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acute myocardial infarction (mi) is a major cause of mortality and morbidity in western hemisphere. annually, around 15 million patients in the united states and europe are presented to the emergency department with chest pain or other symptoms indicative of mi [1, 2 ]. early diagnosis and treatment of patients with acute mi could prevent or reduce ischemic damage to the myocardium and, therefore, could prevent subsequent cardiac remodeling and failure. mi reflects cardiomyocyte death (necrosis) as a consequence of prolonged ischemia, which results from acute thrombotic occlusion of a coronary artery. myocardial cell death can be detected by release of multiple proteins from the damaged cells into the circulation. cardiac troponin (tn) i and t are structural proteins predominantly expressed in the heart, and are currently considered as the gold standard for acute mi (ami). their detection in peripheral blood indicates cardiomyocyte necrosis, and in combination with 12-lead electrocardiogram (ecg) and creatine kinase isoenzyme mb (ck - mb) they form the diagnostic cornerstones for mi [2, 5, 6 ]. however, although elevated biomarkers in the blood indicate myocardial damage, they do not diagnose the underlying mechanisms. for example, elevated values of the biomarkers in the absence of clinical evidence of ischemia could also occur as a consequence of other causes of cardiac injury, such as myocarditis, sepsis, cardioversion, or ablation [710 ]. cardiac troponins are superior to all other biomarkers that have been clinically available in the diagnosis of mi [1114 ] and they directly correlate with the size of mi [15, 16 ]. a major drawback of the contemporary tn assays is their inadequate sensitivity during the first few hours after the onset of mi, as they are released slowly from damaged cardiomyocytes and do not peak until 6 to 12 h after the onset of symptoms. therefore, sampling is needed every 6 h. however, high - sensitive tn assays are already on the market, and recent multicenter studies [18, 19 ] have shown that these assays improved the early diagnosis of mi, even if patients are presented within 3 h after the onset of chest pain. micrornas (mirs) are evolutionary conserved, short, non - coding (~ 22 nucleotides) rna molecules involved in post - transcriptional gene regulation [2022 ]. their binding potency to the 3 untranslated region (utr) of messenger rnas (mrnas) determines their mode of action : translational inhibition and/or augmented mrna degradation, both pathways resulting in endogenous gene silencing. around 1500 human mirs (http://www.mirbase.org/ v 18.0) have been cloned and sequenced and it is estimated that they regulate up to 50% of the protein - coding genes [2325 ]. mirs are generally considered to act as intracellular mediators essential for normal cardiac function and their deregulated expression profiles have been associated with cardiovascular diseases [2629 ]. recent studies have revealed existence of freely circulating mirs in human peripheral blood that are stably expressed [3032 ]. this has raised the possibility that micrornas may be probed in the circulation and can serve as novel diagnostic markers for cardiovascular diseases. an important characteristic of mirs is their tissue and cell specificity providing unique signature with diagnostic opportunities for diverse diseases. in line with this, several studies have demonstrated circulating mirs reflecting pathologic condition, such as cancer and liver injury indicating that mirs could be used as sensitive and specific biomarkers of various pathologies and tissue injuries [35, 36 ]. in addition, another advantage is the relative ease with which mirs can be measured : polymerase chain reaction (pcr) allows being more sensitive and specific than classic antibody - based assays. this could become feasible against reasonable costs, comparable to the costs of measurement of only two or three protein markers. this induces a completely novel way of designing and using biomarkers in heart disease : instead of seeking one or two gold standard diagnostics, a more complete profile can be routinely measured to allow sensitive characterization of subtypes of disease. it is already known for classical biomarkers that adding a novel marker to the existing ones adds information and increases diagnostic power but also sharply increases the associated costs. designing oligonucleotides for mirs and performing subsequent quantitative reverse transcriptase (qrt)-pcrs are much less time- and cost - consuming processes when compared to the development and production of new and specific antibodies and enzyme - linked immunosorbent assays (elisas). therefore, circulating mirs as biomarkers for mi could increase the number of markers measured and continue to increase diagnostic power without increasing costs to a larger extent. ideal blood biomarkers of mi should comprehend the following characteristics : they should be 1) abundant and preferably exclusively expressed in the tissue of interest, 2) expressed at low levels in the blood under normal / healthy circumstances, 3) released into the circulation after tissue injury, 4) stable in the circulation, and 5) easily detected with high sensitivity and specificity. here we will review the recent findings of circulating mirs that fulfill the mentioned criteria and discuss their potential to be used as biomarkers for mi (table 1).table 1circulating micrornas in acute myocardial infarction studiesmicrornafold change, increase or decreaseroc (auc)correlationtime of blood samplingno. samplesspeciesreferencemir-13000.98tnt 12 h, day 2, day 3, > 1 month25 stemi, human[39]mir-133a700.8611 healthypigmir-208b30001.006 closedmir-4992500.99chest mimir-208andmir-1600, 30, 60, 90, 120 and 150 minmir-133a60mir-208b80mir-49960mir-1all four significantly 0.854.8 3.8233 ami, human[40]mir-133a0.8717 non - chd, 16 chdmir-208a0.976 non - op, mir-4990.826 sham - op, mir-15001, 3, 6, 12, and 24 h after the ligation6 coronary artery ligationratmir-133a750mir-208a850mir-49940mir-1ns < 12 h32 ami, human[44]mir-133a436 controlsmir-208b16000.94tntmir-4991000.92tntmir-116517 309 min33 stemi, 17 healthy donorshuman[38]mir-133a140mir-133b60mir-499 - 5p100mir-208andmir-1615 min to 5 d45 coronary artery ligation and sham operatedmousemir-133a13mir-133b5mir-499 - 5p60mir-208amir-49962ck - mb48 h9 ami, 5 uap, 9 chf_iii, chf_ii, normal controlshumanmir-161%0.77qrs widening93 ami, 66 controlshuman[41]mir-133nsmir-120100ck - mb8.5 3.8231 ami, 20 controlshuman[42]200infarct size in vivo0, 1 h, 3 h, 6 h, 12 h, 24 h, 3 d, 7 d, 14 d, 21 d, and 28 d812 lad ligation, 812, sham operatedratcell damage in vitromir-1334.40.895.24 1.38 h, 20 h, 7 d51 ami, humanmir-32810.916.10.8128 controlsmir-1452hs - tnt3.0 2.3 h, 3 d, 4 d20 stemi, human[52]mir-30c1.3 hs - tnt20 controls, mir-12914.5 0.9120 internal controlmir-663b2 0.94mir-1egfr, hs - tnt117 uap, human[43]mir-133aegfr, age, hs - tnt131 nstemi, mir-133bnshs - tnt196 stemimir-208ans - mir-208bage, smoking, hs - tntmir-499nsmale genderami acute myocardial infarction ; auc area under the curve ; chd coronary heart disease ; chf_ii congestive heart failure, nyha class ii ; chf_iii congestive heart failure, nyha class iii ; ck - mb creatine kinase isoenzyme mb ; egfr estimated glomerular filtration rate ; hs - tnt high - sensitive tnt ; lad left anterior descending artery ; mi myocardial infarction ; mir microrna ; nd not detected ; non - op non - operated ; ns not significant ; nstemi non - st elevation mi ; roc receiver operator characteristics ; sham - op sham operated ; stemi st elevation myocardial infarction ; tni troponin i ; tnt troponin t ; uap unstable angina pectoris. circulating micrornas in acute myocardial infarction studies ami acute myocardial infarction ; auc area under the curve ; chd coronary heart disease ; chf_ii congestive heart failure, nyha class ii ; chf_iii congestive heart failure, nyha class iii ; ck - mb creatine kinase isoenzyme mb ; egfr estimated glomerular filtration rate ; hs - tnt high - sensitive tnt ; lad left anterior descending artery ; mi myocardial infarction ; mir microrna ; nd not detected ; non - op non - operated ; ns not significant ; nstemi non - st elevation mi ; roc receiver operator characteristics ; sham - op sham operated ; stemi st elevation myocardial infarction ; tni troponin i ; tnt troponin t ; uap unstable angina pectoris. mir-1 - 1/mir-133a-2 and mir-1 - 2/mir-133a-1 are two bi - cistronic mir clusters expressed in both skeletal and cardiac muscle. the sequence of mature mir-1 - 1 and mir-1 - 2 is identical and the same holds true for mir-133a-1 and mir-133 - 2. mir-133b differs from mir-133a in the 2 nucleotides at the 3 terminus and is specifically expressed in skeletal muscle. both mir-1 and mir-133 are anti - hypertrophic mirs, involved in regulation of signaling cascades and sarcomeric organization. six hours after coronary artery occlusion in mice, mir-1 and mir-133 levels were decreased in infarcted and border zone area of the heart whereas their levels were increased in the peripheral blood, indicating release of these mirs into the circulation after tissue injury. also, in ami patients, significantly higher circulating levels of mir-1 and mir-133 levels could be detected in the circulation [38, 39, 40, 41, 42, 43, 44 ] as soon as 5 h after the onset of symptoms [40 ]. in addition, when plasma samples were obtained 156 min after the onset of symptoms, mir-1 and mir-133 levels had a significantly higher peak than tni, indicating their early release after mi. however, one study showed no significant differences in mir-1 expression between ami and controls [44 ], which could be explained by possible renal elimination of mir-1 [39 ] or by conceivable presence of higher skeletal muscle turnover in the control patients [40 ]. in addition, enhanced mir-1 and mir-133 levels in the circulation were detected after sham operated hearts [40 ], demonstrating that damaged muscle could also contribute to induced levels of mr-1 and mir-133 in the circulation. both mir-1 and mir-133 levels augment in the circulation of the st - elevated mi (stemi) patients compared to the healthy controls within the first 12 h after the onset of the symptoms, followed by a return to adjacent baseline levels after 12 to 24 h [39 ]. in contrast, one study demonstrated significantly high levels of mir-133 in the plasma after 20 h of the onset of the symptoms, although not precisely describing which mir-133 isoform was studied. in 8 of 25 stemi patients, mir-1 and mir-133a could be detected in the urine within 24 h after the onset of symptoms indicating renal elimination. this is in line with another study showing a correlation between mir-1 and mir-133a and estimated glomerular filtration rate (egfr) in stemi patients but not between mir-133b and egfr [43 ]. furthermore, both mir-1 and mir-133 correlated with tnt [43 ] implying myocardial damage. moreover, mir-1 also positively correlates with creatine kinase - mb [42 ] and qrs widening in ami patients, with infarct size in an animal model of mi and with cell damage in vitro [42 ]. receiver operator characteristics (roc) curves for mir-1 demonstrate area under the curve (auc) varying from 0.77 [41 ] to 0.98 [39, 40 ] indicating fair to excellent accuracy of this mir for the use of a biomarker in mi. for mir-133, the roc analysis reveal auc values between 0.86 and 0.89 indicating good sensitivity and specificity distinguishing between stemi patients and healthy controls [39, 40 ]. together with their high expression in the heart, and increased levels in the blood after the mi, mir-1 and mir-133 could be suitable candidates as biomarkers for the mi. however, other injuries to the skeletal muscle should be preferably excluded to rule out the non - specific effects. in addition, the oscillations of these mirs after mi should be studied accurately to reveal their exact times of increase and decrease in the circulation and to study to which extent they could contribute to the already existing biomarkers. finally, larger study populations are essential to make correct conclusions if mir-1 and mir-133 could be used as biomarkers in mi. mir-499 is an evolutionary conserved muscle - specific mir that is located in an intronic region of the myh7b gene and plays a role in myosin gene regulation [45, 46 ]. although mir-499 is highly expressed under normal conditions in the heart [45, 47 ], its expression decreases in the ischemic heart, suggesting its release from the damaged tissue. indeed, mir-499 levels could be detected in plasma of ami patients [38, 39, 40, 44, 49 ], whereas one study did not detect differences in mir-499 expression between stemi, non st - elevated mi (nstemi), and patients with unstable angina [43 ]. although mir-1 and mir-133a peaked around 156 min after the onset of mi symptoms, mir-499 is at its highest point around 12 h [38, 39 ], suggesting possible slower kinetics compared to mir-1 and mir-133. however, similar to mir-1 and mir-133, mir-499 levels increase in the circulation in the sham operated hearts, indicating that damaged muscle contributes to enhanced circulating mir-499 levels. besides parallel expression of mir-499 and tni in an animal model of mi [38 ], mir-499 correlates with tnt [44 ] and ck - mb in ami patients. roc analysis demonstrates auc values varying between 0.82 and 0.99, indicating good to excellent sensitivity and specificity [39, 40, 44 ]. other mirs encoded by the myosin genes are mir-208a and mir-208b, which are located in myh6 and myh7 genes, respectively. mir-208a and mir-208b are abundantly and exclusively expressed in the heart, making them most suitable candidates to be used as biomarkers for the mi. indeed, mir-208a [40 ] and mir-208b [39, 43, 44 ] levels are increased in the circulation after the mi. two studies showed that mir-208b was the most abundantly elevated mir in the plasma, with 1600 [44 ] and 3000 [39 ] times more expression in the ami patients than in the controls. furthermore, mir-208b levels correlated with tnt levels reflecting myocardial damage [39, 43, 44 ]. moreover, the amount of mir-208b in blood inversely correlates with left ventricular ejection fraction, raising the possibility in using this mir not only for diagnostic purposes but also for potential prognostic use in long - term cardiac function and probability of developing heart failure. nevertheless, one larger population study of 444 subjects revealed that although circulating levels of mir-208b were associated with all - cause mortality at 6 months, after adjustment for tnt levels, its association was lost with the outcome. this implies that mir-208b does not add prognostic information to an already sensitive necrosis marker. however, it should be taken into account that this does not exclude the potential of mir-208b to augment its prognostic value when measuring at an earlier or later time point. only one study detected significantly higher circulating mir-208a levels in the ami patients [40 ] in contrast to the others [38, 39, 49 ]. the first is related to the time point of sampling and could explain why mir-208a was not detected in the circulation. in this study, blood sampling occurred within 48 h after the onset of symptoms, although mir-208a peaks around 3 h after the ami [38, 40 ] and is restored to the baseline after 24 h [40 ]. the second explanation is related to the myosin isoform expression between a mouse and a human heart. mir-208a is abundantly and exclusively expressed in the adult mouse heart, whereas mir-208b is more expressed during development and in response to stress. however, myh7, harboring mir-208b, is the primary isoform in the human cardiac muscle [50, 51 ], providing an explanation why mir-208b is abundantly present in the human heart. this could explain why two studies could not detect mir-208a in the circulation, although sampling the blood within the appropriate time line [38, 39 ]. specificity and sensitivity analysis for mir-208b reveal aucs between 0.94 and 1.00, indicating excellent accuracy for discrimination between ami patients and the controls [39, 44 ]. in combination with its exclusive expression in the heart, and high increase in blood after the mi nevertheless, the kinetics of mir-208a should be studied extensively and in bigger populations to reveal its exact incline and decline after the mi and to decide what its contribution could be to the already existing biomarkers such as hs - tnt and tni. in theory, candidate biomarkers for mi should display cardiac specific expression patterns. recently, one study revealed mir-663b and mir-1291 as the most predictive mirs for the mi with 92.5% and 85% accuracy, respectively [52 ]. in addition, mir-30c and smooth muscle cell - enriched mir-145 were significantly increased in these stemi patients and correlated with hs - tnt, indicting their potency to reflect cell death. moreover, the same study reported an mirs signature, which could serve as a new class of biomarkers [52 ]. this signature represents a combination of 20 most up- and down - regulated mirs in 20 stemi patients in contrast to the controls and could enhance diagnostic discrimination of stemi patients from the control with 96% specificity, 90% sensitivity, and the auc of 0.99, indicating excellent accuracy. these results are promising, but because these mirs are not cardiac specific, other pathologic processes could underlie the effects seen in this study. furthermore, the patient population is too small to drawn correct conclusions about this particular mirs signature and because these mirs are isolated from the whole peripheral blood, circulating cells could also contribute to the mirs expression levels. nevertheless, such a multi - marker approach could be of interest to increase diagnostic power and to gain more information about the time line of mi, which in turn could lead to faster and accurate treatments and prevent subsequent cardiac remodeling and failure. extracellular mirs have been found recently in multiple human body fluids, such as blood plasma, urine, and saliva [30, 53, 54 ]. mirs are surprisingly stable in the plasma regardless of high rnaase activity, contributing to the possibility that that mirs could be used as new class of blood - based biomarkers. circulating mirs can be carried in different types of vesicles, such as exosomes [55 ] and microvesicles [56 ]. exosomes are small (5090 nm) vesicles that are released into the extracellular environment by fusion of the multivesicular bodies and the plasma membrane. one study showed that 121 mirs are associated with exosomes and that several mirs were abundantly expressed in exosomes compared to the cells, suggesting packing regulation [55 ]. these mirs are not localized on the external structures or macromolecules, but instead are restricted to the inner part [55 ]. besides exosomes these particles are larger (up to 1um) and shed from the cell membrane. in addition, microvesicles are enriched in bio - active molecules and contain nucleic acids and/or proteins. mirs located in exosomes or microvesicles are extremely resistant to rnaase - dependent degradation with necessity to first destroy the lipid bilayer of the vesicles before mirs can become accessible for rnaase degradation [55 ]. previous studies have shown that nucleic acids can bind and form stable complexes with specific lipids found on lipoproteins [5860 ] by divalent cation bridging. high - density lipoproteins (hdl) are used as carrier to deliver lipophilic anti - tumor drug to human hepatocellular carcinoma cells in vitro. in animal models, liposomes containing apolipoprotein a - i, which is the main protein component of hdl, have been used to the delivery of several sirnas to the liver. recently, one study revealed that hdl (812 nm) isolated from patients with hypercholesterolemia contained small rnas including mirs [64 ]. the exact mechanism of how mirs are loaded into the hdl particles and how they are protected from the external rnaases has to be elucidated. one study demonstrated that endothelial cell - derived apoptotic bodies, which are produced during atherosclerosis, carry mir-126 as well as other mirs. the majority of extracellular mirs in blood plasma are bound to protein complexes protecting these mirs from degradation. in line with this, one study has shown that a rna binding protein, nucleophosmin 1 (npm1), and nucleolin were highly abundant in the fibroblast medium after serum deprivation and that especially npm1 could bind and protect mir-122 against degradation. however, it needs to be investigated if npm1 is present and operative in binding and protecting mirs in the human circulation. very recently, two studies have demonstrated that vesicle - encapsuled mirs represent only a minor fragment of circulating mirs and that over 90% of circulating mirs are exosome free and bound to argonaute proteins [67, 68 ]. these proteins are naturally occurring within the cell and are a part of rna - induced silencing complex. argonaute 2 (ago2) plays an especially important role in stabilizing mirs in the plasma as ago2/mirs complexes are extremely nuclease and protease resistant. in conclusion, several mechanisms are described for mirs export and their subsequent stabilization in the circulation. export of most mirs is energy dependent and active transport, suggesting complex regulation of this process. however, mir release after an ami would reflect a rather passive form due to necrosis. mature mirs in the cells that are ago2 bound and necessary for the stabilization are localized in the cytoplasm or in the p - bodies, suggesting that extracellular mirs detected in the plasma after an ami would represent ago2-bound mirs. myocardial - derived mirs, such as mir-1, mir-133, mir-499, and mir-208, might be useful as potential biomarkers for mi. these mirs are abundantly expressed in the heart but less so in the circulation under normal / healthy circumstances. in addition, they are released into the circulation after the mi, where they are in a stable conformation and can be detected with high accuracy. larger study populations and exact time points of blood sampling are required to study the kinetics of these myocardial - derived mirs in ami patients and to investigate their potential to be used as new biomarkers in mi. it is already known for classical biomarkers that adding a novel marker to the existing ones adds information and increases diagnostic power but also sharply increases the associated costs. designing primers for mirs and performing subsequent qrt - pcrs are much less time- and cost - consuming processes when compared to the development and production of new and specific antibodies and elisas. in this way, a multi - mirs marker approach also becomes attractive, which could lead to increased diagnostic power and provide more information about the time line of mi, with the faster and accurate treatments with the subsequent improvement of the clinical outcome. to promote the use of mirs as biomarkers, it is essential to develop new techniques that can provide quick detection of mirs in the circulation.	micrornas (mirs) are short non - coding rna molecules involved in post - transcriptional gene regulation by binding to the 3 untranslated region of a messenger rna (mrna), thereby inhibiting the translation or inducing mrna destabilization. mirs are generally considered to act as intracellular mediators essential for normal cardiac function, and their deregulated expression profiles have been associated with cardiovascular diseases. recent studies have revealed the existence of freely circulating mirs in human peripheral blood, which are present in a stable nature. this has raised the possibility that mirs may be released in the circulation and can serve as novel diagnostic markers for acute or chronic human disorders, including myocardial infarction (mi). this review summarizes the recent findings of mirs that fulfill the criteria of candidate biomarkers for mi.
it commonly affects young and middle - aged patients and shows a predilection for adults under age 40 years. the incidence of radiographically evident osseous involvement is between 1% and 13%, with an average of 5% on conventional imaging. the majority of osseous lesions occur in the short tubular bones of the hands and feet and the abnormalities can be either unilateral or bilateral. technetium-99 m labeled pyrophosphate or diphosphonate compounds, gallium-67 citrate and f-18 fdg are the agents which are used for imaging of osseous sarcoidosis.[46 ] we present a case of sarcoidosis with unusual skeletal bone involvement and review the literature. a 52-year - old man with back pain and right hip pain was referred to our hospital. he had a history of lung sarcoidosis for 21 years. as his disease was considered inactive, corticosteroids were discontinued approximately 2 years after diagnosis. laboratory tests were normal except alkaline phosphatase which was elevated (448 u / l, normal 40 - 150 pelvic radiographs showed sclerosis and radiolucency on multiple areas mimicking bone metastases [figure 1 ]. anterior view of the pelvis and proximal femurs showing widespread lesions mimicking sclerotic bone metastasis a computed tomography (ct) of thorax showed sclerotic and lytic lesions on 1 and 10 thoracic vertebras in addition to the findings of stage 4 sarcoidosis in both lungs [figure 2 ]. (a) sagittal section of computed tomography images show osteolytic lesions and sclerosis on 1 and 10 thoracic vertebras and osteolytic lesion on 7 cervical vertebra with corresponding increased tracer uptake in bone scan ; (b) transaxial images of 10 thoracic vertebra show multipl sclerotic and lytic lesions ; (c) lung parenchyma image shows diffuse fibrosis consistent with stage 4 sarcoidosis furthermore, cranial ct and radiographs were obtained to investigate the involvement of cranium. although radiographs were normal, ct of the cranium revealed lytic lesions in addition to extensive sclerosis [figure 3 ]. (a) no lesion on the lateral radiograph of the skull ; (b) although cranial computed tomography showed sclerotic and lytic lesions on bones, brain parenchyma was normal the patient was referred for bone scintigraphy to scan the whole skeleton. bone scintigraphy was performed 3 h after intravenous injection of 20 mci (740 mbq) tc99 m methylene diphosphonate. bone scan demonstrated multiple areas of increased tracer accumulation at the calvarium, upper - to - lower thoracic spine, pelvic bones, sacrum, both sacroiliac joints, and both proximal femurs [figure 4 ]. after scintigraphy, bone marrow biopsy was done at 10 thoracic vertebra and demonstrated non - caseating granulomata consistent with skeletal sarcoidosis. bone scintigraphy showed diffusely increased tracer accumulation at the calvarium, pelvic bones, both sacroiliac joints and proximal femurs and posterior left 7. additional focal uptakes were observed upper and lower thoracic spine, 7 cervical vertebra and sacrum osseous sarcoidosis is most often seen in the short tubular bones of hands and feet but has been reported in the spine, sternum, calvarium, pelvis, ribs, nasal bones, tibia, and femur.[2712 ] our patient had no involvement of his hands or feet bones. however, he had involvement of his calvarium, thoracic spine, pelvis, and femurs which are rare sites. spinal lesions may be seen at any level, but the thoracic spine usually affected. bone involvement is reported to indicate a worse prognosis with a mortality rate 4 times higher in patients who are detected to have abnormalities on bone radiography than in those with normal findings. the radiographic, ct, and magnetic resonance (mr) findings of the bone lesions are non - specific. our patient 's lesions were sclerotic on pelvis radiography and were lytic - sclerotic on thoracic and cranial ct. the mr usually demonstrates multifocal lesions within the vertebrae that are hypointense on t1-weighted images, hyperintense on t2-weighted images, and enhance following contrast medium administration. the differential diagnosis includes metastatic lesions, lymphoma, myeloma, infectious processes including tuberculosis. nuclear medicine studies including technetium-99 m labeled pyrophosphate or diphosphonate compounds, ga-67, and f-18 fdg have been used for imaging of osseous sarcoidosis.[46 ] bone involvement is much more commonly detected with the use of bone scan than with routine radiography. bone scan findings may not be distinguished from other pathologies such as bone metastasis and osteomyelitis. the sensitivity of ga-67 for detecting osseous sarcoidosis is less than that of bone scintigraphy. however, the advantage of gallium scan over diphosphonate bone scan is that besides demonstrating bone involvement, it also detects pulmonary and extrapulmonary foci of sarcoidosis., suggested that fdg pet is more accurate and contributes to a better evaluation of extrapulmonary involvement in the patients with sarcoidosis. although fdg pet imaging is highly sensitive for skeletal involvement of sarcoidosis, it can mimic widespread skeletal metastases. in conclusion, bone scintigraphy is a sensitive imaging modality in order to demonstrate the sarcoid lesions of bones including the uncommon axial skeletal involvement. but, because of the low specificity of the imaging techniques, radiological correlation and histopathological confirmation should be done.	sarcoidosis is a systemic, granulomatous disorder that affects multiple organ systems, but most often the lungs and the skin. the incidence of radiographically evident osseous involvement is between 1% and 13%, with an average of 5% on conventional imaging. sarcoidosis generally involves the peripheral skeleton with the phalanges, metacarpals, and metatarsals being most frequently affected. the majority of osseous lesions occur in the phalanges of the hands. involvement of the axial skeleton is rather uncommon. sarcoid bone lesions are usually asymptomatic. nuclear medicine studies, in particular bone scintigraphy, gallium-67 (ga-67) and f-18 fluoro-2-deoxyglucose positron emission tomography (f-18 fdg pet) have been used in staging of sarcoidosis, including assessment of extrapulmonary involvement. here, we present a case of osseous sarcoidosis in a man whom the disease presented with multiple lesions in the axial skeleton and the long bones.
periodontal diseases in general and gingival recession, in particular, have been subject of intense analysis in terms of classification. various classifications of periodontal diseases as well as gingival recession have been proposed over a period of time. gingival recession is defined as the displacement of marginal tissue apical to the cemento - enamel junction (cej). it is one of the signs of the periodontal disease and has been found to be existing at most of the ages, beginning early in certain populations. gingival recession is important not only from an esthetic point of view, but loss of gingival tissue may result in root sensitivity, cemental erosion, and root caries. sullivan and atkins classified soft tissue defects in mandibular incisors into four classes : narrow, miller in 1985 proposed a classification system which is probably the most commonly employed system to classify gingival recession and divided it into four categories. class i marginal tissue recession does not extend to the mucogingival junction (mgj) with no periodontal loss (bone or soft tissue) in the interdental area, and 100% root coverage can be anticipated. class ii recession extends to or beyond the mgj with no periodontal loss (bone or soft tissue) in the interdental area, and 100% root coverage can be anticipated. class iii recession extends to or beyond the mgj with bone or soft tissue loss in the interdental area, or there is malpositioning of the teeth which prevents the attempting of 100% root coverage. class iv recession extends to or beyond the mgj with the bone or soft tissue loss in the interdental area and/or malpositioning of teeth is so severe that root coverage can not be anticipated. other classifications, which have been proposed to classify recession defects, include smith 's index for gingival recession, mahajan 's classification, cairo. 's classification, and kumar and masamatti 's classification. nordland and tarnow proposed a classification system for the papillary height. despite being extensively used, pini - prato stated the noncritical and widespread use of the miller classification should be evaluated carefully with sound clinical trials on gingival recessions and root coverage. new classification systems of gingival recessions should be provided on the basis of the characteristics of suitable taxonomy, on the basis of information from more recent scientific evidence and then validated by reliability studies for appropriate application in clinical practice. considering the limitations of miller 's classification, kumar and masamatti proposed a new classification system in 2013 for gingival and palatal recession [table 1 ]. the level of the interdental papilla (idp) in relation to the cej and the level of the marginal recession were used as the basis for this new classification. the new classification system is more comprehensive, informative, and tries to overcome the limitations of miller 's classification. kumar and masamatti 's classification the aims of the present study were : to find the percentage of recession cases that could be classified by application of miller s classification of gingival recessionto find the percentage of recession cases that could be classified by application of kumar and masamatti s classification of gingival recessionto compare the percentage of the clinical applicability of miller 's criteria and kumar and masamatti 's criteria to the total recessions present. to find the percentage of recession cases that could be classified by application of miller s classification of gingival recession to find the percentage of recession cases that could be classified by application of kumar and masamatti s classification of gingival recession to compare the percentage of the clinical applicability of miller 's criteria and kumar and masamatti 's criteria to the total recessions present. before the start of the study, all the examiners were informed, instructed and trained to use miller s as well as kumar and masamatti s classification system by the first author. twenty - five cases of the gingival recession were assessed by each examiner individually (authors 26) along with the first author. each case was evaluated twice ; once by the first author and then by one of the examiners. the results were evaluated, and comparisons were made between the recordings of the first author and each of other examiners. any differences in the recordings were discussed with the individual examiner (in the presence of all other examiners). all necessary explanations were provided individually to each examiner (in the presence of all other examiners) before the study. the presence of other examiners was mandatory at the time of discussion between the first author and any of the other examiners (26) so that conversation would be helpful to each examiner in diagnosing the cases. all the authors also contributed to concept and design of the study, revising the manuscript critically for intellectual content and finally approving it. the applicability of the recession classifications was examined clinically without the help of any radiographic aids. all the patients reporting to the outpatient department of department of periodontics were examined for the presence or absence of gingival recession (buccal / palatal / lingual the subjects who agreed to be a part of the study signed a written informed consent. an ethical clearance was taken from institutional review board before the start of the study. the inclusion criterion was the presence of gingival recession (b / p / l). the cases were classified using both the classification systems (miller 's and kumar and masamatti s) by six examiners. the results of all the groups have been mentioned in percentages for simplicity of data presentation and understanding. the results of all the groups have been mentioned in percentages for simplicity of data presentation and understanding. a total of 104 patients (61 males and 43 females) with an age range of 1460 years (mean age of 34.175 years) were examined. table 2 provides the details of the demographic data and number of recession cases recorded by each examiner. distribution of recession cases on the basis of demographic data by the examiners of 1089 recession cases, a total of 458 cases (42.05%) were recorded in the maxillary arch and 631 cases (57.94%) were recorded in the mandibular arch. tables 3 and 4 provide the details of the recession cases recorded on the basis of the arch (maxillary / mandibular) and tooth type (incisors / canines / premolars / molars) by each examiner. the incisors accounted for 39.76% (433), canines 14.04% (153), premolars 23.78% (259), and molars 22.40% (244) of recession cases. arch and tooth wise distribution of recession cases percentage distribution of recession cases on the basis of arch and tooth type table 5 provides the details of applicability of miller 's classification. 377 out of 1089 (34.61%) cases of the recession could be classified by miller 's classification. the data collected by examiners in respect of miller 's classification has been segregated into completely classified cases and incompletely classified cases. complete application of miller 's classification included those cases that had only buccal recession confirming to miller 's criteria. the incompletely classified cases were those, which had both buccal and lingual / palatal recessions but only buccal recessions (confirming to miller 's criteria) could be classified and included in this data (as miller 's classification does not include palatal / lingual recession). complete application of miller 's criteria was possible in 208 cases (19.10%), and 169 cases were incompletely classified (15.51%). the number of cases in each category of miller 's classification has been detailed in table 5. buccal recession cases, which did not confirm to miller 's criteria, e.g., cases with interdental bone loss with the buccal recession not extending up to mgj, were not categorized in this group. details of applicability of miller 's classification the data collected for each of the 1089 cases were also categorized on the basis of surfaces involved, details of which are presented in table 6. the cases with only buccal recessions were categorized under buccal (b), only palatal recessions under palatal (p) category and only lingual recessions under lingual (l) category. the cases having marginal recessions on buccal and palatal aspect with / without idp involvement were categorized under buccal and palatal category and cases with marginal recessions on buccal and lingual aspect with / without idp involvement were categorized under buccal and lingual category. only p / l marginal recession were categorized under idp with marginal p / l recessions. recessions with idp involvement and b marginal recession were categorized under idp with marginal b recessions. details of recession cases on the basis of surfaces involved all the 1171 cases were classified by kumar and masamatti 's classification. table 7 gives the details of the applicability of this classification in 1089 cases of gingival recession. the table also provides the percentage of cases recorded in each class and its sub - class. it was observed that maximum number of cases belonged to class ii category (48.32%) of kumar and masamatti 's classification. it is important that all the cases of gingival recession get recorded and classified at the time of case - history recording to arrive at a correct diagnosis. a classification system immensely helps in easy chronicling and communication among the clinicians and patients. the gingival recession has been frequently classified using miller 's classification system. from the time of its introduction in 1985, miller 's classification has been exclusively applied on labial / buccal aspects of teeth. in the recent past the limitations of miller 's system and inability to classify certain defects have been highlighted. the limitations of miller 's classification result in the insufficient depiction of the clinical condition. pini - prato in 2011 suggested careful evaluation of the noncritical and widespread use of the miller classification. it was suggested to devise new classification systems of the gingival recession on the basis of the characteristics of taxonomy and scientific evidence. kumar and masamatti proposed a new classification system to overcome the limitations of the miller 's classification and to include or help the clinicians to classify those cases, which can not be categorized into a particular class with any of the current classifications. both the classification systems (according to the defined criteria of each system) were applied on each case recorded. although all the cases recorded (1171) were categorized on the basis of kumar and masamatti 's criteria, only 1089 cases were included in the study. eighty - two cases were excluded during the interpretation of results. it was observed that all the cases in a clinical setting could nt be categorized by miller 's criteria. the class i - a (f) of new classification and miller 's class i, class i - b (f) and miller 's class ii, class ii - c (f) and miller 's class iii, class iii - b (f) and miller 's class iv are similar. very few typical miller 's class ii, iii, and iv were observed in our study. there were few cases, which actually extended up to mgj with or without idp loss. hence, in majority of subjects, the recessions which were observed, remained coronal to mgj with or without idp loss. all the cases having p / l recession could not be classified according to miller 's criteria. maximum numbers of cases were recorded under class ii of the new classification. in class ii, the tip of the idp is located between the interdental contact point and the level of the cej midbuccally / mid - lingually. ii - b deals with idp recession with gingival margin on f / l aspect lies apical to cej but coronal to mgj with attached gingiva present between the marginal gingiva and mgj. hence, the cases with either idp recession only or idp recession with marginal tissue recession coronal to mgj form a substantial group that remained uncategorized by miller 's criteria. another frequently observed gingival recession was class iii - a (according to new classification). class iii - a deals with cases where the tip of the idp is located at or apical to the level of the cej mid - buccally / mid - lingually, and marginal tissue recession is coronal to mgj. this category of recession has a severe interproximal bone loss, but the marginal recession is coronal to mgj. miller has not defined any such criteria where cases of severe interproximal loss and marginal recession coronal to mgj could be classified. another important aspect of the new classification is that idp position is defined in each category of classification. hence, all the cases belonging to class ii will have the tip of idp located between the interdental contact point and the level of the cej midbuccally / mid - lingually and class iii cases have the tip of idp located at or apical to the level of the cej mid - buccally / mid - lingually. cases with open contacts where idp was nonexistent, the position of the crest of gingiva in the interdental region was used to distinguish between class i, ii, and iii. furthermore, the cases having only idp loss can also be categorized by this new classification and need of another classification gets minimized. in our study, we found 1912 sites of recession in 1089 teeth. also cases having idp loss with the marginal loss only on one aspect (either buccal or lingual / palatal) were marked for idp loss on both the aspects. the cases with idp loss can be considered as one entity (without splitting the cases into buccal and lingual). cases demonstrating idp loss with marginal tissue loss on either buccal or palatal / lingual side can also be considered as one category and categorized on the basis of idp and marginal tissue level. as this is probably the first study, where the applicability of two classification systems has been compared, we recorded each idp case separately into buccal and palatal / lingual category. the recording of idp separately as buccal and palatal / lingual is one major limitation of our study. considering idp loss on only one aspect, the total no of cases in class ii - a would be 69 and would have reduced number of sites of recession from 1912 to 1653. another observation of substantial interest was the number of cases with both b and p / l (51.33%) recession. this confirms the fact that most of the cases of recession have p / l component that remains uncategorized in miller 's classification. the results are similar to the study conducted by vehkalahti and wilson. in our study, the maximum recession was observed in mandibular anterior teeth, as was mentioned by albander and kingman. it was also observed that incisors as tooth type demonstrated more recession as compared to other tooth types. at the site level, the buccal recessions had a higher rate of prevalence than lingual / palatal recession. eighty - two cases recorded by examiners were excluded from the study at the time of data interpretation. the examiners had categorized these cases on the basis of kumar and masamatti 's classification. but none of these excluded cases could be categorized on the basis of miller 's criteria. the cases were excluded because of the difference in the levels of papilla observed on buccal and palatal / lingual aspects. this difference of level of idp prohibited the categorization of these cases on the basis of the level of idp. all the examiners had categorized buccal and palatal / lingual recessions into different groups on the basis of the level of buccal and palatal / lingual papilla. radiographic evidence, although not used in our study, would have helped in such cases. this fact was not discussed before the start of the study, as we did not encounter any such case in our prestudy period. this study suggests that the kumar and masamatti 's classification system may be used to classify gingival recession defects, so that the cases, which can not be categorized on the basis of miller 's classification, also find their relevant place.	background : the aims of the present study were to (i) find the percentage of recession cases that could be classified by application of miller 's and/or kumar and masamatti 's classification of gingival recession, and (ii) compare the percentage of clinical applicability of miller 's criteria and kumar and masamatti 's criteria to the total recessions present.materials and methods : a total of 104 patients (1089 recession cases) were included in the study wherein they were classified using both miller 's and kumar and masamatti 's classification systems of gingival recession. percentage comparison of the application of both classification systems was done.results:data analysis showed that though all the cases of the recession were classified by kumar and masamatti 's classification, only 34.61% cases were classified by miller 's classification. 19.10% cases were completely (having only labial / buccal recession) classified. in 15.51% (out of 34.61%) cases, only buccal recession was classified according to miller 's criteria and included in this category, although these cases had both buccal and lingual / palatal recessions. furthermore, 29.75% cases of recession with interdental loss and marginal tissue loss coronal to mucogingival junction (mgj) remained uncategorized by miller 's classification ; categorization of palatal / lingual recession was possible with kumar and masamatti 's classification.conclusion:the elaborative evaluation of both buccal and palatal / lingual recession by the kumar and masamatti 's classification system can be used to overcome the limitations of miller 's classification system, especially the cases with interdental loss and having marginal tissue loss coronal to mgj.
ambulatory surgery was initially limited to procedures performed under local or regional anaesthesia, which required minimal postoperative monitoring. anesthesia techniques and perioperative management have evolved ever since such that low risk surgeries performed under general anaesthesia can now also be performed in the ambulatory setting. patients undergoing breast cancer surgery seldom develop serious complications and most return to their preoperative function soon after the surgery, making them ideal candidates for ambulatory surgery. in spite of this, many patients have been managed in the past as inpatients due to concerns about drain care and the lack of structured outpatient follow - up care. after sentinel lymph node biopsy (slnb) was adopted as the standard of care, full axillary lymph nodal dissection (alnd), and consequently the use of surgical drains, became less common. this, together with the establishment of specialised breast units in many centres to provide continuity of care after hospital discharge, has led to a greater push towards ambulatory breast cancer surgery. early discharge has been shown to contribute to greater healthcare efficiency without compromising the quality of care. studies have consistently affirmed the safety and benefits of ambulatory surgery, even in patients discharged with surgical drains in situ [13 ]. despite this, ambulatory surgery is not as readily accepted in asia as compared to western countries, where there is greater emphasis on patient empowerment [4, 5 ]. older women, in particular, are reluctant to be discharged home early as they perceive cancer surgery to be major surgery and believe that specialised care in a hospital setting during the postoperative period will prevent complications and even future disease relapse. in march 2004, the as23 unit functions as an independent facility from the inpatient wards, with its own bed capacity and staff complement. strict admission criteria ensure that only patients undergoing low risk surgeries under general anaesthesia and who require only basic postoperative monitoring and care are admitted. patients are typically admitted to the unit after surgery and are monitored until they are discharged home, either later on the same day or the following morning. patients who develop perioperative complications requiring more intensive monitoring or who can not be discharged by the following morning are transferred to the inpatient wards. women undergoing breast cancer surgery were among the first to be included in this service. prior to this, all women were admitted to the inpatient wards after breast cancer surgery. in this study, we reviewed the outcomes of women who underwent breast cancer surgery at our institute over a 7-year period, starting from the implementation of the as23 service. in order to determine the safety and feasibility of ambulatory breast cancer surgery, we evaluated the frequency of postoperative complications, the frequency of unplanned prolonged hospital stays, and the readmission rate within 30 days of surgery. a retrospective review was performed of 1742 women who underwent definitive breast cancer surgery at our institute from 1 march 2004 to 31 december 2010. these included 18 bilateral procedures (bilateral mastectomy or wide local excision (wle), with or without slnb or alnd) and 62 repeat surgeries for mastectomy or alnd. those who underwent immediate breast reconstruction were also included. surgeries were performed either as a day surgery (ds) procedure (with patients being discharged on the same day of surgery), an as23 procedure (patients were discharged the following morning), or as an inpatient procedure (patients were discharged more than 24 hours after surgery). a single drain would be inserted under the skin flaps following a mastectomy, and another into the axilla following alnd. surgical drains were not inserted if a wle or a slnb was performed. following surgery, after the discharge criteria were satisfied, patients were then transferred to the as23 unit or inpatient wards. diet and long - term medications, with the exception of anticoagulants and antiplatelet agents, were resumed once the patients were fully awake. oral analgesia was prescribed for pain relief, while antiemetics were given on a pro re nata basis. prior to discharge, patients were reviewed by the surgical team, and specialist breast care nurses would reinforce instructions on wound, drain care, and arm physiotherapy. the nurses would also schedule another review in the outpatient clinic 3 to 4 days later. patients were scheduled for ambulatory surgery unless they had existing medical conditions that necessitated more intensive postoperative monitoring or if they were undergoing immediate breast reconstruction (breast reconstruction with autologous myocutaneous flaps is standard at our institute). those with poor family or social support and who were residents of nursing homes or mental institutes were also managed as inpatients. specialist breast care nurses would then engage patients and their families in preoperative counselling sessions, where the surgical process, postoperative recovery, and concerns regarding early discharge were discussed. thereafter, patients were evaluated by the anaesthesia team to assess the suitability for ambulatory surgery and to optimise the control of any existing comorbidities. data collected included age, ethnicity, preexisting medical conditions, tumor characteristics, surgical procedure, and postoperative outcomes including complications, length of hospital stay, and readmissions within 30 days of discharge. correlation analyses were performed using the chi - square test or fisher 's exact test where appropriate ; the mann whitney u test was used to compare median age and median length of stay. a 2-tailed p value test was used in all analyses and a p value of less than 0.05 was considered statistically significant. a total of 1742 women underwent definitive breast cancer surgery at our institute in the 7-year period. median patient age was 54 years (ranging from 20 to 94 years) and ethnic distribution reflected that of local population demographics (table 1). eighty - three percent of surgeries were performed for invasive carcinoma, and 73.1% were classified as stage i or stage ii disease. of the 1822 surgical procedures, 1277 (70.1%) were done in the ambulatory setting, either as day surgery procedures or as23 procedures (table 2). those who underwent wle were 6 times more likely to undergo surgery in the ambulatory setting compared to those who underwent mastectomy (p 0.05). there has been an increasing trend towards ambulatory surgery over the 7 years (p 0.05). median length of stay during the readmission episode was also similar between the two groups. hospital stays after breast cancer surgery were shortened after it became apparent that early discharge, even with the surgical drains in situ, was safe and did not compromise recovery [3, 611 ]. while ambulatory surgery has become well accepted in many western countries, it is less commonly practiced in asia [2, 3, 6, 7, 10, 1216 ]. early discharge after surgery involves a major change in patient mindset, requiring them to be confident of recovery outside of what is often perceived as a more controlled and specialised environment. more importantly, early discharge is possible only when there is adequate home and social support and a well - organised infrastructure to provide professional and comprehensive postoperative outpatient care. our institute was one of the first in singapore to actively push for breast cancer surgery to be done as an ambulatory procedure. since the first implementation of the as23 service in 2004, more than 70% of all breast cancer surgeries are now being performed as ambulatory procedures in our institute. only 6% of those initially scheduled for ambulatory surgery were not discharged as planned ; even so, most stayed only for an additional 2 days. most of our patients had adequate home support, and only 2% of patients opted for inpatient admission because of social reasons. similar to other published reports, we have observed that patient safety was not compromised by early discharge [13 ]. readmission within 30 days of surgery was not more common among those who had undergone ambulatory surgery. wound complications, such as wound haematoma and infection, were the most common reasons for readmission but were not more frequent nor more severe in those who had ambulatory surgery, implying that wound care in the outpatient setting was comparable to that provided in the hospital wards. even when wound complications developed, most resolved with conservative management and very few patients required further surgery. of note, none of the patients who had been discharged with drains in situ were readmitted for drain - related complications. psychological benefit and improved patient outcomes have been said to be among the main advantages of ambulatory surgery [3, 12 ]. many asian women, the elderly in particular, are apprehensive about early discharge because cancer surgery is thought to take such a physical toll on the body that recovery would be slow and difficult. on the other hand, advising that surgery be done as an ambulatory procedure can instead give the impression that the surgery is likely to be straightforward and uncomplicated. this may in turn promote better emotional and psychological adaptation and a faster return to normal activities, explaining why better outcomes are observed. yet another major benefit of ambulatory surgery is the significant cost savings resulting from shorter hospital stays [8, 1013, 1720 ]. the initiative for an as23 service to facilitate ambulatory surgery was largely driven by increasing pressure on hospital beds. faced with an ageing population, local hospitals are seeing a significant increase in the number of elderly patients being admitted. elderly patients are more often frail and, with multiple medical problems, they take longer to recover and are particularly vulnerable to deconditioning [22, 23 ]. loss of functional independence further slows bed turnover as families are not always able to cope with the additional care needed and these patients then have to remain in hospital until transfer to a step - down care facility. in recent years, it has become increasingly common to have to reschedule elective surgeries because the hospital bed capacity has been exceeded. rescheduling is psychologically frustrating and inconvenient for the patient and their families and also adds to the workload of the healthcare staff involved. the as23 service was set up to provide additional resources to allow surgery to proceed as scheduled regardless of inpatient bed availability. its implementation has led to more streamlined workflows to allow more patients to undergo ambulatory surgery without having to be admitted to the inpatient wards. an integrated workflow involving the surgeon, anaesthetist, and the breast care nurse specialist ensures proper patient selection and realistic management of patient expectations. surgical and anaesthetic reviews ensure patient safety by selecting only the patients who are not expected to be at an increased risk of perioperative complications. while these medical and administrative aspects are essential, we would not have been able to achieve such a high uptake of ambulatory surgery among our patients without the active involvement of our specialist breast care nurses. the breast care nurses are primarily responsible for counselling and postoperative wound and drain care and are instrumental in providing the necessary support for postoperative outpatient care. the rare occurrence of serious complications and the continuity of care provided by the breast care nurses have undoubtedly contributed towards reassuring our patients and their family of the safety and feasibility of ambulatory surgery. our study has shown that ambulatory breast cancer surgery can be successfully implemented in an asian population. an integrated workflow involving the surgeons, anaesthetists, and breast care nurses is fundamental to the success of ambulatory surgery. such a workflow minimises the occurrence of adverse events through proper patient selection and ensures continuity of care upon discharge.	introduction. ambulatory surgery is not commonly practiced in asia. a 23-hour ambulatory (as23) service was implemented at our institute in march 2004 to allow more surgeries to be performed as ambulatory procedures. in this study, we reviewed the impact of the as23 service on breast cancer surgeries and reviewed surgical outcomes, including postoperative complications, length of stay, and 30-day readmission. methods. retrospective review was performed of 1742 patients who underwent definitive breast cancer surgery from 1 march 2004 to 31 december 2010. results. by 2010, more than 70% of surgeries were being performed as ambulatory procedures. younger women (p < 0.01), those undergoing wide local excision (p < 0.01) and those with ductal carcinoma - in situ or early stage breast cancer (p < 0.01), were more likely to undergo ambulatory surgery. six percent of patients initially scheduled for ambulatory surgery were eventually managed as inpatients ; a third of these were because of perioperative complications. wound complications, 30-day readmission and reoperation rates were not more frequent with ambulatory surgery. conclusion. ambulatory breast cancer surgery is now the standard of care at our institute. an integrated workflow facilitating proper patient selection and structured postoperativee outpatient care have ensured minimal complications and high patient acceptance.
menopause and increasing age are associated with a decrease in calcium absorption that can contribute to the pathogenesis of osteoporosis. calcium supplements appear to be effective in reducing bone loss in women, but there is a considerable uncertainty about the effectiveness of calcium supplementation in preventing bone loss at the early stage of the menopause [24 ]. several adjunctive therapies with calcium supplementation were attempted earlier to prevent osteoporosis in early stage of menopause [5, 6 ]. of these different adjunctive therapies, hrt, serms, and vitamin d were mostly advised [7, 8 ], but with controversial results [5, 9, 10 ]. in addition, hrt and serms are associated with well - known adverse health effects like breast cancer, thromboembolism [9, 12 ], stroke, and cardiovascular disease early in the course of therapy [13, 14 ]. however, black tea (camellia sinensis) has been reported as a medicinal plant with rich flavonoid content and plethora of health - promoting effects including phytoestrogenic efficacy [1820 ], but with no reported adverse health effect. earlier, it was reported that phytoestrogens have affinity for estrogen receptors (ers), both and with a more preference for er-. a recent report has further indicated that, in rat intestine, the predominant estrogen receptor population is beta - type. earlier, it was also reported that changes of bone metabolism in female rats after ovariectomy are similar to the alterations in early postmenopausal women. in addition, hormone therapy is considered as first - line therapy for preventing bone loss and fractures in early postmenopausal women who are symptomatic [9, 25 ]. keeping in view the controversial role of the adjuncts in regulating bone loss in early menopausal condition, the present study was undertaken with black tea to examine its role on intestinal absorption of calcium and its association, if any, with preservation of bone mass in the early phase of menopause. furthermore, to examine its (bte) efficacy as an adjunct, a comparative study was undertaken with 17-estradiol. all animal experiments were performed according to the ethical guidelines suggested by the institutional animal ethics committee (iaec) and the committee for the purpose of control and supervision of experiments on animals (cpcsea), government of india. rats were housed in an environmentally controlled animal laboratory and maintained on a 12-hour light / dark schedule at 25 2c throughout the experimental period. they were acclimated for seven days in laboratory environment and fed with a standard laboratory diet containing 67.36% carbohydrate, 22.7% protein, 5.7% fat, 0.4% calcium, 0.3% phosphorus, and 0.195 nmol vitamin d3/g of diet. they were then randomly divided by initial body weight into four groups consisting of six animals in each group : (a) sham - operated control ; (b) bilaterally ovariectomized ; (c) bilaterally ovariectomized + bte ; (d) bilaterally ovariectomized + 17-estradiol. as the changes of bone metabolism in ovariectomized rats are similar to the alterations in early postmenopausal women, bilateral ovariectomy was performed to simulate the early menopausal condition. under light ether anaesthesia, bilateral (dorsolateral) ovariectomies were performed in groups b, c, and d, and animals of group a were subjected to sham - operation. after seven days of recovery from surgical convalescence, animals of group c were treated orally with 2.5% aqueous black tea extract (bte) at a single dose of 1 ml/100 g of body weight daily for 28 days. animals of group d were treated with 17-estradiol intraperitonially at a single dose of 10 g / kg of body weight. animals of other groups were given only deionized water and solvent vehicle as placebo. during the period of bte and 17-estradiol treatment, group a was pair - fed with experimental groups b, c, and d so as to overcome the impact of any altered food intake in the experimental groups. the black tea extract (bte) was prepared from ctc (curl, tear, and crush) bop (broken orange pickoe) grade black clonal tea. it was processed and supplied by tocklai experimental station, jorhat, assam, india, to the drug development division, indian institute of chemical biology, jadavpur, kolkata. we received a generous gift from that institute, and a fresh 2.5% aqueous bte was prepared everyday following the method of wei.. after the experimental period was over, the animals of all groups were fasted for 16 hours. the preparation of animals and intestinal loops for the study of calcium transference in situ was made by following the method as described elsewhere by islam.. briefly, the animal was anaesthetized (urethane, 1.7 mg / kg body wt.), the abdomen of each animal was opened through a midline longitudinal incision, the bile duct was ligated and duodenal, and jejunal and ileal segments were located. two ligatures, one proximal and the other distal, were applied tightly in each loop measuring about 68 cm in all duodenal, jejunal, and ileal segments. loops were so selected that each contained 810 vessels, and care was taken so that no major blood vessels were occluded by the ligature. for the measurement of intestinal calcium transference, 1 ml of prewarmed (37c) tris - hcl buffer solution containing 0.2 mmol cacl2 was injected with a 25-gauge needle in each ligated segment. the intestinal loops were placed in their usual position, and the abdomen was closed. after one hour, animals were sacrificed, the preselected loops were removed and the fluid from each loop was collected separately, together with a few washings of the lumen with triple - distilled water. the calcium content of the collected fluid was then estimated according to the method described by adeniyi.. the difference between the amount of ca introduced and the amount left unabsorbed was used to estimate the amount of ca absorbed. the intestinal part constituting the loop was dried at 90c to attain a constant weight, which was recorded as the weight of the dried loop. after sacrificing the animal and opening of the abdomen, the whole of the small intestine was quickly removed. the portion comprised of the duodenum, jejunum, and ileum were separated and chilled in ice. intestinal mucosa was collected as described by maenz and cheeseman, and the scrapings were homogenized according to the method of koyama.. the activity of alkaline phosphatase of intestinal mucosa was estimated using p - nitrophenyl phosphate as substrate. the protein content of the homogenate used for the study was determined using the method of lowry.. the activity of the enzyme ca atpase was also studied from the mucosal extract using the method of rorive and kleinzellar. phosphate liberated during ca atpase enzyme activity was estimated by the method of lowry and lopez. serum was obtained by using standard laboratory protocol. serum estrogen level (pg / ml) was determined by using the elisa eiagen estradiol kit (adaltis italia, italy), and pth was determined by elisa technique by using anti - rat pth monoclonal antibody (bio trend, germany), goat anti - mouse igg - hrp and tmb / h2o2 (genei, india). the intra - assay coefficients of variation were 9.08% in case of estradiol and 9.45% in pth. fasting urine was collected for 24 h (9 a.m. to 9 a.m.) according to the standard laboratory procedure as described elsewhere by chanda.. excretion levels of urinary calcium, phosphate, creatinine, and hydroxyproline were measured according to the methods of adeniyi., lowry and lopez, r. l. nath and r. k. nath, and bergman and loxley, respectively. serum tartrate - resistant acid phosphatase (trap) activity was estimated spectrophotometrically (double - beam spectrophotometer, shimadzu 160a ; shimadzu corporation, kyoto, japan) by using the kit (labkit, spain). alkaline phosphatase activity was measured by the hydrolysis of p - npp (para nitrophenyl phosphate) at ph 10.8 using glycine - naoh buffer at 37c. for bone density determination, the right femur, eighth thoracic rib, eighth thoracic vertebra, and fourth lumbar vertebra were freed of soft tissue and cleaned. bone density (g / cm bone volume) was measured as described by arjmandi. by using archimedes ' principle. briefly, each bone was put in an unstoppered vial filled with deionized water, and the vial was placed under a vacuum for 90 min to ensure that all the trapped air diffused out of the bone. each bone was removed from the vial, blotted with gauze sponge, weighed and returned to the vial containing deionized water. the bone was reweighed in water, and density was calculated (g / cm bone volume). to estimate the mineral content of bones, the whole bone was extracted two times with a 1 : 1 mixture of ethanol and diethyl ether for 48 h and one time with diethyl ether for 24 h. the dehydrated and defatted bones were ashen for 48 h at 600c and hydrolyzed in 6 n hcl for determination of calcium and phosphate. calcium and phosphate were estimated according to the method as described, respectively, by adeniyi. and lowry and lopez. after sacrifice of the animal, left femur was carefully cleaned off adhering adhesive muscles, tendons, and other soft tissues. bone breaking force was measured as described by shapiro and heaney using a hand - held force meter (excel enterprises, india). before testing the loading point was determined as the midpoint of the distance between the greater trochanter and the lateral condyle in the femoral midshaft. this point was exactly at the middle of the distance between the two supports apart from each other by 1.0 cm. the force was applied with the plunger (at a constant rate of 1 mm / min) directly to the femoral midshaft until the sample fractured. the left proximal tibia and third lumbar vertebra were removed, dissected free of soft tissue, and fixed with 4% paraformaldehyde for 1618 h at 4c. then, specimens were washed for 12 h at 5c in each of the following series of solutions : 0.01 m pbs containing 5% glycerol, 0.01 m pbs containing 10% glycerol, and 0.01 m pbs containing 15% glycerol. the specimens were then decalcified in edta - g solution (14.5 g edta, 1.25 g naoh, and 15 ml glycerol, ph 7.3) for 1014 days. the decalcified tissues were washed sequentially at 5c for 12 h in (a) 15% sucrose and 15% glycerol in pbs, (b) 20% sucrose and 10% glycerol in pbs, (c) 20% sucrose and 5% glycerol in pbs, (d) 20% sucrose in pbs ; 10% sucrose in pbs, (e) 5% sucrose in pbs, and (f) 100% pbs. then, the tissues were washed with pbs and dehydrated in a graded series of alcohols, followed by clearing in xylene, and finally embedded in paraffin. the specimens were cut into 5 - 6 m sections and stained with hematoxylin - eosin. representative sections were observed, and photomicrography was performed with the help of a bright - field microscope equipped with a digital camera (carl zeiss, germany). the kruskal - wallis nonparametric anova test was performed to find whether or not scores of different groups differ significantly. to test intergroup significant difference, intestinal calcium transference profiles of sham - operated control, ovariectomized, and ovariectomized supplemented with bte are shown in table 1. mucosal transference of calcium was reduced significantly in the ovariectomized rats in all segments when compared with the sham - operated control group (p < 0.001 for duodenum ; p < 0.01 for jejunum and ileum). bte supplementation could significantly recover such alterations in mucosal transference of calcium in ovariectomized rats (p < 0.01 for duodenum and ileum ; and p < 0.05 for jejunum). results of intestinal alkaline phosphatase activity of different groups of rats are shown in table 1. table 1 indicates that, compared with sham - operated group, ovariectomized rats showed significant segment - wise reduction in the activity of alkaline phosphatase in all segments of small intestine (p < 0.01 for duodenum, jejunum, and ileum). bte supplementation was found effective in restoring this enzyme activity in all these segments (p < 0.01 for duodenum, jejunum, and ileum). results of intestinal mucosal calcium atpase activity of different segments of small intestine of rats are shown in table 1. it was observed that bilateral ovariectomy caused significant reductions in the activity of this enzyme (p < 0.01 for duodenum, jejunum, and ileum) when compared with sham - operated control group. but, bte supplementation in ovariectomized animals could significantly increase this enzyme activity (p < 0.01 for duodenum, jejunum, and ileum). results of serum estradiol titer of sham - operated control rats, ovariectomized rats, and ovariectomized rats supplemented with bte are shown in table 2. as expected, assay indicated that, compared with sham - operated control, a significant decrease in estradiol level was seen in ovariectomized animals (p < 0.01).. however, serum pth level did not show significant variation between sham - operated control, ovariectomized, and ovariectomized supplemented with bte (table 2). the urinary calcium, phosphate, creatinine, and hydroxyproline excretion profiles together with ca : cr ratio of sham - operated control group (group a), bilaterally ovariectomized (group b), ovariectomized rats supplemented with aqueous bte (group c), and ovariectomized rats supplemented with 17-estradiol (group d) are shown in table 3. compared with sham - operated group, animals of ovariectomized group showed a significant increase in all of the urinary parameters studied, namely, calcium, phosphate, creatinine, and hydroxyproline and ca : cr ratio (p < 0.01). elevated responses of all these parameters were significantly counter - regulated in both groups of rats receiving either aqueous bte (group c) or 17-estradiol (group d). the serum alkaline phosphatase activity profiles of rats of sham - operated control, ovariectomized, ovariectomized + bte, and ovariectomized + estradiol groups are shown in table 3. rats of ovariectomized group (group b) showed a significant increase in serum ap activity when compared with animals of sham - operated control group (p < 0.01) (group a). this increase in ap activity was significantly lowered (p < 0.01) in rats receiving either bte (group c) or 17-estradiol (group d). however, compared with bte, 17-estradiol supplementation was found more effective in reducing the activity of serum ap in ovariectomized rats. likewise, the significant increase (p < 0.01) in trap in ovariectomized animals (group b), compared with control (group a), could be effectively reduced by either aqueous bte treatment (group c) or 17-estradiol (table 3). animals in the ovariectomized group (group b) had significantly lower densities of the right femur (p < 0.01), eighth thoracic rib (p < 0.01), eighth thoracic vertebra (p < 0.01), and fourth lumbar vertebra (p < 0.01), compared with the sham - operated control group (group a). except thoracic vertebra, bte supplementation, like 17-estradiol, could produce significant increase (p < 0.01) in bone density of all bones : right femur (p < 0.01), eighth thoracic rib (p < 0.01), eighth thoracic vertebra (p < 0.05), and fourth lumbar vertebra (p < 0.01) (table 4). animals of ovariectomized group (group b), compared with sham - operated control group (group a), showed a marked decrease in calcium and phosphate levels of right femur (calcium : p < 0.01 ; phosphate : p < 0.01), eighth thoracic rib (calcium : p < 0.01 ; phosphate : p < 0.01), eighth thoracic vertebra (calcium : p < 0.01 ; phosphate : p < 0.01), and fourth lumbar vertebra (calcium : p < 0.01 ; phosphate : p < 0.01). significant recovery of both mineral contents of these bones was seen when ovariectomized animals were supplemented with either bte (group c) or 17-estradiol (group d). table 4 shows the results of bone strength of sham - operated control (group a), ovariectomized (group b), and ovariectomized rats supplemented with either bte (group c) or 17-estradiol (group d). compared with sham - operated control (group a), femurs of ovariectomized rats (group b) required less force (p < 0.01) to fracture. however, a significant recovery (p < 0.01) of femur breaking force was noticed when ovariectomized rats were supplemented with either bte (group c) or 17-estradiol (group d) alone. compared with bte, 17-estradiol supplementation in these ovariectomized rats showed a better recovery of breaking force of femur. histological study reveals that ovariectomized rats showed a decreased cortical thickness and empty bone marrow at the proximal tibia (figure 1(b)) as compared to control (figure 1(a)). additionally, there was a decrease in trabecular bone volume and thickness at the 3rd lumbar vertebra (figure 1(f)) compared to control (figure 1(e)). however, on bte administration, bone remodeling action was observed in these ovariectomized animals (figures 1(c) and 1(g)). as expected, similar observation was also found in tibia and lumbar vertebra of the estradiol supplemented group (group d) (figures 1(d) and 1(h)). in the present study, ovariectomized rat was used as an animal model of ovarian hormone deficiency in postmenopausal women. the aim of this study was to examine the prospective role of bte as an adjunct, because efficacy of several compounds as adjunct with calcium supplementation in the early phase of menopausal bone loss is still uncertain and remains controversial [5, 9, 10 ]. additionally, many of these adjuncts are reported to be linked with serious adverse health effects [9, 1114 ]. precisely, this study has demonstrated that supplementation of bte in an ovariectomized rat model could significantly restore the reduced intestinal absorption of calcium, bone mineral contents, bone histological features, and bone breaking force, with a simultaneous increase in serum estrogen titer, but with no change in pth level. results of in vivo intestinal loop transference studies with the classical technique of wills indicated that, irrespective of groups, intestinal absorption of ca was reduced in ovariectomized rats. also, there was a segmental variation in calcium absorption which was in the order of descending gradient from duodenum to ileum. results of this study furthermore indicated that deficiency of estrogen in ovariectomized rat possibly had a negative influence upon intestinal absorption of calcium, as these animals showed a greater decrease in absorption of calcium than sham - operated control animals (table 1). such speculation finds its support from the results of earlier observations with ovariectomized rats [44, 45 ]. in addition, it has also been reported earlier that estrogen may have direct role via the ers in regulating intestinal calcium absorption in vitro and in vivo [46, 47 ]. in the present study, bte supplementation was found effective in correcting such reduction in calcium absorption, indicating that bte possibly has positive influence upon mucosal transference of calcium. to ascertain the mechanism of such decrease in intestinal absorption of calcium, we examined the activities of the two most relevant mucosal calcium transferring enzymes, namely, alkaline phosphatase and calcium - atpase. activities of both of these enzymes were found inhibited in ovariectomized group of animals (table 1). this agrees well with the earlier observations that both enzymes are linked with calcium absorption as the activities of these enzymes correlate with the degree of calcium absorption in different parts of the intestinal tract under different circumstances [29, 47, 48 ]. bte supplementation could well restore the activities of both of these enzymes indicating that the observed positive influence of bte upon intestinal absorption of calcium was possibly mediated through modulation of the activities of these transferring enzymes. to verify whether such ovariectomy - induced alteration in calcium absorption could in any way influence calcium homeostasis and bone functional profiles, a series of parameters were tested. results revealed that, compared with sham - operated control, ovariectomized rats showed an increase in urinary loss of calcium and phosphate (table 3). such an increase in renal excretions of minerals was significantly corrected in ovariectomized animals on receiving bte, suggesting that bte has efficacy in retaining bone minerals possibly by preventing bone resorption and/or increasing bone formation or both. results of studies of the two other specific marker parameters of bone turnover, namely, serum alkaline phosphatase (ap) and urinary calcium to creatinine (ca : cr) ratio (table 3) further support our suggestion. it was observed that bte was effective in reducing ovariectomy - induced increase in serum ap and urinary ca : cr ratio. since a rise in ap and ca : cr ratio is linked with collagen degradation, bone resorption, and osteoporosis, it was presumed that this phytoestrogenic compound (bte) possibly has effective role in reducing bone loss. the markers of bone resorption and osteoclastic activity measure circulating or urinary concentrations of fragments of bone matrix that are released during bone resorption, or enzymatic activities associated with osteoclasts. the close association of increased serum concentrations of trap and urinary hydroxyproline, respectively, as a potential index for osteoclastic activity and degradation of type i collagen are well established. in the present study, compared with the control, these responses were found well regulated by bte supplementation indicating that bte possibly is effective also in controlling osteoclastic activity and collagen degradation to prevent skeletal health. results of histological studies with cortical and cancellous bones (figures 1(a)1(d), 1(e)1(h)) strongly supported this speculation. such speculation was further cross - examined in our studies by measuring parameters like bone density and bone mineral content (table 4). compared with the control group of animals, bilateral ovariectomy showed low bone densities suggesting that high rate of bone turnover occurred in ovariectomized animals. bte supplementation could effectively reduce such high rate of bone turnover confirming that bte has protective action against ovarian hormone deficiency - related bone loss in early menopause. further confirmatory results were obtained in our experiments with bone mineral content where ovariectomized animals showed lower bone calcium and phosphate content (table 4), which could be revived significantly after supplementation with bte. this was further supported by the results of the breaking force of femur (table 4) because ovariectomy - induced decrease in breaking force of femur was restored by bte supplementation. to assess the efficacy of bte as a possible agent for adjunct therapy, the results of urinary test parameters of calcium homeostasis and bone functional profiles with bte were compared with 17-estradiol, the most widely used hormone to prevent early menopausal bone loss. as the results, potency - wise, indicate a close similarity of responses between these two agents (tables 3 and 4), it may be suggested that bte has a promise to be an effective adjunct for calcium supplementation in the early phase of menopause, which possibly may be attributed to its phytoestrogenic efficacy in regulating the intestinal absorption of calcium, the mechanism of which may be either by modulating theactivities of mucosal calcium transferring enzymes or by a direct action on intestinal er or both. further detailed study on direct intestinal er activation by bte is underway. in summary, bte has a promise to be an effective adjunct for calcium supplementation in the early phase of menopause. additionally, it may be used as an anti - osteoporotic naturopathic agent in subjects who are clinically advised not to receive hormone replacement therapy.	the present study was undertaken to find out the ability of black tea extract (bte) as a suitable alternative of adjunct for calcium supplementation in treating an ovariectomized rat model of early osteoporosis. female wistar rats weighing 140150 g were divided into four groups consisting of six animals in each group : (a) sham - operated control ; (b) bilaterally ovariectomized ; (c) bilaterally ovariectomized + bte ; (d) bilaterally ovariectomized + 17-estradiol. results suggest that bte could promote intestinal absorption of calcium significantly (p 0.05). a comparative study with 17-estradiol, a well - known adjunct for calcium supplementation, indicated that efficacy of bte in maintaining skeletal health is close to that of 17-estradiol. this study suggests that simultaneous use of bte is promising as a prospective candidate for adjunctive therapies for calcium supplementation in the early stage of menopausal bone changes.
bulking agents were first described for treating stress urinary incontinence (sui) more than 100 years ago as a minimally invasive method to treat urinary incontinence (ui). since that time, many bulking agents of different makes and tissue interactions have been used to treat ui. however, in the intervening period, many substances have been tried and abandoned due to concerns with both efficacy and safety [39 ]. one hypothesis is that intraurethral bulking produces coaptation of the urethral edges during the storage phase of the micturition cycle and particularly during periods of increased abdominal pressure. studies describing increased abdominal pressure transmission in the first quarter of the urethra and increased abdominal leak - point pressure as a result of successful urethral injection outcomes would support this hypothesis. injectable agents therefore were thought to restore continence by increasing urethral resistance only at rest and allowing the urethra to funnel and open during micturition. this theory was challenged by the publication of an alternative explanation of the mechanism of action [11, 13 ] whereby it was suggested that if positioned appropriately, the bulking agent could create a zone of increased contractility of the rhabdosphincter by creating an increased stretch of the muscle fibers. this theory proposed a possible mechanism whereby increase in the active component of the urethral pressure profile after bulking was explained. these two theories are not, of course, mutually exclusive, and there may be a combined mechanism. the first description of the use of polyacrylamide hydrogel (pahg) for urethral bulking was in 2006. bulkamid is a polymer gel consisting of 2.5 % cross - linked polyacrylamide and 97.5 % water for injection. it is atoxic [15, 16 ] and resistant to degradation [17, 18 ]. pahg has been used in aesthetic plastic and reconstructive surgery in europe for the past 7 years, and long - term as well as experimental studies show that the gel is gradually integrated through a fine network of vessel - bearing connective tissue, with no capsular fibrosis or calcification. tissue integration starts immediately after implantation and is completed approximated 12-months post - injection depending on the bulk size [1720 ]. the 12-month results of this study were previously presented, and they confirmed a 67 % subjective response rate, with a reduction in both incontinence episodes and 24-h pad weights. the aim of the paper presented here was to assess the effectiveness and safety 2 years post pahg injection in women with sui or mixed ui (mui) as a secondary endpoint to the initial presentation of the 1-year data. the study was an open, noncomparative, multicenter, multinational study involving ten centers from five countries : two in denmark, two in sweden, one in finland, four in the uk, and one in germany. inclusion criteria were duration of symptoms (12 months) and incontinence episode frequency (1 per 24 h). additional requirements were a maximum flow rate of 15 ml / s, a postvoid residual urine volume (pvr) of 100 ml, a bladder capacity of 300 ml, and normal diuresis (4 weeks) ; treatment with systemic corticosteroids ; active autoimmune or connective tissue diseases ; or pregnancy. all women had a screening visit involving full medical history, physical and pelvic examination including genital prolapse (pop quantification) score, uroflowmetry, pvr measurement, and urine dipstick test (if positive with culture and sensitivity). the screening visit was followed by a baseline visit for collecting data from the 24-h pad - weighting test and 3-day micturition diary, including bladder capacity, number of micturitions, number of sui and urge incontinence (uui) episodes, and estimated renal output. participants were also asked to complete the international consultation on incontinence questionnaire (iciq) [21, 22 ] and a patient quality of life (qol) and visual analogue scale (vas) score. treatment with pahg was performed at the baseline visit or within 3 days and no later than 8 weeks from the screening visit. pahg injection was performed under local anesthesia (10 ml, 5 % lidocaine injected into the urethral wall). pahg was injected under urethroscopic control transurethrally into the submucosa (three deposits of 0.20.8 ml each, 0.51 cm distal to the bladder neck) using a 23-gauge 120-mm needle with 1-cm markings to ensure correct depth of injection placement. after satisfactory urethral occlusion women were discharged after successful voiding (pvr 4 weeks) ; treatment with systemic corticosteroids ; active autoimmune or connective tissue diseases ; or pregnancy. all women had a screening visit involving full medical history, physical and pelvic examination including genital prolapse (pop quantification) score, uroflowmetry, pvr measurement, and urine dipstick test (if positive with culture and sensitivity). the screening visit was followed by a baseline visit for collecting data from the 24-h pad - weighting test and 3-day micturition diary, including bladder capacity, number of micturitions, number of sui and urge incontinence (uui) episodes, and estimated renal output. participants were also asked to complete the international consultation on incontinence questionnaire (iciq) [21, 22 ] and a patient quality of life (qol) and visual analogue scale (vas) score. treatment with pahg was performed at the baseline visit or within 3 days and no later than 8 weeks from the screening visit. pahg injection was performed under local anesthesia (10 ml, 5 % lidocaine injected into the urethral wall). pahg was injected under urethroscopic control transurethrally into the submucosa (three deposits of 0.20.8 ml each, 0.51 cm distal to the bladder neck) using a 23-gauge 120-mm needle with 1-cm markings to ensure correct depth of injection placement. after satisfactory urethral occlusion, the bladder was emptied via the endoscope. during injection, women were discharged after successful voiding (pvr 6 months before the 24-month assessment, and one patient was included as a responder who missed the 12-month analysis but attended the 2-year follow - up. one hundred and thirty - five patients were initially included in the intention to treat analysis. following the rules described for last observation carried forward (locf) and imputation results for subjective outcome are presented for 124 patients at 12 months and 116 patients at 24 months. table 1 shows the subjective success rates as both per protocol and intention to treat. on the intention to treat analysis at 24 months, the success rate was maintained at 64 % (which was a nonstatistically significant reduction from 67 % at 12 months). twenty (17 %) were cured and 54 (47 %) improved. of the responding patients at 12 months, 94 % (116/124) had sustained success rates at the 24-month follow-up.table 1subjective success rates at 1, 6, 12, and 24 months after treatment1 month6 months12 months24 monthsitt (patients)135129124116responders % 87 % 71 % 67 % 64 % ci8192 % 6278 % 5872 % 5572 % itt intention to treat, ci confidence interval subjective success rates at 1, 6, 12, and 24 months after treatment itt intention to treat, ci confidence interval the slight deterioration seen with the subjective outcome is not recorded in either the iciq (fig. 2) scores, where the improvement measured 12 months after treatment is maintained. similarly, in the objective outcomes, both incontinence episode frequency and pad - weight test results retained the initial improvement. results show a significant decrease in the number of incontinence episodes (p < 0.0001, table 2) as well as a significant reduction in leakage from baseline to 24 months (p < 0.0001, table 3). consistent with the 12-month results, there was a nonstatistical trend toward a slightly better outcome for patients with pure 1international consultation on incontinence questionnaire (iciq) scores at 1, 6, 12, and 24 months [intention to treat (itt) analysis ]. question 2 : how much urine do you usually leak ? (range 06). question 3 : how much does leaking urine interfere with your everyday life ? (range 010)fig. 2patient quality of life visual analog scale (vas) scores over timer in the intention to treat (itt) analysis settable 2urinary leakage (pad test) over timebaseline1 month6 months12 months24 monthsmean leakage (g) mean (sd)54.3 (78.1)20.8 (49.2)14.6 (23.3)10.9 (19.0)13.2 (28.1) median28.54.05.33.83.0leakage is the 24-h leakage measured as mean over 2 dayssd standard deviationtable 3summary of daily incontinence episodes over timebaseline1 month6 months12 months24 monthsmean (sd)3.68 (2.5)1.78 (2.7)1.81 (2.6)1.55 (2.7)1.53 (2.8)median3.000.670.670.670.50daily number of incontinence episodes is the mean over 3 days registered in the patient s diarysd standard deviation international consultation on incontinence questionnaire (iciq) scores at 1, 6, 12, and 24 months [intention to treat (itt) analysis ]. question 2 : how much urine do you usually leak ? (range 06). question 3 : how much does leaking urine interfere with your everyday life ? (range 010) patient quality of life visual analog scale (vas) scores over timer in the intention to treat (itt) analysis set urinary leakage (pad test) over time leakage is the 24-h leakage measured as mean over 2 days sd standard deviation summary of daily incontinence episodes over time daily number of incontinence episodes is the mean over 3 days registered in the patient s diary sd standard deviation in terms of response to repeat treatment, the estimated responder rate was 69 % in patients receiving only one treatment and 53 % in patients receiving two treatments. compared with the responder rates at 12 months follow - up (one treatment 72 % ; two treatments 57 %), the differences were borderline statistically significant (p = 0.081) at the 24-month follow - up, 16 new nonserious aes and four new serious adverse reactions were reported, none of which was thought to be related to the treatment. no woman had signs of impaired bladder emptying based on postvoid residuals at 24 months. at the 24-month follow - up, 16 new nonserious aes and four new serious adverse reactions were reported, none of which was thought to be related to the treatment. no woman had signs of impaired bladder emptying based on postvoid residuals at 24 months. the evidence of medium / long - term durability and safety of different bulking agents is limited. we report the first systematic 2-year follow - up of pahg (bulkamid) injection for sui and mui. the response rate of 67 % after 12 months (cured or improved) was sustained, which was supported by objective findings of 24-h pad - weighting test, number of incontinence episodes, and qol data. the findings are in good agreement with a study by ghoniem. that showed 88 % durability success and few complications after 2 years. in contrast, two studies on transurethral injection of hyaluronic acid / dextranomer (nasha / dx gel) showed poor durability of effect and a high incidence of complications [28, 29 ]. after injection with these gels, the foreign - body reaction is mild (pahg) to modest (silicone). silicone, however, is hydrophobic and has a tendency to migrate from the implantation site via circulating phagocytic cells [30, 31 ] in contrast to pahg, which is hydrophilic. moreover, pahg will interact with the surrounding tissue as a consequence of the high water content and structure and integrate into the surrounding tissue through a fine network of vessel - bearing connective tissue, thus being firmly anchored into the surrounding tissue. analysis of cost effectiveness suggests that the decline in the use of bulking agents may be premature and that there may be a valid economic argument to consider their use. advantages of bulking agents are ease of application, which can be achieved in the office setting, and a reduced side effect / complication risk for patients, where this is the more pressing part of the risk benefit ratio. this study, as with many others, has continued to supply evidence demonstrating that pahg in pure sui tend to achieve better outcomes than those for mui [14, 33 ]. however, as with other surgeries, this treatment method may lead to improvement in qol as a result of decreased symptoms, rather than cure, and as such may have a valuable therapeutic role within stress - predominant mui. in our previous paper, the potential mechanisms of action were discussed. no attempt at site assessment or implant action was made during this study, and so no conclusion can be made as to the mode of action. it would seem logical, given the length of the urethra, that both mechanisms may be involved, and further assessment of placement site and success may be of value. safety concerns have been raised regarding specific bulking agents because of migration, hypersensitivity, urethral erosion, pseudocysts / abscess, granuloma formation, and obstruction [39, 28, 29 ]. no treatment - related aes were reported by the investigators during follow - up from 12 to 24 months. indications for using bulking agents to treat ui remain a contentious issue. in one publication, the cure rate of a repeat midurethral sling was significantly lower and the incidence of de novo urgency and uui was significantly higher in repeat procedures. this report presents the largest series in a powered study demonstrating medium - term longevity of pahg treatment of ui, with no trend toward a decrease in efficacy at 2 years. the reason for this is almost certainly related to the properties of the polyacrylamide implant, in which the bulking substance is made up predominantly of water held within the polyacrylamide gel matrix. the substance is unlikely to migrate, and indeed, there are no reported cases of this in any of the applications in medicine in over 20 years of use. secondly, there are no reported aes, such as granuloma or abscess formation, suggesting that the substance remains inert, does not metabolize, and as such maintains shape and size. we therefore conclude that these properties are central to the results achieved in this series 2 years posttreatment. these results confirm pahg to be an easy and safe bulking agent for treating uncomplicated sui or mui, with favorable durability 2 years after injection. the trial was sponsored by contura international a / s (sydmarken, dk-2860 soeborg, denmark), which also provided the bulkamid for the trial. p. toozs - hobson : paid travel expenses or honoraria by astellas and pfizer in the last 2 years ; g. tegerstedt : none ; m. fynes : none ; w. al - singary : consultant, western sussex nhs trust ; g. lose : paid travel expenses or honoraria, payment for research, workshops, consultancy by contura, johnson & johnson, pfizer, astellas. this article is distributed under the terms of the creative commons attribution license which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.	introduction and hypothesispolyacrylamide hydrogel (pahg, bulkamid) is a promising urethral bulking agent. this article presents the 2-year follow - up results of a multicenter study of pahg injections for treating stress and stress - predominant mixed urinary incontinence.methodssubmucosal injection of pahg was performed in 135 women with urinary incontinence, with subjective and objective assessment of the efficacy and safety 24 months postinjection.resultsat 24 months, the subjective responder rate was 64 % (a statistically non - significant reduction from 67 % at 12 months). the decreased number of incontinence episodes and urine leakage were maintained compared with the result from the 12-month evaluations, as were objective result rates and quality of life data. no safety issues occurred.conclusionspahg is an effective and safe treatment option for women with stress - predominant mixed urinary incontinence, with maintained medium - term responder rates.
since pain is an inherently subjective phenomenon, it is often said that the gold standard for pain assessment in both children, and adults is verbal - reporting [13 ]. that is to say that patient 's verbal reporting of pain is the only way to determine its presence, intensity, and quality however, reliable description of pain may be difficult for certain individuals including infants, very young children, individuals with intellectual disabilities (i d) and adults with severe dementia. reliable description of pain may be difficult for children due to their cognitive immaturity and their inability to separate pain from fear and anxiety. children with i d frequently have the added challenge of not being able to express their pain or verbalize it. this explains why these children were believed not to experience pain and were forced to undergo painful procedures on occasions without the proper control of pain. it also explains why improper pain assessment and management have been practiced with individuals with i d. intellectual disability which affects nearly 2.5% of the population is currently defined by the american association on intellectual and developmental delay (aaidd) as significantly subaverage intellectual functioning, existing concurrently with related limitations in two or more of certain adaptive skills including communication, self - care and social skills manifesting before the age of 18 years [8, 9 ]. research shows that children with developmental disabilities generally experience more pain than normal children [10, 11 ] due to comorbid conditions associated with their disability or due to medical interventions necessary to monitor or treat the disability. their pain however may not be properly appreciated because of their lack of verbal communication and because many of these children often have idiosyncratic behaviors such as moaning, grunting, and grimacing which may lead to overestimation of pain by those unfamiliar with the child. (2003) showed that children who experienced the most pain were those who were least able to verbally describe it, that is, children with greater physical and cognitive disability. studies show that children with disabilities have higher dental caries experience, unmet treatment needs, and prevalence of malocclusion, than those without disabilities [1517 ]. in their study of special needs children, hennequin. (2000) showed that there was a significant underestimation of dental treatment needs by parents and dentists suggesting that dental pain in this population was also underestimated. this study was aimed at assessing the ability of caregivers of children with i d to evaluate the presence of dental pain through behavioral changes in their children. this study recruited a total of 86 children among those visiting the dental clinics at the faculty of dentistry, and the hospital dental clinics at king abdulaziz university (kau), jeddah, saudi arabia, during the years 2009 and 2010. cases fulfilled the following criteria.both boys and girls ages range from birth to 16 years.a history of i d as verified by the child 's medical file or caregivers ' reporting. child may be verbal or nonverbal.caregivers were able to understand spoken arabic or english.included children could be diagnosed with medical problems other than i d. a history of i d as verified by the child 's medical file or caregivers ' reporting. controls included age - matched children recruited from the dental clinics at the faculty of dentistry, and the hospital dental clinics at king abdulaziz university (kau). they basically fulfilled the same criteria only they had normal intellectual development without i d. before the beginning of the study, ethical approval was obtained from the faculty of dentistry, kau, to conduct the study. the investigators developed a questionnaire containing a mixture of closed- and open - ended questions regarding participants ' demographical, medical, and dental data. reliability of the two examiners was assessed in measuring dental caries on 10% of the sampled children with kappa = 0.85, the investigators reviewed the children 's medical records to determine their medical diagnosis, prescribed medications and degree of i d (if present in medical record). the nature of the study was explained to the caregivers and signed written consents were obtained. the investigators pretested the questionnaire prior to the study to check its readability and caregivers ' understanding of questions. this questionnaire was originally developed by versloot. to measure possible dental pain - related behaviors in very young children and behaviors such as crying with meals or at night, problems of chewing or brushing, excessive salivation, or putting hands inside the mouth were among those included in the ddq+ (table 2). studies show that the behaviors included in the questionnaire were observed more often in children suffering from caries and toothache than in children with only caries or in children without caries or toothache. the investigators used arabic or english languages to describe the items of the questionnaire according to the native language of the caregiver. for each question in the ddq+, parents ' rated the occurrence of the behavior as never coded as zero, sometimes coded as 1, or frequently coded as 2. a total numeric ddq+ for each child and a mean ddq+ for the group were calculated. caregivers and investigators were also asked to rate the children 's dental pain on a ten - point scale where zero represented the absence of pain and ten the maximum pain imaginable. this scale was converted to an ordinal scale with mild, moderate, and severe categories. gentle removal of soft debris on teeth was done when needed without forceful use of dental explorer. caries examination was based on the who criteria where the dft / dft scores were measured. dental caries was considered at the level of frank cavitation which was defined as a break of 0.5 mm or more into enamel. filled teeth were defined as those with any type of restorations excluding sealants. because many included children were at the age of mixed dentition, the missing the severity of dental treatment needs was determined for each child based on the following categories. none : no restorative treatment required ; simple : preventative treatment required such as scaling, prophylaxis, oral hygiene instructions, topical fluoride application, or sealants ; moderate : one or more teeth requiring one- or two - surface restorations ; complex : one or more teeth requiring a three- or four - surface restorations, stainless steel crowns, endodontic therapy, or extractions. bivariate analyses were done to study associations between demographic, medical, and dental data and the ddq. regression models were also used with the main outcome variable being the average total ddq score. the level of significance () the study sampled a total of 86 children of which 33 were cases diagnosed with i d and 53 were healthy age - matched controls. the sampled children were categorized into three age groups ; birth to 6 years (n = 29), 6 to 12 years (n = 53), and 12 to 16 years (n = 4). no significant difference was found between cases and controls with regards to demographical data including age (x = 3.92, p = 0.4), gender (x = 0.25, p = 0.62), and mother 's education (x = 2.05, p = 0.56) or occupation (x = 0.37, p = 0.54). a significant association however was found between father 's education and having a child with i d (x = 8.46, p = 0.04). mothers were the caregivers answering the questionnaires in the majority of participating children (73.6% in healthy children and 84.4% in children with i d). among children with i d, 31% were perceived by their parents to have mild delays, 37.9% moderate, and 24.1% severe delays, while 6.8% of parents did not recognize the presence of delay despite the presence of a confirmed diagnosis in the child 's medical record. the majority of children with i d were diagnosed at birth (84.8%). the results showed that 97% of children with i d had other associated medical problems compared to 26.4% of healthy children (p = 0.0001), and that 57.6% were taking medications compared to 13.2% of healthy children (x = 18.89, p = 0.0001). when asked about the child 's verbal abilities, 57.6% of parents with children having i d replied that their children could speak. among those, 74% (n = 14) were perceived to have partly intelligible speech and 26% (n = 5) very intelligible speech. children with i d had significantly higher prevalence of heart (p = 0.05) and respiratory (p = 0.05) conditions, growth problems (p = 0.03), seizures (p = 0.0001), and verbal and physical limitations (p = 0.0001 and 0.007, resp.). table 1 shows a comparison of the dft and dft scores in healthy controls and those with i d. it can be seen that the dft score was significantly higher in children with i d (p = 0.04) and that these children had higher d component compared to that in healthy children (p = 0.03). the dft score on the other hand, was higher in healthy children (p = 0.04) and so was its d component (p = 0.05). the results essentially show that caregivers ' perception of dental pain was comparable in healthy children and those with i d. the investigators also showed no significant difference in their perception of dental pain among the two groups. the investigators however, perceived more children to have dental pain compared to caregivers and rated the pain higher on the pain scale. when caregivers were asked about specific behaviors related to dental pain (ddq+) results showed that the mean ddq+ in healthy children was 4.19 3.26 and in children with i d was 4.3 3.5 with no significant difference (mann - whitney = 0.08, p value = 0.94). also, the mean ddq+ in children with i d who had dental caries (4.55 3.46) was not significantly different from that in healthy children (4.19 3.26) with dental caries (p = 0.7). table 3 shows elements of the ddq+ in children with dental caries (healthy versus those with i d). the table shows that children with i d had significantly more salivation (p = 0.01) and were putting their hands inside their mouths more often (p = 0.003). healthy children with dental caries ; however, tended to chew more on one side of the mouth (p = 0.04). the assessment of dental pain by caregivers and investigators was compared in healthy children and those with i d to check for agreement (table 4). the results showed significant agreement between caregivers and investigators in both groups of children being more statistically significant in healthy children (p = 0.0001). the analysis looked at the correlation between the average ddq+ and the dft / dft scores. the results showed a significant positive correlation in healthy children between the ddq+ and dft score (spearman rho = 0.47, p = 0.02), but no correlation was found between the ddq+ and dft (p = 0.11). on the other hand, in children with i d, a significant positive correlation was found between the ddq+ and dft (spearman rho = 0.47, p = 0.01), but no correlation was found between the ddq+ and the dft (p = 0.3). the association between caregivers ' perception of the presence of dental pain and the complexity of dental treatment needs was evaluated (table 4). the results showed a stronger association between the ability of caregivers to determine pain and the complexity of treatment in children with i d than healthy children (p = 0.003) as seen in table 5. additionally, caregivers of children with i d were more able to determine severity of pain (mild, moderate, severe) with relation to complexity of treatment than caregivers of healthy children (p = 0.003). the regression model with the ddq+ as the outcome variable did not show any of the predictors (gender, mother 's education or occupation, father 's education, child 's medical condition, and medication intake) to be significant. reliable description of pain is generally difficult in children, and more so in those with i d due to their cognitive immaturity, lack of verbal skills, and their demonstration of idiosyncratic behaviors [5, 13 ]. research shows that children with disabilities are generally at higher risk of having their pain underestimated or undertreated. due to communication difficulties, children with i a study by hennequin. in 2003 showed that parents had more difficulties recognizing pain in children with down syndrome than healthy children. also, children with i d often attended dental visits when symptoms of acute dental pain arise [24, 25 ]. research shows a relationship between certain pain - associated behaviors and the presence of toothache and dental caries with children having dental caries or tooth ache displaying these behaviors more often. our results show that the dft score was significantly higher in children with i d whereas the dft was higher in healthy children. this was surprising as children with i d seemed to have more caries risk factors such as associated medical conditions and intake of medications. our assumption is that children with i d had both higher dft and dft scores compared to healthy children. however, because of their often uneasy behavior in dental settings, it is can be difficult to provide them with good quality dentistry which may at times make extractions a common treatment. because our calculations of the dft / dft scores did not include the missing teeth component, we may have underestimated the actual caries severity (dft score) in children with i d. this underestimation was not seen in the dft score because children in our sample were mostly 612 years old and may not have had extractions of permanent teeth yet. our data indicate that caregivers can recognize pain - related behaviors in children with i d such as excessive salivation and putting hands inside the mouth more often. this finding is in accord with previous research and indicates that parents become experts in their children 's behavior ; hence there are useful tools to healthcare providers in making diagnoses. the positive correlation between average ddq and dft / dft shows that the greater the severity of dental caries, the more likely the child will display pain - related behaviors, again a finding in accord with previous literature. these findings should alert both caregivers ' and dentists that determining the presence of dental pain in children with i d is a joint process between the two. some of these limitations included the lack of a professional assessment of the level of intellectual disability. however, a professional psychological assessment of their level of disability was not always present. future research in this area can be modified to include only children with professional psychological assessment and to stratify the analysis by level of intellectual disability. based on the investigators findings from this research, the following can be concluded.there is no significant difference in the general display of dental pain - related behaviors among healthy children and those with i d. children with i d display more salivation and putting hands inside the mouth in response to dental pain. the more severe the carious process the more the child will display pain - related behaviors. caregivers of children with i d who had complex dental treatment needs were more able to detect dental pain in their children compared to those of healthy children. there is no significant difference in the general display of dental pain - related behaviors among healthy children and those with i d. children with i d display more salivation and putting hands inside the mouth in response to dental pain. the more severe the carious process the more the child will display pain - related behaviors. caregivers of children with i d who had complex dental treatment needs were more able to detect dental pain in their children compared to those of healthy children.	purpose. description of pain is generally difficult in children, and more so in those with intellectual disabilities (i d). aim. this study is aimed at evaluating dental pain from caregivers ' perspective in children with i d. methods. the study sampled 86 children (33 with i d, 53 normally developing) ages ranges from birth to 16 years old among those visiting the school of dentistry, king abdulaziz university, saudi arabia. caregivers were asked about their awareness of dental pain in their wards using the dental discomfort questionnaire (ddq+). the children were examined for dental caries and periodontal status. results. the mean ddq+ in children with i d (4.55 3.46) was not significantly different from that in healthy children (4.19 3.26, p = 0.7). children with i d had more salivation (p = 0.01) and were putting their hands inside their mouths more often (p = 0.003). conclusions. caregivers can recognize dental pain - related behaviors in children with i d such as excessive salivation and putting hands inside the mouth more often.
interval mapping and multiple interval mapping are the most commonly used methods for qtl mapping. these methods are developed in the maximum likelihood framework, which has limitation in terms of handling large saturated models. bayesian mapping [37 ] deals with large models more efficiently through the reversible jump markov chain monte carlo (rjmcmc), the shrinkage analysis [8, 9 ], or the stochastic search variable selection (ssvs). shrinkage mapping and ssvs are more efficient in terms of whole genome evaluation because they are statistically easy to understand and also provide better chance to evaluate the entire genome. these two methods are related to the lasso method for regression analysis. rather than deleting nonsignificant qtl explicitly from the model, these methods use a special algorithm to shrink estimated qtl effects to zero or close to zero. a qtl with zero estimated effect is treated the same as being excluded from the model. no statistical test is required because genome regions bearing no qtl often show no bumps (qtl effects) in the qtl effect profile (plot of qtl effects against genome location). the visual inspection on the qtl effect profile is not optimal because small qtl may come and go during the mcmc sampling process. it is desirable to provide some kind of statistical confidence on these small qtl. permutation test itself is not a method of qtl mapping ; rather, it is a method to find the critical value used to declare the significance of qtl for any method of qtl mapping. a new resampling method was developed by zou. for significance test under the composite interval mapping or other multiple effect based qtl mapping schemes. the new resampling method is computationally less intensive and may perform better than the permutation test. the reason for this is perhaps due to the fact that the theory behind the method is not straightforward to most qtl mapping experimentalists. the permutation test, people tend to trust a simple method they understand, rather than a comprehensive method they do not, even if the simple method is suboptimal. therefore, the permutation test remains the most popular method for finding the critical value of a test statistic for qtl detection. kopp. applied the permutation test to determine empirical thresholds for bayesian shrinkage mapping. the problem with such a test for the mcmc implemented bayesian mapping is the heavy computational burden. each mcmc run may take one or a few hours to complete for a reasonable sample size of the mapping population. performing thousands of permutation analyses broman and speed treated multiple qtl mapping as a model (variable) selection problem and developed a new method called bic. more recently, manichaikul. extended the broman and speed model selection by allowing epistatic (nonallelic interaction) effects to be included in the model. they called the extended model selection method the penalized lod score method (plod). two versions of the penalized lod score method were investigated ; one is called the heavy penalized lod score (plodh) and the other is called the light penalized lod score (plodl). with this new notation, the original bic of broman and speed was renamed as ploda, penalized lod score for additive effects only. the authors compared these methods along with two other bic - based methods and the bayesian model selection method of yi. using both simulated data and real data. they concluded that the plod methods including epistatic effects and the bayesian model selection method outperformed other methods in most cases they evaluated. they can not replace the bayesian shrinkage analysis because the two have quite different purposes. we realized that if the bayesian shrinkage analysis is accompanied with a significance test, it can serve both qtl detection and genome selection. the original bayesian shrinkage analysis [8, 9 ] has no significance test associated with the method because the entire genome was evaluated simultaneously in a single model. more recently, researchers, especially animal and plant breeders, became interested in genome selection [18, 19 ] using the bayesian method. genome selection does not require statistical tests because qtl of the entire genome, regardless the sizes, are included to predict the genomic effect of individuals. however, there is no report so far to investigate whether inclusion of small qtl will benefit genome selection. cross - validation can be used to determine how large a qtl should be included in genome selection. for the paper to be self contained, we briefly introduce the bayesian shrinkage model here. let yj be the phenotypic value of a quantitative trait measured from individual j for j = 1, suppose that the individual is genotyped for m markers, which are more or less evenly distributed across the genome. let xjk be the genotype indicator variable for individual j at marker k for k = 1,, m. the linear model describing the relationship between the phenotype and the genotypes of markers is (1)yj = b0+k=1mxjkbk+ej, where b0 is the intercept, bk is the qtl effect for marker k, and ej is the residual error with an assumed n(0,) distribution. the reason that the bayesian shrinkage method can handle a large m is the prior distribution assigned to each qtl effect : (2)p(bk)=n(bk 0,k2), where k is a qtl specific prior variance. this prior alone is not sufficient to generate the desired shrinkage estimate of qtl effect. a hierarchical model with a higher level of prior assignment is necessary, in which the prior variance k is further assigned a scaled inverse chi - square distribution : (3)p(k2)=inv2(k2,). in the original shrinkage analysis, xu set = = 0, leading to p(k) = 1/k. ter braak. claimed that this prior is improper and leads to an improper posterior distribution. they revised the prior so that the posterior distribution becomes proper. their revised prior is (4)p(k2)=inv2(k2 2,0)1(k2)1, where 0 k20bkbk(12rkk)=19.6034. the proportion of the genetic variance contributed by each qtl is 0.5bk / vg for the kth qtl (given in the column headed with prop - g in table 1). the corresponding proportion of the phenotypic variance contributed by the kth qtl is 0.5bk / vp and given in the column headed with prop - p in table 1. all 241 markers were included in the model, leading to the dimensionality of the model of n (m + 1) = 500 (241 + 1). the burn in period was 1000. the chain was thinned by keeping one observation out of 10 iterations until the posterior sample size reached 5000. the true values of the qtl effects and the locations of the simulated qtl are depicted in table 1. say qtl numbers 19 and 20, the estimated effects are also small with values no larger than the bumps in the no qtl regions (noises). we calculated the equal tail credible interval at = 0.05, that is, the 2.5%97.5% percentile range, for each marker. only one (the largest) qtl was detected because the interval excluded 0 (data not shown). the equal - tail credible intervals of all other qtl covered zero, and thus, they are not significant in terms of statistical testing. using the equal tail credible interval at = 0.10, two more qtls were detected in addition to the largest qtl (data not shown). certainly, the equal tail credible interval is not a good criterion for significance test. the posterior distributions for most estimated qtl effects have a special distribution with a spike at zero, which is the cause for the failure of equal tail credible interval as the criterion for significance test. the reason is that almost all qtls have an equal - tail interval covering the null value, for example, zero. even the largest qtl in our simulation had a high probability mass at zero (see figure 2). this spike - shaped or zero - inflated posterior distribution for qtl effect is typical in bayesian shrinkage mapping. if we had used the equal tail interval at = 0.05 as the significance test criterion, only one qtl (the largest one), out of the 20 simulated qtl, would have reached the statistical significance level. the permutation test, however, detected many major qtl, as demonstrated next in the permutation test sections. each permuted sample was subject to the same mcmc analysis as the original data (51000 iterations). the sas / iml program took approximately 20 days in a dell pc (2.5 ghz and 3.25 go of ram). for each marker, the 2.5%97.5% and 5%95% intervals (corresponding to = 0.05 and = 0.10) were calculated. the profiles of these percentiles along with the estimated qtl effects are given in figure 3(a). using the 2.5%97.5% interval, we can detect 15 qtl out of the 20 simulated qtl. a few more qtls with small effects the results here are more reasonable than those when the equal tail credible interval was used. this permutation strategy only requires running one more chain in addition to the mcmc run of the original data. we first evaluated the performance of h = 1, that is, reshuffling the phenotype in every iteration. the 2.5%, 5%, 95%, and 97.5% percentiles plotted against the genome location are shown in figure 3(b) to compare with the result of permutation outside the chains. these intervals (the within - chain permutation) appear to be wider than the intervals of the between - chain permutation analysis. therefore, the tests for the within - chain permutation are more conservative than the between - chain permutation. using the within - chain permutation, 13 qtls were detected for = 0.05 and 19 qtls were detected for = 0.10, not too much different from the result of the between - chain permutation. a more conservative test is better than a more liberal test, as long as the statistical power is not compromised (examined later in the power study section). the 2.5%, 5%, 95%, and 97.5% percentiles plotted against the genome location are shown in figure 4. these intervals appear to be similar to h = 1 except that the higher h 's tend to generate rougher percentile profiles. therefore, h = 1 is more preferable than other values of h. hereafter, we chose h = 1 for all subsequent analysis. using the same parameters given in table 1, we simulated 100 more independent samples to investigate the statistical power of the bayesian shrinkage method. one run was the mcmc sampler on the original data to estimate qtl effects and the other run was the mcmc sampler on the within - chain reshuffled data to generate the critical values for qtl detection. the statistical power for each qtl was calculated based on the proportions of samples in which the qtl fell outside the empirical intervals. we observed that if a true qtl failed to be detected at the locus where it was placed, the effect was often picked up by a marker nearby (10 cm away). therefore, a true qtl was claimed to be detected if one or more of the triplets (three loci) covering the true qtl (20 cm range) was detected. the powers seem to be reasonable ; seven out of the 20 simulated qtl have a power reached 80% at = 0.10. therefore, the conservative within - chain permutation significance test does not sacrifice much statistical power. for the 241 marker effects included in the model, were reserved for the true qtl (20 true qtl plus 40 flanking markers), leaving 241 60 = 181 model effects as false qtl. if a false qtl was detected in a particular sample, it was counted as one false positive. for each false qtl the proportion of false positive (false positive rate or type i error) was recorded for each false qtl simulated. the observed false positive rate is indeed less than 0.05, confirming our previous conclusion that the within - chain permutation approach is conservative. the average false positive rate for all these markers was about 0.05, again confirming the conservativeness of the within chain permutation approach. using the original data simulated in the beginning of the experiment (not a sample from the power study), we performed the fivefold cross - validation study to determine how large a qtl should be included in the model to predict the total genetic value of an individual. the pe (squared prediction error) values are plotted against the value in figure 7. the decrease of the pe from = 0.0 to = 0.2 is very sharp, but after = 0.2, the pe value tends to be stabilized or slightly increased. the conclusion is that in genome selection, we should choose the value around 0.2. of course we recommend such a cross - validation test for each data analysis to determine how many qtl should be included. from the pe profile, including all qtls (= 1) into the prediction model (regardless the sizes of the qtl) does not lead to any significant loss in the precision of genome selection compared to the optimal number of qtl determined by the cross - validation test. therefore, a robust choice is to include all qtls in the model for genome selection. we now use three sample data to demonstrate the application of the permutation test - associated bayesian shrinkage analysis. these data were collected from qtl mapping experiments in model plants and agricultural crops. the first dataset is the recombinant inbred line data of arabidopsis data, where the two parents initiating the line cross were bay-0 and shahdara with bay-0 as the female parent. the recombinant inbred lines were actually f7 progeny of single seed descendants of the f2 plants. flowering time was recorded for each line in two environments : long day (16-hour photoperiod) and short day (8-hour photoperiod). a couple of lines did not have the phenotypic records and their phenotypic values were replaced by the population mean for convenience of data analysis. these markers are more or less evenly distributed along five chromosomes with an average 10.8 centimorgan (cm) per marker interval. we inserted a pseudomarker in every 2 cm of the genome. including the inserted pseudomarkers, the total number of loci subject to analysis was 200 (38 true markers plus 162 pseudomarkers). therefore, the model for the short day flowering time trait is (9)y = b0+k=1200xkbk+, where xk is a 420 1 vector coded as 1 for one genotype and 0 for the other genotype for locus k. if locus k is a pseudomarker, xk = pr(genotype = 1), which is the conditional probabilities of marker k being of genotype 1. finally, bk is the qtl effect of locus k. for the original data analysis, the burn - in period was 1000. the posterior sample size was 10000, and thus the total number of iterations was 1000 + 10000 10 = 101000. the posterior sample size of the within - chain permutation analysis was 80000, that is, 1000 + 80000 10 = 801000 iterations in total. the estimated qtl effects and the permutation generated 2.5%97.5% and 5%95% intervals are plotted in figure 8(a). chromosomes 1 and 4 each has one qtl and chromosome 5 has two qtl. when = 0.10 was used, one more qtl on chromosome 1 was detected. the fivefold cross - validation shows that the optimal strategy of genome selection for this dataset was to include all qtls in the model, regardless the significance of the estimated qtl effects (see figure 8(b)). the general pattern of the pe profile remains the same as that of the simulated data. below = 0.2 the decrease of pe was dramatic but after = 0.2 the pe values approached a stable value. the second data are the doubled haploid (dh) data obtained from luo.. this dataset consists of 150 doubled haploids (dhs) derived from the cross of two spring barley varieties, steptoe and morex, designated as the s m cross. the phenotype was the spot blotch (a fungus cochliobolus sativus) resistance measured as the lesion size on the leaves of barley seedlings. the total number of markers was 495 distributed along seven chromosomes of the barley genome. because of the small sample size, we could not analyze all the 495 markers simultaneously (high collinearity). therefore, we placed one pseudomarker in every 5 cm and overall obtained 225 pseudomarkers for the entire genome. therefore, the model for the disease resistance trait is (10)y = b0+k=1225xkbk+, where xk is a 150 1 vector coded as 1 for one genotype and 0 for the other genotype for locus k. if locus k is a pseudomarker, xk = pr(genotype = 1), which is the conditional probabilities of marker k being of genotype 1. finally, bk is the qtl effect of locus k. the parameters of the mcmc experiment (e.g., burn - in period, thinning rate, etc.) the estimated qtl effects and the permutation - generated 2.5%97.5% and 5%95% intervals are plotted in figure 9(a). these two are major qtl because their estimated values are way over the critical value. when the critical values at a = 0.10 were used, five more qtls were declared as significant. the cross - validation analysis shows that the optimal strategy of genome selection for this dataset was to include all qtls that are significant at = 0.15 (see figure 9(b)). below = 0.15 the decrease of pe was dramatic but after = 0.15 the pe values increased slightly until they reached a plateau at = 0.3 (see figure 9(b)). this example demonstrated the usefulness of using cross - validation to select qtl for inclusion for prediction of genomic effect. this example demonstrates the application of the bayesian shrinkage analysis to qtl mapping for the number of seeded spikelets (a female fertility trait) in wheat. the experiment was conducted by dou. who made the data available to us for this analysis. a female sterile line xnd126 and an elite cultivar gaocheng 8901 with normal fertility were crossed for genetic analysis of female sterility measured as a quantitative trait. the f1 and f2 progeny of the parents were planted at the huaian experimental station in china for the 2006 - 2007 growing season under the normal autumn sowing condition. these markers covered 5 chromosomes of the wheat genome with an average genome marker density of 15.5 cm per marker interval. these chromosomes were scanned for qtl of the fertility trait using the mcmc implemented bayesian method. the dependent variable was the fertility phenotype while the independent variables were numerically coded genotype indicator variables for the part of genome under investigation. we placed one pseudomarker in every 5 centimorgan (cm) of the genome. therefore, we have a total of 75 independent variables. for each independent variable, the numerically coded value was the difference between the conditional probabilities of the two homozygote genotypes. let a1a1, a1a2, and a2a2 be the three genotypes for the kth pseudo marker of the genome. the numerically coded value for the locus is (11)xjk = p(gjk = a1a1 marker)p(gjk = a2a2 marker) for k = 1,, 75. the map of the 75 pseudomarkers, the phenotypic values of the 243 plants, and the 75 numerically coded independent variables can be obtained from the authors. the parameters of the mcmc experiment (e.g., burn - in period, thinning rate, etc.) the estimated qtl effects and the permutation - generated 2.5%97.5% and 5%95% intervals are plotted in figure 10(a). when we lowered the critical value to = 0.10, one more qtl was detected on chromosome 5. the cross - validation shows that the optimal strategy of genome selection for this dataset was to include all qtls that are significant at = 0.1 (see figure 10(b)). below = 0.1, the decrease of pe was dramatic but after = 0.1 the pe values increased slightly until they reached a plateau at = 0.3. in general, the optimal gamma value is somewhere between 0.1 to 0.2, but it varied from one experiment to another. the last two data analyses did indicate that including small qtl can be detrimental to genome selection. cross - validation is an experimental specific approach and is useful to decide how large a qtl should be included in the model for genome selection. qtl mapping aims to detecting qtl with large effects while genome selection tries to predict the total genetic values of individuals using markers of the entire genome. in qtl mapping, significance test is important, but bayesian inference usually does not mix with significance test. this is because bayesian inference focuses on the probability statement of a parameter given the information drawn from the current data and it does not intend to extend the statement beyond the data. significance test, however, assumes a null distribution and tries to compare the statistics against the null distribution. the null distribution is purely hypothetical and, therefore, significance test gives conclusion that applies to hypothetical future experiments. the permutation test adopted in the bayesian analysis is a convenient way to connect significance test with bayesian analysis. permutation analysis is a way to draw the null distribution. if a statistics, for example, estimated qtl effect, is far away from the null distribution, we are confident that this qtl is true. this type of significance test provides different conclusion from the bayesian credible statement. in bayesian analysis, people often report the -equal - tail interval or -highest posterior density (hpd) interval. the reason is that almost all qtls have an equal - tail interval covering the null value, for example, zero. even the largest qtl in our simulation had a high probability mass at zero (see figure 2). this zero - inflated posterior distribution for qtl effect is typical in bayesian shrinkage mapping. if we had used the equal tail interval at = 0.05 as the significance test criterion, only one qtl (the largest one), out of the 20 simulated qtls, would have reached the statistical significance level. in the simulation experiment, we observed that the percentile profiles for the 0.5 100% (1 0.5) 100% interval were pretty much constant across the entire genome (see figures 3 and 4). we simulated 241 markers covering the entire genome evenly with 10 cm per marker interval. in contrast, the three real data analyses showed that the percentile lines varied dramatically across the genome. the intervals were narrow at marker positions and wide when the positions are away from the markers. the lengths of marker intervals also varied across the genome, making the information content much uneven across the genome. the location specific empirical threshold values in real data analysis mean that different locations of the genome should use different criteria for qtl detection. we have two qtl with the same estimated effect but located in different regions of the genome, one may be declared as significant but the other may not be significant due to the variation in information content. this actually justifies the use of estimated qtl effects, not some kind of test statistics, for significance test. in classical qtl mapping experiments, investigators always use some kinds of test statistics (e.g., t - test, f - test, likelihood ratio test, or lod score) to decide whether a qtl is significant or not. a permutation test also draws critical values for the test statistic under consideration, not the critical values for the qtl effects. this merely reflects the tradition or convention of people who do statistical analysis and does not mean that a test statistic is the only quantity that can be used in qtl mapping. the reason for using test statistics is that one can compare the observed test statistic (calculated values) with the critical values of some distribution, for example, normal distribution, f - distribution, t - distribution, and chi - square distribution. the critical values of these standard distributions can be found from statistical tables or calculated from statistical analysis software. with the permutation test therefore, there is no need to use the test statistics. directly comparing the estimated qtl effects with the critical values the significant qtl will be the targets for further study, for example, cloning or marker assisted selection. what do we do with those qtls whose effects do not reach the significance level ? these qtls may not be significant individually, but collectively they may contribute to a large proportion of the phenotypic variance. this implies that they are perhaps useful to predict the total genetic effects of individuals, a technology called genome selection. our cross - validation experiments showed that qtls should be used to predict the total genetic effects once they reached a certain critical value. common sense tells us that estimated effects of small qtls are most likely caused by noises rather than by true signals and inclusion of the many small qtls to predict the genetic effects may be even worse than inclusion of only the significant qtl. we developed two permutation tests, one is called permutation outside markov chain and the other is called permutation inside markov chain. the latter is recommended because it is (1) faster computationally and (2) slightly more conservative. cross - validation studies showed that small qtls should be excluded from the model for prediction of the total genetic effects for individual plants. the criterion of exclusion is experimental specific and should be decided by cross - validation.	bayesian shrinkage analysis is the state - of - the - art method for whole genome analysis of quantitative traits. it can estimate the genetic effects for the entire genome using a dense marker map. the technique is now called genome selection. a nice property of the shrinkage analysis is that it can estimate effects of qtl as small as explaining 2% of the phenotypic variance in a typical sample size of 300500 individuals. in most cases, qtl can be detected with simple visual inspection of the entire genome for the effect because the false positive rate is low. as a bayesian method, no significance test is needed. however, it is still desirable to put some confidences on the estimated qtl effects. we proposed to use the permutation test to draw empirical thresholds to declare significance of qtl under a predetermined genome wide type i error. with the permutation test, bayesian shrinkage analysis can be routinely used for qtl detection.
the anterior cruciate ligament (acl) is a commonly injured knee ligament in the united states population, and accordingly, there are more than 150,000 acl surgical reconstruction procedures performed per year.1 the acl can be torn completely, which has a clear natural history of resulting in long - term instability in 15%66% of patients, or be partial torn, in which the natural history is less clear.2 a review by pujol showed that partial acl tears could have a favorable short- to medium - term outcome with nonoperative management if sports activity was limited. surgical indications for both partial and complete acl ruptures are continued instability, or ligament injury and returning to pivoting sport.3 although acl reconstruction can have good success rates, one of the potential adverse outcomes following acl reconstruction is lack of restoration of full physiologic movement, due to the biomechanically disadvantageous location of the graft placement.4 this leads to increased tibial rotation and impaired neuromuscular control during high - demand activities among patients with acl grafts,4 potentially resulting in increased cartilage load and thus higher risk of reinjury. for these and other reasons, although surgical reconstruction remains a standard and common procedure for management of acl tears, alternative treatments are needed that can both address the integrity of the ligament and at the same time maintain the biomechanical competence of the knee joint. regenerative therapy, a cell - based technique directed at improved healing of the injured ligament, carries with it the potential of preserving the proprioceptive nerve fibers and collagenous architecture of the ligament, which in turn, preserves the normal biomechanics of the knee joint.5 several experimental techniques aimed at regeneration of the acl have been described in the literature. the application of extracellular matrix derived from pigs, combined with suture repair of torn acls, has been shown to accelerate healing of the ligament versus suture repair alone in a goat model.6 in other animal studies, partial acl tears treated with platelet rich plasma (prp) combined with a collagen matrix demonstrated healing and improved function in comparison with a control group.7 seijas reported high return to sport in professional football players with a partial acl tear treated with surgical reconstruction of the torn bundle and intraligamentous placement of platelet - derived growth factors into the intact bundle.2 other in vitro and in vivo animal models have demonstrated neovascularization, granulation, and stimulation of mesenchymal stem cells (mscs), such as fibroblasts, following the application of various growth factors and prp to torn acls.7 additional animal studies have described intra - articular (ia) injection of bone marrow derived mscs, with promising results for repair of acl tears.7,8 there may be merit in healing acl tears in situ. first, acl grafts are placed in a nonphysiologic position, which could impact normal biomechanics.9 second, osteoarthritis is common after acl reconstruction.10 one possible benefit from mscs is that they may aid in cartilage repair.11 finally, proprioception is often lost after acl repair,12 which may be due to the fact that the ligament graft does nt contain viable or intact proprioceptors. an in site repair may allow for retention of intact proprioceptors, leading to better neuromuscular control. these principles may translate to an effective clinical application, but as of the date of the present study, there is a paucity of evidence for msc therapy as a treatment for acl injuries in humans. adult mscs have been demonstrated as safe, multipotent therapeutic agents in regenerative medicine, including for a number of orthopedic applications.13 we have previously published longitudinal studies demonstrating the safety and efficacy of mscs to treat knee osteoarthritis.14,15 in the present study, we describe a treatment for magnetic resonance imaging (mri)-documented acl tears in a case series of adults using bone marrow concentrate (bmc) rich in mscs, in which therapeutic efficacy was assessed by pain and functional outcome measures as well as mri evidence of structural alteration of the acl. this is an introductory study on the use of regenerative therapies for treatment of different acl injuries. we believe there is a subset of patients who may benefit from regenerative therapies, such as patients with nonhealing grade 12 tears for whom surgery may not be recommended or patients with grade 3 tears without retraction of the ligament fibers who are surgical candidates but wish to avoid surgery. based on the work of rudolph, patients with more normal knee kinematics after acl rupture (copers) may perform better than those with functional instability (noncopers).16 with this study, we hoped to gain insight on the safety and preliminary efficacy for this treatment, as well as provide the foundation for future more rigorous investigation. consecutive patients presenting to an interventional pain practice with complaints of knee instability with or without pain and with an acl tear previously documented with mri, and laxity with lachman testing17 on exam were enrolled in a case series of the first set of acl treatments, under a larger registry of all msc treatments tracked by our clinic. patients were included in the analysis of prospectively collected registry data if they had a grade 1, 2, or 3 acl tear without greater than 1 cm retraction, and agreed to participate. we defined retraction as any visible area of increased mri signal intensity within the substance of the ligament that was full thickness and resulted in a discontinuity of the course of the structure. to our knowledge, this is the first study of acl tears to focus on the extent of the separation of torn acl fragments, thus this delineation has not been validated previously. patients treated during acute (6 months postinjury)18 were eligible for inclusion. exclusion criteria were grade 3 acl tear with > 1 cm retraction, active neoplasm within the past 5 years, a history or presence of anemia, or age younger than 16 years. were recorded at baseline and prospectively at 1 month, 3 months, 6 months, and annually following treatment. additionally, percentage improvement on a likert scale was recorded at 1 month, 3 months, 6 months, and annually following treatment. institutional review board oversight and approval acl injuries were graded as follows:19 grade 1 sprain : the ligament is partial torn, with less than half of the ligament substance disruptedgrade 2 sprain : the ligament is partially torn, with more than half of the ligament substance disruptedgrade 3 sprain : the ligament is completely torn. grade 1 sprain : the ligament is partial torn, with less than half of the ligament substance disrupted grade 2 sprain : the ligament is partially torn, with more than half of the ligament substance disrupted grade 3 sprain : the ligament is completely torn. the first step of the treatment was a preinjection of a hypertonic dextrose solution into the acl 25 days prior to injection of the bmc. the purpose of this preinjection procedure was to introduce a chemical irritant to the acl in order to prompt a brief inflammatory response. a 25 gauge 3.0 inch quinke needle (catalog number 405170 ; bd biosciences, franklin lakes, nj, usa) was inserted through the skin overlying the patellar tendon and directed through the inferior patellar tendon to a location just anterior to the tibial spine, on lateral fluoroscopy. iodixanol (visipaque ndc 0407 - 2223 - 06 ; ge healthcare, little chalfont, uk) radiographic contrast was injected to confirm flow in the acl sheath traveling between the radiographic origin and insertion landmarks, in both views (figure 1). this was followed by injection of 35 ml of 12.5% dextrose (ndc 0409 - 6648 - 02) and 0.1% lidocaine (ndc 0409 - 4276 - 02) in normal saline (ndc 0409 - 4888 - 50). the next step of the treatment was to harvest bone marrow and isolate the portion containing mscs from each patient, in preparation for reinjection. prior to the procedure, the patients were restricted from taking corticosteroids and nonsteroidal anti - inflammatory drugs (nsaids) for at least 2 weeks, as these medications can impair soft tissue healing.2024 whole bone marrow isolate was harvested from the patients iliac crest, under ultrasound or fluoroscopic guidance. approximately 1015 ml of bone marrow aspirate was drawn from six sites into heparinized syringes. there were 1,000 units of heparin (ndc 25021 - 403 - 01 and 25021 - 404 - 01) per 1 ml of volume collected in the syringe. the aspirate was processed by hand, in a sterile iso-7-class clean room and in iso-5-class laminar flow cabinets, to isolate the buffy coat through centrifugation. this isolation produced 15 ml of bmc, which was then taken via sterile transport over the short distance back to the operating room. the nucleated cell count of the injectate was counted and recorded by lab staff with a cell counter (tc10 ; bio - rad laboratories, hercules, ca, usa) via light microscopy. coincident with this harvest procedure, approximately 60 ml of heparinized venous blood was drawn to be used for isolating prp and platelet lysate (pl). to prepare the prp, plasma was prepped via centrifugation at 200 g to separate the plasma and buffy coat layers from the red blood cells. the resultant liquid lying above the concentrated solids (supernatant) was red cell / white cell poor. to prepare the pl, prp was drawn off and stored at 20c overnight ; platelets were recentrifuged, and the supernatant drawn off. all samples were processed in a current good manufacturing practice (cgmp) air - handling lab, using off the shelf equipment and a dedicated lab staff. needle placement into the acl was accomplished utilizing the same procedural protocol described for the preinjection. the injectate consisted of 23 ml of bmc, prp, and pl, and was injected directly into the ligament. the needle was withdrawn from the ligament approximately 1 cm, and while still in the joint, approximately 24 ml of a mixture of 1 ml of prp, 1 ml of pl, and any remaining bmc were injected into the joint. after the procedure, the patients were given instructions to participate in activity as tolerated. all of the patients were encouraged to participate in physical therapy, but this was not required nor controlled. patients were enrolled in a treatment registry and tracked prospectively via an electronic database system using clincapture software (clinovo clinical data solutions, sunnyvale, ca, usa) (http://www.clinovo.com/clincapture). the program includes an automated emailing system to send patients clinical outcome questionnaires to complete. a pain visual analog scale (vas) and the lower extremity functional scale (lefs)25 were recorded preoperatively (same day as the procedure) and postoperatively. postoperatively, matching questionnaires were sent at 1 month, 3 months, 6 months, 12 months, and 24 months. in addition, a subjective percentage - improvement likert - scale (from 90% worsened to 100% improved) was recorded postoperatively. the functional questionnaires for gathering patient follow - up data were updated as part of changes to the treatment registry, and thus for the first two patients, the pretreatment functional data were recorded using the functional rating index (fri),26 whereas posttreatment function was recorded using the lefs. however, the likert improvement and the complications questions were the same throughout the study period. patients received a mri scan of the knee, prior to treatment and at a minimum of 3 months posttreatment. the model and field strength of the mri scanners as well as the available sequences varied between patients ; however, for each patient, the pre- and posttreatment scans were always performed on the same scanner and with the same sequences. to quantify and reduce the variability in the interpretation of the changes in the mri appearance of the acl ligaments imagej is a public java image processing and analysis program developed at the national institutes of health (nih) (http://rsbweb.nih.gov/ij/). we chose metrics that the software could produce that objectively measure gray scale. the metrics used to assess the appearance of the acl were the mean gray value, modal gray value, median, skewness, and raw integrated density. once both pre- and posttreatment images were obtained, the image selected for the pretreatment ligament integrity assessment was the sagittal t1 weighted, proton density (pd)-weighted acl sequence, pd sequence, pd fast spin - echo sequence, or the pd fat saturation sequence whichever image visualized the greatest cross - sectional area of the acl. for the posttreatment analysis, the closest matching image was selected from the same imaging sequence type. if a single sagittal image was insufficient to capture a complete slice through the acl, two adjoining images were used for analysis. the examiner manually outlined the acl to create a region of interest (roi). the examiner was instructed to only outline where he believed the acl to be on the image and that the posterior extent of the roi should not be more posterior than the posterior margin of the femoral condyle. imagej created a histogram of pixel frequency and intensity as a proxy for ligament integrity (figure 2). it also calculated the selected metrics : mean gray value, modal gray value, median, skewness, and raw integrated density. three random acl images were selected and then measured by three examiners (cjc, bn, and jp), with each examiner manually outlining the acl using the protocol stated above. the examiners were blinded both as to whether the image was pre- or posttreatment, as well as regarding the results of the other examiners. the resulting three images were measured serially three times each, and then the results were assessed for intra- and interexaminer agreement. the averages of the three serial measurements for each image were used for the statistical analysis. procedures of sas software (sas 9.4 ; sas institute inc., cary, nc, usa) were used to perform all statistical analyses and calculations. to test the differences between pretreatment and posttreatment scores, we performed the signed - rank test, a nonparametric test for dependent samples, utilizing the univariate procedure. the first step of the treatment was a preinjection of a hypertonic dextrose solution into the acl 25 days prior to injection of the bmc. the purpose of this preinjection procedure was to introduce a chemical irritant to the acl in order to prompt a brief inflammatory response. a 25 gauge 3.0 inch quinke needle (catalog number 405170 ; bd biosciences, franklin lakes, nj, usa) was inserted through the skin overlying the patellar tendon and directed through the inferior patellar tendon to a location just anterior to the tibial spine, on lateral fluoroscopy. iodixanol (visipaque ndc 0407 - 2223 - 06 ; ge healthcare, little chalfont, uk) radiographic contrast was injected to confirm flow in the acl sheath traveling between the radiographic origin and insertion landmarks, in both views (figure 1). this was followed by injection of 35 ml of 12.5% dextrose (ndc 0409 - 6648 - 02) and 0.1% lidocaine (ndc 0409 - 4276 - 02) in normal saline (ndc 0409 - 4888 - 50). the next step of the treatment was to harvest bone marrow and isolate the portion containing mscs from each patient, in preparation for reinjection. prior to the procedure, the patients were restricted from taking corticosteroids and nonsteroidal anti - inflammatory drugs (nsaids) for at least 2 weeks, as these medications can impair soft tissue healing.2024 whole bone marrow isolate was harvested from the patients iliac crest, under ultrasound or fluoroscopic guidance. approximately 1015 ml of bone marrow aspirate was drawn from six sites into heparinized syringes. there were 1,000 units of heparin (ndc 25021 - 403 - 01 and 25021 - 404 - 01) per 1 ml of volume collected in the syringe. the aspirate was processed by hand, in a sterile iso-7-class clean room and in iso-5-class laminar flow cabinets, to isolate the buffy coat through centrifugation. this isolation produced 15 ml of bmc, which was then taken via sterile transport over the short distance back to the operating room. the nucleated cell count of the injectate was counted and recorded by lab staff with a cell counter (tc10 ; bio - rad laboratories, hercules, ca, usa) via light microscopy. coincident with this harvest procedure, approximately 60 ml of heparinized venous blood was drawn to be used for isolating prp and platelet lysate (pl). to prepare the prp, plasma was prepped via centrifugation at 200 g to separate the plasma and buffy coat layers from the red blood cells. the resultant liquid lying above the concentrated solids (supernatant) was red cell / white cell poor. to prepare the pl, prp was drawn off and stored at 20c overnight ; platelets were recentrifuged, and the supernatant drawn off. all samples were processed in a current good manufacturing practice (cgmp) air - handling lab, using off the shelf equipment and a dedicated lab staff. needle placement into the acl was accomplished utilizing the same procedural protocol described for the preinjection. the injectate consisted of 23 ml of bmc, prp, and pl, and was injected directly into the ligament. the needle was withdrawn from the ligament approximately 1 cm, and while still in the joint, approximately 24 ml of a mixture of 1 ml of prp, 1 ml of pl, and any remaining bmc were injected into the joint. after the procedure, the patients were given instructions to participate in activity as tolerated. all of the patients were encouraged to participate in physical therapy, but this was not required nor controlled. the first step of the treatment was a preinjection of a hypertonic dextrose solution into the acl 25 days prior to injection of the bmc. the purpose of this preinjection procedure was to introduce a chemical irritant to the acl in order to prompt a brief inflammatory response. a 25 gauge 3.0 inch quinke needle (catalog number 405170 ; bd biosciences, franklin lakes, nj, usa) was inserted through the skin overlying the patellar tendon and directed through the inferior patellar tendon to a location just anterior to the tibial spine, on lateral fluoroscopy. iodixanol (visipaque ndc 0407 - 2223 - 06 ; ge healthcare, little chalfont, uk) radiographic contrast was injected to confirm flow in the acl sheath traveling between the radiographic origin and insertion landmarks, in both views (figure 1). this was followed by injection of 35 ml of 12.5% dextrose (ndc 0409 - 6648 - 02) and 0.1% lidocaine (ndc 0409 - 4276 - 02) in normal saline (ndc 0409 - 4888 - 50). the next step of the treatment was to harvest bone marrow and isolate the portion containing mscs from each patient, in preparation for reinjection. prior to the procedure, the patients were restricted from taking corticosteroids and nonsteroidal anti - inflammatory drugs (nsaids) for at least 2 weeks, as these medications can impair soft tissue healing.2024 whole bone marrow isolate was harvested from the patients iliac crest, under ultrasound or fluoroscopic guidance. approximately 1015 ml of bone marrow aspirate was drawn from six sites into heparinized syringes. there were 1,000 units of heparin (ndc 25021 - 403 - 01 and 25021 - 404 - 01) per 1 ml of volume collected in the syringe. the aspirate was processed by hand, in a sterile iso-7-class clean room and in iso-5-class laminar flow cabinets, to isolate the buffy coat through centrifugation. this isolation produced 15 ml of bmc, which was then taken via sterile transport over the short distance back to the operating room. the nucleated cell count of the injectate was counted and recorded by lab staff with a cell counter (tc10 ; bio - rad laboratories, hercules, ca, usa) via light microscopy. coincident with this harvest procedure, approximately 60 ml of heparinized venous blood was drawn to be used for isolating prp and platelet lysate (pl). to prepare the prp, plasma was prepped via centrifugation at 200 g to separate the plasma and buffy coat layers from the red blood cells. the resultant liquid lying above the concentrated solids (supernatant) was red cell / white cell poor. to prepare the pl, prp was drawn off and stored at 20c overnight ; platelets were recentrifuged, and the supernatant drawn off. all samples were processed in a current good manufacturing practice (cgmp) air - handling lab, using off the shelf equipment and a dedicated lab staff. needle placement into the acl was accomplished utilizing the same procedural protocol described for the preinjection. the injectate consisted of 23 ml of bmc, prp, and pl, and was injected directly into the ligament. the needle was withdrawn from the ligament approximately 1 cm, and while still in the joint, approximately 24 ml of a mixture of 1 ml of prp, 1 ml of pl, and any remaining bmc were injected into the joint. after the procedure all of the patients were encouraged to participate in physical therapy, but this was not required nor controlled. patients were enrolled in a treatment registry and tracked prospectively via an electronic database system using clincapture software (clinovo clinical data solutions, sunnyvale, ca, usa) (http://www.clinovo.com/clincapture). the program includes an automated emailing system to send patients clinical outcome questionnaires to complete. a pain visual analog scale (vas) and the lower extremity functional scale (lefs)25 were recorded preoperatively (same day as the procedure) and postoperatively. postoperatively, matching questionnaires were sent at 1 month, 3 months, 6 months, 12 months, and 24 months. in addition, a subjective percentage - improvement likert - scale (from 90% worsened to 100% improved) was recorded postoperatively. the functional questionnaires for gathering patient follow - up data were updated as part of changes to the treatment registry, and thus for the first two patients, the pretreatment functional data were recorded using the functional rating index (fri),26 whereas posttreatment function was recorded using the lefs. however, the likert improvement and the complications questions were the same throughout the study period. patients received a mri scan of the knee, prior to treatment and at a minimum of 3 months posttreatment. the model and field strength of the mri scanners as well as the available sequences varied between patients ; however, for each patient, the pre- and posttreatment scans were always performed on the same scanner and with the same sequences. to quantify and reduce the variability in the interpretation of the changes in the mri appearance of the acl ligaments, we used computerized pixel analysis, using imagej software. imagej is a public java image processing and analysis program developed at the national institutes of health (nih) (http://rsbweb.nih.gov/ij/). the metrics used to assess the appearance of the acl were the mean gray value, modal gray value, median, skewness, and raw integrated density. once both pre- and posttreatment images were obtained, the image selected for the pretreatment ligament integrity assessment was the sagittal t1 weighted, proton density (pd)-weighted acl sequence, pd sequence, pd fast spin - echo sequence, or the pd fat saturation sequence whichever image visualized the greatest cross - sectional area of the acl. for the posttreatment analysis, the closest matching image was selected from the same imaging sequence type. if a single sagittal image was insufficient to capture a complete slice through the acl, two adjoining images were used for analysis. the examiner manually outlined the acl to create a region of interest (roi). the examiner was instructed to only outline where he believed the acl to be on the image and that the posterior extent of the roi should not be more posterior than the posterior margin of the femoral condyle. imagej created a histogram of pixel frequency and intensity as a proxy for ligament integrity (figure 2). it also calculated the selected metrics : mean gray value, modal gray value, median, skewness, and raw integrated density. three random acl images were selected and then measured by three examiners (cjc, bn, and jp), with each examiner manually outlining the acl using the protocol stated above. the examiners were blinded both as to whether the image was pre- or posttreatment, as well as regarding the results of the other examiners. the resulting three images were measured serially three times each, and then the results were assessed for intra- and interexaminer agreement. the averages of the three serial measurements for each image were used for the statistical analysis. procedures of sas software (sas 9.4 ; sas institute inc., cary, nc, usa) were used to perform all statistical analyses and calculations. to test the differences between pretreatment and posttreatment scores, we performed the signed - rank test, a nonparametric test for dependent samples, utilizing the univariate procedure. all of ten enrolled patients completed both pre- and post - treatment mris, six males and four females. the mean time from injury to treatment was 3.45 months (range 111 months). nine of ten patients received the injection during the subacute injury phase (range 14 months), and the tenth patient was treated 11 months after injury. there were five patients with grade 3 lesions, three with grade 2, and two with grade 1 acl injuries, based on mri assessment. the mean time to posttreatment mri follow the median time of the last observed outcome follow up was 6 months (range 324 months). the mean nucleated cell count and volume of the bmc was 694 million and 2.7 ml, respectively (table 1). the intra- and interrater correlations for the selected metrics were excellent, ranging from 0.9 to 1. based on the histogram analysis of the pre- and posttreatment sagittal acl mris, eight of ten patients demonstrated a decrease in the mean gray value, eight of ten had a decrease in the modal gray value, seven of ten had a decrease in the median, and ten of ten had a decrease in the raw integrated density. the decrease in the mean, median, and raw integrated density were all significant (table 2). five of ten patients had a more positive skew (histograms leaned toward darker pixels), but this was not significant for the group (p=0.6) (figures 35). nine of ten completed the percentage change, seven of ten completed pain vas, while seven of eight completed the lefs, and one of two completed the fri. the mean pretreatment vas score was 2.6, and the mean vas score posttreatment was 0.9, with a mean pain change of 1.7 (range 6 to + 2). the mean lefs change was an improvement of 23.3 (range 538) points, and the single patient who completed the fri reported an improvement of a decrease of 20 points (table 3). lefs score changes (n=6, difference = 23.3) were statistically significant (signed - rank p - value = 0.0313) ; however pain changes (n=7, difference = -1.7) did not show statistical significance (signed - rank p - value = 0.25). the fri is a scale of 0100, with lower score indicating better function,26 and the lefs is a scale of 080, with a higher score indicating better function.27 the meaningful detectable change in the fri is 12.3,28 and the minimal detectable change and the minimally important change for the lefs is 9,27 thus based on those criteria, our reported changes in function are meaningful. the normal acl ligament appears with a low signal (dark), and an injured or disrupted ligament shows a high signal (bright) on most mri sequences.25,29 however, interpreting some findings on an mri can be a subjective process, with substantial variability between readers.30 the sagittal image was selected for the roi evaluation because it is most helpful in the evaluation of the linear integrity of acl fibers.25 the roi protocol and imagej software analysis demonstrated excellent inter- and intrarater reliability. the five metrics used to quantify the gray scale of the roi are explained as follows : 1) the mean gray value is the sum of the gray values of all the pixels in the selection divided by the number of pixels ; 2) the modal gray value is the most frequently occurring gray value within the selection and corresponds to the highest peak in the histogram ; 3) the median is the middle value of all of the pixels in the selection, with lower value correlating with a darker image ; 4) skewness describes how the histogram is distributed to one or the other side of the mean, with a positive skewness indicating the right tail is longer than the left, and the mass of the distribution concentrated on the left indicating a darker image ; and 5) the raw integrated density is the sum of the values of the pixels in the selection, with a lower value an indication of a darker image. in this case study, seven of ten patients demonstrated improvement in at least four of five measures of pixel intensity in their acls from pre- to posttreatment, in the expected direction of a darker and more normal appearing acl. all five of the objective measures showed a trend toward darker acls, from pre- to posttreatment mris (more normal appearing), and three of five reached 95% statistical significance. all seven patients that completed both pre- and posttreatment functional scales (lefs or fri) reported improvement that was clinically meaningful per the metrics of those individual functional questionnaires. the functional scales changed through the course of the study because these patients were tracked as part of an ongoing registry that tracks many patients with various orthopedic injuries. the decision to change the questionnaire was based on the evolution of the needs of that larger data collection project, and the lefs is one of many validated lower - extremity functional questionnaires. this only affected one patient, so it is unlikely to have affected the interpretation of the results. however the use of two different functional questionnaires may skew interpretation of the functional data due to lack of a consistent method of functional data reporting. in this study, the majority of patients (five of ten) had grade 3 tears. we believe the significance of this finding is that grade 3 tears are more likely to be disabling due to the complete rupture, and thus, these patients are more likely to seek help. we did treat two injuries that were classified as nonhealing grade 1 tears, which theoretically could have spontaneously healed without treatment. we excluded patients with ligament retraction greater than 1 cm because of concern that the mscs might not be able to bridge a gap between a tear any larger, which was inferred from clinical experience gained in treating these cases. we believe that a 1 cm or greater gap would not respond to biological treatment and would be a better candidate for surgical treatment. it should be noted that the specific treatment protocol used in this study is not standard or widely accepted and is unique to this specific center and affiliated clinics. the rationale for the treatment protocol chosen was to provide a proinflammatory and proliferative stimulus with a preinjection and then several days later, once the cellular phase of healing had been initiated, to augment the area with bmc rich in mscs and prp / platelet growth factors, and then finally, to provide prp / platelet growth factors to promote msc survival. research has suggested that hypertonic dextrose can provide a proliferative stimulus to ligaments and tendons.31,32 it has also been shown through multiple in vitro studies that prp can stimulate and provide supportive nutrients to mscs.3335 similarly, pl, the product of stripping viable growth factors from whole platelets, can also promote msc proliferation and support these cells in culture.3537 we used both intraligamentous and intraarticular sites due to the common finding of osteoarthritis after acl injury,38 theorizing that the ia injection may help undetected chondral injury. given that there are many variables that remained uncontrolled, this limits the validity and generalizability of these results outside of the use of this specific protocol. our goal was to allow the patient to load the ligament, depending on pain. past study has shown that loading is essential for ligament healing.39 physical activity and physical therapy programs were not controlled. we are unaware of any specific physical therapy or physical activity research demonstrating a benefit for healing acl tears. given that bracing rarely allows full healing of an acl on mri, based on our clinical experience, we did not feel that bracing (or not bracing) would make a difference. however, being the traditional approach for conservative care in acl injuries, some would argue that we might have seen better results had we required bracing, and the lack of standardized rehabilitation is a limitation of the study. for imaging analysis, we searched for an open - source imaging analysis program that had histogram capabilities. it should also be noted that despite excellent inter- and intrarater reliability, the nih imagej software has not been used in a similar study involving ligament changes on mri. however, it has been used in other areas of medicine requiring histogram - based analysis of medical imaging.4044 at the time of this manuscript was being prepared, there were 789 studies that came up under the search term imagej on pubmed. finally, the use of two different functional questionnaires may have skewed interpretation of the functional data, due to lack of a consistent method of functional data reporting. major limitations of the study include the lack of a control group and the lack of randomization. as a result, we can not conclude that the observed effects were due to the msc treatment versus natural healing or the pretreatment protocol. there is good reason to believe that the results were probably due to the msc therapy, however. grade 23 acl injuries rarely heal spontaneously.4547 it is possible that patients could have reported improved symptoms because they adapted to the instability, because of placebo effect, or because of some natural healing that may have reduced symptoms. given the specific treatment protocol with multiple variables, any component of the protocol could have accounted for the observed effects. we do not believe that the pretherapy injection routine, which was meant to cause a proinflammatory healing environment, was the cause of the observed changes. there is no evidence that we are aware of that the injection of a 12.5% hypertonic dextrose solution can result in objective improvements in the mri appearance of ligaments. while it could be argued that it was the prp component of the msc injectate that was the cause of the observed effects, the results of prior studies do not support such a conclusion. prp use has been previously studied in patients undergoing acl reconstruction, with one study showing increased cortical bone formation on mri48 and another describing faster remodeling of the grafts.49 a third study did not report any differences on posttreatment mri.50 we were unable to find any studies demonstrating a benefit of prp for acl injuries, however. lastly, the lack of pre- and posttreatment objective measurements of acl laxity, such as kt1000 testing,51 is a major limitation in accessing treatment outcomes. for future study, we suggest recorded clinical findings before and after treatment, such as the lachman, pivot shift test, an objective ligament integrity test (eg, rolimeter or kt1000 test), and a validated functional questionnaire. postprocedural rehabilitation should be standardized as well. in addition to the objective review of acl integrity on mri with software, review of pre- and posttreatment mris should ideally be performed at one center, with a blinded musculoskeletal radiologist reviewing the images and reporting findings. with a larger number, perhaps statistical analysis of the difference in outcome between patients with varying grades of acl tear would shed light on which patients may be more or less likely to benefit from this type of treatment. based on this small case series, the precise injection of autologous bone marrow - derived nucleated cells into the acl ligament under fluoroscopic guidance, utilizing this specific protocol shows promise as a treatment for symptomatic grade 1, 2, and, possibly, nonretracted grade 3 acl tears., controlled trial is needed to determine whether our results are repeatable and due to the intervention used in this study.	introductionthis was a prospective case series designed to investigate treatment for anterior cruciate ligament (acl) tears using an injection of autologous bone marrow concentrate.methodsconsecutive adult patients presenting to a private outpatient interventional musculoskeletal and pain practice with knee pain, acl laxity on exam, and magnetic resonance imaging (mri) evidence of a grade 1, 2, or 3 acl tears with less than 1 cm retraction were eligible for this study. eligible patients were treated with an intraligamentous injection of autologous bone marrow concentrate, using fluoroscopic guidance. pre- and postprocedural sagittal mri images of the acls were analyzed using imagej software to objectively quantify changes between pre- and posttreatment scans. five different types of measurement of acl pixel intensity were examined as a proxy for ligament integrity. in addition pain visual analog scale (vas) and lower extremity functional scale (lefs) values were recorded at baseline and at 1 month, 3 months, 6 months, and annually postinjection. objective outcomes measured were pre- to post - mri measurement changes, as analyzed by the imagej software. subjective outcomes measured were changes in the vas and lefs, and a self - rated percentage improvement.resultsseven of ten patients showed improvement in at least four of five objective measures of acl integrity in their postprocedure mris. in the entire study group, the mean gray value, median, raw integrated density, and modal gray value all decreased toward low - signal acls (p=0.01, p=0.02, p=0.002, and p=0.08), indications of improved ligament integrity. seven of ten patients responded to the self - rated metrics follow up. the mean vas change was a decrease of 1.7 (p=0.25), the mean lefs change was an increase of 23.3 (p=0.03), and mean reported improvement was 86.7%.conclusionbased on this small case series, autologous bone marrow concentrate shows promise in the treatment of grade 1, 2, and possibly grade 3 acl tears without retraction. further investigation using a controlled study design is warranted.
pleural effusion and pneumothorax are common conditions presenting to secondary care and accounted for over 29 000 admissions in the period 20112012 within the national health service in england. first line management is usually conservative, needle aspiration or by medical teams utilizing small bore seldinger intercostal chest drains. small bore tubes are more comfortable for patients and can be therapeutically equivalent to larger drains. the national patient safety agency issued a rapid response report in 2008 due to concerns about the risks associated with chest drains. this case demonstrates that damage to intercostal drains can occur during difficult insertion and that appropriate checks of equipment during clinical procedures can avoid the a gentleman in his 80s presented to hospital with a 3-week history of chest pain and shortness of breath brought on by an episode of sneezing. he had a notable past medical history of previous right anterior thoracotomy and wedge resection of a lower lobe nodule that was found to be benign. on assessment, he was found to have a right - sided pleural effusion and a right - sided 18 fr seldinger drain was inserted for diagnostic and therapeutic purposes. at this point, it was noted that the tip of the drain was missing. the patient was informed about this, retrieval of the foreign object was not attempted and the event was clearly documented. a new seldinger drain was placed and the missing drain tip was visible on chest radiograph, see fig. 1. cessation of fluid output prompted drain removal and a computed tomography (ct) scan of the thorax was performed to further assess the pleural disease ; this showed a large persistent multiloculated effusion. the patient was transferred to a specialist thoracic surgery unit and proceeded to urgent right thoracotomy and decortication for the loculated effusion with removal of the foreign body. video - assisted thoracoscopic surgery (vats) was not possible due to the presence of adhesions from previous surgery. the 1-cm drain tip was identified medial to the 12th rib in the posterior cardiophrenic angle and removed. the patient required transfusion of two units of packed red cells for a low haemoglobin post - operatively but otherwise made a good recovery. iatrogenic foreign bodies in the chest have been formally reported as early as 1964 when a rubber chest drain was retained in the pleural space. it was believed at this time that management should usually be that of thoracotomy and decortication and a further case series confirms that surgical removal is almost always performed [3, 4 ]. with the advent of vats, it is usually possible and indeed preferable to retrieve a foreign body before pleural sepsis occurs, averting thoracotomy ; however, surgery is still likely to be necessary. the retained foreign material following seldinger chest drain insertion has been reported once before and the patient required surgery to remove the item, in the form of a vats decortication. in this paper, the distal portion of the drain had sheared off, most likely due to repeated withdrawal and advancement of the drain over the guidewire. correct insertion technique is important in preventing damage to the drain and avoiding inadvertent introduction of free foreign bodies into the pleural cavity, specifically repeated insertion and withdrawal of a seldinger drain over a guidewire. surgeons and physicians should perform a count of instruments during clinical procedures that they undertake in ward or clinic environments. should a foreign object be retained inside a patient then prompt recognition, as occurred in our case, is important for patient safety. a plan for further management, including whether to remove the object can then be made. unusual problems may be encountered during procedures but a retained foreign object that is not recognized would constitute a never event. chest drains inserted at the end of cardiothoracic procedures commonly require removal of the proximal end prior to connection with the drainage system ; we now also include these drain ends in the instrument count for operative cases. patients with chest drains should be cared for on a ward where specialist staff are trained in their management. these staff should be familiar with the normal appearance of equipment upon removal ; any discrepancies at this point are a further step in ensuring patient safety. review of a chest radiograph after drain insertion or removal is a routine step that should enable prompt identification of a foreign body in correlation with review of any equipment which is causing concern.	pleural effusions are commonly drained with seldinger intercostal drains. one uncommon but serious risk of drain insertion is that of a foreign body being retained in the pleural cavity following removal. we report a case in which the tip of the drain was retained in the pleural space following difficult insertion of a seldinger intercostal drain in a district general hospital. prompt recognition and clear patient communication are important at the occurrence of an unusual complication. surgical removal of the foreign body was performed following transfer. we report this case to raise awareness that insertion and withdrawal of drains over the guidewire during insertion may damage the drain and highlight the need for doctors who insert chest drains to perform a count of instruments during ward or clinic - based procedures as well as those performed in theatres. we now include removable parts of chest drains in our theatre instrument count.
the electrophilicity of (e)-2-aryl-1-cyano-1-nitroethenes is not sufficient to induce a zwitterionic course of their [4 + 2 ] cycloaddition to cyclopentadiene. the one - step mechanism of these reactions is indicated by the activation parameters, and the substituent and solvent effects on the reaction. for the last decade, reactions of conjugated nitroalkenes have aroused our considerable interest [19 ], and we have exerted considerable effort to examine their reactivity in thermally allowed cycloaddition reactions. especially the [4 + 2 ] cycloaddition reactions of cyclopentadiene (1) with strong electrophilic (e)-2-aryl-1-cyano-1-nitroethenes 2a2 g were investigated [46, 9 ]. by means of hplc and noesy experiments, it has been unequivocally established that this reaction leads to the corresponding 6-endo - aryl-5-endo - cyano-5-exo - nitronorbornenes 3a3 g and 6-exo - aryl-5-exo - cyano-5-endo - nitronorbornenes 4a4 g as the only reaction products. the stereospecific reaction course suggests that, independently of the nitroalkene electrophilicity, this reaction occurs according to a concerted mechanism (paths a and b). however, stereospecificity can not be regarded as sufficient evidence of the concertedness [1012 ]. nevertheless, it can not be ruled out that the reaction proceeds by a zwitterionic mechanism [10, 1315 ] (paths c and d), while preserving the original nitroalkene configuration in cycloadducts, when the activation barrier for the carbocyclic ring closure in postulated intermediates is lower than the corresponding barrier for rotation around the c c bond of the cyanonitroethyl moiety. the aim of this work was to evaluate the nature of the transition complexes and to confirm or exclude the presence of zwitterionic intermediates on the reaction paths. for this purpose, the kinetics of the title reaction were investigated, and the substituent and solvent effects as well as activation parameters were determined. kinetic studies (table 1) showed that the rate constants of the reactions leading to the nitronorbornenes 3a3 g and 4a4 g (scheme 1) increase with the nitroalkene electrophilicity. in particular, the rate constants ka and kb for the reactions involving the least electrophilic (e)-2-(p - methoxyphenyl)-1-cyano-1-nitroethene (2a) (= 3.14 ev) are 0.08 10 dm mol s and 1.66 10 dm mol s, while those for the reactions with the most electrophilic (e)-2-(p - methoxycarbonylphenyl)-1-cyano-1-nitroethene (2 g) (= 3.80 ev) are 13.35 10 dm mol s and 83.42 10 dm mol s, respectively. we investigated the substituent effect on the reaction using classical correlation analysis, seeking the best match between substrate reactivity (log ka and log kb) and the values of the hammett constants, p, r, i of the substituents. the best linear correlations (r = 0.995 for the reaction 1 + 2 3 and 0.991 for the reaction 1 + 2 4) were obtained with the hammett constants p (fig. 1). this means that the substituent effect is transferred to the reaction centers via both induction and resonance, and the transition complexes are similar regardless of the nitroalkene electrophilicity. \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \log k_{\text{a } } = 2.93 \cdot \sigma_{\text{p } } - 3.20\quad (r = 0.995) $ $ \end{document}2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \log k_{\text{b } } = 2.31 \cdot \sigma_{\text{p } } - 2.20\quad (r = 0.991) $ $ \end{document}table 1results of kinetic measurements of [4 + 2 ] cycloaddition of cyclopentadiene with (e)-2-aryl-1-cyano-1-nitroethenes 2a2 g nitroalkenesolvent [(30) ] t/c k total 10/dm mol s isomer ratio / k a 10/dm mol s k b 10/dm mol s r sdnrrp /ev 2a och3 0.273.14nitromethane (46.3)251.740.050.081.660.9980.01 2b ch3 0.173.31nitromethane (46.3)253.280.090.273.010.9980.02 2c h0.003.42nitromethane (46.3)256.230.120.675.560.9980.02 2d f0.063.50nitromethane (46.3)257.260.120.786.480.9970.03 2e cl0.233.68nitromethane (46.3)2522.460.142.7619.700.9980.03 2f br0.233.71nitromethane (46.3)2525.130.143.0922.040.9980.04 2 g cooch3 0.453.80nitromethane (46.3)535.150.154.5830.570.9980.05 2 g cooch3 0.453.80nitromethane (46.3)1559.990.157.8252.170.9980.05 2 g cooch3 0.453.80nitromethane (46.3)2596.770.1613.3583.420.9970.07 2 g cooch3 0.453.80dichloroethane (41.9)2534.460.123.6930.770.9990.04 2 g cooch3 0.453.80dichloromethane (41.1)2533.230.123.5629.670.9990.05 2 g cooch3 0.453.80chloroform (39.1)56.260.100.575.690.9990.03 2 g cooch3 0.453.80chloroform (39.1)1510.050.111.009.050.9970.05 2 g cooch3 0.453.80chloroform (39.1)2520.280.122.1718.110.9990.04 2 g cooch3 0.453.80tetrachloromethane (32.5)257.450.110.746.710.9970.07scheme 1 fig. 1plot of log k versus hammett constants p and global electrophilicity index for the [4 + 2 ] cycloaddition of cyclopentadiene with (e)-2-aryl-1-cyano-1-nitroethenes 2a2 g (at 25 c in nitromethane) results of kinetic measurements of [4 + 2 ] cycloaddition of cyclopentadiene with (e)-2-aryl-1-cyano-1-nitroethenes 2a2 g plot of log k versus hammett constants p and global electrophilicity index for the [4 + 2 ] cycloaddition of cyclopentadiene with (e)-2-aryl-1-cyano-1-nitroethenes 2a2 g (at 25 c in nitromethane) it is noteworthy that the values of the reaction constants in eqs. 1 and 2 (2.93 for the reaction 1 + 2 3 and 2.31 for the reaction 1 + 2 4) are almost two times higher than those for concerted [2 + 4 ] cycloadditions of cyclopentadiene with (e)-2-aryl-1-nitroethenes, while still being within the range typical of one - step cycloadditions. furthermore, the positive sign of the constant shows that charge transfer within the transition complexes of both competing reactions occurs from the cyclopentadiene substructure towards the nitroalkene substructure. moreover, the relationships between log ka and log kb and the nitroalkene global electrophilicity indexes are similar. in this case, however, the correlations are slightly lower.3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \log k_{\text{a } } = 3.04 \cdot \omega - 13.64\quad (r = 0.986) $ $ \end{document}4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \log k_{\text{b } } = 2.37 \cdot \omega - 10.34\quad (r = 0.971) $ $ \end{document } in order to gain more insight into the nature of the transition complexes, we studied the effect of solvent polarity on the reaction kinetics. this was done for the reaction of the most electrophilic nitroalkene 2 g (table 1). the cycloadditions 1 + 2 g 3 g and 1 + 2 g 4 g were found to occur more rapidly in polar than in non - polar solvents. this suggests that the transition complexes of the reactions tested are more polar than the substrates. this is consistent with the magnitude of dipole moments (= 7.36/8.54 d) of the respective critical structures located on the reaction potential energy hypersurface using b3lyp/631 g(d) calculations. to obtain a quantitative description of the solvent effect on the reaction rate, we studied correlations between substrate reactivity and the solvent polarity constants such as dielectric constant, dipole moment, berson constant, and reichardt - dimroth et(30) constant [18, 19 ]. 2).5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \log k_{\text{a } } = 0.09 \cdot e_{\text{t } } - 6.08\quad (r = 0.987) $ $ \end{document}6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \log k_{\text{b } } = 0.08 \cdot e_{\text{t } } - 4.76\quad (r = 0.994) $ $ \end{document}fig. 2plot of log k versus reichardt dimroth e t(30) constants for the [4 + 2 ] cycloaddition of cyclopentadiene with (e)-2-(p - methoxycarbonylphenyl)-1-cyano-1-nitroethene (2 g) (at 25 c) plot of log k versus reichardt dimroth e t(30) constants for the [4 + 2 ] cycloaddition of cyclopentadiene with (e)-2-(p - methoxycarbonylphenyl)-1-cyano-1-nitroethene (2 g) (at 25 c) these data prove that solvent polarity affects the rate of 1 + 2 g cycloaddition to a much higher extent than that for the reaction involving (e)-2-phenyl-1-nitroethene. however, the sensitivity constants in eqs. 5 and 6 do not exceed 0.1 and are below the typical range observed for ionic reactions [18, 20 ]. hence, the nature of the hammett and reichardt dimroth relationships suggests that the reactions 1 + 2 3 and 1 + 2 4 do not proceed according to a zwitterionic mechanism (paths c and d). these parameters were determined from the rate constants of the reactions 1 + 2 g 3 g and 1 + 2 g 4 g at 5, 15, and 25 c using the eyring equation in the form:7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \log (k / t) = 10.319 + \updelta s^ { \ne } /4.576 t $ $ \end{document}table 2activation parameters for [4 + 2 ] cycloaddition of cyclopentadiene with (e)-2-(p - methoxycarbonylphenyl)-1-cyano-1-nitroethene (2g)nitromethanechloroform 1 + 2 g 3 g 1 + 2 g 4 g 1 + 2 g 3 g 1 + 2 g 4 g h /kj mol k 165.4157.4149.9153.2 activation parameters for [4 + 2 ] cycloaddition of cyclopentadiene with (e)-2-(p - methoxycarbonylphenyl)-1-cyano-1-nitroethene (2 g) in particular, the activation enthalpies (h) were estimated from the slopes of the plots of logarithm of the ratio of ka or kb by the absolute temperature (t) versus reciprocal of the absolute temperature (1/t), while the entropies of activation (s) were determined from the intercepts of these plots. it follows from the data in table 2 that the h values for the cycloadditions 1 + 2 g 3 g and 1 + 2 g 4 g do not exceed 46 kj mol. they are typical for the reactions, where the energy changes within the transition complexes resulting from breaking of bonds existing in the substrates and formation of new bonds present in the cycloadducts compensate one another. on the other hand, the s values are high and negative (150 to 165 j mol k), both in the moderately polar chloroform and the highly polar nitromethane. this is typical for one - step cycloadditions proceeding through highly rigid transition complexes [2224 ]. by comparison, s for a two - step cycloaddition of 1,1-dimethoxy-1,3-butadiene with tetracyanoethene is 26 j mol k. it is noticeable that the entropy changes in the transition state are much higher in our case than those for a similar reaction involving (e)-2-phenyl-1-nitroethene (s = 102.5 to 120 j mol this is probably due to the polarization and dispersion of the substrate interactions within the transition complexes, which should be much stronger in the case of more electrophilic (e)-2-aryl-1-cyano-1-nitroethene than in the case of less electrophilic (e)-2-aryl-1-nitroethenes [26, 27 ]. this is indirectly confirmed by the depths of the pre - reaction local minima located on the potential energy hypersurface found for the reactions involving (e)-2-aryl-1-cyano-1-nitroethenes [5, 9 ] and (e)-2-aryl-1-nitroethenes. in summary, the electrophilicity of (e)-2-aryl-1-cyano-1-nitroethenes is not sufficient to induce an ionic course of their [4 + 2 ] cycloaddition to cyclopentadiene. the activation parameters and the substituent and solvent effects on the reaction studied indicate its non - ionic mechanism. cyclopentadiene (1) was prepared by pyrolysis of commercially available dicyclopentadiene (aldrich) at 180200 c, according to a known method. before use (e)-2-aryl-1-cyano-1-nitroethenes 2a2 g were obtained by condensation of appropriate aromatic aldehydes with nitroacetonitrile, according to a reported procedure. kinetic experiments were carried out in a glass reactor supplied with a thermostatically controlled jacket, magnetic stirrer, thermometer, reflux condenser, and sampling device. liquid chromatography (hplc) was done using a knauer apparatus, equipped with a uv vis detector (= 254 nm). for monitoring of the reaction progress a lichrospher 100rp column (4 240 mm) thermostated at 5 c was used, and 70% methanol at a flow rate of 1.1 cm min was applied as the eluent. the overall rate constants (ktotal) were determined by monitoring the decrease of the hplc peak area (a) corresponding to cyclopentadiene. it was found that the product composition was controlled kinetically, since the ratio of the products (= /) was constant throughout the reaction course. the kinetic experiments were carried out at 5, 15, and 25 c up to 7080% completion, using in each case equimolar quantities of substrates. in definite time intervals 250-mm samples were taken out of the reactor with a microsyringe and analyzed immediately by hplc. in this way 15 series of measurements it showed excellent linear relationships (r > 0.99) between 1/a and time t for all kinetic runs, using the second - order kinetic eq. 8 of the form:8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ 1/a = - k_{\text{total } } \cdot t + { \text{const } } $ $ \end{document } the ktotal and values were then applied for calculation of the rate constants ka and kb according to eqs. 9 and 10:9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ k_{\text{a } } = \gamma \cdot k_{\text{total } } /(\gamma + 1) $ $ \end{document}10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ k_{\text{b } } = k_{\text{total } } /(\gamma + 1) $ $ \end{document } the second - order rate constants for different temperatures and solvents obtained by this method are summarized in table 1. the overall rate constants (ktotal) were determined by monitoring the decrease of the hplc peak area (a) corresponding to cyclopentadiene. it was found that the product composition was controlled kinetically, since the ratio of the products (= /) was constant throughout the reaction course. the kinetic experiments were carried out at 5, 15, and 25 c up to 7080% completion, using in each case equimolar quantities of substrates. in definite time intervals 250-mm samples were taken out of the reactor with a microsyringe and analyzed immediately by hplc. in this way 15 series of measurements it showed excellent linear relationships (r > 0.99) between 1/a and time t for all kinetic runs, using the second - order kinetic eq. 8 of the form:8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ 1/a = - k_{\text{total } } \cdot t + { \text{const } } $ $ \end{document } the ktotal and values were then applied for calculation of the rate constants ka and kb according to eqs. 9 and 10:9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ k_{\text{a } } = \gamma \cdot k_{\text{total } } /(\gamma + 1) $ $ \end{document}10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ k_{\text{b } } = k_{\text{total } } /(\gamma + 1) $ $ \end{document } the second - order rate constants for different temperatures and solvents obtained by this method are summarized in table 1. this article is distributed under the terms of the creative commons attribution license which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.	abstractthe electrophilicity of (e)-2-aryl-1-cyano-1-nitroethenes is not sufficient to induce a zwitterionic course of their [4 + 2 ] cycloaddition to cyclopentadiene. the one - step mechanism of these reactions is indicated by the activation parameters, and the substituent and solvent effects on the reaction.graphical abstract
cardiovascular complications are more common among diabetic patients and are usually associated with a significantly greater risk of morbidity and mortality than in nondiabetic subjects. the relative risk of myocardial infarction (mi) is 50% greater in diabetic men and by 150% greater in diabetic women compared to age - matched nondiabetic subjects. sudden cardiac death is 50% more frequent in diabetic men and 300% more frequent in diabetic women compared to age - matched nondiabetic controls. diabetes also influences outcomes following acs, and therefore, secondary prevention in diabetic individuals is equally critical. consequently, the diabetic patient needs special management and monitoring, with a view to the prevention, control, and treatment of the various manifestations of coronary artery disease. all patients with acs require evidence - based treatments such as lifestyle advise and drugs including antiplatelets, angiotensin converting enzyme inhibitors (aceis), or angiotensin receptor blockers (arbs), beta - blockers, and lipid - lowering medications (statins) therapy to prevent death, and secondary complications. this study was conducted to assess clinical characteristics, management, and prognosis in acs patients with or without diabetes presenting to a tertiary care hospital. the study protocol was approved by the institutional ethics committee, and individual written consent was obtained from all the patients. demographic details, clinical characteristics, lifestyle factors, and prescribed treatment were obtained in successive patients presenting to the hospital with a diagnosis of acs. diagnosis of acs was confirmed by the presence of typical chest pain or uneasiness, combined with electrocardiographic changes, and cardiac enzyme elevation. physical activity was classified as mild, moderate, or severe based on the who criteria. overweight was defined as body mass index (bmi) 25.029.9 kg / m, obesity as bmi 30 kg / m, abdominal obesity as waist circumference > 90 cm in men, and > 80 cm in women according to the who guidelines. subjects were defined as hypertensive when a person was either a known hypertensive or had multiple readings over the course of hospitalization of 140/90 mmhg. the patients were classified as diabetics if they had been previously diagnosed were receiving hypoglycemic therapy or repeatedly has high fasting blood glucose levels (> 126 mg / dl at > 2 measurements) during admission. details of medical and interventional coronary management were prospectively recorded for each patient by interview of patients and treating physicians. chicago, usa ; version 13.0 for windows) was used for the data analysis. differences in continuous variables have been determined by independent t - test and test as used for ordinal variables. chicago, usa ; version 13.0 for windows) was used for the data analysis. differences in continuous variables have been determined by independent t - test and test as used for ordinal variables. the consort statement and flowchart of patients in the study are shown in figure 1. majority of these patients were transferred from other hospitals more than 5 days after the acute event (n = 389) and 15 (2.7%) died and not included in the study. one hundred and forty - six patients were eligible, and 46 did not agree to participate in the study or had screen failure, and finally, 100 patients were included in this study. mean age was 59.0 10.8 years ; it was 59.3 11.6 years in diabetics and 58.9 8.5 years in nondiabetics. overall, 49 patients had st - elevation mi (stemi) while 51 had nonstemi or unstable angina. in diabetics as compared to nondiabetics, there was the similar prevalence of stemi (50.0% vs. 48.6%) as well as nonstemi (50.0% vs. 51.4%). among diabetics versus nondiabetics, there was greater prevalence of hypertension (78.6% vs. 44.4%), obesity (25.0% vs. 8.3%), abdominal obesity (85.7% vs. 69.4%), and physical inactivity (89.2% vs. 77.8%) (p < 0.05). lifestyle factors in diabetics versus nondiabetics were - smoking and/or tobacco use (10.7% vs. 25.0%), high fat intake (78.6% vs. 76.4%), high salt intake (53.6% vs. 59.7%), high calorie intake (35.7% vs. 48.6%), low fiber intake (64.3% vs. 54.9%), low fruits and vegetables intake (53.6% vs. 52.8%), and alcohol use (3.6% vs. 6.9%) [p = nonsignificant, table 1 ]. compared to patients without diabetes, diabetic patients had greater mean heart rate and bmi (p < 0.05). prevalence of obesity, abdominal obesity, and hypertension was also greater in diabetic patients [p < 0.05, table 1 ]. consort statement depicting the study flow demographic, lifestyle, and clinical characteristics in diabetic and nondiabetic patients with acute coronary syndrome in stemi, patients 28 (57.1%) were thrombolysed (diabetics 17.8% vs. 31.9%). in diabetics versus nondiabetics, percutaneous coronary intervention (pci) was in 67.8% versus 84.7% (p < 0.05) and coronary artery bypass grafting (cabg) surgery was in 21.4% versus 8.3%. at discharge, 91 patients were on dual antiplatelets, aspirin, or thienopyridine (clopidogrel, prasugrel, or ticagrelor) (diabetics 85.7%, nondiabetics 95.8%), 71 patients were on aceis or arbs (diabetics 71.4%, nondiabetics 70.8%), 71 patients were on beta - blockers (diabetics 64.3%, nondiabetics 73.6%), 99 patients were on lipid - lowering agents (diabetics 100%, nondiabetics 98.6%), 19 patients were on diuretics (diabetics 21.4%, nondiabetics 18.1%), and 7 patients were on calcium channel blockers (diabetics 14.3%, nondiabetics 4.2%) [table 2 ]. this study shows that diabetic patients with acs have a higher prevalence of cardiometabolic risk factors (obesity, abdominal obesity, and hypertension) than nondiabetics, and there was the lower use of pci. there was the lower use of dual anti - platelet therapies and beta - blockers in patients with diabetes. previous studies from india including large registries such as create, kerala - acs and demat, have reported on patterns of acs and therapies. these studies reported that stemi was responsible for about half of all hospital admissions similar to this study. these studies also report a low use of various cardioprotective agents, especially beta - blockers, during hospitalization and at discharge. these studies did not study diabetic subgroups and our study, although small, is unique to address this question. long - term follow - up studies from india have reported similar low use of beta - blockers in acs patients. studies from developed countries such as multicountry global registry of acute coronary events (grace) registry as well as british and north american acs registries have shown similar results. grace registry reported that beta - blocker use in diabetics (75.0%) was significantly lower than in nondiabetics (80%). this indicates a physician - level barrier in prescribing beta - blockers to diabetics due to the popular misconception of greater side effects in this group. lower use of pci and greater cabg surgery indicates greater prevalence of multivessel disease patients. this is a single - center study with a small sample size, and this is a major study limitation. moreover, we evaluated patients at a tertiary care hospital, and more than 90% patients underwent some coronary intervention, and the findings may not reflect the general situation in india. larger and multicentric studies are required to identify patterns of acs in diabetes, management strategies, outcomes, and secondary prevention therapies. long - term studies to assess adherence to therapies and lifestyle measures as well as long - term outcomes are also required. other limitations of the study include lack of assessment of prehospital phase of acs, details of symptoms, and in - hospital management. our study shows that diabetic patients with acs have greater prevalence of obesity, abdominal obesity, and hypertension.	objectives : to compare clinical characteristics, treatment, and utilization of evidence - based medicines at discharge from hospital in acute coronary syndrome (acs) patients with or without diabetes at a tertiary care cardiac center in india.methods:we performed an observational study in consecutive patients discharged following management of acs. we obtained demographic details, comorbid conditions, and cardiovascular risk factors, physical and biochemical parameters, and management. descriptive statistics are reported.results:we enrolled 100 patients (diabetics = 28) with mean age of 59.0 10.8 years (diabetics 59.3 11.6, nondiabetics 58.9 8.5). forty - nine patients had st - elevation myocardial infarction (stemi) (diabetics = 14, 28.7%) while 51 had nonstemi / unstable angina (diabetics = 14, 27.4%) (p = nonsignificant). among diabetics versus nondiabetics there was greater prevalence (%) of hypertension (78.6% vs. 44.4%), obesity (25.0% vs. 8.3%), abdominal obesity (85.7% vs. 69.4%) and sedentary activity (89.2% vs. 77.8%), and lower prevalence of smoking / tobacco use (10.7% vs. 25.0%) (p < 0.05). in stemi patients 28 (57.1%) were thrombolysed (diabetes 17.8% vs. 31.9%), percutaneous coronary interventions (pci) was in 67.8% diabetics versus 84.7% nondiabetics and coronary bypass surgery in 21.4% versus 8.3%. at discharge, in diabetics versus nondiabetics, there was similar use of angiotensin converting enzyme inhibitors (67.9% vs. 69.4%) and statins (100.0% vs. 98.6%) while use of dual antiplatelet therapy (85.7% vs. 95.8%) and beta - blockers (64.3% vs. 73.6%) was lower (p < 0.05).conclusions : diabetic patients with acs have greater prevalence of cardiometabolic risk factors (obesity, abdominal obesity, and hypertension) as compared to nondiabetic patients. less diabetic patients undergo pcis and receive lesser dual anti - platelet therapy and beta - blockers.
the characterisation of bone microarchitecture has been essential in elucidating the pathogenesis of osteoporosis, and alterations in bone microarchitecture have been recognised as playing an important role in the susceptibility of bone to fracture [1, 2 ]. the ability to quantify the degree to which measures of bone microarchitecture predict bone strength, ex vivo, has provided valuable insights into the mechanisms of fragility fractures. the advent of high - resolution micro - computed tomography (micro - ct) and its increasing accessibility to researchers have ushered in a new era in bone histomorphometry. the constraints of extrapolation from two - dimension (2d) to three - dimension (3d) have now been broken enabling the measurement of bone quality indicators derived from accurate 3d voxel - based representations of cancellous bone structure. the classic model - based histomorphometry espoused by parfitt [3, 4 ] and universally adopted can now be applied to 3d datasets with or without correction factors related to 2d histological sections. however, the model is a simplified representation of the complex cancellous bone structure. vijayapalan. have shown by direct measurement of trabecular rod thickness from 3d anaglyphs that the true dimensions of trabecular rods are different to trabecular thickness derived from parfitt 's model. this means that the magnitude of derived dimensions such as trabecular thickness and trabecular separation may not be accurate measures but were useful for demonstrating relative differences between study groups or tracking temporal changes within a study series. there have been numerous protocols published describing the adaptation of three - dimensional descriptors of cancellous bone architecture for use with two - dimensional histological sections [610 ]. these include connectivity density, degree of anisotropy, and trabecular bone pattern factor but their utility has been limited because of large systematic errors related to the extrapolation from 2d to 3d. the accurate utilisation of these 3d descriptors of cancellous bone structure has been possible with micro - ct datasets. micro - ct imaging provides a series of 2d tomographs, which enable accurate reconstruction of the specimen as a voxel - based dataset. each tomograph is equivalent to a histological section, whereby in 2d the bone matrix is clearly delineated from marrow spaces. importantly, the series of tomographs enable an accurate 3d representation of the bone to be constructed. it is possible from this voxel - based dataset to measure trabecular dimensions using a sphere - fitting algorithm and to apply other model - independent algorithms for bone structure, such as connectivity density, structural model index, and degree of anisotropy. datasets derived from micro - ct imaging provide a comprehensive suite of descriptive parameters for bone structure in 3d as well as accurate measurement of the amount of bone. the increased focus on the functional properties of cancellous bone it has been shown, theoretically, that the magnitude and variability of trabecular dimensions affect the functional properties of the cancellous structure and that trabecular rods may be the critical structures in determining cancellous bone strength. measurement of individual trabeculae can be achieved by decomposition of micro - ct datasets ([17, 18 ], which also enables classification of trabeculae as rods or plates. recent work has shown that apparent mechanical properties of cancellous bone are compromised, to a greater or lesser degree, when groups of trabeculae with defined spatial orientation are removed in micro - ct derived datasets. together with location data these algorithms provide the tools to identify the critical structures in cancellous bone that contribute to its strength. it has been shown that individual trabeculae fails, in compression, by euler buckling [16, 19, 20 ] and sutton - smith. have shown that using 3d anaglyphs a load to buckling index (buckling index) can be derived from measurements of trabecular rod thickness and trabecular rod length. it is hypothesised that the distribution of the buckling index for trabecular rods indicates a potential failure mechanism for the cancellous bone structure. while it has been shown that the distributions of trabecular rod thicknesses and trabecular rod lengths are normally distributed, the distribution of the buckling index for trabecular rods is a log normal distribution. the shape of this distribution indicates that a force of a critical magnitude will cause a significant number of trabecular rods to fail simultaneously resulting in failure of the entire cancellous structure. the aim of this study is to determine the degree to which measures of bone architecture, derived from micro - ct imaging of human vertebral body cancellous bone, explain the variability in a surrogate of bone strength (buckling index). it is hypothesised that measures of bone architecture in addition to the amount of bone will explain significantly more of the variability in the buckling index than the amount of bone alone. the degree to which these parameters explain the variability in the buckling index will be determined using a statistical multiple regression approach. it is expected that the approach used in this study will further identify model - independent descriptors of bone structure, which may determine bone strength. thoracolumbar vertebral bodies (t6, t8, t10, t12, l1, l3, and l5) were obtained from four embalmed male cadavers. the donor spines were sourced from a forensic science facility and some vertebral bodies were used in other studies, hence there were not adjacent vertebral bodies. the ages of the cadavers were not known, however an estimate of the age was made using a grading protocol based on the amount of osteophytic lipping. the age estimates were 2 cadavers under 60 years of age and 2 cadavers over 60 years of age. a sagittal slice 2.5-mm in thickness was taken adjacent to the mid - line from all 28 vertebral bodies, which ranged in size from 15 mm to 25 mm crano - caudally to 30 to 40 mm antero - posteriorly. three - dimensional anaglyphs were obtained by scanning electron microscope (sem) imaging of nine contiguous fields, each 35 mm and covering most of the vertebral body slice, at a spatial resolution of 7.83 m. each anaglyph was constructed from two digitised sem images (the second image tilted through 5) and viewed on a computer screen with red - green stereo glasses. from the 3d anaglyphs, trabecular rod thickness (tb.th(rods)) and trabecular rod length (tb.le(rods)) were measured and the load to buckling index (buckling index) was calculated for a total of 2225 randomly selected trabecular rods (buckling index r / l, r = radius of rod and l = length of rod). for measurement, the 3d anaglyphs were viewed on a computer screen with red - green stereo glasses using a quantimet 500mc image analyser (leica, cambridge, uk) with a grid at a random angle overlaid to enable random sampling of trabecular rods. the 3d anaglyphs enabled clear identification of trabecular rods as smooth, roughly cylindrical structures, with a length at least three times their width. measurement of trabecular rod thickness was made at the point of intersection of the rod with the grid and perpendicular with the long axis of the rod. the ends of a trabecula for measuring trabecular length were defined as the midpoints of an arc formed between the trabecular rod and the adjacent trabecular structures. micro - ct imaging was subsequently performed on the specimens at a spatial resolution of 15.83 m with a skyscan 1072 (skyscan, kontich, belgium). cone - beam reconstruction software yielded up to 100 tomographs for each specimen, in the sagittal plane. using a custom - written routine in matlab (mathworks ltd, natick, mass, usa), a binary image, discriminating bone from marrow in each tomograph, this was followed by a binary closing (one cycle of erode followed by one cycle of dilate), to remove small unconnected artifacts. ct analyser software (ctan) provided by skyscan uses the marching cubes method to generate a surface rendering of the bone (figure 1). the volume of interest for each micro - ct dataset was 243 mm. to establish the efficacy of the buckling index as an appropriate surrogate of cancellous bone strength, the strength of cancellous bone cubes taken from the centre of 28 vertebral bodies was measured as ultimate failure stress (ufs). the cancellous bone cubes were obtained from the l2 and l3 vertebral bodies of 14 fresh - frozen spines. prior to mechanical testing, micro - ct imaging was performed using the same machine settings as for the sagittal bone slices and which resulted in a micro - ct dataset for each 10 10 10 mm bone cube consisting of 640 tomographs, which were each 640 pixels by 640 pixels. trabecular rods were identified using the volumetric spatial decomposition algorithm, where each discrete trabecular element was classified as a trabecular plate or a trabecular rod. the volumetric decomposition algorithm yields length and thickness, from which the buckling index was calculated, as above. mechanical testing was by uniaxial compression in the supero - inferior direction at 0.1 mm / sec to failure (hounsfield h25 km, hounsfield ltd, uk). using the 3d voxel - based datasets, the following model - independent parameters were obtained : bone volume per total volume (bv / tv) and trabecular thickness (tb.th) and trabecular separation (tb.sp) using a sphere fitting algorithm, trabecular bone pattern factor (tbpf) as the ratio of convex to concave surfaces, structural model index (smi) as an index of how rod - like or plate - like the structure, the connectivity density (conn.d) as an indicator of connectivity within the structure, and degree of anisotropy (da) from mean intercept length analysis as an index of the degree of preferred orientation of the structure. from the mechanical testing study, ultimate failure stress (ufs) was calculated from the stress / strain curve, as a measure of bone strength and the buckling index was calculated for trabecular rods. for all parameters, a mean value was calculated for each of the 28 vertebral body specimens and used in subsequent statistical analyses. the geometric mean was calculated for the buckling index from each vertebral body due to the log normal distribution of this parameter. the correlations between anaglyph - derived parameters and micro - ct - derived parameters were determined by least - squares linear regression analysis. the degree to which these descriptive parameters explain the variability in the buckling index was determined by least - squares linear regression analysis. multiple regression analysis was performed to determine what combination of parameters best explains the variability in trabecular rod buckling index. this entails sequential addition to the statistical model of measures of bone architecture to determine the best combination of predictors of the buckling index. from the mechanical testing study, the buckling index for each trabecular rod was calculated as per methods outlined above. the correlation between the buckling index and ufs was determined by least - squares linear regression analysis. all statistical tests were performed using pc - sas (sas institute, cary, nc). within the study sample, the vertebral body bv / tv ranged from 8% to 18% with descriptive statistics for all parameters shown in table 1. buckling index significantly correlates with bv / tv, tb.th, tb.sp, tbpf, smi, and conn.d (table 2). it is noteworthy that the correlations of tb.th(rods) and tb.le(rods) with bv / tv and conn.d, while statistically significant, are not as strong as the correlations of the buckling index with bv / tv and conn.d. least squares linear regression shows that bv / tv explains 59% of the variability in the buckling index (r = 0.59 ; p <.0001) (table 2). of the other model - independent parameters, conn.d is the best individual explanatory parameter of the buckling index (r = 0.52 ; p <.0001) (table 2). multiple regression analysis, with r selection, determines what combinations of parameters explain best the variability in the buckling index of trabecular rods (table 3). there is a statistically significant (p <.05) improvement in explanatory power for trabecular rod buckling index from 59% of the variability for bv / tv alone to 77% of the variability when conn.d, tb.sp, and smi are added to bv / tv in the multiple regression model. buckling index this study showed that measures of bone architecture, derived from micro - ct imaging of human vertebral body cancellous bone, can explain a statistically significant proportion of the variability in a surrogate of bone strength (buckling index). the data supports the hypothesis that measures of bone architecture in addition to the amount of bone explain significantly more of the variability in the buckling index than the amount of bone alone. the study sample consisted of vertebral bodies, where bv / tv of the vertebral body cancellous bone encompasses the range found in healthy adults (8% to 18%). using a 3d - based direct method of measuring trabecular rod dimensions, a surrogate of trabecular rod strength was derived, the buckling index. rendering of the tomographs into a 3d voxel - based dataset enables the application of algorithms, which describe model - independent 3d structural characteristics of the cancellous bone (bv / tv, tb.th, tb.sp, tbpf, smi, conn.d, and da). the analysis of anaglyphs enables both trabecular rod thickness and trabecular rod length measurement and the derivation of a functional descriptor of the trabeculae, the buckling index. it is assumed that buckling is the most likely mode of failure for trabecular rods given that they experience predominantly compressive loads and are fixed at both ends. also, this index of trabecular rod buckling strength is calculated in the direction of the trabecular long axis. to date, it has not been possible to directly measure the strength of individual trabeculae in an intact trabecular network, although the strength of isolated trabeculae has been measured [19, 25 ]. the strong statistical relationship between the buckling index and bv / tv translates to a coefficient of determination (r) of 59%, which is within the range of previously published data showing the ability of the amount of bone to explain the variability in vertebral body strength. while the correlations of tb.th(rods) and tb.le(rods) with bv / tv and conn.d are statistically significant, they are not as strong as the correlations of the buckling index with bv / tv and conn.d. the parameters derived from the 3d voxel - based dataset show that individually tbpf and conn.d correlate strongly with the buckling index but not with each other. this indicates that the form of individual trabeculae and the connectivity of the bone structure are independently important in determining the strength of the vertebral body. these model - independent algorithms when applied to histological sections were notoriously unreliable because the assumptions made to extrapolate from 2d to 3d were sources of large systematic errors. micro - ct imaging allows application of the theory underpinning these algorithms with greatly reduced systematic errors. the multiple regression analysis shows that the amount of variability in the buckling index attributable to bv / tv can be improved from 59% to 77% with the addition of model - independent 3d morphological parameters (conn.d, tb.sp, and smi). these 3d microarchitecture - related factors explain a further 18% of the variability in the predicted buckling strength of trabecular rods. addition of more morphological parameters to the statistical model does not significantly improve the prediction of the buckling index. this statistical model quantifies the contribution of the 3d architecture of the cancellous bone to trabecular rod strength in addition to the contribution of the amount of bone. specifically, connectivity of the trabecular structure, as measured by conn.d, would be expected to be an important determinant of structural integrity. in fact conn.d is almost as strong as an independent determinant of the buckling index as bv / tv (r = 0.52 versus r = 0.59, resp.). measures of trabecular form and structure such as smi and tbpf contribute significantly to prediction of the buckling index, and individually they are both negatively correlated with the buckling index. this supports the view that more rod - like structures are weaker than more plate - like structures. the degree of anisotropy (da) does not contribute to the buckling index because it is a measure of orientation of the entire cancellous structure, whereas the buckling index of individual trabeculae is orientation independent. thomsen. report a statistically significant improvement in the prediction of bone strength at l3 from 74% to 79% when 2d histomorphometric indices measured at l2, such as tb.th, tb.sp, and star volume, were added to bv / tv. hence, bone strength can be more reliably predicted when the contribution of 3d - bone structure is added to the contribution of bv / tv alone. a limitation of this study is that there are multiple vertebral levels from a small number of individuals. this study made use of human cadaveric material that was sourced for other studies but was used in this study to maximize the use of this difficult - to - access material. the use of multiple vertebral levels from each individual reduces the statistical power if conclusions as to an individual 's risk of fracture were made but in this cross - sectional study where each vertebral body is an independent observation, it is appropriate to make conclusions as to the ability to explain the variability in bone strength within the group of vertebral bodies. another limitation of this study is that mechanical testing was not performed on the sagittal slices. however, the use of the buckling index as a surrogate of bone strength provides a morphological framework by which functional aspects of cancellous bone structure can be studied. specimen handling or processing prior to micro - ct imaging can compromise the ability to perform mechanical testing. other recent studies have quantified the role of microarchitecture and the effect of regional variability in determining vertebral body cancellous bone strength and whilst these studies have utilised mechanical testing as the outcome variable, their results are not inconsistent with the results of this study. namely, that cancellous bone per se is an important determinant of vertebral body strength and that the contribution of microarchitecture of individual trabeculae to bone strength can be quantified. therefore, a purely morphological approach, such as the one presented in this study can provide useful data on the functional characteristics of cancellous bone. in this study, the morphological descriptors of vertebral body cancellous bone derived from micro - ct images provide a composite explanatory model of trabecular rod strength (buckling index). the literature does not provide many theoretical alternatives to the buckling index as a descriptor of the functional properties of trabeculae. while in this study a relatively small sample of vertebral bodies are studied, the strength of the relationships outlined above give grounds to further investigate the use of a functional descriptor of cancellous bone derived from 3d morphological parameters of cancellous bone.	high - resolution micro computed tomography has enabled measurement of bone architecture derived from 3d representations of cancellous bone. twenty - eight vertebral bodies were obtained from four embalmed male cadavers. from 3d anaglyphs, trabecular rod thickness and length were measured and the trabecular rod buckling index was calculated. from 3d voxel - based datasets, bone volume density, trabecular thickness, and trabecular separation were measured. also, trabecular bone pattern factor, structural model index, connectivity density, and degree of anisotropy were calculated. bone volume density alone explains 59% of the variability in trabecular rod buckling index. the addition of connectivity density, trabecular separation, and structural model index, in a multiple regression statistical model, improves the explanatory power to 77%. the relationships between measures of cancellous bone architecture and a derived measure of trabecular rod strength were investigated. morphological descriptors of cancellous bone provide a composite explanatory model of trabecular rod strength.
it is well recognized that hearing loss is the most common global disability. as reported by davis (british mrc institute of hearing research) the numbers with hearing impairment in developing countries hearing loss is the third most common disability and the prevalence of moderate to severe hearing loss is reported to be 6.3 percent by who. the majority of india 's population lives in rural areas with poor facilities and accesses for evaluation of hearing and treatment being seldom available. also, due to the paucity of trained audiologists, the burden of hearing handicap is magnified. the primary goal of the study was to determine the degree and nature of benefit that could be expected in the population by using trained community workers to fit semidigital hearing aids which can be programmed easily by trained workers and quantifying benefit using aphab which was translated in to the local language. deafness is an expensive handicap, leading to loss of work and active participation in social activities of the community. the measurement of outcomes in rehabilitative audiology has received more attention because of the need to demonstrate efficacy of treatment for consumers, carry out cost - benefit analyses. traditionally, the outcomes of hearing aid intervention have been demonstrated using objective measures such as the functional gain speech recognition testing, and real ear responses. an alternative to the use of objective hearing aid benefit measurements is the use of self - report methodology. in the 1980s and 1990s several other subjective measurement instruments were developed to assess hearing aid benefit [10, 11 ]. one of the most common subjective benefit measures in use today is the abbreviated profile of hearing aid benefit (aphab) developed by cox and alexander. aphab is a questionnaire which has 24 predetermined questions regarding various situations of hearing aid usage. it also gives the patient a realistic estimation of the use of a hearing aid. the difference in scores with and without the use of hearing aid(s) is considered the measure of benefit. there are four categories in which benefit is calculated : ease of communication (ec), listening in background noise (bn), listening in reverberant conditions (rv), and aversiveness of sounds (av). in a large developing country like india with significant rural population, the use of community health workers to identify hearing loss and provide hearing aids and associated services and evaluate the use and satisfaction of has by self - report measure aphab and its stability was studied over a period of six months. community hearing workers (chws) were selected with educational qualification of graduate with preference for science subjects. they were given a six week training program on basic hearing health care, which included performing pure tone audiometry (pta) using a portable audiometer, impression taking, and ear mould making, performing hearing aid trial and hearing aid fitting, maintenance of hearing aids, minor repairs of hearing aids, and maintenance of ear moulds. they were also trained to administer aphab, translated into the local language (tamil) and validated by the audiologist. camps were conducted with the help of the local government organization and nongovernmental agencies (ngos), using local propaganda machine which included television broad casting and advertising in the regional language papers. patients were screened by an ent specialist and hearing assessment was done in the field using arphi-500 mki dual channel portable audiometers programmed to ansi standards in a quiet room. a three frequency (0.5 khz, 1 khz, 2 khz) average was taken and hearing loss was classified as moderate, moderately severe, severe, and profound hearing loss. hearing loss bilateral moderate to severe sensorineural hearing loss (4190 db). children below 14 years and adults above 70 years.mild hearing (90 db) hearing loss.. hearing loss bilateral moderate to severe sensorineural hearing loss (4190 db). hearing loss which can be surgically corrected. children below 14 years and adults above 70 years.mild hearing (90 db) hearing loss. mild hearing (90 db) hearing loss. the aphab is a hearing aid outcome measure that reflects the impact of hearing loss and benefit of hearing aid in daily communication. the subscales are as follows.ease of communication (ec) : the strain of communicating under relatively favorable conditions.reverberation (rv) : communication in reverberant rooms such as class rooms.background noise (bn) : communication in settings with high background noise levels.aversiveness (av) : the unpleasantness of environmental sounds. ease of communication (ec) : the strain of communicating under relatively favorable conditions. there are 24 items ; each item contributes to only one subscale and there are six items for each subscale distributed randomly with in the inventory. each item is answered for without my hearing aid and with my hearing aid so that each subscale produces a score for unaided listening and a score for aided listening. in addition, the difference between these two scores can be obtained to give a score for benefit. the difference in the rating between the initial and the final visit is scored as the amount of benefit in each of four general categories. the first three subscales, ec, rv, and bn, are known as the speech communication subscales. these subscale benefit scores are reported in the form of a positive percentage (i.e., 10%, 20%, 30%). the fourth subscale, aversiveness (av), quantifies an individual 's negative reaction to aversive environmental sounds. this subscale is reported in a negative percentage (i.e., 10%, 20%, 30%). hearing aids (siemens 213) were dispensed based on the formula (1)nh=3 khz500 hz, mpo60 decibel=0.33 fa+89 25 in all the three speech subscales (figure 7). the main purpose of this study was using trained community workers to provide hearing aids in the community and to assess the self - report measure aphab in the community and its stability over a period of six months. it was found that most hearing aid users were above the age of 40, which was similar to the other studies. hearing aids were fitted for patients with moderate to severe hearing loss which had a high acceptance level (98%). thus during followup patients were called together in the village and they could discuss the problems they faced and how to resolve them. the issues regarding usage of hearing aid and the stigma of not being able to hear were sorted out among themselves. a feeling of community helped them to overcome the shyness of using a hearing aid and thus it led to increased acceptance and the despondency of being a deaf person was overcome which led many other people with similar problem to seek help for their problem. in aphab, in contrast to the report by hojan., there was no significant difference in responses between male and female ha users. the missing responses to bn and av scales could be explained from the fact that the mentioned situations were not encountered immediately after hearing aid fitting and may have been due to rural living, which did not expose them to situations where background noise and aversive sounds were present. this likely reflects the increased audibility of certain environmental sounds that even individuals with normal hearing may find aversive. the small, statistically nonsignificant change in the av scores indicates that the hearing aid provided acceptable listening comfort and sound quality for most of the participants. there was correlation between the aided condition and pta thresholds as reported by cox. which confirms that while fitting hearing aids in the community, clients with moderately severe and severe hearing loss suffer more problems which can be alleviated with provision of hearing aids suited for that range that is, the hearing aid has to be compatible or provide significant gain with respect to speech communication subscales (ec, rv, bn). aphab picked up significant improvements in communication especially in reverberant rooms and in settings with high background noise levels. the limitation of this study was the lack of responses to questions, as in a rural community the provision of modern amenities is lacking when compared to an urban setting. aphab picked up significant improvements in communication especially in reverberant rooms and in settings with high background noise levels. though the questionnaire is long the questions take into consideration most of the situations the patients face in their daily life. however in a rural community with lack of basic amenities some of the questions may not be assessed for a considerable time period till the patient comes into contact with such situations. however it is the most comprehensive questionnaire and gives the patient a realistic expectation from a hearing aid. it can also be used at a later time when the patient upgrades his hearing aid and thus the benefits can be compared.	hearing loss is a major handicap in developing countries with paucity of trained audiologists and limited resources. in this pilot study trained community health workers were used to provide comprehensive hearing aid services in the community. one hundred and eleven patients were fitted with semi - digital hearing aid and were evaluated over a period of six months. they were assessed using self - report outcome measure aphab. results show that trained chws are effective in detecting disabling hearing loss and in providing has. aphab can identify and pick up significant improvements in communication in daily activities and provides a realistic expectation of the benefits of a hearing aid. the model of using trained chws to provide rehabilitative services in audiology along with self - report outcome measures can be replicated in other developing countries.
colorectal cancer (crc) is the third - ranked neoplasm in order of incidence and mortality worldwide.1 it is more frequent in industrialized countries, where it is the second cause of cancer death and is a social and health care issue of major importance.1,2 in 2009, 146,970 new cases of crc and 49,920 crc - related deaths occurred in the united states.3,4 in the european union, approximately 220,000 new crc cases per year are estimated to be diagnosed annually.5 the likelihood of developing crc increases from the second to the ninth decade. crc is rare before the age of 40 years, and most cases are diagnosed after the age of 50. the incidence of crc is slightly higher in males than in females, especially for rectal carcinoma. in developing countries crc has a lower, yet growing incidence.1 although the etiology of crc is still unknown, epidemiological and biomolecular studies have unveiled environmental and genetic risk factors that favor the onset of crc.3 one of the most important environmental risk factors for crc is a western - type diet, which is characterized by a low fiber and high fat content. crc - related mortality is directly correlated with per - person calorie intake, high consumption of meat proteins and fat, and high blood cholesterol levels. conversely, in african populations, in which consumption of fruit and vegetables is higher than in western countries, the incidence of crc is much lower. moreover, it has been observed that people who have migrated to industrialized countries tend to acquire the same crc incidence rates of those countries, supporting the hypothesis that geographic differences in crc incidence are due to dietary habits rather than different genetic patterns.6 research has shown that up to 25% of patients with crc have inherited mutations that increase their risk for crc. a fraction of these patients are affected by well - characterized hereditary syndromes, which can be classified as polyposis and nonpolyposis crc : familial adenomatous polyposis (fap ; also known as familiar polyposis of the colon) is a rare autosomal dominant condition that causes the growth of hundreds or thousands of adenomatous polyps in the large bowel. it is associated with a deletion in the long arm of chromosome 5, which contains the apc oncosuppressor gene. there is a classic form (in which patients typically harbor between 500 and 2500 colonic adenomas) and an attenuated form of fap, characterized by a lower number of polyps (typically 30) that are predominantly located in the proximal colon. affected patients may also have associated extracolonic diseases, such as desmoid tumors and gastroduodenal carcinomas.7 gardner syndrome is characterized by a combination of intestinal polyposis (identical to classic fap) and numerous osteomas, epidermal cysts, fibromatosis, as well as thyroid and duodenal carcinomas and dental abnormalities. turcot syndrome is a rare condition characterized by the association of fap and tumors of the central nervous system (such as medulloblastoma and glyoblastoma).3 hereditary nonpolyposis colorectal cancer (hnpcc, also known as lynch syndrome) is an autosomal dominant hereditary syndrome, associated with germline mutations in genes that result from dna repair mechanisms (in particular, msh2, mlh1, pms, and pms2), causing microsatellite dna instability and impaired mismatch repair, and leading to higher risk of cancer development. a strong association exists between hnpcc and endometrial and ovarian carcinomas.8 in hnpcc patients, crc typically occurs earlier than 50 years, that is, 10 to 15 years before the average onset of crc in the overall population. moreover, crc in hnpcc patients is more frequently located in the cecum or ascending colon.3,9 chronic inflammatory bowel diseases : crc is more frequent in patients with ulcerative colitis, in whom the absolute risk of cancer increases at a rate of 0.5% to 1% per year after 10 years of disease, with cancer development typically in 8% to 30% of cases after 25 years from disease onset. conversely, in patients with crohn disease, crc risk is not significantly higher than in the normal population.3 other conditions that increase the risk for crc development are : bacteremia related to streptococcus bovis : for unknown reasons, individuals with a history of s. bovis - induced endocarditis or sepsis have a higher incidence of crc.3 tobacco smoke, which is associated with the development of colorectal adenomas : several studies have investigated the potential procarcinogenetic role of tobacco smoke, and a dose response relationship with crc has been found for cigarette pack - years, smoking duration, smoking intensity, smoking history in the distant past, and younger age at initiation of smoking.10,11 alcohol abuse : epidemiological studies have demonstrated that another risk for crc could arise from the malnutrition caused by lower folate concentrations in heavy alcohol drinkers. however, recent studies have yielded inconsistent findings concerning such a relationship.1214 despite multiple risk factors increasing the likelihood of crc development, the majority of individuals (about 75%) who develop crc do not have specific risk factors.15 therefore, the population should be stratified into averagerisk individuals (age greater than 50 years, no personal risk factors, no familial history of crc) and those with moderate (first - degree relative with a history of adenoma or carcinoma, or personal history of large adenoma or carcinoma) and high crc risk (inflammatory bowel disease or a family history of an inherited crc syndrome).16,17 the onset of crc is triggered by a well - defined sequence of genetic alterations that form the etiological basis of the adenoma - carcinoma sequence hypothesis and justify the rationale for crc screening. the validity of this hypothesis is supported by several findings:18 populations with a high incidence of adenomas have a high prevalence of crc, and vice versa. there is a substantial overlap between the topographic distribution of colorectal adenomas and that of crc. the risk of crc is directly proportional to the number of adenomas, and the substantial certainty of developing crc in patients with fap is an extreme case of this rule. follow - up programs to detect adenomas with resection of suspected lesions are associated with a reduced incidence of crc. polyps with the highest risk of neoplastic degeneration are those characterized by a large size (20 mm), villous histology, and sessile morphology. the time of progression from adenomatous polyp to carcinoma is about 10 years. more than 90% of crcs arise from adenomatous polyps,19 while a small fraction of crcs develop without evidence of an adenomatous precursor, suggesting that some lesions may undergo malignant transformation without passing through a polypoid intermediate phase. evidence exists that polypectomy can reduce crc incidence and mortality by interrupting the adenoma - carcinoma sequence in the colonic segment where the polyp has been excised.20 from a genetic point of view, carcinogenesis is characterized by the accumulation of multiple, consecutive mutations in oncogenes and oncosuppressor genes, starting the adenoma - carcinoma sequence at the level of the colonic mucosa. the genes most often involved (and the most widely known) are apc, k - ras, p53, smad2, and smad4. moreover, mutations involved in dna mismatch repair may account for the development of 10% to 15% of sporadic crc and hnpcc.18 the knowledge of the biological mechanisms underlying crc carcinogenesis and their slow progression over time make this neoplasm ideal for the development of screening programs. furthermore, crc is a common disease with a high mortality rate, for which risk conditions can be detected and treatment is more effective in early than in advanced disease stages. to be effective, screening needs to be performed on a large population, and be sensitive, specific, and well tolerated by patients.. finally, screening must be cost - effective. based on the guidelines of the american cancer society, techniques currently available for the secondary prevention of crc (after the age of 50 years) can be classified as follows:21 tests for detecting polyps and crc : flexible rectosigmoidoscopy (every 5 years) ; optical colonoscopy (oc, every 10 years) ; double contrast barium enema (dcbe, every 5 years) ; computerized tomography colonography (ctc, also known as virtual colonoscopy : every 5 years). tests that can make crc diagnosis : fecal occult blood test (fobt, every year) ; fecal immunochemical tests (every year) ; fecal dna test (uncertain time interval). it must be pointed out that individuals with an increased/ high crc risk should undergo screening with a higher frequency based on their individual risk level. in western countries, the most common and readily available tests for crc screening are rectosigmoidoscopy, oc, and fobt. since the introduction of fobt as a crc screening test in the 1990s, crc mortality has been reduced ; however, the main limitations of fobt are a low specificity and a low sensitivity in polyp detection (about 10%), as polyps seldom manifest with bleeding, although, for this same reason, immunochemical fobt has a sensitivity and specificity of 95% for detection of advanced colonic neoplasms.22 therefore, it is necessary to shift attention earlier along the adenoma - carcinoma sequence : this task is accomplished by rectosigmoidoscopy and colonoscopy (either optical or virtual). the rationale for rectosigmoidoscopy (also referred to as left colonoscopy if exploration is extended to the descending colon) is related to different frequency of crc in the various colonic segments ; in fact, crc arises in the rectum and sigmoid colon in 55% of cases, in the descending colon in 6% of cases, in the transverse colon in 11% of cases, and in the cecum / ascending colon in 22% of cases.18 one limitation of this technique is, of course, the lack of evaluation of the entire colorectum, that may cause a considerable number of colonic adenomas and carcinomas to be missed. in recent years proximal for those reasons, whenever a colonic mass, or an advanced adenoma, or 3 or more adenomas, or a villous polyp larger than 10 mm is found, rectosigmoidoscopy must be followed by complete colonoscopy.17 oc is today regarded as the gold standard technique for crc screening, because it allows lesion detection with both high sensitivity (90%100% also for small size polyps, ie, less than 6 mm diameter) and high specificity. moreover, like recto - sigmoidoscopy, oc is not only useful for diagnostic purposes, but has also a therapeutic role as it enables polypectomy by means of a diathermic loop or hot bioptic pliers (hot biopsy), allowing lesion removal and biopsy. it is usually performed with light sedation or conscious sedation and therefore requires an in - patient setting. however, the main limitation of oc is patient intolerance, which can be due either to anatomical conditions (for example, presence of short colonic mesentery or a redundant colon may cause pain with progression of the oc probe), adhesions, or neoplastic and/or fibrotic narrowing/ obstruction of the bowel lumen. other limitations of oc are the need for a cathartic preparation for optimal bowel cleansing, its high cost, and invasiveness. moreover, some lesions of the colonic mucosa can be missed even by oc.23 finally, oc is not completely free from potentially life - threatening complications, such as bowel perforation or bleeding. dcbe is often used whenever conventional endoscopy fails to explore the entire colon, eg, because of patient intolerance and/or inability to reach the cecum on oc (which is not uncommon in cases of redundant bowel) (figure 1). compared with oc, dcbe has the advantage of less invasiveness, and therefore it is usually better tolerated by patients. however, the visualization of colonic lesions is heavily dependent on the radiographic projections made to obtain dcbe images, and therefore polyps (especially smaller ones) could be masked by extracolonic structures or even by nearby colonic segments or folds, thus causing dcbe to be less sensitive than oc. sensitivities as low as 42% for 10 mm polyps have been reported.24,25 another important issue of dcbe is radiation dose, that can be quite unpredictable due to the different radiographic technique used for each patient (for example, particular anatomic conditions and/or the different time and maneuvers needed to ensure arrival of barium and air up to the cecum), but it tends to be quite high, in the order of 5 to 8 msv. larger patients usually require even higher radiation doses to be imaged, owing to their higher x - ray absorption. this may be a serious limitation in case of younger patients and especially for young females, who are more sensitive to ionizing radiation due to the presence of gonads inside the abdomen. ctc has recently been included by the american cancer society among tests recommended for crc screening, and has evolved from an experimental technique to a universally recognized, accurate, and validated diagnostic tool that is gaining widespread use and acceptance among both patients and referring clinicians. it is based on the acquisition of spiral ct data of the abdomen after adequate distention of the colon through rectal insufflation of gas (either air or carbon dioxide).2628 the ct acquisition is usually performed twice : once in the supine position and then in the prone position (or vice versa) : this is to optimize distention of the various colonic segments depending on gravitational compression by the surrounding abdominal structures, as well as to distinguish polyps (that are fixed to the bowel walls) from fluid and/or fecal residues (that tend to fall down due to gravitation) (figure 2). as ctc image formation is based on the x - ray attenuation of low - density, high - intrinsic - contrast objects (such as the air contained in the colonic lumen vs the large bowel walls, acting as an interface between intraluminal air and the extraluminal compartment), low x - ray energy is sufficient to achieve diagnostic ctc images, resulting in a low radiation dose. in other terms, if ctc is aimed at the sole examination of the colon (eg, for crc screening purposes), use of low - radiation - dose ct acquisition protocols is warranted. conversely, regular - dose ct protocols will be used if ctc is part of a ct examination in which all abdominal organs are to be investigated. this is the case, for example, of ctc in patients with known crc and incomplete oc, in whom ct plays a role both for complete assessment of the colonic lumen and for oncological staging (including search of lymph node and liver metastases, or peritoneal carcinomatosis) (figure 3). for adequate colonic distention to be achieved, air or carbon dioxide is usually delivered into the patient s colon by means of a thin rectal catheter prior to ctc data acquisition. insufflation can be accomplished either manually or via automatic insufflators, which allow a continuous and reliable measurement of the volume and pressure of insufflated gas. air has the advantage of no cost and ease of administration, but is less tolerated because it is not absorbed by the colonic mucosa. conversely, carbon dioxide is more comfortable (after completion of the exam) as it is gradually absorbed by the colonic walls, although larger volumes must be supplied compared with air. in practical terms, administration of 1 to 1.5 l of air or 3 to 4 l of carbon dioxide is usually sufficient.28 colonic distention is also favored by parenteral administration of spasmolytic agents, such as glucagon or hyoscine - n - butyl bromide, which inhibit peristalsis and reduce the tone of the parietal musculature. contraindications to hyoscine - n - butyl bromide are glaucoma, prostatic hypertrophy, and heart rhythm disorders, whereas glucagon is contraindicated in patients with elevated blood glucose levels. in the united states (where glucagon is the only option) most experts have stopped using it. by orally administering positive contrast material into the large bowel (barium or iodine), fecal and fluid tagging can be performed, helping to distinguish fecal / fluid residues from parietal polyps. tagged residual fluid can then be electronically removed from ctc images by means of dedicated software. research is in progress on subtracting solid tagged stool in patients who undergo no cathartic cleansing. in contrast with the 2d plain film nature of dcbe images, one great advantage of ctc is the fact that cross - sectional images of the abdomen are acquired, which allows analysis of the various anatomical structures (eg, colonic lumen and content vs bowel walls, and the several components of the extracolonic compartment) without the superimposition of the surrounding organs and tissues. ctc also allows quantitative measurements of ct densities (such as that of fat for the normal ileo - cecal valve, or parenchymal density for extracolonic tissues or colonic lesions), as well as quantitative geometrical information, such as distances, areas, or volumes, without the projective distortion that is typical of radiographic images. in order to achieve a high spatial resolution (which is essential for detection of small polyps and for high quality 2d and 3d image processing), thin slice acquisition is mandatory for ctc examinations. with current multidetector row ct equipment (64 detector rows and beyond), the entire abdomen can be scanned in less than 10 seconds with submillimeter slice thickness, resulting in voxel isotropy and elimination/ minimization of motion artifacts.29 2d and 3d image reconstructions are necessary components of any ctc exam, as they allow assessment of large bowel anatomy on multiple planes (either orthogonal [axial, coronal, and sagittal planes ], or user - defined oblique planes), as well as provision of a volumetric depiction of normal anatomy and polyp morphology. 3d reconstructions enable accurate quantification of polyp volume, which can be helpful in a follow - up to assess growth of the polyp. with modern multidetector ct equipment and powerful image processing workstations, 2d (multiplanar reformation) and 3d reconstructions (volume rendering) it is also possible to perform volume rendering from an intraluminal perspective, thus simulating endoscopic navigation (virtual endoscopy), or to open up the colon and generate dissected views (virtual dissection) (figures 4 and 5). in the systematic review by halligan in 2005,30 who assessed 24 studies for a total of 4181 patients, average ctc sensitivity for large polyp (1 cm or above) detection was 93% with an average specificity of 97%. for large and medium size polyps (6 mm or above), average sensitivity and specificity were both 86%, while for polyps of all sizes, sensitivity ranged from 45% to 97% and specificity from 26% to 97%. ctc sensitivity for detection of crc was 96%.30 in another systematic review of the literature by mulhall,31 results were substantially similar to those by halligan.30 in summary, ctc has been shown to be highly sensitive for crc detection, as well as highly specific for detection of small size polyps, which makes it suitable as a crc screening tool.32,33 few studies have compared the radiation dose of ctc and dcbe.34,35 hirofuji measured a dcbe effective dose value of 12.7 msv (decreasing by 12% when digital radiography equipment was used), while an effective dose of ctc performed with a low - dose protocol was 5.7 msv. neri managed to perform successful ctc examinations for crc screening purposes with an even lower radiation dose (2.2 msv) by using a combination of low exposure settings and tube current modulation.36 the lower radiation dose of ctc compared with dcbe is due to the fact that with ctc, fluoroscopy is not needed and data acquisition is performed in the supine and prone positions only. single - acquisition protocols are currently discussed (eg, in the pediatric population), which would actually halve radiation exposure.37 according to the radiological risk figures of the international commission on radiological protection,38 an average effective dose value of around 2 msv for ctc is associated with a theoretical 0.005% risk of lethal radiation - induced malignancy, which further decreases with increasing patient age. thus, ctc is an important crc screening tool especially in middle - aged and elderly patients, in whom the potential benefit of early diagnosis of crc far outweighs the risk associated with radiation exposure. computer aided diagnosis (cad) tools are software applications designed for assisting the radiologist in the diagnosis of several conditions ; for detection of lung nodules on chest x - ray or ct, breast nodules in screening mammography, and polyps in ct colonography.3941 as ctc continues to evolve and improve, its use is shifting from highly specialized academic centers to community hospitals and nonacademic radiology practices.42,43 thus, many radiologists are experiencing pressure from clinical colleagues to offer ctc as part of the routine services provided in their practices. in parallel, modern multidetector ct scanners can generate data with unprecedented spatial resolution (down to 0.5 mm), with a consequent enormous increase in image number ; therefore, manual reading of ctc images may become error - prone due to reader fatigue.44 moreover, image interpretation is subject to reader bias, and no systematic method has been devised so far for lesion visualization (either 2d or 3d).45 cad systems have been developed in an attempt to overcome the limitations of software - unassisted reading of ctc datasets. such applications include dedicated algorithms for automatic detection of geometrical properties of image objects (such as the shape of colonic polyps and their relationship with the bowel wall) and ct density of the normal colon and polyps. candidate polyps, which are shown on 2d and 3d reconstructions and native ctc images, and which must be validated or rejected by the user. cad systems can be used in a first - reader or second - reader paradigm, depending on cad being switched on before or after examination of ctc datasets by the human reader. several studies have addressed the issue of evaluating the diagnostic performance of cad systems for ctc. they are valuable for assisting radiologists in the detection of polyps, especially for detection of small lesions. however, such an increase in sensitivity is usually paralleled by a decrease of specificity, suggesting that results of cad systems need to be adjusted to maximize overall diagnostic accuracy.3941 the impact of cad as second reader on experienced readers has recently been investigated in a multireader, multicase trial.46 thus, it would be of interest to evaluate the impact of cad systems on the diagnostic performance of readers without dedicated ctc experience, such as readers working in nonacademic centers, involved in reading a large amount of ctc generated by a screening program. in this respect, baker have concluded that application of a cad system for ctc is advantageous for assisting diagnostic performance of inexperienced readers, with decreased specificity being offset by a higher increase in sensitivity. however, cad power can not compensate for reader inexperience, as it has been shown that use of cad software for ctc image evaluation by inexperienced readers does not significantly increase sensitivity of individual raters, thus stressing the importance of adequate training.48 colorectal cancer is one of the most frequent malignancies in the western world, for which genetic and environmental risk factors have been identified and form the basis for disease prevention. several imaging techniques are currently available that allow detection of colorectal polyps at an early stage of development. among those, ct colonography is one of the most promising and will likely gain increasing importance for crc screening purposes.	colorectal cancer (crc) is the third - ranked neoplasm in order of incidence and mortality, worldwide, and the second cause of cancer death in industrialized countries. one of the most important environmental risk factors for crc is a western - type diet, which is characterized by a low - fiber and high - fat content. up to 25% of patients with crc have a family history for crc, and a fraction of these patients are affected by hereditary syndromes, such as familial adenomatous polyposis, gardner or turcot syndromes, or hereditary nonpolyposis colorectal cancer. the onset of crc is triggered by a well - defined combination of genetic alterations, which form the bases of the adenoma - carcinoma sequence hypothesis and justify the set - up of crc screening techniques. several screening and diagnostic tests for crc are illustrated, including rectosigmoidoscopy, optical colonoscopy (oc), double contrast barium enema (dcbe), and computed tomography colonography (ctc). the strengths and weaknesses of each technique are discussed. particular attention is paid to ctc, which has evolved from an experimental technique to an accurate and mature diagnostic approach, and gained wide acceptance and clinical validation for crc screening. this success of ctc is due mainly to its ability to provide cross - sectional analytical images of the entire colon and secondarily detect extracolonic findings, with minimal invasiveness and lower cost than oc, and with greater detail and diagnostic accuracy than dcbe. moreover, especially with the advent and widespread availability of modern multidetector ct scanners, excellent quality 2d and 3d reconstructions of the large bowel can be obtained routinely with a relatively low radiation dose. computer - aided detection systems have also been developed to assist radiologists in reading ctc examinations, improving overall diagnostic accuracy and potentially speeding up the clinical workflow of ctc image interpretation.
a sample of 103 voluntary male participants, based on their response to the following question from the whoqol - bref questionnaire : are you currently ill ? (yes - no), were divided into two groups : unhealthy (n=55) and healthy (n=48) respectively. whoqol - bref and the depression anxiety stress scale (dass-42) were used to assess satisfaction rating and negative emotions of depression, anxiety and stress. the who 's qol scale (whoqol - bref) was used to assess satisfaction with four domains of life : physical health, psychological, social relationships and environment support. the four domain scores are scaled in a positive direction, with a score range of 0 - 20, and with higher scores denoting higher satisfaction. the 42-item format of dass was used to measure the negative emotional states of depression, anxiety and stress. in order to find how rating the whoqol - bref and dass scales is combined to produce an overall measure of quality of life and satisfaction with health rating, a qol - dass model was designed ; and the strength of this hypothesized model of qol - dass was examined using structural equation modeling (figure 1). in this model, ellipses represent latent variables ; whoqol, and dass, and rectangles represent measured variables ; satisfaction in four different domains of life, physical health, psychological well - being, social relations, and environment, as measured with whoqol - bref and depression, anxiety, stress as measured with dass-42. a sample of 103 voluntary male participants, based on their response to the following question from the whoqol - bref questionnaire : are you currently ill ? (yes - no), were divided into two groups : unhealthy (n=55) and healthy (n=48) respectively. whoqol - bref and the depression anxiety stress scale (dass-42) were used to assess satisfaction rating and negative emotions of depression, anxiety and stress. the who 's qol scale (whoqol - bref) was used to assess satisfaction with four domains of life : physical health, psychological, social relationships and environment support. the four domain scores are scaled in a positive direction, with a score range of 0 - 20, and with higher scores denoting higher satisfaction. the 42-item format of dass was used to measure the negative emotional states of depression, anxiety and stress. in order to find how rating the whoqol - bref and dass scales is combined to produce an overall measure of quality of life and satisfaction with health rating, a qol - dass model was designed ; and the strength of this hypothesized model of qol - dass was examined using structural equation modeling (figure 1). in this model, ellipses represent latent variables ; whoqol, and dass, and rectangles represent measured variables ; satisfaction in four different domains of life, physical health, psychological well - being, social relations, and environment, as measured with whoqol - bref and depression, anxiety, stress as measured with dass-42. for analysis, a confirmatory factor analysis (cfa),using maximum likelihood, was conducted on a sample of 103 participants whose scores derived from two rating scales of whoqol - bref and dass-42. using amos and maximum likelihood estimation, the relationships were examined between the whoqol, a latent variable with 4 indicators of four domains concerning with satisfaction with physical health (dom1), psychological well - being (dom2), social relationships (dom3) and environment support (dom4);and another latent variable, dass, with 3 indicators of depression, anxiety, and stress ratings. the most commonly used goodness - of - life index is the chi - square statistic. the results of the run of the data obtained from the proposed model of qol - dass is presented in table 1. fit indices of the model of qol - dass for different healthy and unhealthy groups = chi - square ; df= degrees of freedom ; cfi= comparative fit index ; nnfi= non - normed fit index ; nfi= normed fit index ; rmsea = root mean square error of approximation ; pclose = probability of close fit as observed in table 1, for both the healthy and unhealthy groups, the 2 statistic for the proposed model of qol - dass indicates that the proposed model fit the data well, which seemed to suggest an inadequate fit of the model. moreover, other indicators indicated much more favorable results. table 2 provides estimated regression weights (b) and standardized regression weight () coefficients of all pathways for the hypothesized model of qol - dass. regression weights (b) and standardized regression weight () coefficients in order to find whether or not components of the measurement model (qol - dass model) are invariant across particular groups (here scores are derived from healthy and unhealthy persons),a multiple group analysis was conducted on the two groups. these groups varied depending on their response to a question (do you feel ill now ?- the no response for the healthy group ; and the yes response for the unhealthy group. the question is whether the relation between specific items (satisfaction with physical health (dom1), psychological well - being (dom2), social relationships (dom3), environment support (dom4), depression, anxiety, and stress ratings.) and the underlying construct or factor (whoqol and dass) tapped by the item is the same across groups. unconstrained model does not specify any constraints on groups of healthy and unhealthy participants, and the measurement weights model specifies all the factor loadings as equal on the different studied groups. fit indices as well as the change in 2, and change in degrees of freedom between unconstrained and measurement weights models are presented in table 3. goodness - of - fit statistics for tests of invariance across different groups cfi= comparative fit index ; rmsea = root mean square error of approximation as demonstrated in table 3, the comparison of models, unconstrained and measurement weights, was found to be non - significant across healthy / unhealthy groups (2 = 7.95, df = 9, ns. given this finding, it can be concluded that the factor loading is shown invariant across the studied groups. in order to find whether or not components of the measurement model (qol - dass model) are invariant across particular groups (here scores are derived from healthy and unhealthy persons),a multiple group analysis was conducted on the two groups. these groups varied depending on their response to a question (do you feel ill now ?- the no response for the healthy group ; and the yes response for the unhealthy group. the question is whether the relation between specific items (satisfaction with physical health (dom1), psychological well - being (dom2), social relationships (dom3), environment support (dom4), depression, anxiety, and stress ratings.) and the underlying construct or factor (whoqol and dass) tapped by the item is the same across groups. unconstrained model does not specify any constraints on groups of healthy and unhealthy participants, and the measurement weights model specifies all the factor loadings as equal on the different studied groups. fit indices as well as the change in 2, and change in degrees of freedom between unconstrained and measurement weights models are presented in table 3. goodness - of - fit statistics for tests of invariance across different groups cfi= comparative fit index ; rmsea = root mean square error of approximation as demonstrated in table 3, the comparison of models, unconstrained and measurement weights, was found to be non - significant across healthy / unhealthy groups (2 = 7.95, df = 9, ns. given this finding, it can be concluded that the factor loading is shown invariant across the studied groups. george & bearon, (16), define quality of life in terms of four underlying dimensions, two of which are objective (general health and functional status and socioeconomic status) and two of which reflect the personal judgment of the individual (life satisfaction and related measures and self - esteem and related measures), and believe that these dimensions are especially central for assessment of quality of life of older people (16). although the boundary between subjective and objective components of qol is not always clear in practice, measures of quality of life are based on both objective and subjective variables (17). meeberg (1) referring to subjective and objective components of quality of life, claimed that the subjective aspect is essential because a sense of personal satisfaction is intrinsic to qol and the objective component is also necessary. a person living in poverty and squalor who has never known any other way of life may feel satisfied with his or her life. yet, a person from outside those living conditions can see the prevailing health hazards and would evaluate that individual 's qol as less than ideal (1). in order to find how subjective evaluation of satisfaction with life domains and feeling negative emotions affect perceived general health and quality of life, a hypothesized model of qol - dass was designed, and the strength of this hypothesized model was examined using structural equation modeling (see fig. our results of running the hypothesized model of qol - dass indicated that the proposed model of qol - dass fitted the data well for both healthy and unhealthy groups (see table 1). we continued our analysis by testing for multiple group invariance to find whether or not components of the measurement model, qol - dass model, are invariant across the two groups. our results of conducting multiple group invariance across the groups of healthy and unhealthy indicate that all factor loadings across different groups used to be invariant (table 3). as the baseline (unconstrained) and regression weights models are not significantly different, it is concluded that the hypothesized model of qol - dass is invariant between the two groups. our findings to evaluate the hypothesized model of qol - dass indicate that the different satisfaction domain ratings obtained from whoqol - bref and the negative emotions (depression, anxiety and stress measured using dass-42 as the observed variables) can represent the underlying constructs of general health and quality of life on both healthy and unhealthy groups.	objectivein order to find how rating the whoqol - bref and dass scales are combined to produce an overall measure of quality of life and satisfaction with health rating, a qol - dass model was designed ; and the strength of this hypothesized model was examined using the structural equation modeling.methodparticipants included a sample of 103 voluntary males who were divided into two groups of unhealthy (n=55) and healthy (n=48). to assess satisfaction and negative emotions of depression, anxiety and stress among the participants, they were asked to fill out the whoqol - bref and the depression anxiety stress scale (dass-42).resultsour findings on running the hypothesized model of qol - dass indicated that the proposed model of qol - dass fitted the data well for the both healthy and unhealthy groups.conclusionour findings with cfa to evaluate the hypothesized model of qol - dass indicated that the different satisfaction domain ratings and the negative emotions of depression, anxiety and stress as the observed variables can represent the underlying constructs of general health and quality of life on both healthy and unhealthy groups.
the simplicity of maternal inheritance without bi - parental recombination, high rates of variability and large copy numbers are key features that make mitochondrial dna (mtdna) highly useful in resolving questions related to phylogenetic and phylogeographic relationships (avise., 1987 ; zhang., 1995). however, certain processes such as heteroplasmy, nuclear integration of mitochondrial genes, polymerase errors, dna contamination, ancestral polymorphisms and recombination may lead to incongruence using independent loci. for example, heteroplasmy, the presence of more than one haplotype within a single organism (boyce., 1989 ; frey and frey, 2004) may result in incongruence of phylogenetic topologies and hence negatively impact the resolution of evolutionary relationships of organisms. heteroplasmy has been reported in insects and other arthropods (frey and frey, 2004 ; fontaine., 2007 ; magnacca and brown, 2010 ; nunes., 2013). another source of potential phylogenetic ambiguity is the nuclear integration of mitochondrial gene fragments, which has been suggested to occur in triatominae (dotson and beard, 2001) and other arthropods (zhang and hewitt, 1996a ; parfait. triatomines of the genus mepraia (mazza., 1940) are blood - sucking insects that play an important role in the transmission of trypanosoma cruzi, the etiologic agent of chagas disease in the sylvatic cycle (rozas., 2007 ; botto - mahan., 2008). mepraia is endemic to semiarid and arid regions, and is distributed in coastal and interior valleys of northern and central chile (fras., 1998 ; their distribution in wild and peridomestic habitats, their opportunistic feeding behavior and human settlement in risk areas are features of high epidemiological significance as potential vectors for humans (cattan., 2002 ; toledo., 2013) three species are currently included in the genus : m. spinolai porter 1943, m. gajardoi and m. parapatrica (fras., 1998 ; fras, 2010). mitochondrial gene sequences also support three lineages congruent with the three described species (campos., 2013a) however, the specific status of m. parapatricaremains controversial, given the widely recognized morphological plasticity within the subfamily triatominae (dujardin., 1999) and the presumptive introgression / hybridization processes acting within mepraia (calleros., 2010 ; campos., 2011). the genus mepraia belongs to the spinolai complex together with triatoma eratyrusiformis del ponte 1929 and triatoma breyeri del ponte 1929. the last two taxa are geographically separated from mepraia species by the andes range (lent and wygodzinsky, 1979). t. eratyrusiformis is closely related to mepraia (lent and wygodzinsky, 1979 ; hypa., 2002 ; moreno., 2006 ; fras, 2010 ; campos., 2013b). the monophyly of the spinolai complex is supported by mitochondrial gene sequences (campos., 2013b ; justi., 2014), and their divergence on both sides of the andes from the common ancestor probably occurred after the uplift of the andes during the miocene (moreno., 2006 ; although several studies have reported heteroplasmy in arthropods, this phenomenon seems to be underestimated because conventional (automated) sequencing may fail to detect it (dos., we found incongruence in phylogenies inferred with two mitochondrial markers, cytochrome b (cyt b) and cytochrome oxidase subunit - i (coi) in mepraiaspecies and t. eratyrusiformis and discuss the origin that may have produced this pattern. we used 66 mitochondrial gene sequences of coi and cyt b of mepraia (genbank accession numbers kc236913-kc236978, campos., 2013a). ten additional sequences of both genes were also included : m. spinolai from til til, metropolitan region (330619 s ; 705553 w, n = 2) and los andes, valparaso region (320133 s ; 700416 w ; n = 2) ; t. eratyrusiformis from salinas de bustos, departmento of independencia, province of la rioja, argentina (n = 4) ; triatoma infestans (n = 2) was used as outgroup, (accession numbers km258433-km258442). insects were manually collected by trained people as follows : a person wearing safety clothes and carrying collecting tools (forceps, brush, plastic can) was used as bait, waiting 1520 min in areas with ecological attributes to harbor kissing - bugs ; if no kissing - bug appeared, the investigator moved to another sampling site. bugs were transported to the laboratory ; the limbs were dissected and kept in 70% ethanol at 20 c. tissue dna (omega biotec, georgia) according to manufacturer s instructions. a 636-bp fragment of the mitochondrial cytochrome oxidase subunit - i (coi) gene, and a 682-bp fragment of the cytochrome b (cyt b) gene were amplified via polymerase chain reaction (pcr) using platinum taq dna polymerase (invitrogen, brazil) and the primers 7432 (forward) (5-ggacgwggwatttattatggatc-3) and 7433 (reverse) (5-gcwccaattcargttartaa-3) for cyt b (monteiro., 2003) and the primers lco1490 (forward) (5-ggtcaacaaatcataaagatattgg-3) and hco2198 (reverse) (5-taaacttcagggtgac caaaaaatca-3) for coi (folmer., 1994). the following conditions were used to amplify both cyt b and coi : an initial denaturation at 94 c for 3 min, 30 cycles of 1 min at 94 c, 45 c for 1 min and 72 c for 1 min, followed by a final extension of 10 min. sequences were edited using bioedit 7.0.8.0 (hall, 1999) and aligned using clustal w (thompson., 1994) as implemented in bioedit (hall, 1999). after the alignments, sites that showed nucleotide substitutions were re - examined by visual inspection of each individual s raw chromatogram. for phylogenetic reconstruction (see below) ambiguous bases were coded using the nucleotide ambiguity code (iupac). non - synonymous substitutions and stop codons were checked using dnasp 5.1 (librado and rozas, 2009). the resulting coi data was 508 bp in length and cyt b was 514 bp. phylogenetic reconstructions were performed separately for each mitochondrial gene by maximum likelihood (ml) and maximum parsimony (mp). ml analyses were performed using the online platform phyml 3.0 (guindon., 2010). the best - fitting model of nucleotide substitution was selected using the akaike information criterion (akaike, 1974) on cyt b (trn + i, i : 0.66) and coi (hky+g, g : 0.068) implemented in the program jmodeltest 0.1.1 (posada, 2008). mp analyses were performed using paup 4.0b10 (swofford, 2002) with the heuristic search option and tree bisection reconnection (tbr) branch swapping. nodal supports were estimated by the bootstrap method (felsenstein, 1985) with 1000 replicates. triatoma infestans, was used as outgroup based on its phylogenetic proximity to the spinolai complex (hypa., 2002 ; de paula., 2005 this research was undertaken with approval from the bioethics committee of the pontificia universidad catlica de valparaso, chile. we used 66 mitochondrial gene sequences of coi and cyt b of mepraia (genbank accession numbers kc236913-kc236978, campos., 2013a). ten additional sequences of both genes were also included : m. spinolai from til til, metropolitan region (330619 s ; 705553 w, n = 2) and los andes, valparaso region (320133 s ; 700416 w ; n = 2) ; t. eratyrusiformis from salinas de bustos, departmento of independencia, province of la rioja, argentina (n = 4) ; triatoma infestans (n = 2) was used as outgroup, (accession numbers km258433-km258442). insects were manually collected by trained people as follows : a person wearing safety clothes and carrying collecting tools (forceps, brush, plastic can) was used as bait, waiting 1520 min in areas with ecological attributes to harbor kissing - bugs ; if no kissing - bug appeared, the investigator moved to another sampling site. bugs were transported to the laboratory ; the limbs were dissected and kept in 70% ethanol at 20 c. tissue dna (omega biotec, georgia) according to manufacturer s instructions. a 636-bp fragment of the mitochondrial cytochrome oxidase subunit - i (coi) gene, and a 682-bp fragment of the cytochrome b (cyt b) gene were amplified via polymerase chain reaction (pcr) using platinum taq dna polymerase (invitrogen, brazil) and the primers 7432 (forward) (5-ggacgwggwatttattatggatc-3) and 7433 (reverse) (5-gcwccaattcargttartaa-3) for cyt b (monteiro., 2003) and the primers lco1490 (forward) (5-ggtcaacaaatcataaagatattgg-3) and hco2198 (reverse) (5-taaacttcagggtgac caaaaaatca-3) for coi (folmer., 1994) the following conditions were used to amplify both cyt b and coi : an initial denaturation at 94 c for 3 min, 30 cycles of 1 min at 94 c, 45 c for 1 min and 72 c for 1 min, followed by a final extension of 10 min. sequences were edited using bioedit 7.0.8.0 (hall, 1999) and aligned using clustal w (thompson., 1994) as implemented in bioedit (hall, 1999). after the alignments, sites that showed nucleotide substitutions were re - examined by visual inspection of each individual s raw chromatogram. for phylogenetic reconstruction (see below) ambiguous bases were coded using the nucleotide ambiguity code (iupac). non - synonymous substitutions and stop codons were checked using dnasp 5.1 (librado and rozas, 2009). the resulting coi data was 508 bp in length and cyt b was 514 bp. phylogenetic reconstructions were performed separately for each mitochondrial gene by maximum likelihood (ml) and maximum parsimony (mp). ml analyses were performed using the online platform phyml 3.0 (guindon., 2010). the best - fitting model of nucleotide substitution was selected using the akaike information criterion (akaike, 1974) on cyt b (trn + i, i : 0.66) and coi (hky+g, g : 0.068) implemented in the program jmodeltest 0.1.1 (posada, 2008). mp analyses were performed using paup 4.0b10 (swofford, 2002) with the heuristic search option and tree bisection reconnection (tbr) branch swapping. nodal supports were estimated by the bootstrap method (felsenstein, 1985) with 1000 replicates. we considered branches receiving > 70% bootstrap support to be well - supported (hillis. triatoma infestans, was used as outgroup based on its phylogenetic proximity to the spinolai complex (hypa., 2002 ; de paula., 2005 ; campos., 2013b). this research was undertaken with approval from the bioethics committee of the pontificia universidad catlica de valparaso, chile. ambiguous bases in the chromatograms were observed in the samples tera1 and 53til for the coi gene, and in the tera2 sample for the cyt b gene. samples tera1 and tera2 were reamplified and sequenced for both genes. the sample 53til was not again amplified due to limited genomic dna availability. the second round of sequencing for the cyt b gene did not show differences with the first round for the tera2 sample, however some nucleotide differences were observed in the tera1 sample. the second round of sequencing for the coi gene amplification did not show differences in the tera2 sample, while six sites showed nucleotide differences in the tera1 sample. all nucleotide differences between the first and the second round of sequencing for the tera1 and tera2 samples were observed in sites with ambiguous bases (figure 1). the cyt b and coi alignments showed 155 and 125 variable polymorphic sites, respectively. both phylogenetic topologies showed similar results, although incongruence was observed with four haplotypes intermingled among lineages. the cladogram reconstructed with the coi gene sequence (figure 2b) showed two haplotypes of m. spinolai (53til and 21la) grouped within the m. gajardoi lineage, and two haplotypes of t. eratyrusiformis (tera1 and tera2) were also included within the m. gajardoi lineage (81% bootstrap support). these haplotypes were grouped as expected according to their taxonomy and geography in the cladogram reconstructed with cyt b, leading to conclude that the samples tera1 and tera2 represent sister species of mepraia and the samples 53til and 21la belong to m. spinolai (figure 2a). in the incongruent sequences (53til, 21la, tera1, and tera2) non - synonymous mutations the extent of topological incongruence between two molecular markers depends both on evolutionary and methodological processes. for example, incongruence of mitochondrial and nuclear gene trees may be due to differential lineage sorting, introgression or heteroplasmy (sota and vogler, 2001 ; magnacca and brown, 2010 ; barnab and brenire, 2012 ; messenger., 2012). however, due to the linked nature of mtdna genes it is expected that different mitochondrial genes show similar topologies. our phylogenetic analyses using two mtdna genes (cyt b and coi) showed incongruent topologies that were evident for four haplotypes placed in different lineages. this unexpected result may be explained by : i) contamination of samples with foreign or neighboring dna ; ii) taq polymerase may yield errors during the amplification process due to lack of fidelity ; and iii) nucleotide intra - individual variation. in this study, samples were extracted and amplified in different periods and were processed as groups of the same species, which rules out contamination errors. similarly, it is highly unlikely that polymerase errors would be the primary source of the phylogenetic incongruence, as we used the same conditions for the platinum taq dna high fidelity polymerase in all our pcr reactions (gyllensten, 1989 ; frey and frey, 2004). hence, it is highly probable that the main cause of the incongruence is related to nucleotide intra - individual variation. nuclear integration of mitochondrial gene fragments has been reported in triatominae (dotson and beard, 2001) and other arthropods (zhang and hewitt, 1996a ; parfait., 1998 ; bensasson., 2001), but this process seems not to represent a major issue due to the larger number of mtdna copies (lightowlers., 1997 ; scheffler, 2001). however, the presence of multiple nuclear copies of coi and other mitochondrial genes has been reported in aphids and other insects (gellissen and michaelis, 1987 ; sunnucks and hales, 1996 ; bensasson., 2000), and therefore we do not rule out this phenomenon until further analyses are performed. these copies behave as pseudogenes that are not correctly expressed, because they accumulate nonsense mutations resulting in stop codons in the reading frame and a greater number of non - synonymous changes, thus tending to diverge from the homologous mitochondrial genes (zhang and hewitt, 1996a ; frey and frey, 2004). we found no sequences with stop codons in the two genes, and found no non - synonymous mutations in the coi gene. finally, the co - amplification of more than one mitochondrial haplotype (i.e. heteroplasmy ; solignac., 1986 ; boyce., in the samples tera1 tera2 and 53til we observed ambiguous bases after the nucleotide amplification (figure 1) ; the sample 21la did not show ambiguous bases. the retention of ancestral polymorphisms through incomplete division of lineages may also explain the misplacement of the t. eratyrusiformis and m. spinolai haplotype. however, due to the linked nature of mitochondrial genes, the same results (incongruence) should have been observed in both topologies, contrary to our results. in addition, incomplete lineage sorting has a higher chance to occur in recently diverged groups and with high population sizes (maddison and knowles, 2006), which contrasts with the lineages in our study that are highly structured and show deep divergences (campos., 2013a). therefore, we suggest that heteroplasmy is the most parsimonious explanation for the incongruence observed in our study. heteroplasmy of mtdna may result from two or more haplotypes within a single mitochondrion, or different mitochondria with different haplotypes, either within one cell or in different cells (lightowlers. processes that may explain the latter are paternal leakage, recombination, and segregating mutations (white., 2008). paternal leakage or inheritance of mitochondrial dna has been described in insects including hemiptera (kondo., 1990 ; fontaine., 2007 ; nunes., 2013 ; wolff., 2013), and may have occurred between t. etratyrusiformis and mepraia.this incongruence between mitochondrial genes due to heteroplasmy may lead to erroneous phylogenetic hypotheses resulting in wrong taxonomic classification and population genetic structure (zhang and hewitt, 1996b ; white., 2008). the presumptive phenomenon of heteroplasmy suggests that mtdna from t. eratyrusiformis can be found within the mepriaia genome, which supports their close systematic relationship and is independent evidence of common ancestry. due to the non - overlaping distribution of mepraia and t. eratyrusiformis (separated by the andes), the processes that may have caused the heteroplasmy seen within the spinolai complex probably occurred a long time ago, before or at early periods during the uplifting of the andes ranges, and reveal that heteroplasmy may persist in populations even though new lineages are subsequently formed. in conclusion, after performing phylogenetic reconstructions with two mitochondrial genes, we found incongruent topologies for some haplotypes of m. spinolai and t. eratyrusiformis. we suggest as explanation of our results that there is intra - individual variation likely due to heteroplasmy. the mitochondrial incongruence within the spinolaicomplex requires further investigation to determine more accurately the extent and probable causes of this pattern.	mitochondrial dna (mtdna) is widely used to clarify phylogenetic relationships among and within species, and to determine population structure. due to the linked nature of mtdna genes it is expected that different genes will show similar results. phylogenetic incongruence using mtdna genes may result from processes such as heteroplasmy, nuclear integration of mitochondrial genes, polymerase errors, contamination, and recombination. in this study we used sequences from two mitochondrial genes (cytochrome b and cytochrome oxidase subunit i) from the wild vectors of chagas disease, triatoma eratyrusiformis and mepraia species to test for topological congruence. the results showed some cases of phylogenetic incongruence due to misplacement of four haplotypes of four individuals. we discuss the possible causes of such incongruence and suggest that the explanation is an intra - individual variation likely due to heteroplasmy. this phenomenon is an independent evidence of common ancestry between these taxa.
selective serotonin reuptake inhibitors (ssris) and exposure with response prevention for treatment of obsessive - compulsive disorder (ocd) have demonstrated empirical support ; however, a substantial number of patients remain with clinically significant ocd symptoms after these treatments (1 - 6). in recent years, one of the promising novel treatment strategies developed to improve the efficacy of treatment for patients with ocd is acceptance and commitment therapy (act) (7, 8). act is a third - wave behavior therapy, which specifically focuses on decreasing experiential avoidance (ea), increasing activity in the chosen valued life direction and increasing psychological flexibility. the goal of act is not symptom reduction per se, but helping patients to accept aversive inner experiences (e.g. thoughts, images, emotions and bodily sensations) in the service of engaging in values - guided behavior (9). ea is defined as unwillingness to remain in contact or experience aversive private thoughts or experiences to avoid or escape from these experiences (7). ea has been suggested to play an important role in the development and maintenance of ocd (10). ea has been hypothesized to manifest as compulsions in ocd (10). in ea perspective, patients with ocd engage in compulsions to control or reduce their unwanted obsessional thoughts, because they want to reduce negative affect associated with them (10). in support of this perspective, correlational studies have found that high levels of ea were positively associated with high levels obsessive - compulsive (oc) symptoms (11 - 13). in treating ocd act teaches patients to create a new relationship with obsessive thoughts and anxious emotions, for example, helping patients notice that a thought is just thought and anxiety is an emotion to be felt. act also helps patients commit to act in the service of their valued life goals rather than spending large amounts of time trying to decrease the obsession or avoid anxious feelings. act helps patients to accept their obsessional thoughts and negative feelings and commit to acting in service of their valued life directions whether obsessions were occurring. thus, these constructs would increase psychological flexibility, which is the ability to act inconsistent with patient s meaningful life directions regardless of unpleasant inner experiences (14, 15). act has demonstrated large and clinically significant improvements in ocd symptoms in adults (14 - 17) and adolescent (18). however, according to the best of our knowledge, there are no randomized controlled studies directly comparing the relative effectiveness of act with ssris and combination of act and ssris in the treatment of ocd. therefore, the aim of this study was to compare act with ssris and combination of act and ssris in improvement of oc symptoms and ea. in an experimental design using convenience sampling, adults with ocd were recruited from outpatients of clinics in tehran, iran, from february 2012 to march 2013. a written informed consent was obtained from patients and received complete descriptions regarding the study procedures. inclusion criteria for the sample were : a) a primary diagnosis of ocd according to the diagnostic and statistical manual of mental disorders, fourth edition, text revision (dsm - iv - tr) (19) ; b) age between 18 and 50 years ; and c) oc symptoms duration of at least one year. patients were excluded from the study if they : a) had a current or past psychotic disorder ; b) had suicidal tendencies ; c) had a medical illness ; d) had a substance abuse disorder ; e) had a personality disorder and f) had been treated with either pharmacotherapy or psychotherapy in the last one month. forty - four patients were referred for treatment, of which 40 met the dsm - iv - tr criteria for ocd. they were interviewed by a structured clinical interview for dsm - iv axis i disorders, patient edition (scid - i / p version 2) (20) and a structured clinical interview for dsm - iv axis ii disorders (scid - ii) (21) to verify the diagnosis by an independent evaluator (phd level clinical psychologist). two patients with bipolar disorder, two patients with psychotic disorder, one patient with avoidant personality disorder, two patients with oc personality disorder and one patient with borderline personality disorder were excluded from the study. in all, 32 patients fitted the inclusion / exclusion criteria and were randomly assigned to receive act (n = 10), ssris (n = 11) or combination of act and ssris (n = 11). three patients dropped out the drug group ; one because of not tolerating adverse effects of sertraline and two for irregular use of fluoxetine. the mean age of the final sample (n = 27) was 26.96 6.83 years ; 55.6% (n = 15) of the patients were men and 44.4% (n = 12) women. the yale - brown obsessive - compulsive scale (y - bocs) total and acceptance and action questionnaire (aaq) scores (mean standard deviation) for the sample were 24.40 3.07 and 36.20 6.08, respectively. all patients met the criteria for ocd as their primary diagnosis with 33.3% receiving one additional diagnosis. the frequency of comorbidities in each treatment condition was as follows ; major depressive disorder and dysthymia, two act, two ssris, two act plus ssris ; anxiety disorders, one act, one ssri, one act plus ssris. ocd subtypes for the sample were washing, checking, ordering, religious / sexual / aggressive thoughts and hoarding. act was based on an act manual for ocd developed by twohig (14). a phd clinical psychologist (the first author) the act program included evaluating patient s obsessions and compulsions (session 1) ; the man in the hole metaphor was used to illustrate how patient s efforts to regulate obsessions are ineffective (session 2) ; the tow scales metaphor was used to illustrate possible benefits of acceptance of obsessions and anxiety rather than attempting to control or reduce them (sessions 3 and 4) ; using defusion, contact with present or mindfulness, and self as context exercises (sessions 5 and 6) ; recognize his values ; and preventing relapse (sessions 7 and 8). patients in drug group received a different ssri (sertraline = 50 - 200 mg / d ; fluoxetine = 20 - 80 mg / d) for 10 weeks, which was monitored by two psychiatrists. the combination group received both act and a different ssri (sertraline = 50 - 200 mg / d ; fluoxetine = 20 - 80 mg / d) for 10 weeks. studies showed equivalent efficacy for different ssris in ocd (2). in this study, we did not compare the efficacy of sertraline with fluoxetine on each outcome measure at the end of treatment. scid - i / p and scid - ii are widely used as gold standard instruments for diagnosing axis i and axis ii disorders (22). all participants were initially assessed using the scid - i / p and scid - ii. clinical symptoms were assessed using the y - bocs (23, 24), aaq (25) at the beginning and end of treatment. y - bocs is a 10-item clinician administered scale used for the assessment of severity of oc symptoms. it has good psychometric properties and has shown excellent treatment sensitivity (23, 24, 26, 27). each item is rated on a 7-point scale with higher scores indicating greater ea (range : 7 - 63). it has demonstrated good internal consistency (= 0.70) and test - retest reliability over a 4-month interval (rs = 0.64) (24). the reliability and validity of the iranian version of the y - bocs (28), aaq (29), scid - i / p and scid - ii (30) have been evaluated and shown to be as good as their original versions. the criteria for clinically significant change (csc) considered a y - bocs total score reduction of eight scores or more from pre - treatment to post - treatment and a final y - bocs total score 14. patients were classified as improved if they achieved reliable change that is eight score or more, but scoring above 14 on y - bocs total. patients who did not achieve reliable change on the y - bocs were defined as unchanged (6). full remission was defined as post - treatment y - bocs total score 8 (31). group differences were evaluated using chi - square, one - way analysis of variance (anova) and one - way analysis of covariance (ancova) statistical procedures on each of the outcome measures using spss for windows (version 16.0, spss inc., chicago, il, usa) statistical package. using anova and chi - square test, no significant difference was found between the groups regarding demographic characteristics and pre - treatment measures (tables 1 and 2). abbreviations : act, acceptance and commitment therapy ; ocd, obsessive - compulsive disorder ; and ssris, selective serotonin reuptake inhibitors. abbreviations : aaq, acceptance and action questionnaire ; act, acceptance and commitment therapy ; ssris, selective serotonin reuptake inhibitors ; and y - bocs, yale - brown obsessive - compulsive scale. patients achieving csc at post - treatment were 44.4%, 40.0% and 12.5% for act, combined and ssris groups, respectively. two patients (22.2%) in act and two patients (20.0%) in combined group and two patients (25.0%) in ssris group met the criteria for reliability improved. three patients (33.3%) in act, four patients (40.0%) in combined and five patients (62.5%) in ssris groups were classified as unchanged. regarding the criteria for oc symptoms full remission (y - bocs score 8), two patients (22.2%) in act and two patients (20.0%) in combined group achieved this improvement level, but none of the patients in ssris group had full remission. table 3 displays the mean scores, standard deviation and ancova results on each outcome measure at the end of treatment. the assumptions of homogeneity of variance and homogeneity of regression were met for each of the comparisons. pair - wise post - hoc comparisons of the means of the groups showed significant differences between act and ssris as well as between combined treatment and ssris on the y - bocs and aaq at post - treatment. however, there were no significant differences between act and combined treatment on the measures. effect sizes using cohen s d (difference between adjusted means divided by the pooled standard deviation (32) was calculated to evaluate the size of differences between the treatment groups. large effect sizes were found for differences between the act group and the ssri group as well as between the combined group and the ssri group on the y - bocs and aaq. however, there were no significant differences between act and combined groups on oc symptoms and ea at post - treatment (table 4). abbreviations : aaq, acceptance and action questionnaire ; act, acceptance and commitment therapy ; ssris, selective serotonin reuptake inhibitors ; and y - bocs, yale - brown obsessive - compulsive scale. abbreviations : aaq, acceptance and action questionnaire ; act, acceptance and commitment therapy ; ssris, selective serotonin reuptake inhibitors ; and y - bocs, yale - brown obsessive - compulsive scale. d = cohen s effect size (0.2 = small effect, 0.5 = medium effect, 0.8 = large effect). patients achieving csc at post - treatment were 44.4%, 40.0% and 12.5% for act, combined and ssris groups, respectively. two patients (22.2%) in act and two patients (20.0%) in combined group and two patients (25.0%) in ssris group met the criteria for reliability improved. three patients (33.3%) in act, four patients (40.0%) in combined and five patients (62.5%) in ssris groups were classified as unchanged. regarding the criteria for oc symptoms full remission (y - bocs score 8), two patients (22.2%) in act and two patients (20.0%) in combined group achieved this improvement level, but none of the patients in ssris group had full remission. table 3 displays the mean scores, standard deviation and ancova results on each outcome measure at the end of treatment. the assumptions of homogeneity of variance and homogeneity of regression were met for each of the comparisons. pair - wise post - hoc comparisons of the means of the groups showed significant differences between act and ssris as well as between combined treatment and ssris on the y - bocs and aaq at post - treatment. however, there were no significant differences between act and combined treatment on the measures. effect sizes using cohen s d (difference between adjusted means divided by the pooled standard deviation (32) was calculated to evaluate the size of differences between the treatment groups. large effect sizes were found for differences between the act group and the ssri group as well as between the combined group and the ssri group on the y - bocs and aaq. however, there were no significant differences between act and combined groups on oc symptoms and ea at post - treatment (table 4). abbreviations : aaq, acceptance and action questionnaire ; act, acceptance and commitment therapy ; ssris, selective serotonin reuptake inhibitors ; and y - bocs, yale - brown obsessive - compulsive scale. abbreviations : aaq, acceptance and action questionnaire ; act, acceptance and commitment therapy ; ssris, selective serotonin reuptake inhibitors ; and y - bocs, yale - brown obsessive - compulsive scale. d = cohen s effect size (0.2 = small effect, 0.5 = medium effect, 0.8 = large effect). all three treatments were effective in reducing total scores in the y - bocs and aaq at post - treatment. nevertheless, act and combined treatment presented significantly greater improvements in severity of oc symptoms and ea and a higher rate of complete remission of oc symptoms than those resulting from ssris use. furthermore, act and combined treatment produced greater rates of recovery in oc symptoms compared to ssris alone. regarding oc symptoms improvement, our results were similar to those found in previous studies with act (14 - 18) and ssris (1, 2). the rate of reduction in the y - bocs in the study of twohig. (15), was 47.3% at post - treatment, which is virtually the same as 41.3% reduction at the end of act observed in the current study. a possible explanation for the effectiveness of act may be the fact that reductions in ea and cognitive fusion produce greater psychological flexibility. act aimed at helping patients to accept their obsessional thoughts and negative feelings and commit to acting in service of their valued life goals regardless of obsessions were occurring. increasing psychological flexibility and values - based action in the presence of obsessional thoughts and negative emotions could be a core process of change in oc symptoms (15). in support of this perspective, evidence (14 - 18) show that reduction in oc symptoms is due to specific processes used in act (i.e. acceptance and cognitive defusion). act processes (e.g. reductions in ea) changed prior to changes in ocd severity. in the present study, ea decreased in act and combined treatment groups, without significant difference between the groups. these results are inconsistent with studies of twohig (14), twohig. (15, 16) and armstrong (18), which demonstrated reductions in ea in patients with ocd following act. from an act perspective, increasing one s willingness to experience obsessional thoughts and negative feelings and not engage in behaviors to decrease them produces reductions in ea and compulsions (15). the finding that act is more effective in improving oc symptoms than ssris is consistent with previous findings by foa. (35) who found the superiority of psychotherapy over anti - obsessive drugs alone in the treatment of adults with ocd. similarly, foa. (36) observed that the combination of psychotherapy and pharmacotherapy reached better results than pharmacotherapy alone. eddy. (3) by reviewing 18 randomized controlled trials found that the combination treatment is more effective in reducing oc symptoms than either psychotherapy or medication alone. however, in the present study, the combined treatment was only more effective in reducing total scores in the y - bocs than medication alone. in this study, no significant differences were found between act and combined treatment regarding the oc symptoms improvement. these findings support the results of two studies (37, 38), which found no advantages of combining medication and psychotherapy compared to psychotherapy alone in treating patients with ocd, although, unlike the present study, these studies found no significant differences between combined treatment and medication alone. the results of the present study demonstrated that combining ssris with act does not increase the effectiveness of act in improving oc symptoms and ea in patients with ocd in short - time. moreover, patients treated with act and combined treatment experienced further improvement in oc symptoms and ea at post - treatment compared to those treated with ssris alone. the sample size was small, and thus, these findings should be considered preliminary until replicated in larger sample sizes. further researches are necessary to examine whether our results are applicable to patients with ocd and other comorbid conditions excluded from this study. therefore, future research is needed to evaluate long - term effects of all three mentioned treatments for ocd. it is recommended to perform further studies to compare the effectiveness of act with other empirically supported treatments and combination of these treatments with ssris for ocd. despite these limitations, our results provided supporting evidence for effectiveness of act in helping patients with ocd to experience significant improvements in ea and oc symptoms, as well highlighted the importance and need for additional studies in this area.	background : selective serotonin reuptake inhibitors (ssris) and exposure with response prevention for treatment of obsessive - compulsive disorder (ocd) have demonstrated empirical support ; however, a substantial number of patients remain with clinically significant ocd symptoms after such treatments. objectives : the aim of this study was to compare the effectiveness of acceptance and commitment therapy (act), selective serotonin reuptake inhibitors (ssris) and combination of act and ssris in the treatment of adults with obsessive - compulsive disorder (ocd).patients and methods : thirty - two outpatients meeting diagnostic and statistical manual of mental disorders, fourth edition, text revision criteria for ocd were randomly assigned to one of the three treatment conditions : act, ssris and combined treatment. the yale - brown obsessive - compulsive scale and acceptance and action questionnaire were administered at pre - treatment and post - treatment. twenty - seven patients completed the study. data was analyzed using one - way analysis of variance (anova) and one - way analysis of covariance (ancova), clinically significant change (csc) and complete remission status.results:ancova revealed that patients treated with act and combined treatment experienced a significantly greater improvement in obsessive - compulsive (oc) symptoms and experiential avoidance (ea) at post - treatment compared to those treated with ssris alone. however, there were no significant differences between act and combined treatment on oc symptoms and ea. csc and complete remission status results showed that unlike ssri, act and combined treatment led to more improvement in oc symptoms.conclusions:act and combined treatment are more effective than ssris alone in treating oc symptoms and ea. however, it appears that adding ssris to act does not increase the effectiveness of act in the treatment of adults with ocd in the short - term.
birth weight is one of the most important indicators of infant 's health, and it also indicates mother 's health condition, hygiene, and prenatal care. at present, birth of low weight infants is one of the most serious health problems in the world. scientific and technological advances in obstetrics and neonatal care during recent years have increased the rate of survival for premature and low weight infants. global prevalence of low birth weight is reported to be 15.5%, and 96.5% of these infants have been born in developing countries. in addition, the prevalence of low birth weight and very low birth weight in iran have been reported to be 71 and 1.30%, respectively ; the prevalence of low birth weight in isfahan province has been reported to be 9.5%. results of some studies have shown that, despite long and costly intensive care and the survival of some low birth weight infants, outcomes are not promising ; these infants would encounter complications in neurodevelopment and congenital malformations three times more than infants with normal birth weight. these children are prone to many disabilities including cerebral palsy, cognitive disorders, blindness, deafness, impaired short - term memory, ocular deviation, delayed language development, learning, and behavioral disorders. some researchers believe that these disabilities would remain until school age and even afterwards ; hence, most of these children need special and constant care. results of the study by wang. showed that cognitive functioning in children with very low birth weight is lower than term infants, and that there was a significant relation between growth and cognitive ability. further, the results of gick fan. revealed a significant relation between behavioral disorders and birth weight. santo in a study reported that premature and low weight infants would encounter more cognitive and behavioral disorders at pre - school age ; however, in the study of gurka., that was conducted on 52 healthy infants with congenital age of 3436 weeks and compared to term infants, no significant difference was found between the two groups regarding behavioral disorder. therefore, further studies in this field are required. because of the ever increasing birth rate of premature and low weight infants, their parents would care regarding the physical and mental health of their children in the future. considering the controversial results of previously conducted studies, the fate of these children is ambiguous. previously conducted studies in iran have only evaluated the growth of these children, and simultaneous evaluation of their growth and behavioral disorder has not been considered before. pre - school is the official start of children 's educational activity, and the assessment program before elementary school is performed during this period. in this assessment program,, diagnosis of behavioral disorders in children and referring them to specialized centers at early ages and recognizing their abilities and disabilities would be a great help to families and society. therefore, this study was conducted to comparatively evaluate the indicators of growth and behavioral disorder in children with normal, low, and very low birth weight at pre - school age in isfahan during 2015. study population included all the children with normal, low, and very low birth weight who were enrolled in pre - school centers of isfahan. with a 95% confidence interval and 80% statistical power, data collection tools were digital scale in kilograms, standard standing stadiometer in cm made by seca, germany, which was accurately calibrated before each measurement, and a two - part questionnaire including parents and children 's demographic characteristics such as age, gender, educational level and job, and the rutter children behavior questionnaire for parents. this questionnaire consists of 31 three - choice questions about children 's behavioral characteristics, which parents answered according to the rating scale. the rating scale was in the form of no problem or issue, few problems or some issues, and a lot of problems or many issues, which were scored as 0, 1, and 2, respectively. the total score of the test varies between 0 and 62 for each individual, and if children scored 13 or more (cut - off point) they were determined to be a person with behavioral disorders. validity and reliability of this questionnaire was first evaluated in iran by mehryar in 1996. according to agha yousef (2012) using split - half method, and its durability was reported to be 85% and its cut - off point for parents version was set at 13 by mehryar. the inclusion criteria for all three groups were being enrolled in pre - school, not having a birth weight more than 4600 g, not having any congenital malformation, obviously or according to the mother, not having any chronic diseases, being of iranian nationality, living with both of the parents, and no experience of any severe mental stress during the past 6 months according to the parents. children with normal birth weight should not have had a long hospitalization during infancy due to any disease. child 's birth specifications were completely be mentioned in their birth card, and parents providing full consent for participation of their child in the study. moreover, unwillingness to participate in the study and not completing the questionnaire were the exclusion criteria. the researcher referred to pre - school centers from january 2015 to june 2015 and performed the sampling. after obtaining necessary permissions for this study from all the pre - school centers in isfahan, 25 were selected using random cluster and quota sampling methods (14 centers under the supervision of ministry of education and 11 under the supervision of welfare organization). from all the children who were present at pre - school centers, appropriate to the population of each center, children with normal birth weight children with low birth weight and very low birth weight were selected based on enumeration method. the information of children who had the inclusion criteria were recorded in the questionnaire from their birth cards by the researcher. to complete other questions and to answer the questions of the rutter children behavior questionnaire for parents, the questionnaire and the consent form they were asked to answer the questions and return the forms to the pre - school teacher. after the parents completely answered the questionnaire, the researcher accurately measured the growth indicators of the child. at the end data were analyzed using descriptive (mean and standard deviation) and inferential (variance analysis, chi square, kruskal wallis tests) statistics. aims of the study were explained to the parents and written informed consent was obtained from them. with regards to ethical consideration, aims of the study were explained to the parents and written informed consent was obtained from them. in this study, 236 children (126 children with normal birth weight, 100 with low birth weight, and 10 with very low birth weight) who were enrolled in pre - school centers under the supervision of ministry of education and welfare organization were selected as participants. the mean age of fathers and mothers at the time of their child 's birth was 31.40 and 26.90 years, respectively. most of the mothers were housewives, most of the fathers were freelancers, and the educational level of most of the parents was diploma. chi square and kruskal wallis tests showed no significant difference between the gender and age of children as well as their parents job and educational level along with other demographic characteristics of the three groups. the means of weight, height, and body mass index (bmi) are shown in table 1. results of one - way analysis of variance (anova) showed a significant difference between the mean of weight and height of three groups at pre - school age (p = 0.009, f = 4.802). in addition, post - hoc least squares difference (lsd) test showed that the mean of weight and height in the group with very low birth weight was significantly lower than the normal (p = 0.004, p = 0.001) and the low birth weight (p = 0.003) groups. however, the difference between the mean of weight and height of the normal and low birth weight groups had no significant difference (p = 0.10, p = 0.66). mean and standard deviation of growth indicators of normal birth weight, low birth weight, and very low birth weight children in preschool age furthermore, one - way anova showed that the mean of bmi of three groups had a significant difference among each other (p = 0.03, f = 3.61). moreover, post - hoc lsd test showed that the bmi of the group with normal birth weight was significantly higher than the group with low birth weight (p = 0.02) and the group with very low birth weight (p = 0.04). however, the difference between low birth weight group and very low birth weight group was not significant (p = 0.52). the results also revealed that 63.2% of the normal weight group, 52% of low birth weight group, and 60% of very low birth weight group had behavioral disorders. wallis test showed that the condition of behavioral disorder had no significant difference between all three groups (p = 0.13, = 4.11). the mean score of behavioral disorders in the normal weight group was 9.10, in the low weight group was 10.2, and in the very low weight group was 10.10. furthermore, one - way anova showed that the difference between the three groups regarding behavioral disorders was not significant (p = 0.49, f = 0.70). results of the present study showed that there was a significant difference between the three groups regarding the indicators of growth at pre - school age ; comparing the weight and height of three groups at pre - school age revealed that the mean of weight and height in the group with very low birth weight was lower than the other two groups. the mean of bmi of three groups had a significant difference with each other, and the bmi of group with normal birth weight was significantly higher than the group with low birth weight and the group with very low birth weight. growth disorders could have many complications including increased number of referrals for medical treatment, having longer school periods, growth disorders during childhood, and decreased capacity and volume of lungs. (2009) showed that the mean of weight and height in 5-year - old children with a history of low birth weight was lower than children with normal birth weight. the results of the study by mahram. revealed a significant difference between 6-year - old children with normal birth weight and low birth weight regarding their weight and height. a study by kato. showed that low birth weight children, despite their high rate of growth until the age of 3.5 compared to children with normal birth weight, have a lower bmi and height at the age of 5 than children with normal birth weight. darendeliler. showed that, although the bmi of premature children was within normal range, it was lower than the bmi of children with normal birth weight.. also showed that the mean of bmi in children with low birth weight and children with normal birth weight at the age of 5 had a significant difference and the group with low birth weight had a lower mean of bmi than the normal group. in the present study, participants were divided into three groups of normal birth weight, low birth weight, and very low birth weight, however, in the abovementioned studies, only two groups existed normal birth weight and low birth weight. in the present study, children with low birth weight showed no significant difference with normal weight children regarding their weight and height growth ; this implies that these children have the potential to recover from their low birth weight and have a growth spurt, and also due to their low birth weight, their parents would have more sensibility and concern regarding their nutrition and health care, which is another reason for them to have a desirable growth. however, children with very low birth weight were lower than the other two groups regarding indicators of growth ; this could imply that very low birth weight is a predicting factor for growth during childhood. regarding the bmi of the participants in the present study, results were similar to the studies of kato. and darendeliler. in these studies, children with low birth weight also had a lower bmi than normal children, which could be due to children 's growth rate and nutritional factors. comparing behavioral disorders between the three groups showed no significant difference between the mean score of behavioral disorder of the groups. in this regard crombi. and gurka. have reported that premature children and children with low birth weight are more prone to behavioral problems and have weaker emotional signs and mental health compared to healthy term infants, and these problems would occur in preterm infants more than term infants at the age of pre - school. in contrast, results of guellec. revealed that birth weight had no significant relation with children 's cognition, behavior, and academic performance, which is similar to the results of the present study. it seems that the reason for no significant difference between these three groups regarding behavioral disorder is the high prevalence of these disorders among children of pre - school age in this study. in addition, many other factors including parents educational level and economic, social, and environmental status would also affect children 's behavior. another reason for the differences between the present results and the results of other studies could be the differences in data collection tools. results of this study revealed that children with very low birth weight have lower indicators of growth than children with normal and low birth weight. it appears that birth weight could be an important and effective factor in the growth of these children and serious follow - ups should be conducted to monitor their growth. in addition, regarding the high prevalence of behavioral disorder among participants of this study, it is recommended that monitoring and educational programs regarding behavioral disorders should be conducted before the start of elementary school. in this study, children with low birth weight and very low birth weight who were enrolled in pre - school were evaluated. probably some of these children were not able to go to pre - school due to the complications caused by their low birth weight, hence if all of the children with low and very low birth weight have been evaluated, the results would have been different. it is necessary to conduct long - term prospective studies with larger sample sizes all over the country to evaluate the growth of these children more accurately. one of the limitations of this study was the individual differences in children and their parents and how parents react to their child 's behavior, which was controlled as much as possible through random sampling. another limitation was not studying children with extremely low birth weight due to their little number. this study was financially sponsored by the research deputy of isfahan university of medical sciences. this study was financially sponsored by the research deputy of isfahan university of medical sciences.	introduction : birth weight is one of the most important indicators of infant 's health and could predict their health condition in future. this study was conducted to determine and compare indicators of growth [weight, height, and body mass index (bmi) ] and behavioral disorders in children with normal, low, and very low birth weight at pre - school age.materials and methods : in this descriptive analytical study, 236 children (126 with normal weight, 100 with low birth weight, and 10 with very low birth weight) at pre - school age were investigated in three groups. data collection tools were a two - part questionnaire including the rutter children behavior questionnaire for parents, and parents and children 's demographic characteristics questionnaire, scale, and stadiometer. data were analyzed using descriptive statistics, variance analysis, chi square, and kruskal wallis tests.results:the mean of weight, height, and bmi at pre - school age in three groups had a significant difference (p = 0.009) and it was lower in the group with very low birth weight than the other two groups ; however, the difference between the group with normal birth weight and the group with low birth weight was not significant (p = 0.10). the mean score of behavioral disorder had no significant difference between groups (p = 0.49).conclusions : results showed that children with very low birth weight grew less than the other two groups. therefore, this group needs special attention and long - term follow - up for taking care of them to ensure better growth. it is recommended to conduct more extended studies to evaluate behavioral disorders in these children.
sources and dilutions of primary antibodies used in immunoblotting, immunoprecipitation, and immunostaining are listed in supplementary table 1. aea, 2-ag, aea - d8, 2-ag - d5, win55,212 - 2, arachidonyl-2-chloroethylamide (acea), am251, and am630 were obtained from cayman chemical (ann arbor, mi). the human ir and gi3 cdna were amplified by rt - pcr from a human pancreas rna (stratagene, la jolla, ca), with oligo - dt (18 bp) for the reverse transcription. the ir cdna was incorporated into a 3flag vector and the gi3 cdna was incorporated into an mvenus - c1 vector. ir mutant (ir-3ya), whose tyr1158/1162/1163 residues were substituted to ala, was generated from wild - type ir (ir - wt) using a quikchange ii xl site - directed mutagenesis kit (stratagene). cb1r mice and their wt littermates were developed and backcrossed to a c57bl/6 j background, as previously described (16). am251 (10 mg / kg) was administrated by daily intraperitoneal injection to 1-month - old normal c57bl/6 j mice for 4 weeks. benjamin cravatt (scripps research institute), and 1-month - old db / db mice were from the jackson laboratory (bar harbor, me). dmso, am251 (10 mg / kg), and am630 (10 mg / kg) were injected daily for 4 weeks. then, the pancreata were dissected and blood was collected in the ad lib state of eating. blood glucose concentration was measured using an elite glucometer (bayer healthcare, tarrytown ny), and plasma insulin was measured using a rat / mouse insulin elisa kit (crystal chem, downers grove, il). all animal care and experimental procedures followed national institutes of health (nih) guidelines and were approved by the national institute on aging animal care and use committee. pancreata were fixed in 4% paraformaldehyde, immersed in 20% sucrose before freezing, and sectioned (7-m thickness). human and mouse paraffin - embedded pancreatic sections were immunostained as before (17). after antigen unmasking, the slides were incubated with primary antibodies (supplementary table 1), followed by secondary antibodies (invitrogen, carlsbad, ca) along with to - pro-3 (invitrogen), in some cases, for nuclear staining. slides were viewed using a lsm-710 confocal microscope (carl zeiss microimaging, mnchen - hallbergmoos, germany). multiple sections from 3 to 5 mice per group, separated by at least 200 m from each section, were assessed for signal intensity and the number of nuclear p27- or proliferating cell nuclear antigen (pcna)-positive (pcna) -cells with lsm image browser software (carl zeiss). to determine -cell area and mass, the cross - sectional areas of pancreata and -cells were determined from multiple sections (n = 5 mice per group), separated by at least 200 m from each section, using lsm image browser software. the relative cross - sectional area of -cells was determined by quantification of the cross - sectional area covered by insulin - positive cells divided by the cross - sectional area of total pancreas tissue. the -cell mass per pancreas was estimated as the product of the relative cross - sectional area of -cells per total tissue and the weight of the pancreas. mouse islets were isolated using collagenase digestion, as we previously described (18). human or mouse islets in dulbecco s modified eagle s medium (dmem) containing 4 mmol / l glucose and 1% bsa were pelleted and incubated with dmem containing 4, 10, or 15 mmol / l glucose, with or without kcl (30 mmol / l) for 10 min. lipid extraction and tandem mass spectrometry analysis were done next, and details are described in the supplementary data. we used the pixcell ii workstation (arcturus engineering, carlsbad, ca) to perform laser capture microdissection and image acquisition of -cells in pancreas sections, as we previously described (17). total rnas were isolated using the picopure rna isolation kit (arcturus), according to the manufacturer s instructions. after reverse transcription, the resulting materials were used for quantitative (q)rt - pcr amplification using gene - specific primer pairs and sybr green pcr master mix (applied biosystems, carlsbad, ca). -irwt, -irko, min6, -tc6, and -tc1 cells were maintained in dmem with 10% fbs (invitrogen). cho - k1 and cho - ir (cho - k1 cells stably transfected with ir) cells were maintained in dmem / f-12 with 10% fbs. transfections of small interfering (si)rnas (santa cruz biotechnology, santa cruz, ca) for gi3 and cb1r and the expression vectors for cb1r, gi3, and ir were done using lipofectamine rnaimax or 2000 (invitrogen). scramble sirna (silencer negative control # 1 ; ambion, austin, tx) or empty vector was transfected as the negative control. for exogenous insulin treatment, cells starved overnight in low - glucose dmem containing 0.1% fbs were incubated for 2 h in glucose - free dmem with 0.1% bsa, followed by pretreatment with cb1r agonists for 15 min and insulin stimulation for 10 min, with or without cb1r agonists. for cell proliferation studies, after treatment with cb1r antagonist or agonists, or both, the proliferation rate was determined after 48 or 72 h using the celltiter 96 aqueous one solution cell proliferation assay (promega, madison, wi), according to the manufacturer s instructions. cell lysates extracted using radioimmunoprecipitation assay buffer (ripa) containing protease and phosphatase inhibitor cocktails were incubated with the appropriate antibody and subsequently incubated with protein a / g beads. beads were washed three times with ripa buffer and underwent western blot analysis with the primary antibodies and with an hrp - conjugated secondary antibody. blots were visualized by enhanced chemiluminescence (ge health, berne, switzerland). for gi activation assay, we used the gi activation assay kit (neweast biosciences, malvern, pa) as previously described (21). cells treated with or without acea were lysed with ice - cold 1assay / lysis buffer and underwent immunoprecipitation with anti - gi guanosine triphosphate (gtp) antibody and western blot analysis with anti - gi3 antibody. for positive control, -irwt cell lysate, not treated with acea, was incubated with guanosine 5'-o-[-thio ] triphosphate (gtp-s) for 90 min before immunoprecipitation. comparisons were performed by using graphpad prism (graphpad software, la jolla, ca). a p value of < 0.05 was considered statistically significant. sources and dilutions of primary antibodies used in immunoblotting, immunoprecipitation, and immunostaining are listed in supplementary table 1. aea, 2-ag, aea - d8, 2-ag - d5, win55,212 - 2, arachidonyl-2-chloroethylamide (acea), am251, and am630 were obtained from cayman chemical (ann arbor, mi). the human ir and gi3 cdna were amplified by rt - pcr from a human pancreas rna (stratagene, la jolla, ca), with oligo - dt (18 bp) for the reverse transcription. the ir cdna was incorporated into a 3flag vector and the gi3 cdna was incorporated into an mvenus - c1 vector. ir mutant (ir-3ya), whose tyr1158/1162/1163 residues were substituted to ala, was generated from wild - type ir (ir - wt) using a quikchange ii xl site - directed mutagenesis kit (stratagene). cb1r mice and their wt littermates were developed and backcrossed to a c57bl/6 j background, as previously described (16). am251 (10 mg / kg) was administrated by daily intraperitoneal injection to 1-month - old normal c57bl/6 j mice for 4 weeks. benjamin cravatt (scripps research institute), and 1-month - old db / db mice were from the jackson laboratory (bar harbor, me). dmso, am251 (10 mg / kg), and am630 (10 mg / kg) were injected daily for 4 weeks. then, the pancreata were dissected and blood was collected in the ad lib state of eating. blood glucose concentration was measured using an elite glucometer (bayer healthcare, tarrytown ny), and plasma insulin was measured using a rat / mouse insulin elisa kit (crystal chem, downers grove, il). all animal care and experimental procedures followed national institutes of health (nih) guidelines and were approved by the national institute on aging animal care and use committee. pancreata were fixed in 4% paraformaldehyde, immersed in 20% sucrose before freezing, and sectioned (7-m thickness). human and mouse paraffin - embedded pancreatic sections were immunostained as before (17). after antigen unmasking, the slides were incubated with primary antibodies (supplementary table 1), followed by secondary antibodies (invitrogen, carlsbad, ca) along with to - pro-3 (invitrogen), in some cases, for nuclear staining. slides were viewed using a lsm-710 confocal microscope (carl zeiss microimaging, mnchen - hallbergmoos, germany). multiple sections from 3 to 5 mice per group, separated by at least 200 m from each section, were assessed for signal intensity and the number of nuclear p27- or proliferating cell nuclear antigen (pcna)-positive (pcna) -cells with lsm image browser software (carl zeiss). to determine -cell area and mass, the cross - sectional areas of pancreata and -cells were determined from multiple sections (n = 5 mice per group), separated by at least 200 m from each section, using lsm image browser software. the relative cross - sectional area of -cells was determined by quantification of the cross - sectional area covered by insulin - positive cells divided by the cross - sectional area of total pancreas tissue. the -cell mass per pancreas was estimated as the product of the relative cross - sectional area of -cells per total tissue and the weight of the pancreas. mouse islets were isolated using collagenase digestion, as we previously described (18). human or mouse islets in dulbecco s modified eagle s medium (dmem) containing 4 mmol / l glucose and 1% bsa were pelleted and incubated with dmem containing 4, 10, or 15 mmol / l glucose, with or without kcl (30 mmol / l) for 10 min. lipid extraction and tandem mass spectrometry analysis were done next, and details are described in the supplementary data. we used the pixcell ii workstation (arcturus engineering, carlsbad, ca) to perform laser capture microdissection and image acquisition of -cells in pancreas sections, as we previously described (17). total rnas were isolated using the picopure rna isolation kit (arcturus), according to the manufacturer s instructions. after reverse transcription, the resulting materials were used for quantitative (q)rt - pcr amplification using gene - specific primer pairs and sybr green pcr master mix (applied biosystems, carlsbad, ca). -irwt, -irko, min6, -tc6, and -tc1 cells were maintained in dmem with 10% fbs (invitrogen). cho - k1 and cho - ir (cho - k1 cells stably transfected with ir) cells were maintained in dmem / f-12 with 10% fbs. transfections of small interfering (si)rnas (santa cruz biotechnology, santa cruz, ca) for gi3 and cb1r and the expression vectors for cb1r, gi3, and ir were done using lipofectamine rnaimax or 2000 (invitrogen). scramble sirna (silencer negative control # 1 ; ambion, austin, tx) or empty vector was transfected as the negative control. for exogenous insulin treatment, cells starved overnight in low - glucose dmem containing 0.1% fbs were incubated for 2 h in glucose - free dmem with 0.1% bsa, followed by pretreatment with cb1r agonists for 15 min and insulin stimulation for 10 min, with or without cb1r agonists. for cell proliferation studies after treatment with cb1r antagonist or agonists, or both, the proliferation rate was determined after 48 or 72 h using the celltiter 96 aqueous one solution cell proliferation assay (promega, madison, wi), according to the manufacturer s instructions. cell lysates extracted using radioimmunoprecipitation assay buffer (ripa) containing protease and phosphatase inhibitor cocktails were incubated with the appropriate antibody and subsequently incubated with protein a / g beads. beads were washed three times with ripa buffer and underwent western blot analysis with the primary antibodies and with an hrp - conjugated secondary antibody. blots were visualized by enhanced chemiluminescence (ge health, berne, switzerland). for gi activation assay, we used the gi activation assay kit (neweast biosciences, malvern, pa) as previously described (21). cells treated with or without acea were lysed with ice - cold 1assay / lysis buffer and underwent immunoprecipitation with anti - gi guanosine triphosphate (gtp) antibody and western blot analysis with anti - gi3 antibody. for positive control, -irwt cell lysate, not treated with acea, was incubated with guanosine 5'-o-[-thio ] triphosphate (gtp-s) for 90 min before immunoprecipitation. comparisons were performed by using graphpad prism (graphpad software, la jolla, ca). a p value of < 0.05 was considered statistically significant. cb1rs are present in - and -cells in both mouse (fig. 1a) and human (fig. 1b) islets. to confirm specificity of staining, we used pancreata from cb1r mice (16) (fig. for added proof of our findings, we microdissected -cells from islets of cb1r and cb1r mice by laser - capture microscopy, and using qrt - pcr of the captured cells, we confirmed cb1r expression (fig. western blot analysis showed that cb1rs are expressed in mouse insulinoma (-tc6 and min6) and glucagonoma (-tc1) cells (supplementary fig. cb2rs are absent in - and -cells, but cellular cb2r staining is evident in islets (fig. 1d). of general interest, and as previously reported (10), the cb2rs are present in somatostatin - containing cells in islets (fig. the specificity of anti - cb1r antibody was assessed by antigen preabsorption with the corresponding blocking peptides. c : qrt - pcr amplification of insulin (ins2) and cb1r mrna from laser - captured -cells of cb1r and cb1r mice. the upper panel shows a representative image of islets before and after laser capture of -cells. the specificity of anti - cb2r antibody was assessed by antigen preabsorption with the corresponding blocking peptides. (a high - quality digital representation of this figure is available in the online issue.) also as previously reported (11), ec - synthetic enzymes, n - acyl - phosphatidyl ethanolamine phospholipase d (nape - pld ; fig. 2b) are present mainly in -cells, with little if any expression in -cells in both human and mouse islets. the ec - degrading enzyme, fatty acid amide hydrolase (faah), is present mainly in -cells (fig. 2c), and monoacyl glycerol lipase (magl) is present in both - and -cells (fig. 2d) in human and mouse islets. in agreement with published literature (9,10), increasing glucose concentrations increased 2-ag and aea levels in human (fig. 2f) islets, and membrane depolarization with kcl also increased 2-ag and aea levels in human islets (fig. consistently, glucose and kcl also increased 2-ag levels in insulin - secreting immortalized -cells (-irwt) established from mice (2,19,20) (supplementary fig. thus, these results favor the presence of an entire self - contained ecs in mouse and human islets. a and b : immunostaining for nape - pld (a) and dagl (b) in human and mouse islets. the specificity of anti - nape - pld and anti - dagl antibodies was assessed by antigen preabsorption with the corresponding blocking peptides. c : immunostaining for faah in pancreatic sections of human and faah and faah mice. the specificity of anti - magl antibody was assessed by antigen preabsorption with the corresponding blocking peptides. e : levels of aea and 2-ag extracted from one lot of human islets exposed to the indicated glucose concentrations with or without kcl for 10 min. the amount of aea and 2-ag in the samples were determined by calculating the ratio of abundance of aea or 2-ag to the internal standard abundance. f : levels of aea and 2-ag extracted from mouse islets exposed to the indicated glucose concentrations for 10 min. the amount of aea and 2-ag in the samples were determined by calculating the ratio of abundance of aea or 2-ag to the internal standard abundance. (a high - quality digital representation of this figure is available in the online issue.) the cb1r mice are known to have a lean phenotype and are resistant to weight gain, even when fed a high - fat diet, and to have improved glucose tolerance and insulin sensitivity compared with wt mice (22,23). consistently, treatment with cb1r ligands leads to glucose intolerance and insulin resistance in rodents (2325) and conversely, peripheral, but not central, cb1r antagonism caused weight - independent improvement in glucose tolerance, insulin sensitivity, fatty liver, and plasma lipid profile even in mice with genetic or diet - induced obesity (23,25,26). we found that the amount of phosphorylated ir at y1162/1163 (p - ir), irs1/2 at y612 (p - irs1/2), and akt at s473 (p - akt) were all higher in the islets of cb1r mice compared with age - matched cb1r mice (fig. 3a). because ir signaling is a key regulator of -cell proliferation (14,2729), we investigated -cell area and islet size in cb1r mice. in keeping with enhanced ir signaling in isolated islets of cb1r mice, -cell area (fig. 3c), but not pancreas wet weight of cb1r (0.216 g 0.02 g) versus cb1r(0.196 0.02 g, n = 6 per genotype, p = 0.48), were increased, and the distribution of islet size (fig. pcna, a marker of cell proliferation, was more readily apparent in nuclei of -cells in cb1r mice than in cb1r mice (fig. ki-67 staining, another marker of cell proliferation, generated similar results (data not shown). concordant with these observations, cb1r blockade by am251 also led to increases in -cell area (fig. a : western blot analysis for the indicated proteins in isolated islets from overnight - fasted cb1r and cb1r mice. c and d : mean islet size (c) and islet size distribution (d) of cb1r and cb1r mice. e : pancreatic sections of cohorts in panel b, showing pcna -cells as indicated by arrows. the number of cells that are positive for both pcna and insulin were quantified as a percentage of the total number of insulin - positive cells in the sections. g : pancreatic sections of cohorts in f, showing pcna -cells as indicated by arrows. the number of cells that are positive for both pcna and insulin were quantified as a percentage of the total number of insulin - positive cells in the sections. data are shown as the mean sem in all panels (n = 35 animals per group). p < 0.05 ; p < 0.01. (a high - quality digital representation of this figure is available in the online issue.) genetic and pharmacologic blockade of cb1r in mice with diet - induced obesity results in improved glucose tolerance and insulin sensitivity (23,25,26,30). because genetic cb1r ablation resulted in larger islets and enhanced -cell proliferation, despite improved insulin sensitivity, we investigated if cb1r modulation would be beneficial to -cells in a mouse model of type 2 diabetes. we injected dmso, am251, or am630 (a cb2r antagonist) daily into 4-week - old db / db mice for 4 weeks (fig. consistent with previous reports (23,25,26,30), am251-treated mice had lower body weight (fig. 4d) levels than dmso - treated mice, whereas am630 had no obvious effects (fig. pancreatic sections showed increased intraislet insulin content and total -cell mass in am251-treated mice compared with dmso - treated mice (fig. 4h). increased -cell mass and proliferation in am251-treated db / db mice. a : experimental timeline for dmso, am251, or am630 (cb2r antagonist) treatment in 4-week - old db / dmso, am251 (10 mg / kg), or am630 (10 mg / kg), were administrated by daily intraperitoneal injection for 4 weeks. b : body weight of dmso-, am251-, or am630-treated db / db mice. c and d : blood glucose (c) and plasma insulin (d) levels at the end of the 4-week period. e : representative images for insulin in db / db mice injected with dmso or am251 for 4 weeks. f : quantification of insulin intensity in islets of cohorts in e. g : morphometric assessment of -cell mass of cohorts in e. h : representative images of pcna -cells in islets of cohorts in e. arrows denote pcna -cells. the number of cells that are positive for both pcna and insulin were quantified as a percentage of the total number of insulin - positive cells in the sections. i : representative immunostaining for insulin and p27 in islets of cohorts in e. scale bar = 50 m. j : representative immunostaining for ir and p - ir in islets of cohorts in e. scale bar = 50 m. intensity for p - ir was normalized to that for total ir-. data are shown as the mean sem in all panels (n = 5 animals per group). p < 0.05 ; p < 0.01. (a high - quality digital representation of this figure is available in the online issue.) given that akt regulates p27 (p27) activity, an inhibitor of cell cycle progression, by affecting its abundance and subcellular localization (31,32), and that accumulation of p27 in the nuclei of -cells contributes to deficient -cell mass and proliferation during the development of type 2 diabetes in irs2 and db / db mice (28), we examined the expression and subcellular localization of p27. immunostaining of pancreatic sections from am251-treated mice showed a significant decrease in the total amount and nuclear localization of p27 in -cells, with most of the protein being localized in cytoplasm, compared with dmso - treated mice (fig. 4i). to evaluate whether the increased -cell proliferation seen by cb1r antagonism associates with any changes in ir signaling, we examined phosphorylation levels of ir. the amount of p - ir was significantly increased in am251-treated mice compared with dmso - treated mice (fig. because pharmacologic and genetic blockade of cb1rs led to enhanced ir signaling and -cell proliferation, we next investigated the potential role of irs as a / the mediator of cb1r - controlled -cell proliferation. we used -cells established from control (-irwt) and -cell specific ir knockout (-irko) mice (2,19,20) in which expression levels of cb1rs are similar (fig. the selective synthetic cb1r agonist acea slowed proliferation of -irwt cells that was prevented by am251 (fig. a : levels of cb1r, p27, and ir in -irwt and -irko cells exposed to a selective synthetic cb1r agonist acea for 40 h. b : proliferation rates of -irwt cells exposed to acea with or without am251 for 48 h. data are shown as the mean sem from three independent experiments. p < 0.01. data are shown as the mean sem from three independent experiments. p < 0.01. data are shown as the mean sem from three independent experiments. p < 0.05 ; p < 0.01. e : effects of acea (48 h) on the proliferation of -irwt and -irko cells. data are shown as the mean sem from three independent experiments. p < 0.01. f : levels of the indicated proteins in -irwt and -irko cells exposed to acea for 40 h. g : representative immunostaining of insulin and p27 in pancreatic sections from dmso- and am251-injected mice. quantification of p27 intensity and nuclear p27 -cells in islets is shown on the bottom. data are shown as the mean sem (n = 3 per group). p < 0.01. (a high - quality digital representation of this figure is available in the online issue.) as previously reported (19), -irko cells already had significantly reduced proliferation rates compared with -irwt cells (fig. 5e), whereas both compounds had no or a lesser effect on -irko cells (fig. 5d and e). consistently, acea dose - dependently decreased levels of p - irs1/2, p - akt, and p - foxo1 (fig. cb1r blockade by am251 in normal mice also resulted in a significant decrease in both the amount and nuclear localization of p27 (fig. these results suggest that cb1r signaling functions as a negative regulator of the proliferative effects of endogenously secreted insulin from -cells. to test the direct effect of cb1r activation on ir activity, isolated mouse islets (fig. 6b) were pretreated with aea or 2-ag and acea, respectively, before addition of exogenous insulin. this was also true in be(2)-m17 human neuroblastoma cells (data not shown), which express cb1rs (supplementary fig in addition, acea diminished exogenous insulin - stimulated ir autophosphorylation and levels of p - irs1/2, p - pdk1, p - akt, and p - foxo1 in min6 cells (data not shown). the inhibitory effects of cb1r agonists on ir autophosphorylation and p - akt were not observed in cho cells (cho cells lack cb1rs, see supplementary fig. stably transfected with ir) cells transfected with the expression vector containing gfp - ha tagged cb1r led to reduced ir autophosphorylation (fig. regulation of ir activity by cb1r. a : phosphorylation levels of p - ir in isolated mouse islets exposed to insulin (10 nmol / l) for 10 min with vehicle, aea or 2-ag. b : western blot analysis of the indicated proteins in -irwt cells exposed to insulin (10 min) with acea or vehicle. c : levels of p - ir and p - akt in cho - k1 cells exposed to insulin with or without acea. cho - ir cells transiently transfected with the indicated plasmids were preincubated with acea for 20 min before insulin treatment (10 min), after which their lysates were subjected to western blot analysis for the indicated proteins. f : western blot analysis for the indicated proteins in -irko cells exposed to acea for 40 h after transfection with empty vector, ir - wt, or ir-3ya. (a high - quality color representation of this figure is available in the online issue.) to directly confirm the inhibitory effects of cb1r on ir autophosphorylation, we transfected flag - tagged ir - wt and ir mutant (ir-3ya), whose tyr1158/1162/1163 residues were substituted to ala (fig. 6e), into -irko cells and then treated the cells with acea for 40 h (fig. ir autophosphorylation was detected in ir - wt transfected -irko cells, presumably due to endogenous insulin secretion, and acea reduced levels of p - ir and p - akt in those cells, but in ir-3ya transfected -irko cells, p - akt levels were not altered (fig. 6f). in sum, these results suggest a functional interaction between cb1r and ir signaling upon ir autophosphorylation. activated cb1rs mediate their intracellular actions through gi proteins (5). gi3 appears to be expressed mainly in -cells of both human and mouse (fig. 7a), and its activity was increased by cb1r activation in both -irwt and -irko cells (fig. furthermore, exogenous insulin treatment led to a decrease in gi3/ir association, which was impeded by acea (fig. lysates of -irwt and -irko cells treated with or without acea (10 mol / l) for 20 min were subjected to immunoprecipitation with anti - gi - gtp and to immunoblotting with anti - gi3. for positive control, -irwt cell lysate not treated with acea was incubated with gtp-s for 90 min before immunoprecipitation. c : endogenous gi3/ir interaction in -irwt cells exposed to acea or vehicle (15 min) after transfection of the indicated sirnas. d : endogenous gi3/ir interaction in -irwt cells exposed to insulin (10 nmol / l) with or without acea (10 mol / l). e : western blot analysis for the indicated proteins in -irko cells exposed to acea (10 mol / l) for 15 min after transfection with empty vector, ir - wt, or ir-3ya. f : increase of gi3/ir interaction by acea in -irko cells of cohorts in e. g : reduction of gi3/ir interaction by am251 in -irko cells transfected with empty vector, ir - wt, or ir-3ya. (a high - quality digital representation of this figure is available in the online issue.) we further confirmed an ec - mediated gi3/ir association using ir - wt and ir-3ya transfected -irko cells with or without acea treatment for 15 min. 6e, acea reduced levels of p - ir and p - akt in ir - wt transfected -irko cells, but p - akt was not reduced in ir-3ya transfected -irko cells (fig. 7e), and most interestingly, gi3 association with ir-3ya was actually increased compared with ir - wt (fig. 7f and g). these results suggest that gi mediates the inhibitory effect of cb1r activation on ir autophosphorylation by its association with ir. furthermore, over - expression of gi3 in -irwt cells led to decreased levels of p - ir and p - irs1/2 (fig. 2b) by sirna in -irwt cells abolished the ability of acea to inhibit insulin - stimulated phosphorylation of ir, irs1/2, and akt (fig. consistently, knockdown of cb1rs or gi3 resulted in an increase in -cell proliferation (fig. these results suggest that the cb1r - mediated effects on ir and downstream signaling involve ec - induced activation of gi. inhibitory effects of cb1r on ir signaling require gi. a : effects of gi3 over - expression on p - ir and p - irs1/2 in -irwt cells. b : phosphorylation of ir, irs1/2, and akt in -irwt cells exposed to insulin (10 nmol / l) with acea (10 mol / l) or vehicle after transfection of the indicated sirnas. c : level of p27 in -irwt cells exposed to acea for 40 h after transfection of the indicated sirnas. d : comparison of the proliferation rate between -irwt cells transfected with scramble, cb1r, or gi3 sirna data are shown as the mean sem from three independent experiments. p < 0.01. e : effects of acea on the proliferation of -irwt cells transfected with the indicated sirnas. data are shown as the mean sem from three independent experiments. p < 0.01 vs. vehicle ; # p < 0.05 ; # # p < 0.01 vs. scramble sirna. cb1rs are present in - and -cells in both mouse (fig. 1a) and human (fig. 1b) islets. to confirm specificity of staining, we used pancreata from cb1r mice (16) (fig. for added proof of our findings, we microdissected -cells from islets of cb1r and cb1r mice by laser - capture microscopy, and using qrt - pcr of the captured cells, we confirmed cb1r expression (fig. western blot analysis showed that cb1rs are expressed in mouse insulinoma (-tc6 and min6) and glucagonoma (-tc1) cells (supplementary fig. cb2rs are absent in - and -cells, but cellular cb2r staining is evident in islets (fig. 1d). of general interest, and as previously reported (10), the cb2rs are present in somatostatin - containing cells in islets (fig. the specificity of anti - cb1r antibody was assessed by antigen preabsorption with the corresponding blocking peptides. c : qrt - pcr amplification of insulin (ins2) and cb1r mrna from laser - captured -cells of cb1r and cb1r mice. the upper panel shows a representative image of islets before and after laser capture of -cells. the specificity of anti - cb2r antibody was assessed by antigen preabsorption with the corresponding blocking peptides. (a high - quality digital representation of this figure is available in the online issue.) also as previously reported (11), ec - synthetic enzymes, n - acyl - phosphatidyl ethanolamine phospholipase d (nape - pld ; fig. 2b) are present mainly in -cells, with little if any expression in -cells in both human and mouse islets. the ec - degrading enzyme, fatty acid amide hydrolase (faah), is present mainly in -cells (fig. 2c), and monoacyl glycerol lipase (magl) is present in both - and -cells (fig. 2d) in human and mouse islets. in agreement with published literature (9,10), increasing glucose concentrations increased 2-ag and aea levels in human (fig. 2f) islets, and membrane depolarization with kcl also increased 2-ag and aea levels in human islets (fig. consistently, glucose and kcl also increased 2-ag levels in insulin - secreting immortalized -cells (-irwt) established from mice (2,19,20) (supplementary fig. thus, these results favor the presence of an entire self - contained ecs in mouse and human islets. a and b : immunostaining for nape - pld (a) and dagl (b) in human and mouse islets. the specificity of anti - nape - pld and anti - dagl antibodies was assessed by antigen preabsorption with the corresponding blocking peptides. c : immunostaining for faah in pancreatic sections of human and faah and faah mice. the specificity of anti - magl antibody was assessed by antigen preabsorption with the corresponding blocking peptides. e : levels of aea and 2-ag extracted from one lot of human islets exposed to the indicated glucose concentrations with or without kcl for 10 min. the amount of aea and 2-ag in the samples were determined by calculating the ratio of abundance of aea or 2-ag to the internal standard abundance. f : levels of aea and 2-ag extracted from mouse islets exposed to the indicated glucose concentrations for 10 min. the amount of aea and 2-ag in the samples were determined by calculating the ratio of abundance of aea or 2-ag to the internal standard abundance. (a high - quality digital representation of this figure is available in the online issue.) the cb1r mice are known to have a lean phenotype and are resistant to weight gain, even when fed a high - fat diet, and to have improved glucose tolerance and insulin sensitivity compared with wt mice (22,23). consistently, treatment with cb1r ligands leads to glucose intolerance and insulin resistance in rodents (2325) and conversely, peripheral, but not central, cb1r antagonism caused weight - independent improvement in glucose tolerance, insulin sensitivity, fatty liver, and plasma lipid profile even in mice with genetic or diet - induced obesity (23,25,26). we found that the amount of phosphorylated ir at y1162/1163 (p - ir), irs1/2 at y612 (p - irs1/2), and akt at s473 (p - akt) were all higher in the islets of cb1r mice compared with age - matched cb1r mice (fig. 3a). because ir signaling is a key regulator of -cell proliferation (14,2729), we investigated -cell area and islet size in cb1r mice. in keeping with enhanced ir signaling in isolated islets of cb1r mice, -cell area (fig. 3c), but not pancreas wet weight of cb1r (0.216 g 0.02 g) versus cb1r(0.196 0.02 g, n = 6 per genotype, p = 0.48), were increased, and the distribution of islet size (fig. pcna, a marker of cell proliferation, was more readily apparent in nuclei of -cells in cb1r mice than in cb1r mice (fig. ki-67 staining, another marker of cell proliferation, generated similar results (data not shown). concordant with these observations, cb1r blockade by am251 also led to increases in -cell area (fig. a : western blot analysis for the indicated proteins in isolated islets from overnight - fasted cb1r and cb1r mice. c and d : mean islet size (c) and islet size distribution (d) of cb1r and cb1r mice. e : pancreatic sections of cohorts in panel b, showing pcna -cells as indicated by arrows. the number of cells that are positive for both pcna and insulin were quantified as a percentage of the total number of insulin - positive cells in the sections. g : pancreatic sections of cohorts in f, showing pcna -cells as indicated by arrows. the number of cells that are positive for both pcna and insulin were quantified as a percentage of the total number of insulin - positive cells in the sections. data are shown as the mean sem in all panels (n = 35 animals per group). p < 0.05 ; p < 0.01. (a high - quality digital representation of this figure is available in the online issue.) genetic and pharmacologic blockade of cb1r in mice with diet - induced obesity results in improved glucose tolerance and insulin sensitivity (23,25,26,30). because genetic cb1r ablation resulted in larger islets and enhanced -cell proliferation, despite improved insulin sensitivity, we investigated if cb1r modulation would be beneficial to -cells in a mouse model of type 2 diabetes. we injected dmso, am251, or am630 (a cb2r antagonist) daily into 4-week - old db / db mice for 4 weeks (fig. consistent with previous reports (23,25,26,30), am251-treated mice had lower body weight (fig. 4d) levels than dmso - treated mice, whereas am630 had no obvious effects (fig. pancreatic sections showed increased intraislet insulin content and total -cell mass in am251-treated mice compared with dmso - treated mice (fig. 4h). increased -cell mass and proliferation in am251-treated db / db mice. a : experimental timeline for dmso, am251, or am630 (cb2r antagonist) dmso, am251 (10 mg / kg), or am630 (10 mg / kg), were administrated by daily intraperitoneal injection for 4 weeks. b : body weight of dmso-, am251-, or am630-treated db / db mice. c and d : blood glucose (c) and plasma insulin (d) levels at the end of the 4-week period. e : representative images for insulin in db / db mice injected with dmso or am251 for 4 weeks. f : quantification of insulin intensity in islets of cohorts in e. g : morphometric assessment of -cell mass of cohorts in e. h : representative images of pcna -cells in islets of cohorts in e. arrows denote pcna -cells. the number of cells that are positive for both pcna and insulin were quantified as a percentage of the total number of insulin - positive cells in the sections. i : representative immunostaining for insulin and p27 in islets of cohorts in e. scale bar = 50 m. relative signal intensity for p27 in islet is shown on the right. j : representative immunostaining for ir and p - ir in islets of cohorts in e. scale bar = 50 m. intensity for p - ir was normalized to that for total ir-. data are shown as the mean sem in all panels (n = 5 animals per group). p < 0.05 ; p < 0.01. (a high - quality digital representation of this figure is available in the online issue.) given that akt regulates p27 (p27) activity, an inhibitor of cell cycle progression, by affecting its abundance and subcellular localization (31,32), and that accumulation of p27 in the nuclei of -cells contributes to deficient -cell mass and proliferation during the development of type 2 diabetes in irs2 and db / db mice (28), we examined the expression and subcellular localization of p27. immunostaining of pancreatic sections from am251-treated mice showed a significant decrease in the total amount and nuclear localization of p27 in -cells, with most of the protein being localized in cytoplasm, compared with dmso - treated mice (fig. 4i). to evaluate whether the increased -cell proliferation seen by cb1r antagonism associates with any changes in ir signaling the amount of p - ir was significantly increased in am251-treated mice compared with dmso - treated mice (fig. because pharmacologic and genetic blockade of cb1rs led to enhanced ir signaling and -cell proliferation, we next investigated the potential role of irs as a / the mediator of cb1r - controlled -cell proliferation. we used -cells established from control (-irwt) and -cell specific ir knockout (-irko) mice (2,19,20) in which expression levels of cb1rs are similar (fig. the selective synthetic cb1r agonist acea slowed proliferation of -irwt cells that was prevented by am251 (fig. inhibitory effects of cb1r on -cell proliferation depends on ir. a : levels of cb1r, p27, and ir in -irwt and -irko cells exposed to a selective synthetic cb1r agonist acea for 40 h. b : proliferation rates of -irwt cells exposed to acea with or without am251 for 48 h. data are shown as the mean sem from three independent experiments. data are shown as the mean sem from three independent experiments. p < 0.01. data are shown as the mean sem from three independent experiments. p < 0.05 ; p < 0.01. e : effects of acea (48 h) on the proliferation of -irwt and -irko cells. data are shown as the mean sem from three independent experiments. p < 0.01. f : levels of the indicated proteins in -irwt and -irko cells exposed to acea for 40 h. g : representative immunostaining of insulin and p27 in pancreatic sections from dmso- and am251-injected mice. quantification of p27 intensity and nuclear p27 -cells in islets is shown on the bottom. data are shown as the mean sem (n = 3 per group). p < 0.01. (a high - quality digital representation of this figure is available in the online issue.) as previously reported (19), -irko cells already had significantly reduced proliferation rates compared with -irwt cells (fig. 5e), whereas both compounds had no or a lesser effect on -irko cells (fig. 5d and e). consistently, acea dose - dependently decreased levels of p - irs1/2, p - akt, and p - foxo1 (fig. cb1r blockade by am251 in normal mice also resulted in a significant decrease in both the amount and nuclear localization of p27 (fig. these results suggest that cb1r signaling functions as a negative regulator of the proliferative effects of endogenously secreted insulin from -cells. to test the direct effect of cb1r activation on ir activity, isolated mouse islets (fig. 6b) were pretreated with aea or 2-ag and acea, respectively, before addition of exogenous insulin. this was also true in be(2)-m17 human neuroblastoma cells (data not shown), which express cb1rs (supplementary fig in addition, acea diminished exogenous insulin - stimulated ir autophosphorylation and levels of p - irs1/2, p - pdk1, p - akt, and p - foxo1 in min6 cells (data not shown). the inhibitory effects of cb1r agonists on ir autophosphorylation and p - akt were not observed in cho cells (cho cells lack cb1rs, see supplementary fig. stably transfected with ir) cells transfected with the expression vector containing gfp - ha tagged cb1r led to reduced ir autophosphorylation (fig. regulation of ir activity by cb1r. a : phosphorylation levels of p - ir in isolated mouse islets exposed to insulin (10 nmol / l) for 10 min with vehicle, aea or 2-ag. b : western blot analysis of the indicated proteins in -irwt cells exposed to insulin (10 min) with acea or vehicle. c : levels of p - ir and p - akt in cho - k1 cells exposed to insulin with or without acea. cho - ir cells transiently transfected with the indicated plasmids were preincubated with acea for 20 min before insulin treatment (10 min), after which their lysates were subjected to western blot analysis for the indicated proteins. f : western blot analysis for the indicated proteins in -irko cells exposed to acea for 40 h after transfection with empty vector, ir - wt, or ir-3ya. (a high - quality color representation of this figure is available in the online issue.) to directly confirm the inhibitory effects of cb1r on ir autophosphorylation, we transfected flag - tagged ir - wt and ir mutant (ir-3ya), whose tyr1158/1162/1163 residues were substituted to ala (fig. 6e), into -irko cells and then treated the cells with acea for 40 h (fig. ir autophosphorylation was detected in ir - wt transfected -irko cells, presumably due to endogenous insulin secretion, and acea reduced levels of p - ir and p - akt in those cells, but in ir-3ya transfected -irko cells, p - akt levels were not altered (fig., these results suggest a functional interaction between cb1r and ir signaling upon ir autophosphorylation. gi3 appears to be expressed mainly in -cells of both human and mouse (fig. 7a), and its activity was increased by cb1r activation in both -irwt and -irko cells (fig. furthermore, exogenous insulin treatment led to a decrease in gi3/ir association, which was impeded by acea (fig. lysates of -irwt and -irko cells treated with or without acea (10 mol / l) for 20 min were subjected to immunoprecipitation with anti - gi - gtp and to immunoblotting with anti - gi3. for positive control, -irwt cell lysate not treated with acea was incubated with gtp-s for 90 min before immunoprecipitation. c : endogenous gi3/ir interaction in -irwt cells exposed to acea or vehicle (15 min) after transfection of the indicated sirnas. d : endogenous gi3/ir interaction in -irwt cells exposed to insulin (10 nmol / l) with or without acea (10 mol / l). e : western blot analysis for the indicated proteins in -irko cells exposed to acea (10 mol / l) for 15 min after transfection with empty vector, ir - wt, or ir-3ya. f : increase of gi3/ir interaction by acea in -irko cells of cohorts in e. g : reduction of gi3/ir interaction by am251 in -irko cells transfected with empty vector, ir - wt, or ir-3ya. (a high - quality digital representation of this figure is available in the online issue.) we further confirmed an ec - mediated gi3/ir association using ir - wt and ir-3ya transfected -irko cells with or without acea treatment for 15 min. 6e, acea reduced levels of p - ir and p - akt in ir - wt transfected -irko cells, but p - akt was not reduced in ir-3ya transfected -irko cells (fig. 7e), and most interestingly, gi3 association with ir-3ya was actually increased compared with ir - wt (fig. 7f and g). these results suggest that gi mediates the inhibitory effect of cb1r activation on ir autophosphorylation by its association with ir. furthermore, over - expression of gi3 in -irwt cells led to decreased levels of p - ir and p - irs1/2 (fig. 2b) by sirna in -irwt cells abolished the ability of acea to inhibit insulin - stimulated phosphorylation of ir, irs1/2, and akt (fig. consistently, knockdown of cb1rs or gi3 resulted in an increase in -cell proliferation (fig. these results suggest that the cb1r - mediated effects on ir and downstream signaling involve ec - induced activation of gi. a : effects of gi3 over - expression on p - ir and p - irs1/2 in -irwt cells. b : phosphorylation of ir, irs1/2, and akt in -irwt cells exposed to insulin (10 nmol / l) with acea (10 mol / l) or vehicle after transfection of the indicated sirnas. c : level of p27 in -irwt cells exposed to acea for 40 h after transfection of the indicated sirnas. d : comparison of the proliferation rate between -irwt cells transfected with scramble, cb1r, or gi3 sirna. data are shown as the mean sem from three independent experiments. p < 0.01. e : effects of acea on the proliferation of -irwt cells transfected with the indicated sirnas. p < 0.01 vs. vehicle ; # p < 0.05 ; # # p < 0.01 vs. scramble sirna. in contrast to adipose tissue, liver, and muscle, the presence and effects of cb1rs in -cells have been inconclusive. here, we demonstrate that -cells contain all of the components of a self - contained ecs : cb1r, the necessary enzymes for catalyzing ec biosynthesis and degradation, and the capacity to generate ecs in response to glucose stimulation and depolarization, even when isolated from the pancreas. we used several antibodies (33,34), including the l15 antibody to the c - terminus of cb1r that exhibits the expression pattern most consistent with in - situ hybridization (33) as well as qrt - pcr of laser - captured -cells from islets, to affirm that cb1rs, but not cb2rs, are present on -cells of mice and men. bermdez - silva. (14) have very recently written that using the l15 antibody they also found a cb1r signal with insulin costaining ; after using a commercially available cb1r antibody, they reported that cb1rs were mainly expressed in -cells (10). most reports, including this study, found that -cells contain ec synthetic and degrading enzymes (911,14). since at least 1979, insulin mediators, also referred to as insulin second messengers, are known to be generated from lipid precursors present on plasma membranes in response to ir activation and consequent downstream phospholipase activation (35). we are now suggesting that ecs, also generated from lipid precursors in -cells, influence ir activation (supplementary fig. 3). this is especially relevant because insulin concentrations would be expected to be at their highest levels surrounding -cells and therefore the islet ecs potentially evolved to prevent an over - exuberant -cell ir signaling cascade. favoring this view are our findings that in various -cell lines, isolated islets and a mouse model of diabetes cb1r signaling counteracts the effects of insulin on -cells by preventing ir autophosphorylation and downstream signals. this finding was not unique to pancreatic -cells because activation of cb1rs also impeded exogenous insulin - stimulated ir autophosphorylation in non - insulin - secreting cells. we also found that gi, which is involved in the regulation of insulin secretion (36) and -cell proliferation (37), mediates the inhibitory effect of cb1r activation on ir activity by its association with ir. cb1r activation increased gi3 activity and gi3/ir association was strengthened by cb1r activation and substitution of tyr1158/1162/1163 residues of ir with ala, which, conversely, was weakened by suppression of cb1r activity and by insulin. furthermore, knockdown of gi3 by sirna abolished the ability of cb1r to inhibit exogenous insulin - stimulated ir autophosphorylation and -cell proliferation. these results suggest a functional and physical crosstalk between cb1r and ir signaling upon ir autophosphorylation in a gi - dependent manner. given that binding of insulin to the extracellular -chains of ir causes a change within the quaternary structure of ir that places the phosphorylation sites of one -chain within reach of the active site of the other -chain and that results in autophosphorylation at tyr1158/1162/1163 residues in the activation loop of the -chains (38,39), we propose that gi3 activated by cb1r associates with unphosphorylated ir at the tyr1158/1162/1163 residues, preventing a conformational change that secures the activation loop in a catalytically competent configuration upon ligand binding. because these receptors are found to be present within caveolae, a cholesterol - rich microdomain that performs a number of signaling functions (40), it is possible that the closeness of the cb1rs causes them to be involved in modulating ir - mediated signaling. although the detailed molecular mechanism underlying cb1r and gi3 as regulatory components in the ir signaling pathway awaits further exploration, collectively, our results imply that the alteration in ir activity by cb1rs is a reflection of a direct inhibition of ir autophosphorylation in a gi - dependent manner and that ecs directly regulate proliferation through activation of cb1rs expressed in -cells. through these actions, cb1rs are likely to set a threshold level for ir - mediated responses, which depends on the level of expression or activation of cb1r. alteration in ir activity by cb1rs may additionally be due to the change in autocrine activation because of altered insulin secretion. cb1r - mediated suppression of insulin secretion in a ca - dependent manner has been reported (12,41) ; however, there are also reports to the contrary (9,10,15,42,43). inhibition of cb1r activity in db / db mice led to reduced blood glucose and increased -cell proliferation, coupled with enhanced ir signaling. there is also evidence that insulin itself reduces glucose - stimulated ec synthesis in -cells, which would serve as a negative feedback loop to reduce intraislet ec levels (9). this would logically mean that when ir function is reduced, as in type 2 diabetes, such a robust feedback would also be impaired, leading to nonphysiologic ec levels in islets (in addition to in fat and liver) and consequent cb1r - mediated -cell dysfunction through further impeding ir activity. blocking cb1rs would therefore be expected to improve -cell function in db / db mice, as we found. an inadequate expansion of -cell mass or failure of the existing -cell mass to compensate for the changing insulin demand are hallmarks of type 2 diabetes, and these prominent features may result from defective ir signaling (14,2729). therefore, our data should result in the resumption of attention being paid to ecs as a key factor in -cell physiology and may lead to development of a new therapeutic strategy aiming to preserve better functioning -cells. ec levels, not only in the circulating blood but also in the pancreas, are said to be elevated in diabetes and obesity (9,11,44,45), and elevated ec levels are associated with increased dagl and decreased faah levels in -cells (11). thus, it is possible that increased ec tone (due to increased ec synthesis, receptor expression or activity) affects the well - described glucose - unresponsiveness of -cells and the development of insulin resistance by impeding ir autophosphorylation in insulin - sensitive tissues. indeed, aea was recently found to impair insulin - stimulated akt phosphorylation and decrease glucose uptake in skeletal muscle cells (7), and cb1r antagonism enhanced insulin responsiveness of skeletal muscle (8). in addition, pharmacologic blockade of cb1r in obese fa / fa zucker rats decreased blood glucose levels and preserved -cell mass (46), and eliminating cb1rs in liver protected against fatty liver and improved glucose tolerance and insulin sensitivity in high - fat diet peripheral, but not central, blockade of cb1r was recently reported to improve overall insulin sensitivity and glucose homeostasis (26) and a non - brain - penetrant cb1r antagonist improved glucose homeostasis, insulin sensitivity, and fatty liver in a weight - independent manner (25). this is a very important point, because a centrally acting cb1r antagonist, rimonabant, used for treating obesity, was removed from patient use because of potentially life - threatening psychiatric problems (47). therefore, cb1r antagonists with poor brain penetrance might be useful therapies in type 2 diabetes where they would be expected to lessen insulin resistance in skeletal muscle and liver, ameliorate or prevent fatty liver, and improve -cell function / proliferation.	objectiveoptimal glucose homeostasis requires exquisitely precise adaptation of the number of insulin - secreting -cells in the islets of langerhans. insulin itself positively regulates -cell proliferation in an autocrine manner through the insulin receptor (ir) signaling pathway. it is now coming to light that cannabinoid 1 receptor (cb1r) agonism / antagonism influences insulin action in insulin - sensitive tissues. however, the cells on which the cb1rs are expressed and their function in islets have not been firmly established. we undertook the current study to investigate if intraislet endogenous cannabinoids (ecs) regulate -cell proliferation and if they influence insulin action.research design and methodswe measured ec production in isolated human and mouse islets and -cell line in response to glucose and kcl. we evaluated human and mouse islets, several -cell lines, and cb1r - null (cb1r/) mice for the presence of a fully functioning ec system. we investigated if ecs influence -cell physiology through regulating insulin action and demonstrated the therapeutic potential of manipulation of the ec system in diabetic (db / db) mice.resultsecs are generated within -cells, which also express cb1rs that are fully functioning when activated by ligands. genetic and pharmacologic blockade of cb1r results in enhanced ir signaling through the insulin receptor substrate 2-akt pathway in -cells and leads to increased -cell proliferation and mass. cb1r antagonism in db / db mice results in reduced blood glucose and increased -cell proliferation and mass, coupled with enhanced ir signaling in -cells. furthermore, cb1r activation impedes insulin - stimulated ir autophosphorylation on -cells in a gi - dependent manner.conclusionsthese findings provide direct evidence for a functional interaction between cb1r and ir signaling involved in the regulation of -cell proliferation and will serve as a basis for developing new therapeutic interventions to enhance -cell function and proliferation in diabetes.
assistive technologies (ats), defined as electrical or mechanical devices designed to help people recover movement, have demonstrated clinical benefits in upper - limb stroke rehabilitation1. at has the potential to reduce the effects of occupational performance limitations on everyday life activities by facilitating and enhancing work performance and social interactions2, 3. an assistive device may compensate for decreased or lost physical function and ability to manage the activities of daily life, increase or maintain function and ability, and prevent future loss of function and ability. assistive technologies may provide ; an increased intensity of therapy without a corresponding increase in clinical contact time, motivating relevant activities, either functional or impairment based, and can be used outside the hospital1. also, a health - care professional must provide an assistive technology intervention that enhances the user s self - reliance while maintaining quality in a cost - effective manner4. the imperative of adopting the philosophy and methods of evidence - based practice confronts many elds of human service, assistive technologies among them5. therapists are under increasing pressure to base their practices more firmly on evidence and to verify the effects of certain interventions. evidence - based practice should form the basis of all rehabilitation, including the application of assistive devices. an empirical and useful evidence base for the outcomes of assistive device use requires research based on relevant and valid evaluation tools as well as a client - centered working model. this is a challenge, as few measures are available that target the measurement of assistive device use outcomes7. the quebec user evaluation of satisfaction with assistive technology (quest) was developed by an occupational therapist in canada during the 1980s. it can be either self - administered or completed with the help of an evaluator8. it is designed to evaluate a person s satisfaction with his or her assistive device and can be used with adolescents, adults, and elderly people who have acquired an assistive device because of physical or sensory impairments. quest 2.0 has been amended to focus on satisfaction with specific features of an assistive device as well as certain characteristics of services related to the device9. it is important to analyze the benets and limitations of a device from the user s point of view. this information supports policy makers in the identication of optimal health - care procedures in this eld7. despite the increasing need for, interest in, and use of assistive devices among clients, the aim of this study was to evaluate client satisfaction concerning products and service related to assistive devices and their serviceability and usefulness. a cross - sectional follow - up study design was used. two assistive device centers in seoul, south korea, participated. the regional councils for the areas in which the centers were locate decided the selection of assistive devices available for prescription. was sent to a random sample of 448 users of assistive devices during the winter of 2011. one questionnaire included items concerning demographic data and questions about usage as well as the user s opinion of how the device had influenced activity engagement. the other questionnaire was a korean - quebec user evaluation of satisfaction with assistive technology (k - quest 2.0) form. user sampling was performed from the regional councils registries of all prescriptions for assistive devices in 2011. an addressed reply envelope and a letter explaining the objectives of the study the k - quest 2.0 scale investigates two dimensions : satisfaction with the device (eight items) and satisfaction with the assistive device service (four items)8. evaluation related to the characteristics of the device includes user satisfaction with dimensions, weight, adjustment, safety, durability, simplicity of use, comfort, and effectiveness. each item is scored on a 5-point satisfaction scale, where 1 denotes not satisfied at all and 5 denotes very satisfied. evaluation associated with the related services includes user satisfaction with service delivery, repairs and servicing, professional services, and follow - up. a checklist with the 12 satisfaction items is presented on the quest 2.0 form, and the user is asked to select the three most important items. a total of 142 (72%) users out of 448 originally queried returned the questionnaires. included in the data analysis are 138 users with several types of assistive devices. in all, 75 men and 63 women participated (table 1table 1. demographic data of responding users of assistive devices (n=138)gender n (%) male75 (54.3)female63 (45.7)age n (%) under ten years28 (20.3)10 19 years31 (22.5)20 29 years25 (18.1)30 39 years16 (11.6)40 49 years11 (8.0)50 59 years16 (11.6)60 69 years8 (5.8)over 70 years3 (2.2)disability severity n (%) first119 (86.2)second13 (9.4)third6 (4.4)other67 (48.6)residence n (%) apartment67 (48.6)single family15 (10.9)multi - family20 (14.5)other36 (26.1)highest level of education n (%) none23 (16.7)elementary school23 (16.7)middle school34 (24.6)high school34 (24.6)college19 (13.8)graduate school5 (3.6)). user satisfaction levels for both assistive device (ad) and assistive device service (ads) were high, as illustrated in table 2table 2. item by item analysis of aspects affecting user satisfaction and their relative importance to users of assistive devicesitemssatisfaction(n=138)>most important itemsnot satisfied at allnot very satisfiedmore or less satisfiedquite satisfiedvery satisfiedassistive devicedimensions2 (1.4)20 (14.5)39 (28.3)42 (30.4)35 (25.4)35 (25.7)weight13 (9.4)26 (18.8)40 (29.0)36 (26.1)23 (16.7)23 (16.7)adjustment9 (6.5)24 (17.4)35 (25.4)40 (29.0)30 (21.7)30 (21.7)safety2 (1.4)17 (12.3)40 (29.0)44 (31.9)35 (25.4)35 (25.4)durability6 (4.3)15 (10.9)34 (24.6)43 (31.2)40 (29.0)40 (29.0)ease of use5 (3.6)12 (8.7)37 (26.8)41 (29.7)43 (31.2)43 (31.2)comport5 (3.6)16 (11.6)42 (30.4)47 (34.1)28 (20.3)28 (21.3)effectiveness3 (2.2)5 (3.6)40 (29.0)51 (37.0)37 (26.8)37 (26.8)assistive device serviceservice delivery12 (8.7)18 (13.0)36 (26.1)31 (22.5)40 (29.0)40 (29.0)repairs & services14 (10.1)33 (23.9)35 (25.4)26 (18.8)30 (21.7)30 (21.7)professional service7 (5.1)26 (18.8)41 (29.7)28 (20.3)36 (26.1)36 (26.1)follow up15 (10.9)22 (15.9)46 (33.3)27 (19.6)28 (20.3)28 (20.3) n (%) ; multiple responses. a majority of users marked the ratings more or less satisfied or quite satisfied for most items. satisfaction ratings for weight and repairs & services were the lowest for the ad and ads dimensions, respectively. ease of use and durability in the ad dimension and service delivery in the ads were deemed the three most important factors. n (%) ; multiple responses participants used their assistive devices for personal activities of daily living (n = 72/209, 34.45%), education (n = 12/209, 5.74%), cultural, leisure, and sports activity (n = 8/209, 3.83%), communication (n = 5/209, 2.39%), religious and spiritual activities the most desired activities to engage in and most difficult to perform were activities of daily living followed by social participation comparison of user ratings of activity difficulty and desirabilityactivity found difficult to engage inactivity desirable to engage inactivities of daily livings96 (28.1) 134 (30.2)rest and sleep19 (5.6)16 (3.6)education45 (13.2)57 (12.9)work37 (10.8)39 (8.8)play43 (12.6)69 (15.6)leisure35 (10.2)58 (13.1)social participation67 (19.6)70 (15.8) n (%) ; multiple responses). assistive devices improve the ability of the user to perform activities of daily living15, 16. they also decrease users dependence on human assistance, especially assistance from informal caregivers, that is, friends, family, and community members who provide unpaid assistance to ill or disabled recipients17. evaluation of success must be based on evidence obtained from the user s point of view. for the at field, this entails an emphasis on outcomes research that speaks to the contributions that devices and related services make to users daily lives5. this is the rst experimental study to examine the impact of assistive technology - focused interventions on users satisfaction with and the usefulness of assistive devices in south korea. the results from k - quest 2.0 showed that the overall satisfaction for both ad and ads was high. the level of satisfaction with weight and adjustments in the ad dimension and repair & services in the ads dimension were relatively lower than with other categories. users most often considered ease of use and durability in the ad dimension and service delivery in the ads dimension to be the most important items. therefore, health - care providers need to have information about various types of assistive devices to enable them to educate users on how to utilize and adapt their prescribed devices, and need to provide follow - up service. overall, k - quest 2.0 showed that satisfaction with ads was lower than with the ad. the two device centers in seoul, south korea, began to prescribe assistive devices a few years ago. similar results may be found in other centers that have opened recently outside of seoul. one plausible explanation for obtaining lower satisfaction with ads is that user - centered assistive device delivery systems have not been established. professional services and support should be based on individual needs and promote community participation18. only a few suppliers provide assistive devices for many consumers without full consideration of needs, body fit, and follow - up service19. assistive device suppliers and centers therefore need to develop effective assistive device delivery models for users. determining the outcomes of assistive device interventions designed to improve participation of users provides critical feedback to clients, therapists, and agencies that provide funding for equipment20. as social participation reflects the fulfillment of personal roles21, appraisal of participation outcomes for each individual is critical22. most participants use assistive devices to improve their personal activities of daily living and mobility. nevertheless, they express difficulty in engaging in both activities of daily living and social participation while declaring a desire to perform these activities. these results indicate that continuous social and economic support derived from the users points of view must be provided to secure basic activities and social engagement.	[purpose ] the objective of this study was to determine users satisfaction with assistive devices, and their serviceability and effectiveness. [methods ] a random sample of 138 users participated in this study. the korean - quebec user evaluation of satisfaction with assistive technology 2.0 and an additional questionnaire were used for data collection. data were analyzed by using descriptive statistics. [results ] overall client satisfaction was high. respondents most commonly reported use of their device for personal activities of daily living and mobility, and considered engagement in activities of daily living, and social participation to be most desirable, respectively. [conclusion ] this study will provide rehabilitation professionals with valuable information about client satisfaction with assistive devices.
it is generally accepted that a fundamental process for distant metastasis formation is epithelial - mesenchymal transition (emt), during which tumour cells lose their epithelial properties and acquire a fibroblast - like phenotype. as a consequence, reduced intercellular adhesion, enhanced invasiveness and increased apoptotic resistance of cells have been observed [1, 2 ]. in developmental and cancer emt, signals promoting and regulating this process originate from a tissue microenvironment and usually act as transcriptional repressors. moreover, they are associated with the zinc - finger factors including snail1, snail2 (slug), zeb1/2 and the basic helix - loop - helix proteins (bhlh) with twist1/2. among signalling molecules there are also goosecoid and foxc2, which repress transcription indirectly via unknown mechanisms [3, 4 ]. snail1 as a transcription factor has been shown to be crucial for cellular movement during cancer progression and metastasis. therefore, in colon cancer patients, enhanced levels of snail1 are usually associated with poor clinical outcome, probably due to down - regulation of e - cadherin expression [5, 6 ]. there is evidence showing that the e - cadherin gene (cdh1) is also inactivated in diffuse type cancers such as breast lobular carcinoma and diffuse gastric carcinoma. in these cases, neoplastic cells have lost their epithelial properties and display a highly invasive pattern [7, 8 ]. the question arises regarding which factors are responsible for e - cadherin silencing during emt. the continuous expression of this epithelial marker during developmental emt in snail1-deficient mouse embryos clearly supports the idea that transcriptional repression of e - cadherin is mostly related to snail1 activity. as mentioned earlier, snail1 plays a significant role in the development of human malignancies, including those of the gastrointestinal tract. one of these is colorectal cancer, which is one of the most aggressive cancers and a common cause of cancer - related death in westernised countries. the high mortality, accounting for over 90% of colorectal cancer - related deaths, is linked to the ability of cancer cells to spread beyond the large intestine to distant locations. these metastatic abilities of colon cancer cells are closely associated with emt and transcription factors which are responsible for its activation. therefore, in this paper we will discuss the role of snail1 in colorectal cancer progression and metastasis. adenoma is also the first stage of colorectal cancerogenesis exhibiting snail1 expression, which is inversely correlated with expression of e - cadherin. this finding is in agreement with the statement that snail1 acts as a transcriptional repressor of cdh1. it is well demonstrated that repression of cdh1 is a consequence of molecular reaction cascade, during which a major role is played by interactions with regions containing the consensus core sequence (caggtc), a motif that is a subset of e - box (canntg) [12, 13 ]. the expression of snail1 in adenoma tissue suggests that the emt process promoting cancer metastasis is already active in precancerous lesions. studies with the use of transgenic mouse models such as her-2 and pymt have revealed that tumour cells may disseminate in morphologically benign hyperplastic lesions. metastatic mammary epithelial cells have been observed in the lungs of mice carrying the pymt oncogene at the early stage of breast tumorigenesis when the mammary tree appears to be at normal or at premalignant stage. it would appear that cells acquire invasive abilities even before the process of malignant transformation is completed. additionally, rcken suggests that neoplastic cells may spread during the initial steps of cancer development. it is worth noting that the main class of cells, which possess the ability to inhibit the growth of disseminated tumour cells, are cd8 t cells. they restrain the growth of neoplasias not by cytotoxic effects, but in a cytostatic manner. found that in the presence of cd8 t cells, the proliferating ki67 cells were characterised by a very low level. moreover, the number of ki67 tumour cells grew significantly in the absence of cd8 t cells. thus, the cytostatic inhibition of cancer cell growth may be responsible for keeping disseminated tumour cells from spreading to different distant organs. in humans, tumour cell dissemination could take place in morphologically non - invasive lesions as well. for example, in patients with ductal carcinoma in situ (dcis), cytokeratin - positive disseminated tumour cells have been detected in their bone marrow. moreover, cytokeratin - positive as well as epcam - positive disseminated tumour cells have also been observed in the bone marrow of patients with colorectal adenomas. it should be stressed here, that in the case of colon tissue, snail1 expression has also been revealed in normal mucosa in close vicinity to the tumour lesions. interestingly, some differences between non - pathological tissue and cancer tissue have been demonstrated at the subcellular level, where the shift of the protein to the cytoplasm with mixed cytoplasmic and nuclear or pure cytoplasmic expression has been detected. regarding snail1 expression in colorectal mucosa with no specific pathological changes, some published results suggest that snail1 is not connected with malignant transformation only. in normal tissue, this factor may be involved in the maintenance of homeostasis by regulation of cellular proliferation, differentiation and apoptosis. moreover, up - regulation of snail1 both at the level of mrna and protein has been reported to promote regional and distant lymph node metastasis [21, 22 ]. snail1 expression as a marker for lymph node metastasis has also been reported in the case of breast cancer and oesophageal squamous carcinoma [2224 ]. their study showed that about 77% of colon cancer samples display snail1 immunostaining in the stroma, especially in the stromal cells with fibroblast - like phenotype [2527 ]. expression of this factor in the stroma will indicate how many cells possess the ability to escape from the primary tumour mass, and how many cells may invade the basal lamina to colonise distant target organs. therefore, the presence of snail1 immunoreactive cells in the stroma may serve as prognostic marker in patients with colon cancer. it is worth noting that intratumoural injection with snail1-specific monoclonal antibody inhibits tumour growth and metastasis followed by an increased number of stromal tumour infiltrating lymphocytes (tils). it is not surprising then, that snail1 immunoreactivity in tumour stroma is related to distant metastasis (p = 0.006) and lower specific survival of patients (p = 0.011). importantly, this correlation was also detected in stage i and ii, which are known to display a variable prognosis. the capability to predict recurrence at these stages would be important from a clinical point of view because markers for them have not yet been identified. thus, the expression of snail1 would help to distinguish patients with an unfavourable clinical outcome and would be applicable for chemotherapy, which is not a treatment commonly used in the early stages of colorectal tumours. although kroepil. did not find a prominent correlation between snail1 expression and e - cadherin loss in cancer tissue, they did observe a significant correlation between the expression of snail1 in the tumour and snail1 expression in the corresponding lymph node metastasis. interestingly, they did not find any evidence of snail1 expression with clinical parameters such as n - stage, grading, age or sex. it is worth noting that also no association between snail1 expression and clinical parameters has been revealed in adenocarcinomas of the upper gastrointestinal tract. probably there are other more powerful mechanisms which are implicated in emt triggering during colon cancer development, e.g. factors associated with the wnt signalling pathway. revealed that wnt signalling and snail1 may be interconnected by multiple positive loops, possibly adding to the robustness of both. some published results indicate that vitamin d intake shows a protective effect against colorectal carcinogenesis [3234 ]. also analogues of vitamin d such as 1,25-dihydroksyvitamin d3 [calcitrol, 1,25(oh)2d3 ] demonstrate, additionally to its classical role in regulation of calcium and phosphate homeostasis, an anti - proliferative, pro - apoptotic and pro - differentiation impact on a wide array of cancer cells in vitro [35, 36 ]. therefore, it is not surprising that vitamin d derivatives with reduced hypercalcaemic activities are under clinical investigation for use against some malignancies, including colon cancer. nevertheless, only a small population of colon cancer patients respond to this therapy, because of the loss of vitamin d receptor (vdr) expression, which belongs to the nuclear hormone receptor superfamily [37, 38 ]. a study by palmer. demonstrated that human colon cancer cells such as sw480-adh with expression of vdr are sensitive to the anticancer action of calcitrol. in this case, the ligand - activated vdr induces the process of differentiation and expression of e - cadherin. interestingly, e - cadherin and vdr expression were down - regulated in sw480-adh and macat cells when they were co - cultured with snail1-expressing cells. this leads to the question regarding which factors are responsible for the loss of vdr expression during colon cancer progression. therefore, the high expression of this factor may be responsible for the failure of therapy with vitamin d analogues in patients with colon cancer [37, 38 ]. revealed that snail1 and snail2 bind to the promoter region of exon 1a of the human vdr gene and repress its expression. the repressive effects of snail1 and snail2 on vdr gene promoter are similar and are mediated by three e - boxes (caggtg / cacctg), which are present in the first 400 nucleotides of human vdr promoter (fig. 1). both snail1 and snail2 cooperate to repress vdr promoter, showing an additive effect because the level of vdr expression has is decreased in tumours co - expressing snail1 and snail2 compared to those expressing only one of these genes [40, 41 ]. the association between snail factors expression and down - regulation of vdr is absent in tumours in which p300 and ctbp (c - terminal binding protein) are strongly expressed. this suggests that the level of co - repressor protein ctbp and nuclear acetylase p300 are critical for snail1 activity. a growing number of studies have revealed that calcitrol and other non - hypercalcaemic analogues suppress cellular proliferation and provoke differentiation in the case of colon cancer. this is probably due to the formation of vdr/-catenin complexes and subsequent expression of e - cadherin, which take a part in redistribution of -catenin to the plasma membrane. -catenin is one of the most significant factors implicated in the canonical wnt signalling pathway, which plays a major role in colonic epithelial differentiation. in the case of constitutive expression of this pathway, -catenin is accumulated in the nuclear cell compartment and binds to the proteins of the t - cell factor / lymphoid enhancer factor (tcf / lef) family (fig. these nuclear complexes regulate expression of genes involved in proliferation, invasiveness and angiogenesis. in this context, it should be noted that neoplastic cells located at the invasive front of colorectal cancer tissues frequently demonstrate increased levels of nuclear -catenin. these cells gain a mesenchymal - like phenotype as a result of mutations occurring in adenomatous polyposis coli (apc) or -catenin genes, which are also the major mechanisms responsible for aberrant activation of wnt signalling [11, 45, 46 ]. therefore, colon cancer cells with an invasive phenotype may show both the aberrant wnt signalling and expression of emt - activating transcription factors such as snail1. the canonical wnt signaling pathway as indicated earlier, both snail1 and snail2 are responsible for the low level of vdr receptors in colon cancer cells. re - expression of this receptor may restore the inhibitory effect of calcitrol associated with the formation of vdr/-catenin complexes interfering with those of -catenin / tcf. these molecular events are responsible for the partial inhibition of wnt signalling in colon cancer cells. but for the completed inhibition of this pathway, stable redistribution of -catenin from the nucleus to the plasma membrane is required. such redistribution is associated with induction of e - cadherin expression, and, therefore, the poorer induction of e - cadherin in snail1 + vdr cells is observed. importantly, this weaker e - cadherin expression activated by calcitrol is related to repression of cdh1 promoter mediated by snail1 [31, 40 ]. a growing number of studies have demonstrated that tumour - associated macrophages (tams) provide soluble factors promoting the growth and metastasis of colon cancer cells [48, 49 ]. for example, macrophage - derived il-1 promotes wnt signalling in colon cancer cells and therefore regulates their growth and survival [50, 51 ]. kaler. revealed that macrophage - induced activation of wnt signalling in colon cancer cells not only initiates a plethora of cell - intrinsic changes in epithelial cells but also influences communication between epithelial cells and macrophages. inactivation of the mt -catenin allele in hct116 cells prominently changes their interactions with macrophages, which fail to promote wnt signalling or protect hct116wt cells from trail - induced apoptosis. this factor enhances -catenin / tcf transcriptional activity both in parental hct116 cells and in hct116 cells retaining the wt -catenin allele. it seems that activation of pathways such as wnt not only triggers changes in cell structure but also induces stromal modifications, which are needed for cancer development. macrophage - derived factors are also associated with stabilisation of snail1 transcription factor in colon cancer cells. in contrast to the parental hct116 cells, macrophage - derived factors fail to stabilize snail1 in hct116 cells with deleted mt -catenin allele. the ability of macrophages and il-1 to suppress trail - induced apoptosis and to generate clonogenic growth of tumour cells is also inhibited in tumour cells with silenced snail1 expression. more so, active wnt signalling in colon cancer cells and wnt - dependent stabilization of snail1 are needed for tumour cells to provoke macrophages to produce il-1. this suggests that macrophage - derived factors protect colon cancer cells from trail (tnf - related apoptosis - inducing ligand)-induced apoptosis through stabilisation of snail1. therefore, the lack of snail1 stabilisation in hct116 cells with inactivated mt -catenin allele underlies the inability of macrophages to protect these cells from trail - induced apoptosis. the results of these studies clearly confirm that induction of wnt signalling and subsequent stabilisation of snail1 in colon cancer cells influence their interactions with macrophages, and indicate a fundamental role of snail1 in the crosstalk between colon cancer cells and macrophages. moreover, cells which are characterised by high levels of wnt signalling display colon cancer stem cell (csc) features [55, 56 ]. silencing -catenin in hct116 cells has been shown to reduce the number of colonospheres which have a great number of cscs. macrophages and il-1-enhanced wnt signalling and stabilised snail1 in tumour cells have been demonstrated to equip cells with stem cell - like characteristics, suggesting that macrophage - derived factors contribute to heterogeneity of colon tumours by broadening the population of cells which possess cancer stem cell features [51, 58 ]. in summary, snail proteins, as a major factor responsible for emt induction, are promising targets for development of pharmaceutical agents. blocking snail protein activity has the potential to avert cancer cell metastasis by interfering with such cellular processes as remodelling of the actin cytoskeleton, migration and invasion, which are clearly associated with the aggressive phenotype of the disease. furthermore, the link between snail and cancer stem cells suggests that inhibitory agents may prove potent as inhibitors of cancer recurrence. some recent studies revealed that snail1 might modulate several steps in rna maturation including polyadenylation, splicing and translation. also, this factor may regulate gene expression post - transcriptionally by modulation of protein levels involved in pre - mrna processing and location. it is also worth noting that, additionally to their anti - cancer potential, inhibitors of snail1 activity would be significant tools to simplify the study of these transcriptional factors in model cell types associated with embryonic development such as neural crests.	snail1 is a zinc - finger transcription factor, which plays a role in colorectal cancer development by silencing e - cadherin expression and inducing epithelialmesenchymal transition (emt). during emt tumour cells acquire a mesenchymal phenotype that is responsible for their invasive activities. consequently, snail1 expression in colorectal cancer is usually associated with progression and metastasis. some studies revealed that about 77% of colon cancer samples display snail1 immunoreactivity both in activated fibroblasts and in carcinoma cells that have undergone emt. therefore, expression of this factor in the stroma may indicate how many cells possess the abilities to escape from the primary tumour mass, invade the basal lamina and colonise distant target organs. blocking snail proteins activity has the potential to avert cancer cell metastasis by interfering with such cellular processes as remodelling of the actin cytoskeleton, migration and invasion, which are clearly associated with the aggressive phenotype of the disease. moreover, the link between factors from the snail family and cancer stem cells suggests that inhibitory agents may also prove their potency as inhibitors of cancer recurrence.
pancreatic neuroendocrine tumors (pnets) are uncommon neoplasms which originate from diffuse neuroendocrine cells. they have an incidence of approximately 1 in 100,000 and account for 1% to 2% of all pancreatic tumors. although they may manifest at any age, they most often occur in the sixth to eighth decades of life. most of the pnets are sporadic ; however, some of them can be associated with inherited genetic syndromes such as multiple endocrine neoplasia type 1 (men1) or von hippel lindau syndrome. the clinical presentation varies depending on whether the tumor is functioning or nonfunctioning, and which hormones are produced. in recent years, there has been a significant increase in the number of incidentally discovered nf - pnets because of the widespread use of high - quality cross - sectional imaging and ultrasound, with one of the studies showing a greater than 2-fold increase from 1986 to 2002. surgical resection is the treatment of choice for patients with functional tumors, and it is preferred for most of the nf - pnets. it is debatable whether all of the small and asymptomatic lesions should be routinely resected. there is insufficient information about the natural history of nf - pnets, especially when they are small. one of the studies showed that most of the neoplasms 2 cm are likely to be benign or intermediate - risk lesions, and only 6% of nf - pnets 2 cm are malignant when incidentally discovered. in this setting, a nonoperative approach could be advocated in selected cases for tumors 2 cm that are discovered incidentally. another recent study also suggested that nonoperative management of asymptomatic sporadic nf - pnets 2 cm however, the natural history of pnets is not well - established, and the proper management and follow - up strategy of incidentally discovered small pnets are controversial. some authors suggest that for nf - pnets measuring 2 cm, a nonoperative management can be considered, and the risks and benefits of surgical resection should be carefully weighed in patients with small lesions. an analysis of the nationwide inpatient sample from 1998 to 2006 demonstrated an in - hospital mortality rate of 1.7% and an overall complication rate of 29.6% after pancreatectomy for pnets. the majority of complications involved postoperative infections (11.1%), digestive system complications (8.8%), or pulmonary compromise (7.3%). in - hospital other authors suggest that surgery is indicated in any case because nf - pnets should always be considered as potentially malignant tumors, and a proper histological examination of the tumor (including the mitotic and ki-67 indexes) is possible only on the resected specimen. previous studies have reported that several factors are associated with survival, including age, tumor size, grade / differentiation, ln status, presence of distant metastases, and surgical resection. age has consistently been found to be a powerful predictor of survival in patients undergoing resection of pnets. similarly, the present study demonstrated that older age (55 years) was associated with net g2 or g3. most of the neoplasms measuring 2 cm are likely to be benign or intermediate - risk lesions. when incidentally discovered small tumors were considered, only 6% of the nf - pnets measuring 2 cm were malignant and none of the patients died of the disease. in the present study, none of the nf - pnets (< 1.5 cm) and only 3.9% of nf - pnets (2 cm) that were discovered incidentally and confirmed pathologically were classified as net g3. interestingly, only 1 patient in our study who was observed by an ongoing follow - up was diagnosed as having net g3. because this patient also had nonsmall cell lung cancer, net g3 there were no cases of disease - related death or recurrence during the clinical follow - up in our study. in the ongoing follow - up group, most of the patients therefore, a nonoperative approach could be advocated for nf - pnets measuring 2 cm (especially < 1.5 cm) that are discovered incidentally. this more conservative approach would be the most suitable for higher - risk patients with significant medical comorbidities. an intensive 3-month follow - up for the first year and then a 6-month follow - up up to 3 years could be recommended in these patients. the present study showed that a meaningful tumor growth was associated with more malignant lesions (net g2 or g3). if a meaningful increase in the tumor size is detected during the follow - up, prompt operative resection should be considered. only 5.5% of patients in the ongoing follow - up group had confirmation regarding the diagnosis of a nf - pnet by a biopsy, which raises the question of the accuracy of the diagnosis in the remaining patients. despite high - quality imaging of the pancreas, radiologists can not always distinguish between different pancreatic pathologies or accessory splenic tissue. one of the studies reported that 17% of patients in the nonbiopsy - confirmed, operative nf - pnets group had false - positive preoperative imaging findings, and one might expect a similar percentage of patients in the nonoperative group. similarly, in our study, 13 patients (18%) in the nonbiopsy - confirmed, operative group had false - positive preoperative imaging finding of nf - pnets, but later confirmed to have other tumors. first, there is a possibility that patients in the ongoing follow - up group did not have nf - pnets. the reason for this possibility is the difficulty in distinguishing nf - pnets from other pancreatic lesions. we included patients with a high probability of being diagnosed as having nf - pnets. second, our conclusions are limited by the retrospective nature of the data. a randomized trial comparing between the observation and resection groups would provide more definitive results, but it would be difficult to perform such a study due to the low incidence of nf - pnets. in conclusion, older age (55 years), larger tumor size (1.5 cm) and a meaningful tumor growth (20% or 5 mm) during follow - up were associated with net g2 or g3. especially, larger tumor size (1.5 cm) is a significant independent risk factor for net g2 or g3.. the choice of the appropriate management for these small tumors should be well - balanced after considering the short- and long - term sequelae following pancreatic resection procedures. therefore, an intensive follow - up could be an acceptable approach in small (especially < 1.5 cm in size), asymptomatic nf - pnets.	abstractsmall nonfunctioning pancreatic neuroendocrine tumors (nf - pnets) usually exhibit minimal or no growth over many years. however, there is a controversy regarding the optimal management of incidentally discovered, small nf - pnets. this study aimed to gain insights into tumor behavior and potential strategies for clinical management.we retrospectively reviewed a total of 202 patients with a suspected pnet (size 2 cm or smaller) at samsung medical center from january 1, 1995 to april 30, 2012. among these patients, 72 patients were excluded and 145 patients were enrolled in our study. patients were included if the size of the tumor was 2 cm without familial syndrome, radiographic evidence of local invasion or metastases.among the 145 patients, 76 patients (52.4%) had pathologically confirmed pnets. eleven (14.5%) and 3 (3.9%) of these 76 patients were diagnosed with net g2 and g3, respectively. pnets measuring 1.5 cm or more in size had a higher probability of being classified as net g2 or g3 compared with pnets measuring < 1.5 cm (p = 0.03). older age (55 years) and a meaningful tumor growth (20% or 5 mm) were significantly associated with net g2 or g3 (p < 0.05).older age (55 years), larger tumor size (1.5 cm), and a meaningful tumor growth (20% or 5 mm) were associated with net g2 or g3. intensive follow - up could be an acceptable approach in small (especially < 1.5 cm), asymptomatic, nf - pnets.
conventionally, aldosterone antagonists and loop diuretics have been used for the management of ascites associated with liver cirrhosis (1,2). however, some patients show poor responses or adverse reactions, such as renal dysfunction and electrolyte abnormalities, especially with high - dose administration and combined use. tolvaptan is a vasopressin receptor antagonist and it shows a diuretic effect without na excretion. it was approved as a concomitant medication for fluid retention in liver cirrhosis patients that insufficiently respond to existing diuretics for the first time in the world in september 2013 (3). reportedly, there are some non - responders to tolvaptan, while others exhibit a delayed response after sustained administration. we herein report a patient in whom tolvaptan began to show an effect at least 2 months after the start of its administration. the patient was a 67-year - old female. during outpatient treatment for hepatitis c at a local clinic, hepatpcellular carcinoma (hcc) was detected in the right lobe of the liver, and percutaneous radiofrequency ablation was performed at our hospital on november, 2012. the patient was thereafter followed up at our hospital as an outpatient, but she developed anorexia and abdominal fullness in april 2014. despite the administration of furosemide at 20 mg / day and spironolactone at 25 mg / day, the ascites did not decrease. she also had no history of interferon therapy for hepatitis c. the blood chemical analyses at the time of admission are shown in table 1. the child - pugh score was 10 with grade c. hyponatremia and marked renal dysfunction were also noted. the platelet count was markedly reduced at 37,000 cells / ml. both alpha - fetoprotein (afp) and des - gamma carboxyprothrombin (dcp) were mildly increased. abdominal computed tomography (ct) before admission showed an irregularity of the liver surface, and the marked accumulation of ascites was observed. wbc : white blood cell, hb : hemoglobin, plt : platelet, pt : prothrombin time, na : sodium, k : potassium, bun : blood urea nitrogen, cr : creatinine, egfr : estimated glomerular filtration rate, alb : albumin, t - bil : total bilirubin, alt : alanine aminotransferase, n.e. : not evaluate, crp : c - reactive protein, tm : tumor marker, hcc : hepatocellular carcinoma (a) abdominal ct performed 2 months before tlv administration showed cirrhosis with massive ascites. (b) a comparison of the abdominal ct performed 2 months before tlv administration with that performed 1 month after tlv administration revealed an increase in ascites. (c) a comparison of abdominal ct performed 1 month after tlv administration with that performed 2 months after tlv administration showed no significant increase or decrease in ascites. (d) abdominal ct performed 6 months after tlv administration showed a significant decrease in ascites. her body weight was 37.6 kg, and the daily urine volume was around 900 ml. while the administration of tolvaptan was initiated at 3.75 mg on the 1st hospital day, the urine volume did not increase with a slight increase in her body weight ; the dose of tolvaptan was thene increased to 7.5 mg on the 7th hospital day. thereafter, although the urine volume showed no marked increase, the increase of her body weight became mild, and the patient was discharged on the 12th hospital day at her request. the water intake underwent a change of approximately 1,000 ml / day, but the intake of fluids included with her meals clearly increased because the symptoms of anorexia also improved after admission. a comparison of the abdominal ct performed 2 months before tolvaptan administration with that performed 1 month after tolvaptan administration showed an increase in ascites (fig. 1b), and the patient had clearly gained weight from 37.6 kg immediately before tolvaptan administration to 52.2 kg. however, she continued to receive only tolvaptan at 7.5 mg / day without any other additional treatment, such as cell - free and concentrated ascites reinfusion therapy (cart) because the worsening of her subjective symptoms was mild and she wished to do so. a comparison of the ct performed 1 month after tolvaptan administration with that performed 2 months after tolvaptan administration revealed no significant increase or decrease in ascites (fig. however, she showed a weight loss from 52.2 to 49.9 kg ; therefore, she continued to receive tolvaptan at 7.5 mg / day. the abdominal ct performed at 6 months revealed the ascites to have markedly decreased (fig. therefore, the oral administration of furosemide was discontinued, and the administration of only spironolactone at 25 mg and tolvaptan at 7.5 mg was continued. after the beginning of tolvaptan administration, no adverse reaction such as liver and kidney dysfunction was observed. clinical course after admission ; despite the administration of tolvaptan at 3.75 mg on the 1st hospital day, no increase in urine volume or decrease in body weight was noted. therefore, the dose of tolvaptan was increased to 7.5 mg on the 7th hospital day, but there was still no increase in urine volume or decrease in body weight. in cirrhotic patients, splanchnic vasodilation, an increase of portal vein pressure and portosystemic shunt result in a decreased effective arterial blood volume. a consequent decrease in the renal blood flow is considered to induce fluid retention through the activation of the renin - angiotensin - aldosterone system (4,5). although, aldosterone receptor antagonists are effective for the treatment of fluid retention associated with liver cirrhosis (1), it is insufficient in many patients. fluid retention is also caused by the activation of vasopressin (vp) through the decrease in the circulating plasma volume (4,5), but there is still no available medication that has been shown to be effective for the activation of vp. tolvaptan is an antagonist for vasopressin v2 receptors in the collecting ducts of the kidney, and it exerts a diuretic effect by suppressing the expression of aquaporin 2. in japan, a phase 3 comparative study of tolvaptan was carried out in cirrhosis patients who responded poorly to the administration of loop diuretics and anti - aldosterone agents (3). according to the phase 3 study, the urine volume significantly increased on days 1 and 7 in the tolvaptan group, but no significant change was observed in the placebo group. thirst, constipation, kidney dysfunction, hepatic encephalopathy, and itching were noted as major adverse reactions, but their severity was only mild to moderate. zhang. investigated 39 liver cirrhosis patients accompanied by refractory ascites, and observed the effect of tolvaptan to be attenuated in the patients with hepatorenal syndrome (hrs) (6). the effect of tolvaptan has also been reported to decrease as the estimated glemerular filtration rate (egfr) decreased in renal failure patients in a single - dose study of tolvaptan (7). in a phase 3 clinical study in japan, the effect of tolvaptan decreased when blood urea nitrogen (bun) was high in a sub - analysis (8). in addition, the effect of tolvaptan decreased in patients demonstrating renal parenchymal disorder with hrs and a decreased egfr and those with high bun, i.e., patients suggested to have intravascular volume depletion and prerenal renal dysfunction. the present patient had severe anorexia and prerenal renal dysfunction with 72 mg / dl bun and 2.19 mg / dl creatinine (cr) before admission, and these conditions may have been the cause of the poor effect of tolvaptan early after the initiation of administration. however, the symptoms of anorexia improved after admission, where the renal function markedly improved and bun and cr on day 14 after the initiation of tolvaptan administration were 27 and 1.34 mg / dl, respectively (table 2). on the other hand, no reduction in the body weight or ascites was observed for at least 2 months after the initiation of tolvaptan administration. thus, it is difficult to explain the cause of the poor effect of tolvaptan early after the initiation of administration with renal dysfunction alone. however, although both the ascites and the body weight (37.652.2 kg) clearly increased one month after the introduction of tolvaptan compared with those before introduction, no change in body weight was observed over the next month (52.249.9 kg). therefore, it is conceivable that the renal dysfunction caused by dehydration could have blocked the effect of tolvaptan in the early course of tolvaptan treatment. wbc : white blood cell, hb : hemoglobin, plt : platelet, pt : prothrombin time, na : sodium, k : potassium, bun : blood urea nitrogen, cr : creatinine, egfr : estimated glomerular filtration rate, alb : albumin, t - bil : total bilirubin, alt : alanine aminotransferase, crp : c - reactive protein we herein presented a case showing a delayed effect of tolvaptan. therefore, even when the initial responses of the urine volume and body weight are not remarkable, the continuation of tolvaptan administration should be considered if there is no exacerbation of ascites. although the safety of long - term tolvaptan therapy has been reported (9),	the patient was a 67-year - old female with liver cirrhosis due to hepatitis c. she was administered furosemide at 20 mg / day and spironolactone at 25 mg / day, but the ascites did not improve. despite the additional administration of tolvaptan at 3.75 mg / day, the response to ascites was still poor. while the dose of tolvaptan was thereafter increased to 7.5 mg / day on the 7th hospital day, the ascites still persisted. however, she continued to receive tolvaptan (7.5 mg / day) because the worsening of her subjective symptoms was mild and she wished to do so. the ascites was later found to have almost completely disappeared on computed tomography (ct) at 6 months.
study subjects were selected from participants in a nutrition genomic study conducted by the national research laboratory of clinical nutrigenetics / nutrigenomics (program r0a-2005 - 000 - 10144 - 0) at yonsei university. study subjects were recruited from the health service center or the outpatient clinics at yonsei university severance hospital in seoul, korea, and national health insurance corporation ilsan hospital in goyang, korea. subjects with a diagnosis of type 2 diabetes or ifg who consented to the dietary intervention program were included. hyperglycemia was defined according to the american diabetes association criteria (9), in which diabetes is defined as fasting glucose 126 mg / dl, and ifg is defined as fasting glucose between 100 and 125 mg / dl. exclusion criteria included 1) previous history of diabetes, 2) abnormal liver or renal function, 3) history of cardiovascular disease and cancer, 4) unstable weight loss / gain (2 kg) over the previous 6 months, and 5) thyroid or pituitary disease. a total of 430 koreans were recruited, and 67 participants discontinued the study for personal reasons. all subjects gave their written informed consent for the study, which was approved by the institutional review board of yonsei university. detailed methods for the genotyping of adipoq snps (11377, 45, and 276) and clinical laboratory tests were described previously (7). the subjects ' usual diets were assessed using a semiquantitative food frequency questionnaire and a 24-h recall method at baseline. all subjects were given written and verbal instructions by a registered dietitian on how to complete a 3-day dietary record every 4 weeks. individualized and nutritionally balanced diets were planned for each subject at the initial visit. the dietary intervention program consisted of the replacement of refined rice with whole grains three times a day as a carbohydrate source and an increase in vegetable intake to at least 6 units (3070 g / unit) per day for sufficient dietary fiber intake. the subjects were told to drink no more than one alcoholic beverage drink (15 g alcohol) per day and were assigned physical activity, comprising a regular 30-min walk after dinner each day. the energy values and nutrient content of dietary intake were calculated using the computer aided nutritional analysis program (can - pro 2.0, korean nutrition society). total energy expenditures were calculated from activity patterns including basal metabolic rate (by the harris - benedict equation), physical activity for 24 h, and the specific dynamic action of food. hardy - weinberg equilibrium, linkage disequilibrium (ld), and haplotype frequencies were determined by haploview (version 3.32 ; http://www.broad.mit.edu/mpg/haploview/), and subject - specific haplotypes were estimated using the hapanalyzer program (http://hap.ngri.go.kr) with the em algorithm. we also used spss (version 12.0 for windows ; spss, chicago, il). anova with the bonfer - roni method or a general linear model was used to test the genotype or haplotype effects. pearson and partial correlation coefficients were used to examine the relationship between adiponectin levels and other metabolic variables. finally, a multiple linear regression analysis was performed to find independent effects on changes in adiponectin levels after dietary intervention. each variable was examined for normal distribution, and the skewed variables were tested after logarithmic transformation. for descriptive purposes, mean the subjects ' usual diets were assessed using a semiquantitative food frequency questionnaire and a 24-h recall method at baseline. all subjects were given written and verbal instructions by a registered dietitian on how to complete a 3-day dietary record every 4 weeks. individualized and nutritionally balanced diets were planned for each subject at the initial visit. the dietary intervention program consisted of the replacement of refined rice with whole grains three times a day as a carbohydrate source and an increase in vegetable intake to at least 6 units (3070 g / unit) per day for sufficient dietary fiber intake. the subjects were told to drink no more than one alcoholic beverage drink (15 g alcohol) per day and were assigned physical activity, comprising a regular 30-min walk after dinner each day. the energy values and nutrient content of dietary intake were calculated using the computer aided nutritional analysis program (can - pro 2.0, korean nutrition society). total energy expenditures were calculated from activity patterns including basal metabolic rate (by the harris - benedict equation), physical activity for 24 h, and the specific dynamic action of food. hardy - weinberg equilibrium, linkage disequilibrium (ld), and haplotype frequencies were determined by haploview (version 3.32 ; http://www.broad.mit.edu/mpg/haploview/), and subject - specific haplotypes were estimated using the hapanalyzer program (http://hap.ngri.go.kr) with the em algorithm. we also used spss (version 12.0 for windows ; spss, chicago, il). anova with the bonfer - roni method or a general linear model was used to test the genotype or haplotype effects. pearson and partial correlation coefficients were used to examine the relationship between adiponectin levels and other metabolic variables. finally, a multiple linear regression analysis was performed to find independent effects on changes in adiponectin levels after dietary intervention. each variable was examined for normal distribution, and the skewed variables were tested after logarithmic transformation. for descriptive purposes, mean snp 11377 contained 56.5% c / c, 36.6% c / g, and 6.9% g / g (c frequency = 0.748). snp 45 contained 49.3% t / t, 41.9% t / g, and 8.8% g / g (t frequency = 0.703). snp 276 contained 52.3% g / g, 39.4% g / t, and 8.3% t / t (g frequency = 0.720). ld was calculated between the three snps : low ld between snp + 45t > g and snp 11377c > g (d = 0.692 ; p t and snp 11377c > g (d = 0.003, p = 0.092) and complete, strong ld between snp + 45t > g and snp + 276g > t (d = 1 ; p g combined with snp 276g > t. estimated 45276 haplotype frequencies were 17.8% for tg / tg, 22.8% for tg / tt, 25.7% for tg / gg, 8.1% for tt / tt, 16.3% for tt / gg, and 9.2% for gg / gg. in subsequent statistical analyses, subjects were divided into three haplotype groups : homozygous for the tg haplotype (tg / tg ; n = 65) and heterozygous for the tg haplotype (tg / x ; n = 177) and non - tg haplotype (x / x ; n = 121). the 12-week dietary intervention increased hdl cholesterol but significantly decreased blood pressure and serum concentrations of glucose, triglycerides, total cholesterol, and ldl cholesterol (table 1). there were significant increases from baseline in both fiber intake and the polyunsaturated fatty acids to saturated fatty acids ratio and a slight increase in total energy expenditure but a decrease in total energy intake. the mean body weights of the study participants were slightly but significantly reduced after dietary intervention (table 1). effects of a 12-week dietary intervention on anthropometric and biochemical markers data are means se. homa - ir = (fasting insulin [microunits per milliliter ] fasting glucose [millimoles per liter])/22.5. there were no significant differences in terms of sex distribution, age, and bmi associated with snp 45 (table 2), snp 276 (table 2), and snp 11377 (data not shown) genotypes. at baseline, we did not observe any significant genotype - related associations between the three snps and plasma adiponectin levels. however, for snp 276, baseline serum glucose levels were significantly higher in gg subjects than in tg and tt subjects (table 2). for the 45t > g/276g > t haplotypes, tg / tg subjects showed higher insulin levels and homa - ir indexes than did non - tg subjects (x / x) (table 2). effects of adipoq snp 45 and snp 276 on insulin resistance indices and plasma adiponectin levels before and after dietary treatment data are means sem. g/276g > t regarding the change in adiponectin levels (p = 0.03). tg / tg subjects had a mean adiponectin increase of 0.71 0.35 g / ml (16.3%) after the 12-week trial, whereas x / x subjects showed a mean adiponectin decrease of 0.19 0.21 g / ml (3.8%). the subjects were divided into two subgroups according to individual bmi values, either 25 kg / m (overweight - obese). we evaluated the effects of bmi on changes in homa - ir indexes and plasma adiponectin levels in association with the 45276 haplotype. as expected, the two bmi subgroups differed significantly in baseline values of adiponectin (5.1 0.26 vs. 4.4 0.25 g / ml, p g/276g > t haplotype according to bmi (a : normal - weight ; b : overweight - obese). p1, unadjusted p value ; p2, baseline - adjusted p value. p g and snp 11377c > g (d = 0.692 ; p t and snp 11377c > g (d = 0.003, p = 0.092) and complete, strong ld between snp + 45t > g and snp + 276g > t (d = 1 ; p g combined with snp 276g > t. estimated 45276 haplotype frequencies were 17.8% for tg / tg, 22.8% for tg / tt, 25.7% for tg / gg, 8.1% for tt / tt, 16.3% for tt / gg, and 9.2% for gg / gg. in subsequent statistical analyses, subjects were divided into three haplotype groups : homozygous for the tg haplotype (tg / tg ; n = 65) and heterozygous for the tg haplotype (tg / x ; n = 177) and non - tg haplotype (x / x ; n = 121). the 12-week dietary intervention increased hdl cholesterol but significantly decreased blood pressure and serum concentrations of glucose, triglycerides, total cholesterol, and ldl cholesterol (table 1). there were significant increases from baseline in both fiber intake and the polyunsaturated fatty acids to saturated fatty acids ratio and a slight increase in total energy expenditure but a decrease in total energy intake. the mean body weights of the study participants were slightly but significantly reduced after dietary intervention (table 1). effects of a 12-week dietary intervention on anthropometric and biochemical markers data are means se. homa - ir = (fasting insulin [microunits per milliliter ] fasting glucose [millimoles per liter])/22.5. the 12-week dietary intervention increased hdl cholesterol but significantly decreased blood pressure and serum concentrations of glucose, triglycerides, total cholesterol, and ldl cholesterol (table 1). there were significant increases from baseline in both fiber intake and the polyunsaturated fatty acids to saturated fatty acids ratio and a slight increase in total energy expenditure but a decrease in total energy intake. the mean body weights of the study participants were slightly but significantly reduced after dietary intervention (table 1). effects of a 12-week dietary intervention on anthropometric and biochemical markers data are means se. homa - ir = (fasting insulin [microunits per milliliter ] fasting glucose [millimoles per liter])/22.5. there were no significant differences in terms of sex distribution, age, and bmi associated with snp 45 (table 2), snp 276 (table 2), and snp 11377 (data not shown) genotypes. at baseline, we did not observe any significant genotype - related associations between the three snps and plasma adiponectin levels. however, for snp 276, baseline serum glucose levels were significantly higher in gg subjects than in tg and tt subjects (table 2). t haplotypes, tg / tg subjects showed higher insulin levels and homa - ir indexes than did non - tg subjects (x / x) (table 2). effects of adipoq snp 45 and snp 276 on insulin resistance indices and plasma adiponectin levels before and after dietary treatment data are means sem. g genotype, the dietary intervention significantly decreased serum glucose levels without any significant increase in insulin levels (table 2). after the dietary intervention, homa - ir indexes were reduced in subjects carrying the snp 45 t allele. snp 45tt subjects also had a mean adiponectin level increase of 0.48 0.17 g / ml (10.9%). there was a significant genotype - related difference in insulin levels (p = 0.005), homa - ir indexes (p = 0.049), and adiponectin levels (p = 0.037) for snp 45. after the 12-week trial, serum glucose levels were reduced in subjects carrying the snp 276 g allele, and homa - ir indexes were reduced in snp 276gg subjects (table 2). there was initially a significant decrease in homa - ir indexes among the genotypes (p = 0.025), but this difference disappeared after adjustment for the baseline levels (p = 0.074). in the 45276 haplotype analysis (table 2) after the 12-week trial, serum glucose levels were significantly decreased in all haplotype groups. however, homa - ir indexes were reduced only in subjects carrying the tg haplotypes (tg / tg + tg / x) but not in the non - tg subjects (x / x). there were significant differences in insulin levels (p g/276g > t regarding the change in adiponectin levels (p = 0.03). tg / tg subjects had a mean adiponectin increase of 0.71 0.35 g / ml (16.3%) after the 12-week trial, whereas x / x subjects showed a mean adiponectin decrease of 0.19 0.21 g / ml (3.8%). the subjects were divided into two subgroups according to individual bmi values, either 25 kg / m (overweight - obese). we evaluated the effects of bmi on changes in homa - ir indexes and plasma adiponectin levels in association with the 45276 haplotype. as expected, the two bmi subgroups differed significantly in baseline values of adiponectin (5.1 0.26 vs. 4.4 0.25 g / ml, p g/276g > t haplotype according to bmi (a : normal - weight ; b : overweight - obese). p1, unadjusted p value ; p2, baseline - adjusted p value. p g, 276g > t, and 11377c > g. for the 11377c > g snp, we found no genotype - dependent differences in homa - ir indexes and adiponectin levels in response to dietary intervention. although previous findings have suggested that snps 11391 and 11377 are associated with adiponectin levels and contribute to the development of type 2 diabetes, these studies were performed in caucasians (3,5). in fact, the 11391 snp at the adipoq promoter region was monomorphic in this study population (g : a = 1:0), and it is also not found in either the japanese or chinese population (4,14). fasting serum glucose levels were decreased in all three snp 45t > g genotypes after dietary intervention. however, subjects with the snp 45tt genotype had an increase in adiponectin levels and a decrease in homa - ir indexes. similarly, in haplotype analysis for adipoq 45t > g/276g > t, subjects with the tg / tg haplotype had a 21% decrease in homa - ir indexes and a greater increase in adiponectin levels after dietary intervention. (15) also found that the snp 45 g allele predicts the conversion to type 2 diabetes in subjects with impaired glucose tolerance despite regular exercise and either a weight - reducing or a weight - maintaining diet. furthermore, the combined effects of the 45 g allele and the 276 t allele on the conversion from impaired glucose tolerance to type 2 diabetes were observed to be stronger than that of each snp alone (15). the high homa - ir index observed among subjects with the tg / tg haplotype was only seen in the overweight - obese subgroup. this result is in accordance with jang. (7) who found an association between the tg haplotype and increased homa - ir index in overweight - obese but not in normal - weight nondiabetic koreans. in response to dietary intervention, overweight - obese subjects carrying the tg / tg haplotype exhibited an increase in adiponectin levels (1.09 g / ml) and a 25% decrease in homa - ir index. this finding might indicate that a genotypic - specific effect is heightened in overweight - obese individuals in terms of altered adiponectin levels and homa - ir index after dietary intervention compared with normal - weight individuals. they do not mean that normal - weight subjects have a relatively poor outcome after dietary intervention compared with overweight - obese subjects. moreover, it remains unclear whether adiponectin levels change according to adipoq snps and whether this change is clinically relevant for long - term dietary intervention programs. even though several reports have shown an interaction between adipoq snps and obesity (5,8,16), the mechanism is not yet known. one possible explanation might be that obesity with increased adipose tissue predispose individuals to altered adipocytokine levels (e.g., decreased adiponectin and increased tumor necrosis factor-) and, thus, the effect of the adiponectin gene is more exaggerated in obesity. another possibility is that lower baseline adiponectin levels in obesity might induce additional changes in adiponectin levels and amplify the diet - intervention effect. indeed, a multiple regression analysis in this study showed that baseline adiponectin levels were independent factors to determine changes in adiponectin levels. also, there are several reports to suggest the relationship between adiponectin levels and treatment responses (17,18). however, further experimental studies are needed to clarify the mechanism underlying these responses. our findings that a small increase in adiponectin (1.09 g / ml) along with a decrease in both fasting insulin and glucose levels in overweight - obese subjects carrying the tg / tg haplotype confirm a previous report of the insulin - sensitizing effects of adiponectin (19). in the tg / tg haplotype group, the insulin - sensitizing effects of adiponectin compensate for insulin and result in a decreased demand for insulin and improvement in the insulin resistance index. this hypothesis might be supported by the observation that overweight - obese subjects carrying non - tg alleles showed a decrease in adiponectin levels (0.33 g / ml) and a slight, although insignificant, increase in homa - ir indexes. thus, we also observed an inverse relationship between changes in homa - ir indexes and adiponectin levels similar to previous findings (18,20). 21) found that variations at the adiponectin locus affect body weight gain, onset of hyperglycemia, and adiponectin levels in a 3-year prospective study in french caucasians. specifically, gg carriers of snp 45t > g were found to gain more weight and have a higher risk of hyperglycemia. in this study, however, 45gg subjects or subjects who did not carry the tg haplotype of 45t > g/276g > t showed no significant changes in body weight, fasting glucose and insulin levels, homa - ir indexes, or adiponectin levels either before or after dietary intervention. this discrepancy might be due to either the ethnic specificity or the short - term effects of dietary changes. (22) in which adiponectin levels are altered independently of adipoq polymorphisms after dietary supplementation with -linolenic acid. because small sample size (n = 57) in the previous study can not allow interpretation of the genetic effect, a possible assumption can be made that specific effects of dietary factors (i.e., high -3 fatty acids) can mask the genetic effects in relatively insulin - sensitive healthy subjects. in addition, direct comparisons between studies are difficult, considering the differences in the characteristics of the subjects studied (healthy, young, primarily female, and caucasian versus hyperglycemic patients, middle - aged, fewer female, and korean) and study intervention program. however, measurement errors from self - reported dietary intake and lifestyle variables have been shown to be relatively small (23). in this study, well - controlled, fasting glucose concentrations reflected the compliance of the subjects to dietary intervention. second, because of the small sample size, the results of genetic analyses should be interpreted with caution. finally, heterogeneity in the causes of predisposition to diabetes and diabetes itself must be considered because metabolic differences can be present between subjects with ifg and type 2 diabetes. however, our patients with newly diagnosed type 2 diabetes did not show different metabolic characteristics, including plasma adiponectin levels, compared with those in subjects with ifg (data not shown). further additional studies with a larger sample size are needed to confirm our findings. despite these limitations, we showed that adipoq genetic variants can be factors in the interindividual differences in adiponectin levels and insulin resistance indexes after dietary intervention in hyperglycemic patients. we found the greatest decrease in homa - ir indexes and greatest increase in adiponectin levels in overweight - obese subjects with ifg or newly diagnosed type 2 diabetes who carry the tg / tg haplotype of adipoq 45t > g/276g > t. however, long - term prospective studies are needed to examine the relationship between adiponectin levels and clinical outcomes in these subjects. with a more complete understanding of these factors, our results provide good evidence for tailoring of dietary intervention programs to individuals on the basis of their genetic patterns.	objectivethe aim of this study was to determine the effect of common adiponectin gene polymorphisms on dietary intervention - mediated changes in adiponectin levels and homeostasis model assessment of insulin resistance (homa - ir) indexes.research design and methodsa total of 363 subjects with impaired fasting glucose (ifg) or newly diagnosed type 2 diabetes followed a dietary intervention (replacement of cooked refined rice with whole grains and an increase in vegetable intake) and regular walking for 12 weeks without any medication. adiponectin gene single nucleotide polymorphisms (snps) (45, 276, and 11377) were examined in these subjects.resultsafter this dietary intervention, fasting glucose levels decreased in all three snp 45t > g genotype groups. subjects with the snp 45tt genotype showed increased adiponectin levels and decreased homa - ir indexes. haplotype analysis revealed that homozygous carriers of the tg haplotype (45tt and 276gg) and heterozygous carriers of the tg haplotype (tg / x) showed a reduction in the homa - ir index after adjustment for baseline levels. significant differences were observed in changes in homa - ir indexes and adiponectin concentrations according to the 45 - 276 tg haplotype in overweight - obese, but not in normal - weight subjects : the greatest decrease in homa - ir indexes and the greatest increase in adiponectin levels were shown in overweight - obese subjects with the tg / tg haplotype.conclusionsadipoq genetic variants can affect circulating adiponectin levels and insulin resistance indexes in subjects with ifg or newly diagnosed type 2 diabetes in response to dietary intervention.
research in the wide field of empirical aesthetics demonstrates highly reliable and consistent assessments of aesthetic judgments, especially in regard to what we like and what we hate from a visual aesthetic perspective. most studies investigating the psychological basis of aesthetic judgments have been conducted in the specific domain of the aesthetics of human faces (for a meta - analysis, see langlois 2000 ; for an overview, see perrett 2010). we will therefore first inspect the findings and resulting theories in this particular area of aesthetic research to subsequently extend our view to a more general approach of universal psychological principles that are responsible for creating aesthetic preferences. the specific aim will be to develop a model of how and on what basis such aesthetic preferences are susceptible to changes, the so - called dynamics of aesthetic appreciation (carbon 2006). in the domain of facial attractiveness, studies continuously indicate high internal consistencies (eg, cronbach 's >.87, carbon 2010) as well as high inter - rater reliabilities of attractiveness ratings (eg, r >.88, carbon 2010 ; for a meta - analysis, see langlois 2000), even if persons with different face recognition abilities or belonging to different cultures (perrett 1994) or morphological groups (cunningham 1995) are included in the study. when people are asked to simply categorize faces into four different attractiveness groups, even higher inter - rater reliabilities of >.97 (buhlmann 2008) are quite common. a recent paper by hnekopp (2006) reveals the routine usage of cronbach 's alpha for demonstrating the supremacy of commonly shared aesthetic standards as a misconception. using new indices for personal versus shared taste based on separate scores for judges and faces, he could demonstrate that both sources account for equally high amounts of explained variance of facial attractiveness. although this methodological approach stresses the importance of private taste, it still obtains a very high amount of shared taste. langlois and colleagues (2000) cite several possible explanations for such high amounts of shared assessments of attractiveness, including (a) social expectancy theories proposing social stereotypes created by cultural similarities in standards of attractiveness, (b) fitness - related evolutionary theories assuming that morphological properties are direct indicators of fitness and the probability of reproductive success, (c) theories emphasizing the possibility of assessing the quality of genes by mere phenotypical properties (the so - called good genes theory), and (d) theories linked to the theories (b) and (c) raising the point that attractiveness might be an important visual signal in the context of mate selection. obviously, for a more comprehensive or even general theory of the dynamics of aesthetic appreciation a mechanism has to be proposed that is relatively independent of a strictly hard - wired, universal biological program that relies on clear and objective properties such as hip measurement, a cue for increased probability of unproblematic child delivery, or the observed health status. such a general theory should also deal with aesthetic phenomena evoked by consumer products, artworks, or landscapes (the sublime, erhabenheit), which are not necessarily if at all linked to biological advantages. as a matter of principle the above - mentioned social expectancy theories (langlois 2000) have the capability to be quite adaptive towards cultural and social circumstances because they are based on the assumptions (a) that cultural norms and experience shape the perception of aesthetic objects and (b) that social stereotypes create their own reality. this adaptive capability conversely lowers the possibility of gaining trans - age and trans - cultural congruency in aesthetic judgments. this, in turn, can hardly be reconciled with results of developmental studies (eg, johnson and pittenger 1984 ; zebrowitz 1993) or cross - cultural studies (eg, cunningham 1995 ; maret and harling 1985 ; rhodes 2001, 2002) showing high congruency of aesthetic appreciation at least for biologically relevant stimuli such as faces or bodies. at first glance, this theory also seems to be hardly reconcilable with the fact that even babies show a strong preference for attractive faces (langlois 1991), categorized so by adult experimenters. a closer look, however, reveals that the documented evidence for such cross - cultural and early operating attractiveness universalities is much weaker than often supposed. for instance, there are many indications for culturally dependent aesthetic phenomena such as extreme tattoo practices of the maori often covering large parts of their bodies and faces or aesthetic optimization by teeth filling, scarification, or massive enlargement of lips or ears (for illustrations, see chapter 6 in zebrowitz 1997). additionally, the notoriously reported fact that babies already show the same preferences as adults is rather problematic : first, because only vague indications for attractiveness (eg, habituation - dishabituation paradigms or inspection times) can be used due to a lack of valid verbal responses and, second, because such measures, no matter how valid they are, can not be compared with rating scale data due to their obviously incommensurable scale quality. nevertheless, it is undeniable that we share taste with others to a certain degree. it is clear that such a mechanism has to be quite flexible and should not be (exclusively) based on hard - wired processes. this requirement seems mandatory when aesthetic appreciation is analysed in a historical context and not only as a one - off measurement at one specific point of time. although aesthetic appreciation is a true topic of psychological research, we have to refer to approaches stemming from the context of art history to get deeper insights into the dynamics of specific styles. art history typically develops theories on how different artistic styles evolved, how they inspired each other, and how they replace out - dated ones. notwithstanding, art history mainly tries to explain how and sometimes why certain artistic streams emerged, but not whether and why beholders appreciate the objects generated from these streams. figure 1 illustrates how similar some of pierre - auguste renoir 's masterpieces were in terms of high saturation of the colour blue during his period of work when he visited italy in the early 1880s. this idiosyncratic period shows a clear difference compared with earlier as well as later styles where he strongly relied on his manner referred to as diffuse reflection and ingres period, respectively (gombrich 2005). note : it can be assumed that, around the early 1880s, renoir shifted from the so - called cobalt blue, a cobalt(ii) aluminate of pure blue colour that was a very costly and stable pigment, to cheaper and less light - resistant blue pigments ; this change can be observed, for example, by closely inspecting the different shades of blue in his painting the umbrellas (les parapluies), painted in the same period. as bazin (1958) noted already, the highly saturated blue that can nowadays be observed in renoir 's paintings like those shown in figure 1 might be owed to the aging of these cheap pigments. even if so, this would indicate that renoir was reliably using these specific pigments in that creative period which already expressed a specific colour style back to the date of the paintings origins. with such characteristic usage of specific styles, artists as well as designers create their own brand, which is important to be recognized by and associated with (for art, see augustin 2008 ; for consumer products, see kreuzbauer and malter 2005 ; ulrich and smallwood 2007). importantly, artists as well as designers often change their style after a while, mostly due to further developing or revolutionizing their techniques by integrating new procedures, changing their themes or the focus of their work in response to new insights and experiences made in the meantime (panofsky 1955). paintings by pierre - auguste renoir (18411919) from a narrow period of time of his artistry show a high consistency of the specific usage of the colour blue. from left to right : (a) sleeping girl (1880), (b) venezia, the doge 's palace (1881), (c) bay of naples, evening (1881), (d) child with a bird (1882). the change of style, though, does not necessarily imply a change of aesthetic appreciation for it. for instance, we can not be sure that gothic art was preferred to the romanesque style when it was initially established as a widely known architecture style in the 13th century. it is, nevertheless, highly implausible that a vast majority of people still preferred the ancient style after a while. instead, processes such as mere exposure (zajonc 1968) or cognitive mechanisms like understanding and appreciation induced by repeated evaluation (carbon and leder 2005 ; faerber 2010) may have led to an increase of aesthetic appreciation towards certain exemplars frequently available to the beholders. although the change in preferences is an important topic of aesthetic research, systematic data on such effects, especially concerning longer periods of time spanning several decades (cutting 2011), are rather sparse. pioneering psychological research contributing to this topic was conducted by martindale (1990). in his noteworthy book a clockwork muse, martindale gathered strong evidence for cyclic changes of artistic style in a variety of fields, such as music, visual arts, and poetics. carbon (2010) replicated the general cyclic character of the evolution of aesthetic dimensions in the area of consumer product design, particularly when referring to the curvature of car exteriors. importantly, he extended the mere analysis of historical data by testing adaptation mechanisms as a plausible explanation for cyclic changes of appreciation as well. when confronted with highly innovative designs of so - called concept cars, presumably containing many future design features, the participants adapted their aesthetic appreciation towards the style of these designs, while their liking for previously preferred cars introduced over the last 15 years decreased a significant degree. this study demonstrated the power of adaptation towards frequently or, particularly, recently inspected exemplars and the associated style, which is quite compatible with the strong and reliable effects of adaptation in the domain of faces (rhodes 2003). this strong cognitive mechanism of adaptation proved to be very powerful in explaining the shift of prototypes (carbon 2009 ; carbon and leder 2006 ; leopold 2005), normality / veridicality assessments (carbon and ditye 2011 ; rhodes and jeffery 2006), and preferences (carbon 2010 ; carbon 2007 ; farber 2007 ; rhodes 2003) and makes it improbable that concepts such as classic aesthetic norms, constant prototypes, or standards of beauty exist at all. although notions like timeless design (vandenbroecke 1992) or eternal standards of beauty (vitruvius 1999) and even advertisements suggestively propagating high - priced brands that allegedly embody classical perfection and are thus a sign for good taste (figure 2) imply stability in aesthetic appreciation, such preferences actually seem to be quite flexible. thus, taking the psychological mechanism of adaptation into account, gaultier 's implication of eternal aesthetic perfection and stable taste shown in figure 2 seems incorrect. jean - paul gaultier 2011 advertising campaign for the gaultier perfume brand classique, first introduced in 1993, which directly refers to the concept of classic or (good) taste comparable to a perfect body shape which stays rather constant. photograph made by ccc. only by assuming a highly flexible mechanism we seem to fit with and adapt to an ever - changing world. psychological mass phenomena of dynamic changes of aesthetic appreciation, detectable in fashion (majima 2007), artistic style (sorokowski 2010) and design (carbon 2010), and even in body shape (pettijohn and jungeberg 2004), documented as very old in cultural history, would be inconceivable if such an adaptive mechanism was not postulated. what we need to explain phenomena of dynamic changes of aesthetic appreciation is a flexible cognitive mechanism that is relatively independent from fixed norms or hard - wired principles. if we analyse the dynamics of aesthetic appreciation over the years, across different object categories and for different aesthetic domains, it becomes quite obvious that we can identify streams, movements, or periods. for instance, art history is mainly concerned with studying objects of art in a historical context by explicitly identifying the developments, the emergence, and the change of stylistic properties. typically, art history can explain how such changes emerge as a result of political, religious, geographical, or sometimes even of weather - related factors. the adaptation mechanism identified by previous research (eg, carbon 2010) could be a promising candidate for explaining mass phenomena of changes of aesthetic appreciation towards new design or art features in a simple but psychologically relevant way. documented phenomena of desynchronization of preferences can be concordantly explained with this approach by insufficient or non - existing exchange of information about the objects of desire, for instance, fashion items (figure 3). illustration of desynchronization of fashion styles in former times : the picture shows a drawing by albrecht drer from 14961497 depicting a typical style of a bourgeois from nuremberg (left) and from venice (right) emphasising long legs by the usage of high chopines plus a skirt cut with the waist edge just beneath the female bust. source : wikimedia commons. synchronization, on the other hand, leads to concordant, similar or shared processes of aesthetic appreciation and can even have behavioural consequences as documented by humans synchronized behaviour when encountering, for instance, depictions of saints or holy persons, producing constant abrasion of certain spots of the artistic objects (figure 4). detail of the bronze gates of the duomo di milano (milan cathedral ; lower part of the major door, planned by l pogliaghi) demonstrating frequent touching of specific parts of the holy scenes, which results in burnishing these parts over the years. the highly selective burnishing indicates synchronized visitors behaviour, here the touching of the central and significant mutual soft touch of jesus and maria 's hands. adaptation also seems to be an elegant way to dissolve some inconsistencies in the literature concerning the degree of shared taste. vessel and rubin (2010) have found high amounts of shared taste for real - world images but not for abstract ones. the more abstract an image is the lower is the chance to create associations to objects that we can explicitly refer to. this lowers the possibility of creating an image of such an item a classical cognitive finding dating back to paivio (1969). this means that items of high abstractness are also susceptible to being evaluated on a rather idiosyncratic basis without a great chance of being a source of adaptation in a general way. real - world images, in contrast, are a perfect starting point for such adaptation effects ; consequently, they are also potential objects of triggering shared taste. beside the mechanism of adaptation being a plausible source of synchronization of aesthetic appreciation, we essentially need a further ingredient for explaining the sources of the changes of artistic style (augustin 2008) or the formensprache (carbon 2010). actually, we need a clue for the cognitive principle which triggers innovation itself. artists and designers belonging to the creative sector evidently have to innovate, or else they will only copy previous work and just prefer (and thus create) what they have already generated due to mere exposure or other familiarity phenomena (martindale 1988). martindale (1990) solves the problem by assuming inherent conservatism on the side of typical beholders (see carbon and leder 2005), but high innovative power and flexible taste on the side of the artists (martindale 1994). such trendsetters can start a period of synchronization with a more conservative audience just as illustrated by figure 5. first, people often get bored by too familiar exemplars after a while, a phenomenon ascribed in the 19th century by gller (1888), which he termed formermdung (form fatigue) ; second, creative people compete with each other, creating ongoing innovations ; third, partly nonconscious, adaptation to innovative and distinctive parts from other cultures, domains, or exemplars re - shaping the creative one 's visual habits themselves. clearly, the feature beholders adapt to varies across different classes of (consumer) products and might, even within one class, change over time ; probable candidates for the automobile sector, for example, are colour, shape (formensprache), and the specific material used. so, over time, the most important feature for which beholders are synchronized can change from, eg, colour to shape and then, some more years later, to material or other design features. model of trendsetters triggering starting points of synchronisation : from a certain point of time, the characteristic feature, which can vary among many different aspects such as material, form, colour, etc, indicated by item 0 which is generated by a highly creative and publicly renowned trendsetter is appreciated via adaptation. competing items (works of art, consumer products, design) oriented towards this innovative feature, even copying or imitating it, increase the availability and consequently the appreciation of the new feature. research in the wide field of empirical aesthetics demonstrates highly reliable and consistent assessments of aesthetic judgments, especially in regard to what we like and what we hate from a visual aesthetic perspective. most studies investigating the psychological basis of aesthetic judgments have been conducted in the specific domain of the aesthetics of human faces (for a meta - analysis, see langlois 2000 ; for an overview, see perrett 2010). we will therefore first inspect the findings and resulting theories in this particular area of aesthetic research to subsequently extend our view to a more general approach of universal psychological principles that are responsible for creating aesthetic preferences. the specific aim will be to develop a model of how and on what basis such aesthetic preferences are susceptible to changes, the so - called dynamics of aesthetic appreciation (carbon 2006). in the domain of facial attractiveness, studies continuously indicate high internal consistencies (eg, cronbach 's >.87, carbon 2010) as well as high inter - rater reliabilities of attractiveness ratings (eg, r >.88, carbon 2010 ; for a meta - analysis, see langlois 2000), even if persons with different face recognition abilities or belonging to different cultures (perrett 1994) or morphological groups (cunningham 1995) are included in the study. when people are asked to simply categorize faces into four different attractiveness groups, even higher inter - rater reliabilities of >.97 (buhlmann 2008) are quite common. a recent paper by hnekopp (2006) reveals the routine usage of cronbach 's alpha for demonstrating the supremacy of commonly shared aesthetic standards as a misconception. using new indices for personal versus shared taste based on separate scores for judges and faces, he could demonstrate that both sources account for equally high amounts of explained variance of facial attractiveness. although this methodological approach stresses the importance of private taste, it still obtains a very high amount of shared taste. langlois and colleagues (2000) cite several possible explanations for such high amounts of shared assessments of attractiveness, including (a) social expectancy theories proposing social stereotypes created by cultural similarities in standards of attractiveness, (b) fitness - related evolutionary theories assuming that morphological properties are direct indicators of fitness and the probability of reproductive success, (c) theories emphasizing the possibility of assessing the quality of genes by mere phenotypical properties (the so - called good genes theory), and (d) theories linked to the theories (b) and (c) raising the point that attractiveness might be an important visual signal in the context of mate selection. obviously, for a more comprehensive or even general theory of the dynamics of aesthetic appreciation a mechanism has to be proposed that is relatively independent of a strictly hard - wired, universal biological program that relies on clear and objective properties such as hip measurement, a cue for increased probability of unproblematic child delivery, or the observed health status. such a general theory should also deal with aesthetic phenomena evoked by consumer products, artworks, or landscapes (the sublime, erhabenheit), which are not necessarily if at all linked to biological advantages. as a matter of principle the above - mentioned social expectancy theories (langlois 2000) have the capability to be quite adaptive towards cultural and social circumstances because they are based on the assumptions (a) that cultural norms and experience shape the perception of aesthetic objects and (b) that social stereotypes create their own reality. this adaptive capability conversely lowers the possibility of gaining trans - age and trans - cultural congruency in aesthetic judgments. this, in turn, can hardly be reconciled with results of developmental studies (eg, johnson and pittenger 1984 ; zebrowitz 1993) or cross - cultural studies (eg, cunningham 1995 ; maret and harling 1985 ; rhodes 2001, 2002) showing high congruency of aesthetic appreciation at least for biologically relevant stimuli such as faces or bodies. at first glance, this theory also seems to be hardly reconcilable with the fact that even babies show a strong preference for attractive faces (langlois 1991), categorized so by adult experimenters. a closer look, however, reveals that the documented evidence for such cross - cultural and early operating attractiveness universalities is much weaker than often supposed. for instance, there are many indications for culturally dependent aesthetic phenomena such as extreme tattoo practices of the maori often covering large parts of their bodies and faces or aesthetic optimization by teeth filling, scarification, or massive enlargement of lips or ears (for illustrations, see chapter 6 in zebrowitz 1997). additionally, the notoriously reported fact that babies already show the same preferences as adults is rather problematic : first, because only vague indications for attractiveness (eg, habituation - dishabituation paradigms or inspection times) can be used due to a lack of valid verbal responses and, second, because such measures, no matter how valid they are, can not be compared with rating scale data due to their obviously incommensurable scale quality. nevertheless, it is undeniable that we share taste with others to a certain degree. it is clear that such a mechanism has to be quite flexible and should not be (exclusively) based on hard - wired processes. this requirement seems mandatory when aesthetic appreciation is analysed in a historical context and not only as a one - off measurement at one specific point of time. although aesthetic appreciation is a true topic of psychological research, we have to refer to approaches stemming from the context of art history to get deeper insights into the dynamics of specific styles. art history typically develops theories on how different artistic styles evolved, how they inspired each other, and how they replace out - dated ones. notwithstanding, art history mainly tries to explain how and sometimes why certain artistic streams emerged, but not whether and why beholders appreciate the objects generated from these streams. figure 1 illustrates how similar some of pierre - auguste renoir 's masterpieces were in terms of high saturation of the colour blue during his period of work when he visited italy in the early 1880s. this idiosyncratic period shows a clear difference compared with earlier as well as later styles where he strongly relied on his manner referred to as note : it can be assumed that, around the early 1880s, renoir shifted from the so - called cobalt blue, a cobalt(ii) aluminate of pure blue colour that was a very costly and stable pigment, to cheaper and less light - resistant blue pigments ; this change can be observed, for example, by closely inspecting the different shades of blue in his painting the umbrellas (les parapluies), painted in the same period. as bazin (1958) noted already, the highly saturated blue that can nowadays be observed in renoir 's paintings like those shown in figure 1 might be owed to the aging of these cheap pigments. even if so, this would indicate that renoir was reliably using these specific pigments in that creative period which already expressed a specific colour style back to the date of the paintings origins. with such characteristic usage of specific styles, artists as well as designers create their own brand, which is important to be recognized by and associated with (for art, see augustin 2008 ; for consumer products, see kreuzbauer and malter 2005 ; ulrich and smallwood 2007). importantly, artists as well as designers often change their style after a while, mostly due to further developing or revolutionizing their techniques by integrating new procedures, changing their themes or the focus of their work in response to new insights and experiences made in the meantime (panofsky 1955). paintings by pierre - auguste renoir (18411919) from a narrow period of time of his artistry show a high consistency of the specific usage of the colour blue. from left to right : (a) sleeping girl (1880), (b) venezia, the doge 's palace (1881), (c) bay of naples, evening (1881), (d) child with a bird (1882). the change of style, though, does not necessarily imply a change of aesthetic appreciation for it. for instance, we can not be sure that gothic art was preferred to the romanesque style when it was initially established as a widely known architecture style in the 13th century. it is, nevertheless, highly implausible that a vast majority of people still preferred the ancient style after a while. instead, processes such as mere exposure (zajonc 1968) or cognitive mechanisms like understanding and appreciation induced by repeated evaluation (carbon and leder 2005 ; faerber 2010) may have led to an increase of aesthetic appreciation towards certain exemplars frequently available to the beholders. although the change in preferences is an important topic of aesthetic research, systematic data on such effects, especially concerning longer periods of time spanning several decades (cutting 2011), are rather sparse. pioneering psychological research contributing to this topic was conducted by martindale (1990). in his noteworthy book a clockwork muse, martindale gathered strong evidence for cyclic changes of artistic style in a variety of fields, such as music, visual arts, and poetics. carbon (2010) replicated the general cyclic character of the evolution of aesthetic dimensions in the area of consumer product design, particularly when referring to the curvature of car exteriors. importantly, he extended the mere analysis of historical data by testing adaptation mechanisms as a plausible explanation for cyclic changes of appreciation as well. when confronted with highly innovative designs of so - called concept cars, presumably containing many future design features, the participants adapted their aesthetic appreciation towards the style of these designs, while their liking for previously preferred cars introduced over the last 15 years decreased a significant degree. this study demonstrated the power of adaptation towards frequently or, particularly, recently inspected exemplars and the associated style, which is quite compatible with the strong and reliable effects of adaptation in the domain of faces (rhodes 2003). this strong cognitive mechanism of adaptation proved to be very powerful in explaining the shift of prototypes (carbon 2009 ; carbon and leder 2006 ; leopold 2005), normality / veridicality assessments (carbon and ditye 2011 ; rhodes and jeffery 2006), and preferences (carbon 2010 ; carbon 2007 ; farber 2007 ; rhodes 2003) and makes it improbable that concepts such as classic aesthetic norms, constant prototypes, or standards of beauty exist at all. although notions like timeless design (vandenbroecke 1992) or eternal standards of beauty (vitruvius 1999) and even advertisements suggestively propagating high - priced brands that allegedly embody classical perfection and are thus a sign for good taste (figure 2) imply stability in aesthetic appreciation, such preferences actually seem to be quite flexible. thus, taking the psychological mechanism of adaptation into account, gaultier 's implication of eternal aesthetic perfection and stable taste shown in figure 2 seems incorrect., first introduced in 1993, which directly refers to the concept of classic or eternal (good) taste comparable to a perfect body shape which stays rather constant. photograph made by ccc. only by assuming a highly flexible mechanism we seem to fit with and adapt to an ever - changing world. psychological mass phenomena of dynamic changes of aesthetic appreciation, detectable in fashion (majima 2007), artistic style (sorokowski 2010) and design (carbon 2010), and even in body shape (pettijohn and jungeberg 2004), documented as very old in cultural history, would be inconceivable if such an adaptive mechanism was not postulated. what we need to explain phenomena of dynamic changes of aesthetic appreciation is a flexible cognitive mechanism that is relatively independent from fixed norms or hard - wired principles. if we analyse the dynamics of aesthetic appreciation over the years, across different object categories and for different aesthetic domains, it becomes quite obvious that we can identify streams, movements, or periods. for instance, art history is mainly concerned with studying objects of art in a historical context by explicitly identifying the developments, the emergence, and the change of stylistic properties. typically, art history can explain how such changes emerge as a result of political, religious, geographical, or sometimes even of weather - related factors. the adaptation mechanism identified by previous research (eg, carbon 2010) could be a promising candidate for explaining mass phenomena of changes of aesthetic appreciation towards new design or art features in a simple but psychologically relevant way. people get effectively synchronized in their behaviour and preferences via this mediating adaptation mechanism. documented phenomena of desynchronization of preferences can be concordantly explained with this approach by insufficient or non - existing exchange of information about the objects of desire, for instance, fashion items (figure 3). illustration of desynchronization of fashion styles in former times : the picture shows a drawing by albrecht drer from 14961497 depicting a typical style of a bourgeois from nuremberg (left) and from venice (right) emphasising long legs by the usage of high chopines plus a skirt cut with the waist edge just beneath the female bust. synchronization, on the other hand, leads to concordant, similar or shared processes of aesthetic appreciation and can even have behavioural consequences as documented by humans synchronized behaviour when encountering, for instance, depictions of saints or holy persons, producing constant abrasion of certain spots of the artistic objects (figure 4). detail of the bronze gates of the duomo di milano (milan cathedral ; lower part of the major door, planned by l pogliaghi) demonstrating frequent touching of specific parts of the holy scenes, which results in burnishing these parts over the years. the highly selective burnishing indicates synchronized visitors behaviour, here the touching of the central and significant mutual soft touch of jesus and maria 's hands. adaptation also seems to be an elegant way to dissolve some inconsistencies in the literature concerning the degree of shared taste. vessel and rubin (2010) have found high amounts of shared taste for real - world images but not for abstract ones. the more abstract an image is the lower is the chance to create associations to objects that we can explicitly refer to. this lowers the possibility of creating an image of such an item a classical cognitive finding dating back to paivio (1969). this means that items of high abstractness are also susceptible to being evaluated on a rather idiosyncratic basis without a great chance of being a source of adaptation in a general way. real - world images, in contrast, are a perfect starting point for such adaptation effects ; consequently, they are also potential objects of triggering shared taste. beside the mechanism of adaptation being a plausible source of synchronization of aesthetic appreciation, we essentially need a further ingredient for explaining the sources of the changes of artistic style (augustin 2008) or the formensprache (carbon 2010). artists and designers belonging to the creative sector evidently have to innovate, or else they will only copy previous work and just prefer (and thus create) what they have already generated due to mere exposure or other familiarity phenomena (martindale 1988). martindale (1990) solves the problem by assuming inherent conservatism on the side of typical beholders (see carbon and leder 2005), but high innovative power and flexible taste on the side of the artists (martindale 1994). such trendsetters can start a period of synchronization with a more conservative audience just as illustrated by figure 5. first, people often get bored by too familiar exemplars after a while, a phenomenon ascribed in the 19th century by gller (1888), which he termed formermdung (form fatigue) ; second, creative people compete with each other, creating ongoing innovations ; third, partly nonconscious, adaptation to innovative and distinctive parts from other cultures, domains, or exemplars re - shaping the creative one 's visual habits themselves. clearly, the feature beholders adapt to varies across different classes of (consumer) products and might, even within one class, change over time ; probable candidates for the automobile sector, for example, are colour, shape (formensprache), and the specific material used. so, over time, the most important feature for which beholders are synchronized can change from, eg, colour to shape and then, some more years later, to material or other design features. model of trendsetters triggering starting points of synchronisation : from a certain point of time, the characteristic feature, which can vary among many different aspects such as material, form, colour, etc, indicated by item 0 which is generated by a highly creative and publicly renowned trendsetter is appreciated via adaptation. competing items (works of art, consumer products, design) oriented towards this innovative feature, even copying or imitating it, increase the availability and consequently the appreciation of the new feature. to sum it up, having adaptation as a basic cognitive mechanism, we are equipped to fulfil many demands of an ever - changing world. by mere exposure to (zajonc 2001) and, even stronger, by active elaboration (carbon and leder 2005) of highly innovative material such as fashion, design, and art objects, we automatically integrate the perceived exemplars into our visual habits. as the compatibility with these visual habits, mostly operationalized by prototypicality (blijlevens in press), is a key factor in explaining aesthetic appreciation, our preferences for such exemplars change dynamically. when different people perceive the same objects iteratively, for instance, because they belong to the cultural canon or refer to frequently promoted products of famous brands, shared preferences for these items will emerge. importantly, this mechanism alone can only explain dynamic changes as a reaction to given streams of design or art innovations. consequently, we have to propose an initial mechanism started by highly creative sources that generate and trigger such trends, taking the role of so - called innovators (rogers 2003). such sources may be individuals from the creative industries with high publicity, for instance, famous designers or artists, celebrities merely wearing or using such products, or influential events or media presentations such as the milan design week or a typical trend journal such as vogue promoting innovative goods to a broader mass (sproles 1981). if we take these two mechanisms together, the first one promoting innovative objects and the second one allowing people to adapt to these innovations, we can explain the often documented high amounts of shared preferences as well as idiosyncratic tastes of desynchronized (sub)cultures. the idea of two subsequent sub - processes leads to a dynamically adapting two - step model of aesthetic appreciation (figure 6). the result of the whole process will not be the establishment of a final kind of appreciation space, where all the most appreciated exemplars are represented, but only a temporary state which we might call taste. according to this conception, (see figure 6), which has the power to establish and stabilize our appreciation not longer than for a certain period of time after which new, innovative exemplars will be encountered and, so, start the process again. essentially, this process is a dynamic one that never reaches full stability, as experience with new stimuli will always start and revive the process again (carbon 2010). figure 6 illustrates this two - step model with the historical change in art history from impressionist paintings to post - impressionist / expressionist streams of art. two - step model of the dynamics of aesthetic appreciation, illustrated with appreciation changing from impressionist to post - impressionist paintings. taste is (a) established and stabilized until beholders are (b) intensively confronted with innovative exemplars (step # 1). in a subsequent adaptation phase (step # 2) beholders (c) adapt towards the new stimuli integrating them into their visual habits. the appreciation space is (d) continuously retuned, establishing and stabilizing taste again. paintings depicted here are from the wikimedia commons showing (a) typical impressionist works by claude monet and pierre - auguste renoir and (b) distinctive and, in the period of impressionism, highly innovative post - impressionist / expressionist works by vincent van gogh. image is from the wikimedia commons, originally from life magazine from 1913 describing fashion as an evil force which explicitly dictates fashion rules that people have to follow. with such a cognitive mechanism in mind, we can also understand typical mechanisms moving fashionista, people who slavishly follow current fashion trends as they intensively consume fashion media and permanently adapt their taste. additionally, it is also possible to explain why even elderly people are susceptible to being influenced by design innovation if they elaborate sources referring to such material (carbon and schoormans in press). in conclusion, we should be aware that adaptation is a very helpful mechanism to adjust to a highly dynamic world. it is, though, also a potentially dangerous mechanism that brings us to adapt to ideals we have not chosen on a conscious basis but which change our geschmacksapparat (system for validly assessing our taste) incidentally, without explicit reference. in contrast to figure 7, which portrays the view of an almighty entity explicitly dictating fashion rules that people have to follow, we seem to be biased much more indirectly, nonconsciously by such a mechanism. therefore, it does indeed matter which media we consume : whether these media show lean or chubby bodies, attractive or ugly faces, or arousing or calm art these images will change our aesthetic appreciation, thus our preferences, and consequently, our view onto the world as a whole.	for many domains aesthetic appreciation has proven to be highly reliable. evaluations of facial attractiveness, for instance, show high internal consistencies and impressively high inter - rater reliabilities, even across cultures. this indicates general mechanisms underlying such evaluations. it is, however, also obvious that our taste for specific objects is not always stable in some realms such stability is hardly conceivable at all since aesthetic domains such as fashion, design, or art are inherently very dynamic. gaining insights into the cognitive mechanisms that trigger and enable corresponding changes of aesthetic appreciation is of particular interest for psychologists as this will probably reveal essential mechanisms of aesthetic evaluations per se. the present paper develops a two - step model, dynamically adapting itself, which accounts for typical dynamics of aesthetic appreciation found in different research areas such as art history, philosophy, and psychology. the first step assumes singular creative sources creating and establishing innovative material towards which, in a second step, people adapt by integrating it into their visual habits. this inherently leads to dynamic changes of the beholders aesthetic appreciation.
although plants have an effective immune system, plant pathogens have evolved mechanisms to overcome plant resistance, and are thus able to invade and cause disease. vital to the success of plant pathogens is their ability to secrete so - called effectors that manipulate host plant defense (reviewed by stassen and van den ackerveken, 2010 ; de jonge., 2011). ergo, the pathogen can colonize plant cells with less hindrance. in the arms race this recognition leads to effector - triggered immunity (eti) that blocks further growth of the pathogen and that is often associated with a hypersensitive response (hr ; jones and dangl, 2006). oomycete plant pathogens are notorious for causing devastating diseases in a large variety of plants. the well - known and best studied species are the potato late blight pathogen phytophthora infestans, the soybean stem and root rot pathogen phytophthora sojae, and the downy mildew hyaloperonospora arabidopsidis that infects arabidopsis. plant resistance to oomycetes is mostly governed by r proteins comprising a n - terminal nucleotide - binding site (nbs) domain and a c - terminal leucine - rich - repeat (lrr) domain. since these nbs lrr proteins are located intracellular, it was hypothesized that oomycete avirulence (avr) proteins should be translocated to the cytoplasm of the plant cell. indeed, it has been shown that oomycetes secrete effectors that they deliver inside host cells. these intracellular effectors include avr proteins that are recognized by their cognate r proteins (rehmany., 2005 ; whisson., 2007). they all share a signal peptide and a conserved n - terminal rxlr motif with often an deer motif in its vicinity. these rxlr and deer motifs were detected in 2004 during a phytophthora genome annotation jamboree (govers and gijzen, 2006), a discovery that marked the beginning of a new episode in plant pathogen interaction research. at that time, after many years of labor - intensive map based cloning procedures, only a few oomycete avirulence (avr) genes were identified. as no conserved motifs were known for fungal avr proteins the presence of a conserved motif in oomycete avr proteins was unexpected. the number of rxlr deer effector genes in each of the three sequenced phytophthora genomes is remarkably high, with 563 predicted for p. infestans (haas., 2009) and 385 in p. sojae (jiang., 2008). significantly fewer rxlr deer effector genes were found in the genomes of two obligate oomycete pathogens sequenced so far, 115 in h. arabidopsidis and 25 in the white rust albugo laibachii (baxter., 2010 ; kemen., 2011), whereas very few or none (2) were found in two species with a necrotrophic life style, pythium ultimum and aphanomyces euteiches (gaulin., 2008 ;, is the clear resemblance of the rxlr motif with the rxlx host cell targeting motif in effector proteins of the malaria parasite plasmodium falciparum known as the plasmodium export element (pexel). transfer into the erythrocyte cytoplasm enables the pexel effectors to remodel and reprogram the host cell to establish successful asexual replication of the malaria parasite (templeton and deitsch, 2005 ; tilley. the rxlx motif was shown to be crucial for effector translocation across the parasitophorous vacuole membrane, which surrounds the malaria parasite in red blood cells (hiller. this led to the hypothesis that oomycete rxlr effectors are also targeted into host cells, and stimulated many researchers to address the question how oomycete effectors are delivered into plant cells (haldar., 2006). whisson. (2007) were the first to show that the rxlr deer domain is indeed required for effector translocation into host plant cells. infestans transformants expressing avr3a in which either the rxlr or deer motifs were mutated, were shown to be no longer detected by the resistance protein r3a, and as a result did not trigger an hr. transient co - expression of the r3a gene with variants of avr3a lacking the signal peptide sequence and with a mutated or deleted rxlr deer domain showed, however, that r3a - mediated cell death was not affected, and therefore it was concluded that the rxlr deer domain in avr3a is needed for effector translocation to the host cytoplasm (whisson., 2007). a similar approach was used for p. sojae avr1b-1, the avirulence counterpart of the soybean r gene rps1b and the first oomycete avr gene that was cloned (shan., the phenotype of a p. sojae strain changed from virulent to avirulent on rps1b plants when complemented with the full length avr1b-1 gene, but remained virulent when transformed with avr1b-1 with mutations in rxlr and deer. on the other hand, delivery of avr1b-1 by particle bombardment for transient expression of the avr gene in rps1b - leaves triggered cell death even when the rxlr or deer motifs were mutated or absent, and hence, a role for these motifs in delivery of avr1b was apparent (dou., 2008). although the two studies described above provided convincing evidence that the rxlr deer domains of avr3 and avr1b are crucial for uptake, the mechanisms underlying effector uptake by host cells remained unclear. to gain further insight in this process dou. (2008) investigated what happens when avr1b, produced in planta upon bombardment with an avr1b-1 construct, has a signal peptide. whereas secreted avr1b triggered rps1b - mediated cell death with the same efficiency as avr1b lacking its signal peptide, secreted avr1b with mutated rxlr and deer motifs triggered no response. this suggested that the motifs mediate re - entry of avr1b into the cell and, more strikingly, that the entry does not require the presence of the pathogen (dou., 2008). using similar approaches and supported by immunolocalization, rafiqi. (2010) showed that also internalization in host cells of two avr proteins produced by the flax rust fungus can occur in the absence of the pathogen. as with avr1b, uptake is dependent on features in the n - terminal region of the avr proteins ; but unlike avr1b, the rust avr proteins have no obvious conserved motifs. for p. sojae avr1b autonomous uptake in plant cells was also demonstrated with avr1b gfp fusion proteins synthesized in escherichia coli (dou., 2008). soaking root tips of soybean seedlings for 12 h in a solution containing partly purified fusion protein resulted in uptake of the avr1b gfp protein into the root tip cells up to 10 cell layers. these experiments suggested that the rxlr deer domain is all that is needed from the pathogen to enable translocation of oomycete effectors into host cells (dou. this is quite different from the situation in certain gram - negative bacteria that make use of the type iii secretion system (t3ss). these bacteria provide a suite of building blocks for a complex translocation machinery consisting of a multi - protein needle - like structure through which effectors are injected into the host cell cytoplasm (figures 1a, b). (a) oomycete plant pathogens secrete rxlr deer effector proteins from haustoria into the extrahaustorial matrix (em). how these effectors cross the plant plasma membrane (ppm) to reach the host cell cytoplasm is unknown. (b) bacterial pathogens exploit the type iii secretion system (t3ss) to inject effectors into the host cell cytoplasm. t3ss is a pathogen - derived multi - protein needle - like structure that penetrates the plant cell wall (pcw) and the plasma membrane. (c) malaria parasites (plasmodium spp.) invade red blood cells and secrete effector proteins into the parasitophorous vacuole (pv). pexel effectors are translocated to the red blood cell cytoplasm (rbcc) via a pathogen - derived translocon complex (ptex), which resides in the parasitophorous vacuole membrane (pvm). effectors are shown in different colored bars [in (a, c) ] and hexagons [in (b) ]. in the colored bars the red box is the signal peptide (sp) and the blue box the rxlr or rxlx motif. the next question to be answered is how rxlr deer effectors orchestrate their own translocation. if it is true that no other pathogen components are required, an issue that is still under debate, are the pathogens then actively recruiting host - derived components to support effector entry into host cells ? or do they possibly exploit an existing uptake machinery ? according to kale. (2010) the rxlr motif is capable to interact with phosphoinositides (ppis) at the host plasma membrane (pm), in particular phosphatidylinositol-3-phosphate (pi3p). in filter - binding and liposome - binding assays they showed interaction of the avr protein avr1b and two other members of the p. since mutations in rxlr resulted in loss of binding it was concluded that the interaction with ppis is specific. a puzzling issue, however, was the dogma that ppis always reside on the cytoplasmic site of eukaryotic cell membranes whereas the rxlr effectors approach the pm from the outside. to address this kale. (2010) exploited highly specific ppi biosensors, proteins composed of lipid - binding domains that target either pi3p or pi4p fused to a fluorescent tag. when exposed to soybean root suspension cultures they observed fluorescence with the pi3p biosensor, but not with the pi4p biosensor, on the extracellular leaflet of the pm, suggesting that pi3p is externally exposed. in a recent study yaeno. (2010), they found that mutations in the rxlr motif of avr1b do not interfere with pi3p binding. instead they observed that a positively charged lysine - rich patch in the c - terminus of avr1b, that is conserved in the p. infestans homolog avr3a, is responsible for the binding to ppi. the ppi binding to the c - terminus of avr3a, which is thought to take place inside host cells, seems to be required for accumulation of avr3a and for suppressing host immunity mediated by avr3a via interaction with the e3 ubiquitin ligase cmpg1 (yaeno., 2011). to what extent ppi binding plays a role in the activity of other rxlr effectors remains to be investigated. taken together the data that are currently available on the role of ppi binding are conflicting and fragmented, and the frontier - crossing journey of rxlr effectors is still a black box. the fact that the rxlr motif is highly similar to the rxlx translocation motif present in plasmodium effectors and the observations that the rxlr and rxlx motifs are functionally interchangeable (bhattacharjee. although pexel motif - containing proteins are shown to be taken up in the absence of the pathogen (kale., 2010), a plasmodium translocon complex (ptex) is described to work as an active export machine to deliver pexel effectors into the cytosol of the erythrocyte host cell (figure 1c ; de koning - ward., 2009). components comprising the ptex complex are an aaa - atpase, the exported protein exp2, thioredoxin 2, and two novel plasmodium proteins, ptex88 and ptex150. in the model so far, blast searches did not reveal obvious homologs of ptex components in phytophthora genomes (k. bouwmeester and h. j. g. meijer, unpublished results). this makes it less likely that plasmodium and phytophthora share highly conserved translocon machineries, although the existence of an analogous translocon complex for shuttling phytophthora effectors is still conceivable. intriguingly, it was reported that the pexel motif is not involved in a direct interaction with the ptex, but is functioning as a recognition site for cleavage (figure 2 ; chang., 2008 ; boddey., cleavage of the rxlx motif was shown to occur after the third residue (rxl), and when the conserved l residue was mutated, export was prevented (boddey., 2009). plasmepsin v, an endoplasmic reticulum (er) aspartic protease (ap), was found responsible for pexel cleavage (boddey., 2010 ; russo., 2010 a recent study showed that each of the three sequenced phytophthora species has 1112 ap genes distributed over five clades (kay., 2011). one clade with phytophthora ap10, ap11, and ap12 clusters with plasmepsin v, but whether or not these are functional rxlr or pexel proteases remains to be tested. are proteolytically processed by a signal peptidase and the er - localized aspartic protease plasmepsin v that cleaves the rxlx motif after the leucine (l). phytophthora effectors have an rxlr motif that resembles the plasmodium rxlx motif and the motifs are functionally interchangeable. what happens to rxlr effectors after removal of the signal peptide is not known. as indicated by the question marks, there is as yet no evidence for further processing, neither for cleavage within the rxlr motif nor for n - acetylation. an additional step in pexel processing is n - acetylation of the new n - terminus, which could well be a crucial step to facilitate translocation to the red blood cell (chang., 2008 ; boddey., 2009 ; figure 2). both cleavage and n - acetylation are executed early in the trafficking pathway within the er of plasmodium, but not in the parasitophorous vacuole or during export to the erythrocyte, as was concluded based on inhibition of er - to - golgi protein transport with brefeldin a. as yet, the n - acetyltransferase in plasmodium remains to be identified. so far, there are no indications for cleavage of oomycete rxlr effectors or n - acetylation and it is therefore questionable whether rxlr deer effectors from oomycetes are processed in a similar way. one of the 563 predicted p. infestans effectors that carries an rxlr motif is ipi - o. the encoding gene ipio (with ipi referring to in planta induced) was one of the first oomycete genes that was isolated and studied in detail (pieterse., 1993). at that time, the assignment of a putative function of ipio in pathogenesis was solely based on its expression pattern, i.e., high expression in planta versus low or no expression in in vitro culture. attempts to determine a function of ipi - o by gene silencing or overexpression failed (van west, 2000). the finding that ipi - o is a potential avr factor came many years later when ipio was tested in a high - throughput effector genomics screening aimed at identifying novel pairs of p. infestans avr genes and host plant r genes (vleeshouwers., subsequently, we confirmed that ipio is avr - blb1, the counterpart of the late blight r gene rpi - blb1 which originates from the nightshade solanum bulbocastanum (champouret., 2009). in the pre - genomic era comparative analysis that could have led to the discovery of the rxlr deer domain in ipi - o was not yet feasible ; at that time the only potentially interesting feature that could be distinguished in ipi - o was the presence of a cell attachment motif (pieterse., 1994). this motif consisting of the tripeptide sequence arg - gly - asp (rgd) partly overlaps with the rxlr motif in ipi - o, i.e., rxlrgd. the rgd motif is found in a large number of extracellular proteins, such as the mammalian glycoproteins fibronectin and vitronectin that reside in the extracellular matrix (ecm). a wide variety of these proteins binds to integrins, a family of transmembrane receptors that function in inside - out and outside - in signaling and regulate many cellular processes. the fact that membrane - to - matrix interactions can be easily disrupted by adding synthetic rgd peptides shows that these interactions are important for the ecm integrins are also chosen by pathogens as a target for binding and their role in animal innate immune responses has been emphasized in several reports (isberg and van nhieu, 1994 ; pribila., 2004 ; stewart and nemerow, 2007). integrins, for instance, interact with the rgd - containing penton base proteins of adenoviruses (reviewed in mercer., 2010). following this interaction it was demonstrated, however, that the rgd motif is not necessary for initial virus attachment per se ; adenoviruses with deleted rgd motifs could still attach but were impaired in endosomal internalization and in escape to the host cytoplasm (shayakhmetov., 2005). another example can be found in helicobacter pylori, a bacterium causal to stomach inflammation, that exploits integrins for effector translocation into the host cell (kwok., 2007). this process is dependent on cagl, a rgd - containing protein that is situated on the surface of the pilus and interacts in an rgd - dependent manner with the integrin 51. upon interaction, other potent effectors of integrin function are the disintegrins from snake venoms of various viper species (calvete., 2007 ; disintegrins are usually short cysteine - rich peptides that contain a rgd motif on an exposed hairpin loop. disintegrins selectively bind and activate integrin receptors, and hence compete with their natural rgd - containing ligands, such as fibrinogen and von willebrand factor, subsequently disturbing platelet aggregation and cell ecm adhesion. several rgd - disintegrins such as contortrostatin and jarastatin have been shown to activate the focal adhesion kinase fak that regulates actin dynamics, and to trigger phosphoinositide 3-kinase and mapk pathways upon integrin - binding (coelho., 2001 ; oliva., 2007). it is conceivable that also plant pathogens take advantage of the rgd motif for manipulating their hosts. one example comes from the rgd - containing toxa protein produced by pyrenophora trititci - repentis and stagonospora nodorum, two fungi that cause leaf spot disease on wheat. these pathogens require the host - selective toxin toxa to induce necrosis in host cells (sarma., 2005 ; the toxa gene encodes a pre - pro - protein of which the signal peptide (pre - region) and the n - terminal pro - region are both cleaved by the fungus prior to secretion of the mature 13.2 kda protein. the rgd motif in toxa was found to be required for internalization into wheat mesophyll cells (meinhardt., 2002 ; manning., 2008) and it was proposed that internalization occurs by means of receptor - mediated endocytosis. this is in line with the finding that a number of genes encoding endocytosis - related proteins and lectin receptor kinases were significantly upregulated in wheat treated with toxa (pandelova., 2009). other studies reported that exogenously applied rgd peptides can change the structure and behavior of plant cells. when added, adhesions between the cell wall (cw) and plasma membrane (pm) were easily disrupted, and resulted in reduced levels of phytoalexins and phenolic compounds (schindler., 1989 ; moreover, mellersh and heath (2001) showed that rgd - treated cowpea plants were attenuated in their defense response, which correlated with enhanced fungal penetration efficiency. infestans effector ipi - o we also obtained strong evidence that the rgd motif is a functional determinant. similar to rgd peptides, exogenously added ipi - o has the capacity to disrupt cw plasmolysed arabidopsis cells from a suspension culture showed convex pockets when exposed to recombinant ipi - o protein but concave pockets when exposed to recombinant ipi - o protein with rgd mutated into rge or rga (senchou., 2004 ; figure 3a). transgenic arabidopsis plants with constitutive expression of the ipio gene develop normally but at the microscopic level we again observed concave forms of plasmolysis in hypocotyl cells reminiscent of a disrupted adhesion between the cw and pm, this in contrast to wild - type plants and transgenic plants expressing a mutant form of ipio (rgd > rge ; bouwmeester., 2011 ; figure 3b). intriguingly, the ipio expressing plants responded in a different manner to infection and elicitor treatment. they displayed gain of susceptibility to phytophthora brassicae, and showed less callose deposition, thus suggesting that ipi - o functions as a suppressor of defense (bouwmeester. since also these phenotypes are rgd dependent, we conclude that rgd is essential for ipi - o to function at the host we imagine that plants perceive ipi - o either directly via the rgd motif or via a structural feature imposed on ipi - o by the rgd motif. the phytophthora infestans rxlr effector ipi - o and its putative host cell receptor arabidopsis lecrk - i.9. (a) observed changes in arabidopsis suspension cells upon exposure to ipi - o. the cw pm continuum (shown as green vertical bars) is disturbed (dotted vertical bars) when ipi - o has an intact rgd motif (left panel) but when rgd is mutated into rga or rge the continuum is not disturbed (middle panel ; senchou., model depicting the membrane - associated lecrk - i.9 in interaction with a putative rgd - containing extracellular ligand (right panel). under normal circumstances (wild - type) the cw (b) observed changes in the arabidopsis phenocopy lines 35s - ipio1 and lecrk - i.9 (top left and right panel, respectively). both show gain of susceptibility to phytophthora brassicae (the gray area on the leaf depicts lesion growth) and reduced callose deposition (shown as blue dots at the cell boundaries). arabidopsis lines expressing ipio with a mutated rgd motif (35s - ipio1 or 35s - ipio1) behave as the wild - type col-0 accession (lower left and right panel, respectively) ; no lesion formation upon infection with p. brassicae (the brown dot represents a hypersensitive response) and efficient callose deposition (bouwmeester., 2011). the model predicts that ipi - o competes with the endogenous ligand of lecrk - i.9 leading to less adhesions between cw and pm, and gain of susceptibility. a mutation in the rgd motif abolishes the competition resulting in a wild - type phenotype. in the absence of lecrk - i.9 the proper balance between endogenous ligand and receptor is disturbed and results in gain of susceptibility. the fact that several animal and human pathogens exploit integrin - mediated - uptake for host cell entry or effector delivery raises the question whether plant pathogens use similar strategies. plants however, lack integrins. in the 1980s and 1990s several researchers attempted to identify plant integrins using immunological and biochemical approaches but failed. also the first plant genome sequence did not reveal the occurrence of integrin homologs (the arabidopsis genome initiative, 2000). it is conceivable though that plant pathogens use other types of host receptors for cell entry. in a search for functional homologs of integrins in plants (1998) showed that arabidopsis contains high affinity rgd - binding sites in the pm. consistently, one of these binding sites had a very high affinity for ipi - o. since this affinity was lost when rgd in ipi - o was mutated into rga or rge, the rgd motif seemed to be crucial for binding (senchou., 2004). (2006) performed a phage display screen to identify proteins that potentially interact with ipi - o via the rgd motif. this resulted in two heptamers that could inhibit the rgd - binding activity to the arabidopsis pm and disrupt cw pm membrane adhesions, possibly by competing with naturally occurring rgd - based ligand - receptor contacts. screening the arabidopsis proteome for the occurrence of these two peptide sequences led to the identification of the arabidopsis legume - like lectin receptor kinase lecrk - i.9, which has two amino acid motifs in the n - terminal region (asyy and phpr) that are implicated in binding ipi - o via its rgd motif (gouget., 2006). lecrk - i.9 belongs to a family of 45 members divided over nine clades and a few singletons (bouwmeester and govers, 2009). they are typical transmembrane receptors with an extracellular lectin domain and an intracellular kinase domain. as yet, little is known about the functions of lecrk - i.9 and other legume - like lectin receptor kinases, neither about their natural ligands. they are likely involved in monitoring cw integrity and it is conceivable that lecrk - i.9 interacts with endogenous rgd - containing proteins to balance cw pm adhesions (figure 3a). to elucidate the role of lecrk - i.9 at the phytophthora host interface we analyzed expression during compatible and incompatible interactions with p. brassicae isolates and determined the phenotypes of arabidopsis lecrk - i.9 knock - out lines (lecrk - i.9) in disease assays. these studies convincingly demonstrated that lecrk - i.9 plays a role in phytophthora disease resistance. 2008) and two independent knock - out lines showed gain of susceptibility to p. brassicae and the bacterium pseudomonas syringae. strikingly, the lecrk - i.9 lines behaved as perfect phenocopies of the arabidopsis lines that constitutively express the rxlr effector gene ipio suggesting that an overload of ipi - o disables lecrk - i.9 to properly guard the frontier (figure 3b). p. brassicae does not have an ipio homolog and hence can not effectively disable lecrk - i.9. in contrast, ipio is highly expressed in p. infestans hyphae invading potato leaves (van west., 1998). competition of ipi - o with potential endogenous rgd - containing ligands of lecrk - i.9 may destabilize the cw pm adhesions might facilitate the formation of haustoria, feeding structures that are not only important for nutrient uptake but also for effector translocation to host cells. we hypothesize that phytophthora uses lecrk - i.9 as a gateway to establish infection and that one of the functions of ipi - o is to mediate this early step in the infection process. other anticipated functions of ipi - o are more downstream in the process when ipi - o has reached the cytoplasm. in recent years comparative genomics and high - throughput functional analyses of phytophthora effectors have boosted the discovery of translocation motifs but the molecular machinery that is used by oomycetes to deliver their armory into the host cell is still a black box. the recent identification of host components at the cell surface that bind translocation and adhesion motifs opened new avenues and the search for additional components is on - going. insight into similarities and dissimilarities in transport mechanisms exploited by different groups of pathogens will help to shed light in the black box. the ultimate challenge is to find ways to block the uptake machinery thereby strengthening the frontier at the phytophthora the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.	plants are constantly beset by pathogenic organisms. to successfully infect their hosts, plant pathogens secrete effector proteins, many of which are translocated to the inside of the host cell where they manipulate normal physiological processes and undermine host defense. the way by which effectors cross the frontier to reach the inside of the host cell varies among different classes of pathogens. for oomycete plant pathogens like the potato late blight pathogen phytophthora infestans it has been shown that effector translocation to the host cell cytoplasm is dependent on conserved amino acid motifs that are present in the n - terminal part of effector proteins. one of these motifs, known as the rxlr motif, has a strong resemblance with a host translocation motif found in effectors secreted by plasmodium species. these malaria parasites, that reside inside specialized vacuoles in red blood cells, make use of a specific protein translocation complex to export effectors from the vacuole into the red blood cell. whether or not also oomycete rxlr effectors require a translocation complex to cross the frontier is still under investigation. for one p. infestans rxlr effector named ipi - o we have found a potential host target that could play a role in establishing the first contact between this effector and the host cell. this membrane spanning lectin receptor kinase, lecrk - i.9, interacts with ipi - o via the tripeptide rgd that overlaps with the rxlr motif. in animals, rgd is a well - known cell adhesion motif ; it binds to integrins, which are membrane receptors that regulate many cellular processes and which can be hijacked by pathogens for either effector translocation or pathogen entry into host cells.
fifteen consecutive patients underwent cervical odl by one surgeon (t.e.m.) for the treatment of cervical spondylotic myelopathy. in each patient, a 3-mm carbide bur was used on the side to be opened to transect the laminae at the laminae lateral mass junction (fig. 1). next, the ligamentum flavum along this trough was meticulously ligated, as was the ipsilateral ligamentum flavum between the supra- and subjacent levels to facilitate opening. next, the hinge side was then drilled and each surgical - level lamina was elevated in the typical fashion for an odl. all spinous processes and supraspinous ligaments were preserved within the operative levels and between supra- and subjacent levels in all patients with the exception of the most caudal and rostral aspects of the exposure ipsilateral to the open side (where the ligamentum flavum was transected to facilitate the opening of the laminae). the presence of these structures did not hinder or prevent adequate opening of the laminae as compared with expansion without preservation of the posterior elements. the extensor muscles are elevated (but not removed) and then allowed to fall back after the exposure. in all patients but one, the laminae on the open side were reconstructed using miniplates with either an 8-mm or a 10-mm cortical allograft strut bone graft affixed to the plate. the laminar portion of the plate was secured with two 5-mm screws, and the lateral mass portion of the plate was secured with one 7-mm (cranial) and one 5-mm (caudal) screw. although the patients ' spines were placed in a neutral to slightly flexed alignment to facilitate the procedure, great care was taken to avoid placing the plate too far rostrally on the lateral mass to ensure there would be no abutment with the rostral lateral mass during subaxial extension. once to the ward where soft collars are available, they are transitioned to a soft collar. on postoperative day 1, the surgical team (i.e., residents, fellows, attendings, or physician assistants) instructed the patient on active lateral bending, rotation, flexion, and extension. the patients were instructed to perform these exercises at least three times a day during the hospital stay and a minimum of ten times per day upon discharge. the soft cervical collar was discontinued as soon as possible, typically by postoperative day 3. (a) posterior exposure of the spine showing the ligamentum nuchae (1) and the burr path on the open side (2). (b) an oblique view of the spine with an idealized exposure of the cord demonstrating the interspinous muscles, which remain intact where possible (3), and the terminal end of the construct with an intact ligamentum nuchae (4). postoperative radiographs were taken at 1.5, 3, 6, and 12 months with an average terminal follow - up of 41.2 months and included neutral, flexion, and extension positions. postoperative measurements were based on the final follow - up radiograph at a mean of 12.9 months, and preoperative measurements were based on radiographs taken closest to the date of surgery., t.y.) utilized the posterior tangent method to estimate preoperative and postoperative angles in the neutral, flexion, and extension positions electronically using sienet magicview 1000 ve 42 (siemens medical systems, erlangen, germany). the posterior tangent method involves the measurement of the angle created by the vertex of two lines : the posterior body of c2 and the posterior body of c7 (fig. this method was originally described by gore and has since been reported to provide good reliability and a lower standard error of measurement than that of the cobb angle.6 after assessment, intraclass correlation coefficients (icc) were calculated to determine agreement between raters.7 icc for all measurements ranged from 0.85 to 0.93, indicating that there was excellent correlation between observers. computerized tomography (ct) scans with sagittal reconstructions and 2-mm axial ct images of each surgical level were obtained at a mean of 12.9 months (range : 9 to 26) in all patients to assess the construct integrity, hinge healing, and bone graft consolidation on the open side. on the open side, grafts were deemed consolidated if there was (1) bridging bone on both sides of the graft to the lamina and lateral mass, and (2) absence of instrumentation loosening. a hinge was deemed to be healed if bridging bone was evident on axial images. all statistics were calculated using jmp 8.0 (2008, sas institute, carey, nc). descriptive statistics including mean, standard deviation, standard error, and confidence intervals were computed for all measurements. p values were calculated using student t test and were considered nominally significant at 0.05). the preoperative angle of the patient in full extension was 20.5 7.3 degrees (ci : 16.4 to 24.6) and at postoperative analysis was 17.2 11 degrees (ci : 11.2 to 23.3). the preoperative angle of the patient in full flexion was 19.2 8.0 degrees (ci : 14.7 to 23.6), and analysis postoperatively was 16.4 10.4 degrees (ci : 10.7 to 22.2), p > 0.05. the arc rom (measure of extension subtracted by flexion) was 39.6 7.5 degrees (ci : 33.3 to 43.8) preoperatively and 33.7 12.3 degrees (ci : 26.9 to 40.5) postoperatively (p = 0.004). the difference between the preoperative and postoperative arc rom was 5.96 11.9 degrees (ci : 0.62 to 12.5). the average percentage loss of motion was 3.5% (0.1 to 6.9%). overall, four patients had an increased rom postoperatively, and 11 patients had a decreased rom as determined by changes in arc rom. this 52-year - old man presented with a several - year history of progressive bilateral arm pain and numbness, along with gait and fine motor skills difficulties over the past year. (a c) plain films demonstrating preoperative neutral, flexion, and extension views, and (d f) postoperative comparison films. additionally, there were no significant correlations between any preoperative values of the neutral, flexion, or extension positions and rom outcomes. based upon the review of all radiographs and ct scans, there were no cases of construct failure. specifically, there was no lucency or migration of the screws or plate fracture or migration, and all laminae were maintained in the open position at terminal radiographic follow - up (average 14.8 months). the strut allograft consolidation rate was 54.5%, and the hinge healing rate was 87.2%. it is typically associated with loss of rom, and certain studies have documented loss of sagittal alignment.1 2 3 8 9 10 the precise reason for the loss of motion remains elusive ; however, there are several potential causes including postoperative axial pain, facet joint arthrosis / facet joint autofusion, and paraspinal muscle dysfunction. similarly, it is not entirely clear why many patients lose sagittal alignment to varying degrees postoperatively. most published studies on odl suggest that there is disruption of the posterior tension band (i.e., removal of intervening supraspinous and interspinous ligaments, spinous processes, etc.).1 2 3 8 9 10 it is most logical to presume that removal or disruption of the posterior tension band during laminoplasty underlies the kyphogenic potential of cervical laminoplasty. therefore, the hypothesis of this study was that preservation of all posterior structures would positively influence postoperative alignment (i.e., preservation) and possibly rom (i.e., smaller loss). odl with preservation of all posterior structures provided greater mean preservation of motion than has been previously described. additionally, this is the first report showing that an increased mean rom is also possible following odl, as was seen in 4 of the 15 patients in the study. the average 3.5% loss of motion (5.96 degrees) that was identified in the present study is less than previously reported estimates, which include a study from 2009 that reported a loss ranging between 17% and 80%,11 and a study by kang in 200712 that reported a mean loss of 9.54 degrees of motion (table 2). several studies have shown that preserving the ligamentous structures in the cervical spine can potentially reduce postoperative pain levels.13 14 15 16 it follows from these studies that preserving posterior structures may possibly assist in the maintenance, and potentially even a gain, of physiological rom. recently, sakaura reported that preservation of muscles attached to c2 and c7 spinous processes was more important than preserving the subaxial deep extensors in reducing unfavorable outcomes after laminoplasty. particularly with regard to rom, they showed no statistical difference between those patients with deep muscle preservation versus those undergoing traditional odl (26% versus 24.3%, respectively). the authors concluded that the temporally demanding preservations of deep structures were unnecessary when preserving superficial attachments from c2 to c7. the study by sakaura is not necessarily in conflict with the present study data, as the presented technique preserves both deep extensors and more superficial muscle groups.17 the preservation of the extensor muscles is accomplished by elevating, instead of ligating, them during the procedure. the interspinalis muscles are likely denervated during this procedure, yet the clinical implication of this is not known. it is difficult to tease out the necessity of the deep extensors, but when provided with the preservation of rom found in the present study, it can be argued that some combination of both approaches may facilitate better outcomes. in the present study, the loss of rom in four - level laminoplasty (0.53 degrees) was significantly lower than that of five - level laminoplasty (13.4 degrees). when taking into account that many previously reported rom values are done on the analysis of surgeries involving four levels or less, these results are particularly salient. the value we obtained for the five - level laminoplasty matches that of at least one other study by kang in 2007.12 the nearly 13-degree difference in loss of rom between these two procedures may be due to the fact that the five - level laminoplasty often involves c7, which is thought to play an important role maintaining cervical spine stability and preserving muscle / ligament attachments. as there is limited research on this area, further studies to examine the use of this technique in four- and five - level laminoplasty will be necessary. although lateral radiographs have comprised the basis of these results, we have utilized ct and magnetic resonance imaging in a subset of patients to determine the results of laminoplasty in regard to distribution and settling of posterior tissues. it is important to note that a study by tani in 200218 reported a loss of rom of only 7.26 degrees among patients undergoing laminoplasty for cervical myelopathy. first, only 3 of their 30 patients underwent five - level laminoplasty, and the majority underwent three- and four - level laminoplasty.19 none of their patients underwent laminoplasty to c7. ceramic spacers were only placed at two levels and then fixed. in the present study, allografts were applied to all levels and then fixed by miniplates. the first is the value of loss of rom between the preoperative and postoperative period. however, it is necessary to include the four patients who gained motion in the analysis. the authors are also aware of the inherent difficulty in interpreting pre- and postoperative images in the flexion and extension positions. these images may be affected by patient compliance with the radiographic technician and the presence of neurological symptoms with neck motion and neck pain. all patients in this study transitioned to a soft collar and started on active flexion, extension, and rotation exercises on postoperative day 1. not all surgeons utilize this postoperative regimen, and currently there is limited literature of the effects of perioperative motion on the eventual rom following cervical laminoplasty. although this rehabilitative aspect is not the main focus of this study, it may have a substantial bearing on the patients ' eventual outcomes. the lack of literature on this subject leads to a surgeon - dependant movement regimen postoperatively, and it is difficult to tease out the benefits of motion preservation techniques that are emphasized in the operating room versus those conducted on the surgical ward and in the perioperative period. the limitations of this study are well recognized. it is a retrospective review of a small number of male patients, with no control group, treated by one surgeon. the results, however, indicate that this variation of odl may have merit, especially in the context of the preservation of motion reported when compared with previous studies. a prospective study is indicated to further assess the clinical and radiographic outcomes of this procedure. kalil g. abdullah, none takayuki yamashita, none michael p. steinmetz, consulting : biomet spine daniel lubelski, none jeffrey c. wang, royalties : medtronics, stryker, seaspine, osprey, aesculap, biomet, amedica, zimmer, synthes ; stock ownership : fziomed ; private investments : promethean spine, paradigm spine, benevenue, nexgen, k2 medical, pioneer, amedica, vertiflex, electrocore, surgitech, axiomed ; board of directors : north american spine society, cervical spine research society, ao spine / ao foundation ; scientific advisory board : vg innovations, corespine, expanding orthopaedics, syndicom, osprey, amedica, bone biologics, curative biosciences, pearldiver, inc., pioneer, seaspine edward c. benzel, none thomas e. mroz, stock option : pearldiver, inc. ; consultant : globus medical ; speaking honorarium : ao spine : fellowship support : oref, nref, ao spine kalil g. abdullah, none takayuki yamashita, none michael p. steinmetz, consulting : biomet spine daniel lubelski, none jeffrey c. wang, royalties : medtronics, stryker, seaspine, osprey, aesculap, biomet, amedica, zimmer, synthes ; stock ownership : fziomed ; private investments : promethean spine, paradigm spine, benevenue, nexgen, k2 medical, pioneer, amedica, vertiflex, electrocore, surgitech, axiomed ; board of directors : north american spine society, cervical spine research society, ao spine / ao foundation ; scientific advisory board : vg innovations, corespine, expanding orthopaedics, syndicom, osprey, amedica, bone biologics, curative biosciences, pearldiver, inc., pioneer, seaspine edward c. benzel, none thomas e. mroz, stock option : pearldiver, inc. ; consultant : globus medical ; speaking honorarium : ao spine : fellowship support : oref, nref, ao spine	objective to demonstrate that preservation of all posterior structures during open - door laminoplasty (odl) is associated with a significant preservation of motion. methods fifteen patients underwent cervical odl by one surgeon for treatment of cervical spondylotic myelopathy. an open - door technique was employed, and the laminae on the open side were reconstructed using miniplates with allograft strut bone graft. all spinous processes and interspinous and supraspinous ligaments were preserved within the operative levels and between supra- and subjacent levels in all patients. postoperative radiographs were obtained 1.5, 3, 6, and 12 months. computed tomography scans were obtained at 12 months. results there were no significant intraoperative or perioperative complications. postoperatively, the neutral angle was 6.8 11.5 degrees (95% confidence interval : 0.5 to 13.1), representing a loss of lordosis of 3 degrees (not significant). the difference between the preoperative and postoperative arc range of motion was 5.96 11.9 degrees (confidence interval : 0.62 to 12.5). the average percent loss of motion was 3.5% (0.1 to 6.9%). four patients had an increased range of motion postoperatively. conclusion open - door laminoplasty with preservation of all posterior structures provides greater preservation of motion than has been previously described.
extraskeletal ewing 's sarcoma (es) is a small round cell tumor that occurs in extraskelectal tissue. this has the same genetic origin as es, which shows up as an (11;22) (q24;q12) translocation (ewing 's sarcoma gene / friend leukemia intergration 1 transcription factor gene [ews / fli-1 ] fusion ; which fuses the es gene of chromosome 22 to friend leukemia intergration 1 transcription factor gene of chromosome 11), so that it is generally categorized as an ewing 's family tumor together with es.(1,2) every part of the human body could be affected such as the small bowel, esophagus, vagina, pancreas or kidney. three cases of gastric es / peripheral neuroectodermal tumor have been previously reported.(3 - 5) this is the fourth reported case of gastric es. a 35-year - old female who had no special symptoms or past history of cancer was referred for surgical intervention of a protruding mass in her gastric antrum which was incidentally found by esophagogastroduodenscopy, while she was at a routine medical checkup. endoscopic examination showed a protruding mass of about 2.5 cm size with a central depression and an erythematous change in the anterior wall of the proximal antrum (fig. a laparoscopic gastric wedge resection was done since there was no lymph node enlargement or invasion to other organs. the pathologic gross finding showed a 5.5 cm sized granular tissue with a grape - like appearance (fig. strong membranous cd 99 was noted by immunohistologic study, which is essential for diagnosis of es. synaptophysin was also strong by immunohistologic study but chromogranin a, cytokeratin, and desmin were negative (fig. 3). fluorescent in situ hybridization for detecting the t(11;22) ewsr1 gene was negative. the patient did not receive any adjuvant chemotherapy or radiotherapy and she remains symptom free and without recurrence for eleven months. es is a neoplasm of undifferentiated small round cells, which generally affects the bone and deep soft tissue of children and adolescents. the most commonly affected site is skeletal tissue but it has been reported that it could be present in extraskeletal tissues such as small the bowel, esophagus, vagina, pancreas or kidney. this case of es occurs in the stomach, which is very rare. to date, the number of reported cases of es in the stomach in the literature remains small with fewer than three being described, to the authors ' knowledge. we herein report the fourth known case of gastric es.(6 - 8) es histologically is a small round cell tumor and it is cd99 positive by immunohistochemistry. it has a genetic mutation t(11;22)(q24;q12) translocation (ews / fli-1 fusion) that can be seen by fluorescence in situ hybridization (fish) or reverse transcription polymerase chain reaction (rt - pcr). but there is a small chance to be negative for the t(11;22)(q2;q12) translocation by fish or rt - pcr because both of them have chance to be negative in es, about 3% of time in fish and 19% in rt - pcr.(9,10) the mainstay of es 's treatment is surgery and chemotherapy.(2) chemotherapy has become the mainstay of es treatment due to the high recurrence rate and difficulty in obtaining a resection negative margin when it has occurred in skeletal tissue and deep soft tissue. but based on its toxicity, compliance and effectiveness in the patient, it could be excluded if it is sure that there is no metastasis to other sites and a negative resection margin is possible. most of the cases mentioned chemotherapy in addition to surgery and had better prognosis than surgery only table 1. in this case first, the impression of this case was gastrointestinal stromal tumor of stomach, which was diagnosed by endoscopic ultrasonogram before the pathological diagnosis was made. third, we could perform just a wedge resection to obtain free margin because it was well demarcated. finally, the patient wanted to have a child. to further refine our search we selected similar cases that involved the gastrointestinal tract and compared these with each other table 1. the total number of cases was seven, which we classified into 3 different groups and are listed in table 1. group a was defined as those whom had chemotherapy with mention of metastasis and vascular invasion to the adjacent bowel (no 1, 2, 3).(3,4,6) group b was those who had chemotherapy without mention of metastasis (no 4, 5).(7,8) group c, the third and last group, was those who had no chemotherapy and no metastasis (no 6, 7).(5) in group a, despite the fact that they underwent chemotherapy additionally, there was only one survivor among three patients. b, there was no distant metastasis and invasion but they underwent an additional chemotherapy like group a. unlike group a however, there were no deaths during the follow - up period. in group c, patients had no metastasis and invasion, nor did the undergo chemotherapy. although 4 cases were in groups b and c, it suggested that there was no death after proper surgical management, regardless of an additional chemotherapy. therefore it is necessary to consider the efficacy of the chemotherapy in groups b and c (no distant metastasis nor invasion) whether it is essential or not. 7 did not receive any adjuvant chemotherapy or radiotherapy and remains symptom free and without recurrence for eleven months. it is required to review prospective data of these patient groups (b and c) to speak about prognosis of each group, regretfully there is n't more data regarding these groups. in this case, our patient has regular medical check ups and computed tomography or esophago - gastro - duodenoscopy is done in every visit to evaluate for recurrence. if a recurrence in this patient is found during regular medical check up, additional chemotherapy will be mandatory for treatment.	ewing 's sarcoma is a neoplasm of the undifferenciated small round cells, which generally affects the bone and deep soft tissues of children and adolescents. we present a case of gastric ewing 's sarcoma ; a 35-year - old female who had no symptoms. while she was at a routine medical checkup, a protruding mass in her gastric antrum was incidentally found on esophagogastroduodenoscopy. endoscopic ultrasonogram showed a submucosal mass on the same lesion and a laparosopic wedge resection was done. pathologic gross findings showed a granular grape appearance tissue and histoloigc examination revealed a small round cell tumor with cd 99 immunoexpression positive. in general, a combined modality therapy for ewing 's sarcoma such as surgical resection with chemotherapy, is accepted as an effective method. however, this patient had no adjuvant chemotherapy after surgery and she has no recurrence for eleven months.
hyperhomocysteinemia and its association with vaso - occlusive disease and thrombosis are well - established and may be an independent risk factor for cardiovascular disease and venous thrombosis. hyperhomocysteinemia is due to the disrupted metabolism of homocysteine a sulphur containing aminoacid ; leading to the accumulation of homocysteine with its varied symptomatology. in cushing 's syndrome endogenous glucocorticoid excess is associated with a prothrombotic state that put patients at increased risk of vascular thrombosis that is responsible for the increased mortality of these patients. the hypercoaguable state produced by homocysteinemia and cushing 's syndrome and subsequent anticoagulation therapy, titration of the glycaemic levels and theoretical risk of using nitrous oxide create a unique anaesthetic challenge that must be carefully addressed. medline search showed paucity of literature in the management of perioperative hypercoagulable state in the presence of these dual risk factors. hence, we present a case of homocysteinemia with cushing 's syndrome and describe the anaesthetic implications in the perioperative period. permissions were obtained from the patient and institutional ethics committee to report this case. a 25-year - old male with features of cushing 's syndrome he had a history of superior sagittal sinus and left transverse sinus thrombosis 2 years back for which he was evaluated and found to have hyperhomocystenemia. he received anticoagulation therapy (tablet acenocoumarol 4 mg / day) and folic acid, vitamin b6 and b12 for 7 months, and the anticoagulation therapy was discontinued. two years later he presented with pulmonary thromboembolism and deep vein thrombosis of left lower limb. he was immediately thrombolysed with injection streptokinase and inferior vena caval filter was placed and anticoagulation therapy was instituted with low molecular weight heparin (lmwh) and later with oral anticoagulants (tablet acenocoumarol 4 mg od). echocardiogram showed right atrial and ventricular dilatation, ejection fraction of 60%, trivial tricuspid regurgitation, moderate pulmonary regurgitation and pulmonary arterial hypertension with normal biventricular function. patient was evaluated for cushing 's syndrome in view of physical appearance and presence of thrombotic tendency despite controlled homocysteine levels. general examination revealed puffy moon face with facial plethora, red purple striae over thighs with body mass index of 26 kg / m and american society of anaesthesiologists physical status of grade i. his airway examination revealed short thick neck, and mallampati grade iii suggesting difficult airway. laboratory investigations oral anticoagulant was stopped 5 days prior to surgery and was started on injection heparin 5000 iu 6 hourly. his premedication consisted of tablet pantoprazole 40 mg, and alprazolam 0.5 mg and rest of the medications were continued. morning dose of heparin was not administered. on the day of surgery injection hydrocortisone 100 the patient was pre - medicated with injection glycopyrrolate 0.2 mg and fentanyl 120 g iv. anaesthesia was induced with thiopentone 300 mg and after confirming mask ventilation injection atracurium was administered, and the airway was secured with 8 mm oral endotracheal tube. on laryngoscopy the cormack lehane grading was iiib and intubation was performed using mccoy laryngoscope and gum elastic bougie. anaesthesia was maintained with oxygen, air, isoflurane and titrated doses of atracurium ; nitrous oxide was avoided. epidural analgesia was activated with 0.25% bupivacaine and fentanyl in a ratio of 1:1 (1 ml containing 0.25 mg bupivacaine and 1 g fentanyl) infusion and continued intra - operatively. the intra - operative course was uneventful with minimal blood loss and lasted for 4 h. iv fluids administered were normal saline and ringer 's lactate, one litre each. recovery was unremarkable, and trachea was extubated after reversing the residual neuromuscular blockade and after meeting the criteria for extubation. post - operatively, analgesia (epidural), fluid, glucose electrolyte and acid - base imbalance were managed meticulously. hydrocortisone 100 mg iv was administered on day 0 and day 1 and after that 50 mg 8 hourly for 2 days. lmwh was started 6 h after surgery and epidural catheter was removed on 3 post - operative day (pod) 2 h before the scheduled dose of heparin and heparin was stopped on 6 pod. patient was mobilised from 3 pod and was discharged on 10 pod with an advice to continue his routine medication. the patient 's hyperhomocysteinemic state was compounded with cushing 's syndrome with its complex derangement in haemostatic parameters. together they confer an increased risk of coronary heart disease and cerebrovascular accidents, which are important determinants of the increased mortality. the normal role of homocysteine in the body is to control growth and support bone and tissue formation. plasma levels of 1530 mol / l are considered indicative of mild to moderate hyperhomocysteinemia and in severe hyperhomocysteinemia, levels exceed 100 mol / l and are because of genetic mutation. high levels of plasma homocysteine are usually associated with vascular injury by mechanisms of oxidative damage, vascular smooth muscle proliferation, promotion of platelet activation and aggregation. it also disrupts the normal procoagulant anticoagulant balance favouring thrombosis, stimulate growth of arteriosclerotic plaque, which leads to heart disease. homocysteine plasma levels above 10 mol / l are associated with a doubling of vascular risk. initiation of therapy with b12, folic acid, and b6 tends to normalize homocysteine levels in 48 weeks. although vitamin supplementation is successful in decreasing total plasma homocysteine levels, anticoagulation therapy is required to prevent vascular occlusive pathology. in the present case, the patient had anticoagulation therapy for 7 months and later was put on supplemental therapy. this patient initially had a history of superior sagittal sinus thrombosis and found to have hyperhomocysteinemia of 59.7 mol / l and 2 years later despite normal range of homocysteine levels had pulmonary thromboembolism and deep venous thrombosis. at this time, cortisol levels were very high with features of cushing 's syndrome. intra - operative measures like monitoring central venous pressure for maintenance of euvolemia, elastocrepe bandage application were instituted. post - operatively, lmwh and oral anticoagulants were started 6 and 24 h after surgery respectively and the patient was mobilized early to prevent thromboembolism., the patients will suffer from hypoglycaemia, the mechanism being increased methionine leading to increased insulin release resulting in hypoglycaemia. insulin resistance is also described with homocysteinemia. however in cushing 's syndrome, the patients will be effected by hyperglycaemia. in the present case, blood glucose and electrolytes were carefully monitored perioperatively, and the glucose levels were well within the normal range despite cushing 's syndrome and steroid therapy. patients with cushing 's syndrome are also the candidates for potential airway difficulties because of obesity. literature shows that the use of n2o - based anaesthesia increases plasma homocysteine and significantly impairs endothelial function and is a risk factor for post - operative cardiovascular morbidity in patients undergoing non - cardiac surgery. duration of anaesthesia is also important because prolonged exposure to n2o causes a significant elevation of homocysteine levels than short term exposure. this case report discusses several important anaesthetic issues relevant to the management of patients with hyperhomocysteinemia and cushing 's syndrome including meticulous titration of anticoagulation therapy, glycaemic control, airway management and the potential risk of using nitrous oxide. understanding the anaesthetic implications and the pathophysiological interactions of multiple metabolic disorders with a potential for multisystem involvement is a key to the successful management of these patients as seen in the present case with hyperhomocysteinemia and cushing 's syndrome.	maintenance of homeostasis during anaesthesia in the patient with two major metabolic disorders whose systemic effects either compliment or contradict each other is a challenge to the anaesthesiologist. a 25-year - old male patient with cushing 's syndrome and known hyperhomocysteinemia was scheduled for open adrenalectomy. both these disorders compound the hypercoagulable state and differ in glucose metabolism. in addition, obesity, difficult airway, electrolyte and metabolic derangements that accompany cushing 's syndrome warrant special attention. he was on anticoagulant therapy and inferior vena cava filter following an episode of pulmonary thromboembolism with deep vein thrombosis. perioperative hydrocortisone was administered. thoracic epidural catheter was placed at t10t11 interspace, standard general anaesthesia was administered without nitrous oxide. patient was extubated following an uneventful procedure and discharged home on 10th post - operative day. understanding the anaesthetic implications and the pathophysiological interactions of multiple metabolic disorders with a potential for multisystem involvement is key to the successful management of these patients.
egypt has long been considered as a very attractive country to foreign students for several reasons, e.g., the diversity of educational institutions, educational systems and curricula ; a very pleasant environment ; and its great historical background. zagazig faculty of medicine is an egyptian college that offers medical degree programs in english. about 5% of the student population is malaysian students (cultural affairs and student mission section - ministry of higher education, 2010) (1). a steady flow of foreign students have chosen to study medicine in egypt over the past few decades (2). the traditional undergraduate medical program is composed of two phases, i.e., the pre - clinical phase and the clinical phase. effective communication involves the patients, clinical staff (tutors / mentors), and other paramedical staff (3). in the clinical training at zagazig faculty of medicine, the arabic language is used to communicate with the patients / nurses, while english is used in theoretical teaching. this creates a significant challenge for malaysian students who have inadequate proficiency in the arabic language. in prior years, researchers have conducted studies related to the experiences of foreign students in clinical practice (36), and they observed that the students had both positive and negative experiences (3). the studies indicated that one of the major problems the students encountered was their poor proficiency in the language of the country in which they chose to study and obtain their clinical practice (7, 8). these researchers noted that the clinical experiences of non - native speakers created significant challenges for them, including finding suitable methods for coping with the difficulties and getting to the point that they felt accepted by their peers. the literature indicates that communication skills are vitally important for doctors in establishing effective relationships with their patients. the physicians must be able to provide information effectively to patients and their families so they can understand both the severity of their medical problems and the treatments that will be required (12). there is a significant amount of information in the literature concerning foreign students experiences during clinical studies, but few such studies have been conducted in egypt concerning the experiences of foreign medical students during their clinical placements. thus, the aim of this research was to investigate the experiences of malaysian students in the clinical environment in zagazig faculty of medicine (egypt), identify their problems and issues, and identify approaches for improving the situations they face. to those ends, answers were sought for the following questions : do malaysian students encounter communication difficulties during their clinical placement ? what are the best ways of minimizing the difficulties for these students during their clinical placement ? we systematically collected and analyzed in - depth information, attitudes, behaviors, and opinions of malaysian students in their clinical years. we reviewed the literature concerning the experiences of international students, and we used the information and data acquired to develop the questionnaire that was used in the study. the questionnaire addressed the following areas : 1) communication between mentors, the nursing staff, and the students ; 2) communication between the students and their patients ; 3) the attitudes toward foreign students in the clinical environment and the level of acceptance they experienced ; 4) clinical guidance by supervisors ; and 5) educational and practical activities in the clinic. the participants in the study represented a random sample of malaysian medical students in faculty of medicine - zagazig university. the students in the sample consisted of fourth-, fifth-, and sixth - year malaysian students enrolled in the bachelor s degree program. the selection of students who had undergone some clinical placements and constraining factors was purposeful, since the first - year students had just made the transition into the university and had no clinical practice. the undergraduate curriculum in this traditional medical school is divided in two phases, i.e., the pre - clinical curriculum and the clinical phase. a questionnaire with open - ended questions the study was explained to each of the participants before they were given the questionnaire in an effort to enhance the return rate. the questionnaire had two parts, i.e., part i, which gathered the students background information, and part ii in which data were acquired concerning the students experiences during their clinical practices. quantitative descriptive statistics were used to analyze part 1 of the questionnaire to describe and synthesize averages and percentages. qualitative content analysis was used to analyze the data collected in part 2 (13). the researchers read the students answers to the open - ended questions thoroughly in order to understand as completely as possible what the participants had written. interpretative data analysis was performed, and the themes that emerged were communication, clinical guidance and feedback, and clinical teaching environment, ethical approval was obtained from the faculty of medicine (zagazig university). the descriptions of the study s purpose and data processing were communicated to them, and they were told that they were not obligated to participate. protecting the identity and confidentiality of the participants was maintained in that the questionnaires were not numbered and they did not include the names of the participants. we systematically collected and analyzed in - depth information, attitudes, behaviors, and opinions of malaysian students in their clinical years. we reviewed the literature concerning the experiences of international students, and we used the information and data acquired to develop the questionnaire that was used in the study. the questionnaire addressed the following areas : 1) communication between mentors, the nursing staff, and the students ; 2) communication between the students and their patients ; 3) the attitudes toward foreign students in the clinical environment and the level of acceptance they experienced ; 4) clinical guidance by supervisors ; and 5) educational and practical activities in the clinic. the participants in the study represented a random sample of malaysian medical students in faculty of medicine - zagazig university. the students in the sample consisted of fourth-, fifth-, and sixth - year malaysian students enrolled in the bachelor s degree program. the selection of students who had undergone some clinical placements and constraining factors was purposeful, since the first - year students had just made the transition into the university and had no clinical practice. the undergraduate curriculum in this traditional medical school is divided in two phases, i.e., the pre - clinical curriculum and the clinical phase. the study was explained to each of the participants before they were given the questionnaire in an effort to enhance the return rate. the questionnaire had two parts, i.e., part i, which gathered the students background information, and part ii in which data were acquired concerning the students experiences during their clinical practices. quantitative descriptive statistics were used to analyze part 1 of the questionnaire to describe and synthesize averages and percentages. qualitative content analysis was used to analyze the data collected in part 2 (13). the researchers read the students answers to the open - ended questions thoroughly in order to understand as completely as possible what the participants had written. interpretative data analysis was performed, and the themes that emerged were communication, clinical guidance and feedback, and clinical teaching environment, the descriptions of the study s purpose and data processing were communicated to them, and they were told that they were not obligated to participate. protecting the identity and confidentiality of the participants was maintained in that the questionnaires were not numbered and they did not include the names of the participants. the age range of the participants was 2125, with 36 males and 40 females responding. among the participants, 69.7% had undergone one clinical practices period (4th year students), 17.1% had undergone two clinical periods (5th year students), and 13.2% had undergone three clinical periods (6th year students). the data from the questionnaire were analyzed and classified into the following themes and categories : communication and its barriers, clinical guidance and feedback, and clinical teaching environment. malay was the mother tongue of all participants, but 50% of the participants spoke fluent english. the other 50% of the respondents stated that they had an average to satisfactory level of english language skills. communication barriers due to their limited language skills were experienced by the students who were not proficient in either english or arabic. the students mentioned difficulties in communicating with patients / nurses / staff members (mentors). communication barriers were experienced by the majority of the students (56.6%, 43 students) because of their level of understanding of the arabic language. the participants indicated that talking with patients was more difficult than talking with mentors because most of the patients were from villages and were illiterate. some of the participants had difficulty understanding the medical terms or finding the right words to use in the clinical situation. some of the participants found the egyptian accent of english to be different from their own accent. one student said, the way our mentor speaks english took a bit of time for me to become easily understood. the majority of the students (85.5%, 65 students) indicated that their communication with their mentors was very good, and, if they misunderstood information in arabic, it was either translated into english or another method was used to explain. one student stated that explanation and repetition from mentor made communication with patients easy through translation. their mentors encouragement and support gave them the opportunity to improve their skills during the clinical practice. other positive experiences as stated by students included a sense of appreciation, acceptance, and becoming a member of team. clinical environment was always stressful for most of the students (89.5%, 68 students) with no time to try to speak slowly. i did nt have any chance to talk to the patient by myself, all the patient condition was explained by the doctors. another student said that i ca nt take patient history by myself, i ca nt speak arabic and the patient ca nt speak english, the patient was non cooperative. some students experienced loss of important information during talking patient s history which affected their learning experience. they could not take data from the patients or sometimes misunderstood what was said by their teaching staff. if i needed something, i could not get it for the patient even after thorough explanation by our staff, some issues remain vague. another student said that even reviewing the patient history could not be helpful to get the missed information ; history taking is conducted in arabic. students reaction of the clinical experience was influencing by ways of dealing with situation during their placement. the inability to effectively communicate can lead feeling of unease, discomfort, feeling of rejection, frustration and isolation was experienced by some participants due to their limited language skill which made the student feel they were being avoided. during practice, i need to be effective, i found my collogues who are good in english more effective with our staff and they rather isolate me. the students provided some recommendations for overcoming these communication barriers : using standardized patients was mentioned by 72.15% (57 students). students said that patients should be trained before clinical class, and another student stated that a dictionary of medical terms in arabic and english was mentioned by 49.37% (39 students). a student said arabic important medical terms translated in english, and other students said we need medical dictionary contain terms used patients history. an arabic orientation course before clinical placements was mentioned by 26.58% (21 students), and some students stated specify lecture to explain terms in arabic before commencing each clinical placement. statistical comparison between these improvement suggestions by chi square test revealed a very high significant difference (x=32.25, p<0.001). the age range of the participants was 2125, with 36 males and 40 females responding. among the participants, 69.7% had undergone one clinical practices period (4th year students), 17.1% had undergone two clinical periods (5th year students), and 13.2% had undergone three clinical periods (6th year students). the data from the questionnaire were analyzed and classified into the following themes and categories : communication and its barriers, clinical guidance and feedback, and clinical teaching environment. malay was the mother tongue of all participants, but 50% of the participants spoke fluent english. the other 50% of the respondents stated that they had an average to satisfactory level of english language skills. communication barriers due to their limited language skills were experienced by the students who were not proficient in either english or arabic. the students mentioned difficulties in communicating with patients / nurses / staff members (mentors). communication barriers were experienced by the majority of the students (56.6%, 43 students) because of their level of understanding of the arabic language. the participants indicated that talking with patients was more difficult than talking with mentors because most of the patients were from villages and were illiterate. some of the participants had difficulty understanding the medical terms or finding the right words to use in the clinical situation. some of the participants found the egyptian accent of english to be different from their own accent. one student said, the way our mentor speaks english took a bit of time for me to become easily understood. the majority of the students (85.5%, 65 students) indicated that their communication with their mentors was very good, and, if they misunderstood information in arabic, it was either translated into english or another method was used to explain. one student stated that explanation and repetition from mentor made communication with patients easy through translation. their mentors encouragement and support gave them the opportunity to improve their skills during the clinical practice. other positive experiences as stated by students included a sense of appreciation, acceptance, and becoming a member of team. clinical environment was always stressful for most of the students (89.5%, 68 students) with no time to try to speak slowly. i did nt have any chance to talk to the patient by myself, all the patient condition was explained by the doctors. another student said that i ca nt take patient history by myself, i ca nt speak arabic and the patient ca nt speak english, the patient was non cooperative. some students experienced loss of important information during talking patient s history which affected their learning experience. they could not take data from the patients or sometimes misunderstood what was said by their teaching staff. if i needed something, i could not get it for the patient even after thorough explanation by our staff, some issues remain vague. another student said that even reviewing the patient history could not be helpful to get the missed information ; history taking is conducted in arabic. students reaction of the clinical experience was influencing by ways of dealing with situation during their placement. the inability to effectively communicate can lead feeling of unease, discomfort, feeling of rejection, frustration and isolation was experienced by some participants due to their limited language skill which made the student feel they were being avoided. during practice, i need to be effective, i found my collogues who are good in english more effective with our staff and they rather isolate me. the students provided some recommendations for overcoming these communication barriers : using standardized patients was mentioned by 72.15% (57 students). students said that patients should be trained before clinical class, and another student stated that patients better to know medical terms in english. a dictionary of medical terms in arabic and english was mentioned by 49.37% (39 students). a student said arabic important medical terms translated in english, and other students said we need medical dictionary contain terms used patients history. an arabic orientation course before clinical placements was mentioned by 26.58% (21 students), and some students stated specify lecture to explain terms in arabic before commencing each clinical placement. statistical comparison between these improvement suggestions by chi square test revealed a very high significant difference (x=32.25, p<0.001). the body of research on the nature and extent of problems faced by foreign students in egypt is still insufficient. research efforts were either from the perspective of teaching staff members or on behalf of policy making bodies (cultural affairs and student mission section- ministry of higher education, 2010) (1). foreign students face many problems, such as cultural differences, differences in teaching and learning styles, and communication barriers (14). teaching in the clinical environment is defined as teaching and learning focused on patients health problems. the clinical environment is one in which students learn skills, such as taking histories, conducting physical examinations, communicating with patients, and professionalism, i.e., the things that a real doctor must be able to do (15). for effective patient care, students should integrate each patient s biological, psychosocial, and cultural background. to reach this, a physician must use communication skills, biomedical knowledge, and clinical judgment to perform clinical reasoning (16). most of the malaysian students experienced a communication barrier because of their limited language skills in arabic, which is different from their mother tongue. moreover, the clinical environment was always stressful with no time to try to speak slowly. speaking in a language different from the mother tongue is often cited by foreign students as the most anxiety - producing experience ; they experienced feelings of fear and frustration as well as difficulties in interacting with the nursing staff (17). so teaching staff (tutor/ mentor) should keep their english language in the clinical environment as simple as possible, avoiding the use of idioms, slang, and ambiguous vocabularies (18). (19) described twelve tips to help guide medical schools, educators and healthcare institutions in developing comprehensive cultural diversity educational programs. among these tips also, they mentioned that tutors should provide a safe learning environment that allows both students and teachers to feel free to express their critical views and opinions. similar to previous studies that addressed learning environment for students lacking domestic language proficiency, students behaviors varied : some chose to withdraw and to stay in the placement for the credits, only, whereas others kept on trying to make contact with staff and patients with considerable persistence (3, 4). international students tend to be passive recipients of knowledge that is transmitted during the lectures. the use of interactive methods of learning and teaching especially within small group help teachers discover weak points (20). their understanding can be communicated by using teaching scripts to convey their accumulated experience on various health - related topics, thereby enabling students to view medical problems in a larger context (18). a major barrier experienced by students during their study is their lack of basic skills in understanding and speaking arabic. students had problems understanding medical terms in arabic when taking patients histories and updating their records. in exploring the students perceptions of their difficulties during clinical practice, linguistic problems have been suggested as a major obstacle, and similar findings have been made in various research studies, including studies in australia (21), the usa (7), and finland (3). the use of slang, having a strong accent, and using complicated terminology without explanation contribute to the students poor comprehension. language barriers are evident in diverse clinical situations, including giving and receiving instructions and understanding information in reports (21). it has been recommended by the academic staff at the university of queensland that foreign students be made aware of the differences between educational systems and approaches to learning in various environments. also, training courses were suggested to help international students to learn to deal with these adjustments more easily (e.g., language skills) (20). positive experience was also noted by the students, with the majority of students describing the communication between them and their mentors as very good. they stated that if they did not understand information in arabic, it was translated to english or another method was used to explain. students felt support and sufficient guidance from the mentor and improved their language proficiency (5). although students should be encouraged to take responsibility for their learning, samuelowicz (20) recommended that academic staff should facilitate learning of international students through management of the learning context and efforts to change students perception of learning. the students provided some recommendations to overcome these communication barriers, including using standardized patients (sps), a dictionary of medical terms in arabic and english, and an arabic orientation course before clinical placements. it has been known for a long time that an orientation period for diverse healthcare students in a clinical environment is very important (22) ; familiarizing students with patients, staff members, and facilities of the placement can help them with their diverse daily activities in the clinical environment. several models exist for preceptorship in health professionals education (6, 23, 24). however, saarikoski. (6) concluded all types of preceptorships were significant and that students should have the essential language competence to ensure appropriate interpersonal relationship / learning within the clinical environment. although, using standardized patients (sps) is costly and requires faculty effort, the literature supports that the use of sps is generally helpful and allows students to enjoy the experience. exposing students to sps can help them manage their feelings of anxiety and increase their ability to manage anxiety in the clinical setting (25). poor command of the clinical environment language can lead to positive or negative experiences and insufficient learning outcome which may cause challenges for the students. the staff members and clinical supervisors should encourage student to focus on their positive experiences which could help contribute to learning the language. arabic medical dictionary, standardized patients, and successful orientation course can contribute to a significant role in facilitating clinical experiences. intensive language courses in arabic language before commencing clinical placement can be effective in limiting communication barrier that student might experience during their placements. although the sample of the study was small, it could be used towards the development of malaysian medical undergraduate program in zagazig faculty of medicine.	introductioneffective communication in a clinical environment plays a vital role in patient assessment and treatment. the aim of this study was to understand the experiences of malaysian medical students concerning communication barriers during clinical practice. the goal was to provide answers for three important research questions, i.e., 1) are communication barriers an impediment to malaysian students during clinical teaching ? 2) what is the nature of the language barriers that the students encounter ? and 3) what are the best ways of reducing these barriers during clinical teaching?methodsthe qualitative method was used to conduct the research, and open - ended questionnaires were used to collect the data. the study was conducted on 95 fourth-, fifth-, and sixth - year students, 80% of whom completed the study.resultsmedical students from malaysia who have limited knowledge of the arabic language experience some difficulties in communicating with staff members, patients, and nurses during their clinical practices.conclusionsuccessful orientation of students to the language used in the clinical environment will help the students overcome the communication barriers they encounter during their clinical practices.
statins are inhibitors of 3-hydroxy-3-methyl - glutaryl coenzyme a (hmg - coa) reductase that can reduce the plasma level of total cholesterol, low density lipoprotein cholesterol, and glycerin trilaurate and increase high density lipoprotein cholesterol. in the past two decades, more than 170,000 participants in a number of large - scale clinical trials demonstrated that statins reduce the incidence of cardiovascular events ; thus, statins are now widely used for the primary and secondary prevention of both atherosclerotic cardiovascular disease (ascvd) and stroke [36 ]. however, in contrast to its clear benefit to cardiovascular disease prevention, a substantial number of meta - analyses of large, randomized controlled clinical trials showed that statins also increased the risk of new onset diabetes, in comparison to the placebo group [79 ]. the mechanism by which statins cause this increase in type 2 diabetes risk is unknown. accordingly, in this study, we investigated the effects of statins on the two known mechanisms that cause type 2 diabetes : insulin resistance and the dysfunction of islet beta cells. thus, we tested the effects of simvastatin on insulin secretion in the mouse islet beta cell line, min6, and explored the mechanism of this function. the mouse islet beta cell line, min6, was a gift from the ruijin hospital, affiliated to shanghai jiao tong university school of medicine, institute of endocrinology. simvastatin was obtained from merck ; antibodies against inward rectifier potassium channel 6.2 (kir6.2) and glut2, as well as the hrp - conjugated rabbit polyclonal antibody, were purchased from abcam. the antibody against cav1.2 was obtained from novus ; the antibody against glyceraldehyde-3-phosphate dehydrogenase (gapdh) was obtained from cell signaling. the formulation of the extracellular fluid for the katp channel currents (ikatp) was as follows : 80 mm nacl ; 60 mm kcl ; 10 mm hepes ; 1 mm mgcl2 ; 0.1 mm cacl2 (ph 7.4, naoh). the electrode internal fluid for these katp channel currents contained the following : 15 mm nacl ; 92 mm kcl ; 33 mm koh ; 10 mm hepes ; 1 mm mgcl2 ; 1 mm cacl2 ; 10 mm egta ; 0.2 mm mgatp (ph 7.2, koh). the formulation of the extracellular fluid for the l - ca channel currents (ica - l) was as follows : 30 mm bacl2 ; 99.3 mm cscl ; 16.7 mm glucose ; 10 mm tetraethylammonium chloride (teacl) ; 1.2 mm mgcl2 ; 10 mm hepes (ph 7.4, csoh). the electrode internal fluid for these l - ca channel currents contained the following : 130 mm cscl ; 10 mm teacl ; 5 mm mgcl2 ; 4 mm atp - na2 ; 0.4 mm gtp - na2 ; 10 mm egta ; 10 mm hepes (ph 7.2, csoh). min6 cells were cultured in dmem supplemented with 15% fbs, 100 u / ml penicillin, 100 g / ml streptomycin, 2 mm l - glutamine, and 70 mol / l beta - mercaptoethanol. cells were divided into nc, a, b, and c groups at 6 10/ml ; medium was changed every two days, and a, b, and c groups were treated with 2, 5, and 10 m simvastatin, respectively, for 48 h. nc was the normal control group. cells were cultured in 12 wells at 5 10/ml for 48 h and then washed with krbb (115 mm nacl, 4.7 mm kcl, 1.2 mm mgso4, 1.2 mm kh2po4, 1.3 mm cacl2, 24 mm nahco3, 0.1% bsa, and 10 mm hepes, ph 7.4) twice ; glucose - free krebs - ringer buffer was added for 2 h at 37c, and then the medium was removed. krebs - ringer buffer with 2.8 mm and 16.7 mm was added into each group for 1 h at 37c ; then the supernatant was removed, and the cells were centrifuged at 1000 rpm for 5 min at 4c ; then the supernatant was used for immediate testing or stored at 70c. equal acid alcohol (75% ethanol, 1.5% hydrochloric acid, and 75% ultrapure water) was added to extract the cells, and then they were stored at 4c. in the following day, insulin was measured in the supernatant, according to the kit, and the results were adjusted with total protein concentration. cells were cultured in 12 wells ; 100 l cell lysis buffer was added to each well, and then the lysed cells were centrifuged at 12,000 g for 8 min. the supernatant was collected ; 100 l atp working solution was added to the tube at room temperature for 5 min, and then 20 l sample buffer was added and mixed quickly ; then the rlu was read with a luminometer. total rna was isolated using the trizol reagent, according to the manufacturer 's instructions. the cdna was synthesized from 2 g of total rna, using mmlv transcriptase with random primers. cells were lysed with lysis buffer (200 mm tris - hcl (ph 7.5), 1.5 m nacl, 10 mm edta, 25 mm sodium pyrophosphate, 10 mm glycerol phosphate, 10 mm sodium orthovanadate, 50 mm naf, and 1 mm pmsf, in combination with a protein inhibitor cocktail). 30 g of protein lysates from each sample was subjected to sds - page and transferred onto nitrocellulose membranes. after washing them three times for 15 min each with tbst (tbs + 0.1% tween20), blots were incubated with a horseradish peroxidase - conjugated secondary antibody (pierce, rockford, il, usa) and visualized by chemiluminescence. band densities were quantified by densitometry using the scion image software and normalized to -actin levels. a coverslip containing adherent cells was mounted in a recording chamber on an olympus ix 51 microscope (olympus, tokyo, japan). glass capillaries (sutter, usa) were used to fabricate patch electrodes through a micropipette puller (p97, sutter, usa), which had resistances of 2 to 5 m, when filled with an intracellular solution. the cells were washed with extracellular solution for 2 min, and the patch electrodes, which were attached to the min6 cell (well - grown, smooth - faced, and tile - shaped), were moved using a motorized micromanipulator (mp-225, sutter, usa). first, the liquid junction potential was eliminated ; then the electrode capacitance transients were compensated, and, after gigaseals (> 1 g), fast capacitance was established by negative pressure. using further suction to break the membrane, a whole - cell recording was formed. the ikatp was elicited from a holding potential of 70 mv, with a prepulse to 40 mv for 500 ms, and with test potentials ranging from 120 mv to + 80 mv in 20 mv increments during 3000 ms. the ica - l was elicited from a holding potential of 70 mv, a prepulse to 70 mv for 500 ms, and depolarized from 40 to + 50 mv in 10 mv increments during 3000 ms. all recordings were performed at room temperature (~23c), with a 100 s sampling interval, and digitally filtered at 1 khz. leak subtraction for gating currents all patch - clamp data were acquired with a patch - clamp amplifier (axon 700b, usa), digitized with a digidata 1440a analog - digital converter (molecular devices, usa), and analyzed with pclamp9+clampfit software. data were analyzed using one - way anova with tukey 's posttest or nonparametric tests in spss 15.0. comparing with the normal control cells, the cells treated with simvastatin had less insulin content, less insulin secretion, and less total amount of insulin induced from a low level of glucose (p < 0.05). furthermore, simvastatin decreased insulin secretion in a concentration - dependent manner (table 2 and figure 1). comparing with the normal min6 cells, cells treated with simvastatin had less insulin content, less insulin secretion, and less total amount of insulin induced at high glucose levels (p < 0.05). furthermore, simvastatin reduced the total amount of insulin in a concentration - dependent manner. the group treated with 10 m simvastatin had a greater decrease in cellular insulin content, insulin secretion, and total insulin, in comparison to the group treated with 2 m simvastatin under high glucose levels (p < 0.05, table 3 and figure 2). atp levels decreased in min6 cell lysates after simvastatin treatment, in comparison to the normal control cell lysates (p < 0.05). furthermore, simvastatin caused this decrease in atp in a concentration - dependent manner (p < 0.05, table 4 and figure 3). compared with the normal control cells, the mrna levels of kir6.2 increased (23.97 4.09, p < 0.001) in cells treated with 10 m simvastatin, while the mrna levels of cav1.2 and glut2 decreased (0.63 0.22, p = 0.002 and 0.50 0.21, p = 0.023, resp.) in cells treated with 5 m simvastatin and were further reduced in cells treated with 10 m simvastatin (0.42 0.10, p < 0.001 and 0.26 0.11, p < 0.001, resp.). kir6.2 mrna levels were significantly higher in the 10 m treatment group than in the 2 m and 5 m groups (p < 0.01). the cav1.2 mrna levels were significantly lower in the 10 m treatment group than in the 2 m and 5 m groups (p < 0.001, p = 0.038). furthermore, cav 1.2 mrna levels were more significantly downregulated in the 5 m group than in the 2 m group (p = 0.036). glut2 mrna levels were more significantly reduced in the 10 m group than in the 2 m group (p = 0.013, figure 4). as shown in figure 5, 5 m and 10 m simvastatin increased the protein expression of kir6.2, while the expression of cav1.2 decreased, in comparison to the control min6 cells. the protein expression of glut2 decreased in cells treated with 10 m simvastatin, in comparison to the control min6 cells. simvastatin increased ikatp in a concentration - dependent manner. at 80 mv electric voltage, ikatp increased by 118.36 3.30 pa / pf, 154.16 10.72 pa / pf, and 311.37 7.06 pa / pf at 2 m, 5 m, and 10 m simvastatin, respectively (figure 6). as shown in figure 7, at 10 mv, simvastatin significantly decreased the ica - l current density in a concentration - dependent manner. pa / pf, 10.11 0.59 pa / pf, and 4.71 0.19 pa / pf in min6 cells treated with 2 m, 5 m, and 10 m simvastatin. statins inhibit cholesterol synthesis, reduce plasma cholesterol levels to stabilize the atherosclerotic plaque in blood vessels, and inhibit the activity of hmg - coa reductase in the cholesterol synthesis process. statins are widely used in a great number of medical fields, including cardiology, neurology, nephrology, and endocrinology. recently, there has been a great deal of attention on the effect of statins, especially in enhancer dosages, on glycometabolism, but there have been few studies on the mechanism of how statins increase diabetes risk. the mechanism of statins on glycometabolism, decreased insulin sensitivity, and diabetes risk increase is still unknown. nakata. found that atorvastatin reduces insulin sensitivity by inhibiting adipose cell maturation and the expression of glucose transporter-4. the side effect of statins on glycometabolism may be related to a decrease in metabolite production. coenzyme q10 plays a critical role in energy metabolism ; it not only is important for antioxidation and cell membrane stabilization, but also participates in mitochondrial oxidative phosphorylation. some statins have been shown to significantly reduce plasma coenzyme q10 level, leading to a reduction in mitochondrial oxidative phosphorylation and decreased atp production [1215 ]. furthermore, statins can inhibit the mevalonate pathway, thus preventing the production of mevalonate metabolites, such as isoprenoid and coenzyme q10, and affecting glycometabolism and insulin sensitivity. insulin synthesis and secretion are a complete and orderly process in pancreatic islet beta cells. at present, the consensus on glucose - stimulated insulin secretion is that beta cells are electrically excitable ; glut2 transports glucose into the cells, and then glucokinase phosphorylates it to atp. these events result in atp - sensitive potassium channel (katp) closure and membrane depolarization, voltage - dependent calcium channel (cav) opening, calcium influx, intracellular calcium concentration increase, and activation of a series of enzymes and proteins, which are necessary for insulin secretion. our study shows that, after 48 h of treatment, simvastatin significantly inhibited glucose - stimulated insulin synthesis and secretion at basal (2.8 mm) and high glucose levels (16.7 mm), in a dose - dependent manner in min6 cells. in this study, we investigated the mechanism of how simvastatin inhibits insulin secretion in islet beta cells. atp is a very important regulator of insulin secretion in islet beta cells ; reduction of atp inhibits the katp channel closure, membrane depolarization, and subsequent cav channel opening, thus inhibiting insulin secretion. our results demonstrated that simvastatin significantly inhibited atp production in a dose - dependent manner in islet beta cells. katp is an octamer, which consists of four kir6.2 subunits and four thiourea receptor-1 (sur1) subunits. katp plays a critical role in the insulin secretion process by coupling glucose metabolism and electrical activity in beta cells. in addition, cav, in its relation to the l - type non - l - type calcium channels, plays an important role in insulin secretion. our study showed that simvastatin significantly increased kir6.2 expression, while it decreased cav1.2 and glut2 expression at 5 m and 10 m treatment for 48 h. the patch - clamp technique results showed that simvastatin increased the katp current, while it reduced the l - ca current in min6 cells. kir6.2 is an inward rectifier potassium channel, which plays important roles in membrane resting potential maintenance and insulin secretion in beta cells. thus, by increasing kir6.2, while decreasing cav1.2, and subsequently inhibiting depolarization and calcium influx, simvastatin is able to reduce insulin secretion. furthermore, simvastatin can also indirectly inhibit insulin secretion by decreasing glut2 expression, resulting in reduced glucose uptake. collectively, although it is clear that statins have beneficial effects on cardiovascular disease prevention, our study supports the evidence that long - term use of large doses of statins has adverse effects on glucose metabolism and insulin sensitivity. therefore, patient blood glucose levels should be monitored to reduce these adverse events caused by statins. we found that simvastatin inhibits the synthesis and secretion of insulin through a reduction in saccharometabolism in beta cells.	the aim of this study was to investigate the effects of simvastatin on insulin secretion in mouse min6 cells and the possible mechanism. min6 cells were, respectively, treated with 0 m, 2 m, 5 m, and 10 m simvastatin for 48 h. radio immunoassay was performed to measure the effect of simvastatin on insulin secretion in min6 cells. luciferase method was used to examine the content of atp in min6 cells. real - time pcr and western blotting were performed to measure the mrna and protein levels of inward rectifier potassium channel 6.2 (kir6.2), voltage - dependent calcium channel 1.2 (cav1.2), and glucose transporter-2 (glut2), respectively. atp - sensitive potassium current and l - type calcium current were recorded by whole - cell patch - clamp technique. the results showed that high concentrations of simvastatin (5 m and 10 m) significantly reduced the synthesis and secretion of insulin compared to control groups in min6 cells (p < 0.05). atp content in simvastatin - treated cells was lower than in control cells (p < 0.05). compared with control group, the mrna and protein expression of kir6.2 increased with treatment of simvastatin (p < 0.05), and mrna and protein expression of cav1.2 and glut2 decreased in response to simvastatin (p < 0.05). moreover, simvastatin increased the atp - sensitive potassium current and reduced the l - type calcium current. these results suggest that simvastatin inhibits the synthesis and secretion of insulin through a reduction in saccharometabolism in min6 cells.
in september 2008, a 70 years old female was diagnosed with a lobular carcinoma after a core biopsy of a mass in her left breast. a month later, she underwent skin sparing mastectomy and axillary clearance, the histological examination confirmed a grade 2 invasive lobular carcinoma, estrogen receptor positive (er+), progesterone receptor negative (pr), and human epidermal growth factor receptor 2 negative (her2). there was no vascular space invasion, no in situ component and the axillary tissue included eight lymph nodes, three of those contained metastatic lobular carcinoma with extracapsular spread. a ct scan to chest and abdomen and subsequently, she had radiotherapy and was started on adjuvant hormonal treatment, but because of her past medical history which includes previous nephrectomy for pelvic ureteric junction (puj) obstruction 10 year ago, arthritis and essential hypertension, chemotherapy was not considered. in early 2010, the patient was referred to the department of plastic surgery for breast reconstruction, as she continued to show no clinical evidence of any recurrence, she underwent reconstruction with a pedicle transverse rectus abdominis myocutaneous (tram) flap. one month later she had multiple episodes of abdominal pain and constipation which needed hospital admission. a ct scan showed evidence of small bowel obstruction (figure 1) ; a laparotomy and resection of a thickened and narrowed terminal ileum were carried out followed by bowel anastomosis. the histological analysis confirmed the presence of widely infiltrated small bowel by well - differentiated epithelial cells, some of which contained intracytoplasmic lumina and formed a single file pattern of infiltration (figure 4). the appearances were those of metastatic lobular carcinoma of breast, which was er positive and e - cadherin & her 2 negative (figure 5), invaded the mucosa, the mesentery, and appeared to extend beyond the mesenteric margins of excision. a subsequent chest / abdomen ct scan and a bone scan showed no signs of other metastasis. unfortunately, 3 months later, the patient had new episodes of diarrhoea with fresh blood ; sigmoidoscopy showed a narrowed lumen and firm swollen mucosa at the upper rectum (figure 3) ; biopsies revealed the presence of metastatic lobular carcinoma of breast, the tumour foci stained positive with cytokeratin 7 (ck7), pancytokeratins, and er. staging ct scan identified left hydronephrosis caused by compression on the ureter from the metastasis in the rectum (figure 2). no extraintestinal spread reported ; the patient had a ureteric stent put in, and she was subsequently started on palliative therapy. the most common metastatic sites from breast cancer are well known, the lungs, bones, liver, and brain whereas the involvement of the gastrointestinal tract remains very unusual. borst published a large series that analyzed more than 2500 cases of breast cancer with metastatic disease over a period of 18 years ; he found that only 17 patients (less than 1%) had metastasis to the gi tract. metastasis to the stomach and small bowel have been more frequently reported than colonic and rectal ones. however, in a retrospective study, mclemore reported 23 cases of gi metastasis, he found that the disease is more frequent in the mid- and hindgut than in the foregut (oesophagus 8%, stomach 28%, small intestine 19% and colon - rectum 45%). despite the greater prevalence of infiltrative ductal breast carcinoma among women (90%), lobular breast cancer has a specific metastatic pattern and more frequently metastasizes to the gi tract and the retroperitoneal tissue than ductal cancer. it has also been reported that even in patients with a mixed ductal and lobular primary breast carcinoma, the lobular type is the most likely to cause metastatic disease. the presenting symptoms and signs of metastatic disease to the gi tract are usually nonspecific, often mimicking other gastrointestinal disorders ; this patient presented with multiple episodes of intermitted abdominal pain and restrain of stool ; the initial thought was that she might had adhesional obstruction or incarcerated incisional hernia as a complication of the pedicel tram, but clinically there was no evidence of hernias. diagnosis of gi metastasis from breast cancer could be difficult prior to surgical intervention ; the findings of diagnostic imaging are frequently not specific for gi metastasis ; the picture can also mimic crohn 's disease. gi endoscopy with deep biopsy is widely used for accurate diagnosis depending on the patient status. santini. reported an interesting case where an increase in cancer antigen ca 19.9 was used to diagnose metastatic lobular breast cancer to ileocecal valve in a symptomatic patient. establishing the histological origin could be challenging, metastases of lobular carcinomas have a typical fashion of spread with intramural infiltration growing within the serosal, muscular, and submucosal layers with small cells in cords throughout the tissue. compared to primary adenocarcinoma of bowel, the metastatic lobular carcinoma is usually positive for ck 7, er, pr and gcdfp 15 and negative for vimentin, while ck 20 and cea are almost invariably present in primary gastrointestinal tumours and absent in breast carcinomas [9, 10 ]. in this case, the metastatic disease was positive for er, and negative for pr and her2, which matched the immunohistochemical findings of the primary breast cancer ; this finding, along with the unique histological pattern of the disease, favoured the diagnosis of metastatic lobular breast carcinoma.. it may involve systemic hormonal or chemotherapy either alone or combined with surgery since patients usually present with the involvement of multiple organs, despite not detecting extensive disease in preoperative investigation. patients with intestinal obstruction, perforation, or bleeding require palliative surgical intervention but that does not extend the patient survival. interestingly, tang. reported 2 cases when the intestinal obstruction due to metastatic lobular carcinoma was settled with a parenteral endocrine hormonal agent (fulvestrant). long - term survival rate has been reported after resection of an isolated gi metastatic lesion whilst surgery combined with chemotherapy may result in long - term remission [7, 13 ]. bowel surgery in postmastectomy patients who have undergone transverse rectus abdominis myocutaneous (tram) flap would require careful preoperative planning of surgical incision, and the need for stoma can not be ruled out [14, 15 ]. in summary, metastasis to gi tract from breast cancer is a rare condition and not easy to diagnose. this diagnosis should be suspected when the patient has a history of breast cancer especially of lobular type. not surprisingly, in most cases, gi metastasis is part of widespread disease, and early diagnosis may help towards the improvement of the outcome. this case also emphasizes the importance of patients ' suitability for breast reconstruction, despite the patient herself making the decision to go for tram flap as a means of delayed reconstruction after knowing the pros and cons, less invasive procedures and techniques should be made available and must be considered in patients with a kind of cancer that carries high risks of recurrence.	breast cancer is the most frequent malignancy in women accounting for approximately 32% of all cancers, with a lifetime risk of 1 in 10. it causes considerable morbidity and mortality. recently, the survival rate has dramatically increased due to early detection of the disease and improvement in the treatment measures. however, more than 30% of the patients develop metastatic diseases following surgical treatment, radiotherapy, hormonal therapy, or chemotherapy. distant spread is usually found in bones, lungs, liver, brain and skin. rarely, it spreads to bowel, spleen, gallbladder, pancreas, urinary bladder, and eyes. breast cancer is the second commonest primary tumour responsible for gastrointestinal metastases after malignant melanoma. we report a case of a caucasian female who developed an intestinal obstruction secondary to metastatic deposits to the small bowel and later to the rectum from breast lobular carcinoma 2 years after mastectomy, axillary clearance, radiotherapy, hormonal therapy, and transverse rectus abdominis myocutaneous (tram) flap for reconstruction.
at the core of social cohesion among mammals is the communication of affective or emotional states between individuals. when individuals respond to others emotional states as if they were their own, the result is a bond, thereby building social cohesion within the larger group. defined in this way, empathy is an umbrella term that includes a large range of interactions in which an emotional or affective response is elicited by the emotional or affective state of another individual. moreover, according to this definition, empathy is neutral in that responding to another s affective state, mood, or emotion does not constrain the actions taken, if any, as a result. we may hope that an individual reacts with helping behavior to a member of his or her own species in distress the social instincts and sympathies darwin suggested. yet, inaction and even targeted cruelty aimed at exacerbating a victim s distress are also possible reactions. the perception - behavior link, an automatic function that links our behavior to the behavior of another, is critical to affective communication between two individuals. many of us are familiar with the phenomenon of adopting the physical stance of a person or people with whom we are talking ; soon after one member of a group crosses his or her arms, another person does the same. simply viewing another individual s actions increases the probability that the viewer will perform the same actions even if the individuals are strangers.6 similarly, people in conversation with each other modify their fundamental speech frequencies to more closely match each other.7 these social adjustments make the actions of two interacting people more similar to each other and serve as an affiliative signal, or a kind of social glue. they also influence affect the interaction between emotions and outward expressions is two - way.8 in other words, just as our emotions lead to actions, our motor actions are re - experienced as affect. affect and emotions are expressed through voluntary muscles responsible for posture, facial expression, breathing, and gaze, as well as autonomic processes such as a rise in heart rate or perspiring, blanching, and blushing. people report emotions commensurate with artificially arranged facial expressions.9 feeling happy makes us smile and smiling can make us feel happy, or at least happier. when you re feeling good and laughing with friends, just try to feel angry or sad. as long as you keep your face in a smile or laugh, feeling an incongruent emotion is nearly impossible. deriving emotion from action, often termed embodied emotion, is the essence of the stanislavski system of method acting in which the affects that emerge from movements provide the emotive force of a performance. the links between perception and action and between action and affect set up a cascade whereby one person s perception by another s actions ultimately results in the first person feeling the second one s mood. this cascade results in matching affects.10 the affect the viewer experiences is vicarious in nature, caught from the other individual. the process by which one individual catches the affect or emotion of another is called emotional contagion, and it is a fundamental building block of more complex forms of empathy. emotional contagion is required but not sufficient to elicit empathically motivated helping. in humans, personal distress must be suppressed in order to move from emotional contagion to helping, to choose action over immobility and panic. high levels of personal distress are detrimental to helping.11 suppressing personal distress allows someone to focus on the other over the self and leads to empathic concern, an other - oriented emotional response elicited by and congruent with the welfare of an individual in distress. the response s congruence with the welfare of the other precludes antisocial actions so that the action taken by someone feeling empathic concern is always prosocial in nature. by helping a distressed individual, a helper resolves not only the distressed individual s predicament but also his or her own uncomfortable affective state, providing an internal reward.12 thus, helping dissipates the distress of both the helper and the beneficiary. that the helper benefits does not diminish the prosocial action or its effect. the empathy - helping connection is so effective precisely because empathy gives individuals an emotional stake in the welfare of others. the feeling of empathy may drive behavior such as a facial expression. in humans, nonhuman animals, however, do not have the control over, and variety of, facial expressions that humans possess. therefore, one has been to test for emotional contagion. a typical experiment using rodents tests the influence of one rodent s expression of either fear or pain on another rodent s behavior. the second approach asks whether, given the opportunity, animals will engage in prosocial behavior, such as sharing food (in primates) or working for the relief of another from foot shock or confinement (in rodents). mounting evidence suggests that emotional contagion and prosocial behavior are present in nonhuman primates and rodents and likely are widespread among mammals. for example, a useful paradigm to test for helping behavior is to place an object out of the reach of an experimenter but within reach of the test subject. the question is whether the subject will hand the object to the experimenter even though the subject gains no reward by doing so. in one study using this paradigm, chimps handed the object to the experimenter in about 40 percent of the trials ; human infants helped in about 60 percent of trials.13 the proportions of children (60 percent) and chimps (50 percent) that helped at least once in 10 trials were similar. the subsets of children and chimps that had helped were then tested on the same task but with physical obstacles placed between them and the object, adding to the cost of helping. just over half of the subjects of both species helped even when helping required significant effort. since emotional contagion is automatic, from perception to action to embodied emotional cascade, it is not surprising that chimps and other primates also appear to experience empathy.12 moreover, because the pathways involved in linking perception to action to embodied emotional cascade are shared across mammals and the perception - action model for empathy does not depend on conscious deliberation or higher cognition, there is no reason to expect that empathy and prosocial behavior are exclusive to primates.12, 14 indeed, emotional contagion has been documented in a number of mammalian species, including rodents.15 a mouse that views another mouse experiencing foot shock, for example, shows fear by freezing in place.16 in another example, pairs of mice that receive a noxious stimulus show more pain behavior than a single mouse that receives the same noxious stimulus ; this finding has been interpreted as emotional contagion of pain.17 several years ago, we designed a behavioral helping test for rats.18 in this test, one rat is restrained in a plastic tube located in the center of an arena while a second rat is free to roam in the arena. the restrainer door can be opened only from the outside only by the free rat. within a few sessions, most rats begin to open the door consistently, releasing the other rat. by the final session the fact that rats opened the restrainer door repeatedly and consistently is remarkable in light of rats strong preference to remain close to walls and avoid open areas. the motivation to approach the trapped rat in the arena center evidently is sufficient to overcome rats natural avoidance of open space. we would expect that emotional contagion would lead the free rat to experience at least some of the distress felt by the trapped rat. the most common reaction of a rat to personal distress is freezing or immobility such as that which occurs in response to foot shock. yet instead they act intentionally to open the restrainer door.18 this behavior suggests that the helper rat recognizes that its distress is vicarious in origin. in other words, the rat is able to attribute personally felt distress to the trapped rat s condition and distinguish that from its own condition. such recognition of the distinction between self and other is unexpected in a rat. the helping behavior test is not the first scientific demonstration of helping, but it is the first tractable paradigm for studying prosocial behavior in a mammal. already, the test has been used to demonstrate that helping is socially selective.19 rats help a stranger rat but only if that rat is from a familiar strain. in other words, an albino rat that has never before seen a black - hooded rat will not help it. however, an albino rat that has lived with a black - hooded rat will open the restrainer door for black - hooded strangers. remarkably, albino rats raised since birth with black - hooded rats do not help other albino rats, although they do help black - hooded strangers. this suggests that rats do not inherit genetic instructions to help others of their own kind. the test result tells us that environmental experiences trump genetics when it comes to targeting helping, resolving one piece of the nature - nurture debate. moreover, because the fostering - from - birth experiment is easy to perform in rodents but would be impossible in most other mammals, this result shows the power of an experimental model for prosocial behavior in rodents. the finding that rats help strangers of a familiar type but not strangers of an unfamiliar kind may appear, at first glance, to suggest a biological basis for a social bias, a kind of strainism. however, the results are more consistent with a biological basis for groupism through social experience. humans readily form strong affiliations to groups that are based on minimal - group criteria such as an arbitrary assignment to one of two meaningless markers (e.g., red or blue wrist bands).20 the finding that rats raised without experience with their own strain do not help strangers of their own strain demonstrates that group affiliation, with respect to helping, is fluid, based on experience, and not genetically determined. we still need to understand more about the rat s motivation to help another in distress and to discover the underlying brain mechanisms that support helping behavior. while the motivation for prosocial behavior looks like empathic concern, a rat may open a restrainer door for other reasons. one commonly raised possibility is that the rat finds some part of the trapped rat s behavior so aversive that it opens the door to terminate this aversive experience. since rats do not open restrainer doors for rats from unfamiliar strains, including strangers from their own strain if they were fostered with a different one, escaping aversion is a possible but unlikely motivation for door - opening in the helping behavior test.19 nonetheless, empathic concern must start with an individual showing distress. because an individual s demonstration of distress is as critical to empathic concern as another s noticing and responding to that distress, biology has left little to chance. crying works babies and others get attention when they need help. facial expressions and posture also work because they are universal, with commonalities across populations and across species. conversely, in the absence of an individual displaying distress, nonhuman animals and young humans are never moved to help. a second common theory is that rats may be motivated by a desire to interact socially with the trapped rat. social reward is a fundamental underpinning of social behavior, and all rodent behavior involving more than one individual is, at the very least, influenced by social reward. rats will opt to be together when given the opportunity. using a modification of the helping behavior test in which rats were repeatedly retrapped, a free rat that could not release the trapped rat opted for physical proximity.21 in contrast, when given the chance, rats continue to open the door for a trapped cagemate even when subsequent interactions are prevented.18 this finding suggests that the opportunity to play or interact with the trapped rat is not a requirement for prosocial behavior. we are nt alone : about 25 percent of the rats that we have tested in standard conditions do not exhibit helping behavior. the predominant reason for not helping appears to be an excessive amount of personal anxiety. similarly, bonobo apes who show more anxiety (measured by how much they scratch themselves) and take longer to recover from a stressful event show less consolation behavior toward other bonobos in distress.22 this finding dovetails beautifully with research in humans suggesting that in order to use empathy for helping or caring, an individual must overcome personal distress, a process typically termed self- or down - regulation. people with a specific genetic variation who show greater social anxiety also demonstrate less helping behavior.23 this finding suggests that rather than lacking empathy, many individuals who do not help may be unable to suppress the anxiety associated with catching another s feeling of distress. in professions that involve repeated exposure to human suffering, such as medicine, strong down - regulation physicians have a down - regulated response to noxious events that are common in medical practice. for example, an image of a needle stick evokes a lower assessment of pain intensity and unpleasantness by physicians than controls.24 finally, human psychopaths appear to lack empathy and exhibit a callous disregard for others suffering. whether psychopathic individuals exist in other mammalian species a particularly instructive approach has been to compare brain activation in humans, using functional magnetic resonance imaging (fmri), to compare when an emotion is experienced by the self versus when it is experienced by another. when one person views another in pain, the activated brain areas are similar and overlapping, but not identical, to those activated by a personal experience of pain.25 the overlapping regions of activation evoked by self- and other - pain can breed confusion so that an individual experiences another s distress as their own. conflation of the distress originating with the self and the other may explain why vicarious distress can be as immobilizing as personal distress. it appears that the prefrontal cortex allows us to make the distinction between ourselves and others by promoting down - regulation.25 for example, the medial and dorsolateral prefrontal cortex was activated as physician acupuncturists viewed images of needle insertions, an activation that was not observed in control subjects.26 moreover, the degree of activation in the prefrontal cortex was inversely correlated to the ratings of pain intensity made by the subjects so that those with the greatest prefrontal activation judged the needle insertions with the lowest pain ratings. these studies show that viewing others pain engages ascending affective pathways, while top - down regulation arising from the prefrontal cortex is critical to stemming personal distress so that empathy can serve as a call for action. indeed, fear contagion in mice appears to require the anterior cingulate cortex as well.16,25 a full elucidation of the similarities and differences in brain circuits involved in empathy and down - regulation between humans and other mammals is an exciting challenge for the future.	editor s note : why are certain individuals born with a brain that is wired to help others ? what daily habits or life experiences reinforce compassion but also selfishness, narcissism, and psychopathy ? social neuroscience models have assumed that people simply rely on their own emotions as a reference for empathy, but recent studies suggest neurobiological underpinnings for how the brain processes empathy. a better understanding of these processes, says the author, could lead to more social cohesion and less antisocial harm in society.
fixed drug eruption to ornidazole has been infrequently reported. fixed drug eruption (fde) is a special variety of allergic drug reaction where the reaction areas become reactivated with well - demarcated erythematous area whenever the same drug is administered. the lesions may be multiple and may occasionally be surmounted by a bulla in the center. in the majority of cases brocq in 1894 was the first to coin the term fixed eruption to describe a pattern of skin eruption due to antipyrine. since then, numerous drugs including pseudoephedrine, trimethoprim, tetracycline, barbiturates, salicylates, phenolphthalein, ibuprofen oxyphenbutazone, fluconazole, azithromycin, levofloxacin, levamisole, and colchicine have been implicated among many others as a causative agent. fde due to ornidazole, a relatively new 5 nitro - imidazole derivative has been very infrequently reported, though it is very commonly used for amoebic dysentery in developing countries. a 56-year - old male developed pain, redness, and tenderness of his right index finger since 2 days for which he had been treated elsewhere with cephalexin 500 mg twice daily ; ofloxacin 200 mg twice daily ; and ornidazole 500 mg twice daily orally. by third day, when i first saw, the infection in the index finger improved but he developed an ulcer on his glans penis [figure 1 ] and the mucosae of his upper and lower lips. at this juncture, all the medicines were discontinued and he was started on dexamethasone 2 mg daily orally which led to the clearance of all the lesions during the next 3 days. superficial ulcer of fixed drug eruption on glans penis one week later, oral provocation test was started after obtaining informed consent by giving one drug each day in full therapeutic dose. there was no reactivation of the lesions with cephalexin, ofloxacin, ciprofloxacin, tinidazole, paracetamol, diclofenac sodium, amoxicillin and clavulanic acid. however, he developed reactivation of itching, redness and occasional erosions on his glans penis, upper and lower lips 4 hours after taking ornidazole 500 mg. he was immediately started dexamethasone 2 mg orally twice daily with clearance of all the symptoms and lesions during the next 24 hours. recurrence of pruritus, erythema and occasional erosions on the lips and glans penis 4 hours after administration of 500 mg ornidazole confirm that the reaction was due to ornidazole and not due to the other medicines used in the provocation test. among the nitro - imidazoles, fde seems to be a form of delayed hypersensitivity reaction mediated by cd8 + t cell. the causative drug is supposed to act as hapten which binds to basal keratinocytes resulting in the release of lymphokines and antibodies which damage the basal cell layers. on drug intake, cd8 + cells are reactivated to release interferon and cytotoxic granules into the local micro - environment. mast cells are also believed to contribute to the activation of intraepidermal cd8 + cells through the induction of cell adhesion molecules. provocation tests are still the only reliable method to find the causative agents and are often essential or even mandatory in the patient 's interest. though provocation is very safe especially in fixed eruption, if history suggest severe reaction, then it may be safer to start with half the dose on first day, followed by full therapeutic dose on the next day if there is no reaction to the first dose.	a 56-year - old male developed an ulcer on his glans penis and mucosae of upper and lower lips 3 days after taking ofloxacin, cephalexin, and ornidazole. clinically, a provisional diagnosis of fixed drug eruption was made. the causative drug was confirmed by an oral provocation test which triggered a reactivation of all lesions only with ornidazole.
development of hepatocellular carcinoma, in common with other types of tumor, is considered to be a multistep process. genetic and epigenetic alterations accumulate in regulatory genes, leading to activation of oncogenes and inactivation or loss of tumor suppressor genes. the concept that hepatocellular carcinoma is a disease of epigenetic as well as genetic alterations has been validated in the past two decades. the epigenetic pathway is, in contrast to the genetic events, a reversible alteration that is not caused by primary dna sequence changes. there are three main epigenetic mechanisms : (i) hypermethylation of cpg islands in promoter sequences leading to silencing of tumor suppressor genes ; (ii) dna hypomethylation, which causes genomic instability or induction of genes involved, for example, in cell growth and invasion ; and (iii) histone modification, which affects chromatin conformation. because epigenetic mechanisms may function as an interface between environmental factors and the genome, deregulation of the epigenome by environmental stressors (for example, hepatitis b and hepatitis c viruses, chronic alcohol intake, and aflatoxins) is believed to disrupt cellular processes and contribute to the risk of hepatocellular carcinoma. moreover, drugs to reverse the epigenetic abnormalities are under development and some have already been approved. cancer epigenetics is continuously translating into clinical practice and will help to optimize cancer diagnostics and treatment. the recent observation by schaeffeler. in genome medicine that uptake transporters for anticancer drugs are epigenetically regulated in hepatocellular carcinoma adds an important piece of information to the growing body of research on cancer epigenetics. the potential implications of these findings for biomarker development and pharmacotherapy of hepatocellular carcinoma will be discussed. whereas previous work has focused mainly on the role of cpg hypermethylation for regulating oncogenes, tumor suppressor genes or dna repair genes in hepatocellular carcinoma, schaeffeler. investigated the role of dna methylation in the expression of specific members of the solute carrier (slc) gene superfamily, namely the organic cation transporter genes slc22a1 to slc22a3 (encoding proteins oct1, oct2 and oct3, respectively). in normal hepatocytes, slc22a1 (solute carrier family 22 member 1), which encodes oct1 (organic cation transporter 1), is highly expressed and is responsible for the uptake of nutrients, metabolites and xenobiotics. whereas lipophilic antineoplastic agents can enter cells by passive diffusion, uptake of charged hydrophilic agents, such as the anticancer compounds imatinib, cisplatin, oxaliplatin, picoplatin, irinotecan and paclitaxel, requires active transport. indeed, oct1 transports these six drugs and thereby contributes to the susceptibility of cancer cells to these antineoplastic drugs. cdna microarray studies have demonstrated decreased expression of slc22a1 in hepatocellular carcinoma compared with normal liver tissue. however, the mechanisms of downregulation have remained unclear. based on their findings, scheffeler., most likely impairs uptake of some anticancer drugs into hepatocellular cancer cells and thus is likely to impair their efficacy. this might, in part, explain the low response rates and uncertain survival benefit of, for example, cisplatin - based chemotherapy for hepatocellular carcinoma. in contrast, the tyrosine kinase inhibitor sorafenib, which does not appear to require active transport to enter cells, induces better disease control, significantly extends survival, and has become a standard first - line option for systemic treatment of advanced hepatocellular carcinoma. given the important role of oct1 in cancer cell uptake and thus treatment efficacy of several anticancer drugs, methylation of slc22a1 might be used as a marker for predicting therapeutic response. the work by schaeffeler. has demonstrated substantial interindividual variability in the degree of slc22a1 methylation and slc22a1 production, as revealed by histochemical analysis. it is tempting to speculate that patients with a high slc22a1 methylation phenotype and silenced transporter expression may exhibit chemoresistance, whereas patients with a low methylation phenotype and enhanced transporter expression may respond to chemotherapy. most recently, ma. provided another example of the involvement of epigenetic mechanisms in determining chemoresistance and chemosensitivity in hepatocellular carcinoma. mir193a promoter hypomethylation is associated with transcriptional induction of the microrna 193a-3p, which targets the serine / arginine - rich splicing factor 2 (srsf2). srsf2 sensitizes hepatocellular cancer cells to the chemotherapeutic drug 5-fluorouracil via upregulation of the proapoptotic splicing form of caspase 2. accordingly, its repression by microrna 193a-3p reduces sensitivity of hepatocellular cancer cells to 5-fluorouracil. this experimental observation suggests that the dna methylation state of mir193a could function as a marker to predict the therapeutic response to 5-fluorouracil. as well as using dna methylation as a biomarker for personalizing chemotherapy, evidence has emerged that detection of abnormal promoter cpg island hypermethylation is also a potential biomarker for risk of developing cancer. dna methylation in adjacent histologically normal liver tissue of hepatocellular carcinoma has been reported from several groups and is now accepted as an early event in cancer development. thus, aberrant dna methylation might be useful for early cancer detection and for predicting prognosis. comparisons of cpg island methylation in hepatocellular carcinoma with that in adjacent tumor - free tissue or normal control livers have produced a continuously expanding list of potential marker genes. interestingly, schaeffeler. demonstrated that slc22a1 belongs to a group of genes with progressively increasing cpg methylation, from normal liver to hepatocellular carcinoma, with intermediate methylation in precancerous tissues (that is, in adjacent tumor - free tissue). similar methylation changes related to the progression of malignant transformation have been observed previously for other genes, including rassf1a, prdm14 and tbx4, which encode ras association domain family member 1, pr domain containing 14 and t - box 4, respectively. therefore, the cpg methylation phenotype of these genes may serve as a marker for early cancer detection or assessing cancer risk. as biomarker strategies move towards actual clinical practice, these proof - of - principle findings should be validated in larger patient cohorts. for detection of other tumors, such as colorectal and lung cancers, assays that test methylation patterns of marker genes in dna from the stool, blood or bronchoalveolar lavage the results reported by shaeffeler. also highlight the clinical implications of epigenetic modulation of transporter expression. dna methyltransferase inhibitors, such as decitabine, have already been approved for treatment of lymphomas and are being tested as a therapeutic option against various solid tumors. if these agents are concomitantly administered with classical anticancer drugs, the resulting induction of transporter expression may influence the disposition and effect of these anticancer drugs. specifically, repression of oct1 might be reversed by treatment with the dna methyltransferase inhibitor decitabine, enhancing uptake of cisplatin into hepatocellular tumor cells. however, the fascinating possibility of overcoming the problem of chemoresistance with an epigenetic therapy awaits proof of concept. oct1 : organic cation transporter 1 ; srsf2 : serine / arginine - rich splicing factor 2.	epigenetic alterations, such as aberrant dna methylation, are a hallmark of cancer. dna hypermethylation of the promoter region affects, for example, the expression of tumor suppressor genes and is associated with their transcriptional silencing in tumors. a recent report has provided evidence for epigenetic silencing of the multispecific organic cation transporter slc22a1 in hepatocellular carcinoma. given the role of this transporter in the cellular uptake of several anticancer drugs, the study provided a novel mechanism to explain the substantial variability in treatment response, and it might provide a new strategy for optimization of pharmacotherapy of hepatocellular carcinoma.see research article http://www.genomemedicine.com/content/3/12/82
autism is a complex condition that lacks an established method of prevention ; thus there is a need for research to identify its possible causative factors. identification of environmental factors is the first step in preventing and mitigating risks by primary prevention. statistical estimates show that one out of every 88 individuals in the united states is diagnosed with autism. the condition may be defined as a chronic neurodevelopmental disorder that first becomes prevalent in an individual during his / her early childhood. consequently, autism results in a patient experiencing a life that is marked by the ongoing challenge of its negative effects on communication and interaction with others. the disorder is debilitating for the patient and immensely challenging for his / her family. an initial diagnosis of autism occurs as an individual is noticeably unable to achieve certain early childhood milestones in terms of communication and socialization. furthermore, while the incidence and the prevalence of autism also tend to raise on an ever - increasing basis, public health concerns over the disorder have also been coupled with concerns over its etiology, which is still in the investigative stage of clinical research. initial research efforts, which were embarked upon during the 1950s, assert that complications in a given pregnancy, the birthing process, and the incidence of autism are linked. over the past several decades, continuing research has been conducted, and much literature has been published that has supported the existence of the links between autism and factors that relate to pre-, peri-, and neonatal care. such commonly identified, prenatal risk factors may include advanced - aged parents, a short gestation period, low birth weight, hyperbilirubinemia, and breech presentation. another key factor may be the use of any prenatal, prescription medication [46 ]. autism, to reiterate, impairs an individual 's ability to use language to effectively communicate and navigate social situations. the assertion that autism patients engage in repetitive, behavioral patterns has arguably become stereotyped. the symptoms of autism become notable in patient in the first three years of life and may necessitate lifelong care that must be provided by family members, guardians, or healthcare professionals. autism and autism spectrum disorders (asds), including asperger 's syndrome and pervasive developmental disorder, have become increasingly diagnosed over the past decade. accordingly, population studies, which have been conducted by the united states centers for disease control and prevention, have reported a prevalence of asds of 1 of 68 children at risk. it should be noted, however, that, from a stand - alone perspective, each of the factors (as listed above) that link autism with pre-, peri-, and neonatal care does not correlate consistently as the incidence of autism is reviewed in terms of its cross - reference amongst various research studies. it should also be noted that, despite ongoing research efforts, relatively little has yet been divulged regarding delivery - room attributes that may be directly or indirectly associated with the incidence of autism. furthermore, the nature of the precise catalyst of autism has not been revealed as of yet. early diagnosis of autism and any subsequent related intervention(s) may serve to alleviate the challenges that are faced by those individuals, who must manage the effects of autism on an ongoing basis, including patients, their guardians and/or families, and related healthcare professionals. the following narrative review aims to better understand the existing evidence for perinatal and labor - related associations with autism. the etiology of autism is unknown, although perinatal and neonatal exposures have been the focus of recent epidemiologic research. exposure to certain medications such as perinatal medications, antiepileptic drugs, supplements, and ssris has been studied as well as drug exposure to cocaine and smoking. artificial insemination and fertility medications, meconium aspiration syndrome, and birth weight have also all been subjects of focus and the respective association with pregnancy and neurodevelopmental complications such as autism. several studies have suggested the association between perinatal factors and the onset of autism. a recent case control study done in new south wales, australia, focused on determining risk factors for autism. four factors were shown to be significantly associated with autism : being male, premature birth, maternal age greater than or equal to 35 years, and mothers born outside of australia, with highest risk found in mothers from north - east or south - east asia. a separate study from western australia concluded that autistic children generally had parents who were older and had greater frequencies of threatened abortions, epidural anesthesia, labor induction, and labor duration of less than 1 hour. these children were more often born first, had a low apgar score, and tended to have more complications. similar studies using a swedish medical birth registry observed different factors that affected autism spectrum disorders. the researchers found that prematurity, low apgar scores, growth restriction, or macrosomia, and if the mother was from sub - saharan africa or east asia had a positive correlation with autism spectrum disorders. information obtained from the finnish prenatal study of autism and autism spectrum disorders correlated advanced paternal age with an increased risk of asd. a group in norway assessed the association between interpregnancy lengths and asd and found that interpregnancy intervals for less than 9 months and interpregnancy between 9 and 11 months carried an increased risk for asd. subjects used in an australian study were diagnosed with autism spectrum disorder and compared to control subjects. the mothers of autistic children were studied, and it was demonstrated that the cases with autism had more pregnancy complications. the mothers had greater frequencies of threatened abortions, epidural caudal anesthesia use, labor induction, and labor duration of less than one hour. they were more likely to have experienced fetal distress, been delivered by an elective or emergency cesarean section, and had an apgar score of less than 6 at one minute. it was concluded that although autism is unlikely to be caused by a single obstetric factor, there was an increased prevalence of obstetric complications among autism cases that is most likely due to underlying genetic factors and an interaction of these factors with the environment. another australian study evaluated maternal conditions and perinatal characteristics associated with asd and intellectual disability. although a weak correlation was found between poor intrauterine environments and asd with intellectual disabilities, there was no relationship found between asd without intellectual disabilities and perinatal factors. a recent study illustrates that the neuropathology of some individuals who are diagnosed as having asd may actually also have focal cortical dysplasias, which could explain the high incidence of sensory irregularities and seizures in asd diagnosed patients. no specific article examined specific aspects such as room temperature, lack of equipment, time spent with the mother, or proximity to other neonates. however, some sources did cite other factors that could potentially be associated with the delivery room itself. these include the type of birth, such as whether it was breech, a prolonged birth, or an induced birth, along with various forms of fetal measures such as birth cry, fetal distress, low heart rate, or high fetal blood pressure [2, 3, 1315, 60 ]. feeding practices have also been looked at, as well as maternal states such as high temperature or an unhappy maternal emotional state [1719, 58 ]. lastly, the environmental impact of perinatal air pollutants has also been observed [20, 21 ]. because these studies have pointed to all of these various factors which in and of themselves can all occur within a delivery room atmosphere, they warrant a further look into even more specific delivery room attributes to see if there is a more direct association between them and autism. beyond the cases conducted to study the relationship between pregnancy complications and augmented childbirth to autism, several studies have been done to investigate the association between prenatal exposures to 2 adrenergic receptor agonists to autism spectrum disorders. these agents include terbutaline, which is commonly used as a tocolytic and a bronchodilator in obstetrics. terbutaline can cross the placenta and potentially affect the developing neonatal brain. in a study of neonatal rats, zerrate. found neuroinflammatory and behavioral changes, similar to that in autism, after administering terbutaline to subjects. the results demonstrated that terbutaline increased microglial activation in the subjects ' cerebral cortex, cerebellar, and cerebrocortical white matter. nucleotide polymorphism of the 2 adrenergic receptor gene has also been implicated as risk factors for autism, namely, gly 16 and glu 27 [24, 25 ]. the researchers of independent subjects and dizygotic twins found that activation of receptors with either polymorphism has been link with increased signaling, secondary to decreased downregulation of the 2 adrenergic receptor gene. critics of these genetic predisposition argued that since many genes are involved in the etiology of autism, the role of any single gene polymorphism in causing autism is small. the effect of each gene is further diluted by variable penetrance. in clinical studies, researchers from kaiser permanente northern california hospitals examined 291 children born from 1995 to 1999, compared to 284 controls. they showed that there was no evidence linking 2 adrenergic receptor agonist exposure in pregnancy with an increase in autism risk, except exposure to terbutaline during the third trimester for more than two days [26, 62 ]. the authors suggested that future studies with larger sample sizes would be helpful to endorse or challenge their findings. proposed that permanent changes in autonomic tone could result in neurological changes in the fetus, especially after two or more weeks of high dose 2 adrenergic receptor agonist exposure. in addition to environmental factors, infection and disease may play a role in the development of autism. children born to mothers with antiphospholipid syndrome were observed to have a higher frequency of autism spectrum disorders in comparison to mothers without antiphospholipid syndrome, which further supports findings that perinatal complications increase the risk of autism in children. another study was organized to determine asd in children born preterm and the role of exposure to perinatal inflammation. it was a review that covered the evidence supporting the idea that prenatal infection on the central nervous system may increase the incidence of autism spectrum disorders. an increase in c - reactive protein indicates inflammation, which is common in infection. this increase in c - reactive protein has been found to have a correlation with a risk for autism spectrum disorders. although it has been shown that autism is unlikely to be caused by a single factor, there have been many studies conducted studying the relationship between the use of certain medications during pregnancy and the associated risk of autism. for example, the increased prevalence of the use of anticonvulsant medications during pregnancy is correlated with an increased risk of autism. valproate is used for the treatment of epilepsy and other neuropsychological disorders and is considered one of the only available treatment options for women of childbearing potential. recent studies, however, have shown that exposure to valproate during pregnancy indicates a significantly increased risk of autism spectrum disorder, even after adjusting for maternal epilepsy. in fact, valproate use during pregnancy showed an 11.3% increased risk in developmental malformations, and it relied heavily on the dosage prescribed. women who were prescribed increased doses of valproic acid showed a twofold increase in risk of developmental malformations and significantly reduced intelligence. studies exposing pregnant rats and mice to valproate demonstrated increased autistic - like behaviors in the offspring. this behavior included social behavior deficits, increased repetitive behaviors, and deficits in communication. the animal studies performed by roullet have been used as a model to ameliorate and improve upon studies that focus on the association between the risk of autism and the maternal need for anticonvulsants. the current recommendation of health care professionals is to avoid anticonvulsant drug use such as valproate during pregnancy. it is important, however, to assess the intrinsic iatrogenic risk of birth defects or perinatal complications and the general safety for the expectant mother before discontinuing anticonvulsants. prenatal folic acid supplements have been shown to reduce the risk of neural tube defects in children and are associated with a lower risk of autistic disorder. in this study following 270 children, of the children whose mothers took folic acid, 0.1% developed autistic disorder. those children whose mothers were not exposed to folic acid comprised 0.21% of the group. this study also analyzed the use of fish oil supplements, which showed no association with autistic disorder. although no causality was found, it was determined that prenatal folic acid supplements taken during pregnancy were associated with a decreased risk of autistic disorder. vitamin d, which has a unique role in brain homeostasis, embryogenesis and neurodevelopment, neural differentiation, and gene regulation, has recently been proposed as a possible environmental risk factor for autism during early childhood. children with autism had significantly lower serum levels of 25-hydroxy vitamin d than healthy children. on the other hand, recent studies have shown that a wide range of disorders, including neuropsychiatric disorders and autism, are associated with increased homocysteine levels in biological fluids. various b vitamins such as b6 (pyridoxine), b12 (cobalamin), and b9 (folic acid) are required as cofactors by the enzymes involved in homocysteine metabolism. therefore, it is crucial to monitor the homocysteine levels in body fluids of autistic children. it can provide information on genetic and physiological diseases, improper lifestyle (including dietary habits), and a variety of pathological conditions. a study was conducted to evaluate the association between autism and developmental abnormalities in children with perinatal cocaine exposure. it was concluded that most of the cases had language delays and difficulties with expressive skills and communication disorders. significant neurodevelopmental abnormalities were observed and a high frequency of autism was found in children exposed to cocaine, especially in comparison to those exposed to alcohol or opiates alone. studies have been done determining the effects of smoking in a pregnant patient and the risk of autism in the mother 's offspring. epidemiological data were obtained from children with autism, including residential history of the mother, which was compared to the traffic - related air pollution assigned to each location using measures from the environmental protection agency 's air quality system data. it was assessed that maternal smoking was related to a modest increase in the risk of pervasive developmental disorders, however, there was no known association between maternal smoking and childhood autism. interestingly, the amount of air pollution that a mother and child were exposed to including traffic related air pollution and nitrogen dioxide, both during pregnancy and during the first year of life, was associated with autism. selective serotonin reuptake inhibitors (ssris) are a class of drugs currently and increasingly prescribed for anxiety disorders and depression. these drugs, such as fluoxetine, sertraline, and citalopram, work by inhibiting the reuptake of the neurotransmitter serotonin, resulting in a greater amount of serotonin at the synapse. much like anticonvulsants and other drug exposures, the use of ssris in pregnant women and its association to autism have been explored. previous studies have shown an increased blood level of serotonin in individuals affected with autism spectrum disorder, warranting the exploration of a possible association. the risk of autism spectrum disorders was shown to be doubled with maternal use of ssris in a recent case controlled study of 298 children, with prenatal exposure reported for 20 of those children. this was seen by evaluating medical records, considering both the ssri exposure and the mental health history of the mother, and pointing to a high association with ssri treatment and autism during the first trimester. however, the sample size for this study is small. a recent study conducted in sweden by rai. it demonstrated an association between in utero exposure to both ssris and nonselective monoamine reuptake inhibitors with an increased risk of autism spectrum disorders, particularly without intellectual disability. however, there is again a limitation with interpreting medical records, such as prescribed versus actual use of ssris. further, other studies using animal models show that preclinical findings in rodents exposed to ssris during development point to an increase in depression and anxiety, exhibiting alteration in social behaviors in the offspring. the data show adverse effects of maternal mental illness on pregnancy outcomes and infant neurodevelopment but are not robust enough to discourage the use of ssris during human pregnancy. in a study by gur., it was shown that the manipulation of serotonin during early development in both in vitro and in vivo models disturbs characteristic chemoarchitectural and electrophysiological brain features. this includes changes in raphe and callosal connections, sensory processing, myelin sheath formation, neophobia, and disrupted juvenile play behavior. these findings indicate that serotonin homeostasis is necessary for proper brain maturation and fetal development. although ssris are not contraindicated for use during pregnancy, the effects should be examined more carefully. in a similar study done by simpson., animal models were used to study the importance of serotonin homeostasis for the development of rat pups ' brains. it was demonstrated that when the serotonin levels were altered by ssris during early development of brain structure, the functions were altered. conversely, other recent studies have suggested that there may actually not be a strong association between ssri intake and the subsequent development of autism. a recent cohort study of live births in denmark from 1996 to 2005 involving 3,892 cases of asd found no significant association, as only 52 cases of ssri use were found. larger observational studies are required to establish a true link between the use of ssris and autism. furthermore, it must be noted that the effect of depression itself as a factor in autism separate from the administration of ssris is a possibility. thus, it may be that, in some cases, depression itself or other concomitant factors have a combined effect that is not necessarily due to the use of ssris. there is an increase in prevalence in the number of women utilizing fertility measures, thus calling for studies to determine the association between assisted reproductive therapy and neurodevelopmental complications, and more specifically autism. shows that assisted reproductive therapy and a history of infertility do not increase the risk for autism spectrum disorders. however, the age of the mother needs to be taken into consideration, as artificial insemination among older mothers has been studied to lead to more complications. women under the age of 35 using fertility therapies such as ovulation inducing drugs and artificial insemination did not increase the risk for having a child with autism, while researchers suggest that the rise in fertility treatments in older women above 35 may increase the risk of having a child with autism for those using artificial insemination and/or ovulation inducing drugs. while the study is strong in that it uses a large national sample, it is important to note that both diagnoses and fertility treatments were self - reported. conversely, a recent cohort study conducted by bay. showed a statistically significant increase in autism and other mental health disorders following ovulation induction. this was done through a cohort study using 555,828 children in denmark using information from a birth register and evaluated with statistical analyses. there were no significant associations found with in vitro fertilization / intracytoplasmic sperm injection (icsi) or with cryopreserved embryos or gametes, types of hormones, or cause of infertility. the strength in this study includes long followup and low risk of selection bias, but numerous subgroup analyses may lead to risk of chance influencing significant associations. in an epidemiological study conducted in india by mamidala., maternal hormonal intervention was a significant risk factor for asd, possibly due to maternal hormone disturbances causing obstetric complications, which in turn are a risk for asd. while there was a large sample size, thus, with differing results it is evident that more specific studies need to be conducted on infertility treatments keeping in mind specific subtypes and their effect on autism in children. controls for age can also be accounted for, and adverse effects can be more accurately studied in the future. meconium aspiration syndrome, which occurs when a fetus who is under stress and not getting enough oxygen inhales waste products inside the womb, was linked to a sevenfold increase in the likelihood that a child would later develop autism. a study examined the incidence of autism in the neonatal intensive care unit and risk factors connected with autistic development. findings in this study indicate that children with autism had a significantly higher history of meconium aspiration syndrome than the controls. however, there is insufficient evidence to implicate any one perinatal or neonatal factor in autism etiology, although there is some evidence to suggest that exposure to a broad class of conditions reflecting general compromises to perinatal and neonatal health may increase the risk. therefore meconium aspiration syndrome can not be the cause of autistic disorder in children, but it can be a contributing factor. a significant fourfold increased risk was observed in low birth weight females for autism accompanied by mental retardation. in low birth weight males though this implies a certain etiological premise of difference in gender, it is also important to note that while the population cohort design is strong, there was not a large number overall of the lower birth weight categories in females. an earlier study by wilkerson supports this finding as it determined that birth weight was a factor for the risk of autism spectrum disorders, with significant associations between autism and gestational age, maternal morphology and intrauterine stress. specifically, it is the prescriptions taken during pregnancy, the length of labor, viral infection, abnormal presentation at delivery, and finally low birth weight that were associated with autism. in this study only 183 autistic children were considered and medical conditions of the mother based on a maternal self - report. their studies suggest that the abnormal growth in children with autism spectrum disorders may be related to the causal factors that also increase the risk for asd, as it was found that children with asd had proportionally smaller head circumference compared to height during their first year of life, which was absent in premature / low birth weight children with asd. even more importantly, the body grew faster than the head for those with asd by the end of the first year of life, suggesting a closer following of length to circumference during development. however, there may have been some recall bias and head circumference could only be measured on the first year of life all within a modest sample size, indicating a need for replicating the study in a larger cohort. similar studies were conducted illustrating that relative microcephaly was more frequent in children with asd. however microcephaly can also be caused by other factors such as low intelligence and heredity, which could not be controlled for, and also only focused on measurements obtained at birth. expanding on this, a matched case control studies of children in stockholm county showed a correlation between low growth for gestation age and preterm birth with asd combined with intellectual disabilities. the strength of this study lies in its comprehensive sample and size but did not measure some other maternal factors such as substance abuse and nutritional influence. several other studies have focused on maternal changes during pregnancy and their association with autism. a population - based study in utah (us) looked at prenatal variables of autistic children and focused on gestational weight. it was found that maternal prenatal weight gain, but not prepregnancy body mass index, is directly related to an increased risk of asd. because of use of cohort and control groups, it not only allowed a representative sample of utah children, but also as a surveillance study could not directly assess cases and controls. however, a recent longitudinal study using birth data from 4800 children from the united states showed that children born to over-/underweight mothers during prepregnancy had increased incidences of being born with a low birth weight. the group noted that low birth weight increased chances of developing asd, as well as rapid head growth. this study concluded that abnormal maternal gestational weight indirectly caused an increased chance of developing asd. in addition to a lower birth weight population, a secondary analysis longitudinal design study using cranial ultrasound evidence of 14 study participants diagnosed with asd identified that children with ventricular enlargement were strong indicators of the potential risk for autism spectrum disorders. overall, these birth weight studies illustrate that more research with larger populations needs to be conducted to determine whether autism etiology is genetic in relation to birth weight, whether birth weight is a risk factor or a cause of autism, how much mother 's gestational age plays a role, and whether pre- or postpregnancy weight of the mother has a larger effect on prevalence of autism and asd as research currently supports a variety of findings. since the early 1990s, a number of studies have suggested the link between asd and labor induction, such as the use of oxytocin. the research that ensued found significantly different results between population - based studies and clinic based studies. 's analysis showed induction increase in the risk of autism by 72% in three clinic based studies, while the selected population - based studies reported no association. recently, however, labor induction was positively correlated with autism in a large population study comparing birth records in north carolina (19901998) and corresponding school records of autistic children (19972007). the study controlled for environmental factors such as socioeconomic status, maternal health, pregnancy - related events and conditions, and birth year. the lack of differentiation of underlying complications in pregnancy, which lead to eventual use of induction, could be the cause of these conflicting findings. fetal malposition, fetopelvic disproportion, excess sedation, inadequate contraction, and rupture of fetal membranes before the onset of active labor. however, these complications differ in severity and may require varied quantity of induction agent used at distinctive stages of labor. further research is necessary to differentiate between the underlying obstetric complications that lead to the eventual use of induction and their respective potential association with autism. a review of literature found limited number of researches on the topic of labor time and autism. the research done by glasson. purports that labor duration of less than 1 hour carries an increased risk of developing asd. on the other hand, wilkerson. found that a prolonged labor (greater than 1824 hours) resulted in an increased risk for autism. one of the latest cohort studies on the topic that involved 268 subjects also suggests positive association between prolonged labor and autism. 's meta - analysis of nine studies on this subject, five have neutral results, only three have positive association, and one has negative association. current understanding suggests that hypoxia is one of the complications of prolonged labor, along with infection, or head trauma from extended pressure. all of these complications could lead to permanent damages in the neonates ' developing brain. neonatal oxygen deprivation can be quantified by several measurements, including low apgar score, fetal distress, caesarian delivery, threatened abortion, and hemorrhage during pregnancy. four large cohort studies conducted across sweden, australia, and denmark found that an apgar score of 7 has a strong predictive value of autism [18, 5456 ] ; similar findings were reported by garderner.. hypothesize that the increased prevalence of autism may be in part due to higher success in obstetric and neonatal care, which resulted in an increased survival rates of hypoxic neonates with brain damage. in summary, some investigations have shown an association between length of labor and autism, but findings have been inconsistent owing to differences in sampling and methods. while there is still room for much further research, current understanding suggests that length of labor is possibly associated with autism. prolonged delivery due to oversedation, inappropriate position of the fetus, and unbalanced fetopelvic size and oversedation, which results in induction, can play a role. it is also possible that children who become autistic may also be slow to deliver that the prolonged delivery is a symptom of, rather than a cause of, autism. in summary, several attributes of the perinatal and labor experiences may be associated with the development of autism. yet, importantly, these factors may work separately or may work together with other stimuli. table 1 provides a summary of the findings in the studies explored in this article. while association between 2 adrenergic receptor agonists and autism spectrum disorders is not strong, exposure to terbutaline during the third trimester has shown to increase incidence. furthermore terbutaline has been shown to increase microglial activation associated with behavioral abnormalities akin to autism. labor induction and augmentation appear to be an area of association. while one study showed that children whose mothers received augmented and/or induced childbirth showed a greater risk for autism compared with controls, further research is needed to prove an association. the role of maternal disease can not be ignored as antiphospholipid syndrome and perinatal inflammation may have important roles in the eventual development of autism. cocaine has been associated with an increase in the onset of neurodevelopmental abnormalities, but the direct influence on autism development is still lacking. while a link between smoking and autism could not be established, air pollution may have a role. several studies have implicated ssris in autism, but a larger recent cohort study showed no association. artificial insemination and fertility medications were largely found to have to link, yet reports are conflicted. attributes of the delivery room and birthing experience also may have a role in autism. while some evidence suggests that meconium aspiration syndrome may have a link as a contributing factor, more studies are needed. birth weight, gestational age, and preterm birth may correlate with autism and intellectual disabilities. the identification of prenatal factors that are linked to autism may serve as a paradigm shift in our current view and management of autism. the fact that several risk factors during the prenatal and labor periods are implicated in autism should prompt the medical community to focus on the pregnancy and labor periods as preventive measures to curb the incidence of autism.	autism is a neurodevelopmental disorder marked by severe deficits in social communication and interactions. it is a complex condition that lacks an established preventive method, warranting a need for research to identify possible environmental triggers. the identification of external factors particularly perinatal risk factors forms the initial critical step in preventing and alleviating risks. we conducted a literature review to assess evidence suggested in the worldwide literature. perinatal risk factors that have a suggested association include 2 adrenergic receptor agonists, labor induction and augmentation, maternal infection and disease (i.e., antiphospholipid syndrome), antiepileptic drugs, cocaine use, and oral supplements. smoking has not been found to have a direct association. pollutants, selective serotonin reuptake inhibitors, artificial insemination, and fertility medications may have a link, but results are often conflicted. factors related to the delivery room experience may be associated with meconium aspiration syndrome, birth weight, and labor time. several risk factors during the pregnancy and labor periods have been associated with autism ; yet further studies with large populations are needed to establish definitive associations. the fact that several risk factors during the prenatal and labor periods are implicated in autism should prompt the medical community to focus on the pregnancy and labor periods as preventive measures to curb the incidence of autism.
benign prostatic hyperplasia (bph) is a common problem in aging males and the most common cause of bladder outlet obstruction (boo) in men over 50 years old. bph is known to be associated with lower urinary tract symptoms (luts) and acute urinary retention. however, several studies have suggested that it is not the prostate volume alone that causes luts, but rather the extent of protrusion of the enlarged prostate lobe into the bladder. intravesical prostatic protrusion (ipp) is a gross structural morphologic change due to the development of prostatic adenoma. bph with a mildly enlarged prostate and high ipp can cause obstruction by creating a ' ball - valve ' effect and abnormal movement of the bladder due to the inhibition of the funnel effect of the bladder neck at voiding [6 - 8 ]. some studies have implied the correlation of ultrasonically measured ipp and luts related to bph. for example, tan and foo discovered that the outcomes of a voiding trial correlated with the degree of ipp. suggested ipp to be a parameter for predicting boo because of its good correlation with the results of conventional pressure - flow study. the current gold standard of removing a boo due to bph is a transurethral resection of the prostate (turp). recently, however, various lasers and advanced techniques have been applied. as a result, laser photoselective vaporization of the prostate (pvp), a minimally invasive surgical technique, has been replacing conventional turp. pvp has been reported to be a safe and effective technique for removing an enlarged prostate. in short- and long - term follow - up studies, the postoperative results, including symptom improvement, were similar to turp, and reoperation risks and complications such as retrograde ejaculations were similar to or lower than turp. because of its safety and effectiveness, pvp can be used with patients who take oral anticoagulant medications. as mentioned before, however, technical difficulty remains, especially with bph patients who have severe ipp or a high bladder neck. the aim of this study was to determine the efficacy of pvp using the 120 w greenlight high performance system (hps) laser for the treatment of bph groups according to the degree of ipp. from april 2009 to august 2011, this study was conducted on patients who visited a urologic outpatient clinic because of luts and underwent trus (transrectal ultrasonography), uroflowmetry, and measurement of postvoid residual volume (pvr), prostate - specific antigen (psa), and international prostate symptom score (ipss). operation indications were persistent luts regardless of proper medication with an a - blocker or 5a - reductase inhibitor, refusal of medical treatment, and persistent hematuria originating from the prostate. patients with urologic tumors, uncontrolled diabetes, neurogenic bladder including detrusor overactivity, urinary tract infection, or bladder stones that could affect voiding were excluded. patients with a psa value over 4.0 ng / ml underwent a trus - guided extended prostate biopsy. in these patients, only patients with pathologically proven bph the prostate was assessed by trus by moving the sagittal scan of the ultrasound probe both horizontally and longitudinally and examining the bladder neck for protrusion of the prostate into the bladder. ipp was measured from images of the prostate obtained from trus by using a midline sagittal image and drawing a line from the anterior to posterior intersections of the bladder base and tip of the ipp. for more precise ipp measurement, the vertical distance from the tip of the protrusion to the circumference of the bladder at the base of the prostate gland was assessed by one urologist. according to the grading system suggested by nose., cutoffs of ipp were set at 5 mm and 10 mm or greater. bladders with ipp of 5 mm or less were assigned to group 1, 5 to 10 mm to group 2, and greater than 10 mm to group 3. ipss with quality of life (qol), maximal urinary flow rate (qmax), and pvr were used for pre- and postoperative comparative variables. during the operation, operation time, laser exposure time, and the amount of laser energy were evaluated. all patients underwent assessment of ipss, qmax, and pvr at 1, 3, 6, and 12 months after the operation. statistical analysis was performed by using ibm spss ver. 18.0 (ibm co., armonk, ny, usa). a paired t - test was used to analyze the pre- and postoperative variables in the patients. a repeated - measures analysis of variance was used to analyze the correlations between groups. a p - value of less than 0.05 was set as the threshold for statistical significance. from april 2009 to august 2011, this study was conducted on patients who visited a urologic outpatient clinic because of luts and underwent trus (transrectal ultrasonography), uroflowmetry, and measurement of postvoid residual volume (pvr), prostate - specific antigen (psa), and international prostate symptom score (ipss). operation indications were persistent luts regardless of proper medication with an a - blocker or 5a - reductase inhibitor, refusal of medical treatment, and persistent hematuria originating from the prostate. patients with urologic tumors, uncontrolled diabetes, neurogenic bladder including detrusor overactivity, urinary tract infection, or bladder stones that could affect voiding were excluded. patients with a psa value over 4.0 ng / ml underwent a trus - guided extended prostate biopsy. in these patients, only patients with pathologically proven bph the prostate was assessed by trus by moving the sagittal scan of the ultrasound probe both horizontally and longitudinally and examining the bladder neck for protrusion of the prostate into the bladder. ipp was measured from images of the prostate obtained from trus by using a midline sagittal image and drawing a line from the anterior to posterior intersections of the bladder base and tip of the ipp. for more precise ipp measurement, the vertical distance from the tip of the protrusion to the circumference of the bladder at the base of the prostate gland was assessed by one urologist. according to the grading system suggested by nose., cutoffs of ipp were set at 5 mm and 10 mm or greater. bladders with ipp of 5 mm or less were assigned to group 1, 5 to 10 mm to group 2, and greater than 10 mm to group 3. ipss with quality of life (qol), maximal urinary flow rate (qmax), and pvr were used for pre- and postoperative comparative variables. during the operation, operation time, laser exposure time, and the amount of laser energy were evaluated. all patients underwent assessment of ipss, qmax, and pvr at 1, 3, 6, and 12 months after the operation. statistical analysis was performed by using ibm spss ver. 18.0 (ibm co., armonk, ny, usa). a paired t - test was used to analyze the pre- and postoperative variables in the patients. a repeated - measures analysis of variance was used to analyze the correlations between groups. a p - value of less than 0.05 was set as the threshold for statistical significance. the patients ' mean age was 72 years (range, 52 to 88 years). the men studied, 216 had an ipp of 0 to 5 mm (group 1), 135 had an ipp of 5 to 10 mm (group 2), and 38 had an ipp of 10 mm or more (group 3) (table 1). the mean age of each group was 70.817.99 years in group 1, 70.888.64 years in group 2, and 74.899.66 years in group 3. there were no significant differences in age or psa level between the groups (table 1). the prostate volumes of group 1, group 2, and group 3 were significantly different. the preoperative total ipss score, voiding symptom score, storage symptom score, and qol score were analyzed and there were no significant differences except for the storage symptom score. there was a significant difference in the storage symptom scores between group 1 (8.55.1) and group 3 (10.84.9) and between group 2 (9.04.5) and group 3 (10.84.9) (p0.05) (table 1). the operation time, laser exposing time, and total energy were analyzed, and there were no significant differences between the groups (table 1). there were no intraoperative or immediate postoperative complications due to uncontrollable gross hematuria. in the time frame of 1, 3, 6, and 12 months after the operation, all variables were reevaluated and compared with the preoperative data. in group 1, the total ipss score, the voiding symptom score, and the storage symptom score had significantly improved at 1 month postoperatively compared with the preoperative data (figs. 1 - 3). qmax, pvr, and the qol score at 1 month following the operation also showed similar significant improvement when compared with the preoperative data (figs. these improvements were maintained for 12 months after the operation. in the analysis of group 2 and group 3, there were similar postoperative improvements compared with the preoperative data (p0.05) (table 1). the operation time, laser exposing time, and total energy were analyzed, and there were no significant differences between the groups (table 1). in the time frame of 1, 3, 6, and 12 months after the operation, all variables were reevaluated and compared with the preoperative data. in group 1, the total ipss score, the voiding symptom score, and the storage symptom score had significantly improved at 1 month postoperatively compared with the preoperative data (figs. 1 - 3). qmax, pvr, and the qol score at 1 month following the operation also showed similar significant improvement when compared with the preoperative data (figs. these improvements were maintained for 12 months after the operation. in the analysis of group 2 and group 3, there were similar postoperative improvements compared with the preoperative data (p<0.05). at 1 month after the operation, the clinical indexes of group 1 were compared with the clinical indexes of group 2 and group 3. no significant differences were found in the intergroup analysis, including for the total ipss score, storage symptom score, voiding symptom score, qol, qmax, and pvr. these results were sustained at 3, 6, and 12 months after the operation (p<0.05). during the 12-month follow - up period, none of patients in the three groups developed hematuria, urethral stricture, infection, or any other complication. small numbers of patients (n=14) complained of mild dysuria, which improved with conservative care and proper medications. in the preoperative comparison of the ipss, the storage symptom scores were significantly different between groups 1 and 2 and group 3. group 3, which had more severe ipp, had a higher ipss storage symptoms score. our study demonstrated that voiding symptoms, storage symptoms, and qol were improved after 120 w greenlight hps pvp in bph patients with ipp. also, these improved states were maintained for 12 months after the operation regardless of degree of ipp. recently, several studies have reported the importance of anatomical factors in evaluating men with luts. ipp represents the median and lateral lobes of a prostate that have protruded into the bladder and causes a ball - valve type of obstruction and disruption of the funnel effect of the bladder neck, which increases urethral resistance. in addition, the presence of median lobe enlargement causes dyskinetic movement at the time of micturition. with severe ipp, the prostatic enlargement causes the bladder outlet to narrow and changes the shape of the internal urethral orifice. to open a compressed urethra, detrusor overactivity and low bladder compliance are the manifestation of an impaired detrusor as a result of boo. moreover, there is a higher risk of detrusor impairment and lower bladder compliance with more severe ipp. ochiai and kojima emphasized the importance of bladder weight and prostatic configuration in the determination of intravesical obstruction. these anatomical configurations of the prostate, in particular the grade of ipp, could affect voiding. in more detail bladder wall thickening increases the extracellular collagen and nerve thickening, thereby inducing storage symptoms by activating the unmyelinated c nerve fibers, which are absent in normal bladder. after the greenlight hps pvp, these storage symptom scores decreased in all groups. these findings are underpinned by the results of other reports showing that laser therapy is effective for reducing overactive bladder symptoms because the laser can vaporize the entire urothelium and submucosal tissue of the prostate urethra and bladder neck. furthermore, in our study, the preoperative difference between group 1, group 2, and group 3 was improved similarly. these results suggest that proper elimination of boo could induce favorable outcomes, even in patients with severe ipp. also, these overactive bladder symptoms have been found to be related to storage symptoms rather than voiding symptoms, especially after turp. they measured ipp through abdominal sonography and concluded that ipp affects voiding symptoms more than storage symptoms. in our study, however, ipp had a relationship with the storage symptom score rather than the voiding symptom score. this would be the case because the storage symptom score of the ipss is underestimated compared with the voiding symptom score. we measured ipp through trus, and ipp showed a significant correlation with the storage symptom score. it may be that the ipp not only created the boo and voiding symptoms but also aggravated the storage symptoms by lengthening the prostatic urethra and irritating the bladder trigonal area. transabdominal ultrasonography can be used for patients who have a history of anal surgery or rectal disease. too much urine in the bladder can compress the ipp and too little urine can make ipp measurement difficult. however, it does not require bladder filling ; thus, more accurate ipp measurement is possible. in this study, ipp was measured with trus and the relation with the preoperative storage symptom score and ipp was affirmed. we suggest that ipp measurement with trus would complement the deficiencies of the ipss in a bph patient with luts symptoms. to verify this, a prospective study would need to be carried out rather than a retrospective study. in our study, in addition to the storage symptom score, voiding symptom scores, total ipss scores, and qol scores were significantly improved after the operation. these improvements were compared among each group and there were no intergroup differences after the operation. according to our results, preoperative ipss storage symptoms were affected by ipp and postoperative ipss storage symptoms were significantly improved in all ipp groups. we suggest that these findings were the result of the method used to resolve the boo, that is, laser techniques. the technical issue of hps laser pvp is that it is difficult to effectively vaporize the prostate, especially in patients with a high bladder neck, severe ipp causing difficulty in recognizing the ureteral orifice, and bladder neck bleeding during the vaporization. hence, hps laser pvp is generally recognized to be more difficult for effectively removing ipp than is conventional turp. in the hps laser pvp technique and training recommendation, gentle fingertip movements of the laser fiber and cystoscope are recommended, and progressive smoothening of the bladder neck contour is needed to avoid ureteral orifice injury. effective bladder neck vaporization can be achieved through the use of these techniques and can lead to favorable results. qmax and pvr are objective parameters that can be monitored after the elimination of boo. in group 1, the results at postoperative 1 month showed marked improvement compared with the preoperative state, and the improvements remained over 12 months. pvr improved over time after the operation, and the difference was significant in all groups (p<0.05). according to these results, we can assume that proper elimination of boo will improve the subjective and objective parameters, regardless of the volume of prostate. it did not have a long follow - up time and was a retrospective study. although our findings are statistically significant, they may not satisfactorily explain the actual mechanism. we propose that this study serve as the initiation of a larger and longer validation study of the importance of prostatic configuration. however, there is no significant correlation between the degree of ipp and postoperative variables after prostate vaporization. also, the degree of ipp does not affect the short- and long - term results of pvp.	purposeto evaluate the efficacy of photoselective vaporization of the prostate (pvp) with the 120 w greenlight high performance system (hps) laser for the treatment of benign prostatic hyperplasia (bph) with intravesical prostatic protrusion (ipp).materials and methodsthis study was conducted on 389 bph patients who underwent pvp with the 120 w hps laser from april 2009 to august 2011. the patients were divided into groups according to ipp : group i was defined as ipp of 0 to 5 mm (n=216), group ii as ipp of 5 to 10 mm (n=135), and group iii as ipp above 10 mm (n=38). prostate volume, prostate - specific antigen, international prostate symptom score (ipss), maximal urinary flow rate (qmax), and postvoid residual volume (pvr) were assessed and checked at postoperative 1, 3, 6, and 12 months.resultsthere was a significant difference in the mean prostate size in each group (p<0.05). the preoperative total ipss score, ipss voiding symptom score, and quality of life score were not significantly different. however, the ipss storage symptom score was significantly different between groups 1 and 2 and group 3. ipss scores, qmax, and pvr at postoperative 1, 3, 6, and 12 months showed significant improvement compared with preoperative values.conclusionsthe degree of ipp can affect storage symptoms. however, there is no significant correlation between the degree of ipp and postoperative results. also, the degree of ipp does not affect short- and long - term pvp results. proper elimination of bladder outlet obstruction is important for symptomatic relief.
the use of hydrogen peroxide to treat discolored and stained teeth is older than the technique of night guard vital bleaching with 10% carbamide peroxide. for in - office treatment, the bleaching agent indicated is hydrogen peroxide at high concentrations (3040%), which is a strong oxidizing agent. hydrogen peroxide at high concentrations must not come into contact with soft oral tissues, and patients have reported both severe and mild tooth hypersensitivity during and after bleaching. bleaching with 10% carbamide peroxide using a mouthguard was suggested by heymann and haywood in 1989. the concentration of hydrogen peroxide in 10% carbamide peroxide is 10 times less than that in 35% hydrogen peroxide, resulting in low levels of hypersensitivity and lower surface morphology changes. in this technique, patients use a 10% carbamide peroxide gel for 38 h daily, whereas in the hydrogen peroxide technique, 35% hydrogen peroxide is applied to the buccal surfaces of the teeth for short periods of time. nightguard vital bleaching with carbamide peroxide is considered an effective and safe technique for bleaching teeth. in contrast, the use of high concentrations of hydrogen peroxide remains questionable, and some researchers have expressed concerns regarding the widespread use of this technique. several studies have evaluated different types of peroxides at various concentrations, and have expressed concerns about such bleaching techniques and pointed out the limitations of those techniques. these concerns are related to changes in the enamel morphology and mineral content, and to effects on the organic components of dentin and on the dentin however, few investigations have aimed at studying and comparing the effects of carbamide peroxide and hydrogen peroxide on the mechanical and physical properties of dentin, even though these effects are clinically important. the purpose of this study was to compare the biaxial flexural strength and modulus of bovine dentin using dentin disks treated with hydrogen peroxide, disks treated with carbamide peroxide, and disks kept unbleached as a control group, simulating 1 day of a bleaching treatment. the null hypothesis tested was that the two peroxides would not affect the biaxial flexural strength or the flexural modulus of the dentin. thirty sound bovine incisors free from structural cracks and defects were selected for this study. after pumicing, the incisors were stored in a 0.1% thymol solution at 4c for 30 days. bovine dentin disks (0.5 mm thick 6.0 mm diameter) were obtained from the coronal buccal surfaces by use of a hole saw. the disks were polished with 1200- and 2000-grit silicon carbide paper (norton, guarulhos, sp, brazil) and randomly divided into three groups (n = 10) : a control group (unbleached). the disks in this group were kept at 100% humidity for 8 h at 37cdentin disks bleached with 10% carbamide peroxide (opalescence pf, ultradent products inc., salt lake city, ut, usa). each disk in this group was immersed in 0.2 ml of peroxide gel for 8 h at 37c. after bleaching, the disks were thoroughly rinsed with an air / water spray and then testeddentin disks bleached with 38% hydrogen peroxide (opalescence boost pf, ultradent products inc., each disk in this group was immersed in 0.2 ml of peroxide gel for 10 min at 37c and then rinsed with hydrogen peroxide (10 volume). this protocol was performed three times and then, after the last repetition, the specimens were thoroughly rinsed with an air / water spray and tested. a control group (unbleached). the disks in this group were kept at 100% humidity for 8 h at 37c dentin disks bleached with 10% carbamide peroxide (opalescence pf, ultradent products inc., salt lake city, ut, usa). each disk in this group was immersed in 0.2 ml of peroxide gel for 8 h at 37c. after bleaching, the disks were thoroughly rinsed with an air / water spray and then tested dentin disks bleached with 38% hydrogen peroxide (opalescence boost pf, ultradent products inc., salt lake city, ut, usa). each disk in this group was immersed in 0.2 ml of peroxide gel for 10 min at 37c and then rinsed with hydrogen peroxide (10 volume). this protocol was performed three times and then, after the last repetition, the specimens were thoroughly rinsed with an air / water spray and tested. both of the protocols used in this study simulated only one session of bleaching treatment (either a mouthguard technique or an in - office technique). composition and batch number of bleaching agents used in this study are listed in table 1. characteristics of bleaching agents used in this study the disks were individually placed in a custom - made testing jig and tested in biaxial flexure on a universal testing machine (instron 5844, instron corp., canton, ma, usa) at 1.27 mm / min until failure occurred. the maximum load was recorded for each specimen, and the elastic modulus was determined from the linear portion of the stress / strain curve. the formula used for the biaxial flexural strength was : = - 0.2387p(x - y)/b, where is the maximum tensile stress in the center (in mega pascals), p is the total load at fracture (in newtons), x = (1 +) ln (r2 / r3) + [(1) / 2](r2 / r3), y = (1 +)[1 + ln (r1/r3) ] + [(1)(r1 / r3), and b is the specimen thickness (in millimeters) at the origin of the fracture. in the above equations, is poisson 's ratio (a value of 0.25 was used), r1 is the radius of the support circle (in millimeters), r2 is the radius of the loaded area (in millimeters), and r3 is the radius of the specimen (in millimeters). the flexural strength and modulus were calculated using the srs biaxial - testing software package (instron corp., for each test parameter, the data were analyzed statistically using fisher 's protected least significant difference test (with a predetermined = 0.05). statistically significant differences were found among the groups for the flexural strength (p = 0.0309) and for the modulus (p = 0.0003). the dentin disks treated with 38% hydrogen peroxide exhibited a lower flexural strength and modulus than did the control group (unbleached), whereas bleaching with 10% carbamide peroxide resulted in no significant difference in the flexural strength and modulus from the control group (p > 0.05). the 38% hydrogen peroxide produced a significantly lower flexural modulus than did bleaching with 10% carbamide peroxide (p = 0.0002), whereas no significant difference was found between the two bleaching materials for the flexural strength (p = 0.1517). biaxial flexural strength (in mpa) and modulus (in gpa) means (sd) of the bovine dentin some studies have reported changes in the fracture toughness, tensile strength, and flexural strength of human dentin and enamel after bleaching ; however, the type of bleaching agent and the duration of the bleaching treatment were not standardized. although many techniques and bleaching agents were used, the authors of these studies agree that caution is necessary when peroxides are used for prolonged treatment times, since the action of hydrogen peroxide is not specific against only pigments that discolor or stain teeth. the current study was aimed at comparing the effects of 10% carbamide peroxide and 38% hydrogen peroxide on the mechanical properties of bovine dentin, simulating only one appointment or session. in most previous studies, bar - shaped specimens were used and a three - point flexural test was performed to evaluate the flexural strength and modulus of the dentin. in contrast, the present study used a biaxial flexural test and disk - shaped specimens, and it seems to be the first investigation in which the biaxial flexural strength and modulus of dentin have been studied. our biaxial test has the advantage of utilizing small specimens, which allowed the peroxide to be in contact with the entire sample. when the stress exceeds the flexural strength, the loading may lead to dental fracture, which compromises the integrity of the tooth. the biaxial test also provided another important property, the flexural modulus, which is the ratio between stress and strain in the elastic regime : in other words, it tells us how much stress is required to deform the material before the proportional limit is reached. according to the results of this study, one session in which bovine dentin was exposed to 38% hydrogen peroxide (three 10 min applications at 37c) was enough to significantly reduce the flexural strength and modulus compared with the unbleached control group, whereas exposure to carbamide peroxide did not change the mechanical properties of the dentin. thus, the null hypothesis stating that the two peroxides (carbamide peroxide and hydrogen peroxide) would not affect the biaxial flexural strength and flexural modulus of dentin was rejected, since 38% hydrogen peroxide altered both of these mechanical properties. although 10% carbamide peroxide used for just 8 h did not change the biaxial flexural strength and modulus of bovine dentin, some other authors have shown that, depending on the bleaching time, carbamide peroxide may affect the strength of teeth as hydrogen peroxide does. studies have shown that the flexural strength and modulus of dentin can be reduced after 2 weeks or prolonged use (2 months) of 10% carbamide peroxide applied directly to the dentin. in those studies, when the carbamide peroxide application was on enamel, the mechanical properties of underlying dentin was not affect. found that mouthguard and in - office bleaching gels induced collagenolityc activity when applied to human dentin. in that study, 21 carbamide peroxide applications with duration of 8 h / day can induced higher collagen degradation than three applications of 38% hydrogen peroxide for 20 min each. however, seghi and denry investigated the effects of 10% carbamide peroxide on the fracture toughness, hardness, and abrasion characteristics of enamel and reported that the abrasion resistance and toughness decreased after only one 12 h session of bleaching. hydrogen peroxide is a binary compound of hydrogen and oxygen that is a highly reactive oxygen species in aqueous solution. the color of the dentin disks treated with 38% hydrogen peroxide changed to white, demonstrating the visual effect of the oxidizing effects of hydrogen peroxide. however, the carbamide peroxide bleaching did not modify the color of the dentin samples, nor did it change the mechanical properties measured. the dentin disks from the control (untreated) group showed higher values of the flexural modulus than the disks that were bleached with 38% hydrogen peroxide. a study reported that six applications during 3 weeks (1 h each application and 2 days / week) of 35% hydrogen peroxide did not cause a decrease in dentin flexural strength and modulus. it used dentin bars (2 mm 2 mm 20 mm), while this study tested very thin and small dentin disks (0.5 mm thick 6.0 mm diameter), which may be responsible, in part, for differences in the findings of studies. thus, higher concentration of hydrogen peroxide and the dimension of dentin disks that facilitated the contact with entire sample and peroxide reaction caused the reduction of the two dentin mechanical properties. some authors have suggested that peroxides should not be applied over exposed dentin, in order to avoid the effects of bleaching on the mechanical properties of dentin. the main hypothesis for why peroxides affect the mechanical properties of dentin is that peroxides can degrade or disorganize type i collagen fibrils, denature proteins, and cause loss of mineral components attached to the organic matrix. demineralization has been observed and measured by several different methods, such as scanning electron microscopy, polarized light microscopy, microhardness measurements, atomic absorption spectrometry, and fourier transform raman spectroscopy. jiang. described the dissolution of dentin in hydrogen peroxide. according to these authors, the destruction of the organic components was due to the oxidizing ability of hydrogen peroxide while the changes in the mineral components were caused by its acidity. high concentrations of hydrogen peroxide, such as 30 or 40%, can cause alterations in the surface morphology of human enamel and promote an increase in the permeability of dentin. furthermore, as reported by costa., bleaching with 38% hydrogen peroxide for 45 min may lead to irreversible pulp damage in the lower incisors, because of the high permeability of dentin to hydrogen peroxide. however, despite the changes in the properties of dentin induced by hydrogen peroxide, is important to note that in the present study the bleaching agents were applied directly to dentin, whereas in clinical use they should be applied to dental enamel. carbamide peroxide seems to produce less severe side effects than hydrogen peroxide, and this must be considered in relation to clinical indications for the use of bleaching techniques. the exposure of bovine dentin to 38% hydrogen peroxide significantly reduced both the flexural strength and the flexural modulus compared with the no - treatment control, whereas exposure to 10% carbamide peroxide did not significantly affect either of these parameters.	objective : this study evaluated the effects of carbamide peroxide and hydrogen peroxide on the biaxial flexural strength and flexural modulus of bovine dentin.materials and methods : thirty coronal dentin disks (0.5 mm thick 6.0 mm diameter) were prepared from bovine teeth. the disks were randomly divided into three groups (n=10) : a control group (unbleached), a group bleached with 10% carbamide peroxide (8 h at 37c), and a group bleached with 38% hydrogen peroxide (three 10 min applications at 37c). the specimens were tested in a biaxial flexural apparatus held in a universal testing machine at 1.27 mm / min until failure occurred, and the biaxial mechanical properties were calculated. for each test parameter, the data were statistically analyzed by fisher 's plsd test (predetermined = 0.05).results : the group bleached with 38% hydrogen peroxide demonstrated significantly lower flexural strength than the unbleached control group. hydrogen peroxide treatment resulted in a significantly lower flexural modulus compared with the control group and with carbamide peroxide bleaching.conclusion:exposure of dentin to hydrogen peroxide significantly reduced both the flexural strength and the flexural modulus compared with the no - treatment control, whereas exposure to carbamide peroxide did not significantly affect either parameter.
trapp is a cutaneous pseudolymphoma occurring in a wide range of age group from 16 - 71 year. t - cell rich angiomatoid polypoid pseudolymphoma (trapp) of the skin is a recently described unique form of t cell rich cutaneous pseudo lymphoma. it falls within the spectrum of a specific form of pseudolymphoma that presents with rich vasculature amidst a dense inflammatory infiltrate. no recurrence has been seen in any case after a mean follow - up period of 46 months. we report a case of a 13-year - old girl who presented with an asymptomatic skin - colored papule over the medical aspect of the right elbow for the past 6 months. microscopic examination revealed a thinned out epidermis with basket weave keratin [figure 1a ]. the polypoidal nature of the lesion could not be made out as the biopsy did not include adjacent normal skin. the superficial and mid - dermis showed a dense diffuse infiltrate of lymphocytes, plasma cells, and histiocytic collections [figures 1b and 2a ]. grenz zone was present focally [figure 1b ]. however, no epidermotropism was observed. prominent vessels lined by plump endothelial cells were present amidst the dermal infiltrate [figure 2b ]. special stains geimsa and fites were negative for organisms. with the above morphological features, the possibilities we considered were acral pseudolymphomatous angiokeratoma, trapp, and angioplasmacellular hyperplasia. (b) dermal lymphoplasmacytic infiltrate, grenz zone, absence of epidermotropism (h and e, 400) (a) histiocytes admixed with plasma cells (h and e, 400). (h and e, 400) a panel of immunohistochemical markers comprising of cd3, cd20, cd4, cd8, and cd68 were done on the biopsy. there was a predominance of cd3 over cd20 positive lymphocytes [figure 3a and b ]. an admixture of cd4 and cd8 t cells along with collections of cd68 positive histocytes were present [figure 3c ]. (a) t lymphocytes (cd3), (b) few b cells (cd 20) in the dermal infiltrate (h and e, 100), and (c) histiocytes (cd68) (h and e, 400) thus, considering the morphological features and immunohistochemical studies the diagnosis of trapp was favored. cutaneous pseudolymphoma refers to a heterogeneous group of benign reactive t or b cell lymphoproliferative processes of diverse causes that simulates cutaneous lymphomas clinically and/or histologically. the inflammatory infiltrate is band - like, nodular or diffuse and is composed predominantly of lymphocytes with or without other inflammatory cells. depending on the predominant cell type in the infiltrate, they are divided into t and b cell pseudolymphomas. trapp is a distinctive recently described variant of cutaneous pseudolymphoma affecting a wide age group ranging from 16 to 71 years with a female predominance. lesions are solitary and present as small (less than 1 cm), polypoid, erythematous papule with predilection for head and trunk ; knee, shoulder and thigh being less common sites. in the present case, a dense dermal infiltrate of lymphocytes with variable numbers of admixed plasma cells and histiocytes is prototyical of trapp. epidermal collarette grenz zone, prominent vessels lined by plump endothelial cells reminiscent of endothelial venules of lymph nodes, and some degree of telangiectasia are also commonly observed in trapp. our case also exhibited similar histopathological features except for the epidermal collarette which was not present. immunohistochemical studies revealed a predominance of cd3 over cd20 and the cd4 t cells were greater than cd8 cells. the possibilities of acral pseudolymphomatous angiokeratoma of children (apache) and angioplasmacellular hyperplasia were considered in the differential diagnosis as the lesion occurred in the acral site and microscopic examination revealed a lymphohisocytic infiltrate with plasma cells. the possibility of apache was excluded by immunohistochemical studies which showed a predominance of cd3 over cd20 cells, unlike equal numbers of cd3 and cd20 as seen in apache. another distinguishing feature is the presence of thick walled blood vessels in apache in contrast to the vessels lined by plump endothelial cells in trapp as was seen in our cases. apart from these features, trapp presents as a solitary papule and in apache multiple papules are noted. angioplasma cellular hyperplasia is a rare lesion with blood vessel proliferation and a reactive plasma cell infiltrate.. lymphocytes and eosinophils are present in a small proportion and histiocytes have not been described in this lesion. the proliferating blood vessels were capillaries and small venules scattered throughout the lesion lined by endothelial cells exhibiting vacuolization. in the present case, plasma cells constituted approximately 30 - 40% of the infiltrate and the remaining were lymphocytes and histiocytes. the absence of epidermotropism, morphologically atypical lymphocytes with convoluted cerebriform nuclei, and the presence of good number of plasma cells, histiocytes, prominent blood vessels excludes the possibility of early stages of mycosis fungoides. further, clinically the lesion was a single papule rather than multiple scaly atropic lesions seen in mycosis fungoides. simple excision would suffice. thus, although trapp and apache fall within the same spectrum of a specific form of pseudolymphoma that presents with rich vasculature amidst a dense inflammatory infiltrate, trapp differs from apache in that the former are t cell rich solitary polypoidal skin lesions while the latter is a pseudolymphoma of variable t and b lymphocytes that occurs predominantly in acral sites in children and may be multiple. the new entity of trapp needs to be distinguished from its close mimicker apache with clinical features and immunohistochemistry to demonstrate the predominance of t lymphocytes.trapp in the present case has manifested at a much earlier age of 13 years compared to the earliest reported case at 16 years. the new entity of trapp needs to be distinguished from its close mimicker apache with clinical features and immunohistochemistry to demonstrate the predominance of t lymphocytes. trapp in the present case has manifested at a much earlier age of 13 years compared to the earliest reported case at 16 years.	cutaneous pseudolymphomas refer to a group of benign reactive t or b cell lymphoproliferative processes that simulate cutaneous lymphomas. t cell rich angiomatoid polypoid pseudo lymphoma (trapp) of the skin is a unique form of t cell rich cutaneous pseudolymphoma. a 13-year - girl presented with an asymptomatic skin colored papule on the right elbow, the biopsy of which revealed features consistent with trapp. the dermis showed an infiltrate of lymphocytes, plasma cells, and histiocytes along with prominent vascular channels lined by plump endothelium. an immunohistochemical study showed a predominance of t cells over b cells. the morphological and immunohistochemical features pointed towards the diagnosis of trapp. we report this rare entity, only 17 cases of which have been described so far in literature. the differential diagnoses have also been discussed.
papillary (ptc) and follicular (ftc) thyroid carcinomas are follicular cell - derived carcinomas. they are differentiated forms of thyroid carcinoma and are characterized by slow growth and an indolent biological behavior. it accounts for 1020% of all thyroid malignancies and is most often seen in patients over 40 years of age. these neoplasms tend to metastasize hematogenously, with lung and bone most commonly affected, while distant metastases at the time of diagnosis are reported in 1120% of patients. less than 1% of these cases are seen in patients younger than 45 years of age. there have been a few case reports of follicular carcinoma causing unusual bony metastases, such as skull, mandible, maxilla, spine, and orbit. when combined with distant especially widespread metastases, the quality of life is compromised and the overall survival rate significantly decreases. hence management of follicular carcinoma requires special attentions and knowledge about its metastatic potential and its effect. eighty percent of differentiated thyroid carcinoma (dtc) patients respond to total thyroidectomy, radioiodine-131 (131i) ablation and levothyroxine suppression treatment. we discuss here the unusual presentations of these cases of follicular thyroid carcinoma and their difficult management. we have described three cases of follicular carcinoma of thyroid with unusual presentations. first case presented as thyroid abscess, second case presented with a large skull swelling in a pre - exiting goiter, and the third case presented with a swelling in the sternum. the first case was a 25 year old lady having a pre - existing goiter for 5 years (fig. she was diagnosed as follicular adenoma by fine needle aspiration cytology (fnac) 2 years back but refused for surgery. she came with sudden onset of pain, swelling, fever with chill & rigor, redness, erythema and tenderness over the swelling and dysphagia. she was treated with a complete course of antibiotic based on culture sensitivity. on ultrasonography only cystic component was found and subsequently underwent hemithyroidectomy which came out to be follicular carcinoma of thyroid. second case was a 40 yr old lady, had a pre - existing goiter for last 10 years. she developed a swelling on the left parietal region of the skull for last one year, gradually increasing in size for which she reported to us. on examination, a globular hard swelling of size about 2 cm 4 cm 4 cm situated on left parietal bone of her skull (fig. the histopathological examination revealed thyroid follicles filled with colloid material which is positive for pas stain. x - ray and magnetic resonance imaging (mri) of skull showed bone destruction with intracerebral extension (fig. she underwent total thyroidectomy and the histopathology of the specimen showed follicular carcinoma with lymphovascular invasion (fig. 2c). third case was a 45 year old lady presented with a solitary thyroid nodule (stn) on left lobe of thyroid, left hemithyroidectomy was done based upon fnac report as follicular adenoma. two years later patient presented with a swelling on right lobe of thyroid with a swelling on the sternum (fig. first case was a minimally invasive follicular carcinoma and hemithyroidectomy was appropriate extent of surgical excision. she was on suppressive dose of thyroxine and doing well in 3 years follow up. in the 2nd case after total thyroidectomy and radioiodine treatment the skull swelling disappeared, on follow up of 1 year she had hemoptysis. the patient is now on chest external beam radiation therapy (ebrt). in the third case after total thyroidectomy and radioiodine therapy follicular carcinoma is the second most prevalent of the thyroid carcinomas with an incidence of approximately 1015%. blood borne metastasis is common with spread to lung, bone and other solid organs. in less than 10% cases of follicular carcinoma patients with differentiated thyroid carcinoma (dtc) have a 10-year survival rate of 8095%. however, when distant metastases are present, the overall 10-year survival rate is 40%. thyroid abscess and acute supporative thyroiditis are not common, presenting only 0.10.7% of surgically treated thyroid pathologies. infection of the thyroid gland are rare due to its isolated anatomic location, fibrous capsule, rich blood supply, generous lymphatic drainage, and high content of iodine. however, various factors like congenital pyriform sinus, thyroid / neck injury, blood - borne infections, surrounding infections and intrinsic gland pathology like nodular goiter, thyroid cyst, and papillary thyroid cancer may predispose to suppurative thyroiditis. infection in pre - existing thyroid pathology may present with localized thyroid abscess. in the first case, when bone metastases are present, the overall survival at 10 years was reported to range from 13 to 21%. they are more frequent in follicular thyroid carcinoma (728%) compared with papillary thyroid carcinoma (1.47%). the bones most often involved in metastasis of follicular carcinoma are the long bones, such as the femur and flat bones, particularly the pelvis and sternum. it is not unusual for a pathological break of a long bone to be the first clinically significant manifestation of follicular thyroid carcinoma. the second case was being followed up by an endocrinologist who aggressively worked up the patient for the benign lesions of the thyroid over a period of two - years, but had significant metastasis in the left parietal region of the skull that measured about 2 cm 4 cm 4 cm in size. research indicates that treatment with radioactive iodine improves survival for patients with thyroid cancer that has spread to nearby lymph nodes or to distant locations in the body. the staging of follicular thyroid carcinoma is differentiated from the standpoint of the age of the patient. there are two categories : older than 45 years of age and younger than 45 years of age. staging of patients who are younger than 45 years of age is quite simple ; if the carcinoma is confined to only the thyroid with no lymph node involvement or metastasis, it is stage i. in our patient, who had distant metastasis, the carcinoma would be considered stage ii. this method of staging is the tumor, node, metastasis method (tnm method) and is the official method of staging adopted by the american joint commission on cancer. in cases of metastatic disease to lung and/or bone, adverse prognostic factors are said to include multiplicity of sites, older patient age at the time of discovery of the metastases, and absence of radioactive iodine uptake by the metastases. the extent of metastatic disease to bone and its response to radioactive iodine are associated with survival. a retrospective study evaluated therapeutic outcome, total administered radioiodine activities, and side effects (blood count alterations grades i iv, who classification, and acute leukemia) in 107 patients with initial bone metastases. this study concluded that initial bone metastases in selected dtc patients up to 45 years and especially in those with less than three bone metastases can be treated with curative intent. although the incidence of papillary thyroid carcinoma is much higher than of follicular carcinoma, the later accounts for more deaths. follicular carcinoma differs from papillary in that it occurs at an older age, exhibits hematogenous spread rather than lymphatic dissemination, and has a more aggressive behavior. so total thyroidectomy with adjuvent radioiodine therapy remains the standard of treatment for follicular carcinoma thyroid. disseminated metastases from follicular carcinoma at the time of diagnosis in a young patient remain exceedingly rare. even if it is advanced and metastatic, can be managed adequately with proper modalities of treatment. meher study design ; byomokesh patro writing ; manas ranjan samantaroy data analysis and writing.	introductionfollicular carcinoma of thyroid usually behaves in an indolent manner with low metastatic potential. distant metastases as initial presentation is rare in follicular carcinoma ; especially in young patients.presentation of casewe report the clinical, pathological features and the management of three different cases of follicular carcinoma of the thyroid with unusual presentations at the time of diagnosis. first case presented as thyroid abscess, second case with a large skull swelling in a pre - exiting goiter and the third case with a swelling in the sternum.discussionfollicular carcinoma of thyroid is the second category of well - differentiated thyroid cancer that constitutes about 10% of all thyroid malignancies. blood borne metastasis is common with spread to lung, bone and other solid organs. in less than 10% cases of follicular carcinoma, there is evidence of lymphatic involvement. the patients presentations above are highly unusual.conclusionrecognizing these cases has a significant impact on clinical decision - making and prognosis of the patients. treatment in these patients should be individualized and an alternative therapeutic approach should be considered.
benidipine is a triple calcium channel blocker, simultaneously blocking l, t, and n type channels. it is reported that the effect on t channel is stronger than that on l channel, making it a great potential protection for kidney. a number of studies explored the rho signaling pathway, renal interstitial fibrosis, and tubular epithelium cell transdifferentiation (emt) [24 ]. the blocking of t calcium channel (tcc) was reported to inhibit the activity of rho kinase, and this is essential in podocyte effacement in immune complex - mediated glomerular disease and other kidney injuries. furthermore, under cellular stress, rho kinase activation results in cytoskeletal rearrangement, stress fiber formation, and loss of cellular integrity and function. these suggested that blocking t channel may have a protective effect on diabetic kidney and reduce epithelium - mesenchymal transdifferentiation and fibrosis via inhibiting rock1 (rho kinase 1) activity. it was suggested that fasudil, a rho kinase inhibitor, may attenuate emt through reduced activation of rhoa / rock signaling and be a renoprotective agent for the treatment of dn. based on that, in this study, we proposed that by inhibiting rho kinase activity, benidipine reduces epithelium - mesenchymal transdifferentiation and protects kidney in rats with type 1 diabetes (t1 dm). by treating type 1 diabetic rats with benidipine and using rho kinase inhibitor fasudil as positive control, we studied the effects of benidipine on the activity of rho kinase and emt in diabetic nephropathy in vivo. eight - week old male wistar rats weighed at 180200 g (spf class) were supplied by the center for animal experiment of wuhan university (produce permission no. rabbit antibody p - mypt1 (p853) and e - cadherin antibody were purchased from bioworld technology, usa, rock1 antibody was purchased from santa cruz, usa, rabbit antibody -sma from sigma, usa, secondary antibody for internal control protein from santa cruz, usa, enzyme - labeling secondary antibody from sigma usa, streptozocin (stz) from sigma usa, hydrochloride fasudil injection from tianjin hongri pharmaceutical inc. (lot : 070525), benidipine from japanese kyowa hakko kogyo co., ltd. (lot : 119afi), anti - rabbit / rat universal immunohistochemistry kit from denmark (dako), protein extraction buffer from shanghai xinghan (dbi), real - time pcr master mix from japanese toyobo biotech, real - time fluorescence pcr equipment from biorad usa, and the analysis software for fluorescence quantitation was purchased from icycler (version 3.1.7050). fifty - four spf male wistar rats were fed with normal chow diet, had free access to water, with room temperature of 20~25c and relative humidity of 40%~70%, and were in the 12 h light - dark cycle. the rats were randomly assigned into normal group (n = 8) and diabetic model group (n = 46). after a one - week adaption, the model group was injected intraperitoneally with a single dose of streptozocin (stz) 60 mg / kg (dissolved in 10 mmol / l citrate buffer, ph 4.5), after a 12-hour fasting. seventy - two hours after the injection, blood glucose was tested with the samples from tail vein for 3 consecutive days. the criteria for diabetic models were as follows : nonfasting blood glucose is 11.1 mmol / l (all was 16.7 mmol / l in this study), urine output exceeds the controls over 50%, and urine glucose is strongly positive. during the procedure, 3 rats died and 6 did not meet the criteria. thirty - seven diabetic rats were randomly assigned into three groups : diabetic without treatment (d, n = 13), diabetic treated with fasudil (f, n = 12), and diabetic treated with benidipine (b, n = 12). fasudil was injected intraperitoneally with 10 mg / kg / d ; benidipine was dissolved in 0.3% carboxymethyl cellulose solution and given via gastric tubing with 3 mg / kg / d. after three months, 8 rats in n group, 9 in d group, 9 in f group, and 8 in b group survived and were sacrificed accordingly. one day prior to the sacrifice, 24-hour urine was collected in metabolic chamber. on the same day of sacrifice, tail artery blood pressure was measured with noninvasive blood pressure meter and blood samples were collected. after rinsing with normal saline, some of the kidney tissues were fixed with 10% neutral formalin, embedded with paraffin, made into 3 m slides, and stained with he for pathological analysis. twenty - four - hour urine protein quantification was measured with sulfosalicylic acid method ; serum creatinine (scr) was tested with picric acid method ; blood glucose was tested with glucose oxidase method ; and nag activity was measured with colorimetry as described previously. deparaffin the slides routinely, heat repair with microwave, incubate in 3% peroxide at room temperature for 15 minutes, and rinse with pbs (ph 7.4) for three times, 5 minutes for each. add rabbit antibody p - mypt1 (1 : 50), -sma (1 : 50), and e - cadherin (1 : 100) antibody, respectively, and incubate at 4c overnight. incubate with horseradish peroxidase - labeled chemmatetmenvision secondary antibody at room temperature for 45 minutes, detect with dab, repeat staining with he, dehydrate, and seal the film with transparent plastic membrane. the total proteins of kidney tissues were extracted with total protein extraction kit. the protein concentration was analyzed with uv spectrophotometry at 260 nm wavelength. thirty g of total protein was loaded for sds - page electrophoresis, then was transferred to nitrocellulose membrane, and then was observed with ponceau s staining. wash with pbs, then add rabbit anti - rat p - mypt1 (1 : 1000), rock1 (1 : 400), -sma (1 : 400), e - cadherin (1 : 1000), and -actin (1 : 1000), respectively, and incubate overnight. and then incubate with 1 : 2000 hrp - labeled goat anti - rabbit igg. detect with chromogenic agent and expose the film. scan the image and analyze absorbance with computer software. take the kidney cortex tissue 0.1 g from each rat, extract total rna with trizol, and remove genomic dna with dnase i. reverse rna and obtain cdna. the fluorescence pcr quantification of cdna was performed with sybr green, with triplets for each sample per protocol. the total volume of each reaction was 30 l, with the following condition : 95c for 3 min for predenature, then 95c for 20 s, 60c for 20 s, and 72c for 30 s and repeat for 35 cycles, and then 72c for 5 min. take the ct ratio of each sample to internal control as the relative value of the gene expression of this sample. normal distributed quantitative variables were presented as mean sd. comparison among groups was performed with anova, snk, and lsd tests. abnormally distributed variables were log - transformed into normal distributed variables and then analyzed thereafter ; data were presented with median. as shown in table 2, at 12 weeks, compared with n group, d group had elevated 24-hour urine protein, nag activity, scr (p < 0.05), and decreased ccr (p < 0.05). compared with d group, f group had decreased 24-hour urine protein, nag activity, and scr (p < 0.05). there was no significant difference between f and b groups. there was no significant difference for blood pressure or glucose among groups, as shown in table 3. compared with n and f groups, d group had significant expanded glomerular mesangial matrix, increased cell number, thickened basement membrane, with multiple inflammatory cells infiltrated in interstitial space, dilated renal tubular, and fibrosis in interstitial space. there were mild proliferation of glomerular mesangial matrix, inflammatory infiltration, tubular dilatation, and fibrosis in f and b groups, as shown in figure 1. the expression of rock1 showed trace in tubular epithelium cells in n group, was enhanced in d group which mainly distributed in dilated renal tubular, and was reduced in f and b groups. -sma was presented in the smooth muscle cells of renal small artery in n group and visible in epithelium of renal tubular in d group with majority expressed in medullar area but no expression in f or d group. e - cadherin was mainly presented in the epithelium cells of renal tubular in n group, especially in the cell conjunction area, with partial expression for f and b groups which was enhanced at the cell junction area but no expression on tubular epithelium in d group, as shown in figure 2. as shown in figure 3, compared with n group, the rats in d group had enhanced protein expressions of p - mypt1, rock1, and -sma in renal cortex but reduced e - cadherin. compared with d group, f group and b group had reduced protein expressions for p - mypt1, rock1, and -sma and enhanced e - cadherin which was still less than that of the normal group. there was no significant difference between f and b groups, as shown in figure 3 and table 4. compared with n group, mrna expression of rock1 in renal cortex was increased in d group. compared with d group, there was less mrna expression of rock1 in f and b groups, lower than normal, as shown in figure 4. in this study, by treating rats with type 1 diabetic nephropathy with benidipine, a triple channel blocker, and fasudil, a rho kinase inhibitor, we successfully investigated the effect of benidipine on epithelium - mesenchymal transdifferentiation and its possible mechanism via inhibiting rho kinase activity. these results were consistent with some previous studies. rock directly affects myosin light chain (mlc) or indirectly affects the target subunit of myosin phosphatase (mypt1) and thus increases the phosphorylation of mlc in plasma and controls the attachment, chemoattractant, contraction, and so forth. fasudil is a rock - specific inhibitor and inhibits rock activity by competitively combining atp sites of rock catalytic domain. rocki and rockii were both reported. in kidney tissues, rocki is the major one presented. recent studies revealed that abnormal activation of rock signaling pathway played a very important role in the pathophysiology of all kinds of complications of diabetes [13, 14 ]. our study confirmed that there was rock activation in renal tubular epithelium cells of 12 weeks of diabetic rats, and the effects of rock on diabetic renal interstitial space were through rock1. further study found that the protein expression of e - cadherin, the marker protein of renal tubular epithelium cells, was downregulated in diabetic rats, while the protein expression of -sma, the marker protein of myofibrillar cells, was upregulated, indicating that there was emt in diabetic nephropathy of rats. benidipine or fasudil can significantly inhibit nag activity, reduce urine protein and scr level, decrease the expression of p - mypt1, rock1, and -sma, and increase the expression of e - cadherin without affecting blood glucose or pressure. benidipine has protective effect on diabetic nephropathy of rats, independent of its effect of lowering blood pressure. tcc is a low - voltage activated channel, mainly located in renal efferent arterioles and pacing cells of the heart. through its strong blocking effects on tcc, benidipine dilates renal afferent and efferent arterioles equally and thus effectively reduces the resistance of renal vessels and intrarenal pressure. it is showed that, besides its effects on adjusting capillary pressure of glomerular, tcc has many nonhemodynamic effects [1619 ]. it regulates the activity of nf - k and thus inhibits inflammation, promotes the secretion and release of aldosterone, improves the remodeling of the heart and kidney, and anti - oxygenize and anti - proliferate [2023 ]. it is found that in the subremoval kidney models, by inhibiting rho kinase activity, selective t channel blocker improves renal interstitial fibrosis and emt. however, there are limitations of our study ; the sample size was rather small, and it is a study in animals instead of human being. in summary, our study is the first study suggesting that benidipine protects kidney in rats with type 1 diabetes, possibly through its effect of inhibiting rho kinase activity and thus reducing epithelium - mesenchymal transdifferentiation (emt). this may guide further animal studies, clinical trials on the importance of benidipine in diabetic emt development especially in type 1 diabetic, and the possible mechanism involved. from the long run, it may direct the clinical use of benidipine in treating patients with diabetic nephropathy especially those in t1 dm.	we investigated the protective effect of benidipine, by testing the changes of the activity of rho kinase and transdifferentiation of renal tubular epithelium cells in vivo. wistar rats were randomly divided into two groups : normal (n) and diabetes. stz were used to make the rats type 1 diabetic and were randomly assigned as diabetes without treatment (d), diabetes treated with benidipine (b), and diabetes treated with fasudil (f) and treated for 3 months. immunohistochemistry and western blotting were for protein expressions of rock1, -sma, and e - cadherin and real - time pcr for the mrna quantification of rock1. compared with n group, d group had significant proliferation of glomerular mesangial matrix, increased cell number, thickened basement membrane, widely infiltrated by inflammatory cells and fibrosis in the renal interstitial, and dilated tubular. those presentations in f and b groups were milder. compared with n group, d group showed elevated mypt1 phosphorylation, increased expression of rock1, -sma protein, and rock1 mrna and decreased expression of e - cadherin protein. b group showed attenuated mypt1 phosphorylation, decreased rock1, -sma protein, and rock1 mrna expression and increased expression of e - cadherin protein. in conclusion, benidipine reduces the epithelium - mesenchymal transdifferentiation and renal interstitial fibrosis in diabetic kidney by inhibiting rock1 activity.
it is mostly considered as a reversible stricture in airway tracts, which could be followed by some viral respiratory infections such as respiratory syncytial virus (rsv) in children. although, the exact cause of asthma has not been identified, yet a combination of environmental factors, biological genetic tendencies, inhaled allergens, viral infections, and chemical - biological pollutants such as tobacco play an important role in this respect. the immunological response could be a stimulant for chronic inflammation and inappropriate healing in damaged tissue of airway tracts. these pathological processes in growing - up lungs at an early age cause negative effects upon airway tracts growth and evolution (1). in the united states of america, childhood asthma is one of the most common reasons of reference to the emergency departments and hospitalization. asthma was responsible for 12.8 million absent days of school in 2004, 750000 episodes of referring to urgency services, 198000 episodes of hospitalization and 186 episodes of death in children. it has been pointed out that a combination of biological, environmental, economical, and mental risk factors could increase the possibility of severe ablaze of asthma (2). during the last decades, it seems that prevalence of childhood asthma is increasing in spite of remarkable improvement in management and pharmacotherapy (3). several published studies in different countries have reported a 50% increase in prevalence during the recent decade (4 - 7). it has been demonstrated that there is a correlation between asthma prevalence and allergic rhino conjunctivitis and atopic eczema (8). it is assumed that childhood asthma is more common in modern capitals and crowded nations and correlates extremely with other allergic reactions (9). in comparison, there is less possibility for children who live in rural areas of developing countries to become asthmatic (10). in the recent years, many studies have been conducted to determine the correlation between allergic reactions and infectious diseases (11 - 14). helicobacter pylori is a urease - positive gram - negative microaerophilic bacteria that grows in the stomach s mucosa and stimulates the human immune system (15), which helps release bacteria and host s inflammatory mediators in the blood (16). recent studies showed that h. pylori could cause chronic gastritis (3), peptic ulcer disease (pud) (6) and stomach cancer (15). in addition, there is a direct correlation between h. pylori infection and b - cell lymphoma (17), and cardiac, dermal, hepatic and rheumatologic diseases (18). it has been identified that h. pylori could produce pre - inflammatory mediators such as cytokines, eicosanoids and acute phase proteins in stomach mucosa, which can explain the correlation between h. pylori and inflammatory diseases (19) beside the similarity of antigens of the bacterium and the host (20). as the correlation between h. pylori and some inflammatory diseases has been proved in the recent years, this study was conducted to study the correlation between h. pylori infection and childhood asthma. three hundred children (5 to 18 years old) undergoing endoscopy owing to gastro - intestinal problems at shahid beheshti hospital were observed for childhood asthma by the gina 2010 questionnaire, which includes 24 questions with yes and no answers. next, the patients were referred to an allergy and asthma specialist for clinical examinations, spirometry, and also post bronchodilator test (post bd). finally, results of the questionnaires, clinical examinations, spirometric findings, and pathological reports were analyzed by the spss version 16.0 software, via fisher and chi square tests. the youngest examined child was five years old while the oldest was 18 years old. one hundred and forty - three children (47.7%) were male and 157 (52.3 %) were female. children with h. pylori had a mean age of 11.73 4.3 while those without this infection had a mean age of 10.95 3.78 ; no significant difference was observed (p = 0.312). the mean age of children with asthma was 11.23 4.07 while the mean age of those without asthma was 10.72 3.7, no significant difference was indicated (p = 0.481). forty - three of the cases (14.3%) had an asthmatic family history. amongst the 138 h. pylori positive patients, eight cases (5.8 %) were asthmatic while of the 162 h. pylori negative patients 28 cases (17.3%) were asthmatic, with the difference being statistically significant (p value = 0.002) (table 1). the spirometric findings considered as mild, moderate and severe stages in obstruction of lower - respiratory tracts were consistent with the questionnaire ; all cases of disease were confirmed by an asthma and allergy specialist. p value = 0.002 ; or 1 = 3.39 ; ci 2 = 1.49 - 7.7. was studied after controlling for confounding variables, including gender, age, and family history. the obtained results for the above - mentioned variables were significant (p values of 0.004, 0.005 and 0.002, respectively) (tables 2, 3 and 4). also, the odd - ratio mantel haenszel (ormh) of all of the three comparisons were obtained (ormh gender = 3.38, ormh age = 3.24 and ormh familial history = 4.06). p value = 0.004 (mental - haenszel) ; or = 3.38 ; ci = 1.2 - 7.6. p value = 0.005 (mental - haenszel) ; or = 3.24 ; ci = 1.4 - 7.37. p value = 0.002 (mental - haenszel) ; or = 4.06 ; ci = 1.7 - 9.6. among the 300 children under study, 36 (12 %) were asthmatic and 138 (46 %) were infected by h. pylori. the prevalence of the asthma has increased across the world for all ages, both genders, and all races from 7.3 to 8.2% during years 2001 to 2009 (8, 4). also, in 2011, 14% of children under 17 years of age had been described as asthmatic (22). nowadays, attention is being paid to exogenous contacts with environmental microorganisms and their anti - genes, which are able to make harmful changes in the human immune system (11). hygiene hypothesis is a hypothesis, which states that a lack of early childhood exposure to infectious agents, symbiotic microorganisms (e.g. gut flora or probiotics), and parasites increases susceptibility to allergic diseases by suppressing the natural development of the immune system causing change in the th1/th2 ratio, which leads to an increase in allergic disorders (21). it is believed that asthma could appear due to uncontrolled immunological response to environmental anti - genes by th2 cells. overcoming the number of th2 to th1 cells resulting from less contact with environmental microorganisms causes development of asthma. according to delios and bernard, lack of th1 cells stimulation causes hyper - activity in th2 cells, which could lead to asthma (7). the h. pylori is a urease - positive gram - negative microaerophilic bacteria in the stomach that has spread in most populations. the h. pylori remains in the mucosal layer of the stomach for decades and people under the age of ten could become infected and transfer the infection to other family members as well (14). the anti - body response following h. pylori infection would remain stably with h. pyloric gastric colonization for decades or for an entire lifetime. it has been determined that colonization of h. pylori in the mucosal layer of the stomach would increase different pre - inflammatory mediators such as cytokines and acute phase proteins (23). therefore, there is a pathogenic correlation between h. pylori infection and the diseases, which would appear by action of inflammatory mediators and/or auto - immune induction (12). helicobacter pylori infection decreases th2 cells leading to a reduction of th1 cells number (24). it can also decrease the prevalence of severe allergic responses by induction of regulatory t cells (2, 25). in the current study, 138 cases (46%) out of 300 were h. pylori positive as illustrated by their biopsy samples. several studies have been performed on the correlation of oral - fecal infections and asthma with controversial results. helicobacter pylori has been introduced as an allergy stimulant by some researches (26), while others claim that it has a protective role against allergies (27) and a number of studies suggest its miscorrelation with allergic diseases (13). among the 138 h. pylori positive patients, eight cases (5.8 %) and among the 162 h. pylori negative, 28 cases (17.3 %) were asthmatic, showing a statistic difference with odds ratio of 3.39. the obtained results are compatible with several other studies in which an inverse correlation between h. pylori anti - bodies and asthma has been revealed. for instance, results obtained by arram. pointed out that there is a significant difference in h. pylori infection in asthmatic patients compared with the control group (9). the study of chen and blaser indicated that h. pylori infection could significantly reduce the risk of asthma and emphasized that performing more researches in order to orientate the exact mechanisms of action are necessary (5). 6959 children indicated that there is an inverse correlation between childhood asthma and h. pylori infection (28). however, tsang. (29) and jaber (30) showed that there is no correlation between h. pylori infection and asthma. there is little evidence on the inverse correlation between h. pylori and asthma as indicated by wang. performed on 2437 patients showed that there is no correlation between h. pylori serology and pulmonary function tests (10). some used h. pylori serology tests in which sensitivity and specificity related to variables play an important role (50 to 100 %) ; and this could be a strong confounding factor and also a justification for the obtained incommensurable results in the above - mentioned researches. on the other hand, some factors such as gender, age, and family history of asthma could be considered as strong confounding factors, which affect h. pylori and asthma leading to incommensurable results. the advantage of the present study is that some features such as age, gender, and family history were considered in the assessment of patients and that diagnosis of h. pylori infection was made by the biopsy method, which has a higher sensitivity and specificity than serology tests. there is an inverse correlation between h. pylori and asthma as indicated by our findings. obviously, precise recognition of the correlation between h. pylori infection and asthma could play an important role in the recognition of the physiopathology of not only asthma but also other allergic diseases, which would offer potentially helpful new treatments for allergic diseases.	background : asthma is a chronic inflammatory air - way disease with increasing prevalence rate during the recent years. there are studies about the relationship between asthma and infectious diseases, including the association between asthma and helicobacter pylori. according to the latest studies, there is an epidemiological correlation between asthma prevalence and prevalence of h. pylori.objectives:the aim of this research was to study the correlation between h. pylori and asthma by biopsy in five to eighteen year - old children who had undergone endoscopy at shahid beheshti hospital.patients and methods : three hundred children (5 to 18 years old) undergoing endoscopy owing to gastro - intestinal problems at shahid beheshti hospital were observed for childhood asthma using the gina 2010 questionnaire which included 24 questions with yes and no answers to identify asthmatic patients with five positive answers. next, the patients were referred to an allergy and asthma specialist for clinical examinations, spirometry and post bronchodilator test (post bd).results : among 138 h. pylori positive patients, eight cases (5.8 %) were asthmatic while of the 162 h. pylori negative patients 28 (17.3%) were asthmatic. this difference was statistically significant (p value = 0.002). the correlation between h. pylori and asthma was studied after controlling the confounding variables including, gender, age and family history. the results obtained for the above - mentioned variables were significant (p values of 0.004, 0.005 and 0.002, and odd - ratio mantel haenszel (ormh) of 3.38, 3.24 and 4.06, respectively).conclusions : our findings showed that there is an inverse correlation between h. pylori and asthma. performing more studies with larger sample sizes is necessary to confirm these results.
genetic analysis in polyploids has received considerable interest in recent years because of the biological and economic importance [13 ]. genetic linkage maps constructed from molecular markers have been published for several major polyploids [410 ]. statistical models for linkage analysis and map construction that consider unique biological properties of polyploids have been developed [1114 ]. [15, 16 ] incorporated the so - called chromosomal pairing preference into the linkage analysis framework, to increase the biological relevance of linkage mapping models. there have been several statistical models developed to map quantitative trait loci (qtls) in bivalent polyploids [18, 19 ]. there is also a group of polyploids, called multivalent polyploids, in which chromosomes pair among more than two homologous copies at meiosis, rather than only two copies as like in bivalent polyploids. the origin of multivalent polyploids is mostly from the duplication of similar genomes and, for this reason, they are called autopolyploids [20, 21 ]. the consequence of multivalent pairing in autopolyploids is the occurrence of double reduction, that is, two sister chromatids of a chromosome sort into the same gamete. fisher proposed a conceptual model for characterizing the individual probabilities of 11 different modes of gamete formation for a quadrivalent polyploid in terms of the recombination fraction between two different loci and their double reductions. wu. used fisher 's model to derive the em algorithm for the estimation of the linkage between fully informative markers. wu and ma extended this model to analyze any type of markers, regardless of their informativeness and dominant or codominant nature. the significant advantage of the models by wu and colleagues directly lies in their generality, flexibility, and robustness. in this paper, we develop a statistical method for qtl mapping in multivalent tetraploids by considering fisher 's 11 classifications of gamete formation. the method allows the estimation and test of not only the qtl - marker linkage, but also the extent of double reduction of the qtl. because of the inherent complexity of classification analyses of gamete formation, we will focus on the modeling and analysis of one - marker / one - qtl associations. a two - stage hierarchical model is derived to estimate the probabilities of gamete formation modes and therefore double reduction in the upper hierarchy and estimate the marker - qtl recombination fraction in the lower hierarchy within the maximum likelihood context implemented with the em algorithm. the method is used to analyze a simulated data set, with results demonstrating statistical properties of the method and its analytical and biological merits. consider a heterozygous multivalent tetraploid line crossed with a homozygous line to generate a so - called pseudotest backcross population. for such a population, the genotypes of progeny are consistent with the genotypes of gametes produced by the heterozygous parent and, therefore, the derivation of mapping models can be based on the segregation of gametes. a panel of codominant markers is typed for each individual, with which a linkage map is constructed. all the pseudotest backcross individuals are phenotyped for a quantitative trait that is assumed to be controlled by qtls. to simplify our analysis, m4 be the four alleles at a marker m, and let q1, the marker and qtl are linked with a recombination fraction of r. because of the double reduction, the multivalent tetraploid generates 10 diploid gametes for locus, which are arrayed as (m1m1, m2m2, m3m3, m4m4, m1m2, m1m3, m1m4, m2m3, m2m4, m3m4) for the markers, and (q1q1, q2q2, q3q3, q4q4, q1q2, q1q3, q1q4, q2q3, q2q4, q3q4) for the qtl. in each case, the first four gametes are derived from the double reduction, whereas the second six gametes are derived from the chromosome paring. let and be the frequencies of double reduction at the marker and qtl, respectively. the frequency of double reduction is a constant for any given locus, with the value depending on its distance from the centromere. when the marker and qtl are co - segregating in a multivalent tetraploid, a total of 136 diploid gamete formation mechanisms are generated although there are only 100 gamete genotypes that are observable. based on the presence / absence of double reduction and the number of recombinant events, fisher classified these 136 formation mechanisms into 11 gamete modes. of these 11 gamete modes, however, only nine can be observed each with a frequency denoted by gh (h = 1,, 9). these 9 observable gamete modes were rearranged by wu. in matrix form expressed as (1) q1q1q2q2q3q3q4q4q1q2q1q3q1q4q2q3q2q4q3q4 m1m1 m2m2 m3m3 m4m4g = m1m2m2m1 m1m3m3m1 m1m4m4m1 m2m3m3m2 m2m4m4m2 m3m4m4m3 [14g1112g2112g2112g2112g5112g5112g5112g6112g6112g6112g214g1112g2112g2112g5112g6112g6112g5112g5112g6112g2112g214g1112g2112g6112g5112g6112g5112g6112g5112g2112g2112g214g1112g6112g6112g5112g6112g5112g5112g3112g3112g4112g416g7124g8124g8124g8124g816g9112g3112g4112g3112g4124g816g7124g8124g816g9124g8112g3112g4112g4112g3124g8124g816g716g9124g8124g8112g4112g3112g3112g4124g8124g816g916g7124g8124g8112g4112g3112g4112g3124g816g9124g8124g816g7124g8112g4112g4112g3112g316g9124g8124g8124g8124g816g7 ], where g1 and g2 are associated with double reductions at both the marker and qtl, g3 and g4 with double reductions only at qtl q, g5 and g6 with double reductions only at marker m, and g7 g9 with nondouble reductions. from matrix (1), we see that there are no, one and two recombinant events in the cells (g1), (g3, g5), and (g2, g4, g6, g9), respectively. the cells (g7) and (g8) are each a mixture of two different gamete formation mechanisms or configurations (a and b), that is, g7 = g7a + g7b and g8 = g8a + g8b, with relative proportions determined by r. because different configurations contain different numbers of recombination events, the expected number of recombination events in each cell, that is, an observable gamete genotype, should be the weighted average of the number of recombination events for each configuration. wu. used a matrix form (e) to count the expected number of recombination events for each observable gamete genotype expressed as (2)c= q1q1 q2q2 q3q3 q4q4 q1q2 q1q3 q1q4 q2q3 q2q4 q3q4 m1m1m2m2m3m3m4m4m1m2m2m1m1m3m3m1m1m4m4m1m2m3m3m2m2m4m4m2m3m4m4m3 [0 2 2 2 1 112222 0 2 2 1 221122 2 0 2 2 121212 2 2 0 2 212111 1 2 2 2 1+1+1+1+21 2 1 2 1+ 21+1+21+1 2 2 1 1+ 1+221+1+2 1 1 2 1+ 1+221+1+2 1 2 1 1+ 21+1+21+2 2 1 1 2 1+1+1+1+2 ], where (3)=r210r218r+9,=r32r. based on matrices (1) and (2), the expressions for the frequencies of double reduction (and) and the recombination fraction r can be expressed in terms of gi as (4)=g1+g2+g3+g4,=g1+g2+g5+g6,r=12[g3+g5 + 2(g2+g4+g6+g9)+2g7+(1+)g8 ]. for a given qtl, there are 10 different qtl gamete genotypes in the multivalent tetraploid, whose values can be partitioned into additive and dominance genetic effects of different types, expressed as (5)11=+a1, for q1q1,22=+a2, for q2q2,33=+a3, for q3q3,44=a1a2a3, for q4q4,12=+a1+a2+d12, for q1q2,13=+a1+a3+d13, for q1q3,14=a2a3+d14, for q1q4,23=+a2+a3+d23, for q2q3,24=a1a3+d24, for q2q4,34=a1a2+d34, for q3q4, where is the overall mean, a1, a2, and a3 are the additive genetic effects of alleles q1, q2, and q3 relative to allele q4, and d12, d13, d14, d23, d24, and d34 are the dominant genetic effects due to interactions between different alleles q1 and q2, q1 and q3, q1 and q4, q2 and q3, q2 and q4, and q3 and q4, respectively. from expression (5), we can solve the overall mean and additive and dominant effects as (6)=14(11+22+33+44), (7)a1=14(311223344), (8)a2=14(322113344), (9)a3=14(333112244), (10)d12=1214(333 + 3441122), (11)d13=1314(322 + 3441133), (12)d14=1414(322 + 3331144), (13)d23=2314(311 + 3442233), (14)d24=2414(311 + 3332244), (15)d34=3414(311 + 3223344). ignoring the effects of other covariates, the phenotypic value, yi, for individual i in the pseudotest backcross can be expressed in terms of the qtl effect and residual error as (16)yi=j1j2=14j1j2 ij1j2+ei, where j1j2i is the indicator variable that is defined as 1 if individual i has a qtl genotype j1j2 (j1 j2 = q1, q2, q3, q4), and 0 otherwise, j1j2 is the genotypic value of qtl genotype j1j2 as defined in (5), and ei is the residual error assumed to be normally distributed with mean zero and variance. we use to denote the unknown vector (11, 22, 33, 44, 12, 13, 14, 23, 24, 34,). for a qtl mapping experiment, let nl1l2 be the observation of marker genotype l1l2 (l1 l2 = m1, m2, m3, m4). the likelihood of the phenotypic (y) and marker data (m) is constructed, within the mixture model framework, as (17)l(y, m)=i=1n l1=m1m4 l2=m1m4 j1=q1q4 j2=q1q4j1j2 l1l2fj1j2(yi), l1l2,j1j2, where j1j2l1l2 is the conditional probability of qtl genotype j1j2 given marker genotype l1l2, and fj1j2(yi) is assumed to follow a normal distribution with mean j1j2 and variance. prior conditional probability j1j2l1l2 is calculated as the frequency of joint marker - qtl genotype l1l2j1j2, expressed in terms of nine g probabilities in matrix (1), divided by the frequency of marker genotype l1l2. marker genotype frequencies are /4 for each of double reduction gametes m1m1, m2m2, m3m3, and m4m4, and (1)/6 for each of nondouble reduction gametes m1m2, m1m3, m1m4, m2m3, m2m4, and m3m4. the estimates of unknown parameters that maximize the likelihood (17) can be obtained by implementing the em algorithm. in step e, we calculate the posterior probability of a qtl genotype given a specific marker genotype of individual i by (18)j1j2 l1l2i=j1j2 l1l2fj1j2(yi)j1=q1q4j2=q1q4j21j2 l1l2fj1j2(yi). in step m, we calculate the frequencies of nine observable gamete modes based on the calculated posterior probabilities using the following : (19) which lead to the estimates of the frequencies of double reduction as (20)^=g^1+g^2+g^3+g^4=1n(n11+n22+n33+n44),^=g^1+g^2+g^5+g^6,r^=12[g^3+g^5 + 2(g^2+g^4+g^6+g^9) + 2g^7+(1+)g^8 ]. the genotypic value of qtl genotype j1j2 and residual variance are estimated by (21)^j1j2=i=1nl1=m1m4l2=m1m4j1j1 l1l2iyil1=m1m4l2=m1m4j1j1 l1l2i, l1l2;j1j2, (22)^2=1ni=1n l1=m1m4 l2=m1m4 j1=q1q4 j2=q1q4j1j1 l1l2i(yi^j1j2)2, l1l2;j1j2. the iteration is repeated between the e step, (3) and (18), and m step, (19)(22), until stable estimates are obtained. the hypothesis about the presence of a qtl segregating in the pseudotest backcrosses is formulated as (23)h0 : a1=a2=a3=d12=d13=d14=d23=d24=d34=0,h1 : at least one of them is not equal to zero. the difference between the log - likelihood functions under the null and alternative hypotheses are calculated. but the distribution of this log - likelihood ratio (lr) is not known because of the violation of regularity conditions for the mixture model (1). for this reason, a commonly used empirical approach based on permutation tests by reshuffling the relationships between the marker genotypes and phenotypes is used to determine the critical threshold, in order to judge whether there is a qtl for the trait. after a significant qtl is detected, the next hypothesis is about the additive genetic effect of the qtl. this can be tested by formulating the null hypothesis, (24)h0 : a1=a2=a3=0, under which the estimates of genotypic values of qtl genotypes can be obtained with the em algorithm as described above, but posing three constraints derived from (7), (8), and (9). similarly, the dominant genetic effects can be tested with the null hypothesis, (25)h0 : d12=d13=d14=d23=d24=d34=0, with estimates of genotypic values under the constraints derived from (10)(15). a pseudotest backcross for a multivalent tetraploid was hypothesized, in which a marker is assumed to be linked with a qtl that affects a quantitative trait. marker and qtl genotypes were simulated for the pseudotest backcross of different sample sizes (n = 100,200,400) based on a range of double reduction (0.05, 0.15, 0.30) and recombination fraction (0.05, 0.25). we assume the same frequency of double reduction between the marker and qtl. the phenotypic value of an individual is expressed as the summation of genotypic values of a qtl genotype carried by this individual and a normally distributed error. the genotypic values of a qtl genotype are calculated by (5), where the overall mean is assigned as 1, and the additive and dominant effects assigned as a1 = a2 = a3 = 0.6 and d12 = d13 = d14 = d23 = d24 = d34 = 0.5. the error variance is determined according to the heritability of h = 0.1 and 0.4, respectively. in this simulation study, fully informative markers and qtl are assumed and, thus, the double reduction at the marker can be estimated analytically. the estimates of the parameters converge to stable values at a rapid rate given that there are closed forms for parameter estimators in the em framework. we evaluate the estimation of the other parameters related to qtl segregation, effects, and position. the means of the mles of the qtl - related parameters and their standard errors based on 1000 simulation replicates are illustrated in tables 1, 2, and 3. with a small sample size (100), the double reduction of the qtl was accurately estimated, with the precision of estimation relatively independent of the magnitude of heritability and the degree of qtl - marker linkage (table 1). the most significant factor that affected the estimate of qtl position (in terms of its recombination with the marker) was the heritability, followed by sample size and the degree of qtl - marker linkage. in general, at least a sample size of 200 was required to reliably estimate the qtl position for a major gene that explains about 20%30% of the phenotypic variance. the estimation precision of the qtl effects depended on the heritability, sample size, and degree of qtl - marker linkage. as heritability, sample size, and linkage degree increased, as compared with the dominant genetic effects, the estimates of the additive genetic effects required a larger sample size, more precise phenotypic measurements (leading to a higher heritability), and a denser linkage map (with a stronger degree of qtl - marker linkage). we found that the estimates of qtl effects were influenced by the frequency of qtl double reduction. at low frequencies of double reduction, the effects of qtl were more accurately estimated than at higher frequencies. for a qtl undergoing a strong double reduction (say = 0.3), a sample size of at least 400 is required even if the qtl explains a large proportion of the phenotypic variance (0.4). we performed a simulation study to test how the misspecification of double reduction affects the estimate of qtl - related parameters. this was done by using traditional mapping models (without considering double reduction) to analyze the simulated data of qtl genotypes with different degrees of double reduction. when a qtl undergoes double reduction, traditional models that do not consider double reduction provided misleading results about the estimates of qtl effects and position (data not shown). a statistical method for genetic mapping of quantitative trait loci (qtls) in a multivalent tetraploid undergoing a double reduction process is described. as an important cytological characteristic of polyploids, double reduction may play a significant role in plant evolution and maintenance of genetic polymorphism in natural populations. also, because double reduction affects the result of linkage analysis through the crossing - over events between different chromosomes [24, 25 ], it is important to incorporate double reduction into a qtl mapping framework. this method provides a powerful tool for qtl mapping and understanding the genetic control of a quantitative trait in a multivalent tetraploid. the method capitalizes on 11 different classifications of two - locus gamete formations, derived by fisher, during multivalent tetraploid meiosis and has proven to be powerful for simultaneous estimation of the frequencies of double reduction and the recombination fraction between different loci. although a couple of statistical approaches have been proposed to map multivalent tetraploid qtls [26, 27 ], this method has for the first time incorporated fisher 's tetrasomic inheritance into the mapping framework, thus enhancing the cytological relevance of qtl detection. results from simulation studies showed that the method can be used to map qtls in a controlled cross of multivalent tetraploids when the mapping population is adequately large (say 400). when a qtl undergoes double reduction, traditional mapping approaches will incorrectly estimate the position and effects of the qtl, proportional to the degree of double reduction. the new method can estimate the double reduction of a qtl, an important parameter related to the genetic diversity and evolution of polyploids [28, 29 ]. because of the high complexity of the mixture model implemented with tetrasomic inheritance, we only considered a one - marker model for qtl mapping. interval mapping, which localizes a qtl with two flanking markers, has proven to be more advantageous in parameter estimation over the one - marker model. it will be worthwhile to integrate components of our model into the interval mapping framework to fully explore the statistical merits of interval mapping for qtl mapping in multivalent tetraploids. furthermore, the model proposed in this article assumes the segregation of fully informative codominant loci, each with 10 distinct genotypes, in a controlled cross of multivalent tetraploids. for partially informative codominant markers, a two - stage hierarchical mixture model will be needed to model the different allelic configurations for a phenotypically identical genotype. although molecular marker technologies have improved in recent years, dominant markers may still be used in genetic mapping projects of some underrepresentative species including polyploids. thus, it is also important to extend our model to map qtls with dominant markers. for partially informative loci, the number of qtl genotypes may be unknown and, thus, a model selection procedure should be incorporated to determine the optimal number of genotypes at a qtl. sophisticated statistical models are required to tackle genetic problems hidden in the polysomic inheritance of polyploids. currently, there are some debates on the optimal modeling of tetrasomic inheritance in linkage analysis [13, 25 ] and qtl mapping [18, 31 ] partly because of our limited knowledge about these fascinating species. before a detailed understanding of the cytological mechanisms for meioses in multivalent polyploids, the development of powerful statistical models for polyploid mapping continues to be a pressing need. the application of these models to real - world data will not only test their usefulness, but also provide an unprecedented opportunity to understand the genetic differentiation among polyploid genomes and characterize the genetic architecture of quantitatively inherited traits for this unique group of species.	multivalent tetraploids that include many plant species, such as potato, sugarcane, and rose, are of paramount importance to agricultural production and biological research. quantitative trait locus (qtl) mapping in multivalent tetraploids is challenged by their unique cytogenetic properties, such as double reduction. we develop a statistical method for mapping multivalent tetraploid qtls by considering these cytogenetic properties. this method is built in the mixture model - based framework and implemented with the em algorithm. the method allows the simultaneous estimation of qtl positions, qtl effects, the chromosomal pairing factor, and the degree of double reduction as well as the assessment of the estimation precision of these parameters. we used simulated data to examine the statistical properties of the method and validate its utilization. the new method and its software will provide a useful tool for qtl mapping in multivalent tetraploids that undergo double reduction.
vertebral artery dissection (vad) is among the most common identifiable etiologies of stroke in young adults aged 1845 years, with an estimated annual incidence between 1 and 1.5 per 100,000.1 compared to carotid dissection, detection of vad is challenging due to nonspecific symptoms and substantial variability of imaging results. intramural pathology can be captured by noninvasive techniques such as fat - saturated magnetic resonance imaging (mri) and duplex ultrasound. although duplex ultrasound provides hemodynamic information in real - time, the technique is limited by the low rate of clear findings suggestive of intramural hematoma in patients with vad.2 here, we present a case of unspecific neck pain as isolated symptom of bilateral vad with unusually compelling evidence on duplex ultrasound. a 28-year - old woman with a history of amyotrophic neuralgia one year ago with electroneurographic evidence of neurogenic paresis of the right serratus anterior muscle experienced acute neck pain radiating in both shoulders. an elective cervical mri was performed 21 days later which showed no radiculopathy but suspicion of left vad. neither physical examination nor medical and family history revealed any indications of connective tissue disease or trauma. duplex ultrasound showed luminal narrowing of the left vertebral artery (va) along its extracranial course and hypoechoic thickening of the vessel wall suggestive of intramural hematoma (figures 1 and 2) fat - saturated t1 mri confirmed dissection of the left va and additionally showed dissection of the right extracranial va (figures 3 and 4). antiplatelet therapy with oral aspirin (100 mg / day) was initiated and the patient was discharged. at follow - up visit duplex ultrasound showed a patent left va with only slight residual luminal narrowing and decreased blood flow velocity of its intracranial segment compared to the contralateral va. aspirin was prescribed for another six months after which another follow - up visit was scheduled. our report of isolated pain in the neck as the sole symptom of spontaneous bilateral va dissection has two clinically relevant implications. first, the absence of any neurological symptoms in our patient highlights the necessity of considering cervical artery dissection in patients presenting with unspecific symptoms such as neck pain, even if isolated.3 second, our image of intramural hematoma on duplex ultrasound has educational value, as pathology is captured in an unusual, clear and distinct fashion and can be a useful supplement to other neurovascular reference images in the clinical assessment of patients with a suspicion of cervical artery dissection. while in our patient, dissection of the left va was clearly detected on both duplex ultrasound and mri, contralateral vad was missed by duplex ultrasound most likely due to its distal manifestation. direct b - mode imaging of the v3-va is challenging, and stenotic lesions caused by intramural hematoma are commonly suspected on the basis of upstream flow disturbances (eg, high - resistance pulsatile flow). thus, we may speculate that luminal narrowing of the right v3 segment was too slight to produce corresponding pre - stenotic flow changes. in concordance with best available evidence, our patient was prescribed antiplatelet treatment.4 improving accuracy in detection of vad is important, since patients have an increased risk of acute ischemic stroke and early recurrent stroke due to embolism from thrombus formation at the dissection site.57 data from observational and nonrandomized studies indicated efficacy of oral anticoagulation therapy applied for three or six months after dissection to prevent recurrent stroke, an observation which has been widely translated into clinical practice.8 although this treatment regimen has been recommended by the american heart association (aha) guidelines, similar evidence was found for antiplatelet therapy, which is why these guidelines also acknowledged that the relative efficacy of anticoagulation versus antiplatelet therapy remained unknown at the time of publication.9 a recent multicenter trial compared the two treatment forms in a randomized fashion but was stopped prematurely due to failure of recruitment. however, subsequent analysis showed no difference between the antiplatelet and anticoagulant treatment arms in preventing stroke and death in patients with symptomatic cervical artery dissection.4 although the rate of strokes was slightly lower in the group of patients receiving anticoagulant therapy than in the antiplatelet group, this difference was counterpoised by one severe subarachnoid hemorrhage in the anticoagulant group. our clinical case of vad with unusually clear evidence on duplex ultrasound, confirmed by fat - saturated mri, and presenting with neck pain as the only symptom does not only highlight the diagnostic value of ultrasound as supplement to fat - saturated mri but also emphasizes the importance of thorough clinical assessment in patients with nonspecific symptoms such as neck pain.	vertebral artery dissection (vad) is among the most common identifiable etiologies of stroke in young adults and poses a diagnostic challenge due to nonspecific symptoms and substantial variability of imaging results. here, we present a case of unspecific neck pain as isolated symptom of vad with unusually compelling evidence on duplex ultrasound. this observation has clinical relevance as the absence of any neurological symptoms in our patient highlights the necessity of considering cervical artery dissection in patients presenting with unspecific symptoms such as neck pain, even if isolated. furthermore, our image of intramural hematoma on duplex ultrasound has been captured in an unusual, clear and distinct fashion and might therefore be a useful reference image in the clinical assessment of patients with a suspicion of cervical artery dissection.
abdominal sacrocolpopexy is considered the gold standard in the repair of symptomatic high grade vaginal vault prolapse, secondary to high success rates and durable long - term results (1, 2). recently, multiple series have shown similar excellent long - term outcomes in patients managed with a robotic approach to sacrocolpopexy (3 - 5). however, while replicating the anatomic principles of the open sacrocolpopexy and potentially decreasing length of hospitalization and blood loss, one issue unique to a minimally invasive approach (whether laparoscopic or robotic) to sacrocolpopexy is that of the potential for requiring conversion to an abdominal sacrocolpopexy (4, 6). prior series on rsc have demonstrated a conversion rate ranging from 0 to 11% (3, 6 - 8). furthermore, in other surgeries performed with robotic assistance, multiple potential risk factors for conversion such as surgeon experience (9), technical difficulty / failure to progress / injury to adjacent organs (9, 10), patient risk factors (prior abdominal surgery, obesity, etc.) (9 - 11) and equipment malfunction (12) have been proposed. however, there is a paucity of data regarding potential predictors of conversion specifically for rsc. notably, compared to other robotic pelvic surgeries, rsc presents unique technical challenges such as dissecting in the retroperitoneal fat and potential for hemorrhage from presacral veins. thus, recognizing specific factors associated with conversion during rsc may aid in patient selection as well as pre - operative patient counseling. therefore, in a large cohort of rsc patients we sought to evaluate for clinical predictors of intraoperative conversion from rsc to an open procedure. following institutional review board approval, 83 consecutive patients undergoing rsc at our institution between 2002 and 2012 were identified. rsc was performed for patients with high - grade (baden walker - grade 3 to 4 or pelvic organ prolapse quantification - stage 3 to 4) symptomatic post - hysterectomy vaginal vault prolapse. all patients were treated by a single surgeon via our previously reported technique for rsc (3, 13). briefly, we utilize the da vinci - s system (intuitive surgical, sunnyvale ca, usa) and set up our operative approach with a periumbilical trocar, two standard laparoscopic ports for retraction, and two robotic ports. the sacral promontory is exposed with the use of retraction suture placed through the sigmoid mesentery. the posterior peritoneal reflection is incised and a polypropylene y - graft is sutured to the sacrum and vagina. following fixation to the sacral promontory we place the posterior vaginal sutures and then the anterior sutures. clinical variables recorded for evaluation included age, body mass index (bmi), pertinent medical comorbidities (hypertension, diabetes and tobacco use), operative time, estimated blood loss, concurrent procedures performed and post - operative complications. bmi (weight in kilograms divided by height in meters squared) was examined using the national institutes of health definitions of normal weight (bmi 30) (n=23)p valueage at surgery (years), median (iqr)70.5 (59, 74)69 (58, 73)65 (59, 74)0.70operative time (min), median (iqr)155 (130, 173)167.5 (150, 195)180(135, 235)0.06hospital stay (days), median (iqr)1 (1, 1)1 (1, 2)1 (1, 2)0.18estimated blood loss (cc), median (iqr)50 (25, 100)50 (25, 100)50 (25, 100)0.52intraoperative conversion1 (3.8%)5 (14.7%)8 (34.8%)0.004 we found, in a large cohort of patients treated by rsc for symptomatic high - grade vaginal vault prolapse, that only obesity (bmi > 25 kg / m) was associated with a significantly increased risk of intra - operative conversion from rsc to an abdominal sacrocolpopexy. furthermore, the risk associated with conversion rose with increasing bmi values. to our knowledge this represents the first report evaluating such risk factors for conversion during rsc. with regard to conversion from a minimally invasive approach to sacrocolpopexy, previous series on laparoscopic and robotic sacrocolpopexy have demonstrated conversion rates between 0 - 11% (3, 6 - 8). our overall conversion rate (16.9%) is somewhat higher, which may be secondary to early adoption of the technique (3) and broad patient inclusion (median bmi 26.4 kg / m, range 18.2 - 47.3 this occurred as we have attempted to further application of rsc, while acknowledging that conversion may be necessary when discussing management options with patients pre - operatively. in other laparoscopic and robotic surgeries, multiple risk factors for conversion have been proposed. for instance, when evaluating patients undergoing laparoscopic colectomy, chew. noted an increased conversion rate with increased patient age at the time of surgery, obesity and more advanced pathologic stage (11). other potential factors such as surgeon experience (9), technical difficulty / failure to progress (9, 10), injury to adjacent organs (9, 10), prior abdominal surgery (9, 14) and device malfunction (12) have been reported. bmi was the only identified predictor of conversion and roughly one - third of the conversions were due to difficulty with the presacral dissection specifically. notably, this dissection is unique to sacrocolpopexy and can be significantly more technically difficult when a large volume of presacral retroperitoneal adiposity is encountered. this may explain why conversely, some studies on other robotic female pelvic surgeries (for instance, hysterectomy) have reported no association between bmi and conversion (15 - 17). furthermore, in our series, year of surgery (a marker for surgeon experience over time), route of hysterectomy, prior prolapse repair and prior abdominal surgery were not associated with conversion. notably, there was a trend toward increased conversion among patients with prior abdominal versus transvaginal hysterectomy, and the absence of statistical significance may be secondary to the limited number of conversions. recognizing specific challenges of the rsc and identification of potential risk factors for conversion is needed to assist with accurate pre - operative counseling and appropriate patient selection. one potential area of future study is in pre - operative weight loss and pelvic organ prolapse surgery. it has previously been shown that obesity is associated with an increased risk of pelvic organ prolapse progression over time (18, 19). interestingly, several reports demonstrate no difference in surgical success rate in obese versus normal weight patients (18, 20). that being said, bradley. reported a higher serious adverse events rate among obese patients (22.4% versus 36.5% ; p=0.02) (18). notably, in the bariatric literature, enhanced post - recovery has been noted in patients with a 5 - 10% weight reduction preoperatively (21). thus, in addition to overall health benefits from weigh reduction, highlighting the importance pre - operative weight loss to patients may be beneficial regarding successful completion of rsc and helping to avoid peri - operative complications. limitations of our study should be noted including its retrospective, non - randomized design. as such, it is subject to bias from patient selection and alterations in technique over time. additionally, as expertise with the procedure developed over the timeframe of the study, the complexity of cases including patient bmi increased. this likely contributed to the higher conversion rate reported in the later years of the study. furthermore, given the retrospective nature of this study, there is potential for additional confounding variables / missing data (such as indication for hysterectomy and in some cases route of hysterectomy) which could not be accounted for. likewise, many of the conversions were not directly related to issues other than obesity (for instance intra - abdominal adhesions), suggesting that while no other factor was associated with conversion on univariate analysis, the risk of conversion may be multifactorial. however, given the sample size in our series, some differences in the cohorts may not be detected. additionally, our results represent those of a tertiary care center with a relatively high - volume rsc practice, and thus may not be able to be extrapolated to all surgical practices. a higher bmi was the only clinical factor associated with a significantly increased risk for intra - operative conversion during robotic sacrocolpopexy. while external validations of these results are needed, recognition of this may aid in pre - operative counseling and in surgical patient selection.	objective to evaluate for potential predictors of intraoperative conversion from robotic sacrocolpopexy (rsc) to open abdominal sacrocolpopexy.patients and methods we identified 83 consecutive patients from 2002 - 2012 with symptomatic high - grade post - hysterectomy vaginal vault prolapse that underwent rsc. multiple clinical variables including patient age, comorbidities (body - mass index [bmi ], hypertension, diabetes mellitus, tobacco use), prior intra - abdominal surgery and year of surgery were evaluated for potential association with conversion.results overall, 14/83 cases (17%) required conversion to an open sacrocolpopexy. patients requiring conversion were found to have a significantly higher bmi compared to those who did not (median 30.2kg / m2 versus 25.8kg / m2 ; p=0.003). other medical and surgical factors evaluated were similar between the cohorts. when stratified by increasing bmi, conversion remained associated with an increased bmi. that is, conversion occurred in 3.8% (1/26) of patients with bmi 25 kg / m2, 14.7% (5/34) with bmi 25 - 29.9 kg / m2 and 34.7% (8/23) with bmi 30 kg / m2 (p=0.004). when evaluated as a continuous variable, bmi was also associated with a significantly increased risk of conversion to an open procedure (or 1.18, p=0.004).conclusions higher bmi was the only clinical factor associated with a significantly increased risk of intra - operative conversion during robotic sacrocolpopexy. recognition of this may aid in pre - operative counseling and surgical patient selection.
ten healthy subjects (male, 5 ; female, 5 ; average age, 24 4 years) were included in this study. all were university students with no history of neurological or psychiatric disorder, and gave informed written consent. the subjects received detailed instructions regarding the experimental conditions and had a brief practice session before entering the scanner. working memory tasks consisted of verbal and visual stimuli. during the activation period of the verbal working memory task, randomized sequences of five phonologically dissimilar consonants of the korean language were displayed on the screen every second. in order to detect whether a target constant appeared 1 second after a series of five consonants was presented, subjects were instructed to rehearse the stimuli silently and to remember them serially. during the activation period of visual working memory task, serial diagrams or symbols that could not be phonologically transcoded for both types of tasks, each sequence of stimuli was repeated five times during the activation period. for the identification of each sequence of stimuli, one blank slide was presented for 1 second after one sequence of stimuli during an activation task period. subjects responded by pressing a squeeze ball. under control conditions of visual or verbal working memory task, the subjects were instructed to visually fixate on a crosshair. mr images were generated on a 1.5-t mr (general electric medical systems, milwaukee, wi). a series of echoplanar gradient echo images was acquired at intervals of 3 seconds (tr / te, 3000/60 msec ; flip angle, 90 ; matrix, 6464 ; slice thickness, 5 mm ; no slice gap ; fov, 2424 cm). twenty brain slices were obtained parallel to the plane of the anterior commissure (ac) and posterior commissure (pc) line. before the acquisition of functional images, t1-weighted images (tr / te, 417/9 msec ; matrix, 256256 ; slice thickness, 5 mm ; no slice gap ; fov, 2424 cm) were obtained as anatomic reference images. during the acquisition of functional images, there were two periods of task performance and three resting periods (off - on - off - on - off). each period of activation or rest was 39 sec and 13 series of functional images were acquired during each period. dummy scans were obtained during an initial 12-second period of echo - planar imaging, and during a verbal or visual task the total time for acquisition of the whole functional image was 207 sec. each subject 's echoplanar functional images were corrected for possible head movements using automated image reconstruction (air) software (10). functional image analysis was performed using statistical parametric mapping (spm) software 96 (spm96, mrc cyclotron unit, london, u.k.). to smooth each image a gaussian filter was applied, and to suppress the low frequency physiologic noise in the functional images, a high - pass filter was used. functional maps were created overlapping of functional echoplanar images with anatomic t1-weighted images, and the activated signals were thresholded at p 3.1). brodmann areas (bas) of activated signals in the two working memory tasks were visually determined by means of a standard stereotactic space (11). for the activated regions, ten healthy subjects (male, 5 ; female, 5 ; average age, 24 4 years) were included in this study. all were university students with no history of neurological or psychiatric disorder, and gave informed written consent. the subjects received detailed instructions regarding the experimental conditions and had a brief practice session before entering the scanner. working memory tasks consisted of verbal and visual stimuli. during the activation period of the verbal working memory task, randomized sequences of five phonologically dissimilar consonants of the korean language were displayed on the screen every second. in order to detect whether a target constant appeared 1 second after a series of five consonants was presented, subjects were instructed to rehearse the stimuli silently and to remember them serially. during the activation period of visual working memory task, serial diagrams or symbols that could not be phonologically transcoded were presented instead of consonants. for both types of tasks, each sequence of stimuli was repeated five times during the activation period. for the identification of each sequence of stimuli, one blank slide was presented for 1 second after one sequence of stimuli during an activation task period. subjects responded by pressing a squeeze ball. under control conditions of visual or verbal working memory task, the subjects were instructed to visually fixate on a crosshair. mr images were generated on a 1.5-t mr (general electric medical systems, milwaukee, wi). a series of echoplanar gradient echo images was acquired at intervals of 3 seconds (tr / te, 3000/60 msec ; flip angle, 90 ; matrix, 6464 ; slice thickness, 5 mm ; no slice gap ; fov, 2424 cm). twenty brain slices were obtained parallel to the plane of the anterior commissure (ac) and posterior commissure (pc) line. before the acquisition of functional images, t1-weighted images (tr / te, 417/9 msec ; matrix, 256256 ; slice thickness, 5 mm ; no slice gap ; fov, 2424 cm) were obtained as anatomic reference images. during the acquisition of functional images, there were two periods of task performance and three resting periods (off - on - off - on - off). each period of activation or rest was 39 sec and 13 series of functional images were acquired during each period. dummy scans were obtained during an initial 12-second period of echo - planar imaging, and during a verbal or visual task the total time for acquisition of the whole functional image was 207 sec. each subject 's echoplanar functional images were corrected for possible head movements using automated image reconstruction (air) software (10). functional image analysis was performed using statistical parametric mapping (spm) software 96 (spm96, mrc cyclotron unit, london, u.k.). to smooth each image a gaussian filter was applied, and to suppress the low frequency physiologic noise in the functional images, a high - pass filter was used. functional maps were created overlapping of functional echoplanar images with anatomic t1-weighted images, and the activated signals were thresholded at p 3.1). brodmann areas (bas) of activated signals in the two working memory tasks were visually determined by means of a standard stereotactic space (11). for the activated regions, in all subjects, errors in both verbal and visual working memory tasks were less than 20%. aactivated regions of the brain are summarized in table 1. during the two working memory tasks, activated signals were seen in the frontal, parietal, temporal, and occipital lobes, and the insula, cingulate gyri and cerebellum. activated regions of the frontal lobe were the supplementary motor area (ba 6), motor and premotor (ba 4, 6), prefrontal cortex (ba 9, 10, 46), and inferior frontal gyri (ba 44, 45, 47). activated regions of the parietal lobe were the superior parietal lobe (ba 7) along the intraparietal sulci, the supramarginal gyrus (ba 40), and the angular gyrus (ba 39). when the visual working memory task was compared with the verbal task, common regions activated by the two tasks were the supplementary motor area, the motor and premotor area, the prefrontal cortex, the superior parietal gyri, and the posterior fusiform gyri. although these common regions were activated by both working memory tasks, there was a difference in the distribution of activated signals between each hemisphere. in the verbal working memory task, bilaterally activated signals that were similar between each hemisphere were seen in the prefrontal cortices (8 of 10 subjects), fusiform gyri (7 of 8 subjects), and occipital cortices (all 10 subjects). activated signals in the supplementary motor area (9 of 10 subjects), premotor area (all subjects), and superior parietal cortices (7 of 10 subjects) were lateralized to the left hemisphere during the verbal working memory task. in the visual task, signals activated in the prefrontal cortex and occipital lobe were similar in both hemispheres. in the visual task, signals activated in the fusiform gyri were stronger in the right hemisphere in 3 of 8 subjects and were similar between each hemisphere in other subjects. although bilaterally activated cerebellar signals were found in both visual and verbal working memory tasks, those activated by the visual task were less strong than those activated by the verbal task. in activated regions which included the inferior frontal gyri, inferior parietal cortex, temporal lobe and insula, there was a difference between the verbal and visual working memory task. in the former, these activated regions were, in most subjects, lateralized to the left hemisphere. in the visual task, however, signals in these regions were found in only a few subjects and they were not distinctly lateralized. during the verbal working memory task, activated signals of the inferior frontal gyri were seen in all ten subjects and these were lateralized to the left hemisphere in eight. meanwhile, during the visual task, activated signals were seen in the inferior frontal gyri in only two subjects. in the inferior parietal gyri, activated signals were noted in nine subjects during the verbal working memory task, but in only two during the visual task. in this same area, activated signals were seen mainly in the left supramarginal gyri (ba 40) during the verbal working memory task. in the temporal lobe, activated signals were seen in the middle temporal gyri in all subjects during the verbal working memory task, but in only three during the visual task. in nine subjects, signals were also activated in the insula during the verbal working memory task, but in only one during the visual task. a widely recognized distinction within memory is a dichotomy based upon temporal duration, namely short - term and long - term. according to the accessibility of the retrieved elements to consciousness, long - term memory can be subdivided into declarative, explicit memory, and procedural, implicit memory (12). short - term memory is a system that provides temporary storage and active manipulation of limited information. at present, the concept of working memory has increasingly replaced the concept of short - term memory. after atkinson and shiffrin (13) proposed the modal model that postulated a unique short - term store, this is a temporally and spatially limited capacity that includes multicomponents of short - term storage systems and is capable of simultaneously storing and manipulating the information used in complex cognitive functions (2). working memory also invloves the cognitive function of continuously updating and actively maintaining information, as well as the temporary storage of digits or words. working memory is, therefore, a system necessarily required for complex cognitive tasks (3), and is known to have three subsystems : the central executive, the visuospatial sketch pad and the phonological loop. the visuospatial sketch pad manipulates visuospatial information, and the phonological loop stores and rehearses verbal informations. the central executive control and integrates the other two subsystems (1 - 3). in the present study, the paradigm of working memory task was modified from the item - recognition task described by paulesu (14, 15). the experimental design of this study was based on a subtraction format in which both visual and verbal working memory tasks were designed as activation tasks, and visual fixation as a control task for the avoidance of subtraction of common activated signals. in this study, the verbal stimuli of korean consonants activated the function of verbal working memory of the phonological store and subvocal rehearsal of verbal information, and the visual stimuli of symbols or diagrams activated the function of maintenance of visual images. the central executive function may be activated by both visual and verbal working memory tasks. the results of this study showed that common regions activated by both verbal and visual stimuli were the prefrontal cortices (ba 9, 10, 46) and secondary visual cortices (ba 18, 19), in which the activated signals were similar between each hemisphere during both verbal and visual working memory tasks. these common regions are consistent with the brain regions activated during previous pet and fmr studies (16 - 20). previous studies (16 - 20) also showed that the prefrontal cortex was activated by a task that stimulates the updating or active maintenance of verbal or visual information. the lateral occipital cortex is an area of visual pathway that is activated by various visual stimuli (21, 22). in this study, bilateral activation of lateral occipital cortices seems to have been caused by the perceptual processing of visual stimuli of the verbal or visual working memory task. according to a study of visual working memory (19), occipitotemporal areas in the ventral object vision pathway showed mostly transient responses to visual stimuli, thus indicating their predominant role in the perceptual process, whereas prefrontal areas demonstrated sustained activity, indicating their predominant role in working memory. the prefrontal cortices activated by the visual and verbal working memory tasks may, therefore, play a predominant role in the executive control of working memory. in our study of verbal working memory, signals which activated in the superior parietal cortex were dominant in the left hemisphere. this left dominance seems to related to the neural network of the phonological loop of verbal working memory (20). however, signals which activated in the superior parietal cortex during the visual task were similar between each hemisphere. activation of the bilateral superior cortex during the visual working memory task may be explained by the findings of a pet study (12) showing that bilateral superior parietal cortices are activated by a visual working memory task, whereas simple visual stimuli activate only bilateral occipital cortices. activation of the right parietal cortex may also be related to the neural network of sustained attention involved in the function of working memory (23). in the present study, the cerebellum was activated by both tasks and activated cerebellar signals were stronger in the verbal working memory task than in the visual task. activation of the cerebellum is known to be related to motor function, and its activation may be related to the internal articulation involved in the verbal working memory task (20). the mechanism by which the cerebellum is activated during the visual working memory task is not clear, but it may be that in some subjects, a diagram or symbol is partly phonologically transcoded. bilateral activation of the posterior fusiform gyri might be due to the existence of a ventral visual pathway that is involved in the perceptual processing of visual objects and the visual recognition of verbal stimuli (22, 24). the activation of premotor and motor areas during the verbal working memory task may be related to the motor function of the phonological loop, and the activation of these areas during the visual task might be related to the use of phonological loop or secondary activation originating in the prefrontal cortex during the visual task (20). activated areas which were distinct between verbal and visual working memory tasks were the inferior frontal gyrus, inferior parietal cortex, superior and middle temporal gyri, and the insula. in most subjects, these areas were strongly activated by verbal working memory task, whereas during the visual task, weak activated signals were seen in these same areas in just a few subjects. this result shows that these areas have a predominant role in the phonological loop of the working memory task. according to a previous pet study (14), the inferior frontal gyri (broca 's area) may play a role in subvocal rehearsal, and the supramarginal gyrus may play a role in phonological store. in summary, fmr was used to investigate the functional brain anatomy associated with visual and verbal working memory. based on converging results in this and previous studies, prefrontal cortices activated by both visual and verbal stimuli appear to play a role in the central executive function of active maintenance of information. the left inferior frontal gyri and inferior parietal cortex were dominantly activated by verbal task, forming part of the phonological loop of verbal working memory. the bilateral prefrontal and superior parietal cortices, activated by visual stimuli, seem to be related to the neural pathway involved in the visual working memory task. our study demonstrates that functional areas of working memory could be identified by fmr and that different regions of the brain involved in visual and verbal working memory.	objectivein order to investigate the functional brain anatomy associated with verbal and visual working memory, functional magnetic resonance imaging was performed.materials and methodsin ten normal right handed subjects, functional mr images were obtained using a 1.5-t mr scanner and the epi bold technique. an item recognition task was used for stimulation, and during the activation period of the verbal working memory task, consonant letters were used. during the activation period of the visual working memory task, symbols or diagrams were employed instead of letters. for the post - processing of images, the spm program was used, with the threshold of significance set at p <.001. we assessed activated brain areas during the two stimulation tasks and compared the activated regions between the two tasks.resultsthe prefrontal cortex and secondary visual cortex were activated bilaterally by both verbal and visual working memory tasks, and the patterns of activated signals were similar in both tasks. the superior parietal cortex was also activated by both tasks, with lateralization to the left in the verbal task, and bilaterally without lateralization in the visual task. the inferior frontal cortex, inferior parietal cortex and temporal gyrus were activated exclusively by the verbal working memory task, predominantly in the left hemisphere.conclusionthe prefrontal cortex is activated by two stimulation tasks, and this is related to the function of the central executive. the language areas activated by the verbal working memory task may be a function of the phonological loop. bilateral prefrontal and superior parietal cortices activated by the visual working memory task may be related to the visual maintenance of objects, representing visual working memory.
breast cancer is now the most common cause of female cancer and leading cause of cancer deaths among women in the united states and many other parts of the world [1, 2 ]. over the past several decades human epidermal growth factor receptor 2 (her2) and estrogen receptor (er) play critical roles in the development and progression of breast cancer. about 80% of breast cancers are hormone - receptors positive and express estrogen receptors. about 20% of breast cancers therefore, breast cancer that negative for er and her2 does not respond to hormonal therapy. current therapies for the treatment of breast cancer may result in drug resistance or toxicity. there is growing interest in the use of herbs to aid in the maintenance of women 's health ; it is interesting to use of herbs for the women 's health care. plants contain a wide variety of chemicals that have potent biological effects, including anticancer activity. natural cucurbitacins are highly oxygenated, tetracyclic triterpenes containing the cucurbitane nucleus skeleton and are predominantly found in plants of the family cucurbitaceae, members of which have long been used in oriental medicines because of the wide range biological activity they exhibited in plants and animals. among the various cucurbitacins, the most abundant is cucurbitacin b. cucurbitacin b (scheme 1) extracted from the thai herb trichosanthes cucumerina l. has been shown to have anticancer, antimicrobial, and anti - inflammatory activities [7, 8 ]. several studies reported that cucurbitacin b and its relatives inhibit the growth of human malignant cells both in vitro and in vivo including breast cancer, head and neck squamous cell carcinoma, pancreatic cancer, hepatocellular carcinoma, osteosarcoma, and myeloid leukemia. our previous report has shown that cucurbitacin b exerts anticancer effect by inhibiting telomerase via downregulating both the htert and c - myc expression and arrest of the cell cycle at g2/m phase in breast cancer cells. some studies have reported that cells are most sensitive to radiation in g2/m and most resistant in s phase. for example, synchronized chinese hamster cells were most sensitive to irradiation during mitosis and in g2 phase and less sensitive in g1 and the latter past of s phase [17, 18 ]. drugs, including docetaxel, that arrest cells cell in g2/m phase of cell cycle have been demonstrated as radiosensitizer agent. the aims of this study were to determine the radiosensitizing potential of cucurbitacin b in human breast cancer cells and to elucidate the cellular mechanism of the radiosensitization. in the present study, we demonstrated that cucurbitacin b sensitizes human breast cancer cells to radiation by inducing them to accumulate in g2/m phase of the cell cycle. human breast cancer cell lines (skbr3 (er/her2 +), mda - mb-231 (er/her2), and hormone - independent mcf7:5c (er/her2)) was cultured at 37c under a 5% co2 atmosphere. this compound was dissolved in 10% dimethylsulfoxide (dmso) and diluted with dmem / f12 medium and mccoy 's 5a medium to the desired concentrations prior to use. a cesium machine was used to radiate cells with a dose ranging from 0 to 10 gy. cells were seeded in a 100-mm culture plate and treated with the indicated concentration of cucurbitacin b for 48 hr prior to radiation exposure. after exposure, cells were then trypsinized and seed on the basis of difference density in a 60 mm culture plate with 5 ml of medium. surviving fractions were calculated by normalization to the plating efficiency of appropriate control groups. for cell cycle analysis, cells were treated with cucurbitacin b at various concentrations for 48 hr and harvested. one million cells were centrifuged and suspended in 0.5 ml of krishan reagent (0.1% na citrate, 0.03% np-40, 0.05 mg / ml pi, 0.02 mg / ml rnase a) before analysis. the stained cells were subjected to dna content / cell cycle analysis using an lsr flow cytometer. for apoptosis, the annexin v - fitc apoptosis detection kit (bd bioscience, bedford, ma), fresh cell preparations were incubated with 1x annexin binding buffer and annexin v - fitc- (2.5 g / ml) conjugated primary antibody for 15 min on ice. after incubation, propidium iodide (pi ; 10 g / ml) was added to the suspension and the cells were analyzed by flow cytometry using an lsr flow cytometer. cells (5 10 cells / well) were seeded into 6-well plate and treated with various concentration of cucurbitacin b for 48 hr. total rna was isolated from each cell line using the qiagen rneasy mini kit (qiagen, valencia, ca). two micrograms of total rna were reverse - transcribed with random primer according to the manufacture 's protocol using high - capacity cdna reverse transcription kit (applied biosystems, foster city, ca). real - time pcr was performed using fast sybr green master mix (applied biosystems) with the applied biosystems 7500 fast real - time pcr system (applied biosystems). the pcr primers set were as follows : sense5-tgagccgcgactgtgatg-3 and anti - sense5-gtctcggtgacaaagtcgaagtt-3 for p21 and sense5-gaaggtgaaggtcggagtc-3 and anti - sense5-gaagatggtgatgggatttc-3 for gapdh. the relative ratio of p21 was then calculated using the formula : 2 = 2, where ct = ct(p21)ct(gapdh). after cucurbitacin b treatment, cell pellets were collected and lysed with 100 l ripa cell - lysis buffer (50 mm tris ph 8.0, 150 mm nacl, 0.1% sds, 0.5% na deoxycholate, 1% tx-100) plus 1 mm naf, 10 mm navo4, 10 mm pmsf, and 1/100 protease inhibitor cocktail (sigma). total protein was determined using bio - rad protein assay (life science, hercules, ca). equal amount of proteins were separated by 12.5% sds - polyacrylamide gels and electrotransferred onto nitrocellulose membranes and treated with anti p21 (santa cruz biotechnology, inc., santa cruz, ca) overnight. equal protein loading was confirmed on all immunoblots using beta - tubulin antibody (developmental studies hybridoma bank, university of iowa, iowa city, ia) at a dilution 500. goat anti - rabbit igg (bd transduction laboratories, san diego, ca) was used as a secondary antibody against all primary antibodies. bands were visualized by chemiluminescence with ecl plus reagent (pierce, rockford, il) on a typhoon fla 7000. statistical analysis was performed using one - way anova to compare the effect among control (without cucurbitacin) and treated cells. the inhibitory effect of cucurbitacin b on colony formation in human breast cancer cells was evaluated by clonogenic assay. cells were incubated with cucurbitacin b alone for 48 hr and then allowed to form colonies in fresh medium. the average 50% (ic50) inhibitory concentrations for clonogenic cell death in three cells was 3.2, 2.4, and 1.9 m for mcf7:5c, and mda - mb-231, and skbr-3 respectively. the results are the average from three independent experiments for each cell lines. in the clonogenic assay skbr-3, was the most sensitive cell to cucurbitacin b under the same condition for other cells. effect of cucurbitacin b on cell cycle progression in mcf7:5c, mda - mb-231, and skbr-3 cells were analyzed according to the principle of the dna content in each phase of, cell cycle. cells were treated with cucurbitacin b for 48 hr, and dna content was analyzed via flow cytometry. mcf7:5c and mda - mb-231 cells after treated were arrested at g2/m phase of cell cycle with a decrease of cells population in g1 and s phase of cell cycle, as was observed in several cancer cell lines. however, in contrast to the effect of cucurbitacin b on mcf7:5c and mda - mb-231 cells, cucurbitacin b did not contribute to g2/m phase arrest in skbr-3 cells (figure 2). apoptosis effect of cucurbitacin b was evaluated by using annexin v - fitc and propidium iodide staining. this assay revealed that the negatively charged phospholipids phosphatidylserine found on the interior surface of the plasma membrane of the cells is trans - located to the cell surface during apoptosis. after 48 hr incubation with 0 m, 2.5 m, and 5 m of cucurbitacin b, cells were stained and subjected to bivariate flow cytometric analysis. as shown in figure 3, untreated cells did not show any significant apoptosis, whereas cells become apoptotic with cucurbitacin b treatment at the indicated concentration in all cells. to determine the effect of cucurbitacin b on p21 mrna expression, all three cell lines were incubated with cucurbitacin b for 48 hr, in parallel with untreated cell. exposure of mcf7:5c, mda - mb-231, and skbr-3 cell lines to 2.5 m and 5 m of cucurbitacin b resulted in the progressive increase p21 mrna level. the expression p21 mrna in skbr-3 was increased up to 20 times when compared with untreated cell while mcf7:5c and mda - mb-231 was increased 3 - 4 times as shown in figure 4(a). the real - time pcr products were applied on 0.8% agarose gel containing ethidium bromide (etbr) to scrutinize that the pcr reaction was specific and that cucurbitacin b induced gene expression of p21. we examined the effect of cucurbitacin b on the expression of cell cycle regulated protein by western blot analysis. cells were incubated with the indicated concentration of cucurbitacin b for 48 hr and total protein were extracted for western blot analysis. as shown in figure 5, protein expression of cyclin - dependent kinase inhibitor p21 was significantly increased following cucurbitacin b treatment in all study cells. skbr-3 cells, which showed the highest mrna accumulation in response to cucurbitacin b, also showed the greatest induction of the p21 protein ; however, this was not necessarily linked to g2/m arrest or radiation sensitization by cucurbitacin b. to determine whether cucurbitacin b sensitized human breast cancer cells to ionizing radiation, all three cell lines were treated with 5 m cucurbitacin b for 48 hr following irradiation with a cs gamma - irradiator at doses ranging 08 gy. figure 6 shows the radiation survival curves derived from clonogenic assays of the three cell lines irradiated after 48 hr incubation with cucurbitacin b. the latter slope of survival curve of mcf7:c and mda - mb-231 cells for radiation and cucurbitacin b treated were greater than radiation only, especially at the 6 and 8 gy radiation doses, indicating that cucurbitacin b treatment augmented the effects of radiation in both cell lines where g2/m arrest was observed to occur. however, skbr-3 did not show to augment the effects of radiation, consistent with the cell cycle distribution shown in figure 2 and the absence of a g2/m arrest in skbr-3 cells. the cucurbitacins are highly oxygenated, tetracyclic triterpenes containing the cucurbitane nucleus skeleton and are of great interest because of the wide range biological activity they exhibited in plants and animals. many reports have shown that cucurbitacin b has potent antiproliferative effect on breast cancer cells. for instance, cucurbitacin b, extracted from root and fruit juice of t. cucumerina has been reported to exert the cytotoxicity on human breast cancer cell lines. reported that cucurbitacin b exerts the anticancer activity against er, her2/neu amplified, and p53 mutant breast cancers both in vitro and in vivo. similarly, cucurbitacin b has been reported to inhibit wnt signaling pathway through reduction of wnt - associated protein and reduced translocation of galectin-3-mediated -catenin to the nucleus. in this study, we analyzed the anticancer activity of cucurbitacin b in human breast cancer cells lines : mcf7:5c, mda - mb-231, and skbr-3 using clonogenic survival assay. among these three cells lines, skbr-3 express high levels of her2/neu receptor, whereas mcf7:5c and mda - mb-231 do not express these receptor. skbr-3 is the most sensitive to cucurbitacin b when compared with other two cell lines. we further determined the effect of cucurbitacin b on cell cycle progression and apoptosis induction in breast cancer cell lines. the results showed that apoptotic cells were induced by cucurbitacin b treatment in all cell lines, and cell cycle is arrested at the g2/m phase in mcf7:5c and mda - mb-231 cells but not in skbr-3 cells (figure 2). several authors have shown that the radiosensitizing effect of anticancer drug is due to cell cycle alteration. paclitaxel, a microtubule - stabilizing drug, has been shown to enhance radiosensitivity by blocking cells in g2/m phase of the cell cycle [24, 25 ]. since cells in the g2/m phase have been reported to be more radiosensitive than in other phases of cell cycle, and cucurbitacin b treatment increases the number of cells in g2/m phase of the cell cycle and thus enhanced the effects of radiation on breast cancer cell line. in our study, cucurbitacin b exerted the radiosensitivity when administered 5 m of cucurbitacin b before radiation on mcf7:5c and mda - mb-231 cells. however, no radiosensitization occurred when skbr-3 cells were exposed to cucurbitacin b at 5 m and radiation, and no accumulation of cells in the g2/m phase was observed prior to the start of irradiation under these conditions. p21 and cell cycle checkpoints were shown to modulate the nucleotide excision repair process to facilitate the repair of dna damage even in the absence of wild - type p53. our results indicate that p21 expression level by cucurbitacin b treatment mostly showed upregulation in all cell lines especially in skbr-3 which showed the highest induction when compared with other cell types (figures 4 and 5). therefore, no radiosensitization was manifest using 5 m cucurbitacin b in skbr-3 likely because cucurbitacin b did not induce a g2/m cell cycle arrest. taken together, the radiosensitizing effect by cucurbitacin b was dependent on the induction of g2/m arrest in breast cancer cells but not necessarily on the induction of p21. in summary, cucurbitacin b can enhance the effect of radiosensitization on breast cancer cells and studies in vivo are required to evaluate the biological efficacy of cucurbitacin b treatment.	purpose. to explore the effects of cucurbitacin b on the radiation survival of human breast cancer cells and to elucidate the cellular mechanism of radiosensitization if any. materials and methods. human breast carcinoma cell lines were treated with cucurbitacin b before irradiation with 010 gy of 137cs gamma rays. the effect of cucurbitacin b on cell - survival following irradiation was evaluated by colony - forming assay. cell cycle distributions were investigated using flow cytometry. real - time pcr and western blots were performed to investigate the expression of cell cycle checkpoints. results. cucurbitacin b inhibited breast cancer cell proliferation in a dose - dependent manner. only mda - mb-231 and mcf7:5c cells but not skbr-3 cells were radiosensitized by cucurbitacin b. flow cytometric analysis for dna content indicated that cucurbitacin b resulted in g2/m arrest in mda - mb-231 and mcf7:5c but not skbr-3 cells. moreover, real - time pcr and western blot analysis demonstrated upregulated p21 expression before irradiation, a likely cause of the cell cycle arrest. conclusion. taken together, these findings suggest that cucurbitacin b causes radiosensitization of some breast cancer cells, and that cucurbitacin b induced g2/m arrest is an important mechanism. therefore, combinations of cucurbitacin b with radiotherapy may be appropriate for experimental breast cancer treatment.
the rarely described cytological features of chondroid syringoma included a mucoid aspirate, abundant chondromyxoid matrix material and epithelial components. chondroid syringoma or cutaneous mixed tumors are rare benign skin adnexal tumors accounting for only about 1% of commonly encountered skin tumors. it is now widely accepted that fine needle aspiration cytology (fnac) is useful in diagnosis of skin adnexal tumors. a mucoid aspirate, abundant chondromyxoid matrix material, and epithelial components were suggested as diagnostic criteria. the reported case is one of chondroid syringoma confirmed by histopathology, the fnac of which yielded thin fluid - like material and microscopy revealed epithelial clusters with admixed smaller myoepithelial cells, background cyst macrophages, and only scanty chondromyxoid stromal elements. a 31-year - old male patient presented with a solitary 3 cm 2.5 cm slowly growing, non - tender, cystic left supraorbital swelling [figure 1a and b ]. fnac yielded a thin fluid - like aspirate from which leishman and pap stained smears were prepared. microscopic examination revealed moderately cellular smears composed of cohesive clusters of medium - sized epithelial cells with moderate to abundant amount of cytoplasm, peripheral cytoplasmic processes, and bland appearing monomorphic centrally placed or slightly eccentric nucleus with fine chromatin. smaller hyperchromatic myoepithelial cells were seen intimately admixed with the epithelial cells in most of the clusters [figure 2 ]. fair number of cyst macrophages, a thin proteinaceous material, and only rare chondromyxoid stromal fragments were detected in a predominantly clean background. a cytological diagnosis of benign cystic neoplasm possibly benign skin adnexal tumor was made and excision biopsy suggested. (b) left supraorbital swelling (closer view) epithelial myoepithelial cell clusters in a clean background of thin proteinaceous material and cyst macrophages (inset ; seen elsewhere) (pap ; 100) excision biopsy was performed 2 weeks later. low power showed multiple well - circumscribed cystic spaces lined by tissue having biphasic growth pattern [figure 3 ] with epithelial elements in the form of non - branching bilayered tubules as well as solid nests embedded within a predominantly fibrous and focally chondromyxoid stroma. differential diagnoses included eccrine hydrocystoma and syringocystadenoma papilliferum both of which lacked the distinctive chondromyxoid stromal element. well - circumscribed cystic spaces lined by tissue having biphasic growth pattern (h and e, 100). the term chondroid syringoma, introduced in 1961, may be preferred to the alternative designation - mixed tumor of the skin because of the recognition that the tumor is epithelial with merely secondary changes in the stroma. the world health organization has defined chondroid syringomas as benign adnexal tumors of skin composed of epithelial and stromal elements with a wide spectrum of patterns histologically analogous to mixed tumors of the salivary gland. they most commonly occur as solitary asymptomatic slowly growing nodules on the head and neck of adults, although other sites may be affected. there is a male predilection and most lesions are between 1 and 3 cm in diameter, although examples as large as 10 cm have been reported. it is generally accepted that there are both apocrine and eccrine variants of mixed tumors. ultrastructural studies confirm that myoepithelial cells surround the epithelial cells and appear to produce the stromal components of the lesions. histologically, two types of chondroid syringomas can be recognized : one with tubular and cystic, partially branching lumina and the other with small tubular lumina. the former type is much more common than the latter and shows marked variation in the size and shape of the tubular lumina ; it also shows cystic dilatation and branching. embedded in an abundant stroma, the tubular lumina are lined by two layers of epithelial cells : a luminal layer of cuboidal cells and a peripheral layer of flattened cells. among the published literature on cytological features of chondroid syringomas, predominance of chondromyxoid elements and presence of only epithelial cell clusters the present case had extensive cystic changes which according to the published literature is unusually seen in cytology but consistent with the more common histological type. the cystic change and presence of both epithelial and myoepithelial cells including the cytological features of the present case among the diagnostic criteria for cytodiagnosis of chondroid syringomas might increase the chances of a definitive preoperative diagnosis and help in planning the extent of surgery.	chondroid syringoma is a rare benign skin adnexal tumor, the cytological features of which have been published very rarely in the literature. a mucoid aspirate, abundant chondromyxoid matrix material, and epithelial components were suggested as diagnostic criteria. the reported case is one of chondroid syringoma confirmed by histopathology, the fine needle aspiration cytology of which yielded thin fluid - like material and microscopy revealed epithelial clusters with admixed smaller myoepithelial cells, background cyst macrophages, and only scanty chondromyxoid stromal elements. including these cytological features of the present case among the diagnostic criteria for cytodiagnosis of chondroid syringomas might increase the chances of a definitive preoperative diagnosis and help in planning the extent of surgery.
a 50-year - old female was referred to the department of oral and maxillofacial surgery for treatment of tmj ankylosis. the patient had experienced a fall, but she could not remember the time and the specifics of the associated trauma accurately. clinical examination revealed facial asymmetry and limitation of the mouth opening (2 mm). on a panoramic radiograph, bony fusion of the right tmj, a prominent right antegonial notch, and severe deviation of the mandible to the right were noted, along with multiple dental caries, retained roots, and generalized severe alveolar bone loss. a well - defined outline of the condyle was noted in the left tmj (fig. 1). ct images revealed bony ankylosis with a large mass in the right tmj area, the loss of air cells in the temporal squama and mastoid area, and a thickened right zygomatic arch. the normal relationship of the articular fossa and the condyle with the articular space was revealed in the left tmj (fig. 2). magnetic resonance imaging (mri) showed a large fused bone mass with normal bone marrow in the right tmj (fig. 3) and flattening of the condyle with a thin disk in the left tmj. irregularities of the cortical bone of the articular fossa and the condyle were also detected in the left tmj (fig. 4). a three - dimensional (3d) rapid prototyping (rp) model was fabricated using the ct images. a narrowed articular space between the condylar head and the articular fossa was detected in the left tmj (fig. radiographically, the differential diagnoses for the right tmj included benign bone tumors such as osteochondroma, ostoma, chondroma, chondroblastoma, benign osteoblastoma, fibro - osteoma, fibrous dysplasia, myxoma, and giant cell tumor. however, careful history taking and radiological and histological examination for this patient would be useful for the differential diagnosis.12 on the basis of the clinical and radiological examinations, the patient was diagnosed with bony ankylosis in the right tmj and suspected adhesion in the left tmj. a preauricular incision with a temporal extension on the right side was made. in order to create an approximately 15 mm wide gap, excision of the bony mass an intraoral incision was made on the left side, and the left coronoid process was removed. surgical opening of the joint, discectomy, and high condylectomy were performed on the left tmj. an occlusal stop (height : 30 mm) was inserted between the dental arches and maxillomandibular fixation (mmf) and was maintained for 5 days. postoperative pain medications were administered, and vigorous postoperative physiotherapy was performed to preserve the range of motion and to prevent postoperative hypomobility due to recurrence.513 after the 3-month follow - up, the mmo was 25 mm. four months after dental implant surgery, she had full mouth rehabilitation (fig. 7). two years after surgery, the mmo was approximately 20 mm, and the masticatory function was normal (fig. 8). in the past, conventional radiographies such as panoramic, lateral and posteroanterior skull, submentovertex, and transcranial radiography were frequently used for the diagnosis of tmj pathologies. these radiographies had a limitation with respect to the analysis of anatomical structures because of the superimposition of these structures.3 nowadays, ct imaging provides more detailed information to radiologists and surgeons, such as the shape of the mandibular condyle and pathological changes in the joint outline. the osseous changes in the condyle such as erosion, osteophyte, and sclerosis are easily detected on the coronal and sagittal views of ct images. moreover, 3d ct and rp models are useful to radiologists and surgeons in the pre - operative period. they show an accurate, realistic structure of the patient 's joint, which is very useful for diagnosis and surgical planning.314 mri has good tissue contrast resolution and performs better in the differentiation of soft tissue structures than ct images. fibrous adhesion is the most common form of an anteriorly displaced disc.9 in our case, radiological findings revealed that the right tmj definitely had bony ankylosis with a loss of air cells in the temporal squama and mastoid area. mri showed flattening of the condyle and a thin disk in the left tmj in this case. adhesion of the tmj is usually diagnosed on the basis of an immobile disk during mouth opening as revealed by mri.8915 because our patient had severe bony ankylosis of the right tmj, the left tmj also did not move. the mandible did not move after right gap arthroplasty ; therefore, the left tmj could be diagnosed with severe adhesion. further, a patient with unilateral severe bony ankylosis of the tmj might have adhesion at the opposite side, which would limit mouth opening. roychoudhury.16 recommended at least a 15 mm gap between the recontoured glenoid fossa and the mandible for preventing reankylosis. in our case, we obtained a 15 mm gap ; this gap was well maintained after surgery. in order to prevent reankylosis, it is important to place an interpositional graft in the surgically formed gap.17 to achieve mmo, ipsilateral and/or contralateral coronoidectomy is necessary.18 in our case, the temporal fascia was used for preventing recurrence, and coronoidectomy of the left mandible was also performed. because the tmj area is a complex region, the risk of complications after tmj surgery is high ; complications include facial scars, facial nerve palsy, gustatory sweating, perforation of the middle cranial fossa, and damage of the maxillary artery. 1619 our patient had pain and a facial scar immediately after surgery, but no other complications. two years after surgery, although the mmo decreased from 35 mm to 20 mm, ankylosis did not recur, the patient was satisfied with surgery, and her body weight increased due to proper nutrition. in conclusion, a patient with severe bony ankylosis of the tmj on one side might have adhesion on the opposite side, which would limit mouth opening. therefore, radiologists and surgeons should use a more careful approach for diagnosis and treatment.	bony fusion between the mandibular condyle and skull base involves temporomandibular joint (tmj) bony ankylosis. this condition might originate from trauma, infection, or systemic disease. tmj adhesion can develop after synovial damage. both tmj ankylosis and adhesion lead to functional impairment and pain. here, we present a case of a 50-year - old female who had bony ankylosis of the right tmj and adhesion of the left tmj. she had otitis media in the right ear. a large mass in the right tmj was observed on computed tomograph. magnetic resonance image showed a large fused bone mass with normal bone marrow in the right tmj and flattening of the condyle with a thin disk in the left tmj. gap arthroplasty with temporal fascia was performed on the right tmj, and discectomy, high condylectomy, and coronoidectomy were performed on the left tmj. during a 2-year follow - up after surgery, the patient had no recurrence.
solid organ transplantation has become the mainstay of treatment for children with end - stage heart, kidney, lung and liver diseases, improving survival and life expectancy in up to 80% of patients. the history of solid organ transplantation dates back to 1952, when the first pediatric kidney transplant was performed (1), followed by the first liver transplant in 1963 (2) and the first heart transplant in 1967 (3). since these early successes, the development and constant improvement of surgical techniques, anesthetic procedures (4 - 6) and post - transplantation care, especially immunosuppressive therapy, have greatly increased the rate of graft success and have improved recipient outcomes (7 - 10). despite the progress made in the field of pediatric transplantation in recent years, the clinical development of this therapeutic approach remains a fundamental challenge, mainly due to inherent issues related to conducting clinical transplantation research in children. these issues can be categorized as those associated with the study of surgical techniques and anesthetic procedures, those related to post - transplant care, including the study of immunosuppressive therapy, and challenges specific to the disease type and population group. research exploring surgical techniques for pediatric solid organ transplantation poses several burdens on researchers. for example, the use of placebo or sham procedures is both ethically and logistically unviable, making it impossible to include a control group in clinical trials. it is also difficult to establish a clear definition for standard of care due to the constant evolution of surgical techniques, innovation in operative instruments, changes in the criteria for donor and recipient selection, improvements in perioperative management and the continuing perfection of anesthetic procedures. consequently, the comparability and translation of the results is limited. due to the low number of cases per year, most clinical trials resort to the use of multiple research centers to meet their participant accrual and enrollment requirements, which can introduce bias to the study related to the variability in surgical procedures among centers and the grade of expertise among surgeons. conducting observational studies is valuable ; however, similar confounders, mainly those associated with variability in donor and recipient selection criteria and in surgical technique and expertise among centers, can obscure and limit the results. post - surgical management is an essential part of transplant care, and a high percentage of the success of solid organ transplants can be attributed to the improvement in peri- and post - operative care of these patients, including immunosuppression therapy. research studies evaluating the efficacy and safety of immunosuppressive therapies for solid organ post - transplant care are difficult to design and conduct (11). most of the challenges associated with this type of trial are related to ethical concerns of using placebo in the control group. only a few randomized clinical trials have been conducted, and most of these compare a new intervention combined with standard treatment to standard treatment alone (12,13) or opt for a non - inferiority design, comparing the new intervention to standard treatment (14). the main limitations of these two types of designs are the large sample size required to achieve good power and poor generalizability of the results because of the strict selection criteria and lack of a true placebo - control group. due to these limitations, most researchers opt for observational studies ; however, this type of design has inherent restrictions associated with the effects of potential confounders on the results. the lack of consensus and standardization of post - transplant care in pediatric patients is also a major limitation for the design of clinical trials and for drawing conclusions that can be used in the clinical setting. (15) reported a wide range of definitions for inclusion criteria and high variability among measurements in the 18 trials included, demonstrating that these disparities can be a significant barrier to the improvement of clinical research in pediatric transplantation. similar findings were reported by rothbaum and colleagues (16), emphasizing the lack of strong evidence in the field. results from in vitro studies and animal models can not always be translated to in vivo outcomes, mainly because they are unable to accurately predict the effects of new therapeutic molecules in the dynamic donor - recipient relationship or reflect their influence on disease type and severity. extrapolation of the results from research exploring similar diseases is not always possible because of the specific effects of this therapeutic procedure on the pathophysiological mechanisms of each condition. most clinical trials and observational studies in this field include only adult participants ; these studies are commonly used as guidelines for decision - making related to transplantation in children. however, the generalizability of results obtained with adults to pediatric populations is limited and should be assessed carefully. the effects and success of transplantation in children can significantly differ from that in adults, mainly because of specific aspects such as growth and development, differences in the classification of severity in end - stage diseases and criteria for recipient selection, which can highly influence graft viability and success. the small number of solid organ transplantations performed in pediatric patients per year remains an important challenge for research in this field, and it is even more evident when the analysis is focused on organs such as the heart and lung. 17) reported that 125 pediatric lung transplantations were performed annually worldwide, with an average of 2 - 3 transplants per center per year. randomization of children in the context of a clinical trial poses major ethical concerns, especially when new interventions are being tested. obtaining informed consent either from the subjects themselves or from parents and legal guardians is a major barrier for randomized clinical trials (rcts) in pediatric populations (18,19). the zelen design was implemented as an alternative to conventional rcts, allowing researchers to seek consent from the patient or parents after randomization is completed. therefore, participants are asked to consent to receiving a specific treatment and not to being randomized in the trial, which increases the accrual rate given that subjects are aware of their allocation at the moment of consent. the first example of this randomization approach was the extracorporeal membrane oxygenation trial, which tested the effects of extracorporeal membrane oxygenation in neonates with severe pulmonary hypertension (20). to date, no adaptations of the zelen design have been reported in the transplant literature. given all of these challenges, researchers in pediatric transplantation should be aware of potential alternatives that can aid in overcoming the barriers to clinical research in this field. these studies should be carefully designed, and researchers must be aware of and actively think about potential confounders that may obscure the results. these potential cofounders should be accounted for both at the moment of design and during data analysis. the development of pragmatic trials can also be an alternative to research in pediatric transplantation, as they allow the effects of an intervention to be studied in real - life routine practice conditions and can be extended to more complex and robust explanatory trials. as previously mentioned, some of the limitations of rcts, both in surgical and clinical scenarios of pediatric transplantation, are associated with the inability to translate the results obtained from adult studies and from pediatric studies examining similar diseases and procedures. for researchers to overcome these barriers, it is necessary to have a clearer understanding of the mechanisms that determine success or failure of transplantation in children and how they differ from those in adults and in other diseases. another major challenge is related to the small number of pediatric transplantation cases per year. several initiatives have been created to aid this purpose, with the aim of consolidating a strong network of transplant centers with unified criteria for patient selection, standard surgical procedures and post - operative care. these initiatives also aim to create a large registry of pediatric solid - organ transplant recipients for future observational research in the field. for example, the international pediatric lung collaborative (ipltc) (17) and the pediatric heart transplant study group (phts) (21) were created for this purpose and have been successful in terms of consolidating a strong database for observational studies in this area. the national institute of allergies and infectious diseases designed a helpful strategy that aims to increase the number of trials conducted in pediatric transplant recipients, referred to as clinical trials in organ transplantation in children (ctot - c) 22. the goal of this initiative is to improve the understanding of the immune factors that play a role in transplant success. to date, approximately 10 clinical trials have been approved and are actively recruiting. more than 70 research centers in 50 different academic institutions are involved in this initiative. in conclusion, there are fundamental challenges related to the design and execution of clinical trials and observational studies for pediatric solid - organ transplantation. efforts should be made to develop alternatives that can help researchers overcome some of these challenges so that evidence in this field can be strengthened and clinical practice can be based on robust results from well - designed studies.	solid organ transplantation has greatly improved survival in children with end - stage disease, becoming one of the main treatment options in this population. nonetheless, there are significant challenges associated with validating and optimizing the effects of these interventions in clinical trials. therefore, we reviewed the main issues related to conducting clinical transplantation research in children. we divided these challenges into three different categories : (i) challenges related to surgical techniques and anesthetic procedures, (ii) challenges related to post - transplant care and (iii) challenges specific to a particular population group and disease type. some of the observed burdens for clinical research in this field are related to the limitations of conducting studies with a placebo or sham procedure, determining the standard of care for a control group, low prevalence of cases, ethical concerns related to use of a placebo control group and lack of generalizability from animal studies and clinical trials conducted in adult populations. to overcome some of these barriers, it is necessary to utilize alternative clinical trial designs, such as observational studies or non - inferiority trials, and to develop multicenter collaborations to increase the recruitment rate. in conclusion, the lack of robust data related to pediatric transplantation remains problematic, and further clinical trials are needed to develop more efficacious and safer treatments.
superficial acral fibromyxoma (saf) is a slowly growing soft tissue tumor that tends to appear in the acral sites., reported its clinicopathologic features and immunohistochemistry (ihc) findings, and since then, around 170 cases have been reported. clinically, it appears as a slow growing, well - circumscribed neoplasm of fingers and toes in middle - aged adults. pathological findings include a dermal or subcutaneous tumor with spindled and stellate - shaped cells that are embedded in myxoid or collagenous matrix. it stains positive for cd34 and focally positive for epithelial membrane antigen (ema) and cd99. there have been a few reports of cases that were negative for cd34, however, to our knowledge, none of the cases reported before showed the distinct immunophenotype seen in our case. herein, we report a case of cd34 negative saf with clinical pictures and ihc investigation. an 18-year - old female presented to our dermatology clinic with the complaint of a slowly growing subungual nodule since a few months. the lesion was painless, she did not mention any history of trauma in the area, and the past medical history was negative for any relevant disease. the physical examination showed a nontender subungual, nodular lesion with 12 mm diameter in the right big toe [figure 1a ]. (a) nontender subungual, nodular lesion in the right toe ; (b) after excision of the lesion punch biopsy of the lesion revealed a fairly circumscribed dermal proliferation of spindled and stellate cells with loose storiform and fascicular pattern embedded in fibrocollagenous stroma with foci of myxoid change [figure 2a and b ]. the lesion was completely excised by a plastic surgeon with free margin and did not recur in 6 months follow - up [figure 1b ]. ihc staining of our case revealed positive staining with vimentin, focal reaction with smooth muscle actin, negative reaction with cd34, and positive staining pattern with cd99 [figure 3a+d ]. (a and b) high power view of bland spindle cells in a fibromyxoid stroma. note the normal looking epidermis overlying the tumor (h and e 40) immunohistochemical (ihc) study ; low power view of neoplastic spindle cells which show ihc positive staining with vimentin (a), focal reaction with smooth muscle actin (b), negative reaction with cd34 (c), and positive staining pattern with cd99 (d). saf is a relatively rare dermal or subcutaneous myxoid tumor, which is well - circumscribed and unencapsulated. the size of the lesions could be between 0.6 to 5.0 cm (mean : 1.75 cm). in a series of cases with saf, the lesion existed from 3 months to 30 years (median duration : approximately 3 years) it habitually arises on the fingers and toes of middle - aged adults but unusual sites of occurrence such as heels have also been reported. the frequent deformity of the nail plate necessitates the removal of the nail plate during surgical procedure. our case did not show involvement of the underlying bone, however, the nail bed was involved and the nail unit was completely excised during the surgical procedure. saf is a benign neoplasm and there have been no reports of malignant transformation or metastasis ; however, there are a number of recurrent cases that have been related to incomplete resection. histological appearance of saf is more or less consistent with virtually all tumors presenting with spindle cells. it shows an indistinct storiform and fascicular pattern embedded in a myxoid / fibromyxoid / collagenous stroma often with mildly accentuated vasculature and increased numbers of mast cells. atypia and mitotic figures are generally absent ; however, there are reports of cells with atypical features found in this tumor. neoplastic cells in saf usually present with immunoreactivity for cd34, cd99, and ema and negative staining for actin, desmin, keratins, s100 protein, and hmb45. our case exhibited positive staining for cd99, vimentin and sma and negative staining for cd34, and s100. immunoreactivity to cd34 is a common feature of saf ; however, there are tumors with negative staining for this marker. in fact, in a detailed characterization of 124 cases with saf, approximately one - third of lesions were negative for cd34. therefore, a diagnosis of saf should be considered even in the absence of reactivity for cd34. benign lesions include those with proliferation of spindle cells and myxoid lesions with spindle - shaped cells such as myxoid neurofibroma, superficial angiomyxoma. angiomyxoma stains positive for cd34 but has a predilection for head, neck, and trunk and has prominent hyalinized vessels. malignant myxoid lesions include myxoid dermatofibrosarcoma protuberans, acral myxo - inflammatory fibroblastic sarcoma, and low - grade fibromyxoid sarcoma. focal storiform pattern in this tumour should be differentiated with dermatofibrosarcoma protuberans, which shows cd34 reaction but occurs very rarely in acral sites. acral myxoinflammatory fibroblastic sarcoma has a predilection for subcutaneous soft tissues of extremities and shows prominent inflammatory cell component and bizarre tumor cells and cd34 is diffusely positive in most cases. the other differential diagnosis of our case is onychomatricoma in which there are similar stromal cells, however, the epidermal changes in onychomatricoma makes it quite different. glomus tumor is another differential diagnosis which is almost always painful and usually stains positive for vimentin and smooth muscle antigen and negative for desmin and cd99. since this tumor was cd99 positive, it should be distinguished from monophasic synovial sarcoma, which usually shows areas of calcification and at least focal reaction to cytokeratin. in conclusion a diversity of lesions can occur in the subungual area, and proper diagnosis and treatment of these lesions requires a meticulous histopathological and ihc evaluation.	superficial acral fibromyxoma (saf) is a slow growing soft tissue tumor that mainly appears in the acral areas. here, we report a case of a saf with distinctive immunophenotype charachteristics. an 18-year - old female was referred to our clinic with the complaint of painless subungual nodule of great toe for a few months. the diagnosis of saf was made according to histopathology and immunohistochemical (ihc) study, however, the ihc assessment showed positive staining with vimentin, focal reaction with smooth muscle actin, negative reaction with cd34, and positive staining pattern with cd99. these ihc findings are unusual for saf. this reported case of saf supports the fact that, although cd34 expression is characteristic for saf, it is not always present.
acute renal failure is an important disturbance in many patients and renal ischemia - reperfusion (rir) injury is the most common cause of acute renal failure. rir injury induces renal dysfunction characterized by increasing levels of creatinine (cr) and blood urea nitrogen (bun). in addition, studies have shown that the incidence of renal diseases is gender - related, and males exhibit a more rapid reduction in renal function than females. mature male rats are more vulnerable to rir injury than female rats, and females have higher survival rates than males. studies on experimental animal models have shown that male and female sex hormones affect renal injury. in addition, it is reported that estradiol replacement therapy attenuates renal dysfunction in diabetic ovariectomized female rats. estradiol therapy in brain injury in rats provides a protective effect against ischemia and improves spatial learning and memory. in addition, administration of estradiol ameliorates liver dysfunction in aging male rats while estradiol did not protect the kidney against confers protection nephrotoxicity. to the best of our knowledge, the role of estradiol in rir injury in males and females was not compared yet. accordingly, this study was designed to determine whether estradiol could prevent rir - induced renal injury in male and ovariectomized female rats. sixteen adult female (weighing 175 5.2 g) and 19 adult male (weighing 222.4 8.6 g) wistar rats (animal center, isfahan university of medical sciences, isfahan, iran) were used in this study. prior to the experiment, the protocols were confirmed to be in accordance with the guidelines of animal ethics committee of the isfahan university of medical sciences. one week before estradiol administration, all female rats were anesthetized, and an incision was made in the abdominal middle line above the urinary outlet. the animals were randomly divided into six experimental groups ; group 1 (n = 5, the sham - operated group) : male rats received sesame oil (intramuscularly) and after 3 days underwent the surgical procedure without rir surgerygroup 2 (n = 8) : male rats received regimen the same as group 1 and underwent rir surgerygroup 3 (n = 6), male rats received single dose of estradiol (500 g / kg ; intramuscularly) and 3 days later underwent rir surgerygroups 4, 5, and 6 (n = 5, 6, and 5) : ovariectomized female rats received treatment / regimen the same as groups 1, 2, and 3, respectively. group 1 (n = 5, the sham - operated group) : male rats received sesame oil (intramuscularly) and after 3 days underwent the surgical procedure without rir surgery group 2 (n = 8) : male rats received regimen the same as group 1 and underwent rir surgery group 3 (n = 6), male rats received single dose of estradiol (500 g / kg ; intramuscularly) and 3 days later underwent rir surgery groups 4, 5, and 6 (n = 5, 6, and 5) : ovariectomized female rats received treatment / regimen the same as groups 1, 2, and 3, respectively. to induce the rir model, the animals were anesthetized by ketamine (75 mg / kg, i.p.) and xylazine (10 mg / kg, i.p.). two small incisions were made on the flanks and when the kidneys were visible, the kidney arteries and veins on both sides were clamped to stop the renal blood circulation. serum samples were removed and stored at 20c until measurement. finally, all animals were killed, and kidneys and testes or uterus were removed and weighed immediately. in addition, the left kidney was fixed in 10% formalin solution for pathological assessments. serum levels of cr and bun were measured using quantitative kits (pars azmoon, iran) and automatic analyzer ra-1000 (technicon, ireland). the tissue slices were stained by hematoxylin and eosin to examine the tissue damage based on the presence of tubular atrophy, hyaline cast, ischemic necrosis, vacuolization, and debris. according to the damage intensity the uterus, kidney, and testis weights ; as well as bun and cr levels in the ischemia group were compared with the values obtained for the ischemia plus estradiol group or the sham - operated group using the student 's t - test. to compare the groups with regard to the kidney tissue damage score (ktds), the mann sixteen adult female (weighing 175 5.2 g) and 19 adult male (weighing 222.4 8.6 g) wistar rats (animal center, isfahan university of medical sciences, isfahan, iran) were used in this study. prior to the experiment, the protocols were confirmed to be in accordance with the guidelines of animal ethics committee of the isfahan university of medical sciences. one week before estradiol administration, all female rats were anesthetized, and an incision was made in the abdominal middle line above the urinary outlet. the animals were randomly divided into six experimental groups ; group 1 (n = 5, the sham - operated group) : male rats received sesame oil (intramuscularly) and after 3 days underwent the surgical procedure without rir surgerygroup 2 (n = 8) : male rats received regimen the same as group 1 and underwent rir surgerygroup 3 (n = 6), male rats received single dose of estradiol (500 g / kg ; intramuscularly) and 3 days later underwent rir surgerygroups 4, 5, and 6 (n = 5, 6, and 5) : ovariectomized female rats received treatment / regimen the same as groups 1, 2, and 3, respectively. group 1 (n = 5, the sham - operated group) : male rats received sesame oil (intramuscularly) and after 3 days underwent the surgical procedure without rir surgery group 2 (n = 8) : male rats received regimen the same as group 1 and underwent rir surgery group 3 (n = 6), male rats received single dose of estradiol (500 g / kg ; intramuscularly) and 3 days later underwent rir surgery groups 4, 5, and 6 (n = 5, 6, and 5) : ovariectomized female rats received treatment / regimen the same as groups 1, 2, and 3, respectively. to induce the rir model, the animals were anesthetized by ketamine (75 mg / kg, i.p.) and xylazine (10 mg / kg, i.p.). two small incisions were made on the flanks and when the kidneys were visible, the kidney arteries and veins on both sides were clamped to stop the renal blood circulation. serum samples were removed and stored at 20c until measurement. finally, all animals were killed, and kidneys and testes or uterus were removed and weighed immediately. in addition, the left kidney was fixed in 10% formalin solution for pathological assessments. serum levels of cr and bun were measured using quantitative kits (pars azmoon, iran) and automatic analyzer ra-1000 (technicon, ireland). the tissue slices were stained by hematoxylin and eosin to examine the tissue damage based on the presence of tubular atrophy, hyaline cast, ischemic necrosis, vacuolization, and debris. according to the damage intensity the uterus, kidney, and testis weights ; as well as bun and cr levels in the ischemia group were compared with the values obtained for the ischemia plus estradiol group or the sham - operated group using the student 's t - test. to compare the groups with regard to the kidney tissue damage score (ktds), the mann the uterus, kidney, and testis weights ; as well as bun and cr levels in the ischemia group were compared with the values obtained for the ischemia plus estradiol group or the sham - operated group using the student 's t - test. to compare the groups with regard to the kidney tissue damage score (ktds), the mann renal ischemia - reperfusion injury increased the serum cr and bun levels in both genders (p < 0.05). however, estradiol decreased these parameters toward normal values in female, but not in male rats (p < 0.05) [figures 1 and 2 ]. serum creatinine (cr) and blood urea nitrogen (bun) levels, testis weight (tw) and kidney weight (g)/100 g bodyweight (kw), kidney tissue damage score (ktds) in the male groups. abbreviations of isch and isch + es were applied for ischemia and ischemia + estradiol. and # indicates significant differences between the sham - operated and isch groups, and between the isch and isch + estradiol groups, respectively serum levels of creatinine (cr) and blood urea nitrogen (bun) levels, uterus weight (uw) and kidney weight (g)/100 g body weight (kw), kidney tissue damage score (ktds) in female groups. abbreviations of isch and isch + es were assigned to ischemia and ischemia + estradiol. and # indicates significant differences between the sham - operated and isch groups, and between isch and isch + estradiol groups, respectively renal ischemia - reperfusion increased kidney weight in both genders (p < 0.05) and estradiol intensified kidney weight in male (p < 0.05), but not in female rats. in addition, estradiol ameliorated ktds induced by rir in female, but not in male rats (p < 0.05) [figures 13 ]. abbreviations of isch and isch + es were assigned to ischemia and ischemia + estradiol administration of estradiol increased uterus weight (p < 0.05). however, it did not alter the testis weight [figures 1 and 2 ]. renal ischemia - reperfusion injury increased the serum cr and bun levels in both genders (p < 0.05). however, estradiol decreased these parameters toward normal values in female, but not in male rats (p < 0.05) [figures 1 and 2 ]. serum creatinine (cr) and blood urea nitrogen (bun) levels, testis weight (tw) and kidney weight (g)/100 g bodyweight (kw), kidney tissue damage score (ktds) in the male groups. abbreviations of isch and isch + es were applied for ischemia and ischemia + estradiol. and # indicates significant differences between the sham - operated and isch groups, and between the isch and isch + estradiol groups, respectively serum levels of creatinine (cr) and blood urea nitrogen (bun) levels, uterus weight (uw) and kidney weight (g)/100 g body weight (kw), kidney tissue damage score (ktds) in female groups. abbreviations of isch and isch + es were assigned to ischemia and ischemia + estradiol. and # indicates significant differences between the sham - operated and isch groups, and between isch and isch + estradiol groups, respectively renal ischemia - reperfusion increased kidney weight in both genders (p < 0.05) and estradiol intensified kidney weight in male (p < 0.05), but not in female rats. in addition, estradiol ameliorated ktds induced by rir in female, but not in male rats (p < 0.05) [figures 13 ]. however, it did not alter the testis weight [figures 1 and 2 ]. the present study was performed to compare the protective effect of estradiol against rir - induced renal injury in male and female rats. the results obtained show that rir induces renal dysfunction in both male and female rats ; characterized by increasing serum bun and cr levels. rir - induced renal failure increases oxidative stress, inflammation, and apoptosis by increasing the malondialdehyde, interleukines, and tumor necrosis factor - alpha levels, respectively. estrogen may protect renal and cardiovascular systems by affecting glomerular mesangial cells in the kidney and smooth muscle cells in the vasculature and improve renal and vascular damage induced by renal ischemia in atherosclerotic female mice through antiinflammatory cytokine, interleukine-6. furthermore, administration of estradiol decreased albuminuria, glomerulosclerosis and tubulointerstitial in spontaneously hypertensive stroke - prone female rats. in addition, the protective effects of estrogen were exhibited via nitric oxide synthase system, antioxidant, and inflammatory properties. it is possible that the estrogen ameliorates renal blood flow diminished by ischemia due to activation of endothelial nitric oxide synthase and nitric oxide production. estrogen protects kidney against rir through suppression of endothelin 1 overproduction. also, it is possible that the presence of progesterone in the female gender plays a role in ameliorating rir injury. it has been reported that progesterone has neuroprotective effects and in combination with estradiol improves the brain ischemia - induced injury and ameliorates bone loss in female rats. in the present study, administration of estradiol not only it is demonstrated that administration of estradiol (10 g / kg) attenuated renal damage induced by bilateral rir, while this was not the case at the doses of 20 and 100 g / kg. in another study, high mortality rate in male pretreated with estradiol was reported, while the course of ischemia - induced renal failure improved. administration of estradiol also attenuates vascular responses against acetylcholine in women but not men, and estrogen supplementation improves endothelium - related dilation through increasing level of nitrite / nitrate in women, but not in men. our study showed that normalized kidney weight was increased by rir injury in both genders. this observation was consistent with the findings of other studies, which is probably due to edema. administration of estradiol intensified normalized kidney weight in male gender ; confirmed by renal dysfunction and pathological findings. it has been demonstrated that estrogen increases uterus weight through increasing the glucose metabolism, activity of rna polymerase, dna content, and finally cell proliferation. it was concluded that administration of estradiol could attenuate renal injury induced by rir in female rats ; however, as an antioxidant agent, it was not efficient in preventing renal dysfunction induced by rir in male rats.	background : renal ischemia - reperfusion (rir) is the main cause of renal failure. the incidence of rir injury seems to be gender - related due to female sex hormone ; estrogen. this study was designed to investigate the protective role of estrogen against rir injury in male and ovariectomized female rats.methods:thirty-nine wistar rats were used in this study as male and ovariectomized female rats in the sham - operated, rir, and estradiol - treated plus rir groups. the rir was induced by clamping the renal vessels for 45 min and then 24 h of reperfusion. all animals finally were sacrificed for the measurements.results:the serum levels of creatinine and blood urea nitrogen and kidney tissue damage score significantly increased in both male and female rir rats (p < 0.05). estradiol however significantly attenuated theses parameters (p < 0.05) toward normal levels in female (p < 0.05), but not in male rats. kidney weight increased in both genders and estradiol intensified it in the male rats (p < 0.05). uterus weight was increased by estradiol in female rats (p < 0.05) and testis weight did not alter in male rats.conclusions:estradiol demonstrated a protective role against rir injury in female rats ; however, estradiol as an antioxidant could not protect the male kidney from rir injury.
magnetic resonance imaging studies are done in children with epilepsy with the aim of identifying any structural lesions that may be responsible for the same. the aim is both to prognosticate outcomes and guide proper management (either medical or surgical). while imaging may be normal, often multiple abnormalities are seen which may be part of a developmental syndrome. the challenge in these cases is to try to isolate if any one of these is responsible for the epilepsy so that a decision can be taken if surgical treatment of the same will be beneficial. a 7-year - old male child, born at term, of a nonconsanguineous marriage presented with a history of poorly controlled generalized tonic - clonic seizures that started 3 years ago. he had a younger brother aged 4 years who was stated to be normal. a computed tomography scan done previously showed a left temporal arachnoid cyst (ac) and the child was referred for considering the possibility of surgery. he had a soft nonfluctuant swelling at the root of his nose due to which he appeared to have hypertelorism. this swelling, according to his parents, was noticed at 1-year after birth and was slowly increasing in size [figure 1 ]. clinical photograph of the patient showing swelling at the root of and over the bridge of the nose causing apparent hypertelorism a magnetic resonance imaging (mri) scan was done that showed left temporal ac with left hemispheric periventricular nodular (pvn) heterotopia that was extending to the frontal horn of the left lateral ventricle. the cortex on the side of the heterotopia was thin compared with the contralateral side [figures 2 and 3 ]. we were not convinced regarding the contribution of the ac in seizure genesis in the presence of pvn and callosal abnormalities. surgery was offered for the encephalocele only, but the family refused any further investigations and treatment. coronal t2-weighted and axial t1-weighted magnetic resonance imaging sections showing heterotopic grey matter seen in the left hemisphere (closed arrows), abutting the ventricular ependyma of the frontal horn. a left temporal arachnoid cyst (open arrow) is also seen sagittal t1-weighted magnetic resonance imaging section showing frontonasal encephalocele protruding outwards through a defect in the basal part of frontal bone and extending inferiorly over the nasal bone. grey matter heterotopias are congenital errors of neuronal migration that normally takes place sequentially outwards from the subependymal layer to the cerebral cortex. they are of three types band, focal subcortical and subependymal or periventricular. in the latter, clumps of normal looking (on mri) islands of grey matter lie within the white matter. they lie near the ventricle and occasionally may project below the ependymal lining and are the most common variant of heterotopias. various genetic anomalies have been found in these patients, but males with x - linked disease have been noted to have other associated developmental disorders too and this was found in our patient as well though no genetic studies could be done. describe two series the first of six patients treated for posterior encephalocele in whom heterotopias were found in 3 on mri and a second series of 8 autopsy cases of encephalocele in which heterotopias were found in 4- and state that the combined occurrence of the two may not be accidental. balaji. have reported a 6-year - old girl with frontonasal encephalocele and subependymal nodular heterotopias. have also described a case of anterior encephalocele in a 15-year - old girl presenting with epilepsy who was found to have subependymal nodular heterotopia with partial agenesis of corpus callosum. martnez - lage. had found two patients with heterotopias in 46 children treated for cephalocele (both anterior and posterior) and stated that one of the predictors of good outcome was lack of disorders of neuronal migration. an association between heterotopia and ac with sensorineural deafness has also been reported in the chudley - mccullough syndrome. we found only one such case (nadkarni.) reported from india. the disease is supposed to be due to a mutation in the g - protein signaling modulator 2 gene on chromosome 13. most but not all cases reported have had hydrocephalus usually due to block at the foramen of munro. our patient did not have hydrocephalus, but did have heterotopias, callosal dysgenesis, sensorineural deafness and an ac as well as an anterior encephalocele. whether this was a variant of the same syndrome can not be commented on as genetic studies could not be done. arachnoid cyst 's of the temporal fossa are associated with partial agenesis of the temporal lobe. whether the latter is responsible for the cyst formation or is a consequence of compression by the cyst is a matter of debate. arroyo and santamaria examining the relationship between ac 's and seizure foci concluded that ac 's are an incidental finding in epilepsy and do not necessarily reflect the location of the seizure focus. in a study of eight patients with ac 's and epilepsy, yalin. found that eeg abnormalities corresponded to cyst location in only one patient. analyzing the association between intracranial ac 's and seizures, murthy has suggested that in presence of identifiable epileptogenic factors ac 's may be incidental whereas in cases where no other cause can be found the association between ac 's and epilepsy is more likely. as our patient had pvn seen on imaging, we did not consider that ac was causal in his seizures and did not proceed with surgery for the same. to conclude, all patients with seizures and ac 's must be meticulously searched for other structural abnormalities that may be present as well as undergo a detailed workup before proceeding on any surgery. secondly, patients with encephaloceles must also be worked up for underlying structural abnormalities, like heterotopias, in the brain. faulty neural tube closure (resulting in encephalocele) may affect the subsequent process of neuronal migration, and the combination of these may not be just incidental. finally, a better understanding of the constellation of abnormalities will help to identify children with particular syndromes and aid in counseling the parents about prognosis and risks of subsequent pregnancies.	a 7-year - old male child presented with poorly controlled generalized tonic - clonic seizures. on examination, he was mentally retarded, deaf and had a swelling at the root on the nose. computed tomography scan done previously revealed a left temporal arachnoid cyst (ac) due to which he was referred for surgery. however, magnetic resonance imaging revealed a constellation of abnormalities all of which could be responsible for his seizures. the combination of periventricular nodular heterotopias with encepaholcele is rarely described in the literature, and more infrequently so its combination with ac and callosal dysgenesis the chudley - mccullough syndrome. we describe the case and review relevant literature on this subject.
inflammatory response has been confirmed to play a detrimental role in the pathogenesis of cerebral ischemia / reperfusion (i / r) injury. the relationship between inflammation and cerebral i / r outcome has assured the considerable and continued interest in the development of anti - inflammation oriented therapies to mitigate i / r - induced brain damage. the inflammatory process involves activation and interaction of a broad spectrum of immune factors pioneered by the upregulation of proinflammatory cytokines. these cytokines, including members of the interleukin (il)-1 family such as il-1, are detected in the infarct area and their imbalance is associated with brain tissue damage. a previous study has demonstrated that mice deficient in il-1 show improved survival, reduced the infarct volume, and improved neurological functions as compared with wild - type mice after middle cerebral artery occlusion (mcao). recent evidence suggested that the downstream processing of il-1 is regulated by some cytosolic factors such as inflammasomes, a family of protein complexes that were recently identified as the cellular machinery responsible for recognizing pathogen - associated molecular patterns and reacting to these through activation of inflammatory processes. among different types of inflammasomes is the nalp3 inflammasome, which has been well characterized in a variety of mammalian cells. it is characterized as a proteolytic complex mainly composed of the nacht domain-, leucine - rich repeat-, and pyrin domain (pyd)-containing protein 3 (nalp3), the adaptor protein apoptosis - associated speck - like protein (asc), and caspase-1. the nalp3 inflammasome can be activated by bacterial toxins or pathogen - associated molecular patterns, such as muramyldipeptide, and other stimuli. nalp3 can also detect and respond to endogenous stress - associated danger signals, such as atp, ros, monosodium urate crystals, low intracellular potassium concentrations, sodium overload, or -amyloid by activating caspase-1, and active caspase-1 subsequently matures the proinflammatory il-1 family cytokines by cleaving their proforms into biologically active cytokines in turn [11, 12 ]. active il-1 then triggers the il-1 receptors on surrounding tissues [13, 14 ], leading to the activation of multiple cytokines involved in the inflammation cascade, including il-8, tnf, and il-17 [15, 16 ] (figure 1). however, the role of nalp3 inflammasome in cerebral i / r inflammatory disorders has not been well explored [17, 18 ]. chrysophanol (chr), a member of the anthraquinone family, was originally extracted from plants of rheum genus (figure 2). chr has been shown to have multiple pharmacological effects, including its anti - inflammation activity, for which the underlying mechanisms remain to be elucidated. previous studies have shown that chr inhibits caspase-1 and its downstream cytokines expression in colitis. however, little is known on the relationship between chr and nalp3 inflammasome during cerebral i / r. using mouse transient middle cerebral artery occlusion (tmcao) model, this study was designed to examine the cellular distribution and dynamic expression of nalp3 inflammasome during cerebral i / r and investigate whether chr has any neuroprotective effect and what are the underlying mechanisms mediating this protection if it does have. male cd1 mice (25~30 g) were purchased from the vital river laboratory animal technology co., ltd., all mice were given at least 2 days to acclimatize ahead of any experimentation. during this period, animal houses were maintained in a 12/12 h light / dark cycle with humidity of 60% 5% and ambient temperature of 22c 3c. the experimental procedures were approved by the local experimental ethics committee as well as the institutional animal care and use committee. the mouse tmaco model was established by following the surgical procedures as previously described [20, 21 ]. in brief, animals were anesthetized with chloral hydrate (350 mg / kg, intraperitoneally). the right common carotid artery (cca), external carotid artery (eca), and internal carotid artery (ica) were then exposed through blunt separation. branches of eca were then cauterized and the eca was ligated and cut off at the distance of 2 mm from bifurcation of cca, while middle cerebral artery (mca) was occluded by inserting a heparin - dampened monofilament nylon suture (beijing sunbio biotech co., ltd., beijing, china) into the ica, which was moved forward until the flow of right mca was reduced to 20%~30% of basal flow, which was monitored by a blood flow monitor (moor vms - ldf, moor instruments ltd., uk). the animals ' body temperature was also monitored and maintained at 36.5c to 37.5c during experiment [2224 ]. all animals were divided into two groups : the control group and the drug treatment group. for the control group, a total of 63 mice were randomly divided into 7 subgroups, 9 mice each, including the sham controls and untreated controls for six time points after tmcao (3 h, 6 h, 12 h, 24 h, 48 h, and 72 h). for the treatment group, 162 mice were randomly divided into 6 groups : (1) the sham group : animals received sham operation and equal volume of 0.9% nacl ; (2) tmcao group : animals received tmcao and equal volume of 0.9% nacl ; (3) vehicle group : animals underwent tmcao and equal volume of 1% dmso and 1% tween-80 prepared with 0.9% nacl ; and (4) three chr groups : animals underwent tmcao and were treated with a high dose of chr, (10 mg / kg, chr - h) or middle dose of chr (1 mg / kg, chr - m) and low dose of chr (0.1 mg / kg, chr - l). mice were reanesthetized and sacrificed at 24 h after tmcao. the chr product (nanjing zelang medical technological co., ltd., jiangsu, china) with purity of more than 98% was first dissolved in 0.9% nacl including 1% dmso and 1% tween-80 prior to the treatments. the infarct volume, brain edema, and blood brain barrier (bbb) permeability were determined by an examiner blinded to the experimental grouping at 24 h post - tmcao for the mice from the drug treatment group. meanwhile, a neurological test was administered on these animals following a scoring system adapted from the one developed by longa. as follows : 0, no deficits ; 1, difficulty in fully extending the contralateral forelimb ; 2, unable to extend the contralateral forelimb ; 3, mild circling to the contralateral side ; 4, severe circling ; and 5, falling to the contralateral side. infarct volume of brain was measured at 24 h after tmcao. after the brains (n = 6 in each group) were dissected, each brain sample was cut into five slices with 2 mm thickness, incubated by a 2% solution of 2, 3, 5-triphenyltetrazolium chloride (ttc) at 37c for 15 min, followed by fixation with 4% paraformaldehyde. with ttc staining, all normal tissue was stained in dark red, while the infarct area was stained in a pale gray color. all ttc - stained slices were then photographed and analyzed using image - pro plus 5.1 software (media cybernetics, inc., bethesda, usa), and the infarct volumes were calculated as follows : percentage hemisphere lesion volume (% hlv) = { [total infarct volume (volume of intact ipsilateral hemisphere volume of intact contralateral hemisphere)]/contralateral hemisphere volume } 100%. mice (n = 6 in each group) were decapitated under deep anesthesia at 24 h post - tmcao, while the cerebrum was removed and placed on a preprepared dry tray as quickly as possible. the frontal pole with 2 mm thickness and cerebellum were removed ; only coronal slices with approximately 4 mm in thickness were left. the slices were divided into ischemic and nonischemic hemispheres. at the same time, the two hemisphere slices were packaged, respectively, with tin foil, and their wet weights were evaluated with electronic balance and then dried for 24 h at 100c to get dry weights. brain water content (bw) was calculated as follows : bw = (wet weight dry weight)/wet weight 100%. at 2 h before the mice were anesthetized, 4 ml / kg of 2% evans blue (sigma) prepared with 0.9% nacl was administered by tail vein injection. then, they were perfused into heart with 0.9% nacl. for quantitative measurement of evans blue extravasation, the ischemic and nonischemic hemispheres were removed and homogenized in 1 ml of trichloroacetic acid and then centrifuged at 21,000 g for 20 minutes. evans blue concentration was quantitatively determined by measuring the absorbance of the supernatant at 610 nm using spectrometer (infinite m200 pro, tecan, austria). representative coronal brain sections of mice were obtained at 24 h after sham operation or tmcao (n = 3 for each group). mice were perfused with 0.9% nacl quickly followed by 4% paraformaldehyde in pbs. fixed frozen cerebral sections (30 m thick) were blocked with 10% goat serum for 1 h and then incubated overnight at 4c with primary antibodies from abcam including anti - nalp3 antibody (1 : 100) ; anti - asc antibody (1 : 100) ; anti - caspase-1 antibody (1 : 100) ; and anti - il-1 antibody (1 : 100), together with anti - neun (1 : 200, sternberger monoclonal incorporation, lutherville, md, usa). after 3 washes with pbs, they were incubated with secondary antibodies (1 : 100, zhongshan biology technology company, china) at 37c for 2 h. immunofluorescence was visualized using a laser scanning confocal microscope (olympus fv10-asw, japan). all proteins were obtained from the cortex of the ischemic hemisphere using a total protein extraction kit from applygen technologies inc., (beijing, china) following the manufacturer 's protocols. the protein concentrations of the extracts were determined using a bca protein assay reagent kit (novagen, madison, wi, usa), while 50 g of proteins (n = 3 per group per time point) was separated by sds / page electrophoresis and transferred to pvdf membranes. the membranes were blocked with 5% nonfat milk in 0.01 m pbs at room temperature for 1 h and then incubated overnight at 4c with the primary antibodies obtained from abcam, including anti - nalp3 antibody (1 : 1000), anti - asc antibody (1 : 1000), anti - caspase-1 antibody (1 : 500), and anti - il-1 antibody (1 : 1000). polyclonal rabbit anti - beta actin antibody (1 : 500) from zhongshan biotechnology was used for internal control. after three washes with tpbs, all membranes were incubated with irdye 800-conjugated goat anti - rabbit or anti - mouse secondary antibodies for 1 h at room temperature. an imaging densitometer (li - cor bioscience) was used to analyze the relative density of each band. for immunohistochemical staining, brain tissues were removed and immediately immersed in 4% paraformaldehyde with pbs for 24 h at 4c. brain sections (5 m thick) were blocked in 3% h2o2, 3% normal goat serum, and then incubated overnight with anti - mouse nalp3, asc and il-1 rabbit polyclonal antibodies (1 : 100, abcam biotechnology), and anti - mouse caspase-1 rabbit polyclonal antibody (1 : 100, sigma biotechnology). the secondary antibodies, secondary biotinylated conjugates, and diaminobenzidine were from the vect abc kit (zhongshan biology technology company, china). in the end the cause is four factors will be observed altogether with each bearing 5 sections. meanwhile, five visual fields of ischemic region from the infarct area were selected and the immunoreactive cells were counted under a 400x light microscope (n = 3 per group per time point). total rna from ischemic region cortex was isolated using trizol reagent (invitrogen, carlsbad, ca, usa) and was reverse - transcribed into cdna using revert aid first strand cdna synthesis kit (fermentas international inc., burlington, canada) for quantitative pcr (mx 3005p, usa) in the presence of a fluorescent dye (sybr green i ; cwbio). relative abundance of mrna from the perspective target genes was calculated after normalization to -actin rna and was calculated by the 2 method. real - time pcr was used to analyze the levels of nalp3, asc, caspase-1, and il-1 mrna at 24 h after tmcao. all data were analyzed using proper tools from spss 13.0 package, while all quantitative data were expressed as mean sd. statistical comparisons were conducted using one - way anova followed by snk and lsd tests for intergroup comparisons. for neurological deficit, mann - whitney u test was applied for comparisons between groups. confocal microscopy was utilized to detect the presence and the cellular distribution of nalp3 inflammasome components as well as il-1 in the ischemic penumbra zone of the cerebral cortex. the neuronal karyopyknosis and chromatin margination could be observed and there was a small amount of neutrophils in the marginal zone of infarction. in the normal brain (sham), caspase-1 and nalp3 were mainly localized in the nucleus of neurons, whereas il-1 was mainly present in the cytoplasm. however, after tmcao, nalp3 and caspase-1 were redistributed predominantly to the cytoplasm, while asc and il-1 locations remained unchanged (figure 3). immunohistochemistry, qrt - pcr, and western blotting were used to examine expression of nalp3, asc, caspase-1, and il-1 in brain tissue after tmcao. the results showed that nalp3 and active caspase-1 were both upregulated as compared with sham group (p 0.05). remarkably, mice from the chr - h and chr - m groups showed significantly improved neurological function scores as compared with tmcao and vehicle groups after tmcao (p 0.05). infarct volume of each group was measured by ttc at 24 h after tmcao (figure 5(b)). normal tissue was stained in dark red, while the infarct area appeared pale gray. an extensive lesion was found in both cortex and striatum in animals from tmcao and vehicle groups (p 0.05). the neuroprotective effects of chr at different doses were clearly observed by both ttc staining (figure 5(b)) and examining the infarct volume at 24 h (figure 5(c)). a significant protective effect from chr was observed as the infarct volumes from animals in the chr - h and chr - m groups but not the chr - l group are significantly different from the tmcao and vehicle groups (figure 5(c)). there was a significant reduction in infarct volume in chr - h (29.80% 1.15% versus 49.87% 1.99% and 51.31% 1.57%, p 0.05). following chr treatment, as compared with tmcao and vehicle groups, there were significant reductions in brain water content in the chr - h group (80.60% 0.58% versus 83.75% 0.48% and 83.72% 0.35%, p 0.05). to evaluate bbb permeability at 24 h after tmcao, bbb leakage was measured using evans blue extravasation (figures 5(e) and 5(f)). as expected, an extensive bbb disruption was found in animals from the tmcao and vehicle groups (p 0.05) (figure 5(f)). the protective effects of chr with different doses were also observed by examination of evans blue content at 24 h and, as compared with the tmcao and vehicle groups, significantly lower evans blue extravasation was observed in the chr - h and chr - m groups (p 0.05). to further investigate the impact of chr on the endogenous expression of nalp3 inflammasome and il-1 in ischemic hemisphere, the expression of nalp3, asc, caspase-1, and il-1 was also investigated in situ by immunohistochemical staining of tissue slices from the brain tissue slices from all experimental groups. the positively stained cells for the examined proteins were counted manually and intergroup comparisons were performed. the immunohistochemical staining of nalp3, asc, caspase-1, and il-1 of each group at 24 h after operation was shown in figure 6. few cells were expressing nalp3, caspase-1, or il-1 in the cortex in sham group indicating a low baseline of nalp3, caspase-1, and il-1 expressions in the nonischemic cortex. in contrast, tissues from the tmcao group showed an augmented expression of all three examined proteins. the number of cells expressing nalp3, caspase-1, or il-1 in the ischemic cortex of tmcao mice was significantly increased as compared with sham group (p 0.05) (figure 7(e)). in chr - h and chr - m groups, the number of positive cells for nalp3, caspase-1, or il-1 was significantly lower than the tmcao and vehicle groups (p < 0.05) (figure 7(e)). then we further analyzed the protein expression of nalp3, asc (figure 7(a)), caspase-1 (figure 7(b)), and il-1 (figure 7(c)) with western blotting. it was found that the expressions of nalp3, caspase-1, and il-1 at protein level were upregulated in tmcao and vehicle groups compared with sham group (p < 0.05). high dose and middle dose chr significantly decreased the nalp3, caspase-1, and il-1 protein expression compared with tmcao and vehicle groups (p < 0.05) (figure 7(d)). in agreement with the results of immunohistochemistry and western blotting, the mrna expression of nalp3, caspase-1, and il-1 was upregulated in tmcao and vehicle groups compared with sham group (p < 0.05). the overexpression of those factors was significantly decreased in chr - h and chr - m groups compared with tmcao and vehicle groups (p < 0.05) (figure 7(f)). although several treatments have been claimed to promote neuronal survival, reduce infarct volumes, and improve behavioral responses after experimental cerebral ischemia, the recombinant tissue plasminogen activator (rtpa) is the only fda - approved drug for treatment of ischemic stroke clinically [2830 ]. however, rtpa has a narrow window of efficacy of 3 h after the onset of stroke, which limits the use of this medicine only to a small portion of stroke patients. since reperfusion after prolonged ischemia exaggerates tissue injury, in which inflammatory damage plays a critical role, anti - inflammatory oriented treatments may have the potential to mitigate the i / r - induced, inflammation - mediated secondary damage and improve the treatment outcome in stroke patients [3135 ]. in this study, we have investigated the role of the nalp3 inflammasome in the inflammatory response induced by i / r using mouse tmcao model, the most matured animal model that mimics human stroke. meanwhile, we examined the anti - inflammation and neuroprotective effects of chr, a member of the anthraquinone family, in the mice who underwent tmcao. previous pharmaceutical studies have shown that derivatives of anthraquinones exert multiple biological effects, including anticancer [37, 38 ], hepatoprotection, and antimicrobials. a number of studies have also implied that extracts [4143 ] containing chr have a wide range of pharmacological activities, including anti - inflammation, antidyslipidemia, and antioxidant activities. it has been proved that chr inhibits the nitric oxide production and activation of proinflammatory cytokines, such as tumor necrosis factor (tnf)-, interleukin (il)-6, nf-b, and caspase-1 in vivo and vitro. our results further confirmed the association between nalp3 inflammasome and the i / r induced brain damage. furthermore, we firstly demonstrated that chr could significantly suppress the inflammatory response after ischemic stroke, which is consistent with the previous studies reporting the effects of chr on dextran sulfate sodium (dss)-induced colitis and lipopolysaccharide (lps)-induced inflammatory responses in mouse peritoneal macrophages. nalp3 inflammasome is pivotal in the processing of active caspase-1 and downstream maturation of il-1. il-1 can then promote the increase of endothelial permeability and the expression of a number of adhesion molecules [32, 44 ]. these effects further result in the recruitment of inflammatory cells into the brain parenchyma, amplifying the inflammatory reaction within the ischemic brain. our work focused on nalp3 since it is apical in the inflammation cascade and potentially an ideal target for anti - inflammation oriented therapies. a previous study has confirmed that nalp3 signaling is involved in liver i / r and silencing nalp3 can protect the liver from i / r injury. the same results were observed during inflammation and immune responses in renal i / r as well. furthermore, nalp3 expression was elevated in the spinal cords during experimental autoimmune encephalomyelitis (eae), and nalp3 (/) mice had a dramatically delayed course and reduced severity of disease. in our study, we detected nalp3 inflammasome in both normal and ischemic brain tissues while the expression of nalp3 inflammasome was activated after cerebral i / r. we observed that nalp3, caspase-1, and il-1 were all upregulated starting at an early stage after tmcao and could remain at a high level for at least 72 h. for asc, the adapter protein in the nalp3 inflammasome, its activation was relatively lagging behind and its expression would not be upregulated significantly until 72 h post - tmcao. furthermore, we demonstrated that systemic administration of chr was effective in containing neurological impairment and tissue injury during cerebral i / r in a dose - dependent manner. we have shown that the neurological deficits, infarct volume, brain edema, and bbb disruption could all be significantly improved while the upregulation of nalp3, caspase-1, and il-1 was significantly suppressed. all these are suggestive that the neuroprotective effect of chr might involve inhibition of nalp3 inflammasome activation. although it is still premature to conclude that inhibition of nalp3 inflammasome is the sole or foremost mechanism mediating the observed protective effects of chr, our findings support the therapeutic effect of chr in the acute phase of ischemic stroke.	the most effective way to contain cerebral ischemic injury is reperfusion ; however, reperfusion itself may result in tissue injury, for which inflammatory damage is one of the main causative factors. nalp3 inflammasome is a multiprotein complex. it consists of nalp3, asc, and caspase-1, whose function is to switch on the inflammatory process. chrysophanol is an extract from plants of rheum genus and it possesses many pharmacological effects including its anti - inflammation activity. in this study, the effects of chrysophanol in cerebral ischemia / reperfusion and the potential mechanisms were investigated. male cd1 mice were subject to transient middle cerebral artery occlusion (tmcao). the nalp3 inflammasome activation status and its dynamic expression during the natural inflammatory response induced by tmcao were first profiled. the neuroprotective effects of chrysophanol were then assessed and the potential mechanisms mediating the observed neuroprotection were then explored. physical parameters including neurological deficit, infarct size, brain edema, and bbb permeability were measured at 24 h after tmcao. confocal microscopy, western blotting, immunohistochemistry, and qrt - pcr techniques were utilized to analyze the expression of nalp3 inflammasome and il-1. our results indicated that the brain tissue damage during cerebral ischemia / reperfusion is accompanied by nalp3 inflammasome activation. chrysophanol could inhibit the activation of nalp3 inflammasome and protect cerebral ischemic stroke.
as one of the groups iii v nitride semiconductors, gan is a highly attractive material because of its great potential for development of optoelectronic devices in short - wave length, semiconductor lasers, and optical detectors. the compound exhibits some interesting characteristics, such as larger band gap, strong interatomic bonds, and high thermal conductivity, which make it to be fine materials for optoelectronic, high - temperature and high - power devices. nevertheless, the successful fabrication of devices based on epitaxial gan thin films requires better understanding of the mechanical characteristics in addition to its optical and electrical performances, since the contact loading during processing or packaging can significantly degrade the performance of these devices. consequently, there is a growing demand of investigating the mechanical characteristics of materials, in particular in the nanoscale regime, for device applications. the practical use of materials requires a knowledge of their mechanical behaviors and the effect of mechanical damage on their desired characterizations, because through the fabrication or handling processes the materials may be subjected to mechanical loads which cause damage, or the mechanical loads arise from shock loading in mobile systems. in this respect, the advent of nanoindentation instruments and the subsequent development of underlying science may potentially address the scaling issue and the scientific evaluation of all contact loading - related phenomena. since the deformation occurred during the test is controlled locally on the nanometer - scale, nanoindentation has, recently, been widely used to investigate the deformation mechanisms of various semiconductors [3 - 6 ] ; as well as the mechanical characteristics of surfaces of solids and thin films, such as the hardness and young s modulus, have been extracted using this technique [7 - 9 ]. we present herein the berkovich nanoindentation - induced mechanical deformation mechanisms of the metal - organic chemical - vapor deposition (mocvd)-derived gan thin films by means of the cathodoluminescence (cl) microscopy in a scanning electron microscopy (sem / cl) and the cross - sectional transmission electron microscopy (xtem) techniques. gan thin films used in this work were grown on (0001)-sapphire substrates by using the mocvd method with an average thickness of about 2 m. the details of growth procedures in preparing these gan thin films can be found elsewhere. the nanoindentation measurements were performed on a nanoindenter mts nanoxpsystem (mts cooperation, nano instruments innovation center, tn, usa) with a diamond pyramid - shaped berkovich - type indenter tip, whose radius of curvature is 50 nm. the cyclic nanoindentation tests were performed in a way with loading to the maximum load and unloading by 90% ; reloading to the maximum load and unloading by 9% ; holding for 10 s at 10% of the maximum load for thermal drift correction and complete unloading, as illustrated in fig. the thermal drift was kept below 0.05 nm / s for all indentations considered in this work. the same loading / unloading rate of 10 mn / s was used. results show the clear multiple pop - in events (indicated by the arrows) during loading, while no pop - out event is evident in the unloading segments, indicating that no phase transition is involved. the inset is an sem micrograph of an indent at an applied load of 100 mn. no apparent material pile - up around the indented area is evident in addition, the hardness and young s modulus of gan thin films were measured by nanoindentation with a continuous stiffness measurements technique, and the indentations were made using a constant nominal strain rate of 0.05 s. frequency of 45 hz was used to avoid the sensitivity to thermal drift and the loading resolution was 50 nn. in this technique this oscillated force with a known phase and amplitude interacts with the material, which responds with a displacement phase and amplitude characteristic of the stiffness and damping of contact with the indenter. hardness and young s modulus of gan thin films, calculated from the load - displacement (ph) records by using the analytic method developed by oliver and pharr, are 19.3 1.1 and 286.1 25.3 gpa, respectively. in our measurements, firstly, a 10 10-indentation array with each indentation being separated by 100 m to avoid inter - indent interactions was produced with an indentation load of 100 mn. in each indentation tests, the berkovich diamond indenter was operated with the same loading / unloading rate (10 mn / s) and was held at the peak load for 10 s. after that, xtem samples were prepared by the lift - out technique using a dual - beam focused ion beam (fib) station (fei nova 220). the technique for material preparation using the fib consisted of first milling two crosses alongside the indented area, acting as markers and then depositing a 1-m thick layer of pt to protect the area of interest from gaion beam damage and implantation. material was therefore removed from both sides of the selected area using an ion current of 5 na, followed by successive thinning steps using decreasing currents from 3 na to 300 pa until the lamella was about 1-m thick. subsequently, the bottom and one side of the lamella were cut - free while tilting the specimen at an angle of 45 to the ion beam. a central area containing the indentation apex of a few micrometers in length was then chosen and thinned further to a thickness of 100 nm, leaving at the sides thicker areas that prevented the lamella from collapsing. finally, a small area of interest was selected and thinned until electron transparency was achieved. the transfer of the lamella from the sample holder to the tem grid with a carbon membrane was made ex situ using the electrostatic force of a glass needle. the xtem lamella was examined in a jeol 2010f tem operating at 200 kv with a point - to - point resolution of 0.23 nm and a lattice resolution of 0.10 nm. the room temperature cl measurement was performed using a gata monocle equipped on the jeol jsm-7000f field - emission sem. the typical cyclic nanoindentation ph curve of gan thin films subjected to a maximum indentation load of 100 mn is illustrated in fig. 1. the curve exhibits irregularities during loading characterized by small jumps in the penetration depth (see the arrows), referred to as the multiple pop - in events in what follows. the fact that multiple pop - in events are observable over such a wide range of indentation load and penetration depth indicates the close relations to the plastic deformation of gan thin films. furthermore, since the phenomena are randomly distributed on the loading curve and each curve is associated with a different stress rate which increases with the maximum indentation load, it is suggestive that the first pop - in event is not thermally activated. dislocation - induced pop - in event is usually associated with two distinct deformation behaviors before (pure elastic behavior) and after (elastoplastic behavior) the phenomena. in this work, it can not be provided for such indentation load any evidence of dislocation activity and crack features at the free surface around the indentation from sem observations, as illustrated in the insert of fig. 1. also, no reversal discontinuities during unloading segment of ph curves, commonly called the pop - out events are observed here ; such phenomena are observed for instance in si and related to phase transformation just beneath the diamond indenter. one possible mechanism that would explain the observed behavior is the formation and propagation of dislocations in gan thin films. after berkovich nanoindentation, the residual indentations were directly imaged by using secondary electron (se) and cl. a series of cl images of near - gap emission from gan thin films indented to the maximum indentation loads of 10, 50, and 100 mn is shown in figs. we clearly observe an indentation pattern called rosette, with a structure symmetry reflecting the hexagonal symmetry of gan thin films subjected to berkovich nanoindentation. and, the evolution of cl impressions of such defects with increasing the indentation loads is displayed in fig. the central zone becomes larger and more perturbed, accompanied by the shorter and very dark arms displaying between the double arms. the sizes of dark regions in cl images are about 2, 4, and 7 m for the indentation loads of 10, 50, and 100 mn, respectively. room temperature cl images (right figures) of indentation at loads of (a) 10, (b) 50, and (c) 100 mn. cl imaging conditions : electron beam energy = 20 kev, cl wavelength = 366 nm (d) schema of an indentation figure as imaged by sem / cl from cl observations, it displays as two well - separated zones : (i) a strongly perturbed central zone, appearing as a very dark zone with non - uniform distribution damage and (ii) six double arms, nearly parallel to axes separated by an angle of 60, confirming the hexagonal symmetry. from fig. 2d, the cl images of berkovich nanoindentations showed a very well - defined rosette structure with the hexagonal symmetry, which extends to the distance as double arms in directions at 60 intervals (namely ;, and). the dark spots are visible in each arm ; they correspond to the emergences of dislocations on the surface which act as non - radiative recombination centers and therefore quench locally the luminescence in the deformed areas. in addition, the intensity of the near - gap cl emission from gan thin films is dramatically suppressed after berkovich nanoindentation due to the induced defects / dislocations. despite the star - of - david - like rosettes have been investigated around the larger indentation loading ; however, such details could not be clearly resolved for the lower condition in our present work. it is interesting to note that cl imaging on the indented area of gan thin films corresponding to the critical pop - in load revealed slightly detectable reduction in the cl emission intensity. indeed, under such a lower indentation load of 10 mn, for which only a small percentage of plastic deformation was induced, cl imaging was able to distinguish between the indentations that had undergone certain degrees of plastic deformation (after pop - in event) and those that were purely in the elastic regime (before pop - in event). consequently, an observable cl impression is only detected after the pop - in event. it is thus evidenced that the pop - in event involves the nucleation of slip in the deformation mechanisms. in addition, according to the previously studies, we note here that the critical applied indentation load for direct identification of the multiple pop - in events in ph curve is not only dependent on the type of indenters used, but also even very much dependent on the test systems and the maximum indentation loads applied. consequently, we reasonably deduce that these discrepancies are mainly owing to the various indentation methods used. for instance, the tip - surface contact configuration and stress distribution for the berkovich indenter tip can be drastically different from that for the spherical one or vickers - type indenters. returning to the multiple pop - in events, it should be noted that the phenomena have been observed previously in hexagonal structured sapphire, single - crystal bulk zno, and gan thin films. on the other hand, nevertheless, the above discussions do suggest that multiple pop - in events indeed are specific features of materials with the hexagonal lattice structure and the geometry of the indenter tip may play an important role in determining the nanoindentation - induced mechanical responses. as a result, in order to identify the deformation mechanisms specific to the berkovich nanoindentation directly microstructures characteristics in the vicinity of indented area are needed. this plastic deformation process is complex and further studies will be necessary to delineate what happens to the material as the indenter penetrates into the surface. the nanoindentation - induced deformation mechanisms will be discussed in more detail with the aids of xtem techniques in the following. a bright - field xtem image of gan thin film after indented with an indentation load of 100 mn it clearly displays that, within this thin film, the deformation features underneath the indented spot are primarily manifested by dislocation activities. namely the slip bands are aligning parallel to the { 0001 } basal planes all way down to the gan thin film - sapphire substrate interface. it is also interesting to note that the heavily strained features in the vicinity of interface may not be just accidental artifacts resulted from sample preparation. it might be direct evidence for displaying that, because of the excellent interface epitaxy between thin film and substrate, the effects of nanoindentation have, in fact, extended into the sapphire substrate. nevertheless, the slip bands (dark thick lines in the photograph) clearly indicate that during the indentation the rapidly increasing dislocations can glide collectively along the easy directions. here, in addition to those aligning parallel to the interface of gan thin film - sapphire along the (0001) basal planes, slip bands oriented at 60 to gan surface can also be observed. the 60 slip bands, which are believed to originate from dislocations gliding along the pyramidal planes, however, are distributing in much shallower regions near the contacting surface. it is indicative that much higher stress level is needed to activate this slip system as compared to the one along the basal planes. (a) xtem images of specimen after an indentation load to 100 mn : a lower magnification photograph showing the regions beneath the indent and part of the substrate. notice that the basal plane slip bands propagate all way to the film - substrate interface and the stress field appears to extend into the substrate. (b) a close - up view of xtem image of the heavily deformed region of gan thin film, displaying that despite of being deformed heavily there is no evidence of crack formation in order to have a closer look at the dislocation activities immediately beneath the berkovich indenter tip, a more detailed microstructures near the intersections of the two sets of slip bands are displayed in fig. it clearly displays a typical microstructure of a heavily deformed material, characterized by features of very high density of dislocations. the distorted slip bands and the extremely high dislocation densities at the intersections indicate highly strained state of gan thin films. nevertheless, even at the submicron scale, no evidence of subsurface cracking and film fragmentation was observed. furthermore, the selected area diffraction (data not shown here) of heavily damaged regions did not show evidence of newly formed phases either. from the above - mentioned observations and discussion, it is apparent that, in the berkovich indentation scheme, the primary deformation mechanism for gan thin film is dislocation nucleation and propagation along the easy slip systems. since the multiple pop - in events are usually observed after the permanent plastic deformation has occurred and two of the possible mechanisms, the deformation - induced phase transition and fracture of thin films, were basically ruled out, the most likely mechanisms responsible for the multiple pop - in events appear to be associated with the activation of dislocations sources. in this scenario, the plastic deformation prior to the multiple pop - in events is associated with the individual movement of a small number of newly nucleated and pre - existing dislocations. as the number of dislocations is increased and entangled to each other, large shear stress is quickly accumulated underneath the indenter tip. when the local stress underneath the tip reaches some threshold level, a burst of collective dislocation movement on the easy slip systems is activated, resulting in a large release of local stress and a pop - in each of these collective dislocation movements is reflected as a slip band in the indented microstructure as displayed in fig. 3. notice that, although the slip bands appeared to stop near the interface of gan thin film - sapphire substrate, the released stress owing to this effect could extend deep into the sapphire substrate as mentioned above. moreover, the narrow spacing of dense bands of defects and/or dislocations along the basal planes near gan surface suggests that, in the later stage of indentation, a larger indentation load, such as the 100 mn applied in the present work, starts to activate the extensive slip bands along the 60 pyramidal planes. the extensive interactions between the dislocations slipping along the two slip systems, therefore, confined the slip bands in a shallow regime, which, in turn, resulted in a heavily deformed and strain - hardened lattice structure. finally, we note that the so - called slip - stick behavior, characterized by material pile - ups phenomena caused by interactions between the as - grown defects and the nanoindentation - induced dislocations, is not significantly here. whether it is owing to the insignificant grown - in defect density of our gan thin films or is related to the specific geometric shape of indenter tips used is not clear at present and further studies may be required to clarify this issue. to summarize, a combination of nanoindentation, cl, fib, and tem techniques has been carried out to investigate the contact - induced structural deformation mechanisms in mocvd - derived gan thin films. cl observations of nanoindentation show the very well - defined rosette structures with the hexagonal system. the berkovich nanoindentation - induced deformation acts as non - radiative recombination centers, which is confirmed by the reduction in the intensity of cl emission. in addition, cl imaging of indents displays to be a sensitive measure of the onset of slip since cl contrast of indents correlates well with the observation of multiple pop - in events. xtem observations show that the prime deformation mechanisms in gan thin films are the nucleation of slip on both the basal and pyramidal planes, rather than otherwise proposed stress - induced phase transformation or crack formation events. finally, this study has significantly implications for the extent of contact - induced damage during fabrication of gan - based optoelectronic devices. this work was partially supported by the national science council of taiwan, under grant no. :	in this study, details of berkovich nanoindentation - induced mechanical deformation mechanisms of metal - organic chemical - vapor deposition - derived gan thin films have been systematic investigated with the aid of the cathodoluminescence (cl) and the cross - sectional transmission electron microscopy (xtem) techniques. the multiple pop - in events were observed in the load - displacement (p h) curve and appeared to occur randomly by increasing the indentation load. these instabilities are attributed to the dislocation nucleation and propagation. the cl images of nanoindentation show very well - defined rosette structures with the hexagonal system and, clearly display the distribution of deformation - induced extended defects / dislocations which affect cl emission. by using focused ion beam milling to accurately position the cross - section of an indented area, xtem results demonstrate that the major plastic deformation is taking place through the propagation of dislocations. the present observations are in support to the massive dislocations activities occurring underneath the indenter during the loading cycle. no evidence of either phase transformation or formation of micro - cracking was observed by means of scanning electron microscopy and xtem observations. we also discuss how these features correlate with berkovich nanoindentation produced defects / dislocations structures.
endometriosis is a clinical entity characterised by the presence of functional endometrial tissue in an anatomical location outside the uterine cavity. this condition is sometimes seen in the scars of caesarean section where it can be confused with incisional hernia. it is rarely (0.2% of ectopic endometrium) seen in the perineum where it is usually localised in episiotomy scars. a 35-year - old lady presented as an emergency to different hospitals on three occasions with a history of severe pain in the perianal area. there was no change in the severity of symptoms in relation to the menstrual cycle during the first two episodes. she had had two deliveries, the first one a normal delivery without episiotomy and the second one caesarean section. at the time of first consultation she was noted to have a 2 cm tender nodule in the perianal area at the 2 o'clock position which was thought to be a perianal abscess. this was incised and drained and as no pus was seen, she was treated as suffering from cellulitis. she presented with similar symptoms a few months later to a different hospital and was treated for recurrent perianal abscess. during her third presentation some blood clots were seen in the nodule and hence an excision biopsy was done which showed endometrial tissue (fig. her white blood cell count was normal and c - reactive protein was mildly elevated. she was further investigated with flexible sigmoidoscopy and an mri scan of the pelvis and perineum which did not show any pelvic endometriosis. she was referred to a gynaecologist for hormonal treatment as nothing was shown on mri scan. it is rarely suspected by the clinician as a differential diagnosis of perianal problems, and the characteristic cyclical pain is uncommon in endometriosis. as perianal sepsis is more commonly dealt with by junior surgeons, unusual presenting features of this condition may not be recognised. a high degree of suspicion is the key to diagnosis, particularly when a patient presents with recurrent perianal pain and no pus is found. anal endosonography can help to know the precise anatomical location and involvement of the external anal sphincter. involvement of senior surgeons and biopsy of unusual lesions in patients presenting with symptoms of recurrent perianal pain would establish the diagnosis early. wide local excision sparing anal sphincter cures the condition and hormonal treatment can be used when it involves multiple sites or when excision is not possible. painful perianal lesions / swellings raise the suspicion of perianal abscess. in cases of recurrence of such lesions and fistulation, crohn 's disease is often suspected. in females such lesions can be endometriosis, particularly when no pus is found or the problem is cyclical.	endometriosis of the perianal region is an extremely rare condition usually seen in episiotomy scars and can involve the septum separating vagina and anal canal. the clinician is unlikely to suspect it if there is no scar in the perineum and patient the does not give a history of episiotomy. moreover it is difficult to suspect when the patient does not report cyclical pain and therefore it is likely to be treated as perianal sepsis. we report the rare case of a 35-year - old woman who presented with recurrent painful nodule of endometriosis of the perianal area without previous episiotomy.
tissue - specific transcription factors play a pivotal role in regulating expression of tissue - specific genes, thereby controlling the function, homeostasis, and differentiation of tissue where they are expressed. their altered expression due to gene mutation, deletion, amplification, and/or epigenetic modification, and/or posttranslational modification can change the cell fate and perturb metabolism and differentiation status, leading to various clinical conditions. since both cell proliferation and differentiation are involved in the process of normal and cancer development, it is not surprising that genes critical for development play an important role in oncogenesis. transcription factors, containing the homeobox [1, 2 ], forkhead domain (fox), and paired domain (pax), that are among those expressed tissue - specifically that play a critical role in tissue homeostasis and development, can also have roles in carcinogenesis. thyroid is an organ in which the homeodomain, forkhead domain, and paired domain - containing transcription factors all play major roles in tissue - specific gene expression and thyroid development. the current view on the roles of thyroid - specific transcription factors in thyroid cancer will be summarized below. the three distinct thyroid - specific transcription factors are critical for the function of thyroid : nkx2 - 1 (also called ttf1, titf1, t / ebp, or nkx2.1) [5, 6 ], foxe1 (also called ttf2 or titf2), and pax8 (table 1). they are members of the homeodomain, forkhead box, and paired box family of transcription factors, respectively, and regulate genes encoding thyroglobulin, thyroid peroxidase, thyrotropin receptor, and sodium / iodide symporter, proteins critical for thyroid hormone synthesis [57, 914 ]. they are also essential for thyroid development [1517 ] ; nkx2 - 1-null mice are born without the thyroid (agenesis), while pax8-null mice are severely hypothyroidism with rudimental thyroid remnant. these transcription factors are responsible for the athyreosis, hypothyroidism, and/or ectopic thyroid, which provide crucial clues to their roles in thyroid dysgenesis in humans. in addition to thyroid, nkx2 - 1 is expressed in lung primordium and ventral forebrain, pax8 in developing kidney, and foxe1 in the floor of the foregut and the craniopharyngeal ectoderm including rathke 's pouch during development. at later stages, foxe1 is expressed in the secondary palate, definitive choanae, whiskers, and hair follicles. thus, nkx2 - 1-null mice also have severely hypoplastic lung, defective hypothalamus, and pituitary agenesis. similar to the defects found in the nkx2 - 1-null mice and sometimes more manifested in humans, various mutations in the nkx2 - 1 gene result in the brain - thyroid - lung syndrome, which is characterized by benign hereditary chorea, congenital hypothyroidism, and respiratory diseases [2022 ]. mutations in the foxe1 gene are responsible for syndromic congenital hypothyroidism dysgenesis, cleft plate, and spiky hair [2224 ]. due to the nature of tissue - specific expression, nkx2 - 1 is expressed in human thyroid and lung cancers [2528 ]. in particular, nkx2 - 1 is highly expressed in human lung adenocarcinomas and small cell carcinomas (~6090%) [25, 26, 29 ]. nkx2 - 1 has been widely used as a marker for the diagnosis of primary and metastatic lung cancer and as a prognostic indicator for survival [26, 31, 32 ]. in fact, nkx2 - 1 is a lineage - specific oncogene amplified in lung cancers and the survival of a subset of adenocarcinoma cells depends on the sustained expression of nkx2 - 1 [3335 ]. however, no mutations in the nkx2 - 1 gene are described in any adenocarcinomas examined in these studies. patients with adenocarcinomas that lack nkx2 - 1 expression or have nkx2 - 1 expression accompanied by nkx2 - 1 gene amplification tend to have a significantly worse prognosis than patients with nkx2 - 1 expression and no nkx2 - 1 gene amplification. in contrast to the expression in lung, nkx2 - 1 is expressed at lower levels in malignant thyroid as compared to normal thyroid. the level of expression is significantly correlated with the progressive dedifferentiation and increase of malignancy of thyroid tumors. thus, the expression is generally found in the order of follicular thyroid adenoma > follicular thyroid carcinoma > papillary thyroid carcinoma > medullary thyroid carcinoma > anaplastic thyroid carcinoma [27, 3739 ]. these studies use immunohistochemical analysis of primary thyroid tissues, and low or no expression of nkx2 - 1 is found in anaplastic thyroid carcinomas. using rt - pcr, nkx2 - 1 expression is reported in some anaplastic thyroid carcinoma - derived cell lines [40, 41 ]. the latter studies present different results for the expression of nkx2 - 1 within the same cell line, suggesting the controversial nature of nkx2 - 1 expression. in order to explain the loss of nkx2 - 1 expression in most of undifferentiated thyroid carcinomas and cell lines, epigenetic silencing of the nkx2 - 1 gene through dna hypermethylation and further studies are required to obtain clear understanding of the relationships in between expression of nkx2 - 1, differentiation status of tissues and primary carcinomas versus cell lines, and the mechanisms underlying the loss of nkx2 - 1 expression in malignancy. a genome - wide association study (gwas) revealed the predisposition of common variants on 9q22.33 and 14q13.3 to both papillary and follicular thyroid cancers. the gene nearest to the 9q22.33 is foxe1, and among the genes located at the 14q13.3 locus is nkx2 - 1, suggesting potential roles for these two thyroid - specific transcription factors in thyroid cancers. a germline mutation of nkx2 - 1 gene leads to a mutant nkx2 - 1 protein (a339v) that has impaired transactivation of thyroid - specific genes such as thyroglobulin, thyrotropin receptor, and pax8, while the expression is associated with the increased cell proliferation, thyrotropin - independent growth, and enhanced activation of survival signaling molecules such as stat3 and akt as compared to wild - type protein. a population study demonstrated that the nkx2 - 1 a339v mutant contributes to predisposition of maltinodular goiter and/or papillary thyroid carcinomas and to the pathogenesis of papillary thyroid carcinomas. nkx2 - 1(fl / fl);tpo - cre thyroid - specific conditional knockout mouse provides an animal model to study the role of nkx2 - 1 in adult thyroid, which circumvents the problem of immediate neonatal lethality of nkx2 - 1-null mouse. in the nkx2 - 1(fl / fl);tpo - cre mouse, the recombination of nkx2 - 1 floxed gene occurs at the rate of ~50%, resulting in nkx2 - 1 thyroid - specific conditional hypomorphic mouse. these mice exhibit either atrophic / degenerative thyroids with frequent presence of adenomas and extremely high tsh levels, or thyroids with reduced numbers of extremely dilated follicles having more number of follicular cells than usual within a follicle. the atrophic / degenerative thyroid mostly consists of atrophic / degenerative follicles, in which many follicular cells frequently have lost nkx2 - 1 expression, suggesting that the loss of nkx2 - 1 may be the cause of atrophic / degenerative follicular cells. these findings further suggest that nkx2 - 1 is required for the maintenance of ordered architecture and function of the differentiated thyroid. in chemical carcinogenesis bioassays using the genotoxic mutagen n - bis(2-hydroxypropyl)-nitrosamine (dhpn) followed by sulfadimethoxine (sdm) as a promoter, the nkx2 - 1(fl / fl);tpo - cre mice developed significantly higher incidence of adenomas as compared with wild - type or nkx2 - 1-heterozygous mice. in contrast, with the non - genotoxic carcinogen amitrole (3-amino-1,2,4-triazole), all three genotype groups of mice developed adenomas at similar incidence. the increased incidence of adenomas in the nkx2 - 1(fl / fl);tpo - cre mice after genotoxic carcinogen exposure may be partially explained by more than a twofold higher cell proliferation rate found in these mouse thyroids as compared to those of wild - type or nkx2 - 1-heterozygous mice. these results may be analogous to human exposure to genotoxic mutagens or radiation, which could cause somatic mutation of nkx2 - 1 gene inactivation of nkx2 - 1 gene degeneration of thyroid follicular cells increased cell proliferation augmentation of the damage occurred in dna, and/or chromosomes by genotoxic mutagens or radiation exposure, ultimately leading to cancer. the loss of heterozygosity of marker d9s180 from this chromosomal area is frequently observed in squamous cell carcinomas of skin, suggesting the presence of tumor suppressor gene in this genomic region. the common variant rs965513 on 9q22.33 contributes to an increased risk of papillary and follicular thyroid cancer. further, a high incidence of foxe1 gene promoter methylation is found in cutaneous squamous cell carinomas (scc), pancreatic cancers, and breast cancers. foxe1 protein has a polyalanine tract starting at the 13th amino acid residue from the end of the forkhead domain, which stretches from 12 to 17 residues with the 14 alanine stretch at the highest frequency. the less common variant (allele 16) is associated with scc, suggesting that the more common variant (allele 14) may be protective against developing scc. similar to nkx2 - 1, foxe1 expression is found in various thyroid cancers [38, 53 ]. the level of expression correlates with their differentiation status as seen with nkx2 - 1, and anaplastic thyroid carcinoma has very little expression of foxe1 [38, 53 ]. the candidate gene association study revealed that the variant rs1867277 (283 g > a) located in the foxe1 5 utr is associated with papillary thyroid cancer susceptibility through recruitment of usf1/usf2 transcription factors to the 283a allele, which affects gene expression. foxe1 is required for thyroid cell precursors to migrate into the underlying mesenchyme from the thyroid bud [15, 55 ]. although the exact mechanism for the enhanced transcription of foxe1 gene leading to increased susceptibility to papillary thyroid cancer remains unknown, the enhanced expression of foxe1 in thyroid carcinomas could be related to a motile advantage of malignant thyroid cells. radiation exposure causes papillary thyroid cancer as revealed by various studies after the chernobyl accident. genome - wide association studies (gwas) employing belarusian patients and control subjects demonstrated that the variant rs965513 on 9q22.33 is significantly associated with the radiation - induced papillary thyroid cancer. this variant was identified together with nkx2 - 1, as those having the strongest rink to papillary and follicular thyroid cancers. although foxe1 thyroid conditional null mice are currently not available, they would be a useful model to understand the role of foxe1 in the pathogenesis of thyroid cancer. pax8 is a crucial transcription factor for organogenesis of the thyroid, kidney, and mllerian system [8, 58 ]. pax8 is expressed in normal as well as neoplastic renal tissues, and in wilms ' tumors [58, 59 ]. pax8 is a useful marker for mllerian carcinomas and ovarian cancer [61, 62 ] and can be used to distinguish ovarian serous tumors from malignant mesothelioma [6164 ] or from other metastatic tumors such as breast and colon [63, 64 ]. pax8 is expressed in various thyroid cancers ; however, the pattern of expression is somewhat controversial ; one study showed that the nuclear pax8 staining is correlated with the thyroid differentiation phenotype as seen with nkx2 - 1 and foxe1, while others demonstrated that pax8 is a useful marker for the diagnosis of anaplastic carcinomas. more studies are required to determine the expression pattern and the role of pax8 in thyroid cancers, including the use of pax8 thyroid - conditional null mice. the chromosomal translocation of the 2q13-qter region to 3p25 results in an in - frame fusion protein (ppfp) between most of the coding sequence of pax8 and the entire translated reading - frame of the nuclear receptor - family member peroxisome proliferator - activated receptor gamma (ppar) (reviewed in [66, 67 ]). the ppfp has several different pax8 breakpoints while the ppar breakpoint seems to be constant [65, 67, 68 ]. this fusion protein is more prevalently expressed in follicular thyroid carcinomas (36%, reviewed in) ; however, follicular adenomas (11%), follicular variant of papillary carcinoma (16%), and hrthle cell carcinoma (2%) also express ppfp [6870 ]. ppfp has been proposed to be an early follicular thyroid carcinoma - specific oncogene [65, 71 ]. several in vitro studies demonstrated that ppfp has oncogenic activity such as increased cell cycle transition, reduced apoptosis, and enhanced growth, which is partly due to ppfp 's dominant negative activity to suppress wild - type transcriptional activities of ppar [65, 71, 72 ], the suggested tumor suppressor [73, 74 ]. ppfp can also work as a dominant negative inhibitor of wild - type ppar in vivo. normal embryogenesis is believed to share many of the same pathways as neoplasia, such as wnt/-catenin, hedgehog, and notch pathways. these signaling pathways are also involved in the maintenance and/or activity of stem cells, while their dysregulation plays a role in tumorigenesis (reviewed in [7680 ]). it is increasingly recognized that homeobox proteins including pax proteins play a critical role in stem cell maintenance [4, 81 ]. pax3 or pax7 is essential for generating the cell pool of muscle progenitors from which satellite cells derive. overexpression of pax3 and 7 is frequently found in pediatric soft - tissue malignant tumor rhabdomyosarcomas [83, 84 ]. pax6 is essential for maintenance of the multipotency of retinal progenitor cells. on the other hand, hox genes are expressed in hematopoietic cells in a stage- and lineage - specific manner, and are implicated in leukemogenesis ; for instance, hoxa10 is a critical regulator for haematopoietic stem cells, and erythroid and megakaryocyte development, while hoxa9 is required for normal hematopoietic stem cell function. the involvements of other homeobox genes in the maintenance of stem cells are described in various tissues including brain and kidney. in the prostate, nkx3 - 1, another member of the nkx gene family, the targeted deletion of pten, a tumor suppressor gene in castration - resistant nkx3 - 1-expressing cells, results in rapid carcinoma formation after androgen - mediated regeneration. the three transcription factors, nkx2 - 1, foxe1, and pax8, are critical for normal embryogenesis and appear to play a role in tumorigenesis in various tissues where they are expressed, including the thyroid. by analogy to other homeodomain / pax proteins, it is likely that nkx2 - 1 and pax8 may be involved in the maintenance and/or activity of stem cells in the thyroid, dysregulation of which may lead to thyroid cancer (figure 1). currently, it is not clear whether fox transcription factors are involved in stem cell maintenance / activity. knockout mouse studies demonstrated that in the absence of nkx2 - 1, primordium cells to both thyroid follicular and c cells disintegrate during thyroid organogenesis [16, 91 ], while pax8 is required for the survival of follicular cells. it would be interesting to determine whether nkx2 - 1 and/or pax8-expressing stem / progenitor cells exist that can rapidly form cancers upon targeted disruption of a tumor suppressor gene in cell pools, similar to that seen with nkx3 - 1. in this regard, nkx2 - 1 in lung cancers may be more analogous to this scenario since nkx2 - 1 is a lineage - specific oncogene and is required for survival of a subset of adenocarcinoma cells [3335 ]. it appears that most transcription factors, if not all, that are critical for developmental process are involved in the maintenance and/or activity of stem cells, whose dysregulation results in cancers. currently, it is entirely unknown whether and/or how the thyroid - specific transcription factors nkx2 - 1, foxe1, and pax8 are related to stem / progenitor cells of the thyroid that may lead to cancer when dysregulated. identification / characterization of thyroid stem / progenitor cells, their relation to the expression of nkx2 - 1, foxe1, and/or pax8, and more detailed characterization of various thyroid cancers and/or cancer cells, particularly in relation to the expression of these transcription factors, are urgently required in order to better understand the roles of nkx2 - 1, foxe1, and pax8 in thyroid cancer.	homeodomain, forkhead domain, and paired domain - containing transcription factors play a major role in development, tissue - specific gene expression, and tissue homeostasis in organs where they are expressed. recently, their roles in stem cell and cancer biology are emerging. in the thyroid, nkx2 - 1, foxe1, and pax8 transcription factors are responsible for thyroid organogenesis and expression of thyroid - specific genes critical for thyroid hormone synthesis. in contrast to their known roles in gene regulation, thyroid development and homeostasis, their involvement in stem cell, and/or cancer biology are still elusive. in order to further understand the nature of thyroid cancer, it is critical to determine their roles in thyroid cancer.
worldwide, thyroid cancer is the most common endocrine malignancy making up about 15% of all cancers in both males and females. over the past several decades papillary thyroid carcinoma (ptc) is the most common type of thyroid cancer constituting 7585% of all cases and therefore represents a major contributor to the worldwide increase of thyroid cancer. the braf mutation is the most common genetic alteration in ptc, found in 45% of cases. threonine protein kinase that plays a critical role in cell signaling as an activator within the mitogen - activated protein kinase pathway. the most common braf mutation is a valine - to - glutamic acid change in codon 600, creating the so - called braf v600e mutation, which elevates the serine threonine activity and causes a constitutive kinase signaling pathway in ptc. the braf mutation confers a worse prognosis for ptc such as a higher risk of lymph node metastasis, extrathyroidal extension, advanced stage, vascular invasion, impaired iodine uptake, recurrence, and mortality. in addition, ptcs that are < 1 cm, but harbor braf mutations have been reported to be associated with adverse prognostic features such as extrathyroidal extension and lymph node metastasis, conveying a more ominous prognosis than their size would predict. braf v600e detection has also been a helpful diagnostic marker for ptc, and it has been shown to be a possible follow - up step to evaluate potential malignant thyroid nodules, particularly when the cytomorphological findings of the initial fine - needle aspiration (fna) are equivocal. studies have found that at least 20% of fnas of thyroid nodules show indeterminate cytological findings. in these cases, detection of the braf v600e mutation in the fna specimen would favor a diagnosis for ptc and patients may be considered candidates for surgical intervention at higher rates than with fna diagnosis alone. recent studies have indicated that an average of 17% of thyroid cancers, which were initially considered indeterminate on fna biopsy, were able to be diagnosed with braf mutation analysis as malignant. to maximize the diagnostic utility of fna specimens, the goal of the current study is to explore the diagnostic implication of detecting braf mutations in the supernatant fluid from cytocentrifugation of fna specimens of thyroid lesions. the current practice for the detection of braf mutations is primarily done using the cellular dna extracted from fna specimens, while the supernatant is routinely discarded. however, at times, paucicellular or acellular fna specimens may fail to provide adequate material for mutation testing. we hypothesize that enough dna may be present in the fna supernatant to conduct mutation testing and that braf mutation analysis using supernatant may increase the diagnostic yield of the fna. a total of 78 thyroid fna samples from 72 patients (59 females, 13 males, mean : 56 years, range : 1391 years) were used for the current study. samples were submitted to a reference laboratory (asuragen, austin, tx, usa), primarily due to indeterminate cytology results, for genetic mutation profiling (including braf) between may 2014 and january 2015 [table 1 ]. samples were obtained, during fna procedures performed under ultrasound guidance, from thyroid nodules. following the fna, some patients had thyroidectomies and corresponding surgical pathology diagnosis. braf mutation testing results within fine needle aspiration cytology classification groups both the cellular dna and supernatant dna from each individual sample were analyzed for braf v600e mutation. routinely, four passes of fna were performed during each fna procedure. from the first three passes of the fna, the supernatant was removed and a thinprep slide was prepared from the cells remaining in the cytolyt tube. a fourth pass containing cellular dna was collected directly into rnaretain media for molecular testing at the reference laboratory (asuragen, austin, tx, usa) using allele - specific polymerase chain reaction (pcr) with sensitivity of detecting 2% mutant braf alleles. of note, the asuragen laboratory also tested other mutations such as kras and nras in these samples. however, the results of the other mutations were not the focus of the current study and were not used for analysis. supernatant dna was extracted using the qiagen biorobot ez1 (qiagen, hilden, germany) with 200 l of supernatant according to the manufacturer 's instruction. dna then underwent braf mutation testing using an in - house developed pyrosequencing - based method with a limit of 10% detection of mutant alleles. this assay has been used for more than 3 years to diagnose braf mutations in melanoma and colorectal cancers. pcr amplification was performed by applied biosystems 9700 pcr system (applied biosystems, foster city, ca, usa) using primers (braf - f : 5-tct tca tga aga cct cac agt aaa aa-3 ; braf - r : 5-cca caa aat gga tcc aga ca-3). the amplicons were immobilized on streptavidin sepharose high - performance beads (ge healthcare bio - sciences, uppsala, sweden) as instructed by the manufacturer. codons 594 - 601 of braf were sequenced with a sequencing primer (5-gga ccc act cca tcg aga-3) in the reverse direction using pyromark q24 system (qiagen, venlo, the netherlands). in addition, braf mutation data were correlated with clinical, cytology, and/or surgical pathology findings. of the 78 samples evaluated, 63 (80%) samples had amplifiable dna from both the supernatant and cellular components for braf mutation analysis. about 68 (87%) samples had amplifiable dna from the supernatant and 73 (94%) samples had amplifiable dna from the cellular. all samples had dna that was either amplified in the cellular or acellular supernatant [table 1 ]. cytological fna diagnosis was rendered using the six diagnostic categories of the bethesda system for reporting thyroid cytopathology : nondiagnostic or unsatisfactory, benign, atypia of undetermined significance (aus) or follicular lesion of undetermined significance, follicular neoplasm or suspicious for a follicular neoplasm, suspicious for malignancy, and malignant. after cytological evaluation of our samples, 0 (0%) were classified as nondiagnostic or unsatisfactory, 5 (6%) were benign, 50 (64%) were aus or follicular lesion of undetermined significance, 16 (21%) were follicular neoplasm or suspicious for a follicular neoplasm, 6 (8%) were suspicious for malignancy, and 1 (1%) was malignant [table 1 ]. two (3%) of the 68 supernatant samples with amplifiable dna were positive for braf mutations [table 1 ]. of importance, the cellular dna counterparts of these two samples showed no braf v600e mutations. the first case was a 29-year - old female who had an fna diagnosis of aus [figure 1a ] with 27% mutant v600e allele by pyrosequencing [figure 2b ]. the second case was a 52-year - old female who had an fna showing findings suspicious for ptc [figure 1b ] which was confirmed in the resected thyroid [figure 1c ]. this case had 34% mutant v600e allele by pyrosequencing in the supernatant [figure 2c ]. these samples were checked for validity against a pyrosequenced wild type control [figure 2a ] and reagent (negative) control [figure 2f ]. in addition, the resected thyroid specimen showed the identical v600e mutation [figure 2e ] by the same method used for supernatant, confirming that the supernatant result was truly positive. (a) fine - needle aspiration showing a cluster of atypical follicular cells characterized by slight nuclear enlargement and nuclear overlapping (pap, 400). (b) fine - needle aspiration shows clusters of atypical follicular cells displaying slight nucleomegaly and nuclear overlapping (diff - quick, 400). (c) surgical resection reveals 1.2 cm focus of papillary thyroid carcinoma (200) braf mutation site comparison between wt and v600e samples. (a) wild type control ; (b and c) two samples with braf v600e mutations in the supernatants and wild type in the cellular counterparts ; (d) an example of braf v600e mutation in the cellular counterpart but wild - type allele in the supernatant ; (e) braf v600e mutation as identified in the thyroidectomy specimen of the case shown in c ; (f) reagent (negative) control five samples were positive for braf mutations using cellular dna [table 1 ]. two of the samples had no amplifiable dna in the supernatant, and the remaining three showed no mutation in the supernatant [figure 2d ]. these five samples showed aus (n = 3), suspicious for malignancy (n = 1), and malignant ptc (n = 1) by fna. of note, one of the aus cases had follow - up surgical resection specimens revealing ptc in which the v600e mutation was again confirmed by the in - house pyrosequencing assay (data not shown). among the eleven samples that showed fna and/or surgical pathology findings suspicious for or diagnostic of ptc (suspicious for malignancy [n = 6 ], malignant [n = 1 ], and/or surgical pathology diagnostic of ptc [n = 4 ]) [table 1 ], 6 (55%) samples (one supernatant and 5 cellular) were positive for the braf v600e mutation. this suggests that testing the supernatant dna in fna specimens may increase the diagnostic yield by 1/11 (9%) in this setting. however, in the setting of indeterminate cytology, the yield of testing supernatant was less and increased the braf mutation detection by only about 2.4% (1/42 supernatant samples with amplifiable dna). our results indicate that the vast majority (87% of our samples) of routinely discarded fna supernatants contain amplifiable dna that could be used for molecular analysis. in addition, some samples of the supernatant contained the mutated braf v600e allele which was not present in the cellular counterpart of the same individual patient sample. therefore, this routinely discarded material could be critical in some cases to diagnose and properly treat ptc patients. in the current study, all of the braf v600e mutations detected in the supernatant were paired with wild - type braf results in the cellular component of the same fna sample. of interest, all patients with cellular components showing braf v600e mutations had the corresponding supernatants revealing either wild - type braf or no amplifiable dna. the higher sensitivity (detecting 2% of mutant alleles) of the method used to detect braf mutations in the cellular component may have contributed to the increased detection of mutated braf genes in the cellular component as evaluation of the supernatant component used a method with sensitivity of detecting only 10% of alleles. using a more sensitive method to test the supernatant, similar to the sensitivity of the one performed on the cellular component at the reference laboratory, may detect more mutations in these samples. however, this is beyond the scope of this exploratory study where the goal is to preliminarily show the diagnostic utility of the fna supernatant. however, the differences in sensitivity between the two different assays used can not explain the discrepancies within the samples with mutant alleles in the supernatant and wild type alleles in the cellular component. it is conceivable that some tumors may have a higher cellular turnover (i.e., increased mitosis, apoptosis, and necrosis), resulting in the shedding of dna (cell - free dna) in the interstitial fluid of the tumor, which is then caught only in the supernatant. therefore, the lack of braf mutations in the cellular component of these samples could be likely due to sampling issues (i.e., cancer cells with braf mutations were missed by the fna needle in the fourth pass, which is the sample submitted to the reference laboratory, resulting in a negative cellular dna analysis while the tumor cells with the braf mutations were obtained from the first three fna passes and therefore were present in the supernatant fluid leading to a positive supernatant dna analysis). another possibility, although less likely, is that the supernatant contained a few tumor cells that remained after centrifugation. to the best of our knowledge, the current study represents the first attempt to use fna thyroid specimen supernatants to test for braf mutations in ptc. our findings of detecting braf mutations in fna supernatants of ptc are in agreement with the results of a recent study, which demonstrated that the use of fna supernatant specimens from patients with pancreatic cancers for molecular testing may outperform cellular dna analysis in certain situations. we attempted to quantify the dna amounts in the supernatant ; however, the majority of samples showed the amount of dna was below the linear range of nanodrop 1000 (thermo scientific, wilmington, de) even though 87% of our samples had enough amplifiable dna for the detection of braf mutation. of note, we only used 200 l of about 2550 ml supernatant for dna extraction ; therefore the yield of dna could potentially be increased if a greater amount of supernatant was used for dna extraction. the percentage of braf mutations among aus cases in the current study is lower than in the literature (8% or 4/50 combining both supernatant and cellular samples vs. the 1520% found in other studies). however, this braf mutation rate is compatible to that of a recent study using residual thinprep material from thyroid fnas for braf testing. our results indicate that profiling the mutations of braf and other genes using dna from routinely discarded fna supernatants may provide valuable diagnostic information to assist with the diagnosis of ptc in a substantial percentage of patients, particularly if clinical / morphologic findings are suspicious for malignancies and no mutations are identified in cellular dna samples. in the current study, testing supernatant dna in the fna specimens increases the diagnostic yield by 1/11 (9%). based on the findings of the current study, we suggest that the fna supernatants should be kept until the cellular molecular testing results are available. if the molecular test results using the cellular dna are inconclusive (i.e., negative or no amplifiable dna), the supernatants can be then submitted for molecular testing to maximize the diagnostic yield of an fna when clinically indicated. this may prevent patients from being subjected to unnecessary further work - ups such as repeat fna procedures. gc performed the mutation study. at and mh made the diagnosis and provided the photographs. aus : atypia of undetermined significance dna : deoxyribonucleic acid fna : fine - needle aspiration pcr : polymerase chain reaction ptc : papillary thyroid carcinoma wt : wild type genotype. 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.	objective : braf mutations using cellular dna from fine - needle aspiration (fna) specimens are commonly used to support the diagnosis of papillary thyroid carcinoma (ptc). the goal of this study was to preliminarily evaluate the diagnostic utility of detecting braf mutations in the routinely discarded fna specimen supernatant fluid.materials and methods : seventy - eight fnas of thyroid lesions were evaluated for braf mutations using both cellular and supernatant dna. braf mutation data were correlated with cytology and surgical pathology.results:of the 78 samples evaluated, 68 (87%) had amplifiable dna in the supernatant with 2 (3%) positive for braf mutations. these two samples showed no mutations in the cellular counterpart. among the 11 samples showing morphologic findings (fna / surgical pathology) suspicious / diagnostic of ptc, 6 (55%) samples (one supernatant and five cellulars) were positive for braf mutations. this suggests that testing supernatant dna in fna specimens may increase the diagnostic yield by 1/11 (9%) in this setting.conclusions:the vast majority of routinely discarded fna supernatants contain amplifiable dna. in addition, profiling the mutations of braf and other genes using supernatant dna may provide valuable diagnostic information to assist the diagnosis of ptc in patients with clinical / morphologic findings suspicious for malignancies and cellular dna showing no mutations.
tumor thrombus into major neck veins in medullary carcinoma thyroid is as yet an unreported event. despite an absent gross local tumor in the neck, such metastatic veno invasive phenomenon has occurred in the post operated follow - up period. outlining the tumor thrombus and its extent by fluorine-18 fluorodeoxyglucose positron emission tomography computed tomography (f18 fdg pet / ct) and the subsequent management by pet - ct planning based intensity modulated radiotherapy (imrt) with successful eradication of the tumoral metabolic activity and regression of the thrombus highlights this rare clinical phenomenon. a 44-year - old male presented with a mass on the right side of the neck known to have been stable for more than 10 years with a history of the increase in size from past 1 month associated with pain. there were no compressive symptoms and no history of flushing, cardiovascular disturbances, or diarrhea. physical examination revealed a nodule of 5 cm 4 cm in the right lobe of thyroid gland with enlargement of level iii cervical lymph nodes on the left side. high - resolution ultrasonography of neck showed a 4.4 cm 3.5 cm heterogeneous echo textured nodule right lobe thyroid with multiple calcific specks within. left lobe also showed multiple small nodules with areas of coarse calcifications. few lymph nodes noted along the left jugular vein largest measuring 1 cm 0.7 cm, one of them showing calcific specks. fine needle aspiration cytology (fnac) performed showed features suggestive of anaplastic carcinoma with brownish - black pigment. a diagnosis of poorly differentiated malignancy with melanin pigment, possible malignant melanoma was made from the intraoperative frozen section of the tumor and hence a total thyroidectomy with removal of enlarged cervical lymph nodes was performed in one sitting. gross findings showed enlargement of both lobes of thyroid with blackish discoloration of right lobe [figure 1a ]. microscopic examination of right lobe thyroid lobe revealed nodularity comprised of polygonal to spindle cells, showing organoid pattern with clumped chromatin, moderate to abundant cytoplasm, with areas of necrosis, focal hemorrhage, and abundant extracellular brownish black melanin pigment. sections from left lobe of thyroid showed features of medullary carcinoma of thyroid with amyloid production, without melanin pigment. immunohistochemistry showed positivity with hmb45 in right lobe [figure 1c ] calcitonin in left lobe [figure 1d ]. (a) gross specimen showing nodular enlargement of thyroid with blackish discoloration of the right lobe. (b) microscopic features showing polygonal to spindle cells, in an organoid pattern with clumped chromatin, abundant cytoplasm, and areas of necrosis, focal hemorrhage, copious extracellular brownish black melanin pigment (arrow). (c) immunohistochemistry right lobe shows tumor cells with brownish - black pigment showing strong cytoplasmic positivity with hmb45. (d) immunohistochemistry left lobe shows tumor cells with brownish - black pigment showing strong cytoplasmic positivity with calcitonin in view of medullary carcinoma with melanin production, further work - up was done with serum calcitonin whole body iodine 131 meta iodo benzyl guanidine (i 131 mibg) scan to outline any residual or metastatic foci with an intent to treat with high dose of i 131 mibg. ng / l and i 131 mibg scan did not reveal any residual or metastatic localization. thus, due to nontherapeutic options with mibg and known radio resistance to radiotherapy of melanotic medullary carcinoma thyroid, patient was kept on close follow - up. one year later, patient presented with diffuse boggy swelling in the right side of neck associated with severe pain radiating to the right ear, however, there was no puffiness of face, engorged veins on the neck or chest wall. a f-18 fdg pet / ct was performed which revealed an intensely hypermetabolic hypo dense intraluminal filling defect with an standardized uptake value maximum of 16.61 in the internal jugular vein (ijv), external jugular vein (ejv), right subclavian vein confluencing at the right innominate vein with inferior extension into the superior vena cava (svc) falling just short of the right atrium suggestive of tumoral thrombus. there was no residual thyroid in the thyroid bed, however, a small metabolically active pretracheal lymph node was noted [figure 2 ]. serum calcitonin was elevated to 300 ng / l fnac from the solitary pretracheal node revealed metastatic deposit of medullary carcinoma. with limited therapeutic options and impending cardiovascular catastrophe due to large tumor thrombus load, an immediate blunderbuss salvage radiotherapy the field included the metabolically active mass in the ijv and svc extending up to right atrium along with neck, sparing the adjoining trachea and carotid vessels to prevent carotid artery stenosis and blow out. a hypofractionated technique was employed delivering a cumulative dose of 50 gy in 20 fractions instead of the conventional 2825 fractions of 1.82 gy / fractions. the patient tolerated the entire course without any hematological or vascular complications and became symptom - free by the end of the radiation treatment. patient was kept on clinical follow - up and assessment with f18 fdg pet//ct done subsequently revealed complete metabolic regression in the intravascular tumor with significant reduction in the tumor thrombus and better contrast passage through the svc [figure 3 ]. follow - up at 3 months patient continued to be symptom - free with complete disappearance of the boggy neck swelling. color doppler of the neck revealed significantly shrunken thrombus in the right ijv with flow void and normal flow through the left innominate vein [figure 4 ]. a contrast ct chest showed non - enhancing hypodense filling defect reduced in extent with tiny calcifications in the right ijv, ejv, subclavian veins suggesting chronic bland venous thrombus [figure 5 ]. a patient being disease free is on replacement therapy with thyroxine and on follow - up. coronal and axial section of fluorine-18 fluorodeoxyglucose positron emission tomography / computed tomography showing intensely hypermetabolic hypo dense intraluminal filling defect right internal jugular vein, external jugular vein, subclavian vein confluencing at the right innominate vein with inferior extension into superior vena cava (arrow) and a small metabolically active pretracheal lymph node (dotted arrow) coronal fluorine-18 fluorodeoxyglucose positron emission tomography / computed tomography postradiotherapy showing complete metabolic regression in the intravascular tumor (arrow) with significant reduction in the tumor thrombus load and better contrast passage through the superior vena cava (dotted arrow) color doppler of the neck showing grossly shrunken thrombus in the right internal jugular vein with absent flow (arrow) and normal flow through the left innominate vein (dotted arrow) coronal and sagittal contrast computed tomography chest showing hypodense filling defect in the right internal jugular vein reduced in extent with tiny calcifications, suggesting chronic thrombus (arrows), and patent left innominate vein filled with contrast streaking past the thrombus into superior vena cava (dotted arrows) medullary carcinoma of thyroid (mtc) is a tumor of parafollicular c - cells present in thyroid, which are derived from neural crest. melanotic variance is a rarity and also adds aggressiveness to the pathology. though the prognosis of a melanocytic variant of medullary carcinoma of thyroid is not known due to the scarcity of numbers it appears to be more aggressive. increased mitotic activity and cellular primitivity may contribute to bad prognosis. on electron microscopic examination, the presence of both neurosecretory granules and melanosomes in the same tumor cells were demonstrated and suggested tumor arising from a common precursor cell with the potential for both melanocytic and c - cell differentiation. surgical excision by total thyroidectomy and local nodal clearance is the preferred treatment of choice. because cancer often is multifocal, surgery includes total thyroidectomy with complete resection of nodes in the central compartment and an ipsilateral modified radical neck dissection if the patient presents with palpable lymph node disease. the c - cells in medullary thyroid cancer do not concentrate iodine and are not part of the thyroid follicular apparatus ; hence remnant ablation with high dose radioiodine is of no indication. the effectiveness of external irradiation for medullary cancer is still controversial, but recent data indicate that it can be used in the curative treatment of patients with microscopic residual or gross disease. the radiosensitivity of medullary cancer probably falls between that of differentiated cancers and anaplastic cancers. patients with detectable calcitonin concentrations after initial treatment and who have no other evidence of disease are followed up regularly. recurrence is usually local and nodal, however, metastasis to lungs, liver, and bones is known. intravascular extension as tumoral thrombosis into major neck veins in papillary and follicular carcinoma thyroid though rare has been reported. surgical intervention in the presence of svc syndrome, however, remains controversial because of the treatment dilemma between perioperative morbidity and mortality with aggressive surgery and the poor prognosis with palliative therapy. the preponderance of the tumoral thrombus predominantly in the right sided major neck veins is hypothesized by the migration of the tumor cells from the neck through the lymphatic channels in the right side of the neck draining through the right lymphatic duct also called the right thoracic duct which drains lymphatic fluid from the right thoracic cavity, the right arm, and from the right side of the neck and the head directly opening into the right subclavian ijv junction, unlike thoracic duct which opens at the left jugulo subclavian junction. the presented clinical status was one of extensive tumoral thrombus with compromised venous lumens of the major right cervical veins. patient 's retrieval with an immediate hypofractionated imrt approach not only salvaged the patient from impending svc syndrome but also contained the tumor thrombus making it metabolically bland and regresses morphologically. f-18 fdg pet / ct outlined hypermetabolic region inside the neck veins was principally taken as the guideline for carotid artery sparing imrt planning of gross tumor volume and planning tumor volume. hypofractionated schedule of 20 fraction 50 gy instead of conventional 2 gy, 25 fraction method was adapted. this will reduce the treatment duration and also deliver higher dose per fraction with better therapeutic effect by overcoming radioresistance and achieving faster and better control of intravascular tumor load which was sanctified with gratifying clinical outcome. the case had the distinction of more than year 's record of events from the onset of disease, initial treatment, and the catastrophic tumor thrombus occurring 1 year later. pet / ct - aided diagnosis and radiotherapy planning with the rewarding response from a metabolically active tumor thrombus to a bland nontumoral thrombotic disease free status is the quintessence of this rare case.	the authors report an extremely rare occurrence of a massive tumor thrombus involving right internal and external jugular veins extending into superior vena cava from a still rarer melanotic medullary carcinoma thyroid in the postoperative follow - up. the case was managed by hypofractionated intensity modulated radiotherapy technique with gratifying results.
today, medical laboratories play a significant role in the healthcare system and the decision - making of clinical doctors about their patients. according to official data, 6070% of clinical decisions about hospitalization, discharge, and prescription are based on laboratory results. clinical tests enjoy a high status in screening, treatment follow - up, and assessment of response to treatment. given this high percentage and the significant role, the quality of laboratory tests is of high importance. in other words, quality is the cornerstone of management in a laboratory and it needs to conform to the highest standards. the errors are categorized into three stages depending on whether they are done before, during, or after analysis : the preanalytic, analytic and postanalytic phase errors. since there are no definite methods of sampling, most errors take place before the test, just before the sample is being prepared (it includes specimen collection, handling and processing variables, physiological variables, and endogenous variables). some of the preanalytical variables such as specimen collection can be controlled, while knowledge of uncontrollable variables needs to be well understood to be able to separate their effects from disease - related changes affecting the laboratory results. since the quality is interconnected, precision and accuracy are not the only guarantors of the quality. from the very beginning, all three stages need to be under monitoring and quality control with precision and accuracy. the objective of this study is to examine the frequency of errors before, during, and after analysis in a referral laboratory so that proper approaches could be developed for minimum errors. this descriptive, cross - sectional study was conducted at imam teaching hospital throughout 2012 (january december 2012). the hospital, affiliated to tehran university of medical sciences, is a referral hospital for the entire country. it has a department for every medical specialty (surgery, internal medicine, pediatrics, intensive and coronary care units, etc.). the sections in the hospital 's laboratory include hematology, urine biochemistry, parasitology and microbiology, general and specialized biochemistry, hormonology, serology, coagulation, and flow cytometry (fcm). two associate professors and an assistant professor, as well as 106 employees are working in the laboratory, which provides laboratory testing for inpatients and outpatients round the clock. the laboratory testing process starts either through a requisition from the hospital 's departments or by physicians in private practice. for inpatients, the laboratory clerks process the patients identification data including their name, age, sex, file number, and physician 's name and register them in the hospital information system (his). if the requisition is not stat order or emergent, the following morning a trained laboratory staff collects blood samples from all hospital departments. in stat and emergent occasions, nurses or medical students collect the blood samples, which are then transported by couriers to the laboratory department. for outpatients, the physicians in private practice fill out the appropriate requisition form, which serves as a referral to the laboratory department. then, the laboratory clerk registers the patient 's identification data into the laboratory information system (his). all samples were collected in 2012 and there was no exclusion criterion. after transporting specimens to the laboratory department, a laboratory technologist evaluates the samples for misidentification errors, inappropriate container, improper labeling, inadequacy of sample collection, and inadequacy of sample / anticoagulant ratio volume. any probable errors are reported to the ordering department and request is sent for a new proper sample. also, the errors are recorded in a roster and a copy is sent to the laboratory 's quality control committee (the quality control committee comprises of a laboratory director, who is a professor of pathology ; a supervisor, who is a graduate of laboratory sciences with at least 15 years of experience ; and two laboratory technologists with at least 10 years of experience in quality control). the specimen preparation, including preparing suitable samples for analytic procedures like centrifugation, aliquoting, and sorting specimens are performed. the test results data are entered to his both manually and automatically, depending on the test type. after monitoring and checking by a quality manager who is a technologist or a laboratory physician, the test results are allowed to be observed by medical staff for evaluation, interpretation, and appropriate action. the physicians and nurses of hospital 's departments are trained to be observant of test results and are asked to report any questionable result to the quality control committee of the laboratory. if any result is labeled clinically questionable, the quality manger checks the testing process and usually the specimen is retrieved or in some circumstances asks for a new sample. errors are recorded by quality manager on a special roster and the date, time, source of the test, type and detail, and signature are recorded. the reported case was discussed in the committee based on clinical and laboratory standards institute (clsi) instruction and classified in database. the quality control committee comprised the laboratory director, faculty member, qc manager as well as supervisor and sub - directors. the laboratory error definition that has been incorporated in the international organization for standardization (iso) technical report 22367 is defined as a defect occurring at any part of the laboratory cycle, from ordering test to reporting, interpreting, and reacting to results. since studies on laboratory errors are diverse and there is no general acceptance on definitions used especially on the types of errors, it is an arduous task to categorize variable types of errors. however, in this study it is tried to utilize more general and practical categorization as da rin used in his article. preanalytical errors : these errors are defined as the errors occurring from physician order to analytic phase, including test request, patient identification, collection, transportation, and preparation for analysis. for better classification and monitoring of preanalytical phase, it is subdivided to errors that occur outside the laboratory and errors that occur under the control of laboratory as da rin has mentioned in his study. outside laboratory : inappropriate test request, order entry errors, misidentification of patient, inappropriate container, improperly labeled container, inadequate sample collection and transportation, specimen collected from infusion route, inadequate sample / anticoagulant volume ratio, and insufficient sample volume. within laboratory : 2. analytical errors : these errors occur during the test, and include equipment malfunction, sample mix - ups, interference, undetected failure in quality control, and procedure not followed. postanalytical errors : these errors happen after the test is conducted, and include failure in reporting, erroneous validation of analytical data, improper data entry, and excessive turnaround time. their relative frequencies when compared with the total specimens were also calculated and presented as percentage. the statistical significance level accepted in this study was set at p value equal to or less than 0.05. the difference between relative frequencies of mistakes observed in the departments considered was tested by proportional z test. their relative frequencies when compared with the total specimens were also calculated and presented as percentage. the statistical significance level accepted in this study was set at p value equal to or less than 0.05. the difference between relative frequencies of mistakes observed in the departments considered was tested by proportional z test. a total of 303,866 samples, 2,430,928 tests were received for analysis (230,938 samples, 1,847,504 tests were from inpatients and 72,928 samples, 583,424 tests were from outpatients). total number of errors was 153,148 (6.3% 62999.8 parts per million, ppm) (11639262999.5 ppm for inpatients and 3675663000 ppm for outpatients). number and frequency of three main groups of errors in inpatients and outpatients are shown in table 1, and type and subtype of three main groups of errors in inpatients and outpatients are shown in tables 2 and 3, respectively. number, frequency and 95% ci of errors in preanalytical, analytical, postanalytical phases error and sub - error type tests in inpatients error and sub - error type tests in outpatients some studies have found that about 70% of clinicians decisions are based on laboratory results. therefore, the quality, accuracy, and precision of laboratory results are indispensable in clinical care. according to a definition acknowledged by international organization for standardization, laboratory error is defined as failure of planned action to be completed as intended, or use a wrong plan to achieve an aim, occurring at any part of the laboratory cycle, from ordering examinations to reporting results and appropriately interpreting and reacting to them. the aim of this descriptive study is to evaluate type and frequencies of errors in three phases of laboratory tests to reduce preventable errors. the analysis of results of previous studies shows that despite heterogeneity in methods, the error distribution across the different phases of testing process appears to be similar with most errors occurring in the preanalytical phase. in one review article written by howkins in 2012, he mentioned that the proportion of errors associated with pre- and postanalytical phases of testing is 45 times higher than that seen in analytical phase with preanalytical phase representing over half of the errors in published studies. in another study, preanalytical factor consists of 4668.2% of total errors while a high error rate 18.547% of total errors has been found in postanalytical phase. in a retrospective study performed by plebani. they found out that despite a 34% reduction in error rate, the pattern of 62% preanalytical, 15% analytical, and 23% postanalytical phase errors remained basically unchanged. analysis of results of this study shows that about 65.09% of errors occur in preanalytical phase, while about 23.2% and 11.68% occur across analytical and postanalytical phases, respectively. according to different studies, there is a considerable difference between in- and outpatients with the 0.60% versus 0.039% for the two categories, respectively. it seems that most of these differences are related to human factors including personal skills in venipuncture (drawing blood) and the sheer volume of laboratory tests carried out for inpatients. the results of this study also revealed that errors in hospitalized patients were more numerous than in outpatients. that might be due to the grave conditions of hospitalized patients and variety of staff involved in the total testing processes. ambiguities about the standard methods and appropriate transporting times for different laboratory tests are also of great importance in decreasing preventable errors. therefore, nurses and other laboratory workers need to be more specialized in taking blood samples and other interventions. the vacutainers (glass or plastic tubes) could be prevented from being broken if they are carried in special vessels. although the importance of preanalytical phase has been acknowledged for many years, laboratories have often overlooked this area in their quality management programs, focusing instead on analytical quality and the associated activities within their direct control. in the analytical stage of this study, errors for inpatients and outpatients did not differ meaningfully and were mainly due to factors that could be prevented by an accurate and precise quality control procedure. training special staff to conduct quality control or even offering a specialized university course is recommended for this purpose. in the postanalytical stage, the errors did not show any meaningful difference between these two groups and they resulted mainly from human errors in reporting procedures. to that effect, laboratories with high volume of work postanalytical errors are indicative of the significance of thorough control on the results of the tests by the laboratory 's chief technician before being confirmed by the laboratory director. when compared with other studies, this study shows more analytic errors, most likely because the aging equipment 's state - of - the - art technology is not used for financial restrictions, or possibly, inadequate training of laboratory workers. the limitations in this study were that registration of the errors during the evening, night - shifts, and holidays were not completely performed. more studies are recommended to be carried out to examine differences between day and night shifts separately and at multi centers. hospital and laboratory directors can minimize errors and boost the quality of healthcare services based on the results of these studies. since more than half of the laboratory errors occur during preanalytical phase, proper training and knowledge of the intervening factors that can influence laboratory results are essential to minimize laboratory errors. it should be reminded that all three stages of laboratory tests need to be under thorough monitoring to improve the quality of results.	background : according to official data, 6070% of clinical decisions about hospitalization and discharge are based on laboratory results.aims:the objective of this study is to examine the frequency of errors before, during, and after analysis in a major medical laboratory.materials and methods : this descriptive, cross - sectional study was conducted throughout 2012 (january december 2012). errors are recorded by the quality control committee in a specially designed record.results:a total of 303,866 samples, 2,430,928 tests were received for analysis. the total number of errors was 153,148 (6.3%) (116,392 for inpatients and 36,756 for outpatients). analysis of the results revealed that about 65.09% of the errors occur across preanalytical phase, whereas 23.2% and 11.68% are related to analytical and postanalytical phase, respectively.conclusion:more than half of the laboratory errors are related to preanalytical phase ; therefore, proper training and knowledge of intervening factors are essential for reducing errors and optimizing the quality.
nosocomial infections cause prolonged duration of hospitalization and increased treatment costs with high morbidity and mortality. acinetobacter species have become the most common etiology of hospital acquired infections due to the present frequent use of wide spectrum antibiotics (1). acinetobacter species cause pneumonia, skin and wound infections, bacteremia, and urinary tract infections while they have shown resistance to various antibiotics (2). carbapenems, sulbactam and colistin seem to be the most effective antibiotics for treatment (3). sulbactam shows high efficacy against acinetobacter species in vitro and in vivo (4 - 6). previously in turkey, sulbactam could not be found alone ; therefore sefoperazone and sulbactam combination was widely used for the treatment of multi - resistant acinetobacter species. today, however, commercially available sulbactam antibiotics are being used thus, the determination of the minimum inhibitory concentration (mic) of sulbactam is necessary. the aim of this study was to determine the susceptibility rate of genotypically different a. baumannii species, isolated from various clinical samples of patients with hospital acquired infections, to sulbactam, amikacin, netilmicin, meropenem, tigecycline and colistin. the isolates are defined as the agent of hospital acquired infections, if they emerged after 72 hours of hospitalization., 300 a. baumannii were isolated from various clinical samples and detected by the vitek 2 compact system (biomerieux, france) at the microbiology laboratory of our hospital between january 2010 and march 2012. the isolates were stored at -80c in brain heart infusion broth including 10% glycerol until susceptibility tests and genotypic evaluations were completed. according to the instructions of the manufacturer, these isolates were subjected to pcr analysis using the appropriate diversilab dna fingerprinting kit (biomerieux, france). an analysis of the bands was completed using the diversilab software 3.3 gel images, while percentage similarity rates and dendrogram reports were formed for each dna sample. the samples were classified as different (similarity 2 band difference), similar (similarity in between 95 - 97%), and indistinguishable (similarity > 97%, 1 - 2 band difference) (7). the susceptibilities of genotypically different isolates (similarity 2 band difference), to sulbactam, amikacin, netilmicin, meropenem, tigecycline and colistin were tested. antibiotic susceptibilities were studied using the e - test method for sulbactam and tigecycline (biomerieux, france), while the bouillon broth microdilution method was used for meropenem (tumekip, turkey), colistin, amikacin and netilmicin (sigma aldrich, usa). for the e - test method, bacterial suspensions with turbidity equivalent to 0.5 mcfarland standard were prepared and spread onto mueller hinton agar (mha) medium (himedia, mumbai, india), followed by the addition of e - test strips. after the plates were incubated at 37c for 24 hours, the e - test mic value was read as the concentration at which the growth on the plate intersected the e - test strip. according to the food and drug administration (fda) criteria ; if the e - test mic value of tigecycline was 8 g / ml, it should be accepted as resistant, if the value was 4 - 6 g / ml, it should be accepted as intermediate, and if the value was 2 g / ml it should be accepted as susceptible (8). the cut - off value for sulbactam alone was not defined for a. baumannii therefore, we determined the cut - off value of sulbactam considering the values of ampicillin / sulbactam for a. baumannii isolates in the clsi document. accordingly, if the value was 4 g / ml, it was accepted as susceptible, if the value was 8 g / ml it was accepted as intermediate and if the value was 16 g / ml it was accepted as resistant. for the bouillon broth micro dilution method, cation - adjusted mueller - hinton broth (bbl, becton dickinson, usa) was used according to clsi recommendations (9). serial dilutions of each of the four antibiotics (32 - 0.062 g / ml) were prepared. a. baumannii isolates were suspended to 0.5 mcfarland turbidity and added to micro dilution plates with 96 wells. escherichia coli (atcc 25922) and pseudomonas aeruginosa (atcc 27853) strains were used as quality control strains for all mic determinations. the lowest antimicrobial drug concentration at which there was no growth was considered as the minimum inhibitory concentration (mic). the isolates are defined as the agent of hospital acquired infections, if they emerged after 72 hours of hospitalization., 300 a. baumannii were isolated from various clinical samples and detected by the vitek 2 compact system (biomerieux, france) at the microbiology laboratory of our hospital between january 2010 and march 2012. the isolates were stored at -80c in brain heart infusion broth including 10% glycerol until susceptibility tests and genotypic evaluations were completed. according to the instructions of the manufacturer, these isolates were subjected to pcr analysis using the appropriate diversilab dna fingerprinting kit (biomerieux, france). an analysis of the bands was completed using the diversilab software 3.3 gel images, while percentage similarity rates and dendrogram reports were formed for each dna sample. the samples were classified as different (similarity 2 band difference), similar (similarity in between 95 - 97%), and indistinguishable (similarity > 97%, 1 - 2 band difference) (7). the susceptibilities of genotypically different isolates (similarity 2 band difference), to sulbactam, amikacin, netilmicin, meropenem, tigecycline and colistin were tested. antibiotic susceptibilities were studied using the e - test method for sulbactam and tigecycline (biomerieux, france), while the bouillon broth microdilution method was used for meropenem (tumekip, turkey), colistin, amikacin and netilmicin (sigma aldrich, usa). for the e - test method, bacterial suspensions with turbidity equivalent to 0.5 mcfarland standard were prepared and spread onto mueller hinton agar (mha) medium (himedia, mumbai, india), followed by the addition of e - test strips. after the plates were incubated at 37c for 24 hours, the e - test mic value was read as the concentration at which the growth on the plate intersected the e - test strip. according to the food and drug administration (fda) criteria ; if the e - test mic value of tigecycline was 8 g / ml, it should be accepted as resistant, if the value was 4 - 6 g / ml, it should be accepted as intermediate, and if the value was 2 g / ml it should be accepted as susceptible (8). the cut - off value for sulbactam alone was not defined for a. baumannii isolates in the clinical and laboratory standards institute (clsi) document. therefore, we determined the cut - off value of sulbactam considering the values of ampicillin / sulbactam for a. baumannii isolates in the clsi document. accordingly, if the value was 4 g / ml, it was accepted as susceptible, if the value was 8 g / ml it was accepted as intermediate and if the value was 16 g / ml it was accepted as resistant. for the bouillon broth micro dilution method, cation - adjusted mueller - hinton broth (bbl, becton dickinson, usa) was used according to clsi recommendations (9). serial dilutions of each of the four antibiotics (32 - 0.062 g / ml) were prepared. a. baumannii isolates were suspended to 0.5 mcfarland turbidity and added to micro dilution plates with 96 wells. escherichia coli (atcc 25922) and pseudomonas aeruginosa (atcc 27853) strains were used as quality control strains for all mic determinations. the lowest antimicrobial drug concentration at which there was no growth was considered as the minimum inhibitory concentration (mic). among the 300 a. baumannii isolates the susceptibilities of 30 isolates were determined as genotypically different (figure 1). twenty - one (70%) of the 30 genotypically different a. baumannii isolates included in the study were isolated from tracheal aspirates, one (3.3%) from blood, four (13.3%) from the catheter, one (3.3%) from a wound infection, two (6.6%) from urine samples, and one (3.3%) from peritoneal fluid samples. the susceptibility results of 27 carbapenem resistant isolate (including the one with intermediate susceptibility) to antibiotics other than carbapenem are shown in table 2. the sensitivity rates for netilmicin, tigecycline, sulbactam, amikacin, and meropenem were 66.6%, 50%, 36.6%, 30% and 10%, respectively. the rates determined for carbapenem resistant isolates were 66.6%, 51.8%, 33.3%, and 25.9% for netilmicin, tigecycline, sulbactam, and amikacin, respectively. the mic50 values for netilmicin, tigecycline, sulbactam, amikacin, meropenem and colistin were 4 g / ml, 3 g / ml, 8 g / ml, 128 g / ml, 64 g / ml, and 1 g / ml, respectively, while the mic90 values were 512 g / ml, 8 g / ml, 12 g / ml, 1024 g / ml, 128 g / ml, and 1 g / ml, respectively. a. baumannii has become an important health problem due to the fact that it is a pathogen that causes hospital acquired epidemics and due to treatment failures caused by multiple antibiotic resistances (2, 10). the same bacteria obtained from different patients in a clinic shows that there is cross contamination between the patients originating from the same source. in such a case, the source of the microorganism causing the hospital acquired infection must be investigated. through determination of the clonal relationship between the isolates, the source of the infection, the porter, and the way of spread can be revealed, and proper prevention methods can be chosen. the aim of genotyping studies is to determine whether there is an epidemiological relationship between isolated and genotyped strains and to grade this relationship if one exists. rep - pcr is performed with primers specific to a repeated sequence in the genome and can be used in phylogenetic relationship studies for providing reliable results and repeatability (11). in a study by caretto., genotyping of acinetobacter isolates was carried out and they emphasized that rep - pcr is an expensive but a rapidly performed and repeatable method (12). therefore in this study rep - pcr method (diversilab, biomrieux, france) was used for genotyping the isolates as it is a rapid and easy to use method. only genotypically different isolates were evaluated for antimicrobial susceptibilities as the genotypically related isolates could give the same susceptibility patterns. therefore, in the current study the antimicrobial susceptibilities were evaluated for only 30 isolates that were genotypically different and not for the 300 isolates that were collected in a two year period. monotherapy with sulbactam is not recommended for severe acinetobacter infections. however, wood., reported that the use of sulbactam for the treatment of 14 patients with ventilator associated pneumonia caused by multi drug resistant acinetobacter was successful and that there was no difference between 63 patients treated with sulbactam or imipenem in terms of clinical results (13). in the study of rodriguez - hernandez., which was carried out on 150 a. baumannii isolates, it was indicated that sulbactam could be a good treatment option for multi resistant a. baumannii infections (6). they tested ampicillin, piperacillin, ticarcillin, and beta lactamase inhibitors, such as clavulanic acid, sulbactam, and tazobactam, alone or in combination. it was emphasized that beta lactamase inhibitors, with the exception of sulbactam, had no effect and ampicillin sulbactam was the most effective combination. applied intravenous treatment of sulbactam to 18 patients and intravenous treatment of ampicillin sulbactam to 24 patients out of 42 patients in which multidrug - resistant acinetobacter was isolated. they found that the effect of ampicillin - sulbactam combination is deriven by sulbactam and this combination does not produce a synergistic effect (14). furthermore, in an experimental model of pneumonia in which sulbactam sensitive a. baumannii was used, sulbactam was found as effective as imipenem (6, 15). in the current study, we found sulbactam susceptibility in a lower ratio, discordant from the literature. the sensitivity ratio of sulbactam in vitro was found to be 36.6% in all acinetobacter isolates and 34.6% in carbapenem resistant isolates. this ratio was determined when the mic value of sulbactam was accepted as sensitive if it was 4 g / ml. if the mic value 8 g / ml was accepted as sensitive, the sensitivity ratio would be 73.3%. furthermore, the low sulbactam susceptibility of acinetobacter isolates in the current study may be due to selecting clones different from each other and that the isolates were multidrug resistant. although the carbapenems are the most effective antibiotics for the acinetobacter species, recently in studies from turkey and worldwide, it was reported that acinetobacter isolates resistant to carbepenems were isolated with an increasing frequency (2, 16 - 21). there are studies from turkey demonstrating that there is an increasing resistance to imipenem (50 - 84%) and meropenem (63 - 80.3%) (18 - 21). in the current study, there was 86.6% resistance to meropenem and3.3% of the isolates were intermediate. the high levels of carbapenem resistance may be due to the multidrug resistance of the isolates and the prolonged duration of empirical treatment with carbapenem at our studied hospital. this study is the first study from turkey where the mic value of sulbactam in acinetobacter was determined alone in vitro. further clinical studies are needed to determine the efficacy of sulbactam on a. baumannii. in the current study, the mic value of sulbactam 4 g / ml was accepted as susceptible, the value of 8 g / ml was accepted as intermediate and the value of 16 g / ml was accepted as resistant. if we had determine the cut - off mic value two folds higher it would have considerably affected the susceptibility results. therefore, it is important to determine the resistance profile for hospital settings in order to choose the proper antibiotic for empirical treatment.	background : the treatment of acinetobacter baumannii infections is difficult. carbapenems, sulbactam, and colistin are the most effective antibiotics.objectives:the aim of this study was to evaluate the susceptibilities of genotypically different a. baumannii isolates to sulbactam, amikacin, netilmicin, meropenem, tigecycline and colistin.patients and methods : isolates from various clinical samples of patients with hospital - acquired infections that were identified by the vitek 2 compact system in our hospital s microbiology laboratory between january 2010 and march 2012 were included in the study. to determine genetic relatedness of the isolates, the rep - pcr method was used. the broth microdilution method was used for amikacin, netilmicin, meropenem and colistin, while e - test was used for sulbactam and tigecycline.results:among the 300 isolates, 30 were found to be genotypically different and were evaluated in terms of their antimicrobial susceptibilities. all isolates were susceptible to colistin. the susceptibility rates were 66.6%, 50%, 36.6%, 30%, and 10% for netilmicin, tigecycline, sulbactam, amikacin, and meropenem, respectively. for carbapenem resistant isolates, the susceptibility rates were 66.6%, 51.8%, 33.3%, and 25.9% for netilmicin, tigecycline, sulbactam, and amikacin, respectively. the sulbactam minimum inhibitory concentration (mic) 50 and mic 90 were 8 g / ml and 12 g / ml, respectively.conclusions:in this study, it was concluded that determining the cut - off value for mic breakpoints for sulbactam alonehas a critical impact on the susceptibility results.
unpasteurized fruit juice is defined as the product produced by pressing or squeezing of the fruits. consumption of fresh juices increased dramatically due to their freshness, high vitamin content, and low caloric consumption. extracted juices from fruits contain most substances which are found in the original ripe and sound fruit from which the juice is made. the high potassium and low sodium characteristic of most juices help in maintaining a healthy blood pressure. vitamin c is naturally present in juices which are essential for the body to form collagen, cartilage, muscle, and blood vessels. fruit juices contain a microflora which is normally present on the surface of fruits during harvest and postharvest processing which include transport, storage, and processing. many microorganisms such as acid tolerant bacteria and fungi (moulds, yeasts) use them as a substrate for their growth. the major genera include candida, dekkera, hanseniaspora, pichia, saccharomyces, and zygosaccharomyces. penicillium, byssochlamys, aspergillus, paecilomyces, mucor, cladosporium, fusarium, botrytis, talaromyces, and neosartorya are filamentous fungi most frequently isolated from fresh fruits and juices. among bacteria, lactic acid bacteria and acetic acid bacteria the critical factors affecting the spoilage of juices include juice ph, oxidation reduction potential, water activity, availability of nutrients, presence of antimicrobial compounds, and competing microflora. among these factors, ph and water activity are the most influential factors affecting the spoilage of juices. the spoilage caused by microorganisms in juices includes cloud loss, development of off - flavours, co2 production, and changes in colour, texture, and appearance resulting in degradation of product [6, 7 ]. the most commonly reported bacterial genera include acetobacter, alicyclobacillus, bacillus, gluconobacter, lactobacillus, leuconostoc, zymomonas, and zymobacter. among yeasts pichia, candida, saccharomyces, and rhodotorula are commonly encountered genera responsible for spoilage of juices., aspergillus sp., eurotium, alternaria, cladosporium, paecilomyces, and botrytis have also been reported in spoilage of fruit juices [5, 6 ]. fruit juices have ph in the acidic range (< 4.5) serving as important barrier for microbial growth. however, food borne pathogens such as e. coli and salmonella survive in acidic environment of fruit juices due to acid stress response. therefore, in the last two decades a number of food borne outbreaks associated with unpasteurized fruit juices have been documented in many countries [1, 9 ]. the source of entry of microorganisms into fresh fruit juices from environment exposure and soil. in developing country like india, a large population of all income and age groups consume freshly squeezed fruit and vegetable juice, but the presence of pathogenic microorganisms in street vended fruit juices has also been reported in various parts of india such as vishakhapatnam, mumbai, amravati, and nagpur. in view of the demand for fresh fruit juices throughout the year and threat of emerging food borne outbreaks associated with consumption of fruit juices, the aim of present study was to investigate the microbiological examination of freshly prepared juices commonly consumed in kurukshetra. three juices commonly consumed in kurukshetra such as orange (citrus reticulata blanco), sweet orange (citrus sinensis), and carrot (daucus carota) were selected for microbiological study. sweet orange, carrot, and orange were purchased from the local markets of kurukshetra from october 2011 to february 2012. each sample was washed, peeled, and cut into pieces and juice was extracted through sterile hand blender and poured into sterile beaker. ten ml of juice sample was diluted with 90 ml of 0.1% sterile peptone water (1 g peptone, 1l distilled water) and plated on nutrient agar (ph 5.5) for enumeration of bacteria and pda supplemented with antibiotic (ph 5.5) for enumeration of fungi in duplicates. mould and yeast isolates were purified on potato dextrose agar, bacteria on nutrient agar, and further subcultured for microscopic examination and identification. for bacterial identification, 24-hour - old culture of bacteria was observed under microscope by gram stain method and further various biochemical tests were performed for the identification of bacteria such as catalase test, oxidase test, starch hydrolysis test, sugar fermentation test, imvic test, and methods described in bergey 's manual of systematic bacteriology. further identification of bacteria was performed on the basis of methods described in the methods adopted for identification of yeasts include morphological characteristics, fermentation of sugars, germ tube test and cycloheximide resistance test, and methods described in fungi and food spoilage [17, 18 ]. moulds were identified on the basis of morphological and cultural characteristics such as colour of the colony, surface, appearance, presence, and absence of cross walls, and asexual and sexual reproductive structures. further identification of moulds was carried out according to the methods described in fungi and food spoilage. moulds were cultured on czapek yeast extract agar (ph 6.7), malt extract agar (ph 5.6), and glycerol nitrate agar (ph 7.0) at 25c. in the present study, 30 samples of freshly prepared juices (10 samples each of orange, sweet orange, and carrot) were examined for microbiological analysis. factors which determine the colonization of juices by microorganisms include ph, redox potential, water activity, nutrients, structures, antimicrobial agents, temperature, relative humidity, and atmosphere. in the present study the frequencies of occurrence of moulds and yeasts were more as compared to bacterial genera which is attributed to low ph values and high sugar content. a total of 34 bacterial, 12 yeast, and 25 mould isolates were isolated from juices classified by grouping them into 9 bacterial species, 5 yeast species, and 11 mould species on the basis of phenotypic characteristics. yeasts and moulds are capable of growth at ph values of 1.5 and at water activity values below 0.89. the minimum ph values allowing the growth of lactic acid bacteria (ph 2.93.5), acetic acid bacteria (ph 3.04.5), and enteric bacteria (ph 3.64.5) are higher than those for growth of yeasts and moulds. the frequency of occurrence of bacteria, yeasts, and moulds are summarized in tables 8, 9, and 10, respectively. bacillus cereus was also observed in 64.91% of samples of unpasteurized street vended fruit juices. leuconostoc and lactobacillus were also reported as important group of spoilage microorganisms in acidic products. these microorganisms produce acetic and formic acids along with ethanol and carbon dioxide which can alter the flavor of juice. leuconostoc, lactobacillus, and acetobacter were detected in tested juice samples (table 8). the presence of e. coli, salmonella, and s. aureus in fruit juices is primarily concern because these pathogens were implicated in a number of outbreaks associated with fruit juices. in our study, the presence of e. coli and s. aureus was detected in a smaller number of samples. the survival of pathogens in acidic environment of juices is attributed to their ability to regulate their internal ph and maintained at neutral ph by combination of passive and active homeostasis mechanisms. the acid survival mechanisms of enteric bacteria are due to induction of enzymes that are involved in raising the internal ph and activation of enzymes devoted to the protection and repair of proteins and dna. yeasts genera responsible for spoilage of fruit juices include candida, pichia, rhodotorula, torulopsis, saccharomyces, zygosaccharomyces, hansenula, and trichosporon. in our study, the dominant yeasts isolated from juices were rhodotorula, pichia, and saccharomyces (table 9). rhodotorula was found in maximum number of juice samples tested followed by pichia and saccharomyces. candida parapsilosis and c. krusei were only detected in orange and sweet orange juices, not detected in carrot juice. rhodotorula, pichia, candida, and saccharomyces have also been reported as spoilage causing organisms in pasteurized fruit juices [4, 27 ]. yeasts also produced pectin esterases which degrade pectin causing spoilage ; organic acids and acetaldehyde, which contribute to a fermented flavor, may also be formed [5, 6 ]. cladosporium sp., aspergillus niger, a. fumigatus, botrytis sp., and aureobasidium pullulans. rhizopus and mucor are also associated with spoilage of fresh fruits and vegetables. in the present study, the most frequently encountered moulds were aspergillus flavus, a. terreus, and penicillium islandicum (table 5). p. digitatum, colletotrichum, and curvularia were isolated from orange and sweet orange juices. spoilage by moulds in fruit juices is characterized by loss of juice cloud. among these, major mycotoxins associated with fruit juices are byssochlamic acid (byssochlamys fulva, b. nivea), patulin (b. fulva, b. nivea, and p. expansum), ochratoxin (aspergillus carbonarius), and citrinin (penicillium expansum, p. citrinum) [29, 30 ]. juices squeezed from fresh fruits and vegetables contain microorganisms which are potentially hazardous to public health. juices were spoiled with high level of moulds and yeasts which is attributable to low ph of juices. the selling and consumption of juices are never stopped on nutritional grounds as well as livelihood of street vendors. it is alarming situation for suitable agency to take some necessary action, make guidelines to prevent potential food poisoning from juices that contain pathogenic bacteria, and find natural antimicrobials from plants that control spoilage and pathogenic microorganisms in juices.	fruit juices are popular drinks as they contain antioxidants, vitamins, and minerals that are essential for human being and play important role in the prevention of heart diseases, cancer, and diabetes. they contain essential nutrients which support the growth of acid tolerant bacteria, yeasts, and moulds. in the present study, we have conducted a microbiological examination of freshly prepared juices (sweet lime, orange, and carrot) by serial dilution agar plate technique. a total of 30 juice samples were examined for their microbiological quality. twenty - five microbial species including 9 bacterial isolates, 5 yeast isolates, and 11 mould isolates were isolated from juices. yeasts and moulds were the main cause of spoilage of juices. aspergillus flavus and rhodotorula mucilaginosa were observed in the maximum number of juice samples. among bacteria bacillus cereus and serratia were dominant. escherichia coli and staphylococcus aureus were detected in few samples. candida sp., curvularia, colletotrichum, and acetobacter were observed only in citrus juice samples. alternaria, aspergillus terreus, a. niger, cladosporium, and fusarium were also observed in tested juice samples. some of the microorganisms detected in these juice samples can cause disease in human beings, so there is need for some guidelines that can improve the quality of fruit juices.
multiple myeloma (mm) is a hematologic malignancy characterized by the accumulation of monoclonal plasma cells in the bone marrow (bm), over 10% by definition. in almost all cases, mm is preceded by a premalignant disease well known as monoclonal gammopathy of undetermined significance (mgus) [2, 3 ]. mgus affects 2% of the population above the age of 50 and it progresses to overt mm at a rate of 1% per year. the biologic transition from normal plasma cells to mgus and smm to mm consists of many oncogenic events.an early event described in mgus as well as secondary translocations, sometimes involving an ig locus, can occur at any stage of plasma cell dyscrasia. activating mutations of nras and kras constitutive activation of the nuclear factor b (nfb) pathway is mediated by mutations in some tumors during progression.in addition to these oncogenic events, the tumor cells are strongly dependent on the bone marrow microenvironment. substantial advances have been made in understanding the biology of mm through the study of the bm microenvironment. indeed, the bm niche appears to play an important role in differentiation, migration, proliferation, survival, and drug resistance of the malignant plasma cells providing the preclinical evidences for targeting mm cells and bone marrow stromal cells (bmsc) as an antitumor strategy in this disease. the cellular compartment is composed of hematopoietic cells and nonhematopoietic cells including fibroblasts / bmsc, endothelial cells (ecs), osteoclasts, and osteoblasts. the non - cellular compartment is composed of the extracellular matrix (ecm) and the liquid milieu including cytokines, growth factors, and chemokines. however the new host microenvironment is not well adapted to the cancer cells that metastasized into it, leading to the new concept of premetastatic niche. indeed significant changes occur in the microenvironment even before the first tumor cell homes to the bone marrow, as already described in solid tumor models. in this paper tumor cells bind to ecm proteins, such as type i collagen and fibronectin via syndecan 1 and very late antigen 4 (vla-4) on mm cells and to bmsc vcam-1 via vla-4 on mm cells. adhesion of tumor cells to bmsc activates many pathways resulting in upregulation of cell cycle regulating proteins and antiapoptotic proteins. specifically, the interaction between mm cells and bmscs triggers nf-b signaling pathway and interleukin-6 (il-6) secretion in bmscs. in turn the existence of this paracrine loop optimizes the bm milieu for mm tumor cell growth. the interaction notch - notch ligand leads to activating notch - signaling pathways both in mm cells as well as in bmsc, with induction of il-6, vascular endothelial growth factor (vegf), and insulin - like growth factor (igf-1) secretion and is associated with mm cell proliferation and survival [11, 12 ]. moreover, bmsc from mm patients expresses several proangiogenic molecules, such as vegf, basic - fibroblast growth factor (bfgf), angiopoietin 1 (ang-1), transforming growth factor (tgf)-, platelet - derived growth factor (pdgf), hepatocyte growth factor (hgf), interleukin-1 (il-1). recently, bmscs from mm patients have been shown to release exosomes, which are transferred to mm cells, thereby resulting in modulation of tumor growth in vivo, mediated by specific mirna. this finding suggest that exosomes might constitute a novel mechanism for intercellular transfer of genetic information in the form of mirna in clonal plasma cell disorders. bm angiogenesis represents a constant hallmark of mm progression, partly driven by release of pro - angiogenic cytokines from the tumor plasma cells, bmsc, and osteoclasts, such as vegf, bfgf, and metalloproteinases (mmps). indeed, the adhesion between mm cells and bmscs upregulates many cytokines with angiogenic activity, most notably vegf and bfgf. in mm cells, these pro - angiogenic factors may also be produced constitutively as a result of oncogene activation and/or genetic mutations. evidence for the importance of angiogenesis in the pathogenesis of mm was obtained from bm samples from mm patients. the level of bm angiogenesis, as assessed by grading and/or microvessel density (mvd), is consistently increased in patients with active mm as compared to those with inactive disease or mgus, a less advanced plasma cell disorder. comparative gene expression profiling of multiple myeloma endothelial cells and mgus endothelial cells has been performed in order to determine a genetic signature and to identify vascular mechanisms governing the malignant progression. twenty - two genes were found differentially expressed at relatively high stringency in mm endothelial cells compared with mgus endothelial cells. functional annotation revealed a role of these genes in the regulation of ecm formation and bone remodelling, cell adhesion, chemotaxis, angiogenesis, resistance to apoptosis, and cell - cycle regulation. the distinct endothelial cell gene expression profiles and vascular phenotypes detected in this study may influence remodelling of the bone marrow microenvironment in patients with active multiple myeloma. overall, these evidences suggest that ec presents with functional, genetic, and morphologic features indicating their ability to induce bm neovascularization, resulting in mm cells growth, and disease progression, providing preclinical evidences for using antiangiogenic compounds in the treatment of mm. the usual balance between bone resorption and new bone formation is lost in many cases of mm, resulting in bone destruction and the development of osteolytic lesions. bone destruction develops adjacent to mm cells, yet not in areas of normal bone marrow. there are several factors implicated in osteoclast activation, including receptor activator of nf-b ligand (rankl), macrophage inflammatory protein-1a (mip-1a), interleukin-3 (il-3), and il-6. rank ligand is a member of the tumor necrosis factor (tnf) family and plays a major role in the increased osteoclastogenesis implicated in mm bone disease. mm cell binding to neighboring bmsc within the bone marrow results in increased rankl expression. this leads to an increase in osteoclast activity through the binding of rankl to its receptor, on osteoclast precursor cells, which further promotes their differentiation through nf-b and junn - terminal kinase pathway. blocking rankl with soluble form of rank has been shown to modulate not only bone loss but also tumor burden in mm in vivo models. it has been reported that osteoblasts may contribute to mm pathogenesis by supporting mm cells growth and survival. this could potentially result from the ability of osteoblasts to secrete il-6 in coculture system with mm cells, thus increasing il-6 levels within the bm milieu and therefore inducing mm plasma cells growth. other mechanisms include the possible role of osteoblasts in stimulating mm cells survival by blocking trail - mediated programmed mm cell death, by secreting osteoprotegerin (opg), a receptor for both rankl and trail. in addition, it is clear that suppression of osteoblast activity is responsible for both bone destructive process and progression of myeloma tumor burden. dkk1 is a wnt - signaling antagonist secreted by mm cells and it inhibits osteoblast differentiation. myeloma - derived dkk1 also disrupts wnt - regulated opg and rankl production by osteoblasts. studies have shown that blocking dkk1 and activating wnt signaling prevents bone disease in mm but is also associated with a reduction in tumor burden [2729 ]. il-6 is primarily produced by bmsc and osteoblasts and mediates paracrine mm cell growth and is also secreted by mm tumor cells in an autocrine manner. il-6 secretion from bmsc is upregulated by many molecules / cytokines (i.e., cd40, tnf-, vegf, il-1, tgf-) and mm cell adherence. il-1 appears to be one of the major cytokines responsible for the paracrine production of il-6 by the bmsc. the aberrant production of il-1 by the mm cells induces il-6 production by bmsc, which in turn supports the growth and survival of the myeloma cells. importantly, nf-b plays a central role in cytokine- and adhesion - mediated il-6 upregulation, and specific inhibition of nf-b blocks il-6 secretion. after binding with its receptor, il-6 triggers activation of mek / mapk, jak / stat3, and pi3k / akt signaling pathways. il-6 induces proliferation of the tumor plasma cells by activating the ras / raf / mek - erk signaling pathway. il-6 is also able to inhibit the antiproliferation effects of cyclin - dependent kinase (cdk) inhibitors p21 and p27 through the pi3k / akt pathway. il-6 activation of the jak / stat3 pathway induces tumor cells survival by up - regulation / activation of anti - apoptotic proteins mcl-1 and bcl - xl and c - myc. clinically, elevated serum il-6 levels are associated with a poor prognosis and reflect the proliferation fraction of mm cells within patients. otherwise il-1 receptor targeted therapies have shown activity in increasing pfs of patients with smoldering disease. insulin - like growth factor 1 (igf-1) is involved in tumorigenesis of several solid cancers. in mm, igf-1 is secreted by the bmsc and osteoblasts and induces growth, survival, and migration of mm cells. the phosphorylation of igf-1 receptor (igf-1r), after igf-1 binding, leads to activation of mapk and pi3k / akt signaling pathway. activation of akt leads to activating the anti - apoptotic proteins bcl - xl, bcl-2 and downregulating the proapoptotic protein bim, thereby promoting cell survival. interestingly, igf-1 could be involved in the pathophysiology that relates obesity and diabetes to neoplasia. vegf represents a well - known proangiogenic factor : its levels increase in several hematologic malignancies including mm. in mm, vegf is produced both by mm cells and bmsc, and its secretion is stimulated by different cytokines and cell growth factors such as il-6, bfgf, tgf- or tumor necrosis factor (tnf)-. it is an important factor in the formation of new vasculature ; upon binding with vegf receptor-1 and -2 (vegfr-1 and vegfr-2), it triggers proliferation, migration, differentiation, and survival of bmsc and ec, through several signaling pathway such as ras gap, fak, pi3k / akt, mek / erk, and stat. blood vessels are required for tumor growth and progression for provision of vital oxygen and nutrients. it has been shown that increased microvessel density (mvd) in bm of mm patients is associated with poor prognosis. the initiation of mm is likely from long - lived plasma cells that develop in germinal center of lymphoid tissues and home to the bm. oncogenic events along with support of the microenvironment allow the growth, survival, and proliferation of these cells in the initial sites of the bm niche. furthermore, some studies showed the presence of a small number of circulating plasma cells in mm and its association with a poor prognosis. migration of cells through the blood to the bone marrow niches requires active navigation, a process termed homing. the first step in the homing process is the rolling of the mm cell along the ec through selectin. the adhesion and extravasation are induced by activation of integrin expressed by mm cell such as lfa-1 and vla-4. the sdf-1/cxcr4 axis plays a critical role in homing of mm cells to the bm. studies to identify expression of chemokine receptors in mm have shown large variations in cxcr4 expression ranging from 10 to 100%. sdf-1 induces migration of mm cells in vitro and homing into the bone marrow in vivo. moreover, cxcr4 knockdown led to significant inhibition of migration to sdf-1 in mm cell lines and primary cd138 + cells. mm is characterized by the disseminated involvement of the bm, and its progression involves a continuous circulation of the mm cells in the peripheral blood and homing back to the bm. mobilization or egress of cells out of the bone marrow could be enhanced by disrupting the sdf-1/cxcr4 axis. this may occur by decreasing sdf-1 by protease in the bm milieu, or by upregulation of cxcr4 expression by hypoxia. it has also been shown that hypoxia leads to inactivation of e - cadherin and activation of transcription factors regulating epithelial - mesenchymal transition, including snail and twist, indicating that this mechanism can participate to the egress process. although preparation of the premetastatic niche has not been studied in mm, several works have shown the importance of the premetastatic niche in solid cancer metastasis to the bone marrow. indeed, the new host microenvironment is not well adapted to the cancer cells that metastasized into it. therefore, significant changes in the stroma, endothelial cells, ecm constituents, cytokines, and chemokines need to occur to allow for the growth and survival of these metastastic cells. preparation of the metastatic niche occurs even before the first metastastic cell arrives. evidence has emerged that growth factors and cytokines secreted by the tumor prepare tissues for tumor cell engraftment. for example, bone marrow - derived hematopoietic cells that express vegfr1 as the fibronectin receptor vla-4 are localized to the premetastatic sites before the arrival of tumor cells. moreover, microvesicles such as exosomes have been shown to alter the premetastatic niche in different studies. exosomes are small vesicles (30100 nm) of endocytic origin, which are released in the extracellular milieu by several cell types. recently, melanoma - derived exosomes have been shown to induce neoangiogenesis at pre - metastatic niche sites. rab1a, rab5b, rab7, and rab27a, regulators of membrane trafficking and exosome formation, are highly expressed in melanomacells. rab27a rna interference decreased exosome production, preventing bone marrow education and reducing, tumor growth and metastasis. these data show that exosome production, transfer and education of bone marrow cells support tumor growth and progression to the bone marrow. several reports have clearly indicated that mm pathophysiology is supported by a strong interaction between the clonal plasma cells and the surrounding bone marrow microenvironment ; indeed, there are several autocrine or paracrine circuits of growth that support the transformation from an mgus stage to an active mm stage. by understanding the interaction occurring between bmsc and mm cells, and vice versa, we have now available the preclinical rational for testing novel therapeutical approaches in order to better target not only the mm cell clone, but also the bm milieu, thus preventing mm disease progression.	substantial advances have been made in understanding the biology of multiple myeloma (mm) through the study of the bone marrow (bm) microenvironment. indeed, the bm niche appears to play an important role in differentiation, migration, proliferation, survival, and drug resistance of the malignant plasma cells. the bm niche is composed of a cellular compartment (stromal cells, osteoblasts, osteoclasts, endothelial cells, and immune cells) and a noncellular compartment including the extracellular matrix (ecm) and the liquid milieu (cytokines, growth factors, and chemokines). in this paper we discuss how the interaction between the malignant plasma cell and the bm microenvironment allowed myeloma progression through cell homing and the new concept of premetastatic niche.
pemphigus vulgaris is a potentially life - threatening disease that causes blisters and erosions of the skin and mucous membranes. the immune reaction against these autoantibodies primarily affects the mucous membranes of patients over the age of 50 years, resulting in the formation of intraepithelial bullae and mucosal ulceration. these autoantibodies, usually of the igg class, are directed against desmosal glycoproteins present on the cell surface of the keratinocyte. the specific protein target has been identified as desmoglein 3, one of the several proteins in the desmosomal cadherine family. the first sign of the disease appears in the oral mucosa in approximately 60% of cases and eighty to ninety percent of patients with pemphigus vulgaris develop oral lesions sometime during the course of the disease. it is common for the oral lesions to be present up to 4 months before the skin lesions appear. if treatment is instituted during this time, the disease is easier to control, and the chance for an early remission of the disorder is enhanced. the lesions of pemphigus vulgaris may involve any mucosal surface, but they most frequently involve the oral mucosa. most commonly the lesions start on the buccal mucosa, often in areas of trauma along the occlusal plane. the management of a patient with pemphigus is complicated by the chronic nature of the disease and the fact that affected individuals tend to be elderly and have multiple medical problems. in addition several forms of therapy have been advocated for the management of oral lesions of pemphigus vulgaris. when the lesions are confined to the oral mucosa, systemic corticosteroids will suppress their formation, but the clinician must weigh the benefits against the hazard from side effects of the durg. pemphigus is a fatal disease, and long - term use of steroids for this purpose must be carefully evaluated, particularly because most cases are chronic, most patients are elderly, and treatment is required for a long period of time. when steroids must be used for long periods of time, adjuvants such as immunosuppressive drugs, antimetabolites and dapsone are added to the regimen to reduce the complications of long - term corticosteroid therapy. prednisone is used initially to bring the disease under control, so the clinicians should weigh the potential benefits of adding adjuvant therapy against the risks of additional complications such as blood dyscrasia and hepatitis. it is effective in a variety of infectious diseases, including malaria and leprosy. dapsone also has anti - inflammatory properties.in this capacity, it has been used as a corticosteroid sparing drug to treat a number of diseases, including bullous pemphigoid, rheumatoid arthritis, and dermatitis herpetiformis. in the previous studies, dapsone has shown promise in treating conjunctival, oral, and laryngeal lesions of mucous membrane pemphigoid and pemphigus.5 - 9 the purpose of this study was to investigate the efficacy of dapsone, when used in combination with systemic corticosteroids, to treat the oral lesions of pemphigus vulgaris. twenty patients referring to the department of oral medicine, university of jundishapur ahwaz, iran, from 2002 to 2005, subsequently diagnosed with pemphigus vulgaris were selected. each patient included in this study fulfilled the following criteria : (1) the patient had oral mucosal ulcers, vesicles, orbulla ; (2) a biopsy specimen was obtained for routine histologic analysis and demonstrated intraepithelial cleavage characteristic of pemphigus vulgaris observed by oral pathologist. the clinical oral features and site involvement at the time of initial presentation were recorded. these features were then graded according to severity of the disease based on the classification used by rogers & mehregan in 1988. severity of each lesion was assigned a grade from 1 to 3 ; 1 representing mild, localized disease ; 2 representing generalized desquamative gingivitis only ; and 3 representing generalized severe disease (involvement of multiple sites). all patients were treated initially with systemic corticosteroids (prednisone 40 mg / day) according to ciarrocca & greenberg. when a patient had more than 50% healing of lesions, systemic therapy was reduced and tapered for another 4 weeks, after which the patient was re - evaluated. those who responded minimally less than 50% healing of lesions to corticosteroid treatment began a trial of dapsone after glucose 6-phosphate dehydrogenase (g6pd) screen and baseline hemoglobin were taken. we began dapsone therapy at a low dosage gradually increasing it at 3-day intervals as outlined by rogers & mehregan. treatment lasted for 4 weeks : 25 mg daily for 7 days, 25 mg twice daily for 7 days, 25 mg 3 times daily for 7 days, and 50 mg twice daily for 7 days. each patient, regardless of treatment modality, was assigned to one of the following 4 groups according to his or her response to therapy : complete resolution, substantial resolution (more than 75% improvement), partial resolution (more than 50% improvement), and no noticeable change. resolution was defined as complete clearance of lesions and absence of clinical disease in oral cavity. at least two investigators determined the response to therapy at each visit.6, 7 a complete blood count as well as alanine aminotransferase (alt) and aspartate aminotransferase (ast) tests were obtained at each visit preceding the initiation of dapsone therapy and then repeated at each of subsequent follow - ups. if no new lesions developed, the taper was continued ; however, if the patient developed new ulcerations, the dosage was increased by 25 mg / day at weekly intervals until the patient was once again lesion - free. after tapering the dose of dapsone for 4 weeks and the 3-month follow - up, no new lesion was seen ; therefore, addition of dapsone to the treatment regime was not continued. a total of 20 patients including 15 women and 5 men (34 to 68 years old, mean age 52.6 years) were studied. 15 patients showed desquamative gingivitis, 18 had buccal mucosal lesions, 9 had palatal lesions, 14 had lip lesions, and 13 had tongue and floor of mouth lesions. initial therapy for each of the 20 patients consisted of the use of systemic corticosteroids. five patients with mild to moderate disease were treated with systemic corticosteroids alone (prednisone 40 mg / day) for the duration of the study ; 1 of these 5 patients had complete resolution, 1 had more than 75% improvement, and 3 had more than 50% improvement. 15 patients with moderate to severe disease were treated initially with systemic corticosteroids, and since they had less than 50% improvement, dapsone therapy was then added to the regimen. 10 patients had significant benefits : 7 of 15 patients had complete resolution and 3 had more than 75% improvement. the remaining 5 patients who had less than 50% improvement continued with conventional treatment and were referred to a hospital center. an immunosuppressive agent has also been used in some patients as a primary agent or as a supplement to systemic corticosteroids. dapsone, originally an anti - leprosy agent, has become a valuable therapeutic agent in many inflammatory diseases and is currently being used as the first line therapy for the treatment of dermatitis herpetiform (dh) and ocular pemphigoid. it has also been found to be an effective adjunct to topical or systemic corticosteroid therapy in treatment of some vesiculobullous conditions, including mucous membrane pemphigoid (mmp) and pemphigus. the results of our study was in agreement with some of the previous studies.5, 12, 13 lpez - jornet & bermejo - fenoll found that dapsone could decrease the signs and symptoms of pemphigus patients. stoopler stated that corticosteroid therapy is the best method for treatment of mmp and pemphigus and that immunosuppressive agents and dapsone can not be effective in resolving ulcerative lesions of pemphigoid and pemphigus. the reason of this difference could be intolerance of patients in that research to dapsone. dapsone has been used for nearly 50 years to treat infectious diseases such as leprosy and malaria, and its antimicrobial effects involves the inhibition of the conversion of para - aminobenzoic acid to folic acid. more recently, however, dapsone has been used to treat noninfectious inflammatory diseases characterized by neutrophil - rich infiltrates, though the mechanism of its anti - inflammatory action remains unclear. dapsone appears to inhibit the migration of polymorphonuclear leukocytes (pmns) by inhibiting lysosomal enzyme activity, interfering with the leukocyte cytotoxic system, or preventing the cells from responding to chemotactic stimuli. clues to the mechanism of the anti - inflammatory action of dapsone are available from studies of dh, a skin disease caused by sensitivity to the protein gluten. the skin lesions are characterized by patches of small, itchy blisters, usually found on the arms and back.14, 15 dapsone as also been shown to suppress neutrophil migration by blocking integrin - mediated adherence function. for neutrophils to be directed to an extravascular lesion, they must first adhere to vascular endothelial and then penetrate the vessel wall. on the basis of these theories, the effectiveness of dapsone in the treatment of mmp and pemphigus is better understood by reviewing the pathogenesis of the disease. the pathogenesis of the intraepithelial lesion of pemphigus is believed to be an intricate cascade of events characterized initially by antigen / antibody complexes at the intraepithelial zone. this complexity is followed by complement activation and elaboration of chemotactic factors, which attract pmns to the area. our study showed that systemic corticosteroid and dapsone can decrease signs and symptoms of pemphigus vulgaris.	background and aims oral pemphigus vulgaris is a chronic autoimmune mucocutaneous intraepi - thelial disease that primarily affects patients over the age of fifty, resulting in mucosal ulceration and is a potentially life - threatening disease. the purpose of this study was to investigate the efficacy of dap - sone in combination with systemic corticosteroids to treat the oral lesions of oral pemphigus. materials and methods twenty patients diagnosed with oral pemphigus were selected. oral mani - festations were graded according to the severity of disease from 1 to 3. all patients were treated initially with systemic corticosteroids. each was assigned to one of 4 groups according to their response to ther - apy. patients who responded less than 50% healing of lesions began a trial of dapsone. after 4 weeks, signs and symptoms were recorded, and if a patient was lesion - free, the dapsone dosage was gradually tapered. results five patients with mild to moderate disease were treated with systemic corticosteroids alone. 15 patients with moderate to severe disease were treated with systemic corticosteroid and dapsone ther - apy. of these, 10 patients had significant benefits, while 5 patients did not respond to dapsone adjuvant. conclusions. the use of dapsone in combination with systemic corticosteroids is a useful method for treatment of oral pemphigus. conclusions the use of dapsone in combination with systemic corticosteroids is a useful method for treatment of oral pemphigus.
this study was approved by the institutional review boards at our hospitals, and an informed consent in respect to the pft and cardiac mdct was obtained from all the patients. the data were collected prospectively. from june 2004 to april 2005, 33 patients with copd underwent the pft and cardiac mdct examinations. the group of the patients consisted of 29 men and four women, aged from 50 to 71 years (mean age, 62 years). the diagnosis of copd was made by the clinical examination, chest radiographs and lung function parameters based on the global initiative for chronic obstructive lung disease (gold) workshop report (11). the following patients were excluded from our study : patients who had severe primary cardiac disease, diffuse or focal parenchymal abnormalities affecting more than one segment, and pleural effusions. pulmonary function test was performed in a dedicated pulmonary function laboratory, under the supervision of a certified pulmonary technologist, before the cardiac mdct examinations. for all the patients, the interval between the pft and cardiac mdct examinations was less than one week. the pft indexes were measured using a spirometer (vmax 229, sensormedics, yorda linda, ca). the pft parameters included the vital capacity (vc), the forced expiratory volume in 1 sec (fev1), the ratio of the forced expiratory volume in 1 sec to the forced vital capacity (fev1/fvc) and the diffusing capacity of the lung for carbon monoxide (dlco). the values of vc, fev1, fev1/fvc and dlco were expressed as the percentages of the predicted values (percentage of predicted). all the patients were divided into three groups, according to the disease severity, based on the gold classifications (11) ; stage i (mild, fev1/fvc 0.8), stage ii (moderate, fev1/fvc 0.8), stage ii (moderate, fev1/fvc 0.05). in concordance with this, the rvef was significantly lower in stage iii than in stage i and ii (p 0.05). correlation was excellent between the mdct results and the fev1 (r = 0.797 for rvef and r = -0.769 for rv mass) (fig. 3) and fev1/fvc (r = 0.745 for rvef and r = -0.718 for rv mass) (fig. the interobserver agreement was good, with a correlation coefficient of 0.907 for rvef and a correlation coefficient of 0.894 for rv wall mass. there were no significant differences between the rvef and rv mass (p = 0.74 for rvef, and p = 0.71 for rv mass, respectively) (table 3). this study shows that the mean rv wall mass and ef, as measured by mdct scan, correlate with the copd disease severity. it is known that rv hypertrophy occurs in numerous disorders and conditions, including valvular disease, lung disease, and chronic left - sided heart failure (12, 13). chronic hypoxemia, which is not unusual in patients with copd, leads to hypoxic pulmonary vasoconstriction and later to structural changes in the pulmonary vasculature. thus it contributes to the development of pulmonary arterial hypertension and causes an increased rv afterload (4). rv hypertrophy may results from a response to this rv pressure overload (14, 15). in a recent study by vonk - noordegraaf. (16), rv hypertrophy is described as the earliest sign of the rv pressure overload in copd patients with mild hypoxemia. however, rvef is well preserved in most patients with advanced emphysema during rest (4, 17 - 19). (17) have described that rvef is not decreased in copd patients, and it is well preserved in most stable copd patients. (19) also demonstrated that in patients with stable copd, despite the presence of pulmonary hypertension, the rvef remained relatively normal. in our study, the rvef between stage i (mild copd patients) and stage ii (moderate copd patients) was not significantly different (473% vs 466%, p > 0.05), whereas it was significantly lower in stage iii (severe copd patients). these results may reflect that with a persistent rv pressure overload, the compensated rv function falls, which eventually results in rv failure (4). in addition, it has been strongly suggested that even in a hypertrophied rv, the ef is preserved until relatively late in the course of copd (19, 20). however, if rv hypertrophy progresses and the rvedv decreases, the compensated rv function can eventually fall. in our study, the rv mass was significantly different among the three stages of the disease severity, and it gradually increased with preservation of rvef in stage i and stage ii (mild and moderate copd patients) ; however, in stage iii (severe copd patients), the rvef fell, although the rv mass increased. several imaging modalities, including echocardiograph, angio - cardiography and radionuclide imaging, have all been used to evaluate rvef and the mass ; however, the success of these techniques is limited. because of the complex shape and substernal location of the rv, it is difficult to estimate the rv function and mass with most of the imaging techniques (21). magnetic resonance imaging (mri) has recently been introduced for more accurate measurements of the rv function and the wall mass in copd patients (17, 18). (17) used mri to evaluate the rv function and the wall mass in 17 patients with moderate copd. they concluded that detection of rv myocardial hypertrophy with mri may aid in the early diagnosis of cor pulmonale that complicates copd. the advantages of cardiac mri as compared with mdct are the lack of radiation exposure, avoidance of iodinated contrast media and an improved temporal resolution in the evaluation of the rv volumes and the mass. furthermore, short - axis images are readily available, and the secondary reformations required in cardiac mdct are not needed with cardiac mri. however, cardiac mri can not evaluate lung parenchyma, and it is difficult to perform mri in patients with dyspnea and heart failure due to a much longer examination time. the recently introduced mdct scanners have demonstrated their ability to assess the rv function and the wall mass (9, 10). koch. (9) demonstrated that the mean rvedv, rvesv and rvsv determined with mpr by 16-detector - row ct correlated well with those parameters measured by cardiac mri. however, to our knowledge, there have been no studies that have evaluated the rv function with using mdct in copd patients. although cardiac mdct can be used as a modality for evaluating the rv volume, function and the wall mass in copd patients, it has certain limitations in routine use. cardiac mdct requires contrast media to discriminate the ventricular cavity, and its temporal resolution is inferior to either electron beam ct or mri (22). (23) have reported that a temporal resolution of about 20 milliseconds is needed to completely avoid any motion artifacts in cardiac imaging by ct. thus, the limited temporal resolution used in our study may have been insufficient to acquire a precise end systolic volume. furthermore, in copd patients, dyspnea often makes it difficult to perform breath - holding more than 10 seconds long, and the stair step artifact can affect evaluation of the ventricular function. it is hoped that with technological developments, the image quality will further improve and the imaging time will be shortened. the total radiation dose was calculated to be approximately 4 to 6 msv, depending on the scan range and the patient 's body weight. first, the study population was too small to generalize the results, especially in respect of the stage i patients (mild copd patients). second, we did not include normal control subjects for correlation of the rv function. third, the mdct results were not compared with the cardiac mri results, which is the accepted gold standard modality. cardiac mdct has a potential to become a useful modality for assessing the rv function and rv myocardial hypertrophy in copd patients. our results show that the mean rv wall mass measured by cardiac mdct correlates well with the disease severity of copd as determined by pft, and that the mean rvef is significantly lower in patients with severe copd than it is in those patients with mild or moderate copd.	objectivewe wanted to assess the relationship between measurements of the right ventricular (rv) function and mass, with using cardiac multi - detector computed tomography (mdct) and the severity of chronic obstructive pulmonary disease (copd) as determined by the pulmonary function test (pft).materials and methodsmeasurements of pft and cardiac mdct were obtained in 33 copd patients. using the global initiative for chronic obstructive lung disease (gold) classification, the patients were divided into three groups according to the severity of the disease : stage i (mild, n = 4), stage ii (moderate, n = 15) and stage iii (severe, n = 14). the rv function and the wall mass were obtained by cardiac mdct. the results were compared among the groups using the student - newman - keuls method. pearson 's correlation was used to evaluate the relationship between the right ventricular ejection fraction (rvef) and the wall mass results with the pft results. p - values less than 0.05 were considered statistically significant.resultsthe rvef and mass were 473% and 412 g in stage i, 466% and 465 g in stage ii, and 355% and 556 g in stage iii, respectively. the rvef was significantly lower in stage iii than in stage i and ii (p < 0.01). the rv mass was significantly different among the three stages, according to the disease severity of copd (p < 0.05). the correlation was excellent between the mdct results and forced expiratory volume in 1 sec (r = 0.797 for rvef and r = -0.769 for rv mass) and forced expiratory volume in 1 sec to the forced vital capacity (r = 0.745 for rvef and r = -0.718 for rv mass).conclusionour study shows that the mean rv wall mass as measured by cardiac mdct correlates well with the copd disease severity as determined by pft.
population structure was assessed by examining the principal components (pc) of the variance of the genotype profiles, using eigenstrat software6. initial examination revealed several clusters of siblings and other close relatives (cousins or similar) whose similarity skewed the axes ; where data were available, these identities were in agreement with participant records. after removal of these relatives, analysis of 163 unrelated individuals revealed seven significant eigenvectors. none of these explain more than 5% of the variance, and pc3 through pc7 are heavily weighted by large clusters of snps on one or a few chromosomes. as described by others, such axes are commonly observed and do not provide reliable genome - wide estimates of population structure7,8, but it is interesting to note that pc3 distinguishes ighrem from the other locations (supplementary fig. pc1 separates just a dozen individuals, and we inferred that this axis represents a sub - saharan african contribution, consistent with expected levels of admixture in morocco, by performing an analysis including 21 yoruban individuals (supplementary fig. pc2 is highly correlated with both location and self - reported ethnicity, so is inferred to capture the major component of arab - amazigh ancestry. a surprising aspect of this analysis is the positioning of ighrem arabs between boutroch amazigh and half of the agadir arabs along pc2. this was confirmed by structure analysis9 of 16,000 randomly chosen autosomal snps assuming admixture of two ancestral populations (fig. 2b), which indicates that ighrem residents tend to be a mixture, while most amazigh are derived from one population, and only a handful of agadir arabs represent the other. there has thus likely been considerable admixture between these two groups over an extended period of time, possibly with movement of arabs from other locations into agadir recently. a slight shift of ighrem arabs toward the amazigh pole of pc2, relative to agadir arabs, would also be consistent with some genetic exchange between the villages over 50 generations. further sampling of villages in the region may reveal subtle population structure across southern morocco1013. next we asked whether region, location and ethnicity impact gene expression profiles, and if they do so in a gender - specific manner. since location and ethnicity are confounded in the villages, several parallel analyses were undertaken to tease apart these influences. transcript abundance data was transformed by median centering on the log base 2 scale (supplementary fig. 2 online), which results in maximal overlap of profiles without altering their variance. gene specific analysis of variance14 with expression as a function of region, gender, and their interaction discovered 1,521 probes significant at a 1% false discovery rate (fdr ; p 0.95) between both of these models and the original correlation test. furthermore, figure 5d shows that there is no evidence for significant genotype - by - location interactions in any of the association trend tests. neither the ethnicity nor the relatedness variance components explain an appreciable amount of the expression variation for any of the transcripts (supplementary fig. 8 online). the absence of interaction effects is readily visualized by plotting expression as a function of genotype with color coding of each location, for each association. an example of a trans association in supplementary figure 9 online shows the clear trend of increased expression of amy1a (chromosome 1) in homozygotes for the a allele of actg1 gamma actin (chromosome 17), consistently across the three locations despite slight overall location effects. expression of amy1a is highly correlated with that of amy1b (r > 0.8) as well as of dozens of other genes in a co - expression module, but the esnp only regulates amy1a, because it increases expression of the gene two - fold in an additive manner. a similar plot for a representative gene that shows highly significant location and genotype effects in cis, c21orf57, is provided in figure 6a and further discussed below, and further examples can be seen in supplementary figure 9c online. gwas - expression associations detected in one tissue can identify regulatory variants that may be active in other tissues that are directly engaged in the etiology of disease23,25,26. one example is the cis - linkages in peripheral blood with the t1d susceptibility locus at chromosome 12q13. the strongest expression association is with transcription of the rps26 ribosomal protein gene, and network analyses have been employed to argue that this is the more likely diabetes candidate gene than the initially reported erbb328. however, the strongest t1d association involves a different snp than that associated with expression and/or splicing24 of rps26. we further find that the same linkage group of esnps, centered on the rs10876864 in the suox gene 35 kb from rps26, is also associated in trans with half a dozen other rp26 paralogs (probably due to cross - hybridization), and with ccdc4 on chromosome 4, albeit at the suggestive significance level ofp = 3.510. intriguingly, expression of rps26 is only weakly correlated with that of the module of ribosomal proteins that differentiate locations (supplementary fig. 4b online), so this association does not contribute to the environmental effect on transcription of ribosomal protein genes. another trans - association of interest involves rs11987927 in myom2 at 8p23 with znf71 at 19q13, but also with its own myom2 transcript. logic suggests that the cis - association likely affects the abundance of the myom2 myomesin protein, which in turn regulates znf71, but the trans association is actually significantly stronger and conditional dependence analysis29,30 points in the opposite direction, namely that the myom2 regulatory site influences znf71, which then feeds back on the myom2 transcript (supplementary fig. it is worth mentioning that four of the seven strongest trans associations involve regulation by loci that include genes that encode structural proteins, the others being the lama5 laminin (20q13) with osbpl2, and the plekhm1 plekstrin homology domain protein (17q21) with mapk8ip1. we found association of two probes that detect both hbg1 and hbg2 transcripts from 11p15 with rs766432 in the second intron of the bcl11a zinc finger proto - oncogene at 2p16. this same snp has previously been associated with the fraction of erythrocytes that contain measurable fetal hemoglobin31, and alteration of bcl11a activity was recently shown drive differences in globin switching between mice and humans32. another snp in bcl11a, rs4671393 has been associated with abundance of two bcl11a transcript isoforms in ceu and yri hapmap lymphoblast cell lines33, but is not associated with bcl11a transcript abundance in our leukocyte data, suggesting that regulation of bcl11a translation or protein activity is more likely to be affecting hbg expression in our sample. we scanned the gwas association database for overlap between our study and established disease associations at p 0.95) between both of these models and the original correlation test. furthermore, figure 5d shows that there is no evidence for significant genotype - by - location interactions in any of the association trend tests. neither the ethnicity nor the relatedness variance components explain an appreciable amount of the expression variation for any of the transcripts (supplementary fig. 8 online). the absence of interaction effects is readily visualized by plotting expression as a function of genotype with color coding of each location, for each association. an example of a trans association in supplementary figure 9 online shows the clear trend of increased expression of amy1a (chromosome 1) in homozygotes for the a allele of actg1 gamma actin (chromosome 17), consistently across the three locations despite slight overall location effects. expression of amy1a is highly correlated with that of amy1b (r > 0.8) as well as of dozens of other genes in a co - expression module, but the esnp only regulates amy1a, because it increases expression of the gene two - fold in an additive manner. a similar plot for a representative gene that shows highly significant location and genotype effects in cis, c21orf57, is provided in figure 6a and further discussed below, and further examples can be seen in supplementary figure 9c online. gwas - expression associations detected in one tissue can identify regulatory variants that may be active in other tissues that are directly engaged in the etiology of disease23,25,26. one example is the cis - linkages in peripheral blood with the t1d susceptibility locus at chromosome 12q13. the strongest expression association is with transcription of the rps26 ribosomal protein gene, and network analyses have been employed to argue that this is the more likely diabetes candidate gene than the initially reported erbb328. however, the strongest t1d association involves a different snp than that associated with expression and/or splicing24 of rps26. we further find that the same linkage group of esnps, centered on the rs10876864 in the suox gene 35 kb from rps26, is also associated in trans with half a dozen other rp26 paralogs (probably due to cross - hybridization), and with ccdc4 on chromosome 4, albeit at the suggestive significance level ofp = 3.510. intriguingly, expression of rps26 is only weakly correlated with that of the module of ribosomal proteins that differentiate locations (supplementary fig. 4b online), so this association does not contribute to the environmental effect on transcription of ribosomal protein genes. another trans - association of interest involves rs11987927 in myom2 at 8p23 with znf71 at 19q13, but also with its own myom2 transcript. logic suggests that the cis - association likely affects the abundance of the myom2 myomesin protein, which in turn regulates znf71, but the trans association is actually significantly stronger and conditional dependence analysis29,30 points in the opposite direction, namely that the myom2 regulatory site influences znf71, which then feeds back on the myom2 transcript (supplementary fig. it is worth mentioning that four of the seven strongest trans associations involve regulation by loci that include genes that encode structural proteins, the others being the lama5 laminin (20q13) with osbpl2, and the plekhm1 plekstrin homology domain protein (17q21) with mapk8ip1. we found association of two probes that detect both hbg1 and hbg2 transcripts from 11p15 with rs766432 in the second intron of the bcl11a zinc finger proto - oncogene at 2p16. this same snp has previously been associated with the fraction of erythrocytes that contain measurable fetal hemoglobin31, and alteration of bcl11a activity was recently shown drive differences in globin switching between mice and humans32. another snp in bcl11a, rs4671393 has been associated with abundance of two bcl11a transcript isoforms in ceu and yri hapmap lymphoblast cell lines33, but is not associated with bcl11a transcript abundance in our leukocyte data, suggesting that regulation of bcl11a translation or protein activity is more likely to be affecting hbg expression in our sample. we scanned the gwas association database for overlap between our study and established disease associations at p<10. of 1,628 entries, 10 involve cis associations observed in our dataset that explain between 15 and 55% of the transcript variance (supplementary table 4 online). five of the associations are with disease conditions (rheumatoid arthritis, celiac disease, t1d, ulcerative colitis, and sle) and five are with endophenotypes (pafah1b2 and icam-1 protein levels, triglycerides, ldl cholesterol, and hip bone mineral density). the two serum protein associations34,35 are with the same snps as we detect and hence suggest that protein abundance is largely regulated at the transcriptional level. our geographical genomic survey of gene expression variation in southern morocco has highlighted two parallel and for the most part non - overlapping insights. on the one hand, it is evident that as much as half of the transcriptome is influenced by the environment in a highly coordinated manner such that where a person lives explains up to a quarter of the variation for a substantial fraction of the transcripts. the environmental influences are likely a combination of biotic and abiotic factors, as well as cultural and behavioral ones, while genetic differences between the two north african ethnicities are relatively minor. on the other hand, the genome is littered with strong genetic associations, mainly in cis, that explain between 15 and 60 percent of the variance of 5% of the transcripts. impressive as these associations are, particularly since they are discovered in a sample of just under 200 individuals, they have essentially no bearing on the vast majority of the transcriptional variation, and are not informative of the genetic basis of the environmental response. the robustness of the observed associations to the environmental effect raises the issue of whether genotype - by - environment interactions influence the peripheral blood transcriptome at all. genome - wide significant interaction effects are generally unlikely to occur in the absence of significant main genotype effects36. the only circumstances in which they will are if the genotype effect is in the opposite direction in two locations, and if the genetic effect in these locations is at least the same magnitude as the main effects detected in this gwas, namely explaining over 30% of the variance of a particular transcript. while a few such interactions may exist, it would take a study comparing several thousand individuals from each location to reveal weaker genotype - by - environment interactions. if the genetic architecture of transcription is generally similar to that of visible phenotypes like height and body mass37,38, even such a study will be underpowered to explain the vast majority of transcriptional variance. a related question is whether or not genotype - by - environment interactions at the level of transcription are necessary to explain genotype - by - environment interactions for disease. it is possible the small interactions beneath the level of detection of gwas are prevalent, or alternatively that disease arises primarily as a result of rare alleles of major effect, whose penetrance may be modulated in an environment - specific manner. however, transcriptional interactions are not required to explain the increased incidence of chronic disease. it is not difficult to imagine that individuals that fall into the major categories of transcriptome profiles (such as those implicated in fig. 4 and supplementary fig. 4 online) have different distributions of disease susceptibility that alter the genotype - disease association matrix genome - wide, thereby inducing environment - by - genotype interactions for disease. transcription of some genes that contribute to this expression component may also correlate with disease directly, effectively uncovering cryptic variation and resulting in environment - specific esnp disease associations without any interaction effect at the level of transcription (fig. a corollary of this is that gene expression profiling might be used to stratify individuals at elevated risk for disease, thereby increasing the resolution of genome - wide association studies by focusing attention on the subset of individuals where genetic effects on disease are most pronounced. our geographical genomic survey of gene expression variation in southern morocco has highlighted two parallel and for the most part non - overlapping insights. on the one hand, it is evident that as much as half of the transcriptome is influenced by the environment in a highly coordinated manner such that where a person lives explains up to a quarter of the variation for a substantial fraction of the transcripts. the environmental influences are likely a combination of biotic and abiotic factors, as well as cultural and behavioral ones, while genetic differences between the two north african ethnicities are relatively minor. on the other hand, the genome is littered with strong genetic associations, mainly in cis, that explain between 15 and 60 percent of the variance of 5% of the transcripts. impressive as these associations are, particularly since they are discovered in a sample of just under 200 individuals, they have essentially no bearing on the vast majority of the transcriptional variation, and are not informative of the genetic basis of the environmental response. the robustness of the observed associations to the environmental effect raises the issue of whether genotype - by - environment interactions influence the peripheral blood transcriptome at all. genome - wide significant interaction effects are generally unlikely to occur in the absence of significant main genotype effects36. the only circumstances in which they will are if the genotype effect is in the opposite direction in two locations, and if the genetic effect in these locations is at least the same magnitude as the main effects detected in this gwas, namely explaining over 30% of the variance of a particular transcript. while a few such interactions may exist, it would take a study comparing several thousand individuals from each location to reveal weaker genotype - by - environment interactions. if the genetic architecture of transcription is generally similar to that of visible phenotypes like height and body mass37,38, even such a study will be underpowered to explain the vast majority of transcriptional variance. a related question is whether or not genotype - by - environment interactions at the level of transcription are necessary to explain genotype - by - environment interactions for disease. it is possible the small interactions beneath the level of detection of gwas are prevalent, or alternatively that disease arises primarily as a result of rare alleles of major effect, whose penetrance may be modulated in an environment - specific manner. however, transcriptional interactions are not required to explain the increased incidence of chronic disease. it is not difficult to imagine that individuals that fall into the major categories of transcriptome profiles (such as those implicated in fig. 4 and supplementary fig. 4 online) have different distributions of disease susceptibility that alter the genotype - disease association matrix genome - wide, thereby inducing environment - by - genotype interactions for disease. transcription of some genes that contribute to this expression component may also correlate with disease directly, effectively uncovering cryptic variation and resulting in environment - specific esnp disease associations without any interaction effect at the level of transcription (fig. a corollary of this is that gene expression profiling might be used to stratify individuals at elevated risk for disease, thereby increasing the resolution of genome - wide association studies by focusing attention on the subset of individuals where genetic effects on disease are most pronounced.	studies of the genetics of gene expression reveal expression snps that explain variation in transcript abundance. here we address the robustness of esnp associations to environmental geography and population structure in a comparison of 194 arab and amazigh individuals from a city and two villages in southern morocco. gene expression differed between pairs of locations for up to a third of all transcripts, with notable enrichment for ribosomal biosynthesis and oxidative phosphorylation. robust associations were observed in the leukocyte samples with cis - esnps (p < 1008) for 346 genes, and trans - esnps (p < 1011) with 10 genes. all of these were consistent across the three sample locations and after controlling for ethnicity and relatedness. no evidence for large - effect trans - acting mediators of the pervasive environmental influence was found and instead genetic and environmental factors acted in a largely additive manner.
we searched the national center for biotechnology information (ncbi) database of genomic sequences from l. gigantea and found 9 potential members of the tgf- superfamily. in addition, examination of the ncbi genomic database of the polychaete annelid capitella sp. phylogenetic analyses included the newly determined deduced amino - acid sequence of nodal of l. gigantea, b. glabrata, and capitella sp. i, as well as the nodal sequences of other deuterostomes and other tgf - superfamily members available from genbank. sequence data were analyzed with macclade version 4.05 osx, macvector version 7.2.3, and paup version 4.0b10. the deduced amino - acid sequences were aligned using clustal x version 1.62b followed by refinement by eye in an effort to maximize positional homology. the aligned amino acid sequence was subjected to bayesian inference (bi) based methods of phylogenetic reconstruction. prottest version 1.3 was used to estimate the evolutionary model that best fit the amino - acid data set. the akaike information criterion (aic) implemented in prottest selected the wag+i+g evolutionary model. bi analyses were performed with mrbayes 3.12 by simulating a metropolis - coupled markov chain monte carlo (mcmcmc) with four simultaneous chains, each of 2 million generations (sampled every 100 generations) under the wag+i+g model. trees sampled before the cold chain reached stationarity (as judged by plots of ml scores) were discarded as burn - in. robustness of the resulting bi tree was evaluated using bayesian posterior probabilities (bpps). sexually mature l. gigantea were collected in los angeles, california during the breeding season and in vitro fertilizations were performed according to gould. a breeding population of b. glabrata is maintained in freshwater tanks at 25c and embryos and larvae are regularly collected and raised in the lab. pcr reactions were performed with gene - specific (for l. gigantea) and degenerate (for b. glabrata) primers sequences (available from the authors upon request). 5 and detailed phylogenetic methods are available in supplementary methods. in situ hybridizations were performed with digoxigenin and fluorescein - labeled rna probes as previously described by price and patel 26. nodal inhibition was performed by placing egg masses of b. glabrata in freshwater containing 1% dmso and sb431542 (tocris bioscience) at a concentration of 5 or 10 m (diluted from a 1 mm stock of sb431542 in dmso) and treatment with rapamycin was performed by placing egg masses in freshwater containing 1% dmso and rapamycin (eton bioscience inc.) at a concentration 10 m (diluted from a 10 mm stock of rapamycin in dmso). all controls and drug treated embryos were kept in the dark during the treatment period.	many animals display specific internal or external features with left - right asymmetry. in vertebrates, the molecular pathway that leads to this asymmetry utilizes the signaling molecule nodal, a member of the tgf- superfamily 1, that is expressed in the left lateral plate mesoderm 2, and loss of nodal function produces a randomization of the left - right asymmetry of visceral organs 3,4. orthologs of nodal have also been described in other deuterostomes, including ascidians and sea urchins 5 - 6, but no nodal ortholog has been reported in the other two main clades of bilateria : ecdysozoa (including flies and nematodes) and lophotrochozoa (including snails and annelids). here we report the first evidence for a nodal ortholog in a non - deuterostome group. we isolated nodal and pitx (one of the targets of nodal signaling) in two species of snails and found that the side of the embryo that expresses nodal and pitx is related to body chirality : both genes are expressed on the right side of the embryo in the dextral (right handed) species lottia gigantea and on the left side in the sinistral (left handed) species biomphalaria glabrata. we pharmacologically inhibited the nodal pathway and found that nodal acts upstream of pitx, and that some treated animals developed with a loss of shell chirality. these results suggest that the involvement of the nodal pathway in left - right asymmetry might have been an ancestral feature of the bilateria.
it is well recognized that anticoagulation is required during haemodialysis to maintain the patency of the extracorporeal circuit. traditionally, unfractionated heparin (ufh) has been used due to the uncertainty surrounding the safety and accumulation of low - molecular - weight heparins (lmwh) in patients with end - stage renal failure. it is now widely accepted that the use of lmwh in haemodialysis has an equal safety and efficacy as ufh [15 ]. lmwh have been consistently reported as easier to use than ufh, and there are some suggestions of the beneficial effect on lipid and bone profiles. lmwh, such as tinzaparin, are now widely used for anticoagulation during haemodialysis. here a 50-year - old male with end - stage renal failure secondary to mesangioproliferative glomerulonephritis commenced on haemodialysis in 2005. in 2009 there was a poor perfusion at the time of implantation, and delayed graft function was expected. he was commenced on cyclosporin and mycophenolate mofetil, but following an episode of biopsy - proven acute rejection, he was converted to tacrolimus. he was re - admitted for a planned transplant graft biopsy for persistent graft dysfunction. he had his planned haemodialysis for 4 h with 2500iu of lmwh (tinzaparin) as anticoagulation, and 1 l of fluid was removed. post dialysis, he had a full examination, including his abdomen ; all of which was unremarkable. thirty minutes later, he developed excruciating right upper quadrant pain, his blood pressure dropped from 140/80 to 90/50 and he became tachycardic. he was not known to have a pre - existing coagulopathy, and his coagulation screen at this time is normal. an ultrasound scan 2 weeks earlier demonstrated a normal liver and biliary tree, although it was not a focused scan. his haemoglobin dropped from 10.1 to 8.4 g / dl over 2 h. all the remaining blood tests were normal. the pain did not settle, and an urgent ct abdomen and pelvis was arranged (see figures 1 and 2).the ct scan revealed acute gall bladder haemorrhage. he was treated expectantly with analgesics and a blood transfusion (2 units), but no intervention was required. this was the second time that the patient had dialysed using lmwhs. prior to this, he had been anticoagulated with unfractionated heparin with a 2000-iu loading dose and then 1200iu across dialysis. it has therefore been concluded that this was a spontaneous gallbladder haemorrhage secondary to lmwh. figures 1 and 2 show ct abdomen and pelvis following intravenous (iv) contrast enhancement. there is a high density content within the gall bladder and biliary tree consistent with acute haemorrhage. acute spontaneous gall bladder haemorrhage is a rare occurrence with a few cases presented in the literature. the majority of cases reported to date have been in relation to trauma, known liver or gallbladder pathology, or underlying coagulopathy [68 ]. in this case, there was no known history of gall bladder pathology, and ultrasound scan, including the biliary tree, was normal 2 weeks previously, although this does not fully exclude biliary pathology. there was also no clinical or biochemical evidence to suggest an infective or inflammatory process at presentation. haemorrhagic acalculous cholecystitis has been reported in haemodialysis patients, and ischaemia due to vascular disease in conjunction with uraemia is the postulated mechanism [911 ]. these previous cases were older patients with concurrent vascular disease ; all of whom required cholecystectomy. to our knowledge, a spontaneous gallbladder haemorrhage has not previously been reported as a consequence of lmwh in the absence of other pathology, or treated conservatively in a haemodialysis patient. there have been a few reported cases of major bleeding episodes in patients dialysed using lmwh, including tinzaparin. bramham. found no major bleeding episodes in 1823 dialysis sessions in 108 haemodialysis - dependent patients. all patients received a minimum of 2500 unit bolus at the beginning of dialysis session. lord. found similar results in a crossover study looking at a selection of haemodialysis patients. these patients received tinzaparin (35004500iu) at the start of dialysis for 4 weeks followed by a further 4 weeks of ufh. one major bleeding episode was reported with tinzaparin, and this related to a fistula - needling site. there have been no reported interactions between the immunosuppressive drugs tacrolimus and mycophenolate mofetil and lmwh to suggest that this patient was at any increased risk of bleeding. drug safety information suggests that bleeding with lmwh may be increased with concomitant use of anti - platelet drugs. the duration of action of tinzaparin is longer than ufh, with a time - to - peak action of 45 h post - administration ; while this allows a single dosage at the onset of dialysis, it would suggest that the majority of patients will remain anticoagulated for a prolonged period post - dialysis in contrast to ufh. due to the timing of the haemorrhage in relation to dialysis, which occurred 5 h after the administration of lmwh, the spontaneous bleeding was felt to be related to the use of lmwh. it is possible that the bleeding may have been exacerbated by an abdominal examination by the admitting doctor given that symptoms arose within minutes of this examination. this represents a rare but serious complication of lmwh use in a haemodialysis patient. as the use of lmwh in dialysis patients increases, we may encounter more rare bleeding episodes, and physicians should keep this in mind when faced with unexplained symptoms in a dialysis patient.	spontaneous gall bladder haemorrhage is a rare and serious occurrence with a few cases reported in the literature in haemodialysis patients. this report describes this complication following dialysis with a low - molecular - weight heparin (lmwh) tinzaparin. this patient presented with acute right upper quadrant pain and intermittent haematemesis following 4 hours of haemodialysis. despite being well established on dialysis, lmwh had only been used once previously. there was no history of trauma or pre - existing gall bladder pathology and no clinical or biochemical evidence of inflammation or infection. computed tomography (ct) scan revealed an extensive gall bladder haemorrhage. the patient was treated conservatively with analgesia, and blood transfusion and symptoms settled without intervention. this case report highlights a rare site of bleeding following lmwh use in a haemodialysis patient.
patients with coronary artery disease (cad) or multiple risk factors for cad are at a higher risk of perioperative myocardial infarction and mortality. an association between anomalous origin of coronary arteries and fatal cardiac events has been reported. the proposed mechanism is stress induced mechanical compression of anomalous coronary arteries between great vessels. we report a case of an elderly man with double vessel disease, poor left ventricular (lv) function and concomitant anomalous origin of left anterior descending (lad) artery from the right coronary artery (rca) for repair of irreducible, obstructed paraumbilical hernia. a 62-year - old, 180 cm, 90 kg male with a history of unstable angina, old inferior wall myocardial infarction and irreducible, obstructed paraumbilical hernia presented for resection of gangrenous loop of small bowel with anatomical repair of hernia. he also had long - standing diabetes, hypertension and was on multiple medications such as regular insulin, aspirin 75 mg, clopidogrel 75 mg, metoprolol 25 mg, atorvastatin 20 mg daily and nitrocontin 2.6 mg twice daily. on examination, he was obese with poor effort tolerance. there was no pallor or edema feet and no signs of heart failure. his resting heart rate (hr) was 70/min and blood pressure measured 130/80 mmhg. most of his preoperative blood investigations including coagulation profile were within normal limits except random blood sugar which was 164 mg / dl. electrocardiography (ecg) showed q waves in leads ii, iii and avf and st segment depression in leads v1 -v6. two - dimensional echo done on admission revealed akinetic inferior and mid - posterior wall ; hypokinetic basal, posterior wall, mid and distal interventricular septum ; mildly dilated lv and left ventricular ejection fraction (lvef) < 25%. two months ago, a detailed cardiac work up of the patient had been done wherein a coronary angiogram revealed anomalous origin of the lad from rca and absent left main coronary artery (figure 1). thallium scan also showed lvef of 25% at rest tomography angiography showed a common ostium for rca and lad (figure 2). coronary angiography : total occlusion of right coronary artery (rca) and left circumflex artery. left anterior descending artery originating from rca computed tomography coronary angiography : common ostium for right coronary artery (rca) and left anterior descending artery ; absent left main artery. left circumflex artery short in length and significantly narrow in caliber ; completely blocked rca in proximal segment due to dense calcification chronic total occlusion of rca (after giving off lad) as well as a short, narrowed out left circumflex (lcx) artery resulted in complete loss of viable myocardium in their corresponding territories. thus, the myocardium had its blood supply from lad and partly from lcx artery. the patient was on medical management as coronary revascularization could not be considered owing to the semi - emergent nature of surgery and that most of the myocardium was already compromised. the patient 's relatives were initially reluctant to give their consent for the procedure, thereby causing delay in surgical intervention. until then, the patient was managed conservatively on continuous ryle 's tube aspiration, intravenous fluids and antibiotics. all the prescribed medications were continued preoperatively except aspirin and clopidogrel, which were stopped at admission. therefore, sequential epidural combined with local infiltration of the surgical site with 0.25% bupivacaine was selected as the anesthetic technique. central venous pressure (cvp), arterial blood pressure (abp), blood gas analysis and continuous cardiac output (co) monitoring (edwards life sciences flotrac monitor) were used along with routine monitors. the baseline abp, hr, cvp, co were 122/72 mmhg, 90/min, 7 mmhg and 7 l / min respectively. an epidural catheter was inserted in t10 -t11 interspace with the patient in sitting position. total fluid administered was 1.5 l (1 l of normal saline ; 500 ml of colloid). it was guided by cvp, stroke volume (sv), stroke volume variation (svv) and co on the edwards sciences flotrac vigileo monitor. the patient remained hemodynamically stable until about 50 min of the start of surgery when abp was 86/54 (65mmhg) and co was 3 l / min probably due to last epidural bolus of bupivacaine. ionotropic support (nor - adrenaline 0.07 g / kg / min) was then started. the surgical procedure included resection of gangrenous segment of bowel, anastomosis and anatomical repair of hernia, which lasted approximately 2 h. postoperatively, 12 lead ecg was done, which showed no fresh changes. intensive monitoring (abp, cvp, ecg, and co) was continued for 48 h postoperatively. the ionotropic support was tapered and discontinued after 36 h of surgery to maintain a mean abp of 75 - 80 mm hg. analgesia was maintained with epidural infusion of 0.125% bupivacaine with100 mcg fentanyl in 50 ml of normal saline titrated to effect and hemodynamics, intravenous analgesics (diclofenac 75 mg 8 hourly and tramadol whenever required). all preoperative medications including enoxaparin (60 mg sc twice daily for 2 days) and aspirin were restarted on the 1 postoperative day (pod) and review done by a cardiologist and an endocrinologist. the course of stay in the surgical intensive care unit (48 h) and later in the ward was uneventful. coronary artery anomaly refers to a wide range of congenital abnormalities involving origin, course and structure of epicardial coronary arteries. it is an incidental finding during 1.3% of conventional cardiac catheterizations. in our patient, lad arose from rca along with double vessel disease (complete block in rca and thinned out lcx) and poor lv function. patients with cad undergoing intermediate to high - risk noncardiac surgery are at increased risk of perioperative cardiac complications and death. most of them presenting for emergency noncardiac surgery can not undergo prophylactic cardiac catheterization or coronary revascularization. care of these patients focuses on a thorough preoperative evaluation ; stratifying risks ; optimizing medical treatment ; intensive monitoring and choosing the appropriate anesthetic technique and drugs in the perioperative period. as per the revised american college of cardiology / american heart association guidelines for cardiac patients undergoing noncardiac surgery, our patient was a high cardiac risk scheduled for a high - risk surgery. the anesthetic goal in these patients should be to minimize hemodynamic instability and interference with myocardial oxygen supply. hypotension and volume overload may precipitate myocardial ischemia owing to poor cardiac reserve. a meta - analysis by beattie. reports a reduction in pmi with thoracic epidural analgesia (tea), which attenuates neuro - humoral stress response through adequate pain management.. showed decreased hypercoagulability with epidural anesthesia that may reduce the incidence of coronary artery occlusion and deep vein thrombosis.. provided insight into mechanisms by which epidural anesthesia may be a therapeutic measure in preventing and treating myocardial ischemia. prevention, early detection and treatment of myocardial ischemia are crucial for a favorable cardiovascular outcome. computerized analysis has become standard in modern monitors, which have an average sensitivity and specificity of 74% and 73% respectively. perioperative fluid management in our patient was guided by cvp, sv, svv, co. high variations of svv and co were not observed as our patient was on spontaneous ventilation with mild sedation of fentanyl and midazolam. the approach to preventing perioperative cardiac events entails a careful risk assessment and modification of patient and procedure related risk factors as far as possible. the most important determinants of cardiovascular stability are an anesthetic technique that obtunds the pathophysiological responses induced by surgery, combined with excellent control of pain, fluid balance and oxygenation under close monitoring.	anesthetic management of patients with coronary artery disease undergoing noncardiac surgery is quite challenging. such patients are at increased risk of perioperative cardiac complications and death. we report an illustrative case of a 62-year - old male with ischemic heart disease and anomalous coronary arteries for obstructed paraumbilical hernia repair.
nonlinear problems have posed a challenge to the scientific world since long and many scientists and researchers have been working hard to find methods to solve these problems. quite a remarkable progress has been made to achieve qualitative as well as quantitative solutions of some tough nonlinear problems of significance in the field of physical and biological sciences, as well as in engineering and technology. there are several analytical methods, such as homotopy analysis method (ham), homotopy perturbation method (hpm), adomian decomposition method (adm), variational iteration method (vim), and a new iterative method, are available to solve nonlinear fractional partial differential equations. the aim of the present paper is to propose an optimal variational asymptotic method (ovam) to solve the following initial value nonlinear time fractional pde : (1)ly+ny = g(x, t), x=[a, b]r, t>0, with the initial conditions (2)y(x,0)=f0(x),y(x,0)=f1(x),,y(n1)(x,0)=fn1(x), where l = /t, n 1 1. to determine the optimal value of lagrange multiplier via variational theory we use the following proposition. taking = n in (4) and using proposition 1, we obtain (6)yn+1(x, t)=yn(x, t) + (1()0t(ts)1 iiiiiiiiiiiiiiii{j=0n{j hj(x)lynly~nkkkkkkkkkkkkkkkki+j=0n{jhj(x)kkkkkkkkkkkkkkkkkkkkkk(ly~nj(x, s)kkkkkkkkkkkkkkkkkkkkkk + ny~nj(x, s)iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiig~nj(x, s))}1()ef}ds)=yn(x, t) + (1(n)0t(ts)n1 dnyndsn ds)=(1+(s)\|s = t)yn(x, t) 0t(s)yn(x, s)ds, giving 1 + (s)\|s = t = 0 and (s)\|s = t = 0. = 1 and discarding the added and subtracted terms lyn in (3) we get (7)yn+1(x, t)=yn(x, t) jt[j=0n{jhj(x)(lynj(x, t) iiiiiiiiiiiiiiiiiiiiiiiiiiiii + nynj(x, t) iiiiiiiiiiiiiiiiiiiiiiiiiignj(x, t))}j=0n{j hj(x)(lynj(x, t)],mmmmmmmmmmmmn=0,1,2,. in our proposed algorithm, computing (7) for a given problem writing (8)yn(x, t)=m=0num(x, t), where (9)u0(x, t)=y(x,0)+ty(x,0)+t2y(x,0) + +tn1y(n1)(x,0)+jt(g(x, t))=j=0n1fj(x)+jt(g(x, t)), we get the series representation of the solution y(x, t) as (10)y(x, t)=limnyn(x, t)=limnm=0num(x, t). the nonlinear term nyn(x, t) is expanded in terms of adomian 's polynomials as (11)nyn(x, t)=n(m=0num(x, t))=m=0nam(u0,u1,,um), where am 's are adomian 's polynomials which are calculated by the algorithm (12) constructed by adomian : (12)an(u0,u1,,un)=1n![dndnn(k=0nkuk)]=0, n0. evaluation of the nonlinear term nyn(x, t) by (12) will be referred to as the second step. combining the first and second step, the new generalized correction functional (7) becomes (13)un+1(x, t)=jt[j=0n{jhj(x)(gnj(x, t)l(m=0njum(x, t))iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii+m=0njam(u0,u1,,um)lllllllllllllllllllllllllllllllllllliiiiiiiignj(x, t)(m=0njum(x, t)))j hj(x)(l}j=0n{j hj(x)(l],iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiin=0,1,2,. equation (13) can also be written as (14)u1(x, t)=0h0(x)jt [lu0(x, t)+a0(u0)g0(x, t)],un+1(x, t)=un(x, t)jt [j=0n{jhj(x)iiiiiiiiiiiiiiiii(lunj(x, t)+anjiiiiiiiiiiiiiiiiiiiiiii(u0,u1,,unj)iiiiiiiiiiiiiii iiiiii(gnj(x, t)iiiiiiiiiiiiiiiiiiiii iimmnj+1gnj1(x, t)))}j=0n{j hj(x)],iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiin=1,2,. combining the above two equations, we get (15)un+1(x, t)=n+1un(x, t)jt [j=0n{nj+1gnj1(x, t)))jhj(x)iiiiiiiiiiiiiii(lunj(x, t)iiiiiiiiiiiiiiiiiii+anj(u0,u1,, we calculate the various un(x, t) for n 1 and substituting these values in (10), we obtain the analytical solution of (1). truncating the solution series (10) at level m = n, the approximate solution at level n is given by (16)y~n(x, t,0,1, the values of j 's are still to be found. substituting (16) into (1), one gets the following residual : (17)rn(x, t,0,1, there are several methods like galerkin 's method, ritz method, least squares method, and collocation method to find the optimal values of 0, 1, 2,. we apply the method of least squares to compute the optimal values of these auxiliary parameters. at the nth - order of approximation, we define the exact square residual error jn(0, 1,,,n1)dx dt. thus, at the given level of approximation n, the corresponding optimal values of convergence control parameters 0, 1,, n1 are obtained by minimizing the jn which corresponds to the following set of n algebraic equations : (19)jn0=0,jn1=0,,jnn1=0., n1 so obtained, when substituted in equation (16) gives the approximate solution at level n. the novelty of our proposed algorithm is that (1) a new generalized correction functional (15) is constructed by introducing auxiliary parameters 0, 1, 2,, auxiliary functions h0(x), h1(x), h2(x),, and expanding the nonlinear term as series of adomian polynomial in the correction functional of the standard vim and (2) the values of auxiliary parameters 0, 1, 2, now we apply our proposed method to solve the following two problems in sections 3.1 and 3.2. advection - diffusion equation (ade) describes the solute transport due to combined effect of diffusion and convection in a medium. it is a partial differential equation of parabolic type, derived on the principle of conservation of mass using fick 's law. due to the growing surface and subsurface hydro environment degradation and the air pollution, the advection - diffusion equation has drawn significant attention of hydrologists, civil engineers, and mathematical modellers. its analytical / numerical solutions along with an initial condition and two boundary conditions help to understand the contaminant or pollutant concentration distribution behaviour through an open medium like air, rivers, lakes, and porous medium like aquifer, on the basis of which remedial processes to reduce or eliminate the damages may be enforced. it has wide applications in other disciplines too, like soil physics, petroleum engineering, chemical engineering, and biosciences. in 2002, inc and cherruault applied adomian decomposition method to solve nonlinear convection- (advection-) diffusion equation. the most important advantage of using fractional order differential equation in mathematical modelling is their nonlocal property. it is a well - known fact that the integer order differential operator is a local operator whereas the fractional order differential operator is nonlocal in the sense that the next state of the system depends not only upon its current state but also upon all of its proceeding states. in the last decade, many authors have made notable contribution to both theory and application of fractional differential equations in areas as diverse as finance, physics [11, 12 ], control theory, and hydrology [14, 15 ]. several papers have been written [16, 17 ] to show the equivalence between the transport equations using fractional order derivatives and some heavy - tailed motions, thus extending the predictive capability of models built on the stochastic process of brownian motion, which is basis for the classical ade. the motion can be heavy - tailed, implying extremely long - term correlation and fractional derivatives in time and/or space. in recent past several papers [8, 14, 15, 18, 19 ] momani proposed an algorithm to solve the following fade with nonlinear source term : (20)yt=2yx2cyx+(y)+f(x, t), iimm00, 00. taking hi(x) = 1, i = 0,1, 2,, n, t0 = 0, t1 = 1, gj(x, t) = 0, j = 0,1, 2,, m, and u0(x, t) = (1/10)sin(x / l) and applying the proposed algorithm, we obtain the correction functional (15) for (21) as (38)un+1(x, t)=n+1un(x, t)+jt [i=0n{+ani(u0,u1,,uni)tuni)i(tuni(x, t) iiiiiiiiiiiiiiiiiii+22x2uni(x, t) iiiiiiiiiiiiiiiiiii+4x4uni(x, t) iiiiiiiiiiiiiiiiiii+(1)uni(x, t) iiiiiiiiiiiiiiiiiii+ani(u0,u1,,uni)tuni)}ef22i=0n{i ], where (39)an(u0,u1,,un)=1n![dndn{(k=0nkuk)3}]=0,iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiin0. solving (38) and using (39), we get the various un(x, t) as (40)u1(x, t)=0(15l22sin(xl)+110l44sin(xl) m+110(1)sin(xl)+11000sin(xl)3) t(1+),. substituting the above iterations in (16) and taking n = 3, the 3rd order approximate solution for (35)(37),i). from (17), the 3rd order residual is (42)r3(x, t,0,1,2)=ty~3 + 22x2y~3 + 4x4y~3+(1)y~3+y~33. as discussed in section 3.1, we first replace x and t by l(1 + x)/2 and (1 + t)/2, respectively, and then approximate (18) using gaussian legendre quadrature with twenty nodes. the optimal values of 0, 1, 2 for all the values of, l, and considered are obtained by minimizing (18) using the mathematica function minimize and are given in table 3. from table 3 we see that the square residual error j3 for l = 10, = 0.6, and = 1, 0.75, 0.5, respectively, obtained by our algorithm is smaller than that obtained by vishal. using eighth order approximate solution of ham as given in tables 13. figures 5(a)5(d) show the solution profile y~3(x, t) versus x for l = 3, l = 6, l = 8, and l = 10, respectively, at = 1, = 0.3 on the corresponding optimal values of 0, 1, 2 given in table 3. from figure 5 we see that, for l = 3, the value of y~3(x, t) increases as we increase the value of t, whereas for l = 6, l = 8, and l = 10, the value of y~3(x, t) decreases as we increase the value of t. figures 6(a)6(d) show the solution profile y~3(x, t) versus x for l = 3, l = 6, l = 8, and l = 10, respectively, at = 0.5, = 0.3 on the corresponding optimal values of 0, 1, 2 given in table 3. from figure 6 we also find the same behaviour of y~3(x, t) as in figure 5. figures 7(a)7(d) show the solution profile y~3(x, t) versus x for l = 3, l = 6, l = 8, and l = 10, respectively, at = 1, = 0.9 on the corresponding optimal values of 0, 1, 2 given in table 3. from figure 7 we see that the value of y~3(x, t) increases as we increase the value of t, for all considered values of l. figures 8(a)8(d) show the solution profile y~3(x, t) versus x for l = 3, l = 6, l = 8, and l = 10, respectively, at = 0.5, = 0.9 on the corresponding optimal values of 0, 1, 2 given in table 3. from figure 8 we also find the same behaviour of y~3(x, t) as in figure 7. we have proposed, for the first time, a new concept which is the generalization of our previous new iterative method by introducing arbitrary number of auxiliary parameters and functions in the correction functional of the previously proposed new iterative method. the method is called the optimal variational asymptotic method. a semianalytic algorithm based on ovam, we apply it to solve two important classes of nonlinear partial differential equations : (1) the fractional advection - diffusion equation with nonlinear source term and (2) the fractional swift - hohenberg equation. only five and three iterations are required to achieve fairly accurate solutions of the first and second problems, respectively. from figures 1, 2, and 3, we see that ovam is better than the methods in [5, 8 ]. also, we get parabolic solution profiles for small and large values of l in 0 t 1.6 similar to the ones obtained in whereas vishal. obtained hat like and nosey profiles for smaller values of l and the corresponding values of.	we propose optimal variational asymptotic method to solve time fractional nonlinear partial differential equations. in the proposed method, an arbitrary number of auxiliary parameters 0, 1, 2, and auxiliary functions h0(x), h1(x), h2(x), are introduced in the correction functional of the standard variational iteration method. the optimal values of these parameters are obtained by minimizing the square residual error. to test the method, we apply it to solve two important classes of nonlinear partial differential equations : (1) the fractional advection - diffusion equation with nonlinear source term and (2) the fractional swift - hohenberg equation. only few iterations are required to achieve fairly accurate solutions of both the first and second problems.
delirium is a common and severe complication among elderly patients and is associated with increased morbidity and mortality, prolonged hospital stay, increased risk of post - discharge institutionalization and dementia. identifying accurate biomarkers for delirium may shed light on the pathophysiology and may help to improve delirium recognition and care. oxidative stress and disturbances in serotonergic and dopaminergic neurotransmission might all be involved in the pathophysiology of delirium and probably act together. within the central nervous system, tetrahydrobiopterin (bh4) functions as an essential cofactor in enzymatic reactions responsible for the production of serotonin and dopamine. in addition, bh4 is a cofactor for nitric oxide synthase (nos) that catalyzes the production of nitric oxide (no) and citrulline from arginine., when bh4 is partially deficient, some cellular sources of nos may generate superoxide (o2) instead of no and citrulline. in patients with delirium, bh4 status has only been investigated after elective cardiac surgery. in order to assess bh4 status, we measured amino acid levels and subsequently calculated the phenylalanine / tyrosine (phe / tyr) ratio. this ratio is an indicator for the bh4 status as it reflects the activity of the enzyme phe hydroxylase, an enzyme that uses bh4 as a cofactor. furthermore, we determined the ratios of tryptophan (trp), phe and tyr to the other large neutral amino acids (lnaas). trp is the precursor of serotonin, while phe and tyr are the precursors of dopamine. the lnaas (trp, phe, tyr, valine, isoleucine and leucine) compete with each other for transport across the blood - brain barrier. therefore, a decreased trp / lnaas ratio is suggestive for a decline in the amount of trp that enters the brain and consequently for reduced synthesis of serotonin. moreover, we measured plasma levels of the dopamine metabolite homovanillic acid (hva), approximately 30% of which is estimated to originate from dopamine neurons in the central nervous system and which is therefore thought to be a reliable indicator for central dopamine activity. finally, we measured plasma levels of arginine and citrulline to investigate the production of no by nos. the aim of the study was to investigate bh4 status, potential disturbances in serotonergic and dopaminergic neurotransmission and the production of no in patients with and without delirium. the present study was performed within the delirium in the old (dito) study in which mean levels of neopterin, interleukin-6 and insulin - like growth factor-1 were compared between patients with and without delirium. in the dito study, a cross - sectional study, we included patients who were admitted to the wards of internal medicine and geriatrics of the erasmus university medical center and the ward of geriatrics of the havenziekenhuis, rotterdam, the netherlands. exclusion criteria were a diagnosis of lewy body dementia, parkinson 's disease, neuroleptic malignant syndrome, tardive dyskinesia, ongoing treatment with antipsychotics or other psychiatric medications except haloperidol and benzodiazepines, aphasia, insufficient understanding of the dutch language and a mini - mental state examination (mmse) score < 10 points out of 30. we excluded patients with a mmse < 10 because it can be quite difficult to distinguish between features of severe dementia and delirium at admission as well as to measure improvement of cognitive function in this group. written informed consent was obtained from all participants. in case of delirium or cognitive impairment at the time of admission, informed consent was obtained from a representative of the patient. all participants were observed daily by the nursing and medical staff and by members of the research team until discharge. to screen for a change in behavior, the 13-item delirium observation screening scale was used during the first 5 days of admission. the diagnosis of delirium was made by a geriatrician, according to the diagnostic and statistical manual of mental disorders, 4th edition (dsm - iv), and was based on the psychiatric examination of the patient, the medical and nursing records, including the delirium observation screening scale scores, and information given by the patient 's closest relative. when the diagnosis of delirium was doubtful, the case was discussed with the geriatric consultation team to gain consensus. cognitive functioning was assessed in absence of delirium using the mmse. when it was impossible to score the mmse during admission because the patient was too ill, cognitive function was discussed with a clinician or assessed with information from the available medical records. when the clinical opinion was that the patient would have a mmse score 10, the patient was not excluded from the study. this index encompasses 19 medical conditions, including dementia, and each condition is weighted with a score of 1 - 6 by severity. physical functionality was assessed using the 6-item katz activities of daily living scale and the barthel index. instrumental functionality was assessed using the 7-item older americans resource scale for instrumental activities of daily living. blood samples of all patients were collected within 48 h after admission. when a patient developed delirium during the hospital stay, new blood samples were collected within 24 h after the onset of the delirium and non - fasting blood was collected preferably between 8 and 10 a.m. in an 8-ml tube containing ethylene diamine tetraacetic acid. after blood sampling, the tubes were stored at room temperature to prevent changes in the transfer of amino acids between plasma and blood cells. within 3 h, plasma amino acid levels were determined by high - performance liquid chromatography with automated pre - column derivatization with ortho - phthalaldehyde as previously described. plasma hva levels were determined by reversed - phase high - performance liquid chromatography and electrochemical detection, as previously described for the determination of serotonin. medians and interquartile ranges were determined for continuous participant characteristics and proportions for categorical characteristics. biochemical parameters with a skewed distribution were logarithmically transformed (all amino acids, amino acid ratios and hva). univariate one - way analysis of variance was used to investigate the association between mean levels of amino acids, amino acid ratios and hva (dependent variables) and the presence of delirium additional analyses were performed for all amino acids, amino acid ratios and hva after also adding mmse score to the models. a two - tailed p < 0.05 was defined as statistically significant. statistical package for the social sciences, version 21.0 (spss inc., the present study was performed within the delirium in the old (dito) study in which mean levels of neopterin, interleukin-6 and insulin - like growth factor-1 were compared between patients with and without delirium. in the dito study, a cross - sectional study, we included patients who were admitted to the wards of internal medicine and geriatrics of the erasmus university medical center and the ward of geriatrics of the havenziekenhuis, rotterdam, the netherlands. exclusion criteria were a diagnosis of lewy body dementia, parkinson 's disease, neuroleptic malignant syndrome, tardive dyskinesia, ongoing treatment with antipsychotics or other psychiatric medications except haloperidol and benzodiazepines, aphasia, insufficient understanding of the dutch language and a mini - mental state examination (mmse) score < 10 points out of 30. we excluded patients with a mmse < 10 because it can be quite difficult to distinguish between features of severe dementia and delirium at admission as well as to measure improvement of cognitive function in this group. written informed consent was obtained from all participants. in case of delirium or cognitive impairment at the time of admission, informed consent was obtained from a representative of the patient. all participants were observed daily by the nursing and medical staff and by members of the research team until discharge. to screen for a change in behavior, the 13-item delirium observation screening scale was used during the first 5 days of admission. the diagnosis of delirium was made by a geriatrician, according to the diagnostic and statistical manual of mental disorders, 4th edition (dsm - iv), and was based on the psychiatric examination of the patient, the medical and nursing records, including the delirium observation screening scale scores, and information given by the patient 's closest relative. when the diagnosis of delirium was doubtful, the case was discussed with the geriatric consultation team to gain consensus. when it was impossible to score the mmse during admission because the patient was too ill, cognitive function was discussed with a clinician or assessed with information from the available medical records. when the clinical opinion was that the patient would have a mmse score 10, the patient was not excluded from the study. this index encompasses 19 medical conditions, including dementia, and each condition is weighted with a score of 1 - 6 by severity. physical functionality was assessed using the 6-item katz activities of daily living scale and the barthel index. instrumental functionality was assessed using the 7-item older americans resource scale for instrumental activities of daily living. blood samples of all patients were collected within 48 h after admission. when a patient developed delirium during the hospital stay, new blood samples were collected within 24 h after the onset of the delirium and non - fasting blood was collected preferably between 8 and 10 a.m. in an 8-ml tube containing ethylene diamine tetraacetic acid. after blood sampling, the tubes were stored at room temperature to prevent changes in the transfer of amino acids between plasma and blood cells. within 3 h, plasma amino acid levels were determined by high - performance liquid chromatography with automated pre - column derivatization with ortho - phthalaldehyde as previously described. plasma hva levels were determined by reversed - phase high - performance liquid chromatography and electrochemical detection, as previously described for the determination of serotonin. medians and interquartile ranges were determined for continuous participant characteristics and proportions for categorical characteristics. biochemical parameters with a skewed distribution were logarithmically transformed (all amino acids, amino acid ratios and hva). univariate one - way analysis of variance was used to investigate the association between mean levels of amino acids, amino acid ratios and hva (dependent variables) and the presence of delirium additional analyses were performed for all amino acids, amino acid ratios and hva after also adding mmse score to the models. table 1 presents the baseline characteristics of the 86 participants who were included in the study. of the 23 patients diagnosed with delirium, 21 the mean levels and corresponding 95% confidence intervals (cis) of the investigated biochemical parameters in patients with and without delirium are presented in tables 2 and 3. in adjusted models, mean levels of arginine were significantly lower in patients with delirium (34.8 mol / l, 95% ci 28.8 - 42.0) than in those without (45.2 mol / l, 95% ci 40.6 - 50.5) (p = 0.022) (fig. mean phe / tyr ratios were significantly higher in patients with delirium (1.34, 95% ci 1.19 - 1.51) than in patients without delirium (1.14, 95% ci 1.06 - 1.22) (p = 0.028) (fig. 1). in addition, mean trp / lnaas ratios were significantly lower in patients with delirium (4.90, 95% ci 4.19 - 5.74) than in those without (6.12, 95% ci 5.58 - 6.71) (p = 0.021) (fig. no associations between the other amino acids and ratios and delirium were found, although citrulline (fig. 1) and trp levels were at the border of significance lower in patients with delirium than in those without (p = 0.052 and p = 0.067, respectively). n = 1 [not enough plasma ] ; no delirium, n = 1 [measurement failed ]). mean hva levels were not statistically significantly different between patients with delirium (123.0 nmol / l, 95% ci 93.3 - 162.6) and patients without delirium (93.3 nmol / l, 95% ci 79.4 - 109.4) (p = 0.098). in the models additionally adjusted for mmse score, the association between arginine and delirium did not reach statistical significance (delirium : mean 36.0 mol / l, 95% ci 28.7 - 45.1 vs. no delirium : mean 44.8 mol / l, 95% ci 39.8 - 50.5, p = 0.107). mean phe / tyr ratios remained borderline significantly higher in patients with delirium (1.34, 95% ci 1.16 - 1.55) than in those without (1.15, 95% ci 1.07 - 1.24) (p = 0.089) and mean trp / lnaas ratios remained borderline significantly lower in patients with delirium (5.00, 95% ci 4.15 - 6.01) compared to those without (6.15, 95% ci 5.58 - 6.78) (p = 0.062). table 1 presents the baseline characteristics of the 86 participants who were included in the study. of the 23 patients diagnosed with delirium, 21 the mean levels and corresponding 95% confidence intervals (cis) of the investigated biochemical parameters in patients with and without delirium are presented in tables 2 and 3. in adjusted models, mean levels of arginine were significantly lower in patients with delirium (34.8 mol / l, 95% ci 28.8 - 42.0) than in those without (45.2 mol / l, 95% ci 40.6 - 50.5) (p = 0.022) (fig. mean phe / tyr ratios were significantly higher in patients with delirium (1.34, 95% ci 1.19 - 1.51) than in patients without delirium (1.14, 95% ci 1.06 - 1.22) (p = 0.028) (fig. 1). in addition, mean trp / lnaas ratios were significantly lower in patients with delirium (4.90, 95% ci 4.19 - 5.74) than in those without (6.12, 95% ci 5.58 - 6.71) (p = 0.021) (fig. no associations between the other amino acids and ratios and delirium were found, although citrulline (fig. 1) and trp levels were at the border of significance lower in patients with delirium than in those without (p = 0.052 and p = 0.067, respectively). hva data were missing for 2 patients (delirium, n = 1 [not enough plasma ] ; no delirium, mean hva levels were not statistically significantly different between patients with delirium (123.0 nmol / l, 95% ci 93.3 - 162.6) and patients without delirium (93.3 nmol / l, 95% ci 79.4 - 109.4) (p = 0.098). in the models additionally adjusted for mmse score, the association between arginine and delirium did not reach statistical significance (delirium : mean 36.0 mol / l, 95% ci 28.7 - 45.1 vs. no delirium : mean 44.8 mol / l, 95% ci 39.8 - 50.5, p = 0.107). mean phe / tyr ratios remained borderline significantly higher in patients with delirium (1.34, 95% ci 1.16 - 1.55) than in those without (1.15, 95% ci 1.07 - 1.24) (p = 0.089) and mean trp / lnaas ratios remained borderline significantly lower in patients with delirium (5.00, 95% ci 4.15 - 6.01) compared to those without (6.15, 95% ci 5.58 - 6.78) (p = 0.062). in the present study we found disturbed serotonergic neurotransmission and an increased status of oxidative stress in patients with delirium when compared to patients without delirium. as far as we are aware, this is the first delirium study investigating bh4 status and levels of arginine and citrulline in acutely ill elderly hospitalized patients. in order to assess the bh4 status, we measured the phe / tyr ratio. in patients with delirium we found an increased ratio, suggesting a deficiency in the essential cofactor bh4 for the production of serotonin, dopamine and no. decreased bh4 availability has already been found in other neuropsychiatric disorders such as alzheimer 's disease, parkinson 's disease and schizophrenia. our finding is not in agreement with the results of a previous delirium study which showed that levels of bh4 and phe / tyr ratios did not differ between patients with and without delirium. however, that study included a relatively younger group of patients undergoing elective cardiac surgery. in the present study, serotonergic neurotransmission was investigated with the trp / lnaas ratio and the phe / tyr ratio. we found that patients with delirium had a decreased trp / lnaas ratio, which might suggest reduced serotonin production in the central nervous system. this hypothesis is strengthened by the finding that patients with delirium had an elevated phe / tyr ratio, which might suggest deficiency in the essential cofactor bh4 in the production of serotonin. in previous studies, controversial results have been reported. several studies found a reduced trp / lnaas ratio during delirium, whereas two studies reported no difference in this ratio between patients with and without delirium. the study performed by flacker and lipsitz included only patients with mild illnesses not requiring hospitalization. therefore, the findings may not be generalizable to acutely ill patients who needed medical care in hospital. the study performed by van der cammen. included delirium patients with alzheimer 's disease. it might be possible that in those patients a disturbance in cholinergic neurotransmission played a more important role in the development of delirium than disturbances in other pathophysiological pathways. furthermore, we found no differences in phe / lnaas ratios, tyr / lnaas ratios and hva levels between patients with and without delirium, suggesting that dopaminergic neurotransmission is not impaired during delirium. the finding that plasma hva levels are not significantly increased in patients with delirium compared to patients without delirium is not in agreement with earlier results. however, those studies were performed in patients with alzheimer 's disease and patients undergoing cardiac surgery, and therefore the results may not be generalizable. ramirez - bermudez. found that cerebrospinal fluid hva levels correlated with psychotic symptoms of delirium (hallucinations and delusions) in neurological patients. it might also be possible that we did not find an association between the dopaminergic markers and the presence of delirium because we included patients both with and without psychotic features. in our study, we also found reduced plasma arginine levels and borderline statistically significantly reduced citrulline levels in patients with delirium. considering the cross - sectional study design first, it is possible that patients with delirium had a pre - existing arginine deficiency which might have resulted in a reduced production of citrulline by nos (fig. second, when bh4 is partially deficient, as our results do suggest, some cellular sources of nos may generate o2 instead of citrulline and no from arginine (fig. if this latter scenario is true for delirium, this would also suggest an increased status of oxidative stress, since it favors peroxynitrite formation (fig. if peroxynitrite is not scavenged by antioxidants, it may cause oxidative damage to cellular macromolecules, which has already been hypothesized to occur in alzheimer 's disease. however, both amino acids have been investigated previously by osse. in patients with delirium after cardiac surgery, but they found no differences in arginine and citrulline levels between patients with and without delirium. since they also reported no difference in bh4 status between patients with and without delirium, first, our findings were obtained in a relatively small group of patients ; therefore, confirmation in a larger population is recommended. second, it might be speculated that the degree of the patients ' cognitive functioning influenced the mean levels of the biochemical parameters. in this study, we adjusted for the charlson comorbidity index, which includes dementia, and our estimates remained statistically significant. however, for our additional analysis mmse scores were not available for all patients ; therefore, we can neither confirm nor deny that the presence of a comorbid cognitive disturbance, not diagnosed as dementia (yet), was a confounding factor. it is possible that the levels of biochemical markers are dependent on delirium duration and severity or even fluctuate during the day in patients with delirium, just like delirium symptoms. in the present study, blood sampling and delirium occurred on the same day, but there is a possibility that the patients had no delirium symptoms at the moment of blood sampling. finally, some potential participants were not included in the study and this may have resulted in some selection bias ; however, since this was random and occurred in both patients with and without delirium, we think that our results are only minimally influenced by this. first, the intensive monitoring of clinical symptoms of patients with delirium until discharge and the dsm - iv diagnosis by a geriatrician makes it less likely that we missed delirium or misdiagnosed symptoms. second, we did not focus on one but on several possible pathways that might lead to delirium as it has been suggested that the pathophysiology is multifactorial. first, our findings were obtained in a relatively small group of patients ; therefore, confirmation in a larger population is recommended. second, it might be speculated that the degree of the patients ' cognitive functioning influenced the mean levels of the biochemical parameters. in this study, we adjusted for the charlson comorbidity index, which includes dementia, and our estimates remained statistically significant. however, for our additional analysis mmse scores were not available for all patients ; therefore, we can neither confirm nor deny that the presence of a comorbid cognitive disturbance, not diagnosed as dementia (yet), was a confounding factor. it is possible that the levels of biochemical markers are dependent on delirium duration and severity or even fluctuate during the day in patients with delirium, just like delirium symptoms. in the present study, blood sampling and delirium occurred on the same day, but there is a possibility that the patients had no delirium symptoms at the moment of blood sampling. finally, some potential participants were not included in the study and this may have resulted in some selection bias ; however, since this was random and occurred in both patients with and without delirium, we think that our results are only minimally influenced by this. first, the intensive monitoring of clinical symptoms of patients with delirium until discharge and the dsm - iv diagnosis by a geriatrician makes it less likely that we missed delirium or misdiagnosed symptoms. second, we did not focus on one but on several possible pathways that might lead to delirium as it has been suggested that the pathophysiology is multifactorial. in this study in older, acutely ill hospitalized patients, we found that patients with delirium had higher phe / tyr ratios, lower trp / lnaas ratios and lower levels of arginine and citrulline than patients without delirium. these findings might suggest that decreased bh4 availability, disturbed serotonergic neurotransmission and an increased status of oxidative stress may have played a role in the pathogenesis of delirium in our patient group. since as far as we know this is the first delirium study investigating bh4 status and levels of arginine and citrulline in acutely ill elderly hospitalized patients, confirmation of our results in a larger, comparable population is recommended. moreover, more research is needed to explore the potential differences in the pathophysiology of delirium in patients with and without cognitive disorders.	backgroundoxidative stress and disturbances in serotonergic and dopaminergic neurotransmission may play a role in the pathophysiology of delirium.aimsin this study, we investigated levels of amino acids, amino acid ratios and levels of homovanillic acid (hva) as indicators for oxidative stress and disturbances in neurotransmission.methodsplasma levels of amino acids, amino acid ratios and hva were determined in acutely ill patients aged 65 years admitted to the wards of internal medicine and geriatrics of the erasmus university medical center and the ward of geriatrics of the havenziekenhuis, rotterdam, the netherlands. differences in the biochemical parameters between patients with and without delirium were investigated by analysis of variance in models adjusted for age, gender and comorbidities.resultsof the 86 patients included, 23 had delirium. in adjusted models, higher mean phenylalanine / tyrosine ratios (1.34 vs. 1.14, p = 0.028), lower mean tryptophan / large neutral amino acids ratios (4.90 vs. 6.12, p = 0.021) and lower mean arginine levels (34.8 vs. 45.2 mol / l, p = 0.022) were found in patients with delirium when compared to those without. no differences were found in hva levels between patients with and without delirium.conclusionthe findings of this study suggest disturbed serotonergic neurotransmission and an increased status of oxidative stress in patients with delirium.
spider silks are renowned for their mechanical properties, but many aspects of how spiders produce these high - performance materials remain elusive. a spider s abdomen contains numerous silk glands that are differentiated into morphologically and functionally distinct types. most gland types express members of a spider - specific gene family that encodes fiber - forming structural proteins called spidroins, a contraction of spider fibroins. after synthesis, spidroins are stored in the lumen of silk glands as part of a liquid spinning dope. as needed, the dope travels through ducts leading to external spigots on the spider s spinnerets. during extrusion, the mechanical properties of fibrous spider silks are related to the amino acid sequences of their spidroin components in that primary structure largely determines secondary and higher - level protein structures. spidroins consist of iterated repeat units that vary in length and complexity depending on spidroin type. the significance of particular spidroin repeat sequences to fiber - specific mechanical properties has been demonstrated through a variety of structural studies. because of the direct relationship between spidroin amino acid sequences and the physical attributes of silk, characterization and recombinant expression of spidroins have been a main focus of spider silk research. despite extensive spidroin research, knowledge of the gene expression and protein profiles that contribute to silk synthesis is far from complete. to further explore how spiders synthesize distinct silk types the western black widow spider, latrodectus hesperus, produces six fibrous silk types from six corresponding silk gland types (major ampullate, minor ampullate, aciniform, flagelliform, pyriform, and tubuliform). here, we focus on major ampullate, minor ampullate, and tubuliform silks. we chose these silk types because of the overlapping functions of major ampullate and minor ampullate silks in orb - weaving spiders, the similar shapes of the major and minor ampullate glands, and evidence that the major ampullate and tubuliform silk glands share expression of some spidroin genes. major ampullate silks emerge from major ampullate glands, which have a long tail that synthesizes proteins, an ampule - shaped sac that both synthesizes and stores proteins, and a z - shaped duct that connects to an external spigot (figure 1a). minor ampullate silks are produced from glands that are morphologically very similar to major ampullate glands but that are smaller, with a shorter tail and lower capacity sac (figure 1b). the combination of a long tail, ampule - shaped lumen, and z - shaped duct are unique to the major and minor ampullate silk glands. bright - field images of l. hesperus silk glands : (a) major ampullate gland, (b) minor ampullate gland, and (c) tubuliform gland. (a, b) anterior tail is at the top right, ampule - shaped lumen is to the left, and posterior z - shaped duct is at the bottom right. anterior tail is pale and to the right ; posterior duct is darker and to the left. l. hesperus major ampullate glands predominantly express the genes for two major ampullate spidroins (masp1 and masp2), and these proteins are the main components of l. hesperus dragline fibers. minor ampullate silk glands express minor ampullate spidroin (misp) genes, and misps are the primary proteins found in minor ampullate silks. minor ampullate silks are generally thought to be used by orb - weaving spiders as the auxiliary spiral during web construction and as support for major ampullate silks in draglines, web radii, and web scaffold. in l. hesperus, which builds a cob - web instead of an orb - web, minor ampullate silks have been found in a composite of silk types used in prey wrapping. cob - webs are a derived type of orb - web, and whether minor ampullate silks have any function during cob - web construction remains unknown. their similar gland morphology and related silk functions suggest that major and minor ampullate spidroins have a close evolutionary relationship, but phylogenetic analysis of spidroins is inconclusive. for example, in garb., misps tended to, but did not always, form a sister group to a large clade of masps. in contrast to major and minor ampullate silks, the thick fibers that wrap l. hesperus egg cases are produced by glands that are unlike any other silk glands in shape : the elongated, worm - like tubuliform glands (also known as cylindrical glands ; figure 1c). the dominant transcripts in tubuliform glands encode tubuliform spidroins (tusp1) and a family of silk proteins unrelated to spidroins, egg - case proteins (ecps). despite having different shapes and producing functionally distinct silks, major ampullate and tubuliform silk glands are known to have overlapping spidroin gene expression. major ampullate glands are dominated by masp1 and masp2 gene expression, but a 3 tag profiling study of transcripts from l. hesperus major ampullate glands found expression of the tubuliform spidroin gene, tusp1. the converse is also true : masp1 and masp2 transcripts were present in l. hesperus tubuliform silk gland cdna libraries. it remains unknown, however, if the presence of nondominant spidroin transcripts in silk glands unequivocally indicates their translation and whether the protein products are incorporated into silk fibers. tubuliform and major ampullate silk glands each express nonspidroin silk - related genes. ecps are members of a nonspidroin gene family that were initially identified in black widow tubuliform glands and fibers and are thought to cross - link with tusp1. further research has also found ecp homologues in a spider species without tubuliform glands, suggesting that ecps have functions across multiple silk types. the recently characterized low - molecular - weight cysteine - rich proteins (crps) are another category of nonspidroin silk constituents. crps appear in l. hesperus major ampullate glands and fibers and are thought to cross - link with masp1 and masp2 to increase silk fiber toughness. other nonspidroin proteins that are vital to the process of synthesizing and assembling spidroins must exist in each gland type, but these are currently unknown. deep transcriptome sequencing studies have made it possible to explore the gene expression profiles of silk glands and to address the dearth of nonspidroin, silk - related genes. a substantial advance to understanding spider silk synthesis was the identification of 647 silk - gland specific transcripts (ssts) from l. hesperus by clarke. ssts are transcripts that were found to be significantly differentially expressed and at least 630 times more abundant in silk glands compared to that in nonsilk gland control tissues. hence, ssts are promising candidates for roles in spidroin production and fiber assembly. previous mass spectrometry studies of protein extracts from l. hesperus egg case wrapping, attachment discs, and gland luminal contents has demonstrated the translation of seven types of silk structural proteins encoded by ssts (aciniform spidroin, ecps, minor ampullate spidroin, major ampullate spidroins 1 and 2, tubuliform spidroin, and pyriform spidroin). it remains unknown whether all or only some of the remaining ssts are translated into proteins. in the present study, we use peptide sequencing to generate protein profiles for the major ampullate, minor ampullate, and tubuliform silk glands as well as the major ampullate fiber of l. hesperus. from these proteomes, we identify proteins arising from the ssts. are from the combined set of l. hesperus silk glands, which makes their localized expression among gland types impossible to discern. knowing which ssts are translated in different glands will identify ssts that are necessary for general silk gland functions (protein products in multiple gland types) and ssts that are necessary for the synthesis and storage of specific silks (protein products restricted to one gland type). additionally, if an sst protein product appears in both the major ampullate gland and fiber, then it may have utility in silk fiber assembly or function rather than having exclusive importance in the gland - specific functions of producing and maintaining silk protein reserves. we predict that major and minor ampullate glands will have similar proteomes, consistent with their similar shape and the overlapping functions of major and minor ampullate silks. we also expect that tubuliform glands will contain tusp1 and ecps and that major ampullate glands will contain masp1, masp2, and crps. tubuliform glands may also express masp1 and masp2, which would be consistent with the discovery of masp1 and masp2 transcripts in tubuliform gland cdna, and, conversely, major ampullate glands may express tusp1, consistent with the detection of tusp1 transcripts in major ampullate glands. comparing major ampullate glands to major ampullate fibers, we expect that only a small subset of the sst protein products identified in major ampullate glands will appear in the fiber. the goal of this research is to combine hypothetical gene products predicted by de novo assembled rna - seq data with proteomic data to gain a broader understanding of how spiders generate silk in vivo. this analysis will extend the framework on how silk dope and fibers are generated by spiders and of gene products that are exceptional candidates for involvement in the synthesis of spider silk. adult l. hesperus females were collected on the university of california, riverside, campus and were fed commercially available crickets. prior to dissection, individuals were not fed for 2 days to minimize contamination from gut contents. spiders were individually anesthetized using carbon dioxide gas, and their abdomens were dissected in 1 ssc. in l. hesperus, major ampullate, minor ampullate, and tubuliform glands are easily distinguished during dissection because of their distinctive morphologies and spatial arrangement in the abdomen (figure 1). silk glands were visually inspected to ensure that there were no breaks or punctures. if damage was detected, no silk glands from that individual were used to avoid cross - contamination. major ampullate, minor ampullate, and tubuliform glands were dissected into separate microfuge tubes and flash frozen in liquid nitrogen or immediately used for protein extraction (below). major ampullate silk fibers were manually drawn from spiders as described in the literature. major ampullate fibers are extruded from large and distinctive silk spigots, enabling visual verification of major ampullate silk collection during fiber harvesting. fibers were drawn onto spools at a rate of 57 cm per second for a total of 23 min. it was sometimes necessary to use up to three individuals to amass the targeted amount of silk fibers. fibers from each individual were stored on separate spools at room temperature in closed containers. two biological replicates were collected for each gland type and major ampullate silk fibers. replicates consisted of one of the following : two major ampullate glands from one individual, six tubuliform glands from one individual, four minor ampullate glands from two individuals, or the combined silk spools of up to three individuals. fresh or frozen silk glands were macerated with a sterile pestle in protein extraction buffer (10% glycerol, 50 mm tris, ph 7.5, 5 mm mgcl2, 2% sds, 150 mm nacl, 0.2% -mercaptoethanol, 0.005 m edta) supplemented with halt protease inhibitor cocktail (thermo fisher scientific, waltham, ma). silk fibers were cut with a clean razor blade, immersed in protein extraction buffer supplemented with halt protease inhibitor cocktail, macerated with a sterile pestle, and incubated at room temperature overnight. samples were then fractionated on an sds - page gel (5% stacking and 6.4% separating) and visualized with bio - safe coomassie stain (bio - rad, hercules, ca). protein concentrations were estimated from these visualizations, with a target of 500 ng / lane. for each sample, multiple lanes (1015 l / lane ; total of 2030 l of sample) were excised and combined for in - gel digest (supporting information figure s1). standard protocols for in - gel tryptic digestion from the arizona proteomics consortium (http://proteomics.arizona.edu/protocols) were followed. briefly, proteins in acrylamide gels were digested overnight at 37 c with 12.5 ng/l trypsin and 1.25 ng/l chymotrypsin in 50 mm ammonium bicarbonate with 10 mm calcium chloride. digested peptides were extracted from gel pieces with a bioruptor standard sonication system (diagenode, denville, nj) for 30 min. after sonication, the supernatant was purified using ziptips c18 pipette tips according to the manufacturer s instructions (millipore, billerica, ma), dried, and stored at 20 c until shipment for analysis at the arizona proteomics consortium. lc ms / ms analysis of trypsin / chymotrypsin - digested proteins was carried out using a ltq orbitrap velos mass spectrometer (thermo fisher scientific) equipped with an advion nanomate esi source (advion, ithaca, ny). 2 cm, thermo fisher scientific) onto an analytical column (75 m i.d solvent b (acetonitrile, 0.1% formic acid) was used for the following concentrations and times : 5%, 5 min ; 57%, 5 min ; 715%, 45 min ; 1535%, 60 min ; 3540%, 28 min ; 4085%, 5 min ; 85%, 10 min ; a return to 5% in 1 min, and another 10 min hold of 5% solvent b. all flow rates were 400 nl / min. ms / ms replicate runs were also performed using a shorter 125 min rp gradient (5% solvent b hold for 10 min ; 520%, 65 min ; 2035%, 25 min ; 35%, 9 min ; 3595%, 5 min ; and finally a 95% solvent b hold for another 5 min). data - dependent scanning was performed by xcalibur, v 2.1.0, software using a survey mass scan at 60 000 resolution in the orbitrap analyzer scanning mass / charge (m / z) 4001600, followed by collision - induced dissociation (cid) tandem mass spectrometry (ms / ms) of the 14 most intense ions in the linear ion trap analyzer. precursor ions were selected by the monoisotopic precursor selection (mips) setting, with selection or rejection of ions held to a 10 ppm window. dynamic exclusion was set to place any selected m / z on an exclusion list for 45 s after a single ms / ms. tandem mass spectra were searched against a protein database made by combining chelicerata proteins downloaded from ncbi on october 17, 2013 and reduced for redundant entries via cd hit (available at http://weizhong-lab.ucsd.edu/cd-hit/) with common contaminant proteins such as keratins (ftp://ftp.thegpm.org/fasta/crap/) and with a nonredundant longest open reading frame translation of an l. hesperus transcriptome (clarke. ; see above). at the time of the search, this combined protein database contained 182 000 entries. all ms / ms spectra were searched against the protein database described above using thermo proteome discoverer 1.3 (thermo fisher scientific) considering tryptic / chymotryptic peptides with up to 2 missed cleavage sites. proteins were identified at 99% confidence with xcorr score cutoffs, as determined by a reversed database search. the protein and peptide identification results were visualized with scaffold, v 3.6.1 and v 4.0 (proteome software inc., portland, or), a program that relies on various search engine results (i.e., sequest, x!tandem, mascot) and that uses bayesian statistics to reliably identify more spectra. proteins were accepted that passed a minimum of two peptides identified at 95% peptide confidence and 99.9% protein confidence by the peptide and protein profit algorithms, respectively, within scaffold. the similarities among the identified protein sets from each sample were quantified in r using the ape package via a binary character matrix, with observed protein presence within a library (protein identification probability > 95%) as the positive result. from the matrix, we calculated the distances between protein libraries using simple matching, from which we built a dendrogram via hierarchical clustering. support for the dendrogram was calculated via bootstrapping with 10 000 replications using a custom r script. replicates were collapsed by taking the higher protein identification probability of the two replicates from the scaffold analysis. the list of identified proteins included ssts. because an sst was not always the top protein identified, we further examined the identified ncbi proteins to see if they were associated with ssts. to do this, we used the identified ncbi nr proteins to build a custom blast database. we searched this database with the longest open reading frame nonredundant translation of the l. hesperus transcriptome in a blastp search. up to five results with an e - value less than 1 10 for each query sequence were retained. the most relevant sst from the set of blast hits was determined by searching for presence of peptide sequences. crps were identified with a blastp search against the longest open reading frame translation of the l. hesperus transcriptome using an e - value cutoff of 1 10. we identified 842 putative proteins in the major ampullate, minor ampullate, and tubuliform glands of l. hesperus (scaffold sample report ; supporting information file s1). the protein identifications in tubuliform gland biological replicates clustered together in a well - supported group (dendrogram bootstrap support = 100% ; supporting information figure s2). major ampullate gland biological replicates also clustered with each other (dendrogram bootstrap support = 92% ; supporting information figure s2). one minor ampullate gland replicate grouped more tightly with the major ampullate gland replicates (bootstrap = 88% ; supporting information figure s2) than it did with the other minor ampullate gland replicate. the difference between the minor ampullate gland replicates may be due to concentration - based variability in detectable proteins. minor ampullate glands are, at most, approximately one - third the length of the other gland types used in this study, and glands from multiple individuals were combined in a single replicate to achieve target concentrations (figure 1 ; see materials and methods). the detectable proteins between the replicates may differ because of variation in protein expression among individuals. despite differences between the minor ampullate gland replicates, consistent with shared ampullate shapes, both minor ampullate gland biological replicates formed a group with the major ampullate gland replicates (bootstrap support = 91% ; supporting information figure s2), with tubuliform gland replicates being more differentiated. the subsets of identified proteins that correspond to ssts also show greater similarity between major ampullate and minor ampullate glands than to tubuliform glands., 48 were predicted by peptide matches in major ampullate, minor ampullate, and tubuliform gland protein extractions (supporting information file s2). some of the identified proteins were fragments from different regions of the same protein ; therefore, the total number of unique proteins predicted by peptide matches to ssts was 39 (figure 2 and supporting information file s2). out of these 39, only 28 had uniprot hits with an e - value less than 1 10. chart of presence (red) or absence (light orange) of peptide - predicted proteins encoded by ssts. predicted proteins from l. hesperus major ampullate, minor ampullate, and tubuliform silk glands with best uniprot hits 19 kb) with a lengthy repetitive region flanked by relatively short amino (n)- and carboxyl (c)-terminal regions. in our analysis, 471 ; figure 2) has peptide matches to the n - terminal and repetitive regions. rare transcript expression would mean that there are few 3 terminal regions available for tagging, whereas enzyme digested acsp1 protein would result in many protein fragments detectable by peptide analysis. an alternative explanation is that the two individuals in the 3 tag profiling study were not expressing acsp1 transcripts at the time of rna extraction. this explanation suggests that ampullate glands may transiently express acsp1 and that accumulated acsp1 protein could be detectable with proteomic approaches. asg2 is a putatively glycosylated protein secreted as part of sticky droplets involved in prey capture. asg2 transcripts and proteins have not previously been detected in major and minor ampullate glands. our finding may be indicative of the specific uses of ampullate silks by black widows. in orb - web weaving spiders, major ampullate silk is used as dragline and as the web frame and radii, and minor ampullate silks are used as temporary spiral during web construction. in black widows, major ampullate silks are thought to be the core fiber of the gumfooted lines that stick to and entangle prey. after ensnarement, minor ampullate silks are incorporated into a composite of different silk types for prey restraint. given the function of major and minor ampullate silks in l. hesperus, black widow ampullate - shaped silk glands may additionally synthesize prey capture glue proteins such as asg2. in the major ampullate gland, peptides identified the translation of an sst sequence that we refer to as masp to distinguish it from masp1 and masp2 (figure 2, scaffold i d no. the masp peptide matches are unique to an sst with a top uniprot hit to masp1 (supporting information file s2), but the sequence is distinct from either masp1 or masp2. the sst that encodes masp appears to be a newly discovered member of the spidroin gene family, and its translation into a protein indicates that masp is functional. other than known silk - related proteins, 11 sst translations with characterized uniprot protein matches were detected in the ampullate glands. four were exclusive to the major ampullate gland, four were identified in the major ampullate and minor ampullate glands, one was found in the minor ampullate and tubuliform glands, and two were in all glands (figure 2). the four proteins exclusive to the major ampullate gland were three enzymes and a protein that have been described in vertebrates (abbreviation, sst to uniprot e - value) : alpha-2 macroglobulin (a2 m, 0), carboxylic ester hydrolase (ceh, 6 10), dimethylaniline monooxygenase (fmo3, 7 10), and putative triacylglycerol lipase (ptl, 3 10). because they are exclusive to major ampullate silk glands, a2 m, ceh, fmo3, and ptl are candidates for having specific roles in the synthesis of major ampullate silks. five uniprot annotated sst proteins beyond silk structural proteins were associated with two gland types (figure 2). major and minor ampullate glands shared four proteins : putative cuticle protein (cuticle, 2 10), putative gamma glutamyltranspeptidase-1 (-gt-1, 4 10), histidine decarboxylase (hdc, 0), and mucin related protein 89f (mur89f, 2 10). minor ampullate and tubuliform silk glands contained one protein in common : scavenger receptor cysteine - rich protein (srcr ; 1 10). cuticle proteins have previously been described in spider silk gland ducts, but the other proteins are undescribed in spiders. on the basis of the glands in which they were found, these five proteins may be involved in either functions exclusive to ampullate glands (cuticle, -gt-1, hdc, mur89f) or functions that are more general to different silk gland types (srcr). two non - silk - related sst proteins were found in all three silk gland types. these were an oxygen transport molecule and a carbohydrate binding protein : hemocyanin subunit d (hemo_d, 0) and a putative lectin (lectin, 6 10), respectively. because of their detection in the three silk glands, these proteins may be common to other types of spider silk glands, such as aciniform, pyriform, and flagelliform. analysis of protein extractions from major ampullate glands and fibers resulted in 527 protein predictions (scaffold samples report is supporting information file s3). of the 527 proteins, 86 were found in the fiber (supporting information file s3). twenty - nine of the 527 proteins had sst encoded protein matches, with 17 in the fiber (supporting information file s4). only nine of the 29 were unique to this major ampullate gland and fiber analysis ; the remaining sst encoded proteins overlapped with those identified in the three - gland analysis of major ampullate, minor ampullate, and tubuliform silk glands. some of the 29 sst predicted proteins were fragments of the same protein ; thus, we identified a total of 25 unique putative proteins (figure 4 and supporting information file s4). these fell into four groups : six matched silk structural proteins, nine were other characterized proteins, three were uncharacterized, and seven had no hits in the uniprot database (figure 4 and supporting information file s4). crp1 was identified and considered a silk structural protein (figure 4, scaffold i d no. chart of presence (brown) or absence (purple) of peptide - predicted proteins encoded by ssts from l. hesperus major ampullate silk glands and fibers. proteins are arranged alphabetically by name of best uniprot hits < 1 10 within groups : silk structural proteins, other characterized proteins, uncharacterized proteins, and proteins with no uniprot hits., protein type has more than one predicted protein. abbreviations : a2 m, alpha-2 macroglobulin ; ac, aciniform ; asg, aggregate spider glue ; ceh, carboxylic ester hydrolase ; crp, cysteine - rich protein ; cuticle, cuticle protein ; fmo3, dimethylaniline monooxygenase ; gt, glutamyltranspeptidase ; hdc, histidine decarboxylase ; hemo_d, hemocyanin subunit d ; lectin, putative lectin ; ma, major ampullate ; mi, minor ampullate ; mur89f, mucin related 89f ; ptl, pancreatic triacylglycerol lipase ; scaffold i d no., scaffold predicted protein number ; sp, spidroin. we predicted that major ampullate fiber sst matches would be a subset of the gland sst matches. indeed, of the 25 unique predicted sst proteins, 12 were found in the fiber and the gland, nine were exclusive to the gland, and four were exclusive to the fiber (figure 4 and supporting information file s4). we did not expect to find any proteins exclusive to the fiber because the fiber is a product of the gland. one of the fiber - exclusive sst proteins matched an uncharacterized uniprot protein, and the remaining three had no hits in the uniprot database. these four fiber - exclusive sst encoded proteins may have been present in the gland, but they were difficult to detect because the gland has a greater variety of proteins than that in the fiber. in addition, the possibility of contamination of major ampullate fibers with products from other silk glands can never be entirely dismissed despite our use of microscopic observation while collecting fibers. consistent with our results from the three - gland analysis, we identified the expected masp1, masp2, and crp1 in the major ampullate gland and we also found acsp1, asg2, and masp. three of these silk structural proteins were identified in the fiber : acsp1, crp1, and masp1. discovery of acsp1 protein in the fiber confirms the protein matches to acsp1 in ampullate glands (figures 2 and 4). similarly, the presence of crp1 and masp1 is consistent with previous peptide studies of l. hesperus major ampullate glands and fibers. the nondetection of masp2 may be due to a greater proportion of masp1 in the fiber. the relative proportions of masp1 and masp2 varies in draglines, and previous evidence suggests a more than 2-fold greater abundance of masp1 compared to that of masp2 in l. hesperus major ampullate fibers (2.5:1 ratio of masp1 to masp2). alternatively, the lack of masp2 detected in the fiber could be due to poor solubility of this spidroin, hampering its inclusion in the protein digests. of the nine sst proteins with uniprot matches that were not silk - related, four were identified in both glands and fibers and five were exclusive to the gland. the four in both glands and fibers were also previously identified in the three - gland analysis : ceh, cuticle, lectin, and mur89f. the identification of these four proteins in major ampullate fibers suggests that they may be crucial to fiber assembly or function. we identified 48 sst encoded proteins in our three - gland analysis and nine additional sst encoded proteins in the major ampullate gland and fiber analysis (supporting information files s2 and s4). thus, we provide evidence for translation of 9% (57/647) of the ssts from clarke. these included known silk - related, non - silk - related, and uncharacterized proteins. we show that silk structural genes that are not known to have dominant expression in a given gland type are translated and that some of these nondominant proteins are incorporated into draglines (e.g., acsp1). peptide analysis also demonstrates translation of putative enzymes, cuticle, and lectin proteins and 18 predicted proteins encoded by ssts that had no uniprot hit. these findings provide clues about the evolutionary history of spider silk types and suggest new ways that spiders may vary silk properties. this result is consistent with the differentiation of major and minor ampullate glands from a more generalized ampullate gland type. we also show that minor ampullate glands contain masp1 and masp2 and that acsp1 is present in dragline silk fibers. inclusion of masp1 and masp2 in minor ampullate silk fibers and inclusion of acsp1 in draglines could modulate the tensile properties of silk fibers. similarly, the expression of asg2 glue proteins in l. hesperus ampullate glands may improve the utility of ampullate silks for prey capture by altering major and minor ampullate silk properties. overall, our proteomic analysis combined with rna - seq - derived transcript predictions provides greater insight into the evolution and synthesis of spider silks.	spider silk research has largely focused on spidroins, proteins that are the primary components of spider silk fibers. although a number of spidroins have been characterized, other types of proteins associated with silk synthesis are virtually unknown. previous analyses of tissue - specific rna - seq libraries identified 647 predicted genes that were differentially expressed in silk glands of the western black widow, latrodectus hesperus. only 5% of these silk - gland specific transcripts (ssts) encode spidroins ; although the remaining predicted genes presumably encode other proteins associated with silk production, this is mostly unverified. here, we used proteomic analysis of multiple silk glands and dragline silk fiber to investigate the translation of the differentially expressed genes. we find 48 proteins encoded by the differentially expressed transcripts in l. hesperus major ampullate, minor ampullate, and tubuliform silk glands and detect 17 sst encoded proteins in major ampullate silk fibers. the observed proteins include known silk - related proteins, but most are uncharacterized, with no annotation. these unannotated proteins likely include novel silk - associated proteins. major and minor ampullate glands have the highest overlap of identified proteins, consistent with their shared, distinctive ampullate shape and the overlapping functions of major and minor ampullate silks. our study substantiates and prioritizes predictions from differential expression analysis of spider silk gland transcriptomes.
the roles of the internal dynamics of protein molecules in supporting their biological functions are becoming increasingly apparent. proteins are not static in living organisms ; rather, their structures have complex dependencies on their environment and are also subject to thermally driven motions. the energy landscapes of proteins are rich and highly variable. the structural rigidity of a protein can range from being able to stay active in boiling water to essentially having no regular structure under any known circumstances. furthermore, some proteins are known to be highly ordered only under certain conditions. with reference to the more ordered proteins, ansari. have talked about fims (functionally important motions) and, by extension, bums (biologically unimportant motions), both of which are present in all proteins, indeed one goal of research in this area is to tell them apart ! since the earliest discussions about allostery, it was easy to accept that a conformational change could occur when a small molecule bound to a protein and that this energy of binding could change the structure, and hence the functional properties, of the protein. conformational change just as molecular biologists invoke trans - acting factors and geneticists say there must be a gene. each discipline has its reductionist arm - waving explanation waiting to be tested. one extreme of this view of changing structures, which is quite reasonable, is that allostery can occur from changes in populations ; this differs from the idea that individual proteins have to undergo some sort of concerted conformational change to achieve a functional difference. this view raises the possibility that all proteins are allosteric at some level, or at least potentially so. crystallographers and other structural biologists have sought out and revealed many such conformational changes, putting forth switching mechanisms to explain protein function. yet the crystallographers are inadvertently guilty of referring to the structure, as if there were only one. even the fluctuations present within the constraints of the crystal lattice are larger than often appreciated. the root mean square deviations for the best behaved part of the protein in a crystal structure are about one - tenth of the nominal resolution, so typically a couple of tenths of an angstrom (). these can range from tiny vibrational states of bonds (or smaller if you want to go into atomic or subatomic physics) to complete unfolding / refolding transitions. natural selection determines which of these matter to the function of the protein. some proteins, such as hemoglobin, appear to be fairly rigid and yet have different functional states, and others have completely different folding patterns depending on the biological environment (for example, prions). the estimated mean square deviations derived from crystallography might be thought of as being close to the lower limits of an ensemble distribution. it is common to refer to proteins without clear heteronuclear single - quantum coherence (hsqc) spectra as unfolded. a better (though overly technical) description might be too broad of an ensemble to satisfy the proper exchange regime for an nmr determination. many times, these proteins can be crystallized, so it is unlikely that they are unfolded in the literal sense. studies of the variation of the structure of proteins crystallized in different forms also point to the existence of conformational ensembles, as they can be correlated with other methods such as nmr. these studies support the notion that crystal lattices do not so much distort the structure as select amongst members of the ensemble that exist in the freely tumbling phase. at first, there were perceived discrepancies between the atomic coordinates that crystallographers determined and those resulting from the application of molecular mechanics force fields. crystallographers were distrustful of the simulations, and molecular dynamicists were quick to blame the crystal environment as an artificial condition. the more recent point of view holds that both camps were right in a sense. simulations are still difficult to run for long enough to get adequate sampling over time periods typical of experiments and the force fields suffer from necessary approximations, and it is now recognized that crystal structures vary to some degree in different crystal forms. these different camps now seem to be converging on a similar view. in molecular dynamics simulations, evidence for tiers of states can also be seen. the concept of a folding funnel also embodies the idea, with a gradual narrowing of the distribution as proteins relax into lower energy states after being synthesized or unfolded. some substates in crystals can be frozen out by lowering the temperature, and some not. this quenching of the structure into different substates also argues for there being many states with quite similar energies. in a more microscopic way, the bond lengths and angles that give rise to atomic arrangements and their diffraction patterns can vary, giving rise to an ensemble with an average electron density distribution that does not conform to any representation of a single structure, even if modeled as one. crystallographers usually recognize this effect only in higher resolution structures where discrete conformations of atoms can be distinguished, but there are promising ways of modeling an ensemble directly from the diffraction data even at lower resolutions. they are necessary, as a stochastic approach is used to generate models that satisfy the experimental observations, typically many more than are selected to represent the final set of models. the extent to which these ensembles represent the true ensemble is debatable as they may be under - constrained by the experimental data. certainly, the depiction of the coordinates as a set instead of a single structure gives a more realistic mental image than a single structure. modern nmr methods also continue to develop new relaxation methods for observing protein dynamics, including hydrogen deuterium exchange experiments (both reviewed by faculty of 1000 biology). models for what the ensemble of protein conformations might be when not constrained by a crystal lattice have recently been developed and used to illustrate likely modes of motions of the proteins (reviewed by faculty of 1000 biology). furthermore, reducing the extent of the ensemble possibilities in proteins by empirically changing the amino acid sequence can result in fewer states and thus a narrower and sometimes more stable folded state. this characteristic is important for developing longer shelf lives for protein - based pharmaceuticals or more robust catalysts for industrial processes. biophysical techniques for observing or describing the flexible nature of proteins and their resulting ensembles continue to improve in both speed and accuracy. x - ray diffraction and magnetic resonance methods both promise to deliver single - molecule structure determinations, the former by diffract and destroy free electron laser illumination and the latter by nanoscale magnetic resonance imaging. if these new ideas come to fruition, scientists would be able to look at individual examples of conformations and at the ensembles directly.	protein molecules are not static but are in varying degrees of motion. of course, the breadth of structural arrangements in an ensemble will vary tremendously. some proteins are like rocks and are very tightly constrained in terms of deviations of their coordinates from their averages. others are writhing snakes, with little recognized persistent structure, and then there is everything in between. through the work of many, there is an increasing awareness of the role of dynamics in the biological function of proteins, and new computational and experimental methods are allowing us to make these connections. a personal perspective on the state of affairs is offered with examples primarily from the author 's own work.
a meningoencephalocele is the extracranial herniation of brain tissue with meninges through a defect in the skull. sincipital and basal encephaloceles present as masses in relation to the nose and may cause external nasal deformity and hypertelorism. congenital lesions arise due to defect in the developing anterior neuropore in the region of foramen cecum. in the asian population, transethmoidal type is the most common basal encephalocele which also includes sphenoethmoidal, transsphenoidal, and sphenoorbital type. pediatric meningoencephalocele has been traditionally addressed with transcranial approaches which have obviously higher complication rates. following the first report by wigand in 1981, the transnasal endoscopic approach has evolved tremendously for repair of cerebrospinal fluid (csf) rhinorrhea. advances in available instruments and surgical expertise have also led to effective management of meningoencephalocele via a single stage exclusively endoscopic approach in select cases. although literature is now emerging, a search on pubmed with the keywords such as children, encephalocele, and endoscopic used in combination revealed about 20 studies (out of 51 hits) addressing this condition. we describe the patient profile, surgical nuances, and outcomes of transnasal endoscopic repair of pediatric intranasal meningoencephalocele in the review of six cases operated at our center. a retrospective study involving six consecutive patients below 12 years of age with intranasal meningoencephalocele treated by endonasal, endoscopic approach at our tertiary center from july 2013 to june 2014 has been carried out. the follow - up period ranged from 6 months to 2 years. a detailed history and clinical evaluation with specific emphasis on duration, mode of onset, associated congenital malformations, features of raised intracranial pressure, meningitis, and nasal endoscopy (if possible) for the radiological evaluation, a spiral computed tomography (ct) scan of the paranasal sinuses with 12 mm sequential sections and a magnetic resonance imaging of the brain with paranasal sinuses with cisternography sequences were carried out in all cases to localize the defect in the anterior skull base, to decide the surgical exposure necessary, and to rule out any associated pathologies, especially hydrocephalus [figure 1 ]. preoperative radiological evaluation of patient 4 showing a transethmoidal variety of encephocele on noncontrast computed tomography and magnetic resonance t1 and t2 imaging nasal fluid evaluation for confirming csf rhinorrhea was however not possible in all cases due to either scanty leaks or for want of the child 's cooperation. following a fitness for general anesthesia and with due consent of the child 's guardian, the child was placed under cover of the 3 generation cephalosporin antibiotic for 7 days starting from the time of surgery. no antiepileptic drug was added for the surgery unless the child was already on treatment. four millimeters endoscopes, 0 and 30, were used for surgery. following topical and infiltration decongestion, an initial attempt was made to localize the site of the fistula between the cranium and the nose, thereby estimating the tissue required for repair. defining the site endoscopically in terms of relation to the skull base and turbinates and correlating with the imaging findings were imperative in delineating the defect in the skull base with maximum precision. after circumferential release of mucosal adhesions, the protruding mass was reduced using bipolar coagulation under constant irrigation flush to the level of skull base. if middle turbinectomy was necessary, only the vertical lamella was fractured and the turbinate preserved for use as a vascularized mucosal flap. the mucosa at the margin of the defect was coagulated to create a raw area and promote healing. defects 1 cm in size were managed with an initial fascia - cartilage gasket (intracranial, extradural) with fascia overlying the bony margins and fat - fascia (extracranial) for support. the entire repair site was covered with either the middle turbinate mucosal flap or nasoseptal mucosal flap harvested from the same side and fibrin glue applied [figures 2 and 3 ]. site of defect in ethmoid roof as seen on sagittal computed tomography sequence with schematic representation of fascia - cartilage cartilage, red vascularized mucosal flap (a) encephalocele in right middle meatus, (b) encephalocele being reduced to identify skull base, (c) encephalocele reduced and bony margins of defect identified, (d) repair with fascia - cartilage gasket seal a polyvinyl acetate nasal pack was placed on the operated side at the end of the surgery. the child was nursed under cover of antibiotics with complete bed rest for 72 h. the nasal pack and lumbar drain were removed 4872 h after the surgery. laxatives, antitussives, and osmotic diuretics were used to avoid elevation in intracranial pressure during the phase of primary healing for the 1 week. following 5 days of care on indoor basis, the child was discharged and followed up at 2 weeks, 1 month, and then monthly for the first 6 months. serial endoscopic evaluation was done on follow - up and imaging reserved for patients with history of rhinorrhea, meningitis, or seizures in the postoperative period. patient profile, causative factors, significant history, radiological findings, surgical technique and follow up of the enrolled cases a detailed history and clinical evaluation with specific emphasis on duration, mode of onset, associated congenital malformations, features of raised intracranial pressure, meningitis, and nasal endoscopy (if possible) were included in the initial workup. for the radiological evaluation, a spiral computed tomography (ct) scan of the paranasal sinuses with 12 mm sequential sections and a magnetic resonance imaging of the brain with paranasal sinuses with cisternography sequences were carried out in all cases to localize the defect in the anterior skull base, to decide the surgical exposure necessary, and to rule out any associated pathologies, especially hydrocephalus [figure 1 ]. preoperative radiological evaluation of patient 4 showing a transethmoidal variety of encephocele on noncontrast computed tomography and magnetic resonance t1 and t2 imaging nasal fluid evaluation for confirming csf rhinorrhea was however not possible in all cases due to either scanty leaks or for want of the child 's cooperation. following a fitness for general anesthesia and with due consent of the child 's guardian, the child was placed under cover of the 3 generation cephalosporin antibiotic for 7 days starting from the time of surgery. no antiepileptic drug was added for the surgery unless the child was already on treatment. four millimeters endoscopes, 0 and 30, were used for surgery. following topical and infiltration decongestion, an initial attempt was made to localize the site of the fistula between the cranium and the nose, thereby estimating the tissue required for repair. defining the site endoscopically in terms of relation to the skull base and turbinates and correlating with the imaging findings were imperative in delineating the defect in the skull base with maximum precision. after circumferential release of mucosal adhesions, the protruding mass was reduced using bipolar coagulation under constant irrigation flush to the level of skull base. if middle turbinectomy was necessary, only the vertical lamella was fractured and the turbinate preserved for use as a vascularized mucosal flap. the mucosa at the margin of the defect was coagulated to create a raw area and promote healing. defects 1 cm in size were managed with an initial fascia - cartilage gasket (intracranial, extradural) with fascia overlying the bony margins and fat - fascia (extracranial) for support. the entire repair site was covered with either the middle turbinate mucosal flap or nasoseptal mucosal flap harvested from the same side and fibrin glue applied [figures 2 and 3 ]. site of defect in ethmoid roof as seen on sagittal computed tomography sequence with schematic representation of fascia - cartilage cartilage, red vascularized mucosal flap (a) encephalocele in right middle meatus, (b) encephalocele being reduced to identify skull base, (c) encephalocele reduced and bony margins of defect identified, (d) repair with fascia - cartilage gasket seal a polyvinyl acetate nasal pack was placed on the operated side at the end of the surgery. the child was nursed under cover of antibiotics with complete bed rest for 72 h. the nasal pack and lumbar drain were removed 4872 h after the surgery. laxatives, antitussives, and osmotic diuretics were used to avoid elevation in intracranial pressure during the phase of primary healing for the 1 week. following 5 days of care on indoor basis, the child was discharged and followed up at 2 weeks, 1 month, and then monthly for the first 6 months. serial endoscopic evaluation was done on follow - up and imaging reserved for patients with history of rhinorrhea, meningitis, or seizures in the postoperative period. patient profile, causative factors, significant history, radiological findings, surgical technique and follow up of the enrolled cases trauma leading to fracture of the anterior skull base is a frequent cause of encephalocele in the pediatric age group, especially for the transethmoidal type of basal encephaloceles (4 out of 6 cases in our series). untreated meningoencephalocele / csf rhinorrhea leads to recurrent episodes of meningitis (5 out of 6 patients). delayed presentation with meningitis has also been reported in literature, and surgery is the treatment of choice with no benefits of prophylactic antibiotic. very few studies addressing endoscopic transnasal management of encephalocele in pediatric population are available in literature. reported a series of 11 cases with congenital encephaloceles managed endoscopically with a favorable outcome. in the present study, a total endoscopic repair was done in all cases with autologous fascia and/or fat harvested from the thigh. the use of endoscope permitted easy localization of the defect in the skull base intraoperatively without the use of fluorescein dye. congenital encephalocele with adherence to the cartilaginous framework of the nose was difficult to dissect intraoperatively (2 out of 6 cases). in these children, inadvertent resection of the nasal cartilaginous framework during surgical repair can lead to cosmetic deformity (1 out of 2 cases). in consistency with excellent results reported in recently published literature, we achieved both successful repair of the defect and avoidance of further episodes of meningitis or seizures in all cases. the repair site was found to be well epithelialized on endoscopy at 2 months postoperatively. the endoscopic approach hence provides an obvious advantage of being a minimally invasive procedure without need for cerebral retraction and a faster recovery than open approaches. the working space available in pediatric patients with an endoscopic approach is restricted ; hence, the procedure may be challenging in children below 5 years of age without use of special instruments such as a 2.7 mm endoscope. we have not yet used this approach in children below 5 years of age ; however, the literature reveals successful endoscopic repair in even younger children. patients with associated soft - tissue deformity of the craniofacial area are definite candidates for transcranial / transfacial / combined endoscopic and external approaches. potential effects on the developing craniofacial skeleton are always a concern for surgery in the pediatric patient, endoscopic, or external. transnasal endoscopic repair of anterior skull base meningoencephalocele is a favorable technique in select cases. being a minimally invasive single stage surgery, results in terms of hospital stay, cost of treatment, and cosmesis are obviously superior to conventional transcranial approaches. noncontrast ct of the peripheral nervous system with magnetic resonance cisternography permits excellent localization of the skull base defect and hence, invasive procedures such as ct cisternography can be avoided. surgery can be performed without image guidance and requires conventional endoscopic instruments only in children over 5 years of age. the size of the defect in skull base is frequently smaller than the size of the encephalocele ; hence, repair following defect localization is feasible endoscopically. the repair technique tailored to the size of defect as adapted by us provides a water - tight seal of the csf fistula and avoids recurrence. prolonged use antibiotics and lumbar drain insertion can be avoided in cases successfully treated by the endoscopic route. careful study of preoperative radiology and meticulous surgery bring about excellent outcomes in transnasal endoscopic repair of pediatric meningoencephalocele. a longer follow - up and a larger number of cases will help us produce consistently favorable results.	introduction : encephaloceles in relation to the nose are rare lesions affecting the skull base. in the pediatric population, majority are congenital lesions manifesting as nasal masses requiring surgical intervention.materials and methods : a retrospective study of 6 consecutive patients below 12 years of age with intranasal meningoencephalocele treated by endonasal endoscopic approach at our tertiary centre was done. the follow up period ranged from 6 months to 2 years. a detailed clinical and radiological evaluation of these cases was done. endonasal endoscopic repair (gasket seal / fat plug) was carried out in all cases.results:out of 6 patients, 4 patients had post - traumatic and rest 2 cases had congenital meningo - encephaloceles. all patients were asymptomatic in post - operative follow up period. one patient had minor complication of nasal alar collapse due to intra - operative adherence of encephalocele to cartilaginous framework.conclusion:transnasal endoscopic repair of anterior skull base meningoencephalocele is a minimally invasive single stage surgery, and has advantage in terms of lesser hospital stay, cost of treatment, and better cosmesis. the repair technique should be tailored to the size of defect to provide a water - tight seal for better outcome.
canine blood samples were obtained from owners, breeders, and blood banks or were made available as residual samples from the clinical pathology laboratory at the ryan veterinary hospital and were studied at the penngen laboratory, university of pennsylvania, from march to december 2015. most samples originated from philadelphia and the surrounding tristate area (pennsylvania, new jersey, and delaware) except for the greyhounds and dalmatians. the > 1 ml ethylenediaminetetraacetic acid (edta)anticoagulated blood samples were kept chilled and typed within 10 days of collection. to standardize results, 20 and 1% rbc suspensions were prepared for each sample as previously described.13 the few dea 7 + and dea 7 blood samples were typed by abri.1 there were no specific selection criteria, but rather the samples that could be made available as previously typed by abri. the antidea 7 antibody reagents were very weak and did not work satisfactorily in our hands. these studies were performed and approved by the institutional animal care and use committee at the university of pennsylvania. for dea 1 typing, a commercially available immunochromatographic strip technique2 was used according to the manufacturer 's instructions and as described previously by the penngen laboratory, adjusting blood samples to a 20% rbc suspension.13, 15 the results were captured by imaging,3 and the dea 1 band strength was assessed on a scale of 0 (no band) to 4 + (as strong as control band) by visual and densitometric analyses by genetools software.4 blood samples were centrifuged to pellet rbcs. the packed cells were washed 3 times with phosphatebuffered saline, each time discarding the supernatant. then, 10 l of packed cells was added to a culture tube5 with 1 ml low ionic strength solution6 to prepare a 1% rbc suspension. we adapted the original tube assay for kai 1 and kai 2 to a gel column technique. thereby, plain saline gel columns7 were used to detect agglutination reactions as previously described.16 briefly, 25 l of antibody reagent (kai 1, kai 2, dal provided by mc blais, montreal university) and dea 3 and 41 for extended typing (as well as saline as autocontrol), and 50 l of 1% rbc suspension were added on top of the gel and incubated at 37c for 15 minutes in the manufacturer 's incubator.8 thereafter, the cards were centrifuged for 15 minutes at 85 g, with the manufacturer 's centrifuge.9 the degree of agglutination strength was graded from negative (0 : all rbc at the bottom of the gel) to positive (4 + : all rbc at the top of the gel). results were interpreted as negative if 1 + with practically all cells pelleted. for detection of alloantibodies in plasma samples, we used the gel as previously described.8, 16 briefly, we prepared 24 plain saline gel columns for each sample by placing 25 l of plasma from kai 1 or kai 2 dogs and adding 50 l of kai 1 + or kai 1, and kai 2 + or kai 2 rbcs in liss, respectively. the results were graded as positive (majority of rbcs at the top of gel) or negative (majority of rbc at the bottom of the gel) as done with gel typing results above. canine blood samples were obtained from owners, breeders, and blood banks or were made available as residual samples from the clinical pathology laboratory at the ryan veterinary hospital and were studied at the penngen laboratory, university of pennsylvania, from march to december 2015. most samples originated from philadelphia and the surrounding tristate area (pennsylvania, new jersey, and delaware) except for the greyhounds and dalmatians. the > 1 ml ethylenediaminetetraacetic acid (edta)anticoagulated blood samples were kept chilled and typed within 10 days of collection. to standardize results, 20 and 1% rbc suspensions were prepared for each sample as previously described.13 the few dea 7 + and dea 7 blood samples were typed by abri.1 there were no specific selection criteria, but rather the samples that could be made available as previously typed by abri. the antidea 7 antibody reagents were very weak and did not work satisfactorily in our hands. these studies were performed and approved by the institutional animal care and use committee at the university of pennsylvania. for dea 1 typing, a commercially available immunochromatographic strip technique2 was used according to the manufacturer 's instructions and as described previously by the penngen laboratory, adjusting blood samples to a 20% rbc suspension.13, 15 the results were captured by imaging,3 and the dea 1 band strength was assessed on a scale of 0 (no band) to 4 + (as strong as control band) by visual and densitometric analyses by genetools software.4 the packed cells were washed 3 times with phosphatebuffered saline, each time discarding the supernatant. then, 10 l of packed cells was added to a culture tube5 with 1 ml low ionic strength solution6 to prepare a 1% rbc suspension. we adapted the original tube assay for kai 1 and kai 2 to a gel column technique. thereby, plain saline gel columns7 were used to detect agglutination reactions as previously described.16 briefly, 25 l of antibody reagent (kai 1, kai 2, dal provided by mc blais, montreal university) and dea 3 and 41 for extended typing (as well as saline as autocontrol), and 50 l of 1% rbc suspension were added on top of the gel and incubated at 37c for 15 minutes in the manufacturer 's incubator.8 thereafter, the cards were centrifuged for 15 minutes at 85 g, with the manufacturer 's centrifuge.9 the degree of agglutination strength was graded from negative (0 : all rbc at the bottom of the gel) to positive (4 + : all rbc at the top of the gel). for detection of alloantibodies in plasma samples, we used the gel as previously described.8, 16 briefly, we prepared 24 plain saline gel columns for each sample by placing 25 l of plasma from kai 1 or kai 2 dogs and adding 50 l of kai 1 + or kai 1, and kai 2 + or kai 2 rbcs in liss, respectively. the results were graded as positive (majority of rbcs at the top of gel) or negative (majority of rbc at the bottom of the gel) as done with gel typing results above. this large typing survey included 503 dogs from north america representing 80 breeds and mixed breed dogs. a larger number of dalmatians (n = 108 related to a separate dal typing study), greyhounds (70, mostly blood donors), and mixed breed (60) dogs were bloodtyped, whereas all other breeds only had 125 dogs (median, 6) represented. overall, there was no significant difference in blood type frequencies among breeds with at least 20 dogs typed, except for dal dogs (s. goulet, u giger, cc euler, mc blais., unpublished data, 2016). patterns of dea 1, and kai 1 and kai 2 typing results among 503 dogs from north america dea, dog erythrocyte antigen. canine typing results of dea 1, 3, 4, and 7 and dal related to kai 1 and kai 2. results < 1 + are graded as negative dea, dog erythrocyte antigen. external dea 7 typing results. utilizing the strip with a monoclonal antidea 1 antibody and grading the dea 1 band strength identified 40.4% dea 1 dogs, with the remainder having varied degrees of dea 1 positivity. adjusting the pcv to 20% and quantitative analyses of the dea 1 band strengths permitted differentiation of the degree of dea 1 positivity (fig 1). a large proportion of the dea 1 + samples showed a very strong band (3+/4 +), and only a small proportion exhibited 1 + and 2 + reactions. there was a close correlation between semiquantitative visual and densitometric assessment of the degree of dea 1 positivity differentiating dea 1 and weakly, moderately, and strongly dea 1 + dogs. none of dea 1 (and not previously transfused) dogs tested had any observable dea 1 alloantibodies by gel. median, range, and extremes for each densitometric dea 1 band strength compared to visual band assignment. monoclonal antibodies against red cell antigens were developed in south korea, and 1 antikai 1 and 1 antikai 2 antibody (j.h. we established a simple blood typing technique utilizing gel saline columns and adjusting the canine rbc quantity (1%) to compare the degree of the agglutination reactions (fig 2). the majority of canine blood samples typed kai 1 + with most showing strong (3+/4 +) and few moderate (2 +) and weak (1 +) agglutination reactions with the antikai 1 reagent. in contrast, nearly all dogs typed as kai 2 with only 5 dogs typed strongly kai 2 +, respectively (table 3). in addition, 5% of the dogs were kai 1 and kai 2, but most notably none were positive for both kai antigens. assessment of the plasma from all kai 1 and kai 2 dogs indicated no alloantibodies against kai 1 and kai 2 cells, respectively. gel column typing results showing the different kai typing patterns for kai 1 and kai 2. (a) kai 1+/kai 2, the most common pattern ; (b) kai 1/kai 2 ; and (c) kai 1/kai 2 +. the degree of agglutination shows grading from negative (0 : all of rbc at the bottom of the gel) to positive (4 + : all of rbc at the top of the gel) to determine whether there was a relationship between the new kai blood types and other blood groups, a subset of dogs were further typed by another laboratory for dea 7 (in our laboratory, the available dea 7 did not identify any agglutination reactions) or further typed for dea 3, 4 and dal with the gel and available polyclonal antisera from negative dogs sensitized with positive red cells (table 2). all dogs typed were dea 4 + with 1 exception ; this sole dea 4 dog was a dalmatian and typed as kai 1, kai 2, dea 1 +, dea 3 +, and dal+. no relationship between dea 1, 3, 4, and dal and kai 1 and kai 2 could be detected. specifically, there were kai 1 dogs that were dea 1 +, dea 3 +, dea 4 + or dal+. similarly, the few dogs that were kai 2 + could be dea 1, dea 3, or dal. utilizing the strip with a monoclonal antidea 1 antibody and grading the dea 1 band strength identified 40.4% dea 1 dogs, with the remainder having varied degrees of dea 1 positivity. adjusting the pcv to 20% and quantitative analyses of the dea 1 band strengths permitted differentiation of the degree of dea 1 positivity (fig 1). a large proportion of the dea 1 + samples showed a very strong band (3+/4 +), and only a small proportion exhibited 1 + and 2 + reactions. there was a close correlation between semiquantitative visual and densitometric assessment of the degree of dea 1 positivity differentiating dea 1 and weakly, moderately, and strongly dea 1 + dogs. none of dea 1 (and not previously transfused) dogs tested had any observable dea 1 alloantibodies by gel. median, range, and extremes for each densitometric dea 1 band strength compared to visual band assignment. monoclonal antibodies against red cell antigens were developed in south korea, and 1 antikai 1 and 1 antikai 2 antibody (j.h. we established a simple blood typing technique utilizing gel saline columns and adjusting the canine rbc quantity (1%) to compare the degree of the agglutination reactions (fig 2). the majority of canine blood samples typed kai 1 + with most showing strong (3+/4 +) and few moderate (2 +) and weak (1 +) agglutination reactions with the antikai 1 reagent. in contrast, nearly all dogs typed as kai 2 with only 5 dogs typed strongly kai 2 +, respectively (table 3). in addition, 5% of the dogs were kai 1 and kai 2, but most notably none were positive for both kai antigens. assessment of the plasma from all kai 1 and kai 2 dogs indicated no alloantibodies against kai 1 and kai 2 cells, respectively. gel column typing results showing the different kai typing patterns for kai 1 and kai 2. (a) kai 1+/kai 2, the most common pattern ; (b) kai 1/kai 2 ; and (c) kai 1/kai 2 +. the degree of agglutination shows grading from negative (0 : all of rbc at the bottom of the gel) to positive (4 + : all of rbc at the top of the gel) to determine whether there was a relationship between the new kai blood types and other blood groups, a subset of dogs were further typed by another laboratory for dea 7 (in our laboratory, the available dea 7 did not identify any agglutination reactions) or further typed for dea 3, 4 and dal with the gel and available polyclonal antisera from negative dogs sensitized with positive red cells (table 2). all dogs typed were dea 4 + with 1 exception ; this sole dea 4 dog was a dalmatian and typed as kai 1, kai 2, dea 1 +, dea 3 +, and dal+. no relationship between dea 1, 3, 4, and dal and kai 1 and kai 2 could be detected. specifically, there were kai 1 dogs that were dea 1 +, dea 3 +, dea 4 + or dal+. similarly, the few dogs that were kai 2 + could be dea 1, dea 3, or dal. two monoclonal antibodies against red cell antigens recently were developed in south korea. based on their original preliminary evaluation, these 2 murine antibodies, named antikai 1 and antikai 2 (kai refers to dog in korea), recognize 2 different canine red cell antigens (j.h lee, u. giger, h.y., unpublished data, 2016). to determine whether kai 1 and kai 2 exist in north america and are novel red cell antigens we documented the presence of both kai 1 and kai 2 in north america. as far as we could determine, neither kai 1 nor kai 2 is associated with each other (albeit we did not find any kai 1+/kai 2 + dogs) nor with any known canine blood group system. both kai 1 and kai 2 dogs as well as dea 1 dogs lacked any naturally occurring alloantibodies against kai 1 and kai 2 and dea 1, respectively. the clinical importance of these new blood groups in transfusion treatment still remains to be determined. in our survey of 503 dogs mostly from philadelphia and the tristate area, including a relatively large number of greyhounds (70 samples) and dalmatians (108 samples) from the usa and canada, 94% of dogs were kai 1 + but only a few (1%) were kai 2 +. therefore, the combination of kai 1 + and kai 2 was most frequently (94%) observed. the kai 1+/kai 2 blood type constellation was found in every blood donor greyhound tested, whereas in other breeds tested, kai 1 + and kai 1 dogs were found. among the few kai 2 + samples, 3 were from lhasa apsos. since completion of these studies, a limited preliminary survey from the united kingdom also found several lhasa apsos with kai 1/kai 2 + blood (watson, c. c. euler and u. giger, unpublished data, 2016). similarly, a recent preliminary survey of mastiffs in south korea indicated a 17% kai 2 + frequency (j.h. lee and h.y. another possible constellation found was kai 1/kai 2, whereas no dog was kai 1+/kai2 +. results of limited family studies are consistent with an autosomal dominant trait for kai 1 (j.h. because the frequency of the 2 phenotypes kai 1+/ and kai 2+/ are < 99% and 1%, respectively, kai 1 and kai 2 should be considered blood groups rather than high or lowfrequency (public or private) red cell antigens.17, 18 dea 4 was thought to be a blood group, although 99.5% of dogs seem to be dea 4 + and thus, it should be considered a highfrequency antigen. in contrast, dal was thought to be a highfrequency red cell antigen with dal originally only found in a few dalmatians, but larger surveys found dal dogs in dobermans and other breeds and thus, it should be recognized as a blood group.8, 9, 10, 11, 12 blood typing for kai 1 and kai 2 was determined by traditional tube agglutination assays in south korea. we adapted the gel column technique for kai 1 and 2 typing and alloantibody studies with neutral nacl gel column cards, which can be better standardized and are simple to perform and interpret, as we have previously shown for dea 113, 19 and other dea types16 as well as dal types.9, 10 the degree of agglutination was very strong (3 + or 4 +) for both kai 1 and kai 2 antigens in nearly all cases with a few samples giving weaker reactions (2 +). moreover, the results were stable during repeat typing, as previously shown for dea 1,13 and results were easy to interpret. however, we did have a few cases in which we observed an unexplained split reaction, with the majority of cells located on top of the gel despite having a few rbcs pelleted ; we designated such cases as 4 +. overall, these monoclonal antibodies and current gel column typing (and tube) techniques seem well suited to detect positive agglutination reactions for screening dogs for the kai 1 and kai 2 blood types in clinical settings. commercial kai typing kits may be developed in the future, if there is more clinical evidence for blood incompatibilities and transfusion reactions. the dea 1 blood group system has not been defined at the protein and molecular levels, but based upon recent studies seems to originate from a single locus with 4 alleles, and thus dea 1 and weakly to strongly dea 1 + dogs rather than dea 1.1, dea 1.2 and dea 1.3 types.1, 2, 13 we have shown previously that dea 1 typing with a monoclonal antibody by gel column, flow cytometry, and chromatographic strip produced identical results.13 in this survey of 503 dogs, we applied the chromatographic strip method with visual and densitometric grading at pcv 20% as previously described.13 the proportions of dea 1 and strongly dea 1 + (3+/4 +) dogs were far larger than those that were weakly (1 +) or moderately (2 +) dea 1 +. among breeds with at least 25 dogs represented, dea 1 and dea 1 + dogs were found, which is consistent with our prior smaller surveys13, 14 and also reports with dea 1.1 and 1.x polyclonal antisera.20 however, our survey included a biased group of dogs, because it included many greyhound blood donors from 2 animal blood banks, where dea 1 dogs are preferred, and many dalmatians related to a separate dal typing survey (s. goulet, u. giger, j. arsenault, a. abramsogg, c. c. euler, and m.c.., unpublished data).9, 10 finally, consistent with prior observations,3 plasma from the previously untransfused dea 1 dogs screened indicated a lack of naturally occurring antidea 1 alloantibodies. results of the dal typing survey of the dogs in our study will be presented elsewhere, but as expected based upon small prior surveys, dal dogs were found among dalmatians and doberman pinschers, shih tzus as well as in few other breeds (s. goulet, u. giger, j. arsenault, a. abramsogg, c. c. euler, and m.c. unpublished data, 2016).9, 10 however, these frequencies of dal dogs may not be representative because the number of dogs tested per breed was small and owners and breeders of dal dogs likely submitted samples from related dogs in order to find potential blood donors for their dal dogs. again, the dal blood group system has no relationship to the new kai 1 and 2 blood group systems. comparison of the dea 1 typing with kai 1 and kai 2 typing results in our survey indicates that these are different blood group systems. reagents are no longer available for all previously reported canine blood group systems, such as dea 5, 6, and 8. these dea canine blood groups and alloantibodies had not well been characterized (mostly based upon agglutination reactions),15, 18 and consequentially, it will be impossible to determine whether the antigens kai 1, kai 2, or both are related to any previously described blood groups. however, based upon our limited extended typing comparisons, there is no relationship to the known canine blood groups for which reagents were available (dea 1, 3, 4, 7, and dal). thus, kai 1 and kai 2 appear to be newly discovered red cell antigens. moreover, a direct relationship between kai 1 and kai 2 could not be determined, although there were no dogs positive for kai 1 and kai 2. however, a few dogs were kai 2 + and based upon immunoblotting studies utilizing the 2 monoclonal antibodies, the sizes of the 2 red cell antigens are different (j.h lee, u. giger, h.y. these dea and kai 1 and kai 2 typing surveys and alloantibody studies have clinical implications. from a practical perspective, it is highly advantageous that most dogs are either clearly dea 1 or strongly dea 1 + and similarly clearly negative or positive for either kai 1 or 2 type. currently, it is not known whether weakly to moderately dea 1 + cells will induce alloantibodies in dea 1 recipient dogs and whether these weakly to moderately dea 1 + dogs would be sensitized by strongly dea 1 + blood. we continue to follow the cautious practice of typing donor and recipient, giving dea 1 and also weakly dea 1 + recipient dogs only dea 1 blood, and labeling weakly or moderately dea 1 + donors as dea 1 + donors.3, 13 recommendations for kai must await demonstration of any alloantibody development after transfusions. preliminary unpublished experimental studies show the development of antikai 1 and antikai 2 alloantibodies in kai 1 and kai 2 dogs, respectively (j.h. lee and h.y kim. the lack of naturally occurring alloantibodies against dea 1 and both kai antigens further supports the practice of not crossmatching before a first transfusion.3 although acute hemolytic transfusion reactions have been documented in previously transfused dogs due to dea 1, dea 4, and dal incompatibilities,2, 8, 11, 12 there is no published evidence of alloantibody production and acute hemolytic transfusion reactions due to kaimismatched transfusions. indeed, we used monoclonal murine antibodies against kai 1 and kai 2 and not antikai alloantibodies from previously transfused dogs for typing. however, unpublished clinical results after kaimismatched transfusions indicate the development of antikai 1 and antikai 2 alloantibodies and hemolytic reactions (j.h. lee and h.y. therefore, it appears appropriate to follow up dogs with incompatible crossmatches for kaitype mismatches and potentially select donors based also on the kai types in previously transfused dogs. in conclusion, we established a simple and accurate gel column typing technique for kai 1 and kai 2 and found both red cell antigens with most dogs to be kai 1+/kai 2 in north america. these blood types represent novel and distinct blood groups with potentially different frequencies among breeds. dogs negative for kai 1, kai 2, or both do not have any naturally occurring alloantibodies in their plasma. the clinical importance of kai 1 and kai 2 in canine transfusion medicine needs to be determined.	backgroundbased upon serology, > 10 canine blood group systems have been reported.objectivewe surveyed dogs for dog erythrocyte antigen (dea) 1 and 2 new blood types (kai 1 and kai 2), and some samples also were screened for dal and dea 3, 4, and 7.methodsblood samples provided by owners, breeders, animal blood banks, and clinical laboratories were typed for dea 1 by an immunochromatographic strip technique with a monoclonal antibody and analysis of band intensity. both new antigens, the dal and other deas (except dea 7 by tube method), were assessed by a gel column method with either monoclonal or polyclonal antibodies. the same gel column method was applied for alloantibody detection.resultsof 503 dogs typed, 59.6% were dea 1 + with 4% weakly, 10% moderately, and 45.6% strongly dea 1 +. regarding kai 1 and kai 2, 94% were kai 1+/kai 2, 5% were kai 1/kai 2 and 1% were kai 1/kai 2 +, but none were kai 1+/kai 2 +. there was no relationship between kai 1/kai 2 and other blood types tested. plasma from dea 1, kai 1, kai 2 dogs, or some combination of these contained no detectable alloantibodies against dea 1 and kai 1 or kai, respectively.conclusions and clinical importancethe new blood types, called kai 1 and kai 2, are unrelated to dea 1, 3, 4, and 7 and dal. kai 1+/kai 2 dogs were most commonly found in north america. the clinical relevance of kai 1 and kai 2 in canine transfusion medicine still needs to be elucidated.
in order to diversify the meat products consumed by the population, the meat industry has implemented marinating technology to help satisfy the psychological need of consumers to broaden their choice of foods and to maintain its market share. several studies have been carried out to optimize this technology in different kind of meats, namely, beef, pork, and poultry [15 ] although, according to the authors of former papers, marinated pork is, perhaps, the most common. the marinating is based on the water - binding capacity of several compounds, such as sodium chloride, sodium lactate, calcium lactate, lactic acid, and calcium chloride. the salt plays several functions and provides some functional properties to the meat. as a bacteriostatic agent, the salt increases the shelf - life of meat and meat products by reducing the growth rate of spoilage bacteria [6, 7 ]. similarly, the salt activates the protein to increase hydration and its water holding capacity, thus increasing the binding properties of proteins, which is an important effect since the meat proteins can swell to twice their original size. additionally, salt improves the texture [8, 9 ] and palatability by increasing its juiciness, and it also improves the tenderness and overall acceptability [11, 12 ]. the addition of sodium lactate was shown to improve color and to help to stabilize this feature during storage [1315 ]. fresh meat presents a highly variable microbiota [16, 17 ] but regardless of the initial number of organisms, the most common spoilage of fresh meat in air - stored conditions is dominated by gram - negative, psychrotrophic, aerobic rod - shaped bacteria. although a wide range of genera are present on meat, only pseudomonas, acinetobacter, and psychrobacter (formerly some acinetobacter and moraxella) species are normally considered to be important.. typically account for > 50% of the total microbiota and sometimes even up to 90%. the spoilage is manifested by the release of off - odors when the number of bacteria reaches the level of about 5 10 cfu / cm and the appearance of slime when the bacterial load reaches the value 10 cfu / cm [21, 22 ]. marinated meat products have some of the benefits discussed above, but they may have an added problem and limited food safety, since there may be pathogenic cross - contamination during processing and storage, as well as from raw materials such as fresh meat, sauce, and fresh vegetables [23, 24 ]. marinated pork is mainly distributed by the industry as whole pieces but they are usually displayed as slices packed in family - sized portions exposed on the refrigerated shelves of supermarkets so that consumer can freely choose the product, which is then cooked at home. the slices preparation involves manipulation during which the product may be potentially contaminated with pathogen bacteria from the environment, handlers, equipment, and so forth. an additional problem is the temperature abuse (e.g., increase to 710c), which could occur during distribution. among the pathogens that may be encountered, those of the greatest concern are listeria monocytogenes and salmonella spp. they can be considered ubiquitous. however, l. monocytogenes is the most dangerous of the two organisms since it is a facultative anaerobic, which grows even under strict refrigeration [25, 26 ], while salmonella spp. are not able to multiply at refrigerated temperatures but they can at > 7c [26, 27 ]. another highly dangerous pathogen is escherichia coli o157:h7 since it is responsible for a severe disease process [26, 28 ]. however, it is less frequent and unable to grow under refrigerated conditions but, like salmonella spp. thus, the risk of the food - borne pathogens growth may be higher when there is a temperature abuse by retailers and consumers. it may be considered that the marinated pork also has a short shelf - life since this is an uncooked product. considering to the current trade situation of perishable foods over long distances, it is obvious that any action applied to increase the shelf - life of this product would be very useful from a commercial point of view. the present work proposes the treatment with accelerated electrons (e - beam) to extend its shelf - life. this treatment is also useful to substantially reduce the potential pathogens that may be present since that e - beam is an effective procedure to kill vegetative pathogen bacteria [3136 ]. salmonella spp. resistance against e - beam radiation is similar to that of l. monocytogenes [37, 38 ], and it has been repeatedly demonstrated that e. coli o157:h7 is more radiosensitive than l. monocytogenes [31, 33, 37 ]. therefore, the results obtained for the l. monocytogenes may also be extended to two other species with a high confidence range (95%) due to the psychrotrophic character of l. monocytogenes. likewise, in previous works, we have observed that l. innocua nctc 11288 is more radioresistant than five strains of l. monocytogenes. accordingly, l. innocua, as a surrogate of l. monocytogenes, was used as the target organism for experiments. l. innocua nctc 11288 was supplied by the spanish type culture collection (cect, valencia university, c. dr. fresh cultures were prepared by removing a piece of frozen culture and inoculating it into trypticase soy broth, then incubating at 32c for 24 h. the culture was subsequently centrifuged at 4c and the pellet suspended in a sterile test tube with 10 ml sterile saline, which yielded a bacterial load that was close to 10 cells / ml. the handling, subculture, and inoculum preparation of l. innocua and sample contamination have been previously described for other meat product slices [34, 35 ]. marinated pork loins were prepared in a local industry under supervision of one of the authors. whole loins were placed in a rotating drum, in which they were massaged for 15 min in a brine consisting of (w / v) salt (1.6%), nitrates and nitrites (0.025% of kno3/nano2 (2/1) (w / w)), sodium ascorbate (0.080%), and spices (1.4% of a mixture of white pepper / paprika (2/12) (w / w)). slices (46 mm thickness) were cut from freshly marinated pieces (3 - 4 kg in weight) using an electric machine, whose rotating blade and contact surfaces were previously deeply cleaned with hot water (around 90c) and ethanol 70. following this, 24 slices were packaged in low permeability plastic (60 m copolymer of poliamide / polietilene) bags (diffusion coefficient of 35 cm/24 h m bar to oxygen and 150 cm/24 h m bar to carbon dioxide) and heat - sealed without air exclusion. the gases ratio for filling bags was air / meat 4/1 (volume bag / sample weight). they were treated in an industrial electron beam radiation source, which operates at 10 mev located in tarancn, cuenca, spain, 60 km from the laboratory. the radiation doses employed were between 0.2 and 3 kgy, namely, 0.2, 0.5, 1, 1.5, 2, 2.5, and 3 kgy and the dose absorbed by samples was verified considering the absorbance of cellulose triacetate dosimeters simultaneously irradiated. to determine the inactivation of l. innocua, slices were contaminated by immersion for 10 sec in the cell suspension (higher than 10 cells / ml formerly described, which yield an initial load of approximately 10 cfu / cm. a large number of cells (> 10 cells / cm) were used to determine the death kinetic parameters accurately. the contaminated (one slice per trial for microbial analysis) and uncontaminated (four - five slices for physicochemical or sensorial analysis) samples were packaged as mentioned above. following the irradiation treatment, samples were transported (less than 1 h) in insulated boxes to the laboratory and they were stored in termostated chambers at 4 and 8c, the latter was used as an example of temperature abuse during product distribution. to count the survivors, an aliquot (about 1 g) were homogenized with 10 ml of a sterile saline solution in a stomacher bag. total viable counts (tvc) were determined by the pour - plate method using plate count agar (pca ; difco, detroit, mi, usa) containing 2% (w / v) nacl [40, 41 ] as the culture medium. lactic acid bacteria (lab) were enumerated in double layer mrs agar (conda - pronadisa, madrid, spain) at ph 5.5 [42, 43 ]. the incubation was carried out at 32c for 48 h. enteriobacteriaceae were counted in violet red bile glucose agar (oxoid, basingstoke, hampshire, uk) after an incubation of 24 h at 37c. counts were determined after incubation at 25c for 48 h in pseudomonas agar base, supplemented with cetrimide, fucidin, and cephalosporin (oxoid), brochothrix thermosphacta was enumerated in staa (oxoid) at 25c for 48 h. the selective palcam medium (oxoid) for listeria spp. was elected to assay the survival counts of this organism and to avoid the growth of endogenous microbiota. the growth curves were constructed according to the baranyi model. in shelf - life experiments, irradiated and nonirradiated loin slices were removed from the trays, in which several groups of organisms were periodically determined. from a microbiological point of view, the end of shelf - life was established when the total viable counts exceeded the value of 5 10 cfu / cm. analyses were performed just after e - beam treatment (0 days) and at various times during storage until the end of the shelf - life. the dry matter was analyzed by the oven air - drying method (aoac, 1995). the marinated loin ph was determined in a homogenate of the sample with distilled water (1 : 10) (w / v), using a crison digit-501 ph meter (crison instruments ltd, barcelona, spain). the meat sample (0.3 g) was placed on a piece of filter paper (whatman no. 1, 125 mm), then set between two plexiglas plates, and subjected to a mechanical force of 345 kpa for 5 min. the whc values were calculated as the percentage of water retained based on water content in the product before pressing. the tpa and tensile test were performed with a ta.xt2i sms stable micro systems texture analyser (stable microsystems ltd., surrey, england) using a cylindrical probe p/25 for tpa or a tensile grip (a / tgt) for the tensile test. the tpa was assayed in cylinders (1.5 cm high by 2 cm wide), and the tensile test was carried out on prismatic pieces (6 cm long, 2 cm wide, and 0.4 cm thick) of marinated loin samples. the resulting textural parameters were calculated as previously described by herrero.. the measurements were performed using a tristimulus colorimeter (minolta chroma meter cr300, minolta corporation, nj). the values of the lightness (l), redness (a), and yellowness (b) parameters were periodically (0, 5, and 10 days of storage at 4c) measured 58 times on the surface of the e - beam treated and control (nontreated) slices at three different analyse times (0, 4, and 24 h to air exposure after opening the packaged bags). after the first color measurement, samples were kept at 6 2c and about 64 2% relative humidity, without protection (similar conditions to a refrigerated display or a domestic refrigerator). color parameters were determined in non - treated and treated samples at 0, 5, and 10 days of storage at 4c. the sensory analyses involved a panel of twenty tasters (ten females and ten males) selected from the members of the departamento de nutricin, bromatologa y tecnologa de los alimentos. slices treated at 0, 1, and 2 kgy were used for sensory analysis. the following tests were performed : a triangular analysis, a rank order test, and a descriptive trial. the tests were carried out in individual booths built according to the international standards organization dp 66.58 criteria. for the flavor analysis, pork loin steaks with a thickness of 0.5 cm were cooked during 2 min on each side using a grill - pan previously heated to 150c. the temperature inside the steaks reached approximately 70c, as measured by a portable digital thermometer (testo model 735 ; testo, s.a., the range order test was performed until the end of the shelf - life of untreated samples. triangular and descriptive tests were performed until the end of the shelf - life of the untreated and treated samples. survival curves were obtained by plotting the logarithm of the number of survivors against the dose assayed. decimal reduction dose (d - values) were calculated from the linear regression equation of survival curves. regression equations, coefficients of determination (r) and the error bars were calculated by excel (microsoft, redmond, wa, usa). in the case of the data obtained with the physicochemical analysis, the fresh pork loin used in this research had average moisture content values of 74.16 1.56%, an ash concentration of 1.88% 0.19%, dry matter content of 27.3 1.6%, aw = 0.992 0.005, and ph = 5.71 0.037. the effect of e - beam treatment (1 and 2 kgy) on the former parameters was negligible, except for whc (table 1). these results, excluding the latter feature, are in total agreement with previous data obtained for e - beam treated fresh pork loin. there were not observed effects in pork loin [50, 51 ] nor in ground beef patties even when ascorbic acid was added to samples and they were treated with a dose as high as 10 kgy. the effect of e - beam irradiation (1 and 2 kgy) on the whc of the marinated product is recorded in table 1. the slices showed a significant increase of the whc (p no regulation has been set by the ec for l. monocytogenes in relation to this kind of product. however, as the shelf - life is extended by e - beam treatment, there is, if present, an opportunity for l. monocytogenes to grow due to its psychrotrophic condition thereby increasing the risk of dissemination of this organism through cross - contamination. therefore, it seems convenient to be aware of its potential increase in numbers during the storage period. when trying to optimize any process (in this case e - beam treatment) to sanitize a meat product, taking into account the growth of l. monocytogenes during its shelf - life is necessary in order to eliminate it to ensure a level that guarantees the product safety. the resulting treatment will be enough to reduce the number of salmonella, if present, since the latter bacterium does not grow (at 4c) or grow more slowly (at 8c) than l. monocytogenes. in fact, the previous literature reports a g - value of 22 h at 10c for s. enteritidis and 57 h at 9.3c for l. monocytogenes. for these reasons, in this work the optimization with e - beam treatment has been performed with l. monocytogenes, using l. innocua as a surrogate. the response of l. innocua to the e - beam treatment was fitted to first - order inactivation kinetics, following the equation : log cfu / cm = 7.0277 2.1918 dose (r = 0.9944), from which a decimal reduction value (d - value) of 0.46 kgy was calculated. this value validate the death kinetic of this bacterium in meat products since values of 0.49 kgy and 0.44 kgy were previously determined in cooked ham and fresh pork loin. among nonsporeforming pathogens, l. monocytogenes is one of the most radioresistant bacterium [62, 63, 77, 78 ]. several authors have reported increased numbers of l. monocytogenes in various products stored at 4 - 5c. the data have been collected in a fda report from which an average increase of 0.2 log units / day may be estimated in fresh meat and 0.35 log units / day when storage is at 8c. so, assuming a contamination in the raw marinated loin of 10 cells / cm (log = 1), as suggested by the icmsf, the load of the nonirradiated loin slices will be 250 cfu / cm at 4c at the end of shelf - life (7 days) and 562 cfu / cm at 8c (shelf - life of 5 days). nevertheless, e - beam treatment provokes a 2.17 d and 4.35 d reduction with the application of 1 and 2 kgy, respectively. therefore, the e - beam treatment will reduce the level of listeria to 6.76 10 cfu / cm and 4.46 10 cfu / cm, respectively. as this bacterium is able to grow under refrigeration, its numbers will increase during storage in such a way that, assuming the same growth rates, the levels will be around 107 cfu / cm at the end of the shelf - life at 4c (16 days) with a dose of 1 kgy and 4.57 cfu / cm if 2 kgy was applied. in a temperature abuse situation (8c), with similar reasoning, the l. monocytogenes load at the end of the shelf - life may be estimated in 214 cfu / cm and 182 cfu / cm. thus, the e - beam treatment has led to an important improvement of the hygienic status. although more than 2,000 serovars of salmonella enterica are known, most infections in humans are caused by only a few serovars, the most common of which corresponds to s. enteritidis and s. typhimurium. publications [37, 61, 82 ] have repeatedly confirmed that the resistance of s. typhimurium to irradiation is significantly higher than that of s. enteritidis, and the d - value for the former organism is in the level of 0.450.50 kgy. assuming a contamination similar to that of l. monocytogenes (i.e., 10 cfu / cm) and a d - value of 0.47 kgy for s. typhimurium, the application of 1 kgy or 2 kgy would reduce the number of salmonella 2.13 d and 4.25 d, respectively. these figures mean that salmonella loads posttreatment would be 0.05) with increased storage irrespective of the dose applied. in addition, in each sample (treated with 0, 1, or 2 kgy and stored at 4c postprocessed) this parameter increased with more time of air - exposure once the package was opened. all these differences were minimized as the storage was extended in such a way that after 10 days, regardless of the dose, the e - beam treated samples at the beginning (0 day to the air exposure) were slightly more red but after exposure to air for 4 or 24 h the differences were practically eliminated. in the fresh loin, the differences in the a, b, and l parameters between control and e - beam treated samples were clearer than in marinated loin. it could be attributed to the marinating substances (mainly the paprika) overlooked the oxidizing effect of the radiation, which may be due to the availability of oxygen during the e - beam treatment. besides this, free radicals, ozone, and oxygen peroxide are produced by radiolysis of water. these compounds are strong oxidizing agents which, in turn, could work together with the oxygen to oxidize several meat compounds, in this case the myoglobin (red), yielding traces of metmyioglobin (brown) responsible for the deeper red color of the meat. minor changes were detected in the parameters l and b (figure 3). all values were similar except those that corresponded to the control samples of 10 days, already discussed previously (spoiled on the 7th day) and in those treated with 2 kgy, in which the increase of the yellowness could be noted in samples stored for 10 days. the effects of the e - beam application (1 and 2 kgy) on selected textural attributes (hardness, adhesiveness, springiness, cohesiveness, gumminess, chewiness, and the breaking strength) were explored. since this product was conceived to be eaten after being cooked, the former attributes are of less concern. briefly, no differences were found in adhesiveness, springiness, and breaking strength between control samples and those treated by e - beam. a significant difference (p 0.05) for appearance between untreated and treated samples when they were cooked. in relation to the odor, in the descriptive analysis, both immediately after treatment (0 days) and after 7 days of storage at 4c, in the raw samples treated at 1 kgy, the typical fresh marinating odor was slightly lost and a negligible odor like scalded feather was detected. this odor was clearer in the samples treated at 2 kgy and, additionally, slight off - odor defined as scalded feather, poultry, metallic and sulfur taints were identified. these off - odors were detected when samples were air exposed after opening the packaged bags. more than 7% of the volatiles found in irradiated foods are hydrocarbons commonly found in thermally processed and unprocessed foods. the off - odors detected in the e - beam treated samples would be responsible for the lower scores assigned to the treated samples versus those that were untreated (data not shown) in the rank order text. despite this effect, the radiated samples, even at 2 kgy, were qualified as acceptable for trading. moreover, after 7 days of storage at 4c, the untreated raw samples showed a slight off - odor associated with the growth of spoilage organisms and the aging of meat (pungent, sour, unpleasant). irradiation can slightly increase levels of dimethyl disulfide, dimethyl trisulfide, s - methyl ester, and ethanoic acid. these sulfur compounds are highly volatile and can be eliminated by storing the irradiated meat under aerobic conditions. after cooking, a slight off - odor was detected only in the samples treated at 2 kgy. these results are in agreement with the findings of other authors, who reported that cooking can reduce or eliminate irradiation - induced odor [92, 93 ]. in cooked samples, the flavor analysis by both the triangular and the rank order tests, significant differences (p < 0.05) were only found when untreated and treated samples at 2 kgy were compared just after e - beam application (data not shown). in the descriptive analysis, samples treated at 2 kgy were judged to be less juicy and had a very slight taint of burnt, hot culture medium, acids and metallic notes and negligible, astringent feel aftertaste. it has been reported [94, 95 ] that postirradiation storage can allow flavor to return to the near normal features of the untreated products as the volatiles are lost. much of the work on irradiated meat odor and flavor has targeted selected constituents, particularly lipids [96, 97 ]. the reactions of sulfur - containing amino acids with water radiolytic products appear to be the source of hydrogen sulfide and other volatile sulfur - containing compounds which contribute to off - flavor. the literature reports that irradiation increases the concentration of 3-methylbutanal and 2-methylbutanal, mainly in vacuum packaged samples. however, dimethyl disulphide levels did not differ between irradiated and untreated samples in aerobic packaging. in irradiated cooked meat, a slightly higher volatile content has been found than in irradiated meat that was subsequently cooked. the shelf - life of marinated pork loin slices at 4c may be extended from 7 to 16 and 20 days with the application of 1 and 2 kgy, respectively. likewise, there is a mild temperature abuse (increase to 8c), the shelf - life will be extended from 5 to 10 and 16 days, respectively, when applying the same dose without compromising the sensory quality. from a hygienic point of view, e - beam treated marinated loin that is stored under refrigeration (4c) practically guarantees a pathogen - free product during its shelf - life. minor changes are produced in the main sensory characteristics, including the flavor of the coked product.	the effectiveness of e - beam radiation to extend the shelf - life of marinated pork loin slices stored at 4 and 8c (temperature abuse) has been studied. the shelf - life was extended from 7 to 16 and > 20 days after the application of 1 and 2 kgy, respectively. in the event of a temperature abuse occuring during the product distribution (e.g., increase to 8c), the shelf - life would be extended from 5 to 10 and 16 days, respectively, when applying the doses mentioned previously. from a public health point of view, the irradiation of marinated pork loin may be marketable for a longer period of time of up to two weeks, and guarantees a practically salmonella and listeria - free product. minor changes are produced by the e - beam treatment in the main sensory and rheological characteristics. the odor was the most affected feature, but the off - odors diminished with increased storage. in any case, testers judged the samples to be adequate for marketing.
the ongoing ' hide and seek ' between pathogens and their host immune systems has led to a molecular arms race which makes immunology one of the most interesting, but also one of the most complex, areas of research. by contrast with the harmless children 's game, the outcome of this molecular race determines health versus disease and, in many cases, survival versus death. a prerequisite for immunogenomic research is the availability of comprehensive and fully informative sequence variation and gene maps. a high - quality sequence map of the human genome and successive generations of annotation have been available since 2001. using this sequence as a reference, global and population - specific variation maps of increasing resolution because of the extreme polymorphism encountered in some immune regions, separate efforts were focused on regions such as the major histocompatibility complex (mhc ; reviewed by allcock.), the leukocyte receptor complex (lrc ; reviewed by yawata.) and others, to complement the global variation map. in addition, several specialised databases, including the immuno polymorphism database and the international immunogenetics information system, catalogue many of the classical immune genes and their allelic variants. using immune ontology definitions, the first comprehensive gene map and database of the human immune sub - genome was recently reported to consist of 1,562 genes (about 7 per cent of human genes) that are distributed at varying densities across all chromosomes except the y chromosome. in the following review, some of the benefits and caveats of using a holistic approach to immunogenomic analysis will be discussed with respect to genetic and epigenetic variation and linkage disequilibrium (ld). based on both experimental data [11 - 15 ] and inferences from population genetics data [16 - 18 ], there is evidence that recombination is not uniformly distributed across the human genome. in addition to the heterogeneity of recombination rates, it was suggested that the human genome could be subdivided into relatively short fragments with little or no evidence of historical recombination. under this premise, a genome - wide ld map is being constructed by the international hapmap project, which aims to generate a resource of dna variation in human populations with different ancestry. in its first phase, which is close to completion, genotyping data at one single nucleotide polymorphism (snp)/five kilobase (kb) density will provide enough resolution to portray the ld landscape of the genome, including regions of the immune sub - genome. ld maps of complete chromosomes are already available ; [20 - 22 ] however, detailed ld patterns at high snp density covering large chromosomal regions encoding immunerelated gene clusters have long been awaited. ld data have so far been reported for two such clusters : the lrc and the mhc. within the lrc, the haplotypic nature of the killer immunoglobulin - like receptor (kir) gene cluster causes a dna size variation of 120 - 200 kb as a consequence of the presence / absence of genes in some haplotypes [23 - 26 ]. this feature, in addition to intron similarity owing to recent duplications, restricts the availability of informative snps for ld studies and poses particular restrictions on large - scale genotyping protocols. consequently, a detailed ld map for the lrc is not available. for the mhc, a comprehensive ld map is available as a direct consequence of existing snp resources. historically, considerable attention has been paid to the mhc region, resulting in a wealth of valuable information in a variety of areas -- that is, origin and maintenance of nucleotide diversity in classical human leukocyte antigen (hla) genes ; gene conversion, average recombination rates, presence and distribution of the recombination hot spots ; and extended ld in long - range haplotypes [11,14,15,29 - 31 ]. the high - resolution ld map available for the mhc permits the detailed study of the allelic correlation, which can be particularly useful for the selection of tagsnp sets for disease association studies. since the history of recombination between two snps can be estimated using d ', dna segments showing no evidence of historical recombination have been defined as ld - blocks. consequently, haplotypes within such ld - blocks are likely to share a common ancestral haplotype, and the genealogy of an ld - block might be different from the genealogy of neighbouring ld - blocks. phylogenetic analysis of haplotypes observed in a ~110 kb ld - block (rs3115569-rs2022533) at the mhc class ii - iii boundary, the region of lowest recombination rate in the mhc (0.0093 centimorgan [cm]/ megabase [mb ]), surrounded by two recombination hot spots, showed that the pattern of nucleotide differences between haplotypes within this ld - block is difficult to account for by mutation only, but could readily be explained by recombination events (miretti., unpublished). following detailed inspection of the haplotype nucleotide sequence alignment at variable positions (figure 1b), putative recombination sites can be identified. the subsequent splitting of the ld - block at a recombination site (figure 1c) resulted in haplotypic networks presenting distinct clustering in both fragments (figure 1d), which were also dissimilar from that of the complete ld - block. while it needs to be examined in a larger sample of ld - blocks from diverse populations, the presence of recombination within ld - blocks suggests that the occurrence of ld between two snps might not be sufficient to unambiguously detect historical recombination beyond the history of the sample population where ld is being measured. recombination hot spots within ld - blocks -- that is, hot spots that have not left an imprint on ld -- have recently been postulated to be either old hot spots en route to extinction or too young to leave a mark on haplotype diversity in europeans. additional experimental data based on sperm cell recombination analysis would help to confirm the signature of recombination identified here and to determine if it is an example of a potentially evolutionarily very young hot spot. patterns of ld for the complete major histocompatibility complex were obtained for a panel of 180 centre d'etude du polymorphisme humain founder chromosomes and ld - blocks defined according to gabriel. (a) the region selected for the analysis constitutes an ld - block extending over a ~110 kilobase (kb) dna segment (national center for biotechnology information built 35 coordinates : 32321766 - 32431723) containing most of the c6orf10 gene, with an average space between single nucleotide polymorphisms (snps) of 1.6 kb. this region also represents a recombination cold spot -- with recombination rates = 0.00931 centimorgans / megabase, ten times lower relative to that on the neighbouring area -- surrounded by two recombination hot spots identified in notch4 and c6orf10 genes. (b) ten haplotypes (rows) observed in this sample, which were recognised based on alleles at 68 variable positions (snps, in columns) within the selected ld - block. (c) a break in the haplotype alignment suggesting the presence of ancestral recombination within the ld - block. haplotypes were then split at a potential recombination site into two sub - regions (with 41 and 27 snps, respectively) aiming to construct a phylogenetic network in order to check for consistency. (d) the distribution of variation among haplotypes as represented by median - joining trees obtained for both sub - regions employing the network program. haplotypes f and d are grouped closely related within each network but belong to different clusters when comparing networks from the two sub - regions. the number of substitutions involved in these branching differences could preferably be explained by recombination events rather than owing to mutations occurring between haplotypes. the distribution pattern of these differences adds consistency to this view and strongly suggests the presence of recombination. the circle size is proportional to the haplotype frequency, and the number of substitutions between nodes is represented by the number of lines between them. the site where the haplotypes were split can be changed to few positions on either side of the current site without significantly modifying the topology of the network. overall recombination rates within the mhc are known to be lower than across chromosome 6 and the genome average, and regions of relatively high recombination rates (hot spots) and low recombination rates (cold spots) have been described in males based on microsatellite typing. the fine - scale experimental recombination analysis in a 200 kb segment within the mhc class ii region revealed that a few hot spots shape the distribution of ld throughout the segment, that hot spots tend to be delimited to short dna segments (usually < 5 kb) and that a portion of the recombination is contributed by gene conversion. the question therefore arises as to whether the recombination pattern observed in this segment can be extended to the complete mhc and, ultimately, if it represents a general characteristic of the genome. the heterogeneous model of recombination seems to be a general feature of the genome, where the landscape is moulded by the local distribution and intensity of recombination. according to the recombination rates inferred from population genetic data, the experimentally verified recombination hot spots in the 200 kb segment of the mhc class ii region might not represent a paradigm for the entire mhc, as their intensities are ~ 2 - 100 times higher than hot spots in the rest of the mhc. figure 2 shows the uneven distribution of hot spots across the complete mhc -- integrated with gene annotation, ld - blocks and tagsnp distribution, as represented by the glovar genome browser http://www.glovar.org -- and also that the hot spot intensity is far from being uniform. additional large - scale experimental evidence of hot spot intensity and distribution is required to more accurately assess the concordance with predictions based on population genetic data. importantly, hot spot features will significantly influence the selection of tagsnps for disease association studies involving any particular region of the immune sub - genome. data integration in the major histocompatibility complex (mhc) region as represented by the glovar genome browser www.glovar.org showing the distribution and intensity of recombination hot spots relative to gene annotation, linkage disequilibrium blocks and tag single nucleotide polymorphism distribution. note that recombination hot spots within the mhc class ii region are comparatively more intense than in the rest of the mhc. most gene names were excluded owing to space constraints. from the detailed distribution of recombination and ld across the mhc, it would be possible to exploit the common variation observed in approximately 80 per cent of the mhc sequence, which is in high ld, to conduct association studies. even with the current snp density (1 snp/1.9 kb), however, between 10 - 27 per cent of the dna sequence in the mhc sub - regions is contained in regions of low ld, where tagsnp efficiency -- the number of genotyped markers divided by the number of tagging snps -- would not entail any benefit, meaning that almost every snp would need to be typed. figure 2 illustrates the dependence of the tagsnp distribution on the snp density and ld pattern, which is particularly variable across the mhc. long regions of strong ld are common in the extended class i sub - region, where 75 tagsnps are sufficient to recover haplotypic information provided by 408 snps. conversely, 204 tagsnps are necessary to recover the equivalent information from 466 snps in the class ii sub - region, where the ld pattern is more interrupted and the decay of ld is more pronounced. also, the tagsnp sets are derived from studies which mostly dismiss loci of minor allele frequency (maf ; < 5 per cent), which seem to be present at increased frequency in low ld regions, thus excluding rare variants from the analyses. this raises the question of whether failure to detect disease variants simply results from the exclusion of snps with rare alleles from the analysis (discussed below). furthermore, tagging effectiveness -- the proportion of ' hidden ' snps being detected in ld with snps from the tagging set -- can be substantially increased by incorporating snps with rare alleles (maf < 5 per cent) ; this effect was even more prominent in low ld regions. much less is known about epigenetic variation, both in terms of causality and function. dna methylation, which was first discovered in 1948, occurs naturally at the carbon-5 position of cytosine (5-methyl cytosine) at cpg dinucleotides. in subsequent years, it was proposed that dna methylation played an important role in the regulation of gene expression and disease aetiology, particularly cancer. the discovery of cpg islands (sequences enriched in cytosine - guanosine dinucleotides) suggested candidate regions in the genome for epigenetic modulation. dna methylation occurs predominantly at cpg sites and, in addition to gene regulation, is also involved in phenomena such as x - chromosome inactivation in female mammals, parent - of - origin - specific, mono - allelic gene expression (imprinting) and epigenetic reprogramming in mammalian development. the key technology for detecting 5-methyl cytosine by dna sequencing was developed in 1992, and is known as bisulphite sequencing. the need to consider epigenetic variation alongside genetic variation in the context of disease has been highlighted by the finding of high discordance rates in monozygotic twin studies and numerous other studies, confirming that epigenetic factors play a decisive role in the aetiology of virtually all human pathologies (reviewed by robertson and wolffe). therefore, when the snp consortium announced in 1999 that it would generate a first - generation genetic variation map of the human genome, the time and opportunity seemed right to generate a first - generation epigenetic (methylation) variation map alongside -- which is what the human epigenome consortium announced that it would do. one aspect of the human epigenome project (hep) that is of particular interest in the context of this review, is the aim and ability to identify methylation variable positions (mvps), which, together with snps, promise to significantly advance our ability to understand and diagnose human disease. mvps are defined as differentially methylated cpg sites that have the statistical power to discriminate, for example, between biological states such as active versus inactive or healthy versus diseased. as a result of the hep and other studies, a detailed genomic methylation map is already available for the mhc and models for the epigenetic control of the kir expression repertoire have been proposed (reviewed by uhrberg). for the mhc, methylation profiles have been generated essentially for all expressed genes, demonstrating tissue - specificity (eg for the c2 locus) and inter - individual heterogeneity (eg for the tnf locus). previously, it has been shown that transcription profiles can be associated with specific haplotypes, with epigenetic states or even with specific epi - alleles in the absence of dna variation. overall, the methylation profile of the mhc appears to be strongly bimodal, with good correlation between hypo- and hypermethylation within the upstream regions of genes and their transcriptional activity (figure 3). for the lrc, kir genes exhibit a high degree of polymorphism and are expressed in a clonally restricted fashion. each cell expresses a mono - allelic repertoire which is highly variable with respect to number and combination of receptors. kir genes are regulated by three types of promoters, corresponding to the three different modes of expression : one for kir2dl4, which is constitutively expressed ; one for kir3dl3, which is expressed at a very low level ; and one which is common to all clonally distributed kir genes. given the absence of promoter variability among the clonally distributed kir genes, it is, in fact, likely that the variegated expression of these genes is mainly regulated epigenetically, and a model has been proposed to this effect. the model proposes four stages of sequential dna and histone modifications leading to the observed mosaicism of clonally restricted kir expression but requires some still unproven processes, including locus- and allele - specific dna demethylation. dna methylation data derived from the human epigenome pilot project were grouped according to 5'-utr being methylated or unmethylated and the body of the gene being methylated or unmethylated. each group was then plotted against the corresponding genomics institute of the novartis research foundation (gnf) http://symatlas.gnf.org/symatlas expression level, which is set to anything below 200 as not being expressed and anything above 200 as being expressed. this cluster analysis shows that genes with methylated 5'-utrs are silent and genes with unmethylated 5'-utrs are expressed. in this study, no correlation was found between expression and the methylation level within the body of the gene. reproduced from rakyan compared with genetic data, the ability to analyse and interpret epigenetic data is still fairly simplistic, although enormous progress has been made, particularly in the past few years. what is becoming increasingly clear, however, is that the two are inextricably intertwined, and efforts towards an integrated (epi)genetic approach to common disease are well underway. major progress in genotyping technology has improved our ability to harvest genotypic information from a comprehensive number of loci and has laid down the foundation for the genome - wide common variation survey, enhancing our understanding of human genetic variation. current genotyping costs, however, make genome - wide association studies still challenging for the large sample sizes that will be required for adequately powered studies. the interdependencies of marker numbers, allele frequency, ld, cohort size and power in disease association studies have recently been reviewed [60 - 62 ] and will therefore not be discussed in depth here. essentially, genome - wide studies are still restricted by the allelic spectra underlying complex diseases. the frequency distribution of the disease variants and the proportion of the trait variance for which they are responsible determine the potential power of genetic association studies and, therefore, the feasibility of the study. disease susceptibility variants associated with mendelian disorders tend to be modelled by purifying selection and present low population frequencies (< 1 per cent). thus, rare alleles -- also including lowfrequency, mildly deleterious variants together with those alleles in high ld -- might be under - represented in genomic ld surveys and tagsnp sets excluding loci with maf < 5 per cent. it has been proposed, however, that genetic risk for common diseases and most of the clinically important traits are determined by the joint contribution of diseasepredisposing high - frequency alleles that are shared between unrelated affected individuals in the population : the common - disease / common - variant (cd / cv) model. a less extreme model based on the divergence of the allelic spectra of disease susceptibility variants relative to that of all variants has been proposed for evaluating the impact of allele architecture on common diseases. ultimately, the applicability of the cd / cv model in disease association studies will eventually rely on the selected markers and on the relationship between the four parameters that affect the apparent size at the marker locus, namely the odds ratio of the disease allele, the disease allele frequency, the marker allele frequency and the ld between the marker and the disease locus (reviewed by zondervan and cardon). diseases related to defective immune response, such as autoimmune disorders, might comply with the cd / cv model of allelic frequency distribution for susceptibility loci, showing increased allele frequency after being under positive selection for infectious disease resistance or heterozygote advantage. direct extrapolations to the immune sub - genome and autoimmune diseases could be elusive, however ; that is, it would be difficult to prove that alleles having an effect on the function of an immune gene -- and also contribute to the risk of autoimmune diseases -- are beneficial on a population level because they increase the diversity in the immune response. in fact, the overall level of polymorphism in immune genes is, in general, similar to that of non - immune genes, and only a few loci among the genes constituting the immune sub - genome largely contribute to the diversity of the immune response -- disregarding loci with somatic recombination. in addition, modes of selection maintaining extreme allelic diversity levels -- heterozygote advantage, balanced selection -- have been proposed for these few loci, including the classical hla class i and ii and kir genes.. a recent study shows, for instance, that multiple disease loci with predominant disease variants are not necessarily required to develop a complex immune disorder. it has been shown that sarcoidosis -- a multi - systemic immune disorder initially associated with several classical hla markers -- is associated with a single disease locus independently responsible for most of the predisposing influence. the authors of this study demonstrated that a single transition in exon 5 of the btnl2 gene, which alters a splice site and causes a premature stop codon, has a profound effect on the mature protein structure and function. a number of autoimmune disease - susceptibility loci have been mapped to the mhc and lrc regions [70 - 72 ]. autoimmunity and cancer are associated with the progressive decline in immune functions, such as decline in t cell function, dysregulation of t cell apoptosis and immune senescence during ageing. since the term haplotype was coined in 1967, a vast amount of literature has been published employing haplotypes to construct genetic and ld maps, which have enabled the emerging field of immunogenetics to make major contributions to immunology, population genetics and medicine. for the mhc, a haplotype hierarchy has been defined based on pedigree and population genetic data, where physically linked classical hla genes constitute ' blocks ' of relatively short dna segments containing alleles in ld at different loci, namely hla - b / c, tnf, complotype and hla - dqb / drb. combinations of these four basic blocks are frequently found in ld, constituting ' conserved extended haplotypes ' (ceh) or ' ancestral haplotypes '. more recently, local fine - scale ld blocks based on high - density snp typing have been described and meiotic recombination hot spots inferred from population genetic data. the integration of long - range ld in ceh and local fine - scale ld data will provide new and exciting opportunities to explore the evolution and disease associations of the immune sub - genome. while the jury is still out on the validity of the cd / cv model, the recently announced wellcome trust case control consortium (wtccc) is likely to provide the answer to this ongoing debate. the wtccc proposes to analyse 19,000 samples for common genetic variants in eight common diseases using data generated by the hapmap consortium. integration of the genetic and epigenetic datasets discussed here is a necessary next step towards a more holistic approach to immunological research, which is one of the aims of the recently formed international immunomics society, immis http://research.i2r.a - star.edu.sg / iimms/. the immis was formed with the main objective of promoting the science of immunomics, which is the interdisciplinary field spanning immunology, immunoinformatics, genomics, proteomics, bioinformatics and related scientific fields. we wish to thank v. rakyan for reading the manuscript and c. tyler - smith for suggestions.	similar to other classical science disciplines, immunology has been embracing novel technologies and approaches giving rise to specialised sub - disciplines such as immunogenetics and, more recently, immunogenomics, which, in many ways, is the genome - wide application of immunogenetic approaches. here, recent progress in the understanding of the immune sub - genome will be reviewed, and the ways in which immunogenomic datasets consisting of genetic and epigenetic variation, linkage disequilibrium and recombination can be harnessed for disease association and evolutionary studies will be discussed. the discussion will focus on data available for the major histocompatibility complex and the leukocyte receptor complex, the two most polymorphic regions of the human immune sub - genome.
compulsive sexual behaviour (csb) is characterized by repetitive and intense pre - occupation with sexual thoughts, urges, and behaviours causing clinically significant distress or impairment in occupational, interpersonal, or social domains of functioning. it has also been called by other names such as sexual addiction, sexual compulsivity, and hypersexuality. hypersexuality is usually seen in mania, but can also be seen in depression and anxiety disorders. persons afflicted with these conditions are currently diagnosed as sexual disorder not otherwise specified on diagnostic and statistical manual iv edition text revision (dsm iv - tr). however, the upcoming dsm-5 is more inclusive and proposes hypersexual disorder as a new diagnostic category in its latest edition to include such diagnoses. in this paper, i present a case report of a patient with depression who had underlying csb in the form of compulsive frottage. x, a 25-year - old man, migrant from uttar pradesh to mumbai, presented to the psychiatry out - patient department with chief complaints of persistent sadness of mood and decreased interest in work since the last 3 months. he complained of easy fatigability, body aches, headaches and feelings of guilt, hopelessness and helplessness over the last few weeks. he had decreased sleep but no appetite disturbances. on further probing, he reported of not being able to control urges to rub his genitals in crowded trains. x reported that after work he would board the mumbai local trains and travel for 2 - 3 hours per day in crowded trains. in his description of the act, he recollected that he chose to climb only those coaches in the train that had middle aged women in them. once he boarded the train, he would then stand near the unsuspecting woman passenger and start rubbing his genitals against her body. in case there was no resistance by the woman, it was taken as a positive signal by him and the act was then continued until orgasm and ejaculation, without actual genital touching or contact. however, if the woman would resist or show anger, he would immediately stop and move away. he specifically reported that he has never removed or exposed his genitals to any woman in the crowd. in case he would not find any woman in the train - coach, then he would get down at another station and board a different coach to search for another victim. initially the frequency was about 2 - 3 times in a week but since 1 year it had increased to once twice everyday spending almost 2 - 3 h in a day in this activity. on public holidays and sundays he would then release his sexual tension by spending the whole day (starting at 10 am in morning and stopping by 1 am at night) in genital rubbing in trains. on a few occasions, he had also gone with 9 - 10 other men to indulge in group genital rubbing against women in the crowd. tekaa bharna. over a period of time, his liking for such genital rubbing had increased to an extent that now his interest in actual sexual intercourse had reduced compared to his interest in genital rubbing. sometimes, he also avoided sexual intercourse for the fear of contracting any sexually transmitted disease or hiv. often he travelled ticketless while indulging in these acts and was caught. on one occasion, he was suspected to be a terrorist element, when he was found frequently changing the train coach in search of a better partner for genital rubbing, and was imprisoned for almost a week. sometimes, he was also beaten by other men when any woman in the crowd raised alarm on his behavior. although the patient said that he enjoyed this act, but since recent past, it was becoming excessive and interfering with his work. he was always preoccupied with the thoughts of genital rubbing and wanted to repeatedly go and do it in trains. there was history of multiple sexual contacts (heterosexual) in the past, all of which were unprotected and involved sex in risky situations such as open public areas, or in group with commercial sex workers. in addition, even though he was depressed since the last 3 months, his sexual behavior was not affected at all and he was continuing with his genital rubbing behavior in the same way as before. there was no history suggestive of mania, hypomania, anxiety, obsessive compulsive disorder, or psychotic disorder. there was occasional alcohol use, around once in a few days, but never to the point of intoxication. the patient was married since 6 years but was staying separate from his wife due to work - issues. he would visit his home once in 8 - 9 months when he would have normal sexual intercourse with her. his average reported frequency of intercourse with his wife (and other sexual partners) was about 5 - 6 times in one night. in the past too, mr. x reported of having masturbated at a higher frequency (about 3 - 4 times every day) than his friends. he described himself as a social, outgoing and extrovert person who was very religious. he feared that god might punish him for this sin that he was indulging in. his general and systemic examination was normal and on mental status examination he was alert and fully oriented. there were ideas of hopelessness, helplessness, and worthlessness, but no suicidal ideations. his routine blood investigations, thyroid function tests, electroencephalogram (eeg) and magnetic resonance imaging (mri) brain were normal. he was started on oral fluoxetine 20 mg which was increased to 60 mg over a period of 2 months. patient reported gradual improvement in his mood symptoms and also claimed that his sexual urges had reduced after the treatment. he was motivated to make a contract about this behavior that he wanted to change. he was advised to keep himself busy with work and spend time with his co - workers and friends, especially when the thoughts of indulging in genital rubbing came to his mind. he was encouraged to avoid acting out by postponing going to the railway stations to board the train. in every session, his contract was reviewed and he was further motivated to resist his urges. he was taught orgasmic reconditioning strategy wherein he masturbated to the point of orgasmic inevitability, when he switched his fantasy to a more socially desirable one, hoping thereby to increasingly associate orgasm and later erection with the desirable stimulus. the patient reported almost 80% reduction in his genital - rubbing behavior over a period of 8 months. he was now able to concentrate more on his work and postpone acting out on his sexual urges. after about 10 months of being on 60 mg of oral fluoxetine, his dose was reduced to around 20 mg daily. the patient discussed here is a case who presented with major depressive disorder as a primary complaint. on further probing it was found that he also had issues with his sexual urges, especially problems with controlling his urges to rub genitals in crowds. human sexual behavior can have a wide range both in terms of types of behaviors and their frequency. thus, it is difficult to define what is hypersexual behavior or compulsive sexual behavior. however, in this case the patient himself reported feeling distressed about his repeated sexual feelings and urges that were intrusive in his daily - life. an important characteristic of sexual compulsivity is the difficulty to regulate sexual impulses despite negative consequences. in the index patient, risk - taking behavior related to sexual experiences (in the form of unprotected sexual intercourse, group - sex, ticketless travelling) had increased in response to these urges and were continuing despite being jailed in this context. csb can be divided into paraphilic (un - conventional sexual behaviors) and non - paraphilic (conventional sexual behaviors) csb. the patient described in this case report had the paraphilia of frotteurism and paraphilias by their very nature are known to be compulsive in nature. symptoms of hyper - sexuality are well documented in mania, substance use disorders and certain medical conditions such as parkinson 's disease, kleine - levine syndrome, and kluver - bucy syndrome. csb may also be seen in organic brain lesions for example in lesions of medial prefrontal cortex. the patient in this report did not have any of these including mania, but had depression since past 3 months. anxiety and depression have been linked to hypersexual behavior and have been reported as the most common diagnoses among hypersexual individuals. 1997) reported major depression or dysthymia in almost 14 out of 36 subjects reporting compulsive sexual behavior. loneliness, presence of interpersonal problems and increased vulnerability to stress has also been observed in association with hypersexual behavior. though the index patient did not report any interpersonal problems with his wife, he was a migrant and was staying alone in mumbai away from his family. as far as treatment of csb is concerned, medications and psychotherapy are effective. various medications have been tried including selective serotonin reuptake inhibitors (ssris), mood stabilizers, antipsychotics and anti - androgens. ssris are useful in reducing sexual obsessions and compulsions as well as the associated anxiety and depression. this case highlights the importance of probing cases of depression for possible hypersexual behavior to the point of sexual compulsivity especially when there is underlying paraphilic tendency. often such cases are under - recognized and under - diagnosed in depression in clinical practice.	compulsive sexual behavior consists of sexual obsessions and compulsions that are recurrent, distressing, and interfere with daily functioning. it has been called hypersexual disorder in the upcoming diagnostic and statistical manual 5th edition. though hypersexuality is commonly seen in mania, it can also be seen in depression and anxiety disorders. this case report describes a case that presented with depression and had underlying compulsive sexual behavior in the form of frottage.
vagal schwannomas are benign, rare peripheral nerve sheath tumors in the head and neck region. some physicians opt to closely observe cases of schwannoma of the neck on an outpatient basis rather than to perform radical surgery. however, there is a possibility, albeit rare, of malignant transformation of a benign schwannoma. here, we are reporting the first case from the indian subcontinent, which was transformed into the angiosarcoma from benign vagal schwannoma over a long period. a 47-year - old male patient presented with a left sided neck swelling since last 12 years, insidious in onset, and slowly progressive. in last 2 months, there was a firm, nodular, well - defined, nontender swelling of approximately 6 cm 6 cm size, in the left lateral part of the neck. fine - needle aspiration (fna) cytology revealed paraganglioma and magnetic resonance imaging (mri) demonstrated a tumor thought to be a vagal schwannoma with its morphology, and its relation to the surrounding structures [figure 1 ]. contrast - enhanced magnetic resonance imaging, t1-weighted fat saturated coronal image shows a large isointense signal intensity mass admixed with hyperintense signal intensity during surgery, the tumor was found to be arising from the vagus nerve and adherent to the common carotid artery through the artery was unaffected by the tumor. pathologic findings showed a solid gray - white tumor with large areas of hemorrhage, necrosis, and cystic changes. microscopic examination showed verocay bodies (antoni a region), focal admixed hypocellular areas with edematous vascular stroma and haphazardly arranged tumor cells (antoni b region). multiple areas revealed a vasoformative tumor composed of numerous, irregular, anastomosing vascular spaces with dissecting pattern and with high mitotic activity, and the presence of atypical mitosis. the vascular channels were lined by pleomorphic, endothelial cells showing large ovoid nuclei, heterogenous chromatin, and prominent nucleoli [figure 2 ]. section showing a well - encapsulated benign spindle cell tumor with characteristic verocay bodies, consistent with schwannoma (h and e, 5) on immunohistochemistry - spindle cell tumor (nerve sheath tumor) component was positive with s-100 and negative for cd31. ki-67 showed the high proliferation index in vasoformative tumor areas (4050%) and was only about 1% in nerve sheath tumor areas. the final histopathological diagnosis was angiosarcoma arising in a benign schwannoma [figure 3 ]. cd 31 positive staining of atypical endothelial cells in view of high - grade malignancy, whole body positron emission tomography - computed tomography was done, which revealed liver metastasis for which liver biopsy was done, which revealed angiosarcoma. the patient was given postoperative chemotherapy, but the patient died after 6 months of treatment due to distant metastasis. though rare but benign tumors have the potential for malignant transformation. angiosarcoma transformation in peripheral nerve sheath tumors usually such tumors transform into neurofibromas or malignant peripheral nerve sheath tumors, especially in von reckinghausen disease. the process of malignant transformation from schwannoma to angiosarcoma was postulated by rckert. although fna is clearly a good diagnostic tool that can be performed easily in the outpatient clinic once the mass had completely changed to malignant cells, fna for diagnosis of schwannoma is difficult and the accuracy reported was only 20%. thus, our physicians should be cognizant of the need for careful follow - up including mri and fna of benign neurogenic tumors of the neck because of this potential for transformation. the accumulation of more cases in the literature will help physicians decide on future diagnostic and treatment strategies. because neurogenic tumors sometimes undergo malignant transformation, we recommend that for neck masses suspected to be benign, surgery should be performed if possible. however, if follow - up observation is chosen, mri and fna should be regular, and patients should sign a statement acknowledging awareness of the potential for malignant transformation.	schwannomas are benign, rare peripheral nerve sheath tumors that occur in the head and neck region. some physicians opt to closely observe cases of schwannoma of the neck on an outpatient basis rather than to perform radical surgery. however, there is a possibility, albeit rare, of malignant transformation of a benign schwannoma. here, we are reporting the first case from the indian subcontinent which was transformed into the angiosarcoma from benign vagal schwannoma over a long period. a 47-year - old male patient complaining of left sided neck swelling since last 12 years, swelling was insidious in onset, gradually progressive very slowly. in last 2 months, the size of the swelling was suddenly increased. on examination, there was an approximately 6 cm 6 cm of size, firm, nodular, well - defined, nontender swelling in the left lateral part of the neck. fine - needle aspiration cytology (fnac) revealed paraganglioma and magnetic resonance imaging demonstrated very clearly a tumor, its morphology, and its relation to the surrounding structures, the tumor was thought to be a vagal schwannoma. surgery was done, and the whole of the tumor was removed in toto. on final histopathological diagnosis, the tumor was proved to be angiosarcoma developed from vagal schwannoma. postoperative chemotherapy was given but due to distant metastasis, the patient died. long standing neck masses can convert into malignancy as in our case, therefore, work up of the patient should be done properly. multiple fnac should be done because single fnac can give the false negative result as in our case. this was our diagnostic drawback not to do multiple computed tomography guided fnac.
atd was first discovered in 3 patients with copd when it first became possible to quantitatively analyze different globulin peaks in serum. over the years since then investigations have shown that at is the most abundant protease inhibitor in serum and that it is predominantly produced by the liver. these two observations led to the protease - antiprotease imbalance theory for pathogenesis of copd whereby lowering of antiprotease levels or activity rendered the lung connective tissue matrix susceptible to excessive proteolytic damage. the protease - antiprotease imbalance theory was validated by the observation that cigarette smoking could functionally inactivate at, via the effect of smoking on phagocyte - derived active oxygen intermediates. this imbalance theory could thereby explain proteolytic attack on the lung connective tissue matrix in the vast majority of patients affected by copd who were simply cigarette smokers and why patients with atd had a marked increased in incidence and severity of copd when they were also smokers. liver disease was first described in atd by sharp and colleagues who discovered it in a child with cirrhosis in 1969. since that time atd has been widely recognized as the most common genetic diagnosis for children who undergo liver transplantation. eriksson and colleagues showed in 1986 that adults with atd also had a predilection for cirrhosis and hepatocellular carcinoma, and onset of liver disease later in life is now known to be even more common than in childhood. wide variability in the incidence and severity of liver disease among individuals with the classical form of atd was shown by analysis of a cohort of swedish individuals identified in a nationwide screening study by sveger. only ~8% of the cohort had clinically significant liver disease over the first 4 decades of life. a greater proportion of this population will probably be impacted as they reach older ages, but we know that a significant number of affected homozygotes completely escape clinical symptoms of liver disease throughout their lifetime. using a variety of model systems we have come to learn that the liver is damaged in atd by gain - of - toxic function mechanisms activated by accumulation of misfolded atz within the early compartments of the secretory pathway. several theories for how this proteotoxicity leads to excessive collagen deposition and hyperproliferation in the liver have been proposed. putative genetic and environmental modifiers are thought to determine susceptibility to and severity of liver disease among homozygotes. we have postulated that these modifiers target proteostasis mechanisms that are designed to mitigate the proteotoxic effects of atz accumulation in the er. furthermore, we have postulated that these proteostasis mechanisms fall into two general categories : cellular pathways for degradation of misfolded atz and signaling mechanisms that permit cellular adaptation to the presence of misfolded atz. indeed, several novel concepts for therapeutic intervention that capitalize on the pathways for intracellular degradation of the atz variant are currently being investigated. although liver disease can be first diagnosed in infancy, childhood or adolescence, recent studies show that it is more common in adults with peak age 5065 years. more than 88% of all liver transplants done in the us for this diagnosis are adults. some of these entries are not true homozygotes for the atz allele but, based on interrogating several different types of liver transplant databases, we ascertained that severe progressive liver disease occurs more than 2.5-fold more in adults than children with true homozygosity for atz. because the peak onset of 5065 years correlates with the known age - dependent decline in autophagy and probably other proteostasis machinery, we suspect that the adult form of this liver disease is likely to reflect the normal aging process whereas onset in infancy, childhood and adolescence will be related to particularly powerful genetic and/or epigenetic modifiers. recent studies have shown that the fibrotic response is characteristic of other protein accumulation disorders, including fibrosis in skeletal muscle in inclusion - body myositis, cardiac fibrosis in desminopathy and lung fibrosis in surfactant protein c deficiency and hermansky - pudlak syndrome [1114 ]. affected patients are also predisposed to hepatocellular carcinoma and there is growing evidence that this pathological characteristic relates to heterogeneity in accumulation of misfolded atz among liver cells such that some cells evolve to a selective proliferative advantage and are trapped in a chronic hyperproliferative state. the diagnostic hallmark of the disease is inclusions within some of the hepatocytes and these inclusions are known to contain atz but apparently not other proteins. these inclusions have long been characterized as representing rough er. however, recent studies using ips - derived hepatocyte like - cells and liver specimens from atd patients indicate that the inclusions correspond to both er and non - er compartments. the nature of the non - er compartments is not yet known but they do not appear to be autophagosomes even though the liver cells have a marked increase in number of autophagosomes. the peak age range is also fifth or sixth decade but very severe cases can be found in the late 20 s or 30 s. cigarette smoking is a powerful modifier of copd and it is well established that at can be functionally inactivated as a protease inhibitor by active oxygen intermediates, presumably released by phagocytes in response to cigarette smoking. nevertheless there are other modifiers of lung disease in atd even when cigarette smoking is taken into consideration. recently we found evidence for fibrosis in the lungs of atd patients who had undergone lung transplantation and in the piz mouse model of atd. our data suggests that excess collagen deposition in the lung is caused by expression of the at gene in respiratory epithelial cells and accumulation of the misfolded atz variant in pneumocytes can elicit fibrogenesis in the same way that it occurs in the liver in atd and in the lung in other respiratory epithelial cell proteinopathies. modifiers that promote fibrogenesis could explain a subgroup of patients with atd that have both lung and liver disease. the atz variant is characterized by a point mutation that substitutes lysine for glutamate 342 and this leads to reduced secretion and intracellular accumulation of atz molecules [16, 1922 ]. immunostaining and biochemical analyses show that mutant atz accumulates in early compartments of the secretory pathway, including most notably the rough er, and perhaps other as yet undefined compartments that do not stain with classical markers of the rough er. it is important to note that in these model systems the secretion of atz is reduced whereas it is completely absent for a variety of null variants when they are expressed in similar model systems [2325 ]. this reflects what is seen in humans with serum levels of atz at 1015% of normal while those of the null variant are undetectable. together, these observations make it possible to conclude that these model systems faithfully recapitulate the human disease and definitively prove that the primary abnormality is defective biogenesis. furthermore, site - directed mutagenesis studies have shown that the single amino acid substitution of lysine for glutamate 342 is sufficient to cause the cellular defect in which the mutant atz is inefficiently transported through the intracellular secretory pathway. it is still not entirely clear how the amino acid substitution leads to diminished secretion and intracellular accumulation of atz. these authors demonstrated polymers and insoluble aggregates in liver biopsy specimens and plasma from patients with atd and provided evidence for a loop - sheet insertion mechanism of polymerization. in this conceptualization of polymerization, the characteristic lysine substitution is at the hinge of the mobile reactive - site loop and because it has more bulk than the glutamate that is ordinarily at that site it prevents the reactive - site loop from relaxing into a known gap in the a sheet that is part of the flexible conformational changes of the at molecule. this permits the reactive - site loop on another atz molecule to insert into the gap and begin the oligomerization and ultimately polymerization process. in an important study sidhar showed that the secretory defect that characterizes atz was partially corrected by introducing a second mutation that suppressed polymerization. more recent studies by huntington and colleagues have suggested a different mechanism for polymerogenic and aggregation - prone properties of atz that involves at least 2 different domain - swapping phenomena (2830) and this mechanism seemed most consistent with recent characterization of a putative atz monomer crystal structure. several lines of evidence suggest that misfolding is the primary defect responsible for impaired secretion / intracellular accumulation of atz and in this conceptualization polymerization / aggregation is a result rather than the primary defect itself. first, only 18% of the intracellular pool of atz is in polymers at steady state in a mammalian cell line model that faithfully recapitulates the intracellular accumulation / fate of atz. second, a naturally occurring variant of atz which has the same e342k mutation as atz and a carboxyl - terminal truncation accumulates in the er even though it does not form polymers suggesting that misfolding is sufficient to lead to intracellular accumulation of atz (23). third, the results of sidhar do not exclude the possibility that diminished secretion of atz is partially corrected by the second engineered mutation because this second mutation also prevents the primary misfolding defect. furthermore, in a very interesting study a small molecule that prevents polymerization in vitro does not correct the secretory defect of atz in vivo but rather leads to enhanced degradation. it is hard to know for certain about this applicability of this last study because there was no analysis of whether polymerization was prevented by the small molecule in vivo. taken together, we believe that misfolding is the primary defect and that polymerization and aggregation are time - dependent effects of the accumulation of atz that result from misfolding. this conceptualization is also consistent with the domain - swapping mechanism of polymerization described by huntington in which polymerization is viewed as a kinetic result, or delayed folding, of monomeric atz and explains how some atz gets secreted. in one of the most interesting studies on the mechanism for atz accumulation in the er, nyfeler. provided evidence for the lectin er golgi intermediate compartment 53 kd protein (ergic-53) as an export receptor for at. this study did not address whether polymerization prevents atz from being presented to ergic-53 or that the altered folding pathway of atz prevents an essential ligand domain from being available to bind to ergic-53 and there has been no follow - up of these important studies. investigations of the mechanisms of atz degradation have shown that the proteasomal and autophagic pathways play critical roles. early studies using yeast and mammalian cell lines showed that the proteasomal pathway participates in intracellular disposal of atz by a process that is now known as er - associated degradation [erad ] in which the substrate is extracted retrograde from the er to the cytoplasm [3437 ]. in fact, atz was one of the first identified substrates of the erad pathway. autophagy is an intracellular catabolic pathway by which cells digest subcellular structures and cytoplasm to generate amino acids as a survival mechanism. it is characterized by double membrane vacuoles called autophagosomes which fuse with lysosomes for degradation of the internal constituents. increased numbers of autophagosomes were observed in human fibroblast cell lines engineered for expression of mutant atz, in the liver of piz transgenic mice and in liver biopsy specimens from patients with atd. definitive evidence for the role of autophagy in atz disposal was provided by genetic studies in which atz disposal was delayed in autophagy - deficient [atg5-null ] murine embryonic fibroblast cell lines and atg6-null yeast strains. we have also found that accumulation of atz within the er and early vesicular compartments of the secretory pathway is sufficient to activate the autophagic response by demonstrating increased gfp+ autophagosomes in the liver of a transgenic mouse with liver - specific inducible expression of atz that had been mated to the (gfp - lc3) the importance of the autophagic pathway in intracellular atz degradation has been further validated recently by studies demonstrating that autophagy enhancer drugs promote intracellular atz disposal and attenuate hepatic fibrosis in the piz mouse model of atd in vivo [4345 ]. one of the concepts originating from studies of atz disposal in autophagy - deficient yeast strains is that autophagy becomes particularly important at higher levels of atz expression. these results taken together with the structural constraints of the proteasome have led to the supposition that the proteasomal pathway degrades soluble monomeric forms of atz whereas autophagy is needed for soluble and insoluble polymers. however, it is also possible that autophagy plays a role in the disposal of soluble monomeric atz that accumulates at levels of expression that exceed the capacity of the proteasome. another important result from the studies by kruse in autophagy - deficient yeast also showed that a misfolded fibrinogen variant associated with liver disease in a rare inherited form of hypofibrinogenemia is degraded by autophagy in a manner almost identical to that of misfolded atz. pathways for intracellular disposal of atz other than the proteasomal system and the canonical macroautophagy system are highly likely. for example, a sortilin - mediated pathway from golgi to lysosome has been described to participate in degradation of atz in yeast and mammalian cell line models. another pathway for atz disposal which diverges from the canonical autophagy system was recently identified in a powerful c. elegans model of atd and found to be present in a mammalian cell line model as well. this pathway is particularly interesting because it is suppressed by insulin signaling and when up - regulated by knocking down components of the insulin signaling pathway it can completely mitigate atz proteotoxicity. to determine which signaling pathways are activated when atz accumulated in the er, we developed cell line and mouse model systems with inducible rather than constitutive expression of atz because the latter would potentially permit adaptations that could obscure the primary signaling effects. a series of studies using these kinds of systems have shown that the autophagic response and the nuclear factor b (nfb) signaling pathway, but not the unfolded protein response, are activated when atz accumulates in the er. activation of the autophagic response was shown by investigating the liver of a novel mouse model with hepatocyte - specific inducible expression of atz, the z mouse, bred onto the gfp - lc3 mouse background. lc3 is an autophagosomal membrane - specific protein, so the gfp - lc3 mouse makes green fluorescent autophagosomes. green fluorescent autophagosomes appear in the liver of the gfp - lc3 mouse only after 24 hours of starvation. in the z gfp - lc3 mouse green fluorescent autophagosomes are seen merely by allowing hepatocyte expression of the atz gene to be induced. gfp+ autophagosomes are not seen in the liver of the saar gfp - lc3 mouse, which has hepatocyte - specific inducible expression of the at saar variant that accumulates in the er but does not polymerize. thus, autophagy is activated when atz accumulates in the er and the autophagic pathway then plays a critical role in disposal of atz and in preventing massive intracellular aggregates. one of the most interesting aspects of the nfb signaling pathway under these circumstances is that it is associated with a rather limited set of downstream transcriptional targets. indeed the most significant change in expression that could be attributable to nfb is downregulation of egr-1, a transcription factor that is essential for hepatocyte proliferation and the hepatic regenerative response. our most recent studies have indicated that the downregulation of egr-1 in the liver of the z mouse when atz expression is induced is directly attributable to the action of nfb (a. mukherjee and d. h. perlmutter, unpublished results). furthermore, the complex of proteins that assembles to form nfb when atz accumulates in the er has a profile that is entirely distinct from that which forms when cells are treated with tumor necrosis factor (tnf) or tunicamycin (a. mukherjee and d. h. perlmutter, unpublished results). finally, mating of the piz mouse to a mouse model with conditional hepatocyte - specific deficiency of nfb activity shows more severe inflammation, fibrosis, steatosis, dysplasia, and more hepatocytes with globules (a. mukherjee, t. hidvegi and d. h. perlmutter, unpublished results), indicating that nfb signaling is intended to protect the liver from the effects of atz accumulation. together, the data on nfb suggest that it plays a particularly important role in the effects of atz on cell proliferation, survival, and ultimately the predisposition to hepatic carcinogenesis in at deficiency. although some studies have suggested otherwise, most have been unable to demonstrate changes in gene expression indicative of an unfolded protein response (upr) in systems characterized by intracellular atz accumulation. even when care is taken to eliminate the potential for cellular adaptation to atz accumulation by generating inducible expression of the atz molecule, there is negligible change in expression of the downstream targets of the upr or the changes in expression are minimal compared to positive controls. this means that activation of autophagy and nfb in cells that accumulate mutant atz is independent of the upr, another distinct characteristic of the cellular response in the at deficiency state. with what is known about the mechanism by which the upr is initiated it has always been relatively easy to understand how polymerized and aggregated atz would not elicit the upr. results of recent structural studies by huntington and colleagues provide an explanation for why soluble monomeric atz does not get recognized by the upr apparatus. those results suggest that the monomeric atz intermediate adopts a conformation that resembles the wild - type molecule and therefore would not be recognized as unfolded. transcriptomic analysis of liver when atz is induced in the z mouse has identified other changes in gene expression that are attributable to the cellular response to atz. one of these changes, upregulation of the regulator of g signaling 16 (rgs16), may represent a mechanism by which autophagy is activated in the liver in at deficiency. we have found that the rgs16 response is characteristic and specific for atz. because rgs16 anatagonizes gi3, and gi3 plays a role in inhibiting hepatic autophagy, we have hypothesized that increased rgs16 when atz accumulates in the er leads to reversal of the inhibition of autophagy that would otherwise pertain in resting hepatocytes. there is still relatively limited information about how rgs16 is upregulated and where in the cell it acts to antagonize gi3. the hepatic transcriptomic analysis of the z mouse also shows changes in gene expression indicative of tgf signaling and this is consistent with the fibrotic response that represents the dominant pathological characteristics of the liver in atd. furthermore we have recently found that accumulation of the atz variant in respiratory epithelial cells elicits fibrosis in the lungs with evidence for fibrosis in the lungs of atd patients with very severe copd. the mechanism by which accumulation of misfolded proteins in the er elicits tgf signaling has not been studied. other proteinopathies are known to elicit a fibrotic response including inclusion - body myositis causing fibrosis in skeletal muscle, desminopathy causing cardiac fibrosis and lung fibrosis in several rare proteinopathies that affect respiratory epithelial cells. taken together, these studies show a unique repertoire of signaling pathways activated by accumulation of the atz variant in the er in model systems and, in many cases, validated by investigations of tissue from atd patients. the implication of these results is that the substrate which accumulates elicits specificity in the response of the er. in the case of the atz variant, there is a relatively limited, if any, role of the upr signaling pathway and thus this er storage disease represents an important example of why the upr should be regarded as one of at least several responses to er stress. there is very little known about how accumulation of the atz variant affects the er and elicits structural changes. early ultrastructural studies of livers from patients with atd showed dilation of er with proteinaceous material in the lumen. mostly rough er was described but these studies were done before reagents were available for marker studies or biochemical assays. several observations have come from our recent studies of ips - derived hepatocytelike cells from patients with atd and those investigations led us to look again at liver from atd patients. first, we could conclude from endoglycosidase h digestion assays that atz predominantly localizes to pre - golgi compartments. second, double - label immunofluorescent analyses showed that atz accumulates in rough er but also in one or more compartment that could not be considered rough er. ultrastructural studies showed markedly dilated rough er in many cells but also there were very large vesicular structures enveloping proteinaceous material and only partially covered by ribosomes. these very large vesicular structures are probably what have been traditionally described as the globular inclusions that are the hallmark of atd. the proteinaceous material in the vesicular structures appeared electron - dense in some areas. in some of the cells these vesicular structures were almost completely devoid of ribosomes but, because of the single plane of the image, it was not possible to exclude the presence of ribosome - containing areas. these observations led us to believe that there are structural changes other than simply dilation of the rough er, perhaps including dilation of smooth er, specialized subdomains of the rough er or completely separate subcompartments as has been observed for other misfolded proteins. a number of years ago we, and others, hypothesized that genetic and/or environmental modifiers could explain the differences in onset and severity of liver disease among z homozygotes that were first recognized by the screening / cohort studies of sveger. furthermore it was proposed that these modifiers would target proteostasis mechanisms, such as intracellular degradation pathways (figure 1). this hypothetical paradigm was initially validated by studies showing that atz was degraded more slowly in atd individuals with liver disease than in atd individuals without apparent liver disease, using skin fibroblast line models engineered for expression of atz by retroviral - mediated gene transfer. recently studies by tafalang have provided further validation of this hypothesis by showing slower degradation of atz in ips - derived hepatocytes (iheps) from atd individuals with liver disease as compared to iheps from atd individuals without liver disease. interestingly, the rates of atz degradation in iheps were almost identical to those in skin fibroblasts published 20 years ago with a half - time of disappearance of ~ 4 hours in those affected by liver disease compared to ~2 hours in those without apparent liver disease. furthermore, tafaleng found that large intracellular globular inclusions were only seen in the iheps of the liver disease patients (figure 2). first they validate the hypothetical paradigm that modifiers of disease target proteostasis mechanisms by showing that degradation may be relatively impaired in some atd individuals with liver disease. second, the results show that cell biological mechanisms can explain personalized variations in the clinical effects of a single gene defect and now we can explore the cell biological basis for sequence variants in putative modifier genes. third, the results suggest the iheps may be used to make pre - morbid predictions of liver disease susceptibility. studies designed to identify genetic and environmental modifiers of the liver disease phenotype in human populations have begun to appear in the literature in recent years. in one interesting study, a single nucleotide polymorphism (snp) in the man1b1 gene was found to be over - represented statistically in a series of infants with end - stage liver disease. recent experiments have shown that man1b1 is actually localized to the golgi but it plays a role in regulation of protein secretion as a part of the protein quality control network which is recently recognized to be localized in the golgi. furthermore those experiments have provided a basis for how reduced levels of man1b1 could theoretically lead to greater intracellular atz accumulation. these results for the man1b1 variant would appear to validate our hypothesis that intracellular degradation pathways are targets of liver disease modifiers but further populations studies of this variant would be reassuring. a snp in the upstream flanking region of the at gene has also been implicated in susceptibility to liver disease. however, the nature of variant could not be reconciled with how it might affect liver disease susceptibility and its statistical association with variation in the liver disease phenotype was dependent on a questionable classification of population sub - groups. our hypothesis for variation in liver disease susceptibility also identifies signaling pathways that could increase or decrease atz proteotoxicity as potential targets for disease modifiers (figure 1). as of yet we have not encountered an example of this potential scenario, but we predict that further studies of iheps from patients with different forms of atd liver disease, in terms of age of onset and type of hepatic pathology, will identify such a mechanism in the near future. in the classical form of atd a misfolded variant accumulates in the early part of the vesicular secretory pathway and causes liver disease by gain - of - toxic function. the site of accumulation includes rough er and pre - golgi compartment(s) that are not characteristic of rough er. the misfolded variant has a tendency to polymerize / aggregate and that tendency appears to play an important role in the unique structural and functional consequences. the liver disease is characterized by fibrosis and hyperproliferation but it is variable in how it affects each host. there is growing evidence that this variability is due to genetic and/or environmental modifiers and that the modifiers act on proteostasis mechanisms that are unique for this misfolded variant. these proteostasis mechanisms include the proteosomal and autophagy degradative pathways and a repertoire of signaling pathways that does not include a full - fledged upr but is characterized by activation of autophagy, nfb and tgf signaling in distinct ways. because of this we believe it is important to think of the upr as one form of er stress and that there is substrate specificity in how the er responds to misfolded protein accumulation. exciting new studies using ips - derived hepatocyte - like cell line have provided initial evidence that we can deduce the personalized variations and how they affect the structure and function of the er and other cellular machineries involved in protein quality control.	in the classical form of 1-antitrypsin deficiency (atd) a point mutation leads to accumulation of a misfolded secretory glycoprotein in the endoplasmic reticulum (er) of liver cells and so atd has come to be considered a prototypical er storage disease. it is associated with two major types of clinical disorders, chronic obstructive pulmonary disease (copd) by loss - of - function mechanisms and hepatic cirrhosis and carcinogenesis by gain - of - function mechanisms. the lung disease predominantly results from proteolytic damage to the pulmonary connective tissue matrix because of reduced levels of protease inhibitor activity of 1-anitrypsin (at) in the circulating blood and body fluids. cigarette smoking is a powerful disease - promoting modifier but other modifiers are known to exist because variation in the lung disease phenotype is still found in smoking and non - smoking homozygotes. the liver disease is highly likely to be caused by the proteotoxic effects of intracellular misfolded protein accumulation and a high degree of variation in the hepatic phenotype among affected homozygotes has been hypothetically attributed to genetic and environmental modifiers that alter proteostasis responses. liver biopsies of homozygotes show intrahepatocytic inclusions with dilation and expansion of the er and recent studies of ips - derived hepatocyte - like cells from individuals with atd indicate that the changes in the er directly vary with the hepatic phenotype i.e there is much lesser alteration in the er in cells derived from homozygotes that do not have clinically significant liver disease. from a signaling perspective, studies in mammalian cell line and animal models expressing the classical 1-antitrypsin z variant (atz) have found that er signaling is perturbed in a relatively unique way with powerful activation of autophagy and the nfb pathway but relatively limited, if any, upr signaling. it is still not known how much these unique structural and functional changes and the variation among affected homozygotes relate to the tendency of this variant to polymerize and aggregate and/or to the repertoire of proteostasis mechanisms that are activated.
hypogonadotropic hypogonadism (hh) is the 2 most frequent cause of ovarian failure in young women in whom pulsatile gnrh therapy or replacement of gonadotropin can restore ovulation. the threshold for ovarian response in hh may differ substantially from that established for normal patients. these patients require a combination of follicle - stimulating hormone (fsh) and luteinizing hormone (lh) for inducing ovulation. the success of ovulation induction is reported to be as high 6080% with a high multiple pregnancy rate (2050%). in order to avoid higher order pregnancies these patients may require a longer duration of stimulation with gradually stepping up the dose in order to reach the threshold of fsh and lh. assisted reproductive technology (art) is beneficial in this group of patients to prevent higher order multiple pregnancy or to increase the chance of pregnancy whenever there is poor response on ovarian stimulation. we present 7 cases of hh, who underwent art to evaluate the duration of ovarian stimulation, quality of oocytes and embryo, and the pregnancy outcome in the art cycle in patients with hh. over the period of 4 years, we had 14 patients with hh in whom 21 cycles of ovulation induction were done. 3 of the 7 patients were initially planned for ovulation induction and intrauterine insemination (iui) but later converted to art cycle while 4 patients were planned directly for controlled ovarian hyperstimulation and intracytoplasmic sperm injection (icsi). serum fsh, lh, estradiol, anti - mullerian hormone (amh), ultrasonography (usg) pelvis, and magnetic resonance imaging (mri) were done for all patients and the findings are tabulated in table 1. base line profile of the hh patients who underwent oocyte retrieval before ovulation induction, adequate size of the uterus (at least > 5 cm) was obtained by giving cyclical estrogen and progesterone therapy for a minimal of 3 months. semen analysis of husband was done for all the patients and was found to be normal in all of them. patients planned for ovulation induction and iui were started on injection human menopausal gonadotropins (hmg) 75 iu for 7 days, later the dose was gradually stepped up with injection hmg with or without injection urinary fsh (ufsh) depending upon the response with the aim to obtain 2 or 3 follicles. whenever there was hyper response or poor response during ovulation induction for iui these patients were counseled for conversion to an art cycle. in patients planned for art higher dose of injection hmg 150 iu in combination with injection ufsh 150225 iu was started depending upon the age and amh of the patient. when 3 follicles had reached 18 mm urinary human chorionic gonadotropin 10,000, iu i m trigger was given, and oocyte retrieval was done 36 h later using 17 g needle, under short ga anesthesia under tvs guidance. icsi was done for all patients and grading of embryos was done by veeck 's scoring system. when there was a high risk of ovarian hyperstimulation syndrome (ohss), fresh embryo transfer was avoided, and embryos were frozen. in such patients later frozen embryo transfer was done after hormone replacement therapy. in the fresh cycle, luteal phase was supplemented for all patients with estradiol valerate 2 mg twice daily orally and micronized progesterone 200 mg thrice daily vaginally. in the study group, 4 patients had primary amenorrhea remaining 3 had presented as a case of secondary amenorrhea. ovaries were visualized in 4 patients, and two of these showed polycystic ovarian morphology (pcom) features, but volume was 12 days to respond to stimulation (maximum being 54 days). six patients had > 70% good quality mii oocytes (one patient who had poor response had only 2 mii good quality oocytes). fertilization rate in these patients was 85% (except in one who had 50% fertilization rate), and the cumulative pregnancy rate was 68.6%. the details of the quality of oocyte and embryos and pregnancy outcome are tabulated in table 3. hh is a rare disorder of reproductive function characterized by the absence of normal hypothalamic these patients belong to who group 1 anovulation which account for 10% of cases for anovulation and present with the history of amenorrhea, hypogonadism (3.7 ng / ml suggesting high risk for hyper response. gnrh agonist trigger to avoid ohss can not be used in these patients due to endogenous deficiency of gonadotropin. the third patient was stimulated by step - up protocol but when she did not show any response till the 36 day, serum amh was done which was 0.7 ng / ml. tran. mentioned in the clinical case seminar the role of amh in assessing ovarian reserve that amh has limitations because it only reflects the growing follicular pool that is responsive to gonadotropins. hence, conditions that cause a permanent or sustained interruption of gonadotropin release may lead to a decrease amh levels and therefore an underestimation of the true ovarian reserve suggesting that amh may not be a very good predictor of ovarian reserve in patients with hh. they reported a case with hh in which during the 16-week course of treatment with hmg, follicular development was observed. both amh and antral follicle count (afc) gradually increased during treatment from the initial amh and afc of 0.20 ng / ml and zero, and peaked to 1.26 ng / ml and 6, respectively. considering this we convinced the patient as she was young (26 years) and decided to continue stimulation until the maximum dose of gonadotropin 600 iu / day was used or till response was noted whichever was earlier. the couple agreed to continue the gonadotropins injections and on the 47 day of stimulation with ufsh 225 iu and hmg 225 iu ovaries were seen and the same dose was continued. on the 52 day patient developed 5 follicles of > 17 mm. the couple was counseled for oocyte retrieval as she was a poor responder and had responded after a long course of gonadotropin, details in tables 2 and 3. this patient required higher dose to respond, and she formed relatively less number of follicles compared to patients who had higher amh. last four cases (4 to 7) were planned for art hence these patients were stimulated with a higher dose gonadotropin, and they responded relatively faster. the quality of oocytes and embryo in all these 7 patients were good irrespective of the duration of stimulation as shown in table 3 proving that long duration of stimulation in hh did not affect the quality of oocytes or embryo. lewit and kol has also reported successful stimulation after 29 days of gonadotropin in a patient in whom twice stimulation was cancelled after 20 days due to nonresponse to gonadotropin. they concluded that patients with hh undergoing ovarian stimulation for ivf should be carefully assessed on a trial and error basis for ovarian response before we give up on obtaining fertilizable oocytes. our observation also helps us to confidently continue ovulation induction in patients with hh in step - up protocol till follicular response is seen irrespective of duration of stimulation. juan balasch has suggested to use step - down and step - up approach where patients receive 2 or 3 ampoules of hmg daily (depending upon the patient 's body mass index) for 2 days later one ampoule from day 3 to 7. from day 8 onward, hmg is administered on the principles of step - up regimen as per the requirement of the patient. ovarian reserve is difficult to assess in these patients as amh levels in these patients have to be interpreted cautiously. when planned for ovulation induction we must be prepared for longer duration of stimulation which does not affect the quality of oocytes and embryo, and the pregnancy rate. the treating physician and the patient need a lot of patience and motivation to continue the treatment for longer duration.	context : ovulation induction in patients with hypogonadotropic hypogonadism (hh) is a challenge to the treating physician. the threshold for ovarian response in hh may differ substantially from that of normal patients. to reach that threshold levels of follicle stimulating hormone, in a step - up protocol longer duration of stimulation is required in some cases so as to prevent multiple pregnancy and to eliminate the risk of ovarian hyperstimulation syndrome.aim:to evaluate the duration of stimulation, quality of oocytes, and embryo, and the pregnancy outcome in the assisted reproductive technology (art) cycles in patients with hh.materials and methods : over the period of 4 years, we had 14 patients with hh in whom 21 cycles of ovulation induction were done. of these 7 patients underwent oocyte retrieval and intracytoplasmic sperm injection (icsi). we present a retrospective study of these 7 patients who underwent art to evaluate the duration of stimulation, quality of oocytes and embryo, and the pregnancy outcome.results:in the study group on ovulation induction with gonadotropins, only one patient had the duration of stimulation of the standard 12 days, the remaining 6 patients took 12 days to respond to stimulation (maxium being 54 days). mean et in these patients was 8.9 mm. six patients had > 70% good quality mii oocytes. one patient responded poorly and had only 2 good quality mii oocytes (50%). after icsi procedure, resultant embryos were of grade 1 and 2 in all the patients irrespective of the duration of stimulation. fertilization rate in these patients was 85% (except in one 50% fertilization rate), and the cumulative pregnancy rate was 68.6%.conclusion : in the patients with hh the quality of oocytes and embryos, and the pregnancy rate is not affected even if the duration of stimulation is prolonged.
osteoarthritis (oa) is a leading cause of disability in the elderly and has a significant impact on health care (reviewed in). although the pathogenesis of oa remains unclear, the chronic production of different mediators by articular tissues is believed to contribute to tissue degradation. levels of proinflammatory cytokines such as interleukin- (il-) 1 and tumor necrosis factor- (tnf) are elevated in the inflamed synovium in oa, accompanied by the increased expression of their receptors and decreased levels of inhibitory proteins. these cytokines mediate cartilage destruction through the upregulation of inflammatory or catabolic genes and the downregulation of anti - inflammatory or anabolic genes in articular chondrocytes (reviewed in). in particular, il-1 reduces the expression of type ii collagen and increases the production of matrix metalloproteinases (mmps) [4, 5 ], prostaglandin e2 (pge2), cytokines, chemokines, reactive oxygen species, and nitric oxide (no) [6, 7 ]. chondrocytes are the main source of no in oa articular tissues and the oxidative stress caused by this mediator has been related to degeneration in arthritic joints. therefore, no can play a role in il-1-induced suppression of glycosaminoglycan and collagen synthesis, expression of mmps, and activation of proenzymes. mesenchymal stem cells (msc) are being investigated as a possible cell - based therapy for late stages of oa. some promising results have been obtained in a pilot study of knee oa using autologous bone - marrow - derived mesenchymal stem cells. interestingly, stem cells are able to secrete a wide range of trophic mediators that can exert paracrine effects on other cell types. therefore, stem - cell - conditioned media have shown potential therapeutic applications in neural, myocardial, and osteogenic regeneration or in wound healing (reviewed in). adipose - tissue - derived mesenchymal stem cells (ad - msc) are extensively investigated for tissue regeneration or immunomodulation (reviewed in [13, 14 ]). interestingly, stem cells are able to secrete a wide range of trophic mediators that can exert paracrine effects on other cell types. in this study, we have examined the potential of the conditioned medium from basal ad - msc (cm) to regulate type ii collagen expression and the production of relevant mediators involved in oa articular degeneration using oa chondrocytes in primary cultures as an in vitro model to study inflammatory and degradative responses. adipose tissues were obtained from 11 donors who had undergone abdominoplasty without any underlying diseases. adipose tissue samples were washed with phosphate - buffered saline (pbs), minced, digested at 37c for 1 h with 2% of type i collagenase (gibco, life technologies, madrid, spain), and filtered through a 100 m cell strainer (bd biosciences durham, nc, usa). the cells were washed with dmem / ham f12 containing penicillin and streptomycin (1%), seeded onto tissue culture flasks in dmem / ham f12 medium with penicillin and streptomycin (1%) supplemented with 15% human serum, and incubated with 5% co2 at 37c. human serum was obtained from whole - blood donations of ab - blood - group - typed donors according to the criteria of valencia transfusion center. at 24 h, when the cells reached the semiconfluence, the tissue culture plates were washed to remove any residual nonadherent cells. the phenotype of ad - msc was analyzed by flow cytometry (flow cytometer ii, bd biosciences, san jose, ca, usa) with specific antibodies, anti - cd105-pe, anti - cd90percp - efluo 710, anti - cd34apc (ebioscience, inc., san diego, ca, usa), and anti - cd45-pe (bd pharmigen) and cellular viability with propidium iodide. cm was collected from cells at passages 0 and 1 every 48 h of culture, pooled, centrifuged, and stored at 80c in sterile conditions. the knee specimens were obtained from patients with the diagnosis of advanced oa (22 women and 8 men, aged 72.1 7.8 years, mean s.e.m.) undergoing total knee joint replacement. cartilage was dissected from the femoral condyles and tibial plateau of the knee joint and diced into small pieces. human articular chondrocytes were isolated by sequential enzymatic digestion : 1 h with 0.1 mg / ml hyaluronidase (sigma - aldrich) followed by 1215 h with 2 mg / ml collagenase (type ia) (sigma - aldrich) in dmem / ham f12 (sigma - aldrich) containing penicillin and streptomycin (1%) at 37c in 5% co2 atmosphere. the digested tissue was filtered through a 70 m nylon mesh (bd biosciences), washed, and centrifuged. all experiments were performed with chondrocytes in primary cultures at semiconfluence (270 10 cells / well in 6-well plates or 1.5 10 cells in 3.5 cm plates). chondrocytes were maintained with 5% co2 at 37c in dmem / ham f12 (sigma - aldrich) containing penicillin and streptomycin (1%), supplemented with 10% fetal bovine serum (sigma - aldrich). for cell stimulation, chondrocytes were incubated for 24 h or 5 days in dmem / ham f12 (sigma - aldrich) containing penicillin and streptomycin (1%) supplemented with 10% human serum, in the presence or absence of il-1 (10 ng / ml) and/or cm (1 ml of medium for 6-well plates or 2 ml for 3.5 cm plates). the design of the work was approved by the institutional ethical committees (university of valencia and university clinical hospital, valencia, spain). samples were obtained from donors after they provided informed consent according to the helsinki declaration of 1975, as revised in 2008. the mitochondrial dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (mtt) to formazan was assayed in oa chondrocytes incubated with il-1 (10 ng / ml) or il-1 (10 ng / ml) + cm for 24 h or 5 days. the cells were then incubated with mtt (200 g / ml) for 2 h. the medium was removed and the cells were solubilized in dimethyl sulfoxide (100 l) to quantitate formazan at 550 nm. chondrocytes were seeded at 20 10 cells / well in lab - tek chambers (thermo scientific, rochester, ny, usa) and stimulated with il-1 (10 ng / ml) or il-1+cm for 5 days. cells were fixed with 4% formaldehyde in pbs for 30 min at 4c and incubated with rabbit anti - human type ii collagen polyclonal antibody (chemicon / millipore, schwalbach, germany). finally, chondrocytes were incubated with goat anti - rabbit igg - fitc (r&d biosystems, abingdon, uk). slides were mounted in prolong gold antifade reagent with dapi (molecular probes, invitrogen, life technologies) and examined under a fluorescence microscope (leica dm il led, solms, germany). thereby collagen ii - positive cells were determined as a percentage of total cell number. chondrocytes were stimulated with il-1 (10 ng / ml) or il-1+cm for 24 h or 5 days and supernatants were harvested, centrifuged, and incubated with p - aminophenylmercuric acetate for 12 h at 37c to activate mmps. aliquots of supernatants were then transferred to a 96-well plate and after addition of the 5-fam peptide substrate (anaspec inc., san jose, ca, usa), fluorescence was measured for different times at 490 nm (excitation)/520 nm (emission) in a victor3 microplate reader (perkinelmer espaa, madrid, spain). chondrocytes were stimulated with il-1 (10 ng / ml) or il-1+cm for 24 h or 5 days. tnf, il-6, and il-10 were measured by enzyme - linked immunosorbent assay (elisa) kits from ebioscience (san diego, ca, usa) with sensitivity of 4.0 pg / ml for tnf and il-6 and 2.0 pg / ml for il-10. mmp-3 and mmp-13 proteins were measured by elisa kits (ebioscience) with sensitivity of 8.0 and 18.0 pg / ml, respectively. nuclear factor-b (nf-b) binding to dna was quantified by elisa in nuclear extracts from cells stimulated with il-1 (10 ng / ml) in the presence or absence of cm for 1 h, using the nuclear extract kit active motif for nuclei extraction followed by transam nf-b kit (active motif europe, rixensart, belgium), according to the manufacturer 's recommendations. total rna was extracted using the tripure reagent (roche applied science, barcelona, spain) according to the manufacturer 's instructions. reverse transcription was accomplished on 1 g of total rna using random primers (taqman reverse transcription reagents, applied biosystems, madrid, spain). pcr assays were performed in duplicate on an icycler real - time pcr detection system using sybr green pcr master mix (bio - rad laboratories, richmond, ca, usa). sequences of primers used have been reported previously [1721 ] and were synthesized by eurofins mwg operon (ebersberg, germany). some primer sets were from sa biosciences corporation (tebu - bio, barcelona, spain). for each sample, differences in threshold cycle (ct) values were calculated by correcting the ct of the gene of interest to the ct of the reference gene -actin. chondrocytes were stimulated with il-1 (10 ng / ml) or il-1+cm for 24 h or 5 days. supernatants were used to measure prostaglandin e2 (pge2) by radioimmunoassay and nitrite by a fluorometric method using a victor3 microplate reader (perkinelmer espaa, madrid, spain). the data were analyzed by one - way analysis of variance (anova) followed by bonferroni 's posttest using the graphpad prism 5 software (graphpad software, la jolla, ca, usa). to determine whether cm treatment affected oa chondrocyte proliferation, oa chondrocytes were incubated in the absence or presence of cm and il-1. after 24 h or 5 days, the mtt reaction was performed. as shown in figure 1(a), a significantly higher mtt % was observed for all groups after 5 days with respect to 24 h incubation. mmps are key mediators in cartilage degradation. to characterize the effects of cm on oa chondrocytes, we measured mmp activity as well as the levels of mmp-3 and mmp-13 proteins in cell supernatants. il-1 enhanced the mmp activity present in cell supernatants after 24 h of incubation and to a greater extent after 5 days (figure 1(b)). mmp-3 protein (figure 2(a)) and mrna (figure 2(c)) levels were decreased by cm in il-1-stimulated chondrocytes and this effect was significant after 24 h of incubation. figure 2(b) shows that mmp-13 protein levels were reduced by cm after il-1 stimulation (24 h and 5 days of incubation). mmp-13 mrna expression was also significantly reduced by cm in il-1-stimulated oa chondrocytes (figure 2(c)). figure 3(a) shows a representative image of collagen ii expression in oa chondrocytes in the presence or absence of il-1 and cm after 5 days of incubation. this cytokine reduced the expression of collagen ii, but cm significantly increased the percentage of collagen ii - positive cells either in basal conditions or in chondrocytes stimulated with il-1 (figure 3(b)). il-6 and tnf are key mediators of the inflammatory response and were measured in supernatants by elisa. figure 4(a) shows that il-1 strongly increased il-6 levels in cell supernatants after 24 h or 5 days of incubation whereas cm significantly decreased the production of il-6 at both time points. in addition, the levels of tnf induced by il-1 were reduced by cm after 24 h incubation (figure 4(b)). interestingly, the production of the anti - inflammatory cytokine il-10 was significantly enhanced by cm at both time points (figure 4(c)). therefore, we observed a reduced expression of il-6 and tnf while il-10 mrna expression was significantly enhanced by cm in il-1-stimulated chondrocytes (figure 4(d)). in oa, there is an increased expression of chemokines in chondrocytes under the influence of inflammatory cytokines such as il-1 [24, 25 ]. figure 5 shows that il-1 enhanced the mrna expression of ccl-2, ccl-3, ccl-4, ccl-5, ccl-8, ccl-19, ccl-20, cxcl-1, cxcl-2, cxcl-3, cxcl-5, and cxcl-8 after 24 h of incubation. when cm was included in the incubation media, we observed a significant reduction in the expression of these chemokines. the concentrations of nitrite, as an index of no production, and pge2were measured in the supernatant of oa chondrocytes after 24 h or 5 days of incubation. the presence of cm during the incubation period resulted in a significant reduction in the levels of nitrite (figure 6(a)) and pge2 (figure 6(b)) in the supernatant at both time points in the presence of il-1 stimulation. we analyzed the relative mrna expression of inducible no synthase (inos), cyclooxygenase-2 (cox-2), and microsomal pge synthase-1 (mpges-1) in oa chondrocytes incubated with il-1 and/or cm for 24 h. figure 6(c) shows that cm significantly reduced the expression of inos thus leading to reduced nitrite production, in addition to the downregulation of cox-2 and mpges-1 which would explain the inhibitory effects of cm on pge2. to understand the mechanism involved in the effects of cm on inflammatory and catabolic mediators, we investigated the possible regulation of this key transcription factor. il-1 quickly induces nf-b translocation into the nucleus and dna binding to activate gene transcription. figure 7 shows the enhancement of p65-nf-b - dna binding induced by il-1 in oa chondrocytes. in the presence of cm, p65-nf-b binding to dna was significantly decreased. several lines of evidence have demonstrated in oa articular tissues the production of a wide range of catabolic and proinflammatory mediators leading to cartilage matrix degradation (reviewed in). in addition to other extracellular matrix components, the fibrils of type ii collagen are essential for the integrity and survival of articular cartilage. in oa cartilage, there is an upregulation of collagenases such as mmp-13, the major type ii collagen - degrading enzyme, which initiate the denaturation of fibrillar type ii collagen thus contributing to the initiation and progression of joint damage. in this study, we have demonstrated that cm enhances collagen ii expression in nonstimulated oa chondrocytes and counteracts the negative effects of il-1 on this protein. we have also found that cm downregulates catabolic enzymes which play a key role in cartilage degradation. therefore, cm reduced the production of the collagenase mmp-13 and to a lesser extent of the stromelysin mmp-3 which mediates the direct degradation of extracellular matrix components and the activation of other mmps. proinflammatory cytokines activate chondrocytes leading to the synthesis and release of a wide range of mediators. our study reveals that cm downregulates the proinflammatory cytokines il-6 and tnf but upregulates the anti - inflammatory cytokine il-10. these effects are of relevance for the progression of oa as proinflammatory cytokines induce the synthesis of molecules contributing to the loss of chondrocyte phenotype and cartilage degeneration. in addition, the effect of cm on il-10 may have positive consequences on cartilage metabolism as this cytokine cooperates with other factors to inhibit cartilage breakdown in experimental oa. chondrocytes may amplify inflammatory and catabolic responses through the release of chemokines promoting inflammation, synovial angiogenesis [30, 31 ], and the production of catabolic mediators such as mmps in oa chondrocytes [32, 33 ]. interestingly, cm reduced the expression of a number of chemokines relevant to chondrocyte metabolism. our data also show that cm counteracts no production in oa chondrocytes stimulated by il-1. this effect would be the consequence of inos downregulation which may contribute to the protective actions of cm as no inhibits matrix synthesis. this eicosanoid may contribute to cartilage degradation by promoting the production of mmps and inhibiting the synthesis of tissue inhibitors of these enzymes [34, 35 ]. cox-2 and mpges-1 are functionally coupled and induced by il-1 in oa chondrocytes leading to an increased pge2 synthesis. therefore, the effect of cm on pge2 production by oa chondrocytes may be relevant to cartilage metabolism and would be dependent on the reduction of cox-2 and mpges-1 expression observed in our study. the activation of nf-b plays a key role in the transcription of inos, cox-2, mmps, and different proinflammatory cytokines and chemokines [37, 38 ]. to understand the possible mechanism by which cm downregulates these mediators, we have investigated its influence on nf-b. our results indicate that the observed inhibitory effects of cm on the expression of catabolic and proinflammatory molecules could be related to the reduction of nf-b activation in oa chondrocytes stimulated with il-1. msc have shown cytoprotective properties in experimental models via paracrine mechanisms. the beneficial effects of these cells may be related to the production of different types of mediators such as cytokines and pge2 or a milieu of secreted factors exerting synergistic effects [3941 ]. further studies are needed to elucidate the factors responsible for the chondroprotective effects of cm or to provide additional mechanistic insights. the results of our study have shown the chondroprotective role of cm by targeting catabolic and inflammatory mediators and support the interest of this approach to search for new treatments for inflammatory and/or degenerative conditions of joints.	osteoarthritis (oa) is the most frequent joint disorder and an important cause of disability. recent studies have shown the potential of adipose - tissue - derived mesenchymal stem cells (ad - msc) for cartilage repair. we have investigated whether conditioned medium from ad - msc (cm) may regulate in oa chondrocytes a number of key mediators involved in cartilage degeneration. cm enhanced type ii collagen expression in oa chondrocytes while decreasing matrix metalloproteinase (mmp) activity in cell supernatants as well as the levels of mmp-3 and mmp-13 proteins and mrna in oa chondrocytes stimulated with interleukin- (il-) 1. in addition, cm increased il-10 levels and counteracted the stimulating effects of il-1 on the production of tumor necrosis factor-, il-6, prostaglandin e2, and no measured as nitrite and the mrna expression of these cytokines, ccl-2, ccl-3, ccl-4, ccl-5, ccl-8, ccl-19, ccl-20, cxcl-1, cxcl-2, cxcl-3, cxcl-5, cxcl-8, cyclooxygenase-2, microsomal prostaglandin e synthase-1, and inducible no synthase. these effects may be dependent on the inhibition of nuclear factor-b activation by cm. our data demonstrate the chondroprotective actions of cm and provide support for further studies of this approach in joint disease.
police officers are members of a unique occupational population whose exposure to potentially traumatic and life threatening events is part of their professional duty. additionally, officers routinely encounter organizational stressors, such as court appearances, excessive paperwork, and shift work. higher levels of perceived work stress have been associated with depression among older police officers. police officers have more adverse cardiovascular disease (cvd) risk factor levels and higher rates of cvd morbidity and mortality compared to other populations [36 ]. work stress and depression are risk factors for the development of cvd and diabetes [2, 720 ]. the metsyn is a clustering of abnormalities (abdominal obesity, hypertension, dyslipidemia, glucose intolerance) that has been associated with increased risk for subclinical atherosclerosis, cvd, diabetes, target organ damage, and total mortality. the association between depression and the metsyn has been studied over the past decade [8, 9, 11, 13, 14, 18, 19, 2229 ], and more recently it has been suggested that metsyn may be intermediate in the pathway between depression and cvd [11, 13, 15, 18, 19, 30, 31 ]. one mechanism is neuroendocrine and involves overstimulation of the hypothalamic - pituitary adrenal (hpa) axis which leads to excess cortisol secretion and has been associated with both the development of depression and the metsyn components [32, 33 ]. another indirect pathway is through adverse health behaviors associated with depression, such as cigarette smoking, excessive use of alcohol, and a sedentary lifestyle, which may be used as coping responses and subsequently increase risk of metsyn and cvd [8, 11, 19 ]. the association between depression and metsyn appears to be stronger in women, although the explanation for this gender difference is somewhat unclear [11, 15, 18, 23, 25, 26 ]. overactivation of the hpa axis due to stress or depression may result in decreased levels of sex hormones like estrogen. decreased levels of estrogen, such as that occurs during menopause, can elevate visceral fat mass, a component of metsyn. police officers are considered to be a relatively young and presumably healthy occupational group (i.e., healthy worker effect) ; yet they suffer from high levels of stress and cvd. the purpose of this study was to examine the association between depressive symptoms and the metsyn separately among male and female police officers from two cross - sectional studies. participants are police officers from two geographically different locations, which may make the findings more generalizable to other groups of police officers. based on the literature, the specific study hypotheses were the following : (1) depressive symptoms would be associated with a larger number of metsyn components in male and female police officers, (2) the association of depressive symptoms with metsyn and its components would differ between male and female police officers, and (3) the association between depressive symptoms and metsyn would not differ by geographic location. participants included those from two studies at two separate locations : buffalo, new york and spokane, washington. the buffalo cardio - metabolic occupational police stress (bcops) study, a cross - sectional study of urban police officers, was conducted between 2004 and 2009. the purpose of the bcops study was to examine the association between police stressors and physiological and psychological health consequences. the center for health research at the school of public health and health professions, state university of new york in buffalo, ny served as the data collection site. all 710 active duty police officers from the buffalo, ny, police department were invited to participate and the recruitment was ongoing for five years. no specific inclusion criteria were used for the study, other than that the participant be a sworn police officer and willing to participate in the study. women officers who were pregnant at the time of examination were excluded (n = 2). all participants provided informed consent and all phases, testing, and reports of the study were approved by the state university of new york at buffalo internal review board and the national institute for occupational safety and health human subjects review board. of the 464 officers examined, 54 were removed from analyses (33 retired, 1 missing demographic information, 3 missing depressive symptoms, 17 missing metsyn) leaving a final sample of 410 officers (304 men, 106 women ; age range 2166). participants provided a medical history (including history of cvd) and a 12-hour fasting blood sample was collected by a certified phlebotomist. medication use was ascertained through self - report and by inventory of current medications brought to the clinic. blood parameters for the metsyn were measured by standard laboratory techniques on the beckman coulter lx20 clinical chemistry analyzer and included a blood lipid panel for high - density lipoprotein cholesterol (hdl - c) and triglycerides and chemistry panels for glucose. waist circumference was measured as abdominal girth at the highest point of the iliac crest and the lowest point of the costal margin in the mid - axillary line. blood pressure was determined using the average of the second and third of three separate measurements of resting systolic and diastolic blood pressure obtained with a standard sphygmomanometer. the spokane heart study (shs) was a prospective, longitudinal study investigating the natural history of preclinical atherosclerosis. over 1,000 asymptomatic men and women were enrolled and followed every two years, generating an extensive data set of medical and lifestyle histories, blood and urine measurements, and electron beam computed tomography (ebct) measures of coronary artery calcification. at baseline, all participants completed a comprehensive questionnaire which measured key behavioral, social, and lifestyle domains. fasting blood samples and anthropometric measures all participants provided informed consent and all phases, testing, and reports of the study were approved by the washington state university institutional review board. for the current study, data collection years 20032006, years most comparable to the bcops study, were used. based on the questions, what is the name of your current employer and what is your current job title, four were removed from analyses (two missing depressive symptoms and two missing metsyn) leaving a final sample of 130 spokane police officers (121 men, 9 women ; age range 3562). in both studies, depressive symptoms were measured using the center for epidemiologic studies - depression (ces - d) scale. the ces - d is a short 20-item scale designed to measure symptoms of depression (e.g., poor appetite, restless sleep, sadness) in the general population on a 4-point scale. the 4-point scale represents how much each symptom occurred during the past seven days as follows : 0 (rarely or none of the time, less than 1 day), 1 (some or little of the time, 1 - 2 days), 2 (occasionally or a moderate amount of time, 3 - 4 days), and 3 (most or all of the time, 57 days). the ces - d is scored by reverse coding the appropriate items and summing the scores to obtain an overall score. respondents with scores between 0 and 15 are unlikely to be clinically depressed, scores of 16 to 21 indicate mild to moderate depression, and scores of 22 or greater are associated with major depression. the ces - d has acceptable reliability, cronbach alpha of 0.85, and a split - half reliability ranging from 0.76 to 0.85. for the current study, the continuous ces - d and three categories of ces - d were used : low (08), middle (915), and high (16 or greater). the metsyn criteria were based on the national cholesterol education program adult treatment panel guidelines with recent modifications from the american heart association and the national heart, lung, and blood institute. the individual metsyn components included the following : (1) abdominal obesity (gender - specific waist circumference 102 cm in males, 88 cm in females), (2) hypertension (systolic blood pressure 130 mm hg, diastolic blood pressure 85 mm hg, or reported physician - diagnosed hypertension and antihypertensive treatment), (3) reduced hdl - c (gender - specific fasting hdl - c 49 years, n = 59) versus younger spokane men (49, n = 61), and lower - ranking (patrol officer, n = 39) versus higher ranking spokane men (n = 81) (data not shown). yet, due to the small sample sizes for the stratified analyses the findings are reported with caution. perceived work stress has been associated with depression among older mostly male police officers, and high job strain has been significantly associated with depression among a national sample of working men [2, 43 ]. suggested that chronic stress leads to depression which increases the risk of metsyn and subsequently cvd. for spokane men, the odds of having four of the metsyn components (abdominal obesity, hypertriglyceridemia, hypertension, glucose intolerance) increased by at least 40% for every 5-unit increase in ces - d score. the association with abdominal obesity was slightly attenuated by adjustment for age, smoking status, and marital status, while hdl - c was not associated with depressive symptoms. this finding is supported by previous studies which have found depression to be associated with abdominal obesity, hypertriglyceridemia, hypertension, and glucose intolerance [11, 19, 29, 40, 44, 45 ]. the association between depression and metsyn and its components among these diverse populations suggests that the pathophysiology of both conditions may overlap ; yet the biologic mechanisms may vary by metsyn component. dysfunction of the hypothalamic - pituitary - adrenal (hpa) axis has been the most commonly cited mechanism for the association between depression and metsyn [29, 46 ]. have shown that hyperinsulinemia activates the hpa axis and increases cortisol secretion and this increase has been associated with depression, the metsyn, and its individual components [11, 17, 32, 33, 46, 48 ]. second, inflammation particularly elevated levels of c - reactive protein (crp) have been associated with depressive symptoms in younger men, and in elderly men and women. inflammation has also been acknowledged as a key factor underlying development of metsyn [22, 46 ]. finally, as a means of coping depressed individuals may engage in poor health behaviors, such as a sedentary lifestyle, poor nutrition, cigarette smoking, excessive alcohol intake, or inadequate sleep, and these behaviors may lead to the development of metsyn [11, 19 ]. of these behaviors, only smoking status was available in the current study and it was the buffalo women who had the highest percent of current and former smokers (57.3%) compared to the other groups (all less than 37%). however, consistent with previous studies, smoking status was included as a potential covariate in multivariate analyses although adjustment attenuated the association minimally across all groups. first, the ces - d provides a self - report measure of depressive symptoms as opposed to measuring clinical depression. underreporting depressive symptoms is of concern with this occupational group as officers may not wish to disclose sensitive information. however, the ces - d has been widely used as a measure for assessing depressive symptoms in epidemiological studies. second, this study was cross - sectional ; therefore, we can not determine whether depressive symptoms preceded the development of metsyn and its components or make causal inferences. others have reported that the metsyn may lead to depression, specifically that hyperglycemia increases hpa axis activity causing arousal of the central nervous system thereby promoting development of depression. third, it is possible that participants in these two studies may not be representative of their respective police departments. however, for the buffalo study comparisons between study participants and the buffalo, ny police department resulted in similar frequency distributions for sex, age, and police rank. finally, data are from two studies of police officers, thus potentially limiting the generalizability of the study findings to other police departments and other emergency responder populations. one of the strengths of the current study was the ability to examine the association between depressive symptoms and metsyn separately for men and women officers. female police officers are an understudied occupational group and previous studies of police officers have typically included only male officers. second, the study population was relatively young (mean age 49 for spokane, 41 for buffalo) and we were able to exclude those who have cvd or have received clinical interventions for cvd, enabling us to affirm that the relationship was unlikely attributable to cvd. third, we were able to exclude officers who were taking antidepressant medications and female officers who reported taking exogenous estrogen, although these exclusions did not alter the study findings. finally, we used a highly standardized measure as our outcome, metsyn. in this study metsyn was defined as the count of the number of components for each individual instead of limiting the assessment to presence / absence of metsyn for the current study. this approach provides an interpretable measure of association and is more sensitive in the detection of associations. in summary, depressive symptoms were associated with an increasing number of metsyn components and with higher odds of having the individual metsyn components among spokane male police officers. this is the first study that we are aware of which has examined this relationship in police officers. yet, it is particularly important to understand the association between depression and metsyn among police officers given the high - stress nature of their job and the higher prevalence of cvd. future studies should explore potential mediators for this association, including the role of the hpa axis and cortisol, inflammatory processes, and hyperinsulinemia, as these have been cited as potential mechanisms by others. it is also important to explore the gender - specific differences between male and female police officers. depressive symptomatology and metsyn are early indicators of future chronic health problems and the results could assist in the development of future gender - specific prevention and intervention efforts among police officers.	policing is one of the most dangerous and stressful occupations and such stress can have deleterious effects on health. the purpose of this study was to examine the association between depressive symptoms and metabolic syndrome (metsyn) in male and female police officers from two study populations, buffalo, ny and spokane, wa. depressive symptoms were measured using the center for epidemiologic studies - depression (ces - d) scale. metsyn was defined using the 2005 aha / nhbli guidelines. analysis of covariance was used to describe differences in number of metsyn components across depressive symptom categories. the number of metsyn components increased significantly across categories of ces - d for spokane men only (p - trend = 0.003). for each 5-unit increase in ces - d score, odds increased by 47.6% for having hypertriglyceridemia, by 51.8% for having hypertension, and by 56.7% for having glucose intolerance. exploring this association is important since both are predictors of future chronic health problems and the results could be helpful in developing future gender - specific prevention and intervention efforts among police officers.
early mobilization and stable fixation with anatomic reduction are the main goals in the treatment.1 several surgical methods have been used for treating intertrochanteric fractures. the current widely used treatment methods for intertrochanteric hip fractures include intramedullary nailing or dynamic hip screw.12 external fixation, however, became a valuable alternative method for high risk elderly patients. since, in the external fixation method, operation time is shorter and blood loss and tissue damage are lessened.12 in 1957, scott used an external fixator for the treatment of intertrochanteric fractures 1 time. many complications were reported, such as pin loosening, infection, and mechanical failure of the fixator.2 we have lately started to use a new cannulated compression pin that can be adapted to all fixators. this study investigated the effect of this pin on stability and healing of the fracture. patients with intertrochanteric hip fractures treated by external fixation between january 2009 and july 2014 were retrospectively evaluated. patients considered having a higher operation risk were included in the study, if they were treated with a proximal femoral nail or sliding hip screw. since this study aimed to evaluate the effectivity of compression pin, the patients who could not tolerate partial weight bearing after the operation were excluded. the institutional review board approved this study and an informed consent was obtained from all the patients before treatment. orthopaedic trauma association fracture classification system (ao / ota) was used and the severity of osteoporosis was graded according to the singh index grading system. compression pin is designed for improving stability and it is 9.5 mm in diameter [figure 1 ]. in the compression pin fixation method, one 9.5 mm cannulated compression pin and one standard pin were used for fixation. in the standard pin method, short - segment fixation was aimed in both methods by inserting two additional pins to the femoral shaft as proximal as possible. patients were placed on a translucent table under fluoroscopy control during the operation. a scrubbed assistant fixed the hip in the proper position as traction table was not available. in the compression pin group, first a k - wire was placed in the middle - inferior femoral neck at an angle of approximately 130 [figure 2a ]. then, compression was applied slowly under the control of fluoroscopy [figure 2c ]. then, a 5 or 6 mm self - tapping pin was placed above the compression pin [figures 2d and 3 ]. in the standard pin method, then, in both methods, two 5 mm self - tapping pins were placed on the femoral shaft [figure 4 ]. patients were evaluated according to age, gender, duration of preoperative period, duration of operation time, american society of anesthesiologists (asa) scores, and immediate postoperative and final femoral neck angle measurements. the accuracy of the plain radiographs and whether the angles were properly positioned were determined for each patient. the stability of the fixation was evaluated by calculating the secondary varus angulation after weight bearing. compression pin and nut schematic diagram of application of compression pin (a) application of a k - wire, (b) placement of the pin with the guidance of k - wire, (c) compression procedure, (d) application of second pin above compression pin and two pins on femoral shaft x - ray of hip joint with proximal thigh anteroposterior views left side in a 84 year old patient showing (a) intertrochanteric fracture femur (b) postoperative x - ray after compression pin fixator application (c) after removal of fixator showing union (d and e) clinical photographs of the patient showing range of motion x - ray (r) hip joint with thigh anteroposterior views showing (a) intertrochanteric fracture (b) postoperative x - ray after application of compression pin and fixator (c) after removal of fixator showing union (d and e) clinical photographs of the patient showing range of motion patients were seated in bed or at the bedside on the 1 postoperative day for as long as they could tolerate. after the 2 day, the patients were encouraged to walk with partial weight bearing using a walking frame, and weight bearing was increased progressively every day. we defined healing based on clinical findings, particularly painless full - weight bearing and radiographic findings of trabecular bridging and callus formation. wilcoxon 's signed rank test was used to compare the differences between the immediate and final varus angulations in each group separately. chicago, il, usa) was used. the shapiro wilk test was used to determine whether the data were normally distributed. wilcoxon 's signed rank test was used to compare the differences between the immediate and final varus angulations in each group separately. in the compression pin group, 31 patients (16 females and 15 males) were included and the mean age was 82.1 6.1 years (range 7598 years). in the standard pin group, 36 patients (17 females and 19 males) were included and the mean age was 83.33 6.24 years (range 7599 years). the mean followup period was 18.9 11.4 (950) months in the compressive pin group and 17.8 11.7 (range 953 months) months for the standard pin group. all of the fractures were osteoporotic and were classified as grade 3 or lower according to the singh index. we administered regional anesthesia in 29 of 31 patients and general anesthesia in 2 of 31 patients in the compression pin group. we administered regional anesthesia in 35 of 36 patients and general anesthesia in 1 of 36 patients in the standard pin group. the delay in surgical period (from trauma to surgery) was 2.1 (range 17 days) days in the compression pin group and 2.8 (range 110 days) days in the standard pin group. the operation time was 24.2 (range 1831 min) min in the compression pin group and 22.1 (range 1728 min) min in the standard pin group. at least one comorbid disease was present in all of the patients [table 1 ]. associated of comorbid diseases on the 1 postoperative day, the varus angulation was 3.4 2.8 (range 010) in the compression pin group and 4 2.1 (range 08) in the standard pin group. the immediate postoperative varus angulations were not significantly different between groups (p = 0.13). after fracture healing at the end of the 3 month, the final varus angulation was 4.4 3.1 (range 010) in the compression pin group and 6.5 2.4 (range 012) in the standard pin group (significantly different [p = 0.003 ]). from the time of weight bearing to healing, there was 1 1.25 (range 04) of secondary varus angulation in the compression screw pin group and 2.5 1.8 (range 09) of secondary varus angulation in the standard pin group. six of 31 (20%) patients in the compression pin group and 13 of 36 (36%) patients in the standard pin group had > 5 of varus angulation, while 3 of 31 (10%) in the compression screw group and 7 of 36 (20%) in the standard pin group had > 8 of varus angulation at the final femoral neckangle measurement. with weight bearing, 2 of 31 (6%) patients in the compression screw group and 9 of 36 (29%) in the standard pin group had > 4 of secondary varus angulation. in the compression pin group, 13 patients had displaced fractures considered unstable, and only 2 (15%) of them had > 4 of secondary varus angulation. in the standard pin group, 19 fractures were considered unstable, and 7 (37%) of them had > 4 of secondary varus angulation. we used 4 as the cutoff point because the greatest secondary varus angulation in the compression pin group was 4. pin tract infection was observed in 55% (17/31) of patients in the compression pin group and 58% (21/36) of patients in the standard pin group. these were minor grade infections seen as increased redness of the skin, serous, or purulent discharge. pin tract infections were treated with increased local pin site care and sometimes antibiotics no pin loosening occurred in either group. patients were mobilized during the 1 week (postoperative days 25) in both groups. fracture healing was defined as trabecular bridging and callus formation as observed on plain radiographs and painless movement of the hip joint and painless full - weight bearing. the mean fracture healing time was 11.6 1.7 weeks in the compression pin group and 11.9 1.5 weeks in the standard pin group, respectively. all of the fixators were removed during the 12 to 14 postoperative weeks in the outpatient clinic. refractures were not observed in both groups at least 6 months after the removal of the fixator. treatment of intertrochanteric fractures can be challenging under some conditions, especially for very elderly, severely osteoporotic patients. sliding hip screws and intramedullary hip screws fixation are commonly used for the treatment of intertrochanteric fractures, although the reported high rates of fixation failures can not be ignored.1345 for the treatment selection of the intertrochanteric fracture, age, general condition, comorbid diseases, bone quality, and fracture type should be taken into consideration.6 regularly these fractures are treated with proximal intramedullary nail and sometimes with dynamic hip screw in our clinic. but in very elderly, high - risk patients who could not tolerate long operation times or significant blood loss ; external fixation methods were preferred as an alternative method which reduces surgery stress. the external fixation method fixes a reduced fracture without preventing biological healing and provides early mobilization.7 it has been reported that compressive pins for the fixation of intertrochanteric hip fractures can increase stability.8 therefore, we prefer to use compression pins for the external fixation of intertrochanteric hip fractures. in the compression pin group, the immediate postoperative femoral neckangle reduction was similar between the groups, indicating that there was no difference regarding reduction capability between the methods. however, only two pins were used in the compression pin group, while three pins were used in the standard pin group. intertrochanteric fracture healing time was reported between 11 and 14 weeks for external fixation method.7 in the present study, all patients in the both groups healed up to the end of 14 week. moreover, it has been reported that nonunion is not a commonly seen complication.1910 in addition, fracture healing time was not different between both groups. karn.1 reported that patients treated with external fixator displayed more varus deformities than patients who treated with sliding hip screws. these authors reported > 5 of varus angulation in nine of thirty (30%) patients. we measured > 5 of varus deformity in 6 of 31 (20%) patients in the compression pin group and 13 of 36 (36%) in the standard pin group. vossinakis and badras9 reported that > 10 of varus deformity in 12 of 50 patients (25%). we observed > 8 of varus angulation in 3 of 31 patients (10%) in the compression pin group and 7 of 36 (20%) in the standard pin group. this finding demonstrated that our compression pin maintained fixation better than standard pins after weight bearing and the compression pin offered the advantage of fracture compression, which enhanced healing and increased stability. although the difference in secondary varus angulations between the groups was statistically significant, the 2 mean difference seemed to have little clinical importance. in contrast, beginning with weight bearing, 2 of 31 (6%) patients in the compression pin group had > 4 of secondary varus angulation compared to 9 of 36 patients in the standard pin group (25%). in the compression pin group, 13 patients had displaced, unstable fractures and only 2 (15%) of them had > 4 of secondary varus angulation. in the standard pin group, there were 19 unstable fractures, and 7 (37%) of them had > 4 of secondary varus angulation. hydroxyapatite - coated pins may be used effectively in the treatment of intertrochanteric fractures.11 compression pin can be coated with hydroxyapatite. however, tight integration can occur between the bone and pin. than removing tightly integrated compression pin could be difficult and may require general or regional anesthesia. pin tract infections occur in 3060% of cases.1912 in the present study, although the pin tract infections were observed at a rate of 55% (17/31) in the compression pin group and 58% (21/36) in the standard pin group all of the infections were minor grade and none of them necessitated pin removal. because low - speed drills or t - handles were used while placing the pins, osteolysis around the pins was not observed in either group. since the goal of this study was to evaluate stability of compression pin after weight bearing and to investigate the effect of compression pin on intertrochanteric fracture healing, only the patients who could tolerate partial weight bearing during the 1 week of postoperative period and who returned for followups were included in this study. the patients who could not be mobilized or who died during this period were excluded. patients included in the present study had multiple comorbidities but they were the ones who could be followed at least 6 months after removal of the fixators. treatment of very elderly, high - risk patients with external fixation is an effective method. compression pins maintained stability better than standard pins after weight bearing, particularly in unstable intertrochanteric fractures.	background : external fixation is a well - known procedure for the management of intertrochanteric fractures in very elderly high - risk patients. a new compression pin that can be adapted to all fixators was designed to provide inter fragmentary compression. in the present study, its effects on the fracture stability and healing were evaluated.materials and methods : thirty - one patients treated using compression pin and thirty - six patients treated using standard pins were evaluated retrospectively between january 2009 and july 2014. patients were evaluated according to age, gender, duration of preoperative period, duration of operation time, american society of anesthesiologists (asa) scores, and immediate postoperative and final femoral neck angle measurements. the stability of the fixation was evaluated by calculating the secondary varus angulation after weight bearing.results:thirty one patients (82.1 6.1 years old) comprised the compression pin group, and 36 patients (83.33 6.24 years old) comprised the standard pin group. from the time of weight bearing to healing time, 1.0 1.25 (04) and 2.5 1.8 (09) of secondary varus angulation in the compression pin and standard pin groups were measured, respectively (p = 0.000). with weight bearing, 2 of 31 (6%) and 9 of 36 (25%) patients in the compression and standard pin groups, respectively, had > 4 of secondary varus angulation. in the compression pin group, 13 fractures were unstable, but only 2 (15%) of them had > 4 of secondary varus angulation. in the standard pin group, 19 fractures were unstable, and 7 (37%) of them had > 4 of secondary varus angulation.conclusions:treatment of very elderly, high risk patients with intertrochanteric fractures with external fixation is effective. compression pin maintained stability better than standard pins after weight bearing, especially for unstable intertrochanteric fractures.
the connectome concept was introduced by sporns and his colleagues (sporns., 2005). initially it referred to the global matrix of macroconnections (i.e., axonal connections between gray matter regions seen as black box nodes ; for its complete definition see bams foundational model of connectivity thesaurus, http://brancusi1.usc.edu/thesaurus/definition/connectome/ ; swanson and bota, 2010) for the human brain. the concept has since been refined and today has at least three specific meanings that are applied to the nervous system as a whole, not just the brain (swanson and bota, 2010 ; akil., 2011). besides the macroconnectome between gray matter regions, which includes functional magnetic resonance imaging (fmri) and diffusion tensor imaging (dti) results, there is the global mesoconnectome, which is a matrix of all axonal connections between all neuron types ; and there is the global microconnectome, which is a matrix of all axonal connections between all individual neurons in a particular animal or person (swanson and bota, 2010). the level of abstraction of a connectome is variable, and it depends on the number of experimental variables and metadata (expressivity), associated with connectivity reports in a database. the most abstract levels are those used to construct macroconnection wiring diagrams and square matrices of gray matter regions that show qualitative connection strengths in different visual formats (stephan., 2000b ; bota and swanson, 2007a ; bohland., 2009 ; however, they can be enhanced with other information like the spatial characteristics of injection and labeling sites, axon branching patterns, and routes taken by the axons through associated white matter tracts (swanson and bota, 2010). connection matrix construction requires the use of a single, internally consistent nomenclature for gray matter regions (macroconnectome), neuron types (mesoconnectome), and individual neurons (microconnectome). thus, it is necessary to define relationships within and between the sets of gray matter regions and neuron types defined in different neuroanatomical and neuron nomenclatures, respectively, and individual neurons. the ultimate global connectome would thus consist of an integrated macroconnectome, mesoconnectome, and microconnectome based on an integrated nomenclature for all of them (see figure 1 in bota and swanson, 2007b). first version of the rat cns macroconnectome, defined in the swanson-1998 nomenclature and constructed from data in bams [bota and swanson (2007a) ; bohland. the color code used to produce both macroconnectome versions is as follows : gray absent information ; black evidence that connection is absent ; green evidence that connection exists, without any explicit qualitative strength ; red very strong connection ; pink strong connection ; yellow - to - red medium - to - strong connection ; yellow medium or moderate connection ; blue weak or sparse connection ; light blue very weak (sparse) connection ; or axons - of - passage. see text for details. the brain architecture knowledge management system (bams ; http://brancusi.usc.edu/bkms ; http://brancusi1.usc.edu) was designed to handle neuroanatomical information across multiple levels of vertebrate nervous system organization. it includes five interrelated modules that store and process data about : (1) molecules expressed in different gray matter regions or neurons, (2) neuron types and classes, (3) gray matter regions, (4) relations between gray matter regions defined in different parcellation schemes, and (5) about connections between gray matter regions or neuron types defined in different nomenclatures and species. bams also includes inference engines relating gray matter regions defined in different parcellation schemes neuron populations defined by different authors. its backend database and inference engines currently allow construction of macroconnectomes from manually and semi - automatically inserted data, with several levels of abstraction from simple gray matter region wiring diagrams and 2d connectomes in different graphical formats to more detailed tabular representations of connections, augmented with spatial details that include injection and labeling sites, specific zones within regions (layers and other features), and white matter tract information (bota., 2003, 2005 ; bota and swanson, 2007b). we consider here three bams modules, connections, relations, and cells from the perspective of macroconnectome construction and relationships with mesoconnectomes, as well as the bams inference engine for online translation of connection reports across different nomenclatures (bota and swanson, 2010). the connections and relations modules have been used to construct user - defined connectivity matrices and diagrams (bota., 2003), as well as rat macroconnectomes since the work reported in bota and swanson (2007a). in this paper we present the newest version of the rat bams macroconnectome, which was created by using the inference engine for translation of connections reports. we also discuss the steps that have to be taken to ensure correct translation of connection reports collated from the literature. finally, we discuss the most important challenges related to connectome construction within bams and argue for a collaborative and coordinated effort of the neuroinformatics community. to reiterate : a global mesoconnectome is defined as the complete set of axonal connections between all neuron types in the nervous system of a particular species (swanson and bota, 2010). depending on which methods and classification criteria are chosen, different authors may name the same or overlapping neuron sets (types or classes) differently. therefore, one prerequisite for constructing mesoconnectomes is the construction of a single, internally consistent classification system for neuron types in a particular species. a second prerequisite is a systematic account of relationships between classification schemes used by different authors (bota and swanson, 2007b). the cell module of bams was designed specifically to handle neuron sets (types) reports collated from the literature, as well as criteria for hierarchical classification specified by different authors. each is_a relationship that relates a class to its instances is associated with a complex representation of criteria and subcriteria that are used in the literature. bams also includes an inference engine that relates neuron types and classes defined in different neuron nomenclatures (bota and swanson, 2007b). the relations that are established across neuron populations defined or described by different authors are both qualitatively spatial (i.e., topological ; egenhofer and franzosa, 1991), and in terms of common structural attributes. for example, two neuron sets (classes, or types) are considered identical whenever they share a common space and they have the same attributes as stated by authors, or inferred by collators. to simplify the knowledge maps that can be extracted from the inserted relationships (bota and swanson, 2007b), we use a set of semantic relations that take into account the general spatial relations that can be defined between two neuron populations, and their compared attributes : synonym, includes, partial correspondence, and different. the details of reducing the qualitative spatial relations to a set of semantic relations, and examples of how a knowledge map can be constructed from the information inserted in bams, are discussed in bota and swanson (2007b). because the sources of axonal inputs and the targets of axonal outputs for a specific neuron type or class are explicitly recorded in bams as classification attributes, they can be inferred using the relationships between neuron populations that include hodological criteria (i.e., the set of inputs and targets) in their definitions or descriptions. an inference engine that uses information about axonal inputs and targets for automatic classification of neurons was already implemented in bams. this engine automatically establishes the level of a specific neuron population within bams ' classification scheme as well as the hierarchy of criteria, solely based on the inputs and targets, and their nature (gray matter regions or white matter tracts). examples of such inferences are shown and discussed in detail in bota and swanson (2007b). finally, the backend structures of the cell and connections modules are interrelated such that one or more connection reports about gray matter regions can be associated with the axonal connections of one or more neuron types (bota., 2005). to summarize, axonal connections between neuron types or between neuron types and gray matter regions are encoded in bams in two different ways : first as components of macroconnections, and second as criteria for definition of neuron types and classes. the bams connections module allows insertion of data and metadata at the level of macroconnections. the entity - relationship (er) structure of this module allows insertion of more than 40 qualitative, semi - quantitative, and quantitative attributes associated with a neuroanatomical connection report, as collated from the literature (bota., 2005). the large number of attributes associated with any connection report (i.e., high expressivity) of the bams connections module allows reconstruction of connection patterns between distinct gray matter regions, including connection patterns between sets of neuron types identified in different gray matter regions. it also allows association of macroconnection (as well as mesoconnection) reports with major white matter tracts. thus, the module can be used to reconstruct tracts in terms of contributions from one or more gray matter regions (or neuron types as described above ; for details and examples see bota., 2005). this bams feature becomes increasingly important in the context of global efforts intended to create 2d and 3d connectome maps (hjornevik. each connectivity report can be associated in bams with atlas levels (the serially numbered 2d maps in a brain atlas) and stereotaxic coordinates where the stain was reported. finally, each major white matter tract of a given mammalian species can be associated with a set of neuroanatomical connections, and thus can be reconstructed in terms of the input and output regions. these three features of the bams connections module allow it to be used as a backend data provider for reconstruction of cns roadmaps by future visualization tools. the high expressivity of the backend structure of bams 's connections module is necessary but not sufficient for the complete and correct insertion of connectivity data, and in the end for creating a macroconnectome using connections mapped on neuroanatomical nomenclatures (atlases) other than the standard nomenclature. the construction of any macroconnectome depends on the choice of a standard nomenclature, and it is always ideal to map new data directly onto the standard nomenclature. it also depends on the completeness and correctness of inserted data by the collators or experts. connectivity reports mapped on different parcellation schemes either have to be remapped by collators and curators, or the system can translate topologically the injection and labeled sites. the first option, manual remapping, does not preserve the integrity of original reports, and must be performed whenever a new nomenclature (e.g., atlas) is encountered. to preserve the original integrity of connectivity reports as collated from the associated references, remapping must become as independent as possible from the nomenclature in use. and to avoid unnecessary duplication of reports (e.g., one in the original nomenclature and the second mapped and inserted by the human expert), we employed a second option, automatic translation of connectivity data across nomenclatures. since standard, high - resolution, resliceable 3d computer graphics frames of reference for the rat brain and nervous system are still in the first phases of construction and testing (hjornevik., 2007 ; hawrylycz., 2011), connectome construction has to rely on a qualitative translation engine across different parcellation schemes. for this, we have constructed a special module in bams, relations, that allows encoding of qualitative spatial relations between nervous system parts defined in different neuroanatomical nomenclatures (e.g., atlases) in specific species. this module also includes a large set of metadata associated with the actual process of mapping nervous system parts, performed or inserted by collators (bota and swanson, 2010). after the qualitative spatial relations between gray matter regions of two neuroanatomical nomenclatures defined in the same species have been inserted in bams, connections reports associated with either of the nomenclatures can be translated to the related one. full description of this engine and examples of translations are provided in bota and swanson. besides the problem of choosing a nomenclature for mapping the results of pathway tracing experiments to help establish connections (projections), the second challenge is the process of data entry itself. any connectivity database can be populated from data collated from the literature, or it can be directly inserted by neuroanatomists, or both. the database design of the connections module and its associated interfaces allow both ways of data insertion (bota. bams is used by neuroanatomists to insert their experimental data, and manipulate in different ways the connectivity information. however, the connectivity data inserted in bams is mainly collated from the published literature. the collation procedure is manual and each report inserted in the system is supported by a textual annotation from the associated reference, or by collator 's interpretations. finally, the bams connections module is associated with a set of publicly accessible interfaces that allow construction of user - customized connections matrices. the web interface of bams 's connections module also includes inference engines that construct networks of gray matter regions, defined in specific neuroanatomical nomenclatures (bota., 2003, 2005). the first version of the rat macroconnectome was constructed from ipsilateral connectivity reports inserted in bams (bota and swanson, 2007a ; bohland., 2009) and used the swanson-1998 (swanson, 1998) nomenclature and classification hierarchy. it covered 9.4% of the entire matrix, which has 486 486 cells with each cell representing a gray matter region at the bottom of the region classification hierarchy. the number of cells that are filled with any other color than gray (no data) is 22,178 (figure 1). because the number of connection reports inserted in bams exceeded an internal benchmark value (50,000), we reconstructed the rat bams macroconnectome using the swanson-2004 parcellation scheme (swanson, 2004). the connection reports used in this new macroconnectome were originally mapped onto different nomenclatures recorded in bams ; for example, swanson (1998), fulwiler and saper (1984), and moga. the translation of connections into the swanson-2004 nomenclature was semi - automatic, first using the connections translations inference engine described above, and then using results validation by human agents when the relationships between swanson-2004 nomenclature and the original nomenclature yielded equivocal results. the increase in reports collated in bams 's connections module from the previous connectome version (bota and swanson, 2007a ; bohland., 2009) is 28.20%, from about 39,000 reports to the present value of 52,458. the present version of the rat bams connectome (figure 2) is a matrix of 503 503 cells with 11.2% coverage (i.e., cells filled with any color but gray no data). one percent coverage of the connectome matrix shown in figure 2 corresponds to about five completely filled columns or rows. this connectivity data increase was collated and curated from 15 newly inserted research papers collated from 2009. in addition, the results of pathway tracing experiments from more than 20 references were re - mapped, completed, or corrected. second version of the rat cns macroconnectome, defined in the swanson-2004 nomenclature and constructed from data in bams. the number of filled cells in this new rat connectome is 27,796, which represents more than 25% increase in connection data used to construct the matrix, from the 22,178 labeled cells (figure 1 ; bota and swanson, 2007a) in the first version of the rat connectome. there are two notable differences between the swanson-1998 and swanson-2004 connectomes shown in figures 1 and 2, respectively. first, the swanson-2004 macroconnection matrix is slightly larger than the swanson-1998 macroconnection matrix, mostly because several gray matter regions were remapped and more finely parceled in swanson-2004, especially the lateral hypothalamic area (lha). however, the number of bed nuclei of the stria terminalis (bst) regions is reduced, three of them defined in swanson-1998 nomenclature (bstad, bstav, bstdl) being grouped in a single gray matter region, bstam, in the swanson-2004 parcellation scheme (swanson, 2004). the second and more important difference is the internal organization of the swanson-1998 and swanson-2004 nomenclatures. the main criterion for internal organization of the swanson-2004 rat nomenclature is functional network organization (see table b in swanson, 2003, 2004), whereas that of swanson-1998 is based more on strict topographical relationships. as a result, connections of the same gray matter regions will be displayed in different columns and rows of the matrix, and under different higher - order subdivisions of the rat nervous system. the advantages of using the rat swanson-2004 nomenclature over swanson-1998 is thus three fold : (1) several gray matter regions are refined, (2) the nomenclature is constructed on more criteria, and (3) the nomenclature is applied consistently across the rat cns. hence, the internal consistency of the newest swanson nomenclature is strengthened. finally, the new hierarchical organization of swanson-2004 nomenclature better integrates the structure - function relationships of rat cns gray matter regions. because coverage in the newest version of the rat bams connectome reached a landmark value and the gray matter regions that send or receive at least one connection are not concentrated in a single cns subdivision, we analyzed the results shown in figure 2. the number of connections shown to be absent (black squares) is 22,064, about 80% (79.38%) of the total. the number of regions with data about a connection to at least one other gray matter region is 317, which means that 62% of the regions in the bams rat connectome are associated with at least one output connection. regions with highest numbers of inputs are in the cerebral cortex (prelimbic and infralimbic areas), cerebral nuclei (several bst nuclei), and hypothalamus (lha regions). the highest output connection ratio (i.e., the ratio, matrix cells in a column with data indicating an output : total number of cells in a column) shown by any gray matter region in figure 2 is 34%, and the highest ratio for a cerebral cortical area is 18%. in a second, more stringent and informative, step of this preliminary cns - wide analysis we took into account only those gray matter regions with a complete set of ipsilateral outputs registered in bams (shown as entire filled columns in figure 2). we thus determined the output connection ratio for 44 gray matter regions that include select parts of the cerebral cortex (including the subiculum), amygdalar region, lateral septal nucleus, and hypothalamus and all parts of the bst. the output connection ratios for this subset of 44 gray matter regions range from 2% (for lateral septal nucleus subdivisions) to 34% (for lha regions). in other words, the number of ipsilateral terminal field targets for this subset of gray matter regions ranges from 8 to 150. the average output connection ratio for this subset is 10%, which means that on average each member of the subset has about 4050 ipsilateral gray matter region targets, out of a total of about 500 possible targets. the average output connection ratio for the entire set of rat cns gray matter regions may be significantly less than 10%, however, because the analyzed subset is heavily biased toward gray matter regions with very complex output patterns. a similar situation was found for data about input connection ratios of the gray matter regions shown in figure 2. some 406 (81.2%) gray matter regions from the total of 503 have data about the reception of at least one axonal input. from data available in bams so far, regions that receive the most axonal inputs are mostly located in the cerebral nuclei and hypothalamus. the connectivity data used for constructing the second bams rat macroconnectome is available to the neuroscience community in interactive graphical format in the newest version of bams : http://brancusi1.usc.edu/connections. users can construct it online and export the data in xml or json formats, or as a flat image. a second xml version of the macroconnectome that includes the bams unique i d 's of brain regions is provided in the classic version of bams : http://brancusi.usc.edu/bkms/brain/choose-connection.php. this additional xml version is useful for third party systems that would use the numerical i d 's to bring additional gray matter region data and metadata from bams. thus, the new rat macroconnectome, or parts of it, can be linked to, replicated, analyzed, or enhanced by members of the neuroscience community. connectome construction at all scales (micro-, meso-, or macro-) is important for analyzing and understanding global nervous system wiring diagrams, which in turn may help generate new hypotheses and design the experiments to test them. the most abstract and simplest form of a neuroinformatics - driven connectome is a 2d matrix that shows the presence or absence of connections between gray matter regions (a macroconnectome). obviously, such connectomes can be refined by adding more information about spatial attributes like the route taken by a connection through various white matter tracts, and specific zones (differentiations) within a particular gray matter region. in the following we discuss two of the most important challenges we addressed in macroconnectome construction within bams : nomenclatures and data collation or annotation. the construction of macroconnectomes, even in their simplest and most abstract form, needs to follow a set of rules. first, any macroconnectome must be associated minimally with an internally consistent nomenclature of gray matter regions that can be either based on published parcellation schemes, or can be constructed de novo by an expert or group of experts. for macroconnectomes, internal consistency of the chosen nomenclature is the necessary prerequisite ensuring that the gray matter regions used for matrix construction are distinct and do not overlap. in addition, nomenclature must cover the entire part of the nervous system under consideration and should be species specific. the nomenclatures proposed for nervous system parts in different mammalian species by different authors may or not be hierarchically organized. any nomenclature that is also hierarchically organized according to specific sets of structural or functional criteria allows construction of connectomes that are more informative than those arranged simply alphabetically. thus, the arrangement and size of any connectome in the graphical format of a 2d matrix depends on the nomenclature and internal classification schemes used. second, a macroconnectome based on data collated from the literature is the abstract form of results from many pathway tracing experiments mapped using a variety of non - identical methods and nomenclatures. the translation of connections mapped on parcellation schemes different from the connectome 's standard nomenclature may be performed automatically, but the results must be verified and validated by human experts. results produced by inference engines may be incomplete, or even contradictory, and human experts are necessary for checking them and resolving discrepancies. there are at least three aspects of connectivity data collation that influence the construction and usefulness of connectomes : level of detail, completeness, and correctness of inserted data. the level of detail associated with inserted data depends on the complexity of the computer - readable representation, and on how the results are presented in the literature. the simplest form of a connectivity report is region x connects to y, with no other details. connectomes based on such information provide only a superficial view of cns connectivity patterns in the species of interest, and they will be not as informative as those constructed from reports that include, for example, qualitative assessments and/or quantitative data about connections.. the high degree of abstraction in macroconnectomes that are organized in the format of a 2d matrix allows the construction of relatively simple wiring diagrams. more realistic and functionally relevant wiring diagrams need more structural and functional attributes associated with individual connections. this can be approached by relating each cell of a connectome matrix with the relevant data and metadata, as collated from the literature or inserted by experts. however, the detail level in connectivity reports collated from the literature is constrained by the mode of data presentation within them. because a standard for presenting pathway tracing results in published references is not yet available, usually, an original research article includes images of representative experimental material and more or less detailed descriptions of neuroanatomical connections. thus, without the original results of the published pathway tracing experiments, the collation of connectivity data is best accomplished when displayed on a series of atlas level maps or images. whenever possible, we collate the connectivity data from each atlas level presented in a published reference, and combine this information with the textual description provided by authors. this approach is necessary for qualitatively capturing the topographical details of terminal fields, and the axonal pathways and their routes. it is also useful for any neuroinformatic system that aims to reconstruct macroconnections in visual format (tallis., 2011). regardless of the general procedure used to populate a database with pathway tracing information either collation of the published literature or direct insertion by neuroanatomists ideally, the process of connectivity data insertion should be performed in parallel with the mapping and annotation of pathway tracing experiments performed by neuroanatomists. however, this is not yet possible in an organized and large - scale way, so that collation and curation of the published literature is currently one of the most widely used methods for populating knowledge management systems. examples of such systems include neuroscholar (burns, 2001), cocomac (stephan., 2000b), bams (bota., 2005 ; bota and swanson, 2010), and temporal lobe database (van strien., 2009). the comprehensive collation and curation of connectivity data from published literature is also important from an historical perspective, for establishing novelty (by priority analysis) of current research results, and for future comparisons. while the macroconnectomes of selected gray matter regions or subsytems in few mammalian species may be complete or nearly complete, it is difficult to assess the present degree of coverage for the complete macroconnectome of any particular species. we report here a coverage of about 11% for the rat cns macroconnectome in matrix format. however, the expressiveness (in terms of attributes and associated metadata) of a neuroinformatics system is proportional to the time spent on curation and data entry. minimally, all connectivity reports inserted in a neuroinformatics system should include information about species, standard nomenclature, methods used, and details about injection and labeled sites. pathway tracing experiments rely on many different methods, each with unique advantages and limitations (bota., 2003), so the results of pathway tracing experiments using different methods can be different, or even contradictory. as a result, information about pathway tracing methods used and about injection and labeled sites, respectively, is necessary for future evaluation of connectivity data reliability (bota and arbib, 2004). further details, such as the atlas levels and spatial coordinates of injection and labeling sites, become very important in the context of 3d reconstructions of experimental results. because the prerequisites of connectome construction (pathway tracing data collation and relating gray matter regions across different parcellations) are very time consuming, the order of connectivity matrix filling in species of interest becomes important both for practical and collaborative reasons. thus, the sequential release of updated, more complete macroconnectome versions for a species of interest allows the neuroscience community to perform statistical analyses on the released data, and to integrate it with already existent information. moreover, each release can be seen as a benchmark toward completion of a very large - scale task. because this task can only be done stepwise, the advantage of choosing a hierarchically organized nomenclature is obvious : it can be subdivided and reorganized as needed. completion of the rat macroconnectome both in a timely manner and with high quality data is a task that can only be done collaboratively by multiple neuroanatomy and neuroinformatics groups. several neuroinformatics and neuroanatomy groups can work in parallel toward completion of major structural or functional divisions of the nervous system macroconnectome in a particular species. for example, each group can complete the macroconnectome for one major subdivision of a common, hierarchically organized, nomenclature. collaboration and coordinated efforts of different groups (bota and swanson, 2007a ; akil., 2011) are already underway for neuroscience data integration at different levels of the vertebrate and invertebrate nervous systems. associated with this, bams infrastructure already allows creation of collaborative mouse, rat, or macaque macroconnectomes with several systems, including the ucla mouse connectome project (http://www.mouseconnectome.org/), rodent brain workbench (http://www.rbwb.org/ ; zakiewicz., 2011),, 2009 ; sugar., 2011), cocomac (http://cocomac.org), and the brain architecture project (http://brainarchitecture.org/). for example, the backend structure of bams is compatible with the recorded data and metadata associated with connection and neuron type reports, respectively, in two very important publicly available neuroinformatics applications cocomac and cocodat that were designed, developed, and populated by rolf ktter and his colleagues (stephan. however, whereas the bams - implemented algorithm for qualitatively relating gray matter regions defined in different nomenclatures (for same species) uses the complete set of eight topological relations (egenhofer and franzosa, 1991 ; sharma, 1986) that can be defined for a pair of convex regions, and is thus purely topological (bota and arbib, 2004 ; bota., 2005), the objective relational transformation (ort) algorithm implemented in cocomac uses only five topological relations and a logical inference engine (stephan., 2000a). extensive discussion and comparison of both approaches is provided in bota and arbib (2004). both bams and cocomac are integrated in the neuroscience information framework and provide extensive information about gray matter regions and connectivity to the neuroscience community (nif ; http://www.neuinfo.org ; akil., 2011). a comprehensive comparison of bams with the major publicly accessible neuroinformatics systems developed by other groups was presented in bota and swanson, 2007a. the backend database structure of bams, along with its interfaces and the data collated so far, allow us in principle and practice to construct macroconnectomes for the entire nervous system of any species (including mouse, rat, monkey, and human), using the results of pathway tracing experiments based on different methods and mapped onto different nomenclatures in the same species. the translation of connections across nomenclatures in a species is semi - automatic, and is verified by collators and experts. using this approach, we constructed a new version of the rat macroconnectome that is the most complete connectome available to date for any vertebrate, as far as we know. this second version of the rat macroconnectome contains significantly more data than the first version, and it is based on a complete and internally consistent rat nomenclature and classification scheme for gray matter regions that facilitates network analysis. as discussed in the section above, the amount of connectivity data already inserted in bams allowed us to perform preliminary statistical analysis over the rat macroconnectome, and hypothesize that the average number of targets of any rat gray matter region is a maximum of 50 out of about 500. future work will augment the present matrix - form macroconnectome representation with spatial attributes including pathway tracer injection site and sites of connection labeling resulting from the injection. using these attributes, we will re - implement the inference engine for evaluating connectivity data reliability (bota and arbib, 2004) and we will provide users with a set of tools to construct macroconnectomes that evaluate connections in different ways. in addition, we intend to complete the rat macroconnectome as best as possible from the existing literature and start constructing macroconnectomes for other species, in particular the mouse, monkey, and human. work on the mouse connectome has already begun (mouseconnectome.org), and one novel feature here is the direct transfer of connection data from expert annotators to a reference nomenclature (dong, 2007) in bams. this mouse connectome project is based on a powerful new double coinjection (dci) method that allows to two different stereotaxically placed coinjections of an anterograde and a retrograde pathway tracer in each animal, resulting in data from four separate tracers, each labeled in a different color in each histological section (thompson and swanson, 2010). an example of data from two coinjections in the lha of the rat is shown in figure 3. a growing set of dci experiment results for the mouse brain can be found at mouseconnectome.org. the networks inference engines of bams (bota., 2003, 2005) also will be used to extract functionally relevant gray matter region networks in both rat and mouse that can be further compared. results of a coinjection pathway tracer analysis [hahn and swanson (2010) ] of two nearby regions of the rat lateral hypothalamic area, the lhajp, and lhas, plotted on the connectome matrix for swanson-2004 (see figure 2). the two columns represent anterograde tracer (phal) data from injection sites in the lhajp and lhas (the leftmost and rightmost columns, respectively), whereas the two rows represent retrograde tracer (ctb) data from the same two injection sites (in different animals, in this case, though results were plotted on the same series of reference atlas level plates in the original publication). a large - scale, systematic series of coinjection sites throughout the central nervous system more importantly, we will develop the backend structure and inference engines needed in bams to construct mesoconnectomes and microconnectomes that are fully integrated and interoperable with macroconnectomes thus spanning and integrating the molecular, neuron, neuron type, and gray matter region levels of analysis. this extension will begin with the already implemented molecules and cells modules and their relationships with the connections module. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. the construction of macroconnectomes, even in their simplest and most abstract form, needs to follow a set of rules. first, any macroconnectome must be associated minimally with an internally consistent nomenclature of gray matter regions that can be either based on published parcellation schemes, or can be constructed de novo by an expert or group of experts. for macroconnectomes, internal consistency of the chosen nomenclature is the necessary prerequisite ensuring that the gray matter regions used for matrix construction are distinct and do not overlap. in addition, nomenclature must cover the entire part of the nervous system under consideration and should be species specific. the nomenclatures proposed for nervous system parts in different mammalian species by different authors may or not be hierarchically organized. any nomenclature that is also hierarchically organized according to specific sets of structural or functional criteria allows construction of connectomes that are more informative than those arranged simply alphabetically. thus, the arrangement and size of any connectome in the graphical format of a 2d matrix depends on the nomenclature and internal classification schemes used. second, a macroconnectome based on data collated from the literature is the abstract form of results from many pathway tracing experiments mapped using a variety of non - identical methods and nomenclatures. the translation of connections mapped on parcellation schemes different from the connectome 's standard nomenclature may be performed automatically, but the results must be verified and validated by human experts. results produced by inference engines may be incomplete, or even contradictory, and human experts are necessary for checking them and resolving discrepancies. there are at least three aspects of connectivity data collation that influence the construction and usefulness of connectomes : level of detail, completeness, and correctness of inserted data. the level of detail associated with inserted data depends on the complexity of the computer - readable representation, and on how the results are presented in the literature. the simplest form of a connectivity report is region x connects to y, with no other details. connectomes based on such information provide only a superficial view of cns connectivity patterns in the species of interest, and they will be not as informative as those constructed from reports that include, for example, qualitative assessments and/or quantitative data about connections. the high degree of abstraction in macroconnectomes that are organized in the format of a 2d matrix allows the construction of relatively simple wiring diagrams. more realistic and functionally relevant wiring diagrams need more structural and functional attributes associated with individual connections. this can be approached by relating each cell of a connectome matrix with the relevant data and metadata, as collated from the literature or inserted by experts. however, the detail level in connectivity reports collated from the literature is constrained by the mode of data presentation within them. because a standard for presenting pathway tracing results in published references is not yet available, usually, an original research article includes images of representative experimental material and more or less detailed descriptions of neuroanatomical connections. thus, without the original results of the published pathway tracing experiments, the collation of connectivity data is best accomplished when displayed on a series of atlas level maps or images. whenever possible, we collate the connectivity data from each atlas level presented in a published reference, and combine this information with the textual description provided by authors. this approach is necessary for qualitatively capturing the topographical details of terminal fields, and the axonal pathways and their routes. it is also useful for any neuroinformatic system that aims to reconstruct macroconnections in visual format (tallis., 2011). regardless of the general procedure used to populate a database with pathway tracing information either collation of the published literature or direct insertion by neuroanatomists ideally, the process of connectivity data insertion should be performed in parallel with the mapping and annotation of pathway tracing experiments performed by neuroanatomists. however, this is not yet possible in an organized and large - scale way, so that collation and curation of the published literature is currently one of the most widely used methods for populating knowledge management systems. examples of such systems include neuroscholar (burns, 2001), cocomac (stephan., 2000b), bams (bota., 2005 ; bota and swanson, 2010), and temporal lobe database (van strien., 2009). the comprehensive collation and curation of connectivity data from published literature is also important from an historical perspective, for establishing novelty (by priority analysis) of current research results, and for future comparisons. while the macroconnectomes of selected gray matter regions or subsytems in few mammalian species may be complete or nearly complete, it is difficult to assess the present degree of coverage for the complete macroconnectome of any particular species. we report here a coverage of about 11% for the rat cns macroconnectome in matrix format. however, the expressiveness (in terms of attributes and associated metadata) of a neuroinformatics system is proportional to the time spent on curation and data entry. minimally, all connectivity reports inserted in a neuroinformatics system should include information about species, standard nomenclature, methods used, and details about injection and labeled sites. pathway tracing experiments rely on many different methods, each with unique advantages and limitations (bota., 2003), so the results of pathway tracing experiments using different methods can be different, or even contradictory. as a result, information about pathway tracing methods used and about injection and labeled sites, respectively, is necessary for future evaluation of connectivity data reliability (bota and arbib, 2004). further details, such as the atlas levels and spatial coordinates of injection and labeling sites, become very important in the context of 3d reconstructions of experimental results. because the prerequisites of connectome construction (pathway tracing data collation and relating gray matter regions across different parcellations) are very time consuming, the order of connectivity matrix filling in species of interest becomes important both for practical and collaborative reasons. thus, the sequential release of updated, more complete macroconnectome versions for a species of interest allows the neuroscience community to perform statistical analyses on the released data, and to integrate it with already existent information. moreover, each release can be seen as a benchmark toward completion of a very large - scale task. because this task can only be done stepwise, the advantage of choosing a hierarchically organized nomenclature is obvious : it can be subdivided and reorganized as needed. completion of the rat macroconnectome both in a timely manner and with high quality data is a task that can only be done collaboratively by multiple neuroanatomy and neuroinformatics groups. several neuroinformatics and neuroanatomy groups can work in parallel toward completion of major structural or functional divisions of the nervous system macroconnectome in a particular species. for example, each group can complete the macroconnectome for one major subdivision of a common, hierarchically organized, nomenclature. collaboration and coordinated efforts of different groups (bota and swanson, 2007a ; akil., 2011) are already underway for neuroscience data integration at different levels of the vertebrate and invertebrate nervous systems. associated with this, bams infrastructure already allows creation of collaborative mouse, rat, or macaque macroconnectomes with several systems, including the ucla mouse connectome project (http://www.mouseconnectome.org/), rodent brain workbench (http://www.rbwb.org/ ; zakiewicz., 2011), temporal lobe database (http://www.temporal-lobe.com ; van strien., 2009 ; sugar., 2011), cocomac (http://cocomac.org), and the brain architecture project (http://brainarchitecture.org/). for example, the backend structure of bams is compatible with the recorded data and metadata associated with connection and neuron type reports, respectively, in two very important publicly available neuroinformatics applications cocomac and cocodat that were designed, developed, and populated by rolf ktter and his colleagues (stephan. 2005). however, whereas the bams - implemented algorithm for qualitatively relating gray matter regions defined in different nomenclatures (for same species) uses the complete set of eight topological relations (egenhofer and franzosa, 1991 ; sharma, 1986) that can be defined for a pair of convex regions, and is thus purely topological (bota and arbib, 2004 ; bota., 2005), the objective relational transformation (ort) algorithm implemented in cocomac uses only five topological relations and a logical inference engine (stephan., 2000a). extensive discussion and comparison of both approaches is provided in bota and arbib (2004). both bams and cocomac are integrated in the neuroscience information framework and provide extensive information about gray matter regions and connectivity to the neuroscience community (nif ; http://www.neuinfo.org ; akil., 2011). a comprehensive comparison of bams with the major publicly accessible neuroinformatics systems developed by other groups was presented in bota and swanson, 2007a. the backend database structure of bams, along with its interfaces and the data collated so far, allow us in principle and practice to construct macroconnectomes for the entire nervous system of any species (including mouse, rat, monkey, and human), using the results of pathway tracing experiments based on different methods and mapped onto different nomenclatures in the same species. the translation of connections across nomenclatures in a species is semi - automatic, and is verified by collators and experts. using this approach, we constructed a new version of the rat macroconnectome that is the most complete connectome available to date for any vertebrate, as far as we know. this second version of the rat macroconnectome contains significantly more data than the first version, and it is based on a complete and internally consistent rat nomenclature and classification scheme for gray matter regions that facilitates network analysis. as discussed in the section above, the amount of connectivity data already inserted in bams allowed us to perform preliminary statistical analysis over the rat macroconnectome, and hypothesize that the average number of targets of any rat gray matter region is a maximum of 50 out of about 500. future work will augment the present matrix - form macroconnectome representation with spatial attributes including pathway tracer injection site and sites of connection labeling resulting from the injection. using these attributes, we will re - implement the inference engine for evaluating connectivity data reliability (bota and arbib, 2004) and we will provide users with a set of tools to construct macroconnectomes that evaluate connections in different ways. in addition, we intend to complete the rat macroconnectome as best as possible from the existing literature and start constructing macroconnectomes for other species, in particular the mouse, monkey, and human. work on the mouse connectome has already begun (mouseconnectome.org), and one novel feature here is the direct transfer of connection data from expert annotators to a reference nomenclature (dong, 2007) in bams. this mouse connectome project is based on a powerful new double coinjection (dci) method that allows to two different stereotaxically placed coinjections of an anterograde and a retrograde pathway tracer in each animal, resulting in data from four separate tracers, each labeled in a different color in each histological section (thompson and swanson, 2010). an example of data from two coinjections in the lha of the rat is shown in figure 3. a growing set of dci experiment results for the mouse brain can be found at mouseconnectome.org. the networks inference engines of bams (bota., 2003, 2005) also will be used to extract functionally relevant gray matter region networks in both rat and mouse that can be further compared. results of a coinjection pathway tracer analysis [hahn and swanson (2010) ] of two nearby regions of the rat lateral hypothalamic area, the lhajp, and lhas, plotted on the connectome matrix for swanson-2004 (see figure 2). the two columns represent anterograde tracer (phal) data from injection sites in the lhajp and lhas (the leftmost and rightmost columns, respectively), whereas the two rows represent retrograde tracer (ctb) data from the same two injection sites (in different animals, in this case, though results were plotted on the same series of reference atlas level plates in the original publication). a large - scale, systematic series of coinjection sites throughout the central nervous system would gradually fill in the entire matrix. more importantly, we will develop the backend structure and inference engines needed in bams to construct mesoconnectomes and microconnectomes that are fully integrated and interoperable with macroconnectomes thus spanning and integrating the molecular, neuron, neuron type, and gray matter region levels of analysis. this extension will begin with the already implemented molecules and cells modules and their relationships with the connections module. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.	many different independently published neuroanatomical parcellation schemes (brain maps, nomenclatures, or atlases) can exist for a particular species, although one scheme (a standard scheme) is typically chosen for mapping neuroanatomical data in a particular study. this is problematic for building connection matrices (connectomes) because the terms used to name structures in different parcellation schemes differ widely and interrelationships are seldom defined. therefore, data sets can not be compared across studies that have been mapped on different neuroanatomical atlases without a reliable translation method. because resliceable 3d brain models for relating systematically and topographically different parcellation schemes are still in the first phases of development, it is necessary to rely on qualitative comparisons between regions and tracts that are either inserted directly by neuroanatomists or trained annotators, or are extracted or inferred by collators from the available literature. to address these challenges, we developed a publicly available neuroinformatics system, the brain architecture knowledge management system (bams ; http://brancusi.usc.edu/bkms). the structure and functionality of bams is briefly reviewed here, as an exemplar for constructing interrelated connectomes at different levels of the mammalian central nervous system organization. next, the latest version of bams rat macroconnectome is presented because it is significantly more populated with the number of inserted connectivity reports exceeding a benchmark value (50,000), and because it is based on a different classification scheme. finally, we discuss a general methodology and strategy for producing global connection matrices, starting with rigorous mapping of data, then inserting and annotating it, and ending with online generation of large - scale connection matrices.
the incidence of colorectal cancer (crc) in korea has been markedly increasing in recent years. this epidemiologic change in korea requires an increasing number of colonoscopies, which are highly effective in reducing the incidence and mortality of crc.1,2,3 colonoscopy is currently regarded as the gold standard and preferred screening method for crc. however, evidence suggests that colonoscopy is not as sensitive for the detection of neoplasia as previously believed, as demonstrated in studies analyzing the effectiveness of tandem colonoscopy and ct colonography.4,5 a systemic review of six studies on tandem colonoscopy showed that the pooled miss rate for polyps of any size was 22%.4 using ct colonography as a reference standard, the miss rate of conventional colonoscopy for a large adenoma (10 mm) was 12%.5 as a result, a significant number of colorectal neoplasias, including cancers, may be missed, even by experienced endoscopists. moreover, results from the national polyp study and several other studies6,7,8 suggested that colonoscopy is associated with only a 37 - 65% reduction in mortality from crc, which is much lower than that reported previously. this could be attributed to the limited effectiveness of colonoscopic prevention of crc, especially in the right - sided colon. therefore, the role and limitations of colonoscopic screening must be carefully reconsidered. the problem of the so - called interval cancers or missed cancers has been highlighted recently.9,10,11,12 crcs detected in patients who have received colonoscopies within the surveillance interval are called interval cancers, and if they arise from missed lesions, they are also called missed cancers.13 this raises a question regarding the precise prevalence of interval cancer. early studies have shown that 4 - 5% of crcs may be missed on a single colonoscopic examination. 9,10 however, recent studies reported an increase in the prevalence of interval cancers of up to 8%.11,12 table 1 summarizes the major findings of studies conducted since 2006 reflecting the prevalence of interval cancers.8,11,12,14,15,16,17,18,19,20,21,22,23,24,25,26 based on 57,839 patients from the surveillance, epidemiology, and end results - medicare database, 7.2% of patients developed interval cancers.11 in a manitoba cancer registry database, approximately 7.9% (approximately 1 in 13) of 4,833 crcs were classified as missed cancers.12 in korea, the prevalence of interval cancer was reported to be 6.2% by kim.24 ; however, this study was limited by referral bias, selection bias, and recall bias, as it was based on data obtained by telephone calls from a single tertiary referral center. therefore, well - designed population studies are necessary to determine the prevalence of interval cancer in korea. in a population - based study from a danish group,25 interval cancer (colonoscopies performed 1 - 5 years before diagnosis) was 2.6% among 38,064 crc cases diagnosed between 2000 and 2009. in a population - based study conducted in utah,26 6% of all patients with crc had interval cancers (colonoscopies performed 6 - 60 months before diagnosis) among 2,659 crcs diagnosed between 1995 and 2009. most previous studies on interval cancers may be limited, as they relied on registry or administrative data, which frequently lack detail and might be less appropriate for the identification of underlying causes. previous studies were also limited by the fact that definitions of interval cancer were restricted to the initial 3 - 5 years after colonoscopy and did not specifically focus on a complete negative colonoscopy (i.e., a colonoscopy with no detection of adenoma). to overcome these limitations, a population - based study was performed in germany using the definition of interval cancer as a crc occurring within 10 years after a complete negative colonoscopy.21 using this 10-year definition, 4.0% of interval cancers were identified among 1,945 crc cases diagnosed between 2003 and 2007. to interpret the large variation in the prevalence of interval cancer, several factors should be considered : differences in the study design (retrospective vs. prospective), the different definitions of interval cancers (3 - 5 years vs. 10 years), the use of data (claims - based administrative vs. clinical data), differences in the study population (screening vs. diagnostic indications), and differences in endoscopist specialty.27 however, most studies have shown that colonoscopy offers suboptimal protection against crc, especially in the right colon. we could estimate the magnitude of this risk in our routine practice in korea. assuming that proximately 20,588 new cases of crc were diagnosed (data in 2007) based on the korea national cancer incidence database28,29 and considering an average risk scenario (e.g., 4 - 8% of diagnosed crcs are interval cancers), approximately 823 - 1,647 interval cancers might be expected to occur annually in korea. considering the increasing age of the general population and the number of colonoscopies performed in korea, the incidence of interval cancer is expected to increase. regardless of the magnitude of this problem, the medical and social impact of the failure of colonoscopy to prevent crcs may be devastating.27 therefore, prompt elucidation of the factors implicated in interval cancers is necessary. although the exact etiology of interval cancer is unknown, there are several potential explanations for such interval cancers. first, precancerous or cancerous lesions may have been missed at the time of colonoscopy.30 withdrawal time and technique deserve special consideration in this context, as the detection rate of colorectal neoplasia depends on the withdrawal time during colonoscopy. barclay.31 demonstrated that a withdrawal time 6 min is strongly correlated with an increased adenoma detection rate (adr). in addition, careful inspection of the oral side of the mucosal folds is more important than simple adherence to a standard withdrawal time, especially in the proximal colon.32,33 the adr itself is also an important quality indicator for predicting the risk of interval cancers after screening colonoscopy. kaminski.17 demonstrated that the adr of endoscopist was significantly associated with the risk of interval cancer (p=0.008). endoscopies with an adr of less than 11.0% were associated with a 10.94-fold (95% ci, 1.37 - 87.01) higher risk of interval cancer than endoscopies with an adr of 20.0% or higher. complete colonoscopy, another quality indicator of colonoscopy, is also associated with the risk of interval cancer.9 in a previous study conducted in new zealand, 9 of 17 interval cancers occurred after an incomplete colonoscopy.34 brenner.21 also showed that 18% of interval cancers were associated with the completeness of the preceding negative colonoscopy. in this study, interestingly, incompleteness of colonoscopy was associated with distal interval cancers as well as proximal interval cancers, which was explained by a strong association between incompleteness and other aspects of colonoscopy quality, including the miss rate in the distal colon. non - polypoid lesions, which have a subtle endoscopic appearance, may be more easily missed, especially by endoscopists with lower adr and in inadequate bowel preparations.13 previous several studies showed that non - polypoid lesions and interval cancers share some common features:15,19,35,36 both lesions are common in the proximal colon,15,19,35 and interval cancers have a flat appearance.36 although the relationship between non - polypoid lesions and interval cancers has not yet been clarified, endoscopists should be careful not to miss non - polypoid lesions during colonoscopy, especially in the right - sided colon. second, interval cancers may develop from the incomplete resection of previous neoplastic lesions.37,38,39 the recent complete adenoma resection study showed that complete resection of neoplasia is far less common than previously thought.37 in the complete adenoma resection study, the incomplete resection rate varied broadly (6.5 - 22.7%) among endoscopists, and was significantly higher in large and sessile lesions. robertson.38 showed that 26% of interval cancers occur in the same anatomic segments as those of a previous polypectomy, suggesting a possible role for incomplete polypectomy in the development of interval cancers. a dietary polyp prevention trial showed a similar rate of interval cancers after incomplete polypectomy.36 in this trial, in 4 of 13 patients, interval cancers were associated with incomplete polypectomy. lieberman.39 showed that the 5-year interval cancer rate after a normal colonoscopy without polypectomy is 0.17%, whereas the 5-year interval cancer rate after a previous polypectomy is 1.5%. these findings suggest that interval cancers that develop from lesions are visualized and recognized but are incompletely removed during a prior colonoscopic polypectomy. third, different biological factors associated with molecular carcinogenesis may contribute to the development of interval cancers, especially in the proximal colon.40,41 arain.40 showed that interval cancers are more likely to arise in the right colon and have microsatellite instability (msi) and cpg island methylator phenotypes (cimp) than non - interval cancers. in multivariable logistic regression model, msi (or : 2.7 ; 95% ci : 1.1 - 6.8) and cimp (or : 2.41 ; 95% ci : 1.2 - 4.9) were independently associated with interval cancers. sawhney.41 showed that interval cancers were 3.7 times more likely to show msi than non - interval cancers, and shaukat.42 showed that braf mutation is not associated with interval cancers. however, these three studies were limited as they were conducted by the same author group using the same samples. therefore, additional studies on the molecular characteristics of interval cancers are warranted. finally, the serrated pathway for carcinogenesis of crc has recently been the focus of studies, as sessile, serrated adenomas (ssas) have a malignant potential that is at least as high or higher than the malignant potential of conventional adenomas.43,44 ssas are usually difficult to detect by colonoscopy, as they are often located in the right colon with a sessile configuration and an inconspicuous border. colonoscopists often face difficulties in recognizing and completely removing ssas, and as a result, a subset of these lesions potentially progress to interval cancers. ssas share common features with interval cancers, such as right colon predominance, msi, and a cimp - high phenotype.44 in a multicenter study involving 32 endoscopy centers,45 the detection of proximal serrated lesions varied greatly among endoscopy centers (p<0.0001), and there was also substantial variation among pathologists in the identification of sessile, serrated lesions. fortunately, clinically significantly serrated polyps may be easier to detect with longer withdrawal times.46 the new hampshire colonoscopy registry study showed that a withdrawal time of 9 min resulted in a nearly 30% relative increase in serrated polyp detection.46 therefore, systemic education programs on serrated lesions as well as colonoscopy quality may improve the recognition and diagnosis of serrated lesions. recent evidence suggests that interval cancers are caused by a deficiency in the quality of colonoscopy rather than accelerated tumor biology.12,19 this is good news, as most interval cancers may be prevented by improving colonoscopy quality. first, as described above, interval cancers may develop from suboptimal quality indicators such as withdrawal time, adr, complete colonoscopy, and bowel preparation, as well as incomplete resection of previous neoplastic lesions.37,38,39 in addition, the identification of serrated lesions may be improved with longer withdrawal time.46 second, the grade, stage, histology, and survival patterns do not differ between patients with interval cancer and those with non - interval cancer, which does not suggest an aggressive biology, but rather that the majority represent missed lesions.25,47 in a danish population - based registry,25 982 danish individuals with interval cancers were compared to two reference groups, namely, 358 individuals with crc identified more than 10 years after a colonoscopy and 35,707 individuals with crc but no prior colonoscopy. in this study, no significant differences were found in the characteristics of patients and tumors or survival between interval cancers and cancers arising more than 10 years after colonoscopy.25 the adjusted mortality rate ratios of interval cancers to non - interval cancers were 0.92 and 1.0 after 1 years and 5 years, respectively.25 finally, cases of missed cancer are more common when the colonoscopy is performed by non - gastroenterologists.12,48 a recent canadian registry study12 showed that endoscopist specialty remains a significant predictor of missed cancers despite adjustment for procedural volume. rabeneck.48 also showed that non - gastroenterologists have a significantly higher rate of missed cancers than gastroenterologists, independent of procedural volume. the lack of an association between colonoscopy volume and missed cancers may indicate that even non - gastroenterologists who handle a high volume of procedures continue to miss more crcs than gastroenterologists. it may also indicate that formal endoscopic training generally leads to competency in colonoscopy, whereas providers who do not receive formal training are unable to achieve competence despite the volume of procedures they perform.12,47,48 a higher rate of interval cancers associated with index colonoscopies performed by general practice physicians was also found in studies from ontario and indiana.15,49 emerging evidence suggests a link between endoscopist specialty and colonoscopy quality in korea.50 to the best of our knowledge, there is no formal curriculum or guidelines in colonoscopy training for general practice physicians in korea. therefore, more stringent standards for training and assessment of colonoscopy quality are necessary to cope with the risk of interval cancer. generally, plaintiffs often allege inadequate performance of colonoscopy as the proximate cause of the interval cancer. to date, there is no published data on malpractice claims related with interval cancer. a review of malpractice claims filed with the physicians insurers association of america (database queried between 1985 and 2008) determined that colonoscopy represents the highest frequency (41.5%) of closed claims and the highest total indemnities ($ 54,093,000) among gastrointestinal endoscopy claims.51 indeed, there was an average increase of 15.5% per year in total claim payments associated with colonoscopy, and the majority resulted from inadequate performance of an endoscopic procedure, followed by diagnosis error (may include cases of interval cancer). in korea, a review of malpractice claims reported from the korea consumer agency (database queried between 2009 and 2011) determined an increase of 205% in total claim consultations.52 in addition, the rate of diagnostic error for crcs was 6.8% of all cases of cancers associated with diagnostic errors. these data do not provide detailed information about interval cancer ; however, out anecdotal impression is that lawsuits alleging missed cancers are increasing. given the increased use of colonoscopy, the number of medicolegal litigation related to interval cancers would be logically expected to increase. colonoscopists are encouraged to reduce their risk by documenting the limitations of colonoscopy in the informed consent process and their data of colonoscopy quality (e.g., cecal intubation, bowel preparation, and withdrawal time). colonoscopists commonly discuss the complications of the procedure ; however, they seldom discuss the possibility of missed lesions and failure to prevent interval cancers.16 colonoscopy is not considered a perfect method by colonoscopists or by patients. informed consent may shift the burden of this reality from the colonoscopists to the patients. maintenance of an adequate level of colonoscopy quality indicators and detection of non - polypoid lesions, including ssas, especially in the right colon, may minimize the risk of medicolegal litigation related with interval cancer. considering the significant number of interval cancers that are encountered in daily clinical practice, the importance of adequate training and improvement of colonoscopy quality as causative factors in interval cancers should be highlighted. continuous monitoring of colonoscopy quality, which is amenable to improvement, can not be overstated to prevent the occurrence of interval cancers.	colonoscopy is currently regarded as the gold standard and preferred screening method for colorectal cancer (crc). recently, however, a limitation of colonoscopy in the prevention of crcs has been identified, particularly in the right - sided colon, and the problem of so - called interval cancers has emerged. the prevalence of interval cancer is estimated to be between 4% and 8% of crcs detected. although the exact etiology of interval cancer remains unknown, factors implicated in the development of interval cancers include missed lesions at the time of colonoscopy, incomplete resection of previous neoplastic lesions, different tumor biology, and serrated pathway of carcinogenesis. however, recent evidence suggests that interval cancers are related to the training of the endoscopist and quality of the colonoscopy rather than tumor biology. therefore, the importance of adequate training and continuous monitoring of the colonoscopy quality, which are amenable to improvement, can not be overstated in order to prevent the risk of interval cancers. in this study, the current literature regarding the prevalence and potential factors related to interval cancers and colonoscopy quality - related issues are reviewed.
proteases are enzymes which potentially hydrolyze anything that contains peptide bond, from a dipeptide up to a large protein, containing thousands of amino acids and, thus, it comprises a group of hydrolases that are the most relevant in technological terms. proteolytic enzymes have been the subject of intensive studies for a number of years because proteases have great therapeutic and industrial applications as well. peptidases in which the nucleophile that attacks the scissile peptide bond is the sulfhydryl group of a cysteine residue are known as cysteine - type peptidase. many plant proteases have been isolated from the lattices and most of them belong to this catalytic type. plant cysteine proteases belong to a class which has been widely studied over the years. plant cysteine proteases play a major role in the intracellular and extracellular process such as development and ripening of fruits, degradation of storage proteins in germinating seeds, activation of proteins, and degradation of defective proteins. besides, latex proteases are also involved in the protection of the plants against predator attack. is a wild, thorny, xerophytic, succulent plant, found in boundaries of the agricultural field and also in dry barren areas. phytochemical studies have led to the isolation of ingol diterpenes (3-acetyl-8-methoxyl-7-angolyl-12-hydroxylingol ; 3,12-diacetyl-7-hydroxy-8-methoxylingol ; 3,12-diacetyl-7-angolyl-8-hydroxylingol ; 3,12-diacetyl-8-benzoylingol ; and 3,12-diacetyl-7-benzoyl-8-nicotinylingol) along with three macrocyclic ingol diterpenes derivatives (3,7,12-triacetyl-8-benzoylingol ; 3,12-diacetyl-7-angeloyl-8-methoxyingol ; and 7-angeloyl-12-acetyl-8-methoxyingol). the latex of e. nivulia has been cited for its antioxidant, immunomodulator, cytotoxic, anti - inflammatory, wound healing, haemostatic, and antiproliferative activity. during the course of screening for biochemical constituents, a substantial amount of proteolytic and milk clotting activity recently, an attempt has been made on peptide sequencing of 31-kda, tubulin alpha-1 chain - like protein called nivulian - i, present in the latex of e. nivulia. very recently, a comparative account on proteolytic activity of e. nivulia and other three plants, namely, calotropis procera, carica papaya, and ficus carica, was reported by us. additionally, we characterized the glycosylated cysteine protease called nivulian - ii of the latex of e. nivulia. this paper describes the biochemical characterization, of this cysteine like protease with respect to peptide fingerprinting and n - terminal amino acid sequencing. sigma chemicals : phenyl isothiocyanate (pitc), n - methylpiperidine, methanol, trifluoroacetic acid, dithiothreitol, acetonitrile, 20 amino acid pth standards, etc. were thankfully gifted by the department of biochemistry, indian institute of science, bangalore (karnataka), india. sequencing grade hybond - pvdf membrane (amershyam) was gifted by the department of biochemistry, tmc, advanced centre for treatment, research and education in cancer (actrec), navi mumbai, india. all other chemicals were of the highest purity, analytical hplc grade purchased from himedia laboratories, mumbai ; srl chemicals, bangalore ; qualigen fine chemicals, mumbai ; merck chemicals, india ; and bangalore genie, india. the detail information about identification, collection, preservation, preparation of crude enzyme and its proteolytic activity of euphorbia nivulia latex is described in our previous communication. method of purification of protease was done using acetone precipitation, deae cellulose chromatography, dialysis and followed by rechromatography on deae cellulose column as described in our earlier communication. matrix - assisted laser desorption ionization / time of flight mass spectroscopy (maldi / tof ms) was used for the determination of the molecular mass, as well as the degree of purity of active enzyme. maldi / tof mass spectra were acquired on a bruker daltoncs model ultraflex ii spectrometer, germany, equipped with a pulsed neodymium yttrium - aluminium garnet (nd - yag) smart beam solid state laser (337 nm), in reflectron positive - ion mode, using a 19-kv acceleration voltage. enzyme samples were prepared by mixing equal volumes of a saturated solution of the matrix that is, sinapinic acid (prepared in 0.1% tfa (aq.) and 50% acetonitrile (2 : 1)). 2 l matrix solution and 2 l protein (enzyme) fraction were spotted on the sample plate, mixed them, and allowed to evaporate to dryness. proteins of known molecular mass (insulin, ubiquitin i, cytochrome c, and myoglobin) were used as standards for mass calibration. for peptide mass fingerprinting, the targeted protein band (spot) was manually excised from the gel and was processed for ms and ms / ms analysis as described previously. in brief, gel pieces were washed twice with 50% (v / v) acetonitrile in 50 mm ammonium bicarbonate, shrunk by dehydration in acetonitrile and dried in vacuum centrifuge. disulfide bonds were reduced by incubation with 10 mm dithiothreitol (dtt) in 50 mm ammonium bicarbonate for 45 min at 56c. alkylation was performed by replacing the dtt solution with 55 mm iodoacetamide in 50 mm ammonium bicarbonate. after 30 min incubation at 25c in the dark, the gel pieces were washed with 50% (v / v) acetonitrile in 50 mm ammonium bicarbonate, shrunk by dehydration in acetonitrile, and dried in vacuum centrifuge. the dried gel pieces were incubated with sequencing - grade modified trypsin at 37c overnight. to extract the tryptic digested peptides, 0.1% trifluoroacetic acid in acetonitrile was added, the samples were sonicated for 5 min and the separated supernatant was dried under vacuum. then, the samples (peptides) were desalted using a ziptip c 18 (millipore, usa) according to the manufacturer 's protocol. maldi - ms measurements were performed using maldi tof / ms in reflectron mode using -cyano-4-hydroxycinnamic acid as a matrix. peptide mass fingerprinting (pmf) was carried out using mascot matrix sciences (london, uk) program for protein identification (http://www.matrixscience.com/). maldi - tof / tof fragment ion analysis (see figure s1 in supplementary material available online at http://dx.doi.org/10.1155/2013/569527) of selected individual parent peptide (figure s2) was carried out in the lift mode of the instrument. then, in order to further confirm the identification, all ms / ms data from lift tof / tof spectra were combined with the corresponding ms peptide mass fingerprinting data for database searching. the spectra were analyzed with flex - analysis software (version 3.2, bruker - daltonics, germany) and searched against two taxonomies : (i) other green plants and (ii) species information unavailable in the ncbi (ncbinr) and msdb database using mascot software (version 2.2). the pmf search parameters were : 100 ppm tolerance as the maximum mass error, mh monoisotopic mass values, allowance of oxidation (m) modifications, allowed for 1 missed cleavage, and fixed modification of cysteine by carboxymethyl, that is, carbamidomethylation (c). the purified protease enzyme was adsorbed onto a pvdf membrane and washed several times with deionised water. the n - terminal sequence was determined by pehr edman 's automated degradation method using a procise protein sequencing system (applied biosystem) composed of four integrated modules. this system sequentially cleaves n - terminal amino acids from protein and analyzes the resulting phenylthiohydantoin (pth) amino acid residues. protein homology searches were performed using the blast, indicating the specific residues which are identical identities as well as those which are nonidentical, but nevertheless have positive alignment scores positives. euphorbia nivulia buch.- ham. belongs to the euphorbiaceae family, whose members are characterized by secretory tissues (laticifers) which frequently include proteolytic and milk clotting enzymes. the young stem latex of e. nivulia possesses proteolytic and milk clotting enzyme in more quantity as compared with other investigated laticiferous plants of northern region of maharashtra, india. e. nivulia latex contains thermostable glycosylated cysteine protease having 6.6 and 45c are, respectively, optimum ph and temperature, molecular weight is about 43.42 kda (sds - page), cysteine hydrochloride activator, and inhibitor is mercuric chloride. according to protease nomenclature, this protein is designated as nivulian - ii due to previously nivulian - i (31486.985 da) was already characterized from e. nivulia latex. mass spectroscopy (figure 1) revealed that nivulian - ii had a molecular mass of 43670.846 dalton (43.67 kda), which is relatively matched with molecular mass determined by sds - page, that is, 43.42 kda [5, 12 ]. the resulted molecular mass, that is, 43.67 kda is very close to those of other peptidase belonging to cysteine protease family for example cysteine proteases of vigna mungo, curcuma longa, taenia crassiceps, allomyces aebuscula, triticum arstivum, haemonchus contortus, and gymnophalloides seoi. out of total trypsin - digested peptide fragments (figure 2) of nivulian ii, only thirteen peptides that is, 28.50% sequence was hit in msdb (mass spectrometry protein sequencing database) by mascot peptide fingerprint search engine with maturase k (q52zv1_9magn) of banksia quercifolia (family : proteaceae) with 67.1 score and p < 0.05 (figure 3(a)). an additional match of the mascot peptide mass fingerprint of nivulian - ii with other existing protein sequence from ncbi (national center for biotechnology information) database was also performed. one of the unknown protein (gi\|118485477) of populus trichocarpa (family : salicaceae), matches 7 peptides (i.e., 25.2% sequence) of nivulian - ii as shown in figure 3(b) (http://www.matrixscience.com/). as listed in table 1, the ions at 832.490, 1037.590, 1046.635, 1154.597, 1188.631, 1234.750, 1552.775, 1561.831, 1794.938, 1852.035, 1997.970, 2357.131, and 2872.537 were the thirteen trypsin digested peptides corresponding to residues 115122, 183189, 275282, 283292, 223231, 190199, 287300, 170182, 382397, 350365, 494509, 6786 and 6386. these were designated as n2t1, n2t2, n2t3, n2t4, n2t5, n2t6, n2t7, n2t8, n2t9, n2t10, n2t11, n2t12, and n2t13, respectively. as depicted in table 1, peptide mass profiles were obtained from the database search engine and amino acid sequence of individual peptides were identified from known sequence of maturase k of banksia quercifolia from desired spot of nivulian - ii protein of sds - page. table 2 summarizes the amino acid composition of obtained peptide fragments (n2t1 to n2t13) of nivulian - ii by trypsin digestion. only two residues of oh - group and s - containing amino acid, that is, threonine (t) and cysteine (c), were recorded in sequenced peptides. the content of serine (11.65%) and asparagine (10.52%) was abundant in sequenced peptides of nivulian - ii. the n - terminal amino acid sequence of cysteine protease named as nivulian - ii was deposited in the universal protein resource database, uniprot under the accession number p86837. a comparative study of homology of n - terminal sequences of various cysteine proteases with p86837 using blast algorithm network services (http://blast.ncbi.nlm.nih.gov/blast.cgi) was conducted. the n - terminal sequence of nivulian - ii was aligned with those of other plant cysteine proteases and showed 2550% identity with other cysteine peptidases (tables 3 and 4). the isoleucine (i) is present on the 10th position from n - terminal side of nivulian - ii, which is fully homologous with isoleucine of maturase k, as it is present on the 10th position from amino terminal side (figure 3(a)). n - terminal sequence of nivulian - ii shows only 25% homology sequence with plant origin cysteine proteases, that is, gp - i and gp - ii of zingiber officinale rhizome. it is also homologous with some animal origin cysteine protease, that is, cathepsin f and cathepsin j and bacterial cysteine protease, that is, ides of streptococcus pyogenes. this sequence showed higher identity (50%) with other cysteine endopeptidase isolated from the latex of rubber tree, hevea brasiliensis muell, arg, and a member of the euphorbiaceae family, that is, ricinus communis. it is also homologous (41.66%) with some microbial origin cysteine proteases of naegleria gruberi and polysphondylium pallidum, sulphur - containing protease, that is, methionine protease (map) of mycoplasma hominis and blastula - like protease (bp-10) of saccoglossus kowalevskii. nivulian - ii shared the motifs surrounding the catalytic cysteine (nt and cci) that also occurred in most of the sequences compared (table 4). the asparagine (n) is notably conserved and it is present in all the cases as well as the cysteine residue located in position 8. in the light of all these experimental observations it is envisaged that the present cysteine protease, nivulian - ii, is characterized for the first time. n - terminal amino acid sequence and tryptic digestion profile of nivulian infer the exclusive nature of enzyme and it may be a novel protease of the cysteine family.	a new cysteine protease named nivulian - ii has been purified from the latex of euphorbia nivulia buch.- ham. the apparent molecular mass of nivulian - ii is 43670.846 da (maldi tof / ms). peptide mass fingerprint analysis revealed peptide matches to maturase k (q52zv1_9magn) of banksia quercifolia. the n - terminal sequence (dfppntcccicc) showed partial homology with those of other cysteine proteinases of biological origin. this is the first paper to characterize a nivulian - ii of e. nivulia latex with respect to amino acid sequencing.
a dna ladder is a solution of dna molecules of different lengths used in agarose or acrylamide gel electrophoresis. it is applied as a reference to estimate the size of unknown dna molecules that were separated based on their mobility in an electrical field through the gel. digestion with restriction enzymes of known - length dna fragments from natural sources such as lambda, simian virus 40, and plasmid creates the ladder fragments the lengths of which are dependent on restriction enzyme sites thus, not fully controlled. to overcome this disadvantage and to make dna ladders more flexible, dna engineering was developed perhaps, for commercial purpose, dna manipulation for producing dna ladder fragments became confidential. typically, a dna fragment that contains a tandem repeat units separated by the same unique restriction enzyme sites was cloned into a plasmid and then partially digested to produce a ladder with multimers of the repeats [5, 6 ]. however, the more clear visibility of small size fragments requires the higher amount of plasmid used in cleavage reaction. recently, many laboratory protocols describing the preparation of dna ladders by employing the polymerase chain reaction (pcr) method have been reported [79 ]. this method involves either the simultaneous amplification of a dna target using primer sets or the separate amplification of a different dna targets using specific primers [9, 10 ]. however, using simultaneous primer sets is often difficult to be reproductive because of problematic optimization of pcr conditions, while using separate primer set for a particular fragment of ladders causes a laborious task. here, we describe a method to produce 100 bp dna ladder, which minimizes experimental disadvantages mentioned above. based on our protocol, any laboratory can make its own 100 bp dna ladder instead of purchasing from commercial sources. a dna fragment of length 100 bp was amplified from a known dna sequence by using two specific primer sequences containing a site of recognition enzyme at the 5 ends. since any known sequence could be chosen, pcr conditions were optimized depending on appropriate dna template and designed primers. in our experiment, the specific primers contained a site of smai ; thus, they were named sma-100f and sma-100r, respectively. the 100 bp pcr product was digested completely by the smai enzyme and purified by using qiaquick pcr purification kit and dna was eluted by 50 l of water as recommended by manufactory (qiagen). one hundred ng of smai - treated dna was subsequently self - ligated in the reaction of 16 l from which 1 l was used as a template for pcr with the two primers sma-100f / r. since self - ligated dna templates were mixture of one to multimers of 100 bp repeats, so pcr product was visible as a smear on 1.5% agarose gel after electrophoresis. the smeared dna ranging from 500 bp to 1000 bp was isolated and cleaned using gel elution kit (qiaquick gel extraction - qiagen) and cloned into pgem - t vector system (promega, madison, wi, usa). a clone containing an insert of 800 bp in length was selected, sequenced, and named pgem-800. based on the pgem - t sequences flanking the insert of 800 bp, two specific primers, named gem-1000f and gem-1000r, were designed in order to amplify a dna fragment of 1000 bp in length using pgem-800 as the template (figure 1). these two specific primers were used : gem-1000f 5-ttg taa aac gac ggc cag tga att gta at-3 and gem-1000r 5-cta ttt agg tga cac tat aga ata ctc aag-3. pcr conditions were as follows : 20 l of 5 pcr buffer (gotaq - promega), 5 l of dntps (2.5 mm), 10 l of each primer (2.5 nmoles/l), 1 l of template dna (50 ng/l), and 2.5 u of gotaq dna polymerase in a final volume of 100 l. the thermal cycling profile was 94c for 15 s, 65c for 30 s, and 72c for 1 minute. amplification was carried out for 40 cycles in a gene - amppcr system 9700 thermocycler (applied biosystems, fostercity, ca, usa). the pcr product was 1000 bp like the sum of 800 bp dna insert plus 2 flanking fragments. afterward, this dna fragment was partially digested by smai to yield all the segments of 100 bp dna marker ladder. the smai digestion reaction was as follows : 1 g dna of 1000 bp in length was mixed with 2 l of smai (10 u/l) and incubated at 30c for 5 minutes, then enzyme inactivation was carried out at 65c for 10 minutes. the digested dna was concentrated by ethanol precipitation and dissolved in 200 l of te8 (10 mm tris - hcl, 1 mm edta, ph 8). finally, 5 l of the 100 bp dna ladder was subjected to dna electrophoresis on 2% agarose or 12% acrylamide gels containing ethidium bromide. our study successfully produced 100 bp dna ladder with 10 fragments ranging from 100 to 1000 bp. our procedure contained three steps. in detail, the first step was to make a 100 bp dna fragment from a known sequence by using two specific primers that contain restriction site at the 5 ends. one fragment out of sequenced ones in our experiments that contains smai recognition site at the 5 end was cloned ; thus, we did make the specific primers sma-100f and sma-100r. the selection of sequences and specific primers for preparation of 100 bp dna fragment is easy and flexible in most laboratories working on dna. the second step was cloning pcr products which were reamplified from self - ligated dna of smai - digested 100 bp fragments. using universal primers of pgem - t vector, the largest insert of 800 bp was quickly selected, and recombinant plasmid containing this fragment was named pgem-800. it should be noted that some inserts larger than 800 bp could be selected when the self - ligated reaction was performed by commercial dna ligation kits with a special efficiency in dna ligation. thus, a 100 bp dna ladder with a range outside of 1000 bp could be generated. since the same unique smai restriction site lies at each junction of the 100 bp repeat units, the pgem-800 could be partially digested by this enzyme to produce a 100 bp dna ladder (figure 2(a)). in this case, our dna ladder contained 8 fragments ranging from 100 to 800 bp. we observed that a large amount of pgem-800 plasmid is required for clear visibility of small fragments as 100 bp200 bp. in addition, partial digestion of plasmids was dependent on plasmid conformation (supercoilled, circular and linear forms) ; thus, it was not easy to reproduce. in order to overcome the disadvantages mentioned above, our protocol was used in third step during which a pcr product of 1000 bp was amplified from pgem-800 using two primers gem-1000f and gem-1000r. both primers were designed on the basis of pgem - t sequences which are located at 100 bp far from the upstream and downstream of cloning site. therefore, the size of 1000 bp of pcr product was the sum of the 800 bp insert plus 200 bp. a huge amount of linear formed dna was quickly made by pcr reaction, providing adequate materials for reproduction of dna ladder. in fact, from 1 ml of pcr product we can produce 300 runs of dna 100 bp ladder for electrophoresis on agarose gels or 600 runs on 12% acrylamide gels (figure 2). clearly, this strategy was quite straightforward, time saving, and especially inexpensive. for instance, the price of 100 bp dna ladder for 50 runs is listed 110 usd (g2101-promega) and 53 usd (n3231s - new england biolabs) on available web sites. we estimated that our protocol spends only 3 usd for materials to produce 50 runs of 100 bp dna ladder. for instance, labeled 100 bp dna ladder can be produced by using labeled dntps in the pcr amplification. in fact, we have successfully produced the 60 bp dna ladder and the 500 bp dna ladder supplied for our specific needs (figure 3). we estimated that it takes approximately 2 weeks for laboratory works to carry out this procedure. once self - ligated insert with desirable length was cloned, it took a few hours to produce a huge amount of dna ladder. additionally, size range of each ladder could be broadened by repeating this protocol using pcr product amplified from recombinant plasmid. for instance, the dna fragment of 1000 bp was self - ligated, cloned into a plasmid and dna fragments with favorable size could be selected. a minor disadvantage in using the self- and quickly produced dna ladders is unknown amount of dna in each band. the commercial dna ladders compensate this demand even though this information is not always needed. compared with conventional methods for producing dna ladders, our strategy reported in this paper is simple and flexible for preparation of the 100 bp dna ladder that contains 10 fragments ranged from 100 to 1000 bp. our strategy could be applied for producing different kinds of dna ladders of good quality and could be useful for most laboratories.	dna ladder is commonly used to determine the size of dna fragments by electrophoresis in routine molecular biology laboratories. in this study, we report a new procedure to prepare a dna ladder that consists of 10 fragments from 100 to 1000 bp. this protocol is a combination of routinely employed methods : cloning, pcr, and partial digestion with restriction enzymes. dna fragments of 100 bp with unique restriction site at both ends were self - ligated to create a tandem repeat. once being cloned, the tandem repeat was rapidly amplified by pcr and partially digested by restriction enzymes to produce a ladder containing multimers of the repeated dna fragments. our procedure for production of dna ladder could be simple, time saving, and inexpensive in comparison with current ones widely used in most laboratories.
the onset of a psychiatric disorder in children and adolescents often occurs at a time in life that is characterized by considerable upheavals. in adolescents, detaching oneself from one 's parents, experiencing one 's first love and first heartbreak, finishing school and starting vocational training, initial experiences with alcohol and drugs, as well as many other factors, play an important role, both as potential triggers or amplifiers of a psychiatric disorder and for the adolescents ' subjective quality of life. in addition, there are important factors associated with the disorder itself, including the way adolescents cope with their first experiences with treatment, as well as dealing with the stigma of having a psychiatric disorder and being confronted with the need for potentially lifelong treatment. in addition, symptoms such as affective and cognitive disorders often arise many years before the psychiatric disorder itself becomes manifest, and may have a negative effect on the subjective well - being and everyday lives of those concerned long before the disease is recognised as such and treated. all these factors make it harder for children and adolescents suffering from psychiatric disorders to cope with their age - appropriate development and therefore have serious, long - term consequences for their quality of life. the patients ' subjective perception of the disorder and their living circumstances is subject to complex psychological assessment and processing. the connection between health - related quality of life, psychiatric disorders, and external living circumstances can not be understood without knowledge of these processes. both age - appropriate and disease - related aspects must therefore be taken into account when examining the quality of life of adolescents or patients with psychiatric disorders. in this paper, we will therefore look at age - specific aspects of measuring the quality of life of children and adolescents. when evaluating medical interventions within the medical health services system, not only the quality of life of adults but also that of children and adolescents can be viewed as an important outcome criterion. the use of medical assistance affects not only somatic, but also emotional and social parameters ; it is therefore necessary to shed light on the state of children and adolescents from their own point of view. research into the quality of life of children and adolescents is particularly important also because the number of children and adolescents with chronic disorders, including psychiatric disorders, has increased despite the impressive progress made in medicine. one reason why quality of life is increasingly being taken into account in clinical and health - economic studies is, therefore, the substantial changes in the range of disorders and treatments that have been observed in children and adolescents since the 1980s. this so - called new morbidity is characterized by a shift from acute to chronic diseases and from somatic to psychiatric disorders. this makes it necessary to take into account the long - term quality of life or subjective health of young patients, and to identify the everyday burden due to the disorder, so as to expose potential impairments in well - being and function at an early stage. hence, an understanding of the subjective perception among children and adolescents of their health status and their treatment can be used to assess treatment success, but also on an individual level to optimize the treatment itself. although medical interventions often lead to an objective improvement in the health status of children and adolescents, the frequent hospitalization, multiple operations, and not least the uncertainty about the future can be shown to have a detrimental effect on the course of the disease and on how well patients cope with it. the question of how adolescents feel about their health and their treatment is just as important when assessing treatment success as it is for optimizing treatment in individual cases. so far, the diseases that have been examined most often aside from diseases with high mortality rates are those that may display sudden crises, in some cases on a life - threatening scale, as well as diseases whose treatment is very costly. frequently occurring and less threatening diseases, or diseases that only persist for a limited time, take a back scat by comparison. health - related quality of life (hrqol) has therefore moved into the focus of the assessment of treatment options, particularly for those chronic disorders that do not reduce life expectancy but instead accompany patients throughout their lives, as may be the case with psychiatric disorders. by concentrating on the patients ' needs, additional insights can be gained into the meaningfulness and usefulness of the respective procedures, and subjective differences between different treatment measures can be uncovered. compared with adults, the measurement of hrqol in children and adolescents is still a relatively new field of research. initially, the dimensions of hrqol found in adults were usually simply transferred to children.the available measures primarily involved an external assessment by parents, and new instruments were often developed ad hoc for a specific study and without adequate quality checks. the guidelines for developing hrqol instruments for children issued by the mental health division of the world health organisation (who) demand that such measures should be age - appropriate and child - centred, preferably take into account self - reporting, be usable independently of the health status and cross - culturally, and should include both positive and negative aspects of the relevant domains (eg, family/ social relations, physical function, social and material environment). beyond this, quality of life instruments must meet the quality criteria for psychometric procedures (reliability, validity, sensitivity to change). when measuring the hrqol of children and adolescents, a series of aspects need to be taken into account in terms of contents and method, which will be outlined below. the most important research questions for the measurement of quality of life primarily concern the following key aspects : what dimensions of hrqol are relevant to children and adolescents, and do suitable instruments exist to measure these ? what are the advantages and disadvantages of self - reported and proxy - reported measures of child and adolescent quality of life, and how can hrqol be ascertained in an age - appropriate way ? what are the advantages and disadvantages of disease - specific and generic measures ? answering these questions is of far - reaching importance for the quality of the measurement of quality of life in children and adolescents, and hence for its usefulness as an outcome parameter, and for assessing and choosing between treatment options. there is a general consensus that quality of life should be viewed as a multidimensional construct comprising at minimum a physical, an emotional, and a social component of well - being and function. concerning the question whether children and adolescents describe their quality of life on the same dimensions as adults, the general definition of quality of life given by matza for adults, namely ' an individual 's subjective perception of the impact of health status, including disease and treatment, on physical, psychological, and social functioning, ' also applies to children and adolescents. however, the specific aspects of children 's lives that are included in these three components are different. although it is assumed that those dimensions that are relevant to adults are also significant to children and adolescents, their relative weights and constellations may be expected to differ at different ages. according to schor, for example, the different quality of life dimensions are more closely connected in children than they are in adults. adapting instruments designed for adults to measure child and adolescent quality of life would therefore seem problematic. hrqol is generally considered to be a latent construct, which can not be directly observed, and irrespective of its definition or underlying concept it includes the perception and judgement of one 's own life from the individual 's own subjective perspective, as well as one 's subjective well - being, or affective mood. these two assumptions mean that, when possible, quality of life should be measured using self - reporting. hence the quality of life of children and adolescents, too, should ideally be ascertained by means of self - reporting. the parental assessment of children 's quality of life is an uncertain substitute for self - reporting, based on the assumption that the latter has higher (conceptual) validity, and should only be used as a last resort or as an additional source of information, for example concerning physical or emotional problems, since parental assessment does not represent the way in which the disease is perceived and experienced by the children themselves. research to date has shown that contrary to former doubts, the quality of life of children and adolescents can be measured using self - report questionnaires, provided these instruments are developed with the age, maturity and cognitive development of the subjects in mind. although the understanding of the quality of life concept and the assessment of one 's own health and well - being is determined by the three factors described, children and adolescents over the age of 8 are able to understand and give reliable and valid answers to questions about their quality of life. self - reporting has its limits, particularly in children under the age of 8, so that here an external assessment of a child 's quality of life must be obtained from his or her parents, or else from his or her teachers or the medical health care team. such a proxy assessment can be obtained by asking parents to rate their child 's quality of life from the child 's point of view (proxy - patient perspective) or from their own point of view (proxy - proxy perspective). however, empirical studies have only revealed a low - to - moderate correlation between parental and self - reported ratings. a higher correlation between the self -reported and proxy ratings has been observed for the observable aspects of hrqol, such as physical well - being, than for the non - observable aspects, such as emotional well - being. there is a consensus that in children over the age of 8 parental ratings can supplement self - reporting but not replace it. parents are not objective judges, but have their own perspective, which can serve as an additional source of information. as a matter of principle, when assessing the hrqol of children and adolescents it is necessary to take into account their development and the ongoing changes occurring over time, for example with regard to their perception of their own emotional state. a longitudinal evaluation of hrqol must therefore take into account both the baseline level and the natural changes over time, since the effect of medical treatments can not otherwise be distinguished from normal developmental changes. however, the possibility of an adaptation of internal assessment standards must also be taken into consideration in cross - sectional studies on chronic diseases, due to a phenomenon which is described as the well - being paradox or response shift. some authors offer age - specific versions of their questionnaires in order to take into account the different stages of development. a key requirement for an age - appropriate measurement of quality of life is that the target concerned should be involved when developing an instrument : the contents of a quality of life measure should therefore ideally be obtained directly from the children concerned. disease - specific quality of life measures are designed to determine the quality of life in certain diagnostic groups or patient populations. to do so, they look at those aspects which are particularly relevant to these groups or disorders, such as distinctive features of the treatment options. disease - specific measures are generally appropriate for the clinical examination of specific treatment interventions ; however, they make it more difficult to compare quality of life measurements across different physical and psychological disorders. when studying general health and when comparing the effects and courses of different disorders, non - specific so - called generic these ascertain as wide a range of quality of life aspects as possible and can be used for different diseases, disabilities, situations, patients, and populations. when additional disease - specific information is to be considered on top of generic aspects of quality of life, a number of measures offer additional disease - specific modules, which examine the specific effects of chronic diseases in childhood on top of generic items and scales. when it comes to the cross - cultural measurement of hrqol, it should be noted that quality of life measures for children have often been developed in different countries and cultures, and therefore need to be translated and checked psychometrically before they can be used in another language. multinational measures for children have so far primarily been made available by adapting existing instruments, which have then been confirmed in other countries (sequential approach). in order to ensure a cross - culturally similar measurement of hrqol, the corresponding instruments should ideally be developed, harmonized and tested using a simultaneous, multinational approach (simultaneous approach). the two european quality of life measures for children, kidscreen and disabkids, are examples of this approach. following a review of the literature and discussions by a panel of experts (delphi), focus groups with children were carried out simultaneously in different countries to identify those dimensions of quality of life that were relevant to the children and to formulate the appropriate items. to ensure a cross - culturally comparable measurement the developers checked whether respondents with similar characteristics had the same likelihood of answering the items in a similar way, irrespective of their nationality. the results of this multinational approach show that it is indeed possible to measure hrqol on different dimensions in a way that is comparable across different cultures. although such cross - cultural and simultaneous approaches are the exception, so far, they are desirable in view of their methodological quality. if data on the hrqol of children and adolescents are to be compared cross - culturally, it is advisable to use measures that were developed using such a simultaneous approach (eg, kidscreen, disabkids). hrqol can be determined using profile and index measures. profile measures map the individual dimensions of quality of life (eg, physical well - being, emotional well - being, social support, friends, relationship with parents, school environment). they are particularly suitable for clinical research and for measuring an individual 's quality of life because they allow the effects of a clinical intervention on the different dimensions of quality of life to be examined, and comparisons to be made between the quality of life of different individuals, also for individual dimensions. in index measures, on the other hand, the individual ratings for the quality of life dimensions are collated to form a global aggregate score. however, one disadvantage of this is that they are limited when it comes to representing the multidimensionality of the quality of life construct. there is a general consensus that quality of life should be viewed as a multidimensional construct comprising at minimum a physical, an emotional, and a social component of well - being and function. concerning the question whether children and adolescents describe their quality of life on the same dimensions as adults, the general definition of quality of life given by matza for adults, namely ' an individual 's subjective perception of the impact of health status, including disease and treatment, on physical, psychological, and social functioning, ' also applies to children and adolescents. however, the specific aspects of children 's lives that are included in these three components are different. although it is assumed that those dimensions that are relevant to adults are also significant to children and adolescents, their relative weights and constellations may be expected to differ at different ages. according to schor, for example, the different quality of life dimensions are more closely connected in children than they are in adults. adapting instruments designed for adults to measure child and adolescent quality of life would therefore seem problematic. hrqol is generally considered to be a latent construct, which can not be directly observed, and irrespective of its definition or underlying concept it includes the perception and judgement of one 's own life from the individual 's own subjective perspective, as well as one 's subjective well - being, or affective mood. these two assumptions mean that, when possible, quality of life should be measured using self - reporting. hence the quality of life of children and adolescents, too, should ideally be ascertained by means of self - reporting. the parental assessment of children 's quality of life is an uncertain substitute for self - reporting, based on the assumption that the latter has higher (conceptual) validity, and should only be used as a last resort or as an additional source of information, for example concerning physical or emotional problems, since parental assessment does not represent the way in which the disease is perceived and experienced by the children themselves. research to date has shown that contrary to former doubts, the quality of life of children and adolescents can be measured using self - report questionnaires, provided these instruments are developed with the age, maturity and cognitive development of the subjects in mind. although the understanding of the quality of life concept and the assessment of one 's own health and well - being is determined by the three factors described, children and adolescents over the age of 8 are able to understand and give reliable and valid answers to questions about their quality of life. self - reporting has its limits, particularly in children under the age of 8, so that here an external assessment of a child 's quality of life must be obtained from his or her parents, or else from his or her teachers or the medical health care team. such a proxy assessment can be obtained by asking parents to rate their child 's quality of life from the child 's point of view (proxy - patient perspective) or from their own point of view (proxy - proxy perspective). however, empirical studies have only revealed a low - to - moderate correlation between parental and self - reported ratings. a higher correlation between the self -reported and proxy ratings has been observed for the observable aspects of hrqol, such as physical well - being, than for the non - observable aspects, such as emotional well - being. there is a consensus that in children over the age of 8 parental ratings can supplement self - reporting but not replace it. parents are not objective judges, but have their own perspective, which can serve as an additional source of information. as a matter of principle, when assessing the hrqol of children and adolescents it is necessary to take into account their development and the ongoing changes occurring over time, for example with regard to their perception of their own emotional state. a longitudinal evaluation of hrqol must therefore take into account both the baseline level and the natural changes over time, since the effect of medical treatments can not otherwise be distinguished from normal developmental changes. however, the possibility of an adaptation of internal assessment standards must also be taken into consideration in cross - sectional studies on chronic diseases, due to a phenomenon which is described as the well - being paradox or response shift. some authors offer age - specific versions of their questionnaires in order to take into account the different stages of development. a key requirement for an age - appropriate measurement of quality of life is that the target concerned should be involved when developing an instrument : the contents of a quality of life measure should therefore ideally be obtained directly from the children concerned. disease - specific quality of life measures are designed to determine the quality of life in certain diagnostic groups or patient populations. to do so, they look at those aspects which are particularly relevant to these groups or disorders, such as distinctive features of the treatment options. disease - specific measures are generally appropriate for the clinical examination of specific treatment interventions ; however, they make it more difficult to compare quality of life measurements across different physical and psychological disorders. when studying general health and when comparing the effects and courses of different disorders, non - specific so - called generic these ascertain as wide a range of quality of life aspects as possible and can be used for different diseases, disabilities, situations, patients, and populations. when additional disease - specific information is to be considered on top of generic aspects of quality of life, a number of measures offer additional disease - specific modules, which examine the specific effects of chronic diseases in childhood on top of generic items and scales. when it comes to the cross - cultural measurement of hrqol, it should be noted that quality of life measures for children have often been developed in different countries and cultures, and therefore need to be translated and checked psychometrically before they can be used in another language. multinational measures for children have so far primarily been made available by adapting existing instruments, which have then been confirmed in other countries (sequential approach). in order to ensure a cross - culturally similar measurement of hrqol, the corresponding instruments should ideally be developed, harmonized and tested using a simultaneous, multinational approach (simultaneous approach). the two european quality of life measures for children, kidscreen and disabkids, are examples of this approach. following a review of the literature and discussions by a panel of experts (delphi), focus groups with children were carried out simultaneously in different countries to identify those dimensions of quality of life that were relevant to the children and to formulate the appropriate items. to ensure a cross - culturally comparable measurement the developers checked whether respondents with similar characteristics had the same likelihood of answering the items in a similar way, irrespective of their nationality. the results of this multinational approach show that it is indeed possible to measure hrqol on different dimensions in a way that is comparable across different cultures. although such cross - cultural and simultaneous approaches are the exception, so far, they are desirable in view of their methodological quality. if data on the hrqol of children and adolescents are to be compared cross - culturally, it is advisable to use measures that were developed using such a simultaneous approach (eg, kidscreen, disabkids). profile measures map the individual dimensions of quality of life (eg, physical well - being, emotional well - being, social support, friends, relationship with parents, school environment). they are particularly suitable for clinical research and for measuring an individual 's quality of life because they allow the effects of a clinical intervention on the different dimensions of quality of life to be examined, and comparisons to be made between the quality of life of different individuals, also for individual dimensions. in index measures, on the other hand, the individual ratings for the quality of life dimensions are collated to form a global aggregate score. however, one disadvantage of this is that they are limited when it comes to representing the multidimensionality of the quality of life construct. in recent years, new instruments have increasingly been developed for measuring the hrqol in children. the quality of life instruments database (qolid ; http:// www.qolid.org/) provides a good overview of the existing generic and disease - specific measures. table i summarizes the health - related quality of life measures for children and adolescents, listed in order of the year they were introduced, that are currently most commonly used (internationally) and that satisfy the following criteria : can be used across different diseases and disorders are available internationally in several languages their psychometric quality has been tested and found to be adequate are available as a self - report measure have been published scientifically were primarily designed for children and adolescents measure the three main components of health - related quality of life (physical, emotional and social well - being) as defined by the who. the questionnaire child health and illness profile (chip, www.childhealthprofile.org), originally developed in the us, primarily focuses on measuring functional aspects of quality of life and is available in a child edition chip - ce ; for children between the ages of 6 and 11, and as an adolescent edition chip - ae ; for adolescents between the ages of 12 and 17. the adolescent edition comprises 108 items distributed between five health dimensions : satisfaction, complaints, resilience, achieving social goals, and risk avoidance. the shorter child edition (45 items) comprises the four dimensions satisfaction, complaints, resilience, and risk avoidance and is available as a self - report form and a parental questionnaire. the chip produces a sum score for each of the dimensions as well as a multidimensional profile of the quality of life, based on the different dimensions. the resulting personal profile can be characterized using a taxonomy of health profiles, allowing the subject 's overall situation to be described comprehensively and yet manageably. the chip has good psychometric properties, with a high reliability (cronbach 's >.70) for most of the subscales and satisfactory criterion validity. furthermore, the measure enjoys a high level of acceptance among children and adolescents, as well as among parents. the widely used child health questionnaire (chq), which also originated in the us, was designed for children and adolescents between the ages of 10 and 18. in addition to a self - report version (87 items) there is also a parents ' version for children over the age of 5 (50 items or 28 items). apart from assessing general health, the measure covers 14 different concepts of physical (physical function, physical pain, general health perception), psychosocial health (self - esteem, mental health, behavior, parental burden due to the physical/ mental health of the child) and disabilities associated with these (physical and/or mental impairment of social roles and family activities). the individual scale scores can be combined to form a physical and a psychosocial aggregate score. the chq displays good psychometric properties (cronbach 's >.70) and the parental version has already been validated for more than 21 languages. weaknesses of the measure include the length of the self - report version (87 items) and its focus on those aspects of child health and well - being that are primarily relevant to parents (eg, burden imposed on parents by children 's health). the kindl - k (www.kindl.org) is an originally german - language quality of life measure for healthy and ill children and adolescents, which has been translated into 22 languages. the questionnaire was developed with the help of focus groups involving children and adolescents and is available in age - adapted self - report versions and proxy versions for parents, for the age groups 4 to 7 years (12 items), 8 to 12 years (24 items), and 13 to 16 years (24 items). in the case of 4- to 7-yearold children, self - reporting another possible measure is the computer - assisted cat - screen program, which is available in german and english for self - completion by children from the ages of 6 to 12 years and by adolescents from the ages of 13 to 16 years. it measures the hrqol of children and adolescents in a way that is suitable for children and for their stage of development at the respective age. the kindl - r includes indicators of physical, emotional, family, social, and school - related well - being, as well as of self - esteem. furthermore, the core questionnaire can be extended by means of disease - specific modules (eg, obesity, asthma, diabetes, cancer). when interpreting the kindl - r, a profile consisting of six scores is calculated, as well as an overall score for hrqol. the good psychometric properties of the kindl - r, such as the high reliability (cronbach 's >.70) of most of its subscales and its good ability to discriminate between different clinical diagnoses, have been demonstrated in various studies. the questionnaire pediatric quality of life inventory generic core scales (pcdsql 4.0, www.pcdsql.org) was developed in the us in order to measure the hrqol of healthy and ill children and adolescents (5 to 18 years old) and is available as a self - report and parent - reported version (23 items). the newly developed pediatric quality of life inventory infant scale, allows the quality of life of young children between the ages of 1 and 12 months (36 items) and 13 to 24 months (45 items) to be measured by means of parent reporting. the core measure comprises dimensions for physical, emotional, social, and school - related (or cognitive) function. to evaluate the results, a psychosocial sum score can be calculated (emotional, social and school - related or cognitive function) as well as an overall score. the pedsql subscales can optionally be augmented by various disease - specific modules (eg, asthma, brain tumor, diabetes). the pediatric quality of life inventory has already been used in numerous different research settings, and is characterised by a high reliability of its individual subscales (cronbach 's >.80) and the total score (cronbach 's >.90). numerous studies have shown that healthy children achieve higher scores (indicating greater quality of life) on the pedsql scales than do chronically or acutely ill children. the kidscreen questionnaire (www.kidscreen.org) was developed simultaneously in 13 countries in an international collaboration and is based on probabilistic test theory. it measures physical (physical wellbeing), psychological (psychological well - being, moods and emotions), social (social support and friends, social acceptance), family (relationship with parents, autonomy, financial possibilities), and school - related aspects of the well - being and function of children and adolescents between the ages of 8 and 18 years. in addition to the long version, with 52 items and 10 dimensions, there is also a short version, as well as a 10-item index of general hrqol. the high validity and reliability of the kidscreen subscales have been demonstrated in multinational studies. statistical analyses show that the items of the kidscreen are understood similarly across different cultures, age groups, sexes, and for different diseases and disorders, and allow the principal components of hrqol to be measured in a similar way in children and adolescents. the kidscreen questionnaire therefore fulfils the standards required by the who for a child - appropriate measurement of hrqol. the self - report version for children and adolescents is now available in 38 languages and the proxy version in 31 languages. the disabkids quality of life inventory (www.disabkids.org) was developed in seven european countries using the same cross - cultural approach. it is a disease - specific or chronic - generic measure, which allows the hrqol of children (4 to 16 years old) with various chronic disorders (eg, asthma, cystic fibrosis, cerebral palsy, diabetes, arthritis, and skin disorders) to be determined using so - called chronic - generic items and scales specific to the various diagnoses, either by self - report or by proxy. age - adapted versions are available for children between the ages of 4 and 7 years, and children between 8 and 16 years. the instrument measures mental facets (independence, emotions), social facets (social inclusion, social exclusion), and physical facets (limitations, treatment) using 37 items, whereby a total score can also be calculated. in addition to the chronic - generic module, seven disease - specific modules have been developed, each with 2 to 3 subscales and 12 to 17 items. its psychometric quality has been examined in a multinational study, the results of which indicate high reliability scores (cronbach 's >.70) for the individual scales, as well as cross - cultural validity of the measure. the inventory for measuring quality of life in children and adolescents, ilk, developed in germany, is a screening tool for measuring quality of life in healthy, as well as in psychologically or physically ill children and adolescents (6 to 18 years old). the instrument asks the children, their parents, and their doctors / therapists questions about four different areas of quality of life : school, family, social contact with peers, and interests and recreational activities. beyond this, information is collected about their physical and psychological health, as well as an overall rating of their quality of life. in the case of children and adolescents suffering from a disease or disorder, additional questions are asked about the burden imposed by the current illness and by the diagnostic and therapeutic interventions. overall, the instrument displays satisfactory psychometric properties and representative standard scores are available for germany, as are comparative scores from child and adolescent psychiatric practices and clinics, as well as child and adolescent departments. the health utility index mark 2 (hui2, http://www.healthutilities.com) is a generic measure and is one of the so - called preference - based instruments for children and adolescents. such instruments measure the individual aspects of hrqol and then summarize them in the form of a one - dimensional score, or index. the hui measures the health status along seven dimensions : emotions, perception, mobility, cognitions, self - care, pain, and fruitfulness. for each dimension, 3 to 5 alternative answers (levels) are available for selection, whereby a low level (eg, 1 = able to walk, bend, lift, jump, and run normally for age [mobility dimension ]) indicates good health, and a high level (eg, 5 = unable to control or use arms and legs [mobility dimension ]) indicates poor health. subjects use these seven dimensions to rate their subjective health (eg, 3223242). due to the different possible combinations, 24 000 different heath scores can theoretically be determined, whereby the score 1111111 corresponds to perfect health. an algorithm is used to convert the measured health scores into a unidimensional index, which can then be used in cost - benefit analyses. the eq-5d - y is the children 's version of the eq-5d, a widely used, generic, preference - based instrument that was originally designed by the euroqol group to measure hrqol in adults. the eq-5d - y can be used for self - completion by children and adolescents from the age of eight years upwards, and uses five items to record the dimensions mobility, self - care, general activity, pain / physical symptoms, anxiety and depression. beyond this, children assess their current health using a visual analogue scale (vas) ranging from 0 (worst condition) to 100 (best condition). in developing the children 's version, a first multinational study has shown the eq-5d - y to be easy to use, and a reliable and valid instrument for measuring the hrqol in children and adolescents ; this needs to be confirmed by further population - based and clinical studies. the test scores can be transformed by means of an algorithm into index scores, and can then be used in cost - benefit analyses. examples of quality of life items (kidscreen instrument) can be seen in table ii. in terms of the opening questions about the measurement of hrqol in children and adolescents, it can be stated that international instruments for measuring hrqol in children and adolescents are now available, which allow the most important dimensions of the construct to be measured. although no single quality of life measure can claim to cover the entire universe of hrqol in all its possible facets, the quality of life dimensions that are relevant to a specific subject of investigation can indeed be measured. the quality of life of children and adolescents can and should be measured through self - reporting. in order to do this, however, age - appropriate measures must be used, which take into account the subjects ' maturity and cognitive development. only generic quality of life measures allow hrqol to be measured across different diseases and disorders, and can be used both for ill and for healthy children and adolescents. despite the availability of such instruments, longitudinal studies on the quality of life of children and adolescents are still rare in the field of medicine. in view of the increasing attention being paid to the psychosocial aspects of health, there is still a considerable need for research into the subjective health of children and adolescents. with the help of quality of life measures, studies can examine the well - being and function of large populations, thus providing reference data for ill children and adolescents as well as giving an indication of medical and health - policy interventions that may be necessary. in a clinical context the health of young patients can be improved not only by performing medical procedures but also by teaching psychological strategies, which means that the quality of life of the adolescents is an important target criterion here. future studies ought to focus more on the specific needs of young patients, in order for example to be able to assess how important individual quality of life dimensions are for the further course and for the prognosis of diseases. also, despite the fact that the majority of patients suffering from psychological disorders live with their families, or in close contact with their families, hardly any studies exist that examine the quality of life of the families and relatives of patients with child and adolescent psychiatric disorders. future studies should therefore take greater account of the social environment of child and adolescent patients when examining their quality of life, with a view to developing a better understanding of the interactions and interconnections, and being able to incorporate the results of quality of life research in future treatment concepts. one of the particular challenges facing quality of life research in children and adolescents is the need to place even greater emphasis of self - reporting, and to take into account the way they process and cope with their disease or disorder. by systematically disseminating the empirically based understanding of the quality of life of young patients, as well as the possibility of measuring it and changing it by means of treatment strategies, it can contribute to the quality of life of children and adolescents undergoing treatment not only as the subject of the individual doctor - patient relationship during treatment but also as a target criterion in clinical trials and treatment studies. such a set of tools ultimately serves the goal of assessing the treatment outcomes of child and adolescent patients, and of their families, and thus opens up new paths for further improvements in the medical and psychological care of those concerned. based on the existing findings, the following conclusions can be drawn about measuring the hrqol in children and adolescents : instruments are now available for determining the hrqol of children that allow the relevant dimensions of the construct to be measured. an index instrument on the other hand, converts the ratings of the individual quality of life dimensions into a single, global aggregate score. some instruments take different age groups into consideration and have corresponding versions as age - appropriate measures. the quality of life of children and adolescents from the age of 8 and upwards can and should be determined through self - reporting. if developed and tested accordingly, the dimensions of hrqol can also be measured in a way that is comparable across different cultures. generic quality of life measures allow hrqol to be ascertained across different diseases and can also be used in healthy children and adolescents.	this article provides an overview of the conceptual foundations of measuring health - related quality of life (hrqol) in children and adolescents in child and adolescent psychiatry, and of the current state of research in this field. the available procedures for determining quality of life are presented according to their areas of use and their psychometric characteristics. the internationally available generic instruments for measuring hrqol in children are identified and assessed in terms of their strengths and weaknesses with regard to selected criteria. as a result, seven generic hrqol instruments and two utility procedures have been identified which satsfy the following criteria : (i) psychometric qualty ; (ii) age - appropriate measurement ; (iii) versions for self - reporting and external rating ; and (iv) cross - cultural measurement. the identified instruments satisfy the individual criteria to different degrees. they are increasingly being used in health services research, treatment studies, and epidemiological research ; however, they are not yet widely used as part of the clinical routine in child and adolescent psychiatrics.
seven years prior, the patient underwent a hemiarch descending aortic replacement with a right subclavian artery bypass due to a ruptured acute type b aortic dissection with anomalous origin of the right subclavian artery from the descending aorta. after the operation, the patient was followed regularly at the outpatient clinic and developed a distal aortic aneurysm from the end of the arch graft to both iliac arteries with a maximal diameter of 6.5 cm at the diaphragm level, which caused the symptom of dysphagia (fig. 1). we planned to perform a staged debranching endovascular stent - graft repair of the total remaining aorta including both common iliac arteries., the abdominal aorta and its main branches were dissected and exposed, including both iliac arteries, both renal arteries, the superior mesenteric artery, and the celiac trunk. they were replaced with a custom - designed graft made of a 147 mm and a 168 mm dacron y graft (intervascular, la ciotat, france) (fig. the proximal abdominal aortic anastomosis was made at the true lumen of the infrarenal abdominal aorta after obliterating the pseudolumen, and the other branches were anastomosed end - to - end separately. the total operation time was seven hours and fifteen minutes, and the patient remained in the intensive care unit for four days. the total transfusion required was 17 units of packed cells, 11 units of fresh frozen plasma, and 20 units of platelets. two weeks after the first operation, an endovascular procedure was performed. under local anesthesia three differently sized stent grafts were placed in the true lumen of the dissected remnant of the thoracoabdominal aorta. the first stent graft was a 4036 mm tapered monobody with a length of 20 cm, which was inserted into the proximal descending aorta covering the distal anastomosis of the previous aortic arch graft. the second stent graft was a 3626 mm tapered monobody with a length of 16 cm, which was inserted into the first stent graft with an overlap of 4 cm. finally, the third stent graft, a 26 mm monobody with a length of 16 cm, was inserted distally to cover the graft of the abdominal aorta. all stent grafts were inserted into the true lumen of the dissecting thoracoabdominal aorta and they covered approximately 40 cm of the length of the aorta. there was a small type ii endoleak from the intercostal artery on the immediate postoperative computed tomography (ct) angiography, but subsided by the follow - up ct two months later. the following ct angiography nine months after the operation showed a decreasing aneurysm size without any other abnormal findings (fig. the patient was followed for one year and ten months and no other problem developed during that period. most patients with an untreated thoracoabdominal aortic aneurysm end up dying from its rupture, and mortality risk increases with age. the conventional thoracoabdominal aortic aneurysm repair procedure is a high - risk operation, involving thoracoabdominal exposure, single - lung ventilation, and either partial cardiopulmonary bypass with aortic cross - clamp or full cardiopulmonary bypass with deep hypothermic circulatory arrest. only a few large - volume surgical centers have reported satisfactory results with such a procedure. a study analyzing 1,010 cases of open thoracoabdominal aortic aneurysm repair performed in low - volume centers (fewer than fifteen cases per year) between 1991 and 2002 found that for elective procedures, the 30-day mortality was 19% and the mortality increased to 31% at one year postoperative. mortality risk was shown to increase with age, the number of comorbidities, and more extensive aortic involvement. recently, endovascular aneurysm repair has been used more frequently for risky thoracoabdominal aortic aneurysms, but this approach is restricted by anatomic limits such as the need to establish a safe landing zone without covering the critical end arteries. to overcome these limits, a hybrid strategy has been developed that involves the extra - anatomic bypass of the visceral vessels (a debranching procedure) with either concurrent or staged exclusion of the aneurysm using endovascular devices (such procedures are known as thoracic endovascular aneurysm repair). this hybrid strategy has the advantage of avoiding the thoracic incision, cardiopulmonary bypass, and aortic cross clamp, thereby potentially decreasing postoperative pain and pulmonary complications, intraoperative hemodynamic changes, and bleeding and organ ischemia involving the spinal cord. during the procedure, the spinal cord collaterals may be perfused in a pulsatile manner, which may protect the spinal cord by opening collateral flow. the staged approach is preferred in elective patients to reduce the debranching operation time, transfusion amount, and rates of complications such as respiratory insufficiency and neurologic complications. however, this approach may increase the risk of aneurysm rupture during the waiting period for the second procedure. in our case, the debranching procedure was very tedious, requiring multiple long anastomoses and the endoscopic procedure was not simple, so we chose the staged approach. the design of the graft used in this case was our original design (fig. 2a) ; the branches were all separated and well fitted anatomically without any postoperative perfusion problems (fig. reported debranching techniques are quite diverse and have been mostly applied to high - risk patients such as those who are older, have comorbidities from heart, lung, or kidney disease, and have undergone prior open chest surgery., the patient had already undergone an open thoracotomy to replace the proximal descending aorta, so we were concerned about the potential of subsequent lung injury and/or respiratory difficulty after reopening the adherent left thorax. existing aortic grafts generally provide excellent landing zones, which was the case for our patient : the proximal descending aortic graft from the previous operation and the abdominal aortic graft from the debranching procedure provided an excellent landing zone. in most studies of the hybrid repair strategy, the debranching operation was a variable procedure involving inflow from the iliac arteries, abdominal aorta, or a previous abdominal graft. according to the preliminary results from the north american complex abdominal aortic debranching registry, which includes 163 thoracoabdominal aortic aneurysms (6% type i, 25% type ii, 31% type iii, and 31% type iv) the inflow was based on the iliac arteries in 63% of cases, the aorta or an aortic graft in 29% of cases, and a hepatic / splenic artery in 8% of cases. the extent of visceral reconstruction included one or two vessels in 28% of cases and three or four vessels in 72% of cases. the 30-day or in - hospital mortality rate for hybrid thoracoabdominal aortic aneurysm repair was 16%. the visceral debranching operation is not a simple procedure because a meticulous approach to the abdominal visceral branches with extensive dissection in multiple abdominal areas and careful design of the grafts without kinking or redundancy to prevent organ malperfusion are required. in our case, we did experience a prolonged operation time and an increased amount of transfusions due to multiple anastomoses. the question of whether hybrid thoracoabdominal aortic aneurysm repair leads to improved outcomes compared to conventional thoracoabdominal aortic aneurysm repair is still controversial, and the issue requires larger studies and long - term follow - up. this hybrid procedure does not replace conventional repair strategies, but may provide alternative options for high - risk patients who might otherwise be denied therapy for a potentially fatal aortic rupture.	type ii chronic dissecting thoracoabdominal aortic aneurysms are a surgically challenging disease. the conventional thoracoabdominal aortic aneurysm repair technique using cardiopulmonary bypass is a high - risk procedure. however, a recently developed endovascular technique may be an alternative treatment for the disease, but faces the obstacle of lesional restriction. this new technique uses a hybrid strategy to overcome the limits of endovascular thoracoabdominal aortic aneurysm repair. herein, we report on a successful outcome after performing the hybrid visceral debranching procedure.
dopamine supersensitivity psychosis (dsp) is a type of acute exacerbation of recurrent psychosis in schizophrenic patients with excessive sensitivity to dopamine, caused by long - term treatment with antipsychotics.1) furthermore, it has been estimated that more than half the cases of treatment - resistant schizophrenia might be related to dsp.2) because dsp is associated with tolerance development for antipsychotics and/or vulnerability to minor stress,1) it is exceedingly troublesome for clinicians. nevertheless, no effective treatment has yet been established for dsp. on the other hand, some clinical studies on the treatment of patients with schizophrenia have suggested that aripiprazole, one of the atypical antipsychotics, might lower the risk of relapse and prevent the exacerbation of psychotic symptoms more efficiently than other antipsychotics.3,4) furthermore, in a previous study where we used an animal model of dsp, results suggested that aripiprazole may have not only preventive but also therapeutic effects on dsp through the reduction of excessive sensitivity to dopamine.5) based on these findings, we hypothesize that aripiprazole may reduce the exacerbation of recurrent psychotic episodes in schizophrenic patients with dsp. here incidentally, informed consent was obtained from the patient for publication of this case report. the patient is a 46-year - old married japanese woman with a long history of schizophrenia. her psychotic symptoms, which include delusions of persecution and thought broadcasting, first appeared at age 35 years. at the time, she underwent ambulatory treatment with 4-mg risperidone, and as a result did not experience another psychotic episode for about 10 years. nevertheless, one year ago, her first recurrence of a psychotic episode was triggered by a trifling family problem, and her psychotic symptoms increased in severity to the point that she required hospitalization. on this occasion, an additional dose of risperidone and doses of other antipsychotics barely cured her symptoms, and she was discharged from the hospital after two months. three months later, however, her psychotic symptoms relapsed without any apparent trigger, and this time, neither the maximum dose of risperidone nor other antipsychotics were able to control her psychotic episodes. therefore, she was admitted to chiba university hospital for specialized treatment. after admission, she was mainly treated with a high dose (24 mg or more) of aripiprazole, together with a variable dose of olanzapine (510 mg) or quetiapine (200700 mg) for a short period of time. over approximately two months, similar episodes of acute exacerbation of psychotic symptoms were repeated and continued for about a week each time. three months after the start of sustained treatment with the high dose of aripiprazole, however, the duration of exacerbation episodes became shorter, while the severity of her psychotic symptoms were gradually alleviated. six months after the start of the treatment, episodes of exacerbation ended within half a day, and her psychotic symptoms became insignificant. finally, ten months after the start of treatment, she was discharged from our hospital without any significant psychotic episodes. in order to quantify the severity of acute exacerbation episodes, we used the excited component of the positive and negative syndrome scale (panss - ec).6) we defined the acute exacerbation severity (aes) score as the panss - ec score multiplied by the duration of the episode in 0.5-day units, and examined the aes score of each episode. we recorded the aes scores of the patient during the 305 days of hospital treatment (fig. as can be seen from the figure, the aes score of each episode decreased along with the time course of sustained treatment with aripiprazole. we think that this patient suffered from dsp caused by long - term treatment with risperidone. therefore, she had repeated similar episodes of acute exacerbation without any apparent trigger, owing to her excessive dopamine sensitivity. we consider that sustained treatment with a high dose of aripiprazole gradually reduced the severity of her recurrent psychotic episodes through the reduction of this excessive sensitivity to dopamine. it is known that chronic treatment with d2 dopamine receptor antagonists, such as haloperidol, up - regulates the density of d2 receptors, and leads to dsp.7) based on the results of our animal study and our theoretical findings, we think that aripiprazole can down - regulate the density of d2 receptors and ameliorate dsp, as a result of its partial - agonistic effects on the d2 receptors.8) therefore, we believe that sustained treatment with a high dose of aripiprazole might have down - regulated the density of the patient s d2 receptors, and as a result, reduced the exacerbation of her recurrent psychotic episodes. because aripiprazole yields excessive dopaminergic neurotransmission in patients with dsp due to increased d2 receptor density, a transient worsening of psychosis can appear following the switch to aripirazole.5,9) in fact, the results of a clinical survey suggested that patients with dsp were more likely to experience psychotic worsening following the switch to aripiprazole, especially when the patients had undergone relatively slow titration, i.e., a low initial dose with a gradual increase of aripiprazole.10) however, our patient had undergone a quick titration of aripiprazole. we therefore suspect that the excessive dopaminergic neurotransmission can gradually down - regulate the density of a patient s d2 receptors due to compensatory systems induced over the time course of treatment, without causing any transient worsening of psychosis. in this case, sustained treatment with aripiprazole may have reduced the exacerbation of recurrent psychotic episodes in a schizophrenic patient with dsp. however, well - designed, randomized, double blind, placebo - controlled studies using a large sample would be needed to confirm this.	dopamine supersensitivity psychosis (dsp) is a type of acute exacerbation of recurrent psychosis caused by long - term treatment with antipsychotics in schizophrenic patients. although dsp is exceedingly troublesome for clinicians, effective treatment has not yet been established. based on clinical research and our animal study, we hypothesize that aripiprazole, an atypical anti - psychotic, may reduce the exacerbation of recurrent psychotic episodes. we report the case of a 46-year - old female who suffered from schizophrenia with dsp. in this case, sustained treatment with a high dose of aripiprazole gradually reduced the severity of her recurrent psychotic episodes. in conclusion, sustained treatment with aripiprazole may reduce the exacerbation of recurrent psychotic episodes in schizophrenic patients with dsp, and may be an effective treatment of dsp.
the renormalization group (rg ; wilson, 1971 ; wilson and fisher, 1972) is by now a method found in any classical statistical physics text book (goldenfeld, 1992 ; plischke and bergersen, 1994). it has allowed to categorize broad classes of equilibrium systems into an enumerable set of universality classes, each characterized by discrete features, such as their dimension and the symmetries adhered to by their hamiltonians (goldenfeld, 1992 ; plischke and bergersen, 1994). such universality is made possible through the property of scaling that is an inherent feature near phase transitions (kadanoff, 1966), which these systems undergo in certain regions of the space spanned by their physical parameters (couplings). scaling invariance entails that system - specific details on the microscopic level become irrelevant, as the behavior over many orders in the range of the interactions become self - similar. in this framework, analogous behavior in a surprisingly wide set of phenomena, such as the condensation of fluids, spontaneous magnetization of materials, or the generation of particle mass in the early universe, can be described in a single effective theory ; certainly a major intellectual accomplishment of modern physics across all fields (goldenfeld, 1992). in the past 15 years, statistical physicist have increasingly applied the ideas of critical phenomena and scaling to problems outside of the immediate material realm, in newly emerging fields such as econophysics, sociophysics, etc.. the considered systems typically feature a large number of interacting agents sharing a finite set of intrinsic properties on account of which they interact. but unlike in a euclidean defined arrangement of actors in a physical system, such as atoms in a material, these systems possess a more complex network of mutual interactions (which may even be directed ; watts and strogatz, 1998 ; boccaletti. thus, in many respects, the study of these phenomena is inseparable from the understanding of the geometry of networks (barthelemy, 2011). one major accomplishment of these investigations is the realization that many of the networks that are engineered by some natural or human activity themselves exhibit emergent complex properties, for instance, as found in the scale - free degree distribution of the internet. but while these networks, or dynamical systems on them, may behave critically, many of these phenomena were soon found to be non - universal. for instance, in the preferential attachment model for the world - wide net (barabasi and albert, 1999), the value of the scaling exponent is tied to microscopic details of the attachment rule. in this sense, it would seem unlikely that any sweeping classification scheme could be devised to categorize this amorphous pile of particulars. here, we will attempt a foray into such a scheme, albeit limited to those classical equilibrium phenomena, but on a large set of different networks. we suspect that our discussion might help to explain, for example, a number of similar observations of traditionally obscure critical behaviors, such as infinite - order transitions, in very different network models, in and out of equilibrium (dorogovtsev., 2008). our classification scheme is best introduced with a variety of hierarchical networks on which rg is exact, and the critical phenomena can be studied in great detail. metric version of such networks, such as that introduced in the migdal - kadanoff rg, provide the classical text - book examples of rg and universality (berker and ostlund, 1979 ; goldenfeld, 1992 ; plischke and bergersen, 1994 ; hinczewski and berker, 2006). but in the advent of complex networks, many hierarchical designs with non - metric (i.e., small - world or scale - free) properties have been devised and studied (andrade., 2005 ; hinczewski and berker, 2006 ; hinczewski, 2007 ; boettcher and goncalves, 2008 ; boettcher., 2008) our study shows that criticality in many of these models (percolation, ising, etc.) is definitely non - universal but falls into a few (here, three) generic regimes, each characterized by its degree of singularity in, say, the correlation length at the critical point (boettcher and brunson, 2011). one of these regimes is indeed an infinite - order transition reminiscent of that described by berezinskii and kosterlitz and thouless (bkt ; goldenfeld, 1992 ; plischke and bergersen, 1994). it is flanked on one side by a transition with a weaker, algebraic divergence, similar to the classical ones of second order (but still non - universal), and on the other by a regime with a fully essential singularity. these regimes are defined through the relative strength of (non - euclidean) long - range or small - world bonds in one and the same network, with clear demarcations between these regimes as a function of that coupling strength. in the following section 2, the analysis of the phase diagrams and the rg flow for the ising ferromagnet on these networks is discussed in sections 3 and 4. in section 5, we introduce families of interpolating networks to reveal a more comprehensive set of regimes, each with its own characteristic type of phase transition, and we conclude with a discussion of our results in their implications in section 7. each of the hanoi network possesses a simple geometric backbone, a one - dimensional line of sites n, 0 n n = 2 (k). each site is connected to its nearest neighbor, ensuring the existence of the 1d - backbone. to generate the small - world hierarchy in these networks, consider parameterizing any integer n (except for zero) uniquely in terms of two other integers (i, j), i 0, via here, i denotes the level in the hierarchy whereas j 0 labels consecutive sites within each hierarchy. for instance, i = 0 refers to all odd integers, i = 1 to all integers once divisible by 2 (i.e., 2, 6, 10,), and so on. depending on its level of the hierarchy, any site has also small - world (i.e., long - range) bonds to more - distant sites along the backbone, according to some deterministic rule. for example, we obtain a 3-regular network hn3 by connecting also 1 to 3, 5 to 7, 9 to 11, etc., for i = 0, next 2 to 6, 10 to 14, etc., for i = 1, and 4 to 12, 20 to 28, etc., for i = 2, and so on, as depicted in figure 1. depiction of the 3-regular network hn3 on a semi - infinite line, here drawn suggestively as a model for hierarchically organized transport. many goods and services are distributed in a small - world hierarchical manner before they get delivered in a real - world geometry. while hn3 (and hn4 boettcher., 2008) are of a fixed, finite degree, we introduced here convenient generalizations of hn3 that lead to new, revealing insights into small - world phenomena. first, we can extend hn3 in the following manner to obtain a new planar network of average degree 5, hence called hn5 : in addition to the bonds in hn3, in hn5 we also connect all even sites to both nearest sites within the same level of the hierarchy i(1). the resulting network remains planar but now sites have a hierarchy - dependent degree, as shown in figure 2. it is easy to show that the average shortest path between any two sites increases n in hn3, and logarithmically in hn5, with system size n. depiction of the planar network hn5, comprised of hn3 (black lines) with the addition of further long - range bonds (green - shaded lines). note that sites on the lowest level of the hierarchy have degree 3, then degree 5, 7, etc., of all sites, which makes for an average degree 5 in this network. the rg consists of recursively tracing out spins level - by - level in the hierarchy (boettcher., 2008). (1), we start by tracing out all sites with n odd, i.e., i = 0, then those n which are divisible by 2 only once, i.e., i = 1, and so on. we can always relabel all sites n after any rg step by n n/2, so that we trace out the respective odd - relabeled sites at any level. it is apparent, for instance from figure 1, that odd - labeled sites are connected to their even - labeled nearest neighbors on the backbone, say, by a coupling k0(=j0). at any level, each odd - labeled site xn 1 is also connected to one other such site xn1 across an even - labeled site xn with n = 2(2j + 1) that is exactly once divisible by 2. the basic rg step is depicted in figure 3 and consists of tracing out the two sites xn 1 neighboring the site xn for all j with n the step consists of tracing out odd - labeled variables xn 1 in the top plot and expressing the renormalized couplings (l,k0) on the bottom in terms of the old couplings (l, k0, k1). note that the original network in figure 1 does not contain couplings of type (l, l), but that they certainly become relevant during the process. we can section the ising hamiltonian where contains all coupling terms of higher level in the hierarchy, and each sectional hamiltonian is given by where (k0, k1, l) are the unrenormalized couplings defined in figure 3 and i is a constant that fixes the overall energy scale per spin. (there are effectively 4 spins involved in each graph - let, as those at each boundary are equally shared with neighboring graph - lets.) while couplings of the type l0 between next - nearest even - labeled neighbors emerges that are not part of the network initially in hn3, they do emerge during the rg step (otherwise the system of recursion equations would not close), see figure 3. to simplify the analysis, (plischke and bergersen, 1994), which ensure that the rg flow only contains algebraic functions and, for the ferromagnetic model, remains confined within the physical domain 0,, 1. (3) as tracing out the odd - labeled spins, we have to evaluate for the remaining spins in terms of the renormalized quantities c,,. of the eight possible relations resulting from the combinations xn 2, xn, xn + 2 = 1, only three are independent. after some algebra, we extract from those the rg recursions : note that for couplings in higher levels of the hierarchy it is ki=ki+1 for i 1 ; correspondingly, these couplings, and hence,, will not renormalize. instead, they retain their bare value determined by the temperature, kt / j = 2/ ln. in this sense, we will use as a measure of temperature throughout. only half of the contribution to the renormalized energy scale is originating with the sectional hamiltonian in eq. (5), since at the next level two such sections are combined into one, making c c. while we do not consider the recursions for c in this paper, they are essential to reconstruct the free energy for each system, and will be analyzed elsewhere (boettcher and brunson, unpublished). equation (7) provide recursions order - by - order in the rg for the evolution of the effective couplings characterizing increasingly larger scales of the network. to facilitate this rg flow, we need to specify initial conditions for a particular physical situation realized in the unrenormalized, bare network. here, we restrict ourselves to networks with uniform bonds (although many interesting choices are conceivable, such as distance - dependence ; kotliar., 1983 ; katzgraber, 2003 ; hinczewski and berker, 2006). for hn3 this implies that we chose j = 1 as our energy scale, such that ki = j = and i = l0 = 0 initially, or in terms of eq. (4) ; searching for fixed points k0=k0=k0 and l = l = l, i.e., = = and = = in eq. (7), immediately provides the trivial, high - temperature solution = = 1, i.e., k0=l=0. further analysis yields only a line of (unstable) strong - coupling fixed points, extending from =12 for low temperatures, = 0, to = 1 for t, where = 1. even at t = 0, only the renormalized backbone bonds k0 provide strong coupling, the emerging long - range bonds l0 only exert limited coupling strength. local analysis near the fixed points with the ansatz reveals that the high - temperature fixed point is always stable and corrections decay exponentially, where the exponential contains a factor of 2n2=n. at the low - temperature line of fixed points in eq. (9) we find which is divergent for all t > 0, i.e., 0 < 1, making the fixed point at t = 0 unstable. for any fixed point, there is no linear expansion possible that would yield critical exponents. (8), corresponding to uniform couplings throughout the unrenormalized network, the rg flow always evolves to the high - temperature fixed point. as shown in section 2, hn5 is basically an extension of hn3, created by adding a new layer of links to each level of the hierarchy. as is apparent from the foregoing discussion in 3, these additions correspond precisely to new renormalizable operators (here, the bonds l) that inevitably emerge during the rg of hn3, see figure 3. in hn5, these new operators are simply deemed an original feature of the network, hence, maintaining the rg as an exact procedure. consequently, the rg itself hardly changes, see figure 4 ; it merely differs by one extra link in the graph - let, l1, compared to that for hn3 in figure 3. in eq. 2xn + 2 to the sectional hamiltonian and, like l0 itself, l1 does not get traced over in the calculation in eq. (6). we can introduce these new bonds as yet another free, non - renormalizing coupling in the rg and choose, to wit, depiction of the (exact) rg step for the ising model on hn5. this step is identical to that for hn3 in figure 3 aside from the extra link l1 spanning between xn 2 and xn + 2 (top), which contributes to the renormalization of l0 (bottom). (7) by a factor of. otherwise using the same definitions as in section 3, we obtain the rg recursions for the ising ferromagnet on hn5 : accordingly, due to the bare existence of the l1 bond, we will have to change the initial conditions from eq. (8) to analyzing these recursions for fixed points, = = and = =, we find that the addition of the extra long - range bond has eliminated the high - temperature fixed point found in hn3. at low temperatures, (9) in hn3 a line of fixed points which here extends over the entire domain for the long - range bonds, 0 1 for 0 1. note that although y represents a continuous interpolation between hn3 and hn5, there is a singular limit at y 0 toward an isolated point corresponding to hn3, see eq. we only treat the case of couplings that are homogeneous throughout the unrenormalized network, y = 1. consideration of the rich set of transitions occurring for the family of networks parameterized by interpolating 0 < y 1 is deferred to section 5. dividing out the = 0-solution, further analysis of eq. (13) for y = 1 reveals yet another line of fixed points given by which can be expressed most simply in closed form as by eliminating. as we will see, these relations lead to physical fixed points only within a limited range of the temperature. the phase diagram for the backbone coupling in hn5 at y = 1 can be found in figure 5. (14), which starts on the diagonal, representing all - equal bonds for the homogeneous network. for these initial conditions, the flow always evolves toward smaller values of, i.e., stronger coupling. but there is a notable transition where the attained fixed - point jumps from the low - temperature branch in eq. (15) characterized by = 0, i.e., a solidly frozen backbone, to the branch given eq. (17) on which (as well as) becomes finite. (16, 17) for = 0, which yields = 1/2 = 0.309017, giving a critical temperature of plot of in eq. (19) as a function of for y as defined in eq. the generic cases are represented by y = 0.2 (top), y = 0.45 (middle), and the case of hn5 discussed in section 4 for y = 1 (bottom), for which the branch - point (bp) has the dashed, green - shaded line indicates the initial conditions (ic) for the rg flow in eqs. (13, 14), =. for fixed, the rg flow must proceed vertically, either up or down, to the nearest stable line of fixed points. for low y (top), the ic crosses the unstable branch below bp (see enlargement in the inset) which then can not be reached by the flow. once the ic cross above bp (middle), the flow must pass bp, unless bp sunsets (bottom). for each case, the generic divergence is indicated for the correlation length at t tc, as derived in section 6. where =5 + 12=1.6180339887 is the golden ratio (boettcher and goncalves, 2008). for bare couplings at this temperature, marked by a blue dot in the (y = 1)-plot of figure 5, the rg flow marginally reaches the strong - coupling limit. in this network, for these initial conditions the rg flow never reaches an unstable fixed point such as the unstable portion of eq. (15), marked by a red - shaded line in figure 5. as we will see below, this situation will change when we weaken the impact of long - range couplings. we have already observed in the construction of hn5 in section 4 that it is easy to promote the l - couplings that inevitably emerge during the rg to be associated with an actual bond in the network. here, we will fully exploit this fact to obtain a one - parameter family of problems with various regimes of phase behaviors. in particular, we discover transitions between such regimes as a function of the parameter that will allow us to clarify the connections between the diverse set of behaviors that we have discovered in the previous section. in section 4, we argued for the introduction of small - world bonds with couplings li and developed the rg recursions in (13) assuming a relative strength of these couplings to those germane to hn3 of the form in eq., we will now consider the behavior that results from varying the strength parameter y between the two extremes already explored, y = 0 for hn3 in section 3 and y = 1 for hn5 in section 4. (13) for fixed points, we already found the low - temperature fixed - point line in eq. (15). dividing out this = 0-solution, further analysis of eq. (13) reveals a line of fixed points, abbreviating the discriminant for y 0, this solution degenerates into the high - temperature fixed point of hn3. but for any finite y, these lines of fixed points are non - trivial functions of, as depicted for () in figure 5. the dominant feature in these plots is the root - singularity in with a branch - point - separating the upper stable and lower unstable line of fixed points. essentially, three distinct generic regimes can be discerned : (1) if the branch - point happens to lie below of the physical regime, we observe a phase transition without access to any unstable point (see bottom of figure 5) ; a critical point akin to that for hn5 at y = 1 analyzed in section 4 arises. if the branch - point rises into the physical regime, here for y < yc = [ln(3/2)/ln2 ] = 0.584963, then depending on whether the initial conditions of the rg flow cross the critical line below or above the branch - point, we find (2) a transition seemingly of finite - order on intercepting the unstable lower branch (see top of figure 5) for which the rg flow never accesses the branch - point singularity. if, in turn, the initial conditions cross above, (3) a bkt - like transition results because the rg flow now must pass the singularity (see middle of figure 5), as we will show below. (19) for a coupled set of variables (which can be found in boettcher and brunson, 2011), we rather take a step back and assess the larger picture here. it turns out to be easy to devise a simple theory that reproduces all the previously found features in a generic way, thereby demonstrating the generality of this classification, not only accounting for other hierarchical networks (andrade., 2005 ; hinczewski and berker, 2006 ; hinczewski, 2007 ; boettcher and goncalves, 2008 ; boettcher., 2008) but also for any physical system described by parameter - dependent renormalization group equations. even systems on complex networks that have not been subjected to an rg treatment have been found to exhibit the peculiar infinite - order transitions found here (dorogovtsev., 2008), and may eventually be related to this classification. it is sufficient to consider the rg recursion for a single coupling, say, n with some control parameter, defined as in eq. ; plischke and bergersen, 1994) for a system on a regular lattice leads to recursions that only depend on the evolving coupling itself. there, any parameter dependence, such as on the temperature for spin models or on the bond density for percolation problems, is limited to the initial conditions of the rg, which define the particular model being studied ; they do not affect the properties near the fixed points. in contrast, inserting such a dependence influences the location of fixed points as well as the behavior near them. as the example displayed in figure 5 suggests, even the very fixed point that controls the dynamics may depend on the initial conditions, violating any conventional sense of universality. yet, if we assume that the most elementary, generic fixed - point topology that deviates from the conventional picture is represented by a root branch - point, we can classify all observed critical phenomena into just a few regimes. to wit, we write this model of a generic rg recursion is cubic to ensure that, after extracting the trivial strong - coupling fixed point 0, the remaining fixed - point equations produce a root branch - point, revealing the undetermined constant b as the coupling strength found at the branch - point, which we may choose freely to place the branch - point inside (0 b 1) or outside the physical regime. (regarding section 5, we could view b = b(y) as the quantity that specifies the family of models considered.) the function f () captures the minimal parameter dependence of the model, as expressed through the -dependence of both branches of the fixed points. here, f () is a monotone rising function on the physical interval 0 1, which may contain a zero, f (b) = 0. generically, it would have a simple taylor expansion near b, i.e., f (b) f(c) for small. in light of figure 5, the first two panels correspond to the case where both, b and b, are in the physical regime with a visible branch - point (although the rg recursions there are far more complicated) ; the last panel represents b 0. the decisive difference between those first two panels is whether the location of the branch - point is above or below the line of initial conditions. depending on the model, the line of initial conditions could be any monotone function of, possibly resulting in different critical behaviors in the way they pass by identical branch - points, but it is more convenient to imagine this line as a simple diagonal in the (,)-plane and move the branch - point instead. within the physical regime, the lower fixed - point branch -() is always unstable while the upper +() remains stable, as a local analysis along each branch readily reveals. stable and unstable fixed - point lines merge at the branch - point, where particularly interesting phenomena can arise. in the case that long - range, hierarchical effects are weakest, as for the first panel in figure 5, the branch - point is far on the right, and may even be outside to the right and/or above of the physical regime. then, the initial conditions merely intersect the unstable branch -() at some point c. the rg flow (vertical blue arrows in figure 5) for 0 < c advances toward strong coupling at 0, while for c < 1 it flows toward +(), making c the critical point. note that +()1 only for 1, reflecting the physical phenomenon of patchiness (boettcher., 2009) : hierarchical, long - range couplings enforce some semblance of order between otherwise uncorrelated (sub - extensive) patches of locally connected degrees of freedom even in the disordered regime ; full disorder is often only reached at infinite temperature, dilution, etc. near c, all the critical dynamics of the system is then solely determined by the local properties of the unstable critical point -(c) that has be (non - universally) selected by the particular system via the initial conditions. as in a conventional system, local analysis (goldenfeld, 1992 ; plischke and bergersen, 1994) similar to eq. (10) but near -(c) with an ansatz n-+n for n 1 on eq. (21) provides, e.g., the scaling exponent for the divergence of the correlation length, except that the exponent is non - universal, =(c)=1log2(1 + 2-f) evaluated at the crossing point c (boettcher and brunson, 2011). the rg flow in this case never passes sufficiently near the branch - point singularity to be affected. the other extreme, when long - range couplings dominate, leads to a picture similar to the last panel of figure 5 but with b < 0. no unstable fixed points can be reached for any choice of (physical) initial conditions. the rg flow always advances to the next best stable fixed point, either at strong coupling 0 for 0 < c, or toward patchy order at +() for c < 1, making c again the critical point. at c, where both lines of stable fixed - points intersect, we find an exponentially divergent correlation length to signal the phase transition. (22) near +(c)=0, we get thus, the correlation length diverges as again, the rg flow does not pass the branch - point singularity, as it is located outside (below) the physical domain. only in the intermediate regime, as represented by the middle panel of figure 5, does the rg flow for some critical c pass by the branch - point singularity, which then controls the critical behavior in a novel way. as before, for 0 < c the flow reaches strong coupling at 0 and patchy order at +() for c < 1. a local analysis of the flow just below the critical point, c, with n b(1 + n) for n 1 on eq. (21) yields this relation exhibits a boundary layer, i.e., in the limit 0 the solution drastically changes behavior. with the methods of bender and orszag (1978), we can transform into the inner boundary region by rescaling n n and n n applied to eq. (27), which becomes balanced for 11. accordingly, the characteristic width of the boundary layer scales with which by eq. (26) leads to the divergence in the correlation length characteristic of bkt, clearly, the physical origin of this singularity is not even remotely related to an actual bkt transition. in fact, instead of its rarity, confined to very particular lattice models, we may find it to be one of a few generic types of transition in networks. we have analyzed the fixed - point structure of an ising ferromagnet on a set of hanoi networks with an exact real - space renormalization group. using interpolating families of such networks, with the relative coupling strength between backbone and small - world bonds as the interpolation parameter, we reveal a number of regimes with distinct critical behaviors. while in each such regime the critical transition has non - universal features, the characteristics of the transition in each one has generic, robust features. for increasing strength, we observe that the divergence in the correlation length changes from a non - universal power - law \|x\|, to a bkt - like essential singularity e1x, then to a full singularity e, on approach to the critical point x \|c \| 0. we trace the changes from one regime to the next in terms of the analytic structure of the rg flow. finding an enumerable range of such characteristics suggest a possible classification of critical behavior of statistical models in networks generally, for which we propose a general description. similar critical properties of the kind found here have also been observed in percolation (berker., 2009 ; boettcher., 2009 ; nogawa and hasegawa, 2009 ; hasegawa., the existence of entire regimes that exhibit essential singularities in the divergence of the correlations, as we have found here, might explain the surprising prevalence of typically quite rare bkt - like transitions in otherwise unrelated network models (dorogovtsev., 2008). in the analysis of, say, social interaction networks, which have been found to have a hierarchical structure (trusina the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.	we discuss the behavior of statistical models on a novel class of complex hanoi networks. such modeling is often the cornerstone for the understanding of many dynamical processes in complex networks. hanoi networks are special because they integrate small - world hierarchies common to many social and economical structures with the inevitable geometry of the real world these structures exist in. in addition, their design allows exact results to be obtained with the venerable renormalization group (rg). our treatment will provide a detailed, pedagogical introduction to rg. in particular, we will study the ising model with rg, for which the fixed points are determined and the rg flow is analyzed. we show that the small - world bonds result in non - universal behavior. it is shown that a diversity of different behaviors can be observed with seemingly small changes in the structure of hierarchical networks generally, and we provide a general theory to describe our findings.
amyotrophic lateral sclerosis (als) is the most common degenerative disease of the motor neuron system. it is estimated that at least 3,500 patients and 1,000 new cases per year (http://www.osservatoriomalattierare.it/sla). als is characterized by relentless progression of muscle wasting and weakness until death ensues typically due to respiratory muscle failure. generally, als patients present a number of clinical symptoms, including weakness, spasticity, cachexia, dysarthria and drooling, and pain secondary to immobility, among others (zarei., 2015). the most abundant forms of als are sporadic (90%), but the disease may be also familiar (10%), associated with mutations in the superoxide dismutase-1 gene (sod-1), that encodes for a key antioxidant enzyme, and also in tar - dna binding protein-43 (tdp-43) and fus (fused in sarcoma) which encode proteins involved in pre - mrna splicing, transport and stability (hardiman., 2011). recently, mutation in non - coding hexanucleotide repeat sequence (ggggcc) in the c9orf72 gene was considered as the most common genetic cause of als (matamala., 2016). the exact function of this protein remains undefined ; however, it seems to play a major role in cellular trafficking, mainly in neurons (williams., 2013). the c9orf72 mutation was found also in frontotemporal dementia (ftd) patients (farg., 2014). since 20% of als patients develops dementia with a frontotemporal phenotype, this mutation may explain the link between familial ftd and als (farg., 2014). although the pathogenic mechanisms that underlie als are yet unknown, it is believed that als could have a multifactorial etiology, where environmental factors can greatly contribute to pathology triggering. moreover, several mechanisms including mitochondrial dysfunction, protein aggregation, oxidative stress, excessive glutamate activity, inflammation and apoptosis are involved in als pathogenesis leading to motor neuron cell death in the brain and spinal cord (zarei., 2015). to date, the only therapy available for als is the glutamate - antagonist riluzole that was able to inhibit the presynaptic release of glutamate, most likely by blockade of voltage - gated sodium channels. however, riluzole has limited therapeutic efficacy and also it is able to moderately prolong patient survival (miller., 2007). therefore, new innovative and safer therapeutic approaches are urgently needed, at least aimed at delaying the neurodegenerative processes of the ongoing disease. over the last years, a growing interest has been focused to cannabinoids, the bioactive compounds of cannabis sativa, for their antioxidant, anti - inflammatory and anti - excitotoxic effects exhibited in preclinical models of central nervous system disease (croxford, 2003). here, we provided an overview of the potential usefulness of cannabinoid agents in the management of als. the cannabis plant, also known as marijuana, contains over 500 natural compounds and about 70 of these are classified as cannabinoids (fischedick., 2009). the discovery of - tetrahydrocannabinol (thc) as the major psychoactive principle in cannabis, as well as the identification of numerous non - psychoactive cannabinoids such as cannabidiol (cbd), cannabigerol (cbg), cannabinol (cbn), cannabichromene (cbc),-tetrahydrocannabivarin (-thcv) and cannabidivarin (cbdv), has led to a significant growth in research aimed at understanding the therapeutic effects of these compounds. cannabinoids exert many of their activities by binding cannabinoid (cb) receptors. to date, two types of receptors have been identified to have different tissue distribution and mechanisms of signaling. cb1 receptors are expressed mainly on neurons and glial cells in various parts of the brain, cb2 receptors are found predominantly in the cells of immune system. both cb1 and cb2 receptors belong to the family of g - protein coupled receptors (gpcrs) that, after cannabinoid agonist binding and signaling, exert an inhibitory effect on adenylate cyclaseactivity, activation of mitogen - activated protein kinase, regulation of calcium and potassium channels, and other signal transduction pathways (munro., 1993). moreover, there is increasing evidence supporting the existence of additional cannabinoid receptors (no - cb1 and no - cb2) in both central and peripheral system, identified in cb1 and cb2-knockout mice, involving intracellular pathways that play a key role in neuronal physiology. this kind of receptors includes transient receptor potential vanilloid type 1 (trpv1), g protein - coupled receptor 55 (gpr55), g protein - coupled receptor 18 (gpr18), g protein - coupled receptor 119 (gpr119) and 5-hydroxytryptamine receptor subtype 1a (5-ht1a) (pertwee., 2010).-thc, of which is well - known psychotropic effects, is believed to perform the majority of itsactions in the cns binding cb1 and cb2 receptors. non - psychotrophic phytocannabinoids exert multiple pharmacological effects via cb1/cb2 receptors as well as no - cb1 and no - cb2 receptors (pertwee., 2010). overall, recent studies showed that cannabinoids inhibit the release of pro - inflammatory cytokines and chemokine in neurological preclinical models suppressing in this way the inflammatory response (velayudhan., 2014). they show also a potent action in inhibiting oxidative and nitrosative stress, modulating the expression of inducibile nitric oxide synthase and reducing the production of reactive oxygen species (ros) (velayudhan. moreover, cannabinoids were found to exert anti - glutamatergic action by inhibiting glutamate release and enhancing the effect of the inhibitory neurotransmitter gamma - aminobutyric acid (gaba) (croxford, 2003). just about all these properties exhibited by these compounds, have prompted researchers to investigate their potential therapeutic effects in als, providing interesting results. recent in vivo studies support that cannabinoids may be beneficial as neuroprotective agents in als. the most commonly used murine model for human als is the hsod (g93a) transgenic mouse, which is genetically engineered to develop clinical symptoms similar to those observed in humans with als. treatment with 9-thc in als hsod(g93a) mice, either before or after signs onset, improves motor impairment and increases survival by 5% probably via its anti - glutamatergic and anti - oxidant activity (raman., moreover, it was demonstrated that - thc attenuates oxidative stress in als hsod(g93a) mouse spinal cord primary cultures, that were exposed to the oxidant tert - butyl hydroperoxide (tbh) in the presence of - thc and sr141716a, the cb1 receptor antagonist, as assessed by lactate dehydrogenase (ldh) and sod-1 release. specifically, the antioxidant effect of - thc was not cb1-receptor mediated ; since the cb1 receptor antagonist sr141716a did not diminish the antioxidant effect (raman., 2004).-thc was found also to protect against excitotoxicity produced by kainic acid in primary neuronal cultures, obtained from als hsod(g93a) mouse spinal cord, by activation of cb1 receptor. in this case, the neuroprotective effect was blocked with the cb1 receptor antagonist, sr141716a, indicating a receptor - mediated effect (raman., 2004). therefore, treatment with cannabinoids may reduce elevated glutamate levels observed during als by modulating excitotoxicity events. moreover, treatment with cannabinol (cbn), a non - psychotropic cannabinoid, through its residual affinity to cb1 receptors, is able to delay significantly disease onset in als hsod(g93a) mice subcutaneously implanted with osmotic mini - pumps. likewise, a significant delay in disease progression was found when cb1/cb2 receptor agonist win 55,212 - 2 was intraperitoneally administered to als hsod(g93a) mice beginning after onset of motor impairment and tremor (at 90 days old), however, survival was not extended (bilsland., 2006). genetic ablation of the fatty acid amide hydrolase (faah) enzyme, which results in raised levels of the endocannabinoid anandamide, prevented the appearance of disease signs in 90-day - old to als hsod(g93a) mice. however, elevation of cannabinoid levels with either win55, 212 - 2 or faah ablation had no effect on life span. on the contrary, cb1 deletion had no effects on disease onset in als hsod(g93a) mice, but extend lifespan by 15 days, a 13% increase in survival. therefore, the beneficial effects exhibited by cannabinoids may be mediated by non - cb1 receptors, but presumably by cb2 ones. moreover, the neuroprotective effects of cannabinoids were ascribed to a decrease of microglial activation, presynaptic glutamate release and formation of ros (bilsland., 2006). also, it was demonstrated that mrna, receptor binding and function of cb2, but not cb1, receptors are dramatically and selectively up - regulated in the spinal cords of als hsod(g93a) mice in a temporal pattern paralleling disease progression (shoemaker., 2007). it was found that daily intraperitoneal administration of the selective cb2 agonist, am-1241, initiated after disease onset in als hsod(g93a) mice, delayed motor impairment and increased survival by 56%. the beneficial effects of cannabinoids could potentially be mediated via cb2 receptor - mediated suppression of microglial / macrophage activation in the spinal cords of symptomatic g93a mice and that cb2 receptors are selectively up - regulated in spinal cords as a compensatory, protective measure (shoemaker., 2007). few years ago, the neuroprotective effects of a mixture of two extracts in approximately a 1:1 ratio (2.7 mg of - thc and 2.5 mg of cbd) commercially known as sativex were investigated by using als hsod(g93a) transgenic mice (moreno - martet., 2014). sativex was found to be effective in delaying als progression in the early stages of disease and in animal survival, although the efficacy was decreased during progression of disease. also, it has been demonstrated that changes occur in endocannabinoid signaling, particularly a marked up - regulation of cb2 receptors in sod(g93a) transgenic mice together with an increase of n - acyl phosphatidylethanolamine phospholipase d (nape - pld) enzyme, which is responsible for the generation of anandamide (n - arachidonoylethanolamine), the ligand of cannabinoid and vanilloid receptors (moreno - martet., 2014). therefore, the efficacy of cannabinoids in slowing als progression, in extending life expectancy and in reducing the overall gravity of the disease is mainly due to activation of cb2 receptors. more specifically, it was widely demonstrated that drugs activating cb2 receptors, expressed predominantly in immune cells and non - neuronal tissues, successfully improve the symptoms of several inflammatory diseases (walter and stella, 2004). however, further studies are necessary to assess the neuroprotective effects of cannabinoids that target cb2 receptors. molecular mechanisms underlying cannabinoids - driven neuroprotective effects in als hsod(g93a) mice model are illustrated in figure 1. schematic illustration of the neuroprotective mechanisms of action of cannabinoids into als hsod(g93a) mice. -thc : tetrahydrocannabinol ; als : amyotrophic lateral sclerosis ; cbd : cannabidiol ; cb1r : cannabinoid 1 receptor ; cbn : cannabinol. spinal cord from als patients demonstrate motor neurons damages marked by cb2-positive microglia / macrophages. moreover, a recent study analyzing activated microglia from spinal cord in human als patients demonstrated a cb2 receptors increase. so all these data show how editing cb2-mediated processes could change als progression and how much the endocannabinoid system is potentially involved in reducing neuro - inflammation, excitotoxicity, and oxidative cell damage (yiangou., 2006). the possibility that cannabinoids may provide therapeutic effects in als has been also investigated at the clinical level. however, the small number of people with als that reported using cannabis and the few studies performed on human als, makes difficult the interpretation of the achieved results. nevertheless, it is believed that cannabis could be useful in the symptomatic treatment of als. according to a single observational study of patients with als only the 10% who admitted consuming cannabis, revealed moderate relief of several symptoms, including appetite loss, depression, pain, and drooling was found (carter and rosen, 2001 ; amtmann., 2004). in addition, spasticity is also major problem for als patients, which reported that cannabis can subjectively improve spasticity (amtmann., 2004). moreover, a randomized, double - blind crossover study investigating the safety and tolerability of - thc in als patients revealed that oral - thc administration was well tolerated, but a non - significant attenuation of cramp frequency and intensity were found. other studies confirmed the same results, demonstrating that cannabis is remarkably safe with realistically no possibility of overdose. there are no clinical studies so far that have tried to prove the potential of cannabinoids as disease - modifying therapies as widely supported by experimental studies, so this hypothesis remains a major challenge for future research. in light of the above findings, there is a valid rationale to propose the use of cannabinoid compounds in the pharmacological management of als patients. however, most of the studies that investigated the neuroprotective potential of these compounds in als were performed in animal model, whereas the few clinical trials that investigated cannabinoids - based medicines were focused only on the alleviation of als - related symptoms, not on the control of disease progression. this remains the major challenge for the future and it may be facilitate by the recent approval of the first cannabinoid - based drug (sativex) available for clinical use. in the last years, a growing interest is focused on the combination drug approach with existing medications in order to maximize the therapeutic efficacy and minimize the adverse effects commonly observed with conventional therapies. we strongly hope to have provided a short but important overview of evidences that are useful to better characterize the efficacy as well as the molecular pathways modulated by cannabinoids. we hope that our studies could be an alert to encourage the scientific community to further studies to confirm the therapeutic use of cannabinoids in this devastating disease.	amyotrophic lateral sclerosis (als) is the most common degenerative disease of the motor neuron system. over the last years, a growing interest was aimed to discovery new innovative and safer therapeutic approaches in the als treatment. in this context, the bioactive compounds of cannabis sativa have shown antioxidant, anti - inflammatory and neuroprotective effects in preclinical models of central nervous system disease. however, most of the studies proving the ability of cannabinoids in delay disease progression and prolong survival in als were performed in animal model, whereas the few clinical trials that investigated cannabinoids - based medicines were focused only on the alleviation of als - related symptoms, not on the control of disease progression. the aim of this report was to provide a short but important overview of evidences that are useful to better characterize the efficacy as well as the molecular pathways modulated by cannabinoids.
	we previously reported that cell cultures of human proximal tubule (hpt) cells respond to ionic cadmium in a manner consistent with well - defined cd(2+)-elicited responses reported for in vivo systems. however, one unique finding was that the transepithelial electrical resistance and tight junction sealing strands were altered as a result of cd2 + exposure at micromolar concentrations. these alterations are reexamined in detail in the present report to determine whether the cd(2+)-induced alterations are specific alterations in the tight junction structure or reflect a general alteration in the cell membrane. exhaustive analysis of tight junction sealing strands demonstrated no significant alterations due to cd2 + exposure, even at the concentration that elicited a significant reduction in transepithelial resistance. further analysis of intramembrane particle distribution demonstrated a significant increase in apical intramembrane particles, indicating that cd2 + exposure altered the characteristics of the apical cell membrane. overall, the results were consistent with evidence of cd(2+)-induced alteration in the apical cell membrane of the hpt cell.imagesfigure 1.figure 2.figure 3. afigure 3. bfigure 3. cfigure 3. dfigure 4.figure 5.
a 37-year - old man was admitted to our hospital with long - standing, persistent, lone atrial fibrillation refractory to medical therapy and several attempts of cardioversion. the patient underwent a total thoracoscopic epicardial ablation without any intraoperative or postoperative complications, followed by a percutaneous postprocedural electrophysiologic evaluation that included confirmation of pulmonary vein isolation and a cavotricuspid isthmus block on the tenth postoperative day. two weeks after discharge, the patient was admitted to the emergency room with right - sided motor weakness and paresthesia of both upper and lower extremities. his fever reached 40c, and he had a white blood cell count of 17,690/l. chest computed tomography imaging revealed a collection of loculated air measuring up to 2 cm in diameter between the right side of the left atrium and the esophagus, with no definitive evidence of a fistulous connection (fig. 1). brain computed tomography revealed an acute - onset left middle cerebral artery infarction. an urgent surgical intervention was performed due to our high level of suspicion for a left atrial or pulmonary venous - esophageal fistula. the chest was opened through the fifth intercostal space via a right posterolateral thoracotomy under one - lung ventilation with a double lumen endotracheal tube. after anteriorly retracting the lung, a meticulous dissection of the posterior mediastinum between the esophagus, right upper and lower pulmonary veins, and left atrium was performed. a fistula 2 to 3 mm in diameter was discovered between the right lower pulmonary vein and anterior surface of the esophagus. surprisingly, there was no bleeding from the fistula on the side of the right lower pulmonary vein. the right lower pulmonary vein was repaired with a bovine pericardial patch, covered, and fixed with continuous poly - propylene sutures. after repairing the right lower pulmonary vein, we consulted our hospital s thoracic surgeon about repairing the fistula opening on the esophageal side. he decided to carry out a primary repair of this defect because the defect site was small and relatively clear. the fistula was carefully debrided and repaired with intermittent 4 - 0 black silk sutures. the patient s rhythm was converted to sinus rhythm and sustained after cardioversion one day after surgery. the patient became alert on the third postoperative day, and was extubated on the fifth postoperative day. he was discharged 39 days postoperatively, with a motor power score of three out of five on the medical research council scale after rehabilitation therapy. on his last outpatient visit, two and a half months after surgery, motor power was fully recovered without any sequelae. a hybrid procedure of a total thoracoscopic epicardial ablation and percutaneous transvenous radiofrequency catheter ablation for lone atrial fibrillation, performed either simultaneously as a single stage procedure or as a two - stage procedure, has been developed to overcome the limitations of each technique and result in better outcomes. the success rate of this therapy varies according to the specific goals of the intervention and the duration of atrial fibrillation (paroxysmal or persistent). pison. reported conversion to sinus rhythm after a single stage procedure with a success rate of 83% after one year. muneretto. reported conversion to sinus rhythm after a two - stage procedure with a success rate of 91.6% at a mean follow - up of 30 months. after being performed for the first time in south korea at the samsung medical center in february 2012, we have performed 50 staged hybrid procedures to date with a successful conversion to sinus rhythm in 48 out of 50 patients. unfortunately, we experienced a potentially fatal case of a fistula running from the pulmonary vein to the esophagus fistula despite the high success rate of conversion to sinus rhythm. we believe our case of a right lower pulmonary venous - esophageal fistula after thoracoscopic epicardial and transvenous catheter ablation to be the first reported in the literature. even though the radiofrequency catheter ablation was performed after the total thoracoscopic epicardial ablation in a staged procedure, we think that there may have been some degree of infection or edema at the ablation sites due to the short (ten days) interval between the components of the hybrid procedure. the most commonly reported esophageal complication after radiofrequency catheter ablation are left atrial - esophageal fistulas, with an incidence rate of up to 0.2%. these fistulas result from the usual anatomical relationship of the esophagus to the left atrium and pulmonary veins. however, unusual variations where the esophagus is situated rightward in the posterior mediastinum near the right pulmonary veins have been described, as in our present case.. a median sternotomy or a right or left thoracotomy with or without cardiopulmonary bypass is possible depending on the clinical situation and location of the fistula. surgical exposure in our case was achieved via a right posterolateral thoracotomy to expose the suspicious right pulmonary veins and left atrium. in light of this rare but potentially lethal event, we suggest the development of a better - defined consensus, based on further studies, on whether hybrid thoracoscopic epicardial and percutaneous catheter ablations should be performed simultaneously or as a staged procedure.	a case of a fistula running from the pulmonary vein to the esophagus after a staged hybrid procedure combining total thoracoscopic ablation and percutaneous radiofrequency catheter ablation has not been reported previously. we describe such a case in a 37-year - old man who was successfully treated by surgery.
a single - centered, retrospective study of 198 patients between 2005 and 2012 diagnosed with ie based on modified duke criteria. patients, required to be above age 18, undergone an echocardiogram study and had blood cultures to be included in the study. then we divided the patients into two groups : patients with bmi 25 kg / m and patients with bmi < 25 kg / m. the bmi or quetelet index is a value derived from the mass (weight) and height of an individual. the bmi is defined as the body mass divided by the square of the body height and is universally expressed in kg / m, resulting from mass in kilograms and height in meters. according to bmi scale, the optimal weight is below 25 kg / m and in our study the cutoff number is 25 kg / m. this study was conducted in a major hospital in one of the most diverse communities in the united states, providing a cultural and epidemiologically significant advantage. an approved chart analysis using quadramed computerized patient record (qcpr) among 198 patients, two echocardiographic groups were evaluated as 158 patients obtained a tte, 143 obtained a tee, and 103 overlapped with tee and tte ; 167 patients were included in the study as 109 (65%) were discovered to have native valve vegetations on tee and 58 (35%) with tte. tte findings were compared with tee results for true negatives and positives to isolate valvular vegetations. patients were further divided into two groups with the first group having a bmi 25 kg / m and the subsequent group with a bmi < 25 kg / m. patients with a bmi 25 kg / m that underwent a tte study had a sensitivity and specificity of 54 and 92%, respectively. on the contrary, patients with a bmi < 25 kg / m had a tte sensitivity and specificity of 78 and 95%, respectively. furthermore, we obtained the sensitivities of specific valves diseased from vegetations, visualized by tte (fig. finally, we were able to demonstrate a correlation between the different modalities of echocardiography used in the specific organism identified on blood cultures (fig. with an alarmingly high mortality rates of 1520% in hospitalized patients and a 1-year mortality rate of 40% (3), ie has become a life - threatening infection requiring prompt identification and management. the constant rising rates of ie in the united states have been determined between 2000 and 2011 with an incidence rate of 11 per 100,000 to 15 per 100,000 persons. it is speculated by sadip. that these rising rates may be partly due to the increasing number of invasive instrumentations and procedures performed (4). the combination of rising incidence and high mortality rates of ie postulates a concerning and devastating dilemma to health care. timely identification and diagnosis of ie is imperative to possibly reduce mortality rates and provide appropriate management. delay in diagnosis may possibly cause progressive and irreversible structural heart damage along with compromise to other organs. common complications, such as stroke, embolization, heart failure (hf), the need for surgery, and intracardiac abscess, pose possible debilitating outcomes for patients (3). in 2000, the modified duke criteria provided an important tool to help clinicians better guide the diagnosis of ie. a noteworthy modification to the duke criteria was eliminating echocardiographic findings from minor criteria and proposing it as major criteria with further detail to initial approach between tte and tee studies (5). tee has been well studied to be a highly specific and sensitive study for detection of valvular vegetations. microbiological identification in ie is imperative to identify and isolate the commonality of the organisms as it helps dictate broad spectrum treatment regimens. staphylococcus aureus has been the most common organism identified, among which approximately 30% of the patients had mrsa (methicillin resistant staphylococcus aureus). fowler. identified health care associated ie as the most common form of s. aureus with the highest in - hospital mortality rates (6, 7). similarly, we were able to demonstrate s. aureus as the most common causative organism of ie followed by group d streptococcus and streptococcus viridans. in addition, we illustrated echocardiographic correlation with microorganisms by identifying high rates of s. aureus bacteremia and positive echocardiographic findings when compared to other organisms (fig. 2). tte sensitivity and specificity of ie based on bmi (kg / m). guidelines and recommendations have been well proposed for the role of echocardiography in ie (3, 5). echocardiographic findings have been modified in duke criteria to be part of major criteria for the diagnosis of ie, making the study a significant influence (5). tte has been studied to result in a significantly lower sensitivity rates compared to tee with similarly high specificities. variable low results have been concluded for the percentages of the sensitivities of tte and tee ; however, similar specificities above 90% have been described for both. sensitivities for tte and tee have been shown to range from 4460% to 87100%, respectively. specificities for tte and tee were described to range from 91 to 100% in both imaging modalities (2, 8, 9). in 1984, o'brien and geiser described two - dimensional use of tte in ie for valvular vegetations identified in 80% of patients (10). approximated the sensitivity of tte to be 70% and an increase with tee to approximately 8590% (11). the initial and original duke criteria relied heavily on tte as tee was not systemically evaluated at the time. recommendations have been proposed for the definition of the modified duke criteria with tee as the initial echocardiographic study to be performed in patients with prosthetic valves, possible ie, or complicated ie (paravalvular abscess). in all other cases, although the best method for detection of early abscess formation, tee was found to be a limited study and an underestimation with an abscess detection rate of 48% (12) limitations of tte exist, decreasing the sensitivity as detection of vegetations less than 3 mm were found to be missed. we established similar sensitivities and specificities for tte and proposed to identify a possible limitation to the quality of the study by factoring in patients body habitus, calculated by their bmi. patients with bmis less than 25 kg / m were found to have a higher valvular vegetation detection rates and lower false negatives resulting in a sensitivity of 78%. furthermore, patients with bmis greater than or equal to 25 kg / m were found to have sensitivities of 54% and similar specificities above 90% in both groups (fig. body habitus appeared to have an impact on the detection rates between the two groups with an increased sensitivity difference of 24% in patients with bmi < 25 kg / m. although our study is limited to a small population, we suggest body habitus as a possible factor in the decreased sensitivities of tte. tte provides better detection rates in patients with a bmi < 25 kg / m. we propose using strong clinical judgment before proceeding with an additional, possibly unnecessary tee study. we further suggest proceeding directly to tee as the initial study for ie in patients with a bmi greater than or equal to 25 kg / m, unless a tte is clinically warranted. calculating a bmi in patients with suspicion for ie may provide benefit in reducing further diagnostic imaging. kg / m with a negative tte should refrain from further diagnostic studies with tee, given the findings of increased sensitivities. patients with a bmi 25 kg / m may proceed directly to a tee, possibly avoiding an additional study with a tte given the low sensitivities identified in this population. however, clinician judgment is important in deciding the imaging modalities in patients suspected of ie. the authors have not received any funding or benefits from industry or elsewhere to conduct this study.	backgroundechocardiography has been a popular modality used to aid in the diagnosis of infective endocarditis (ie) with the modified duke criteria. we evaluated the necessity between the uses of either a transthoracic echocardiography (tte) or transesophageal echocardiography (tee) in patients with a body mass index (bmi) greater than or equal to 25 kg / m2 and less than 25 kg / m2.methodsa single - centered, retrospective study of 198 patients between 2005 and 2012 diagnosed with ie based on modified duke criteria. patients, required to be above age 18, had undergone an echocardiogram study and had blood cultures to be included in the study.resultsamong 198 patients, two echocardiographic groups were evaluated as 158 patients obtained a tte, 143 obtained a tee, and 103 overlapped with tee and tte. out of these patients, 167 patients were included in the study as 109 (65%) were discovered to have native valve vegetations on tee and 58 (35%) with tte. tte findings were compared with tee results for true negatives and positives to isolate valvular vegetations. overall sensitivity of tte was calculated to be 67% with a specificity of 93%. patients were further divided into two groups with the first group having a bmi 25 kg / m2 and the subsequent group with a bmi < 25 kg / m2. patients with a bmi 25 kg / m2 who underwent a tte study had a sensitivity and specificity of 54 and 92%, respectively. on the contrary, patients with a bmi < 25 kg / m2 had a tte sensitivity and specificity of 78 and 95%, respectively.conclusionspatients with a bmi < 25 kg / m2 and a negative tte should refrain from further diagnostic studies, with tee strong clinical judgment is warranted. patients with a bmi 25 kg / m2 may proceed directly to tee as the initial study, possibly avoiding an additional study with a tte.
literature was searched on pubmed using combinations of the following two groups of keywords : ocular outcome (visual impairment, blindness, cataract, diabetic retinopathy, glaucoma, eye health, eye care, ophthalmology, and prevalence) and structural determinants of socioeconomic position (socioeconomic status, social class, income, educational status, gender, poverty areas, ethnic groups, race, inequality, disparity, inequity, and access). causes of blindness and visual impairment were included as key words to capture publications that produce secondary results on visual impairment or blindness. the search included original population - based studies, reviews, and meta - analysis from 2000 to 2011 in the english and spanish languages. there were 565 publications found : 101 for gender, 53 for income, 42 for education, 12 for social class, 109 for inequality, 109 for socioeconomic factor, and 95 for race / ethnicity. a total of 312 publications were found for visual impairment and 253 for blindness prevalence outcomes. three reviewers independently examined the title and the abstract of each article, classifying the articles in six fields : gender, income, education, employment status and social class, geographic, and race / ethnicity. full text and tables of all the articles that had results on visual impairment or blindness outcomes were reviewed. two inclusion criteria were evaluated : (1) present empirical findings related to outcomes of prevalence of visual impairment or blindness in population - based studies on adult populations and ; (2) a gender, income, educational level, employment status and social class, or race stratifying measure. a manual search of the references of these studies, an additional 16 articles were identified. given that publication dates included in the review range from 2000 to the present, when the articles found in this review did not fill the gap on the knowledge previously documented additional material was sought from study references and literature collections conducted in earlier years by the authors. a table has been generated that summarizes the literature review of publications in the last 12 years. a total of 23 studies were found that stratified the results of prevalence of blindness or visual impairment with the structural social determinants of health : gender, income, educational level, employment status, social class, and ethnicity / race [table 1 ]. selected articles on social determinants of health and visual impairment or blindness although important inequalities in the prevalence of blindness and sex have been reported, no gender review has been published. a meta - analysis conducted by abou - gareeb ten years ago found that women accounted for nearly two - thirds of the population with blindness. after age adjustment, the overall odds ratio (or) of blind women to men was remarkably consistent by geographical area, being 1.39 for africa, 1.41 for asia, and 1.63 for industrialized countries. later studies on the prevalence of visual impairment and blindness reported a prevalence ratio of more than 1.5 for women in high - income countries, which is surprisingly higher than in low - income countries.[1114 ] women generally have a longer life expectancy than men. since many eye diseases are age - related, we would expect women to have a higher burden of visual impairment and blindness. access to the health care system was an intermediary determinant and played a role in exposure and vulnerability. through the analysis of women 's access to services, differences between countries at low, middle, and high levels of the human development index (hdi) were observed. in high hdi countries such as the united states (us), australia, and germany, women reported more visits to the eye care specialist.[1720 ] all age groups of women in the us had better ocular health care utilization than men for all three racial / ethnic groups. no differences in eye care service utilization were found between men and women in the middle hdi countries of asia, such as oman, and similar access to cataract surgical services was noted in latin america. in the low hdi countries of africa and asia, access to cataract surgical services was lower for women compared with men.[132429 ] reasons for not seeking eye care showed different gender patterns for people with some visual impairment ; no need was the main reason for men and cost / insurance for women in the us. attitudinal differences in seeking health care were also suggested as reasons to explain gender differences in access to eye health care services. studies in france and the us showed that people with low vision had less income. in low hdi countries, such as kenya, the philippines, and bangladesh, multivariate analyses showed that case participants were consistently poorer than controls when assessed using three different measures of poverty even after adjustment for health and social support indicators. only one article evaluated risk of blindness and individual income and found that low income was associated with blindness in india. visual impairment, even unilateral, was associated with household income (> $ 75,000 a year) in the us, and both high rates of blindness and visual impairment were found in the elderly of the us.[3739 ] increased risk for visual problems was documented in the impoverished neighborhoods with the worst economic indicators in the us and australia (we must bear in mind, however, that these data are relatively old). in addition, the prevalence of blindness and visual impairment was higher in low - income countries when compared with high - income countries. there was also a gradient between the gross domestic product (gdp) of a country and its prevalence of blindness. although only a few articles addressed the association of blindness or visual impairment with income, the results were consistent in that lower income was associated with visual problems. not one article, analyzed in this review, stratified association of income with visual outcomes by sex. lower levels of education were associated with higher prevalence of visual impairment in australia, taiwan, and the us, as well as blindness in the us, india, and china.[38394447 ] in 1991, a reverse association with years of education and prevalence of visual impairment and blindness was observed, and prevalence increased at a much faster rate when illiteracy in india, pakistan, nigeria, and the us was taken into consideration. a higher association with the level of education was found for bilateral (as compared with unilateral) visual impairment in the us. reasons for not seeking eye care in the us were based on educational level, with no need as the main reason for the highly educated and cost / insurance for the lower educated. however, highly educated individuals (32%) still reported cost / insurance as a reason for not seeking eye care, although this factor decreased among the high - income population (22%). people with visual impairment in europe were at higher risk of not having a paid job, being unemployed, suffering from permanent disability, belonging to a manual social class (with less job satisfaction), having less opportunity to develop new skills, having less recognition for their work, and having an inadequate salary. in france, individuals without visual problems had a chance of having a paid job five times greater than that of blind people and twice than those with low vision. in india, the concept of social class derived from occupation was also associated with health indicators. for coding social class, each individual was assigned to their occupation, and each occupation was assigned to one of the six social classes. the first three corresponded to nonmanual workers, and the last three to manual workers. social class based on occupation integrated the level of training required for a job, income, and the level of responsibility. in britain, the risk of poor vision was associated with social class (unskilled manual workers). for each increment in social class grade on a scale of i through v, the risk of poor vision increased by 28%, with a prevalence of 1.9% in social class i (professional) and 5% in social class v (unskilled manual workers). additionally, children with manual social class fathers at the moment of birth had an increased risk of far and near visual impairment at adulthood. however, prevalence of low vision and blindness for workers was similar to that of the unemployed in the us. after adjusting for age and socioeconomic position, no association with visual impairments was found among hispanics, african americans, and caucasians in the us. however, hispanics had a higher incidence of visual impairment than that reported in non - hispanic white persons and the highest reported in a population - based study in the us. in australia, bilateral visual impairment and blindness were found to be four to seven times more frequent in the indigenous population. however, it is often difficult to evaluate the prevalence of visual impairment or blindness that might be truly inherent in a racial or ethnic population in addition to the social determinants that a specific racial group were exposed to due to low socioeconomic position and marginalization. many studies in the us, but not all, showed no significant differences between ethnic or racial minorities when compared with caucasian populations, although differences were not always adjusted for socioeconomic position variables. geographic inequalities were found among continents, countries, and regions within a country. in 2000, africa and india bore the highest prevalence of blindness, followed by the rest of asia, china, and latin america. however, asia led the burden of disability - adjusted life years (dalys), and cataract was the principal cause of blindness with 95% of the burden in low - income countries. other studies showed that 87% of the visually impaired and 90% of blind people lived in low - income countries but differences in prevalence persisted between countries in the same region or continent and were inversely correlated with gdp per capita of each country. in france, geographic inequalities were also found between regions within the country for age - adjusted visual impairment and blindness prevalence. geographic inequalities were found after occupational social class adjustment, and they were evident for age - adjusted low vision between regions of nigeria. differences in the prevalence of visual impairment were similarly found for five states in the us. in singapore, an ecologic effect of socioeconomic determinants of the community was found to have an independent association with visual impairment, even when considering individual socioeconomic determinants. in canada, ecologic research found that prevalence of blindness registration correlated with medium household income of districts after evaluating the five geopolitical regions of the country. however, when the model did not consider geopolitical region, medium household income was not statistically correlated. those results suggested that the geopolitical region played a role in blindness independent of district income. moreover, income derived from government transfer payments had a negative correlation with blindness registration prevalence. the review produced four main findings : (1) women had a higher prevalence of visual impairment and blindness, which was not fully explained by age or by access to services, (2) socioeconomic status measured as higher income, higher educational status, or nonmanual occupational social class was inversely associated with prevalence of blindness or visual impairment, (3) ethnicity and race were associated with visual impairment, although other social determinants of health can be associated, and (4) geographic inequalities and visual impairment (of the region, nation or continent) were observed to be related to income, and living in a rural area. results bearing the evidence of the association between socioeconomic position determinants and prevalence of visual impairment and blindness were found in this review, even if this relationship has rarely been addressed in research (23 articles in the last 12 years). however, the effect of an individual 's socioeconomic position on his / her health may not be only direct, but may also emerge from intermediary determinants that remain, pending investigation. future research should be done measuring how exposure or vulnerability explain the pattern of inequalities regarding a specific social stratification such as educational level or income. a few articles analyzed in this review, upon publication, were categorized as social determinant(s) of health, although the tag was an intermediary factor mostly related to accessibility of services. additionally, some of the articles considered only psychosocial factors, which are related to occupational health and environmental factors, as determinants for producing inequalities. although psychosocial consequences of socioeconomic inequality were an important intermediary determinant, interpretation of links between socioeconomic status and health must begin with the structural causes of inequalities. approach is a needed consideration to better understand the mechanism of how differences are produced by integrating social and biological factors in a dynamic, historical, and ecological perspective. a possible effect of the first level of social determinants of health the determinants of socioeconomic and political context might be considered for future research, since the few results produced thus far consistently suggested this concept. a greater awareness of gender discrimination behaviors could explain differences between the outcomes of men and women if the slightly increased gender inequalities in prevalence are confirmed for high - income countries versus low - income countries. in addition, gender discrimination patterns affected the decision - making authority, which not only influenced access to services, but also, differences in psychosocial and environmental risk exposure. women also accumulated more working hours than men, and their additional domestic chores negatively affected their health. this could also influence risk of diabetic retinopathy, glaucoma, and cataract if those health issues were related to stress. more research is needed to identify if perceived gender discrimination, decision - making authority, and working hours are associated with gender inequalities in blindness and visual impairment. a study performed in saudi arabia, a country with significant gender discrimination, found an extremely high gender inequality of visual impairment between men and women attending primary care (lower for women), and there was lower registration for government allowances provided for blindness in kuwait. it is worthy to note that with other pathologies, women generally put less therapeutic effort in seeking treatment with regard to organ transplants, coronary problems, emergency treatments, and pharmaceutical spending.[7175 ] more research would be needed to assess if there are gender differences in therapeutic efforts regarding ophthalmological procedures, and if so, whether those differences could explain why women, despite having had more access to a specialist in high - income countries, had higher prevalence of blindness and visual impairment than men. sex differences in the distribution of pathologies that cause blindness and visual impairment were not broadly described. (it should be clarified that sex refers to the biological construct / characteristics, whereas gender is a social construct concerning behaviors, roles, and interactions between men and women.) genetic, hormonal, and other biological factors associated with ocular pathologies could lead women to greater risk of blindness and visual impairment. reporting sex - stratified data in publications could allow more accurate knowledge on gender inequalities. income, educational level, and social class measure socioeconomic position and act in similar, although not equal, ways to produce visual impairment and blindness. more research should be done to understand performance and how influences vary in relation to visual outcomes. although only a few articles addressed the association of blindness or visual impairment and income, the results consistently showed correlation between lower income and higher risk for visual problems. however, even when considering that women were at a significantly greater risk of developing visual impairment or blindness, none of the articles analyzed in this review stratified association of income and visual outcomes by sex. there were more articles that disaggregated the prevalence of visual impairment and blindness according to level of education, rather than income. educational level was associated with knowledge and awareness of eye conditions and eye care services and poor behavior toward eye care, but education might have a different effect than income in seeking eye care when needed. however, more research is needed to identify whether lack of knowledge and poor behavior explains this association or if other socioeconomic factors can be implicated. complex behavior in seeking and receiving eye care services may be embedded in socioeconomic determinants, and more research needs to be done to confirm those findings.[8082 ] the father 's social class at time of birth of an individual had a direct effect on that individual 's embodiment of social class and affected middle - age risk of developing visual impairment. however, more research is required for measuring the direct or reverse effect of blindness and socioeconomic position, as well as the role of gender. while many studies demonstrated that ethnic or racial groups have a different prevalence of visual impairment, often due to specific eye diseases, it can be difficult to ascertain how much was intrinsic to the race or ethnicity and how much was associated to socioeconomic position or the lack of eye care for various reasons. eye care inequities exist in a variety of ways around the world. while some studies suggested that eye care access is a major barrier even in the presence of national health care systems, substantial numbers of subjects did not utilize services where they were available. in these instances, the lack of education and perhaps more importantly, the lack of basic literacy and/or knowledge of eye diseases provide some explanation. poverty by itself or combined with educational factors (social deprivation) is also another reason why many patients can not access services. as a final note to the discussion, a limitation of the review was that most of the publications were not correctly tagged with adequate keywords when searching for inequalities. as a recommendation, it would be important that future publications should be classified in terms of inequities or social determinants in order to facilitate knowledge - sharing of work that has already been produced. more research and interpretation needs to be done to better understand the social and biological mechanisms that produce the social inequalities patterns in the prevalence of blindness and visual impairment. publications, even those not focused on inequalities, should stratify and interpret findings separately by sex and socioeconomic status to provide better understanding of gender inequalities. associations with determinants of socioeconomic and political context should be further explored.	health inequities are related to social determinants based on gender, socioeconomic status, ethnicity, race, living in a specific geographic region, or having a specific health condition. such inequities were reviewed for blindness and visual impairment by searching for studies on the subject in pubmed from 2000 to 2011 in the english and spanish languages. the goal of this article is to provide a current review in understanding how inequities based specifically on the aforementioned social determinants on health influence the prevalence of visual impairment and blindness. with regards to gender inequality, women have a higher prevalence of visual impairment and blindness, which can not be only reasoned based on age or access to service. socioeconomic status measured as higher income, higher educational status, or non - manual occupational social class was inversely associated with prevalence of blindness or visual impairment. ethnicity and race were associated with visual impairment and blindness, although there is general confusion over this socioeconomic position determinant. geographic inequalities and visual impairment were related to income (of the region, nation or continent), living in a rural area, and an association with socioeconomic and political context was suggested. while inequalities related to blindness and visual impairment have rarely been specifically addressed in research, there is still evidence of the association of social determinants and prevalence of blindness and visual impairment. additional research should be done on the associations with intermediary determinants and socioeconomic and political context.
the circadian clock is an autoregulatory network that regulates behavioral and metabolic programing in the context of a 24 h light - dark (ld) cycle. body temperature is one of the representing benchmarks of circadian patterning, which peaks in animals while awake and troughs while asleep. brown adipose tissue (bat) is a major site for rodent thermogenesis, due to its involvement in controlling circadian thermogenic rhythms and influencing adaptability to environmental temperature challenges. a previous study has revealed rhythmic expression patterns of over 5,000 genes in murine bat, including genes associated with the circadian clock, adipose function, and metabolism. moreover, the inhibitor of dna binding 2 (id2) gene encodes a helix - loop - helix (hlh) transcriptional regulator, which is rhythmically expressed in many mammalian tissues and involved in the input pathway, core clock function, and output pathways of the circadian clock [69 ]. our previous studies have shown that id2/ mice exhibit lower levels of locomotor activity, extended nighttime activity patterns of feeding and locomotor activity, and sex- and age - dependent enhanced glucose tolerance and insulin sensitivity. moreover, an energy - rich diet is able to rescue the disturbances to metabolic homeostasis and survival in the id2/ mice sex - specifically. importantly, id2/ mice show a sex - dependent elevated glucose uptake in interscapular bat (ibat). id2 also plays a role in white adipose tissue (wat) adipogenesis [1012 ]. however, the role of id2 on temperature homeostasis regulation and its influence on bat physiology remain unknown. therefore, we investigated the function of id2 in the regulation of temperature rhythms under normal and thermoneutral conditions in a sex - specific manner and also profiled the expression of genes involved in insulin signaling and adipogenesis in bat of id2/ mice, sex - specifically. the generation of id2/ mice and genotype determination were performed as described previously [7, 10, 11 ]. mice were maintained on a regular chow diet and sterile water containing antibiotic ad libitum [7, 10, 11 ]. all mice were housed in laboratory cages at normal temperature (21c 1c) and humidity of 5065% under a 12 : 12 light : dark (ld) cycle with lights on at zeitgeber time (zt) 0 and lights off at zt12. animal experiments were approved by the university of notre dame animal care and use committee (protocol number 14 - 02 - 1559) and performed in accordance with nih guidelines for the care and use of laboratory animals. temperature measurements were carried out on 2-month1.5-year- (5.5-month median) old male and female id2/ mice and wt littermates, housed individually in a climate - controlled room set to either normal (21c 1c) or thermoneutral (30c 1c) temperature. body temperature sampling was conducted at 3 h intervals over the 24 h ld cycle. for thermoneutral conditions measurement, all wt and id2/ mice used in the studies were allowed to acclimate to thermoneutral temperature for 1 week before temperature measurement. core body temperature was measured using subcutaneously surgically implanted telemetric transmitters positioned proximal to the ibat (iptt 300 transponders, bio medic data systems, seaford, de) following isoflurane anesthetization. after a week of recuperation, ibat tissue was harvested at zt16 (id2 mrna circadian rhythm in ibat has a broad peak phase between zt8 and zt16). id2/ and wt male (wt = 8, id2/ = 6) and female (wt = 6, id2/ = 4) mice from 39 months (6.1-month median) were sacrificed and ibat tissue was frozen in liquid nitrogen and stored at 80c until analyzed. we also measured ibat weight of these and additional mice (310-month - old id2/ mice and wt littermates ; 6.3-month median ; male, wt = 15, id2/ = 7 ; female, wt = 10, id2/ = 9) as described previously [10, 11 ]. rna integrity was assessed using a bioanalyzer 2100 (agilent technologies, santa clara, ca). rna was subjected to a dnase i treatment, and cdna was synthesized by rt first strand kit (sabiosciences). relative mrna expression of 168 genes involved in insulin signaling and adipogenesis pathways was determined by using the mouse pcr arrays (pamm-030zc-24 and pamm-049zc-24, sabiosciences). quantitative real - time pcr was performed using an applied biosystems 7500 system with rt2 sybr green rox qpcr master mix reagent (qiagen). pcr array data were calculated by the comparative cycle threshold method and analyzed by web - based free pcr array data analysis software provided by sabiosciences. normalization of expression was to housekeeping genes provided on each array (actb, b2 m, gapdh, gusb, and hsp90ab1). clock controlled genes (ccgs) were identified from the circa database of mouse 1.ost brown adipose (affymetrix) (http://bioinf.itmat.upenn.edu/circa/) where we defined ccgs as a jtk_cycle algorithm determined q < 0.1 value and a period length of 2028 h as described previously [1315 ]. the linear regression of ibat temperature - body weight relationship was generated and analyzed using prism 5.0 graphpad software. pcr array data were analyzed using the web - based free pcr array data analysis software provided by sabiosciences (student 's t - test). the discovery of a diurnal rhythm of glucose uptake in mice ibat and a sex - dependent elevated glucose uptake in ibat of id2/ mice prompted us to investigate whether id2 contributes to thermoregulation [5, 10 ]. at normal ambient temperature conditions (21c), ablation of id2 reduced core body temperature across the 24 h day, in both male and female mice (figure 1(a)) (males, wild types (wts) = 14, id2/ = 14, anova, time (t), p < 0.001, genotype (g) p < 0.001, interaction (i), n.s. ; females, wts = 18, id2/ = 17, anova, t, p < 0.001, g, p < 0.05, i, n.s.). considering the possibility of any confounding genetic background contribution and partial stimulation of bat activity occurring under normal temperature conditions, id2/ mice core body temperature was also measured under thermoneutral conditions (30c) [3, 16 ]. consistently, at thermoneutrality, id2/ mice displayed a reduced core body temperature (figure 1(a)) (males, wts = 19, id2/ = 20, anova, t, p < 0.001, g, p < 0.01, i, n.s. ; females, wts = 18, id2/ = 17, anova, t, p < 0.001, g, p < 0.01, i, n.s.). under both conditions and in both sexes, no interaction between time and genotype was discovered, suggesting a generalized effect of the null mutation on core body temperature rather than a time - of - day specific contribution of the gene deletion. regression analysis of time - of - day representative core body temperatures (day or night) revealed no significant relationships between temperature and body mass for either id2/ or wt mice. however, id2/ mice of both sexes showed consistently lower y - intercept lines compared to wt mice when examined during either the daytime (zt5.5) or nighttime (zt17.5 or zt20.5), thus confirming the consistently lower temperature of the id2 null mice (figure 1(b) ; supplementary table 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/6785948). lastly, id2/ mice exhibited no statistically significant difference in ibat weight and ibat to body weight ratio compared to wt controls (figures 2(a) and 2(b)) (two - factor anovas, ibat weight, g, n.s., sex (s), p < 0.01, i, n.s. ; ibat / body weight ratio, g, n.s., s, n.s. the mean and sem body mass of wt and id2/ mice for both ibat weight and body temperature experiments are shown in supplementary table 2 : note that both male and female id2/ mice had on average a lower body mass compared to wt counterparts (two - factor anova, g, p < 0.001, s, p < 0.001, i, n.s.). our previous results showed sex - dependent enhanced insulin sensitivity and glucose uptake in ibat of id2/ mice. in the current study we observed a decreased core body temperature in id2/ mice as described above. to fully evaluate the impact of ablation of id2 on bat gene - regulation, we performed a gene expression analysis using rt profiler pcr arrays of bat derived from id2/ mice and their wt littermates collected at the same time of the 24 h day (specifically zt16). deferentially regulated genes involved in insulin signaling and adipogenesis are shown in tables 1 and 2, respectively. thirty of 168 genes examined were identified as differentially expressed when analyzed as a cohort or as individual sex - specific groups. using the circa database as a resource, six genes were identified as clock controlled genes (ccgs), of which four oscillate in proximal phase with the rhythm of peroxisome proliferator activated receptor alpha (ppar), peaking during the middle of the day (~circadian time (ct) 6 ; ct12 = onset of night in prior ld cycle). of importance for insulin signaling, glucose-6-phosphatase, catalytic (g6pc), was upregulated in id2/ females and the related g6pc family member g6pc2 downregulated in id2/ males (p = 0.079, approaching significance) compared to wts. insulin receptor substrate 1 (irs1) was upregulated in both male and female id2/ mice. insulin - like growth factor 2 (igf2) was downregulated in female id2/ mice, while fbp1, a rate - limiting enzyme in gluconeogenesis, and shc1, a component in the igf-1-regulated pathway, were upregulated. for adipogenesis, bone morphogenetic protein 4 (bmp4) was elevated 1.7-fold (n.s.) in male and 1.6-fold in female id2/ mice. nuclear receptor coactivator 2 (ncoa2), pr domain containing 16 (prdm16), ppar, and twist homolog 1 (twist1) were downregulated, in grouped analysis of male and female id2/ mice. fatty acid synthase (fasn), lipase, hormone sensitive (lipe), and peroxisome proliferative activated receptor, gamma, coactivator 1 beta (ppargc1/pgc-1) were all downregulated in male id2/ mice. female id2/ mice displayed a downregulation of proliferative activated receptor, gamma, coactivator 1 alpha (ppargc1/pgc-1). a small 1.2-fold downregulation of peroxisome proliferator activated receptor gamma (ppar) was detected in id2/ males, where the p value was approaching significance (p = 0.061). note that the thermogenic protein, uncoupling protein 1 (ucp1), was present on both the insulin signaling and adipogenesis arrays, but its levels of expression were not significantly altered in the id2/ mice. in the present study, we discovered a reduced core body temperature in id2/ mice, and this effect was not found to be dependent upon the time - of - day. moreover, from the ibat of id2/ mice, genes involved in insulin signaling and adipogenesis were differentially regulated in a sex - dependent manner. these results reveal a role of id2 in the regulation of thermogenesis and bat metabolic functions. our previous study revealed that id2/ mice exhibit less activity as demonstrated by daily counts of general activity and the wheel running activity, which could partially explain the reduced core body temperature, since less physical activity would generate less heat. moreover, id2/ mice show a reduced body mass and less gonadal adipose deposits [6, 10, 11 ]. as the subcutaneous and intradermal fat functions as thermal insulation for mice to preserve heat loss, id2/ mice with low fat content might tend to lose heat more readily than wt mice. furthermore, the reduced body temperature associated with lower fat content might contribute to the high death rate observed previously (mice housed under normal temperature), which was rescued by a high fat diet that resulted in increased total body fat. specifically in male id2/ mice ibat we observed increased glucose uptake and reduced tg accumulation, suggesting alterations in its metabolic programing. interestingly, our results suggest that the role of id2 in thermoregulation is opposite to the function of another member of this hlh family, id1, whose deficiency results in higher thermogenesis and an elevated bat expression of thermogenic proteins. notably, id1 has a distinct and opposite function in wat adipogenesis compared to id2, despite both id1 and id2 null mice exhibiting reduced adiposity [1012, 17 ]. lastly, we examined the relationship between body mass and body temperature in id2/ mice by regression analysis and revealed a limited relationship between the two variables. no significant relationship was observed between body mass and body temperature at any time of the day or in the two sexes. however, as can be seen with the y - intercept of the regression lines, both id2/ male and female mice expressed a consistently lower temperature compared to wt controls, irrespective of body mass, and this feature was observed during both the day and night phases of the ld cycle. these results suggest a role for id2 in the regulation of core body temperature. in this study we also measured ibat mass and ibat / body mass ratio. while there was a tendency for higher ibat / body mass in both id2/ male and female mice, this was not determined to be a significant difference. note that the average body mass of id2/ mice used for both the ibat weight and body temperature experiments was found to be significantly lower, consistent with our previous studies [7, 10 ]. important is the fact that a lower body mass, found for some of the id2/ mice and for males in particular, does not correlate with a lower body temperature, and body mass in this situation is therefore an independent factor when predicting core body temperature. it is important to note that while the objective of examining body temperature using the implanted thermometers was to record core body temperature, the position of the implants may not give an exact measure of true core body temperature. however, in a comparable study of mouse body temperatures, temperature measurements were similar whether derived from similarly subcutaneously implanted thermometers in the interscapular region of wt and rev - erb mutant mice or as determined using dataloggers that were implanted within the abdomen. id2 is rhythmically expressed in bat [4, 15 ] amongst other tissues [6, 7 ]. id2 protein has also been observed to be rhythmic in its abundance over the 24 hr diurnal / circadian cycle within the liver and heart (ward, fernando, hou, and duffield, unpublished data). a role for id2 has been established as a mediator of circadian clock output and control of expression patterns of clock controlled genes (ccgs) within the liver. it is for this reason that we examined whether any of the genes identified as differentially expressed in ibat were in fact known ccgs. using the circa database [14, 15 ], 5 of the 17 differentially genes associated with adipogenesis were found to be ccgs (e.g., ppar and pgc-1), and so a possible role for id2 is in mediating circadian regulatory effects on these genes within bat. the observation that few of the differentially regulated genes involved in insulin signaling are ccgs (1 out of 13 genes) suggests that the contribution of id2 to insulin signaling intrinsic to bat is independent of the role of id2 in mediating circadian clock output. in order to explain how id2 deficiency has an impact on bat insulin signaling and adipogenesis the nuclear receptor ppars are fundamentally important for energy homeostasis and id2 plays a role in interfacing with the molecular pathways upstream or downstream of these transcriptional factors. expression of two members of the ppar subfamily of ligand - activated nuclear receptors, ppar and ppar, was downregulated in our study. ppar is highly expressed in bat and considered a marker of bat ; it also plays an important role in the overall regulation of lipid metabolism ; and its target genes are involved in mitochondrial and peroxisomal -oxidation of fatty acids (fas) [2022 ]. moreover, ppar regulates the expression of uncoupling protein 1 (ucp1), which confers on bat its thermogenic capacity. pgc-1 (downregulated in our study) is a transcriptional coactivator involved in the control of energy metabolism and critical for bat thermogenesis and enhancing overall mitochondrial oxidative activity. ppar can induce pgc-1 gene expression and contributes to the thermogenic activation of brown fat. prdm16 exhibits a brown fat selective expression pattern and regulates the thermogenic gene program in brown and beige adipocytes. the observation of reduced prdm16 expression in id2/ mice is consistent with the role of prdm16 as a transcriptional regulator of pgc-1. ppar is essential for adipocyte differentiation, and ppar alone generates a fat phenotype that is common to both wat and bat. the ccaat enhancer binding protein beta (c / ebp) and pgc-1 are critical for controlling ppar expression in bat and for determining bat - specific programs [29, 30 ]. the ppar thermogenic effect in bat it has been observed that overexpression of id2 associates with ppar expression, id2 acts upstream of ppar, and c / ebp induces id2 expression during the adipogenesis process [12, 31 ]. cofactors such as ncoa2 (downregulated in our study) can interact directly with ppar to initiate its own transactivation. moreover, lipe (downregulated in our study) could modulate adipose metabolism by reducing the availability of ligands for ppar, since gene knockout of lipe in mice attenuates activation of ppar. lipe is also able to hydrolyze stored tgs in adipose tissue and to mobilize free fa from adipose tissue. furthermore, ppar is a direct target of the transcription factor sterol response element binding protein 1 (srebp1), whose transcriptional activity is modulated by id2 and which regulates downstream lipid metabolism genes such as lipe and fasn [35, 36 ]. additionally, irs1 (upregulated in our study) plays essential roles in the differentiation of brown adipocytes and expression of ppar [37, 38 ]. previous studies have revealed irs1-regulated id2 gene expression, although in the current study it is unclear whether this is a direct effect or a feedback response. as for the mechanism by which irs1 is elevated in id2/ ibat, bmp4 (upregulated in our study) is able to induce the white to brown transition of adipose cells, which could indirectly regulate ppar activation [40, 41 ]. the elevated bmp4 expression in the context of reduced ppars is surprising since bmp4 upregulation is associated with increased bat adipogenesis and the wat browning effect. interestingly, the id2 gene promoter has bmp - response elements and has been shown to be a target of bmp signaling. ppar plays an integral role in transcriptional network regulation of fat - burning genes and brown fat metabolism. twist-1 (downregulation in our study) encodes a basic hlh transcription factor, and overexpression of twist-1 is associated with id2 expression. note that the gene encoding the thermogenic protein, ucp1, was present on the pcr arrays, but no difference in its expression was observed between genotypes of either sex. interestingly, in the id1 null mouse, ucp1 gene expression is elevated in ibat, and this is associated with an increased core body temperature phenotype. thus, it is surprising that in the id2/ mouse that exhibits a reduced body temperature phenotype we do not observe a reduction in ucp1 gene expression. of course, this does not exclude, however unlikely, the possibility of an altered ucp1 protein abundance through a posttranscriptional / posttranslational process. hypoxia, while not a focus of the current study, is known to reduce body temperature in mammals and contribute to the thermogenic activity of bat. it is noteworthy that in a recent study of human glioblastoma cells / tissue, an important role was established for id2 in modulating the cellular effects of hypoxia and its activation of the hif2 pathway. the id2 gene is also a target for hif1 and hif2 [47, 48 ], making it part of a positive feedback loop mechanism, at least in models of brain tumor. it is plausible that the hypoxic effects on bat function might also include a contribution from id2, and this would be an important pathway to examine in future experiments in this context. it is a somewhat contradictory finding that elements of the thermogenic pathway are reduced (e.g., pgc1-) or unaltered (ucp1) in id2/ mice but that id2/ male ibat exhibits increased glucose uptake (pet - fdg) coupled with reduced ibat triglyceride levels and a systemwide enhanced insulin sensitivity [10, 11 ] and that core body temperature is consistently reduced in both id2/ male and female mice. id2/ mice also exhibit an altered 24 hr locomotor activity and feeding profile and an overall reduction in nocturnal locomotor activity, the latter of which suggests a reduction in energy expenditure / increased energy conservation. clearly the bat adipogenic program is altered in both male and female id2/ mice, as is wat adipogenesis [1012 ], and it is likely that a change in bat function contributes to the reduced temperature phenotype. due to the nature of whole body knockout of id2, it is possible that the temperature phenotype and ibat gene changes observed are secondary to whole body metabolic changes. clearly additional experiments are required to elucidate the relative contributions of these and other potential components in generating the altered core body temperature phenotype. our previous study showed enhanced glucose uptake in ibat of id2/ mice, and the present study reveals a reduced core body temperature in id2/ mice. this discrepancy could partially be explained by the differential regulation of irs1, lipe, ppars, and pgc-1s. it has been reported that degradation of irs1 leads to impaired glucose uptake in adipose tissue. therefore, upregulation of irs1 might explain the increased glucose uptake we observed before, whereas downregulation of lipe, ppars, and pgc-1s might contribute to reduced fa oxidation, impaired adipogenesis, and a lower body temperature. inactivation of ppars is associated with insulin resistance [50, 51 ], yet paradoxically id2/ mice show enhanced insulin sensitivity with downregulated ppars [10, 11 ]. it has been suggested that mice that lack one allele of the ppar gene are more sensitive to insulin, which could partially explain the enhanced insulin sensitivity we observe in id2 null mice [10, 51 ]. furthermore, the differential regulation of genes specifically in female mutant mice, such as fbp1, a rate - limiting enzyme in gluconeogenesis, shc1, a component in the igf-1-regulated pathway, and igf2, suggests a sex - specific physiological program for id2 in bat. inhibitor of dna binding 2 is rhythmically expressed in bat [4, 15 ], and the observation that few of the differentially regulated genes involved in insulin signaling are ccgs suggests that the contribution of id2 to insulin signaling intrinsic to bat is independent of the role of id2 in mediating circadian clock output. overall, id2 seems to be an important coordinator of energy homeostasis including insulin signaling, adipogenic programing, and thermoregulation. in conclusion, our finding that id2 contributes to the regulation of body temperature and energy homeostasis presents the possibility that id2 could be a potential therapeutic target for metabolic disease. further, these data emphasize the influence of id2 on bat molecular signaling and physiology in a sex - specific manner.	inhibitor of dna binding 2 (id2) is a helix - loop - helix transcriptional repressor rhythmically expressed in many adult tissues. our previous studies have demonstrated that id2 null mice have sex - specific elevated glucose uptake in brown adipose tissue (bat). here we further explored the role of id2 in the regulation of core body temperature over the circadian cycle and the impact of id2 deficiency on genes involved in insulin signaling and adipogenesis in bat. we discovered a reduced core body temperature in id2/ mice. moreover, in id2/ bat, 30 genes including irs1, ppars, and pgc-1s were identified as differentially expressed in a sex - specific pattern. these data provide valuable insights into the impact of id2 deficiency on energy homeostasis of mice in a sex - specific manner.
idiopathic macular hole (imh) is a full - thickness defect of the neuroretina that involves the fovea, and was firstly described by knapp in 1869 with an ocular blunt trauma patient. the condition is frequently used for imhs. also, it may occur as a result of axial elongation in myopic eyes. we used to think that this situation was untreatable before kelly and wendel, who first introduced vitreous surgery for macular holes (mh) in 1991. internal limiting membrane peeling (ilmp) played a crucial role in these modifications and was believed that there is a tangential traction in etiology of mh formation. internal limiting membrane (ilm) plays an important role in the etiology and the enlargement of the imh. as a result of this traction, while posterior hyaloid is detaching perifoveal pseudocysts may occur. younger age, smaller basal diameter of mh, smaller macular hole index (mhi), earlier stage, shorter duration, and better preoperative visual acuity are good prognostic criteria for anatomical closure. in clinical practice, gass classification is used internationally for staging the imhs and used as a predictive factor for the anatomical and functional success. however, this classification needs some modifications, because it does not include the detailed changes like accumulation of intraretinal fluid, tractional foveal cystoid space, or perifoveal pseudocysts, which may be detected via spectral domain optical coherence tomography (sd - oct, heidelberg engineering, heidelberg, germany). on the contrary, the international vitreomacular traction study group introduced an optical coherence tomography (oct) based anatomic classification system for diseases of the vitreomacular interface. in this classification, the authors categorized the vitreomacular interface diseases as vitreomacular adhesion, vitreomacular traction and full - thickness mh. in this oct - based anatomic classification system, staging is also based on aperture size and does not include morphological changes of mh formation. the aim of this prospective study is how these pseudocysts affect the anatomical closure success of the imh surgeries as a prognostic factor. twenty - one eyes of 20 consecutive patients with a gass stage 3 or 4 imh were included in this prospective study. all mhs had been treated with standard 3 port 23 gauge vitrectomy between march 2012 and may 2013. all patients underwent complete ophthalmic examination including measurement of best - corrected visual acuity (bcva) via early treatment diabetic retinopathy study chart, biomicroscopy of anterior segment, dilated fundus examination, and spectral domain optical coherence tomography (sd - oct) preoperatively and postoperative 1 day, 1 week, 1, 3, and 6 month. postoperative sd - oct assessments were made firstly in 1, 3, and 6 month, respectively. all patients had been operated by the same surgeon (aty) in beyoglu eye research and training hospital. exclusion criteria were higher than 6.00 d a refractive error, traumatic mh, macular retina pigment epithelium (rpe) atrophy, history of past ocular surgery except phacoemulsification, and other systemic and ocular diseases. all patients had given written informed consent before surgery, all the procedures were approved by the institutional ethics committee, and the study adhered to the tenets of the declaration of helsinki. all patients underwent standard 3 port 23 gauge pars plana vitrectomy with triamcinolone acetonide - assisted posterior vitreous detachment, if it was not already present. ilm removal were performed using 0.2 ml of dye brilliant blue g (brilliantpeel ; geuder, heidelberg). the area of removal of the ilm was intended to be 2 to 3 disc diameters surrounding the mh. the procedure was completed by an intraocular tamponade with 15% perfluoropropane (c3f8) or 20% sulfur hexafluoride (sf6). anatomic success was defined as the complete closure of the mh and absence of subretinal fluid on sd - oct. based on previous studies, 4 oct parameters were analyzed : mh basal diameter, mh minimum diameter, mh height, mhi, and a new parameter : the area of macular pseudocysts. basal hole diameter was defined as the hole diameter at the level of the rpe (figure 1). the mhi (hole height / basal hole diameter) was calculated according to a previously described method. the area of pseudocysts were calculated by a software option of sd - oct at the widest cross - section of the mh formation (figure 2). the borders of the pseudocysts were marked by the observer and the software programme gives the areas of the pseudocysts. measurements of parameters on oct. basal hole diameter (a) was defined as the hole diameter at the level of the retinal pigment epithelium. hole height (b) was the highest measurement from the rpe to vitreoretinal interface. oct = optical coherence tomography, rpe = retina pigment epithelium. the area of pseudocysts is calculated as in micrometer square at the widest cross section of the macular hole formation. the anatomical success was defined as the complete closure of the mh and absence of subretinal fluid on sd - oct. whitney test in terms of 5 sd - oct parameters : mh base diameter, mh minimum diameter, mh height, mhi, and mh pseudocyst area. the bcva values were converted to logarithm of the minimum angle of resolution (logmar) values for statistical analyses. the patients with preoperative and postoperative bcva were assessed with wilcoxon signed - rank test. spearman rank coefficient was calculated to assess correlation between anatomical closure and preoperative variables such as mh base diameter, mh minimum diameter, mh height, mhi, mh pseudocyst area, preoperative bcva, and age. all patients underwent standard 3 port 23 gauge pars plana vitrectomy with triamcinolone acetonide - assisted posterior vitreous detachment, if it was not already present. ilm removal were performed using 0.2 ml of dye brilliant blue g (brilliantpeel ; geuder, heidelberg). the area of removal of the ilm was intended to be 2 to 3 disc diameters surrounding the mh. fluid - air exchange through an extrusion cannula was performed to flatten the hole. the procedure was completed by an intraocular tamponade with 15% perfluoropropane (c3f8) or 20% sulfur hexafluoride (sf6). anatomic success was defined as the complete closure of the mh and absence of subretinal fluid on sd - oct. based on previous studies, 4 oct parameters were analyzed : mh basal diameter, mh minimum diameter, mh height, mhi, and a new parameter : the area of macular pseudocysts. basal hole diameter was defined as the hole diameter at the level of the rpe (figure 1). the mhi (hole height / basal hole diameter) was calculated according to a previously described method. the area of pseudocysts were calculated by a software option of sd - oct at the widest cross - section of the mh formation (figure 2). the borders of the pseudocysts were marked by the observer and the software programme gives the areas of the pseudocysts. measurements of parameters on oct. basal hole diameter (a) was defined as the hole diameter at the level of the retinal pigment epithelium. hole height (b) was the highest measurement from the rpe to vitreoretinal interface. oct = optical coherence tomography, rpe = retina pigment epithelium. the area of pseudocysts is calculated as in micrometer square at the widest cross section of the macular hole formation. the anatomical success was defined as the complete closure of the mh and absence of subretinal fluid on sd - oct. whitney test in terms of 5 sd - oct parameters : mh base diameter, mh minimum diameter, mh height, mhi, and mh pseudocyst area. the bcva values were converted to logarithm of the minimum angle of resolution (logmar) values for statistical analyses. the patients with preoperative and postoperative bcva were assessed with wilcoxon signed - rank test. spearman rank coefficient was calculated to assess correlation between anatomical closure and preoperative variables such as mh base diameter, mh minimum diameter, mh height, mhi, mh pseudocyst area, preoperative bcva, and age. baseline characteristics and oct parameters of study participants were presented in table 1. between study participants, 6 (30.0%) were male and 14 (70.0%) were female. five cases were stage 3 and 16 cases were stage 4 according to the gass classification. additionally, 3 of 5 stage 3 cases have vitreomacular traction. three patients developed significant cataract during follow - up and underwent phacoemulsification with an intraocular lens implantation. comparing the lens status between study groups, no significant impact on the anatomical outcome thereby no significant influence of a combined surgery on the anatomical outcome was found (p = 0.253, wilcoxon mann perfluoropropane (c3f8) was used in 16 patients and sulfur hexafluoride (sf6) was used in 5 patients as a tamponade. there was not a significant difference between the eyes in which c3f8 or sf6 were used as a tamponade regarding the anatomical outcome (p = 0.897, wilcoxon mann demographic characteristics, ophthalmic characteristics, and oct parameters the mean preoperative bcva was 0.86 0.29 logmar and improved to 0.64 0.28 logmar postoperatively (p = 0.004). in 14 out of 21 eyes (66%), bcva was improved by at least 1 early treatment diabetic retinopathy study line after surgery. in 6 eyes, bcva remained unchanged, and in 1 eye, bcva worsened. among the group of eyes who had unchanged bcva one of them had undergone to second surgery and 1 of them had refused second surgery. the patient who experienced a decrease in her visual acuity had an open mh and had refused the second surgery. primary and final anatomical success rate was 81% (17/21) and 90.5% (19/21), respectively. overall, 4 patients remained open mhs after first surgery and were suggested a second surgery (figure 3). two of them could not maintain prone position for 5 days postoperatively, and did not accept second surgery. case 21 ; mh pseudocyst area was 0.73 m and mh basal diameter was 2627 (top left) and postoperative 7. case 18 ; mh pseudocyst area was 0.25 m and mh basal diameter was 1053 (top right) and postoperative 4. mh = macular hole, sd - oct = spectral domain optical coherence tomography. mh basal diameter and mh pseudocyst area showed statistical significance between anatomical success and failure (p = 0.016 and p = 0.004, respectively). other variables such as mh minimum diameter, mh height, mhi, age, stage, and preoperative bcva and symptom duration showed no statistical significance (table 2). the anatomical closure is correlated with mh basal diameter and mh pseudocyst area (p = 0.01, r = 0.541, and p = 0.001, r = 0.652, respectively). also, there is a positive correlation between mh basal diameter and mh pseudocyst area (p = 0.02, r = 0.493). comparison of clinical characteristics and oct parameters between patients with anatomic success and anatomic failure postoperative bcva is correlated with mh basal diameter and mhi (p = 0.02, r = 0.488 p = 0.02, r = 0.485, respectively), but not with mh pseudocyst area (p = 0.61). also, spearman correlation rank coefficient between postoperative bcva and preoperative bcva is 0.421, but it is not statistically significant (p = 0.05). there is a tangential traction in the etiology of mh formation, induced by vitreous shrinkage as observed by gaudric. as a result of this traction, while posterior hyaloid is detaching, perifoveal pseudocysts may occur in the inner nuclear layer. the objective of this prospective study was to evaluate the effect of these pseudocysts on the anatomical outcome, therefore, we measured the area of the pseudocyst via the software of (spectralis oct, heidelberg engineering, heidelberg, germany). to our knowledge, this is the first study to assess the pseudocyst areas quantitatively and the anatomical outcome (from a pubmed and medline search in december 2013). brockman found that mh with the presence of perifoveal pseudocysts was associated with a 3-fold higher closure rate, but presence of perifoveal pseudocysts was assessed qualitatively. in our study, we found that mh with perifoveal pseudocysts was associated with anatomic failure (p < 0.05). in our study group, in addition, the mean mh basal diameter of these cases was 1794 and mh pseudocyst area was 0.5075 m. however, the mh basal diameter and mh pseudocyst area was 958 and 0.1824 m in the cases with anatomical success (figure 3). also, there was a correlation between mh basal diameter and mh pseudocyst area (r = 0.493 p < 0.05). in the light of these findings, while mh basal diameter was increasing, also mh pseudocyst area was increasing and mh perifoveal pseudocysts were statistically relevant with the anatomical outcome. in the study by brockman, only the presence of perifoveal pseudocysts was qualitatively assessed, and they used both stratus and cirrus oct (carl zeiss meditec, dublin, ca). in stratus oct, perifoveal pseudocyst may not be detected because the stratus oct takes 6 sections of the macula, so the perifoveal pseudocysts may not intersect at the sections that the device provides. as a result of this phenomenon, while evaluating the perifoveal pseudocysts, it may not be appropriate to use time domain oct. in the present study, also, the pseudocysts may be detected in three - dimensional view of the macula, but it is not possible to calculate the volume of these pseudocysts via the present software. in addition to mh perifoveal pseudocysts area, 4 oct parameters were analyzed : mh basal diameter, mh minimum diameter, mh height, and mhi. mh basal diameter and mhi were only 2 oct parameters that showed a statistically significant correlation with postoperative bcva, whereas no significant correlation was found regarding the other 3 oct parameters including mh perifoveal pseudocyst area. kusuhara reported that mhi significantly correlated with the postoperative bcva and also they postulated that mhi represents the preoperative configuration of a mh and is a prognostic factor for visual outcome. furthermore, similar results were reported about the correlation between mhi and postoperative bcva by ruiz - moreno. in our study, we did not find an effect of age, symptom duration, and preoperative bcva on the anatomical outcome. jaycock and ullrich also questioned the symptom duration, and they reported similar results with ours. we think that this variable is a subjective complaint, therefore, no statistically significant effect was found on the anatomical outcome. various studies have shown that ilmp during mh surgery is associated with higher anatomical outcomes. also ilmp had been performed in all our surgeries and our final anatomical success rate is 90.5%, which was similar with the other studies in stage 3 and 4 imhs. limitation of the present study includes the relatively small numbers of patients and mh perifoveal pseudocyst areas were calculated in two - dimensional way. strengths of the study include the fact that these mh perifoveal pseudocysts were first assessed in a quantitative way prospectively. in conclusion, we think that mh perifoveal pseudocysts were positively correlated with mh basal diameter and propose that it may be used as a prognostic factor for the anatomical outcome of mh surgery. we used two - dimensional measurements for the perifoveal pseudocysts, new software programmes may be enhanced for measuring the three - dimensional structure of the foveal pseudocysts with larger number of patients.	abstractto evaluate the effect of perifoveal pseudocysts on the anatomical outcomes of the idiopathic macular hole surgery as a prognostic factor.twenty-one eyes of 20 consecutive patients with a gass stage 3 or 4 idiopathic macular hole were enrolled in this prospective study between march 2012 and may 2013. demographic data, medical history, and ocular examinations were recorded preoperatively and on postoperative day 1, week 1, and month 1, 3, and 6. five spectral domain optical coherence tomography (sd - oct) parameters were analyzed : macular hole (mh) basal diameter, mh minimum diameter, mh height, macular hole index, and a new parameter, the area of macular pseudocysts via the software of sd - oct device at the widest cross section of the mh formation.the mean preoperative best - corrected visual acuity was 0.86 0.29 logarithm of the minimum angle of resolution (logmar) (between 0.4 and 1.3) and improved to 0.64 0.28 logmar (between 0.22 and 1.23) postoperatively (p = 0.004). there was a statistical significant difference between both mh basal diameter and mh pseudocyst area with anatomical success, respectively (p = 0.016 for mh basal diameter, p = 0.004 for mh pseudocyst area). the anatomical closure was correlated with mh basal diameter and mh pseudocyst area (p = 0.01 and p = 0.001, respectively). spearman correlation rank coefficient between with mh basal diameter and mh pseudocyst area was r = 0.493 and statistically significant (p = 0.02).perifoveal pseudocysts seem to be associated with anatomic failure and may be used as a prognostic factor in mh surgery.

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