for 10 min. serum samples for the measurement of tos, taoc, and prolidase activity were stored at 80c until they were used. for histopathological and gastritis scoring standard positive rapid urease test indicated h. pylori infection, and negative result indicated negative h. pylori infection. a positive reaction was dark pink, and a negative reaction was either a yellow or an orange color. the media consisted of 10% urea solution1.5 ml and phenol red indicator (0.2%)2 drops, ph 6.46.8. for measurement of total antioxidant capacity reagent 1 and 2 reagent 1 : 3.17 gm of orthodianisidine dihydrochloride and 0.01764 gm of fe(nh4)2(so4)26h2o were dissolved in 1000 ml of clark and lubs solution ; reagent 2 : 0.641 ml of h2o2 solution (35%) was diluted to 1000 ml with clark and lubs solution. taoc of serum was measured by the use of an automated measurement method developed by erel (2004) [29, 30 ]. reagent 1 : 114 mg of xylenol orange and 8.18 gm of nacl were dissolved in 900 ml of h2so4 solution 25 mm. the final reagent was composed of 150 mm xylenol orange, 140 mm nacl, and 1.35 m glycerol, ph 1.75 ; reagent 2 : 1.96 gm of ferrous ammonium sulfate and 3.17 gm of o - dianisidine dihydrochloride were dissolved in 1000 ml of h2so4 solution 25 mm. tos of serum was measured by a method developed by erel (2004) [29, 30 ]. osi values were calculated according to the following formula : (1)osi (arbitrary unit) = tos (mol h2o2 equiv./l)taoc (mmol trolox equiv./l). chinard 's reagent (25 g of ninhydrin was dissolved in 600 ml of glacial acetic acid and 400 ml of 6 mol / l orthophosphoric acid at 70c), standard proline solution (650 mol / l solution in 0.45 mol / l trichloroacetic acid), and gly - l - pro / sigma chemical co. (94 mmol / l in 0.05 mol / l tris hcl buffer, ph 7.8, containing 1 mmol of mncl2 per liter) were prepared and stored at 4c. plasma was diluted six - fold with buffer mixture containing 0.05 mmol / l tris hcl buffer (ph 7.8) in 2 mmol of mncl2/l and was incubated for 2 hrs at 37c. after incubation, prolidase reaction was initiated by adding 100 l of the preincubated mixture to 100 l of 94 mm gly - l - pro solution. after reaction initiation, it was incubated for 30 min at 37c ; reaction was stopped by adding 1 ml of 0.45 mol / l trichloroacetic acid. the released proline was measured by addon of 0.5 ml of supernatant to 2 ml of 1 : 1 mixture of glacial acetic acid : chinard 's reagent and incubated for 10 min at 90c. a blank and standard were run under the same condition. instead of the supernatant, serum prolidase activity was measured by the method developed by myara., 1984. 50 healthy individuals and 188 consecutive patients with nonulcer dyspepsia who agreed to participate in the study were included in our study. dyspepsia is characterized by upper abdominal pain or discomfort, nausea, vomiting, bloating, or any other symptom related to the upper gastrointestinal tract. nonulcer dyspepsia was diagnosed when dyspeptic symptoms were present for at least 4 weeks unrelated to exercise for which no organic lesion, such as peptic ulcer, reflux esophagitis, and gallstones, or any systemic disease, was found to be responsible. the following patients were excluded from our study : patients with coexistent medical illness like chronic heart failure, chronic renal failure, diabetes mellitus, and so forth, pregnant females, patients with continued use of nonsteroidal antiinflammatory drugs, patients with history of use of h2-receptor antagonist / proton pump inhibitors during the previous 3 months, patients with evidence of any organic gastrointestinal / or hepatobiliary diseases (diagnosed by abdominal ultrasound or upper gi endoscopy), patients that their informed consents are not available, and patients who did not agree on followup at the regular interval. patients with history of alcohol intake and antioxidant supplement consumption were excluded. parametric methods were used ; a p value of less than 0.05 (p < 0.05) was considered significant. most of cases in our study were of moderate inflammation 34 (32.1%) on histopathological examination, while mild inflammation was present in 32 (30.2%) cases. 22 cases (20.8%) had severe inflammation, while 18 (17%) cases had zero grade inflammation of the gastric mucosa. grading index for control-1 and control-2 of chronic inflammation was zero (table 1). activity is denoted by intensity of neutrophilic infiltration. in our study, most of 36 (34%) h. pylori positive subjects show moderate neutrophilic infiltration. while 30 (28.2%), 20 (18.9%), and 20 (18.9%) cases showed mild, severe, and no neutrophilic infiltration activity, respectively. serum prolidase activity was found to be higher in h. pylori positive subjects (37.91 3.19 mmol min l) as compared to h. pylori negative (32.19 3.43 mmol min l) and healthy subjects (31.92 3.10 mmol min l). therefore, serum prolidase activity was significantly higher in cases as compared to controls (p < 0.0001). it was observed that control-1 had increased serum prolidase activity than control-2, but this was nonsignificant (p = 0.6083) (table 2). total antioxidant capacity (taoc) in h. pylori positive, h. pylori negative, and healthy subjects were 1.39 0.36, 1.79 0.37, and 1.88 0.21 mmol trolox eq / l, respectively. thus, the taoc was statistically lower in h. pylori positive cases than controls (p < 0.0001). control-1 had decreased taoc than control-2, which was nonsignificant (p = 0.1186) (table 2). total oxidant status (tos) in cases, control-1, and control-2 were 13.29 1.29, 11.57 1.06, and 10.65 0.21 mol h2o2 equiv./l, respectively. thus, total oxidant status was significantly higher in cases as compared to controls (p < 0.0001). significantly increased tos was observed more in control-1 than in control-2 (p < 0.0001) (table 2). osi was found to be significantly higher in cases (10.25 3.12) as compared to control-1 (6.71 1.47) and control-2 (5.72 0.56) (p < 0.0001). increased osi was observed more in control-1 than in control-2, which was significant (p < 0.0001) (table 2). weak, negative, and nonsignificant correlation was observed between serum prolidase activities and taoc in cases (r = 0.131, p = 0.181), while weak, positive, and nonsignificant correlation was observed between serum prolidase activity and tos and osi (r = 0.029, p = 0.77, and this above type of weak, nonsignificant correlation was also observed for controls (table 3)., the inflammation induced by h. pylori infection has a chronic active gastritis which means that both lymphocytes and neutrophils infiltrate the mucosa in a characteristic manner. apart from the inflammatory infiltrate, foci of intestinal metaplasia with atrophy, lymphatic aggregates, and lymphoid follicles were formed, and the foveolar epithelium was replaced by regenerative epithelium with correspondingly reduced mucus secretion. because it is found throughout the gastric mucosa from pylorus to cardia, h. pylori gastritis is usually pangastritis although in severity it may vary considerably between the antrum and corpus. in the present study, majority of the cases (32.0%) had moderate inflammation, while mild and severe inflammation was seen in 30.2% and 20.8% of cases, respectively. on analyzing activity, mild activity observed in 28.2% of cases, while 34.8% and 18.9% of cases had moderate and severe activity, respectively. 18.9% of cases had no activity (table 1), while in control-1 and control-2, zero grading was observed for both chronic inflammation and neutrophilic infiltration. thus, h. pylori infected nonulcer dyspeptic (nud) patients seem to have more chronic inflammation and neutrophilic infiltration than h. pylori negative nud and healthy subjects. tos, taoc, and osi are used as biomarkers of oxidative stress and reflect the redox between oxidation and antioxidation. it is well known that oxidative stress can be defined as an increase in oxidants or a decrease in antioxidant capacity, and various oxidants and antioxidants have additive effects on oxidative stress. although the concentration of plasma level of oxidants and antioxidants can be measured individually, it may not accurately reflect the oxidative status [3335 ]. here, we observed oxidative stress in the study population by using tos, taoc, and osi. tos in h. pylori infected dyspeptic subjects was found to be higher when compared to h. pylori negative subjects with functional dyspepsia and healthy subjects. in turkey population, taoc was found to be significantly lower in h. pylori positive subjects as compared to control, while oxidative stress index was found to be higher. among 238 eastern indian subjects, mean sd of taoc was found to be significantly lower in h. pylori positive subjects as compared to h. pylori - negative nonulcer dyspeptic and healthy individuals (p < 0.001). oxidative stress index was found to be higher in cases as compared to controls (p < 0.001). here we observed that in h. pylori infected nud as oxidative stress (tos, osi) increases, the taoc value significantly decreases as compared to h. pylori negative nud and healthy subjects. thus, h. pylori infection associated with increased oxidative stress and decreased antioxidant level in nonulcer dyspeptic patients., serum prolidase activity had been showed to increase especially in early stage of fibrosis. it has been suggested that plasma prolidase activity might be useful to evaluate the fibrotic processes in chronic liver disease in human. a study by horoz. from turkey represented that serum prolidase activity is to be significantly higher in patients with nash in comparison to controls (p = 0.016). they also found a significant correlation between serum prolidase activity and fibrosis score (r = 0.661, p < 0.001). increased serum prolidase activity was well documented in certain cancers like pancreatic cancer, lung carcinoma, breast cancer, stage 1 endometrial cancer, stomach cancer, ovarian cancer [3840 ], and so forth. in our study population, increased serum prolidase activity was found in patients with h. pylori positive nud as compared to patients with h. pylori negative nud and healthy subjects. (2007), who also observed that prolidase activity is to be higher in patients with h. pylori positive subjects with mean serum prolidase activity of 44.11 3.71 this study concluded that in patients with nonulcer dyspepsia of eastern indian subjects increased oxidative stress and increased serum prolidase activity may be associated with h. pylori infection and their association may help to grant a better understanding regarding the pathogenesis of h. pylori infection. thus, serum prolidase activity may be used as biomarker for h. pylori infection in patients with nonulcer dyspepsia. it was also observed that chronic inflammation and neutrophilic infiltration of gastric mucosa are significantly related to h. pylori infection in patients with nonulcer dyspepsia.	helicobacter pylori (h. pylori) infection is associated with increased oxidative stress and serum prolidase activity (spa) in many diseases. we aimed to observe spa and oxidative stress in nonulcer dyspepsia (nud) infected with and without h. pylori among eastern indians. 106 patients with h. pylori positive nud, 82 patients with h. pylori negative nud, and 50 healthy individuals were selected. spa, total antioxidant capacity (taoc), and total oxidant status (tos) were measured with the use of spectrophotometer and an automated measurement method. spa, tos, and oxidative stress index (osi) were significantly higher in patients with h. pylori positive than h. pylori negative nud and healthy individuals (all p < 0.0001), whereas taoc was significantly lower (p < 0.0001). nonsignificant, increased spa (p value = 0.6083) and decreased taoc (p value = 0.1186) were observed in patients with h. pylori negative nud than healthy individuals, while increased tos and osi were significant (p < 0.0001). weak, nonsignificant correlations were observed between serum prolidase activity and taoc, tos, and osi in h. pylori positive cases. thus, increased spa along with increased oxidative stress was observed, which seem to be closely associated with h. pylori infection. spa and oxidative stress seem to be used as biomarkers for h. pylori infection in nud.
lasers produce a beam of light that is coherent, monochromatic, and unidirectional, and can converge most of its radiant power over small areas, even at great distances. laser pointers are useful ubiquitous devices used in everyday situations, especially in the educational environment. their potential to cause retinal damage is a matter of concern, and manufacturers warn against injudicious ocular exposure to laser light. low - energy green laser pointers are generally considered to be safe devices and their potential to cause retinal damage is questionable. here we report a case of macular damage caused by a green laser pointer in a teenager, along with a brief review of the literature. a 13-year - old boy presented to our clinic complaining of decreased vision in both eyes 1 day after having intentionally gazed directly into the beam of a green laser pointing device (wavelength 532 nm), that had a maximum power rating of 5 mw (us food and drug administration class 3a or iec class 3r) stated on its labeling. he held the laser 5 cm away from his eyes for an estimated 3060 seconds. current examination revealed best - corrected visual acuity of 20/50 in the right eye and 20/30 in the left eye. fundus examination showed bilateral, yellowish, oval - shaped, drusenoid - like lesions with attenuation of the foveal reflex (figure 1a and b). imaging studies were done on presentation to our practice 18 hours after exposure to the laser device. optical coherence tomography (3d oct ; topcon, tokyo, japan) of both eyes showed disruption of the outer retinal layer with nonspecific retinal thickening (figure 2a and b) ; red - free photographs demonstrated hypopigmented foveal dots bilaterally (figure 1c and d) ; fluorescein angiography showed early foveal hyperfluorescence in both eyes with late ill - defined leakage (figure 1e and f) ; and autofluorescence images showed heterogeneous hyperfluorescence in the macula of both eyes (figure 1 g and h). finally, a computerized 102 visual field threshold test (humphrey automated perimeter ; humphrey instruments, san leandro, ca, usa), showed small pericentral scotomata in the right eye and a normal field in the left eye. harkins company, inc., grover beach, ca, usa) 1 mg / kg for 4 weeks then tapered over 2 months. at 3 months, visual acuity remained impaired but improved to 20/30 in the right eye and 20/25 in the left eye. at 3 months, the hyperreflective line representing the inner segment / outer segment junction was disrupted in the right eye and the left eye (figure 2c and d). this may lead to an increasing number of exposures to this type of laser device. however, there is debate about the ocular risks posed by inadvertent exposure to standard laser pointer devices, with the presence of an actual laser - induced injury often inconclusive or entirely absent in some studies.13 literature supporting laser pointer - induced retinal injury has been limited to only a few articles on class 3a red laser pointers, and even less literature exists on the retinal hazards of class 3a green laser pointers. in the literature, retinal lesions induced by laser pointers (both green and red devices) include foveal granularity, perifoveal drusenoid - like deposits, or foveal ring - shaped hypopigmented lesions, subretinal hemorrhage, vitreous or chorioretinal hemorrhage, retinal edema, scars in the pigment epithelium, and rarely choroidal neovascularization.4 comparison of the retinal findings in our patient with those of other reported cases in the literature caused specifically by green lasers are listed in table 1. according to barkana and belkin, several factors contribute to laser - related retinal damage, and these can be divided into two categories, ie, laser - related factors (wavelength of the radiation ; pulse duration ; and energy level of the beam) and patient - related factors (size of the pupil, with injury being more severe in larger pupil sizes ; degree of retinal pigmentation, with dark - skinned individuals suffering more severe injury than light - skinned ones ; proximity of incident beam to the fovea ; and refraction status, with damage being more severe in emmetropic eyes due to the laser beam being more focused on the retina).5 also, experimental studies that have evaluated the clinical and histopathologic effects of laser pointers in eyes undergoing enucleation for melanoma concluded that green laser pointers (490575 nm) are more damaging to the retina than red laser pointers (635750 nm).1,6 laser - induced damage to the retina is even more concerning in children and infants than in adults. whereas adults terminate accidental laser pointer exposure in less than 0.25 seconds by pupil, blink, and aversion responses,7 children have been reported to display unusual behavior, ie, staring for a prolonged period of time into the laser beam without blinking or averting the eye.8 current medical therapy for retinal injury is mainly limited to corticosteroids on an undetermined regimen and has variable results.5 final visual acuity ranges from 20/20 to 20/60 vision, and this depends on the size and location of the macular lesion.4 in recent years, cheap laser pointers are increasingly being used as toys for children. while shrinking in size, handheld laser pointers are becoming increasingly more powerful, and safety is becoming a public health issue.9 the potential vision - threatening hazards caused by mishandling laser pointers, even class 3a lasers, emphasize the importance of cautious and appropriate use of these devices. further restrictions on their sale and use by the general public will require more than simple recommendations ; legislation will have to be passed and enforced by governmental bodies.	we report the case of a 13-year - old boy who had a bilateral macular injury after playing with a green laser pointer for a duration of 1 minute. clinical examination revealed a decrease in visual acuity and macular injury in both eyes, and imaging investigations revealed a bilateral macular lesion due to exposure to the laser pointer. at 3 months follow up, visual function had improved but remained partially impaired. this case emphasizes the importance of cautious and appropriate use of laser pointer devices because of the potential vision - threatening hazards induced by mishandling of these devices.
harry angelman first described that 3 of his child patients showed severe mental retardation, jerky movements, excessive laughter, and abnormal physical development. he called them puppet children because they resembled puppets with their flat heads. all three showed typically common behavioural features that led him to suggest the possibility of a distinct syndrome. later children with as show developmental delay, lack of speech, ataxia, learning disability, flat occiput, seizures, tongue protrusion, and uncontrollable laughter. individuals suffering from this disorder show hyperactivity and restless behaviour, wide gait, hypotonia, microcephaly, widely spaced teeth, abnormal eeg patterns, hypopigmentation with blond hair and light eyes, love for water, and dysmorphic features like prominent chin and deep set eyes [1, 2 ]. intellectual disability has been described as a feature of as in almost all studies including the first report by dr. many cases of as seem to associate with autism [4, 5 ], which is characterized by reduced social interaction, lack of communication, and stereotypic behavior. high resolution chromosome banding technique revealed that one of the as patient had a deletion of chromosome 15q11 - 12. this was confirmed when a group of children with severe mental retardation, ataxia, and seizures were shown to have a deletion in the proximal long arm of chromosome 15 [7, 8 ]. although this deletion had already been reported in prader - willi syndrome (pws) earlier [9, 10 ], these children showed features suggestive of as rather than pws. the difference in the manifestation of the two syndromes proposed that the genes responsible for both syndromes might be closely associated but definitely distinct. another major breakthrough came when it was found through rflp (restriction fragment length polymorphisms) that deletion in the maternal copy of the chromosome led to as in contrast to the paternal inheritance of pws [12, 13 ]. while 6070% of the as cases showed large (3 - 4 mb) de novo deletions in chromosome 15, less than 5% of cases showed uniparental paternal disomy (upd) [15, 16 ], and 2 - 3% cases occurred due to imprinting defects [1, 2 ]. the remaining 25% of the cases had unknown origin but few of them were observed to be familial. a recent clinical study with 160 as patients suggested that characteristic eeg patterns could be an important biomarker in as and might predict the underlying genetic cause. in 1994, two candidate genes were mapped to the as critical region, e6-ap (e6 associated protein encoded by the ube3a gene) and par-2 for prader - willi / angelman region - gene-2. soon mutations in the ube3a gene were found in around 510% cases of as [19, 20 ]. discovery of point mutations in ube3a gene strongly implicated ube3a as the gene responsible for as [20, 21 ]. although we can not dismiss the involvement of other genes in as, ube3a is the only gene to date whose dysfunction is sufficient to manifest the as phenotype in number of animal models. it is also important to mention that along with various other chromosomal aberrations identified in autism, maternal deletions and duplication in the proximal region of 15q (region deleted in most cases of as) are a common cause of autism [22, 23 ]. ube3a gene was suggested as a strong candidate for autism because of its imprinted nature and maternal dominance [22, 24 ]. a whole genome wide screening for copy number variation revealed ube3a as one of the affected genomic loci in autism. a map of the maternal and paternal human chromosome region 15q11 - 13 containing multiple genes is shown in figure 1. ube3a gene is located within the q11-q13 region on chromosome 15 in humans while it is found on the proximal region of chromosome 7 in mice. it encodes a 100 kda protein known earlier as e6-ap (e6 associated protein) [27, 28 ]. ube3a gene encodes five mrna subtypes generated by alternate splicing that give rise three protein isoforms. e6-ap / ube3a belongs to the hect (homologous to e6-ap c - terminus) domain family of e3 ubiquitin ligases in the ubiquitin proteasome system (ups). these proteins exit with large diversity and promote degradation of short lived or abnormal proteins by transferring multiubiquitin molecules to them as a degradation signal. the members of the hect family share a ~350-residue conserved c - terminal region called the hect domain [31, 32 ]. ube3a is the founding member of the family, discovered based on its interaction with viral e6 oncoprotein to target p53 for proteasomal degradation in cells infected with human papilloma virus (hpv). ube3a is also demonstrated to act as a transcriptional coactivator of steroid hormone receptors [4547 ]. ube3a is shown to interact with number of cellular proteins that indicate its involvement in multiple cellular function including cell cycle regulation [4852 ], synaptic function and plasticity [5358 ], and cellular protein quality control [5961 ]. a list of identified substrates and possible cellular function of ube3a is shown in table 1. this group successfully made a model for pws with maternal duplication in the central region of chromosome 7 but failed to make the same for as with paternal duplication. while the imprinting was expected in the central region on the mouse chromosome 7, (which was considered homologous to the human region 15q11 - 13 deleted in pws / as) the actual imprinting seen in the partial upd mice was more proximal on the chromosome. a few years later, based on detailed investigation by the same group, this mouse model was strongly put forward as a model for as. detailed study in this model suggested that the imprinted proximal region earlier identified in fact should be included in the putative pws / as segment. the mouse model showed various features like gait ataxia, abnormal limb clasping, startle response, and hyperactivity. the cerebral hemispheres did not show any gross abnormality or cell loss but cortical thinning was noticed. abnormal eeg, a typical feature of as [63, 64 ], is also recorded in these mice. soon after the discovery of ube3a mutations in as individuals [65, 66 ], this model was further characterized for the expression of ube3a, and found that the expression of this gene was absent in the hippocampus, cerebellar purkinje cells, and olfactory bulb (mitral - cell layer) of the mice. this shows that majority of the expression observed in these areas is from the maternal allele. using rna in situ hybridization, it was shown that the cortex showed reduced levels of the ube3a transcript, while there was no change in the anterior commissure and optic chiasm. this suggests that the ube3a gene has varied expression in different region of the brain. areas like the cerebral cortex, which show reduced expression, have slight predominance of maternal expression, while optic chiasm and anterior commissure have equal expression from both the maternal and paternal alleles. imprinting in the as brain was reported around the same time [24, 68 ], but albrecht therefore, they looked into different parts of the brain and concluded that ube3a is imprinted only in certain areas of the brain. the absence of ube3a had no effect on the number of purkinje cells or the overall cytoarchitecture of the brain in upd mice. this mouse was generated by a deletion mutation in exon 2 of ube3a gene thereby inhibiting the formation of a functionally active protein. mice generated were termed wild - type ube3a, heterozygous ube3a- or ube3a (depending upon the parental inheritance), and homozygous ube3a (null) for the mutation. the maternal deficient heterozygous mice ube3a exhibited reduced brain weight, ataxia, motor impairment, and abnormal eeg pattern. around 2030% of maternal - deficient and null mice exhibited audiogenic seizures. the maternal deficient mice also showed context - dependent learning and memory impairment and deficits in hippocampal long - term potentiation. ube3a expression was imprinted in hippocampus and cerebellar purkinje cells, and p53 level was increased in the purkinje cells of ube3a mice. this genetic model successfully captured many of the classical features associated with as and provides a tool to discover molecules and pathways affected by the absence of ube3a, mainly the ones responsible for cognitive and motor function. detailed immunohistochemical and immunoblot analysis later revealed that ube3a in these mice is imprinted throughout the brain. various areas of the brain like cortex, striatum, midbrain, and hypothalamus in addition to hippocampus, cerebellum, and olfactory bulbs showed predominant expression from the maternal copy of the chromosome [34, 35, 69 ]. it was reported that along with the neurons, parvalbumin and calretinin positive gabaergic interneurons also expressed ube3a solely from the maternal allele. peripheral tissue like liver, heart, and lungs in as mice showed more than 50% reduction in the levels of ube3a expression, showing that maternal expression was predominant even in the other tissues. further behavioural characterization in this model showed that ube3a mice have motor deficits suggestive of a dysfunctional cerebellum. a novel finding was that these mice had a different licking behaviour than the wild - type mice, with more number of licks at greater intervals. it is possible that the difference in the lick behaviour is due to the loss of synchrony between breathing and swallowing and correlates with the feeding and swallowing difficulties seen in as children [19, 71 ]. although the motor deficits observed in ube3a mice are thought to be due to dysfunction of cerebellar purkinje cells, a recent report indicated probable abnormalities in nigrostriatal pathway [33, 72, 73 ]. the ube3a mice showed reduced number of dopaminergic neurons in the substantia nigra accompanied by poor performance in behavioural paradigms sensitive to nigrostriatal dysfunction. this is further supported by the fact that two patients with as have been shown to manifest typical features of parkinson 's disease like tremors, cogwheel rigidity, and bradykinesia and were responded to levodopa, which is widely used for the symptomatic treatment of parkinson disease. however, similar disabling tremor in as patients also has been treated differently [76, 77 ]. lately, there have been major advancements in understanding the molecular basis of the cognitive deficits associated with as. the level of the inhibitory phosphorylation at thr305 of the calcium / calmodulin - dependent protein kinase ii (camkii) in the hippocampus of the ube3a mice was increased leading to reduction in the activity of the protein. all the behavioural and learning deficits observed were reversed when a mutation was introduced to block the inhibitory phosphorylation of camkii. ube3a mice were shown to have impaired experience - dependent synaptic plasticity in the visual cortex. this impairment is reversible, and late postnatal deprivation of sensory inputs again restores plasticity of the synapses. these observations suggest that absence of ube3a leads to the inability to modify or rearrange synapses as per the requirement in activity - dependent synaptic plasticity. it is hypothesized that this could occur either due to decreased number of excitatory synapses or due to decreased efficiency of neurotransmitter release. the second probability is in turn dependent on the calcium levels and receptor trafficking which very well correlates to the camkii levels. it was observed that the visual cortical circuitry and the retinotopic map are formed normally, but the basal dendrites show reduced spines in ube3a mice. absence of ube3a plays a crucial role in the postnatal experience driven period [35, 37 ]. cognitive development and development of speech are events that depend on the external sensory experience. failure of these important processes in as patients could mean that ube3a is indeed required for remodeling of the circuitry. the work so far emphasizes that ube3a is not directly involved in circuit formation but is crucial in experience - dependent synaptic remodeling. recently, the exact role of ube3a in experience - driven synaptic plasticity was elucidated at the molecular level. ube3a levels are increased after treatments with kainic acid, kcl, nmda (n - methyl - d - aspartic acid), glutamate, and bicuculline in primary neuronal cultured cells, while novel environment increases the levels of ube3a in mice brain compared to standard laboratory caged mice. the promoter of ube3a gene is under the control of activity - dependent transcription factor mef2. the increase in levels of ube3a with glutamate stimulation and decrease with inhibitors of glutamate receptors clearly puts forth the role of ube3a in synapse development. many substrates of ube3a have been discovered but none were directly implicated in the loss of synaptic plasticity. ha - ubiquitin transgenic mice were crossed with ube3a mice, and the proteins that showed reduced ubiquitination were studied. sacsin was one of the substrates of ube3a as it showed reduced ubiquitination in knockout mice as compared to wild type. sacsin is mutated in charlevoix - saguenay spastic ataxia, a disorder similar to as. but sacsin could be one of the causes of the motor deficits seen in as patients, considering its involvement in disorders with ataxia. arc was another substrate discovered, which is responsible at least in part for the rigidity seen at the ube3a deficient synapses. increased arc expression leads to decreased surface ampars while decrease in arc levels leads to increase in the ampars at the surface. lack of ube3a leads to accumulation of arc, which subsequently results in increased internalization of the ampars. there is a reduction seen in the ampa / nmda current ratio, which is due to the loss of ampars as there was no change in nmdars. it has a role in restricting the neuron to form only the required number of synapses [54, 56 ]. mice expressing ube3a - yfp fusion protein exclusively from the maternal copy is a very promising tool to carefully study the microscopic abnormalities in as. study focusing on the cellular localization of ube3a helped to elucidate the probable functions of this protein. ube3a - yfp fusion protein localized mainly in the nucleus with detectable expressions in the cell soma and dendrites. the ube3a protein was found in the pre and postsynaptic compartments and was localized in the growth cones of hippocampal neurons in primary culture [34, 69 ]. this mouse model showed biallelic expression of ube3a in gfap - positive astrocytes lining the ventricular area. in other brain regions although the absence of ube3a did not affect dendritic branching in any of the imprinted neurons, a detailed microscopic study showed that the dendritic spines had abnormal structures. in the absence of any gross cellular or structural changes in the brain, it is hypothesized that absence of ube3a is necessary either for the formation or maintenance of the dendritic spines. this is probable since the activity of phospho camkii is reduced in maternal deficient animals, and camkii is known to help in activity - dependent spine formation. this correlates very well with the observations made in a pathological study in as brain as well. further investigation in this mouse model can give major insights into the role of ube3a during synaptogenesis even at a single synapse level. ube3a is shown to interact with and coactivate nuclear steroid hormone receptors [45, 46, 83 ]. absence of ube3a renders both male and female mice less fertile compared to the wild - type controls. ube3a null male mice show reduced testis size, lesser sperm count, decreased sperm ability to penetrate ova and reduced prostate size. in ube3a knockout female mice, all these findings indicate that coactivator role of ube3a is important in reproductive function. but whether the loss of coactivator function of ube3a is associated with any abnormalities in brain function leading to as are not very clear. recently, we have shown that the defective glucocorticoid hormone receptor signaling in ube3a mice brain could lead to increased stress and anxiety in these mice. these mice also exhibited decrease in the number of parvalbumin - positive gabaergic interneurons in their hippocampus. yet another mouse model of as was generated by inactivating the exons corresponding to the human exons 15 and 16 from the ube3a gene. a lacz reporter was introduced after the deletion site to detect the expressing protein albeit truncated. the expressed ube3a does not show ligase activity, and the -galactosidase activity is seen in the brain wherever maternal copy expresses the truncated protein. this mouse model showed motor deficits, learning and memory impairments, and an abnormal eeg characteristics of as, but seizures were absent in this model. ube3a was imprinted in the hippocampus, basket cells in the cerebellum, as well as in the frontal cortex. cells in the ventricular ependyma showed lacz expression both in maternal and paternal ube3a deficient mice, which is consistent with the observation that the ventricular gfap positive cells express biallelic ube3a. interestingly, it was observed that the progenitor cells do not show imprinted expression, but imprinting is acquired by embryonic day 10 in mouse. neurons specifically expressed the maternal sense ube3a, while the antisense ube3a was expressed only from the paternal copy. surprisingly, there was no imprinted expression in the cerebellar purkinje cells which is a deviation from the other studies [33, 34, 69 ]. as the protein is truncated only in the c - terminal hect domain, the transcriptional coactivator function is still might be active in the animals. absence of imprinted expression in purkinje cells is a major drawback of the model and could be a reason for unaltered p53 levels. interestingly, this mouse model showed disrupted sleep wake cycle seen in most of the as children [2, 86 ]. using this mouse model, another group has shown that the deficiency of ube3a leads to impaired neurogenesis and changes in the hippocampal plasticity. the immediate early genes c - fos and arc, associated with neuronal long - term plasticity and memory formation, showed reduced expression in the maternal deficient mice brain. a knockout mouse model of the gabaa (-amino butyric acid) receptor 3 subunit (gabrb3) showed most of the behavioural features like epilepsy, abnormal eeg pattern, learning deficits, and poor motor coordination. the deletions in gabrb3 are heritable, but since this gene is not imprinted in the brain, gabrb3 only adds to the phenotypic characteristics and is not a direct cause of as. mutation in ube3a is sufficient to show the cardinal features of as, although deletion of gabrb3 might contribute to a more severe phenotype [88, 90 ]. a new mouse model of as, has been reported recently that tries to replicate the most prevalent form of the syndrome. a 1.6 mb region spanning from ube3a to gabrb3 was deleted to generate this mouse model. the maternal deficient mice of this region, on the other hand, showed no developmental abnormality. like the earlier ube3a mice, these mice also showed impairment in motor activity and learning and memory. the anxiety related behavior was assessed in these mice and found that maternal deficient mice spent more time in dark areas as compared to the wild - type or paternal deficient mice. maternal deficient mice with deletion of this region exhibited contextual fear and spatial learning deficits. these may correlate with the lack of speech and impaired communication seen in as patients. another mouse model was generated with an inheritable transgene insertion (epstein - barr virus latent membrane protein 2a, lmp2a) into the central part of chromosome 7 of mouse. the deletion created by transgene insertion led to formation of either pws or as model in a parent - of - origin manner. inheritance of the deletion from the paternal allele led to formation of pws, while maternal transmission led to an as model. around 70% of the cases in humans are due to deletions in the 15q11 - 13 region. this model, therefore, represents the widely prevalent condition of as and, therefore, should be characterized for better understanding of disease pathogenesis and developing therapeutics. several other mouse model have been generated based on as imprinting defect mutation [43, 91 ], radiation - induced mutation removing multiple genes including ube3a, and duplication of the as - pws locus. although all of these mouse models reported reduced expression of ube3a, their neurobehavioral phenotype are not well characterized. a list of as mouse models are shown in table 2. interestingly, mice over expressing triple the dose of ube3a showed autism traits like impaired communication, defective social interaction, and increased repetitive stereotypic behavior. these findings along with others clearly indicate that ube3a plays a very important role in synaptic function, and its altered function could be linked with both as and autism. in addition to these mouse models, human induced pluripotent stem cell model of as or mouse differentiated embryonic stem cell model of as were also developed [93, 94 ]. these models will be useful to understand the developmental timing and mechanism of ube3a silencing in neurons as well as disease biology. drosophila models have also been generated in order to understand the pathogenesis of as. dube3a, the homologue of human ube3a, is deleted imprecisely such that the corresponding protein is not formed. missense mutations analogous to the ones found in as patients were also used to study their effect. the gain - of - function model in this case is particularly informative since the deletion of dube3a does not lead to any morphological abnormality. over activity of dube3a in general promoter specific expression in the eyes and wings leads to aberrant morphology of the organs. the group studied rnai dube3a flies in addition to the deletion mutants. in an interesting approach, they also studied flies by mosaic analysis with a repressible cell marker (marcm) in which a single neuron is injected with gfp labeled genetic mutation while the surrounding neurons continue to have a wild genotype. using these advanced techniques, they found that dube3a is necessary for dendritic arborization in a cell autonomous manner. surprisingly, over expression of dube3a also causes reduction in dendritic branching in the fly, suggesting that the levels of ube3a are critical in formation of the dendrites. the fly, model would be useful in identifying and characterizing the substrates of ube3a and understanding the disease pathogenesis. it is evident from the existing literature that the loss of expression of maternal - inherited ube3a is primarily responsible for as, although we can not completely rule out the possibility of other disease - modifying gene like gabrb3. dysfunction of ube3a is sufficient to produce phenotypes resembling to as in different animal models. most extensively used ube3a - maternal deficient mice replicate many essential features of as including cognitive and motor deficits. clinical features of as like cognitive and motor deficits, sleep disturbance, feeding difficulties, and altered synaptic plasticity have a molecular or electrophysiological correlate due to the studies performed in animal models. a recent clinical study reported that specific eeg pattern could be an important biomarker of as and might indicate the underlying genetic cause. most interestingly, ube3a - maternal deficient mice show significant impairment in activity - dependent synaptic plasticity indicating the role of ube3a in regulation of synaptic function and plasticity. the experience - dependent synaptic plasticity is shown to be modulated by number of ways. therefore, this novel role of ube3a can be exploited further for possible therapeutic intervention of as. in fact one report demonstrated neuregulin - erbb4 signaling is associated with abnormal synaptic plasticity in ube3a mice, and inhibitors of erbb reverse the contextual fear memory deficits. the cognitive deficits observed in ube3a mice were also rescued upon adeno - associated virus vector - mediated expression of ube3a into the brain. since the paternal copy of ube3a is epigenetically silenced in neurons, it is possible that the reactivation of paternal expression could be an exciting therapeutic strategy. clinical trials were conducted in as children using methylation - promoting dietary supplements (creatine, folic acid vitamin b12, metafolin, and betaine) in order to up - regulate the ube3a expression (by suppressing the expression of ube3a antisense transcript). unfortunately, there were no significant improvements of intellectual disabilities or abnormal eeg patterns in as children [99, 100 ]. interestingly, a very recent report has demonstrated that topoisomerase inhibitors activate the dormant expression of ube3a in neurons. however, treatment of such drugs could also alter the expression of other genes and, therefore, lead to other complications. further studies are required to investigate possible role of these topoisomerase inhibitors in the recovery of behavioral abnormalities in animal models. enriched environment or neuronal activity (that can trigger experience - dependent synaptic development) also has been demonstrated to increase the expression of ube3a. therefore, various cognitive training paradigms in early developmental stage could potentially improve cognitive and motor deficits in as children by increasing the expression of ube3a. all together, the field is now passing through an exciting phase, and we all are hoping for a major breakthrough in therapeutic intervention of as.	angelman syndrome (as) is a neurodevelopmental disorder characterized by severe mental retardation, lack of speech, ataxia, susceptibility to seizures, and unique behavioral features such as easily provoked smiling and laughter and autistic features. the disease is primarily caused by deletion or loss - of - function mutations of the maternally inherited ube3a gene located within chromosome 15q11-q13. the ube3a gene encodes a 100 kda protein that functions as ubiquitin ligase and transcriptional coactivator. emerging evidence now indicates that ube3a plays a very important role in synaptic function and in regulation of activity - dependent synaptic plasticity. a number of animal models for as have been generated to understand the disease pathogenesis. the most widely used model is the ube3a - maternal - deficient mouse that recapitulates most of the essential features of as including cognitive and motor abnormalities. this paper mainly discusses various animal models of as and how these models provide fundamental insight into understanding the disease biology for potential therapeutic intervention.
group a streptococcus (strep. pyogenes or gas) is a -hemolytic bacterium often found in the throat and the skin. the two most severe manifestations of gas are streptococcal toxic shock syndrome (stss) and necrotizing fasciitis (nf). annually in the united states there are between 9,000 - 11,500 cases of invasive disease (3.2 to 3.9/100,000 population) ; stss and nf each account for approximately 6 - 7% of invasive cases. the isolation of gas, in a patient with severe sepsis, from a normally sterile site defines severe invasive gas disease. gas produces exotoxins and these superantigens are thought to circumvent traditional immune mechanisms, producing vast discharge of inflammatory cytokines. there is an identifiable need for adjunctive therapy in these cases, as attributable mortality may be as high as 80%, despite prompt antimicrobial therapy. a 40-year - old ex - smoker presented after 2 days of vomiting and severe right - sided chest pain. relevant blood tests were a reduced wcc at 2 10/l, raised crp 361 mg / l, and acute renal failure with urea 14.8 mmol / l and creatinine 358 mmol / l. there were no factors predisposing to the patient being immunocompromised, and a human immunodeficiency virus (hiv) test was negative. blood gases showed ph 7.12, base excess 10, lactate 4.2, po2 17.5 kpa, and pco2 3.1 on 80% inspired oxygen. following intubation he was commenced on renal replacement therapy, at 80 ml / kg for severe sepsis. vasopressin and dobutamine were added, and cardiac output monitoring with pulse contour analysis (lidco) commenced : scvo2 was 80%. hydrocortisone and drotrecogin alfa (activated protein c) (apc) (see footnote) were also instituted. in relation to indications for the use of apc, all four systemic inflammatory response syndrome (sirs) criteria were met ; the patient had more than one sepsis - induced organ failure and there were no specific contraindications. in particular the platelet count was > 30 10/l (at 90 10/l) and the inr < 3.0 (1.5). the patient remained in a refractory septic shock and in multiorgan failure, and an adrenaline infusion was commenced. however muscle layer biopsies and blood cultures soon grew extensive gas and an initial diagnosis of nf was made. the patient was felt to be too unstable to take to theatre for debridement, and a plan was made to observe the chest wall and attempt operative intervention only if there was spreading fasciitis. utilizing the recent centers for disease control (cdc) position statement for the clinical case definition of stss, classifies this patient as a confirmed case of stss. in addition to identifying gas from a normally sterile site (blood), the patient also satisfied the clinical manifestations within the definition. namely these were hypotension (systolic blood pressure 90 mmhg) and multiorgan involvement characterized by the presence of two or more of renal impairment, coagulopathy, liver involvement, acute respiratory distress syndrome, a generalized erythematous macular rash, and soft tissue necrosis, including nf. a decision was made to commence intravenous immunoglobulin (ivig) at 1 g / kg on day 1, followed by 0.5 g / kg on days 2 and 3, as the patient was continuing to deteriorate despite the above maximal medical therapy. the relevant public health authorities were informed, noting that scarlet fever (also caused by gas) remains a notifiable disease in the united kingdom. over the next 24 hours the patient improved, was extubated 3 days later and was subsequently discharged from hospital after 2 weeks. the exact role played by ivig within the improvement of this patient is difficult to quantify within the context of a case report ; however we will discuss the mechanisms and evidence base, which we feel justified its use in this case. ivig is a gamma globulin made from purified pooled plasma of thousands of donors, consisting mainly of igg, and sourced from outside the united kingdom due to the risk of creutzfeldt - jakob disease. ivig is thought to work by a variety of mechanisms including antibody protection against lipopolysaccharides, neutralizing superantigens, modulation of fc - receptor blockade and expression, inhibition of membrane attack complexes (c5b-9), and complement activation and also by facilitating the opsonization of gas bacteria. this concept of immunomodulation of the inflammatory cascade may apply during sepsis, with use of ivig in gas. a multicenter, double - blind rct examining the use of high dose ivig as adjunctive therapy in stss, in addition to clindamycin and penicillin, was published in 2003. placebo control was with 1% albumin, with the primary end point being effect on 28-day mortality with use of ivig (1 g / kg on day 1, then 0.5 g / kg on days 2 and 3). power calculation was for 120 patients, and only 21 were randomized due to recruitment difficulties. an insignificant trend toward lower mortality (10% vs. 36%), as well as a significant decrease in sepsis - related sofa scores at days 2 (p=0.02) and 3 (p=0.04), was observed in the ivig group. further evidence is provided from a canadian observational study comparing 21 consecutive patients who were given ivig for stss. although there was no difference in duration of ventilation or hospital stay (los), survival to 30 days was improved in the ivig group (67% vs. 34%, p=0.02). in contrast a recent paper examining the use of ivig in 84/192 pediatric stss patients found no significant mortality reduction and longer los and higher hospital costs. current uk department of health guidelines for use of ivig in infectious diseases [table 1 ] recommends that ivig may be added to adequate toxin - neutralising antimicrobials, source control and sepsis management when these approaches have failed to elicit a response. united kingdom department of health guidelines for immunoglobulin use in infectious diseases this patient had severe septic shock where use of ivig has also been reported. turgeon. looked at rcts comparing ivig with placebo (or no intervention) in critically ill adult patients with sepsis, and found a 26% survival benefit associated with use of polyclonal ivig (rr 0.74 [95% ci, 0.62 - -0.89 ] : p=0.001 with a number of patients needed to treat to save one life (nnt) of 9. a meta - analysis by laupland. found overall significant reduction in mortality with ivig treatment (or 0.66 (95% ci, 0.53 - -0.83 ; p<0.0005), although this result was not confirmed when only high - quality studies were analyzed. looked at 27 rcts, 15 of which involved 1492 adults and children with trials of polyclonal ivig in septic patients. again reduced mortality was demonstrated, with adult data showing rr 0.79 (95% ci, 0.69 - -0.90). among the authors conclusions in these trials was that, an adequately powered rct was needed to confirm the effects of ivig in septic shock, on the background of previous trials having methodological flaws and heterogeneous data. in 2007 the placebo - controlled rct sbits was published, which examined the use of ivig in patients with sepsis. twenty - eight - day mortality was not reduced significantly, 37.3% versus 39.3% in the ivig group (p=0.6695), and there was also no improvement in 7-day mortality or pulmonary function. one point of note was that this trial used a smaller total dose of ivig (0.9 mg / kg), than the multicenter trial using ivig in stss. a cochrane review update, which found 17 trials in adults (n=1958), concluded that polyclonal ivig had a mortality benefit in adults (rr 0.77, 95% ci 0.68 - -0.87) ; however this was not reproduced when examining trials with low risk of bias. current doh guidance, and recent drugs and therapeutics bulletin advice, do not recommend the use of ivig in general sepsis. ($ 10,000 for a 100 kg (220 lbs) person at 2 g / kg), this will necessitate careful appraisal of its cost benefit by each treating institution. neilson. showed, on the basis of a meta - analysis of studies undertaken in adult septic patients, that the addition of igm - enriched ivig to standard therapy has a positive association with reduced mortality. the increased cost equated to 5715-28443/life saved which compares favorably with other recommendations for adjuvant therapies in sepsis. for example, the addition of apc had a cost implication of 100728-120176/life saved. a further meta - analysis demonstrated favorable comparisons with other current recommendations in sepsis, in relation to nnt and on an economic basis, e.g., ivig in adults ; nnt 9, rr (95% ci) : 0.74 (0.62 - -0.89) compared to apc ; nnt 16, rr (95% ci) : 0.80 (0.69 - -0.94). there are no recommendations for the use of ivig in the current surviving sepsis guidelines for adults, though it may be considered it pediatric septic shock. the omission of ivig from these adult guidelines was recently reviewed, with the conclusion being that small trials of low quality formed the basis of positive results. in addition the largest rct (sbits trial described above) failed to demonstrate benefit, with standard igg ivig in adult septic patients. however the potential benefit of igm - enriched ivig preparations has been shown in experimental sepsis models and meta - analyses, with kreyman. raising the possibility of significant benefit of igm - enriched ivig in adult and neonatal sepsis. it is likely that further larger controlled rct 's concerning igm - enriched ivig in adult sepsis will be needed prior to concrete recommendations in international sepsis guidelines. a uk perspective, we feel the limited worldwide supply and high cost implications, together with an inconclusive evidence base, warrant restricting its use to cases in which conventional therapy has failed. pharmaceutical company eli lilly 's recent decision to withdraw xigris (drotrecogin alfa (activated)) from the market worldwide further to the 28-day mortality results from the prowess - shock study. pharmaceutical company eli lilly 's recent decision to withdraw xigris (drotrecogin alfa (activated)) from the market worldwide further to the 28-day mortality results from the prowess - shock study.	group a streptococcus (gas) is a -hemolytic bacterium often found in the throat and skin. the two most severe clinical manifestations of gas are streptococcal toxic shock syndrome and necrotizing fasciitis. intravenous immunoglobulin (ivig) is a gamma globulin made from purified pooled plasma of thousands of donors, consisting mainly of igg. we report the case of a 40-year - old man admitted after 2 days of vomiting and severe right - sided chest pain. he was hypotensive with a sinus tachycardia, pyrexial, and vasodilated. the only other positive finding was a swollen and erythematous chest wall. muscle layer biopsies and blood cultures soon grew extensive gas, and an initial diagnosis of necrotizing fasciitis was made. the clinical syndrome was of severe septic shock secondary to invasive gas. the patient quickly deteriorated with a worsening metabolic acidosis. despite maximal intensive care therapy including fluids, vasoactive agents, and also activated protein c, the patient continued to remain profoundly hypotensive. a decision was made to commence ivig, with the aim of immunomodulation of the inflammatory cascade seen in sepsis. over the next 24 hours the patient improved, was extubated 3 days later, and subsequently discharged from hospital after 2 weeks. although the evidence for the use of ivig in severe invasive gas disease is limited, we feel that on reviewing the available literature its use in this case was justified. the limited worldwide supply and high costs, together with a limited evidence base, warrant restricting its use to cases in which conventional therapy has failed. the literature for use of intravenous immunoglobulin in invasive gas infection will be reviewed in this article.
walking, which can be defined as moving the body to a target spot or destination using two legs, is a complex and integrated activity requiring low energy consumption1. although it appears to be an easy - to - perform task, it actually requires comprehensive use of all nerves and skeletal muscles. furthermore, learning to walk requires considerable practice and effort and takes at least four years to master2. the gait cycle is divided into stance and swing phases. during the stance phase, the foot is in contact with the ground, whereas during the swing phase, the same foot lifts off the ground and moves forward. the stance phase is divided into heel strike, foot flat, mid - stance, and toe off, whereas the swing phase is divided into acceleration, mid - swing, and deceleration3. the pelvis is a structure located between the hip joint and lumbosacral region. with the use of multiple muscles, the pelvis controls the movements of the hip joint and lumbosacral region. in this respect, the pelvic position is the most important factor for determining the body s sagittal alignment and posture4. with the pelvis in the neutral position, correct posture can be maintained, and movements occurring during activities of daily living and gait can be improved via proper control of the upper and lower extremities in dynamic postures5. in addition, mechanical problems in the musculoskeletal system of the lumbar vertebrae can occur depending on the location of the pelvis. moreover, forward and backward pelvic tilt exercises can influence spinal stability and physical alignment. lumbo - pelvic stabilization and posture have been shown to improve with bridge exercises7 and various mat exercises8. in addition, some studies have examined postures9 or gait variables10 through pelvic adjustment. however, none of the studies have addressed the correlations among pelvic positions and differences in lower - extremity joint angles during walking. thirty female students at the k university were selected as subjects. the average (meanstandard deviation) age, height, and weight of the subjects were 22.33.4 years, 163.25.4 cm, and 53.55.7 kg, respectively. the exclusion criteria were as follows : musculoskeletal or neurological problems ; lumbar and pelvic pain during daily activities ; incorrect posture due to scars related to previous burns or surgeries ; and inability to walk normally. this study was approved by the university s institutional review board, and the subjects were safely protected during all experimental procedures. all the subjects understood the purpose of this study and provided written informed consent prior to their participation, in accordance with the ethical standards of the declaration of helsinki. this study measured the pelvic positions and differences in lower - extremity joint angles during walking. the backmapper (abw, germany), a three - dimensional spinal diagnostic system, was used to measure pelvic positions. the backmapper is an apparatus that obtains precise measurements of the spinal form, location, and degree of distortion, evaluating the spinal form from the backward, forward, downward, and upward directions. it analyzes the inclination of the coronal plane, pelvic position (ppo), left and right inclination of the pelvis, pelvic torsion (pto), degree of declination of the hip bone, and pelvic rotation (pro). the qualisys track manager (qualisys ab, sweden), a three - dimensional wireless motion analysis system with six cameras operating at 100 hz per second, was used to analyze the kinematic motions of lower extremities in the subjects during walking. the l - frame was fixed on the starting point of walking to set the spatial coordinates. in terms of directions, the upper vertical axis was set as the + z - axis, and the direction of motions and the horizontal axis were set as the + y - axis and + x - axis, respectively. based on the l - frame, the space within which movements would be performed was defined using the t - wand to recognize markers clearly. reflective markers for static postures to form basic frames were attached to each side of the sacrum, anterior superior iliac spine, thigh, and shank. four markers were attached to each side of the thigh and shank in a square form to create a three - dimensional space. in addition, reflective markers were attached to the medial and lateral epicondyles, lateral malleolus, and medial malleolus on both sides of the body. in the segments of the foot, reflective markers were attached to specific areas of the foot surface, which corresponded with the calcaneal tuberosity and the first and fifth metatarsal heads. to track the three - dimensional motions of three lower extremity joints during dynamic postures, the markers for static postures, which had been attached to the medial and lateral epicondyles, lateral malleolus, and medial malleolus, were removed prior to filming of the respective motions. to reduce errors, a single well - trained person attached these reflective markers. motion analysis was performed to derive differences between left and right flexion, abduction, and external rotation range of motion (rom) of hip joints, flexion, abduction, and external rotation rom of knee joints, and dorsiflexion, inversion, and abduction rom of ankle joints, according to the x, y, and z - axes. the measured data were analyzed using the spss 12.0 ko (spss, chicago, il, usa) software, and the collected data are presented in terms of means and standard deviations. pearson s correlation coefficient was used to observe correlations among the ppo, pto, pro, differences between left and right hip flexion (hxd), differences between left and right hip abduction (hyd), differences between left and right hip external rotation (hzd), differences between left and right knee flexion (kxd), differences between left and right knee abduction (kyd), differences between left and right knee external rotation (kzd), differences between left and right ankle dorsiflexion (axd), differences between left and right ankle inversion (ayd), and differences between left and right ankle abduction (azd). the ppo, pto, pro, hxd, hyd, hzd, kxd, kyd, kzd, axd, ayd, and azd measurements are presented as meanstandard deviation in table 1table 1.meanstandard deviation of ppo, pto, pro, hxd, hyd, hzd, kxd, kyd, kzd, axd, ayd, and azd (unit - degree) ppoptoprohxdhydhzdkxdkydkzdaxdaydazd2.70.93.61.72.21.55.21.83.51.75.11.46.31.66.41.66.21.45.81.55.51.05.81.4ppo : pelvic position, pto : pelvic torsion, pro : pelvic rotation, hxd : difference between left and right hip flexion, hyd : difference between left and right hip abduction, hzd : difference between left and right hip external rotation, kxd : difference between left and right knee flexion, kyd : difference between left and right knee abduction, kzd : difference between left and right knee external rotation, axd : difference between left and right ankle dorsiflexion, ayd : difference between left and right ankle inversion, azd : difference between left and right ankle abduction. in terms of correlations among these variables, ppo showed positive correlations with hxd, hyd, kxd, kyd, and ayd. in other words, an increase in ppo resulted in corresponding increases in hxd (r=0.51), hyd (r=0.62), kxd (r=0.45), kyd (r=0.42), and ayd (r=0.38). hxd exhibited a positive correlation with axd, as an increase in hxd resulted in a corresponding increase in axd (r=0.64). kxd also showed positive correlations with kyd and ayd ; an increase in kxd led to corresponding increases in kyd (r=0.41) and ayd (r=0.45), as shown in table 2table 2.correlations among ppo, pto, pro, hxd, hyd, hzd, kxd, kyd, kzd, axd, ayd, and azdcategoryppoptoprohxdhydhzdkxdkydkzdaxdaydazdppo1pto0.181pro0.010.121hxd0.510.020.301hyd0.62 0.020.020.261hzd0.370.11 - 0.170.150.081kxd0.45 0.250.030.210.000.341kyd0.42 0.040.020.320.310.000.41 1kzd0.150.280.110.100.040.190.100.151axd0.190.000.120.640.030.060.140.190.101ayd0.38 0.190.150.110.250.220.45 0.090.250.151azd0.100,000.110.240.070.250.00 - 0.110.160.290.131 p<0.01, p<0.05. ppo : pelvic position, pto : pelvic torsion, pro : pelvic rotation, hxd : difference between left and right hip flexion, hyd : difference between left and right hip abduction, hzd : difference between left and right hip external rotation, kxd : difference between left and right knee flexion, kyd : difference between left and right knee abduction, kzd : difference between left and right knee external rotation, axd : difference between left and right ankle dorsiflexion, ayd : difference between left and right ankle inversion, azd : difference between left and right ankle abduction. ppo : pelvic position, pto : pelvic torsion, pro : pelvic rotation, hxd : difference between left and right hip flexion, hyd : difference between left and right hip abduction, hzd : difference between left and right hip external rotation, kxd : difference between left and right knee flexion, kyd : difference between left and right knee abduction, kzd : difference between left and right knee external rotation, axd : difference between left and right ankle dorsiflexion, ayd : difference between left and right ankle inversion, azd : difference between left and right ankle abduction p<0.01, p<0.05. ppo : pelvic position, pto : pelvic torsion, pro : pelvic rotation, hxd : difference between left and right hip flexion, hyd : difference between left and right hip abduction, hzd : difference between left and right hip external rotation, kxd : difference between left and right knee flexion, kyd : difference between left and right knee abduction, kzd : difference between left and right knee external rotation, axd : difference between left and right ankle dorsiflexion, ayd : difference between left and right ankle inversion, azd : difference between left and right ankle abduction this causes them to experience pelvic and lower - extremity misalignment, which in turn influence posture, gait patterns, and balance. the pelvis supports the abdomen and connects the vertebrae and lower extremities. when an individual stands up from a seated position, the pelvis transfers the body weight from the vertebrae to the lower extremities and maintains correct posture, thereby enabling smooth movement of the upper extremities11. with the pelvis positioned in neutral, correct posture can be maintained as well as appropriate motions can be performed during daily living activities ; additionally, gait ability can be improved via proper control of the upper and lower extremities during dynamic postures12. in a similar study on the pelvis and walking, cho and jun selected 30 female university students and randomly divided them into a pelvic adjustment group (n=15) and a stretching group (n=15), which served as the control group, and analyzed their gait. they reported that differences in step length, stance phase, swing phase, single support, and step time were reduced with pelvic adjustment10. moreover, cho selected 30 female university students and randomly divided them into a pelvic adjustment group (n=15) and a stretching group (n=15). after analyzing their postures, the author reported that pelvic adjustment showed statistically significant effects on trunk imbalance, ppo, pto, and the position of the scapulae9. in addition, a study reported that trunk stabilization exercises enhanced normal gait patterns in stroke patients and their perception of the affected lower extremity, promoting normal exercise modes and reducing excessive muscle tension by increasing the symmetry of pelvic alignment13. several existing studies have conducted gait analyses after various interventions in patients with hemiplegia14 or cerebral palsy15 by employing three - dimensional gait analysis techniques. however, few studies have addressed correlations among pelvic positions and differences in lower - extremity joint angles during walking. the present study analyzed correlations among ppo, pto, pro, hxd, hyd, hzd, kxd, kyd, kzd, axd, ayd, and azd. ppo showed positive correlations with hxd, hyd, kxd, kyd, and ayd ; in other words, an increase in ppo resulted in corresponding increases in hxd, hyd, kxd, kyd, and ayd. in addition, hxd showed a positive correlation with axd, in which an increase in hxd led to a corresponding increase in axd. moreover, kxd exhibited positive correlations with kyd and ayd, as an increase in kxd resulted in corresponding increases in kyd and ayd. to summarize these findings, increases in left and right pelvic tilt resulted in corresponding increases in differences between left and right hip flexion, hip abduction, knee flexion, knee abduction, and ankle inversion. this may have resulted from changes in weight support and lower - extremity tilt according to changes in pelvic height on the left and right sides. therefore, both left and right pelvic tilt were important factors that led to differences between the left and right lower - extremity joint angles during walking. when these types of differences are present, pelvic adjustment is considered necessary for balanced walking. additional studies should be carried out to implement pelvic adjustment or lumbo - pelvic stabilization exercises as interventions to reduce differences between the left and right lower extremity joint angles during walking.	[purpose ] this study aimed to identify correlations among pelvic positions and differences in lower extremity joint angles during walking in female university students. [subjects ] thirty female university students were enrolled and their pelvic positions and differences in lower extremity joint angles were measured. [methods ] pelvic position, pelvic torsion, and pelvic rotation were assessed using the backmapper. in addition, motion analysis was performed to derive differences between left and right flexion, abduction, and external rotation ranges of hip joints ; flexion, abduction, and external rotation ranges of knee joints ; and dorsiflexion, inversion, and abduction ranges of ankle joints, according to x, y, and z - axes. [results ] pelvic position was found to be positively correlated with differences between left and right hip flexion (r=0.51), hip abduction (r=0.62), knee flexion (r=0.45), knee abduction (r=0.42), and ankle inversion (r=0.38). in addition, the difference between left and right hip abduction showed a positive correlation with difference between left and right ankle dorsiflexion (r=0.64). moreover, differences between left and right knee flexion exhibited positive correlations with differences between left and right knee abduction (r=0.41) and ankle inversion (r=0.45). [conclusion ] bilateral pelvic tilt angles are important as they lead to bilateral differences in lower extremity joint angles during walking.
schizophrenia is among the most economically costly and severe medical conditions. because onset is in young adulthood and disability can last a lifetime, this illness creates tremendous distress, pain and impoverished quality of life for the affected individual and their family. these additional costs are not measurable in monetary terms but provide compelling reasons for developing treatments that alleviate such suffering. even with best practices, there are limitations to the effectiveness of medications in the treatment of schizophrenia. relapse rates are high and some individuals remain symptomatic and, despite treatment, functional recovery (i.e. social relationships, making friends, finishing school, or attending work) remains a major challenge. all the evidence suggests that individuals with schizophrenia do best with a combination of pharmacological and psychosocial intervention. thus, it is critical that we have effective psychological treatment approaches to complement pharmacology. in the treatment of psychiatric problems, one of the most widely used and effective psychological treatments is cognitive behaviour therapy (cbt). traditionally, cbt has been used to treat depression and anxiety, but over the last twenty years it has been adapted to treat psychosis and is gaining recognition as a potentially effective treatment in schizophrenia at all stages of the illness, including the pre - psychotic phase. cbt aims at modifying the person s non - adaptive thoughts and beliefs in the context of a collaborative working alliance by teaching the link between perceptions, beliefs and emotional or behavioral reactions, questioning the apparent evidence supporting abnormal beliefs, encouraging self - monitoring of thoughts and, teaching effective coping strategies for dealing with distressing symptoms. in fact, most schizophrenia treatment guidelines have specific recommendations about including cbt as an intervention [2 - 4 ] the second is that, since it is unlikely that cbt could be implemented in regular mental health centers, how can it be implemented so that patients can benefit. most work has been carried out in academic research centers where highly trained therapists are available. later it was tested in those at their first episode, and current research has a focus on whether cbt may be the treatment to prevent transition from an at - risk state to full blown psychosis. to date, over 30 randomized controlled trials of cbt for schizophrenia have been published, demonstrating (on average) moderate benefits. some of these trials did have design problems ; however, the main finding by wykes and colleagues was that the more rigorous the study, the weaker the effect of cbt. though most have used a more general cbt for psychosis model, focusing on all positive symptoms (delusions, hallucinations, thought disorder, bizarre behaviour), some have specifically targeted a single symptom (e.g. voices, or paranoid delusions), whereas others have still focused on modifying other symptoms linked to schizophrenia, namely negative symptoms (i.e. apathy, avolition, poverty of content and thought, flat affect) and psychological distress such as depression or anxiety. several studies in the meta - analysis by wykes. have begun to address functional outcome (that is the ability to develop and maintain relationships and to work or attend school) but only as a secondary outcome to symptoms. some excellent work has demonstrated that cbt can be used successfully to prevent relapse and to reduce command hallucinations (i.e. hallucinations that command the patient to perform certain acts), which are among the most distressing and high - risk symptoms, and may be dangerous to the patient and others. despite advocating cbt as a valuable treatment for young people experiencing their first episode of psychosis, there are very few published random clinical trials of cbt with a first episode sample. one of the most methodologically sound and rigorous trials, the socrates trial in the uk [11 - 13 ], demonstrated that both cbt and supportive therapy produced improved symptoms compared with treatment as usual. unfortunately, this trial attempted to deliver the treatment over a 5-week period during the acute phase of the illness. in the acute phase it is difficult to determine the effectiveness of cbt relative to that of medications. similarly, the ace trial from australia demonstrated that although those receiving cbt showed more rapid improvement, this difference was not sustained. however, a group trial comparing cbt for psychosis with social skills training for symptom management and with a wait - list control for individuals with early psychosis discharged from the hospital did demonstrate superior improvements for cbt on overall symptoms, as well as on self - esteem, coping strategies and social support. this suggests that, possibly with these young patients, a group format may be more effective. a large body of recent research has identified individuals who may be at risk for psychosis based on the presence of attenuated psychotic symptoms that are often accompanied by a significant decline in social functioning. there are concerns with using medications in this young group, which make a case for cbt as a treatment for emergent psychotic symptoms as a more acceptable, and presumably safer, first step in preventive intervention. thus, it is possible that cbt might reduce or avoid the need for drug treatment. although cbt seems a logical treatment for those at clinical high risk of developing psychosis, there are actually very few studies examining its effectiveness. early studies had the goal of using cbt to prevent conversion to a full - blown psychotic illness, but later studies began to focus on improving the presenting symptoms and poor functional outcome. in all current studies to date, both the cbt and the control treatment groups appeared to demonstrate some improvement in symptoms. although the control group also improved, the edie trial in manchester did demonstrate that cbt was effective in reducing conversion. however, the german network study reported improvement in both groups in terms of social functioning. the adapt trial in canada was the first to compare two psychological treatments, but the number of conversions was very low and both groups improved. overall interpretation of effectiveness of cbt is difficult in these groups as there are some unique issues studying cbt with this population. firstly, the number of trials is limited and secondly, the numbers converting are low at times and thus many studies are underpowered. first, in examining current studies, cbt seems to offer better results when it is offered at specific stages of recovery and when the treatment delivery is adjusted to the stage. for instance, acute psychosis is not ideal for self - reflection, but following stabilization, individuals with early psychosis might benefit more initially from group cbt that could help normalize their experience by meeting peers with similar issues. multiple studies have shown that individuals with more experience with the illness, and who have some degree of realization of their goals and problems, will benefit from individual cbt for psychosis. older individuals with stabilized psychotic symptoms, who are more likely to be socially isolated with few goal - oriented activities, appear to benefit from a group approach integrating cbt principles with social skills training. the question is no longer whether cbt for psychosis is effective or not, but rather when should it be offered, to who, and which modality. in terms of who should receive cbt for psychosis, currently, it seems to be somewhat random for those who have the opportunity. yet to obtain results above and beyond what, for example, supportive therapy could offer, a certain level of cognitive functioning (memory, attention, problem solving) and of social cognitive functioning (insight, emotion recognition) might be necessary. in terms of modalities, even within the individual and group approaches, several variants exist, making study comparisons at times difficult. even though most cbt therapists agree on the essential elements that should be included in cbt for psychosis, there are a range of treatment manuals available with a variety of techniques and philosophies. for instance, some might be more goal - oriented, whereas others might be problem - focused. individual formulation (i.e. developing a shared comprehension of the person s cognitive biases and problems based on their past experiences) is the basis for some cbt therapists, but not for all. furthermore, there are no guidelines as to what may be an adequate dose of cbt. many studies offer over 9 months of weekly sessions, whereas others offer a few months. in some studies it has been suggested that those in the cbt groups did not receive an adequate dose of cbt. the majority of the trials have been conducted in controlled settings, using highly trained expert cbt therapists. there is emerging evidence suggesting the effectiveness of cbt using non - expert cbt therapists, for example, psychiatrists, community nurses, or other mental health professionals. given the paucity of expert cbt therapists in many mental health settings, training and supervising non - experts in delivering the intervention, albeit perhaps using a structured manual, could be a viable option. there appears to be a need for a typology of cbt approaches and modalities used in studies, in order to ensure replicability of the results by clinicians in real - world settings. cbt for psychosis is currently being adapted according to recovery stages and treatment needs. as such, studies underway are investigating combining cbt with other evidence - based approaches such as supported employment, family psychoeducation, motivational interviewing, social skills training, and third - wave cognitive behaviour therapies, such as acceptance and commitment therapy. more studies are warranted in order to better understand the best moment to offer cbt for psychosis, to whom, and using which modality. researchers are also investigating process issues in real - world settings, and will perhaps help us understand which aspects of the treatment are essential. overall, little is known about the moderators of cbt for psychosis, i.e. for whom or under which conditions cbt may work or about the mediators, that is, how cbt might achieve its effects. baseline patient characteristics, such as cognitive functioning, provide valuable information to help identify which patients might be responsive to a given treatment. understanding the mechanisms through which treatments work is likely to lead to more effective therapies. to date, there is no single treatment (including medication) for schizophrenia that helps 100% of those receiving it. in the meantime,	schizophrenia is one of the major and potentially severe mental illnesses. even with best practices, there are limitations to the effectiveness of treatments that include medications for this disorder. relapse rates are high and often those with the illness remain symptomatic and functionally impaired. all the evidence suggests that individuals with schizophrenia do best with a combination of pharmacological and psychosocial intervention. one psychosocial treatment that has received much attention is cognitive behaviour therapy (cbt). this brief review will address what we know about the use and effectiveness of cbt at all phases of schizophrenia and its strengths, weaknesses and its future.
early loss of primary teeth is most commonly caused by inappropriate oral hygiene, dental injuries, and tooth decay. tooth decay continues to be the main causative factor for the high rate of loss. as a result, those of us in the pedodontics and orthodontics practice frequently encounter malocclusion problems that occur from the premature loss of these primary teeth in children. the premature loss of primary teeth is a major factor that can cause malocclusion in the sagittal, transverse, and vertical planes. studies have shown that the premature loss of primary teeth is associated with the reduction of the dental arch length and migration of the marginal and antagonist teeth, leading to rotation, crowding, and impaction of the permanent teeth. in addition, the reduction of the dental arch length is greater in the mandible than in the maxilla if a primary second molar, rather than primary first molar, is lost. in addition, this effect is also apparent if tooth loss occurs at an earlier age, and if it occurs in crowded dentition as opposed to well - spaced dentition. for instance, early loss of primary second molar, especially in the maxillary arch, results in arch length reduction due to mesial migration of permanent first molars. premature loss in the maxillary arch may require extractions of the permanent teeth to align the dental arch, whereas premature loss in the mandible may require long - term orthodontic treatment in most cases. in yemeni children, early loss of primary teeth correlates to a marked increase in the prevalence of primary teeth extractions that are not further evaluated for spacing treatment needs. because of the severity of the consequences that arise with the premature loss of primary teeth, we decided to study the prevalence of early loss of primary teeth among children in thamar city. a cross - sectional study was conducted involving all the children aged between 5 and 10 years, attending the clinic of child dentistry of thamar university dental school for dental care, during the academic year 2014 - 2015. all the children (n = 185) present in the clinic of child dentistry were invited to participate in the study. all the children who fulfilled the study inclusion criteria, (a) 5 to 10 years of age and (b) a parent or guardian agreeing to participate in the study, were included in the study. the exclusion criteria included : (a) medically comprised children, (b) a parent or guardian not willing to participate, and (c) children with uncooperative behavior to receive a clinical examination. the study was approved by the research ethics committee of faculty of dentistry, university of thamar. all procedures were performed with adequate understanding and written consent of the parents / guardians. detailed information of all participants personal data and general health was recorded through individual interviews conducted by a researcher on the day of the dental check - up. the early loss was classified according to the chronological age table of eruption of the permanent teeth proposed by kronfeld, and decreasing 12 months as proposed by cardoso. we took into consideration age, sex, general health, and type of missing tooth. all statistical analyses were performed with the statistical package for social sciences, version 10 (spss inc., the data were analyzed using descriptive statistics techniques to obtain the absolute and percentage frequency. chi - square tests were applied to verify the existence of significant associations among the variables at a level of significance of 5% (p < 0.05). of the total number of children included in this study group, 75 (40.54%) had prematurely lost primary teeth (49.33% in boys and 50.67% in girls), and the prevalence peak was registered at approximately the age of 8 years [table 1 ]. distribution of children with premature loss of primary teeth according to age and gender we found a total of 170 primary teeth that were prematurely lost, with fdi tooth number 75 (13.5%) [table 2 ] being lost at the highest rate. distribution of primary teeth that were affected the most by premature loss according to gender according to the number of prematurely lost teeth per person, 30 children (40%) had 1 missing tooth, 22 children (29.3%) had 2 missing teeth, 11 children (14.7%) had 3 missing teeth, 4 children (5.3%) had 4 missing teeth, 3 children (4%) had 5 missing teeth, 4 children (5.3%) had 6 missing teeth, and 1 child (1.3%) had 8 prematurely lost primary teeth. according to the distribution of prematurely lost teeth relative to tooth type, the molars (60.6%) were the most commonly prematurely lost teeth followed by the cuspids (27.6%) and the incisors (11.8%) [tables 3 and 4 ]. distribution of primary teeth that were affected by premature loss according to tooth types (central incisors, lateral incisors, cuspids, first molars, second molars) relative to gender distribution of primary teeth that were affected by premature loss according to tooth types (incisors, cuspids, molars) relative to gender according to the distribution of prematurely lost teeth relative to the dental arch, the mandibular arch (53.5%) had more cases than the maxillary arch (46.5%). the mandibular left quadrant had the highest loss of primary teeth (28.8%) [tables 5 and 6 ]. distribution of primary teeth that were affected by premature loss according to dental quadrants relative to gender distribution of primary teeth that were affected by premature loss according to dental arches relative to gender there was a noticeable increase in the prevalence of extractions of primary teeth which were not followed by space maintenance, particularly for cases with the early loss of primary teeth among yemeni children. therefore, the major aim of this cross sectional study to give a comprehensive overview of the premature loss of primary teeth condition in the target sample in order to predict the future health care needs in preventing the disturbances in the development of normal occlusion in children, with no interest in generalizing our findings to the total yemeni population. in addition, this study is, to the best of our knowledge, the first study to explore this issue among the children in yemen. in our study, the status of premature loss of primary teeth in the study group was high with a prevalence rate of 40.54%, which is in accordance to a recent study conducted in saudi arabia. the early loss of primary teeth status in the present study was represented by a sample of children who sought dental care at the clinic of child dentistry of thamar university dental school. therefore, we can expect that these children present with more dental treatment needs than the general population. this supposition may also be made because parents, and occasionally even dentists, do not emphasize the importance of the primary dentition of the child. these parents and dentists may believe that the prevention and treatment of the primary teeth is unimportant because these teeth will eventually be replaced anyhow, and these might explain this high rate of premature loss of these teeth in the present study. the present study also showed that there was not a statistically significant difference in the premature loss of primary teeth between boys and girls. this finding implies that premature loss of primary teeth in the study group is due to poor oral health care, rather than gender. in this study, the majority of children had one or two teeth prematurely lost, similar to that reported in a previous study. the present study revealed that the highest percentage of premature tooth loss was at the age of 8 years, and the primary molars were more prematurely lost than other primary teeth. the most frequently missing teeth were the lower left primary second molars, which is similar to the results reported by previous studies. this finding could be observed because the likelihood of streptococci mutans acquisition in infants increases with age or as the number of erupted teeth increases. the primary molars may be particularly susceptible to initial s. mutans colonization because they emerge into the oral cavity between 16 and 29 months of age and impact both fissured occlusal surfaces and concave approximal surfaces. this can result in caries of the primary molars, that if left untreated, might result in premature extraction and thus contributing to early loss. in addition, by analyzing the distribution of premature loss on the arches, we observed a higher prevalence in the mandibular arch. the greater loss of primary teeth in the mandibular arch may be due to food packing potential and greater plaque accumulation in the mandibular posterior region. in addition, saliva has anticarious properties and is relatively abundant in the maxillary molar teeth, thus reducing the rate of premature loss of these teeth. the premature loss of the primary teeth can cause problems due to loss of function and the increased possibility of migration of other teeth. the incidence of space closure increases with the time that elapses from the moment of extraction. previous studies have demonstrated that the closing rate of a space is higher for the maxillary than for the mandible arch but decreases after the first 6 months. a greater amount of time elapsed because extraction positively correlates with greater space loss, especially in extractions performed for primary second molars. therefore, it is necessary to increase awareness of the importance of oral health in our children and to inform parents of the potential for malocclusion problems caused by the early loss of primary teeth. parents of children with early loss of primary teeth should be advised to bring their children to the dental clinic to have space maintainers if necessary. the main limitation in the present study is the fact that sample size was small. in addition, this study was conducted on children who sought dental care at the clinic of child dentistry of thamar university dental school, hence, we can expect that these children present with more dental treatment needs than does the general population. therefore, generalization must be made carefully as this study population may not reflect the prevelance of premature loss of primary teeth in the children in yemen. further studies are needed to address this issue by observation of large groups of children from different regions in order to form reliable conclusion. the following conclusions can be drawn from the findings of this study : prevalence of early loss of primary teeth was high (40.54%), and was higher at 8 years of age.majority of the children had one missing tooth (40%).more number of teeth was lost in the mandibular arch (53.5%).the lower left primary second molar was the most commonly missing teeth (13.5%). prevalence of early loss of primary teeth was high (40.54%), and was higher at 8 years of age. the lower left primary second molar was the most commonly missing teeth (13.5%). this conclusion is important in light of existing studies that revealed the malocclusion problems associated with early loss of primary teeth. therefore, it is imperative to increase oral health awareness for our children and their parents to make them realize the significance of the primary teeth and how to care for them as well as the deleterious effect of the early loss of primary teeth. parents of children with early loss of primary teeth should be advised to bring their children to the dental clinic to have space maintainers if necessary.	objectives : the premature loss of primary teeth is a potential risk factor for poor arch length development. adequate arch length is important to the progression of the permanent teeth. poor arch length can lead to crowding, ectopic eruption, or impaction of these teeth. this study is designed to assess the prevalence of premature loss of primary teeth in the 5 - 10-year - old age group.materials and methods : the study group included 185 children, that is, 91 boys and 94 girls. the dental examination was conducted by an experienced examiner under sufficient artificial light. data including patient age and missing teeth were collected. descriptive statistics were applied for data analysis, and from the results, chi - square tests were used at a level of significance of 5% (p < 0.05).results : we observed a 40.54% prevalence of premature loss of primary teeth with no statistically significant difference between genders. the lower left primary second molar was the most commonly absent tooth in the dental arch (13.5%).conclusion : the status of premature loss of primary teeth was high in the study group. implementation of efficient educational and preventive programs to promote oral health would help children maintain a healthy primary dentition and eventually prevent the disturbances in the future development of normal occlusion. early detection and management of the space problems associated with the early loss of primary teeth would help in reducing malocclusion problems.
	the influence of electronic symmetry on triplet state delocalization in linear zinc porphyrin oligomers is explored by electron paramagnetic resonance techniques. using a combination of transient continuous wave and pulse electron nuclear double resonance spectroscopies, it is demonstrated experimentally that complete triplet state delocalization requires the chemical equivalence of all porphyrin units. these results are supported by density functional theory calculations, showing uneven delocalization in a porphyrin dimer in which a terminal ethynyl group renders the two porphyrin units inequivalent. when the conjugation length of the molecule is further increased upon addition of a second terminal ethynyl group that restores the symmetry of the system, the triplet state is again found to be completely delocalized. the observations suggest that electronic symmetry is of greater importance for triplet state delocalization than other frequently invoked factors such as conformational rigidity or fundamental length - scale limitations.

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