Datasets:

danieladeeko
/

proccessed_ccdv

Dataset card Data Studio Files Files and versions Community

Split (3)

train · 120k rows

article stringlengths 0 682k	abstract stringlengths 146 3.69k
fundus examination revealed a round - shaped hypopigmented macular lesion including a few yellow spots in the fovea [fig. fa showed a macular transmission hyperfluorescent ring in which patchy hyperfluorescent and normofluorescent areas existed [fig. fundus autofluorescence imaging showed scattered patchy hyperautofluorescent areas matching the hypopigmented fundus lesion [fig. spectral - domain optical coherence tomography (sd - oct, spectralis oct, heidelberg engineering, heidelberg, germany) demonstrated hyper reflective accumulations (humps) involving rpe inner band and photoreceptor outer segments (oss). outer nuclear layer and inner segment / os (is / os) line was distorted because of the accumulations. multifocal electroretinogram (mferg, roland - consult retiscan system, wiesbaden, germany) showed decreased amplitude in the central 2.3. the p1 amplitude in the central response was 50.3 nv / deg [fig. arden ratios in electrooculogram (eog) were 2134 in the right eye and 1650 in the left eye. total error score in farnsworth - munsell-100 (fm-100) hue test was 35 in the right eye and 157 in the left eye. the patient reported a viral (possibly) upper respiratory tract infection 710 days prior to the visual loss. a probable diagnosis of arpe was suggested, and the patient was followed in next weeks. (a) fundus photograph demonstrating round - shaped hypopigmented macular lesion including a few yellow spots in the fovea. (c) fluorescein angiography showing a macular transmission hyperfluorescent ring in which pathcy hyperfluorecent and normofluorescent areas existed. (e) spectral - domain optical coherence tomography image showing hyperreflective accumulations (humps) involving rpe inner band and photoreceptor outer segments. (f) multifocal electroretinogram showing depressed central ring amplitude best - corrected visual acuity improved gradually up to 20/20 in the next 5 weeks with no treatment. there was almost no change in fa and fof imaging compared to the initial imaging [fig. 2c and d ]. sd - oct showed almost entirely restored is / os line almost no distortion in the outer nuclear layer. irregularities and humps decreased, but there were still some persisting hyper reflective spots in the rpe inner band and photoreceptor oss [fig. the p1 amplitude of the central response increased to 114 nv / deg in mferg [fig. arden ratios were 2080 in the right eye and 2071 in the left eye [fig. 3 ]. the patient reported significantly improved central vision in amsler grid testing when compared with the initial testing. total error scores in the fm-100 hue test was 42 in the right eye and 83 in the left eye. (e) spectral - domain optical coherence tomography image showing the decreased hyperreflective accumulations (f). multifocal electroretinogram showing normalized central ring amplitude electrooculogram recordings in the right (a) and left eye (b) during the initial examination and at the 5th week examination (c and d, respectively) the diagnosis of arpe depends on the presence of a characteristic fine pigment stippling in the macular area, at the level of the rpe, surrounded by yellow - white haloes of hypopigmentation. spectral - domain optical coherence tomography findings suggest that the initial lesion in arpe is possibly located at the level of the photoreceptor os and rpe. the disruption of photoreceptor is / os line with a wider disruption of the rpe inner band is almost typical in the reported cases. the rpe inner band is considered to be related with the verhoeff 's membrane that is constituted by tight junctions of rpe cells or with basal infoldings and apical processes of rpe cells. in our case, however, photoreceptor is / os line is discernible throughout the lesion. the main finding in our case was humps in the inner band of the rpe and photoreceptor oss. our findings support the speculation that arpe is a postinflammatory response of the os of photoreceptors and the rpe. eog explores the response of rpe to light and is a function of all rpe cells in the retina. that is probably related with the increased metabolic need of rpe cells in the macula with respect to rpe cells in the peripheral retina. the localized accumulation in sd - oct images likely reflects undigested oss of the photoreceptors. the diminished central amplitudes with progressively increasing amplitudes toward periphery in mferg confirm the functional deterioration of cone photoreceptors in the central retina. the electrophysiologic findings may mean that rpe cells in the peripheral retina can overcome the increased metabolic load although the rpe cells in the macula can not due to the high concentration of photoreceptors. our case showed that mferg recording was more correlated to visual acuity than structural imaging modalities (fa, fof, sd - oct). spectral - domain optical coherence tomography and mferg findings in our case support the hypothesis that rpe is possibly the initial site of involvement in arpe. mferg depression confirmed the functional involvement of cone photoreceptors in the disease process.	a 20-year - old man applied with vision loss in the left eye. right eye examination was unremarkable. best - corrected visual acuity (bcva) in the left eye was 20/200. fundus examination revealed a few yellow spots within a round - shaped macular lesion. autofluorescence imaging showed hyperautofluorescence in the lesion. central amplitudes in multifocal electroretinogram (mferg) were depressed. the patient reported a rhinopharyngitis 710 days before the visual loss. the patient was diagnosed as acute retinal pigment epithelitis. bcva improved gradually up to 20/20 in 4 weeks. mferg amplitudes returned to normal. a slight pigmentary distortion was the only residual fundus finding.
episodic angioedema with eosinophilia (eae), originally described by gleich. in 19841), is a syndrome characterized by recurrent episodes of angioedema, fever, leukocytosis, eosinophilia, elevated serum igm, increased body weight, and a benign clinical course lacking any internal organ involvement. many other cases have been detected since this initial report, and this syndrome has been recognized as a distinct clinical entity2, 3). more than 40 cases with symptomatic presentations and laboratory findings similar to eae have been reported, but these cases have not involved recurrent attacks, and thus have been subsequently designated as nonepisodic angioedema with eosinophilia (neae)4 - 8). the majority of these neae cases have been reported in japanese patients, with the exception of three cases reported in the korean literature7 - 9). this ethnic or regional distribution may be an important characteristic of neae. in this paper, we describe a patient whose clinical and laboratory features were similar to those observed in previous cases of neae, and also present our review of cases thus far reported in korea. a 26-year - old korean woman presented with edema of her hands and lower legs, which had developed 2 weeks prior to her hospital visit. her body weight had increased by 2 kg over those 2 weeks, and this was her first episode of the reported symptoms. the edema was distributed symmetrically on both of the patient 's upper and lower extremities, and was pitted. she denied any febrile sensation and had a pruritic rash on her lower legs (figure 1a-1c), and had also experienced mild joint pain in both knees 1 week after the onset of the edema. she also denied taking any drugs, including oriental medications, with the exception of one acetaminophen tablet (300 mg) 1 week prior to the onset of edema. laboratory tests revealed leukocytosis (12,250/mm ; normal range : 4,000 - 10,000/mm) with 51.5% eosinophils (normal range : 0~7%) in the peripheral blood. serum ige had increased to 1,423 iu / ml (normal range : 0 - 170 iu / ml), but iga, igg, and igm levels were all within normal limits. no abnormal findings were observed in the serum chemistry that would be reflective of renal or hepatic dysfunction. the rash on the patient 's lower legs was examined histologically, and identified as perivascular eosinophil infiltration (figure 2). lymphocyte phenotyping was conducted, but did not indicate the presence of any abnormal lymphocyte clones. we diagnosed the patient with neae, and prescribed an oral antihistamine to control the pruritus. one week after the patient began taking antihistamines, the edema and rash began to evidence improvements. the eosinophil count decreased to 1,530/mm after 2 weeks and to 736/mm after 8 weeks. the patient was free of symptoms at 4 months, and had an eosinophil count of 550/mm. we followed the patient for 18 months, and observed no recurrence of either the symptoms or the eosinophilia (the eosinophil count was 180/mm). our patient presented with eosinophilia with angioedema localized to the upper and lower extremities, and evidenced features typical of the neae cases thus far reported in japan. that is, the patient was a young female exhibiting a mild weight gain, and her condition lacked internal organ involvement, improved without corticosteroids, involved no increase in igm levels, and did not recur over the course of 18 months. eae is characterized by recurrent edema and requires the administration of corticosteroids, and has also been reported principally in europe and the united states. however, in japan and korea, more than 40 cases have been reported with clinical symptoms similar to those observed in eae, but without recurrent episodes ; these patients recovered without corticosteroid treatment4 - 8). first gave this distinct form of eae the designation neae4), which has been loosely identified as a less severe form of eae6). the most notable aspect of neae is the absence of recurrence, but several other differences also exist with regard to the clinical features of neae and eae. the angioedema is normally localized to the hands and the proximal portion of the lower legs, as in the present case, whereas in eae, the angioedema frequently extends farther down the extremities, sometimes including the face10). an increase in serum igm levels is also normally detected in cases of eae, but is seldom observed in neae6, 10). almost all eae patients require corticosteroid therapy, whereas spontaneous remission frequently occurs in patients suffering from neae. additionally, all neae patients thus far have been young females, as in the case described herein4 - 8). the cause of the sudden increase of eosinophils remains unknown, as with idiopathic hypereosinophilic syndrome. the identification of abnormal clonal cd3 cd4 cells in the blood of some eae sufferers11) indicates that eae and neae may be, in some if not all patients, a disease that involves abnormal t cell clones, similarly to the lymphocytic variant of hypereosinophilic syndrome12). the predilection to affect the skin with an absence of internal organ involvement in eae and neae is also a clinical feature reminiscent of the lymphocytic variant of hypereosinophilic syndrome12). in the case described herein, we conducted lymphocyte phenotyping in order to determine the presence of any aberrant clonal lymphocytes, as has been observed in cases of the lymphocytic variant of hypereosinophilic syndrome13). the angioedema of eae and neae is likely associated with a temporal increase in the vascular permeability of local capillaries via some mediators, including antiendothelial cell antibodies and cytokines3, 14 - 16). several reports have shown that histamine levels are within normal limits, and that il-1, il-5, and il-6 generated by monocytes, endothelial cells, t cells, and eosinophils increase during the acute phase of eae3, 5, 11, 15, 16). according to a recent report, il-5 levels were determined to be lower, and tnf-levels were higher in the acute phase of neae than in the acute phase of eae. thus, the cytokine profile may be related with the existence or absence of recurrent attacks5). recently, several cases of eae and neae have been reported in korea7 - 9, 17). table 1 shows these six cases of eae and neae, including the case described in this paper. cases 1, 2, and the present case were readily diagnosed as neae, whereas cases 3 and 5 were thought to be eae. in case 4, certain aspects, such as the elevated serum igm level and the necessity for corticosteroid therapy to control the symptoms, supported a diagnosis of eae. however, the presence of other aspects, including the fact that it occurred in a young female, the absence of weight gain, and the absence of a previous episode or recurrence, was suggestive of neae. this series of neae and eae case reports from korea demonstrates that neae and eae are not rare diseases within the korean population, and also suggests that neae may have a racial predisposition toward asian populations. in this work, we have described the case of a korean patient with neae and have reviewed five other cases of neae and eae occurring in korean patients. as in the japanese population, neae is not an uncommon disease within the korean population.	episodic angioedema with eosinophilia (eae) is characterized by recurrent angioedema, peripheral eosinophilia, elevated serum igm, fever, weight gain, and a benign course lacking any internal organ involvement. dozens of cases of the nonepisodic variant (neae), which is limited to a single attack, have been reported in japan. these neae cases normally have been less severe than the episodic type. in this paper, we describe the case of a korean patient whose clinical and laboratory findings were consistent with neae, and review five other cases of eae and neae reported in the korean literature. the korean neae cases outlined in this paper demonstrate that, as in japan, neae is not uncommon in korea, and also suggest that this disease exhibits a cultural predilection for asian populations.
antimicrobial agents are both a boon and threat to human health, with questions about their proper design, useful application, disposal and regulatory framework looming large for scientists, the medical community, regulators and consumers of antiseptic personal care products. in the late 1930s and early 1940s, it was discovered that the substitution on aromatic rings of hydrogen atoms with chlorine, yielded a novel chemistry of powerful biocides, including antimicrobials. the resultant synthetic organohalides, which are either absent or rare in natural environments, immediately were put to large volume, worldwide use as biocides. however, within a few years, many of these compounds and formulations showed adverse effects, including human toxicity, ecotoxicity, and unwanted environmental persistence and bioaccumulation, quickly leading to regulatory bans and phase - outs. for example, hexachlorophene, introduced in 1948 as a binuclear aromatic organohalide carrying six chlorine substituents, was banned from most uses by the 1970s. curiously, triclocarban (tcc) and triclosan (tcs), two persistent antimicrobials first introduced to commerce in 1957 and 1964, respectively, feature a very similar chemistry (i.e., two benzene rings carrying multiple chlorines) yet continue to be produced and consumed to this day at high volume. indeed, the consumption of tcs and tcc and the abundance of antimicrobial products have increased in the u.s. and abroad over the past two decades, due to relaxed regulation, aggressive and widespread advertising, and media reports driving fears of potent and sometimes lethal microbial infections acquired in everyday - life by unsuspecting victims. this multibillion dollar market has saturated supermarkets worldwide and vastly accelerated the consumption of antimicrobial products ; today, tcc and more so tcs can be found in soaps, detergents, clothing, carpets, paints, plastics, toys, school supplies, and even in pacifiers, with over 2000 antimicrobial products available in 2014s $ 1.4 billion u.s. despite labeling requirements, consumer awareness of harmful active ingredients in household products remains low. by contrast, tcc sees far more limited applications, mostly in bar soap formulated to concentrations of about 2% by weight, higher than the 0.10.5% content of tcs - enabled antimicrobial products. supermarket shelves, likely bring home a product containing either tcs or tcc. in 1999/2000, tcs or tcc were present in 75% of liquid soaps and 29% of bar soaps in the u.s. more than a decade into the accelerated use of polychlorinated aromatic antimicrobials, there now are unmistakable signs of these chemicals taking a toll on the health of the environment and possibly on susceptible human populations. environmental protection agency (epa), food and drug administration (fda), as well as the centers for disease control and prevention, and the european union. on the state - level, efforts have begun to curtail the use of antimicrobials after the discovery of tcs, tcc, and their dioxin - like chemical progeny in minnesota s treasured water resources. in parallel to the discovery of environmental pollution and new health risks of antimicrobials, concerns about the emergence of microbial pathogens resistant to multiple groups of antibiotics of medical import have triggered the need for reassessing the status quo of antimicrobial usage. the present feature article takes a look at the knowledge timeline concerning tcc and tcs, starting with their mid 20th century introduction into commerce and culminating with an assessment of today s information gaps as well as a glimpse of what the future may hold for the age - old chemical war on microbes. enabled by advances in analytical chemistry detection methods, most notably gas and liquid chromatography / mass spectrometry (gc - ms and lc - ms, respectively), tcs and tcc emerged as important environmental pollutants in disparate times and ways. triclosan a broadspectrum bacteriostat and fungicide garnered the attention of environmental chemists soon after its large volume use in the early 1970s. after its patenting in 1964 and worldwide production, tcs was detected within 14 years as an environmental contaminant, first in u.s. wastewater, river water, and sediment, and shortly thereafter in its methylated form, methyl - tcs, in fish from tokyo bay. these early and subsequent environmental detections of tcs were enabled by its amenability to gc - ms analysis. this changed in 2002, however, when the united states geological survey (usgs) reported tcs as one of the top 10 contaminants of american rivers in its first national reconnaissance of 95 pharmaceuticals, hormones, and organic wastewater contaminants. triclocarban a fungicide and bacteriostat with activity against methicillin - resistant staphylococcus aureus (mrsa) and vancomycin - resistant enterococci (vre)emerged as a contaminant of emerging concern (cec) much later, enabled by lc - ms rather than gc - ms detection techniques. contrary to tcs, tcc can not be analyzed by standard gc methods, thereby concealing for decades tcc s presence in environmental samples acquired, extracted and analyzed for the occurrence of anthropogenic pollutants. for tcc to travel through a standard gc column and relief from this conundrum arrived in 2004 with a simple lc - ms technique allowing direct detection of underivatized tcc. use of this tool on samples from baltimore, maryland, showed the presence of tcc in every urban stream monitored. when applied to the city s groundwater, drinking water, wastewater, and sewage sludge, tcc was detected in many of these matrices and consistently in samples also containing tcs. significant co - occurrence of tcc and tcs (r = 0.988) can be easily understood from their similar uses, chemical structures, and down - the - drain disposal mode. upon entering usgs national data on tcs into the forecasting algorithm, tcc emerged in 2005 as a previously unrecognized cec that had been overlooked by environmental analysts for almost half a century ; it was predicted to rank in the top 10 cecs in occurrence rate and in the top 20 in maximum concentration among 96 water pollutants. follow - up research using tandem mass spectrometry (lc - ms / ms) confirmed these predictions and adoption of lc - based analytical tools by laboratories around the world quickly accelerated the discovery of tcc pollution in the environment and in humans. today, tcs and tcc rank in the list of top contaminants of concern worldwide. streams have a 60100% likelihood of containing detectable quantities of tcs and tcc. tcs has been detected in drinking water resources, 75% of urine samples representative of the u.s. breast milk samples, and combined tcs and tcc constitute over 60% of the total mass of 96 pharmaceuticals detectable in municipal sludge using epa method 1694. indeed, the environmental ubiquity of both chemicals has escalated such that tcs, tcc or both compounds are now detectable in house dust worldwide, in ocean water, and locations as remote as the water loop of spacecraft. to understand this phenomenon of ubiquitous pollution, it is important to examine their production rates, distribution mechanisms, and long - term persistence upon environmental release. this behavior may be understood best when viewed through the lens of green chemistry and engineering. sustainably produced green chemicals serve their intended purpose without creating hazardous conditions for either people or the planet during chemical production, use, and following disposal. of particular concern for the epa are chemicals featuring one or multiple of the following characteristics : (i) persistence in the environment, (ii) bioaccumulation in animals and humans ; and (iii) toxicity to humans and ecosystems. as with other problematic chemicals, early warnings existed for decades concerning pbt properties of tcs and tcc, and the unsustainability of their large - volume uses. the cradle - to - grave life cycle of tcs and tcc can be characterized as an open loop that violates multiple principles of green chemistry and engineering. structurally related to highly toxic and carcinogenic dioxins, tcs had been labeled a predioxin as early as 1993 by the u.s. technical grade tcs contains traces of the most toxic member of the dioxin family, 2,3,7,8-tetrachlorodibenzo - p - dioxin (17.2 1,712 ng / kg), and 2,3,7,8-tetrachlorodibenzofuran (0.7 207.3 ng / kg). producers of antimicrobial products to source tcs from tightly monitored european chemical suppliers as opposed to lower - cost competitors in the asian markets. furthermore, mixing of tcs with chlorinated drinking water can result in the formation of carcinogenic chloroform and, upon release into surface water and irradiation with sunlight, of additional toxic polychlorinated dioxins and less toxic dichlorinated dioxins, for example, 2,8-dichlorodibenzo - p - dioxin. similarly, tcc also contains toxic, carcinogenic manufacturing byproducts, such as 4-chloroaniline and 3,4-dichloroaniline, and can release more of these carcinogens upon chemical, physical, and biological attack. durations of utility, i.e., useful lifespans, of tcs and tcc in personal care products are short, on the order of seconds, but their environmental after - lives are much longer, measured at time - scales of up to several decades. upon disposal by consumers, both compounds are washed down the drain and typically are conveyed to municipal wastewater treatment plants (wwtps). these facilities remove both tcs and tcc from raw sewage at a high efficiency of 9798%, leading to low ng / l levels in effluent discharged to surface waters. however, removal from sewage does not necessarily equal degradation. during wastewater treatment, both antimicrobials distribute themselves preferentially into carbon- and lipid - rich sewage sludge, thereby accumulating in this abundant byproduct of biological sewage treatment. during anaerobic sludge digestion, losses can occur as a result of biodegradation of tcs and tcc but concentrations also may increase due to a reduction in volume by gasification of natural organics to methane. levels of tcs and tcc in digested sewage sludge as high as 133 and 441 mg / kg dry weight, respectively, have been reported by the epa ; however, mean concentrations are closer to 16 65 and 39 59 mg / kg dry weight (standard deviation), respectively. wwtps in substantial quantities (227 000454 000 kg / y for tcc and 170 000970 000 kg / yr for tcs) are known to break through wwtps and subsequently can harm algae in surface waters at ng / l concentrations. detected concentrations have been observed to exceed an acute - based predicted no - effect concentration (pnec) of 4.7 ng / l in the river elbe at 75% of monitoring locations, and can accumulate in sediments to mg / kg levels, where they may persist for several decades. in the u.s., sewage sludge is either incinerated (15% of total volume) which can release more carcinogenic dioxins from tcs, or deposited in landfills (30%) and on land (55%), from where antimicrobials and their carcinogenic transformation products may leach into adjacent surface water to impact the composition of microbial communities. antimicrobials applied as sewer sludge on land constitute a pathway for transfer of these chemicals into animal feed and crops destined for human consumption. the volume of antimicrobials reentering the environment in sewage sludge after initial successful capture from wastewater is substantial ; 57 000 233 000 and 140 000 211 000 kg / yr of tcs and tcc, respectively, are applied on u.s. land annually ; for tcc, this is equivalent to a staggering 4.848.2% of its total u.s. crops shown to take up antimicrobials from soil include barley, meadow fescue, carrots, and pinto beans. human exposure to antimicrobials occurs mostly as a result of elective topical application to the human body. showering for 15 min with a 0.6% tcc containing antimicrobial soaps was demonstrated to lead to concentrations in the blood of volunteers sufficiently high to potentially cause local inhibition of enzyme soluble epoxide hydrolase. use of tcs - containing toothpaste, typically formulated to 0.3% by weight, is another important source of human exposure. other known or suspected human exposure routes of lesser importance include the inhalation of antimicrobial - laden house dust, consumption of contaminated drinking water, and ingestion of food contaminated with antimicrobials either during the growing season or postharvesting from antimicrobial - containing packaging materials. unsuspected environmental exposures to tcs and tcc have attracted attention by news media and the general public but the magnitude of these exposures is easily eclipsed by elective, topical use of antimicrobial personal care products. tcs and tcc are known toxicants but there still is a paucity of data on adverse effects in humans from elective and incidental environmental exposures. europe emphasized the need for caution but remain an anomaly, caused by misuse of antimicrobials in conditions not applicable to present day uses. acute and chronic health effects of tcs and tcc observed in humans and animals following exposure include irritation of eyes and skin, sensitization to aeroallergens and food, immunologic reactions such as allergies, developmental and reproductive toxicity, inhibition of muscle function, as well as in vivo genotoxicity. while limited, the number of studies involving human subjects is increasing. an emerging additional toxic outcome of concern is endocrine disruption, meaning an interfering of tcs and tcc with essential signaling systems in animals and humans, thereby adversely affecting development, sexual maturation, metabolism, and behavior. endocrine disruption was observed after exposure of male rats to tcc, of rats to tcs, and of frogs to tcs. of particular human health concern are the adverse effects of tcs on thyroid homeostasis and of tcc on reproductive health. a long recognized potential human health threat of antimicrobials is their ability to induce cross - resistance to medically important antibiotics in human pathogens and commensal microbes, thereby turning environmental microbial communities into a reservoir of antibiotic drug resistance. concerns about tcs - induced cross - resistance to antibiotics used in human medicine were voiced as early as 2001 and have since been substantiated by scientists worldwide. whereas tcs resistance can decrease susceptibility to as many as seven antibiotics simultaneously, the applicability of such data to environmental settings and the actual risk remain uncertain. available studies concentrated on household settings rather than on environmental locales, where the development and proliferation of drug resistance is more likely. one such unexplored locale is sewage sludge, where an abundance of pathogens, multiple antimicrobials and extended contact times creates a large and risky setting for the emergence of drug resistance. ecotoxicological risks also result for other biota enduring antimicrobial contact times that are infinitely longer than the few seconds these persistent antimicrobials reside on consumers hands during their intended use. these unwanted long - term exposures of biota to high concentrations of antimicrobials take place in environments not targeted for disinfection. in the built water environment, for example, inputs and accumulation of antimicrobials in activated sludge units during wastewater treatment are of potential concern, as it may diminish treatment efficacy and microbial diversity while also potentially creating reservoirs of drug resistance. similar risks also exist in soil environments subject to the application of biocide - laden sewage sludge. here, as mentioned earlier, the proximity of large quantities of commensal and pathogenic bacteria with extremely high levels of antimicrobials is of particular concern, as is the uptake of the compounds into higher organisms, such as plants and animals. natural environments also feature multiple compartments where unwanted antimicrobial residues come in immediate and long - term contact with fauna and flora. here, the native, multicellular biota are known to be orders of magnitude more susceptible to the killing power of antimicrobials than are microorganisms. contrary to the situation described for hand washing (exposure times of a few seconds), these environmental toxic exposures are not temporal, but rather extend over the entire lifespan of aquatic and terrestrial organisms and across multiple generations. tcs and tcc are 1001000 times more effective in inhibiting and killing algae, crustaceans and fish than they are in killing microbes. shallow sediments in surface waters receiving treated wastewater inputs are known to contain high g / kg to low mg / kg quantities of tcs and tcc, levels that make impossible the survival and activity of many different species. sediments also represent a latent source of antimicrobials and can release the compounds back into the water column upon disturbance. application of sewage sludge in forestry and nonagricultural settings also can lead to decade long exposure of plants, soil - dwelling biota and their predators over multiple generations. bioconcentration, bioaccumulation and biomagnification of antimicrobials have been observed in multiple organisms, including algae, aquatic blackworms, fish, and even dolphins, whereas affected terrestrial organisms include earth worms and higher species up the food chain. documented accumulation of antimicrobials in worms and plant material and subsequent uptake by higher organisms is a known pathway for ecological risks from exposure of vertebrae, including songbirds. bioaccumulation of antimicrobials also occurs in humans but to a much lesser extent, because well - known detoxification reactions result in the rapid elimination of parental tcs and tcc. despite this, the need for continuous elimination of antimicrobials by the human detoxification machinery has been speculated to potentially prevent expulsion of more harmful agents, such as dioxins, but scientific data are lacking. although tcs and tcc are effective in killing microorganisms when applied judiciously by professionals in health care settings, their proliferating use by the general population, which accounts for the vast majority of the chemicals production volume, lacks convincing data on health benefits, according to epidemiological studies. these seemingly contradictory findings between antimicrobials efficacy in clinical settings and their failure to perform in household settings can be understood easily when considering the contact time between the chemicals and their microbial targets. thoroughly designed clinical studies reproducibly yield favorable results from hand washing times of 30 s to several minutes. however, hand - washing routines of the general population differ significantly from this optimal standard. in real - world settings, the application of soaps on the hands of consumers is followed immediately by rinsing away of the active antimicrobial ingredients. thus, for the majority of household consumers, effective contact times amount to an average of six seconds, too short to provide a measurable impact on antimicrobial efficacy. in 2005, an expert panel convened by the fda had concluded by a vote of 11-to-1 that use of antiseptics does not provide a measurable benefit to consumers. this assessment apparently has not changed in years since, as the fda has issued in late 2013 a notice to industry of its intent to institute tighter regulations in the near future. in the u.s., regulating tcs and tcc has been challenging over the course of the past half century, due in part to the desire to cover multiple uses and multiple compounds under a single umbrella guidance document, namely the topical antimicrobial drug products over - the - counter (otc) drug monograph of the fda (figure 1). this regulation was first drafted in 1974, tentatively finalized in 1978, and updated in 1994 but never finalized. in 2010, the natural resources defense council (nrdc) filed a complaint against the fda in an effort to force the agency to act. this legal action culminated in a consent decree, with the fda agreeing in 2013 to finalize the monograph, at least with respect to tcs. the year 2014 marks the 40th anniversary of issuance of the yet to be finalized initial draft legislation (figure 1). in 1972, in contrast, the fda had acted much more swiftly, by banning the antimicrobial hexachlorophene over concerns of its neurotoxicity. at the time, hexachlorophene - containing personal care products had multiplied in the market similar to tcs - containing formulations today and adverse effects including accumulation in breast milk also had been reported for hexachlorophene. technically, the fda could regulate tcs and tcc over environmental concerns alone but such action would be without precedence ; instead, the fda has deferred to the epa, which regulates tcs but not tcc as registered pesticides under the federal insecticide, fungicide, and rodenticide act (fifra). timeline of scientific and regulatory events concerning the use and occurrence of triclosan (tcs) and triclocarban (tcc) in the united states, with particular emphasis on the tentative final monograph (tfm) of the food and drug administration (fda). so who should use antimicrobials ? for what purpose ? and what is the acceptable extent of collateral damage to ecological health and human populations ? answering these questions should best be left to public health experts, physicians, risk assessors, and sustainability scientists. sixty years into the use of polychlorinated binuclear aromatic antimicrobials, multiple lessons can be learned from the past. hexachlorophene was responsible for the first bloom of antimicrobial products, giving rise to over 400 hexachlorophene - containing personal care products ; this episode lasted only a few years, though, before this active ingredient was banned over concerns of its neurotoxicity. the second bloom in u.s. antimicrobial products from a few dozens to the current count of > 2000 was triggered by the fda s removal of antimicrobial soaps from the drug category of the tentative final monograph (tfm) in 1994 (figure 1). this history suggests that regulatory boundaries are critical in preventing imprudent uses of potentially harmful substances in personal care products. restricting nonmedical uses of tcs and tcc is an approach championed by diverse scholars and health care professionals, including the american medical association (ama), the alliance for the prudent use of antibiotics (apua), an expert group of the american academy for microbiology, and members of the american public health association (apha). any known and potential adverse effects of the usage of antimicrobials should be balanced with immediate and measurable benefits reaped. with respect to tcs and tcc, scientific evidence points to known benefits from their application in health care settings by health care professionals, and possibly from tcs - containing toothpaste used by individuals diagnosed with gingivitis. exclusive sale of tcs / tcc - containing soaps in pharmacies and prescription requirements for tcs in toothpaste may aid in effecting the desirable reduction in unsustainable consumption patterns and with it associated adverse effects. this tiered approach worked well for the now restricted hexachlorophene, whose allowable and prudent applications continue to this date, as a preservative at concentrations of up to 0.1% by weight. regulations proved effective in throttling back hexachlorophene production ; today, the compound is present at levels below the detection limit in u.s. wastewaters, detectable only at low concentrations (0.180.37 mg / kg dry weight) in raw and treated sewage sludge, where it accumulates similarly to tcs and tcc. the question of what collateral damage to people and the planet is acceptable will be informed not only by cost - benefit analyses but also by broader sustainability considerations. evidence abounds for tcs and tcc to represent nongreen chemicals whose current usage volumes are unsustainable, as indicated by large - scale pollution that needlessly places stress on the environment, animals and human populations. these findings suggest the need for next - generation antimicrobials to overcome some of the identified shortcomings of tcs and tcc, while preserving their essential benefits. so what will greener, more sustainable antimicrobials of the future look like ? desirable properties of next - generation antimicrobial include broad - spectrum action and high efficacy toward pathogens but low toxicity to nontarget, multicellular organisms, including aquatic and terrestrial biota and humans. furthermore, future - use antimicrobials should have no or very low potential for fostering antimicrobial drug resistance, should undergo rapid biodegradation in conventional wastewater treatment plants, and pose no risk of bioaccumulation. ideally, the compounds also should be sourced from renewable feedstock and lack occupational hazards during production, storage, and use. upon disposal they should return their benign elemental building blocks to the environment, to complete a more environmentally friendly cradle - to - cradle life - cycle. sustainability considerations already are informing the design of green pharmaceuticals, and adopting this approach for antimicrobials promises to yield important benefits to people and the planet.	the polychlorinated aromatic antimicrobials triclosan and triclocarban are in widespread use for killing microorganisms indiscriminately, rapidly, and by nonspecific action. while their utility in healthcare settings is undisputed, benefits to users of antimicrobial personal care products are few to none. yet, these latter, high - volume uses have caused widespread contamination of the environment, wildlife, and human populations. this feature article presents a timeline of scientific evidence and regulatory actions in the u.s. concerning persistent polychlorinated biocides, showing a potential path forward to judicious and sustainable uses of synthetic antimicrobials, including the design of greener and safer next - generation alternatives.
severe fever with thrombocytopenia syndrome (sfts) is an emerging tick - borne disease that was first reported in china in 2011. there have been 2,047 cases reported in china, while 53 cases and 36 cases have been reported in japan and korea, respectively. it is known that sfts occurs from may to august when the tick 's activity is intense. however, in korea, sfts patients are increasing in september when people are mowing the grass around graves on the mountain. chief complaints include fever, thrombocytopenia, leukopenia, nausea, and vomiting. in a rapidly progressing sfts, the fatality rate becomes 6% to 30%, and it is accompanied by multiple organ failure, bleeding tendency, and altered mentality. supportive treatment, such as transfusion, renal replacement therapy, and empirical antibiotics, has been the basis of treatment for sfts. china has been using intravenous ribavirin as a guideline for the treatment of sfts since 2012. there was a case wherein a patient of advanced age had a high probability of poor prognosis accompanied by clinical features, such as central nervous system (cns) manifestation, bleeding tendency, disseminated intravascular coagulation, and multiple organ failure. we are reporting two patients who showed cns manifestation, and we treated them with ribavirin along with plasma exchange at an early stage. a 60-year - old male mowed the grass near a grave on a mountain 3 weeks before the hospital admission, and then he developed and lower abdominal rash, fever, chills, myalgia, cough, and mucopurulent sputum 1 week before the hospital admission. he visited the local hospital 2 days before the admission, and the blood test showed abnormal liver function test and thrombocytopenia. on admission to the hospital, his blood pressure was 110/70 mmhg, pulse rate was 75/min, respiratory rate was 20/min, and body temperature (tympanic membrane) was 37.3c. there was no eschar on the skin ; however, a lower abdominal popular rash has developed. he had a 1.0 cm right inguinal lymph node, which was movable and non - tender. laboratory findings revealed white blood cell (wbc) 2,510/mm, hemoglobin (hb) 13.6 g / dl, platelet 49,000/mm, na / k 122/3.6 meq / l, c - reactive protein (crp) 1.05 mg / dl, aspartate transaminase (ast)/alanine transaminase (alt) 188/88 u / l, lactate dehydrogenase (ldh) 1,136 u / l, creatinine kinase - mb (ck - mb) 3.4 ng / ml, activated partial thromboplastin time (aptt) 50.1 sec, international normalized ratio (inr) 1.12, and d - dimer 17.06 mg / l. urinalysis revealed red blood cell (rbc), 10 - 20/ high - power - field (hpf), and albumin 2 +. multiple enlarged lymph nodes (size 0.7 - 1.0 cm) on the abdomen ct was observed in the right common iliac area and right inguinal area. clinical features and sftsv real - time rt - pcr was taken by using viral gene - spin viral dna / rna extraction kit (intron, seongnam - si, gyeonggi - do, korea) for rna isolation and using diastar 2x onestep rt - pcr pre - mix (solgent, daejeon, korea) for rna detection. intravenous ceftriaxone (2.0 g qd) injection was started as empirical antibiotics for fever, and doxycycline (100 mg bid) was administered orally. the blood test indicated deterioration, which revealed wbc 1,960/mm, platelet 37,000/mm, ast / alt 1,813/477 u / l, and ldh 2,462 empirical antibiotics have been replaced by intravenous piperacillin / tazobactam (4.0 g/0.5 g tid), and plasma exchange was started due to a suspicion of sfts accompanied by cns manifestation (based on weight and hematocrit, approximately 2800 ml for 120 min, 23 ml / min, lumen size 11.5 fr, cobe spectra apheresis system, lakewood, co, usa). on the 6 day of his hospital we thought of the possibility of acute pulmonary distress syndrome (ards) or pulmonary edema, secondary bacterial pneumonia, thus antibiotics were replaced by intravenous meropenem (1.0 g tid), and ventilator treatment was performed. the sftsv real - time rt - pcr result confirmed positive, and ribavirin 2,000 mg (30 mg / kg) loading dose was started on the 6 day of his hospital stay. on the 7 day of his hospital stay, laboratory findings revealed wbc 6,110/mm, hb 8.8 g / dl, platelet 71,000/mm, and ast / alt 33/27 his fraction of inspired oxygen (fio2) of ventilator remained in stable status, thus the plasma exchange was discontinued and ribavirin 1,000 mg (15 mg / kg qid) was tapered orally. on the 10 day of his hospital stay, ribavirin 500 mg (7.5 mg / kg qd) was tapered orally. on the 13 day of his hospital stay, mental instability has improved, and the patient recovered consciousness and good breathing. the ventilator treatment was discontinued, and antibiotics were de - escalated by intravenous moxifloxacin (400 mg qd). on the 16 day of his hospital stay, on the 18 day of his hospital stay, the patient was discharged in improved condition (fig. a 48-year - old male is a farmer who had a 10-day history of fever, chills, myalgia, and diarrhea, which appeared 10 days prior to his hospital visit. he had diabetes and also had a 45-pack - year smoking history. on hospital admission, the patient 's blood pressure was 130/80 mmhg, pulse rate was 80/min, respiratory rate was 20/min, and body temperature (tympanic membrane) was 38.0c. / k 132/3.6 meq / l, crp 1.01 mg / dl, ast / alt 110/38 u / l, ldh 983 u / l, creatinine phosphokinase (cpk) 268 u / l, prothrombin time (pt) 12.5 sec, aptt 47.7 sec, and inr 1.13. sftsv real - time rt - pcr was conducted on admission day and on hospital day 3, which were both confirmed positive. abdomen ct was conducted as he had a history of diarrhea, and the result showed fatty liver and mild splenomegaly. sftsv real - time rt - pcr were taken using the same method as described above on admission day. intravenous ceftriaxone (2.0 g qd) and oral doxycycline (100 mg bid) were administered as empirical antibiotics for the fever. on the 2 day of his hospital stay, high fever (38.3c, tympanic membrane) has continued and the blood test result indicated deterioration, showing wbc 2,030/mm, hb 16.1 g / dl, platelet 43,000/mm, ast / alt 252/52 u / l, ldh 2,764 u / l, and cpk 592 u / l. the patient experienced a confused mental state, and he was transferred to intensive care unit. we performed brain non enhancement ct and brain diffusion magnetic resonance imaging, and consulted with department of neurology. his antibiotics were replaced by intravenous piperacillin / tazobactam (4.0 g/0.5 g tid). on the 3 day of his hospital stay, the sftsv real - time rt - pcr was confirmed positive. ribavirin 2,600 mg (30 mg / kg) loading dose and 1,200 mg (15 mg / kg qid) maintenance dose were administered orally, and plasma exchange was performed (based on weight and hematocrit, approximately 3400 ml for 150 min, 22 ml / min, lumen size 11.5 fr, cobe spectra apheresis system, lakewood, co, usa). on the 4 day of his hospital his laboratory findings revealed wbc 4,300/mm, hb 13.8 g / dl, platelet 44,000/mm, ast / alt 122/35 u / l, ldh 1,181 u / l, and cpk 461 u / l. on the 6 day of his hospital stay, laboratory findings revealed wbc 3,600/mm, hb 13.3 g / dl, platelet 59,000/mm, ast / alt 101/42 u / l, ldh 768 u / l, and cpk 492 u / l. due to the remarkable improvement of the patient 's clinical course, plasma exchange and ribavirin treatment were all discontinued on the 6 day of his hospital stay. and de - escalation of antibiotics was done by intravenous levofloxacin (750 mg qd). on the 10 day of his hospital stay, the patient showed improved condition ; therefore, the antibiotics were discontinued. on the 14 day of his hospital stay, the patient 's mental state has fully recovered, and he was discharged on the 17 day of his hospital day (fig. a 60-year - old male mowed the grass near a grave on a mountain 3 weeks before the hospital admission, and then he developed and lower abdominal rash, fever, chills, myalgia, cough, and mucopurulent sputum 1 week before the hospital admission. he visited the local hospital 2 days before the admission, and the blood test showed abnormal liver function test and thrombocytopenia. on admission to the hospital, his blood pressure was 110/70 mmhg, pulse rate was 75/min, respiratory rate was 20/min, and body temperature (tympanic membrane) was 37.3c. there was no eschar on the skin ; however, a lower abdominal popular rash has developed. he had a 1.0 cm right inguinal lymph node, which was movable and non - tender. laboratory findings revealed white blood cell (wbc) 2,510/mm, hemoglobin (hb) 13.6 g / dl, platelet 49,000/mm, na / k 122/3.6 meq / l, c - reactive protein (crp) 1.05 mg / dl, aspartate transaminase (ast)/alanine transaminase (alt) 188/88 u / l, lactate dehydrogenase (ldh) 1,136 u / l, creatinine kinase - mb (ck - mb) 3.4 ng / ml, activated partial thromboplastin time (aptt) 50.1 sec, international normalized ratio (inr) 1.12, and d - dimer 17.06 mg / l. urinalysis revealed red blood cell (rbc), 10 - 20/ high - power - field (hpf), and albumin 2 +. multiple enlarged lymph nodes (size 0.7 - 1.0 cm) on the abdomen ct was observed in the right common iliac area and right inguinal area. clinical features and sftsv real - time rt - pcr was taken by using viral gene - spin viral dna / rna extraction kit (intron, seongnam - si, gyeonggi - do, korea) for rna isolation and using diastar 2x onestep rt - pcr pre - mix (solgent, daejeon, korea) for rna detection. intravenous ceftriaxone (2.0 g qd) injection was started as empirical antibiotics for fever, and doxycycline (100 mg bid) was administered orally. the blood test indicated deterioration, which revealed wbc 1,960/mm, platelet 37,000/mm, ast / alt 1,813/477 u / l, and ldh 2,462 empirical antibiotics have been replaced by intravenous piperacillin / tazobactam (4.0 g/0.5 g tid), and plasma exchange was started due to a suspicion of sfts accompanied by cns manifestation (based on weight and hematocrit, approximately 2800 ml for 120 min, 23 ml / min, lumen size 11.5 fr, cobe spectra apheresis system, lakewood, co, usa). on the 6 day of his hospital we thought of the possibility of acute pulmonary distress syndrome (ards) or pulmonary edema, secondary bacterial pneumonia, thus antibiotics were replaced by intravenous meropenem (1.0 g tid), and ventilator treatment was performed. the sftsv real - time rt - pcr result confirmed positive, and ribavirin 2,000 mg (30 mg / kg) loading dose was started on the 6 day of his hospital stay. on the 7 day of his hospital stay, laboratory findings revealed wbc 6,110/mm, hb 8.8 g / dl, platelet 71,000/mm, and ast / alt 33/27 his fraction of inspired oxygen (fio2) of ventilator remained in stable status, thus the plasma exchange was discontinued and ribavirin 1,000 mg (15 mg / kg qid) was tapered orally. on the 10 day of his hospital stay, ribavirin 500 mg (7.5 mg / kg qd) was tapered orally. on the 13 day of his hospital stay, mental instability has improved, and the patient recovered consciousness and good breathing. the ventilator treatment was discontinued, and antibiotics were de - escalated by intravenous moxifloxacin (400 mg qd). on the 16 day of his hospital stay, on the 18 day of his hospital stay, the patient was discharged in improved condition (fig. a 48-year - old male is a farmer who had a 10-day history of fever, chills, myalgia, and diarrhea, which appeared 10 days prior to his hospital visit. he had diabetes and also had a 45-pack - year smoking history. on hospital admission, the patient 's blood pressure was 130/80 mmhg, pulse rate was 80/min, respiratory rate was 20/min, and body temperature (tympanic membrane) was 38.0c. / k 132/3.6 meq / l, crp 1.01 mg / dl, ast / alt 110/38 u / l, ldh 983 u / l, creatinine phosphokinase (cpk) 268 u / l, prothrombin time (pt) 12.5 sec, aptt 47.7 sec, and inr 1.13. sftsv real - time rt - pcr was conducted on admission day and on hospital day 3, which were both confirmed positive. abdomen ct was conducted as he had a history of diarrhea, and the result showed fatty liver and mild splenomegaly. sftsv real - time rt - pcr were taken using the same method as described above on admission day. intravenous ceftriaxone (2.0 g qd) and oral doxycycline (100 mg bid) were administered as empirical antibiotics for the fever. on the 2 day of his hospital stay, high fever (38.3c, tympanic membrane) has continued and the blood test result indicated deterioration, showing wbc 2,030/mm, hb 16.1 g / dl, platelet 43,000/mm, ast / alt 252/52 u / l, ldh 2,764 u / l, and cpk 592 u / l. the patient experienced a confused mental state, and he was transferred to intensive care unit. we performed brain non enhancement ct and brain diffusion magnetic resonance imaging, and consulted with department of neurology. his antibiotics were replaced by intravenous piperacillin / tazobactam (4.0 g/0.5 g tid). on the 3 day of his hospital stay, the sftsv real - time rt - pcr was confirmed positive. ribavirin 2,600 mg (30 mg / kg) loading dose and 1,200 mg (15 mg / kg qid) maintenance dose were administered orally, and plasma exchange was performed (based on weight and hematocrit, approximately 3400 ml for 150 min, 22 ml / min, lumen size 11.5 fr, cobe spectra apheresis system, lakewood, co, usa). on the 4 day of his hospital stay, his condition has improved. his laboratory findings revealed wbc 4,300/mm, hb 13.8 g / dl, platelet 44,000/mm, ast / alt 122/35 u / l, ldh 1,181 u / l, and cpk 461 u / l. on the 6 day of his hospital stay, laboratory findings revealed wbc 3,600/mm, hb 13.3 g / dl, platelet 59,000/mm, ast / alt 101/42 u / l, ldh 768 u / l, and cpk 492 u / l. due to the remarkable improvement of the patient 's clinical course, plasma exchange and ribavirin treatment were all discontinued on the 6 day of his hospital stay. and de - escalation of antibiotics was done by intravenous levofloxacin (750 mg qd). on the 10 day of his hospital stay, the patient showed improved condition ; therefore, the antibiotics were discontinued. on the 14 day of his hospital stay, the patient 's mental state has fully recovered, and he was discharged on the 17 day of his hospital day (fig. sfts is an emerging tick - borne disease which normally occurs in korea from may to august. there have been constant reports after the first patient was identified through the retrospective isolation of the virus in may 2013. laboratory confirmation of sftsv is established through the isolation of sftsv in cell culture, viral identification through the rt - pcr or dh82 cells from the patient 's serum, or serologic detection of 4-fold increase in anti - sfts virus immunoglobulin g titers between acute and convalescent phases. laboratory diagnosis takes times so long from several days to weeks, even if rt - pcr takes approximately minimum 5 - 7 days to know the results from korean centers for disease control and prevention. thus on the basis of the clinical manifestation performing a diagnostic test as soon as possible is most important thing. supportive care, such as transfusion of fresh frozen plasma or platelet for hematologic abnormalities, methylprednisolone for acute lung injury or ards, albumin replacement for hypoalbuminemia, intravenous immunoglobulin for severe infection or encephalitis, granulocyte colony stimulating factor for leukopenia, and antibiotics for bacterial superinfection, would be the most important part of the treatment process. the antiviral effect of ribavirin and plasma exchange can be considered in sfts patients with poor prognosis factor, such as old age, altered mentality, high level of ldh (> 1200 u / l), and cpk (> 800 u / l), and progression to multiple organ failure in spite of traditional supportive care. in korea, two cases reported that early plasma exchange and ribavirin administration were applied for the treatment of rapidly progressive sfts. a 66-year - old farmer and a 62-year - old farmer were transferred from community hospital to tertiary care hospital due to septic shock. positive sftsv real - time rt - pcr and clinical manifestation such as low blood pressure, thrombocytopenia were indication of combination of oral ribavirin and plasma exchange. our treatment is done based on the above cases. in our cases, among clinical manifestations associated with fatal outcome, cns involving symptoms such as confused or altered mentality were remarkably shown. our patients had common history of the exposure to a rural environment by occupation or by accident in endemic season. for the better prognosis, physicians need to have a suspicion from the characteristic clinical manifestations and epidemiologic history, and make early decision when to start antiviral treatment and plasma exchange. since 2012, intravenous ribavirin has been introduced for the treatment of sfts in china because it has been proven that ribavirin is as effective on the crimean - congo hemorrhagic fever (cchf) virus, bunyaviridae, as the sftsv, and ribavirin exerts its antiviral effects through various mechanisms, including the reduction of viral rna - dependent rna polymerase activity, mutagenesis in the viral genome, inhibition of rna capping, reduction of cellular inosine monophosphate dehydrogenase activity, and modulation of the host immune response. since then, ribavirin has been used for the sfts treatment in korea and abroad. intravenous ribavirin has some advantages that time to therapeutic effect is relatively faster than oral ribavirin and it can be used for patients who are not allowed to be administered orally. but it did not show significant difference between intravenous ribavirin and oral ribavirin for treatment cases in other viral infections, such as respiratory syncytial virus, cchf. although antiviral effect of ribavirin on sfts was confirmed only in vitro, because of previously reported positive efficacy of ribavirin and oral ribavirin 's inexpensive price, we are able to use oral ribavirin as treatment for severe sfts and it may be currently the only treatment medication. it has been known that several cytokines (interleukin (il)-1, il-8, macrophage inflammatory protein 1, and interferon - gamma) are elevated in patients with sfts, and the serum levels of cytokines are correlated with serum viral loads and disease severity. as a result, the cytokine - mediated inflammatory response may play an important role in the disease progression in patients with sfts. based on this theory, several cases have reported that the plasma exchange were able to remove the immune complexes correlated with disease severity, such as cytokines, reduce serum cytokines, and improve the clinical courses in patients with ards or hemodynamic shock. however, there are some limitations of the combination of plasma exchange and ribavirin. plasma exchange is not guaranteed by health insurance review and a few hospitals can do this technically. in addition, there are side effects such as hypocalcemia, hypokalemia, severe anaphylactic reactions, and transfusion related acute lung injury. for ribavirin, only oral administration is approved in korea, we can not use for patients who are impossible to be administered orally. the significant adverse effects of the ribavirin, such as reversible hemolytic anemia can occur. moreover, there were only a few cases reported that were treated with both route of administration. in order to establish how ribavirin and plasma exchange works on sfts patients, further in - depth research on its efficacy	severe fever with thrombocytopenia syndrome (sfts) is an emerging tick - borne disease. the primary symptoms associated with sfts are fever, thrombocytopenia, leukopenia, nausea, and vomiting. disease progression shows high mortality rate accompanied with multiple organ failure, bleeding tendency, and altered mentality. however, only supportive care has been the basis for the treatment of sfts. we are reporting two patients who showed central nervous system manifestation, but cured them with ribavirin together with plasma exchange in an early state. the first case is a 60-year - old male, who was admitted to the hospital with a 7-day history of fever, chills, and thrombocytopenia. he was treated with empirical antibiotics ; however, he experienced persistent high fever and an altered mentality has occurred. on hospital day 6, the sfts virus (sftsv) result from a real - time reverse transcription - polymerase chain reaction (rt - pcr) was confirmed positive. therefore, ribavirin (30 mg / kg as initial loading dose, 15 mg / kg qid for 4 days and then 7.5 mg / kg qid as maintenance dose) was administered orally for 11 days and plasma exchange was performed for 5 days. the clinical outcome has improved. the second case is a 48-year - old male, who was admitted to the hospital with a 10-day history of fever, chills, myalgia, diarrhea, and thrombocytopenia. he was treated with empirical antibiotics. on hospital day 3, ribavirin (30 mg / kg as initial loading dose, 15 mg / kg qid as maintenance dose) was administered orally for 4 days and plasma exchange was performed for 4 days due to his high fever and altered mentality after a positive sftsv result from a real - time rt - pcr. the patient had a successful recovery.
interference in the balance between the environmental production of reactive oxygen species (ros), including hydroxyl radicals (oh) and hydrogen peroxide (h2o2), and the ability of biological systems to readily detect and detoxify them, or repair the resulting damage, are defined as oxidative stress. highly reactive radicals cause the oxidative damage of different macromolecules proteins, dna, and lipids leading to loss of function, an increased rate of mutagenesis, and ultimately cell death. in humans, for example, oxidative stress is involved in many diseases, such as rheumatoid arthritis, autoinflammatory diseases, neurodegenerative diseases, and cancer [1, 2 ]. however, the production of some ros (e.g., oh) can also be beneficial, as they are used by the human immune system to attack and kill pathogens, such as the production of ros by macrophages. sensing of ros - mediated signals also plays a crucial role in the biology of microorganisms. bacteria, for example, are in continuous contact with ros generated both endogenously, as a product of aerobic metabolism, or exogenously during ionizing () and nonionizing (uv) irradiation leading to the production of a number of radical and peroxide species through the ionization of intracellular water. industrial contaminants that are widespread in soils and on the surfaces of plants are also sources of ros. iron is earth 's fourth most abundant metal, after oxygen, silicon, and aluminium. its relevance for bacterial cells is emphasized by the fact that it is involved in a wide range of biological processes, including photosynthesis, n2 fixation, h2 production and consumption, respiration, oxygen transport, and gene regulation [4, 5 ]. however, in the presence of oxygen, ferrous ions frequently result in oxidative stress through the generation of hydroxyl radicals via the fenton reaction (fe + h2o2fe + oh + oh). therefore, bacteria have developed a variety of different mechanisms to ensure that iron is sufficiently accessible as well as being maintained in a nontoxic form. they possess high - affinity iron transport systems (i.e., siderophores and membrane iron transporters) that enable iron to be scavenged. intracellular iron can be stored in protein complexes (i.e., in dps and ferritins). thus, the homeostasis of these ions is tightly regulated so that their intracellular concentrations do not reach toxic levels. previously, it has also been suggested that the production of hydroxyl radicals is induced by bactericidal antibiotics to kill bacteria, in which iron ions and the fenton reaction play a role, or during a wide range of plant or human pathogen interactions. moreover, ros can also be produced during the degradation of natural existing biopolymers (cellulose, chitin, or xylan) by microorganisms. to reduce the hazardous effects of iron - based production of oh, bacteria produce proteins with an enzymatic activity to degrade ros (i.e., superoxide dismutases, catalases, peroxidases, and alkylhydroperoxide reductases), other small redox proteins (thioredoxins and glutaredoxins) as well as low molecular - weight thiols (glutathione and mycothiol) [8, 9 ]. all these cell components contribute in maintaining a reducing environment both in the cell and in controlling the extent of the oxidative burst. in analogy to fe(ii) ions, other transition metals ions [i.e., cu(i), co(ii), mn(ii), ti(iii), or cr(v) ] are closely linked with the production of free radicals in cells. although these metal ions can be hazardous for living organisms, they also serve as signal mediators in signalling cascades. it is an essential trace element that is localized in the active center or in a structurally important site of many bacterial proteins. zinc is a cofactor for more than 300 enzymes (i.e., superoxide dismutase and alcohol dehydrogenase). it is also a structural element of at least 40 protein classes (i.e., rna polymerase and trna synthetases). additionally, zinc can protect sulfhydryl groups from free radicals and inhibits free radical formation by competing with redox - active metals such as iron [12, 13 ]. binding of zinc as well as metal - catalyzed oxidation in proteins is closely related with the presence of redox - active cysteine residues (e.g., within cys - x - x - cys motifs) and other metal - sensing amino acids. moreover, bacterial redox sensory proteins can act either as single transcription regulators (e.g., furs or irr) displaying a sensory and response domain within itself [14, 15 ], or as a part of multicomponent systems (e.g., hbps or chrs) in which sensing and response are distributed among each protein component [1618 ]. in bacteria, there are a high number of redox sensory proteins that show different mechanism of function. here, we will give an overview of them and focus further on signalling pathways in which redox - active cysteines as well as iron ions are involved. cysteine residues (cys) in proteins are prominent targets for protein oxidation, as they easily react with h2o2 and free radicals. oxidation of cys by h2o2 involves nucleophilic attack of the cysteinyl thiol group on the electrophilic center of h2o2. deprotonation of the cys thiol group to generate the thiolate anion increases its nucleophilicity, and hence reactivity towards h2o2. these reactions are chemically highly complex and can lead to different sulfur oxidation states, including thiols, sulfenic and sulfinic acids, thiyl radicals, disulfide s - oxides, or disulfides. disulfides can be generated between two cys either intra- or intermolecularly (figure 1). the sensitivity of cys thiol groups to oxidation provides them with redox sensitivity, and hence the ability to sense redox status. for example, the thiolate anion can be stabilised by proximity to hydrogen bond donors, basic residues, and metal ions. in this review, we will focus on intramolecular disulfides that are formed within cys - x - x - cys motifs(x : any amino acid)a motif that is widespread in bacterial sensor proteins (table 1). in addition to redox - sensing properties, these motifs are often involved in zinc binding (i.e., in furs, hsp33, rsra, rsla, trx2, and sbcc) (table 1), and in the stabilization of protein domains that are crucial for function. it can be expected that the molecular environment of cys is in this case also important for zinc binding. table 1 comprises two different types of cys - x - x - cys - containing proteins. proteins belonging to the first group exhibit enzymatic function (hsp33, resa, dsba, sbcc, cytochrome c, trx2, ahpf / ahpc, copa, and hypa). for example, under reducing conditions, the chaperone hsp33 binds a single zinc ion through its c - terminal cys - x - cys - x6-cys - x - x - cys motif. upon oxidative stress, the four cys residues form intramolecular disulfide bridges resulting in release of zinc accompanied by considerable conformational changes that lead to destabilization of its c - terminus. as a consequence, hsp33 dimerises and acquires a chaperone function to prevent protein aggregation similarly, in the dna repair protein sbcc, two monomers are linked via cys - x - x - cys motifs and a zinc ion, forming a functional zinc bridge that is important for sbcc exonuclease activity. an extracellular low potential thiol - disulfide oxidoreductase was shown to maintain cys residues of cytochrome c in their reduced form. the thiolate species within the heme - binding motif (cys - x - x - cys - his) of apo - cytochrome c are required for covalent attachment of heme. contrary to the other cys - x - x - cys - containing proteins described here, resa does not bind zinc through this motif. other proteins with this signature and lacking zinc are listed in table 1 but are not within the focus of this review. the second group of cys - x - x - cys - containing proteins is involved in transcriptional regulation and can be subdivided into two subtypes : proteins that either directly control transcription (i.e., whib3, furs, catr, soxr, and surr) or require further regulatory components (i.e., spx, rsra, rsla, and rsha). the redox - dependent cys - x - x - cys - containing transcriptional regulator whib3, from mycobacterium tuberculosis, has been shown to sense the intracellular redox state in the cell, and to be required for the production of virulence polyketides, including polyacyltrehaloses (pat), sulfolipids (sl-1), di - o - acyl - trehaloses (dats) and trehalose dimycolates (tdms). whib3 directly regulates the expression of the following genes in a redox - dependent manner : pks3 (encoding a polyketide beta - ketoacyl synthase involved in pat and dat anabolism), pks2 (encoding a polyketide synthase involved in sl-1 anabolism), and fbpa (encoding a fibronectin - binding protein for tdms production). whib3 contains an iron - sulfur cluster [4fe-4s ] that under aerobic conditions is oxidized to [4fe-4s ]. further oxidative conversions result in complete loss of this cluster. unlike the superoxide stress sensor soxr, the redox state of the whib3 iron - sulfur cluster does not modify the dna binding affinity to target gene promoters, but rather the redox state of the cysteine residues is critical for dna binding. it was shown that, while the reduction of cys in whib3 abolishes its dna binding activity, oxidation induces it. it is noteworthy to mention that reduction and oxidation in whib3 are reversible processes. in the case of the zinc - containing transcriptional repressor furs from streptomyces reticuli, it was biochemically and spectrophotometrically shown that h2o2-mediated disulfide bond formation between cys93 and cys96 in furs is accompanied by the release of the bound zinc ion. this leads to conformational changes in furs, resulting in a loss of furs binding to its own dna operator sequence within the regulatory region of the target operon, furs - cpeb. as a result, the expression of this operon is no longer repressed, leading to an increase in production of furs and the mycelium - associated catalase - peroxidase cpeb. this enhanced expression in turn detoxifies the surrounding environment of hazardous h2o2 [9, 14 ]. proteins with cys - x - x - cys motifs are also indirectly involved in transcriptional regulation, by interacting with additional components. reversible disulfide formation can regulate the activity of the sigma factor r () of streptomyces coelicolor a3(2). here, the thiol - specific oxidant diamide induces -mediated transcription of > 30 genes, including those encoding thioredoxins and thioredoxin reductases that are involved in antioxidative stress response. however, does not contain any cys and in vitro studies have demonstrated that its transcriptional activity is not dependent on oxidative stress. indeed, researchers have characterised the protein rsra, which interacts with and regulates its transcriptional activity in a oxidative stress - dependet manner. rsra is a zinc - containing and redox - sensitive antisigma factor that possesses seven cys, two of which (cys41 and cys44) are located within a cys - x - x - cys motif that is important for activity. under oxidising conditions, a disulfide bond is formed in rsra, and this oxidized state can not bind or inhibit its transcriptional activity. oxidised rsra can be re - reduced by thioredoxin and in the thiol - reduced state rsra reassociates with and blocks its transcriptional activity. the redox sensing cys in rsra also coordinate zinc ions that provide the protein with a higher structural stability, strengthening its interaction with. upon redox stress and induction of disulfide bond formation, zinc is released and rsra undergoes conformational changes, generating a structure that does not bind. structural studies of an rsra homolog, rsla (table 1) from mycobacterium tuberculosis revealed that the redox sensing and zinc binding cys (cys54 and cys57) are closely located (~3, ; figure 2) to each other and are solvent exposed in the complex, thus, providing a structural basis for the redox sensitivity of rsla. the solvent exposed state of cys within the cys - x - x - cys motif is also an essential feature in redox sensing by the heat - shock protein hsp33 (table 1 ; pdb : 1vq0) from escherichia coli. to assure that iron is sufficiently accessible as well as being maintained in a nontoxic form, bacteria have developed a variety of protein - based protection mechanism. they employ iron - sensing regulatory proteins (i.e., perr and hbps - sens - senr ; see also table 2) that control the expression of proteins (i.e., h2o2-degrading enzymes such as catalases) blocking iron - dependent damage. upon sensing of iron - based stress, these proteins undergo different oxidative modifications, including oxidation of histidines (figure 3), dityrosine formation, or carbonylation. table 2 lists different iron - sensing proteins that mostly control redox stress resistance systems. they can directly regulate the transcription of target genes by dna binding to regulatory regions (i.e., fur, dtxr, rira, irr, perr, dmdr1, ider, sirr, fnr, and tror) or utilize further regulatory elements (hbps - sens - senr ; pmra - pmrb, chrs - chra, and feca - fecr - feci) that mediate the adaptive response. iron - dependent gene regulation has been demonstrated for the diphtheria toxin repressor (dtxr) from corynebacterium diphtheriae and corynebacterium glutamicum. binding of divalent iron ions to the global regulator dtxr results in the transcriptional repression of a number of genes required for iron uptake (i.e., htaa coding for a secreted iron acquisition and transport protein), as well as for usage and storage (i.e., hmuo coding for a heme oxygenase and ftn coding for a ferritin - like protein). mutational analyses have additionally demonstrated that dtxr is also involved in the iron - dependent expression of dna - protecting proteins, including dps - like proteins that bind iron and dna. in bacillus subtilis, the peroxide resistance regulator perr has been shown to sense metal dependent as well as h2o2-based oxidative stress, and to regulate the corresponding adaptive response. the metalloprotein perr is a small dimeric protein that coordinates two metal ions per monomer. one binding site binds a zinc ion, whereas the second one a regulatory metal, either iron or manganese [37, 38 ]. in vivo, the regulatory metal is required for repression of the transcription of target genes, including those ones encoding a catalase, alkylhydroperoxide reductase, and the dps - like dna - binding protein mrga. under h2o2-based oxidative stress conditions, fe catalyzes the oxidation of histidines (either his37 or his91) in the regulatory binding site of perr. analysis of electron maps density in the crystal structure of the oxidised perr protein (perr - zn - ox) showed the presence of a 2-oxo - histidine residue at position 37. further, maldi - tof and tandem esi - ms studies additionally revealed the oxidation of his91 within per - zn - ox [36, 38, 39 ]. it was concluded that the structural conformation of perr is dependent on the oxidation state of the regulatory site. oxidative modifications have also been shown to regulate the activity of the redox sensor and heme - binding protein hbps from streptomyces reticuli. hbps is extracellularly located and interacts with the membrane - embedded histidine autokinase sens from the two - component system sens senr [40, 41 ]. analyses of the crystal structure of hbps combined with size exclusion chromatography and static light scattering [4244 ] allowed the identification of hbps as an octamer. further studies demonstrated that the octameric assembly in hbps is required for an efficient interaction with sens. phosphorylation analyses also revealed that under nonoxidative stress conditions hbps inhibits the autophosphorylation of sens, whereas oxidative stress induces the hbps - mediated activation of sens. after autophosphorylation at a conserved histidine, sens transfers the phosphate group to its cognate response regulator senr, resulting in senr~p. the unphosphorylated form of senr binds to specific sites upstream of the furs - cpeb operon (encoding for the redox regulator furs and the mycelia - associated catalase - peroxidase cpeb), leading to its transcriptional repression. once senr has been phosphorylated, it loses the ability to bind to this operator, leading to a de - repression of the furs - cpeb transcription. comparative physiological analyses have demonstrated that the presence of hbps and sens - senr provides s. reticuli with a defence system against redox - stressing conditions [40, 41 ]. it was proposed that the switching of hbps from its inhibitor to activator state of sens autophosphorylation under oxidative stress conditions is controlled by conformational changes in hbps. indeed, cd spectroscopy as well as fret analyses revealed that after iron - mediated oxidative stress hbps undergoes secondary structure and overall intrinsic conformational changes, which are accompanied by oxidative modifications (i.e., carbonylation and dityrosine formation). while the sites of carbonylation have not yet been determined, the tyrosine residues participating in dityrosine formation have been identified as tyr77. these residues are localized in the interface of hbps subunits and are located in close proximity to each other, over the monomer - monomer interface. interestingly, tyr77 is situated near to a postulated iron - binding site, containing glu78 and glu81 within an e - x - x - e motif (figure 4) that has been previously characterized as an iron - binding motif [4547 ]. the reported oxidative modifications in hbps result in the degradation, either autonomously or protease dependent, of the oxidized protein. a metal - catalyzed protein oxidation accompanied by cross - linking and degradation has been reported for the bradyrhizobium japonicum iron response regulator (irr) protein, which is involved in the regulation of iron transport as well as of heme biosynthesis genes. it is proposed that the iron - catalyzed oxidation with subsequent degradation of irr is the molecular basis for the modulation of the activity of this regulator. the membrane - embedded sensor kinase pmrb from the two - component system pmra - pmrb of the pathogen salmonella enterica serovar typhimurium exhibits two copies of the exxe iron - binding motif. these are periplasmically located and have been proposed to be involved in sensing of extracellular ferric iron. upon iron sensing and autophosphorylation, pmrb transfers the signal to its cognate response regulator pmra, which in turn regulates fe resistance genes : pbgp and ugd both coding for enzymes that modify the lipid a region of lipopolysaccharide (lps) with 4-aminoarabinose. the gene product of pmrc catalyzes the addition of phosphoethanolamine to lipid a, whereas the phosphatase encoded by pmrg targets the phosphate located in the core region of the lps. these modifications result in a less - charged cell surface and diminished binding of ferric iron to the membrane. in contrast to other iron - sensing systems (i.e., perr and hbps - sens - senr), pmra - pmrb seems to protect the cell against iron toxicity independent of the simultaneous presence of oxygen. beside the posttranslational modifications (i.e., oxidation of histidines, carbonylation, dityrosine formation, or s s bonding) that regulate iron - sensing proteins, posttranscriptional regulation of gene expression in response to oxidative stress has been reported for the aconitases acna and acnb of escherichia coli [49, 50 ]. in their holo form, both acna and acnb contain an iron - sulfur cluster, [4fe-4s ], and exhibit enzymatic function. under reducing conditions, they catalyze the isomerisation of citrate to isocitrate. upon oxidative stress and iron depletion, the resulting apo - acna and apo - acnb proteins act as rna - binding proteins that stabilize acna- and acnb - mrna transcripts, leading to increased amount of the respective aconitases that subsequently complex iron - sulfur clusters. moreover, mutational analyses have revealed that the presence of acna provides e. coli with enhanced resistance against superoxide - mediated oxidative stress. additionally, proteomics studies demonstrated that the expression of anti - oxidative stress - working proteins (i.e., soda and trxb) is dependent on acna. as in humans, the exposure of bacteria to ros causes damage to a variety of macromolecules, resulting in mutations and often in cell death. however, ros may also be considered to be beneficial compounds, as they function as signalling molecules that lead to a coordinated response of bacteria under redox - stress conditions. these signals can be sensed by redox - active and zinc - coordinating cys - x - x - cys centres in proteins. under reducing conditions, zinc stabilizes protein structure, but the presence of h2o2 provokes the release of zinc and the formation of s s bridges that significantly alter the conformation and structure of the protein (i.e., furs or rsra). as a result, transcriptional activity is altered (by furs), or the ability to interact with the partner dna - binding protein is lost (by rsra). in both cases, the transcription of genes involved in the anti - oxidative stress response is ultimately activated. the structural basis for the redox sensitivity is given by the location of s s - forming cysteines in the protein, namely, their solvent exposition within the three - dimensional structure. importantly, reduction and oxidation processes within cys - x - x - cys redox centres are reversible and provide bacteria with an elegant switch on / off mechanism. iron - dependent oxidative modifications (i.e., 2-oxo - histidine or dityrosine formation) are also involved in ros - based signalling. under h2o2-based stress, iron catalyzes the oxidation of histidines in the peroxide resistance regulator perr, leading to release of iron and subsequently to de - repression, and expression of target genes. in analogy, iron - based stress activates the hbps - sens - senr - mediated signalling cascade. hbps is a redox sensor that upon oxidative modifications undergoes conformational and structural changes, inducing the phosphorylation cascade between the sensor kinase sens and the response regulator senr. as known for other regulators, oxidative modifications therefore, de novo protein biosynthesis is required to switch off the corresponding signal cascade. the diversity of responsive protein elements among bacteria correlates with the diversity in : ecological niches, life cycles, pathogenic, and nonpathogenic character.	bacteria are permanently in contact with reactive oxygen species (ros), both over the course of their life cycle as well that present in their environment. these species cause damage to proteins, lipids, and nucleotides, negatively impacting the organism. to detect these ros molecules and to stimulate the expression of proteins involved in antioxidative stress response, bacteria use a number of different protein - based regulatory and sensory systems. ros - based stress detection mechanisms induce posttranslational modifications, resulting in overall conformational and structural changes within sensory proteins. the subsequent structural rearrangements result in changes of protein activity, which lead to regulated and appropriate response on the transcriptional level. many bacterial enzymes and regulatory proteins possess a conserved signature, the zinc - containing redox centre cys - x - x - cys in which a disulfide bridge is formed upon oxidative stress. other metal - dependent oxidative modifications of amino acid side - chains (dityrosines, 2-oxo - histidines, or carbonylation) also modulate the activity of redox - sensitive proteins. using molecular biology, biochemistry, biophysical, and structure biology tools, molecular mechanisms involved in sensing and response to oxidative stress have been elucidated in detail. in this review, we analyze some examples of bacterial redox - sensing proteins involved in antioxidative stress response and focus further on the currently known molecular mechanism of function.
the response to interferon - based therapy for patients with compensated cirrhosis due to hepatitis c has been evaluated in several trials, both as the focus of prospective study, as well as in subgroup (post hoc) analyses of large registration trials. despite the improved efficacy of peginterferons, the sustained virological response (svr) rate is suboptimal in cirrhotic patients, relative to non - cirrhotic patients. however, the treatment of patients with compensated cirrhosis has recently been encouraged by the international liver transplant society expert panel in 2003 which concluded that patients with relatively compensated cirrhosis, defined by a meld score of 18 or less, are acceptable treatment candidates. 1 the likelihood of achieving an svr with interferon - based therapy in cirrhotic patients can be deduced from prior studies which focused upon, or incorporated patients with, advanced fibrosis. arguably, the most important trial on this issue was conducted by heathcote and colleagues in patients with advanced stage fibrosis or cirrhosis. 2 patients were randomized to peginterferon -2a, at one of two doses (90 mcg or 180 mcg weekly), or standard interferon. the svr rate was greatest for those treated with peginterferon alpha-2a 180 mcg weekly for 48 weeks (30%) ; this is in comparison to a svr rate of only 8% in those treated with standard, thrice weekly, interferon. in subgroup analysis comparing those with bridging fibrosis to those with cirrhosis, there did not appear to be a statistically significant difference between the groups in terms of achieving an svr. the patients infected with genotype 1 who received the 180 mcg dose of pegylated interferon had a 12% svr rate, in comparison to 51% non - type 1 genotypes treated with this dose. further analysis of data from the heathcote trial revealed that treatment was associated with histological improvement, especially in the group that experienced an svr. comparing the pegylated interferon group (180 mcg) group to standard interferon, histological improvement occurred in 54% and 31% respectively. pertinent to the issue of histological effects of therapy, poynard pooled data retrospectively from 4 large randomized controlled trials that included paired biopsies. 3 this analysis included data on 3,010 patients treated for either 24 or 48 weeks. overall, improvement in both the inflammatory grade and histologic stage of disease were associated with therapy. of the 153 cirrhotic patients with svr included in this analysis, 75 (49%) had significant improvement in fibrosis after treatment. interferon - based therapy may provide additional benefit by reducing the risk of hepatocellular carcinoma in cirrhotic patients as suggested in preliminary studies. in a study among 103 patients with hcv - related cirrhosis, those receiving interferon had a lower incidence of hepatocellular carcinoma and improved survival after 4 years. 4 a reduction in the risk for hepatocellular carcinoma in cirrhotic patients was also detected in a japanese trial of patients on interferon monotherapy for a median of 3 years. 5 therefore, in addition to establishing a target for therapeutic efficacy in patients with advanced fibrosis, early studies suggest potential histological and survival benefits of interferon - based treatment. insight into the efficacy of combination therapy with pegylated interferon and ribavirin in cirrhotic patients can be obtained from the large registration trials for pegylated interferon. in the peginterferon alfa-2b registration trial, the 6% of participants that had cirrhosis demonstrated a lower svr rate compared to those study subjects without cirrhosis. 6 cirrhotic patients treated with pegylated interferon alfa-2b, 1.5 mcg / kg weekly in plus ribavirin 800 mg a day for 48 weeks had a 44% svr ; in comparison, patients treated with standard interferon alfa-2b, 3 million units three times a week plus ribavirin for 48 weeks had a slightly lower response rate of 41%. in the second published pivotal trial of combined therapy with pegylated interferon and ribavirin, cirrhotic patients treated with peginterferon alfa-2a plus ribavirin for 48 weeks had svr rate of 43%, compared to 33% in patients treated with standard interferon combined with ribavirin. non - cirrhotic patients enrolled into this study had svr rates of 58% and 46% respectively. 7 results of two ongoing prospective studies are awaited to answer an important question that remains ; is there any benefit of prolonged treatment of patients with advanced fibrosis or cirrhosis, even in the absence of svr ? the national institutes of health sponsored halt - c trial (hepatitis c antiviral long term treatment against cirrhosis), is a multicenter study of the potential benefit of prolonged peginterferon therapy in mitigating the progression of fibrotic liver disease. 8 in this study, 391 of the 1045 patients enrolled into the initial preliminary results show that cirrhosis alone impaired response to therapy, with lower svr rates compared to non - cirrhotic patients. 9 in the copilot study (colchicine versus peg - intron long term), enrollees are predominantly cirrhotic patients who failed prior treatment. 10 an interim analysis suggested a benefit to pegylated interferon therapy, over colchicine, in reducing complications associated with cirrhosis. long term outcome data from this trial and other suppressive protocols will determine the efficacy of such an approach. 11 in summary, patients with advanced fibrosis or cirrhosis have a lower svr compared to non - cirrhotic patients, even with the latest combination therapy. cirrhotic patients may tolerate therapy less well, given the propensity for thrombocytopenia and leucopenia, as was seen in the three large trials that included cirrhotic patients. 2, 4, 11 in practice, the use of these agents is costly and has not been studied rigorously in a randomized controlled format to assess their value and impact on efficacy of therapy although they are now widely used in patients receiving interferon therapy. chronic hcv infection is the leading indication for liver transplantation (lt) in the united states and europe. given the accelerated progression of hcv following lt, the observation than higher pre - transplant viral load is associated with poorer transplant outcome, and the difficulties in treatment of the infection following lt 12, it is reasonable to emphasize the importance of pre - transplant treatment of hcv. the international liver transplantation society expert panel provided guidelines when considering interferon - based treatment in patients with hcv - cirrhosis. as seen in table 1, clearly, there are major obstacles toward treatment of this group of patients. since the advent of meld - based allocation, with the associated lessened importance of waiting time, those awaiting liver transplant are more ill. the resulting poor synthetic function, presence of hepatic encephalopathy and hypersplenism limit the candidacy of patients for interferon - based therapies. pre - transplant hcv treatment enthusiasm has also been curbed by the lower rates of sustained virologic response in those with fibrotic disease compared to those with less severe histology. several advances in hcv therapy have allowed incremental progress in overcoming these hurdles. as described above, peginterferons have shown better efficacy that unmodified interferons in the treatment of cirrhotic stage hcv. 2 additionally, the use of growth factors, specifically g - csf and erythropoietin, has allowed more aggressive dosing of interferon and ribavirin. 13, 14 finally, growing experience with antiviral therapy in this high - risk group has led to novel approaches and increasing success. table 2 provides details on the published results of antiviral therapy in this difficult - to - treat population. early published results with interferon / ribavirin - based regimens were unfavorable, leading to concern for the safety of such an approach. crippin and colleagues randomized end - stage liver disease patients on the transplant waiting list to three dose regimens : interferon alfa-2b, 1 million units (mu) daily, interferon alfa-2b 3mu t.i.w., and interferon alfa-2b 1mu daily with ribavirin 400 mg po bid. 15 these very sick patients (cpt mean 11.9) were subject to strict eligibility requirements : platelet count > 45,000/ml, hemoglobin > 11 g / dl, and absolute neutrophil count (anc)>1250/ml. dose reductions or study discontinuation were mandated for anc 1500 cells / ml was well tolerated, with temporary dose discontinuation in 3/20 (15%). forns and colleagues used relatively high - dose interferon / ribavirin therapy in their cohort of 30 patients awaiting transplantation in spain in an effort to reduce viral load at the time of lt. 17 when the investigators estimated 4 months remained until transplantation, interferon alfa-2b 3mu / day and ribavirin 800 mg / day were initiated, with a resulting mean treatment period of 12 weeks in 30 patients. the virologic response rate on therapy was 30%, with two - thirds of responders remaining non - viremic after lt. as in other such trials many wait - listed patients did not meet eligibility requirements (38%), and side effects were common. despite the use of g - csf and erythropoietin, dose reduction of interferon was required in 60%, while ribavirin dose was decreased in 23% of study patients. everson and colleagues offer the largest series to date with a unique low accelerating dosage regimen (ladr). 18 one hundred twenty four patients were treated with unmodified interferon or peginterferon at low initial dose, with increases every two weeks toward standard target doses. duration of therapy was intended to be 24 weeks in genotypes 2 and 3 and 48 weeks in genotype 1 infection. hematologic inclusion criteria included anc > 800 cells / ml, hemoglobin > 10 g / dl and platelets > 35,000/ml. one fourth to one sixth of the patients referred to our clinic with advanced liver disease were treated with lad during his period. end of treatment response was seen in 46% while the svr rate was 22%. svr was associated with non-1 genotype, cpt class a, and a complete course of therapy. as expected in this very ill set of patients, adverse drug reaction rates were relatively common, with two such events potentially contributing to patient mortality. of the 15 patients that were virologically negative at the time of transplantation, 12 remained hcv(-) for at least six months following lt. although the data are mixed on the effectiveness and safety of interferon - based therapies in patients with decompensated cirrhosis, the demonstrated possibility of viral eradication in this group with possible improvement in hepatic synthetic function and improved post - transplant outcome provide an important rationale for further studies in this area. future work will focus on pegylated accelerating dose regimens, the increased use of growth factors and non - interferon based antiviral therapies. currently, it is advisable to treat such patients only in experienced centers with close monitoring for adverse events. 1, 19 the morbidity of recurrent hcv following liver transplantation has made pre - transplant antiviral therapy a high priority for research. the low accelerating dosage regimen of everson and colleagues has demonstrated good efficacy but must be replicated in other cohorts and centers. the development of new, non - immunomodulatory antiviral agents promises to be a significant advance in the treatment of this population. ultimately, individualization of the anti - viral regimen chosen in each case may lead to better efficacy rates with less adverse drug reactions and side - effects. international liver transplantation society expert panel consensus conference proposed guidelines for interferon - based treatment of patients with cirrhosis 1 summary of results from interferon - based treatment regimens in patients with advanced liver disease	despite the improved efficacy of peginterferons, the rate of sustained virologic response is suboptimal in cirrhotic patients, relative to non - cirrhotic patients. however, the treatment of patients with compensated cirrhosis has recently been encouraged by expert panels. interferon - based therapy may provide additional benefit by reducing the risk of hepatocellular carcinoma in cirrhotic patients as suggested in preliminary studies. results of two ongoing prospective studies are awaited to answer the important question of the effectiveness of suppressive interferon therapy, even in the absence of sustained virologic response. given the importance of recurrent hcv following liver transplantation, attention has been directed toward the antiviral treatment of patients with advanced liver disease. this approach needs to be pursued with caution given the potential morbidity of the therapy. recently, a low accelerating dosage regimen has provided excellent results and is the subject of additional inquiry.
dermoscopy has become an integral part of diagnosing scalp disorders and differentiating cicatricial from noncicatricial alopecia. discoid lupus erythematosus (dle) is seen in 50 to 85% of patients with cutaneous lupus erythematosus, and scalp involvement is most often the presenting symptom. accurate and rapid diagnosis is critical and if left untreated, scarring and atrophy commonly occur. dermoscopic findings described in scalp dle include follicular red dots, reduced follicular ostia, arborizing vessels, white patches, honeycomb pigmented network, blue - grey dots, and variable scaling.[24 ] follicular keratotic plugging is a typical feature of dle that can be easily appreciated on clinically and dermoscopically. clinically, the keratotic plugs have been referred to as the carpet tack sign since they project up similar to carpet tacks. we present here a case series of three females affected by scalp dle with prominent follicular plugging on dermoscopy and corresponding keratotic plugs on pathology. all dermoscopic images were obtained with the handyscope (handyscope for iphone 4, fotofinder, bad birnbach, bavaria, germany). a 40-year - old african american female with long - standing scarring alopecia presented to our clinic [figure 1 ]. clinical examination revealed scarring alopecia of the vertex of the scalp with complete loss of pigmentation in the involved scalp except for sparing of a few pigmented patches. some areas of alopecia had an atrophic appearance but other areas showed prominent follicular plugging. dermoscopy showed reduced follicular density and numerous yellow keratotic follicular plugs [figure 2 ]. a dermoscopy - guided 4 mm punch biopsy from the scalp area exhibiting the prominent follicular plugging was obtained. horizontal sections were bisected at the level of the lower follicle and multiple sections were examined on hematoxylin and eosin (h and e). at the level of the fat and dermis in the dermis, there were dense nodular collections of lymphoid cells surrounding vessels and adnexal structures. at the level of the infundibulum, many infundibular ostia were filled with keratotic material (follicular plugs) [figure 3 ]. there was an interface lymphocytic infiltrate of the infundibular ostia and around the sweat ducts. in vertical sections, the epidermis showed orthokeratosis with hyperkeratosis and vacuolar changes of the basal layer. case 1 scarring alopecia of the scalp associated with diffuse hypopigmentation, sparse follicular plugs, and diffuse scaling at the borders of the alopecic areas case 1 dermoscopy guided biopsy, the area shows numerous yellow keratotic plugs case 1 horizontal section at the level of the infundibulum demonstrates infundibular ostia filled with keratotic material (follicular plugs) (h and e, 4) a 58-year - old female presented with a five - month history of worsening alopecia on her scalp. physical examination revealed hypopigmented, atrophic plaques on her upper arms, and well - defined, scaly, patches of alopecia on her scalp. vertical sections of her scalp demonstrated loss of hair follicles replaced by follicular scars and few follicles with dilated infundibulums filled with keratin (keratotic plugs). there was an interface infiltrate of lymphocytes and some plasma cells in a perifollicular and perivascular distribution, with some of the aggregations forming germinal - like centers. at the dermoepidermal junction, a 39-year - old female presented with rapidly progressing alopecic patch of the scalp for the past four months [figure 4 ]. dermoscopic examination of her scalp showed polymorphous vasculature, white patches, follicular keratotic plugs, and follicular hyperkeratosis [figure 5 ]. the follicles had a patulous orifice and there was marked vaculolar alteration of the basal layer and a superficial and deep lymphocytic and histiocytic infiltrate, deeper around adnexal structures. case 3 alopecic patch showing erythema and pigmentation case 3 dermoscopic images showing follicular keratin plugs follicular keratotic plugs are a marker of dle and were originally described as a sign of early and active lesions and not in areas of scarring or healed skin. we would like to note that follicular keratotic plugs may still be present in areas of chronic alopecia. their presence can aid in correctly diagnosing scalp dle alone or in conjunction with other described dermoscopic features such as follicular red dots, blue - grey dots, white patches, reduced follicular ostia, and absence of pinpoint white dots [table 1 ]. in conclusion, trichoscopy is critical for diagnosing hair disorders and recognition of follicular keratotic plugging in dle can lead to timely diagnosis and initiation of treatment. a 40-year - old african american female with long - standing scarring alopecia presented to our clinic [figure 1 ]. clinical examination revealed scarring alopecia of the vertex of the scalp with complete loss of pigmentation in the involved scalp except for sparing of a few pigmented patches. some areas of alopecia had an atrophic appearance but other areas showed prominent follicular plugging. dermoscopy showed reduced follicular density and numerous yellow keratotic follicular plugs [figure 2 ]. a dermoscopy - guided 4 mm punch biopsy from the scalp area exhibiting the prominent follicular plugging was obtained. horizontal sections were bisected at the level of the lower follicle and multiple sections were examined on hematoxylin and eosin (h and e). at the level of the fat and dermis in the dermis, there were dense nodular collections of lymphoid cells surrounding vessels and adnexal structures. at the level of the infundibulum, many infundibular ostia were filled with keratotic material (follicular plugs) [figure 3 ]. there was an interface lymphocytic infiltrate of the infundibular ostia and around the sweat ducts. in vertical sections, the epidermis showed orthokeratosis with hyperkeratosis and vacuolar changes of the basal layer. case 1 scarring alopecia of the scalp associated with diffuse hypopigmentation, sparse follicular plugs, and diffuse scaling at the borders of the alopecic areas case 1 dermoscopy guided biopsy, the area shows numerous yellow keratotic plugs case 1 horizontal section at the level of the infundibulum demonstrates infundibular ostia filled with keratotic material (follicular plugs) (h and e, 4) a 58-year - old female presented with a five - month history of worsening alopecia on her scalp. physical examination revealed hypopigmented, atrophic plaques on her upper arms, and well - defined, scaly, patches of alopecia on her scalp vertical sections of her scalp demonstrated loss of hair follicles replaced by follicular scars and few follicles with dilated infundibulums filled with keratin (keratotic plugs). there was an interface infiltrate of lymphocytes and some plasma cells in a perifollicular and perivascular distribution, with some of the aggregations forming germinal - like centers. at the dermoepidermal junction, a 39-year - old female presented with rapidly progressing alopecic patch of the scalp for the past four months [figure 4 ]. dermoscopic examination of her scalp showed polymorphous vasculature, white patches, follicular keratotic plugs, and follicular hyperkeratosis [figure 5 ]. the follicles had a patulous orifice and there was marked vaculolar alteration of the basal layer and a superficial and deep lymphocytic and histiocytic infiltrate, deeper around adnexal structures. case 3 alopecic patch showing erythema and pigmentation case 3 dermoscopic images showing follicular keratin plugs follicular keratotic plugs are a marker of dle and were originally described as a sign of early and active lesions and not in areas of scarring or healed skin. we would like to note that follicular keratotic plugs may still be present in areas of chronic alopecia. their presence can aid in correctly diagnosing scalp dle alone or in conjunction with other described dermoscopic features such as follicular red dots, blue - grey dots, white patches, reduced follicular ostia, and absence of pinpoint white dots [table 1 ]. in conclusion, trichoscopy is critical for diagnosing hair disorders and recognition of follicular keratotic plugging in dle can lead to timely diagnosis and initiation of treatment.	dermoscopy has become an integral part of diagnosing scalp disorders including discoid lupus erythematosus (dle). follicular keratotic plugs are a marker of dle and correlate with the hyperkeratosis and plugging of the follicular ostia with keratotic material. they may be present in acute or chronic lesions and their presence alone or in conjunction with other described dermoscopic features can lead to timely diagnosis and initiation of treatment. we present three cases of scalp dle and discuss the clinical, dermoscopic and histopathologic features.
simulating the dynamics of molecular systems on a digital computer requires that the equations of motion be discretized. the resulting discrete - time integration algorithm that governs the updates of particle positions and velocities will necessarily have properties that differ from the continuous equations of motion on which it was based. to construct such an algorithm, one must decide which properties of the original dynamics should be preserved. even then, a multitude of integration schemes may satisfy these properties and still recover the continuous stochastic equations of motion in the limit of an infinitesimally small time step. for integrating the deterministic classical equations of motion prescribed by newtonian dynamics, explicit symplectic integration schemes such as velocity verlet are now widely regarded as being optimal for condensed matter systems for a number of reasons : they are reversible, simple to implement, preserve phase space volume, require minimal force evaluations, and are generally stable over long integration times. for stochastic equations of motion in which the influence of some system components (often the solvent) are not represented explicitly, but instead by random collisions with fictitious particles no such generally adopted integrator yet exists. in particular, the dynamics produced by existing algorithms differ (in a time step dependent manner) in important respects from the dynamics of the continuous equations of motion. nevertheless, significant effort has been devoted to developing such stochastic integrators due to their utility in simulating many systems of interest to the chemical, biophysical, and physical sciences. driven nonequilibrium systems present their own set of special challenges. for example, a powerful set of nonequilibrium work fluctuation theorems permit the computation of equilibrium properties of systems from their nonequilibrium statistics, but require as input the distribution of work values associated with the ensemble of trajectories. calculations that use naive analogies of the work for continuous dynamics failing to take into account details of the discrete integration scheme here, we consider various choices that could be made in constructing discrete - time integration schemes for langevin dynamics. by examining the various possible strang splittings of the langevin liouville operator, incorporating a novel time step rescaling (eq 15), and comparing their resulting properties to several desiderata that have been enumerated in the literature over recent decades, we show how it is possible to simultaneously satisfy nearly all these criteria with a single integration scheme (eq 7) that is generally applicable, simple to implement, computationally efficient, and produces thermodynamically consistent nonequilibrium statistics. a standard framework for the stochastic simulation of molecular systems assumes that the variables of interest evolve according to langevin dynamics with uncorrelated gaussian noise, which represents interactions with the surrounding environment through frictional drag and stochastic collisions with fictitious bath particles:1a1bhere r and v are time - dependent position and velocity, m is mass, = 1/kbt, kb is boltzmann s constant, t is the temperature of the environment, is a friction coefficient (with dimensions of inverse time), and w(t) is a standard wiener process. the force f(t) is due to the (in general time - dependent) hamiltonian (t) on the system with position r(t), as determined by the derivative of the potential energy, (t)/r, evaluated at r(t). for multidimensional, multiparticle systems, r, v, f, and dw are vectors, and m is a diagonal matrix (see supporting information, multiple dimensions). while these equations of motion can be solved exactly for some simple systems, nearly all complex systems of interest require computational techniques in order to generate dynamical trajectories. on digital computers, the selection of an appropriate discrete time integration scheme is made difficult by the fact that many discretizations may exist that recover the same continuous stochastic differential equations of motion in the limit of an infinitesimally small time step, but these schemes may possess very different properties for finite time steps. finite time step integrators for molecular systems can not hope to exactly reproduce snapshots from the dynamical trajectories of the continuous equations of motion of a real physical system. imprecision of experimental measurements ensures that simulated initial conditions necessarily deviate from true ones, and lyapunov instability of even deterministic dynamics ensures the rapid chaotic growth of such deviations, making the exact reproduction of a particular trajectory impossible even were it desirable. moreover, artifacts are inevitably introduced when one discretizes continuous equations of motion in a straightforward manner : dynamical motion increasingly diverges from that of continuous equations of motion with increasing friction and/or time step. instead, the goal is often to reproduce certain statistical (often observable) properties of the system, especially in terms of correlation functions and (possibly time - dependent) ensemble expectations given a set of initial conditions. thus, a desirable approximation scheme should share certain statistical and dynamical properties of the ensemble of trajectories associated with the exact equations of motion, in lieu of being able to exactly integrate trajectories. pastor,. proposed that a useful discrete - time integrator should reproduce seven quantities associated with the continuous - time equations of motion : for a free particle (zero - force), the mean - squared displacement (msd) as a function of time, the mean - squared velocity (msv), and the velocity autocorrelation function (vac) ; for a uniform external force, the terminal velocity ; and for a harmonic potential (linear force), the msd, msv, and the virial. in table 1, we define these desired dynamical properties and list their analytically computed values for the continuous equations of motion. in the supporting information, under we describe in detail the calculation of these quantities for our family of integrators. one should not expect a discrete algorithm to give meaningful results on time scales less than a single time step. those users who prefer to treat a discrete algorithm as a black box will be most interested in integrators that satisfy given dynamical properties at integer time steps. those willing to look further under the hood would need to know at which specific point within a given time step to measure a given dynamical quantity to recover (or most closely approximate) the continuous - limit value. angled brackets denote an average over the ensemble of phase - space trajectories produced by a given integration scheme. there are several other criteria that one may want an integrator to satisfy, not the least of which being ease of implementation and analysis. additionally, an integrator that is computationally efficient should have an accuracy that scales reasonably with time step length (here, quadratically, the same accuracy order as popular symplectic integration schemes for deterministic dynamics such as velocity verlet), permitting relatively large time steps ; minimize the number of force evaluations (one per time step) so as to minimize computational effort ; and easily incorporate constraints (typically reflecting covalent chemical bonds to light elements such as hydrogen) that push the integrator stability limit to larger time step. path sampling or path reweighting strategies often require an integrator that induces an irreducible markov chain (i.e., it is possible to transition from any phase space point to any other in a single time step through specific choice of the random variables), and that for a given trajectory has a readily evaluated path action that governs the probability of that trajectory within the dynamical ensemble. indeed, the task is further complicated when the integrator must produce thermodynamically consistent nonequilibrium simulations. to facilitate the use of nonequilibrium fluctuation relations and estimators derived from them, the integrator must distinguish between the heat, work, and shadow work, by properly splitting dynamics into stochastic, explicit hamiltonian - update, and deterministic substeps. for the calculated works to be thermodynamically meaningful, the integrator must also have a form symmetric under time - reversal. demonstrate that no member of their family of overdamped integrators can simultaneously satisfy more than four of their seven desired dynamical properties. many other underdamped discretizations of the langevin equation have been proposed that achieve some subset of these enumerated desiderata, yet there still is no widespread agreement on an integrator that performs satisfactorily for a broad set of purposes. drawing inspiration from several popular integrators, in this paper we derive a simple family of integrators that split the different update types to permit the definition of thermodynamically meaningful quantities for work and heat. with a novel rescaling of the time step, the resulting dynamics preserves five of pastor.s seven dynamical properties for any values of friction and time step (six if fractional time step values are also considered), and furthermore satisfies all of the other desiderata enumerated above, including its utility for nonequilibrium simulations and schemes involving path sampling or reweighting. the resulting integrator represents a stochastic generalization of velocity verlet, is simple to implement, and could be a general all - purpose replacement for the various discrete - time langevin integrators now in use. in sampling contexts, this time step rescaling does slow the exploration of conformational space if the raw integration time step t is not concomitantly increased. we believe that such a time step rescaling permits a larger raw time step, but exactly how much larger t can be increased (and hence to what extent sampling efficiency can be recovered or even improved) before reaching the stability limit (or some other integration pathology) remains an open question. addressing this potentially system - specific issue requires further theoretical and numerical investigation, which is beyond the scope of this paper. we derive a family of integrators by splitting the time evolution operator into stochastic and deterministic components and choosing the adjustable parameters to match dynamical quantities from the continuous equations of motion. we write the one - dimensional version here, but the generalization to multiple dimensions is straightforward (see the supporting information, multiple dimensions). the langevin liouville operator (sometimes termed the liouvillian) can be naturally written as a sum of four parts = o + v + r + h. the first operator represents stochastic thermalization via an ornstein uehlenbeck operator,2the next two operators represent deterministic newtonian evolution of velocity and position,3and the last operator represents the time evolution of the system hamiltonian according to the predetermined schedule (or protocol),4where n denotes the time step index and t = nt the simulation clock time for time step t. (note that in the case of a time - independent hamiltonian, e is the identity operator.) similar to several other integrators, we approximate the dynamics over a time t by applying a series of strang (symmetric trotter) operator splittings:5a5b5c5dwhere (a, b, c, d) represents a permutation of 0, v, r, h. there are six strang splittings of the liouville operators 0, r, and v. the hamiltonian update operator h commutes with 0 and with r, so for each of these six splittings, there are only two unique placements of h. for each of the six splittings, one placement of h interleaves the position, hamiltonian, and deterministic velocity updates in such a way as to require multiple force evaluations per step, making the scheme computationally inefficient. thus there are six distinct splittings that each give rise to different finite time step dynamics and require only one force evaluation per step. notably, because the error in each strang splitting is (t), all are identical to the true liouville operator in the limit t 0. one such splitting is a stochastic generalization of velocity verlet that, in analogy to the nomenclature of leimkuhler and matthews, we call ovrvo (to denote the respective ordering of ornstein uehlenbeck (o), deterministic velocity (v), and deterministic position (r) updates),the hamiltonian - update step (d) is placed to minimize the number of force evaluations per time step. for this operator splitting, a single update step that advances the simulation clock by t is given explicitly by7a7b7c7d7e7f7ghere, a = exp(t), and and are independent, normally distributed random variables with zero mean and unit variance (hence, when scaled by (m), distributed according to the equilibrium maxwell the substeps in eq 7 are the finite difference expressions of the corresponding suboperators in eq 6. the initial (a) and final (g) operators randomize the velocity and leave the position unchanged, mixing the old velocity with a maxwell boltzmann random variate (with old velocity weighted according to a = exp[t ]). these operators can be analytically integrated to give the first (7a) and last (7 g) substeps that are stochastic, markovian, and detailed - balanced (with respect to the true canonical measure). the operators (b) and (f) correspond to deterministic velocity updates, while (c) and (e) correspond to deterministic position updates. together they are approximated by the finite difference expressions of eqs 7b, 7c, 7e, and 7f, which together constitute the deterministic and symplectic velocity verlet integrator, but slightly altered by an effective time step rescaling 0 b 1, chosen to maintain the continuous - limit zero - force diffusion coefficient and terminal drift under a uniform external force, regardless of friction coefficient or time step (derived in supporting information, t 0 this rescaling factor b converges to unity and the splitting converges to the continuous - time equation of motion (eq 1). operator (d) and its finite difference expression as eq 7d makes explicit the midpoint hamiltonian update. note that several other popular integrators do not explicitly include the update of the system hamiltonian, presumably because they are not concerned with calculating distributions of the work generated by explicit hamiltonian changes. the alternative splittings with one force evaluation per time step include orvro (a stochastic generalization of position verlet),8rvovr (an explicit hamiltonian - update generalization of leimkuhler and matthew s aboba),9vrorv (an explicit hamiltonian - update generalization of leimkuhler and matthew s baoab),10rovor,11and vorov,12 since for a single time step t the error is o(t) for any of these strang splittings, when applied over n = t/t time steps the global error is o(t). figure 1 confirms that ovrvo errors in the energy are second order in the time step t. standard integrators implicitly set the parameter b to unity in 7b, 7c, 7e, and 7f. however, we show later that a nonunit b recovers, for arbitrarily large time step, the continuous - limit values of important dynamical quantities. the time step rescaling can be most simply derived by noting that in the absence of a potential, for any of the six splittings, a trajectory is isomorphic to a semiflexible gaussian polymer chain : the set of positions correspond to the beads on the polymer chain, and the displacement vectors during single time steps correspond to the intermonomer bond vectors in the chain. in one dimension, the displacement is a normally distributed random variable with zero mean and variance 02 = (bt)/(m) (in accordance with maxwell boltzmann velocity statistics and the time interval bt), and the autocorrelation between velocities separated by n steps decays exponentially as13for this system, the mean - squared displacement in the large time (t 1) limit is the time step rescaling b results from equating this expression to the mean - squared displacement of a freely diffusing particle in one dimension, 2dt = 2t/(m), for a total simulation time over n steps, t = n t. in particular, the time step used in the position update step is rescaled by the factor15ensuring that the effective free - particle diffusion constant is independent of time step length (see figure 2). in the low friction limit t 1, b = 1 o([t ]), and in the high friction limit t 1, b = [2/(t) ]. numerical demonstration that the errors in energy of the ovrvo integrator (eq 7) are second order in t. here, we use a previously described model system of a harmonic potential, with unit spring constant, friction coefficient, temperature, and mass, with initial conditions r(0) = v(0) = 0. the error is the absolute deviation of the estimate of r(1) + v(1) = 0.9796111900... (twice the energy) computed by ensemble averaging over 10 independent realizations. note that even though the position update utilizes an effective time step of bt, the simulation clock is still advanced by the full time step t. the zero - force msv and vac and the uniform - force terminal drift are unaffected by the choice of b. we derive the time step rescaling from a different perspective and in more detail in supporting information, time step rescaling recovers correct force - free diffusion as a function of time step. root mean - squared displacement versus relative time step length t at time t = 64 for a freely diffusing particle in one dimension, with unit mass, temperature, and friction coefficient, subject to the ovrvo integrator (eq 7) without time step rescaling, b = 1 (), or with time step rescaling, eq 15 (). for an explicitly time - independent system hamiltonian, this family of integrators reduces to various other schemes in certain limits or approximations. at zero friction, = 0 and a = b = 1, thus stochastic substeps have no effect, so ovrvo, vrorv, and vorov are identical to the deterministic velocity verlet integrator, whereas orvro, rvovr, and rovor are identical to position verlet. in the high - friction or long - time limit, a = 0 and b = [2/(t) ], and ovrvo reduces to the euler integrator for overdamped langevin dynamics (also known as the euler - maruyama method):16rovor and vorov interpose a velocity randomization substep between the deterministic velocity and position updates. in this t 1 limit, the velocity is completely randomized before each position - update substep, and thus the position updates are completely independent of the hamiltonian. the other four splittings preserve the influence of the hamiltonian on the dynamics even in this limit of large friction (or large time step). ovrvo also reduces to several other popular integrators in other limits or approximations. if the effective time step rescaling for the deterministic substeps is omitted, such that b = 1, then ovrvo is equivalent to an integrator described by adjanor, athnes, and calvo ; and by bussi and parrinello. if we also combine all stochastic and deterministic velocity updates (eqs 7f, 7 g, 7a, and 7b), we recover the integrator of athnes ; and recasting the athnes integrator as a verlet - style integrator (only monitoring position) we converge with the brnger - brooks - karplus (bbk) integrator in the low friction limit. if instead we only combine adjacent pairs of stochastic and deterministic velocity updates (eqs 7a with 7b, 7f with 7 g) (still with no time step rescaling) we produce the low friction limit of the langevin leapfrog integrator of izaguirre, sweet, and pande. there is significant interest in probing the probability distribution of work required during a nonequilibrium driving process, which via the work fluctuation theorems can report on various system properties. the total energy change e during the nth full time step of ovrvo can be cleanly separated into heat q, protocol work wprot, and shadow work wshad:17c17d17ehere, (r, n) is the potential energy for configuration r under hamiltonian (n), and (v) = (1/2)mv is the kinetic energy for velocity v. the five other splittings permit similar decompositions of energy changes into heat, protocol work, and shadow work. constraints, such as rigid bond lengths, can be readily incorporated into the dynamics using standard techniques. the symplectic part of the integrator can be constrained with the standard rattle [7b, 7f ] algorithm. since rattle is symplectic if iterated to convergence, adding constraints does not interfere with the underlying reversibility of the dynamics. similarly, the velocity randomization substeps, eqs 7a and 7 g, can be constrained with rattle, which modifies the heat flow, but preserves detailed balance. consequently, constrained versions of this family of integrators still obey the precepts of nonequilibrium thermodynamics, with the same definitions of heat, protocol work, and shadow work (eq 17a), provided that the definition of free energy is altered to account for the constrained degrees of freedom. for ovrvo, for example, the force in substep 7f is identical to the force in substep 7b of the next time interval. measuring heat requires two evaluations of kinetic energy per time step for all six splittings, for ovrvo just after substep 7a and just before substep 7 g. separately measuring protocol work and shadow work requires two potential energy evaluations per time step, once each just before and after the hamiltonian - update substep. shadow work measurement also requires the kinetic energy evaluations already needed to measure heat, as shadow work and heat are the only processes that change the kinetic energy. however, if only the total thermodynamic work w = e q is of interest, this can be calculated given the heat q during each step n n + 1 (easily accumulated during integration using eq 17c) and the total energy at the beginning and end of the simulation,18 the ovrvo integrator requires two normal random numbers per velocity per time step, one each for the initial 7a and final 7 g velocity randomizations. splitting the velocity randomization across time steps ensures that the dynamics is microscopically reversible and markovian, and that the induced markov chain is irreducible. (the two separate randomization steps permit the independent adjustment of the velocity and the position to arbitrary values.) rvovr, vrorv, and rovor, by contrast, do not generate irreducible markov chains : they effectively agglomerate the two velocity randomizations into a single randomization involving one random number, so for a particular new velocity only one position is possible. irreducibility has utility for path sampling and path reweighting schemes, since any proposed discrete time trajectory through phase space is a valid trajectory of an irreducible markov chain. however, many practical applications do not require strict irreducibility, so one can halve the number of required random variables for ovrvo and orvro by combining the last stochastic substep of one full step with the first stochastic substep of the next full step, and for rvovr and vrorv by combining the two stochastic substeps of a given full step. rovor and vorov separate their stochastic substeps such that they can not be easily combined. leimkuhler and matthews show that in the high friction limit and at medium time step vrorv with this single velocity randomization (and time - independent hamiltonian) is second - order accurate when other integrators become first - order. when only the total thermodynamic work is of interest, we can combine the last two velocity updates of eq 7 with the first two updates from the next step, and combine the two position updates, to give a three substep stochastic leapfrog integrator : under these circumstances, rvovr, rovor, and vorov reduce to similar three substep integrators, but, due to their sequencing of substeps, orvro and vrorv each only reduce to a five substep integrator. the path action s[x ] is a necessary quantity for many path sampling and path reweighting techniques. the conditional path probability functional is a product of single time step probabilities, here, x is a trajectory through phase space between x(0) { r(0),v(0) } at time 0 and x(nt) at nt. each time step probability is determined by the probability of the requisite random variables, which for ovrvo is21the first factor m/[bt(1 a) ] is the jacobian for the change of variables from { r(n + 1), v(n + 1) } to { (n), (n + 1) }, and the probabilities are normal with zero mean and unit variance,22wherethe intermediate velocities can be determined by the initial and final positions and forces:24a24bcombining eqs 2024 gives the action as a function of position and velocity at the unit time steps,25the path probability obeys the expected symmetry under time - reversal, where the work is defined as in eqs 17d and 17e. the path action for orvro additionally requires the evaluation of the force and its derivative at the half - step position, r(n + (1/2)), hence requires paths of twice as many points, and therefore is of lesser utility. rvovr, vrorv, and rovor induce reducible markov chains and thus these splittings have infinite path actions for the vast preponderance of paths. all of our strang splittings lead to seven - substep integration schemes that are time - symmetric ; are second - order accurate in t ; make hamiltonian changes explicit ; distinguish between heat, protocol work, and shadow work ; and easily incorporate constraints. six of the 12 unique splittings require a single force evaluation per time step and thus are computationally efficient. setting a e and b [(2/(t)) tanh (t/2) ] gives for all six one - force - evaluation splittings the continuous - limit msd, msv, and vac in the zero - force case, and the linear - force virial with asymptotic error o(t). as a stochastic generalization of a standard deterministic technique, ovrvo implements what can be considered a form of velocity verlet with velocity randomization (vvvr). it is an integrator of general utility, satisfying five of pastor.s seven dynamical properties (six if fractional time step quantities are considered), as well as the remaining enumerated desiderata. it is well - suited for the study of nonequilibrium thermodynamics : work is easily measured because the hamiltonian changes are made explicit, and these measured works are thermodynamically meaningful because ovrvo distinguishes between heat, protocol work, and shadow work. simple form for the path action facilitates its use in trajectory reweighting or path sampling methods. our novel time step rescaling maintains (for an arbitrary time step) various continuous - limit dynamical quantities, in particular the uniform - force terminal drift and linear - force fluctuations in position and velocity. further research is required to determine to what extent this rescaling permits a larger raw integration time step, and hence how it affects sampling efficiency. finally, ovrvo generalizes several popular integration schemes and thus relates naturally to the existing literature. its extension to a multiple time step integrator is straightforward, requiring only replacement of the inner symplectic step (7b7f) with a corresponding symplectic multiple time step integrator for deterministic dynamics. desiderata are grouped into those satisfied by all six splittings and those where the splittings differ in their performance. by contrast rovor and vorov produce uniform - force terminal drift and linear - force fluctuations of position and velocity that differ from continuous - limit values, lose hamiltonian dependence in the limit of large t, and require two random numbers per time step even for reducible markov chains. rvovr and vrorv induce markov chains that are not irreducible and thus these splittings have limited utility for path - sampling schemes. orvro seems similarly useful to ovrvo, but in path sampling applications requires paths with twice the number of points as ovrvo.	when simulating molecular systems using deterministic equations of motion (e.g., newtonian dynamics), such equations are generally numerically integrated according to a well - developed set of algorithms that share commonly agreed - upon desirable properties. however, for stochastic equations of motion (e.g., langevin dynamics), there is still broad disagreement over which integration algorithms are most appropriate. while multiple desiderata have been proposed throughout the literature, consensus on which criteria are important is absent, and no published integration scheme satisfies all desiderata simultaneously. additional nontrivial complications stem from simulating systems driven out of equilibrium using existing stochastic integration schemes in conjunction with recently developed nonequilibrium fluctuation theorems. here, we examine a family of discrete time integration schemes for langevin dynamics, assessing how each member satisfies a variety of desiderata that have been enumerated in prior efforts to construct suitable langevin integrators. we show that the incorporation of a novel time step rescaling in the deterministic updates of position and velocity can correct a number of dynamical defects in these integrators. finally, we identify a particular splitting (related to the velocity verlet discretization) that has essentially universally appropriate properties for the simulation of langevin dynamics for molecular systems in equilibrium, nonequilibrium, and path sampling contexts.
computed tomography (ct) angiography is an established modality for evaluation of a variety of peripheral vascular disorders including peripheral arterial diseases (pad) and peripheral vascular malformations. besides increasing detector rows allowing shorter acquisition time, an equally important component of this success is the evolution of techniques to trigger the scan at an appropriate time. over the years, bolus tracking technique in ct has become the most popular and is a time - tested technique. in patients with asymmetrical pad, variable transit time of contrast through the diseased vessels can render their opacification inadequate. such pitfalls can also occur in patients with vascular malformations of the extremities which has not been highlighted in the literature to the best of our knowledge. we present a case of lower limb arteriovenous malformation (avm), where bolus tracking technique failed to trigger the scan acquisition as the threshold hounsfield value (hu) could not be attained. a 27-year - old female, a diagnosed case of left foot avm, presented with asymmetrical enlargement of the left lower limb since childhood and recent onset bleeding from the foot following trivial trauma. on examination, there was swelling of left lower limb, predominantly of the left foot and leg. oozing of fresh blood was noted from a small ulcer over the lateral aspect of foot. a ct angiography (cta) was planned to evaluate the avm and contemplate angioembolization. cta was performed on a 64-detector ct scanner (lightspeed vct ; ge healthcare, buckinghamshire, uk) at 120 kvp, 250 ma, collimation of 0.625 mm, and reconstruction interval of 1.25 mm, pitch of 0.5, and scan time of 9 sec. one hundred milliliters of iohexol (omnipaque 300 ; ge healthcare, princeton, nj, usa) was injected without a saline flush at a rate of 4 ml / sec through an 18-gauge cannula placed in the right antecubital fossa. a circular region of interest (roi) was placed covering approximately two - thirds of the abdominal aortic diameter below the level of renal vessels. real - time hu versus time plot revealed gradual increase in enhancement that reached a maximum hu value of 150 at 20 sec after which the plot showed a falling trend, failing to reach the desired hu of 200. evaluation of cta images revealed early opacification of veins in the left lower limb [figures 2 and 3 ]. a fall in the enhancement value following initial increase to a peak of 150 hu was noted, causing a critical lapse in the functioning of automatic triggering of scan acquisition. image from the monitoring phase of cta shows the roi placed in the lumen of abdominal aorta (upper panel). plot of enhancement (y - axis) versus time (x - axis) in the lower panel reveals initial rise followed by a fall in the peak enhancement subtracted coronal volume - rendered (vr) image reveals the abnormal tangle of vessels on the dorsum of foot (arrows) and dilated, tortuous early filling veins (short arrows, compare with the right lower limb) sagittal maximum intensity projection (mip) image reveals a large number of vessels in the leg (arrows) suggestive of early filling veins contrast detection and appropriate timing of scan acquisition is a critical component of cta and an error of a few seconds can render the study non - diagnostic. amongst the various methods of contrast timing (fixed scan delay, test bolus technique, and bolus tracking technique), bolus tracking technique is the most efficient for optimizing contrast enhancement in the peripheral arteries. this technique employs a single low - dose non - contrast ct image at the level of abdominal aorta. a circular roi, 10 - 15 mm, is placed inside the aortic lumen. following a delay of 10 sec after iv contrast injection, serial low - dose monitoring ct scans are acquired at the same table position as the non - contrast scan at a pre - determined interval (usually 2 sec). when the preset enhancement level is achieved within the roi, the table is moved into position for beginning the scan and automatic triggering of scan occurs. this allows a real - time assessment of contrast enhancement characteristics leading to a highly precise timing of scan acquisition with a secondary advantage of reduced contrast requirement compared to the test bolus technique. suggested that the bolus tracking technique yields more homogeneous enhancement than does the test bolus technique. others, however, have found that with the use of appropriate timing, test bolus technique can yield similar results to bolus tracking. the threshold for triggering the scan is rather arbitrary and ranges anywhere between 50 and 150 hu depending on the ct scanner and the operator. however, the mean arterial attenuation values ranging between 232 hu and 281 hu have been found in patients with augmented peripheral arterial flow due to vascular grafts. an arterial attenuation of 200 hu or more has also been suggested for better differentiation of arteries and veins on multidetector cta (mdcta). another issue is the additional delay required between the detection of optimal contrast enhancement and actual initiation of scanning. this delay, varying from 2 to 9 sec, depends on the table position and the ct scanner capabilities. a remedy to long delay in scan initiation is using a lower enhancement threshold. yet another problem with this method of contrast timing if the roi is not placed properly in the center of the vessel, erroneous triggering occurs, yielding non - diagnostic studies. adequate contrast opacification is also dependent on patient - related factors like weight, gender, height, and cardiac output, injection rate and duration, iodine concentration, anatomical area of scan coverage, as well as scanner specifications and duration of scanning. bolus tracking, as employed in our case, substantially overcomes the individual variability of cardiac output. however, the failure to reach the threshold value in the absence of cardiac compromise can be due to various factors. the peripheral high - flow avm can result in a localized hyperdynamic circulation with a rapid run off from the arteries into the arterio - venous - precapillary anomalous connections. this would lead to decrease in local circulation time as well as greater mixing of contrast with unopacified inflowing blood in the volume of interest in the absence of cardiac function derangement, both leading to inability to attain predetermined threshold values., where instead of a uniphasic or biphasic rate of contrast injection, an exponentially decreasing rate is used. however, this needs to have be an inbuilt commercially available tool for implementation at the time of data acquisition. optical sensor based tracking of the contrast bolus has also been recently described which offers the possibility of radiation - free monitoring of contrast kinetics. saline chase is a standard technique in cta as it has been shown to decrease the contrast volume and reduce artifacts. monye reported slightly higher attenuation with the addition of bolus chaser to 80 ml of contrast material in carotid ct angiography. thus, in the present case, lack of saline chase may have contributed to the inadequate cta. in the present case, an interesting pitfall of bolus tracking technique is documented. avm of lower limb causes early filling of the deep veins with rapid mixing of the fresh contrast bolus with unopacified arterial inflow during first - pass flow. the resultant fall in the hu value in the roi leads to erroneous interpretation and failure of scan triggering. unnecessary delay due to such a technical lapse can cost a cta study as arterial washout occurs during this period. we suggest that in case of suspected avm of the periphery where cta is planned, test bolus technique may be a better method of scan timing, though at the cost of a greater contrast and radiation dosage. modification in cta scan timing technique is required in patients with peripheral avm as the bolus tracking technique may fail to achieve adequate scan timing in these patients. the purpose of highlighting this case is to bring to the forefront the often unreported or glossed over suboptimal scans due to suboptimal vascular opacification. close visual monitoring is needed during acquisition to detect a fall in peak enhancement early so that scanning can be immediately initiated manually. the threshold value also needs to be kept low as a localized decrease in circulation time would allow rapid mixing of unopacified blood and contrast dilution. automatic triggering methods should ideally be in place to initiate the scanning in case a fall in peak enhancement is present before reaching the threshold value. instead of a one size fits all approach, each scan must be tailored with meticulous attention to each of the variable factors like patient age, sex, weight, and cardiac output, and the area of interest. there is a need for a commercially available algorithmic approach inbuilt into the scanners, which takes into account these factors before scanning is initiated.	multidetector computed tomography angiography (mdcta) has become a well - established modality for limb angiography for a variety of indications. the technique of mdcta depends on the scanner features including the number of detector rows, rotation speeds and single or dual source energy. integral to a diagnostic quality cta is the acquisition timing. various techniques are available for determining the appropriate timing of scan acquisition which includes fixed delay, test bolus and the bolus tracking technique. the transit times of contrast from the aorta to the peripheral arteries shows a wide variability and is dependent upon the inter individual hemodynamic states. the bolus tracking technique is the most preferred one which allows reliable scan timing with acceptable contrast volume and radiation dose. pitfalls with all these techniques are well described and we report one such technical pitfall in a case of left foot arteriovenous malformation (avm) where the bolus tracking technique employed for scan triggering failed to initiate acquisition.
strains, plasmids, and oligonucleotides used in this study are shown in supplementary table 2. d. dadantii competition assays on chicory were carried out as described in supplementary methods. ec93 and d. dadantii 3937 cdia - ct - cdii deletions and cdia chimeras the 3 end of cdia and all of cdii from upec 536 kpsk15 aracba specrexbad - cdibai (dl5646) were replaced with cdia - ct (sequence immediately following vennx) and cdii from e. coli ec93, y. pestis co92 (accession number q7cgd9), or d. dadantii 3937 region 2 (see supplementary methods). the kpsk15 capsule mutation was used to increase the efficacy of cdi, based on our previous results showing that capsule production blocks cdi 3. immunity plasmids were constructed by ligating pcr - amplified cdii genes into plasmid pbr322 under tet promoter control (fig. the immunity plasmids were constructed by ligating pcr - amplified cdii genes into the minitn7 delivery plasmid puc18r6kt - minitn7 t under tet promoter control (see supplementary methods). 2b) was carried out by cloning specific sequences amplified by pcr into plasmid plac1114 under lac promoter control. all plasmids were propagated in epi100 acrb mutant strain dl5154 to mitigate toxic effects. in vivo interactions between cdia - ct and cdii were determined using a modified bacth bacterial two - hybrid system (euromedex) 8. -galactosidase14 and fluorescence3 analyses were carried out as previously described. in vitro affinity pull - downs with his6-tagged cdii / cdia - ct cdia - ct was released by denaturation in buffer containing 6 m guanidine - hcl, and his6-tagged cdii was released in native buffer supplemented with 250 mm imidazole.	summary paragraphbacteria have developed mechanisms to communicate and compete with one another in diverse environments 1. a new form of intercellular communication, contact - dependent growth inhibition (cdi), was discovered recently in escherichia coli 2. cdi is mediated by the cdib / cdia two - partner secretion system. cdib facilitates secretion of the cdia exoprotein onto the cell surface. an additional immunity protein (cdii) protects cdi+ cells from autoinhibition 2, 3. the mechanisms by which cdi blocks cell growth and cdii counteracts this growth arrest are unknown. moreover, the existence of cdi activity in other bacteria has not been explored. here we show that the cdi growth inhibitory activity resides within the carboxy - terminal region of cdia (cdia - ct), and that cdii binds and inactivates cognate cdia - ct, but not heterologous cdia - ct. bioinformatic and experimental analyses show that multiple bacterial species encode functional cdi systems with high sequence variability in the cdia - ct and cdii coding regions. cdia - ct heterogeneity implies that a range of toxic activities are utilized during cdi. indeed, cdia - cts from uropathogenic e. coli and the plant pathogen dickeya dadantii have different nuclease activities, each providing a distinct mechanism of growth inhibition. finally, we show that bacteria lacking the cdia - ct and cdii coding regions are unable to compete with isogenic wild - type cdi+ cells in both laboratory media and upon a eukaryotic host. taken together, these results suggest that cdi systems constitute an intricate immunity network that plays an important role in bacterial competition.
lipoid pneumonia (lp) is a rare inflammatory disease of the lung parenchyma caused by accumulation of fatty oily material in the alveoli. exogenous lipoid results from aspiration or inhalation of mineral, vegetable, or animal oil into the peripheral lung. children can aspirate large amounts of different oily materials, mineral oil being the most frequently encountered substance, and this happens especially when certain risk factors are present. the aspiration of fatty material elicits a pulmonary inflammatory reaction that produces non - specific clinical and radiologic findings, often similar to those of bacterial pneumonia and tuberculosis, complicating or delaying the diagnosis. thus, it is imperative to identify correctly this entity when there is a high degree of suspicion, through a thorough and focused noting of patient 's medical history and an accurate recognition of its radiologic manifestations. we present a case of acute lp in a 5-month - old infant due to accidental mineral oil aspiration, confirmed by analysis of bronchoalveolar lavage (bal) and focus on the radiologic findings showing evolution of parenchymal lesions over a follow - up period of 7 years. this was a case report of a 5-month - old male infant who was admitted as an emergency case to a secondary care hospital with acute onset of high - grade fever and respiratory distress. the chest x - ray showed bilateral extensive consolidation involving mainly the lower lobes of the lungs. he was placed on broad spectrum antibiotics and bronchodilatory inhalers, in response to which he showed no clinical improvement over a period of 2 days. therefore, and in view of his deteriorating clinical status, he was urgently transferred to our hospital for further management. upon arrival, the infant was in respiratory distress requiring 4 l / min of oxygen to keep his oxygen saturation above 90%. further examination of the infant revealed dysmorphic features such as low set ears, generalized muscular hypotony, and delayed psychomotor development. additional questions on patient history identified that at the age of 5-months the child had accidentally ingested paraffin oil administered by his 3-year - old brother. computed tomography (ct) scan [figure 1a ] revealed extensive bilateral consolidations in the lower lobes. the bal aspirate was opalescent with a supernatant halo of fat and showed an increased number of macrophages. bal was sent for microbiological studies including gram staining, ziehl - neelsen staining, and bacterial and fungal cultures. the cytologic study showed numerous foamy macrophages with intracytoplasmic and extracellular droplets of fat with a positive histochemical confirmation of acute lp with oil red o stain. (oil red o is a fat - soluble dye used for staining of lipids and triglycerides.) total lavage was not performed as it was considered too risky in view of the critical status of the patient and the signs of hypoxia he demonstrated during the procedure. 5-month - old male infant aspirated large amounts of paraffin oil causing respiratory distress diagnosed later as due to lipoid pneumonia (a) chest computed tomography (ct) performed 1.5 month post - paraffin oil aspiration shows diffuse extensive airspace consolidations (arrows) in both lower lobes. (b) high - resolution chest ct taken 7 months after the incident shows persistent imaging findings (arrows) with minimal improvement. a corticosteroid therapy regimen was introduced which led to an improvement of his general condition. the infant remained in our hospital for a total of 5 months showing gradual clinical improvement. before discharge an additional bronchoscopy and bal was performed that showed a clear aspirate with no supernatant fat and fewer lipid - laden macrophages. further, an additional ct scan was performed which showed persistent findings with minimal improvement [figure 1b ]. he underwent spirometry, which revealed a forced vital capacity (fvc) equal to 85% of predicted value, a forced expiratory volume in 1 s (fev1) of 79% of predicted value. fev1/fvc ratio was 81%, which was within the normal range (approximate range : 75 - 80%) for healthy adults. the patient could not undergo further pulmonary function testing (lung volume, diffusing capacity of lung for carbon monoxide). bal revealed far fewer lipid - laden macrophages, while the ct scan showed resolution of the consolidations, but evidence of residual interstitial findings [figure 2 ]. follow - up high - resolution chest computed tomography taken 7-years after the episode of aspiration, shows bilateral thickening of the interlobular septa (open arrow), streaky peripheral infiltrates (arrows) and areas of hyperinflation. lp is an uncommon pulmonary disorder that results from the accumulation of lipids in the alveoli. on the basis of the source of the lipid the exogenous type is due to aspiration of animal fat, vegetable or mineral oil and can be acute or chronic in presentation. acute exogenous lp is a type of pneumonitis associated with an incident of aspiration of a large amount of an oil - based product. most cases of exogenous lp result from aspiration of mineral oil, which is usually used for the treatment of refractory constipation. in early childhood, the factors that increase the risk of aspiration, in both the acute and chronic form, are commonly associated with underlying conditions, such as disruption of the normal physiologic function of the airway and the upper gastrointestinal tract, congenital disorders of the structure and function of the foregut, and impairment of neurologic or neuromuscular control of the process of breathing and/or swallowing. additional predisposing factors to lp in children are gastroesophageal reflux and forced ingestion of oily medication as practiced in a number of geographic areas. our patient showed features of developmental delay along with generalized muscular hypotony, which was hypothesized to have contributed to the aspiration of a large amount of material. acute exogenous lp in children, typically manifests clinically as an acute aspiration condition, which evolves into respiratory failure and, occasionally, death. in the report published by zanetti., 17 children with lp having non - specific clinical presentation, showed air space consolidation on chest radiographs, which suggested bacterial pneumonia. the children showed no improvement with antibiotic therapy and were later investigated with high - resolution ct and bal. only at this stage the time period between the initial radiographs and diagnosis varied from 30 days to 45 days. such a delay was also observed in our case, partially attributed to the initial reluctance of the parents to mention the incident of accidental aspiration, afraid of being accused of lack of proper care of the baby. acute exogenous lp manifests radiologically within 30 min of the episode of aspiration or inhalation. pulmonary opacities can be seen in most patients within 24 h. chest x - ray shows parenchymal abnormalities that include areas of ground - glass opacity or consolidation, bilateral and segmental or lobar in distribution and predominantly involving the middle and lower lobes, similar to those seen in general aspiration pneumonia. high - resolution computed tomography is the imaging technique of choice for investigating lp and, in children, it must be performed with the lowest possible radiation. the principal ct imaging findings include alveolar consolidations, ground - glass opacities, nodular lesions, interlobular septal thickening, and intralobular interstitial thickening. the crazy - paving pattern, consistent with well - defined areas of ground - glass attenuation with superimposed septal thickening, has also been described as a characteristic feature of lp. in addition, exogenous lp may be diagnosed promptly when areas of fat attenuation are identified within a pulmonary air - space consolidation. pure mineral oil has a ct attenuation value of -132 hu and negative density values (between -30 and -150 hu) within the consolidation area. these findings, although non - specific, can suggest the diagnosis of lp. however, in some patients with lp, as in the present case, fat attenuation is not evident on ct images because the fat attenuation values are averaged with the surrounding inflammatory exudates so that the fat component becomes less conspicuous or obscure. according to most studies on pediatric patients, the radiologic manifestations of acute exogenous lp typically improve or resolve over time. sias., evaluated 28 children with lp for 24 months and demonstrated normalization of ct scans in 18 of them right after treatment. in two patients scanned 12 months after treatment, a cystic lesion in the right lung and a discrete area of segmented atelectasia treatment abandonment occurred in 6 cases and no further ct follow - up was reported for the remaining children. sias., also demonstrated the potential role of multiple bals in the treatment of lp, in a prospective study of 10 children with lp. in these cases, lp was secondary to mineral oil aspiration, and the delay between aspiration and treatment was 1 - 60 days. over a follow - up period of 6 months, complete radiologic resolution was demonstrated in 3 out of 6 patients who had a short interval between intake of mineral oil (1 - 4 days) and treatment. in the remaining three cases, all four children with a longer time interval between aspiration of mineral oil and treatment had residual areas of consolidations on ct scans taken at 6-months, but no further follow - up was reported. in our case, the follow - up ct scan that was performed 7 years after the aspiration demonstrated architectural distortion associated with thickening of the interlobular septa. mineral oil (a mixture of inert, long - chain, saturated hydrocarbons obtained from petroleum) is emulsified and phagocytosed by alveolar macrophages, which, filled with oil, reach the interlobular septum through the lymphatic channels. subsequently, with the permanence of the oil, there is development of chronic interstitial inflammation, which can evolve into pulmonary fibrosis and decreased lung volume. in our case, there was no evidence of end - stage lung lesions and functional respiratory tests were normal. the imaging findings can be attributed to the large amount of aspirated oil, the delay in the correct diagnosis and the fact that the patient did not undergo any therapeutic bal, which has been shown to facilitate the removal of lipid - laden macrophages implicated in development of alveolar and interstitial fibrosis. to the best of our knowledge, this is the longest follow - up study of a pediatric patient with acute lp showing imaging features on ct and the only case reported where a single episode of fat aspiration that is associated with residual interstitial changes in the follow - up ct scan. we have described a case of acute exogenous lp induced by accidental aspiration of mineral oil for which the early imaging findings were consistent with published reports. long - term follow - up, however, revealed partial resolution with persistent interstitial lesions seen on ct scan. a finding that has not been reported in the pediatric population and merits consideration when interpreting imaging examinations of patients with a history of acute lp.	acute lipoid pneumonia (lp) in children is a rare disorder caused by the aspiration of oil - based substances and is difficult to diagnose due to non - specific clinical symptoms and radiological findings. we report the case of a 5-month - old male infant with acute lp caused by accidental aspiration of a large amount of mineral oil. we present the imaging findings in the computed tomography scans performed during his hospitalization and focus on the residual abnormalities seen on a scan performed 7-years after the incident. this, to the best of our knowledge, is the longest follow - up report of an acute exogenous lp patient and the only case that demonstrates non - resolving abnormalities in a pediatric patient after a single acute episode of mineral oil aspiration.
x - ray digital subtraction angiography (dsa) has been the gold standard investigation in the evaluation of intracranial aneurysms both before and after surgical clipping [13 ]. two - dimensional (2d) dsa has been used over the years and has been supplemented by three - dimensional (3d) rotational angiography (ra) which has been shown to be superior in evaluation of untreated aneurysms with regard to aneurysm detection rate, morphology delineation, and size estimation, features which have a direct implication in the treatment planning. thus, now 3d ra is being labelled as the new gold standard [49 ]. considering the added advantage of 3d ra over 2d dsa in evaluation of native aneurysms, it follows that the same could also be applicable to evaluation of clipped aneurysms. we undertook the study to assess whether 3d ra compared favourably to 2d dsa in picking up aneurysm remnant / recurrence. 47 consecutive patients referred to the authors for follow up angiograms after surgical clipping were taken into study and underwent both 2d dsa and 3d ra on a neurostar biplane neuroangiography suite (siemens, erlangen, germany). the 2d views taken were anteroposterior (ap), lateral (lat), and right and left anterior obliques (rao, lao) and a view with specific angle determined by preoperative dsa showing the neck of the aneurysm. 3d ra was subsequently performed, acquired data transferred to the work station (inspace, leonardo vb30b, siemens ag, berlin and munchen, 2003) automatically, and reconstruction process started immediately. 3d reconstruction and rendering : various rendering techniques [1014 ] were employed in each case : maximum intensity projection (mip), surface shaded display (ssd), volume rendered transparent (vrt), and two - color coils and clips. the aneurysm residue / recurrence was assessed by two authors (sk and nkm) and classified as per sindou. into 5 grades : grade 1 : less than 50% of neck implantation ; grade 2 : more than 50% of neck implantation ; grade 3 : residual lobe from a multilobulated sac ; grade 4 : residual portion of the sac, less than 75% of aneurysmal size ; grade 5 : residual portion of the sac more than 75% of aneurysm 's size. statistical analysis. wilcoxon signed - rank test was used to compare the different grades of aneurysm residue / recurrence in 2d dsa and 3d ra. a total of 47 patients were evaluated, who underwent angiograms of 51 vessels harbouring 54 aneurysms (4 patients had two aneurysms in the same injected vessel). the patients ' age varied from 14 years to 74 years (maximum were between 45 and 65 years) and the male to female ratio was 1 : 1.85. the aneurysms ' primary distribution was anterior communicating artery20, middle cerebral artery11, paraclinoid internal carotid artery9, posterior communicating artery8, distal anterior cerebral artery3, posterior cerebral artery, anterior inferior cerebellar artery, and basilar tip1 each. figure 1 shows the presence / absence and the different grades of residual / recurrent aneurysm in a head - to - head comparison of 2d dsa and 3d ra. the residual / recurrent aneurysm detection rate was 53.70% (29/54 aneurysms) with 2d dsa and 66.67% (36/54 aneurysms) with 3d ra. the results of the wilcoxon signed rank test were w = 83, n = 17, z = 1.95, area between z = 0.9488, p(1-tail) = 0.0256, and p(2-tail) = 0.0512 (just about significant). in 12 aneurysms, 3d ra upgraded the residue / recurrence, among which nine had been completely not detected on 2d dsa and were found to have grade one or two residual necks on the 3d ra, and, in three aneurysms, a small neck on 2d dsa turned out to be aneurysm sac on 3d ra. in a total of 5 aneurysms, the classification was downgraded by 3d ra, one each from grade 1 to 0, grade 2 to 0, grade 3 to 1, grade 3 to 2, and grade 4 to 3. by simultaneously rotating the x - ray tube and the image intensifier during the intra - arterial injection of contrast media, after having taken a spin of mask images, ra and 3d reconstructed images enable us to obtain accurate information regarding the cerebral arteries and the pathologies they possess by visualizing them from different perspectives and avoiding vessel overlap. the evaluation of the result of aneurysm clip placement is important because the complete exclusion of aneurysms from the circulation is the aim of the operation. the incidence of recurrent aneurysm after successful clip placement has been reported to be 1.52.9% [16, 17 ], while the reported incidences of residual aneurysms are between 4% and 8% [1821 ] and the risk of bleeding from residual aneurysm is believed to be 1 - 2% [16, 18 ]. 2d dsa has been used as the standard for the follow - up imaging of clipped aneurysms. however, the small remnant / recurrent aneurysms are often difficult to detect and evaluate because of the clip mass and overlap of adjacent vessels. kang. studied 71 patients with 88 clipped aneurysms and found 37 residual aneurysms (42%) with 3d ra as opposed to only 18 with 2d dsa. they attributed this to the excellent image quality and the various viewing angles provided by 3d angiography coupled with the ability to avoid and if necessary delete the overlapping vessels. murakamia. have also published a paper wherein they assessed 67 treated (both coiled and clipped) aneurysms and found good results with regard to remnant detection rate. we had a very high residual / recurrent aneurysm detection rate (53.70% and 66.67% with 2d this high percentage was possibly because many of the cases were referred to us and the operating neurosurgeon might have had a suspicion of a residue. however, we do not have correlation with intraoperative findings available for all the patients. 3d ra excellently showed the clip - artery, clip - aneurysm, and artery - aneurysm relationship (figure 2). in fact, most extra residues picked were type 1 or type 2 residual necks. out of the 12 extra aneurysms picked up, 9 were advised a prolonged vigil for aneurysm regrowth and only 3 were advised retreatment (which the patients promptly refused). in our study, we studied ssd, vrt, mip and two - color, coils and clips reconstructed images in all the cases in multiple viewing angles and varied thresholds. as per our day - to - day experience, all of these commonly used techniques, namely, ssd, vrt, mip, and two - color coils and clips have to be viewed for adequate assessment. an example (figure 4) illustrates this fact, wherein two - color and mip could show the artery and clip relationship well, while ssd could not, and vrt could do so only in limited manner. hence, a difference between these various techniques was not evaluated as all of them were found to be complementary to each other and indispensable. the final opinion formed after analyzing all the images was compared with that of 2d dsa findings. 3d ra picks up more residual / recurrent aneurysms than 2d dsa and both 2d dsa and 3d ra should be performed in follow - up of clipped aneurysms.	introduction. 3d rotational angiography (ra) is indispensable for evaluation of intracranial aneurysms, providing infinite viewing angles and defining the aneurysm morphology. its role in follow - up of clipped aneurysms remains unclear. we aimed to compare the aneurysm residue / recurrence detection rate of 3d ra with 2d digital subtraction angiography (dsa). methods. 47 patients harboring 54 clipped aneurysms underwent both 2d dsa and 3d ra. the residual / recurrent aneurysms were classified into five grades and the images of both modalities were compared. results. the residual / recurrent aneurysm detection rate was 53.70% (29/54 aneurysms) with 2d dsa and 66.67% (36/54 aneurysms) with 3d ra (p = 0.05). in 12 aneurysms, 3d ra upgraded the residue / recurrence among which nine had been completely not detected on 2d dsa and were found to have grade one or two residual necks on the 3d ra, and, in three cases, a small neck on 2d dsa turned out to be aneurysm sac on 3d ra. in a total of 5 aneurysms, the classification was downgraded by 3d ra. conclusion. 3d ra picks up more aneurysm residue / recurrence ; hence, both 2d dsa and 3d ra should be performed in follow - up evaluation of clipped aneurysms.
male sprague dawley rats were made diabetic with a single intraperitoneal injection of 75 mg / kg stz (sigma, st louis, mo). insulin implants (two linplant implants ; linshin canada, toronto, on, canada) were placed subcutaneously into stz - diabetic rats after 18 weeks of diabetes and kept in place for 4 weeks. animal procedures followed the guidelines of the university of manitoba animal care committee using the canadian council of animal care rules. immortalized sc16 schwann cells were cultured in low glucose dulbecco 's modified eagle 's medium containing 125 mg / l c6-lysine (k6) and 84 mg / l c6,n4-arginine (r10) (cambridge isotopes, andover, ma), 10% dialyzed fcs (atlas biologicals, fort collins, co), and antibiotics (22). crude mitochondria from labeled cells were obtained by differential centrifugation and purified through a discontinuous nycodenz gradient (23). for quantitative analysis of the drg mitochondrial proteome, the k6r10-labeled mitochondria were used as a source of culture - derived isotope tags to serve as internal standards (20). after assessing the protein concentration of the preparations, the unlabeled mitochondrial protein (k0r0) obtained from each of the control (n = 4), diabetic (n = 3), and diabetic + insulin (n = 4) treatments were mixed in a 2:1 ratio with k6r10 labeled mitochondria. total protein (6070 g) was subjected to sds - page, the gel was stained with colloidal coomassie blue, and lanes were cut into 5 1 cm pieces. a detailed description of these parameters is provided in supplementary table 1 in the online appendix 2 at http://diabetes.diabetesjournals.org/cgi/content/full/db10-0818/dc1. lumbar drg sensory neurons from adult male sprague - dawley rats were isolated and dissociated as described (6,16). cells were plated onto poly - d - l - ornithine hydrobromide and laminin - coated 25-mm glass cover slips (german glass # 1, electron microscopy sciences, hatfield, pa). neurons were grown in defined hams f-12 medium with n2 additives (no insulin), supplemented with neurotrophic factors : 0.1 ng / ml nerve growth factor, 1.0 ng / ml glial cell line - derived neurotrophic factor, and 0.1 ng / ml neurotrophin-3 (all obtained from sigma). neurons from control rats were cultured with 10 mmol / l d - glucose and 10 nmol / l insulin and neurons from diabetic rats were plated with 25 mmol / l d - glucose and no insulin for 1 to 3 days. cultured neurons from control or diabetic rats were either 1) imaged in real time for intracellular ros by loading with 1.2 mol / l 5-(and-6)-chloromethyl-27-dichlorodihydrofluorescein diacetate acetyl ester (cm - h2dcfda), or 2) fixed and stained for adducts of 4-hydroxy-2-nonenal (4-hne) (a product of lipid peroxidation), as previously described (16). to study the role of polyol pathway in ros production, cultures were treated acutely with the specific sorbitol dehydrogenase (sdh) inhibitor, sdi-158 (24) (10 mol / l ; a gift from dr. cultured drg neurons were loaded with 3.0 nmol / l tetramethyl rhodamine methyl ester (tmrm ; molecular probes, eugene, or) for 1 h and the fluorescence signal in the axons detected with a carl zeiss lsm510 confocal inverted microscope (x100 objective ; excitation at 540 nm and emission > 560 nm). the tmrm was used in subquench mode in which decreased fluorescence intensity indicates reduced mitochondrial inner membrane potential (25). antimycin a and oligomycin (sigma) were injected into the culture media to a final concentration of 10 mol / l and 1 mol / l, respectively, at 1 min after baseline fluorescence measurements. all axons in each field were assessed as the average of fluorescence pixel intensity per axon length using the carl zeiss software package (16). intramitochondrial ros generation, mainly superoxide, was detected using the fluorescent mitosox red dye (molecular probes, catalog # m36008). lumbar drg neurons were loaded with 400 nmol / l of mitosox red (in 100% anhydrous dmso ; molecular probes) for 15 min with or without 1.0 mol / l oligomycin at 37c, and then washed three times with f-12 and excited at 514 nm and emission > 560 nm. oxygen consumption was determined at 37c using the oroboros oxygraph-2k (oroboros instruments gmbh, innsbruck, austria) (8). mitochondria from freshly isolated and intact lumbar drg were resuspended in kcl medium (80 mmol / l kcl, 10 mmol / l tris - hcl, 3.0 mmol / l mgcl2, 1.0 mmol / l edta, 5.0 mmol / l potassium phosphate, ph 7.4). various substrates and inhibitors of the mitochondrial respiratory chain complexes were used as described (8). enzymatic activities in lumbar drg mitochondrial preparations were performed spectrophotometrically as previously described (8). when appropriate, data were subjected to one - way anova with post hoc comparison using the tukey test or regression analysis with a one - phase exponential decay parametric test with the fisher parameter (graphpad prism 4, graphpad software, san diego, ca). in all other cases, two - tailed student t tests were performed. to determine the threshold for statistical significance for the proteomic data, proteins showing at least a 20 or 25% increase or decrease were grouped and compared with the entire dataset using a kruskal - wallis nonparametric anova and the dunn multiple comparison test. this analysis indicated that a minimum difference of 25% was necessary for a value to be considered statistically different from the dataset. male sprague dawley rats were made diabetic with a single intraperitoneal injection of 75 mg / kg stz (sigma, st louis, mo). insulin implants (two linplant implants ; linshin canada, toronto, on, canada) were placed subcutaneously into stz - diabetic rats after 18 weeks of diabetes and kept in place for 4 weeks. animal procedures followed the guidelines of the university of manitoba animal care committee using the canadian council of animal care rules. mitochondrial preparations from drg were isolated as described (8,21). immortalized sc16 schwann cells were cultured in low glucose dulbecco 's modified eagle 's medium containing 125 mg / l c6-lysine (k6) and 84 mg / l c6,n4-arginine (r10) (cambridge isotopes, andover, ma), 10% dialyzed fcs (atlas biologicals, fort collins, co), and antibiotics (22). crude mitochondria from labeled cells were obtained by differential centrifugation and purified through a discontinuous nycodenz gradient (23). for quantitative analysis of the drg mitochondrial proteome, the k6r10-labeled mitochondria were used as a source of culture - derived isotope tags to serve as internal standards (20). after assessing the protein concentration of the preparations, the unlabeled mitochondrial protein (k0r0) obtained from each of the control (n = 4), diabetic (n = 3), and diabetic + insulin (n = 4) treatments were mixed in a 2:1 ratio with k6r10 labeled mitochondria. total protein (6070 g) was subjected to sds - page, the gel was stained with colloidal coomassie blue, and lanes were cut into 5 1 cm pieces. a detailed description of these parameters is provided in supplementary table 1 in the online appendix 2 at http://diabetes.diabetesjournals.org/cgi/content/full/db10-0818/dc1. lumbar drg sensory neurons from adult male sprague - dawley rats were isolated and dissociated as described (6,16). cells were plated onto poly - d - l - ornithine hydrobromide and laminin - coated 25-mm glass cover slips (german glass # 1, electron microscopy sciences, hatfield, pa). neurons were grown in defined hams f-12 medium with n2 additives (no insulin), supplemented with neurotrophic factors : 0.1 ng / ml nerve growth factor, 1.0 ng / ml glial cell line - derived neurotrophic factor, and 0.1 ng / ml neurotrophin-3 (all obtained from sigma). neurons from control rats were cultured with 10 mmol / l d - glucose and 10 nmol / l insulin and neurons from diabetic rats were plated with 25 mmol / l d - glucose and no insulin for 1 to 3 days. cultured neurons from control or diabetic rats were either 1) imaged in real time for intracellular ros by loading with 1.2 mol / l 5-(and-6)-chloromethyl-27-dichlorodihydrofluorescein diacetate acetyl ester (cm - h2dcfda), or 2) fixed and stained for adducts of 4-hydroxy-2-nonenal (4-hne) (a product of lipid peroxidation), as previously described (16). to study the role of polyol pathway in ros production, cultures were treated acutely with the specific sorbitol dehydrogenase (sdh) inhibitor, sdi-158 (24) (10 mol / l ; a gift from dr. cultured drg neurons were loaded with 3.0 nmol / l tetramethyl rhodamine methyl ester (tmrm ; molecular probes, eugene, or) for 1 h and the fluorescence signal in the axons detected with a carl zeiss lsm510 confocal inverted microscope (x100 objective ; excitation at 540 nm and emission > 560 nm). the tmrm was used in subquench mode in which decreased fluorescence intensity indicates reduced mitochondrial inner membrane potential (25). antimycin a and oligomycin (sigma) were injected into the culture media to a final concentration of 10 mol / l and 1 mol / l, respectively, at 1 min after baseline fluorescence measurements. all axons in each field were assessed as the average of fluorescence pixel intensity per axon length using the carl zeiss software package (16). intramitochondrial ros generation, mainly superoxide, was detected using the fluorescent mitosox red dye (molecular probes, catalog # m36008). lumbar drg neurons were loaded with 400 nmol / l of mitosox red (in 100% anhydrous dmso ; molecular probes) for 15 min with or without 1.0 mol / l oligomycin at 37c, and then washed three times with f-12 and excited at 514 nm and emission > 560 nm. oxygen consumption was determined at 37c using the oroboros oxygraph-2k (oroboros instruments gmbh, innsbruck, austria) (8). mitochondria from freshly isolated and intact lumbar drg were resuspended in kcl medium (80 mmol / l kcl, 10 mmol / l tris - hcl, 3.0 mmol / l mgcl2, 1.0 mmol / l edta, 5.0 mmol / l potassium phosphate, ph 7.4). various substrates and inhibitors of the mitochondrial respiratory chain complexes were used as described (8). enzymatic activities in lumbar drg mitochondrial preparations were performed spectrophotometrically as previously described (8). when appropriate, data were subjected to one - way anova with post hoc comparison using the tukey test or regression analysis with a one - phase exponential decay parametric test with the fisher parameter (graphpad prism 4, graphpad software, san diego, ca). in all other cases, two - tailed student t tests were performed. to determine the threshold for statistical significance for the proteomic data, proteins showing at least a 20 or 25% increase or decrease were grouped and compared with the entire dataset using a kruskal - wallis nonparametric anova and the dunn multiple comparison test. this analysis indicated that a minimum difference of 25% was necessary for a value to be considered statistically different from the dataset. stz - diabetic rats did not suffer weight loss during the study, but they showed reduced weight gain after 22 weeks of stz - diabetes compared with age - matched controls (table 1). persistence of diabetes was indicated by elevated nonfasting blood glucose and glycated hemoglobin levels (table 1). stz - diabetic rats that received insulin supplementation for the final 4 weeks of a 22-week period of diabetes showed a partial, but statistically significant, recovery of body weight, blood glucose, and glycated hemoglobin levels. body weights, plasma glucose, and glycated hemoglobin (hba1c of treatment groups values are means sd. starting weights for the groups were 293 8.8 g (mean sd ; n = 38). nonfasting blood sugar concentration was measured using the accu - chek compact plus glucometer (roche, laval, quebec city, canada) and blood glycated hemoglobin (hba1c) levels by the a1cnow + system (bayer healthcare, sunnyvale, ca). p < 0.001 vs. other groups ; p < 0.001 vs. diabetic + insulin (one - way anova with tukey 's test). to quantitatively assess the effect of diabetes and insulin therapy on the mitochondrial proteome of lumbar drg, we labeled sc16 immortalized schwann cells with isotopic forms of lysine (k6) and arginine (r10) and isolated labeled mitochondria to serve as internal standards (fig. we examined the quantitative accuracy of this approach by mixing the k6r10:k0r0 mitochondria in ratios of 0.75:1, 1.5:1, and 3:1. the k6/k0 or r10/r0 ratios for individual peptides were obtained from maxquant analysis, and a linear response was observed after plotting the average peptide ratio obtained from each mixture against the known mixing ratio (supplementary fig. 1 in online appendix 1). unlabeled (k0r0) drg mitochondria from the three treatments were then individually mixed in a 2:1 ratio with the k6r10 mitochondria prior to sds - page and ltq - ft ms / ms analysis. from more than 43,600 identified peptides, after culling out contaminants (n = 30), reverse - decoy hits (n = 13), and proteins identified by only a single unique peptide, we identified 672 proteins, of which 334 (49.6%) were quantified by at least one unique peptide identified in samples from at least two animals (supplementary online table 1). the median number of quantified ratios for the three treatments was : control (n = 5), diabetic (n = 8), and diabetic + insulin (n = 7). of the total proteins identified, 206 (30%) were annotated as mitochondrial / glycolytic and 151 were quantified (73%). unlabeled (k0r0) mitochondrial fractions were prepared from the lumbar drgs obtained from each animal in the three treatment groups. each k0r0 mitochondrial fraction was mixed in a 2:1 ratio with k6r10-labeled mitochondria obtained from sc16 cells that had been metabolically labeled with c6 lysine (k6) and c6,n4 arginine (r10) for 10 days. the proteins were resolved by sds - page, digested with trypsin, and analyzed by rp - hplc / ltq - ft ms / ms. for each protein, the ratio of k0r0 to k6r10 quantifies the endogenous protein relative to the internal standard. dividing the protein ratios obtained in the diabetic or diabetic + insulin treatment by those obtained from control animals cancels out the k6r10 internal standard and provides the fold control value. the protein expression ratios from the diabetic and diabetic + insulin treatments were binned and the number of proteins per bin counted. c : to determine the effect of diabetes and insulin therapy on mitochondrial versus nonmitochondrial proteins, the expression ratio for each protein was plotted against each treatment. solid and dotted lines demarcate the threshold necessary for proteins to show a significant change in the diabetic and diabetic + insulin treatments, respectively. proteins in between dotted and solid lines did not change with either treatment. yellow shading indicates proteins that were significantly up- or downregulated by diabetes and normalized by insulin therapy. blue shading indicates proteins that were increased by diabetes but not normalized by insulin therapy. green shading indicates proteins not affected by diabetes but increased by insulin therapy. to provide a global view of the effect of diabetes and insulin therapy on protein expression, insulin therapy induced a rightward shift toward normalizing expression and promoted a significant increase in protein expression. pathway analysis found that the proteins associated with mitochondrial dysfunction, oxidative phosphorylation, and ubiquinone biosynthesis (primarily complex i proteins) were the most significantly over - represented and showed the greatest percentage of proteins that underwent significant downregulation (table 2). consistent with the diabetic phenotype, proteins associated with ketone body biology were also over - represented and diabetes increased the expression of succinyl - coa:3-ketoacid - coa transferase one (scot), which is critical in acetoacetate clearance. over - represented canonical pathways identified in the proteomic analysis significance provides the confidence of the association as identified by the p value of the fisher exact test. the ratio provides the percentage of proteins associated with the pathway that underwent a significant change. the total genes column refers to all known genes to be linked to the pathway (not necessarily identified by the proteomic screen). to determine if diabetes and insulin therapy had a distinct effect on mitochondrial versus nonmitochondrial proteins, the expression ratios for each protein were plotted against each treatment (fig. this analysis indicated that diabetes has little effect on the majority of mitochondrial and nonmitochondrial proteins that were quantified (the region between solid and dotted lines). with rare exception, insulin therapy did not decrease protein expression, but led to a significant increase in the expression of numerous nonmitochondrial proteins (green shading). enrichment analysis of proteins in this region using the biological networks gene ontology (bingo) plugin of cytoscape found that the cluster frequency for proteins annotated to the biologic process of translation was 36.1%, a sevenfold enrichment. we also noted a small group of proteins that were significantly increased by diabetes but whose expression was unchanged by insulin (blue shading). bingo analysis of this subset of proteins indicated that small g protein signaling and protein transport were the enriched processes. diabetes caused a statistically relevant change in 27% of quantified mitochondrial proteins, and insulin therapy had an ameliorative effect that, in general, normalized this decrease (fig. 1c, yellow shading). consistent with another proteomic study of heart mitochondria (14), bioinformatic analysis found that proteins associated with canonical pathways of mitochondrial dysfunction and oxidative phosphorylation were over - represented and mainly decreased in expression (table 3). representative examples from a diabetic animal show a 51% decrease in the complex i protein, nadh dehydrogenase fe - s protein 3 (ndufs3) and a 29% decline of mn superoxide dismutase (mn - sod) (fig. however, insulin therapy improved the deficits in ndufs3 and mn - sod expression (fig. 2c) as previously characterized using western blotting (8,16). on the other hand, diabetes did not alter the expression of atp synthase (supplementary fig. 2 in supplementary online appendix 1). effect of diabetes and insulin therapy on representative proteins annotated to oxidative phosphorylation and mitochondrial dysfunction values shown are the mean and the asterisk () indicates proteins that showed a significant change in expression. percent change represents effect of insulin treatment on the protein expression ratio measured from diabetic rats. representative peptide mass spectra showing the effect of diabetes and insulin therapy on ndufs3 and mnsod. a : the upper spectrum shows the doubly - charged ion of the unlabeled (m / z 743.90) and r10 labeled (m / z 748.90) vvaepvelaqefr peptide of ndufs3 from a control animal. since the peptide is doubly charged, the mass difference is 5 atomic mass units ; and the other peaks represent the isotopic envelope of the monoisotopic peak. the lower spectrum shows the doubly - charged ion of the unlabeled (m / z 720.91) and k6 labeled (m / z 723.91) gdvttqvalqpalk peptide of mn - sod from a control animal. since the peptide is doubly charged, the mass difference is 3 atomic mass units ; and the other peaks represent the isotopic envelope of the monoisotopic peak. b : upper and lower spectra show the same ndufs3 and mn - sod peptides, but from a diabetic animal. the k0r0/k6r10 ratios for each peptide are indicated and the diab / control ratio were obtained after dividing by the control ratios from panel a. c : upper and lower spectra show the same ndufs3 and mn - sod peptide, but from a diabetic + insulin - treated animal. the k0r0/k6r10 ratios for each peptide are indicated and the diab / control ratio were obtained after dividing by the control ratios from panel a. note that the intensity of the k6 and r10 peptides are very similar between the treatments (a c), indicating that the changes in protein expression are minimally influenced by the internal standard. lumbar drg from age - matched control and 22-week - old stz - diabetic rats were analyzed for rates of oxygen consumption as shown in fig. respiratory chain activity, whether coupled or uncoupled, was significantly depressed in diabetic samples. in agreement with the proteomic data and oxygen consumption results, the enzymatic activities of rotenone - sensitive nadh - cytochrome c reductase (complex i) and cytochrome c oxidase (cox ; complex iv), as well as the krebs cycle enzyme, citrate synthase, were significantly decreased in stz - diabetic rats compared with control (supplementary table 2). a : oxygen consumption was assessed in freshly isolated mitochondria from lumbar drg of age - matched control and 22-week - old diabetic rats using an oroboros oxygraph 2k. coupled respiration rates were measured in the presence of pyruvate (p) (10 mmol / l), malate (m) (5.0 mmol / l), and adp (2.0 the addition of fccp (0.5 mol / l) permits a measure of uncoupled respiratory chain activity. addition of ascorbate (asc) (5.0 mmol / l) and tmpd (0.5 mmol / l) permit an analysis of complex iv activity that was verified by specific inhibitors. values are mean sem ; n = 5. p < 0.05 vs. controls ; p < 0.001 vs. controls. b : images of fluorescence confocal microscopy using tmrm in live cultures of drg neurons isolated from control adult rats showing effect of antimycin a and oligomycin. c : trace of tmrm fluorescence signal in the axons of cultured drg neurons isolated from age - matched controls and stz - diabetic rats. d : shows the area under the tmrm fluorescence trace (area under the curve) for control (open bar) and diabetic (filled bar) neurons. the area under the curve was estimated from the baseline (at the point of injection) to a fluorescence level of 0.2 and between time points 1.0 min and 6 min using sums of squares (shown by dotted line). values are the means sem, n = 6580 axons ; p < 0.001 compared with control, t test. the rate constant of decay (k) = 0.013 0.0004 (control) and 0.006 0.0001 (diabetic). half - life of decay = 54.19 s (control) and 108.7 s (diabetic). the fisher parametric (f) ratio = 409.5, p < 0.0001, control vs. diabetic. (a high - quality digital representation of this figure is available in the online issue.) adult sensory neurons were cultured for 1 day from age - matched controls and 22-week - old stz - diabetic rats and loaded with tmrm. this dye was used at a subquench concentration where a decrease in fluorescence signal intensity indicated reduced mitochondrial inner membrane potential (25). the live neurons were exposed to a combination of antimycin a (inhibitor of complex iii) and oligomycin (inhibitor of atp synthase), and the fluorescence signal in axons detected by confocal microscopy. antimycin a blocks electron transfer leading to mitochondrial depolarization, whereas oligomycin inhibits the atpase and prevents reverse pumping of protons and associated generation of a proton gradient. therefore, the mitochondrial membrane potential (and associated proton gradient) is completely dissipated in the presence of both these drugs. in the presence of antimycin a + oligomycin, the rate of mitochondrial depolarization was more rapid in axons of normal neurons compared with diabetic neurons (fig. this suggests that prior to the addition of antimycin a + oligomycin, the axonal mitochondria were more highly polarized in the normal neurons compared with the diabetic neurons. mitochondrial physiology was further investigated by treating cultured neurons from control and diabetic rats with oligomycin alone. blockade of the atpase results in a buildup of the transmembrane proton gradient and induces hyperpolarization of the mitochondrial inner membrane, as indicated by elevated tmrm fluorescence (fig. a transient hyperpolarization was observed, followed by a recovery due to adaption of the respiratory chain. for example, uncoupling proteins become active and dissipate the proton gradient under a high inner membrane potential (27). diabetic neurons exhibited a significantly greater level of hyperpolarization upon oligomycin application and the adaptive response was impaired. impaired respiratory function is associated with reduced ros generation in the mitochondrial matrix of cultured neurons isolated from stz - diabetic rats. a : tmrm fluorescence trace of oligomycin - induced mitochondrial inner membrane hyperpolarization in the axons of control and diabetic neurons. inset shows the area under the tmrm fluorescence trace (auc) for control (open bar) and diabetic (filled bar) neurons. the auc was estimated from the baseline (at the point of injection, dotted line), and between time points of 1.0 min and 4 min using sums of squares. values are mean sem, n = 6580 axons,p < 0.01 compared with control, t test. b e : images of mitosox red fluorescence in cultures of drg neurons showing the effect of 5.0 mol / l fccp. f : quantification of real - time mitosox red fluorescence levels in the axons of cultured drg neurons after 5.0 mol / l fccp treatment. f ratio = 32.48, p < 0.0001 (control vs. diabetic with or without oligomycin by one - way anova). values are mean sem, n = 3573 axons ; p < 0.001 compared with diabetic or diabetic + oligomycin - treated cells by one - way anova. (a high - quality digital representation of this figure is available in the online issue.) we determined if the respiratory chain was contributing to oxidative stress in diabetic neurons by loading cells with the mitochondrially - targeted ros detector, mitosox red. a subset of diabetic neurons was pretreated with oligomycin to hyperpolarize the inner mitochondrial membrane and maximize loading of mitosox red into the mitochondrial matrix. neurons were treated with the uncoupler, carbonylcyanide - p - trifluoromethoxyphenylhydrazone (fccp), to dissipate the transmembrane electrochemical gradient and enhance the rate of electron transfer. increased respiratory chain activity leads to augmented electron leakage and associated generation of ros, primarily superoxide. in normal neurons this was demonstrated with elevated fccp - induced mitosox red fluorescence (fig. 4f and g). diabetic neurons, with or without prior oligomycin treatment, exhibited lower mitosox red fluorescence intensities indicative of reduced levels of superoxide being generated by the respiratory chain. parallel cultures demonstrated elevated oxidative stress in axons of diabetic neurons under 25 mmol / l glucose versus control neurons as exhibited by raised dichlorodihydrofluorescein (dcf) fluorescence and enhanced staining for adducts of 4-hne (fig. 5a subsets of these cultures were treated acutely with the specific sdh inhibitor, sdi-158, to investigate the role of the polyol pathway in ros production. figure 5 g and h shows that blockade of high glucose - dependent sorbitol production results in reduced ros generation in axons of diabetic neurons. axons of sensory neurons from stz - diabetic rats exhibit elevated oxidative stress that is ameliorated by the blockade of sdh. images of ros levels in axons at 24 h in adult drg neuron culture from (a) control and (b) stz - diabetic rats. cultures were stained for ros using cm - h2dcfda dye (dcf is the fluorescent product resulting from oxidation). values are means sem, n = 4457 axons, p < 0.05 by t test. immunofluorescent images of accumulation of adducts of 4-hne in axons in sensory neuron cultures after 3 days ; (c) is control and (d) is diabetic culture. values are means sem, n = 1927 axons, p < 0.01 by t test. g : trace of dcf - fluorescence signal in the axons of cultured drg neurons isolated from age - matched controls and stz - diabetic rats and treated acutely with 10 mol / l sdi-158. k = 0.09 0.02 (control) and 0.13 0.009 (diabetic). half - life of decay = 7.5 min (control) and 5.5 min (diabetic). h shows the area under the dcf fluorescence trace (auc) for control (open bar) and diabetic (filled bar) neurons. the auc was estimated from 0.2 to 1.6 on fluorescence axis and between time points 0 to 22 min using sums of squares (dotted lines show upper and lower limits). values are means sem, n = 4251 axons ; p < 0.01 compared with control by t test. (a high - quality digital representation of this figure is available in the online issue.) the results show that respiratory chain components of the mitochondrial proteome are downregulated in drg in diabetes and this phenotypic alteration was associated with impairment in respiratory chain activity. in addition, for the first time, this study demonstrates that altered mitochondrial proteome expression is linked to altered mitochondrial physiology in axons of diabetic neurons. and finally, although oxidative stress was present in axons of diabetic neurons, the results show that polyol pathway activity, not aberrant respiratory chain function, contributes to generation of ros. we used silac to provide a set of culture - derived isotope tags (20) to serve as internal standards for quantifying the effect of diabetes on the composition of the mitochondrial proteome from drg. one advantage of culture - derived isotope tags is the quantitative accuracy that can be achieved relative to label - free approaches (28), especially for low abundant proteins. despite our small sample size, a 25% change in expression was sufficient to reach statistical significance, and the number of mitochondrial proteins that exceeded this threshold was similar to that previously reported in mitochondria isolated from heart tissue of diabetic akita mice (14). further, the results of our unbiased quantification were in close agreement with those obtained by targeted immunoblot analyses for cox subunit iv, ndufs3, atp synthase, and mn - sod in two prior studies (8,16). similar to a previous gene array study delineating alterations in mrna expression in drg from diabetic rats (29), the magnitude of changes observed in the mitochondrial proteins were rather modest and averaged 0.44 0.16-fold in either direction. interestingly, the gene array study reported that after 2 months of diabetes, no modification occurred in gene expression of enzymes associated with the tricarboxylic acid cycle. we also observed no changes in pyruvate dehydrogenase and in six of eight of the tricarboxylic acid cycle enzymes at the protein level, the exceptions being fumarate hydratase and succinyl co - a ligase. similarly, with the exception of hexokinase 1, the remaining glycolytic enzymes were not altered, in agreement with mrna expression studies at 2 months of diabetes (29). we previously reported that hexokinase 1 localizes to mitochondria of drg (30) and this protein was reproducibly detected in the heavy mitochondrial fraction of all animals. interestingly, although the hexokinase gene and protein expression were not modified after 23 months of diabetes in two studies (2930), expression was decreased after 22 weeks of diabetes in the present study. since this was the only glycolytic enzyme that significantly changed, it is tempting to speculate that its lowered expression also has a function separate from glucose metabolism. in this regard, sensory neurons from diabetic adult animals have an impaired ability to support neurite development (16) and directly inhibiting hexokinase activity blocks neurotrophin - induced neuritogenesis (31). in the cardiac system, diminished mitochondrial respiratory function caused by diabetes has also been identified by proteomic and gene array techniques (14,32). these broad changes in gene expression could be triggered by altered activity of key upstream regulators. for example, in human skeletal muscle in type 2 diabetes, the transcriptional regulator nrf-1 and the transcriptional coactivator peroxisome proliferator - activated receptor- coactivator 1 (pgc-1) were downregulated and corresponded with reduced expression of proteins that regulate cellular energy metabolism, including mitochondrial biogenesis and oxidative phosphorylation (33,34). in fact, our preliminary data demonstrated a significant reduction in activity of amp kinase, a regulator of pgc1-, in drg in type 1 diabetic rodents (manuscript in preparation). initial exposure to high glucose concentration over 1 to 4 weeks in drg in diabetic rats is linked to upregulation of glycolytic pathway expression (mrna) (29). studies in endothelial or schwann cells demonstrate that acute exposure to glucose elevates ros and respiratory chain activity, respectively (2,23). therefore, in the short term, hyperglycemia triggers enhanced glycolysis and associated respiratory chain activity (and possibly ros). however, in the longer term, the high intracellular glucose concentration provides an ample supply of atp via several nonmitochondrial - dependent pathways. consequently, the metabolic phenotype of the cell adapts and functions in the absence of a dependence on the tricarboxylic acid cycle and oxidative phosphorylation for atp production, possibly by initiating a process homologous to the thus, rates of electron donation to the respiratory chain are suboptimal in neurons in long - term diabetic rats and may predispose to lower rates of mitochondrial respiratory chain activity and oxidative phosphorylation. key metabolic activity sensors and/or regulators such as ampk and nrf-1 are putative candidates for this modulation, although it is unlikely that subsequently adapted metabolism of glucose is channeled through glycolysis in isolation. in this regard, elevated glucose flux through the aldehyde and aldose reductase pathway could be critical (24,36). for example, studies in lens (37), retina (5), and cardiac tissue (38) in medium- to long - term animal models of type 1 diabetes show that parts of the glycolytic pathway function are depressed. ros production linked to enhanced electron leakage from the respiratory chain induced by uncoupling, and theoretically comprising superoxide, was lower in the axons of neurons from diabetic rats (fig. 4 and 5 g provide preliminary evidence that the sources of ros in axons of diabetic neurons are not from aberrant respiratory chain function, but in part from polyol pathway activity. the latter pathway has been proposed as a source of ros through a putative sorbitol accumulation - dependent nadph oxidase route in previous studies (24,36). downregulation of the respiratory chain machinery would be predicted to lead to depolarization of the mitochondrial membrane (fig. 3c was not the result of resistance to uncoupling in the diabetic neurons since complementary measures of respiratory chain activity show lower rates of electron transfer in diabetic neurons ; also, see fig. 1 in chowdhury. (8). these findings differ from those in cultured endothelial cells where high glucose concentration enhanced mitochondrial membrane potential and induced elevated ros (23). figure 4a reveals that oligomycin treatment caused a greater level of mitochondrial inner membrane hyperpolarization above baseline in diabetic neurons compared with normal neurons, further highlighting that adult sensory neurons with a history of diabetes and under high glucose concentration behave differently to endothelial cells. the adaption of mitochondria in normal neurons to hyperpolarization was not observed in diabetic neurons, again stressing the aberrant phenotype of mitochondrial physiology. uncoupling proteins such as adenine nucleotide transporters (ant1/2) contribute to the dissipation of a high mitochondrial membrane potential (27) and expression was depressed in diabetic mitochondria (supplementary table 1). in conclusion, our proteomics data reveal a range of altered expression profiles in the mitochondrial proteome of drg from diabetic rats. this modified expression pattern was associated with aberrant mitochondrial respiratory chain physiology and function. under high glucose concentration, the neuron cell body perceives that mitochondrial function can be downgraded ; however, this may ignore the unique high - energy requirements of the nerve ending and contribute to distal axon degeneration. for example, growth cone motility that underpins axonal plasticity and regeneration in the skin has an exceedingly high demand for atp because of significant levels of actin treadmilling (39). impaired respiratory chain function did not elevate ros generation, even though oxidative stress was observed in axons. in fact, the lower rates of respiratory chain activity were linked to mitochondrial membrane depolarization, improper adaption to oligomycin - induced membrane hyperpolarization, and reduced levels of superoxide derived from electron leakage during electron transport. in axons of neurons from long - term diabetic rats, sites of ros production colocalize with the mitochondrial compartment (16,40). for example, nadph oxidase has been localized to the mitochondrial compartment of kidney cortex and mesanglial cells and mediates elevated ros under high glucose concentration (41).	objectiveimpairments in mitochondrial function have been proposed to play a role in the etiology of diabetic sensory neuropathy. we tested the hypothesis that mitochondrial dysfunction in axons of sensory neurons in type 1 diabetes is due to abnormal activity of the respiratory chain and an altered mitochondrial proteome.research design and methodsproteomic analysis using stable isotope labeling with amino acids in cell culture (silac) determined expression of proteins in mitochondria from dorsal root ganglia (drg) of control, 22-week - old streptozotocin (stz)-diabetic rats, and diabetic rats treated with insulin. rates of oxygen consumption and complex activities in mitochondria from drg were measured. fluorescence imaging of axons of cultured sensory neurons determined the effect of diabetes on mitochondrial polarization status, oxidative stress, and mitochondrial matrix - specific reactive oxygen species (ros).resultsproteins associated with mitochondrial dysfunction, oxidative phosphorylation, ubiquinone biosynthesis, and the citric acid cycle were downregulated in diabetic samples. for example, cytochrome c oxidase subunit iv (cox iv ; a complex iv protein) and nadh dehydrogenase fe - s protein 3 (ndufs3 ; a complex i protein) were reduced by 29 and 36% (p < 0.05), respectively, in diabetes and confirmed previous western blot studies. respiration and mitochondrial complex activity was significantly decreased by 15 to 32% compared with control. the axons of diabetic neurons exhibited oxidative stress and depolarized mitochondria, an aberrant adaption to oligomycin - induced mitochondrial membrane hyperpolarization, but reduced levels of intramitochondrial superoxide compared with control.conclusionsabnormal mitochondrial function correlated with a downregulation of mitochondrial proteins, with components of the respiratory chain targeted in lumbar drg in diabetes. the reduced activity of the respiratory chain was associated with diminished superoxide generation within the mitochondrial matrix and did not contribute to oxidative stress in axons of diabetic neurons. alternative pathways involving polyol pathway activity appear to contribute to raised ros in axons of diabetic neurons under high glucose concentration.
the patient can be positioned supine on a radiolucent operating room table or placed in the beach chair position. fluoroscopic images are obtained to confirm visualization with the image intensifier prior to prepping and draping the patient 's shoulder, neck, and arm. full anesthetic relaxation allows for less traumatic retraction of the deltoid and minimizes dynamic forces on the fracture fragments during reduction. a deltopectoral exposure is used to expose the proximal humerus, as described previously by badman and mighell.6) the coracoacromial ligament may be partially or completely released. similarly, the coracohumeral ligament is released. the long head of the biceps brachii tendon is identified at its position medial to insertion of the pectoralis major on the humerus. however, if left in situ, the long head of the biceps brachii can be a source of pain and we often tenodese it at the time of plate fixation in older patients or those with grossly poor tendon quality. the transverse humeral ligament is released with a knife or electrocautery as the biceps is traced superiorly, and as the tendon courses superiorly, it is used to define the rotator cuff interval. after the rotator interval is released to the base of the coracoid process, the long head of the biceps can be released from the supraglenoid tubercle and superior glenoid labrum if there is a plan for tenodesis. heavy nonabsorbable sutures are placed in the subscapularis, supraspinatus, and infraspinatus tendons at the myotendinous junction. temporary traction sutures are often necessary to help mobilize the tendons to obtain better suture purchase more medially. the bone quality in these patients is typically poor, and sutures should be placed in the stronger rotator cuff tendons rather than through the soft, metaphyseal bone of the tuberosities. we have found that unlocked horizontal mattress sutures are adequate. traction sutures should be placed in the tendinous insertions to hold and reduce the fragments securely to the plate (fig. a low - profile, precontoured, peri - articular locking plate with angular stable screws and suture eyelets is selected to provide fracture fixation. separate sutures are passed through the plate eyelets prior to applying the plate (fig. ideally, a superior suture is placed for the supraspinatus tendon, an anterior suture for the subscapularis, and a posterior suture for the infraspinatus tendon. care should be taken to avoid medial dissection, which entails detaching the pectoralis major tendon, and risks injury to the posterior humeral circumflex artery. a provisional reduction of the surgical neck can be held with kirschner wires and confirmed with an image intensifier and by direct inspection. the tuberosities are reduced via the traction sutures with minimal manipulation of the metaphysis to prevent further fracture comminution. this allows compression of the plate against the humeral shaft and allows subsequent reduction of the tuberosities to the shaft via the plate (fig. 3). the plate can be moved caudal or cephalad as needed using the oblong hole in the plate. it is critical not to reduce a fracture in internal rotation, as this will limit the patient 's ability to regain functional external rotation postoperatively. to ensure that this does not occur, reduction and plating are performed with the arm in 30 of external rotation. the position of the plate is then evaluated using fluoroscopy to ensure appropriate placement such that the plate is not too proximal to impinge on the coracoacromial arch with shoulder abduction. similarly, the plate should not be positioned too distal such that fixation into the head is limited. reduction techniques are employed to reduce the head to the shaft by using the sutures in the rotator cuff to gain control of the head. alternatively, the humeral head can be reduced or stabilized by manipulation with blunt elevators or joysticks such as kirschner wires or schanz pins. the use of elevators or joysticks can be problematic when working with osteoporotic bone, due to poor bone quality and further fragmentation at the site of application of these reduction tools. once reduced, fixation into the head is limited to five or more short (32 - 38 mm), fixed angle screws augmented with suture fixation of the rotator cuff (which has control of the head) directly to the plate. screw length is based on the overall size of the proximal humerus. once a single screw is placed and its length is confirmed by an image intensifier to be > 1 - 2 cm from the articular surface, all other holes are drilled and screws of the same length are placed. this is done to prevent cut - out and intra - articular penetration of the screws. in addition, this can lead to decreased operative time. if the long head of the biceps tendon was released, it can be tenodesed to the pectoralis major and the rotator cuff interval prior to a layered closure. patients are placed in a sling for comfort only and are encouraged to perform non - load bearing activities and pendulum exercises immediately after surgery. if there are no signs of reduction loss, patients are increased to full weight - bearing with the arm over the course of 4 - 6 weeks. patients are placed in a sling for comfort only and are encouraged to perform non - load bearing activities and pendulum exercises immediately after surgery. if there are no signs of reduction loss, patients are increased to full weight - bearing with the arm over the course of 4 - 6 weeks. proximal humerus fractures account for 4 - 5% of all fractures.7) the majority of these fractures occur in the elderly population, particularly in those with osteoporosis. approximately 85% of proximal humerus fractures are minimally displaced or stable and can be successfully treated with conservative management and early motion.8 - 10) the remaining 15% of fractures are displaced or unstable and require surgical intervention because of poor results with non - operative treatment.9) plate and screw fixation offer the best chance for stable fixation of multi - fragmented fractures. importantly, no study has described the biomechanical superiority of long versus short screws placed in the humeral head for fixation of these fractures. locking plate constructs have shown promising results for treating displaced and unstable proximal humerus fractures,1 - 3) but the use of this technique has not been without complications.3 - 5) studies evaluating the outcomes of patients treated with locking plates for proximal humerus fractures have shown that one of the most frequent complications (16 - 23%) of this technique is intra - articular penetration of the locking screw.3 - 5) it was noted that this complication is more common in patients > 60 years in whom osteoporotic bone is more likely to be found. we believe that the concept of subchondral screw fixation (as in load bearing joint periarticular fractures such as femoral neck fractures) is a misuse of the locking design for proximal humerus fractures in which rotator cuff tissue integrity often exceeds that of the metaphyseal bone of the humeral tuberosities. for this reason, we use short, divergent locking screws and suture fixation to minimize the risk of varus malunion, plate failure, and intra - articular screw penetration. none have had intra - articular screw penetration or cut - out and only two patients had an asymptomatic varus malunion at an average follow - up of 16 months. it is our belief that such a technique reduces the incidence of screw penetration into the glenohumeral joint and provides stable fixation for healing.	fixation of proximal humerus fractures with precontoured, fixed angle devices has improved operative management of these difficult injuries, particularly in patients with osteoporosis. however, recent data has revealed that fixation with these constructs is not without complications, particularly screw cut - out and loss of reduction. multiple strategies have been developed to decrease the number of complications. we offer a surgical technique combining suture augmentation of the proximal humerus with locked plate fixation utilizing short screws.
cyclin - dependent kinase non - small cell lung cancer pumilio rna - binding family member 1/2 e3 ubitiquitin ligase oncosuppressor mirnas have emerged as powerful post - transcriptional inhibitors of genetic programs controlling cancer cell proliferation, survival, invasion, metastasis, and stemness. moreover, the in vivo inhibition of tumor growth achieved in mouse cancer models by re - expression of well - characterized tumor suppressor mirnas, such as mir-34 and let-7, suggests strong therapeutic potential. experimentally validated bioinformatics analyses show that cell cycle components are highly enriched among targets of the major tumor suppressor mirnas. several g1- and s - phase cyclins (d1, d3, and e2) and cyclin - dependent kinases (cdk4 and cdk6) represent key targets of let-7, mir-15/16, and mir-34 families. conversely, several oncomirs target the expression of cdk inhibitors ; for example members of the mir-1792 and mir-106b25 oncogenic clusters target the p21 transcript. the cdk inhibitor p27 is lost or inactivated in cancer cells by multiple mechanisms, including decreased synthesis, increased proteolysis, and mislocalization. the p27 and p57 transcripts are critical targets of the closely related mir-221 and mir-222 oncomirs, which are overexpressed in multiple solid tumors including non - small cell lung cancer (nsclc). downregulation of mir-340 has been reported in multiple tumors such as breast, colon, neuroblastoma, and osteosarcoma, in which mr-340 expression positively correlates with better prognosis. experimentally validated mir-340 targets include disparate cellular components such as the tyrosine kinase met in breast cancer, the transcription factors sox2 in neuroblastoma and mitf in melanoma, and the cytoskeletal regulator rock1 in osteosarcoma. mir-340 expression inversely correlates with clinical staging in nsclc patients, whereas exogenous mir-340 inhibits proliferation and survival in nsclc - derived cells. mir-340induced growth arrest correlates with p27 accumulation in both lung adenocarcinoma and glioblastoma cells. in a549 cells mir-340 controls p27 at both translational and post - translational levels by directly targeting 3 negative regulators of p27 (pum1, pum2, and skp2) (fig. mir-340 induces p27 at the translational level by targeting the rna - binding proteins (pum1 and pum2) required for mir-221/222mediated inhibition of the p27 transcript. the blue lines indicate both validated (solid lines : pum1, pum2, and skp2) and preliminarily characterized (dashed lines : cyclins d1 and d2) mir-340 target transcripts in a549 cells mir-340 induces p27 at the translational level by targeting the rna - binding proteins (pum1 and pum2) required for mir-221/222mediated inhibition of the p27 transcript. the blue lines indicate both validated (solid lines : pum1, pum2, and skp2) and preliminarily characterized (dashed lines : cyclins d1 and d2) mir-340 target transcripts in a549 cells. human pum1 and pum2 genes encode 2 evolutionary conserved rna - binding proteins related to the pumilio gene products in drosophila and fem-3 in c. elegans. the functional role of the pum1/2 binding sites has been characterized for only a few human transcripts. binding of pum1 to the p27 3-utr induces a conformational switch that positively controls mir-221/222 accessibility. growth factor - induced phosphorylation of pum1 ser714 increases its rna - binding activity, suggesting a role of pum1 post - translational modification in the control of cell cycle re - entry. our results show that mirna - mediated downregulation of pum1 and pum2 antagonizes the mir-221/222mediated inhibition of p27. remarkably, transcriptome - wide analyses of pum1- and pum2-bound mrnas show significant enrichment for multiple cell cycle regulators in addition to p27. therefore, the mir-340pum1/2 axis might control cell cycle progression by targeting multiple transcripts in addition to cdkn1b, which encodes p27. for example, pum1/2 have also been implicated in the mirna - mediated control of the cell cycle regulator e2f3 in bladder cancer cells. pum1 belongs to a growing list of rna - binding proteins including hur, dnd1, crd - bp, and ptb that are implicated in the modulation of mirna targeting in mammalian cells. interestingly, the mir-340 target site in the mitf 3-utr is controlled by the crd - bp rna - binding protein, which interferes with mir-340 binding thus protecting the mitf transcript from mir-340mediated degradation. intriguingly, in addition to pum1/2, mir-340 also targets 2 distinct rna - binding proteins, pbp1/hnrnp1 and hnrnpa2, in colorectal cancer, suggesting a complex interplay between mir-340 and rna - binding proteins in cancer. p27 levels largely depend on protein stability, which is reduced by scf - mediated ubiquitylation. through investigation of the mechanism of p27 stabilization in mir-340-overexpressing cells we have identified s - phase kinase - associated protein 2, e3 ubitiquitin ligase (skp2), the substrate - recognizing component of the scf complex, as a target of mir-340. to our knowledge, this is the first evidence of mirna - mediated regulation of the human skp2 oncoprotein. in summary, in nsclc cells mir-340 induces p27 accumulation by affecting both synthesis (through pum1/2) and degradation (through skp2) of the cdk inhibitor. single nucleotide polymorphisms (snps) or 3-utr shortening events are known to affect the mirna binding sites of transcripts coding for oncoproteins, such as kras. the identification of a skp2 mrna species harboring a short 3-utr lacking the mir-340 target site suggests that, depending on the splicing pattern, some tumors could express a skp2 transcript isoform that is resistant to mir-340mediated repression. interestingly, we have identified cell lines in which p27 is unaffected by mir-340. since mir-340 retains its antiproliferative activity in these cell lines, we investigated other putative mir-340 targets. among various oncogenically relevant target transcripts, our preliminary experiments identified both cyclin d1 and cyclin d2, whose expression shows a significant inverse correlation with that of the mir-340 host gene (rnf130). therefore, mir-340 could influence g1/s transition by affecting the accumulation of cyclins d1/d2 and the activity of cyclin d / cdk4/6 complexes, together with the induction of p27 (via pum1/2 and skp2) and inhibition of the cyclin e / cdk2 complex. in addition, having observed that mir-340 is responsive to serum induction we postulate that mir-340 might participate in the control of cell cycle progression in response to extracellular mitogenic signals. in addition to further studies aimed at the transcriptome - wide identification of target mrnas and oncogenic networks modulated by mir-340, future investigations will address the applications of mir-340. importantly, systemic delivery of pre - mir-340 has recently been shown to inhibit the growth of xenografts of human colorectal cancer cells in mice. therefore, multiple lines of evidence point to mir-340 as a novel, highly promising bullet for mirna - based anticancer therapeutics. this work was supported by funding from airc (associazione italiana per la ricerca sul cancro) grant-10489 and aicr (association for international cancer research, uk) grant-08182 to pasquale verde.	oncosuppressor mirnas inhibit cancer cell proliferation by targeting key components of the cell cycle machinery. in our recent report we showed that mir-340 is a novel tumor suppressor in non - small cell lung cancer. mir-340 inhibits neoplastic cell proliferation and induces p27kip1 by targeting multiple translational and post - translational regulators of this cyclin - dependent kinase inhibitor.
this work was supported by feder funds and fondo social europeo through grants from the junta de andaluca (grants cvi-7335 to t.k. and cvi-7391 to m.eu) and the spanish ministry for economy and competitiveness (grants bio2010 - 16937 and bio2013 - 42297 to t.k ; grant bfu2010 - 17946 to m.eu).	abstractapart from inter - bacteria communication quorum sensing (qs) mechanisms also enable inter - domain interactions. to interfere with bacterial qs, plants were found to secrete compounds ; most of which of unknown identity. we have identified the plant compound rosmarinic acid (ra) to modulate pseudomonas aeruginosa qs by binding to the rhlr qs regulator. ra was found to be a homoserine - lactone (hsl) mimic that caused agonistic effects on transcription, resulting ultimately in a stimulation of several rhlr controlled phenotypes like virulence factor synthesis or biofilm formation. our study was initiated by in silico screening of an rhlr model with compound libraries, demonstrating that this approach is suitable to tackle a major bottleneck in signal transduction research, which is the identification of sensor protein ligands. previous work has shown that plant compounds interfere with the function of orphan qs regulators. our study demonstrates that this has not necessarily to be the case since rhlr forms a functional pair with the rhli synthase. a wide range of structurally dissimilar compounds have been found to mimic hsls suggesting that this class of qs regulators is characterized by a significant plasticity in the recognition of effector molecules. further research will show to what extent ra impacts on qs mechanisms of other bacteria.
over two thirds of us residents are overweight and about one - third are obese.1 approximately 11% of the adult us population has type 1 or type 2 diabetes, and this number is expected to double by 2050.2,3 type 2 diabetes mellitus is the most common form of diabetes, accounting for 90%95% of all cases. it is more common in adults, but can also occur in children and adolescents. the cause of hyperglycemia in type 2 diabetes is usually a combination of insulin resistance and increased hepatic glucose production. patients with type 2 diabetes are at high risk of cardiovascular disease, so it is important to assess each patient s cardiometabolic risk factors. these include overweight / obesity, smoking, hypertension, hyperlipidemia, sedentary lifestyle, inflammation and hypercoagulation, and demographic factors including age, gender, and race. the majority of patients with type 2 diabetes are obese or have increased abdominal fat.2,4 obesity is one factor contributing to insulin resistance. however, it is clear that most overweight individuals do not develop type 2 diabetes. therefore, other risk factors must be involved in the development of diabetes in overweight individuals. little is known in this area, but these risk factors are believed to include inflammatory cytokines (tumor necrosis factor and interleukin-6), insulin resistance, increased free fatty acids, and cellular mechanisms, such as mitochondrial dysfunction.5 the exact importance and relationship of these factors with the development of diabetes is poorly defined. genetic studies may allow us to understand better the complex relationship between these various contributing factors and to identify more precisely individuals who may develop type 2 diabetes. successful management of type 2 diabetes typically involves lifestyle modification (nutrition and exercise) and pharmacological therapy, although lifestyle intervention may suffice initially in highly motivated individuals with mild hyperglycemia.6 weight loss of 5%10% is associated with a significant improvement in glucose, lipids, blood pressure, use of antidiabetic medications, and risk of cardiovascular disease.7 therefore, lifestyle modification and weight loss are not only the foundation of treatment for those with type 2 diabetes, but should also be implemented for those at risk of the disease. in fact, the relevant studies do support lifestyle modification. for example, in the diabetes prevention program, lifestyle intervention resulted in a 58% reduction in risk of developing type 2 diabetes compared with a 31% relative risk reduction using metformin.811 individuals who enter lifestyle modification programs, during which they routinely meet with qualified health care practitioners, generally lose 7%10% of their body weight over 46 months.12,13 those who do not enter formal programs or who do not complete a program, are not as successful in reducing their weight. unfortunately, long - term success with lifestyle modification for weight loss is often poor. some patients are not willing or able to make the necessary lifestyle changes, and for those who are initially successful, many will regain much of the weight lost within a few years. in fact, many regain 30%35% of their lost weight within the first year after completing a treatment program.14 within five years, more than half of patients will have returned to their baseline weight. therefore, it is prudent to consider additional options beyond the traditional nutrition and exercise approach to weight loss, especially if sustained weight maintenance is desired. this article reviews the potential role of lorcaserin, a new antiobesity agent, in the management of weight in obese patients with and without type 2 diabetes. the number of pharmacotherapeutic agents available for the treatment of obesity has decreased in recent years because numerous agents, in particular dexfenfluramine and sibutramine, have been removed from the market as a result of serious side effects.15 in 2012, the us food and drug administration (fda) approved lorcaserin (belviq, arena pharmaceuticals inc, zofingen, switzerland) and a combined phentermine and controlled - release topiramate formulation (qsymia, vivus, mountain view, ca, usa), which were the first new oral antiobesity agents approved in 10 years.15 the phentermine and controlled - release topiramate combination uses two agents with differing mechanisms of action to provide additive weight loss effects while using lower doses of both agents, potentially reducing the risk of side effects. the phentermine - controlled - release topiramate formulation has been shown to decrease weight by an average of 12.2 kg over 52104 weeks of treatment in three separate clinical studies.1618 however, this formulation does not have a clear role in the treatment of obesity because it has not been compared directly with lorcaserin, other obesity treatments, or surgical intervention, such as gastric bypass. given that this formulation is estimated to cost nearly $ 2200 per year,19 its cost may limit its widespread use in the treatment of obesity. although the exact mechanism of action for lorcaserin is not clearly understood, it is believed to act as an agonist at central serotonin subtype 2c (5-ht2c) receptors located on hypothalamic pro - opiomelanocrotin neurons. agonism of the 5-ht2c receptor is believed to reduce food intake and increase satiety, leading to weight loss.2022 lorcaserin shows high selectivity for the 5-ht2c receptor subtype, with minimal activity at 5-ht2b or 5-ht 2a receptor subtypes. earlier antiobesity agents, such as dexfenfluramine, were withdrawn from the market because of an association with valvular heart disease thought to be caused by agonism at the 5-ht2b receptor. time to maximal serum concentrations is approximately 1.52 hours, and the elimination half - life is approximately 11 hours. lorcaserin is approximately 70% protein - bound and undergoes hepatic metabolism to inactive metabolites which are mainly eliminated via the kidneys. no dosing adjustments are required in patients with mild - to - moderate hepatic or renal impairment. however, clinical experience is limited in patients with child - pugh class c hepatic impairment or a creatinine clearance in the range of 3050 ml per minute, so caution is advised. it is not recommended to use lorcaserin in severe renal disease (creatinine clearance 150 mmhg or diastolic blood pressure > 95 mmhg, a diagnosis of type 2 diabetes, use of a selective serotonin reuptake inhibitor within the previous year, use of prescription weight loss medication within the previous three months, and use of over - the - counter weight loss drugs within the previous month. the study enrolled 4008 subjects who were randomly assigned to lorcaserin 10 mg once daily (n = 801), lorcaserin 10 mg twice daily (n = 1602), or placebo (n = 1601) for 52 weeks, in addition to standardized nutritional and exercise counseling, which was provided at each study visit. participants were encouraged to exercise moderately for 30 minutes each day and to reduce their caloric intake by 600 kcal / day. at baseline, the mean age of the patients was 44 years (79.8% women, 67% white). mean baseline body weight, waist circumference, and bmi was 100 kg, 109 cm, and 36 kg / m, respectively. approximately 24% of participants had hypertension, 28% had dyslipidemia, and 1% had cardiovascular disease.20 the primary outcome was the proportion of subjects who lost at least 5% or 10% of their baseline body weight, and the analysis was conducted using a defined intention - to - treat approach with the last observation carried forward. the sample size was based on analysis of echocardiographic endpoints, and the study had 99% power to detect a 15% difference in the primary outcome between placebo and lorcaserin if 720 subjects were enrolled in each group. after one year of treatment, at least 5% weight loss was achieved by 47.2%, 40.2%, and 25% in the lorcaserin twice daily, lorcaserin once daily, and placebo groups, respectively (p 140 mmhg or a diastolic blood pressure > 90 mmhg, diagnosis of type 2 diabetes, and depression or psychiatric illness requiring prescription drug treatment in the preceding two years.21 a total of 3182 subjects were enrolled and randomly assigned to receive lorcaserin 10 mg twice daily (n = 1595) or placebo (n = 1587) for 52 weeks. thereafter, subjects on the active treatment were randomized to either continue lorcaserin or placebo for another 52 weeks. lorcaserin was taken for two years continuously by 573 subjects, while 283 received lorcaserin and placebo for one year each ; 697 received placebo for two years. all subjects received nutritional and exercise counseling at each visit and were encouraged to exercise moderately for 30 minutes per day as well as reduce their caloric intake by 600 kcal / day. at randomization the mean age was 44 years, 67% of subjects were white, 19% were black, 83.5% were female, and the mean weight, bmi, and waist circumference was 100 kg, 36 kg / m, and 109 cm, respectively.21 the primary outcome was the proportion of patients who achieved > 5% weight loss, which was 47.5% in the lorcaserin group versus 20.3% in the placebo group (defined intention - to - treat population with last observation carried forward, p 5% was higher in the group that continued lorcaserin than in the group that switched to placebo (67.9% versus 50.3%, respectively, p 10% weight loss, change in waist circumference, blood pressure change, and bmi. of these, only the proportion achieving > 10% weight loss was statistically significant for the lorcaserin as compared with placebo (p 5% weight loss over 52 weeks, with benefits persisting for an additional 52 weeks. the most common adverse effects of lorcaserin included upper respiratory tract infection, headaches, dizziness, nasopharyngitis, and nausea. the incidence of fda - defined valvulopathy was not different between the groups, although the study only had 60% power to rule out a relative risk ratio of 1.5 due to its low rate of occurrence during the study. other potential limitations include the study being potentially overpowered for the efficacy analysis because the sample size was based on echocardiographic endpoints ; questionable effectiveness of the nutritional and exercise counseling ; and potentially biased data at 104 weeks given that only participants remaining in the study at 52 weeks (ie, those likely obtaining the most benefit) were eligible to continue the study. finally, it is unknown if the marginal difference in mean weight loss (3.6 kg) is of clinical importance.21 the bloom - dm study enrolled subjects aged 1865 years with a bmi of 2745 kg / m, a diagnosis of type 2 diabetes, and a glycosylated hemoglobin a1c of 7%10%.22 at baseline, participants could only be receiving metformin and/or a sulfonylurea for their type 2 diabetes. key exclusion criteria included : use of insulin, exenatide, or pramlintide ; depression or psychiatric illness requiring prescription drug therapy within the last year ; history of cardiac valve disease ; myocardial infarction or stroke within the previous six months ; unstable angina ; and use of selective serotonin or norepinephrine reuptake inhibitors.22 subjects were randomly assigned to one of three treatment groups, ie, lorcaserin 10 mg twice daily (n = 256), lorcaserin 10 mg once daily (n = 95), or placebo (n = 253) and followed for one year. subjects received nutritional and exercise counseling and were advised to reduce their caloric intake by 600 kcal / day and exercise moderately for 30 minutes daily. at baseline, 54% of the participants were female, 61% were white, 21% were black, and the mean age was approximately 53 years. mean baseline body weight and bmi was 104 kg and 36 kg / m, respectively. the proportion of patients achieving at least a 5% weight loss was 45% in the lorcaserin once daily group, 38% in the lorcaserin twice daily group, and 16% in the placebo group (p < 0.001 for both doses compared with placebo). successful weight reduction of 10% was seen in 18.1%, 16.3%, and 4.4% of subjects assigned to lorcaserin once daily, lorcaserin twice daily, and placebo, respectively. the absolute weight loss (using the least squares mean method) for lorcaserin once daily, lorcaserin twice daily, and placebo was 5 kg, 4.7 kg, and 1.6 kg, respectively. improvements in fasting blood glucose and glycosylated hemoglobin a1c were statistically significant in the lorcaserin group (p < 0.05 for both lorcaserin doses as compared with placebo). lorcaserin appears to be more effective than placebo in reducing baseline body weight in those with type 2 diabetes. headache, back pain, nasopharyngitis, and nausea were the most common adverse events noted with lorcaserin. hypoglycemia was more common in the lorcaserin groups, but none of the episodes were categorized as severe. potential study limitations were a small sample size for the lorcaserin once daily group, questionable effectiveness of the nutritional and weight loss counseling, and unknown baseline medication use in patients with high blood pressure or hyperlipidemia.22 lorcaserin is indicated in conjunction with diet and exercise for the treatment of obesity in adults with a bmi 30 kg / m or a bmi of 27 kg / m in presence of one or more comorbid conditions, including hypertension, type 2 diabetes, or dyslipidemia.2022 twelve weeks of treatment should be allowed before efficacy is assessed. patients who have not experienced at least a 5% weight loss after 12 weeks are unlikely to have success with continued therapy. potential side effects include headache, upper respiratory tract symptoms and infection, dizziness, nausea, constipation, fatigue, and dry mouth. the daily cost is approximately $ 4.24 based on an average weight loss of 5.5 kg in the clinical trials, it will cost approximately $ 265 for each kg of weight loss with lorcaserin. the patient s perception of how a new medication affects their overall quality of life has an important role in the success of treatment. compliance and quality of life often comes down to how well the patient feels the medication is working, the simplicity of dosing, and the number and severity of unwanted effects. with many treatments for obesity, discontinuation of therapy is a major factor to overcome and has been one of the factors limiting the evidence for these therapies.25 discontinuation is often because of adverse effects or the patient s dissatisfaction with the efficacy of therapy. aware of this, investigators in the clinical trials of lorcaserin assessed improvement in quality of life from the patient perspective using two questionnaires.2022 the first questionnaire used was the impact of weight on quality of life - lite (iwqol - lite), which is a validated 31-item instrument addressing quality of life specifically in terms of obesity.26 iwqol - lite is sensitive to both weight loss and weight gain, as well as the degree of obesity. the scores range from 0100, with higher numbers correlating with better overall quality of life. the second questionnaire used was the beck depression inventory (bdi)-ii. this instrument is comprised of 21 questions designed to identify the severity of depression in an individual.27 each question can be scored on a 03 scale, and the total score is then used to distinguish between minimal (013 points), mild (1419 points), moderate (2028 points), and severe (2963 points) depression. the blossom trial evaluated quality of life using both the iwqol - lite and bdi - ii questionnaires.20 the surveys were given at randomization and at prespecified intervals thereafter, although the authors did not indicate the exact time point at which the questionnaires were administered. participants were separated into one of three groups (see table 1), and among other exclusions, those taking selective serotonin reuptake inhibitors within the previous year were excluded from this study. the blossom trial reported a significant improvement in iwqol - lite score in all treatment groups. the lorcaserin 10 mg twice daily group showed an 11.8-point increase and the lorcaserin 10 mg once daily group showed an 11.3-point increase versus a 10.0-point increase in the placebo group (p = 0.0057 and p < 0.001 versus placebo, respectively). these numbers were slightly higher in the per - protocol patients (participants who completed the study) at 12.2, 12.6, and 10.4 in the lorcaserin 10 mg twice daily, lorcaserin 10 mg once daily, and placebo groups, respectively (p < 0.001 in both lorcaserin groups compared with placebo). the mean bdi - ii total scores for lorcaserin twice daily, lorcaserin once daily, and placebo decreased by 0.8 3.9, 0.8 4.1, and 0.7 3.8 points, respectively. no statistical analysis was reported for these findings.20 like many weight loss trials, the blossom study reported a relatively high discontinuation rate, with only 55% of the participants actually completing the trial.20 at the end of the study, the number of patients remaining in each group was similar at 917 (57.2%), 473 (59.0%), and 834 (52.0%) in the lorcaserin 10 twice daily, lorcaserin 10 mg once daily, and placebo groups, respectively. the investigators reported the reasons for withdrawal were loss to follow - up, adverse events, or a perception by the patient of a lack of efficacy. the proportion of patients that withdrew because of perceived lack of efficacy was similar between the groups at 2.4% and 3.1% in the lorcaserin 10 mg twice daily and lorcaserin 10 mg daily groups, respectively, and 3.9% in the placebo group. the blossom trial reports that compliance with treatment was monitored, but does not describe how this was assessed. as in the blossom study, the bloom study also used the iwqol - lite and bdi - ii surveys.21 participants were separated into two groups (see table 1), and among other exclusions, those with a diagnosis of depression or any other psychiatric condition requiring medical treatment within two years of randomization were excluded from the study. iwqol - lite was conducted at baseline and at four - monthly intervals thereafter during the trial. there was no significant difference in scores between the lorcaserin 10 mg twice daily group or placebo group at baseline (73.92 0.41 and 73.85 0.42, respectively). at the end of one year, scores were increased by 12.4 0.4 in the lorcaserin group and 10.7 0.4 in the placebo group (p < 0.001) in the intention - to - treat analysis.21 the bdi - ii questionnaire was administered at the screening visit and at nine subsequent visits. there was no difference between the lorcaserin 10 mg twice daily group and placebo at baseline (4.2 0.1 and 4.2 0.1, respectively). after one year of treatment, scores were lower in the lorcaserin group compared with the placebo group (1.1 0.1 versus 0.9 0.1, respectively), although the difference did not reach statistical significance (p = 0.26).21 a large discontinuation rate of nearly 50% was reported in the bloom trial, with a rate of completion of 55.4% in the lorcaserin group and 45.1% in the placebo group. the investigators do not offer any particular reason for this high dropout rate, other than a small percentage of participants reporting an adverse drug effect as the reason for withdrawal. within the study procedures there was no further mention of this in the study nor were there any specifics on how this evaluation was accomplished.21 bloom - dm reported quality of life measures as part of its secondary outcomes, and like the previous studies, used the iwqol - lite and bdi - ii surveys.22 participants were separated into three groups (see table 1) and among other exclusion criteria, potential participants were excluded if they had a history of depression, another major psychiatric disease requiring treatment with prescription medication within the previous year, a bdi - ii score 20, or an individual response suggesting suicidal ideation. the iwqol - lite questionnaire was completed at the baseline visit and again at 24 and 52 weeks. this score was reported to be improved in all treatment groups ; however, only the lorcaserin 10 mg daily defined intention - to - treat / last observation carried forward cohort showed a statistically significant improvement of 12.6 1.1 points compared with placebo (p = 0.042). the bdi - ii survey was administered at baseline and again at weeks 4, 24, and 52. as mentioned earlier, bloom - dm excluded patients with moderate depression, as defined by this questionnaire. there was no difference in scores between any of the groups at baseline and the authors reported a nonsignificant reduction in scores in all groups at the end of the study period.22 based on the findings of bloom - dm, lorcaserin 10 mg once or twice daily may have had a slight benefit in reducing depression and achieving a slight improvement in overall qol, with the 10 mg daily defined intention - to - treat / last observation carried forward cohort showing significant improvement.20 adherence to study treatment was assessed by counting pills at each research visit. patient s reported to be noncompliant to treatment regimen was reported at 3, 1, and 10 in the lorcaserin twice daily, once daily and placebo groups respectively. however, bloom - dm did report a relatively high discontinuation rate of 22.1% for patients in the lorcaserin once daily group compared with 37.9% in the placebo group.22 based on these trials, it appears that the effect of lorcaserin on weight loss may translate into a positive effect on overall quality of life. all three trials reported an improvement in iwqol - lite scores in the treatment groups, and some of the scores were reported to be significant when compared with placebo. however, as with other weight loss medications, the high dropout rates seen in these trials may indicate poor compliance with lorcaserin which will likely affect its overall efficacy in many patients.2022 lorcaserin is a novel antiobesity agent affecting 5-ht2c receptors, and results in decreased food intake and increased satiety. presently it is unclear how this new agent fits into the treatment of obesity. in patients with type 2 diabetes, bariatric surgery has been shown to be more effective than pharmacologic therapy.28 however, those studies did not include the use of phentermine, topiramate or lorcaserin. lorcaserin does appear to be accepted well by patients, and offers clinicians and patients an effective alternative to surgery. weighing the risks and benefits of a new therapy is difficult, and future studies should address this.	type 2 diabetes and obesity commonly occur together. obesity contributes to insulin resistance, a main cause of type 2 diabetes. modest weight loss reduces glucose, lipids, blood pressure, need for medications, and cardiovascular risk. a number of approaches can be used to achieve weight loss, including lifestyle modification, surgery, and medication. lorcaserin, a novel antiobesity agent, affects central serotonin subtype 2a receptors, resulting in decreased food intake and increased satiety. it has been studied in obese patients with type 2 diabetes and results in an approximately 5.5 kg weight loss, on average, when used for one year. headache, back pain, nasopharyngitis, and nausea were the most common adverse effects noted with lorcaserin. hypoglycemia was more common in the lorcaserin groups in the clinical trials, but none of the episodes were categorized as severe. based on the results of these studies, lorcaserin was approved at a dose of 10 mg twice daily in patients with a body mass index 30 kg / m2 or 27 kg / m2 with at least one weight - related comorbidity, such as hypertension, type 2 diabetes mellitus, or dyslipidemia, in addition to a reduced calorie diet and increased physical activity. lorcaserin is effective for weight loss in obese patients with and without type 2 diabetes, although its specific role in the management of obesity is unclear at this time. this paper reviews the clinical trials of lorcaserin, its use from the patient perspective, and its potential role in the treatment of obesity.
the success of restorative procedures depend on effective removal of infected dentin prior to the placement of the restorative material. failure to mechanically remove any carious portion and not being able to achieve complete disinfection may lead to microleakage, increased pulp sensitivity, pulpal inflammation, and eventually may cause secondary caries, thereby making it necessary to replace the restoration. therefore, application of cavity disinfectant before tooth restoration is gaining acceptance. the main objective of adhesive dentistry is to create an effective, durable union between tooth structure and the restorative material. the resin to dentin adhesion occurs through the infiltration into and polymerization of hydrophilic resins within the collagen matrix exposed through acid decalcification of dentin, thus forming a hybrid layer. the durability of the dentine adhesive interface is directly related to the quality of hybrid layer and the hydrophilic nature of the adhesive. in case of etch and rinse adhesives, it is pertinent that the adhesive resin monomer penetrates the acid - etched exposed dentine collagen fibrils. certain mechanisms have been proposed to improve dentin adhesion i.e., adjunctive collagen pretreatment, matrix metalloproteinases (mmp) inhibitors, etc. different types of mmps have been identified from human dentin, including mmps 2, 3, 8, 9, 20. both mmps and cysteine cathepsins contribute to the degradation of denuded collagen within the hybrid layer. chlorhexidine (chx) has been widely used as a cavity disinfectant because of its antimicrobial property. it also has an inhibitory effect on the mmps (against mmps 2, 8, 9) in the dentin. this effect can be useful in preventing collagen degradation and disintegration of the bonding interface over time. in recent years, the potential for the inhibition of mmps by substances derived from natural products has gained increasing attention. a. barbadensis miller (aloe vera) is a short succulent herb resembling a cactus, with green fleshy, spiny, and well marginated leaves filled with a clear viscous gel. a. vera has been used to relieve thermal burn, sunburn, and promote wound healing and has antimicrobial activity and can help stimulate the body 's immune system. recent study has revealed a. vera exhibits mmp inhibitory effect against mmp 2 and 9. conducted a randomized clinical trial in 2015 and advocated the use of a. vera as cavity disinfectant. there have been many researches to prove antimicrobial efficacy a. vera against caries causing microorganisms, i.e., streptococcus mutans. therefore, we intended to use a. vera as a cavity disinfectant in our study. in order to use these agents for extending the longevity of resin - dentin bonds, it is necessary to first evaluate whether these agents interfere with the dentin bond strength following their use to pretreat the acid - etched dentin. the aim of our study was to evaluate a. vera for resin - dentin bond stabilization. this was the first study of its kind to test a. vera for resin - dentin bond stabilization. sixty freshly extracted non carious human mandibular molars were thoroughly cleaned and stored in 0.1% thymol until use. they were horizontally cut using diamond disk (markus ink., michigan, usa) in a high - speed hand piece under air and water spray ; the long axes of the teeth were perpendicular to the surfaces cut. after the removal of enamel, the mid coronal dentin was exposed. the dentine surface was examined for the lack of enamel or pulp tissue under stereomicroscope (olympus ; zoom type, japan). the sections of the teeth including the roots were embedded in auto polymerizing acrylic resin to form cylinders 2.5 cm in diameter and 5 cm high. dentin surfaces were flattened with 1200 grit silicon carbide paper under running water, so that a very smooth surface and to obtain a standardized smear layer. acid etching of the exposed dentine was performed for 15 s with 37% phosphoric acid gel (scotchbond etchant, 3 m espe, st. the teeth in experimental groups were treated with one of the following cavity disinfectants i.e. group 2 : 2% chx solution was prepared from dilution of 20% chx solution using distilled water (basic pharma, gujrat, india). group 3 : a. barbadensis miller (a. vera) solution was prepared using a. vera powder of 99% purity and dissolving 20 mg of a. vera powder in 10 ml of distilled water). the acid etched dentin was pretreated with 2% chx in group 2 and a. barbadensis miller solution in group 3 for 30 s, active application with a brush applicator (microbrush international, wi, usa) and the excess removed with cotton pellet prior to the application of bonding agent (adper single bond 2, 3 m espe, st. adhesive tape with a 3 mm diameter hole in it was used to define the bonding agent. the dentin surfaces of the teeth were then dried with air for 10 s, resin composite was applied in 56 increments (filtek z 350, 3 m espe, st. paul, mn, usa) with the aid of polyethylene tubes (3 mm diameter, 2 mm height and 0.5 mm thickness) and individually light cured for 40 s using light curing unit spectrum 800 (dentsply, caulk, milford, usa) with an output of 600 mw / cm. the teeth were then stored in distilled water at room temperature for 24 h. for shear bond strength testing, each tooth was secured in a specially designed attachment jig to hold the specimens to the universal testing machine (instron, admet, enkay enterprises, new delhi). load was applied by the testing machine through a wire loop adjusted to the bonded interface at a cross head speed of 0.5 mm / min. shear bond strength in mpa was calculated from the peak load at failure divided by the specimen surface area. after testing, the fracture modes were evaluated under a stereomicroscope (olympus, zoom type) and classified according to the predominant mode of fracture as adhesive fracture at the resin - cement dentin interface, cohesive fracture in the resin cement, cohesive fracture in dentin, or mixed adhesive and cohesive fracture in the resin cement. data were analyzed using one - way anova and post hoc tukey test for multiple comparison between the groups. the test was done at a 0.05 level statistical significance [tables 1 and 2 ]. shear bond strength to dentin (meanstandard deviation) comparison of immediate mean shear bond strength values (mpa) of all groups from the results analyzed, it was noted that there was statistically significant difference between the groups control and chx and control and a. vera (p 0.05). hence, the following result for the shear bond strengths to dentin was obtained : control < chx a. vera. fracture analysis indicated that most specimens showed adhesive fracture after 24 h [table 3 ]. acid etching creates a low ph which activates the dentin mmps in the presence of zinc and calcium ions. it has been advocated that to improve the durability of restorations, pretreatment of acid - etched dentin with mmp inhibitors should be carried out. chx increased the immediate shear bond strength in this study, which was in support of the study by brackett., the use of chx after etching prevents collagen fiber degradation and preserves the hybrid layer due to its inhibitory effect on mmps, thereby increasing the bond strength. yet few studies have showed that applying chx before acid etching did not significantly affect the bond strength. this increase in the immediate bond strength can be attributed to the mmp inhibitory action of chx. the present study was conducted from agents derived from natural products that has been recently reported to possess anti - mmp potential, especially against mmp-2 and mmp-9. the results of this study revealed that the application of a. barbadensis miller to acid - etched dentine improves the longevity of resin dentin bonds by inhibiting mmps. the purpose of this study was to introduce new herbal product with possible potential for the inhibition of mmps in order to maintain the dentin adhesive interface. the shear bond testing was performed only after 24 h of storage as this was the first study of its kind and it aimed to determine whether the tested concentration of the herbal product would be really effective and thereafter conduct longitudinal studies. as if immediate bond strength was negatively affected by the use of a. vera, there would have been no point for conducting long duration studies. although the use of a. barbadensis miller on acid - etched dentin prevents the degradation of collagen and improves the longevity of composite restorations, it is imperative that they do not adversely affect adhesive bonding to dentin. as pretreatment with the herbal used was able to maintain immediate dentin bond strength, there is not enough evidence to reject the null hypothesis. the ability of this agent to improve the durability of resin - dentin should be evaluated in future studies. within the limitations of this in vitro study, it may be stated that the use of chx or a. barbadensis miller, as pretreatment agents of acid demineralized dentin collagen, has no adverse effect on the shear immediate bond strength of a two - step etch and rinse adhesive to dentin. further, in vitro and in vivo studies are still warranted to evaluate the effect of a. barbadensis miller for cavity disinfection.	introduction : the main objective of adhesive dentistry is to create an effective, durable union between the tooth structure and restorative material. however, degradation of adhesive dentine interface remains largely responsible for the relatively short lifetime of tooth colored resin restoration.aim:the aim of the study is to compare the dentin collagen stabilization property of chlorhexidine (chx) and aloe barbadensis miller using shear bond strength testing.materials and methods : occlusal reduction was done in sixty extracted human mandibular molars to expose the mid coronal dentin and divided into three groups n = 20. following the surface pretreatment (group 1 = control, group 2 = chx, group 3 = aloevera), dentine bonding agent and composite resin were applied and cured. the specimens were then subjected to shear bond strength testing.results:from the results analyzed, it was noted that there was statistically significant difference between the groups control and chx and control and a. barbadensis miller (p 0.05). hence, the following result for the shear bond strengths to dentin was obtained : control < chx a. barbadensis miller.conclusion:chx and a. barbadensis miller, as pretreatment agents of acid demineralized dentin collagen, has no adverse effect on the immediate shear bond strength of a two - step etch and rinse adhesive to dentin.
current treatment strategies for breast cancer depend on disease stage, tumor grade, tumor hormone receptor status, and whether there is over - expression or amplification of the human epidermal growth factor receptor (her). because of the availability of numerous agents, patients with breast cancer may receive several different lines of treatment throughout the course of their disease. once the disease has reached the metastatic stage, therapeutic strategies are chosen because they have the potential to achieve symptom palliation while significantly prolonging progression - free survival (pfs).17 however, tumors may respond to many available agents by upregulating survival pathways.8 these mechanisms can render the tumor resistant to multiple, sometimes unrelated classes of therapy, presenting an enormous clinical challenge. if tumor resistance develops, the disease may eventually progress despite therapy, necessitating use of agents that are believed to have no cross resistance to previously used drugs, or agents that are able to circumvent such resistance.9,10 furthermore, some patients have innate resistance to a particular drug or regimen and may not respond at all.9 because tumors may adapt to anticancer agents, response rates and survival outcomes tend to decrease with each subsequent line of therapy. given that most patients with recurrent breast cancer have already been exposed to an anthracycline (eg, doxorubicin, epirubicin) and a taxane (eg, paclitaxel, docetaxel) in the adjuvant setting, there is a need for therapies that are noncross - resistant with these two classes of cytotoxic agents. the epothilones comprise a novel class of chemotherapeutic agents that have a low susceptibility to multiple mechanisms of tumor survival that can result in treatment failure.1115 currently, ixabepilone is the only epothilone to receive approval through the us food and drug administration (fda) for the treatment of patients with locally advanced or metastatic breast cancer in combination with capecitabine after failure of an anthracycline and a taxane, and as monotherapy after failure of an anthracycline, a taxane, and capecitabine. similar to taxanes, epothilones like ixabepilone bind to -tubulin and promote tubulin polymerization and microtubule stabilization, leading to cell cycle arrest and tumor cell apoptosis.16 however, epothilones may retain activity where taxanes fail, because they bind to -tubulin in a qualitatively different manner than the taxanes.17,18 importantly, ixabepilone retains activity in tumors that have upregulated their expression of the class iii isotype of -tubulin (iii - tubulin),19 a condition that has been linked to failure of other microtubule - targeting agents such as taxanes and vinca alkaloids.20,21 as a class, epothilones also induce apoptosis through multiple pathways that appear to be distinct for this class of agents, including enhancement of caspase-2 activity and p53-mediated activation of the death effector bax.15 in contrast, taxanes induce apoptosis mainly through caspase 9 activation.15 additionally, in preclinical studies with tumor cell models and xenografts, ixabepilone showed low susceptibility to several important cellular mechanisms that render multiple classes of therapy ineffective, including elevated expression of drug efflux transporter proteins (eg, p - glycoprotein and multiple - resistance protein-1).15 clinically, ixabepilone has exhibited single - agent activity against a broad range of tumors, including breast cancer, nonsmall cell lung carcinoma, pancreatic cancer, renal cell cancer, prostate cancer, and lymphoma.15 furthermore, as a single agent or in combination with capecitabine, ixabepilone has demonstrated clinical efficacy across the spectrum of breast cancer treatment in a number of studies.2229 this article reviews the clinical evidence that supports the use of ixabepilone in the treatment of various stages of breast cancer. neoadjuvant ixabepilone has been evaluated in primary untreated breast cancer in a phase ii trial involving women with previously untreated, invasive, stage iia iiib breast cancer (n = 164).22 patients with tumors at least 3 cm in diameter were administered an intravenous (iv) infusion of ixabepilone at 40 mg / m over 3 hours on day 1 of a 21-day cycle for up to 4 cycles prior to surgery. surgery was followed by anthracycline - based adjuvant chemotherapy, radiotherapy, or tamoxifen, as indicated. a pathologic complete response (pcr) in the breast was achieved in 18% (29/161) of evaluable patients, with a higher response rate (26% ; 11/42) in patients with tumors that were negative for the estrogen receptor (er), progesterone receptor (pgr), and her2 (ie, the so - called triple - negative patients). the pcr rates in the breast and axilla were 11% in all treated patients and 19% in the triple - negative subset. interestingly, a retrospective microarray analysis of tissue samples from patients from this study revealed that the triple - negative patients expressed higher levels of iii - tubulin. moreover, a receiver operating characteristics (roc) analysis showed that, in the overall population, iii - tubulin expression was predictive of response to ixabepilone.30 these results might partially explain the higher response in patients with triple - negative disease, although further characterization of this phenomenon is needed. in this neoadjuvant study, grade 3 or 4 neutropenia was reported by 13% of patients and grade 3 or 4 neuropathy by 2%. although only limited conclusions can be drawn by comparing results from different studies, the pcr rates from this phase ii trial compared favorably with those observed in trials of other regimens, which ranged from 3% to 20% (table 1).22,3135 trials at several institutions are expanding the investigation of ixabepilone in patients with primary untreated breast cancer. for instance, in an ongoing phase ii study, the efficacy and tolerability of neoadjuvant ixabepilone is being compared with paclitaxel when administered after doxorubicin and cyclophosphamide in patients with early breast cancer.36 to date, there is no published data to support the use of ixabepilone in the adjuvant setting. a randomized, open - label, phase iii trial is underway to evaluate the benefit of a sequential regimen of fec 100 (5-fluorouracil, epirubicin, and cyclophosphamide) and ixabepilone in the adjuvant treatment of triple - negative or her2- and pgr - negative node - positive or -negative breast cancer.37 another ongoing phase iii study is comparing single - agent ixabepilone with single - agent paclitaxel administered after doxorubicin and cyclophosphamide for the adjuvant treatment of triple - negative, early - stage breast cancer.38 ixabepilone has been evaluated in patients with locally advanced and metastatic breast cancer in a series of phase ii trials2327,39,40 (as a single agent [table 2]2327 and in combination with various cytotoxic and targeted agents) and phase iii trials (in combination with capecitabine).28,29 several studies have suggested that ixabepilone has significant antitumor activity in patients who have not received prior taxane therapy but have received prior anthracycline therapy. for example, denduluri and colleagues evaluated the use of daily ixabepilone as a single agent in patients with metastatic breast cancer who had not received taxanes in the adjuvant or metastatic setting.23 this study placed no limits on prior therapy. of the 23 patients enrolled in this study, 70% had received prior chemotherapy, including anthracyclines and/or capecitabine, and 61% of those with hormone receptor - positive disease had been treated with prior hormone therapy. ixabepilone at 6 mg / m was administered as a 1-hour iv infusion on the first 5 consecutive days of a 21-day cycle until unacceptable toxic effects or disease progression occurred. patients received between 2 and 22 treatment cycles (median, 8 cycles) ; four patients required dose reductions owing to adverse events. thirteen patients achieved a partial response (pr), giving an objective response rate (orr) of 57%, with a median duration of response of 5.6 months. another six patients (26%) achieved stable disease (sd) for 6 weeks or more. median time - to - progression (ttp) in all patients was 5.5 months. of the five patients who received ixabepilone as first - line metastatic therapy, three achieved a pr and one had sd. of the 12 patients who had received prior anthracyclines, seven had a pr and four had sd. treatment was generally well tolerated, with most adverse events being mild or moderate in severity. grade 3 and 4 adverse events included fatigue (13%), nausea (9%), motor neuropathy (4%), and neutropenia (22%). roch and associates examined the use of first - line ixabepilone monotherapy at 40 mg / m as a 3-hour iv infusion on day 1 of a 21-day cycle among 65 patients with metastatic breast cancer pretreated with adjuvant anthracyclines.26 although patients were receiving first - line treatment for metastatic disease, they had an extensive tumor burden ; most had involvement of at least two tumor sites (77%) and/or visceral metastases (85%). twenty - seven patients achieved a pr, producing an orr of 41.5%, with a median duration of 8.2 months. sd occurred in 23 patients (35%), 11 of whom were progression free for at least 6 months. in the total study population, median ttp was 4.8 months, and median overall survival (os) was 22 months. it should be noted that although no patients had received taxanes for metastatic disease, 17% of patients had been exposed to taxanes as part of an adjuvant regimen. treatment - related adverse events were mostly grade 1 or 2 in severity and included alopecia (92%), sensory neuropathy (71%), fatigue (68%), myalgia (65%), and nausea (54%). hematologic toxicities did not result in treatment discontinuation or death, with only 7 patients requiring a delay in dosing. the main grade 3 and 4 toxicities included sensory neuropathy (20%), neutropenia (58%), and leukopenia (50%). although these phase ii monotherapy studies involved small sample sizes and did not include a control arm, the observed response rates and survival outcomes with ixabepilone are encouraging for the treatment of patients with prior exposure to anthracyclines, even among those with an extensive tumor burden. clinicians will be inclined to ask how ixabepilone performs in comparison to the taxanes, therefore broad comparisons between separate trials suggest that the results with ixabepilone are similar to outcomes achieved in phase iii studies that evaluated docetaxel or paclitaxel in patients with advanced breast cancer that has progressed during anthracycline therapy (orrs, 25% to 59% ; ttp / pfs, 3.6 to 19.0 months ; os durations, 11.4 to 34.0 months).4143 in addition, a recent phase ii trial suggests that, like paclitaxel, ixabepilone is also effective given on a weekly basis (15 mg / m) with trastuzumab (2 mg / kg, after a 4 mg / kg loading dose) and carboplatin (area under curve [auc ], 2) as first - line therapy in her2-positive patients.39 among 59 treated patients, 44% achieved an objective response ; median ttp and os were 8.2 months and 34.7 months, respectively. these results are comparable to phase iii outcomes for the combination of every-3-week paclitaxel and carboplatin plus weekly trastuzumab (orr, 52% ; pfs, 10.7 months ; os, 35.7 months),44 even though approximately one - third of patients in the ixabepilone trial had received taxanes in the neoadjuvant or adjuvant setting compared to none in the paclitaxel trial. data directly comparing ixabepilone to the taxanes in the first - line setting has only recently become available, when a randomized phase ii study evaluated the combination of ixabepilone and bevacizumab at two doses and schedules (ixabepilone 16 mg / m on days 1, 8, and 15 plus bevacizumab 10 mg / kg every 2 weeks [n = 46 ] or ixabepilone 40 mg / m plus bevacizumab 15 mg / kg every 3 weeks [n = 45 ]) in comparison to paclitaxel (90 mg / m on days 1, 8, and 15) plus bevacizumab (10 mg / kg every 2 weeks [n = 32 ]) as first - line therapy for metastatic breast cancer.40 although the numbers of patients in each arm were small, efficacy outcomes for both the weekly (orr, 50% ; 24-week pfs, 75%) and the every-3-week (orr, 71% ; 24-week pfs, 86%) ixabepilone regimens appeared to be at least comparable to those seen with weekly paclitaxel (orr, 56% ; 24-week pfs, 94%) when combined with bevacizumab. toxicity profiles were also similar, although rates of grade 3/4 neutropenia were higher for every-3-week ixabepilone (55%) than for weekly ixabepilone (11%) or paclitaxel (22%).40 results from an ongoing phase iii trial may provide a more definitive comparison of these regimens (calgb 40502 ; nct00785291). options for second and subsequent lines of chemotherapy for metastatic breast cancer are especially limited for patients with innate or acquired taxane resistance. single - agent ixabepilone has proven effective as a second-, third-, or fourth - line option in heavily - pretreated and multidrug - resistant patients. in a registrational study conducted for fda approval, perez and coworkers evaluated ixabepilone at 40 mg / m as a 3-hour iv infusion on day 1 of every 21-day cycle in 126 patients with anthracycline-, taxane-, and/or capecitabine - resistant metastatic breast cancer.25 patients had baseline disease characteristics that included multiple disease sites and visceral disease affecting the liver and lung. in addition, one - third of patients had triple - negative disease. despite the poor prognosis of the population as a whole, 11.5% of 113 evaluable patients achieved an independent radiology review (irr)-assessed objective response, with a median duration of response of 5.7 months. in addition, 50% of patients achieved irr - assessed sd with median pfs and os durations of 3.1 months and 8.6 months, respectively. grade 3 and 4 nonhematologic adverse events included peripheral sensory neuropathy (14%) and fatigue / asthenia (14%). in most instances, neuropathy occurred early on in treatment and was generally reversible with dose reductions within a median time of 5.4 weeks.45 grade 3 or 4 neutropenia and leukopenia occurred in 54% and 49% of patients, respectively, but febrile neutropenia and infection were uncommon (reported in 4 and 3 patients, respectively).25 low and colleagues determined the efficacy and tolerability of ixabepilone in 37 women with locally advanced or metastatic breast cancer previously treated with taxanes in the neoadjuvant, adjuvant, or metastatic setting.24 at baseline, patients were heavily pretreated, with 43% having received between three and nine prior chemotherapy regimens for metastatic disease and all patients having received at least two cycles of a paclitaxel- or docetaxel - containing regimen. ixabepilone was administered as a 1-hour iv infusion of 6 mg / m on the first 5 consecutive days of a 21-day cycle until unacceptable toxic effects or disease progression occurred. one patient achieved a pcr and seven achieved a pr for an orr of 22%, with median response duration of 3.7 months. interestingly, the sd responses were achieved in patients who had experienced disease progression while receiving taxanes and in patients who had been previously treated with doxorubicin, capecitabine, and a taxane, suggesting that ixabepilone was able to circumvent tumor resistance mechanisms in these patients. in the entire study population, grade 3 and 4 toxicities included neutropenia (35%), febrile neutropenia (14%), fatigue (14%), diarrhea (11%), nausea / vomiting (5%), myalgia / arthralgia (3%), and sensory neuropathy (3%). one patient withdrew from the study because of grade 3 sensory neuropathy, and one patient withdrew because of prolonged grade 2 sensory neuropathy. thomas and colleagues investigated the use of ixabepilone in patients with metastatic breast cancer that had progressed during, or within 4 months of taxane therapy (6 months if received in the adjuvant setting).27 in this study, 49 patients were administered ixabepilone at 40 mg / m as a 3-hour iv infusion on day 1 of a 21-day cycle. patients were heavily pretreated (86% had received two or more prior chemotherapy regimens) and had a substantial tumor burden (84% had at least two involved disease sites, and 84% had visceral disease). the orr was 12% (6 prs), with a median duration of response of 10.4 months. forty - one percent of patients achieved sd, with a median ttp of 2.2 months and median os of 7.9 months. the most common adverse events of any grade included fatigue (76%), nausea (57%), pain (65%), and sensory neuropathy (all grades, 63% ; grade 3, 12% ; grade 4, 0%). a combination of ixabepilone plus capecitabine has also been studied in patients with metastatic breast cancer previously treated with anthracyclines or a taxane.28,29 promising efficacy and safety results from a phase i / ii trial46 led to initiation of a pivotal phase iii trial (bms 046) of ixabepilone (40 mg / m as a 3-hour iv infusion on day 1 every 21 days) plus capecitabine (2000 mg / m on days 1 to 14 of a 21-day cycle) compared with capecitabine alone (2500 mg / m on days 1 to 14 of a 21-day cycle) in 752 patients with locally advanced or metastatic breast cancer that had progressed after treatment with anthracyclines and taxanes.28 in its entrance criteria, the study employed a strict definition of taxane resistance, requiring patients to have progressed during treatment or relapsed within 4 months of the last dose in the metastatic setting or within 12 months in the adjuvant setting. in general, patients were heavily pretreated and had a heavy burden of disease ; nearly 50% of patients had received 2 prior regimens in the metastatic setting, 90% of patients had metastases at two or more sites, and 84% had visceral disease involving the liver and/or lung. in this study, adding ixabepilone to capecitabine achieved an irr - assessed orr of 35% compared with 14% for capecitabine monotherapy, and an investigator - assessed orr of 42% compared with 23% for capecitabine monotherapy. median durations of response for the two treatment arms were 6.4 and 5.6 months, respectively. the median irr - assessed pfs was significantly longer for the combined regimen (5.8 months) than for capecitabine alone (4.2 months), resulting in a 25% risk reduction for disease progression with combined therapy (hazard ratio [hr ], 0.75 ; 95% confidence interval [ci ], 0.64 to 0.88 ; p = 0.003 ; figure 1).28 consistent with this result, the investigator - assessed median pfs was longer for the combined regimen than for capecitabine alone (5.3 vs 3.8 months ; p = 0.0011). this improvement in disease progression was maintained in a sensitivity analysis requested by the fda. when patients who received subsequent therapy before disease progression were censored for pfs at the last tumor assessment date, ixabepilone plus capecitabine still prolonged pfs compared with capecitabine alone (5.7 vs 4.1 months ; hr, 0.69 ; 95% ci, 0.58 to 0.83 ; p < 0.0001).47 compared with capecitabine alone, ixabepilone plus capecitabine was associated with a higher rate of grade 3 or 4 sensory neuropathy (21% vs 0%), fatigue (9% vs 3%), and neutropenia (68% vs 11%), as well as an increased rate of neutropenia - related death as a result of toxicity (3% vs 1%, with patients with liver dysfunction [grade 2 liver function tests ] at greater risk).28 a larger confirmatory phase iii trial (bms 048) in patients who were pretreated and/or resistant to anthracyclines and taxanes (n = 1221) provided further evidence that ixabepilone plus capecitabine consistently improved pfs compared with capecitabine alone (6.2 vs 4.4 months).29 notably, 50% of the patients in the confirmatory trial met the resistance criteria utilized in the pivotal trial. neither phase iii study showed a significant difference in median os between the two treatment arms ; however, adjusting for prognostic factors in a preplanned analysis, combination therapy significantly reduced the risk of death by about 15% (p = 0.0803 for bms 046 ; p = 0.0231 for bms 048).29 furthermore, as a part of these phase iii studies, prospectively defined subset analyses revealed statistically significant improvements in pfs when the combination of ixabepilone and capecitabine was used as first - line metastatic therapy (after having received anthracyclines and/or taxanes in the adjuvant setting) and in triple - negative patients.4850 patients who received ixabepilone plus capecitabine as first - line therapy experienced a 54% reduction in the estimated risk of disease progression with combination therapy.48 taken together, these results showed that ixabepilone in combination with capecitabine has superior clinical efficacy to capecitabine alone in patients with metastatic disease pretreated or resistant to anthracyclines and resistant to taxanes, a population with limited effective treatment options. several studies have suggested that ixabepilone has significant antitumor activity in patients who have not received prior taxane therapy but have received prior anthracycline therapy. for example, denduluri and colleagues evaluated the use of daily ixabepilone as a single agent in patients with metastatic breast cancer who had not received taxanes in the adjuvant or metastatic setting.23 this study placed no limits on prior therapy. of the 23 patients enrolled in this study, 70% had received prior chemotherapy, including anthracyclines and/or capecitabine, and 61% of those with hormone receptor - positive disease had been treated with prior hormone therapy. ixabepilone at 6 mg / m was administered as a 1-hour iv infusion on the first 5 consecutive days of a 21-day cycle until unacceptable toxic effects or disease progression occurred. patients received between 2 and 22 treatment cycles (median, 8 cycles) ; four patients required dose reductions owing to adverse events. thirteen patients achieved a partial response (pr), giving an objective response rate (orr) of 57%, with a median duration of response of 5.6 months. another six patients (26%) achieved stable disease (sd) for 6 weeks or more. median time - to - progression (ttp) in all patients was 5.5 months. of the five patients who received ixabepilone as first - line metastatic therapy, three achieved a pr and one had sd. of the 12 patients who had received prior anthracyclines, seven had a pr and four had sd. treatment was generally well tolerated, with most adverse events being mild or moderate in severity. grade 3 and 4 adverse events included fatigue (13%), nausea (9%), motor neuropathy (4%), and neutropenia (22%). roch and associates examined the use of first - line ixabepilone monotherapy at 40 mg / m as a 3-hour iv infusion on day 1 of a 21-day cycle among 65 patients with metastatic breast cancer pretreated with adjuvant anthracyclines.26 although patients were receiving first - line treatment for metastatic disease, they had an extensive tumor burden ; most had involvement of at least two tumor sites (77%) and/or visceral metastases (85%). twenty - seven patients achieved a pr, producing an orr of 41.5%, with a median duration of 8.2 months. sd occurred in 23 patients (35%), 11 of whom were progression free for at least 6 months. in the total study population, median ttp was 4.8 months, and median overall survival (os) was 22 months. it should be noted that although no patients had received taxanes for metastatic disease, 17% of patients had been exposed to taxanes as part of an adjuvant regimen. treatment - related adverse events were mostly grade 1 or 2 in severity and included alopecia (92%), sensory neuropathy (71%), fatigue (68%), myalgia (65%), and nausea (54%). hematologic toxicities did not result in treatment discontinuation or death, with only 7 patients requiring a delay in dosing. the main grade 3 and 4 toxicities included sensory neuropathy (20%), neutropenia (58%), and leukopenia (50%). although these phase ii monotherapy studies involved small sample sizes and did not include a control arm, the observed response rates and survival outcomes with ixabepilone are encouraging for the treatment of patients with prior exposure to anthracyclines, even among those with an extensive tumor burden. clinicians will be inclined to ask how ixabepilone performs in comparison to the taxanes, therefore broad comparisons between separate trials suggest that the results with ixabepilone are similar to outcomes achieved in phase iii studies that evaluated docetaxel or paclitaxel in patients with advanced breast cancer that has progressed during anthracycline therapy (orrs, 25% to 59% ; ttp / pfs, 3.6 to 19.0 months ; os durations, 11.4 to 34.0 months).4143 in addition, a recent phase ii trial suggests that, like paclitaxel, ixabepilone is also effective given on a weekly basis (15 mg / m) with trastuzumab (2 mg / kg, after a 4 mg / kg loading dose) and carboplatin (area under curve [auc ], 2) as first - line therapy in her2-positive patients.39 among 59 treated patients, 44% achieved an objective response ; median ttp and os were 8.2 months and 34.7 months, respectively. these results are comparable to phase iii outcomes for the combination of every-3-week paclitaxel and carboplatin plus weekly trastuzumab (orr, 52% ; pfs, 10.7 months ; os, 35.7 months),44 even though approximately one - third of patients in the ixabepilone trial had received taxanes in the neoadjuvant or adjuvant setting compared to none in the paclitaxel trial. data directly comparing ixabepilone to the taxanes in the first - line setting has only recently become available, when a randomized phase ii study evaluated the combination of ixabepilone and bevacizumab at two doses and schedules (ixabepilone 16 mg / m on days 1, 8, and 15 plus bevacizumab 10 mg / kg every 2 weeks [n = 46 ] or ixabepilone 40 mg / m plus bevacizumab 15 mg / kg every 3 weeks [n = 45 ]) in comparison to paclitaxel (90 mg / m on days 1, 8, and 15) plus bevacizumab (10 mg / kg every 2 weeks [n = 32 ]) as first - line therapy for metastatic breast cancer.40 although the numbers of patients in each arm were small, efficacy outcomes for both the weekly (orr, 50% ; 24-week pfs, 75%) and the every-3-week (orr, 71% ; 24-week pfs, 86%) ixabepilone regimens appeared to be at least comparable to those seen with weekly paclitaxel (orr, 56% ; 24-week pfs, 94%) when combined with bevacizumab. toxicity profiles were also similar, although rates of grade 3/4 neutropenia were higher for every-3-week ixabepilone (55%) than for weekly ixabepilone (11%) or paclitaxel (22%).40 results from an ongoing phase iii trial may provide a more definitive comparison of these regimens (calgb 40502 ; nct00785291). options for second and subsequent lines of chemotherapy for metastatic breast cancer are especially limited for patients with innate or acquired taxane resistance. single - agent ixabepilone has proven effective as a second-, third-, or fourth - line option in heavily - pretreated and multidrug - resistant patients. in a registrational study conducted for fda approval, perez and coworkers evaluated ixabepilone at 40 mg / m as a 3-hour iv infusion on day 1 of every 21-day cycle in 126 patients with anthracycline-, taxane-, and/or capecitabine - resistant metastatic breast cancer.25 patients had baseline disease characteristics that included multiple disease sites and visceral disease affecting the liver and lung. in addition, one - third of patients had triple - negative disease. despite the poor prognosis of the population as a whole, 11.5% of 113 evaluable patients achieved an independent radiology review (irr)-assessed objective response, with a median duration of response of 5.7 months. in addition, 50% of patients achieved irr - assessed sd with median pfs and os durations of 3.1 months and 8.6 months, respectively. grade 3 and 4 nonhematologic adverse events included peripheral sensory neuropathy (14%) and fatigue / asthenia (14%). in most instances, neuropathy occurred early on in treatment and was generally reversible with dose reductions within a median time of 5.4 weeks.45 grade 3 or 4 neutropenia and leukopenia occurred in 54% and 49% of patients, respectively, but febrile neutropenia and infection were uncommon (reported in 4 and 3 patients, respectively).25 low and colleagues determined the efficacy and tolerability of ixabepilone in 37 women with locally advanced or metastatic breast cancer previously treated with taxanes in the neoadjuvant, adjuvant, or metastatic setting.24 at baseline, patients were heavily pretreated, with 43% having received between three and nine prior chemotherapy regimens for metastatic disease and all patients having received at least two cycles of a paclitaxel- or docetaxel - containing regimen. ixabepilone was administered as a 1-hour iv infusion of 6 mg / m on the first 5 consecutive days of a 21-day cycle until unacceptable toxic effects or disease progression occurred. one patient achieved a pcr and seven achieved a pr for an orr of 22%, with median response duration of 3.7 months. interestingly, the sd responses were achieved in patients who had experienced disease progression while receiving taxanes and in patients who had been previously treated with doxorubicin, capecitabine, and a taxane, suggesting that ixabepilone was able to circumvent tumor resistance mechanisms in these patients. in the entire study population, median ttp was 2.5 months. grade 3 and 4 toxicities included neutropenia (35%), febrile neutropenia (14%), fatigue (14%), diarrhea (11%), nausea / vomiting (5%), myalgia / arthralgia (3%), and sensory neuropathy (3%). one patient withdrew from the study because of grade 3 sensory neuropathy, and one patient withdrew because of prolonged grade 2 sensory neuropathy. thomas and colleagues investigated the use of ixabepilone in patients with metastatic breast cancer that had progressed during, or within 4 months of taxane therapy (6 months if received in the adjuvant setting).27 in this study, 49 patients were administered ixabepilone at 40 mg / m as a 3-hour iv infusion on day 1 of a 21-day cycle. patients were heavily pretreated (86% had received two or more prior chemotherapy regimens) and had a substantial tumor burden (84% had at least two involved disease sites, and 84% had visceral disease). the orr was 12% (6 prs), with a median duration of response of 10.4 months. forty - one percent of patients achieved sd, with a median ttp of 2.2 months and median os of 7.9 months. the most common adverse events of any grade included fatigue (76%), nausea (57%), pain (65%), and sensory neuropathy (all grades, 63% ; grade 3, 12% ; grade 4, 0%). a combination of ixabepilone plus capecitabine has also been studied in patients with metastatic breast cancer previously treated with anthracyclines or a taxane.28,29 promising efficacy and safety results from a phase i / ii trial46 led to initiation of a pivotal phase iii trial (bms 046) of ixabepilone (40 mg / m as a 3-hour iv infusion on day 1 every 21 days) plus capecitabine (2000 mg / m on days 1 to 14 of a 21-day cycle) compared with capecitabine alone (2500 mg / m on days 1 to 14 of a 21-day cycle) in 752 patients with locally advanced or metastatic breast cancer that had progressed after treatment with anthracyclines and taxanes.28 in its entrance criteria, the study employed a strict definition of taxane resistance, requiring patients to have progressed during treatment or relapsed within 4 months of the last dose in the metastatic setting or within 12 months in the adjuvant setting. in general, patients were heavily pretreated and had a heavy burden of disease ; nearly 50% of patients had received 2 prior regimens in the metastatic setting, 90% of patients had metastases at two or more sites, and 84% had visceral disease involving the liver and/or lung. in this study, adding ixabepilone to capecitabine achieved an irr - assessed orr of 35% compared with 14% for capecitabine monotherapy, and an investigator - assessed orr of 42% compared with 23% for capecitabine monotherapy. median durations of response for the two treatment arms were 6.4 and 5.6 months, respectively. the median irr - assessed pfs was significantly longer for the combined regimen (5.8 months) than for capecitabine alone (4.2 months), resulting in a 25% risk reduction for disease progression with combined therapy (hazard ratio [hr ], 0.75 ; 95% confidence interval [ci ], 0.64 to 0.88 ; p = 0.003 ; figure 1).28 consistent with this result, the investigator - assessed median pfs was longer for the combined regimen than for capecitabine alone (5.3 vs 3.8 months ; p = 0.0011). this improvement in disease progression was maintained in a sensitivity analysis requested by the fda. when patients who received subsequent therapy before disease progression were censored for pfs at the last tumor assessment date, ixabepilone plus capecitabine still prolonged pfs compared with capecitabine alone (5.7 vs 4.1 months ; hr, 0.69 ; 95% ci, 0.58 to 0.83 ; p < 0.0001).47 compared with capecitabine alone, ixabepilone plus capecitabine was associated with a higher rate of grade 3 or 4 sensory neuropathy (21% vs 0%), fatigue (9% vs 3%), and neutropenia (68% vs 11%), as well as an increased rate of neutropenia - related death as a result of toxicity (3% vs 1%, with patients with liver dysfunction [grade 2 liver function tests ] at greater risk).28 a larger confirmatory phase iii trial (bms 048) in patients who were pretreated and/or resistant to anthracyclines and taxanes (n = 1221) provided further evidence that ixabepilone plus capecitabine consistently improved pfs compared with capecitabine alone (6.2 vs 4.4 months).29 notably, 50% of the patients in the confirmatory trial met the resistance criteria utilized in the pivotal trial. neither phase iii study showed a significant difference in median os between the two treatment arms ; however, adjusting for prognostic factors in a preplanned analysis, combination therapy significantly reduced the risk of death by about 15% (p = 0.0803 for bms 046 ; p = 0.0231 for bms 048).29 furthermore, as a part of these phase iii studies, prospectively defined subset analyses revealed statistically significant improvements in pfs when the combination of ixabepilone and capecitabine was used as first - line metastatic therapy (after having received anthracyclines and/or taxanes in the adjuvant setting) and in triple - negative patients.4850 patients who received ixabepilone plus capecitabine as first - line therapy experienced a 54% reduction in the estimated risk of disease progression with combination therapy.48 taken together, these results showed that ixabepilone in combination with capecitabine has superior clinical efficacy to capecitabine alone in patients with metastatic disease pretreated or resistant to anthracyclines and resistant to taxanes, a population with limited effective treatment options. ixabepilone has a manageable safety profile in patients with all stages of breast cancer, even in those with extensive or heavily pretreated disease (table 3).2328 as mentioned, neutropenia, sensory neuropathy, fatigue, arthralgias, myalgias, and stomatitis are the main toxic effects associated with this agent.2229,46 ixabepilone - related adverse events are usually manageable through dose reductions or delays or with supportive care. importantly, the toxicity profile of ixabepilone does not appear to overlap with that of capecitabine,28, 29 bevacizumab,40 or trastuzumab.39 currently, the approved dose and schedule of ixabepilone (as monotherapy or with capecitabine) is 40 mg / m administered iv every 3 weeks. an interim safety analysis of one such study, a randomized phase ii trial comparing ixabepilone at the approved dose and schedule to ixabepilone 16 mg / m given on days 1, 8, and 15 of a 28-day cycle, suggested that although both schedules were well tolerated among patients (all had advanced metastatic breast cancer and three - quarters had received prior therapy in the metastatic setting), the weekly schedule was associated with a lower overall incidence of adverse events, particularly grade 3/4 peripheral neuropathy.51 although neutropenia is frequently associated with ixabepilone therapy, the rate of febrile neutropenia is generally low. hematologic toxicity is easily managed by dose reduction or by use of hematologic growth factors, but ixabepilone should not be administered until neutrophil counts are at least 1,500 cells / mm and platelet counts at least 100,000 cells / mm.52 furthermore, in the initial phase iii trial of ixabepilone plus capecitabine, a higher rate of neutropenia - related deaths was reported in the combination - therapy group, particularly in patients with pre - existing hepatic insufficiency, than in the capecitabine - alone group.28 for this reason, ixabepilone in combination with capecitabine is contraindicated in patients with signs of moderate - to - severe liver dysfunction at baseline.52 neurotoxicity is a major concern with microtubule - stabilizing drugs like taxanes,53,54 and in clinical trials with locally advanced or metastatic breast cancer, the rates of grade 3 and 4 peripheral sensory neuropathy with ixabepilone therapy ranged from 0% to 21% (table 3). neuropathy was mainly sensory, cumulative, and reversible upon treatment modification or delay.54 for example, in the initial phase iii study of ixabepilone plus capecitabine, 70 of the 79 patients with grade 3 or 4 peripheral neuropathy had complete resolution or improvement of symptoms, with a median time to resolution to grade 1 or baseline severity of 6 weeks (figure 2).54 in patients with grade 2 peripheral neuropathy lasting 7 days, the dose of ixabepilone should be reduced by 20%. patients with grade 3 neuropathy lasting < 7 days should also receive a 20% reduction in the dose of ixabepilone. ixabepilone should be discontinued in patients who experience grade 3 neuropathy lasting 7 days or disabling neuropathy.52 in the initial phase iii study of ixabepilone plus capecitabine, patients with persistent grade 2 or 3 peripheral neuropathy received a median of three additional cycles after dose reduction. after dose reduction, peripheral neuropathy either improved or stabilized in most patients.28 neurologic function tests may be of some value in predicting the risk of sensory neuropathy during ixabepilone therapy.55 as with any chemotherapeutic agent, the benefits associated with ixabepilone therapy should be carefully weighed against the risks. in the setting of locally advanced or metastatic breast, a positive benefit - to - risk profile for combination ixabepilone plus capecitabine was revealed in a quality adjusted time without symptoms or toxicities (q - twist) analysis of the initial phase iii trial.56 this q - twist analysis evaluated the trade - off between toxicity and pfs by taking into account the time spent by each patient with or without grade 3 or 4 toxicity prior to progression and the time from progression to death or end of follow - up. remarkably, quality adjusted mean survival was significantly greater for ixabepilone plus capecitabine than it was for capecitabine alone (p = 0.0227), with a 3.8-week improvement with ixabepilone plus capecitabine over capecitabine alone. the novel chemotherapeutic agent ixabepilone exhibits reduced susceptibility to several important tumor survival mechanisms that limit the efficacy of taxanes and anthracyclines. ixabepilone has demonstrated efficacy in patients across the breast cancer spectrum of early - stage to advanced disease, including patients with extensive, aggressive, and heavily pretreated tumors and those with resistance to several other agents. ixabepilone is approved for the treatment of patients with locally advanced or metastatic breast cancer as monotherapy after failure of an anthracycline, a taxane, and capecitabine, and in combination with capecitabine after failure of an anthracycline and a taxane. ixabepilone is also being investigated in combinations with several other cytotoxic and biologic agents, and preliminary studies point toward efficacy in combination with bevacizumab or trastuzumab that is comparable to that of paclitaxel. preliminary data also suggest promising efficacy with ixabepilone in the neoadjuvant setting, and studies are underway investigating the benefits of ixabepilone in the adjuvant setting. the fact that iii - tubulin appears to be particularly upregulated in triple - negative breast tumors may render ixabepilone more active in this subgroup of patients, an observation in need of additional clinical validation. the manageable safety profile of ixabepilone supports its clinical utility, as adverse events can usually be managed with a dose reduction or delay, or with appropriate supportive care. response appeared to be better and toxic effects were milder when ixabepilone was used earlier in the treatment course (eg, in the first- or second - line setting) in patients who were less heavily pretreated. the phase iii studies reveal that capecitabine does not exacerbate ixabepilone - related toxicities, or vice versa. moreover, adverse events were manageable and responsive to intervention regardless of whether ixabepilone was given alone or with capecitabine. however, as is to be expected with any cytotoxic chemotherapy, the incidence and severity of toxicities was lower when ixabepilone was used as monotherapy. these observations highlight the need to evaluate candidates for ixabepilone therapy on a case - by - case basis to determine whether the patient may experience the greatest overall benefit from monotherapy or from more aggressive combination therapy. importantly, ixabepilone still demonstrated reasonable efficacy when used as monotherapy, even in patients with a heavy disease burden, documented resistance to several other classes of chemotherapy, or aggressive tumors (eg, triple - negative disease). for the reasons discussed above, ixabepilone represents a clinically useful addition to the therapeutic agents available for patients with resistant advanced breast cancer that help to extend pfs in patients with limited treatment options. ongoing investigations in early - stage disease should provide insight as to how this novel agent could be effectively integrated into this setting.	the epothilone analog ixabepilone exhibits reduced susceptibility to several important tumor survival mechanisms that limit the efficacy of taxanes and anthracyclines. as a single agent, ixabepilone has shown promise in metastatic breast cancer when anthracyclines, taxanes, or capecitabine have failed ; and in early - stage breast cancer that is taxane - nave or has previously received taxanes in the adjuvant or metastatic setting. compared with capecitabine alone, ixabepilone used in combination with capecitabine in patients previously treated with and resistant to anthracyclines and taxanes produced a 25% reduction in the risk of disease progression. triple - negative tumors showed particular susceptibility to this doublet. ixabepilone has also demonstrated efficacy as first - line therapy in combination with targeted agents such as bevacizumab and trastuzumab. ongoing investigations should provide insight as to how this agent could be integrated into treatment of early - stage disease. in clinical studies, toxicities with ixabepilone were manageable and reversible through dose reduction or delay, even in patients with extensive or heavily - pretreated disease. thus, ixabepilone represents a useful addition to the therapeutic options available for advanced breast cancer, and it may extend progression - free survival in patients with limited treatment options.
it is caused by a deficiency of both hexosaminidase (hex) a and b, resulting in accumulation of glycosphingolipids and oligosaccharides in the brain. it has three clinical subtypes (infantile, juvenile, and adult forms) and represents around 7% of cases among all the lysosomal storage disorders. the infantile form presents in the first 618 months of age with regression of milestones, developmental delay, startle response, hypotonia, cherry red spots, and convulsions. we report this case as the infant presented with regression without hepatosplenomegaly and confirmed by gene testing. a 1-year - old second born male child, born to a third - degree consanguineously married couple with uneventful perinatal history, was brought with regression of milestones and seizures. the onset of clinical symptoms began at the age of 6 months with gradual loss of the milestones. the child had achieved response to sounds by 2 months ; head holding, social smile, and recognition of mother by 3 months of age. initially, he lost social smile and recognition of mother followed by control of neck. he lost all the milestones by 89 months of age. the child developed exacerbated startle response since 8 months of age and multiple episodes of right - sided focal seizures from 11 months of age. there was a history of sibling death at the age of 18 months with similar complaints. on examination, head circumference was 44 cm (between 3 and 15 centile) with coarse facies. the tone was increased in all the limbs with power of 3/5 (medical research council grade). fundus examination showed cherry - red spot in the macula, and there was no hepatosplenomegaly. magnetic resonance imaging (mri) shows hyperintensity of the basal ganglia (long white arrow) and hypointensity of the ventral thalami (open arrow) on t2-weighted sequences [figure 1 ]. based on above findings suspected to be a case of sandhoff disease, the enzyme assay in leukocytes for hexosaminidase total (a + b) revealed 61 nmhol / h / mg (normal, 9052878). gene testing was positive for homogenous missense substitution p. cys534tyr, c1601g > a, chr5:74016560g > a in exon 13 of hexb gene. axial t2-weighted imaging showing hyperintensity of the basal ganglia (long white arrow) and hypointensity of the ventral thalami (open arrow) our patient had presented with regression of milestones, exaggerated startle response, decreased vision, and seizures. the child had coarse facies, cherry red spot, and normocephaly without hepatosplenomegaly. in a study conducted among 18 gm2 gangliosidosis patients by karimzadeh., seven patients had macrocephaly, three patients had microcephaly, and eight patients had a normal head circumference. a distinguishing feature in standoff 's disease from other gm2 gangliosidosis is presence of hepatosplenomegaly and n - acetylglucosamine - containing oligosaccharides in urine likewise, karimzadeh. also noted, and only two patients of sandhoff disease out of their nine cases had hepatosplenomegaly. similar observation was made by ozkara. in their study of 18 cases affected by sandhoff disease, and hepatosplenomegaly was not found in 11 out of 18 infantile sandhoff disease patients, while the remaining seven had mild hepatosplenomegaly. closest differential diagnosis of sandhoff disease is tay sachs disease, which is characterized by the absence of coarse facies, hepatosplenomegaly, skeletal deformities, and signs of peripheral nerve involvement. axial t2-weighted imaging on mri in our patient revealed hyperintensity of the basal ganglia and hypointensity of the ventral thalami. also reported as bilateral thalamic hyperdensity on computed tomography and hypointensity on t2-weighted mri images as the earliest diagnostic markers of sandhoff disease. the gold standard method for diagnosis of gm2 gangliosidosis is the measurement of -hex activity in plasma, serum, and/or fibroblasts. molecular characterization of the hexa and hexb mutations can also be performed for confirmation by direct sequencing of the entire coding region and intron / exon boundaries using genomic dna. the same mutation was reported previously in a japanese case which showed hepatosplenomegaly unlike our case. prenatal diagnosis advised for next pregnancy. sachs disease if any child presenting with regression of milestones and cherry red spot without hepatosplenomegaly.	sandhoff disease is a neurodegenerative disease caused due to deficiency of hexosaminidase (hex) a and b. a 1-year - old male child presented with regression of milestones, exaggerated startle response, decreased vision, and seizures from 6 months of age. the child had coarse facies without hepatosplenomegaly. serum levels of hexosaminidase total (a + b) were low. genetic testing for sandhoff disease revealed a homozygous missense variant on hexb gene. the case is presented to highlight that the absence of hepatosplenomegaly should not restrain in suspecting sandhoff disease.
implantable cardioverter defibrillator (icd) and cardiac resynchronization therapy (crt) devices have become the mainstay of treating persistent systolic heart failure in addition to guideline directed medical therapy. many of these devices are implanted in patients with congestive heart failure with new york heart association (nyha) class status of ii to iv. while these devices considerably ameliorate patient morbidity and mortality, heart failure remains a significant economic burden costing the us health care system ~$30 billion annually. heart failure hospitalizations (hfhs) account for two - thirds of the total expense. identifying patients at risk of worsening heart failure to allow timely intervention has the potential to prevent hospitalizations and improve long - term patient outcomes while reducing costs of care. in addition to providing life - saving therapies, implantable devices collect a host of continuous physiological patient data (e.g. activity, day and night heart rate, at / af burden, heart rate during at / af, percent crt pacing, number of shocks, and intrathoracic impedance). however, the data collected vary by manufacturer. for example, not all manufacturers have devices with intrathoracic impedance capability. also, while all devices include a single or multi - axis accelerometer, proprietary algorithms to derive daily activity from accelerometer signals vary (see methods for details). many of the diagnostic variables have been shown to be prognostic markers of worsening heart failure and/or mortality risk. for example, nhr is a marker of autonomic tone, and an elevated nhr is associated with higher hfh risk. activity is a reflection of patient functional capacity, and decreasing activity is associated with worsening hf status. a loss of crt pacing compromises cardiac hemodynamics and hence leads to worsening patient status. and finally, a decrease in intrathoracic impedance is associated with an increase in wedge pressure, elevated pre - load, and risk of fluid extravasation into the lungs. however, the clinical adoption of these risk stratification models has been slow because they use thresholds and measurement schemes not yet implemented in the implantable devices. some of these approaches in fact use a fixed 30-day look back window requiring manual sifting of data to identify trends, thus making it cumbersome to use them in day - to - day practice. all crt - d devices have thresholds for various parameters that when crossed trigger a notification, referred to as device observation. the purpose of this study was to examine the performance of the existing device observations for stratifying patients at hfh risk. since the impedance observation is not available in all devices [e.g. optivol observation is not available on carelink (medtronic inc. mn) in the us ], we performed the analysis with and without impedance observation. furthermore, we investigated the relationship between the number of device observations triggered and risk of hfh. we performed retrospective analysis using patient data from fast and partners - hf clinical trials using medtronic devices. both study protocols were approved by institutional review boards and all patients provided written informed consent. fast was a prospective double - blinded observational study in crt - d and icd patients (n = 109) with ef 35% and nyha class iii or iv. partners - hf was a prospective observational study in crt - d patients (n = 1024) with ef 35%, nyha class iii or iv, and qrs duration 130 ms. the two studies combined had 1133 patients and 220 hfhs. only patients with an optivol capable crt - d device were included in this analysis. follow - up sessions with less than 7 days of data before and less than 30 days of data after the evaluation were excluded. furthermore, if there was another follow - up session within 30 days of a previous session, the second session was excluded. after applying above criteria, 186 hfhs in 775 patients and a total of 2276 follow - up sessions were available for analysis. the hfh event rate of 22.2% per year in this cohort is comparable to that in nyha iii and iv device patients. the following diagnostic parameters in medtronic devices have an observation : optivol index, at / af burden, ventricular rate during at / af (vraf), activity, night heart rate (nhr), and percent pacing (% crt pacing) (fig. 1). in addition, an observation is noted if defibrillation shocks are delivered and this was also included in our analysis. impedance (z) is measured intrathoracically across the right ventricular (rv) coil and device - can by injecting a small current pulse (i) and measuring the developed voltage (v ; z = i / v). optivol index is derived as the cumulative difference between expected and actual zs for the duration when expected z is higher than actual z. when actual z exceeds expected z, optivol index is set to zero. since optivol index is integration of z over certain duration, it is measured in units of ohm - days (-days). a higher value of optivol index has been shown to be associated with hfh. several electrophysiological parameters including nhr, af burden, and vraf are derived from atrial and ventricular electrograms (egms) acquired by the device at 10 ms resolution. device algorithms, such as pr logic, are applied to discriminate among different rhythms and derive these electrophysiological parameters. nhr is the average heart rate between midnight and 4 am and is a measure of resting heart rate. af burden is measured as total duration of fast atrial rate during a 24-h period associated with atrio - ventricular conduction ratio 2:1. activity is a quantitative measure of active duration and is a surrogate of functional capacity. it is measured by a single axis accelerometer in the device that is used to detect patient motion and convert it into discrete electrical signals. an algorithm then converts these electrical signals to number of minutes active for the entire 24-h duration during a day (day and night time activities are not reported separately), where a minute is considered active if accelerometer registers signal equivalent to 70 steps / min or greater. the device recorded activity has been shown to have a strong intra - individual correlation with activity measured using a validated external sensor. however, details of the algorithm differ between implantable and external devices (e.g. pedometers and external accelerometers) and absolute duration of reported activity between the two may differ. all of the above parameters have a threshold value, which when exceeded triggers a device observation. fig. 1 shows empirically derived nominal threshold values for various parameters that can be tailored on a patient basis. all nominals (or values close to the nominals) have been shown to be associated with greater hfh or mortality risk. an optivol observation is noted on the device report when a value of 60 -days is exceeded, a threshold shown to predict hfh with optimal sensitivity and false alert rate. at / af burden of 6 h / day for at least one day within the last 30-days this risk is further exacerbated with poor rate control with v - rate > 90 bpm. for example, while there is an optivol observation on the programmer report in the united states, no such observation exists on the carelink hf management report. outside the united states, the optivol observation is available in both the programmer and carelink reports. back was performed until the previous follow - up to evaluate the number of device observations triggered. also, a 30-day look forward was performed to assess if an hfh had occurred within that period. for example, for the follow - up labeled as fu2 in fig. 2, number of observations triggered was evaluated for the duration labeled as risk assessment 2, and a 30-day hfh risk assessment was performed for the duration labeled as risk prediction 2. this was repeated for all the follow - ups for a given patient, and then for all the patients. these data from all risk assessment and risk prediction pairs were then used to compute the relationship between number of observations triggered and 30-day hfh event rate. specifically, raw event rate for 0, 1, 2, and 3 observations was computed as : number of risk prediction windows with1hfhtotal number of risk assessment windows the hfh event rates and odds ratios were estimated using a generalized estimating equations (gee) model for the groups with different number of observations. we made no adjustment for baseline variables (age, gender, nyha, history of coronary artery disease, mi, af, diabetes, and hypertension) and baseline medications (ace - i / arb, diuretics, b - blockers, and anti - arrhythmic drugs). this reflects the real world since device data are not yet fully integrated with clinical and demographic data. sensitivity is defined as the number of evaluations with a given number of device observations in the preceding evaluation window and hfh event in next 30-days divided by the total number of evaluations with hfh in next 30-days. specificity is defined as the number of evaluations without a given number of device observations in the preceding evaluation period and no hfh event in next 30 days divided by the total number of evaluations with no hfh in next 30 days. the sensitivity and specificity computations are adjusted for multiple evaluations in patients using a gee model. all statistical analyses were performed using sas version 9.2 (sas institute inc., cary, nc, usa). the following diagnostic parameters in medtronic devices have an observation : optivol index, at / af burden, ventricular rate during at / af (vraf), activity, night heart rate (nhr), and percent pacing (% crt pacing) (fig., an observation is noted if defibrillation shocks are delivered and this was also included in our analysis. impedance (z) is measured intrathoracically across the right ventricular (rv) coil and device - can by injecting a small current pulse (i) and measuring the developed voltage (v ; z = i / v). optivol index is derived as the cumulative difference between expected and actual zs for the duration when expected z is higher than actual z. when actual z exceeds expected z, optivol index is set to zero. since optivol index is integration of z over certain duration, it is measured in units of ohm - days (-days). several electrophysiological parameters including nhr, af burden, and vraf are derived from atrial and ventricular electrograms (egms) acquired by the device at 10 ms resolution. device algorithms, such as pr logic, are applied to discriminate among different rhythms and derive these electrophysiological parameters. nhr is the average heart rate between midnight and 4 am and is a measure of resting heart rate. af burden is measured as total duration of fast atrial rate during a 24-h period associated with atrio - ventricular conduction ratio 2:1. activity is a quantitative measure of active duration and is a surrogate of functional capacity. it is measured by a single axis accelerometer in the device that is used to detect patient motion and convert it into discrete electrical signals. an algorithm then converts these electrical signals to number of minutes active for the entire 24-h duration during a day (day and night time activities are not reported separately), where a minute is considered active if accelerometer registers signal equivalent to 70 steps / min or greater. the device recorded activity has been shown to have a strong intra - individual correlation with activity measured using a validated external sensor. however, details of the algorithm differ between implantable and external devices (e.g. pedometers and external accelerometers) and absolute duration of reported activity between the two may differ. all of the above parameters have a threshold value, which when exceeded triggers a device observation. fig. 1 shows empirically derived nominal threshold values for various parameters that can be tailored on a patient basis. all nominals (or values close to the nominals) have been shown to be associated with greater hfh or mortality risk. an optivol observation is noted on the device report when a value of 60 -days is exceeded, a threshold shown to predict hfh with optimal sensitivity and false alert rate. at / af burden of 6 h / day for at least one day within the last 30-days this risk is further exacerbated with poor rate control with v - rate > 90 bpm. nhr of > 90 bpm discriminates between hospitalized and non - hospitalized patients. and finally, crt pacing < 90% is associated with increased mortality. for example, while there is an optivol observation on the programmer report in the united states, no such observation exists on the carelink hf management report. outside the united states, the optivol observation is available in both the programmer and carelink reports. back was performed until the previous follow - up to evaluate the number of device observations triggered. also, a 30-day look forward was performed to assess if an hfh had occurred within that period. for example, for the follow - up labeled as fu2 in fig. 2, number of observations triggered was evaluated for the duration labeled as risk assessment 2, and a 30-day hfh risk assessment was performed for the duration labeled as risk prediction 2. this was repeated for all the follow - ups for a given patient, and then for all the patients. these data from all risk assessment and risk prediction pairs were then used to compute the relationship between number of observations triggered and 30-day hfh event rate. specifically, raw event rate for 0, 1, 2, and 3 observations was computed as : number of risk prediction windows with1hfhtotal number of risk assessment windows the hfh event rates and odds ratios were estimated using a generalized estimating equations (gee) model for the groups with different number of observations. we made no adjustment for baseline variables (age, gender, nyha, history of coronary artery disease, mi, af, diabetes, and hypertension) and baseline medications (ace - i / arb, diuretics, b - blockers, and anti - arrhythmic drugs). this reflects the real world since device data are not yet fully integrated with clinical and demographic data. sensitivity is defined as the number of evaluations with a given number of device observations in the preceding evaluation window and hfh event in next 30-days divided by the total number of evaluations with hfh in next 30-days. specificity is defined as the number of evaluations without a given number of device observations in the preceding evaluation period and no hfh event in next 30 days divided by the total number of evaluations with no hfh in next 30 days. the sensitivity and specificity computations are adjusted for multiple evaluations in patients using a gee model. all statistical analyses were performed using sas version 9.2 (sas institute inc., cary, nc, usa). table 1 summarizes clinical and demographic data for the 775 patients in the fast and partners - hf trials selected for our analysis. all patients had a crt - d device, and the majority (87%) of the patients had a heart failure status of nyha iii. a total of 2276 in - clinic follow - up sessions were available for analysis. forty - two follow - up sessions in 37 unique patients had an associated hfh in the following 30 days. the rate of hfh increased with increasing number of observations. for zero device observation, the 30-day event rate was 0.9% and increased to 13.6% for three or more device observations. the odds ratio for three or more observations versus no observation was 17.9 (see table 2 for other odds ratios). also, noteworthy from table 2 is that a vast majority (~71%) of the total follow - up sessions had no device observation. the proportion of follow - up sessions decreased with increasing number of observations (23.5%, 4.3% and 1.3% for 1, 2 and 3 observations, respectively). based on univariate analysis (table 3), hfh rate varied from 3.5% (for optivol) to 11.2% (for vraf). activity, af burden, and decrease in crt pacing triggered observations during a large proportion of follow - up sessions (~10% or more) and the corresponding event rates were 5.1%, 4.7% and 4.6%, respectively. similar to the case of optivol excluded, the hfh rate increased with increasing number of observations. the hfh rate for zero device observation was 0.4% and increased to 13.6% for 3 observations with an odds ratio of 42.4 (see table 4 for other odds ratios). follow - up sessions with zero observation constituted the largest proportion (48.5%) of all follow - up sessions, and the proportion declined with increasing number of observations (36.4%, 12.3% and 2.9% for 1, 2 and 3 observations, respectively). 3 shows hfh event rate during 30 days post - evaluation with optivol excluded (panel a) and included (panel b). the increase in event rate with increasing number of observations is evident in both plots. for example, with optivol excluded, the 30-day hfh rate was less than 1% for 0 observation. the 30-day hfh event rate increased to ~3%, ~7% and ~14% for 1, 2 and 3 observations, respectively. with optivol included, a greater separation between the 3 observations trace and 0 observation and 1 observation traces was observed suggesting a better risk stratification performance. tables 5 and 6 show the sensitivity and specificity in predicting hfh for the parameter set excluding and including optivol for 1, 2 and 3 device observations. with optivol excluded, the sensitivity for 1 observation was 68.9% and decreased to 9.5% for 3 observations. the corresponding specificity for 1 observation was 71.2% and increased to 98.8% for 3 observations. similarly, with optivol included, the sensitivity for 1 observation was 90.5% and decreased to 21.6% for 3 observations. the corresponding specificity increased from 49.1% (1 observation) to 97.4% (3 observations). with optivol included, the relative increase in sensitivity for 3 observations was significant (21.6% versus 9.5% ; see bottom most rows in tables 5 and 6) compared to the decrease in specificity (97.4% versus 98.8%). table 1 summarizes clinical and demographic data for the 775 patients in the fast and partners - hf trials selected for our analysis. all patients had a crt - d device, and the majority (87%) of the patients had a heart failure status of nyha iii. a total of 2276 in - clinic follow - up sessions were available for analysis. forty - two follow - up sessions in 37 unique patients had an associated hfh in the following 30 days. the rate of hfh increased with increasing number of observations. for zero device observation, the 30-day event rate was 0.9% and increased to 13.6% for three or more device observations. the odds ratio for three or more observations versus no observation was 17.9 (see table 2 for other odds ratios). also, noteworthy from table 2 is that a vast majority (~71%) of the total follow - up sessions had no device observation. the proportion of follow - up sessions decreased with increasing number of observations (23.5%, 4.3% and 1.3% for 1, 2 and 3 observations, respectively). based on univariate analysis (table 3), hfh rate varied from 3.5% (for optivol) to 11.2% (for vraf). activity, af burden, and decrease in crt pacing triggered observations during a large proportion of follow - up sessions (~10% or more) and the corresponding event rates were 5.1%, 4.7% and 4.6%, respectively. risk stratification performance with optivol included is shown in table 4. similar to the case of optivol excluded, the hfh rate increased with increasing number of observations. the hfh rate for zero device observation was 0.4% and increased to 13.6% for 3 observations with an odds ratio of 42.4 (see table 4 for other odds ratios). follow - up sessions with zero observation constituted the largest proportion (48.5%) of all follow - up sessions, and the proportion declined with increasing number of observations (36.4%, 12.3% and 2.9% for 1, 2 and 3 observations, respectively). 3 shows hfh event rate during 30 days post - evaluation with optivol excluded (panel a) and included (panel b). the increase in event rate with increasing number of observations is evident in both plots. for example, with optivol excluded, the 30-day hfh rate was less than 1% for 0 observation. the 30-day hfh event rate increased to ~3%, ~7% and ~14% for 1, 2 and 3 observations, respectively. with optivol included, a greater separation between the 3 observations trace and 0 observation and 1 observation traces was observed suggesting a better risk stratification performance. tables 5 and 6 show the sensitivity and specificity in predicting hfh for the parameter set excluding and including optivol for 1, 2 and 3 device observations. with optivol excluded, the sensitivity for 1 observation was 68.9% and decreased to 9.5% for 3 observations. the corresponding specificity for 1 observation was 71.2% and increased to 98.8% for 3 observations. similarly, with optivol included, the sensitivity for 1 observation was 90.5% and decreased to 21.6% for 3 observations. the corresponding specificity increased from 49.1% (1 observation) to 97.4% (3 observations). with optivol included, the relative increase in sensitivity for 3 observations was significant (21.6% versus 9.5% ; see bottom most rows in tables 5 and 6) compared to the decrease in specificity (97.4% versus 98.8%). in this study we presented a novel scheme to stratify patients at risk of hfh using diagnostic parameters available in medtronic 's crt - d devices. the thresholds and corresponding device observations were unmodified from what is available in the device. in addition, the look back period for assessing diagnostic parameters was the entire duration between the follow - up sessions to mirror the real world clinical practice. we found that this relatively simple and easy to implement scheme can stratify patients quite effectively. the risk of an hfh event increases with increasing number of device observations, and a patient with three or more observations is at 18 (optivol excluded) to 42 (optivol included) risk compared to a patient with zero observation (tables 2 and 4). the sensitivity and specificity exhibit a typical trade - off. for example, sensitivity for 3 device observations is lower than that for 2 observations while the specificity for 3 device observations is better. inclusion of optivol improves overall performance. with optivol excluded for 3 device observations, sensitivity and specificity with optivol included sensitivity improves significantly to 21.6% while the specificity drops slightly to 97.4%. in contrast to our dynamic risk assessment scheme, these models are static and use a one - time snapshot of laboratory measurements. furthermore, since the device data are continuously collected, our scheme is amenable to be applied in an ambulatory setting. for example, the device data can be transmitted automatically to a clinic upon an alert (referred to as carealert in medtronic 's carelink system) or on a predetermined schedule. it is instructive to compare the performance of device diagnostics with patient weight in predicting hfh. patient weight increases steadily for ~2 weeks before hfh and is routinely used in clinical practice. however, in a head - to - head comparison, patient weight performed significantly worse than optivol. while optivol had a sensitivity of 76% and an unexplained detection rate of 1.9 per patient - year, patient weight had a sensitivity of mere 20% and an unexplained detection rate of 4.3 per patient - year. given that our scheme combines several device parameters, its performance is better than optivol alone and hence superior to that of weights [e.g. for 1 observation, sensitivity with optivol included is 90.5% (table 6) and an unexplained detection rate at a specificity of 49.1% is 1.5 per patient - year ]. various device diagnostic parameters reflect different underlying physiological processes, and a deviation beyond a certain range may signal a compromise in physiological homeostasis and hence be a marker of patient risk. a drop in impedance and accompanying rise in optivol is indicative of possible fluid overload, while an excessive rise in impedance and drop in optivol might signal dehydration. similarly, elevated nhr is a potential marker of imbalance in autonomic tone, and lower activity can signal compromised functional capacity. while each diagnostic parameter is a risk marker, univariate analysis shows that performance of a single parameter is modest (table 3). for example, 30-day hfh rate following an optivol observation is 3.5%, and hfhs occur even in the absence of an optivol observation at a rate of 0.9% (table 3). corresponding numbers for vraf, the one with highest hfh rate, are 11.2% and 1.5%. prognostic value of device diagnostics is significantly improved when observations from all the parameters are combined. the utility of combining device diagnostic variables for hf risk stratification has been shown earlier. whellan combined device diagnostics for the previous 30 days using a heuristic approach to assess next 30-day hf risk. when two or more parameters exceeded preset thresholds or optivol alone exceeded a very high threshold, the risk was found to be 5.5 higher compared to diagnostic criterion not met. first, while they segmented patients into two risk categories (i.e. high and low), we use a graded approach with 4 risk categories (i.e. 0, 1, 2, 3 observations) in which risk gradually increases with increasing number of device observations. second, while we also use a clinically relevant 30-day risk prediction window, our look back period spans the entire duration between current and previous follow - up. finally, while they modified threshold values for a few parameters and used parameters without an existing observation (e.g. hrv), we only selected parameters with available device observations. these last two differences make our scheme readily implementable (e.g. on carelink) since any alterations in threshold values and ways to combine them makes the implementation cumbersome for health care providers. recently a more sophisticated methodology using a probabilistic bayesian belief network approach has been presented to categorize patients into low, medium, and high risk statuses. while this approach is elegant and more rigorous, it can not be readily applied by health care providers using existing device diagnostics. to improve outcomes using integrated device data has challenges similar to those faced by other management strategies involving a single device parameter (e.g. optivol) or other diagnostic modalities (e.g. intra - cardiac pressure). foremost among these the diagnostic information provided needs to be actionable and must be acted upon. provided device diagnostic data are combined with an appropriate intervention algorithm that is adhered to, it has the potential to improve patient outcomes as shown for intrathoracic pressure. however, given telemonitoring trials have had mixed results, no assertions can be made regarding effectiveness of any novel risk assessment scheme in absence of a prospective study. thus, as is the case for other approaches to manage patients (e.g. weight, blood pressure, temperature, ecg, etc.), it is imperative that a patient 's overall health status be taken into account to devise a management strategy. while an odds ratio for our risk stratification scheme is superior to several clinically used risk stratification tools (tables 2 and 4), it is apparent that the absolute 30-day hf hospitalization rate is relatively low (e.g. ~14% for 3 observations, tables 2 and 4). these low numbers are reflective of hfh being a relatively rare event with a rate of ~12% over a 30-day period. thus, greater number of device observations alone should not trigger an action as it can potentially lead to an increase in health care utilization and hospitalization rate. rather they should be used in conjunction with other clinical data to identify a subset of high risk patients and judiciously allocate resources. the actions may include diet and medication counseling for non - adherence, more frequent tele - monitoring, or medication change. since our proposed scheme uses unmodified device observations, they are already available to clinicians for use. however, whether device diagnostics are integrated into a clinic 's workflow is influenced by several factors such as expertise of staff, proven or perceived value of device diagnostics, resources needed, and ease of use. varying approaches can be used to integrate device diagnostics into a clinic 's workflow, and are influenced by factors such as volume of patients, skill level of staff, and availability of resources. in all cases, presently, hf related observations can only be viewed at a patient level (e.g. clinician must open and view hf management report for each patient in carelink to assess various observations) or they are mingled with other device and electrophysiological observations (e.g. lead failure, low battery voltage). this makes using hf related observations a bit cumbersome. a clinic level view dedicated to hf related observations and the ability to sort patients by the number of these observations could facilitate the triaging process. streamlining the carelink system and then demonstrating that clinic efficiencies, time to clinical action, and perhaps patient outcomes are improved by using a device observations based tri - aging scheme remain topics of future research. although studies demonstrating improvement in time to clinical action with use of device diagnostics are available, none are available demonstrating improvement in outcomes. the device diagnostic parameters and their significance for an icd and crt - d are slightly different. for example, % crt pacing is irrelevant for an icd. instead, a lower rv pacing is desirable. furthermore, since characteristics of icd and crt - d patients differ (class ii and iii for the former versus class iii and iv for the latter), our findings may not translate to an icd patient population. since type of diagnostic parameters, collection method (e.g. time and frequency of sampling), and thresholds for observations can vary among manufacturers, our results are not generalizable to non - medtronic devices. finally, to reflect the current practice of using device data in a standalone fashion, we only used device data and did not include demographic, medication or clinical data (e.g. weight, blood pressure and brain natriuretic peptide) that clinicians have access to. we did not address incremental value of device parameters to the clinical and psychosocial variables. we developed a novel and simplified scheme for stratifying patients at risk of hf hospitalization using existing diagnostic observations available in a crt - d device. the scheme can be readily implemented on a remote management system such as carelink (medtronic inc., mn) and could potentially be another tool in a clinician 's repertoire to help quickly identify patients at risk of hf events. however, whether an appropriately devised intervention strategy when coupled with our stratification scheme improves patient outcomes will require prospective evaluation.	backgroundheart failure hospitalizations (hfhs) cost the us health care system ~$20 billion annually. identifying patients at risk of hfh to enable timely intervention and prevent expensive hospitalization remains a challenge. implantable cardioverter defibrillators (icds) and cardiac resynchronization devices with defibrillation capability (crt - ds) collect a host of diagnostic parameters that change with hf status and collectively have the potential to signal an increasing risk of hfh. these device - collected diagnostic parameters include activity, day and night heart rate, atrial tachycardia / atrial fibrillation (at / af) burden, mean rate during at / af, percent crt pacing, number of shocks, and intrathoracic impedance. there are thresholds for these parameters that when crossed trigger a notification, referred to as device observation, which gets noted on the device report. we investigated if these existing device observations can stratify patients at varying risk of hfh.methodswe analyzed data from 775 patients (age : 69 11 year, 68% male) with crt - d devices followed for 13 5 months with adjudicated hfhs. hfh rate was computed for increasing number of device observations. data were analyzed by both excluding and including intrathoracic impedance. hfh risk was assessed at the time of a device interrogation session, and all the data between previous and current follow - up sessions were used to determine the hfh risk for the next 30 days.results2276 follow - up sessions in 775 patients were evaluated with 42 hfhs in 37 patients. percentage of evaluations that were followed by an hfh within the next 30 days increased with increasing number of device observations. patients with 3 or more device observations were at 42 hfh risk compared to patients with no device observation. even after excluding intrathoracic impedance, the remaining device parameters effectively stratified patients at hfh risk.conclusionavailable device observations could provide an effective method to stratify patients at varying risk of heart failure hospitalization.
although several surgical interventions have been developed, mortality and complications related to surgical management remains high, especially with respect to trans - ventricular approach. ventricular incision also has several disadvantages : increased postoperative bleeding, ventricular malfunction, and ventricular arrhythmia. in the present work, we report a successful case of repair of posterior ventricular septal rupture (vsr) via transatrial approach. a 73-year - old woman presented to a local hospital with shortness of breath and anterior chest pain. the electrocardiogram (ecg) demonstrated st - segment elevations in the inferior leads, but regional wall motion abnormality was not noted on the transthoracic echocardiogram. the coronary angiogram showed severe stenosis at the middle of the right coronary artery (fig. 1). the left coronary catheterization revealed mild stenosis in the middle left anterior descending coronary artery. percutaneous coronary intervention (pci) was performed at the right coronary artery by stent insertion. after pci, sinus tachycardia changed to ectopic junctional rhythm on the ecg, and severe hypotension was noted. the additional transthoracic echocardiogram revealed vsr at the basal inferoseptal wall and severe tricuspid regurgitation with right ventricular dilation (fig. a median sternotomy was performed, and cardiopulmonary bypass (cpb) was established via ascending aortic and bicaval cannulation. vsr was noted at the basal inferior septum measuring approximately 1.5 cm in diameter (fig. the septal and posterior leaflet and a few chordae were resected for more consistent access to the vsr. eleven stitches of pledget buttressed by 2 - 0 prolen were sutured by the method of interrupted horizontal mattress along the ventricular septal defect (vsd) margin. the stitches near the septal leaflet were sutured through the annulus of the septal leaflet, and the stitches for the inferior margin were sutured through the inferior free wall of the right ventricle. these stitches were passed through the trimmed teflon felt and tied into place without difficulty. tricuspid valve replacement was performed with a carpentier edward valve (edwards lifesciences, irvine, ca, usa) by using a supra - annular technique (fig. although pci for right coronary artery lesion was done successfully, we decided to perform a right coronary artery bypass to be prepared for the possibility of acute thrombosis. the distal right coronary artery was bypassed with a saphenous venous graft in an end - to - side fashion. the immediate postoperative transthoracic echocardiogram (tte) revealed right ventricular dysfunction, and the iabp was removed from the patient on postoperative day 4. there was no evidence of residual vsr, and right ventricular function was in the normal range on the tte. several risk factors are associated with post - infarct vsr : first infarction, inferior location, transmural infarction, complete and sudden occlusion of a coronary artery, poor collateral blood flow, and left ventricular hypertrophy.1)2) with the development of percutaneous coronary intervention, the incidence of postinfarct vsr has been reduced ; however, mortality remains high.3) post - infarct vsr is associated with an 87% mortality within 2 months if managed medically.4) previously, many surgeons delay surgical intervention while waiting for myocardial fibrosis to occur because this facilitates surgical repair. more recently, the majority of surgeons have advocated more aggressive management, with immediate start of iabp and urgent repair of vsr. in general, most surgeons conduct the operation via left ventriculotomy or via the infarcted free wall. however, in the case of posterior vsr, access is difficult through left ventriculotomy or infarcted free wall.5) ventricular incision also has several disadvantages : increased postoperative bleeding, ventricular malfunction, and ventricular arrhythmia. in 1986, filgueira.6) described the transatrial approach to repair the vsr in order to avoid some of the problems of ventricular incision. it is difficult to expose the vsr and to accurately identify the vsd due to the trabeculation of the right ventricle. in addition, the placement of the stitch through the tricuspid valve may interfere with the subvalvular apparatus, and tricuspid regurgitation can occur. however, the tricuspid valvectomy method requires tricuspid valve repair after vsr repair. in the present case, we performed a tricuspid valve replacement instead of a repair to avoid the risk of tricuspid regurgitation that might occur if the valvular mechanism is compromised during exposure and patch closure of the vsr. moreover, inferior myocardial infarctions may result in right ventricular dysfunction and low cardiac output syndrome postoperatively, which can worsen due to residual tricuspid regurgitation. accurate identification of vsr is crucial to avoid residual shunt flow and can be achieved by injecting blood - tinged saline solution through a left ventricular vent catheter placed via the right superior pulmonary vein. massetti.7) found that posterior vsr could be successfully repaired via the transatrial approach. by avoiding additional damage to the ventricle lee.8) reported a successful right atrial approach operation for post - infarction rupture of the posterior ventricular septum. however, unlike in our case, the operation was conducted 12 days after infarction. in conclusion, many surgeons advocate the use of ventriculostomy or infarcted tissue for the treatment of postinfarct vsr ; however, posterior septal rupture can be repaired successfully through an alternative transatrial approach, thus avoiding the complications that can occur due to ventriculotomy.	ventricular septal rupture (vsr) is a disastrous mechanical complication of myocardial infarction. although several surgical interventions have been developed, mortality due to surgical management remains high, especially in the case of posterior vsr. we report a successful case of repair of posterior vsr using an alternative transatrial approach to avoid the complications related to ventricular incision.
a 24-year - old male was admitted to our hospital with a 1 week history of progressive headache and fever. his previous medical history was clear. on the neurologic examination, his mental status was normal without focal neurological deficits. lumbar cerebrospinal fluid (csf) analysis showed pleocytosis, increased total protein level (167 mg / dl) and decreased glucose (32 mg / dl). tubercle bacillus was detected in the csf and the adenosine deaminase (ada) level was elevated (12 brain mri revealed small, multiple high signal intensity lesions on the diffusion weighted image. his chest x - ray showed diffuse ill - defined patches and nodules with increased density in both apical lungs. tuberculosis meningitis and pulmonary tuberculosis were suspected and antituberculous treatment with rifampicin, isoniazid, ethambutol and pyrazinamide was started. nine days after the antituberculous treatment, his general condition and headache had improved significantly. however, four weeks after antituberculous treatment, he developed acute sensory disturbance below t10 dermatome as well as urination disturbances. spinal mri showed a long segmentally located soft tissue mass mimicking a meningioma in the intradural, extramedullary space of the posterior spinal canal from t2 to t6 with severe cord compression and displacement (fig. 1). surgical decompression and a biopsy were performed. during the operation, a t3, the dura was thickened and the cord was infiltrated with an adhesive mass. the dura was opened and closed in the normal manner after removing the mass under microscope guidance. grossly, the mass was very soft with a dark green color. the cultures were negative for mycobacterium tuberculosis, and the stains for fungi and acid - fast micro organisms were negative. mri of the thoracic and lumbar areas showed lumbosacral tuberculosis spondylitis with multiple abscesses (fig. anti - tuberculous medication was continued for 18 months. at the one year follow - up spinal involvement in tuberculosis is classified into four categories : potts spine, nonosseous spinal tuberculoma, tuberculous arachnoiditis and tuberculous meningitis. intradural spinal tuberculomas are estimated to be composed of only 2% to 5% of central nervous system tuberculomas.1,3 - 5) compton and dorsch8) reported 11 cases of intratudral extramedullary tuberculoma in 1984. since then, there have been 19 more cases reported in the english literature.1,3,5 - 9) all cases, except for 4 initially presented with tubculous meningitis. most case reviews involved the thoracic spine.1,3,5 - 10) the diagnostic method is generally histopathology, even though afb cultures from the granulomas were positive in only one of the reported cases.7) a myelogram can be helpful but mri is the diagnostic procedure of choice. interestingly, most intradural extramedullary tuberculoma cases were detected after antibuberculous therapy had been initiated, which is known as paradoxical response.2,4,7) tuberculoma can occur at any time but most reported cases showed a paradoxical response during the early course of chemotherapy, which ranged from 3 weeks to 1 year.2,4,7) the mechanism for the paradoxical response is unclear. however, is is believed to be the result of an interaction between the host 's immune response and the direct effects of mycobacterial products.2,3,7,10) this condition has been recognized more frequently. narita.10) reported paradoxical worsening in up to 36% of patients with tuberculosis and aids after antiretroviral therapy. therefore, mental or focal neurological changes during the follow - up must be checked in patients with tubeculous meningitis.2,10) the prognosis for neurological improvement is good with a prompt surgical excison and appropriate antituberculous medication.3,4,6) although intramedullary tuberculoma can be treated with medication alone, an intradural extramedullary tuberculoma is essential for surgery when compression of spinal cord occurs. en plaque meningioma can present with thoracic spinal cord and nerve root compression.1) the case presented herein was initially diagnosed with en plaque meningioma based on the radiological findings. the differential diagnosis between tuberculous patchy meningitis and meningioma in the form of plaque is difficult without obtaining a biopsy specimen. in conclusion, intradural extramedullary tuberculoma can occur in tuberculous meningitis patients as a paradoxical response to antituberculous chemotherapy. although quite rare, intradural extramedullary tuberculomas should be considered in a differential diagnosis of an en plaque meningioma of the spinal cord.	a 24-year - old man with tuberculosis meningitis developed acute paraplegia and sensory disturbances 5 weeks after receiving conventional antituberculous therapy. magnetic resonance imaging revealed an intradural extramedullary long segmental mass mimicking en plaque meningioma at the t2-t6 vertebrae levels. prompt surgical decompression was performed. a histology examination of the mass revealed a tuberculoma. after surgery, the patient showed improved motor power and a normal bladder function. intradural extramedullary tuberculoma of the spinal cord is rare complication of tuberculosis meningitis, which can occur as a response to conventional antituberculous therapy.
such changes may be found in the crown either in the form of anomalous cusps, or in an increased number of roots, which in some instances are associated with an anomalous cusp. the term paramolar tubercle has been applied to any stylar anomalous cusp, supernumerary inclusion or eminence occurring on the buccal surfaces of both upper and lower premolars and molars.1 this was first described in the literature by late prof. l. bolk2 of the anatomical institute of the university of amsterdam in the year 1916. when present in the upper molars and as protostylid when present in the lower molars. parastyle may occur in both deciduous and permanent molars and are usually expressed on the buccal surface of the mesiobuccal cusp (paracone) of the upper molars. in rare instances, it is expressed on the distobuccal cusp (metacone) of the upper molars and the buccal surfaces of the upper premolars. similarly, a double cusp formation is extremely rare.4 with respect to size and shape, paramolar tubercles vary ; the structure can be anything from a mere prominence of the buccal surface, separated from the rest of the tooth by a fossa or a groove, to a well - developed lobulated cusp, separated by a constriction and having the appearance of a fused supernumerary tooth. the lobulated tubercle is often associated with a root that is either rudimentary or fully formed.2,5 it is not always necessary that these tubercles contain pulp tissue. in cases where it is strongly pronounced this may be presumed. root canals in tubercles are often connected with other canals;6 in other cases, they are isolated.7 over the years, many authors have dealt with the problems of supernumerary features of molars. their studies have mainly been restricted to their external morphology only without giving consideration to the internal anatomy of these superstructures. periapical radiographs have been of little significance in assessing the internal structure of the paramolar tubercle as it superimposes on the normal anatomy of the tooth. in these areas, spiral computed tomography (sct) or volume acquisition ct has proved to be a useful diagnostic tool.8 this article reports a rare case of two well - developed lobulated cusps occurring on the buccal surface of maxillary right second molar (tooth # 2) that was examined through the use of sct to ascertain the structure of the tubercles, including the root canal morphology and their relationship with associated tooth roots. also in this article the etiology and the relevance of this structure with respect to different disciplines of dentistry has also been discussed. a 36-year - old male patient reported to the department of conservative dentistry and endodontic with a chief complaint of decayed teeth. the patient s familial and medical history was noncontributory. on clinical examination, in addition to carious teeth, two well - developed lobulated tubercles were found on the buccal surface of tooth # 2 (figure 1a and 1b). the tubercles exhibited asymmetrical prominence ; the mesial tubercles was less pronounced than the distal, while both being more or less expressed on the buccal surface of the mesiobuccal cusp of tooth # 2. the tubercles were cone shaped and were clearly delineated from each other and the associated tooth by a semilunar trench with a groove. the triangular prominence had their base below the gingival margin while their apexes were oriented occlusally but well below the occlusal plane. the buccal aspect of the tubercles was smooth and featureless, descending straight to the cementoenamel junction. gingival recession was observed on the buccal surface due to the projection of the tubercles from the tooth and the alveolus (figure 1b). after obtaining an informed consent from the patient, investigations were carried out to study the internal structure of the tubercles and its relation with the associated tooth. periapical radiograph was of little benefit ; it showed an additional conical tooth like structure superimposed on the mid - surface of the associated tooth. though it was not possible to confirm the presence of pulp chamber or root in the paramolar tubercle, but we could clearly demarcate the enamel overlying the tubercle (figure 2a). a multislice sct (sensation 64, siemens medical solution, forchheim, germany) scan of the maxilla was performed with a tube voltage 120kv and a tube current of 390 ma. the involved tooth was focused, and the morphology was obtained in transverse, axial, and sagittal sections of 0.5-mm thickness at 0.5-mm intervals. axial scans were obtained parallel to the occlusal plane from the level of the maxillary tooth crown to its root apices. these images were processed and evaluated on a separate workstation (siemens medical solution, forchheim, germany). the serial cross - sectional images from the sct scan provided valuable information regarding the canal morphology. sct scan images revealed a single and a distinct pulp chamber in paramolar tubercles (figure 2b). the tubercles had fully formed root that was fused with the mesiobuccal and distobuccal roots of the tooth # 2 along its entire root length. though the roots were fused, we could still demarcate the individual roots by following the constrictions between the convexities of the root outline at different levels. in shape, the canal was almost - round to oval. to accommodate the tubercle and the additional root, the buccal alveolar bone level was seen more apical at the tubercle (figure 3a3c). to understand the etiology of the paramolar tubercles, one need to look into the events that follows during the formation of the dental cusps. developmentally, dental cusps begin their formation during the early bell stage, well before calcification of the tooth has begun. the cells of the inner enamel epithelium proliferate and produce activators and inhibitors while they are being deposited in sequential layers from the cusp apex toward the neck of the crown starting from an enamel knot. enamel knots are sites of nondividing cells that occur in the stellate reticulum as projections from the inner enamel epithelium. the activator produces a primary enamel knot until the concentration reaches a threshold that induces an inhibitor that neutralizes the activator.9 enamel knots have been recognized for over a century, though their function was unknown. recent work by molecular biologists has shown that primary enamel knots produce substances that promote mitotic growth in the adjacent inner enamel epithelium.10,11 since the knots themselves are nondividing, this creates irregularities in the inner enamel epithelium and secondary enamel knots appear.10,12,13 research demonstrates that the primary enamel knot, which is the earliest to form, configures the occlusal table of premolars and molars, while later - forming secondary enamel knots individually constitute the cusps during amelogenesis.14 separate enamel knots seem to coincide with separate centers of enamel formation since amelogenesis invariably progresses gingivally.15 paramolar tubercle seems to arise during the morphogenesis process starting from an accessory enamel knot developed at the surface where the feature s apex forms. furthermore, findings support the hypothesis that most characteristics of tooth shape and pattern can be altered by modulating the signal pathways, organized into complex networks, mediating epithelial - mesenchymal interactions in developing teeth.16 the occurrence of paramolar tubercle is relatively uncommon. the occurrence of this structure is very low in upper first molars (0% to 0.1%) as compared with upper second molars (0.4% to 2.8%) or upper third molars (0% to 4.7%) in all the given populations.4,1719 paramolar tubercles have long been recognized as nonmetric dental traits which are the structural characteristics expressed within certain biological and geographical affiliations. though there is very little information about racial differences in the frequencies of paramolar tubercles, primarily because of their low occurrence, none the less they should not be classified as anomalous structure since they are normal morphological features of the dentition. for example, paramolars are reported to be infrequent among africans, europeans, and their descendants in america, while in a group of native americans from the southwest (pima) paramolars are much more common.1 similarly, no cusp formation was observed among whites, negroes, filipinos, and hawaiians. while as southwestern indians showed a higher occurrence in both deciduous and permanent molars compared with other populations.4 due to its low prevalence there is limited information available about the anatomical and morphological characteristics of these tubercles or its relation with the pulp chamber and root canals of the tooth with which it is associated. bolk2 reported that paramolar tubercles in maxillary molars tended to unite at the root but that those in mandibular molars tended to possess their own roots. he also stated that a paramolar tubercle was always united with the anterior buccal cusp of the molar and its roots were attached to mesiobuccal roots. in addition, he even reported that the paramolar root was often present without the tubercle in lower molars. kustaloglu4 suggested that if the paramolar cusp was large, it might be associated with a separate root. however, it is can not be said with certainty that all well lobulated tubercles have their own canals while non lobulated tubercles do nt. thompson20 reported a case of root canal therapy of maxillary left second molar with a large extra cusp attached with the distobuccal cusp. in this tooth the extra cusp and the distobuccal cusp of a maxillary second molar had widely separated canal orifices but a shared root canal space. while as, friedman treated a maxillary molar that had a projection fused to the mesiobuccal cusp, with an additional canal in the fused root near the mesiobuccal canal. zidan and el - deeb7 reported a case where in the paramolar structure associated with a lower molar was treated with endodontic and restorative means as the canal of the anomalous tooth root was isolated from the main root canal system. ballal reported a case of endodontic management of unilateral fused mandibular second molar with a paramolar with the aid of sct. the images revealed that the mandibular left second molar had 3 root canals and that there was continuity between the root canals of the paramolar and mesiobuccal root canal of the second molar. this continuity started 2 mm below the cement - enamel junction and was extending throughout the length of the mesiobuccal root. the root length of the paramolar was noted to be 2 mm beyond the mesial root length of the molar. ohishi examined the root anatomy of 3 cases with paramolar tuberoles in maxillary second molar with computed tomography. in all the three cases all had their own pulp chamber and canals were combined with the distobuccal canal at various levels. in shape, the canals were almost - round to depressed - round ; the shape resembled the outline form of the roots. unlike to previous reports, in our case the tubercles had its own pulp chamber with its root fused to the mesiobuccal and distobuccal roots while its root canal remained independent from the main root canals. this discrepancy between our findings and previous reports suggests the need for further research into the root anatomy of paramolar tubercles. the paramolar tubercles are clinically relevant as they influence the treatment modalities and associated problems in many dental disciplines. the existence of a tubercle and an additional root canal presents a special problem in endodontic therapy. when pulp is present in a paramolar tubercle, the relationship between the pulp of the tubercle and that of the tooth must be determined. when the canal of the tubercle is connected with the main canals then both should be treated at the same time.2022 these superstructures are potent sites for plaque retention as maintenance of oral hygiene in these areas is difficult and recurrence of dental caries, gingival inflammation, and localized periodontitis is more often possible. the grooves that separated the tubercles from the teeth may extend onto the root surfaces to various depths resulting in vertical bone loss along the groove. in addition, as observed in our case, a tubercle that projects from the tooth or the alveolus may sometimes coincide with recession of the gingiva, a lowered buccal alveolar bone level, or both, leading to deterioration of the surrounding periodontal health.22 during orthodontic treatment, paramolar tubercles interfere with cementation of the brackets and correct alignment of orthodontic archwires and often necessitates it s removed by ameloplasty. these tubercles even pose problem in the preparation of a tooth for the setting of an artificial crown.23 understanding from the facts discussed above, we can conclude that these tubercles exhibit a diverse canal configuration and each case should be investigated properly prior to commencing any treatment. the knowledge of their internal anatomy is not only important when such teeth require endodontic treatment but also it influences alteration in the treatment modalities within various other disciplines of dental practice.	the objective of this article is to increase our understanding of the root canal system of the anomalous structures like paramolar tubercles. the knowledge of the internal anatomy of the paramolar tubercles is very important as they influence the treatment modalities and associated problems in many dental disciplines. this case report investigates the anatomical and morphological characteristics of a rare case of two well - developed lobulated cusps occurring on the buccal surface of maxillary right second molar with the aid of spiral computed tomography. unlike to previous reports, in our case the tubercles had their own pulp chamber with its root fused to the mesiobuccal and distobuccal roots of maxillary right second molar, while its canal remained independent from the main root canals.
piperine, a nitrogenous pungent substance, is an alkaloid found in important and oldest spices, namely, piper nigrum (black peppers) and piper longum (long peppers).. stated that piperine is naturally occurring organic compound that belongs to family piperaceae. the fruits of piperine possess antidepressant effects, hepatoprotective effects, antioxidant activity, antitumour effects, antibacterial effects, and anticonvulsant effects [2, 3 ]. piperine also has the capability of reducing inflammation, relieving pain, improving digestion, and enhancing the bioavailability. piperine is extensively used in medicinal field for years due to various medicinal properties including painkiller, antioxidant, and bioavailability enhancer. molecular imprinting technology (mit) is used to design molecular recognition materials because it is capable of mimicking natural recognition entities like antibodies and biological receptors [514 ]. the original concept of molecular imprinting is developed by linus pauling in 1940s, but wulff and sarhan stimulated the interest in imprinting materials. according to vlatakis., in the early 1980s, the molecular imprinting polymers (mips) were successfully prepared by using noncovalent mit. molecular imprinting is a universal method to produce polymers with high affinity binding sites for organic, inorganic, biological, and chemical molecules or ions. mips allow the functional and crosslinking monomers to copolymerize in the presence of the target compound or known as template. molecular imprinting polymers can be prepared by various methods such as bulk polymerization, electropolymerization, suspension polymerization, emulsion polymerization, two - step polymerization, and precipitation polymerization. zhou. mentioned that the controlled / living radical polymerization (crp) is used to prepare mip microspheres as it permits more precise control over the molecular weight, composition, and end group functionality of the obtained polymers [2427 ]. mips show excellent thermal and chemical stability and can be used in aggressive media. according to yan and row, mips have many advantages over their biological counterparts including inexpensive, simple preparation, storage stability, repeated operations without loss of activity, high mechanical strength, durability to heat and pressure, and applicability in harsh chemical media. lai. stated that mips have been used in important application such as chemical sensors [30, 31 ], capillary electrophoresis and electrochromatography, catalysis, hplc stationary phases [3439 ], and solid - phase extraction (spe). in this research noncovalent imprinting or self - assembly approach the template and a functional monomer interact by noncovalent interactions in the prepolymerization mixture. according to spivak, noncovalent is simpler molecular imprinting method as compared to covalent and semicovalent because it involves synthetic steps toward the prepolymer complex. in this way interaction between the monomer and template this method has been used to produce imprinted polymers of cinnamic acid. in this research as an application these imprinted polymers are used in extraction of piperine from spiked urine sample. (united states), acrylic acid (aa) was bought from nippon shokubai co. ltd. (japan), ethylene glycol dimethacrylate (egdma) was purchased from sigma - aldrich co. ltd. (united states), acetonitrile (acn) was obtained from kunshan yalong trading co. ltd. (china), 2, 2-azobisisobutyronitrile (aibn) was obtained from sigma - aldrich co. ltd. (united states), methanol (meoh) was obtained from nuasa kimia sejati co. (indonesia), acetic acid (ch3cooh) was purchased from alpha chemika (india), potassium bromide (kbr) was obtained from powder pack chem co. (india), and hexane was obtained from seidler chemical co. (united states). scanning electron microscope (jeol jsm-6390la) was used to study the morphology of polymer particles. shaker (nb-101mt) was used to allow the rebinding of polymer particles with template. shimadzu lc-20a, a reversed - phase high performance liquid chromatography (rp - hplc), was used to evaluate the batch binding of polymer particles.. 0.5 mmol of template (piperine), 2 mmol of monomer (aa), 8 mmol of cross - linker (egdma), 75 ml of porogen (acn), and 0.011 g of initiator (aibn) were added into 150 ml conical flask, respectively. the mixture was sonicated for 10 minutes in order to remove bubbles and allow complete dissolution. then, the conical flask containing mixture was placed in a bucket of ice cubes and the reaction mixture was purged with nitrogen gas for 15 minutes. ice cubes were used in this experiment to allow a suitable environment for noncovalent interactions between piperine and acrylic acid. the polymerization was conducted for 6 hours, initially temperature was maintained at 60c for the first three hours, and later temperature was raised up to 80c and maintained for another three hours in order to complete the polymerization. the produced polymer particles were extracted out by using the centrifugation at 5000 rpm for 10 min. the template was removed by washing the mips successively in the mixture of methanol and acetic acid (9 : 1, v / v) until the template was not detected by rp - hplc at 270 nm. the hplc was conducted by using the c18 column (250 4 mm, 5 m) with the mobile phase consisting of acetonitrile, distilled water, and acetic acid in the ratio of 60 : 39.5 : 0.5, v / v / v, respectively. the flow rate was set at 0.6 ml / min with uv detection at 270 nm and injection volume was set at 20 l. the nonimprinted polymeric particles (nips) were prepared in the same way without the addition of the template molecule. the similar procedure was used for the synthesis of different molecular imprinted polymers of piperine with varying composition of aa and egdma (table 1) by precipitation polymerization, namely, mip 2, mip 3, and mip 4 for mips as well as nip. a series of 150 ml five conical flasks containing 0.5 g of the mip (mip 1, mip 2, mip 3, and mip 4) and nip beads were added with a 75 ml of acetonitrile containing 0.5 mmol of piperine. the conical flasks were shaken on the shaker at 100 rpm and the samples were collected at different time intervals (0, 30, 60, 90, 120, 150, 240, and 360 minutes). the collected samples were centrifuged at 5000 rpm for 10 minutes in order to remove any suspended particles and supernatant was used for further analysis. the binding capacity of mips and nip of piperine was calculated by using the following equation:(1)binding capacity%=cicfci100,where ci is the initial piperine concentration in the solution and cf is the final piperine concentration in the solution. a 150 ml conical flask containing 0.5 g of the mip 3 beads was added and a solution of 75 ml of acetonitrile containing equal concentration (0.5 mmol) of piperine and caffeine. both of the conical flasks were shaken on the shaker at 100 rpm and the samples were collected at different time intervals (0, 30, 60, 90, 120, 150, 240, and 360 minutes). after shaking at the appropriate time intervals, all the samples were centrifuged at 5000 rpm for 10 minutes. the distribution ratios (ml g) of piperine between the mips or nip in the porogen (acetonitrile) were determined by the following equation:(2)distribution ratio : kd = cicfvcim, where ci is the initial piperine concentration in the solution, cf is the final piperine concentration in the solution, v is the volume of porogen (acn) used, and m is the mass of mip / nip used. the selectivity coefficients for piperine relative to binding competitor caffeine for mip 3 and nip can be calculated by(3)selectivity coefficient : kpiperine / caffeinesel = kdpiperinekdcaffeine, where kd is the batch binding assay of mip / nip for piperine and kd is the batch binding assay of mip / nip for caffeine. the relative selectivity coefficient (k) was determined by the following equation:(4)k=kmip 3knip. urine sample was first centrifuged and filtered and then spiked with a piperine to get a concentration of 50 ppm. after this 50 ml of spiked urine sample was added in flask containing 0.5 g of mip 3. synthesis of microsphere imprinted polymers is a very crucial step in order to produce uniform shape and size of particles. previous studies revealed that various preparation methods have been carried out for the preparation of polymer microspheres such as the synthesis of polymer microspheres by dispersion and emulsion polymerization, where the surfactants in aqueous solution and stabilizers in organic solution are crucial to stabilize the polymer phase and prevent the aggregation of particles. precipitation polymerization can form polymer microspheres with constant size and shape that can lead to narrow dispersion, without the need for any added surfactant or stabilizer [4649 ]. in this research we have successfully produced imprinted polymer microspheres by precipitation method in the acetonitrile (porogen). ir analysis is an important chemical characterization method to detect the functional groups present in a compound. the ft - ir spectra of different mips and nip are shown in figure 1. based on figure 1, small peak in the range of 3519.29 cm to 3613.06 cm attributed to the vibration mode of o the bands in the range of 2926.75 cm to 2996.02 cm and 2854.76 cm to 2957.24 cm showed the vibration mode of c h stretching of aliphatic compound as well as asymmetric and symmetric ch2 stretching in mips and nip. strong peaks at 1726.11 cm to 1736.42 cm indicated the presence of c = o of acrylic acid. the vibration mode of c = c stretching of aromatic compound can be found within 1635.37 cm to 1636.84 cm. the ch2 bending at 1450.82 cm to 1452.70 cm indicated the presence of alkane group in mips and nip. n stretching of mips and nips after leaching was detected at 1388.44 cm to 1390.06 cm. peaks of mips and nip at 1253.88 cm to 1259.61 cm showed the vibration mode of o ch2o symmetric stretching. small band at 1035.88 cm to 1050.59 cm in mips and nip explained the presence of symmetric stretching of = c o c. the vibration mode of c h stretching of aliphatic compound can be observed at 2954.09 cm to 2985.19 cm. sem analysis is a very important morphological study for polymer particles that provides the idea about the shape and size., uniform size of imprinted polymers can be formed by using a noncovalent imprinting approach by precipitation polymerization. arabzadeh and abdouss stated that interaction between monomer and template could be another factor that contributed to uniform size distribution with clean surfaces. research conducted by park. mentioned that there are various factors that affect the production of uniform polymer microspheres including volume of solvent, reaction of solvent, and presence of template ion. excess solvent or porogen used in the synthesis of polymer particles will produce highly uniform polymer microspheres with imprinted binding sites. rp - hplc was used to evaluate the binding efficiency of mips and nip of piperine. figure 3 depicts the binding capacity of different mips and nip at different time intervals. mip 3 showed the highest binding capacity (84.94%), followed by mip 2 (75.86%), mip 1 (69.40%), and mip 4 (60.80%). mip 3 contains a higher amount of monomer ratio as compared to mip 1 and mip 2 but mip 4 contains a higher amount of cross - linker. in this study increasing amount of monomer would produce specific interaction sites with the piperine and hence rebinding efficiency was also increased. but the increase in amount of cross - linker has produced a reverse effect as can be seen in mip 4. if we compare the mips with nip it is clear from figure 3 the binding capacity is low. this can be conferred that nip does not contain any binding site complimentary with the piperine. in order to evaluate the properties of mip of piperine as a sensing material, two compounds (piperine and caffeine) were tested using both mip 3 and nip. the distribution ratio of piperine in both mip 3 and nip was higher than the distribution ratio of caffeine in both mip 3 and nip, resulting in higher selectivity coefficient of piperine than that of caffeine in both mip 3 and nip (table 2). the results indicate that the imprinted polymer has got complimentary binding sites or cavities with the piperine as compared to caffeine. the extensive use of piperine in medicine and spices has generated this idea to first use the selected mip 3 in the extraction of piperine from urine. this will provide us a way forward to expand the application of these imprinted polymer particles. from this study it was found that about 81.18% of piperine was successfully extracted from the spiked urine sample. the binding efficiencies of mips and nip of piperine were evaluated by batch binding assay. mip 3 exhibited the highest binding capacity (84.94%) as compared to nip (40%).	molecularly imprinted polymer (mip) microspheres for piperine were synthesized by precipitation polymerization with a noncovalent approach. in this research piperine was used as a template, acrylic acid as a functional monomer, ethylene glycol dimethacrylate as a cross - linker, and 2,2-azobisisobutyronitrile (aibn) as an initiator and acetonitrile as a solvent. the imprinted and nonimprinted polymer particles were characterized by using fourier transform infrared spectroscopy (ft - ir) and scanning electron microscopy (sem). the synthesized polymer particles were further evaluated for their rebinding efficiency by batch binding assay. the highly selected imprinted polymer for piperine was mip 3 with a composition (molar ratio) of 0.5 : 3 : 8, template : monomer : cross - linker, respectively. the mip 3 exhibits highest binding capacity (84.94%) as compared to other imprinted and nonimprinted polymers. the extraction efficiency of highly selected imprinted polymer of piperine from spiked urine was above 80%.
choriocarcinoma is a highly malignant gestational trophoblastic disease associated with high urine and serum -hcg levels. to date, few studies have reported pulmonary embolism (pe) and pulmonary hypertension caused by choriocarcinoma of the pulmonary artery. here, we report a young female patient with choriocarcinoma of the pulmonary artery who was clinically misdiagnosed with pulmonary thromboembolism (pte) and died of acute cardiopulmonary failure very soon after embolectomy. a 25 year - old female patient first experienced an episode of sudden dyspnea and frequent coughing after routine physical activity. she was initially diagnosed at a local hospital with acute pneumonia based on a chest x - ray, which showed several small, scattered radio - opaque shadows on both lungs. six months later, the patient was referred to our hospital because of severe orthopnea. upon arrival, arterial blood gas analysis showed that the values of ph, paco2, and pao2 were 7.502, 26.3 mmhg, and 50 mmhg, respectively, under 2 l / minute oxygen inhalation. an emergent contrast - enhanced ct scan showed occlusion of the left pulmonary artery and right inferior pulmonary artery (fig. the scan further revealed multiple patchy, cord - like, nodular fuzzy shadows in bilateral lungs (fig. meanwhile, a diagnosis of pe was made based on echocardiographs, which revealed severe pulmonary hypertension (pulmonary systolic pressure, 105 mmhg) and mild tricuspid regurgitation. although her d - dimer test value was only 0.27 g / ml, a pulmonary angiograph was still performed via the right femoral vein, which showed complete occlusion of the left pulmonary artery and right inferior pulmonary artery. a : ct scan demonstrates occlusion of the left pulmonary artery and right inferior pulmonary artery. b : ct scan shows multiple patchy, cordlike, nodular fuzzy shadows in bilateral lungs. the patient claimed that she was a non - smoker, did not use oral contraception, and had experienced three previous spontaneous abortions (the most recent occurring one year previously). the patient had spent more than 10 hours playing card games every day during the past four years. an angiographic procedure for direct injection of 400,000 iu urokinase failed to dissolve the emboli. since her pe was believed to have derived from deep venous thrombi (sedentary clinical history), a filter was also placed in the inferior vena cava. the condition of the patient continued to deteriorate with progressive tachypnea, cyanosis, non - productive cough, and hemoptysis ; therefore, a pulmonary embolectomy was carried out using a cardiopulmonary bypass procedure. complete obstruction of the openings of the left pulmonary artery and right inferior pulmonary artery by the yellow - pink soft emboli was observed. the emboli were completely removed to re - establish blood flow from the bronchial artery. at the time of re - warming prior to decannulation, the pulmonary artery systolic pressure ranged from 90 - 110 mmhg and surpassed the aortic systolic pressure. the hemodynamic situation was so unstable that a 5 mm hole was made in the atrial septum to mitigate the condition. the patient was then transferred to the cardio - thoracic intensive care unit. in spite of intensive efforts, the patient ultimately succumbed to cardiopulmonary failure and acute renal failure approximately 14 hours after embolectomy. pathologic evaluation of pulmonary emboli showed macroscopic fragments of yellow - pink tissue measuring 4 cm at the greatest dimension (fig. post - mortem histological evaluation showed that the viable tumor constituted only a thin peripheral rim with a central area of necrosis and focal hemorrhage (fig. the tumor cells consisted of an intimate amalgamation of multinucleate syncytiotrophoblasts, mononucleate cytotrophoblasts, and intermediate trophoblasts. there was considerable cytological atypia in the trophoblasts with hyperchromatic nuclei and abnormal mitotic figures (fig. lack of villi and new blood vessel formation comprised the unique morphological features consistent with choriocarcinoma. immunohistochemical staining demonstrated strong reactivity for hcg in syncytiotrophoblasts (fig. 2c) and strong cytokeratin positivity was observed in all the tumor cells (fig. the diagnosis of choriocarcinoma embolism was confirmed. a : macroscopic examination of the yellow - pink soft embolus obtained during pulmonary embolectomy. b : tumor characterized by atypical trophoblasts with polymorphic and hyperchromatic nuclei as well as abnormal mitotic figures. choriocarcinoma of the pulmonary artery has been described as a rare cause of pe and pulmonary artery hypertension (pah). such cases represent a potentially fatal emergency due to cardiopulmonary failure. however, the diagnosis is difficult to establish because of the tendency of these tumors to present characteristics of more common diseases such as pte. almost all choriocarcinomas of the pulmonary artery present with pe, and pah occurs after nonmolar abortions (mostly spontaneous abortions) and rarely after normal pregnancies. the low incidence of choriocarcinoma of the pulmonary artery and the long latency after a live birth or nonmolar abortion make early diagnosis of this condition very challenging. bagshawe first reported choriocarcinoma of the pulmonary arteries, identified at necropsy in patients who died of pah in 1959. he suggested that choriocarcinoma emboli could be the result of a progressive neoplastic obstruction of pulmonary arteries leading to pah and respiratory distress. in addition to the case reported here, we reviewed 10 cases of pulmonary choriocarcinoma presenting with pe and pah reported between 1991 and 2008 ; the findings of these 11 cases are summarized in table 1. almost all of the patients (10/11) had a history of abortion with latency periods ranging from two months to six years and no primary uterine tumor identified. among these cases, seven (7/10) were spontaneous abortion and, only one patient (1/11) a normal delivery three years previously. the most important risk factor, a history of hydatidiform mole, was absent from the cases included in our review ; almost all cases occur after a spontaneous abortion and rarely after normal full term pregnancy. owing to regular check - ups for potential malignant sequelae after hydatidiform molar pregnancies, few of these patients present with advanced disease, such as pulmonary hypertension. therefore, almost all trophoblastic tumors that occur after a normal pregnancy are choriocarcinomas. e : etoposide ; m : methotrexate ; a : actinomycin d / dactinomycin ; v : vincristine ; cyc : cyclophosphamide ; car : carboplatin ; cis : cisplatin ; aned : alive with no evidence of disease ; dod : dead of disease ; na : not available ; pe : pulmonary embolism ; pte : pulmonary thromboembolism ; mcc : metastatic choriocarcinoma ; el : embolectomy.. almost all results of pelvic ultrasound and gynecologic examination of the uteri and ovaries were negative, even by autopsy. rare cases of pe due to choriocarcinoma in women occurring many years after hysterectomy have been reported. moreover, tanimura and colleagues revealed trophoblasts in the pulmonary arteries in autopsies of nine of ten patients who died after delivery or abortion. therefore, it has been speculated that primary choriocarcinoma of the pulmonary arteries in women may be due to malignant transformation of normal villous trophoblasts which entered the pulmonary artery at the time of abortion or delivery ; however, the others consider that pulmonary choriocarcinoma derives metastatically from a regressed tumor previously present in the uterus. similar to our case, patients commonly present with non - specific symptoms such as chest pain, cough and dyspnea, as well as abnormal findings of chest x - rays (diffuse radiopaque shadows in the bilateral lungs). these manifestations are commonly misdiagnosed as pneumonia due to the absence of molar pregnancy history and gynecologic symptoms. as the quantity and volume of the pulmonary artery embolus increases, symptoms of pulmonary hypertension are present. chest ct imaging may show intraluminal contrast filling defects (emboli) in the pulmonary artery down to the segmental and subsegmental levels, as well as multiple peripheral opacities in both lungs. at this stage, thoracic surgeons frequently misdiagnosed the condition as pulmonary thromboembolism and as a result, the duration from onset to treatment ranged from one to 12 months (average 6 months). typically, serum -hcg levels in patients with choriocarcinoma of the pulmonary artery are high, ranging from 1,000 miu / ml to over 1,000,000 miu / ml, and urine pregnancy tests are always positive. in most developed countries, urine and/or serum -hcg evaluation is a routine screening test in female patients regardless of the disorders that bring them to clinics. however, there are still many hospitals around the world (especially in developing countries) in which urine and/or serum -hcg is still not on the list of routine tests. similarly, the clinician responsible for the case described here noted the long sedentary history of the patient but disregarded the three spontaneous abortions ; therefore, urine and/or serum -hcg tests were consistently omitted. thus, urine and/or serum -hcg test should always be a frontline test ordered when female patients present with the symptoms of pe and pah to rule out the possibility of choriocarcinoma. seckl and coworkers reported three cases of young women with complete remission following chemotherapy of choriocarcinoma embolism. the diagnosis was based mainly on high -hcg levels in non - pregnant women, indicating that a definitive histological diagnosis is not essential before initiation of chemotherapy, particularly since surgery is associated with considerable morbidity. recently, fdg - pet / ct has been utilized to assist diagnosis for increased metabolism of the embolus. multiagent chemotherapy includes etoposide, methotrexate, actinomycin d, cyclophosphamide, cisplatin, vincristine, and dactinomycin, leading to a cure rate in excess of 89%. thus, chemotherapy coupled with biochemical and radiological monitoring constitutes an attractive management approach for pulmonary choriocarcinoma embolism therapy. in conclusion, pulmonary choriocarcinoma embolism is now a curable disease which is commonly associated with spontaneous abortion, a long latency and empty uteri. in order to avoid unnecessary deaths, it is very important for the clinician to recognize the non - specific clinical characteristics in the early stages and to use urine and/or serum -hcg as the frontline test in the fertile female patients who are clinically suspected of pe ; the earlier the diagnosis and initiation of chemotherapy, the better the prognosis.	abstractcases of pulmonary embolism and pulmonary artery hypertension caused by choriocarcinoma represent a rare clinical emergency. we report a case of a 25-year - old woman who presented with pulmonary embolism and hypertension and died soon after complete pulmonary embolectomy. a related literature review revealed that almost all of these patients had previously experienced a spontaneous abortion (average, 6 months) and were not pregnant.
wegener 's granulomatosis is an uncommon multi - organ disease first categorized as a distinct syndrome by friedreich wegener in 1936 [1 - 3 ] the hallmarks of this potentially fatal disorder are necrotizing granulomatous inflammation involving the upper and lower respiratory tract, glomerulonephritis, and vasculitis [3 - 6 ]. the limited form of wegener 's granulomatosis runs an indolent course whereas the disseminated disease has a rapid progressive course leading to life - threatening multi - organ failure [2,7 - 10 ]. of the 2 types of wegener 's granulomatosis, patients with the generalized disease are known to have shorter life expectancy than those presenting with the limited disease. wegener 's granulomatosis has an insidious onset and usually develops over a period of time with the mean period from onset of symptoms to diagnosis ranging from 4.7 to 15 months. without treatment it is invariably fatal and most patients do not survive more than a year after diagnosis. delay in the diagnosis of wegener 's granulomatosis is attributed mostly to the nonspecific presenting signs and symptoms associated with the early phase of the disease. the most characteristic oral lesion is hyperplastic gingivitis, which is typically red to purple with many petechiae (strawberry gingivitis). these lesions may remain localized in the oral cavity for unusually long periods of time before multi - organ involvement occurs. therefore, timely recognition of this often overlooked oral finding can help to establish an early diagnosis of this disease. management with appropriate therapy produces a good response in most cases, with only occasional relapses. a 50 year old male was referred to the oral medicine clinic at the department of oral pathology, oral medicine and peridodontology, faculty of dentistry, university of malaya for management of unusual gingival lesions. accordingly, the patient first attended the primary dental care unit here for a routine dental check - up. during the course of intraoral examination, large lobulated purplish - red swellings were found affecting the labial gingival mucosa extending from the distal of the right maxillary first premolar to the distal of the left maxillary central incisor (figure 1a). the patient was unaware of the onset of these swellings, and there were no associated symptoms of pain or bleeding. he was afebrile at the time of examination and his medical history was otherwise unremarkable. the present case consists of large lobulated, purplish - red swellings involving the labial gingival mucosa and extending from the distal of the right maxillary first premolar to the distal of the left maxillary central incisor. a decision was made to perform an incisional biopsy on the gingival growth under local anaesthesia. the specimen obtained was submitted for routine processing followed by staining with haematoxylin - eosin, periodic acid schiff, grocott methenamine silver and ziehl - neelsen. microscopic examination of the lesional tissues showed covering parakeratinized stratified squamous epithelium with an irregular lobular surface and exhibiting pseudoepitheliomatous hyperplasia (figure 2a - b). in the underlying connective tissues, a granulomatous inflammatory response was observed. a diffuse mixed inflammatory cell infiltrate comprising mostly neutrophils which formed subepithelial abscesses (figure 3) and smaller numbers of eosinophils, plasma cells and lymphocytes was present. scattered multinucleated giant cells, some resembling langhan - type giant cells with horse - shoe arrangement of their nuclei, were occasionally seen (figure 4a - b). special stains were negative for fungi and mycobacterial bacilli. based on these histological findings and in correlation with the clinical presentation of strawberry gingivitis, a diagnosis of wegener 's granulomatosis was made. incisional biopsy specimen of present case shows pseudoepitheliomatous hyperplasia (peh) (arrows) and a mixed inflammatory infiltrate with abundant neutrophils and eosinophils in the underlying connective tissues (a, h&e ; b, pas ; original magnification 25). scattered multinucleated giant cells (mgc) (arrows) are present in the lesional connective tissue area, occurring side by side with mixed inflammatory cell infiltrate.(a, h&e ; b, pas ; original magnification 200). investigations yielded normal indices for haemoglobin, complete blood count, erythrocyte sedimentation rate and urine analysis. chest x - rays were also normal. a decision was taken to treat the patient with prednisolone 60 mg daily and cyclophosphamide 100 mg daily. at one and three weeks post - treatment, a dental evaluation showed that the gingival swellings had completely resolved (figure 5a - b). a - b. clinical review of the present case shows regression of the gingival lesions at one week (a) and complete resolution at three weeks (b). the american college of rheumatology (acr) recommended that the diagnosis of wegener 's granulomatosis can be made if two of the following criteria are fulfilled:1) ulcerative lesions in oral mucosa or nasal bleeding or inflammation, 2) nodules, fixed infiltrates or cavities in chest radiograph, 3) abnormal urinary sediment and 4) granulomatous inflammation on biopsy. in the present case, strawberry gingivitis and demonstration of granulomatous inflammation in the gingival biopsy supported the diagnosis of this case as wegener 's granulomatosis. however, there was no history of epistaxis nor pulmonary infiltrates or abnormal urine findings in our patient. furthermore, his routine blood analysis was also noncontributory. in the absence of other systemic findings, majority of these lesions represent reactive hyperplasias due to plaque - related inflammatory gingival disease. drug - induced gingival hyperplasia as encountered in patients on dilantin (figure 6a), cyclosporine or nifidepine therapy formed the other clinical important group of gingival entities. vascular lesions that may occur in the gingiva included haemangiomas, pyogenic granulomas (figure 6b), peripheral giant cell granulomas and the more ominous entities such as kaposi 's sarcoma. therefore histological verification supported by clinical and other investigations are necessary to differentiate the lesions from strawberry gingivitis. a case of strawberry gingivitis presenting as the first sign of wegener 's granulomatosis is described. the clinical lesson from this case report is that early recognition of the characteristic presentation of the gingival lesions of this potentially fatal disorder led to the timely management of the disease.	wegener 's granulomatosis is a rare multi - system disease characterized by the classic triad of necrotizing granulomas affecting the upper and lower respiratory tracts, disseminated vasculitis and glomerulonephritis. oral lesions as a presenting feature are only encountered in 2% of these cases. hyperplastic gingival lesions or strawberry gingivitis, is a characteristic sign of wegener 's granulomatosis. the latter consists of reddish - purple exophytic gingival swellings with petechial haemorrhages thus resembling strawberries. recognition of this feature is of utmost importance for timely diagnosis and definitive management of this potentially fatal disease. a case of strawberry gingivitis as the first presenting sign of wegener 's granulomatosis affecting a 50-year - old malay male is reported here. the differential diagnosis of red lesions that may present in the gingiva is discussed.
ticks were sampled in the villages of diiso and el - humow and at the livestock market and abattoirs in garissa district, north eastern province of kenya, during april may 2008 (figure). garissa district is in a semi - arid to arid ecologic zone that receives sporadic rainfall from march to may ; vegetation consists primarily of acacia - commiphora bushes. its population is largely composed of nomadic herders who travel between districts in northern kenya in search of water and pasture (8). location of garissa district (a, box) in north eastern province, kenya, and tick collection sites (b). ticks were picked by hand from infested livestock, stored in labeled sterile vials, and transported in liquid nitrogen to the kenya medical research institute laboratory. in the laboratory, ticks were washed in sterile water, rinsed first with 70% ethanol, and then rinsed with minimum essential medium containing antimicrobial agents (100 u / ml penicillin, 100 g / ml streptomycin, and 1 l / ml amphotericin b). they were identified to species by using taxonomic keys (9,10) and pooled in groups of 2 to 10 by species, sex, collection date and site, and host. the tick pools were homogenized by using 90-mesh alundum sand in a prechilled, sterile mortar and pestle with 1.6 ml2 ml ice - cold bovine albumin 1 medium (1 medium 199 with earle salts, 1% bovine albumin, 100 u / ml penicillin, 100 g / ml streptomycin, and 1 l / ml amphotericin b) under high containment. the homogenates were clarified by centrifugation at 1,500 rpm for 15 min at 4c, and supernatants were stored at 80c. viral rna was extracted from tick homogenates by using trizol - ls (invitrogen, carlsbad, ca, usa) reagent, according to the manufacturer s instructions. rna was screened by reverse transcription pcr (11) to amplify a 536-bp fragment of the gene encoding for the nucleocapsid protein in the small (s) segment of the cchfv genome by using the following primers (12) : cchf f2 (5-tggacaccttcacaaactc-3) and r3 (5-gacaaattccctgcacca-3), positions 135153 and 653- 670, respectively, on the reference strain cchfv 10200. electrophoresis of the pcr products was performed by using 1% agarose gels in tris - acetate - edta buffer containing ethidium bromide ; product bands were visualized and documented with the canon uvp photodoc - it gel imaging system (uvp, llc, upland, ca, usa) mounted with a digital camera. the pcr products of a subset of 4 of the cchfv - positive homogenates were purified by using the qiaquick pcr purification kit (qiagen sciences, germantown, md, usa), according to the manufacturer s instructions, and sequenced by using the bigdye terminator version 3.1 cycle sequencing kit (applied biosystems, foster city, ca, usa) and the abi 3730 and automated 3130xl genetic analyzer (applied biosystems). the sequences were analyzed by using the basic local alignment search tool (blast ; http://blast.ncbi.nlm.nih.gov/blast.cgi) and the genbank database to confirm the identity of the virus. data (including tick species, collection site, animal host, and virologic test results) were entered into an excel database (microsoft corp., redmond, wa, usa) and analyzed by using pivot tables. a total of 8,600 ticks, of 3 genera and 8 species, were sampled primarily from camels, cattle, goats, and sheep, principally hyalomma rufipes and hy. ticks of the genus hyalomma were sampled 3 more frequently in diiso than in el - humow (table). rufipes (3 from cattle and 1 from a camel), 18 pools of hy. truncatum (14 from cattle and 4 from camels), and 1 unidentified hyalomma species (table) in which single dna bands corresponding to the predicted 536-bp pcr product were detected. cchfv, congo - crimean hemorrhagic fever virus. the detection of cchfv in pools of hyalomma spp. ticks from diiso village and the garissa district slaughterhouse provides strong evidence of cchfv presence in northeastern kenya and indicates that cchfv circulation in kenya is underestimated. livestock play a role in the amplification of the virus because the animals become viremic for 7 days (2,3), during which time they can infect more ticks. our findings indicate that cchfv circulates in northeastern kenya with substantial involvement of camels and cattle. the detection of cchfv in ticks from camels at the slaughterhouse also suggests the potential of exposure for abattoir workers. the presence of cchfv among hyalommid ticks in northern kenya highlights the risk to the resident population and requires the assessment of human exposure. health care workers must therefore help create awareness among the population and take steps to prepare for and prevent outbreaks.	as part of ongoing arbovirus surveillance, we screened ticks obtained from livestock in northeastern kenya in 2008 to assess the risk for human exposure to tick - borne viruses. of 1,144 pools of 8,600 hyalomma spp. ticks screened for congo - crimean hemorrhagic fever virus by reverse transcription pcr, 23 pools were infected, demonstrating a potential for human exposure.
yeastract (yeast search for transcriptional regulators and consensus tracking ; www.yeastract.com) was originally proposed (1) to make publicly available up - to - date information on documented regulatory associations between tfs and target genes, as well as between tfs and dna binding sites, in saccharomyces cerevisiae. additionally, it provides a set of bioinformatics tools that facilitate the full exploitation of the data. although part of the data was obtained from existing yeast data repository like the s. cerevisiae genome database (sgd) (2), the gene ontology (go) consortium (3) and the regulatory sequences analysis tools (rsat) (4), all the data on gene regulation was gathered based on exhaustive literature analysis. the value of yeastract comes from the integration of complete and up - to - date regulatory information, with a number of analysis methods and computational tools. the usefulness of yeastract for the analysis of gene lists, in particular those coming from gene expression analysis by microarrays, also distinguishes this information system from others. although other databases have also made available information about regulatory mechanisms in yeast and other organisms [e.g. mybs (5) and transfac (6) ] or computational tools for the analysis of promoter regions [rsat (4) ], yeastract is the system that most seamlessly integrates extensive regulation data and computational tools for the analysis of this information. the database presently contains more than 30 990 regulatory associations between genes and tfs, based on more than 1000 bibliographic references. these include five papers describing global chip analysis (711), which document 75% of the gathered regulatory associations and one microarray analysis on the effect of the deletion of 55 tfs (12), documenting 15% of these regulatory associations. the results of hundreds of other articles describing more detailed molecular analysis and revealing many regulatory associations that were not detected by global experiments are also included in this version of the system. the explosion of the scientific knowledge in the field of transcriptional regulation led to a 300% increase on the actual number of regulatory associations in the system, with respect to the first release. the total number of tfs in the database is 170, which corresponds to all genes that are identified as tfs at sgd. a comprehensive description of the content and structure of yeastract has been presented in the first publication of this system (1). at a high level, the internal structure of the database is organized around the concept of gene, protein and binding site (consensus) and these three concepts are related by regulation relations. these relations document the associations between tfs and target genes and can be of two types : documented and potential. in the first release, the system made available a set of queries to facilitate the exploitation of the gathered data when solving a number of biological questions, in particular those that involve the analysis of global gene expression results. in the first 6 months of 2007, researchers from more than 300 different institutions, from 70 different countries, have performed over 90 000 queries using yeastract. the number of queries in this period has already reached the total number of queries performed during 2006. in this new release, the available queries and additional utilities were reorganized to simplify their use, maintaining the user - friendly interface and functionality, which were already present in the original release of the system. yeastract has already demonstrated its usefulness as a tool to support research on transcription regulation processes in yeast (13). nonetheless, this release significantly extends the capabilities of the system by connecting it with a number of data processing tools that will significantly increase its usefulness. yeastract now includes discoverer, a system that enables the user to search for common motifs in the promoter region of genes, using efficient algorithms for structured motif discovery and to automatically compare the results with the transcription factor binding sites (tfbs) described in yeastract. pattern matching algorithms were also included to enable the user to search the promoter region of one or more genes, for one or more dna motifs, specified using a number of different representations. another important new feature is the possibility to identify and display transcription regulatory networks (trns) for documented and potential regulatory associations between tfs and target genes. this feature supports the analysis of regulatory mechanisms, based on permanently up - to - date, manually checked, information. such an analysis will, in the future, support mechanisms for the inference of trns in s. cerevisiae, one of the main strategic objectives of this project. the precise coordinated control of gene expression is accomplished by the interplay of multiple regulatory mechanisms. the transcriptional machinery is recruited to the promoter leading to the transcription of the downstream gene through the binding of transcription regulatory proteins to short nucleotide sequences occurring in gene promoter regions. to support the analysis of the promoter sequences in the yeast genome, a set of software tools is available in discoverer. discoverer provides tools for motif extraction, which consists on the identification of de novo binding site consensus sequences from a given set of non - coding dna sequences (such as the promoter regions of a gene). discoverer contains two distinct structured motif discovery algorithms : musa (14) and riso (15). when the algorithms finish, the user receives, by e - mail, a link to a web page (figure 1) where it is possible to download the complete list of motifs found, ordered by their p - value and showing the proportion of sequences containing each motif (the quorum). the motifs identified are also clustered in families, represented by a position weight matrix (pwm) description. this assembling of individual motifs into families of motifs is very useful in reducing the number of motifs, leading to a more tractable output and to a more intuitive motif representation. a new algorithm for the motif assembling problem was developed (16), since this is a very difficult problem in its own right (17,18). from the output page it is also possible to download the list of motifs and the pwm description for each family. figure 1.sample pages showing the motif finder output presenting a pwm for each motif family found and the match output obtained by querying the database for tfs binding sites that match the selected family. these results were obtained for the sample data available in the algorithm 's input page. sample pages showing the motif finder output presenting a pwm for each motif family found and the match output obtained by querying the database for tfs binding sites that match the selected family. these results were obtained for the sample data available in the algorithm 's input page. each pwm can be selected, to be compared with the tfbs that are described in the yeastract database. waterman local alignment algorithm (19) ] with each of the tfbs pwm, using a specific column distance metric from a set of options available. yeastract now makes available pattern matching methods, supporting the search for one or more nucleotide sequences (e.g. tfbs) within the promoter region of chosen genes, thus leading to the identification of putative target genes for specifictfs. however, the tfbs, used for pattern matching, have to be provided by the user. the query string may be a simple nucleotide sequence, a sequence containing iupac nucleotide code or even a sequence containing regular expression elements. this search returns a list of genes in whose promoters the patterns were found, including the number of occurrences in each promoter. the patterns that matched the promoter sequences and their locations in the promoters are also displayed. the queries that were refined, or that are new, using the extended pattern matching capability, are the following ones : search by dna motiffind tfbssearch motifs on motifs search motifs on motifs recent studies in data collection and analysis (7,20,21) have shown that the information needed to understand regulatory networks must come from the integration of different sources, such as genomic sequence data, genome - wide transcription data, structural information and biological literature. the comprehensive data on regulatory associations available in yeastract makes it possible to identify and visualize trns for documented (i.e. described in the literature) and potential (a known binding site is present in the promoter region) regulatory associations between tf and target genes (figure 2). figure 2.sample pages showing the regulation graph obtained from the group by tf query. arrows represent interactions between each tf and target genes. these transcriptional regulatory networks and, in particular, the documented ones, correspond to static regulatory networks in s. cerevisiae, since the evidence for the regulatory associations has been described for different processes and experimental conditions. the generation of trns through the queries group by tf and generate regulation matrix, enables the analysis of regulatory mechanisms, supported by up - to - date, manually checked, information. such an analysis will, in the future, support the inference of mechanisms underlying gene regulatory networks in s. cerevisiae. yeastract now makes available pattern matching methods, supporting the search for one or more nucleotide sequences (e.g. tfbs) within the promoter region of chosen genes, thus leading to the identification of putative target genes for specifictfs. however, the tfbs, used for pattern matching, have to be provided by the user. the query string may be a simple nucleotide sequence, a sequence containing iupac nucleotide code or even a sequence containing regular expression elements. this search returns a list of genes in whose promoters the patterns were found, including the number of occurrences in each promoter. the patterns that matched the promoter sequences and their locations in the promoters are also displayed. the queries that were refined, or that are new, using the extended pattern matching capability, are the following ones : search by dna motiffind tfbssearch motifs on motifs search motifs on motifs recent studies in data collection and analysis (7,20,21) have shown that the information needed to understand regulatory networks must come from the integration of different sources, such as genomic sequence data, genome - wide transcription data, structural information and biological literature. the comprehensive data on regulatory associations available in yeastract makes it possible to identify and visualize trns for documented (i.e. described in the literature) and potential (a known binding site is present in the promoter region) regulatory associations between tf and target genes (figure 2). figure 2.sample pages showing the regulation graph obtained from the group by tf query. arrows represent interactions between each tf and target genes. these transcriptional regulatory networks and, in particular, the documented ones, correspond to static regulatory networks in s. cerevisiae, since the evidence for the regulatory associations has been described for different processes and experimental conditions. the generation of trns through the queries group by tf and generate regulation matrix, enables the analysis of regulatory mechanisms, supported by up - to - date, manually checked, information. such an analysis will, in the future, support the inference of mechanisms underlying gene regulatory networks in s. cerevisiae.	the yeast search for transcriptional regulators and consensus tracking (yeastract) information system (www.yeastract.com) was developed to support the analysis of transcription regulatory associations in saccharomyces cerevisiae. last updated in september 2007, this database contains over 30 990 regulatory associations between transcription factors (tfs) and target genes and includes 284 specific dna binding sites for 108 characterized tfs. computational tools are also provided to facilitate the exploitation of the gathered data when solving a number of biological questions, in particular the ones that involve the analysis of global gene expression results. in this new release, yeastract includes discoverer, a set of computational tools that can be used to identify complex motifs over - represented in the promoter regions of co - regulated genes. the motifs identified are then clustered in families, represented by a position weight matrix and are automatically compared with the known transcription factor binding sites described in yeastract. additionally, in this new release, it is possible to generate graphic depictions of transcriptional regulatory networks for documented or potential regulatory associations between tfs and target genes. the visual display of these networks of interactions is instrumental in functional studies. tutorials are available on the system to exemplify the use of all the available tools.
massive intravascular and intracardiac thrombosis after separation from cardiopulmonary bypass (cpb) is a very rare, but dreaded complication. preexisting thromboembolic diseases or acquired coagulation disorders (related to antifibrinolytic therapy) we report the occurrence of severe acute massive intracardiac and intravascular thrombosis during the immediate post - cpb period in a patient who underwent emergency mitral valve replacement (mvr). a 35-year - old male presented for emergency mvr for severe mitral stenosis with atrial fibrillation (af) and acute heart failure. preoperative transthoracic echocardiography (tte) showed severely calcific and stenosed mv with an area of 0.9 cm in two - dimensional echocardiography and 0.6 cm using pressure half time, and the peak and mean pressure gradients of 11 and 7 mmhg, respectively. associated findings included mild mitral regurgitation, presence of left atrial (la) spontaneous echo contrast, right ventricular systolic pressure of 30 mmhg + right atrial pressure, and an ejection fraction of 60% with no regional wall motion abnormality at rest. preoperative laboratory investigations were within normal limits, except elevated serum glutamic - pyruvic transaminase (152 u / l) levels. the preoperative evaluation revealed a cachectic appearance (weight 48 kg, height 170 cm), irregular pulse with heart rate 102 beats / min, blood pressure (bp) 96/60 mmhg, raised jugular venous pressure, bilateral crepitations in both the lung fields, muffled s1 and a mid - diastolic murmur (grade iii / iv) at the cardiac apex area. warfarin therapy was discontinued. in the operating room standard monitoring was applied, 0.9% saline infusion started and right radial artery cannulation performed. arterial blood gas (abg) analysis revealed metabolic acidosis with a ph of 7.22 and hco3 of 16 mmol / l, na 117 mmol / l, po2298 mmhg and pco240 mmhg. general anesthesia was induced with fentanyl 500 g and thiopentone 50 mg, and rocuronium 60 mg was administered to facilitate tracheal intubation with a cuffed orotracheal tube of size 9. soon after induction, bp decreased to 60/40 mmhg and adrenaline infusion was started, which was titrated up to 0.13 g / kg / min to maintain systolic bp of 90 mmhg. right internal jugular vein cannulation revealed a central venous pressure (cvp) of 18 mmhg. intraoperative transesophageal echocardiography (tee) confirmed the preoperative tte findings of a severely thickened and calcified mv with a valve area of 0.6 cm and the presence of spontaneous echo contrast in la [figure 1 ]. midesophageal long - axis view showing thickened and calcified mitral valve with smoke in left atrium and turbulent flow across the valve (la : left atrium, lv : left ventricle, ao : aorta) standard cpb techniques with crystalloid prime and mild hypothermia (32c) were used. heparin was used as an anticoagulant in the dose of 300 iu / kg and half the initial dose was repeated at hourly intervals with a target activated clotting time (act) of > 450 s. a large thrombus was found on opening the la and was evacuated. the patient underwent mvr with a 33 mm prosthetic valve (medtronic ats medical, inc. the heart was de - aired, and the aortic cross clamp was released (clamp time 56 min). valve prosthesis function was found to be normal on tee and no apparent thrombus was observed in any heart chamber [figure 2 ]. the patient was separated from cpb after a bypass time of 90 min on inotropic support with epinephrine 0.1 g / kg / min, dopamine 5 g / kg / min and nitroglycerin 1 g / kg / min with bp 85/45 mmhg and cvp 7 cm h2o. modified midesophageal four - chamber view showing normal functioning bileaflet prosthesis valve in situ in closed (a) and open (b) position. note the absence of thrombus in left atrium after separation from cpb, the patient was bleeding from suture lines and there was generalized ooze. heparin reversal was commenced at a bp of 96/52 mmhg and a cvp of 7 mmhg. soon after administration (1 min), the bp fell to 60/38 mmhg while the cvp was 6 cm h20. the surgeons were having difficulty in maintaining hemostasis, and there was continuous oozing on closure of la and the aortic cannulation site. still it was difficult to achieve hemostasis and bp decreased to 40/20 mmhg and could not be increased despite increasing inotropic support. suspecting hemodilution and resultant deficiencies to be the cause of continuous oozing from the surgical field and hypotension ; fresh frozen plasma (ffp), platelet concentrate, and pcv transfusion was considered. protamine was re - started in consultation with the surgeon in slow, small aliquots of 10 mg to control microvascular bleeding. administration of three aliquots of 10 mg protamine over next 10 min did not improve hemostasis, and further protamine administration was restricted in the absence of any positive effects. within 2 - 3 min of the last dose of protamine (40 - 45 min from its first dose), st segment elevation was observed on electrocardiography and pulmonary artery could be felt tense. milrinone infusion was started and the patient was transfused with two more units of fresh pcv and ffp. in view of failure to achieve an increase in bp and persistence of st segment elevation, a decision was taken to insert an intra - aortic balloon pump and perform the tee. tee revealed thrombi in all the chambers of the heart and aortic root [figure 3 ]. visual monitoring of the cpb circuitry did not reveal any evidence of thrombosis or red blood cells agglutination at any time. act at this time was > 999 s and the opening of la and right atrium revealed adherent fresh thrombi. thrombus in coronaries could also be observed. despite thrombi removal and high inotropic support, the st segment elevation persisted, and the mean arterial pressure could not be achieved above 30 - 40 mmhg. the patient could not be revived and died after a futile attempt of prolonged resuscitation due to low cardiac output syndrome. modified aortic valve short - axis view showing thrombus in left atrium, aorta and right atrium cpb induces a systemic inflammatory response characterized by activation of the inflammatory, complement, coagulation and fibrinolytic pathways but it rarely leads to pathological thrombosis. the development of new thrombus during or following cpb may be a result of inherited or acquired coagulation defects, which increase the risk of developing a hypercoagulable state. the reports of thrombosis and circulatory collapse associated with protamine reversal of heparin anticoagulation have been observed in patients undergoing complex cardiac surgeries for heart transplantation or mixed aortic and mv surgery and who had received aprotinin anticoagulation. the common features between the present case and these were the preoperative presence of congestive heart failure (chf), af, preoperative warfarin use, pcv transfusion before clot formation, formation of thrombi despite an on bypass act of > 999 s on standard heparinization and high patient mortality. thrombi formation in majority of these patients was observed after full dose administration of protamine, except two cases reported by ramsey. where it was observed after quarter to half dose of protamine. protamine administration was withheld initially in the present case considering a reaction, however, the persistent ooze and unstable hemodynamics despite transfusion led us to re - administer small aliquots of protamine in consultation with surgeon to control microvascular bleed. also there was no evidence of decreased venous return or thrombosis in the circuit / peripheral catheters unlike previous reports, after initial 20 mg of protamine. the aortic and radial pressures were also found to be similar when hypotension occurred. in the present case, no antifibrinolytic medicines were administered and the patient had no known coagulation disorder. warfarin has been reported to deplete protein c levels, potentially causing a hypercoagulable state. the postmortem evaluation for antithrombin deficiency, protein c and s deficiency, factor v leiden, antiphospholipid and cardiolipin antibodies, heparin induced platelet activation assay could not be performed due to lack of testing facilities. the massive and diffuse thrombosis involving the systemic vessels, the cardiac chambers and coronaries along with circulatory collapse during administration of small doses of protamine in the absence of antifibrinolytic medication and elevated baseline act has not been documented before. although highly probable, systemic organ involvement could not be ascertained as the relatives refused for postmortem. in the present case, the precipitating factors could be preoperative chf, preoperative use of warfarin, perioperative hemodilution and antithrombin iii consumption induced by cpb with the potential for acquired antithrombin deficiency, a postprotamine state with loss of heparin anticoagulant effect, perioperative blood product transfusion or any undiagnosed congenital defect. undiagnosed platelet dysfunction, intraoperative heparin use, protein denaturation by cpb, inflammatory mediator release and hyperfibrinolysis during cardiac surgery has been attributed to intraoperative and postoperative bleeding during cardiac surgery. although, cardiac surgery may be associated with an initial hypocoagulable state, hypercoagulability might be manifested later in the postoperative period. delay in identification of initial thrombus formation leading to massive thrombosis patients with chf require careful perioperative monitoring to identify any potential hazards. despite adequate heparinization and more than desired kaolin act values, thrombosis has been observed. such patients usually present in an emergency and the time for evaluation of antiphospholipid syndrome, factor v leiden, protein c and s deficiency may not be there. low cardiac output may cause acute renal and hepatic dysfunction though the preoperative laboratory values may be normal. this will impair endogenous anticoagulant synthesis on one hand from hepatic dysfunction and impair clearance of activated procoagulants on the other hand from renal dysfunction. although, thromboelastography has been suggested to evaluate the coagulation states in such cases, its limited availability may be a hindering factor. moreover, hypocoagulable state has been observed on thromboelastography even in the event of new thrombus formation in la. the authors believe that careful protamine administration guided by early intraoperative tee and vigilance on the part of anesthetic and surgical team whenever there is an unexplained and persistent hemodynamic instability / excessive bleeding after weaning from cpb, even when preweaning tee is absolutely normal, can prove to be beneficial in identifying unexpected thrombosis and prompt institution of therapeutic measures. the authors support the view of reporting such cases and formulation of an international registry to estimate the continuing incidence of this rare, but catastrophic complication. this would provide sufficient data to identify risk factors for such thrombosis so as to have a high degree of suspicion in at - risk patients and avoid any morbidity and mortality.	massive intracardiac and intravascular thrombosis is a rare complication following cardiopulmonary bypass (cpb). most of the cases of the disseminated thrombosis have been reported in patients undergoing complex cardiac surgeries and those receiving antifibrinolytic agents during cpb. we report the occurrence of disseminated intravascular and intracardiac thrombosis after cpb in a patient undergoing mitral valve replacement in which no antifibrinolytic agent was used. the possible pathophysiology and management of the patient is discussed.
a 42-year - old female visited our emergency room (er) with complaints of acute, diffuse abdominal pain. she had been treated for three days at a local clinic because of fever and right upper quadrant pain. the laboratory findings showed mild leukocytosis (11.210 cells/l) and an elevated c - reactive protein level, and the hepatic parameters were markedly elevated. the serum hemoglobin level was 7.8 g / dl and she was transfused with whole blood. the ultrasound examination performed on an atl machine (hdi 5000, bothell, wa) revealed a huge and heterogeneous lesion at the subcapsular area of the right hepatic lobe and also a perihepatic fluid collection (fig. the differential diagnosis was hepatic subcapsular abscess or a hepatic mass such as a cavernous hemangioma draping over the liver. mdct was performed with a 4-channel scanner (volume zoom ; siemens, erlangen, germany) with using following parameters ; 120 kvp, 165 mas, 3 mm collimation and a 1.5 mm reconstruction interval for the arterial and portal venous phases, and 7 mm collimation and a 5 mm reconstruction interval for the unenhanced and equilibrium phase images. after injection of 120 ml of non - ionic contrast material, the arterial phase images were obtained with a 40 sec delay, and 80 sec and 3 min delays were used for the portal venous and delayed phases, respectively. coronal multiplanar reconstruction (mpr) was done at each phase. on the unenhanced images, about a 9 cm - sized, low density, irregular - shaped lesion was seen at the right hepatic dome. most of the lesion was not enhanced, but multiple, irregular and linear, septa - like densities were demonstrated which were of isoattenuation to the adjacent normal parenchyma on all phases (fig. the unenhanced images also revealed a huge, heterogeneous and mainly hyperdense lesion occupying the entire right subcapsular area, and this was suggestive of acute hematoma (fig. most of the lesion was not enhanced, except for multiple small enhancing foci that were gradually enlarged and isodense on all phases as compared with the liver parenchyma. the margin of the lesion at the junction with the normal parenchyma was shaggy and irregular. another 1 cm - sized lesion was seen at the right hepatic lobe, and the lesion was of low density on the arterial phase ; it showed peripheral isodense enhancement on the portal venous phase and it became totally isodense or slightly hyperdense on the delayed phase (fig. the mpr images helped us view all the above - mentioned findings at a glance (fig. angiography was performed for vascular embolization under the impression of rupture of hepatic tumor such as hepatic adenoma after the ct scan. the angiogram revealed a huge hypovascular area that conformed to the entire right subcapsular portion, which was compatible with hematoma, and an irregular parenchymal defect was seen in the hepatic dome without any feeding vessels, definite contrast accumulations or vascular malformations (fig. so, interventional procedures were not done and the diagnosis was not changed even after angiography. the patient was given supportive care after angiography, but she suffered cardiac arrest that necessitated cardiopulmonary resuscitation. finally, emergency right lobectomy was performed about 24 hours after her er admission. at laparotomy, most of the portion of the right hepatic lobe was replaced by hematoma, and some inflammatory patches were present on the liver surface. a mass - like lesion was noted at segment 8, which had ruptured, and this resulted in a large amount of blood and necrotic material in the peritoneal cavity. on microscopic examination, the lesion was diagnosed as peliosis hepatis which was a blood filled cavity lined by fibrinous material and hepatocytes (fig. she was discharged about one month after surgery and has lived healthy without any symptoms or abnormal findings on follow - up ct examinations two years later. the etiology of peliosis hepatis is unknown ; however, a number of causes have been postulated and its pathogenesis remains uncertain (10). peliosis hepatis has been considered as a rare, incidental autopsy finding that 's associated with chronic wasting diseases such as tuberculosis and malignancies. it has been associated with renal transplantation, hematologic disorders and infections such as pulmonary tuberculosis and human immunodeficiency virus. it has also been reported in patients treated with long - term with anabolic steroids, oral contraceptives, hormones or azathioprine (7). in 25 - 50% of the cases, no associated condition has been identified, the same as in this case (9). they range from asymptomatic to signs of acute infection and even to hepatic failure and intraabdominal hemorrhage. the patients with complicated fatal hemorrhage probably could have been successfully managed by surgical intervention such as partial hepatic resection or hepatic dearterialization, interventional selective hepatic arterial embolization or liver transplantation and adequate supportive care. although there were some cases in which the patients died after treatment, the other patients who had survived the disease became healthy without any symptoms (2, 3). two morphologic patterns of hepatic peliosis were described by yanoff and rawson (10). in the phlebectatic type, the blood - filled spaces are lined with endothelium and they are based on by aneurismal dilatation of the central vein ; in the parenchymal type, the spaces have no endothelial lining and they usually are associated with hemorrhagic parenchymal necrosis. senf as reviewed by zak (1) considered the two morphological patterns as one process initiated by focal necrosis of the liver parenchyma, which transforms into an area of hemorrhage (the parenchymal pattern). this pattern may progress to formation of fibrous walls and an endothelial lining around the hemorrhage (the phlebectatic type), or heal by fibrin deposition, thrombosis and sclerosis of the vascular spaces. when reviewing the reports that have described the radiologic findings of peliosis hepatis with using various modalities, including angiography, us, ct and mri the imaging findings were so variable, depending on the histopathologic findings, including lesion size, the extent of communication with the sinusoids and the presence of thrombosis or hemorrhage within a lesion (8). as for the angiographic appearances of peliosis hepatis, multiple small contrast accumulations or puddles have been described (2 - 4). yet such findings were not shown in the present case because the major portion of the affected area was necrotized and replaced by hemorrhage, which attributed to delayed diagnosis and was the reason why radiologic intervention was not performed. hypoechoic or hyperechoic nodule or diffuse heterogeneous hepatic echotexture have been reported as us findings (5, 6). in our case, only a heterogeneous subcapsular mass - like lesion was noted and interpreted as abscess that correlated to the signs of clinical infection, but in retrospect, we could have found a hyperechoic nodule at segment 8, which was considered to be a lesion that was demonstrated on ct scan. the mri findings for peliosis hepatis largely depend on the age or status of hemorrhage, with hypo-, iso-, or hyperintensities on t1-weighted images and hyperintensity on t2-weighted images. the contrast enhancement may be usually slow and late (7 - 9). on the unenhanced ct, the lesions are usually of low density, but the attenuation will vary with regard to the age of the lesion, as well as the presence of hemorrhage. they have been reported to usually become isodense or slightly hyperdense at the late venous or equilibrium phase with or without a central unenhancing portion, although with the presence of hemorrhage they may show little enhancement (8). (7) reported a unique pattern of the early and high enhancement with centrifugal progression on triphasic ct ; kleinig. (5) reported a small lesion of which density was high on the arterial and portal venous phases, respectively. in our case, a small lesion in the right lobe showed gradual and centripetal enhancement, and the attenuation was nearly the same as that of parenchyma. we suggest that this feature might be a more usual dynamic enhancement pattern on triphasic - enhanced ct, including the arterial, portal and delayed phases, in the lesions without necrotic change or other complications. until now, there has been no report describing the contrast - enhanced dynamic ct features in a case with peliosis hepatis with hemorrhagic necrosis, intrahepatic hemorrhage and rupture. here we demonstrated several features that can provide radiologists with clues suggesting that the cause of extensive subcapsular hemorrhage is a peliosis hepatis, or that a low density, mass - like lesion may be peliosis hepatis with hemorrhagic necrosis. the first clue is multiple small enhancing foci that are located at the boundary of the acute subcapsular hemorrhage and enlarge according to the passage of time. these densities are isodense to the parenchyma on all phase images, with the highest hounsfield number on the portal venous phase. this finding is considered to correspond to multiple small accumulations of contrast material on angiography, and this finding was first described by pliskin (4). in our case, the densities were not visualized on us or angiography and it is now clear that visualization of these small foci is attributed to the excellent spatial resolution of mdct. the second clue is a shaggy border of parenchyma with hemorrhage, which may give an impression that the subcapsular high density is not just simple hemorrhage, but it is associated with peliosis hepatis. the last clue is multiple, irregular and linear, septa - like structures inside a low density mass, which show isoattenuation compared to the adjacent parenchyma on all phases. the structures may suggest uncomplicated peliotic parenchyma in contrast to the surrounding, hemorrhagic necrosis. the differential diagnosis of peliosis hepatis may be different according to the presented imaging findings. in the case of hypervascular, well - enhancing lesions, the differential diagnosis includes benign hepatic tumors such as hepatic hemangioma, adenoma, focal nodular hyperplasia, hemangiosarcoma, hypervascular metastasis or arteriovenous malformation. a hypovascular lesion must be differentiated from abscess, hypovascular metastasis or other necrotic masses. yet the addition of the delayed phase imaging can help rule out abscess or metastasis via the finding of disappearance or a decrease in size of the lesion, as compared to the arterial or portal phase images. in conclusion, familiarity with the imaging characteristics and the possible clinical manifestations of peliosis hepatis with or without complications can help radiologists make the early and correct diagnosis. thin - section, triphasic - enhanced mdct can provide quite useful and conclusive information for the diagnosis of peliosis hepatis.	we report here on an uncommon case of peliosis hepatis with hemorrhagic necrosis that was complicated by massive intrahepatic bleeding and rupture, and treated by emergent right lobectomy. we demonstrate the imaging findings, with emphasis on the triphasic, contrast - enhanced multidetector ct findings, as well as reporting the clinical outcome in a case of peliosis hepatis with fatal hemorrhage.
operators hands and knives that are not sterile come into contact with the carcass and transfer bacteria to its surface, during bleeding, pelting, evisceration (hadley., 1997) and post - mortem inspection (walker., 2000). the most frequently involved step among the above mentioned procedures is skinning, especially of unsheared sheep and lambs, when bacteria present in the hide can spread to the carcass not only by cross - contamination but also by direct contact of the carcass surface with the fleece (biss and hathaway, 1996). reducing the possibility of surface contamination is very important and, for this reason, an appropriate de - pelting method is crucial to assure the hygienic status of the carcasses. a number of methods that facilitate the skinning process while ensuring carcass hygiene have been reported (european commission, 2001b), among which de - pelting using inflation of filtered compressed air is found. air inflation is a method commonly used in italy (severini, 1996) allowed by ec directive 95/23 (european commission, 1995) only for skinning of lamb of live weight under 15 kg, when the hygienic conditions of the procedures are checked and approved by the official veterinary inspector. in regulation (ec) 853/2004 (european commission, 2004) the use of air inflation is not mentioned anymore. few data about differences in the hygienic status of carcasses de - pelted with and without air inflation, mainly for lambs, are present in the literature (trevisani., 1996 ; severini., 2000). the aim of this work is to evaluate the hygienic condition of adult ovine carcasses (of live weight over 15 kg) skinned using two different methods with and without the use of filtered compressed air. a comparison between the data obtained and the process hygiene criteria set by commission regulation 1441/2007 (european commission, 2007) is also drawn. the study was conducted in two separate steps. in the first step the hygienic status of carcasses skinned with two different dressing procedures, the pulling down and y cut methods (table 1 ; figure 1) with and without the use of compressed filtered air inflation [exercise pressure 900 kpa for 10 - 15 sec ; 0.01 m aseptic filter mod. ; ethafilter s.r.l., air was inflated from the forelegs before or after the removal of the front hooves. on ten different days, five sheep carcasses per day for each of the four skinning methods were sampled at a local abattoir specialised in ovine slaughtering. a pool of four different sampling sites (brisket, shoulder, thorax and rump) was considered for each animal. wet and dry swab techniques, as described in standard iso 17604 (iso, 2014), were used over a 100 cm area delimited by a sterile template for each sampling site (total area sampled of 400 cm) and swabs obtained for each carcass were collected in a single vial containing 10 ml of maxim recovery diluent (mrd ; oxoid ltd., serial dilutions in mrd were obtained. on them, total viable count (tvc) using plate count agar (oxoid ltd.) aerobically incubated at 30c for 72 h, and enterobacteriaceae count using violet red bile glucose agar (vrbg ; oxoid ltd.) the results were normalised to colony forming unit (cfu)/cm and converted into log10 values. the means of the log values were then calculated for each sampling day for each skinning method. the presence of salmonella spp. was determined following uni en iso 6579:2008 (uni, 2008). the second step aimed at determining whether the use of air inflation could change surface contamination on one of the sampling sites considered. for this reason, 10 animals were randomly selected on 5 different slaughtering days for each of the four skinning techniques. in each animal the four sampling sites were separately swabbed with the above mentioned method and separately analysed. total viable count, enterobacteriaceae count and salmonella spp. were determined as previously described. mean values and standard deviations were calculated for tvc and enterobacteriaceae count. for the latter, when bacteria were not detected at the level of 1.0 cfu / cm, a value of -0.5 log10 cfu / cm was assigned (byrne., 2007). the effect of skinning with the aid of compressed filtered air was evaluated for pulling down and y cut methods separately using the unpaired t test (statview ; sas inst. inc., cary, nc, usa) and the significance level was set at a value of p<0.05. enterobacteraceae were detected in 60 and 50% of the samples for the pulling down method assisted with and without filtered compressed air, respectively, and for both y cut methods in 70% of the samples (data not shown). the results of the first step of the trial are reported in table 1, while those of the second step are reported in tables 2 and 3 for tvc and enterobacteriaceae, respectively. for both tvc and enterobacteriaceae count, no statistically significant differences were recorded between samples obtained from carcasses skinned with and without air for either of the skinning methods used and any of the sites sampled. the results obtained in the first step of the study showed values in compliance with limits set by regulation (ec) 1441/2007 (european commission, 2007) for carcasses sampled using non - destructive method. in this case the italian legislation reports that the limits set by regulation (ec) 1441/2007 have to be reduced to 1/5 (italian republic, 2007), as suggested by commission decision 471/2001 (european commission, 2001a) which indicates that the swab sampling removes only a part (often 20% or less) of the total flora present on the meat surface (m=2.80 log10 cfu / cm and m=4.30 log10 cfu / cm for tvc ; m=0.80 log10 cfu / cm and m=1.80 log10 cfu / cm for enterobacteriaceae). for both parameters the results fell into the satisfactory or acceptable category for enterobacteriaceae and within the acceptable level range for both of the skinning methods with and without air de - pelting. the tvcs were similar to those reported in the literature on ovine carcasses, even though using different slaughtering and sampling procedures (sumner., 2003 ; byrne., 2007 ; nouichi and hamdi, 2009 ; phillips., 2013), or even lower than data reported by other authors (milios., the same was observed as for the enterobacteriaceae count (lenahan., 2010). the use of air inflation in both of the skinning techniques did not affect the hygienic status of sheep carcasses, as previously reported for lambs (severini. this result was evident both for the daily average hygienic level and for the different sampling areas, thus proving that air inflation procedure does not affect the hygiene of the carcass surface. no improvement in the hygienic condition of the carcasses was detected and, for this reason, other aspects should be taken into consideration in choosing the use of gas de - pelting in ovine, such as the time necessary for pelt removal, the appearance of the carcass surface or the reduction of skin damage (severini., 1994). since all the samples were negative, as other authors also reported (lenahan., 2010) the use of gas de - pelting is allowed in other countries (new zealand), where compressed filtered air can be used. those who choose to use this method for industrial production must have an approved hazard analysis and critical control points (haccp) plan with microbiological monitoring for its validation (ministry of agriculture and forestry of new zealand, 2002). the present study gives further evidence that this could be a reliable method to use in europe too, as almost all the checked lots of production obtained with air inflation satisfied the hygiene parameters set by legislation. nonetheless, the official veterinary inspector could request further investigation on the hygiene level of the procedure when unacceptable levels of carcass surface contamination are detected and the food business operators could implement specific prerequisite programme of the haccp system adopted. the use of air inflation could also be evaluated for other dressing systems, such as the inverted one (bell and lovatt, 1999). the use of air inflation to assist ovine skinning did not increase surface contamination in adult ovine carcasses in either of the manual skinning methods used. however, the use of air inflation, in combination with mechanisation, as well as the cleaning level of the fleece in live animals transported to the slaughterhouse, still need further consideration and studies.	the aim of the study was to evaluate the hygienic status of sheep carcasses skinned with two different procedures, the pulling down and y cut methods, with and without the use of compressed filtered air inflation. five sheep carcasses per day for each of the four skinning methods considered were sampled on ten different slaughtering days using wet and dry swab techniques at a local abattoir specialised in ovine slaughtering. a pool of four different sampling sites (brisket, shoulder, thorax and rump) was considered for each animal. furthermore, ten animals were also randomly selected on different slaughtering days for each of the four skinning techniques and the four sampling sites were separately swabbed and analysed in each animal. the total viable count (tvc) and enterobacte riaceae count were performed and the presence of salmonella spp. was also tested. the daily average mean value of each parameter was in compliance with limits set by regulation (ec) 1441/2007, falling into satisfactory or acceptable category for enterobacteriaceae and within the acceptable level range for tvc for both the methods used with and without air de - pelting. for both tvc and enterobacteriaceae count, no statistically significant differences (p>0.05) were recorded between samples obtained from carcasses skinned with and without air inflation for either of the skinning methods used and any of the sites sampled. no salmonella spp. were detected in any of the tested samples. nonetheless, no improvement in the carcass hygiene was detected either and, for this reason, other aspects should be taken into consideration when considering adopting the gas de - pelting method.
tin oxide (sno2) is considered an important wide - bandgap n - type semiconductor which has received a great deal of attention over the past few years due to its high transparency in the visible part of the spectrum and sensitivity to certain gases which make it technologically important for optoelectronic devices [1 - 6 ] and sensors. in addition sn is readily available and cheaper compared to indium (in) which is used for the growth of indium oxide (in2o3). furthermore, in recent years, the field of semiconducting metal oxides has benefited a great deal from the development of one - dimensional nanostructures such as nanowires (nws) and nanorods (nrs) due to their interesting properties arising from their small size and high surface - to - volume ratio. in view of this, there has been growing interest in the synthesis of sno2 nws, the study of their fundamental electronic and optoelectronic properties, and finally device applications [10 - 13 ]. despite the potential applications of sno2 nws there has been no detailed study of the fundamental, ultrafast carrier relaxation mechanisms of the photogenerated carriers in this nanostructured material. consequently, here we investigate the carrier dynamics in sno2 nws and obtain a detailed understanding of the various relaxation mechanisms and the influence of trap states using transient white light absorption spectroscopy [14 - 16 ] with femtosecond resolution. we find that the band gap of the sno2 nws is 3.77 ev and contains two broad absorption bands, the first of which is located below the band edge and is related to shallow trap states while the second is near the center of the band gap due to deep trap states. detailed transient measurements revealed their energetic position, carrier relaxation times, and the importance of auger recombination. the sno2nws were grown using an atmospheric pressure chemical vapour deposition (apcvd) reactor which consists of four mass flow controllers (mfc s) and a horizontal quartz tube furnace, capable of reaching a maximum temperature of 1100 c. initially, approximately 0.2 g of fine sn powder (aldrich, < 150 m, 99.5%) was weighed and loaded into a quartz boat together with a square piece of si(111) which was coated with 0.5 nm of au. the au layer was deposited via sputtering at a slow rate < 5 / s using an ar plasma under a pressure < 10 mbar. the sample was positioned a few mm downstream from the sn and subsequently the boat was loaded into the reactor and positioned directly above the thermocouple used to measure the heater temperature at the centre of tube. after loading the boat at room temperature (rt), ar (99.999%) was introduced at a flow rate of 500 standard cubic centimetres per minute (sccm) for 5 min in order to purge the tube. following this the temperature was ramped to 800 c in an ar flow of 100 sccm at a rate of 30 c / min. upon reachingtg, the flow of ar was maintained at 100 sccm for a further 90 min after which the tube was allowed to cool down over at least an hour in an inert gas flow of ar, 100 sccm. the sample was removed only when the temperature was lower than 100 c. for the optical measurements, nws were grown directly onto square pieces of quartz that were coated with 0.5 nm of au and had an area of 6 6 mm. the morphology of the sno2nws was examined with a tescan scanning electron microscope (sem) while the crystal structure and the phase purity of the nws were investigated using a shimadzu, xrd-6000, x - ray diffractometer, and cu ka source. a scan of 2 in the range between 20 and 80 was performed for the sno2nws that were grown on si(111) and quartz. in this study, we investigate the ultrafast dynamic behavior of carriers in sno2 nws following femtosecond pulse excitation through the temporal behavior of differential absorption [14 - 16 ]. the experimental study was carried out using an ultrafast amplifier system operating at 5 khz. a self mode - locked ti : sapphire oscillator centered at 796 nm and generating 45 fs pulses was the source of short pulses. approximately 1 mj of amplified energy was used to pump an optical parametric amplifier (opa) providing ultrafast pulses in the uv range of the spectrum. the rest of the energy from the amplifier was used to generate 400 nm from a bbo crystal via second harmonic generation and white light super continuum. the uv femtosecond pulses from the opa were used to excite the nanowires given that the expected band gap of this material is around 3.7 ev. a small part of the fundamental 796 nm pulses were used to generate vis - ir super continuum light by focusing the beam on a 1 mm thick sapphire plate. similarly a super continuum light in the uv region of the spectrum was also generated using 400 nm pulses. the white light probe beam was used in a pump - probe non - collinear geometry, with the pump beam been generated from the opa. to minimize the broadening of the laser pulse, optical elements such as focusing mirrors were utilized in the setup. the reflected and transmission probe beams were separately directed onto their respective detectors after passing through a band pass filter and thus selecting the probe wavelength from the broad band white light. the differential reflected and transmission signals were measured using lock - in amplifiers with reference to the optical chopper frequency of the pump beam. the temporal variation in the photo - induced absorption was extracted using the transient reflection and transmission measurements, thus providing a means of monitoring the carrier dynamics within the probing region. tin oxide nws have been grown so far by a variety of methods including thermal evaporation, chemical vapour deposition, and the vls method using carbothermal reduction of stannous oxide sno at 880 c for 90 min. on the other hand, stannic oxide, sno2 nws have been grown by direct oxidation of sn at 900 c under a flow of 10 sccm o2. similarly, yang. obtained sno2 nws at 900 c under a flow of 50 sccm o2 while wan. obtained sb doped sno2 nws by heating up the mixture at 20 c / min up to 900 c under a flow of 500 sccm ar with a trace of o2. a typical scanning electron microscope (sem) image of the sno2 nws grown on quartz is shown in fig. 1 where it is apparent that a large coverage has been obtained. furthermore the diameter of the sno2 nws was found to be uniform along their length. the growth of the sno2 nws occurs via the formation of au nanoparticles (nps) from the thin layer of au and the vls mechanism. in addition we have found that direct oxidation using a flow of o2 during growth hinders the formation of sno2 nws due to the oxidation of the sn upstream, which melts at 232 c and which in turn reduces the vapour pressure, especially at low temperatures i.e., t 800 c. while we obtained sno2 nws at temperatures as low as 700 c we find that the optimum temperature for a high yield and uniform coverage is 800 c. the optimum growth conditions are therefore close to those described by wan. who use only a trace of oxygen under a larger flow of ar i.e., 500 sccm as opposed to 100 sccm used here. in our case the formation of sno2 nws is due to the oxygen admitted into the apcvd reactor at rt prior to the temperature ramp. the sno2 nws grown at the optimum temperature i.e., tg = 800 c on quartz are characterized by the (1 1 0), (1 0 1), (2 1 1), (2 2 0), (3 1 0), and (3 0 1) peaks in the x - ray diffraction spectrum shown in fig. we should point out that the al peaks appearing in the xrd spectrum of fig. sem images of sno2nws grown at 800 c with an average diameter of 50 nm xrd spectrum of the sno2nws grown at 800 c on 0.5 nm au / quartz following the growth of sno2 nws, we performed steady state transmission measurements on the nws grown on quartz. figure 3 shows the optical absorption of the sno2 nws covering a spectral range from the uv near to the ir. given that sno2 is a direct gap semiconductor, a plot of the square of the absorption versus the incident photon energy provides a measure of the bandgap which was determined to be approximately 3.77 ev (see inset of fig. we should point out that there appears to be a broad absorption band around 4.2 ev which we believe to be due to lower lying valance bands. in addition to determining the energy bandgap, the absorption spectrum depicts several features within the energy gap of these nws. there appears to be a broad absorption band below the band edge covering a range from 3.7 ev to 1.8 ev which may be divided into two regions. the first broad absorption band which starts just below the conduction - band edge of the nws is associated with impurity traps and these are therefore commonly referred to as shallow trap states (s.t.s. band generated by defects or / and surface imperfections. both of these absorption bands seem to play a crucial role in the relaxation of photoexcited carriers on a femtosecond timescale. furthermore, there appears to be a weak absorption band centred around 2.2 ev (see fig. 3) which corresponds to the well known surface plasmon resonance (sp.r.) of au nanoparticles that are required as catalysts for the formation of the sno2 nws on quartz. the upper corner inset shows a plot of the square of the absorption versus incident photon energy, providing us with an estimate of the bandgap energy 3.77 ev. there are two broad absorption bands below the bandgap referred to as d.t.sdeep trap states and s.t.s.shallow trap states figure 4shows typical time resolved differential absorption measurements for the sno2nws excited at fluence of approximately 0.5 mj / cmwith uv ultrafast pulses at 4.00 ev (310 nm) and probed at different photon probing energies ranging from uv to near ir. thex - axis on this graph corresponds to the optical delay between the pump and the probe pulse whereas they - axis indicates the induced absorption. for some of the probing wavelengths there is a sharp drop in the absorption reaching a minimum value and then followed by a slower recovery toward equilibrium that takes hundreds of picoseconds, whereas in other cases there is a positive change in the absorption with again a recovery towards equilibrium. time - resolved differential absorption of sno2nanowires excited with 4.00 ev photons (310 nm) at fluence of 500 j / cmand probe at different photon energies ranging from uv to near ir these observed changes in absorption are associated with excitation of the sno2nws by photons whose energy is larger than the bandgap energy which results in the generation of non - equilibrium carriers. these non - equilibrium carriers will distribute themselves along energy states that are normally unoccupied under equilibrium conditions. the occupation of states (referred to as state filling) following an ultrafast laser pulse will appear as a reduction in the absorption at the probing energy states. clearly the observed recovery of this negative absorption change will be a direct measure of the time required by the photogenerated carriers to move out of the occupied states. furthermore, a positive change in the induced absorption is also observed in the transient absorption measurements. this phenomenon is mainly due to secondary excitation of the photo - generated carriers by the probing photons from their initial states to higher energy states. this free - carrier absorption depends on the number of carriers present at the initial states and the coupling coefficient between the two energy bands. the temporal profile of this positive induced absorption is again a direct measure of the presence of the photo - generated carrier at the probing energy states. we will begin the analysis of the data from the degenerate induced absorption measurements where the excitation and probing photon energies were 4.00 ev. it is important to point out that the observed sharp drop reaching a minimum (state filling) is pulse width limited, which is expected since we are probing the same energy states that we are exciting. to obtain a better understanding of the dynamics for the degenerate pump - probe data, we have performed intensity measurements as seen in fig. time - resolved normalized differential absorption intensity measurements of sno2nws excited with 4.0 ev and probe at 4.0 ev. the inset shows the fits (solid lines) to the actual differential absorption data (points) using a simple model which includes multi - exponential decays and auger recombination the normalized induced absorption measurements seen in fig. 5clearly indicate that with increasing fluence there is a faster recovery on the long time scale. this suggests that auger recombination is a contributing factor at the fluence used in this study. with decreasing fluence, auger recombination becomes less important, and for the nws used in this study at fluence less than 50 j / cm, this contribution may be considered negligible. a simple multi - exponential fit to the experimental result at 50 j / cmshows that a minimum requirement of three exponential function is necessary for a good fit to the data. the time constants obtained from this fit were 2.4 ps (18%), 68 ps (22%), and 2.3 ns (60%). a more detailed analysis of the experimental data was performed using a simple differential equation model which incorporated the above three exponential decay mechanisms along with auger recombination. making use of the time constants obtained for the lowest fluence utilized in these experiments, where auger recombination was negligible, it was possible to obtain fits to the differential absorption data at higher fluences. 5) were obtained using an auger coefficient of 7.5 2.5 10 cm / s. a schematic diagram of the various proposed relaxation paths is shown in fig. the first time constant (2.4 ps) listed above, corresponds to mechanism 1, (see fig. 6) whereas the second time constant is associated with mechanism 2 or 3 through carrier saturation in the shallow trap states. given that no direct recombination was observed from the conduction to the valence band which is corroborated by the absence of photoluminescence near 3.75 ev, we believe that the long time constant is associated with the carriers moving through the shallow and deep trap states. saturation of these states from the large number of carriers will result in the decay of paths 4 and/or 5 being effectively seen when probing above the band gap. a schematic diagram of the energy band gap diagram of sno2nws with the various relaxation mechanisms following carrier photoexcitation by an ultrafast pulse we should also point out that the fast decay component, which is associated with mechanism 1 of the photo - generated carriers when they are moving into the shallow traps states, appears to become slower with increasing fluence (fig. furthermore, in the above proposed model, the holes generated near the point will also relax to the top of the valence band. however, the expected relaxation within the valence band is much faster than the multi - picosecond relaxation mechanisms shown in fig. this is expected given the small excess kinetic energy received by the holes during excitation. 4) for probing photon energies below the band gap energy (shallow traps) from 3.54 ev (350 nm) to 3.3 ev (380 nm), we notice that although the maximum state filling occurs very close tot = 0 (within the pulse width), there appears to be a small drop and then a small rise after a few picoseconds. this behavior is due to a small free - carrier contribution which reduces the state filling contribution thus artificially making this feature (dip) appear near the tip of the maximum signal. the free - carrier dip increases with increasing probing wavelength and eventually becomes the main contributing factor at the longer probing wavelengths. furthermore, intensity measurements carried out over a range of 50050 j / cmat the probing photon energy of 3.54 ev (350 nm) indicate that auger recombination has a noticeable effect only at the maximum fluence, however, at fluence as low as ~50 j / cm, this effect becomes negligible. a multi - exponential fit to the data shows a minimum requirement of two exponential function for a good fit with time constants of 72 ps (27%) and 2.08 ns (73%). most likely the fast time constant is associated with carriers moving into the deep traps (path 3 in fig. 6) whereas the long decay is associated with recombination of the carriers (path 5 in fig. 6). here we should point out that differential absorption intensity measurements have also been carried at other probing photon energies within the top shallow trap states with similar results. considering the differential absorption measurements in fig. 4for the longer probing wavelengths, we notice an increase in free - carrier contribution. this contribution becomes dominant for probing photon energies below 1.65 ev (750 nm), where no trap states can be reached from the valence band thus excluding state filling. the initial fast recovery component which is of the order of a few picoseconds seen in these measurements is attributed to free - carrier contributions within the trap states. finally we should point out that due to the presence of au nanoparticles (nps) which are required as a catalysts in the formation of the sno2 nws, transient absorption measurements in the probing region 2.42.1 ev depict the well known surface plasmon resonance of au [28 - 30 ]. time resolved measurements outside the above probing spectral region show no evidence of differential absorption signal from au. furthermore, measurements with excitation photons having energy below the band gap of sno2 show signal only at the probing region of the surface plasmon resonance. identical results were obtained when transient absorption measurements were carried out on just the quartz substrate coated with the 0.5 nm film of the au catalyst. it appears that the au nps required for the formation of the nws have no effect on probing the carrier dynamics in sno2 nws despite the strong plasmon resonance. in conclusion, we have investigated the ultrafast dynamic behavior of sno2nanowires using above band gap excitation uv femtosecond pulses. transmission measurements of the nws provided us with an estimate of the band gap at 3.75 ev and reveal broad absorption bands below the band edge. these absorption bands appear to play an important role in the relaxation of the photogenerated carriers in the nws. transient differential absorption measurements reveal the different pathways and time constants associated with the relaxation of the photogenerated carriers. measurements suggest that the photogenerated carriers take a few picoseconds to move into the shallow traps states whereas it takes ~70 ps to move from the shallow to the deep trap states. furthermore, recombination of electrons from these traps states with holes in the valence band takes ~2 ns. auger recombination has a contribution to the carrier dynamics at the highest fluence used in this study (~500 j / cm), however at fluence of 50 j / cmauger recombination appears to be negligible. transient absorption intensity measurements provided us with an estimate of the auger coefficient for the sno2nws to be approximately 7.5 2.5 10 cm / s. the study in this article was partially supported by the research programs ; epyne/0504/06, eryan/0506/04, and eryne/0506/02 funded by the cyprus research promotion foundation in cyprus.	we have studied the optical properties and carrier dynamics in sno2nanowires (nws) with an average radius of 50 nm that were grown via the vapor liquid solid method. transient differential absorption measurements have been employed to investigate the ultrafast relaxation dynamics of photogenerated carriers in the sno2nws. steady state transmission measurements revealed that the band gap of these nws is 3.77 ev and contains two broad absorption bands. the first is located below the band edge (shallow traps) and the second near the center of the band gap (deep traps). both of these absorption bands seem to play a crucial role in the relaxation of the photogenerated carriers. time resolved measurements suggest that the photogenerated carriers take a few picoseconds to move into the shallow trap states whereas they take ~70 ps to move from the shallow to the deep trap states. furthermore the recombination process of electrons in these trap states with holes in the valence band takes ~2 ns. auger recombination appears to be important at the highest fluence used in this study (500 j / cm2) ; however, it has negligible effect for fluences below 50 j / cm2. the auger coefficient for the sno2nws was estimated to be 7.5 2.5 1031 cm6/s.
a 58-year - old caucasian woman was admitted to the gynecology out - patient department for management of increasing abdominal distension and sustained pain in the right bottom quadrant of her abdomen that had been ongoing for 3 months, was independent of activities, and was without any signs of neurovascular deficit. she also started to suffer from increased frequency of minor urinary incontinence. in the last 48 hours the pain was located in the right iliac fossa. her medical history and family history were unremarkable. her surgical history included only an appendectomy as a child, while her obstetric history was remarkable only for five natural childbirths. a bimanual pelvic exam revealed an agile cervix and a large, solid, ovoid, palpable mass in the right parametrium, which was particularly sensitive to touch. her routine hematological investigation and urine analysis as well as examination of several tumor markers, including carcinoembryonic antigen, -fetoprotein, and carbohydrate antigen 19.9, were all within normal limits. a gynecologic ultrasonography was performed, which revealed an increase in the size of the right ovary (56.6 35 mm) and the possible presence of a cystic formation measuring 32.5 mm. computer tomography confirmed the presence of a mass in the anatomic region of the right ovary, corresponding to the ovary s schwannoma (figure 1). the preoperative findings showed we had to deal with a retroperitoneal tumor of unknown pathology in a menopausal woman. in order to ensure the optimum treatment and survival for our patient the abdomen was opened through a lower midline incision. a large retroperitoneal mass, measuring 6.5 5.5 cm, was noted in the right parametrium and was in close proximity to the interal - iliac vein, which was ligated (figure 2). a frozen section was taken during the surgery before complete resection of the mass, which was ambiguous. because of the possibility of malignancy, complete excision of the mass was performed with pelvic blunt dissection. histological examination showed a benign neoplasm originating from the cells of the peripheral nerve sheath ; diagnosis was a schwannoma. the tumor showed a biphasic pattern consisting mainly of cellular areas with nuclear palisading (antoni a) and few hypocellular areas with loose texture (antoni b) (figure a, b and c). degenerative areas within the tumor were seen, including cystic degeneration, hemorrhagic infiltrations and ischemic foci with pycnotic cells due to vessel infarction and fibrinoid necrosis. immunohistochemical studies were performed with a panel of antibodies including s-100 protein, smooth muscle actin (sma), and cd68. postoperatively, the patient showed a good recovery and was discharged on postoperative day 6. schwannomas are nerve sheath tumors that arise in peripheral, cranial, or visceral nerves at any anatomic site of the human body.5,6 these tumors commonly arise from the cranial nerves as acoustic neurinomas but they are extremely rare in the pelvis and the retroperitoneal area (less than 0.5% of reported cases), unless they are combined with von recklinghousen disease (type 1 neurofibromatosis).6 neurilemomas are nonaggressive, slow - growing, solitary neoplasms with an extremely low possibility of malignant transformation or recurrence after excision.7 macroscopically schwannomas are solitary, well - circumscribed, encapsulated tumors.4 histologically, neurilemomas are characterized by the presence of antoni a and antoni b bodies. antoni a bodies represent a disposition in a verocay body, and antoni b tissue is a loose hypocellular myxoid region of microcysts.8,9 immunohistochemistry is positive for s-100, vimentin, and neuron - specific enolase but negative for sma and cd117.7 as a result of their slow growth rate and anatomic location, pelvic schwannomas remain asymptomatic and are either incidentally discovered during a medical investigation for unrelated symptoms or are discovered as soon as they are sufficiently large to cause a mass effect.7,9 this mass effect can lead to pain in the pelvic area and lower back and a sense of heaviness accompanied with urinary and digestive symptoms caused by bladder and bowel compression.9 pelvic neurilemomas are not easily diagnosed. the clinical signs and symptoms are not pathognomonic for this pathologic situation. on the occasion of our case report, we conducted a literature review and documented the cases of pelvic schwannomas of gynecologic interest. one case was a retroperitoneal pelvic schwannoma located in the right paracolpium;10 another case was a schwannoma present at the l5 vertebral body and causing pelvic pain.11 takeuchi reported eleven presacral and lateral pelvic region neurilemomas, eight of which were located in the right or left presacral area and three in the lateral extraperitoneal region.12 one pelvic schwannoma of the anterior surface of the sacrum mimicking an ovarian cyst was also reported.13 an additional case of a retroperitoneal schwannoma localized in the pelvic cavity with complete cystic degeneration, mimicking an ovarian carcinoma, was reported14 as well as one presacral neurilemoma.15 one pelvic retroperitoneal neurilemoma arose in the fallopian tube,16 and one malignant schwannoma arose in the cervix.17 yadav reported one case of neurilemoma located in the right adnexum,18 and sinha reported two cases of pelvic schwannomas located in the broad ligament, with the clinical expression of a broad ligament myoma.9 one reported case of neurilemoma of the pelvis mimicked a myoma of the uterus.19 pelvic tumors and, more specifically, pelvic schwannomas can cause chronic pelvic pain, as was reported in a case of a femoral nerve schwannoma that was clinically expressed with the symptom of chronic pelvic pain.6 there were two cases of obturator nerve tumors arising from the schwann cells, one expressed as a pelvic tumor and the other as an ovarian tumor.20 nine cases of schwannomas mimicking ovarian malignancies have also been reported.21,22 pelvic schwannomas, as mentioned above, are easily misdiagnosed due to the lack of specific symptoms. their therapy is considered to be complete excision of the tumor either laparoscopically or with open abdominal surgery.23 considering that the vast majority of schwannomas are benign tumors, simple tumor enucleation could also be effective.7 in these surgical approaches it is important that the surgeon not cause neural lesions in the patient after surgery.7 the presacral retrorectal space is considered to be a downward extension of the retroperitoneal space because it communicates superiorly with the latter at the level of peritoneal reflection (s23 vertebrae).23,24 surgical extirpation of presacral and pelvic tumors may have several operative difficulties due to limited access and poor visualization in a narrow pelvis.23,24 schwannomas are solitary, well - circumscribed, encapsulated tumors and do not invade local tissues.4 due to these characteristics they are easily dissected from adjacent tissues, which makes laparoscopic resection possible. laparoscopy might also facilitate dissection due to magnification of the anatomic elements in the narrow pelvis.23 the literature review indicated that laparoscopic excision of these neural tumors is the therapy of choice. of note is that many pelvic schwannomas were misdiagnosed and were discovered during an operation that was considered to be the optimal therapy for the initial diagnosis. laparoscopy is a safe and efficient option for approaching benign pelvic tumors and might offer the advantage of better visualization of structures due to the magnification of the laparoscopic view, especially in narrow anatomic spaces.23	neurilemomas are benign usually encapsulated nerve sheath tumors derived from the schwann cells. these tumors commonly arise from the cranial nerves as acoustic neurinomas but they are extremely rare in the pelvis and the retroperitoneal area (less than 0.5% of reported cases), unless they are combined with von recklinghausen disease (type 1 neurofibromatosis). we report the case of a 58-year - old female with pelvic schwannoma, 6.5 5.5 cm in size, in the right parametrium. this is the first case reported in the literature. based on the rarity of this tumor and in order to ensure optimum treatment and survival for our patient, we performed laparotomy with total abdominal hysterectomy and en - block tumor excision. a frozen section was taken during the surgery before complete resection of the mass, which was ambiguous. because of the possibility of malignancy, complete excision of the mass was performed, with pelvic blunt dissection. histological examination showed a benign neoplasm, originating from the cells of peripheral nerve sheaths ; diagnosis was a schwannoma. there were degenerative areas, including cystic degeneration, hemorrhagic infiltrations, ischemic foci with pycnotic cells, and collagen replacement. pelvic schwannomas are rare neoplasms that can be misdiagnosed. laparoscopy is a safe and efficient option for approaching benign pelvic tumors and might offer the advantage of better visualization of structures due to the magnification in laparoscopic view, especially in narrow anatomic spaces.
insomnia is a highly common disorder, which is experienced by almost everybody, at least at advanced age, and becomes chronic in about 10% of the population. because of the transient nature of its milder forms, its importance is frequently underrated. on the other hand, the treatment of severe sleep disturbances, such as primary chronic insomnia, is challenging and frequently complicated by comorbid symptoms.13 the etiology of insomnia is obviously divergent. it is sometimes related to psychiatric or neurologic diseases that may develop already in younger or middle - aged subjects. moreover, it may be acquired as a consequence of neurodegenerative disorders including alzheimer s disease,4 especially when the circadian pacemaker, the suprachiasmatic nucleus (scn), or its downstream connections are affected.57 circadian rhythm sleep disorders (crsds) may be present or develop independently of neurodegeneration. in particular, familial advanced sleep phase syndrome (fasps) and delayed sleep phase syndrome (dsps) are characterized by exceptionally short or long spontaneous circadian period lengths. other circadian disorders are related to weak coupling with external time cues, eg, in some blind subjects. typically, crsds cause transient or periodically occurring forms of insomnia.810 for the circadian system, a possible mode of intervention is that of favoring synchronization with the environment. apart from bright light in the morning, ie, enhancement of zeitgeber strength to reinforce coupling with light onset, melatonin may be administered in the evening to make use of the re - synchronizing, chronobiotic as well as sleep onset - promoting properties of this molecular mediator of the darkness signal. in fact, melatonin was shown to be effective in the treatment of various forms of crsds.1114 while the use of the chronobiotic melatonin in crsd is plausible for mechanistic reasons, its application in other types of insomnia does not warrant immediate success, but has been worthy of exploration. in neurobiological terms, the actions of melatonin on sleep are largely of a chronobiological nature. high densities of the membrane - bound, g protein - coupled melatonin receptors mt1 and mt2 are found in the scn, where the pineal hormone acts in a dual way, by resetting the clock mainly via mt2 and by suppressing neuronal firing mainly via mt1.1519 leaving aside some complexities of the signaling mechanisms,19 the mt1-mediated effects of melatonin on the scn favor sleep initiation especially, but perhaps not exclusively via the hypothalamic sleep switch. this structure exhibits on - off responses2022 and suppresses, under the influence of melatonin, the wake - related neuronal downstream pathways (off) and promotes the sleep - related ones (on).23,24 however, sleep is a complex phenomenon that involves numerous brain regions. melatonin receptors have been detected in various parts of the brain, but receptor densities are considerably lower than in the scn.2528 the thalamus has been assumed to be also involved in soporific actions of melatonin.29,30 melatonin receptors are expressed in this region, and spindle formation is promoted by the indoleamine.2931 spindles are characteristics of non - rem (rapid eye movement) sleep, and are involved in the transition from stage 2 sleep to deeper sleep stages. however, a major problem for judging the relative importance compared to the primary scn - mediated effects results from the complexity of the neuronal connections. apart from the thalamocortical interplay, which is necessary for spindle formation, the thalamus also influences the scn. inputs to the scn are known from various other brain areas, too, especially from the intergeniculate leaflet,30 which is connected to many parts of the brain and also receives a photic input.32 at the present state of our knowledge, the problem remains as to what extent the thalamus and other brain areas may assist the scn by transmitting melatonin - dependent responses, and whether scn - independent actions of melatonin are sufficient for sleep promotion. in individuals with severe scn dysfunction and melatonin deficiency, exogenous melatonin was found to be insufficient for substantially mitigating sleep difficulties.33 however, scn destruction, which causes sleep fragmentation and losses of circadian rhythmicity, still allows spindle formation.30 another source of complexity results from the necessary integration of primarily chonobiotic and homeostatic components of sleep regulation. the homeostatic mechanism also comprises a circadian component,24,34,35 and the existence of a separate homeostatic oscillator has been proposed.36 the extent of melatonin s influences on homeostatic sleep may deserve further attention. at least, melatonin has been reported to be useful under conditions of an insufficient homeostatic drive to sleep.37 despite the highly complex interplay of brain areas during sleep, and the existence of presumably multiple inputs from melatonin, primary and secondary actions have to be distinguished. the phase - resetting effects are relatively well understood and a participation of the scn in sleep initiation can not be denied. melatoninergic actions in other brain areas and their contribution to sleep require further elucidation. with regard to the high receptor density and the knowledge of scn - mediated actions, the influence of melatonin on the circadian pacemaker will be the focus of our considerations. melatonin differs in its mode of action from other hypnotics such as benzodiazepines and z - drugs (zolpidem, zaleplon, zopiclone, eszopiclone), which lead to a more generalized central nervous depression via gabaa receptors. melatonin is capable of indirectly influencing gabaergic mechanisms involved in sleep - related routes downstream of the scn.2022 indirect gabaergic effects in other brain areas may, possibly, play an additional role. only at strongly elevated pharmacological concentrations can melatonin exert more generalized sedative or even narcotic effects, which are, however, mediated by other mechanisms, such as antiexcitatory suppression of calcium signaling and inhibition of neuronal no synthase.38 moreover, melatonin contrasts with benzodiazepines and z - drugs with regard to sleep architecture, ie, the relative duration of sleep stages (stages 14), which differ in sleep depth and undergo an ultradian rem / nonrem cycle of about 90 minutes duration. while sleep architecture can be considerably changed by gabaergic drugs, the ultradian cycle is usually poorly influenced by melatonin, perhaps because this periodicity is generated by another, the pontine sleep switch,39,40 which does not seem to be a major target of melatonin. however, melatonin was reported to increase rem sleep duration in a subgroup of patients with reduced rem sleep.41 in this context, the scn is, again, not independent of inputs from other brain areas, since certain scn neurons were found to fire more rapidly during rem than nonrem phases,24 notwithstanding the primarily suppressive mt1 signaling. therefore, these changes within the rem / nonrem cycle do not reflect direct melatoninergic actions, although they are relevant to sleep and may be indirectly influenced by the hormone. in crsds, a melatonin surge of relatively short duration can be sufficient for resetting the circadian clock, at least when applied in a suitable phase of the phase - response curve. however, in primary chronic insomnia, the major obstacle for the use of melatonin as a clinically efficient hypnotic drug was assumed to result from its extremely short half - life in the circulation, which is mostly in the range of 20 to 30 minutes, sometimes even less, but maximally about 45 min.2022,42 although a short - acting compound may promote sleep initiation, it can improve sleep maintenance only marginally. theoretically, this problem has two solutions. one is a prolonged - release formulation of melatonin, the other the development of long - acting melatoninergic agonists. both possibilities have been studied and given rise to the production of approved or investigative drugs. their relative advantages will be discussed and, where appropriate, also compared to the nonmelatoninergic, primarily gabaergic, hypnotics that are currently in use. at therapeutic doses, the hypnotic actions of melatonin and synthetic melatoninergic drugs are mediated by the membrane receptors mt1 and mt2, as outlined above. in addition to the first - discovered agonist - dependent decreases in camp, a more complex system of signaling routes has been identified that contributes to the cellular effects.19 these include phospholipase c activation, in the case of mt2, and control of inward rectifier k (kir) channels, with secondary effects on voltage - gated ca channels, by mt1. 19 these last actions may be particularly relevant to the suppression of neuronal firing and, thereby, contribute to sleep induction via the hypothalamic sleep switch. while phase shifting and neuronal suppression in the scn represent a basis of hypnotic actions of all melatoninergic drugs, sleep research literature frequently ignores the fact that the membrane - bound melatonin receptors are not restricted to the scn. even though receptor density may be lower in other target tissues or cells, any melatoninergic agonist has to be expected to exert additional effects via these receptors, eg, in the immune system, the gastrointestinal tract, the vasculature, other central nervous structures and various hormonal subsystems.18,19,4345 therefore, by contrast with other hypnotics, any of the melatoninergic drugs is, for fundamental reasons, not only a soporific agent, but also a regulator of other physiological functions. these additional effects, which are frequently disregarded, may not always be beneficial, especially in patients suffering from autoimmune diseases or parkinson s disease (see following sections). while signaling and distribution of mt1 and mt2 receptors discriminate melatoninergic agonists from gabaergic hypnotics, melatonin also differs from its synthetic analogs in the spectrum of binding sites. several other melatonin - binding sites beyond the g protein - coupled mt1 and mt2 receptors have been identified,19,44,45 which either display negligible affinity to the synthetic analogs, or have not yet been tested. these additional binding sites include quinone reductase 2 (formerly believed to represent a third membrane receptor), nuclear receptors belonging to the retinoic acid receptor superfamily, in particular, ror1, ror2, rzr and rzr, calcium - binding proteins such as calmodulin (presumably requiring pharmacological levels because of low affinity), calreticulin, nuclear calreticulin analogs, and two mitochondrial binding sites, one of which is located at the amphipathic ramp of complex 1 and displays high affinity to the indoleamine.19,21,4345 the majority of synthetic agonists has not been tested for these binding sites, with the exception of ramelteon, which has a low affinity to quinone reductase 2,46 and does not seem to act via calmodulin.21 in addition to its direct actions, melatonin is metabolized to various bioactive compounds, including indolic (eg, 5-methoxytryptamine, n - acetylserotonin) and kynuric [n - acetyl - n - formyl-5-methoxykynuramine (afmk) and n - acetyl-5-methoxykynuramine (amk) ] substances and their derivatives (figure 1).4345 for reasons of chemical dissimilarity, no homologs of these melatonin metabolites can be formed from nonindolic drugs. among the hypnotics tested, only the investigative drug -methyl-6-chloromelatonin might lead to some homologous derivatives (figure 1).47 in conclusion, the full spectrum of actions known from melatonin, which also comprises various beneficial effects,38,44 can not be expected to be found with nonindolic hypnotics. on the other hand, those drugs showing selectivity towards mt1 and mt2 receptors also exert effects beyond sleep promotion. the various melatoninergic agonists tested for soporific effects exhibit substantial differences in receptor affinity, half - life, metabolism, and contribution of metabolites to sleep promotion. melatonin itself has different affinities to human mt1 and mt2 receptors (ki = 80.7 and 383 pm, respectively).21,47 its physiological half - life in the circulation is, as mentioned, usually less than half an hour, mainly because of rapid hepatic 6-hydroxylation by cytochrome p450 monooxygenase subforms, in particular, cyp1a2, but also cyp1a1 and cyp1b1.48,49 6-hydroxymelatonin is conjugated and excreted. in other tissues, especially the brain, melatonin can be metabolized differently.43 no soporific effects are known from any of melatonin s natural metabolites, except for 5-methoxytryptophol.50 the sleep - related effects of this compound are presumably without physiological significance. however, the indolic, partially serotoninergic43 metabolites, 5-methoxytryptamine and n - acetylserotonin, should be tested in more detail for possible interferences with sleep or wakefulness. all these considerations are relevant to immediate and prolonged - release melatonin as well, with a main difference in bioavailability. among the various formulations used in different studies, the brand circadin will be particularly considered, because of its approval by the european medicines agency (emea). circadin has been developed by neurim, israel and uk (marketing authorization holder) and is now also provided by lundbeck and by nycomed. in april 2007, it received marketing authorization by emea for the treatment of insomnia in patients aged 55 years and over. it was licensed for the combination of improvement of sleep quality and next - day feeling. the pharmacokinetics of circadin, which requires more detailed future investigation, has been tested in 8 healthy male subjects receiving 1, 2, 4 or 8 mg of the prolonged - release formulation at 10 am, either in conjunction with fasting or with a standard meal.51 the unusual time of administration was obviously chosen to better demonstrate the efficacy of the slow - release formulation and to avoid interference with the endogenous melatonin peak. normally, melatonin is given shortly (30 minutes) before bedtime, according to the time profile of the natural hormone, which exhibits, at least, in healthy nonelderly subjects a pronounced nocturnal peak. however, the pharmacokinetics of melatonin may differ between daytime and nighttime hours, according to data from rats which received continuous infusions of melatonin.52 this possibility should be considered in humans, too. under the conditions tested in humans, 2 mg led to a shift of tmax from about 4 pm to about 11.30 am (without meal) or 12.30 pm (with meal).51 these values should not be overinterpreted, since peaks resulting from the drug should not be compared with the physiological nocturnal maximum. cmax values, as presented, have to be regarded as preliminary, since they showed considerable variation among the relatively few volunteers [without drug : range 30 to 126 pg / ml (median 51 pg / ml) ; drug and fasting : range 180 to 855 pg / ml (median 393 pg / ml) ; drug with meal : 205 to 1020 pg / ml (median : 390 pg / ml) ]. in the 24 h - auc values, a considerable interindividual variation was, again, observed [basal : range 150 to 1017 pg h / mg (median 375 pg h / mg) ; drug and fasting : range 823 to 4478 pg h / mg (median 2257 pg h / mg) ; drug with meal : range 618 to 5252 pg h / mg (median 2010 pg h / mg) ]. these data merely show that this melatonin formulation causes increases in blood levels, what had to be expected, but the improvements in duration of elevated bioavailability, compared to immediate - release melatonin, are not sufficiently evident from published data. elimination time has been inferred to be the same (t1/2 about 40 to 50 minutes) as with conventional melatonin preparations,51 although this may be dose - dependent. in another study on healthy volunteers of both genders, aged 55 to 69 years, the effect of food on auc after 2 mg circadin revealed only minor changes.51 a major difficulty in interpreting the pharmacokinetic data results from the very high interindividual variability. this is not uncommon with melatonin in general and is usually explained by differences in the rapid first - path hepatic metabolism of the hormone. whether this is really so, especially when authors are claiming a more than 80% elimination via 6-hydroxylation and conjugation,51 additional variation may result from the gut, which is both a source and sink of melatonin,5356 allows enterohepatic cycling of this compound,5558 contains by two orders of magnitude more melatonin than the pineal gland,54 and can release melatonin in terms of a postprandial response.54,55,59 gastrointestinal release of melatonin in response to tryptophan was associated with profound sleep promoting effects.53 because of these complexities and difficulties concerning pharmacokinetics, the advantages of prolonged - release melatonin should be judged rather from the effects on sleep. sustained - release formulations different from circadin have been also tested with regard to their pharmakokinetics, including coated sugar spheres60 and solid lipid nanoparticles.61 however, their clinical use is not sufficiently established, so that they will not be considered here in detail. among the synthetic analogs that have been clinically tested, the investigative drug -methyl-6-chloromelatonin (ly 156735) is that one most related to melatonin. this agonist developed by eli lilly also diplays a high affinity towards mt1 und mt2 receptors (ki = 81 pm for mt1 bzw. the preferential binding to mt2 is typical for the 6-chlorinated melatonin derivatives, and also seen with 6-chloromelatonin.47 because of the substitution at c - atom 6, this drug can not be converted by the respective cyp isoforms to 6-hydroxymelatonin and its half - life in the circulation is, therefore, extended.47,62 although -methyl-6-chloromelatonin was effective in phase - shifting the circadian rhythm and showed sleep latency - reducing properties similar to those of melatonin, its effects on sleep maintenance remained marginal, even at doses of 20 or 100 mg,47,62,63 so that this drug will not be further considered in this review. much more detailed information is available on a structurally dissimilar melatoninergic agonist, ramelteon { = rozerem = tak-375 = (s)-n-[2-(1,6,7,8-tetrahydro-2h - indeno[5,4-b]furan-8-yl)ethyl]propionamide }, produced by takeda pharmaceuticals inc. this compound has been approved in 2005 by the fda for treatment of insomnia in the usa. after a negative recommendation by emea,64 takeda has withdrawn its european marketing authorization application in september 2008.65 the affinities of ramelteon to mt1 und mt2 receptors (ki = 14 pm and 112 pm, respectively) are higher than those of melatonin (cf ki = 80,7 and 383 pm).46,66,67 contrary to melatonin and other indolic analogs, such as n - acetylserotonin, it does not bind to quinone reductase 2, at therapeutic doses (ki = 2.65 m ; cf melatonin : ki = 24 pm).46 it displays very moderate binding to the serotonin receptor 5-ht1a (ki = 5.6 m), but virtually none to other 5-ht receptor subtypes. numerous other receptors, eg, for neurotransmitters, neuropeptides including endorphins have been tested and reported to have no substantial affinity.67 however, other melatonin binding sites, as mentioned above, have not been investigated. it is rapidly taken up, reaching tmax between 0.75 and 0.94 h, over a considerable dose range.68 its half - life in the circulation amounts to about 1 to 2 h and is, therefore, considerably longer than that of melatonin.68 after escalating doses of 4, 8, 16, 32, and 64 mg, cmax values of 1.15, 5.73, 6.92, 17.4, and 25.9 ng / ml, and auc values of 1.71, 6.95, 9.88, 22.5, and 36.1 ng h / ml were obtained.68 because of its structural dissimilarity, the metabolism of ramelteon fundamentally deviates from that of melatonin.21,43,47,68 although it is substrate to cytochrome p450 enzymes, including the melatonin - hydroxylating cyp1a2, the products are substantially different. four metabolites formed by cyp1a2, cyp2c and cyp3a are usually referred to as m - i, m - ii, m - iii und m - iv. apart from cleavage of the tetrahydrofuran ring in m - i, hydroxylations and oxidations take place in positions not accessible in melatonin, either at a c - atom corresponding to the nitrogen in melatonin s pyrrole ring (m - iii, m - iv) or at c - atom 2 of the propionyl residue of the aliphatic side chain (m - ii, m - iv) (figure 1).43,47 because of the absence of a pyrrole ring, no kynuric metabolism is possible in the case of ramelteon.43,47 the properties of metabolite m - ii are rather unusual and substantially contribute to the pharmacological activity of the parent compound. this is not particularly surprising, since m - ii differs from ramelteon only by the hydroxyl group in the aliphatic side chain. on the one hand, this change reduces the affinities to mt1 und mt2 receptors by a factor of about 10, but, on the other hand, m - ii itself is much less metabolized, has a half - life 2 to 5 h longer than the parent compound and can attain concentrations by 20- to 100-fold higher than ramelteon.68 therefore, any pharmacokinetic consideration of ramelteon can not be made without considering the long - lasting contribution of m - ii. consequently, judgments on the time course of action should not be restricted to tmax, cmax and auc values of ramelteon alone. another newly introduced synthetic melatoninergic agonist is tasimelteon (= n-{[(1r,2r)-2-(2,3-dihydro-1-benzofuran-4-yl)cyclopropyl]methyl}propanamide = vec-162 ; earlier research codes : bms-214778 and ma-1). this investigative drug is produced and being further developed by vanda pharmaceuticals, under license from bristol - myers squibb co. binding and pharmacokinetic properties in humans have been disclosed only in part, although the company may possess more detailed information. according to unpublished information cited elsewhere,69 tasimelteon is selective for mt1 and mt2 receptors. in a web appendix, the following affinity data were presented : pki = 9.45 0.04 (0.35 nm) for mt1, and pki = 9.8 0.07 (0.17 nm) for mt2, 70 without experimental details. after single oral doses between 10 and 100 mg, mean tmax values varied from 1.9 to 3.0 h, mean cmax from 59.1 to 417.1 ng / ml, and auc from 171.1 to 1916.1 ng h / ml, however, with considerable interindividual deviations.70 more detailed pharmacokinetic data have been presented for rats and monkeys (macaca fascicularis).71 these data indicate rapid uptake (monkeys, at moderate dose, tmax in the range of 1 h) and longer half - life than melatonin (t1/2 about 2 to 3 h). a longer half - life mentioned elsewhere, without precise values,72 may have referred to the human. the metabolism, studied in rat, monkey and human liver, showed degradation by cyp1a1, cyp1a2, cyp2d6, and cyp2c9, and also some conjugation with glucuronic acid.71,73 most of the metabolites have only been partially characterized and are, again, nonhomologous to those of melatonin (figure 1), for fundamental reasons. agomelatine { = valdoxan = n-[2-(7-methoxynaphth-1-yl)ethyl]acetamide = s20098 }, developed by servier, is also an agonist at mt1 and mt2 receptors (ki = 61.5 pm and 268 pm, respectively), but additionally acts as a 5-ht2c receptor antagonist (ic50 = 270 nm), with low affinities to other 5-ht receptor subforms.21,43,47,74 5-ht2c inhibition is largely responsible for the additional antidepressant action of this drug. the metabolism involving cyp1a1, cyp1a2, and cyp2c9 is partially different from that of melatonin (figure 1). is hydroxylated at the second ring carrying the long side chain, another one demethylated, corresponding to the formation of n - acetylserotonin from melatonin. the demethylated compound (s21517) resembles serotonin with regard to the presence of the hydroxyl group and, in fact, displays affinity to 5-ht2c. 21,43 since agomelatine is not exclusively a melatoninergic drug, it should not be regarded simply as a sleep - promoting compound suitable for treating an average insomniac, but may be of specific value in depressed patients. in february 2009, valdoxan was approved by emea for the treatment of major depressive episodes (mde) in adults,75 but not generally as a hypnotic agent. therefore, it will not be discussed here in any detail, despite its undoubtedly existing soporific actions. first of all, one has to distinguish between sleep - promoting effects in patients with crsds and others suffering from primary chronic insomnia. any of the melatoninergic drugs is effective in phase - shifting circadian rhythms and, thus, seems suitable for treating jet lag and crsds, at least from the hypnotic point of view, but not necessarily under aspects of long - term safety.21,22,47,69 since acute phase shifting and facilitation of sleep onset are also achieved by immediate - release melatonin, advantages of prolonged - release melatonin or longer - acting synthetic analogs should rather be sought in the treatment of primary chronic insomnia. -methyl-6-chloromelatonin was only marginally efficient in sleep maintenance,47,63,63 and, in terms of published evidence, tasimelteon has been only tested after artificial light - dark shifts.69 for reasons mentioned, the use of agomelatine should be only considered in conjunction with depression. therefore, it seems primarily important to compare prolonged - release melatonin, such as circadin, and its synthetic analog, ramelteon (rozerem), with a focus on primary chronic insomnia. studies on prolonged / extended - release melatonin have been conducted in the past using different preparations, sometimes referred to as controlled, sustained or slow release. different doses had been used and some formulations contained combinations fast (1 mg) and controlled - release components (4 mg). 76 the different studies are highly diverse, frequently of exploratory nature, or related to various disorders. trials on larger numbers of subjects were only based on subjective measures. as compared to study design and detailed information on other hypnotics, a conceptual diversity is even apparent in the material summarized by emea on circadin.51 this material includes various exploratory and extended studies, including a phase iii trial. the larger clinical studies on circadin,51,77,78 conducted on several hundred elderly patients (55 years and older) with primary insomnia are randomized, placebo - controlled and double - blind, but not generally with crossover design, and mainly based on questionnaires only (leeds sleep evaluation questionnaire = lseq ; sometimes also pittsburgh sleep quality index = psqi, who-5 well being index, and clinical global improvement scale = cgi). taken together with exploratory studies also using polysomnography (psg) or wrist actigraphy,51,79 the data collectively show that prolonged - release melatonin / circadin significantly reduces sleep onset latency (sol), whereas direct evidence for the support of sleep maintenance and total sleep time is poor. changes in awakenings from sleep are sometimes not statistically demonstrable, but may be deduced from patients reports on improvements of sleep quality.51,77,78 more direct support, based on objective measures, for this important aspect of primary insomnia would be welcome. it should be also mentioned that the percentage of nonresponders to melatonin was substantial.51 in this context, the improvements obtained by the prolonged - release formulation should be decisive. reductions in sleep latency are well known for fast - release melatonin, and have to be also expected for prolonged - release pills, especially as the amounts required for promoting sleep initiation are relatively low.80,81 since the development of prolonged - release melatonin was aiming to support sleep maintenance, especially in patients with primary chronic insomnia, the most relevant parameters should be reductions in number or duration of awakenings from sleep and improvements of total sleep time. to convincingly demonstrate efficacy in sleep maintenance, however, there is a good reason for assuming that improvements in sleep quality and efficiency will be also demonstrable according to hard criteria, insofar as the subjective improvements were particularly evident in patients with severe or very severe forms of primary insomnia as well as in a subpopulation of poor melatonin secretors, as identified by low urinary 6-sulfatoxymelatonin levels.51,78 therefore, circadin or other prolonged - release formulations of melatonin may be suitable for replacement therapy, eg, in patients with age - related decreases in nocturnal melatonin secretion. additional information on prolonged-/controlled - release melatonin is available from studies on treatment jet lag, shift work and various disorders, sometimes including comparisons with fast - release melatonin. in jet lag,8082 not unexpectedly, either formulation proved to be effective. a meta - analysis of 10 studies revealed, however, a superiority of fast - release melatonin.81 a study on aircrews on transatlantic flights, based on both subjective measures and wrist actigraphy, reported a relatively good efficacy of 2 mg sustained - release melatonin.82 in addition to reductions in sleep latency, improvements concerning number and duration of awakenings after sleep onset, quality of sleep and facilitation of returning to sleep were demonstrated. studies on simulated shift work83,84 were affected by the problem of melatonin administration in unfavorable circadian phases and are, thus, difficult to compare. the efficacy of sustained - release melatonin was also studied in children and young adults with crsds76,85 and with neurodevelopmental disabilities.76,8694 improvements were reported, but data on sleep initiation were either not provided or, in part, insufficient. the clearest results were obtained in the most recent study using 5 mg controlled - release melatonin tablets.86 with this higher dosage, reductions in sleep latency and rises in night - time sleep duration were demonstrated by both subjective measures and wrist actigraphy. in children with autism, an open - label study, based on the children s sleep habits questionnaire and diary, improvements were obtained with controlled - release melatonin.95 some circumstantial evidence for sleep improvements were reported for depressed patients,96,97 but without changes in the hamilton rating score for depression.97 positive results were also obtained in intensive - care patients with chronic obstructive pulmonary disease or pneumonia.98 in a subpopulation of schizophrenics, improvements of sleep were reported,99 but not after sleep disturbance by the so - called first night effect in a sleep laboratory.100 although some smaller studies indicated sleep improvements by melatonin in patients with alzheimer s disease, as previously summarized,6 neither 2.5 mg4 nor 6 mg101 sustained - release nor 10 mg immediate - release4 melatonin resulted in statistically significant improvements, perhaps an indication for the heterogeneity of these populations. compared to prolonged - release melatonin, the outcome of trials on ramelteon is much more uniform, as becomes evident from recent summaries.2022,102,103 collectively, all the data unanimously show that ramelteon, at doses of 4 or 8 mg, not only reduces sleep onset latency, but also improves total sleep time and sleep efficiency / sleep quality. this has also been demonstrated in several double - blind, placebo - controlled studies on a total of more than a thousand adult or elderly subjects with primary chronic insomnia.104107 all the effects were statistically significant, but the improvements of sleep maintenance remained moderate. in accordance with the higher receptor affinities of ramelteon compared to melatonin, no further improvements were obtained with 16 or 32 mg daily.105,107 moreover, ramelteon did not worsen sleep apnea,108 in accordance with the lack of generalized central nervous suppression, as would occur with gabaergic agonists. according to the available data for recommended doses (usually 8 mg), ramelteon seems to be somewhat more effective than prolonged - release melatonin in the treatment of primary chronic insomnia, as far as sleep maintenance is concerned. several factors should contribute to this finding : (i) higher receptor affinities to both melatonin receptors, especially to mt1 ; (ii) higher bioavailability because of longer half - life ; (iii) a long - lasting contribution of the metabolite m - ii ; and (iv) the higher recommended doses of 4 or 8 mg ramelteon vs 2 mg circadin. nevertheless, emea found the efficacy of ramelteon in improving sleep maintenance insufficient for a marketing authorization.64 in full agreement with numerous findings on immediate - release melatonin, all studies on the prolonged - release formulation unanimously show that the recommended dose does not cause next - day hangover, but rather favors morning alertness although some exceptions have been described in other investigations using different doses. it does not lead to dependence, early or late withdrawal effects after discontinuation.51,7779 the development of tolerance is usually absent with melatonin, although a few exceptions have been reported, especially in some children with neurological disorders.9194 should the development of tolerance turn out to be a consequence of altered metabolism, which remains to be demonstrated, other melatoninergic agonists might be tested. a recent randomized, double - blind, placebo - controlled crossover study on prolonged - release melatonin confirmed the absence of next - day impairments of psychomotor functions, driving skills and memory recall, in contrast to 10 mg zolpidem.109 controlled - release melatonin (2 mg) was successfully used even for facilitating benzodiazepine discontinuation.110 like melatonin, ramelteon did not cause next - day hangover (as revealed by subjective feeling, psychomotor and cognitive tests, and ability to concentrate),105 rebound insomnia or other withdrawal effects, or development of tolerance or addiction.2022,105 under these conditions, both prolonged - release melatonin and ramelteon appear safe in short - term treatment, as may be assumed for other exclusively melatoninergic drugs in general. for subjective criteria of adverse effects, such as reports of nausea, digestive difficulties, headache or other pain, dizziness, and mood, no substantial differences were detected between circadin and placebo, and frequently trends were detected even towards fewer subjective side effects in the melatonin groups.51 in this context, it should be also noted that considerably higher doses of melatonin, 300 mg / day enterally, were administered for up to 2 years to amyotrophic lateral sclerosis patients and found to be safe.111 subjective reports of adverse effects showed that ramelteon 4 or 8 mg was also well tolerated, with similar outcomes as for placebo.20,21,104107 precautions should be taken with both melatonin and ramelteon for other reasons. first, the use of a melatoninergic agonist should be restricted to appropriate circadian phases in the evening, since it may cause drowsiness when taken during daytime and, in this case, may in fact impair psychomotor functions, including driving skills. while the use of hypnotics should anyway be restricted to bedtime, more specific precautions are related to the pleiotropy19,45 of melatonin. it is of utmost importance to keep in mind that melatonin is not just a hormone transmitting the darkness signal, and not only a regulator affecting the scn, but rather influences numerous additional functions.19,4345,49 even for ramelteon, which may exclusively act on mt1 and mt2 receptors (although this selectivity has not been demonstrated for the newly discovered binding sites), various effects beyond scn modulaton and sleep promotion have to be expected. this would include influences on other hormones, and on the immune system, vasculature, and the gastrointestinal system. the possibility of undesired melatoninergic effects on the reproduction system may be a controversial issue. the respective influences of the hormone are without any doubt not comparable to those in seasonal breeders, but, on the other hand, earlier attempts to use melatonin as a contraceptive,112114 suppressive effects on the gnrh pulse generator115 and deviations of melatonin in reproductive disorders116118 have been seen as a caveat in the opinion of some investigators and also of emea.51 especially in reproductive disorders, changes in melatonin may not be causative, but rather consequences of other anatomical or physiological disturbances. in perimenopausal women, effects of melatonin on lh, fsh and thyroid hormones were observed,119 whereas no changes were detected in lh, fsh, testosterone and inhibin- in normal men subjected to long - term treatment with the pineal hormone.120 however, melatonin was also reported to decrease semen quality in two healthy men,121 but this study was conducted with a very small number of volunteers. concerns because of changes in the reproductive system may be taken as a contraindication for treating children, adolescents and pregnant women with melatonin, as did emea in the case of circadin.51 on the other hand, children, adolescents and young adults have been treated for considerable periods of time with the pineal hormone, without reports of undesired effects in the reproductive system.14,76,85,95,122124 the position of emea, which has approved circadin only for subjects of 55 years and older,51 may appear unduly cautious, but emea intends to be cautious. nevertheless, melatonin formulations or other melatoninergic drugs should be an option for children with severe and otherwise intractable neurological disorders. precautions are necessary in subjects with immunological disorders, since melatonin is also a mainly stimulatory immunomodulator.19,45,49 thus, melatoninergic drugs should generally not be prescribed to patients with autoimmune diseases.51 with both melatonin and ramelteon, another caveat concerns drugs influencing cytochrome p450 enzymes, especially inhibitors of cyp1a2,20,47,48,51 such as fluvoxamine, which would lead to substantial rises in circulating melatonin and ramelteon as well. additional specific precautions are listed for circadin, such as lapp lactase deficiency and glucose - galactose malabsorption,51 and for ramelteon concerning alcohol, high - fat diet and renal impairment.20,125 another disorder that may be regarded as a contra - indication against the use of melatoninergic agonists is parkinson s disease (pd). contrary to findings in various animal models, melatonin has not been generally beneficial in pd patients, especially for disease progression, as summarized earlier.6 more recently, pd has been interpreted as an endocrine disorder characterized by melatonin hyperplasia.126 correspondingly, clinical improvements have been obtained by melatonin antagonist treatment.127 melatonin hyperplasia may also deserve attention in other diseases, eg, irritable bowel syndrome type ii, in which an enhanced proliferation of melatonin and serotonin producing cells is observed, in conjunction with losses of other cell types.128 at recommended doses51 and even higher, melatonin is devoid of mutagenicity or carcinogenicity, but instead appears to be protective in this regard.38,44,49,55,129 the absence of genotoxicity and carcinogenicity is also reported for ramelteon.125 however, some reservations seem appropriate with this drug,20,21,47 since the no - effect level for induction of hepatic tumors in male mice was only three times the concentration of the metabolite m - ii measured after the therapeutic dose.125 moreover, micronuclei formations were observed in chinese hamster lung cells after metabolic activation.125 the naphthalenic compound agomelatine may require further toxicological studies.21,47 in this place, it should be emphasized that safety studies also have to consider the properties of the metabolites, which is not generally sufficiently done. for the metabolites of melatonin kynuric products, which may be relevant in tissues, have been reported to be protective rather than deleterious.38,44,129 more extensive studies on properties of metabolites are necessary for any of the synthetic melatoninergic drugs, including -methyl-6-chloromelatonin, tasimelteon and agomelatine, in the last case also for the serotonin analog s 21517,21 and, most importantly, for the ramelteon metabolite m - ii, because of the high concentrations attained and its long half - life in the circulation. in summary, both prolonged - release melatonin and ramelteon are well tolerated and safe in the populations indicated by the respective approvals, and acceptable for short - term treatment. experience with the extended high - dose melatonin treatment in als patients111 indicates that circadin may be safe even for prolonged treatment, whereas more studies would be required for ramelteon to be sure about this point.20,21,47 melatonin and all synthetic melatoninergic drugs discussed here are capable of phase shifting the circadian pacemaker, and all of them can be expected to reduce sleep onset latency, with the exception of a certain number of nonresponders. in terms of toxicology, beyond the subjective reports on absence or presence of adverse effects, -methyl-6-chloromelatonin, ramelteon, tasimelteon and agomelatine need further investigation for long - term safety, particularly for tasimelteon, which is administered in relatively high doses of 20 or 50 mg,69 compared with the much lower doses of ramelteon (8 mg) or melatonin (2 mg). the nonselective drug agomelatine may be useful in major depressive disorder,21,47 but, alternatively, combinations of classic antidepressants with z - drugs such as zolpidem extended - release may be likewise effective.130 with these reservations, all the chronobiotics, but more in particular, the approved hypnotics circadin (melatonin prolonged release) and rozerem (ramelteon), but presumably also the investigative drug tasimelteon,69 should be suitable for treating jet lag or other phase shifts, and also tractable forms of crsds, such as dsps and fasps. beyond phase resetting, facilitation of sleep onset can be expected in mild types of crds - related insomnia. in this regard, one might, however, ask whether a prolonged - release formulation or a drug of longer half - life and higher receptor affinity is really needed. sleep onset can be even promoted by 0.1 or 0.3 mg immediate - release melatonin,20 so that a higher dose may not be required in these cases, nor prolonged release, longer half - life or higher receptor affinity. the situation is different in primary chronic insomnia, in which a substantial support of sleep maintenance is required. in this disorder, statistically significant but still moderate effects of ramelteon have been reported,2022,47,102107 whereas prolonged - release melatonin would require substantiation of its efficacy. although ramelteon has considerably higher receptor affinities and a relatively longer half - life, 4 or 8 mg are recommended, whereas only 2 mg of melatonin are present in a circadin tablet. it seems inappropriate to be extremely cautious with the natural compound melatonin, which is exceptionally well tolerated in the majority of individuals, but not to apply the same criteria to a longer - acting synthetic analog with higher receptor affinity.47 treatment with melatoninergic agonists seems to be promising in another disorder, smith - magenis syndrome, which is characterized, apart from developmental and neurobehavioral abnormalities, by a largely inverted melatonin rhythm and sleep difficulties.87,88 in this case, a combination of a 1-adrenergic blocker in the morning, to suppress diurnal melatonin secretion, and melatonin in the evening has been applied with some success.89,90 in this congenital disease, a sustained high nocturnal level of melatonin would be of particular importance, which indicates the use of a prolonged - release formulation. whether or not melatonin may be replaced by synthetic agonists such as ramelteon remains to be clarified and may depend on long - term safety. other neurodevelopmental and neuropsychiatric disorders associated with sleep difficulties or crsds, which have been studied in children and young adults and are sometimes otherwise intractable,76,85,86,9194 may be seen as an additional area of treatment, despite the reservations of emea. nevertheless, caution should go beyond the risks mentioned in the previous section, such as autoimmune diseases, parkinson s disease, coadministration of cyp inhibitors, and hepatic and renal diseases. pregnancy would be another condition under which benefits and possible risks have to be weighed. these considerations should equally apply to melatonin prolonged or immediate - release, ramelteon and other melatoninergic drugs. therefore, the decision by emea to approve circadin for subjects older than 54 years, along with a list of specific precautions, is a responsible one, although it may appear unduly cautious. the same criteria should be applied to ramelteon, and to other melatoninergic drugs that may be evaluated for approval. nevertheless, all melatoninergic drugs discussed are well tolerated in short - term treatment, and for the natural compound, melatonin, the same should be valid for long - term administration, except for the precautions mentioned above. melatonin and its synthetic analogs may be helpful even in other disorders, such as relieving sleep difficulties caused by benzodiazepine discontinuation110 and in chronic obstructive pulmonary disorder, in which ramelteon has been shown to be effective,131,132 and for which the same may be valid in the case of melatonin.98,133 in practical terms, sleep difficulties should first be tested for causes related to circadian dysfunction, in which immediate - release melatonin may already be effective, and circadin should be tried if the immediate - release formulation does not suffice. in cases of chronic primary insomnia, ramelteon seems, according to current knowledge, slightly more promising than prolonged - release melatonin. if melatoninergic drugs fail, z - drugs may be the better option. in patients of appropriate age and not belonging to a risk group, the general strategy may be to first test the natural compound, melatonin, because of its remarkable tolerability and safety, before other options are used.	hypnotic effects of melatonin and melatoninergic drugs are mediated via mt1 and mt2 receptors, especially those in the circadian pacemaker, the suprachiasmatic nucleus, which acts on the hypothalamic sleep switch. therefore, they differ fundamentally from gabaergic hypnotics. melatoninergic agonists primarily favor sleep initiation and reset the circadian clock to phases allowing persistent sleep, as required in circadian rhythm sleep disorders. a major obstacle for the use of melatonin to support sleep maintenance in primary insomnia results from its short half - life in the circulation. solutions to this problem have been sought by developing prolonged - release formulations of the natural hormone, or melatoninergic drugs of longer half - life, such as ramelteon, tasimelteon and agomelatine. with all these drugs, improvements of sleep are statistically demonstrable, but remain limited, especially in primary chronic insomnia, so that gabaergic drugs may be indicated. melatoninergic agonists do not cause next - day hangover and withdrawal effects, or dependence. they do not induce behavioral changes, as sometimes observed with z - drugs. despite otherwise good tolerability, the use of melatoninergic drugs in children, adolescents, and during pregnancy has been a matter of concern, and should be avoided in autoimmune diseases and parkinsonism. problems and limits of melatoninergic hypnotics are compared.
to begin this procedure, we ensure that all scuva components have sufficient battery power, recording tape (for the high - definition or hd video camera), and function properly. depending on the flows to be measured, select video camera resolution and frame rates that yield best results for digital particle image velocimetry (dpiv). prepare the laser and camera housings for use by cleaning the o - ring grooves and o - rings with a clean towel or wipe. spread manufacturer provided o - ring grease evenly on the o - rings and replace them in the housing grooves. in addition, clean the laser and camera housing apertures to prevent laser sheet deformation and marks on the camera housing lens. check the o - ring seals by placing both empty housings in a tub full of water. weighted objects will need to be placed on top of the housings to submerge them since the housings float when empty. after 5 to 10 minutes, remove the housings from the tub and towel dry the outside. check whether there is any moisture inside the housings. also consider using disposable, paper moisture strips during the pressure test to indicate whether there is moisture in the housings after the test. after the housings pass the pressure test, place scuva components inside the housings. ensure that the lights are oriented in such a way that they illuminate the area directly ahead of the camera and operator, and do not interfere with maintaining grip on handles and operation of camera controls. in a low light environment, ensure that the laser beam is properly aligned relative to the optical lens installed in the laser housing. when properly aligned, the laser / lens combination will create a vertical sheet of light that is oriented perpendicular to the camera housing. for safety, use a temperature - sensitive sheet of paper to determine laser sheet orientation. using scuva attachments and the rigid, extendable arm, connect the laser housing and the camera housing to each other. ensure that the housings are firmly attached and that the housings can not rotate with respect to each other. it is critical that the laser sheet remains oriented perpendicular to the camera s field of view throughout the measurement. due to the current capabilities of scuva, measurement dives can only be conducted in low - light locations or at nighttime to prevent natural light interference with the laser sheet. the camera housing has a built - in electronic moisture sensor that provides visual warnings (flashing led lights) in case of moisture in the camera housing. once attached to the apparatus, release scuva to determine the buoyancy characteristics of the device. depending on the buoyancy characteristics, attach buoyancy foam or lead weights to one or both housings to ensure neutral buoyancy and prevent rotation of the apparatus in water. position the laser using the extendable arm sufficiently far from the diver to minimize measurement of diver - induced flows. any measurements of diver - induced flows near the target introduce error and are not used for subsequent analysis. adjust the camera zoom until the field of view frames the target and surrounding fluid. while keeping the apparatus stationary, focus the video camera on the laser sheet until particles appear sharp and in focus. once the laser sheet plane is in focus, switch the camera to the manual focus mode. this will prevent the camera from refocusing on any objects that appear in the field of view during measurement, resulting in blurred particles in the laser sheet. to calibrate scuva, place an object with known dimensions in the laser sheet within the video camera 's field of view. after the dive, an image will be extracted from this video sequence to determine a calibration constant that converts the field of view size from units of pixels to cm. if at any time the operator adjusts the field of view size be re - position the extendable arm or changing the camera zoom during the dive, steps 12 and 13 will need to be repeated. the environmental bulk flow properties need to be determined. if present, the current direction will dictate apparatus and diver positioning relative to the target during measurements. the direction of bulk flow surrounding the target can be inferred by observing bubbles exhaled from the diver and noting their lateral motion. in addition to bubbles, a small quantity of fluorescent dye (i.e., fluorescein) can be released to determine the current direction. since diver - generated flow can be a source of dpiv measurement error, the diver should not be located upstream of the target. in addition, the laser sheet should be positioned parallel to the direction of current so as to maximize particle residence time within the laser sheet, thereby minimizing dpiv errors. however, if no current or bulk flow is present, diver and scuva positioning relative to the target are unrestricted. if attempting to record the flow surrounding a moving target first predict the location of the target, and then position scuva to the predicted location while remaining motionless. as the target moves through the camera s field of view, if the target is motionless, frame the target and surrounding fluid in the video camera s field of view and begin recording while remaining motionless. the operator should refrain from rotational and out - of - plane motions during video recording since these motions result in erroneous dpiv results. therefore, measurements collected during rotational and out - of - plane diver motions will not be used for further data analysis. once video collection is complete, turn off all components of scuva and restore the laser arm to its retracted position. remove scuva from the water and detach the camera and laser housings from the arm. rinse or soak the apparatus in fresh water before drying to prevent rusting of the apparatus. once the housings are dried, remove components from the housings, and recharge and replace batteries if needed for another dive. connect the video camera to a computer and extract video from the hd tape by using a hd video software package (i.e., adobe premiere pro or imovie). after the video is extracted, determine the range of video to be converted into a series of images for dpiv analysis. ensure that the pixel aspect ratios and extracted image sizes match the hd video settings. these images are imported to a dpiv processing program (i.e., davis or matpiv). after proper selection of calibration constant and image capture parameters, which are prompted from the dpiv software package, velocity fields can be generated from consecutive particle images. additional post - processing steps, depending on the quality and types of measurements, can also be applied. when the protocol is done correctly, the particle images surrounding the target will be sharp and easy to distinguish. using the particle fields captured in situ by scuva s video camera (figure 1a) and a dpiv processing software package, velocity fields of flow surrounding the target (figure 1b) vectors in the velocity field indicate magnitude and direction of the local flow velocity. if sufficient video is collected to provide a time series of images, a time series of velocity fields can also be determined. figure 1 measured in situ particle fields (a) surrounding aurelia labiata. corresponding velocity field (b) with yellow vectors indicating flow direction and magnitude. red arrow in a indicates a region of high reflectivity, which results in saturation of the image, making it difficult to distinguish between particles and the target. red arrow in b indicates a region of streaking that results when the flow rate is not sampled at a high enough frequency. when the protocol is done correctly, the particle images surrounding the target will be sharp and easy to distinguish. using the particle fields captured in situ by scuva s video camera (figure 1a) and a dpiv processing software package, velocity fields of flow surrounding the target (figure 1b) will be revealed. vectors in the velocity field indicate magnitude and direction of the local flow velocity. if sufficient video is collected to provide a time series of images, a time series of velocity fields can also be determined. figure 1 measured in situ particle fields (a) surrounding aurelia labiata. corresponding velocity field (b) with yellow vectors indicating flow direction and magnitude. red arrow in a indicates a region of high reflectivity, which results in saturation of the image, making it difficult to distinguish between particles and the target. red arrow in b indicates a region of streaking that results when the flow rate is not sampled at a high enough frequency. a potential constraint in the field is the need for particles in the flow, which are necessary to implement digital particle image velocimetry (dpiv). in coastal water, suspended particulate matter exhibits sizes on the order of 10 m in diameter and concentrations between 0.002 and 10 per mm. additional studies using a submersible holocamera for particle detection confirm sufficient presence of seeding particles to perform dpiv in ocean water. during open sea and coastal ocean diving, we have found that particle densities and sizes are not a constraint for conducting in situ dpiv. aside from particle densities and sizes, qualitatively, if a region within an interrogation window has greater particle concentrations than another, the velocity magnitude generated by the dpiv analysis will be biased towards the region with higher particle concentrations. we found thatcle concentrations are relatively constant during particle concentrations are relatively constant during dives where the diver is suspended in the middle of the water column. however, particle fields in benthic environments have the potential for inhomogeneity due to resuspension of particles by environmental or diver - induced flows near the sea floor. care must be taken to minimize disruption of particles during measurements in benthic environments. to the authors ' knowledge, a formal analysis of errors generated by inhomogeneous particle concentration fields has not been conducted in either laboratory or field conditions, and should be a subject for further consideration in a separate publication. several different issues should be considered when preparing and conducting in situ experiments using the protocol. while recording, the operator is instructed to remain stationary and refrain from all out - of - plane and rotational motion. this request is simple in theory but difficult in practice, and these measurements require advanced diving skill to be completed successfully. out - of plane and rotational motions of the operator result in erroneous dpiv data. however, in - plane motions can be corrected by using in - house software. it is recommended to the operator to practice buoyancy control for several dives before using scuva to maximize measurement efficiency. besides buoyancy considerations flows that travel out - of - plane relative to the laser sheet will not yield reliable dpiv results, and the operator should orient scuva to capture these flows most effectively. in addition, the position of the diver relative to the target must be selected so as to minimize diver - induced flow in the measurements. diver - induced flow introduces error to the target flow, and measurements that include diver effects should not be used for further analysis. in the event that the target has a highly reflective surface, the fluid region surrounding the target will be strongly illuminated, making it difficult to distinguish nearby individual particles from surrounding fluid (region indicated by red arrow, figure 2a). filters or polarizers can be added to the laser or camera housings to reduce the intensity of the laser light captured by the video camera sensor. if this is not possible due to logistical constraints and limited access to equipment, post - processing of images using in - house software can provide sufficient correction by subtracting from the images the elevated pixel intensities near the target. another consideration that affects the quality of dpiv data is whether particle streaks are present. if particle fields have regions of streaking (indicated by red arrow, figure 2b), the video camera is recording at a frame rate too low to resolve these high velocities. by increasing the frame rate,, this results in a reduction of light reaching the video camera sensor and makes the particle field look dimmer. if the video camera has the ability to manually set aperture settings, increase the aperture setting to prevent dimming of the particle field. determining the optimal device settings may require multiple dives with scuva before successful data collection.	the ability to directly measure velocity fields in a fluid environment is necessary to provide empirical data for studies in fields as diverse as oceanography, ecology, biology, and fluid mechanics. field measurements introduce practical challenges such as environmental conditions, animal availability, and the need for field - compatible measurement techniques. to avoid these challenges, scientists typically use controlled laboratory environments to study animal - fluid interactions. however, it is reasonable to question whether one can extrapolate natural behavior (i.e., that which occurs in the field) from laboratory measurements. therefore, in situ quantitative flow measurements are needed to accurately describe animal swimming in their natural environment.we designed a self - contained, portable device that operates independent of any connection to the surface, and can provide quantitative measurements of the flow field surrounding an animal. this apparatus, a self - contained underwater velocimetry apparatus (scuva), can be operated by a single scuba diver in depths up to 40 m. due to the added complexity inherent of field conditions, additional considerations and preparation are required when compared to laboratory measurements. these considerations include, but are not limited to, operator motion, predicting position of swimming targets, available natural suspended particulate, and orientation of scuva relative to the flow of interest. the following protocol is intended to address these common field challenges and to maximize measurement success.
heat shock proteins (hsps) are considered part of a family of proteins known as stress proteins since their expression is induced by a wide range of stressors, such as oxidative stress, thermal stress, ischemia, exercise, metabolic stress, and many others. the 72 kda member of the 70 kda family of heat shock proteins, hsp70 (or hspa, encoded by the hspa1a gene in humans), is inducible during cell stress. it is the most abundant of all hsps, accounting for 1 - 2% of cellular protein, and is plentiful in skeletal muscle. as molecular chaperones, the intracellular hsp70 proteins (ihsp70) can interact with other proteins (unfolded, in nonnative state and/or stress - denatured conformations) to avoid inappropriate interactions, formation of protein aggregates, and degradation of damaged proteins, as well as helping the correct refolding of nascent proteins. other functions include protein translocation, antiapoptosis, and anti - inflammatory responses [9, 10 ]. more recently, the hsp roles have been expanded to include control of cell signaling, modulation of immune response, and modulation of chronic disease conditions such as diabetes, obesity, and insulin resistance [14, 15 ]. the heat shock response is regulated by a family of heat shock transcription factors (hsfs) composed of four members (hsf 14), which are maintained in an inactive monomeric form during nonstimulated conditions. hsf-1 is a primary regulator of heat shock response in mammalian cells and a low concentration of it has been associated with a number of human pathologies including t2 dm and obesity - related fatty liver disease. hsf-1 activation is a multistep mechanism that involves its phosphorylation, trimerization, nuclear translocation, and dna binding to heat shock elements (hse) located at the promoter regions of targeted heat shock genes ; nevertheless, hsf-1 activation can be negatively regulated by posttranscriptional modification, such as phosphorylation in specific serine residues and phosphorylation - dependent sumoylation. heat shock proteins were long thought to be exclusive cytoplasmic proteins with functions restricted to the intracellular compartment. however, an increasing number of observations have indicated that they may be released into the extracellular space (ehsp70) having a wide variety of effects on other cells. ehsp70 function is, in general, associated with the activation of the immune system. for example, ehsp70 has been reported to stimulate neutrophil microbicidal capacity and chemotaxis and recruitment of natural killer (nk) cells as well as cytokine production by immune cells [12, 25 ]. in addition, ehsp70 was recently hypothesized to be involved in the inducement of neural cell protection under stress conditions. an intriguing aspect of hsp70 physiology is its versatility to induce antagonistic actions, depending on the location of the protein. for example, ihsp70 exerts a powerful anti - inflammatory effect, while ehsp70 has the opposite role, inducing the activation of several proinflammatory pathways. in fact, chronic exposure to ehsp70 induces the activation of several proinflammatory pathways probably via binding to membrane toll - like receptors (see below) although ehsp70-peptides have also been shown to act as anti - inflammatory and immunosuppressive factors after internalization and antigen processing (see borges., 2012, for review). ihsp70 exerts its anti - inflammatory effect through the interaction with the nuclear factor b (nf-b), blocking its activation. nf-b is a ubiquitous transcription factor originally discovered in b - lymphocytes that is essential for arming inflammatory responses to a variety of signals, immune function, endothelial cell activation, and the control of cell growth. ihsp70 hampers nf-b activation at several levels, by impeding the phosphorylation of inhibitor of b (ibs), by directly binding to ib kinase gamma (ikk), which will result in continued binding (and inactivation of nf-b) thus inhibiting downstream inflammatory signals. this is corroborated by the finding that ihsp70 binds with liver nf-b / ib complex in the cytosol thus hindering transcription of tnf and inducible nitric oxide synthase (nos2) genes which are activated via a nf-b dependent mechanism. stress - induced elevations in ihsp70 inhibit c - jun n - terminal kinase- (jnk-) dependent signal transduction hence promoting cell survival. it can inhibit caspase activation by interfering with apaf-1 and prevent the recruitment of procaspase-9 to the apoptosome. overexpression of hsp70 in lymphoid tumor cell lines inhibited apoptosis by attenuation of caspase activation. the antiapoptotic effects of hsp70 have been reported for mouse brain tissue, where hsp70 overexpression resulted in decreased infarct sizes, improved neurological deficits, and fewer apoptotic cells (determined by reduced dna laddering) after middle cerebral artery occlusion. hsp70s have also been shown to decrease oxidative stress so that they are part of the intracellular antioxidant machinery, making ihsp70 even more important for the inhibition of apoptosis and inflammation. cyclopentenone prostaglandins (cp - pgs), which under certain circumstances may induce hsp70 expression, are consequently powerful anti - inflammatory autacoids [3840 ]. the interplay between ihsp70 and proinflammatory cytokines at gene regulatory level has also been reported. the promoter region of tnf gene contains an hsf1 binding site that represses tnf transcription, and thus loss of this repressor results in sustained expression of tnf ; thus the hsf1 knockout is associated with a chronic elevation of tnf levels and increased susceptibility to endotoxin challenge. regulation of such a network in the opposite directions has also been demonstrated : tnf may transiently repress hsf1 activation. furthermore, jnk1 was unequivocally demonstrated to phosphorylate hsf1 in its regulatory domain causing suppression of hsf1 transcribing activity while hsp70 prevented bax activation both by inhibiting the jnk / bim pathway and by interacting with bax in uv - induced apoptosis. altogether, the above findings explain why the induction of hsp72 (hspa1a) in vitro (by heat shock or hsp72 transgene overexpression) reduces the expression of inflammatory genes such as tnf, il-1, il-12, il-10, and il-18. in contrast to the above findings, ehsp70 proinflammatory actions have been demonstrated to be mediated by myd88/irak / nf-b signal transduction pathway after both toll - like receptor 2 (tlr2) and tlr4 binding, in a cd14-dependent manner [47, 48 ], thus promoting innate immune activation. due to the antagonistic actions of the heat shock proteins within the course of an inflammatory response, it is reasonable to hypothesize that the balance between ehsp70 and ihsp70 might determine the outcome either the induction or the attenuation of inflammation. since low - grade inflammation is involved in several chronic diseases, the management of hsp70 expression and its location can be crucial for the control of inflammatory - related conditions, such as t2 dm. we herein suggest that the ratio of the extracellular medium hsp70 concentration to intracellular hsp70 contents (ehsp70/ihsp70) can determine the progress of insulin resistance and the progression of t2 dm. the incidence of t2 dm has increased dramatically over the last fifty years and this is clearly associated with growing rates of obesity and physical inactivity. obesity is linked to a chronic proinflammatory state, since adipose tissue expansion and adipose associated immune cell activation result in the release of several cytokines, such as tnf, which leads to the activation of serine threonine kinases jnks and ikk. it is known that both jnk and ikk phosphorylate insulin receptor (ir) substrate-1 (irs-1) on ser-307, leading to the inactivation of the insulin receptor downstream response. also, chronic activation of ikk has been reported in diabetic patients, while a reduction in ikk activity prevents the development of insulin resistance in vitro and in vivo. in addition, lipid oversupply and hyperglycemia can lead to increased deposition of lipid species such as diacylglycerols and ceramides, which can also activate jnk and ikk in liver and/or skeletal muscle, leading to insulin resistance, causing sustained hyperglycemia and hyperlipemia. hyperglycemia per se is also known to be involved in inflammation and diabetes - associated vascular complications arising from reactive oxygen species generation and action [53, 54 ]. chronic hyperglycemia induces the production of reactive oxygen species (ros), leading to enhancement of protein oxidation, dna oxidation, and lipid peroxidation. the free radical gas nitric oxide (no) also plays a role in the insulin resistant state generated by proinflammatory cytokines. no is synthesized at high rates by the inducible form of nitric oxide synthase (inos, encoded by the nos-2 gene) which plays a significant role in cell damage associated with obesity and t2 dm. interestingly, a physiological concentration of this free radical is required to stimulate necessary functions such as muscle glut4 expression / translocation and insulin secretion by -cells. however, at high concentrations, no compromises insulin - stimulated glucose transport in skeletal muscle and can also be toxic to -cells inducing death. accordingly, acute treatment with no donors results in a reduction of insulin - stimulated glucose uptake and glycogen synthesis in isolated soleus muscle, inducing decreased ir and irs-1 activity. in vivo, this treatment also promotes insulin resistance in healthy animals by the reduction of irs-1 levels. obese ob / ob mice or rats submitted to high fat diet (hfd) have shown enhanced nos-2 expression associated with insulin receptor and akt s - nitrosylation, which can be dismissed by rosiglitazone treatment by virtue of its nos-2 expression inhibiting activity. expanded adipose tissue triggers the release of interleukin-6 (il-6) in obese subjects that is associated with alterations in glucose uptake by the skeletal muscle. nevertheless, it is possible that there is a dual role of serum il-6 on glucose metabolism, probably related to the exposure time of and concentration of il-6. accordingly, acute il-6 treatment may increase glucose uptake in c2c12 myotubes by stimulating amp - activated protein kinase (ampk) in a serine / threonine kinase 11- (lkb1-) dependent pathway, which induces downstream as160 activation, while il-6 may induce a modest increase in the glucose infusion rate after 4 h of hyperinsulinemic - euglycemic clamp in mice. moderate doses of il-6 stimulate basal and insulin - stimulated glucose uptake in l6 myotubes and 3t3 cells line after 2 h. in addition, physiological concentrations of il-6 were reported to stimulate insulin secretion by isolated pancreatic islets and brin - bd11 clonal -cells through ampk activation. however, chronic treatment (24 h) with this cytokine has been demonstrated to be able to cause insulin resistance in c2c12 murine myotube cell line, due to impairment of insulin signaling (irs / akt cascades) in a jnk1/2-dependent manner. finally, obesity is often associated with a vicious cycle in which adipose tissue expansion increases the levels of free fatty acids (ffa) and proinflammatory cytokines in the circulation, which together with hyperglycemia and altered lipoprotein profiles increase the synthesis and accumulation of intramyocellular triglycerides (imct). sedentary behavior and aging are conditions related to a decreased mobilization of the imct resulting in an increased synthesis of toxic fatty - acid - delivered metabolites (fadm). these metabolites cause, in turn, an elevation in the production of reactive oxygen and nitrogen species (ros and rns), resulting in oxidative / nitrosative stress, mitochondrial dysfunction, and the activation of stress associated transcription factors, such as nf-b, which is followed by increased production and release of proinflammatory cytokines (e.g., tnf). tnf is a major driver of insulin resistance in skeletal muscle and, in addition, it can also induce activation of stress signals in pancreatic -cells, leading to mitochondrial dysfunction that culminates in cell failure and death. it has been reported that ihsp72 mrna levels are decreased in skeletal muscle of t2 dm patients which is correlated with the status of insulin resistance, whereas heat shock - like therapies (i.e., whole body warming, transgenic overexpression, or pharmacological mechanisms to elevate hspa1a protein expression) protect against high - fat - diet- and obesity - induced hyperglycemia, hyperinsulinemia, glucose intolerance, and insulin resistance [14, 65, 66 ]. although the underlying mechanism(s) culminating in lower ihsp70 expression in t2 dm individuals are not fully understood, the observed reductions are likely to be a result of the concerted contribution of (i) reduction in the rate of hsp70 protein synthesis due to attenuation of initiation and elongation phases of translation, (ii) suppression of hsf-1 activation and binding to hse via an increase in glycogen synthase kinase-3 activity (gsk-3) as previously suggested, and (iii) decreased hsf1 expression. in this regard, it has been recently proposed that long - term inflammatory stimuli emanating from the adipose tissue of obese individuals could repress hsf-1/ihsp70 axis because continuous activation of inflammasome nlrp3 may lead to a state of cellular senescence which abolishes the expression and activity of hsf-1. in line with this is the observation that the expression of both hsf-1 and ihsp70 in adipose tissue and the liver of type iii obese patients is dramatically reduced while the activation of jnks is reciprocally enhanced in the same tissues. as discussed above, the anti - inflammatory effect of ihsp70 is attributed mainly to its capacity of interaction with nf-b. in fact, ikk- and nf-b activity have been found to be increased in different obese experimental models. chronic activation of ikk has been reported in diabetic patients, while a reduction in ikk activity prevents the development of insulin resistance in vitro and in vivo. high - fat, high - carbohydrate intake may result in oxidative stress and consequent nf-b activation in obese subjects. on the other hand, nf-b dna binding is suppressed after heat shock, and upregulation of hspa1a can negatively affect nf-b activity in skeletal muscle. in addition, overexpression of hspa1a can restrict no production and release by transfected cells in a mechanism dependent on nf-b dna binding and subsequent inos gene expression. heat treatment is capable of inducing hspa1a expression in several tissues, preventing various obesity - elicited metabolic effects at molecular level, leading to improvement of glucose tolerance, insulin - stimulated glucose transport, and insulin signaling accompanied by the reduction in jnk and ikk activities in skeletal muscle and liver of hfd mice, which is almost completely abolished in transgenic hspa1a mice. furthermore, ihsp70 expression has been shown to decrease jnk activity, irrespective of stress stimulus. this is crucial for insulin sensitivity, since enhanced rate of jnk phosphorylation is associated with glucose intolerance and insulin resistance in skeletal muscle of obese mice, an effect which may be attenuated by long - term (16-week) heat treatment (41.5c) and is also observed in transgenic hspa1a mice. heat treatment also induced improvement of mitochondrial function, increasing citrate synthase and -hydroxyacyl - coa - dehydrogenase (-had) activity in soleus and extensor digitorum longus (edl) muscle. interestingly, a 12-week period of heat treatment is unable to change fasting blood glucose of obese rats, but it does attenuate insulin levels and decrease whole body glucose clearance induced by hfd. in obese hfd animals, phosphorylation of irs1 in tyr612 and the consequent downstream phosphorylation of akt in ser473 and activation of as160 have been found to be reduced in the skeletal muscle, while heat treatment reverted this response in a process associated with hspa1a expression. moreover, jnk inhibition has been demonstrated to rapidly occur in a dose - dependent manner in heat - shocked nih 3t3 fibroblasts via interaction between jnk1 and hspa1a. finally, studies have demonstrated that hsp70 is able to bind to the insulin receptor enhancing its recycling rate after heat shock [75, 76 ], which suggests that heat shock proteins may have direct influence upon insulin receptor function and activity. in contrast to its intracellular proinsulin signaling and anti - inflammatory effect, extracellular hsp70 (ehsp70) when chronic elevated is associated with inflammatory conditions, including t2 dm. other cells (e.g., lymphocytes, macrophages, epithelial cells, dendritic cells, neuronal cells, and hepatocytes) have been reported to release hsp70 proteins via (i) active mechanisms, such as vesicular secretion (classical pathway in rest conditions), (ii) lipid rafts, or (iii) exosomes. with respect to the extracellular compartment, ehsp70 can bind to tlr2 and tlr4 in a variety of cells, leading to the activation of proinflammatory pathways via myd88 and tirap that signal downstream to nf-b via irak4, traf6, and ikk and inducing jnk activation via mekk4/7 [49, 78 ], although high - affinity binding of ehsp70 to other surface receptors has also been described. the signal triggered by ehsp70 promotes typical immunoinflammatory responses directed to the combat of infections and bacterial infiltration through the production and release of no and proinflammatory cytokines, such as tnf and il1. moreover, ehsp70 responses are positively associated with classical inflammatory parameters such as crp, fibrinogen, and monocyte counts being commonly found in clinical situations, in which a danger signalization to immune system must be required. indeed, increased serum hsp70 has been reported in chronic and age - related diseases [8284 ]. interestingly, during conditions in which individuals are chronically exposed to elevated ehsp70 levels, changes in the ihsp70 content are also observed [14, 65 ]. in fact, in t2 dm ihsp70 is reduced in insulin - dependent tissues such as skeletal muscle and adipose tissue [14, 17, 65 ] while the ehsp70 is elevated. this profile is commonly found in t2 dm, where obesity is an aggravating factor for the undesirable high ehsp70/ihsp70 ratio. in addition, serum hsp70 levels were found to be higher in long - term (> 5 years) t2 dm patients as compared to newly diagnosed ones. accordingly, tlr2/4-dependent activation of jnks promotes phosphorylation of irs-1 at ser307 in rodents (equivalent to ser312 in humans) leading to inhibition of akt activation and, consequently, to a reduced glucose uptake by sensitive tissues and to a state of resistance to insulin action. moreover, it has been shown that tlr2 is central to palmitate - induced insulin resistance, via jnk - mediated phosphorylation of irs1/2. on the other hand, loss - of - function mutation in tlr4 prevents hfd - induced obesity and insulin resistance. ser307 phosphorylation of rodent irs-1 may also be elicited by inflammatory cytokines via ikk, in a process that can be inhibited by cp - pgs, which are powerful anti - inflammatory autacoids possessing ihsp70-inducing capacity. indeed, inhibitory ser307 phosphorylation of irs-1 is a physiological mechanism of feedback inhibition of insulin signal that is under the control of both insulin / igf1 and inflammatory cytokines, though via different downstream pathways. tlr4 expression and signaling dependent on ehsp70 is increased in obese and t2 dm subjects, an effect that can explain the high basal rate of mapk phosphorylation and nf-b activation found in these patients [9295 ]. the above findings help to explain why inhibition or absence of tlr4 confers protection against insulin resistance in skeletal muscle, adipose tissue, and liver [26, 97 ]. finally, as recently found, ehsp70 is positively correlated with insulin resistance and inflammation in elderly people. this may indicate a role for ehsp70 in impairment of insulin signaling in the skeletal muscle that occurs with advanced age and in t2 dm. in addition, the same group has shown that chronic exposure of -cells and islets to increased concentrations of ehsp70 results in -cell death and altered cell bioenergetics, a phenomenon that, apparently, is mediated through tlr-2 and tlr-4 activation. since, in t1 dm there is a dramatic increase in ehsp70 and in t2 dm and aging there is a slow chronic increase in the concentration of this protein, we educe that chronic exposure of pancreatic -cells to ehsp70 may lead to -cell failure and loss of functional integrity in vivo. based on the previous discussion, while ihsp70 is clearly protective, antiapoptotic, anti - inflammatory, and associated with normal insulin sensitivity, ehsp70 is related to a proinflammatory response, decreased expression of the anti - inflammatory ihsp70, and reduced insulin sensitivity. because of this, we suggest that the ratio of compartmental distributions of hsp70 between extra- and intracellular locations may determine the outcome of the inflammation and its associated insulin resistance. in a recent study, our group observed that the ratio between plasma ehsp70 and ihsp70 in lymphocytes from rats submitted to different loads of acute exercise can indicate the inflammatory status (heck., manuscript in preparation). accordingly, assuming the ratio r = [ehsp70]/[[ihsp70 ] = 1 for the controls (resting, unstimulated), moderate exercise produces a shift in r to up to ca. 5, which is paralleled by an elevation in inflammatory markers and stimulation of cell proliferation. conversely, r values between 0 and 1 indicate a predominantly anti - inflammatory status. thus, changes in the ratio between extra- and intracellular hsp70 emerge as a potentially new biomarker for inflammation and as a very sensitive indicator of inflammatory status. for instance, yang and collaborators have investigated the correlation between the level of exposure to pollution and ehsp70 in steel workers. from the data obtained in this study, we calculated r (plasma to lymphocyte ratio) as 5.5, 6.5, and 8.8, respectively, for low, moderate, and high exposure, as compared to controls (r = 1.0). we applied the same calculation to the data by rodrigues - krause and colleagues ' obesity - diabetes study, in which hsp70 was investigated in healthy obese (considered the controls herein), nonobese t2 dm and in obese t2 dm patients. in this case, settling controls as r = 1.0, we get 1.8 for t2 dm and 6.0 for obese t2 dm patients in which inflammatory unbalance was found. although healthy lean subjects had not been evaluated in this work, it is likely that such r marks should be even higher if r were taken in comparison with such controls. additionally, changes in r calculation seem to be valid during heat exposure as r values correlated with the heat exposure of peripheral blood mononuclear cells to different temperatures within a physiological range. accordingly, heat - shocked cells concomitantly measured hsp70 at 37c (r = 1.00), 39c (r = 1.45), 42c (r = 0.65), and 43c (r = 0.48). interestingly, at 45c, in which cells are known to trigger jnk - dependent prosurvival inflammatory pathways, r value was calculated as 1.97, confirming proinflammatory expectations. moreover, in a combined protocol of exercise training (60 min during 11 days in a treadmill, 1.692.20 ms, 1% grade) and heat acclimation (rectal temperature elevation by 1c for the duration of the exercise sessions at 40c room temperature), have observed, in human volunteers, hspa1a alterations (in plasma and total leukocytes) that, after conversion to r values, furnish the following picture : before training, r values were 1.11 at rest and 0.51 two days after the primary test ; after training and acclimation, r values were 0.22 at rest and 0.27 two days after the last training session. these values can be explained by the fact that ehsp70 plasma contents raised by ca. 50% in untrained individuals evaluated 48 h after the priming test, while under the same circumstances, ihsp70 was found to be 2.9-fold the remaining values. on the other hand, exercise training combined with heat acclimation evoked a 4.2-fold enhancement in intracellular hspa1a contents (hence, an anti - inflammatory response) which remained unaltered 48 h after the last session, while ehsp70 did not change any more, remaining near the rest of values observed in untrained volunteers. the above observations led us to postulate that r values and, particularly, changes in r values could be used as a predictor gauge for the inflammatory response that culminates in insulin resistance and diabetes, despite the method used to assess intra- and extracellular hsp70 contents. notwithstanding, r values may be also useful for application in several other inflammatory diseases and conditions besides being of value in controlling exercise impact over inflammatory status. furthermore, since ehsp70 and ihsp70 are directly related to insulin sensitivity, r value application in diabetes appears to be straightforward. as discussed above, ehsp70 to ihsp70 ratio (represented by r values) may determine the fate of the insulin sensitivity towards either its improvement (lower ratio) or its reduction / impairment (higher ratio). in this regard, strategies capable of changing the hsp70 contents, in the intra- or extracellular space, or both compartments, are likely to be used as a therapeutic strategy for the prevention or treatment of t2 dm and its efficiency could be precisely followed by the assessment of r values during the course of such treatment. in this sense, physical exercise fulfills all the prerequisites : it is a known modulator of ehsp70 (whose levels drop under exercise training) and ihsp70 (which tends to rise under the same circumstances). in other words, although acute exercise bouts signalize a stressful situation to all physiological systems (please see heck. 2011 for review) leading to augmented but just momentary ehsp70 plasma levels, exercise training or regular physical activity tends to reduce the stressful impact of each exercise session, leading to decreased ehsp70 and enhanced ihsp70 during the course of training. for this reason, exercise can also be used as a tool to decrease or maintain the normal r values and, consequently, optimum insulin sensitivity. in fact, improvement of glucose uptake and storage as well as increase in the oxidative capacity of different muscle fibers has been shown to be associated with increased ihsp70 expression in the skeletal muscle [14, 66 ]. moreover, physical exercise has been proposed as an alternative strategy for t2 dm patient treatment by virtue of its ihsp70 enhancing capacity : patients submitted to acute exercise bouts or moderate training have shown significant increase in muscle hsp70 expression [66, 104 ], which is directly associated with reduction of insulin resistance [14, 72 ]. exercise - induced expression of ihsp70 in skeletal muscle has a major role in restoring muscle metabolic functionality ; besides, it provides cytoprotection to damaged cells. such an ihsp70 inducing ability has been shown as a resultant in different protocols of exercise, including eccentric, concentric not damaging, aerobic, or resistive, all of which are capable of inducing intramuscular hsp70 expression [104106 ]. although time and intensity of the physical effort are determinant factors to increase of intramuscular hspa1a, its rise may be detectable just 2 h after the onset of an acute exercise session, when hspa1a mrna expression peaks. since ihsp70 family members promote the facilitation of protein transport into mitochondria, allowing and improving structural integrity of the organelle during fast energy flow, ihsp70 content has been correlated with an increase in the oxidative capacity of muscle cells. several studies have demonstrated the relationship between high ihsp70 levels in skeletal muscle and increased activity of mitochondrial enzymes after short training period. on the contrary, decreased mitochondrial function is known to be associated with the accumulation of intramyocyte triglycerides (and its byproducts), insulin resistance, and diabetes. for this additional reason, exercise - induced ihsp70 expression can lead to improvements in metabolite oxidation and, consequently, insulin sensitivity. as regarded above, hsf-1 is negatively regulated by gsk3, a serine / threonine kinase that phosphorylates this factor on ser303, keeping it in its inactive form in the cytosol. during acute physical exercise, gsk3 activity has been found to be nevertheless 30% decreased in vastus lateralis muscle, concomitantly with akt phosphorylation in ser473 and glycogen synthesis (gs) activation. in addition, a direct relation between ihsp70 and il-6 has been reported. during physical exercise, il-6 can be expressed and released by the skeletal muscle and, within extracellular space, binds to the il-6 receptor in an autocrine action. interestingly, the myokine il-6 has also been found to induce hsf-1 translocation to the nucleus upregulating heat - induced hsp70 gene, protein expression, and activity in human hepatic cells in a pi3k / akt / gsk3 dependent pathway. on the other hand, absence of il-6 is associated with decreased expression of hspa1a in skeletal and cardiac muscle of mice challenged with lps, although il-6 seems not to be required for exercise - induced hsp expression. the molecular mechanism(s) underlying the connection between ihsp70 expression and increased energy flow are not clear yet. however, changes in atp / adp ratio seem to work as a signal to the activation of different kinases, such as ampk that is capable of decreasing gsk3 activity. ampk is also regulated by ca during muscle contraction that activates ca / calmodulin protein kinase kinase (camkk) in an lkb1-dependent pathway. thus, ampk activation as result of muscle contraction may act as a stimulus for ihsp70 induction in a hsf1-dependent way. exercise is also known to induce ehsp70 release and accumulation into the circulation. while the source of the ehsp70 during the exercise is still in debate additionally, 1 adrenoreceptors in the liver seem to participate [118, 119 ] in a way that is dependent on exercise intensity, type, duration, and training status. in addition, recent data suggests ehsp70 concentration increases once systemic temperature and sympathetic activity exceed minimum endogenous criteria and is likely to be modulated by large and rapid changes in core temperature. however acute physical exercise is an inducer of ehsp70 release into the blood and exercise is not a factor of maintenance of high ehsp70 indefinitely, because chronic exercise (training) suppresses ehsp70 levels. hence, the expected deleterious effects of long - term exposure to high ehsp70 concentrations are never attained. in response to ehsp70, macrophages can release il-1, il-6, and tnf, all cytokines involved in insulin resistance. interestingly, in trained obese zucker rats, the macrophage cytokine release profile in response to ehsp70 is changed. in this case, the macrophages from obese zucker rats released less il-1 and tnf but more il-6 than macrophages from lean animals, indicating that habitual exercise improved the release of proinflammatory cytokines by macrophages. taken as a whole, the above findings clearly indicate that while ihsp70 is protective and anti - inflammatory being associated with normal insulin sensitivity, ehsp70 chronically produced in response to low - grade inflammation (but not to chronic exercise) is related to a proinflammatory response, decreased expression of ihsp70, and reduced insulin sensitivity. therefore, we suggest that the ratio of hsp70 contents between the extra- and intracellular compartments may dictate the outcome of the inflammation and associated insulin resistance (figure 1). since exercise is able to modulate both ehsp70 and ihsp70, it is reasonable to predict that exercise is the most efficient and powerful tool currently available to normalize and/or maintain ehsp70/ihsp70 ratio at appropriate levels, thus preventing t2 dm. the appropriate type, intensity, duration, and frequency of exercise that best fit each condition need to be determined. the versatility of the hsp70 to induce different inflammation - related responses according to its location (intra- versus extracellular) places this protein as a master regulator for the fine - tuned control of the immune system : while ihsp70 induces inactivation of nf-b, ehsp70 induces the opposite effect. thus, we suggest that the ehsp70/ihsp70 ratio may represent a better marker for the immunoinflammatory status of the whole body in exercise as well as in many types of diseases. finally, we advocate that r values could be useful not only in assessing the course of therapeutic approaches in obesity - induced insulin insensitivity and diabetes, but also to evaluate inflammatory status in inflammation - related diseases (e.g., atherosclerosis and other cardiovascular diseases, rheumatoid arthritis, sepsis, and obesity - related nonalcoholic fatty liver disease) as well as in exercise training directed to immunosuppressed and cardiovascular patients. hence, the above propositions may have an important diagnostic value for such patients, since hsp70 status determined through r values may be easily obtained from the ratio of extracellular (plasma) to intracellular (circulating blood leukocytes or mononuclear cells) hsp70 content from a blood sample.	recent evidence shows divergence between the concentrations of extracellular 70 kda heat shock protein [ehsp70 ] and its intracellular concentrations [ihsp70 ] in people with type 2 diabetes (t2 dm). a vital aspect regarding hsp70 physiology is its versatility to induce antagonistic actions, depending on the location of the protein. for example, ihsp70 exerts a powerful anti - inflammatory effect, while ehsp70 activates proinflammatory pathways. increased ehsp70 is associated with inflammatory and oxidative stress conditions, whereas decreased ihsp70 levels are related to insulin resistance in skeletal muscle. serum ehsp70 concentrations are positively correlated with markers of inflammation, such as c - reactive protein, monocyte count, and tnf-, while strategies to enhance ihsp70 (e.g., heat treatment, chemical hsp70 inducers or coinducers, and physical exercise) are capable of reducing the inflammatory profile and the insulin resistance state. here, we present recent findings suggesting that imbalances in the hsp70 status, described by the [ehsp70]/[ihsp70 ] ratio, may be determinant to trigger a chronic proinflammatory state that leads to insulin resistance and t2 dm development. this led us to hypothesize that changes in this ratio value could be used as a biomarker for the management of the inflammatory response in insulin resistance and diabetes.
melioidosis, an infectious disease caused by the tier 1 select agent burkholderia pseudomallei, is notoriously difficult to cure 1. north - eastern thailand is a hot spot for this infection, with an annual incidence of 21.0 per 100 000 population and a case - fatality rate of 40% 2. for patients who survive their first episode of infection, this occurs in approximately 13% of patients followed for 10 years, and half of the recurrences occur within 12 months of the primary episode 3. approximately one - quarter of those with relapse will die as a direct result 4,5. biofilm formation has been described as an important factor associated with persistent infections in a number of infectious diseases, including burkholderia cepacia infection in cystic fibrosis patients 68, but this has not been formally evaluated in relation to relapse of melioidosis. a previous study of a small number of isolates associated with relapse suggested that b. pseudomallei with uncommon lipopolysaccharide (lps) types (smooth type b and rough type) might be associated with relapse 9. here, we evaluated biofilm formation and lps type of b. pseudomallei isolates from patients with primary melioidosis drawn from a cohort described previously 5, and determined their associations with relapse. control study in which both cases and controls were drawn from a cohort of patients with primary melioidosis identified between 1986 and 2004 who survived to receive oral antimicrobial therapy and were observed until july 2005 5. cases were all patients who developed at least one episode of relapse during the study period, with relapse being verified by genotyping of the primary and relapse isolates 5. controls were randomly selected from those patients in the cohort who had not developed relapse by the time relapse was identified in cases. cases and controls were matched for known risk factors for relapse, including choice and duration of oral antimicrobial therapy, positive blood culture, and multifocal distribution of infection at first presentation. primary isolates from cases and controls and relapse isolates from cases were evaluated for quantitative biofilm formation and lps. all isolates were stored at 80c prior to the evaluation. the first isolate cultured and saved from each episode quantitative estimation of biofilm formation was performed with a modified microtitre plate test, as described previously 1012. all experiments were independently conducted twice, and the results reported were the average from those two experiments. lps was extracted, and the type was defined as smooth type a, smooth type b, or rough type, as described previously 9. a conditional logistic regression model was used to evaluate the relationships between independent factors and the relapse outcome. selection of controls and statistical analyses were conducted with stata, version 12.0 (statacorp, college station, tx, usa). the study was approved by the ethical and scientific review subcommittee of the thai ministry of public health 5. of 86 and 202 primary isolates from cases and matched controls, 80 (93%) and 184 (91%), respectively, were available for study and included in the analysis. patients in the case and control groups had comparable characteristics for the matched variables (table1). characteristics of cases and controls na, not applicable, as choice and duration of oral antimicrobial therapy, positive blood culture result and multifocal distribution of first presentation were matched variables ; od, optical density. melioidosis was classified as localized (single focus of infection and a negative blood culture result), multifocal (one or more non - contiguous foci of infection and a negative blood culture result), bacteraemic (a positive blood culture result plus a single or no identifiable focus of infection), and disseminated (a positive blood culture result plus one or more non - contiguous foci of infection). first, we determined whether the quantitative production of biofilm by the primary isolate influenced the likelihood that relapse would occur. biofilm production by primary isolates from patients with relapse was higher than that for primary isolates from matched patients without relapse (median corrected optical density (od)630 nm of 0.95 (interquartile range 0.751.28) vs. 0.79 (interquartile range 0.631.06)). this was independently associated with the relapse outcome (conditional or 2.03 ; 95% ci 1.273.25 ; p 0.003). overall, 99% of primary isolates from cases and 98% from controls had lps smooth type a. lps smooth type b was found in one (1%) case and in two (1%) controls, and rough - type lps was found in one (1%) control. an association between lps type of the primary isolate and relapse was not found (p 0.74). next, we determined whether there was any difference in biofilm formation and in lps type between primary and relapse isolates from the same relapse cases. from 80 relapse cases, biofilm formation of the primary isolate and that of the relapse isolate were not different (mean difference for corrected od630 nm of 0.002 ; 95% ci 0.16 to 0.16 ; p 0.98). the lps type of the primary relapse pair was the same for all 71 isolates (lps smooth type a, n = 70 ; lps smooth type b, n = 1). in this study, we have shown that a quantitative measure of biofilm formation by the primary isolate is associated with relapse in patients with melioidosis. this was independent of known clinical risk factors for relapse, including choice and duration of oral antimicrobial therapy, positive blood culture, and multifocal distribution of infection at first presentation, factors that were matched by the nested case this provides the first evidence to suggest that biofilm formation of b. pseudomallei in vitro is associated with relapse in human melioidosis, and is consistent with findings reported for escherichia coli 6 and other biofilm - producing bacteria 7,8. we also observed that quantitative biofilm formation did not differ between paired primary and relapse isolates. this lack of detectable change between isolates of the same lineage that are separated by the period spanning human infection, quiescence and re - emergence argues against the notion that increased biofilm formation occurs in vivo through positive selection. quantitative biofilm formation by isolates in this study was lower overall than that reported previously for 34 clinical b. pseudomallei isolates (mean corrected od630 nm of 1.98 0.32) 12. possible explanations are that the means used in the previous study were skewed by isolates with exceptionally high biofilm formation. in addition, the isolates in the study described here were only from patients who survived the first episode of acute infection, whereas the isolates in the previous study included those who died during the acute infection. the majority of b. pseudomallei isolates from the primary episode of melioidosis in this study expressed lps smooth type a, and no association between lps type and relapse was found. this contrasts with the findings of a previous study, in which three of 11 (27%) patients with recurrent melioidosis had different lps types in the primary and relapse isolates 9. furthermore, isolates in this previous study were not genotyped, and the possibility that recurrence was attributable to re - infection with a different isolate rather than persistence and relapse with the primary isolate was not excluded 9. the proportions of uncommon, non - type a lps in the primary episode of melioidosis in this study (1% in cases and 2% in controls) were similar to those observed in the previous study (3%) 9. our findings, based on a much larger number of bacterial isolates supported by a robust study design, do not provide evidence for a link between uncommon, non - type a lps and relapse. this study was supported by the thailand research fund (mrg5480172) and the melioidosis research centre, khon kaen university, thailand. s. j. peacock was supported by the national institute for health research cambridge biomedical research centre.	we examined whether quantitative biofilm formation and/or lipopolysaccharide type of burkholderia pseudomallei was associated with relapsing melioidosis. we devised a 1 : 4 nested case control study in which both cases and controls were drawn from a cohort of patients with primary melioidosis. paired isolates from 80 patients with relapse and single isolates from 184 patients without relapse were tested. relapse was associated with biofilm formation of the primary infecting isolate (conditional or 2.03 ; 95% ci 1.273.25 ; p 0.003), but not with lipopolysaccharide type (p 0.74). this finding highlights the importance of biofilm formation in relapsing melioidosis.
chlorophyll and the associated accessory pigments allow plants to maximise use of the visible light spectrum for photosynthesis. by establishing internal gradients in light capture, the green pigments enable plants to respond rapidly to changes in the spectral environment, as well as to exploit niche habitats. equally, they provide some protection against photoinhibition and photooxidation, the damaging effects of excess quanta. given the obvious benefits to their being green, why then do many plant species produce red - pigmented leaves at one or more stages in their life cycles ? red - leafed flora are common throughout all orders of the plant kingdom, from the basal liverworts to the most advanced angiosperms. they occur in habitats as diverse as the antarctic shoreline and the tropical rainforests, are as abundant in arid deserts as in freshwater lakes, and seem equally at home in the light - starved forest understorey as in the sun - drenched canopy. in many red - leafed species the manufacture of red pigments is transient, often associated with a discrete developmental stage such as in the growth flushes of tropical trees [3, 4, 5 ], or in the senescing autumn foliage of deciduous trees [6, 7, 8, 9 ]. in certain other species, however, red pigments can persist throughout the leaf 's entire life span, or else they are induced and retained only after the plant has experienced stress. functional implications of these red pigments in plants have been the focus of a significant research output over the past decade. for most vascular plants, red colouration in leaves is achieved by anthocyanins, predominantly cyanidin-3-o - glucoside, as a solution located in the vacuole of the plant cell. other pigments notably the betalains, certain carotenoids, thiarubrine a, some terpenoids, and the 3-deoxyanthocyanins also impart reddish colours in various species ; these pigments have been less well studied than the anthocyanins, but at least some of them have comparable functions in leaves [12, 13, 14, 15, 16 ]. the synthesis and vacuolar sequestration of anthocyanin molecules represent a considerable metabolic investment for plant cells. first, there are metabolic costs associated with enzyme production and activity ; at least seven enzymes are involved in the biosynthesis of cyanidin from its precursors, 4-coumaroyl - coa and malonyl - coa. then there is a cost associated with the conjugation of each cyanidin molecule to a monosaccharide molecule. finally, there are costs associated with the transport of cyanidin-3-o - glucoside into the cell vacuole via a tonoplast mg - atp - requiring glutathione carrier. this investment suggests that the accumulation of anthocyanins in leaf cells is unlikely to be an extravagancy without a vital function. neither is it likely that these red pigments are simply the default product of a saturated flavonoid biosynthetic pathway, since the timing of anthocyanin production is usually tightly controlled and often occurs in tissues remote from those associated with other flavonoids. on the contrary, a wealth of recent evidence, both empirical and theoretical, ascribes a remarkable diversity of functions to anthocyanins in leaves, many of them associated with stress responses and some potentially critical to a plant 's survival. anthocyanins are arguably the most versatile of all pigments, their multifarious roles in plant stress responses stemming as much from the physicochemical property of light absorption as from their unique combination of biochemical reactivities. recent advances in our understanding of these various functions are the subject of this review. anthocyanins in vivo absorb the green and yellow wavebands of light, commonly between 500 and 600 nm [20, 21, 22, 23, 24, 25 ]. foliage appears red because of the subtraction of yellow - green light from the spectrum of light reflected from the leaf 's surface. interestingly, the amount of red light that is reflected from red leaves often only poorly correlates to anthocyanin content ; leaf morphology and the amount and distribution of chlorophyll are apparently the stronger determinants of red reflectance. the property of anthocyanins to absorb light provides a mechanism for several important functions in leaves. the red colours of anthocyanic leaves have been proposed both to attract and to repel various animal species. burns and dalen postulated that red - orange autumn foliage of canadian shrub species would accentuate the conspicuousness of black - coloured fruits to birds. experimental manipulations of fruit and background foliage colours confirmed that the black - red contrast was indeed an effective enhancer of fruit - removal rates by avian dispersers. certain insects, on the other hand, seem to preferentially avoid eating red - pigmented leaves. california maple aphids, for example, readily colonise yellow - orange leaves of japanese maples, yet they largely ignore red - leafed individuals. similarly, leaf - cutting ants from the tropical forests of panama browse significantly less on red leaves than on green leaves. to these and other insects leaves that are rich in chlorophyll as well as anthocyanin tend to be brown or even black, mimicking dead foliage or else serving to camouflage leaves against the exposed soil and litter of forest floors [30, 31, 32, 33 ]. even brilliant red or scarlet leaves can appear dark to nonmammalian folivores, which lack red light receptors [5, 34 ]. the gains to be had from herbivore deterrence would offset metabolic costs to the plant associated with anthocyanin biosynthesis. by intercepting the high - energy quanta, anthocyanic cell vacuoles can prevent important photolabile molecules from degradation by green light. an elegant example of this was described recently for the silver beachweed (ambrosia chamissonis), a composite that grows at exposed, sunny locations along the california coast. the plant holds large amounts of thiarubrine a, a potent defence compound that is toxic to insects, bacteria, and fungi. thiarubrine a is photolabile ; even short exposures to visible light and/or ultraviolet radiation render it inactive [36, 37 ]. however, the tissues in a chamissonis that contain thiarubrine a are shielded from light by a sheath of cells containing a mix of two anthocyanins, cyanidin-3-o - glucoside and cyanidin-3-o-(6'-o - malonylglucoside). the anthocyanins absorb quanta that would otherwise lead to the destruction of thiarubrine a, and thereby contribute significantly to the defensive armoury of the plant. when leaves receive more light energy than can be used in photochemistry, they show a characteristic decline in the quantum efficiency of photosynthesis, termed photoinhibition. under severe conditions the chloroplasts generate reactive oxygen species, which have the potential to destroy thylakoid membranes, damage dna, and denature proteins associated with photosynthetic electron transport. anthocyanins have been shown in many plant species to reduce both the frequency and severity of photoinhibition, as well as to expedite photosynthetic recovery [23, 39, 40, 41, 42, 43, 44 ]. in red - osier dogwood (cornus stolonifera), for example, a 30-minute exposure to strong white light reduced the quantum efficiency of photosynthesis by 60% in red leaves, but by almost 100% in acyanic leaves. when the plants were returned to darkness, the red leaves recovered to their maximum potential after only 80 minutes, yet their acyanic counterparts had not achieved the pretreatment state even after six hours. anthocyanins protect leaves from the stress of photoinhibitory light fluxes by absorbing the excess photons that would otherwise be intercepted by chlorophyll b. although red leaves absorb more green light in total, their photosynthetic tissues actually receive fewer quanta than do those of acyanic leaves because the energy absorbed by the cell vacuole can not be transferred to the chloroplasts. as a result, under light - limiting environments the photosynthetic efficiencies of red leaves are often slightly lower than those for acyanic leaves [4, 22, 45, 46, 47, 48, 49 ]. under strong light, however, the anthocyanins serve as a useful optical filter, diverting excess high - energy quanta away from an already saturated photosynthetic electron transport chain. chloroplasts irradiated with light that has first passed through a red filter have been shown to generate fewer superoxide radicals, thereby reducing the propensity for structural damage to the photosystems. the anthocyanins are therefore clearly a useful supplement to other nonphotochemical quenching mechanisms such as the xanthophyll cycle pigments. recent studies involving mutants of arabidopsis thaliana indicate that whereas xanthophylls have a greater role in the protection of plants from short - term light stress, the anthocyanins can be the more effective photoprotectants over the long term. the photoprotection hypothesis potentially explains why the leaves of many deciduous trees turn red in the autumn. as leaves senesce, nitrogen associated with their chloroplasts anthocyanins would protect the degrading chlorophyll from damaging light levels, thereby restricting the formation of reactive oxygen that could jeopardize the resorptive process [2, 8, 9, 23, 51, 52 ]. consistent with this hypothesis, nitrogen resorption has recently been shown to be more efficient in wild - type than in anthocyanin - deficient mutants of three woody species. interest in the flavonoid family has increased in recent years following the observation that these compounds act as sunscreens against potentially damaging uv - b radiation. foliar anthocyanins have generally been included with other flavonoids in this uv - b protective role. consistent with this hypothesis, the anthocyanins, particularly when acylated, absorb strongly in the uv region [54, 55 ], are induced or upregulated in plant tissues in response to uv irradiation [56, 57, 58, 59, 60 ], and mitigate dna damage in uv - b - irradiated cell cultures [61, 62, 63 ]. furthermore, certain anthocyanin - deficient mutants of arabidopsis are hypersensitive to uv - b, and red - leafed coleus varieties retain higher photosynthetic efficiencies after uv irradiation than do green - leafed varieties. notwithstanding this body of evidence, there is now a growing conviction that foliar anthocyanins can not be primarily concerned with uv protection. unlike the colourless flavonoids, the anthocyanins are usually located in the internal mesophyll tissue rather than in the epidermis, the optimal site for uv interception [33, 65 ]. for example, an arabidopsis mutant with enhanced sensitivity to uv radiation was found deficient in certain flavonoids, yet it held normal amounts of anthocyanin. similarly, the responses of brassica rapa mutants to supplementary uv - b treatment were for the most part independent of anthocyanin levels in the leaves. indeed, red - leafed plants of petunia (impatiens) and rice have all been observed to perform significantly worse than their green - leafed counterparts under uv - enriched environments [68, 69, 70 ]. noted that dna damage after prolonged uv treatment was substantially greater in purple - leafed rice than in a near - isogenic green line. to repair uv - damaged dna, plants employ photolyase, an enzyme that uses blue / uv - a light to remonomerise the pyrimidine dimers. the anthocyanins in purple rice prevented the photoactivation of photolyase by absorbing some of the blue / uv - a light incident on the leaves. thus, any short - term gain from the absorption of uv - b by anthocyanins would be offset by their property to absorb visible light and thereby limit the rate of dna repair. the red colours of anthocyanic leaves have been proposed both to attract and to repel various animal species. burns and dalen postulated that red - orange autumn foliage of canadian shrub species would accentuate the conspicuousness of black - coloured fruits to birds. experimental manipulations of fruit and background foliage colours confirmed that the black - red contrast was indeed an effective enhancer of fruit - removal rates by avian dispersers. certain insects, on the other hand, seem to preferentially avoid eating red - pigmented leaves. california maple aphids, for example, readily colonise yellow - orange leaves of japanese maples, yet they largely ignore red - leafed individuals. similarly, leaf - cutting ants from the tropical forests of panama browse significantly less on red leaves than on green leaves. to these and other insects leaves that are rich in chlorophyll as well as anthocyanin tend to be brown or even black, mimicking dead foliage or else serving to camouflage leaves against the exposed soil and litter of forest floors [30, 31, 32, 33 ]. even brilliant red or scarlet leaves can appear dark to nonmammalian folivores, which lack red light receptors [5, 34 ]. the gains to be had from herbivore deterrence would offset metabolic costs to the plant associated with anthocyanin biosynthesis. by intercepting the high - energy quanta, anthocyanic cell vacuoles can prevent important photolabile molecules from degradation by green light. an elegant example of this was described recently for the silver beachweed (ambrosia chamissonis), a composite that grows at exposed, sunny locations along the california coast. the plant holds large amounts of thiarubrine a, a potent defence compound that is toxic to insects, bacteria, and fungi. thiarubrine a is photolabile ; even short exposures to visible light and/or ultraviolet radiation render it inactive [36, 37 ]. however, the tissues in a chamissonis that contain thiarubrine a are shielded from light by a sheath of cells containing a mix of two anthocyanins, cyanidin-3-o - glucoside and cyanidin-3-o-(6'-o - malonylglucoside). the anthocyanins absorb quanta that would otherwise lead to the destruction of thiarubrine a, and thereby contribute significantly to the defensive armoury of the plant. when leaves receive more light energy than can be used in photochemistry, they show a characteristic decline in the quantum efficiency of photosynthesis, termed photoinhibition. under severe conditions the chloroplasts generate reactive oxygen species, which have the potential to destroy thylakoid membranes, damage dna, and denature proteins associated with photosynthetic electron transport. anthocyanins have been shown in many plant species to reduce both the frequency and severity of photoinhibition, as well as to expedite photosynthetic recovery [23, 39, 40, 41, 42, 43, 44 ]. in red - osier dogwood (cornus stolonifera), for example, a 30-minute exposure to strong white light reduced the quantum efficiency of photosynthesis by 60% in red leaves, but by almost 100% in acyanic leaves. when the plants were returned to darkness, the red leaves recovered to their maximum potential after only 80 minutes, yet their acyanic counterparts had not achieved the pretreatment state even after six hours. anthocyanins protect leaves from the stress of photoinhibitory light fluxes by absorbing the excess photons that would otherwise be intercepted by chlorophyll b. although red leaves absorb more green light in total, their photosynthetic tissues actually receive fewer quanta than do those of acyanic leaves because the energy absorbed by the cell vacuole can not be transferred to the chloroplasts. as a result, under light - limiting environments the photosynthetic efficiencies of red leaves are often slightly lower than those for acyanic leaves [4, 22, 45, 46, 47, 48, 49 ]. under strong light, however, the anthocyanins serve as a useful optical filter, diverting excess high - energy quanta away from an already saturated photosynthetic electron transport chain. chloroplasts irradiated with light that has first passed through a red filter have been shown to generate fewer superoxide radicals, thereby reducing the propensity for structural damage to the photosystems. the anthocyanins are therefore clearly a useful supplement to other nonphotochemical quenching mechanisms such as the xanthophyll cycle pigments. recent studies involving mutants of arabidopsis thaliana indicate that whereas xanthophylls have a greater role in the protection of plants from short - term light stress, the anthocyanins can be the more effective photoprotectants over the long term. the photoprotection hypothesis potentially explains why the leaves of many deciduous trees turn red in the autumn. as leaves senesce, nitrogen associated with their chloroplasts anthocyanins would protect the degrading chlorophyll from damaging light levels, thereby restricting the formation of reactive oxygen that could jeopardize the resorptive process [2, 8, 9, 23, 51, 52 ]. consistent with this hypothesis, nitrogen resorption has recently been shown to be more efficient in wild - type than in anthocyanin - deficient mutants of three woody species. interest in the flavonoid family has increased in recent years following the observation that these compounds act as sunscreens against potentially damaging uv - b radiation. foliar anthocyanins have generally been included with other flavonoids in this uv - b protective role. consistent with this hypothesis, the anthocyanins, particularly when acylated, absorb strongly in the uv region [54, 55 ], are induced or upregulated in plant tissues in response to uv irradiation [56, 57, 58, 59, 60 ], and mitigate dna damage in uv - b - irradiated cell cultures [61, 62, 63 ]. furthermore, certain anthocyanin - deficient mutants of arabidopsis are hypersensitive to uv - b, and red - leafed coleus varieties retain higher photosynthetic efficiencies after uv irradiation than do green - leafed varieties. notwithstanding this body of evidence, there is now a growing conviction that foliar anthocyanins can not be primarily concerned with uv protection. unlike the colourless flavonoids, the anthocyanins are usually located in the internal mesophyll tissue rather than in the epidermis, the optimal site for uv interception [33, 65 ]. for example, an arabidopsis mutant with enhanced sensitivity to uv radiation was found deficient in certain flavonoids, yet it held normal amounts of anthocyanin. similarly, the responses of brassica rapa mutants to supplementary uv - b treatment were for the most part independent of anthocyanin levels in the leaves. indeed, red - leafed plants of petunia (impatiens) and rice have all been observed to perform significantly worse than their green - leafed counterparts under uv - enriched environments [68, 69, 70 ].. noted that dna damage after prolonged uv treatment was substantially greater in purple - leafed rice than in a near - isogenic green line. to repair uv - damaged dna, plants employ photolyase, an enzyme that uses blue / uv - a light to remonomerise the pyrimidine dimers. the anthocyanins in purple rice prevented the photoactivation of photolyase by absorbing some of the blue / uv - a light incident on the leaves. thus, any short - term gain from the absorption of uv - b by anthocyanins would be offset by their property to absorb visible light and thereby limit the rate of dna repair. anthocyanins diminish the oxidative load in a leaf simply by filtering out yellow - green light, since the majority of reactive oxygen in plant cells is derived from the excitation of chlorophyll. purified solutions scavenge almost all species of reactive oxygen and nitrogen with an efficiency up to four times greater than those of ascorbate and -tocopherol [72, 73, 74 ]. recent experimental evidence indicates that this antioxidant potential is indeed utilised by plant cells. in arabidopsis, for example, strong light and low temperatures caused more lipid peroxidation in anthocyanin - deficient mutants than in wild - type plants. similarly, upon gamma irradiation, only those arabidopsis plants that contained both anthocyanin and ascorbic acid were able to grow and flower normally. microscopic examinations of wounded leaf peels have shown that red - pigmented cells eliminate h2o2 significantly faster than do green cells. it is not clear, however, whether scavenging occurs predominantly by the red tautomers of anthocyanin found inside the cell vacuole, or else by the colourless tautomers in the cytosol. both forms have impressive antioxidant potentials [77, 78, 79 ]. in a model in vitro system, the colourless tautomers of cyanidin 3-(6-malonyl)glucoside were found capable of scavenging up to 17% of the superoxide radicals generated by irradiated chloroplasts. given their proximity to the organelle sources of reactive oxygen, it may be that the cytosolic anthocyanins, rather than those in the cell vacuole, provide the greater contribution to antioxidant defence. the degree to which anthocyanins contribute to the arsenal of low - molecular - weight antioxidants (lmwa) varies among species. in the young, red leaves of elatostema rugosum, an understorey herb from new zealand, anthocyanins are the predominant phenolic component of the lmwa pool. in contrast, the red- and green - leafed morphs of the canopy tree quintinia serrata both hold hydroxycinnamic acids as their most concentrated lmwa. thus it would seem that anthocyanin biosynthesis can enhance but is not usually a prerequisite for protection from oxidative stress. the induction of foliar anthocyanins has been implicated in the acquisition of tolerance to many different kinds of environmental stressors [11, 80, 81 ]. anthocyanins, for example, are associated with enhanced resistance to the effects of chilling and freezing [82, 83, 84, 85, 86 ], to heavy metal contamination [87, 88, 89, 90 ], to desiccation [91, 92, 93 ], and to wounding [76, 94, 95 ]. it is not clear at this stage whether the apparent ameliorative properties stem from one or more types of mechanism. chalker - scott [11, 80 ] provided a compelling case for a generalised role of anthocyanins as osmoregulators in plant cells, since most types of suboptimal environments induce water stress, either directly or indirectly. others have argued that the photoprotective or the antioxidant properties of anthocyanins are paramount. regardless of their mechanism, it is clear that anthocyanins offer multifaceted, versatile, and effective protection to plants under stress.	anthocyanins, the pigments responsible for spectacular displays of vermilion in the leaves of deciduous trees, have long been considered an extravagant waste of a plant 's resources. contemporary research, in contrast, has begun to show that the pigments can significantly influence the way a leaf responds to environmental stress. anthocyanins have been implicated in tolerance to stressors as diverse as drought, uv - b, and heavy metals, as well as resistance to herbivores and pathogens. by absorbing high - energy quanta, anthocyanic cell vacuoles both protect chloroplasts from the photoinhibitory and photooxidative effects of strong light, and prevent the catabolism of photolabile defence compounds. anthocyanins also mitigate photooxidative injury in leaves by efficiently scavenging free radicals and reactive oxygen species. far from being a useless by - product of the flavonoid pathway, these red pigments may in some instances be critical for plant survival.
hemostats, sealants, and adhesives are gaining increasing acceptance in surgical practice in the united states and presently represent a multi - billion dollar industry. a set of definitions and nomenclature are helpful in order to facilitate the safest and most cost effective use of these materials.13 a hemostat is designed to cause blood to clot and requires the presence of blood in the operative field to be effective. they polymerize independently and may work best when blood is not present in the field. finally, adhesives function as self polymerizing glues capable of causing tissue adherence and often work best in a relatively dry area. importantly, although not hemostats, both sealants and adhesives can have major hemostatic effects when used to seal or glue blood vessels together as their application can prevent major blood loss. the three groups : hemostats ; sealants ; and adhesives can be broken down into categories (table 1) which are named to clarify the origin of the agents within each respective category. the hemostat group for example is divided into four categories : mechanical, active, flowable, and fibrin sealant. classes of agents can also be described.2,3 for the purposes of simplification and this review, classes for the categories of the fibrin sealants only have been provided (table 1) to facilitate understanding the role of this material in rhytidectomy. the entire classification system for all hemostats, sealants, and adhesives is available in the literature2 including a recent update.3 fibrin sealant was the first of many new agents approved by the food and drug administration (fda) starting in 1998 and serves as an example for several reasons. first, as the initial approved agent it helped to establish new fda regulatory review pathways for both biologics and devices.3 second, its introduction began to educate surgeons, academics, and marketers that proper use of this type of material actually requires some skill and education.4 third, fibrin sealant is the only product which has achieved fda approval in all three groups as a hemostat, sealant, and adhesive.5 finally, the material has been demonstrated to be effective in randomized prospective multicenter clinical trials in multiple different surgical specialties. at present fibrin sealants are derived from human pooled plasma and manufacturers take multiple steps to increase safety and eliminate the possibility of viral or prion disease transmission. fibrin sealants come in a variety of liquid formulations which may require mixing, thawing, and applicator assembly.13 there is one new form of fibrin sealant approved as a hemostat which combines dry fibrinogen and thrombin on an equine collagen patch and is immediately available for use out of the package.3 fibrin sealants are among the more costly hemostats, sealants, and adhesives and the fibrin sealant patch is the most costly hemostat.3 laboratory68 and clinical9,10 evidence supporting the use of fibrin sealant as a means of simultaneously achieving hemostasis, sealing blood vessels and lymphatics, and attaching skin flaps to eliminate potential space and prevent seroma formation during neoplasm resections was published in the 19801990s. clinical reports of successful use of fibrin sealant for similar indications during rhytidectomy also appeared in the plastic surgical literature at the same time.1114 multiple liquid fibrin sealants are now approved by the fda as broad label hemostats during surgical procedures (tisseel ; baxter, westlake village, ca)15,16 (evicel ; ethicon / j&j, somerville, nj)17 (vitagel ; orthovita, malvern, pa)18 and a patch is approved for hemostasis during cardiac surgery only (tachosil ; baxter).19 one fibrin sealant product is approved as sealant for colon sealing at the time of colostomy closure (tisseel).16 in addition, one fibrin sealant is approved as an adhesive for attachment of skin grafts at the time of burn wound debridement and skin flap adherence at the time of rhytidectomy (artiss ; baxter).20 this latter material will be presented in depth in terms of safety, efficacy, usability, and cost in this review as it is the only agent approved by the fda as an adhesive during rhytidectomy. it is notable, however, that the other liquid fibrin sealants could potentially be used off label in this same indication by first diluting the thrombin concentration in the product to less than 5 iu / ml before combining it with fibrinogen to create a slowly polymerizing adhesive. finally, the ability to use an individual patient s own autologous plasma in the form of platelet rich or poor plasma as a fibrinogen source should be mentioned. this material can then be combined with free standing thrombin of bovine (thrombin - jmi ; king pharmaceuticals, bristol, tn), human pooled plasma (evithrom ; ethicon / j&j) or recombinant (recothrom ; zymogenetics / bms, seattle, wa) origin to make fibrin sealant. although not specifically approved by the fda for producing fibrin sealant or for use in rhytidectomy flap adherence, multiple device separation systems exist for obtaining the autologous plasma from the patient s blood (amicus ; fenwal, round lake, il ; cell saver ; haemonetics, braintree, ma ; harvest ; smith and nephew, memphis, tn ; magellan ; medtronic, minneapolis, mn ; recover ; biomet biologics, warsaw, in ; symphony ; depuy, raynham, ma). advantages of this method include reduced cost and reduction of viral or prion disease transmission risk particularly if used with recombinant thrombin. this choice of thrombin eliminates the risk of immune mediated coagulopathy associated with bovine thrombin and viral or prion disease transmission associated with pooled plasma products. disadvantages of the use of the separation systems include the time and personnel needed to prepare the material and the lower fibrinogen concentration obtained as compared to commercial fibrin sealants. again, it is important to decrease the concentration of the free standing thrombin used to 14.0 for human immunodeficiency virus in thrombin to a low of 3.9 for mice minute virus (a model for parvovirus b19) in fibrinogen. no cases of aids or hepatitis have been documented in the literature secondary to fibrin sealant in 20 years of use.23 there have been two different literature reports of parvovirus b19 transmission secondary to fibrin sealants.24,25 parvovirus infections may cause severe illness in patients who are pregnant, immune - compromised, or with increased erythropoiesis. the illness is characterized by fever, drowsiness, chills, and runny nose followed in two weeks by a rash and joint pains. it may be a source of hypersensitivity reactions and anaphylaxis that are more frequent on re - exposure, but can occur on first use.20 additional safety issues with this fibrin sealant product include : air embolism associated with use of a pressurized spray applicator (recommended pressure 500 iu / ml), the material is indicated for hemostasis of bleeding which is non suturable and non cauterizable. thus, in rhytidectomy, all bleeding that can be treated by conventional methods should be addressed prior to using the fibrin sealant as a slowly polymerizing adhesive employing a low thrombin concentration (500 iu / ml), the material is indicated for hemostasis of bleeding which is non suturable and non cauterizable. thus, in rhytidectomy, all bleeding that can be treated by conventional methods should be addressed prior to using the fibrin sealant as a slowly polymerizing adhesive employing a low thrombin concentration (< 5 iu / ml). any hemostatic benefits to using the fibrin sealant in this setting may be as a result of a sealing effect which may help to keep small vessels closed as vasospasm subsides after the procedure. prior to using the fibrin sealant all preparations should be made to avoid further manipulations of the flaps or skin following adhesive application. all sutures should be in place including anchoring sutures which then should be tied as soon as possible after application of the fibrin sealant. any suture lines should be preplaced as a lattice work (figure 3, top portion) which can be quickly tightened after sealant application. if drains are to be placed, they should be in position prior to fibrin sealant application and should be protected so as not to be clogged by the addition of the adhesive. the drains may become unnecessary as experience and in the method of fibrin sealant use is gained and the drains can then be omitted altogether. during the application of fibrin sealant itself, it is important to assure that all flaps and surfaces are covered with the adhesive using appropriate length cannulas or spray devices (figure 5). the adhesive should be applied as an even thin layer to facilitate the maximum rate of flap healing and to facilitate nutrient diffusion in order to avoid graft tissue necrosis. after the placement of the sealant, the flaps should be quickly positioned and adjusted as necessary so that any manipulation can be avoided after the first 60 seconds of polymerization time. as soon as possible after application of fibrin sealant, gentle pressure should be applied to all flaps (figure 3, bottom portion) for at least 3 minutes to assure bonding between the flap and underlying tissues. it is critical not to do any further flap or skin manipulation after this period of initial bonding. the lifting of skin for placement of additional sutures or manipulation with forceps as well as any further movement of the flaps needs to be strictly avoided. this is necessary because breaking of the fibrin sealant bonds at this point will create a smooth non sticky polymerized fibrin sealant interface between the flap and underlying tissues which has been shown to be an excellent anti - adhesive.44 the placement of an anti - adhesive in this location may actually increase the formation of seromas and fluid drainage. thus clearly arterial hypertension should be minimized and nausea and vomiting should be prevented if possible and treated if present to decrease the potential for venous pressure elevation. fibrin sealant is an important addition to the surgeons armamentarium and is useful in rhytidectomy. this review has characterized the material and provided a nomenclature for understanding the capabilities of this unique hemostat, sealant, and adhesive. the use of fibrin sealant in rhytidectomy and the currently approved product for this indication have been reviewed in depth. the author s suggestions to facilitate the most effective use of the agent have been provided and it should be emphasized that there is a gentle learning curve to master in order to use this powerful multiple capacity product most effectively.	the purpose of this review is to clarify the present use of fibrin sealant in rhyditectomy procedures and help maximize the appropriate and safe application of this material. a set of terms and definitions for hemostats, sealants, and adhesives based on group, category, and class will be employed to highlight the specific capabilities of fibrin sealant. fibrin sealant has now emerged as an example of maximizing the usefulness of a surgical agent and is the only product with food and drug administration approval in all three groupings : hemostats ; sealants ; and adhesives. a variety of manufacturers fibrin sealant products are available including multiple liquids and one patch. a single liquid product is now specifically indicated for skin flap adherence during rhytidectomy. the unique characteristic of this particular two component fibrin sealant adhesive agent is its slower polymerization rate as a result of a low thrombin concentration which when combined with fibrinogen permits adequate time for manipulation of flaps and tissues prior to final fixation. in addition to its flap adherence and potential space elimination capability, fibrin sealant is also an excellent blood clotting agent and can seal tissues to prevent lymphatic leak or serous fluid accumulation. thus, it is almost ideally suited to reduce the occurrence of fluid accumulation, hematomas, ecchymoses, and swelling, as well as to possibly eliminate the need for drains following rhytidectomy. a literature review of fibrin sealant in rhytidectomy is included to help define the current state of its clinical use. the author s recommendations for the best use of this material during facial procedures are also provided.
the united nations children 's fund (unicef) plays a leading role in the collection, compilation, analysis and dissemination of data to inform sound policies, legislation, and programmes for promoting children 's rights and wellbeing, and for global monitoring of progress towards international goals and targets. to this end, unicef maintains a set of global databases on key topics affecting children and women covering nearly 200 countries (table 1). table 1summary of unicef global databasescountries covered197 countries and territories in the following regions : eastern and southern africawest and central africamiddle east and north africasouth asiaeast asia and pacificlatin america and caribbeancentral and eastern europe and central independent statesindustrialized countriespopulation coveredindicators cover a wide range of demographic groups, including primarily : newbornsinfantschildren under fiveschool - age childrenadolescents and young adultswomen of reproductive agedata typeglobal databases compiled from a broad array of sources : nationally representative household surveyscensusesvital registration systemsadministrative recordsreports from national ministries of health / educationestimates calculated by un inter - agency groupsestimates provided by other un agenciesfrequency of data compilationthe databases are regularly updated, with a comprehensive update once per year. for the main source of data, nationally representative household surveys, we expect that many countries will have new data available every 35 yearstopicschild survivalchild healthmaternal healthnutritionimmunizationwater and sanitationhiv / aidseducationchild protectionadolescentsearly childhood developmentfunding sourcesunicef is supported entirely by the voluntary contributions of individuals, foundations, corporations, non - governmental organizations and governments summary of unicef global databases eastern and southern africa west and central africa middle east and north africa east asia and pacific latin america and caribbean central and eastern europe and central independent states industrialized countries adolescents and young adults women of reproductive age nationally representative household surveys vital registration systems administrative records reports from national ministries of health / education estimates calculated by un inter - agency groups estimates provided by other un agencies early childhood development the countries in developing regions are grouped geographically as follows : eastern and southern africa, west and central africa, middle east and north africa, south asia, east asia and pacific, latin america and caribbean, and central and eastern europe and central independent states. data availability varies both across countries and across indicators, but for most indicators used for global monitoring, our databases include estimates for countries covering at least 80% of the developing world. for the standard indicators included in major household surveys, there are updates from about 1525 countries each year. beyond household survey data, administrative data are also included in the global databases, especially in the areas of education and maternal health. these data can often fill in the gaps in countries that do not have data from household surveys, particularly industrialized countries. the inter - agency estimates of child mortality, maternal mortality, immunization and water and sanitation generally cover all countries in both the developing and industrialized world, and are interpolated to a common reference year either annually or every few years. keeping the global databases up to date is an on - going process in unicef s statistics and monitoring section, which is punctuated by a concentrated effort once a year. each spring, unicef undertakes an exercise called country reporting on indicators for the goals (cring) in which field offices are asked to review the existing data for a selection of 80 indicators and provide any updates for their country. updates received by the cring deadline can be reviewed for inclusion in the databases before the statistical tables are finalized for the next edition of unicef s annual flagship publication, the state of the world s children, although new data submissions can be considered at any point throughout the year. in 2012, 152 country offices were invited to participate in cring, and of those, 142 contributed to the exercise. although unicef asks field offices to review the data every year, we expect that for most countries, there will not be annual updates for many of the key indicators. a large share of the data in our databases comes from nationally representative household surveys such as the unicef - supported multiple indicator cluster survey (mics) and the united states agency for international development - supported demographic and health survey, which have a periodicity of 35 years. however, other household surveys focusing on a specific sector such as nutrition, malaria, reproductive health or hiv / aids may be available in the interim, and administrative and vital registration data may be available yearly. beyond the cring exercise, unicef also updates the global databases with inter - agency estimates and data provided by other un agencies. for example, inter - agency estimates of under - five mortality and immunization coverage are produced every year, and harmonized demographic estimates are provided by the un department of economic and social affairs (undesa) population division every 2 years. unicef leads the global mics household survey programme, which supports countries to collect data on a wide range of indicators. experience with the implementation of these surveys, and their analysis, is invaluable in evaluating measurement methods and assessing the data quality of submissions to the global database. this exercise relies on the wide network of unicef field offices, which each draw on their knowledge of local data collection efforts to contribute the latest available data to headquarters. data submissions undergo a rigorous data quality assurance process, whereby each data point is evaluated using a set of objective criteria before it is entered in the database. sector specialists review source documentation for details, including sampling method, indicator definitions and the precise wording used in questionnaires. this review includes consultations with the country and regional offices, colleagues from other un agencies and academic partners. programme specialists within unicef often participate in the review as well by providing context to interpret the data. data compilation for industrialized countries differs from the cring process because there are not field offices to contribute to cring. for these countries, sector specialists need to seek out data from alternative sources, including administrative records, vital registration and censuses. unicef also participates in a number of un inter - agency millennium development goal (mdg) monitoring groups, which contribute estimates to the global databases across a number of sectors. one purpose of these groups is to harmonize estimates across agencies, especially for use in monitoring progress towards the goals. although each inter - agency group has a slightly different methodology, the basic procedure is to collect underlying data, make adjustments if necessary (e.g. for definitional differences, under - reporting and misclassification) and use statistical modelling to generate estimates for all countries for a common reference year. finally, the data for some indicators in the global databases are provided by partner un agencies. this cooperation prevents a duplication of efforts in cases in which another agency already has the expertise and capacity to produce the estimates needed. unaids provides databases with estimates of hiv incidence and prevalence, as well as some indicators related to treatment. for education, the united nations educational, scientific and cultural organization institute for statistics provides databases based on administrative data, including enrolment, survival through primary school, transition to secondary school and youth literacy. below is an overview of the indicators included in unicef s global databases, sorted by compilation method and by sector (tables 2 and 3). table 2overview of indicators : data from primary sources included directly in databasefor the following indicators, estimates from surveys, administrative sources, vital registration or censuses are identified and included directly in the database : child healthhousehold ownership of insecticide - treated netsunder - fives sleeping under insecticide - treated netsanti - malarial treatment of fevercare seeking for suspected pneumoniaantibiotic treatment of suspected pneumoniatreatment with oral rehydration solution for diarrhoeatreatment with oral rehydration therapy or increased fluids and continued feeding for diarrhoeamaternal healthcontraceptive prevalence rateantenatal care (1 + visits and 4 + visits)skilled attendance at birthinstitutional deliveriescaesarean section ratebirths by age 18 yearsintermittent preventive treatment during pregnancy (malaria)nutritionanthropometry (underweight, stunting, wasting and overweight)low birthweightearly initiation of breastfeedingexclusive breastfeedingintroduction to solid, semi - solid or soft foodscontinued breastfeeding (1215 months and 2023 months)households consuming iodized salteducationprimary net attendance ratiosurvival to last grade of primary schoolyouth literacy ratesecondary net attendance ratiohiv / aidscomprehensive knowledge of hivsex before age 15 yearssex with multiple partnerscondom use at last sex among those with multiple partnerscondom use at last higher - risk sexorphan school attendance ratiochild protectionbirth registrationchild labourchild marriagefemale genital mutilation / cuttingchild disabilitychild disciplinedomestic violenceattitudes towards domestic violenceearly childhood developmentattendance in early childhood educationadult support for learningfather s support for learninglearning materials at home (books and playthings)children left in inadequate care table 3overview of indicators : estimates from statistical modelling exercises included in databasesfor the following indicators, primary data sources are identified and used in statistical modelling exercises by inter - agency estimation groups : child survival (igme)under - five mortality rateinfant mortality rateneonatal mortality ratewater and sanitation (joint monitoring programme for water supply and sanitation)use of improved drinking water sourcesuse of improved sanitation facilitiesimmunization (who and unicef estimates of national immunization coverage)tuberculosisdiphtheria, pertussis and tetanuspoliomeasleshepatitis bhaemophilus influenzae type btetanusmaternal health (maternal mortality estimation inter - agency group)maternal mortality ratiolifetime risk of maternal death overview of indicators : data from primary sources included directly in database household ownership of insecticide - treated nets under - fives sleeping under insecticide - treated nets anti - malarial treatment of fever care seeking for suspected pneumonia antibiotic treatment of suspected pneumonia treatment with oral rehydration solution for diarrhoea treatment with oral rehydration therapy or increased fluids and continued feeding for diarrhoea contraceptive prevalence rate antenatal care (1 + visits and 4 + visits) skilled attendance at birth institutional deliveries caesarean section rate births by age 18 years intermittent preventive treatment during pregnancy (malaria) anthropometry (underweight, stunting, wasting and overweight) early initiation of breastfeeding exclusive breastfeeding introduction to solid, semi - solid or soft foods continued breastfeeding (1215 months and 2023 months) households consuming iodized salt primary net attendance ratio survival to last grade of primary school secondary net attendance ratio comprehensive knowledge of hiv sex before age 15 years sex with multiple partners condom use at last sex among those with multiple partners condom use at last higher - risk sex orphan school attendance ratio female genital mutilation / cutting attitudes towards domestic violence attendance in early childhood education adult support for learning father s support for learning learning materials at home (books and playthings) children left in inadequate care overview of indicators : estimates from statistical modelling exercises included in databases under - five mortality rate infant mortality rate neonatal mortality rate use of improved drinking water sources use of improved sanitation facilities diphtheria, pertussis and tetanus haemophilus influenzae type b maternal mortality ratio lifetime risk of maternal death the indicators named in tables 2 and 3 are not meant to represent an exhaustive list, rather an overview of key indicators used for analysis and reporting. the unicef global databases are used extensively for monitoring the situation of children and women at both national and global levels. the data are used in situation analyses at the country level to track progress towards development targets and identify potential areas for intervention. monitoring trends is also done at the global level, and unicef s data are heavily relied on in trend analysis, including mdg monitoring, and the countdown to 2015 initiative. in tracking mdg 4 (reduce child mortality), unicef leads the un inter - agency group for child mortality estimation (igme), which reported that the annual number of under - five deaths dropped from 12 million in 1990 to 6.9 million in 2011. although the average annual rate of reduction has accelerated (from 1.8% a year from 19902000 to 3.2% a year from 20002011), the progress is insufficient to meet the mdg target of reducing the under - five mortality rate by two - thirds by 2015. unicef s databases are also frequently used as the basis for data - driven policy advocacy reports, like the recently published pneumonia and diarrhoea : tackling the deadliest diseases for the world s poorest children. the report featured extensive data analysis on these leading causes of child deaths, including trends in intervention coverage and disparity analysis by residence, household wealth and sex. a key finding was that prevention and treatment of these illnesses remain low, particularly among children in rural areas and from poorer households. for example, in sub - saharan africa and south asia the two regions with the most diarrhoea deaths children with diarrhoea are unlikely to receive solutions made from oral rehydration salts (ors) (coverage of 30 and 33%, respectively) ; the children in the poorest households in the poorest countries are much less likely to receive ors than children in the richest households. this report made the case that preventable child deaths from pneumonia and diarrhoea are concentrated among the most disadvantaged children, and that many lives could be saved with an equity - focused approach. analyses based on the global databases are also often featured in peer - reviewed journal articles. in the area of child protection, the unicef database on child discipline was drawn on for an analysis published in child abuse and neglect focusing on caregivers attitudes towards physical punishment. the analysis of household survey data from 34 countries found that although the majority of caregivers did not think physical punishment was necessary to raise a child, acceptance of physical punishment was common in many countries, and was often associated with background characteristics including rural residence and less household wealth. across most countries, attitudes were correlated with practices : children whose primary caregivers had accepting attitudes towards physical punishment were more likely to experience such discipline. some recent publications have focused on health issues affecting the world s 1.2 billion adolescents, which is an area garnering increasing attention and data collection efforts. analysis of the unicef databases revealed that child marriage (before age 18 years) is common in south asia and sub - saharan africa, with the highest worldwide rate found in niger, where three of four young women were married before age 18 years. rates of early childbearing tend to be higher in these countries where early marriage is common. further, adolescents particularly adolescent girls in hiv - endemic countries are not likely to have a comprehensive knowledge of hiv transmission and are unlikely to use condoms, putting their reproductive health at risk. unicef s global databases include nationally representative data on a broad selection of indicators related to the well - being of children and women. disaggregated data are available by sex, residence and wealth quintile, allowing for easy disparity analysis. the indicators are standardized and aligned with mdg goals and targets ; therefore, trend analysis can be relied on as an indication of progress towards these internationally agreed - on goals. not all countries collect the data used for global monitoring, and in some cases, submissions can not be included, e.g. if the indicator definitions are not aligned with the standard, or if the survey sample is not nationally representative. although unicef s databases include disaggregation by sex, residence and wealth quintile, geographical sub - regions for each country are not yet part of the databases. data coverage for high - income countries also tends to be less complete for many indicators, especially those for which standardized household survey data are the primary source. the compilation of the global databases draws on the strength of unicef s wide network of field offices, where those closest to the local situation can provide context and keep informed about data collection efforts. at headquarters, the statistics and monitoring section also benefits from the vast knowledge of household survey methodology among the staff in the mics survey programme. the unicef statistical website, www.childinfo.org, serves as a repository of data and additional resources. on childinfo, users can view country-, regional- and global - level data in statistical tables either directly on the web page or by downloading the tables in an excel file. figure 1women who live in urban areas are more likely than women in rural areas to be assisted during delivery by a skilled birth attendant. source : unicef global databases 2011, from multiple indicator cluster surveys (mics), demographic and health surveys (dhs), and other nationally representative sources. note : global estimates are based on a subset of 96 countries, covering 80% of births in the world. regional estimates represent data from countries covering at least 50% of regional births figure 2 in india, a greater reduction in underweight prevalence occurred in the richest 20% of households than in the poorest 20%. note : prevalence estimates are calculated according to the national center for health statistics reference population, as there were insufficient data to calculate trend estimates by household wealth according to world health organization child growth standards. estimates are age - adjusted to represent children 059 months in each survey women who live in urban areas are more likely than women in rural areas to be assisted during delivery by a skilled birth attendant. source : unicef global databases 2011, from multiple indicator cluster surveys (mics), demographic and health surveys (dhs), and other nationally representative sources. note : global estimates are based on a subset of 96 countries, covering 80% of births in the world. regional estimates represent data from countries covering at least 50% of regional births in india, a greater reduction in underweight prevalence occurred in the richest 20% of households than in the poorest 20%. note : prevalence estimates are calculated according to the national center for health statistics reference population, as there were insufficient data to calculate trend estimates by household wealth according to world health organization child growth standards. estimates are age - adjusted to represent children 059 months in each survey the website provides a number of additional resources, including methodological information, as well as links to download data - driven publications and statistical country profiles. it also includes links to all of the online resources powered by devinfo, a database management system that facilitates the organization, storage and display of data. these include cmeinfo (child mortality) and mmeinfo (maternal mortality), which are tools for the dissemination of the un inter - agency estimates of child and maternal mortality. unicef data are also available in a number of publications, the most comprehensive of which is the state of the world s children, which includes a set of data - rich statistical tables. this annual compendium presents the most recent key statistics on children for the world s countries, territories and regions. the number of topics covered has grown over the years, and as of 2012, there were 13 tables, each covering key indicators related to a different theme : basic indicators, nutrition, health, hiv / aids, education, demographics, economic indicators, women, child protection, rate of progress, adolescents and equity by both residence and wealth. the tables provide some disaggregated data by sex, residence and wealth quintile, as well as selected trends. each table is accompanied by indicator definitions and main data sources, and there is an extensive section of data notes with methodological information and guidance on data interpretation. the full report, including statistical tables of the state of the world s children, is available for download at http://www.unicef.org / sowc2012/. the tables are also available individually in both pdf and excel formats. further, the website includes a link to sowcinfo, which is a devinfo application featuring the data from the publication. with sowcinfo (www.devinfo.info/sowc), users can create tables, graphs and maps using selected data from the statistical tables.	the united nations children s fund (unicef) plays a leading role in the collection, compilation, analysis and dissemination of data to inform sound policies, legislation and programmes for promoting children s rights and well - being, and for global monitoring of progress towards the millennium development goals. unicef maintains a set of global databases representing nearly 200 countries and covering the areas of child mortality, child health, maternal health, nutrition, immunization, water and sanitation, hiv / aids, education and child protection. these databases consist of internationally comparable and statistically sound data, and are updated annually through a process that draws on a wealth of data provided by unicef s wide network of > 150 field offices. the databases are composed primarily of estimates from household surveys, with data from censuses, administrative records, vital registration systems and statistical models contributing to some key indicators as well. the data are assessed for quality based on a set of objective criteria to ensure that only the most reliable nationally representative information is included. for most indicators, data are available at the global, regional and national levels, plus sub - national disaggregation by sex, urban / rural residence and household wealth. the global databases are featured in unicef s flagship publications, inter - agency reports, including the secretary general s millennium development goals report and countdown to 2015, sector - specific reports and statistical country profiles. they are also publicly available on www.childinfo.org, together with trend data and equity analyses.
in patients with neurogenic bladders as the result of spinal cord injury or spinal disease, complications such as upper urinary tract damage, infection, stone formation, and incontinence commonly occur. in such patients, procedures that are intended to decrease bladder pressure are required to prevent further deterioration of the upper tract. among such procedures, augmentation cystoplasty (ac) incontinence can occur as the result of sphincteric incompetence or bladder factors such as involuntary detrusor contraction (idc) or poor compliance. in patients with sphincteric incontinence, pharmaceutical agents can be prescribed as a first - line treatment. when the incontinence is refractory to medication, surgical treatment such as slings, artificial urinary sphincters (auss), or injection of bulking agents can be considered. in many patients, however, consensus over whether concomitant or staged anti - incontinence surgery with ac is advisable in patients with sphincteric incontinence has not yet been established. the surgical management of neurogenic sphincteric incontinence is still under debate, and an effective method for long - term continence in subjects with neurogenic sphincteric incompetence remains to be found. this patient underwent ac for neurogenic bladder and bladder neck suspension for his incompetent urinary sphincter with satisfactory results. also, chartier - kastler. reported lower complication rates in patients who underwent ac and aus implantation simultaneously than in patients who underwent staged operations. dave and salle analyzed a kropp - salle bladder neck repair performed in 9 children, 6 of whom had myelodysplasia. eighty - nine percent of these patients received concomitant ac, and 88% were dry postoperatively. with concomitant surgery, however, there is a possibility of a higher incidence of morbidities in patients whose incontinence might be resolved with bladder outlet procedures performed as a single procedure. selecting the treatment method for patients with neurogenic sphincter incontinence is a challenging clinical issue. with the goal of obtaining maximal surgical efficacy with the minimum inconvenience to patients, we evaluated patients with sphincteric incontinence who underwent ac as a single procedure at our center. we aimed to determine whether concomitant bladder neck reconstruction was necessary when performing ac in patients with neurogenic bladder. we retrospectively investigated clinical data from 35 patients who underwent ac from january 2006 to september 2010. medical history, preoperative and postoperative fluoroscopic urodynamic study (fuds) parameters, and responses to an incontinence questionnaire (iciq korean version) were reviewed. the iciq questionnaires were previously validated linguistically by bilingual translators and were approved for use by the international consultation on incontinence modular questionnaire advisory board (http://www.iciq.net/index.html). we analyzed these patients to see whether ac performed as a single procedure could improve their continence status. of the patients, 24 with urinary incontinence who filled out both a preoperative and a postoperative iciq questionnaire (17 males and 7 females) were eligible for analysis. of these 24 patients, a final analysis was performed on 17 patients who complained of urinary incontinence preoperatively and showed sphincteric incontinence. sphincteric incontinence was primarily determined by a clinical decision made on the basis of the presence or absence of incontinence according to the patients ' symptoms and signs. it was also supported by a low abdominal leak point pressure (alpp), combined with low maximal urethral closure pressure (mucp), and an open bladder neck observed on fuds. because the initial group of 35 patients underwent ac, we assumed that all of the patients had bladder factors related to their symptoms. fluoroscopic images were simultaneously obtained, and vesicoureteral reflux, bladder neck competence, and bladder trabeculation were evaluated. urodynamic parameters including storage phase characteristics, bladder capacity, bladder compliance, cause of incontinence, alpp, mucp, maximum cystometric capacity, and detrusor sphincter dyssynergia were evaluated. fluoroscopic information regarding the bladder neck at filling, trabeculation, and vesico - ureteral reflux (vur) was also evaluated. a lower midline abdominal incision was made and subcutaneous tissue and the anterior rectus sheath was divided. the rectus muscle was retracted laterally, and the posterior rectus sheath and peritoneum were transected. after the native bladder was exposed, a 45-cm portion of the ileum was selected, and according to the patient 's condition, the sigmoid colon was used in some cases. ileal end - to - end anastomosis was done by use of 3 - 0 vicryl. the native bladder was dissected and bivalved vertically from the anterodome to the posterodome area. the detubularized ileal segment was sutured with the bivalved bladder by use of 2 - 0 vicryl. a 2-sided chi - square test was performed by use of a commercial analysis program pasw ver. 18.0 (ibm co., armonk, ny, usa). this study was approved by the institutional review board of seoul national university hospital (number 1106 - 091 - 366). the average age of the patients was 34.3 years (standard deviation [sd ], 20.76). the average follow - up period was 10.1 months (sd, 3.9), and postoperative fuds was performed a mean of 7.5 months (sd, 5.3) postoperatively. in 15 patients (88.2%) the ileum was used, and in 2 patients (11.8%) the sigmoid colon was used. postoperatively, 9 of the patients (52.9%) did not have vur, 2 had left vur (11.8%), and 1 had right vur (5.9%). among the patients with vur, all of them were grade 2. both preoperative and postoperative urodynamic parameters were compared (table 2). among the urodynamic parameters evaluated that were related to the bladder, bladder capacity, bladder compliance, and idc showed significant improvement postoperatively (p<0.05). the bladder neck was incompetent in all patients preoperatively, but was competent in 83.3% (10/12) of the 12 patients whose postoperative fuds results were available (p=0.004) (table 3, fig. the subjective incontinence status of the patients was evaluated. concerning the pad number, preoperatively, all patients used pads, and the average daily number was 2.2 (0.6). postoperatively, the number of pads used decreased significantly to 0.9 (median ; range 0 to 3) pads a day (p=0.002). significant improvement was shown for questions 1 to 3 on the iciq questionnaire as well as for the total score (p<0.05). in the analyzed group, all patients had incon tinence preoperatively, and the number who answered that their incontinence disappeared showed statistically significant improvement (p<0.001). the number of patients who had incontinence when sleeping, those who had nonspecific incontinence, and those who had constant incontinence showed significant improvement (p<0.05). although postoperatively some patients had incontinence during exercise or when coughing, symptoms disappeared in most patients (table 4). in patients with a small or poorly compliant bladder or detrusor overactivity, ac is recommended to lower intravesical pressure and to preserve the upper tract. persistent incontinence after ac deserves attention, because it is an important problem that negatively affects the quality of life of the patients. borjeson and lagergren reported incontinence to be the main reason for social isolation of patients with neurogenic bladder. herschorn and hewitt reported that 66.6% and 33.8% of patients, respectively, remained incontinent after ac performed as a single procedure. however, in other studies, continence was reported to be achieved after ac as a single procedure. daher. reported improved continence rates after ac performed in a pediatric patient population in a 10-year follow - up study. quek and ginsberg also reported improvement of urodynamic parameters after ac as a single procedure. venn and mundy reported a postoperative continence rate of 78% of neurogenic bladder patients who underwent ac only. some studies support bladder neck closure for continence ; however, this can cause deterioration of a pop - off mechanism of the bladder neck. reported that in some patients with neurogenic sphincteric incontinence, an aus can be considered as a treatment option. in pediatric urology, research about the outcome of bladder outlet procedures on children with urinary incontinence with neuropathic bladder dysfunction or structural deficiency is also rare. evaluation of small numbers of patients undergoing ac does not always show improved status on postoperative questionnaires, despite what generally would be considered to be a good surgical outcome. in our study, we evaluated the outcome of ac performed as a single procedure on incontinence caused by sphincteric incompetence. our data suggested that ac as a single procedure improved both subjective continence proven by the korean iciq questionnaire as well as objective continence proven by decreased pad number and fluoroscopically demonstrated bladder neck competence. when selecting patients for analysis in our study, we reviewed the patient 's fluoroscopic images, urodynamic findings, and most importantly, the patient 's history. also, patients whose bladder necks were partially open on cystourethroscopy but showed leaks when strong idcs occurred on filling cystometrograms were also excluded to avoid ambiguity. the fact that both a preoperative and a postoperative iciq evaluation were available in this study is a strong advantage. although the preoperative iciq evaluation was obtained by a postoperative telephone interview, we considered these data reliable. the reason is that although the evaluation was conducted postoperatively, the patients ' recollection of the number of pads they used and the degree of distress caused by incontinence was accurate. these patients had suffered from preoperative incontinence for a long period of time, and their memory was still fresh and the information they provided reliable at the time of data acquisition. the significant improvement of iciq scores shows that ac as a single procedure improves the subjective symptoms of the patients. when incontinence occurs when a patient coughs or sneezes, we consider the patient to have stress urinary incontinence (sui). postoperatively, 3 patients had sui ; however, their iciq total scores decreased from 16 (sd, 6.2) preoperatively to 7.3 (sd, 6.4) postoperatively. in addition, our study had the advantage of both preoperative and postoperative fuds in 12 of the patients analyzed. fuds was performed according to a standardized protocol, and therefore an objective comparison without variation was possible. also, the operations were all performed by a single experienced surgeon, which provided additional strength to the study. according to our data, vur persisted after surgery in 3 patients, which could be attributed to the chimney of the studer - type enterocystoplasty or to excessive filling during the filling cystometrogram. however, the vur was observable only at maximal capacity on fuds and upper - tract follow - up imaging on kidney ultrasound or nonenhanced computed tomography scans showed no hydronephrosis. patients did not suffer from acute pyelonephritis, either, which led us to conclude that the persistent reflux was caused by overfilling. we performed ac on 2 patients who had normal bladder capacity but severe idc who experienced urine leakage whenever there was an idc on fuds. although some patients showed detrusor overactivity postoperatively, the amplitude and duration of the idcs were negligible. first, it was performed in a retrospective nature, and second, it was a short - term study. due to the limited number of patients, further analysis involving the selection criteria of patients who would require bladder neck procedures either concomitantly or in a staged operation was not possible. however, if more data were accumulated, more valuable information could be provided for patients requiring surgery for incontinence. on the basis of the results of this study, we are able to predict that more valuable information can be obtained and that guidelines for the selection of such patients can be established in the future. our study demonstrated that both objective urodynamic parameters and subjective incontinence symptoms improved significantly after ac as a single procedure, even though the patients had sphincteric incompetence. this implies that in patients whose incontinence persists after ac, anti - incontinence bladder outlet procedures can be considered as a staged operation.	purposein patients with neurogenic bladder due to spinal cord injury or disease who undergo augmentation cystoplasty (ac) for not only bladder dysfunction but also sphincteric incontinence, the need for concomitant bladder neck reconstruction at the time of ac has not yet been established. the aim of this study was to evaluate whether concomitant bladder neck reconstruction is necessary when performing ac.materials and methodswe retrospectively investigated 35 patients who underwent ac from january 2006 to september 2010. medical history, preoperative and postoperative fluoroscopic urodynamic study (fuds) parameters, and responses to an incontinence questionnaire (iciq korean version) were reviewed.resultsa final analysis was performed on 17 patients (9 male, 8 female) who were diagnosed with sphincteric incontinence. continence status, the number of pads used, and the bother score were significantly improved postoperatively in this subpopulation. preoperatively, all patients used pads, and the average daily number was 2.2 (median ; range 0 to 6). postoperatively, the number of pads used decreased significantly to 0.9 (median ; range 0 to 3) pads a day (p=0.002). urodynamic parameters including bladder capacity, compliance, involuntary detrusor contraction, and bladder neck incompetence proven by fuds were also significantly improved.conclusionsour study demonstrated that both objective urodynamic parameters and subjective incontinence symptoms improved significantly after the completion of ac as a single procedure in patients with sphincteric incompetence. this implies that anti - incontinence bladder outlet surgery does not have to be performed simultaneously and can be considered later as a staged operation.
inflammation and altered immune processes are involved in all forms of pulmonary vascular disease underlying pulmonary hypertension, particularly, pulmonary arterial hypertension (pah). perivascular inflammatory elements have been reported in several etiologies of pah, from the idiopathic form to pah associated with autoimmune disorders [24 ]. inflammatory mediators have been investigated in pediatric pah, including pah associated with congenital heart disease (pah - chd) [59 ]. studies generally include patients over a wide age range, from infancy to adolescence, with multiple etiologies of pah. there has been scarce literature on the role of inflammatory mediators in the specific population of young pediatric patients with chd and pulmonary hypertension who are being considered for surgical repair of their cardiac anomalies. these patients generally have communications between cardiac chambers or the great arteries leading to increased pulmonary blood flow and pressure. progressive remodeling of pulmonary arteries causes elevation of pulmonary vascular resistance (pah - chd). structural and functional pulmonary vascular abnormalities are sometimes early, unsuspected, and progressive even in young individuals treated for their cardiac lesions in a timely manner. there are reasons for studying inflammation and immune response in young patients with congenital cardiac communications undergoing surgery. inflammatory cells, mediators, and related molecules have been shown to be closely associated with phenomena that occur early in pulmonary microvasculature, such as medial hypertrophy, muscularization of distal arteries, and intimal proliferative lesions [5, 11, 12 ]. these abnormalities constitute the anatomical basis for increased pulmonary vasoreactivity that occurs postoperatively as a result of vasospastic stimuli. although the incidence of the so - called postoperative pulmonary hypertensive crises has decreased to less than 1% due to contemporary practice in privileged communities, the mortality has been estimated at 2030%. pulmonary hypertension has been identified as a major contributor to prolonged mechanical ventilation and length of stay in hospital. after discharge, some patients remain at a higher risk of persistent pulmonary hypertension despite successful surgery. we therefore planned this study to investigate inflammatory mediators in young pediatric patients being considered for surgical repair of congenital cardiac communications. we wished to investigate how serum cytokines and related molecules correlate with patterns of clinical presentation, pulmonary hemodynamics, and pulmonary vascular abnormalities assessed by intraoperative lung biopsy. the study population consisted of young pediatric subjects (up to the age of 3 years) with congenital cardiac shunts admitted to the heart institute, university of so paulo, brazil, for surgical repair of their anomalies. from march 2012 to march 2015, 44 patients with nonrestrictive cardiac communications were enrolled, as they were suspected to have at least moderate elevation of pulmonary artery pressure. these patients correspond to 2025% of all pediatric subjects with cardiac shunts undergoing surgery in the institution. after complete noninvasive evaluation, 31 patients were considered as suitable for surgery with no need for cardiac catheterization (study group i, clinical features of pulmonary overcirculation with pulmonary congestion). the remaining 13 patients presented with features that suggested higher levels of pulmonary vascular resistance : relatively late presentation with no history of failure to thrive, small - sized heart with no significant enlargement of left cardiac chambers, and absence of pulmonary congestion despite the size of the cardiac communication and bidirectional shunting. patients with these features generally correspond to less than 10% of pediatric subjects with cardiac shunts treated in the institution. subjects with smaller septal defects not associated with pulmonary hypertension and those with advanced pulmonary vasculopathy precluding surgery were excluded. neonates, patients under intensive care, and those with relevant comorbidities or extracardiac syndromes other than down syndrome were also excluded. the study protocol was approved by the institutional scientific and ethics committee, cappesq # 0502 - 11. we used the following parameters to characterize hemodynamic and structural changes in pulmonary circulation : (1) pulmonary blood flow estimated noninvasively by transthoracic echocardiography, (2) pulmonary vascular resistance assessed by cardiac catheterization, and (3) morphology of pulmonary vessels assessed by direct analysis of lung biopsy specimens. transthoracic echocardiography was performed in all patients to define cardiovascular anatomy and assess the severity of pulmonary hypertension. the direction of flow across the cardiac communications and the size of the left cardiac chambers were taken into account when classifying patients as group i or group ii. left - to - right shunting (exclusive or predominant) in the presence of an enlarged left heart was considered as indicative of increased pulmonary blood flow (group i) whereas bidirectional shunting with a relatively small - sized left heart was suggestive of heightened pulmonary vascular resistance (group ii). of the parameters that were recorded, the characteristics of pulmonary venous flow were taken as an indirect estimate of total pulmonary blood flow. at least two pulmonary veins were selected and examined in all patients (same observer, lz). the velocity - time integral of pulmonary venous blood flow was obtained (vtipv, cm), and a mean value per patient was calculated. the pulmonary - to - systemic blood flow ratio (qp / qs) was also estimated, but it was considered less accurate in some cases due to the presence of a communication distal to the aortic and pulmonary valves (patent ductus arteriosus). invasive assessment of pulmonary hemodynamics was considered only for patients suspected to have higher levels of pulmonary artery pressure and vascular resistance on the basis of the clinical history, physical examination, and echocardiographic evaluation. however, to avoid error due to the use of assumed oxygen consumption while calculating blood flow, we decided to express the magnitude of pulmonary hemodynamic abnormalities by calculating the pulmonary - to - systemic vascular resistance ratio (pvr / svr), with no need for considering the level of oxygen consumption. lung biopsy specimens were collected intraoperatively in selected cases : from group ii patients if the surgeon considered the procedure low risk on an individual basis and from group i patients if the elevation of pulmonary artery pressure represented a problem during weaning from cardiopulmonary bypass. thus, lung tissue was available for analysis in 13 of our patients (9 and 4 individuals from group ii and group i, resp.). specimens were collected with the airways distended, fixed in buffered formalin, and subjected to routine histological processing. four - micrometer - thick sections were obtained and stained with hematoxylin - eosin and miller 's elastic stain. vascular abnormalities were graded according to the heath - edward 's classification for pulmonary vascular lesions associated with congenital heart disease. grades i and ii correspond to isolated medial hypertrophy of the pulmonary arteries and intimal nonocclusive proliferation, respectively. grades iii through vi correspond to more advanced vascular abnormalities, including occlusive intimal proliferation, plexiform and dilated lesions, and necrotizing arteritis. peripheral venous blood was collected, and serum samples were obtained and stored at 80c. cytokines and related proteins were analyzed using a human cytokine array (r&d systems, minneapolis, mn, usa). after protein immobilization on nitrocellulose membranes, processing (immunoblotting) was carried out using specific antibodies as previously described. the average signal (pixel density) of the pair of duplicate spots representing each protein was normalized using internal controls. of the 36 inflammatory mediators that were potentially measurable, we analyzed only the proteins that we detected with adequate signal in all patients (figure 1). these included complement component 5/5a (c5/c5a), soluble cd40 ligand (scd40l), growth - regulated oncogene alpha (gro), interleukins 16 and 17e (il-16, il-17e), interleukin-1 receptor antagonist (il-1ra), interferon gamma - induced protein-10 (ip-10), macrophage migration inhibitory factor (mif), regulated on activation, normal t cell expressed and secreted (rantes), serpin e1, and soluble intercellular adhesion molecule-1 (sicam-1). for categorical variables, the number of patients is given, with percentages when appropriate. whitney test for numeric variables or the chi - square family of tests (including fisher 's exact test and the likelihood ratio) for categorical variables. correlations were tested by calculating pearson 's coefficient (r) or spearman 's coefficient (rs). for regression analyses, logarithmic transformation (ln) of the dependent variable was sometimes necessary to obtain adequate closeness to the normal distribution. in all tests a p value lower than 0.05 was considered statistically significant. forty - four patients were enrolled, with an age range of 2.6 to 37.0 months. cardiac anomalies were relatively simple, consisting of pretricuspid and posttricuspid defects in biventricular hearts. demographic and diagnostic data of patients classified as group i or group ii are shown in table 1. group ii patients tended to be a little older and had lower systemic oxygen saturation, probably due to some degree of right - to - left shunting across the septal defects. consistent with this, echocardiographic parameters (qp / qs ratio and vtipv) pointed toward lower pulmonary blood flow in group ii patients compared to group i (table 1). at catheterization, they had a mean pulmonary arterial pressure of 52 (95% ci 4559) mmhg, a pulmonary vascular resistance of 5.7 (95% ci 4.47.4) wood units m, and a pulmonary - to - systemic vascular resistance ratio of 0.39 (95% ci 0.310.50). as shown in table 2, mif chemokine concentration was higher in group ii, where patients were assumed to have higher levels of pulmonary vascular resistance, whereas rantes chemokine was more closely related to group i, corresponding to subjects with clinical features suggestive of increased pulmonary blood flow. the level of gro chemokine was higher early in life and decreased with increasing age (table 2). the tendency toward higher gro levels in group i was probably because group i patients were a little younger compared to group ii. consistent with the observation of higher mif levels in group ii patients was the negative correlation between mif concentration and pulmonary blood flow estimated noninvasively by measuring vtipv. a similar negative correlation was obtained for il-16 (table 2 and figure 2). in group ii, ip-10 chemokine level correlated positively with pulmonary vascular resistance (table 2 and figure 2). nine of the 13 patients who were subjected to intraoperative lung biopsy had pulmonary vascular lesions classified as grade i (5 cases) or grade ii (4 cases). the remaining ones had more advanced lesions classified as grade iii (3 patients) or grade iv (1 patient, figure 3). consistent with the negative correlation of il-16 with pulmonary blood flow (vtipv, table 2 and figure 2) was the observation of higher il-16 levels in the subgroup of patients with more advanced pulmonary vascular abnormalities (figure 3). hemodynamic instability requiring specific therapeutic interventions occurred in 12 patients (6 individuals in each group), and there was a fatal outcome in two of them (group ii). when considering only patients who had a lung biopsy specimen available for analysis (n = 13), clinically relevant postoperative pulmonary vasoconstriction occurred in 4 of 9 subjects with vascular lesions graded as i or ii, and only one of 4 subjects with lesions graded as iii or iv. patients who died had grade i and grade ii pulmonary vasculopathy. preoperative oxygen saturation was lower in patients who had postoperative pulmonary vasoconstrictive episodes compared to those who did not (93% [9195% ] and 95% [9497% ], resp. the 27% incidence of postoperative pulmonary vascular tone instability was considerably higher than the 8% incidence in similar patients from our institution without clinical evidence of pulmonary hypertension. there is general agreement that patients with nonrestrictive cardiac communications and absence of clinical and echocardiographic evidence of pulmonary overcirculation may have pulmonary vascular changes that eventually preclude surgery. in theory, any patients with group ii characteristics, with a low vtipv and a high pvr / svr ratio, would belong to this high - risk population. of note, however, was the observation that postoperative pulmonary vasospastic episodes may occur even in subjects with group i characteristics, or those who have vascular abnormalities considered to be potentially reversible (grade i). there seems to be a potential for occurrence of vascular tone instability leading to life - threatening hemodynamic disturbances all throughout the spectrum of pulmonary vascular changes. we did not test for direct correlations between preoperative cytokine levels and postoperative pulmonary vasoconstriction. actually, pulmonary vascular tone instability is probably more closely related to perioperative than preoperative proinflammatory events. we focused essentially on the analysis of the preoperative scenario where pulmonary vascular remodeling is usually already present to a variable degree. we identified associations between serum inflammatory mediators and patterns of clinical presentation, hemodynamics, and the morphology of pulmonary arteries, with potential pathophysiological significance. mif and rantes chemokines were easily analyzed, as they appeared in all of the membranes with a strong chemiluminescence signal (figure 1). higher concentration of mif was a characteristic of group ii patients, and mif concentration correlated negatively with pulmonary blood flow estimated by transthoracic echocardiography (vtipv). in contrast, rantes was higher in group i, where patients had features compatible with pulmonary overcirculation with congestion. mif is a noncanonical ligand of cxcr2 and cxcr4 chemokine receptors and the cxcr2/cd74 complex. its pleiotropic nature is illustrated by the activation of several signaling pathways that results in inhibition of endothelial cell apoptosis and induction of smooth muscle cell proliferation. its binding to the cxcr2/cd74 complex elicits chemotaxis and the arrest of mononuclear cells, which is central to inflammatory disorders and atherogenesis [2123 ]. in clinical and experimental pulmonary hypertension, mif is associated with a proinflammatory endothelial cell behavior, exaggerated recruitment of peripheral blood mononuclear cells, and increased smooth muscle cell growth [11, 24, 25 ]. rantes is a cc chemokine shown to be involved in a number of biological events including chemotaxis, perivascular inflammation, and vascular response to injury [26, 27 ]. of note, rantes expression has been demonstrated in viral diseases, and it is widely appreciated that infants with pulmonary congestion due to cardiac shunts (group i patients in this study) are predisposed to respiratory infections. thus, a possibility exists that repeated exposures to viral agents contribute to the development of a proinflammatory microenvironment, with expression of molecules known to influence vascular remodeling. levels were higher in subjects with histological grades iii and iv and correlated negatively with noninvasively estimated pulmonary blood flow. il-16 is a lymphocyte chemoattractant factor well known for its role in immune responses, but there has been scarce literature on its role in vascular disorders. il-16 and its receptor cd4 are expressed in vascular smooth muscle cells. they induce migration and invasion of vascular smooth muscle cells in a p21waf1-dependent manner, via the p38mapk / sp-1/mmp-9 pathway. for this reason, they are being considered as potential targets in the treatment of diseases such as atherosclerosis and restenosis. the pathophysiological role of il-16 and downstream signaling molecules in pah have not been investigated yet. we also observed significant correlations of il-17e (positive) and gro (negative) with patient age, considered as an index of disease severity and progression in pah - chd [31, 32 ]. il-17e and mif are capable of eliciting a th2-type immune response [33, 34 ] shown to be involved in experimental pulmonary vascular remodeling. gro is a cxc chemokine known to play a central role in thrombin - induced angiogenesis associated with tumor growth. a potential implication for the age - dependent decrease in gro is that reduction of distal pulmonary vessels does occur in pulmonary hypertension associated with chd and probably precedes obliterative pulmonary vasculopathy. in patients subjected to catheterization ip-10 is a cxc chemokine known to be involved not only in the endothelial - leukocyte interaction but also in the endothelial renin - angiotensin pathway. it is involved in interferon - associated pah and plays a role in idiopathic pah and chronic thromboembolic pulmonary hypertension. we also observed a negative correlation of serpin e1 (formally, plasminogen activator inhibitor-1, pai-1) with pulmonary vascular resistance. despite its function as an inhibitor of fibrinolysis, serpin e1/pai-1 has a paradoxical protumorigenic role in cancer and has been shown to promote angiogenesis [42, 43 ]. furthermore, serpin e1/pai-1 seems to be downregulated in pulmonary artery smooth muscle cells in idiopathic pah. in conclusion, correlations were observed between circulating levels of inflammatory mediators and patterns of clinical, hemodynamic, and histological presentation, suggesting that inflammation has a role in pulmonary vascular remodeling in young pediatric patients with cardiac shunts. however, in view of the relatively small number of patients in the study and so many correlations to be tested, we would like to focus on the observations that, in our opinion, are the most consistent ones. essentially, mif chemokine was elevated in patients for whom cardiac catheterization was considered as necessary, and elevated pulmonary vascular resistance was confirmed. in contrast, rantes chemokine was elevated in patients with congestive heart failure who were assigned to surgery with no need for catheterization. furthermore, there seemed to be a positive correlation between il-16 level and severity of pulmonary vascular lesions. contemporary practice includes therapeutic measures that allow for the management of postoperative pulmonary vascular tone instability, although mortality rates are still high. hopefully, in the future, specific targeted therapies will allow for stabilization or reduction of preoperative pulmonary vascular remodeling in this population.	background and objective. inflammation is central in the pathogenesis of pulmonary hypertension. we investigated how serum cytokines correlate with clinical features, hemodynamics, and lung histology in young patients with pulmonary hypertension associated with congenital cardiac shunts. design. prospective, observational study. methods and results. patients (n = 44) were aged 2.6 to 37.6 months. group i patients (n = 31) were characterized by pulmonary congestion and higher pulmonary blood flow compared to group ii (p = 0.022), with no need for preoperative cardiac catheterization. group ii patients (n = 13) had no congestive features. at catheterization, they had elevated pulmonary vascular resistance (5.7 [4.47.4 ] wood unitsm2, geometric mean with 95% ci). cytokines were measured by chemiluminescence. macrophage migration inhibitory factor (mif) was found to be inversely related to pulmonary blood flow (r = 0.33, p = 0.026) and was higher in group ii (high pulmonary vascular resistance) compared to group i (high pulmonary blood flow) (p = 0.017). in contrast, rantes chemokine (regulated on activation, normal t cell expressed and secreted) was characteristically elevated in group i (p = 0.022). interleukin 16 was also negatively related to pulmonary blood flow (rs = 0.33, p = 0.029) and was higher in patients with obstructive vasculopathy at intraoperative lung biopsy (p = 0.021). conclusion. cytokines seem to be important and differentially regulated in subpopulations of young patients with cardiac shunts.
breast cancer is the most frequently observed type of invasive cancer, affecting approximately 1 million women worldwide and causing bone metastases in 65% to 75% of patients. bisphosphonates are some of the most effective treatments for preventing complications related to bone metastases. zoledronic acid (za) is the most effective molecule in reducing skeletal - related events (sres) in patients with breast cancer. bisphosphonates inhibit bone resorption and protect bone structure by inhibiting the differentiation of osteoclastic precursors, promoting apoptosis of osteoclasts, and stimulating the secretion of osteoclast inhibitory factor from osteoblasts. one of the most important adverse effects that limit its clinical use is osteonecrosis of the jaw (onj). according to the american oral and maxillofacial surgery association, current therapy or a history of therapy with bisphosphonates, no radiotherapy to the head and neck area, and the presence of exposed necrotic bone in the maxilla and/or mandible for at least 8 weeks support the diagnosis of onj. following the first scientific report published by marx in 2003 that pointed to a link between bisphosphonates and onj, the number of studies focusing on this subject has rapidly increased. moreover, numerous risk factors for the development of onj have also been described (cancer and anticancer therapy, dental risk factors, corticosteroids, alcohol and tobacco abuse, anemia, diabetes, obesity, and renal impairment). trastuzumab is one of the most widely used agents for the management of all metastatic breast cancers with human epidermal growth factor receptor 2 (her2) overexpression as indicated by 3 + her2 immunostaining or gene amplification on the fluorescence in situ hybridization test. the development of onj has been reported to have occurred in 2 patients with concurrent use of trastuzumab and bisphosphonates. however, in both these reports, the authors did not correlate the occurrence of onj in their patients with the combined use of these 2 agents. in the present study, we made an attempt to analyze the use of trastuzumab as an independent risk factor for the development of onj in metastatic breast cancer patients undergoing za treatment. patient data were identified retrospectively from the archives of the florence nightingale breast study group, istanbul, between march 2006 and december 2013. in this study, we included 97 consecutive patients with metastatic breast cancer who had bone metastases and underwent treatment with za. patients with < 12 months of follow - up and radiotherapy to the head and neck area were excluded from the study. patients were analyzed according to their characteristics (age, weight, number of za infusions, time of exposure to za [months ], smoking habits, dental procedures, receiving aromatase inhibitors [ai ], receiving chemotherapy [ct ], trastuzumab treatment, and renal dysfunction). the diagnosis of bone metastasis was based on radiologic methods such as direct radiography, bone scintigraphy, and positron emission tomography - computed tomography. the standard therapy involved intravenous infusion of 4 mg every 3 to 4 weeks (in 150 cc of saline within 15 minutes). patients were examined by a dentist every 6 to 12 months and all dental procedures performed before the initiation of therapy with za and during the therapy period were recorded. the diagnosis of the onj was made through clinical and radiologic examinations, and biopsies were performed when necessary. the study was approved by the bilim university ethics committee (decision no : 27 - 200). statistical analysis was performed using the statistical package for social sciences social sciences (spss inc., chicago, il)for windows 17.0 software. during the evaluation of the study data, in addition to the descriptive statistical methods (mean, median, number, and percentage), was employed for the qualitative comparison of the development of onj along with the patient and disease - related characteristics, whereas quantitative comparisons were made through the independent samples t test. evaluation of the independent parameters related to the development of onj was based on the multiple logistical regression (forward stepwise) model. the results were assessed within a 95% confidence interval, and a value of p < 0.05 was accepted as statistically significant. statistical analysis was performed using the statistical package for social sciences social sciences (spss inc., chicago, il)for windows 17.0 software. during the evaluation of the study data, in addition to the descriptive statistical methods (mean, median, number, and percentage), was employed for the qualitative comparison of the development of onj along with the patient and disease - related characteristics, whereas quantitative comparisons were made through the independent samples t test. evaluation of the independent parameters related to the development of onj was based on the multiple logistical regression (forward stepwise) model. the results were assessed within a 95% confidence interval, and a value of p < 0.05 was accepted as statistically significant. the mean time of exposure to za was 37 18 months (range : 1387) and the mean number of za infusions was 35 16 (range : 1073) (table 1). the median age of the patients who developed onj was 61 years (range : 3973). the mean time of exposure to therapy with za was 47 22 months (range : 1387), and the mean number of intravenous za infusions was 41 15 (range : 1566). the 8 (62%) of 13 patients developed onj had received transtuzumab, but 5 (38%) of them had not received. among these patients, 1 patient (7.69%) was asymptomatic, whereas 9 patients (69.24%) were diagnosed through clinical and radiologic examinations. in 4 patients (30.76%), the probability of metastasis was ruled out by biopsy. there were 15 lesions in total ; 11 patients had single lesions whereas 2 patients had double lesions. six of the lesions were detected in the mandible and 5 in the maxilla, whereas 2 involved both the maxilla and the mandible (table 2). when the diagnosis of onj was made, 9 patients (69.20%) were under treatment with ais, 8 patients (62%) were taking trastuzumab, and 3 patients (23%) were receiving systemic ct. the dental procedures included tooth extractions in all patients, root canal treatment in 8 patients, and dentures in 7 patients. following conservative treatment, 4 patients (30.76%) needed surgery (table 2). there was no association of the development of onj with age (p = 0.241), weight (p = 0.218), number of za infusions (p = 0.111), smoking habits (p = 0.989), dental procedures (tooth extraction [p = 0.349 ], root canal treatment [p = 0.393 ], dentures [p = 0.350 ]), diabetes mellitus (p = 0.752), receiving ai (p = 0.510), receiving ct (p = 0.357), or renal dysfunction (p = 0.500). duration of exposure to za and the use of trastuzumab were associated with the development of onj (p = 0.049 and p = 0.028, respectively) (table 3). unless there is an interfering condition, bisphosphonates are currently regarded as the standard therapy for sres in the treatment of bone metastases. onj is one of the most important complications associated with bisphosphonate therapy used in patients who have breast cancer with bone metastases. this condition is due to accumulation of bisphosphonates in great amounts both in the alveolar bone and the surrounding soft tissue. this increases the risk of avascular necrosis, which, in addition to disruption of the mucosal barrier mediated by stimulating the apoptosis of keratinocytes, delays wound healing and tissue repair by inhibiting the formation of blood vessels through antiangiogenic effects. trastuzumab has been demonstrated to inhibit angiogenesis and this effect is believed to occur through the expression of antiangiogenic factors and inhibition of proangiogenic factors. combining bisphosphonates with antiangiogenic agents has been suggested to induce onj more frequently than using bisphosphonates alone. in this article, we focused on the impact of trastuzumab as well as the other factors in the development of onj in metastatic breast cancer patients receiving za. although onj is mostly associated with dental procedures, other factors that play a role in its pathogenesis are listed in some studies : duration of exposure to za, number of infusions, type of bisphosphonate, route of administration (oral, intravenous), concurrent ct, chronic use of corticosteroids, poor oral hygiene, smoking, and poorly fitting dentures. although symptoms including orofacial pain, puffy face, and malodorous discharge during treatment with bisphosphonates support the diagnosis of onj, it may be necessary to rule out any metastases to the orofacial bones. however, because of the risk of diagnostic biopsy of the bone that may lead to a compromise in wound healing, the diagnosis is usually based on clinical and radiologic examinations. in a retrospective study by bamias, the most important risk factors suggested to increase the risk of development of onj were found to be duration of exposure to treatment, the number of infusions, dental procedures, and the type of bisphosphonate used. the duration of bisphosphonate treatment has also been marked as a risk factor for the development of onj in other clinical studies. in our study, duration of za treatment was detected as a significant risk factor in the development of onj, which strengthens the outcomes of the above studies. although a relationship between dental procedures and onj was observed in certain studies, no statistically significant correlation was observed between dental procedures and onj in our study. the fact that the majority of the patients in which no onj occurred had also undergone dental procedures may have led to this result. antiangiogenic agents that are used with increasing frequency may enhance the risk of onj, especially when used concurrently with bisphosphonates. for instance, there have been recent reports of patients with onj caused by antiangiogenic agents such as sunitinib (multikinase inhibitors), bevacizumab (a monoclonal antibody that targets vascular endothelial growth factor), and everolimus (inhibitor of mammalian target of rapamycin), with or without bisphosphonates. there have been no reports in the literature on development of onj solely due to trastuzumab treatment. there have been a few cases of development of onj during concurrent treatment with bisphosphonates and trastuzumab ; however, an association of trastuzumab with the occurrence of onj has not been clearly stated in these reports. moreover, in a study by hoff, a large number of patients were evaluated for onj incidence and risk factors regarding the development of onj. no relationship was observed between onj and treatment with trastuzumab, anthracycline, tamoxifen, taxane, or ais. in agreement with our findings, the rate of treatment with trastuzumab in patients with onj was observed to be significantly higher than in those without onj (p = 0.028). the combination of za and other antiangiogenic agents (sunitinib, everolimus, bevacizumab) has recently been revealed to be associated with an onj rate of up to 16%. in addition, treatment with bevacizumab alone has been correlated to onj in a few case presentations. we suggest that our study brings up the matter of the effect of trastuzumab on onj when it is combined with za, as we observed a 13.6% occurrence rate of onj, which matches the previous reports. the outcomes of our study may indicate that trastuzumab has antiangiogenic potency similar to that of other agents. this study evaluated data obtained from patients with isolated metastatic breast cancer, whose files and treatments were regularly followed up. in this study, the development of onj was associated with longer treatment and higher cumulative doses of intravenous za therapy, and with concurrent treatment of za and trastuzumab in breast cancer patients. the increased risk of onj should be kept in mind and all preemptive measures should be taken, especially when za is used with trastuzumab. the retrospective nature of the study and the statistical analysis of a small number of cases of onj are limitations of our study. we suggest that prospective studies should be performed to confirm these results, and more careful studies are also needed to define the minimum dose and duration of therapy with bisphosphonates necessary to prevent skeletal complications of malignancy.	abstractone of the most important adverse effects of zoledronic acid (za) is osteonecrosis of the jaw (onj). in previous literature, several risk factors have been identified in the development of onj. in this study, we aimed to determine the role of trastuzumab, an antiangiogenic agent, as an independent risk factor for the development of this serious side effect.our study included 97 patients (mean age : 54 10 years) with breast cancer, recorded in the archives of the istanbul florence nightingale breast study group, who received za therapy due to bone metastases between march 2006 and december 2013. we recorded the patients ages, weights, duration of treatment with za, number of za infusions, dental procedures, anticancer treatments (chemotherapy, aromatase inhibitor, trastuzumab), the presence of diabetes mellitus or renal dysfunction, and smoking habits.thirteen patients (13.40%) had developed onj. among the patients with onj, the mean time of exposure to za was 41 months (range : 1382) and the mean number of za infusions was 38 (range : 1556). the duration of treatment with za and the use of trastuzumab were observed to be 2 factors that influenced the development of onj (p = 0.049 and p = 0.028, respectively).the development of onj under za treatment may be associated solely with the duration of za treatment and the concurrent administration of trastuzumab. these findings show that patients who are administered trastuzumab for metastatic breast cancer while undergoing za treatment are prone to developing onj. therefore, we recommend intense clinical observation to avoid this particular condition in patients receiving za and trastuzumab.
it had been studied extensively in two phase iii trials [the apixaban versus acetylsalicylic acid to prevent stroke (averroes) and apixaban for the prevention of stroke in subjects with atrial fibrillation (aristotle) ] as potential alternatives to warfarin for stroke prevention in atrial fibrillation (af) patients.14 these studies are particularly important because af is the most common cardiac arrhythmia and is especially prevalent in the older population. it is also associated with many comorbid conditions including cardioembolic strokes.5 therapeutic anticoagulation is the treatment of choice in moderate- to high - risk af patients because it is the only treatment that has been shown to reduce the risk of embolic phenomena and mortality.6 however, its usage has been limited due to several issues, such as the need for frequent monitoring, multiple drug - to - drug and drug - to - food interactions, and the risk of hemorrhagic complications.7,8 as a result, newer antithrombotic agents have been developed and studied. in this review, we examine the need for new anticoagulants and provide a review of apixaban and its effect in stroke prevention. af patients (both valvular and nonvalvular) are at increased risk of developing cardioembolic strokes, and these af- related complications often result in severe functional deficits or death.9 because these embolic events can be devastating, multiple schemes such as the chads2 risk score index (table 1) and the cha2ds2-vasc score (table 2) have been utilized to classify af patients into different risk groups.6,1012 the chads2 system identified five major risk factors congestive heart failure, hypertension, age > 75 years, diabetes mellitus, and previous stroke or transient ischemic attack (tia). in this scoring system, a previous stroke or tia is assigned two points while the other factors are assigned one point. although it is a useful system, the chads2 scoring system omitted some important risk factors such as thyrotoxicosis, female sex, and coronary artery disease or peripheral artery disease, which may underestimate the cardioembolic risks and end up placing more patients in the intermediate - risk groups. subsequently, researchers evaluated for other risk stratification systems to better stratify af patients. the newer cha2ds2-vasc score added three new risk factors (history of vascular disease, age 6574 years, and female sex) in addition to the five risk factors listed in the chads2 system. in the cha2ds2-vasc score scheme, age > 75 years is assigned two points, on a par with previous stroke and tia.11,12 based on this new scoring scheme, women and elderly patients (75 years) who were previously identified as intermediate risk are now placed into a high - risk category with a recommendation for full anticoagulation. people are placed into different risk groups and prescribed appropriate antithrombotic therapy. in high - risk patients (chads2 score 2), oral anticoagulation with warfarin has been the standard to prevent the cardioembolic complications including stroke until recently.6 in patients with intermediate - risk (score of 1), acetylsalicylic acid (asa) may be used although warfarin is still preferred. on the other hand, asa is recommended in the low - risk patients (score of 0). antithrombotic therapy maybe deferred in patients with lone af or in patients who have contraindication to antithrombotic therapy.6 although therapeutic anticoagulation with warfarin has been proven to reduce the risk of embolic phenomenon and mortality in af patients, it is underutilized in high - risk patients.6,13 warfarin use has been limited by several properties such as slow onset of action, narrow therapeutic window, variable cytochrome p450-dependent metabolism, significant amount of drug - to - food and drug - to- drug interactions, and risk of bleeding complications such as intracranial hemorrhage in the elderly patients (table 3).11,1317 because of all of these issues, patients need to undergo frequent international normalized ratio monitoring and dosage adjustment in order to achieve and maintain appropriate levels. subtherapeutic anticoagulation exposes patients to cardioembolic events, and supratherapeutic anticoagulation subjects patients to bleeding complications. 18 in addition, it is difficult to achieve the desired time in the therapeutic range with warfarin. it is also expensive and impairs quality of life because of frequent doctor and laboratory visits.19 in addition, elderly patients and patients with severe renal impairment are at high risk of developing cardioembolic complications as well as increased risk of bleeding with warfarin use. older patients often have cognitive impairment and physical limitations which place them at increased risk for bleeding. patients with renal impairment can have functional abnormalities within the platelets and other coagulation pathways such as altered von willebrand factor that can place them at a higher risk of bleeding.2022 because of these reasons, the use of warfarin for stroke prevention has been challenging and is associated with significant impairment. therefore, there has been a need to find better therapeutic agents for the prevention of thromboembolic events and strokes in af patients. multiple new drugs have been evaluated as potential alternative anticoagulants for stroke prevention in af patients. active w (atrial fibrillation clopidogrel trial with irbesartan for prevention of vascular events) and active a (a separate arm of the active w) trials evaluated dual antiplatelet therapy of acetylsalicyclic acid and clopidogrel as alternative to warfarin. results from the active trials demonstrated that warfarin is superior to dual antiplatelet therapy and that warfarin is still the antithrombotic agent of choice. in patients who can not tolerate or safely sustain anticoagulation with warfarin, dual antiplatelet therapy may be considered.2325 due to the limitations of warfarin and dual antiplatelet therapy, there has an ongoing search for newer and better oral anticoagulants for stroke prophylaxis. efforts have been directed at finding agents with rapid onset of action, fewer food and drug interaction, and more predictable anticoagulation. as a result, direct thrombin inhibitors, direct and indirect fxa inhibitors, and other vitamin k antagonists have emerged and have been evaluated in various clinical trials. in the following sections, we will discuss briefly some of these agents and focus our primary attention on the newest of these agents, apixaban. dabigatran is a new oral direct thrombin inhibitor which has been approved by the united states food and drug administration (fda) for stroke prevention in nonvalvular af patients. dabigatran was extensively studied in the dabigatran with or without concomitant acetylsalicylic acid compared with warfarin alone in patients with nonvalvular atrial fibrillation (petro study) and the randomized evaluation of long- term anticoagulation therapy (re - ly study).2629 the re - ly study compared dabigatran etexilate with dose - adjusted warfarin in 18,113 patients in a multicenter, randomized, open - label study. results of this study showed that dabigatran 150 mg twice daily was superior to warfarin and dabigatran 110 mg twice daily was noninferior to warfarin in primary outcomes of stroke and systemic embolism. overall, there was a lower risk of bleeding (major, life - threatening, intracranial, and major or minor bleeding) but a higher incidence of dyspepsia compared to warfarin.29 based on the safety and efficacy results from the re - ly study, the fda approved dabigatran for stroke prevention in nonvalvular af patients (150 mg twice daily for patients with normal renal function and 75 mg twice daily for patients with renal impairment) in october 2010. dabigatran was added as a first - line option for anticoagulation in high - risk af patients in the 2011 updated of the acc / aha practice guideline for atrial fibrililation.30 dabigatran has been extensively used by clinicians around the world since its approval and there has been concern regarding an increased risk of gastrointestinal bleeding (especially in the elderly). in addition, the manufacturer has reported 260 fatal bleeding events after reviewing the postmarketing database. these findings have prompted several actions to be taken by different countries, including labeling updates in europe and the united states, safety advisories issued in japan and australia, and an fda investigation to determine whether the reports of bleeding in patients taking dabigatran are occurring more commonly than would be expected.31 an extension of the re - ly study, the rely - able study (rely - able long term multi - center extension of dabigatran treatment in patients with atrial fibrillation who completed re - ly trial nct00808067), is actively ongoing to evaluate the long - term safety of dabigatran. its primary role is to convert prothombin into thrombin which then participates in the thrombus formation. direct fxa inhibitors inactivate both prothrombinase - bound fxa and free fxa.14,32,33 several new agents have been developed as director fxa inhibitors, and these include rivaroxaban, apixaban, and edoxaban. rivaroxaban is the first direct fxa inhibitor that has been evaluated and recently approved for clinical use for stroke prophylaxis in atrial fibrillation. it has a half - life of 59 hours in the healthy individual and 913 hours in elderly patients. it is metabolized by the cyp3a4 enzyme pathyway.33 its use in atrial fibrillation was investigated in the rivaroxaban once - daily oral direct factor xa inhibition compared with the vitamin k antagonism for prevention of stroke and embolism trial in atrial fibrillation (rocket - af) trial as stroke prophylactic agent for af patients. the result of this study showed that rivaroxaban was noninferior to warfarin in preventing stroke and systemic embolism. the rivaroxaban group had fewer intracranial bleeding but more gastrointestinal bleeding events when compared to the warfarin group. these data demonstrated that rivaroxaban is a good alternative to warfarin in af patients who are at moderate to high risk of developing embolic strokes.34 although concern was raised during the approval process for its use as once - daily administration (due to its short half - life), it has been approved by the fda for prevention of stroke in patients with nonvalvular af in november 2011. there has not yet been sufficient substantive clinical exposure with rivaroxaban to evaluate its efficacy and safety in clinical practice. apixaban is the newest selective, reversible, oral, direct fxa inhibitor that has been evaluated for the prevention of strokes in patients with af in multiple trials. it has a rapid onset of action (3 hours) and a mean half - life of 12 hours. it is metabolized by the kidney, liver (primarily by cyp3a4, cyp3a5, and sulfotransferase 1a1), and the intestine. it is excreted 25% renally and 75% through the hepatobiliary system which is different from rivaroxaban (66% renal excretion). its absorption is not affected by food and it needs to be taken in a twice - daily dosing format. it is essential for clinicians to be cautious about concomitant use with cyp3a4 inhibitors and inducers, as the pharmacokinetics of the drug will be altered in such settings.32 a recent crossover study evaluated the pharmacokinetics, pharmacodynamics, and safety of coadministration of enoxaparin and apixaban in 20 healthy subjects. concomitant usage of apixaban and enoxaparin resulted in a 42% increase in peak anti - xa activity. similarly, the peak anti - xa activity was 15% higher when apixaban was coadministrated with enoxaparin compared to apixaban alone. overall, the study showed that the pharmacokinetics of apixaban were not affected by enoxaparin even though there was a modest increase in anti - xa activity when enoxaparin was used concurrently with apixaban. furthermore, the finding also suggested that co - administration of apixaban and enoxaparin was safe and well tolerated in healthy volunteers.35 apixaban has been evaluated in two recent phase iii randomized trials for stroke prevention in af patients. the first study was the apixaban versus acetylsalicylic acid to prevent stroke (averroes trial). it was an international (36 countries), multicenter (522 centers), double - blinded, and double - dummy trial which enrolled 5599 af patients over a 2-year period (september 2007december 2009). the patients enrolled in this trial were those who were not candidates for vitamin k antagonist therapy because of previous intolerance to or ineligibility for warfarin therapy.1,2 patients participating in the averroes trial had to be 50 years old and to have had atrial fibrillation documented in the 6 months before enrollment or by 12-lead electrocardiography on the day of screening. patients also had to have at least one of the risk factors for stroke which included prior stroke or tia, age 75 years, arterial hypertension (on treatment), diabetes mellitus (receiving treatment), heart failure (new york heart association class 2 at the time of enrollment), a left ventricular ejection fraction of 35%, or documented peripheral - arterial disease. patients were excluded if they needed long - term anticoagulation for reasons other than atrial fibrillation, if they had valvular disease requiring surgery, a history of a serious bleeding event in the past 6 months or if they were at high risk for bleeding, current alcohol or drug abuse or psychosocial issues, if they had a life expectancy 2.5 mg / dl or a calculated creatinine clearance of twice the upper limit of the normal range or a total bilirubin > 1.5 times the upper limit of the normal range), and allergy to acetylsalicylic acid.1,2 patients who met the inclusion and exclusion criteria were then randomly assigned through a 24-hour central, computerized, automated voice - response system to receive either acetylsalicylic acid (81324 mg per day) or apixaban (5 mg twice daily or 2.5 mg twice daily in those patients who met two of the following criteria : age 80 years, weight 60 kg, or serum creatinine concentration 1.5 mg / dl). primary efficacy outcome of the study was the occurrence of stroke (ischemic or hemorrhagic) or systemic embolism. the primary safety outcome was the occurrence of major bleeding defined by clinically overt bleeding with one of more of the following : drop in hemoglobin level of 2 g / dl over 24-hour period, transfusion of 2 units of packed red cells, bleeding at critical site, or fatal bleeding. study also included other outcomes such as rates of myocardial infarction, death from vascular causes, death from any cause, and composites of major vascular events. the averroes trial was terminated early by the data and safety monitoring committee when the interim efficacy analysis showed that apixaban was better than acetylsalicylic acid for the primary outcome in february 2010.36 as shown in table 4, both study groups had similar baseline clinical characteristics. of the patients enrolled in the study, 63%64% of patients had a chads2 score 2. the result of the study revealed that patients treated with apixaban had a statistically significant reduction in stroke and systemic embolic rates when compared with the acetylsalicylic acid group (1.6% per year vs 3.7% per year, hazard ratio [hr ] with apixaban 0.45, p 165 mg a day or needed both acetylsalicylic acid and clopidogrel, and severe renal insufficiency (serum creatinine level > 2.5 mg / dl or calculated creatinine clearance of 75 years, diabetes mellitus, and previous stroke or transient ischemic attack (tia). in this scoring system, a previous stroke or tia is assigned two points while the other factors are assigned one point. although it is a useful system, the chads2 scoring system omitted some important risk factors such as thyrotoxicosis, female sex, and coronary artery disease or peripheral artery disease, which may underestimate the cardioembolic risks and end up placing more patients in the intermediate - risk groups. subsequently, researchers evaluated for other risk stratification systems to better stratify af patients. the newer cha2ds2-vasc score added three new risk factors (history of vascular disease, age 6574 years, and female sex) in addition to the five risk factors listed in the chads2 system. in the cha2ds2-vasc score scheme, age > 75 years is assigned two points, on a par with previous stroke and tia.11,12 based on this new scoring scheme, women and elderly patients (75 years) who were previously identified as intermediate risk are now placed into a high - risk category with a recommendation for full anticoagulation. people are placed into different risk groups and prescribed appropriate antithrombotic therapy. in high - risk patients (chads2 score 2), oral anticoagulation with warfarin has been the standard to prevent the cardioembolic complications including stroke until recently.6 in patients with intermediate - risk (score of 1), acetylsalicylic acid (asa) may be used although warfarin is still preferred. on the other hand, asa is recommended in the low - risk patients (score of 0). antithrombotic therapy maybe deferred in patients with lone af or in patients who have contraindication to antithrombotic therapy.6 although therapeutic anticoagulation with warfarin has been proven to reduce the risk of embolic phenomenon and mortality in af patients, it is underutilized in high - risk patients.6,13 warfarin use has been limited by several properties such as slow onset of action, narrow therapeutic window, variable cytochrome p450-dependent metabolism, significant amount of drug - to - food and drug - to- drug interactions, and risk of bleeding complications such as intracranial hemorrhage in the elderly patients (table 3).11,1317 because of all of these issues, patients need to undergo frequent international normalized ratio monitoring and dosage adjustment in order to achieve and maintain appropriate levels. subtherapeutic anticoagulation exposes patients to cardioembolic events, and supratherapeutic anticoagulation subjects patients to bleeding complications. 18 in addition, it is difficult to achieve the desired time in the therapeutic range with warfarin. it is also expensive and impairs quality of life because of frequent doctor and laboratory visits.19 in addition, elderly patients and patients with severe renal impairment are at high risk of developing cardioembolic complications as well as increased risk of bleeding with warfarin use. older patients often have cognitive impairment and physical limitations which place them at increased risk for bleeding. patients with renal impairment can have functional abnormalities within the platelets and other coagulation pathways such as altered von willebrand factor that can place them at a higher risk of bleeding.2022 because of these reasons, the use of warfarin for stroke prevention has been challenging and is associated with significant impairment. therefore, there has been a need to find better therapeutic agents for the prevention of thromboembolic events and strokes in af patients. multiple new drugs have been evaluated as potential alternative anticoagulants for stroke prevention in af patients. active w (atrial fibrillation clopidogrel trial with irbesartan for prevention of vascular events) and active a (a separate arm of the active w) trials evaluated dual antiplatelet therapy of acetylsalicyclic acid and clopidogrel as alternative to warfarin. results from the active trials demonstrated that warfarin is superior to dual antiplatelet therapy and that warfarin is still the antithrombotic agent of choice. in patients who can not tolerate or safely sustain anticoagulation with warfarin, dual antiplatelet therapy may be considered.2325 due to the limitations of warfarin and dual antiplatelet therapy, there has an ongoing search for newer and better oral anticoagulants for stroke prophylaxis. efforts have been directed at finding agents with rapid onset of action, fewer food and drug interaction, and more predictable anticoagulation. as a result, direct thrombin inhibitors, direct and indirect fxa inhibitors, and other vitamin k antagonists have emerged and have been evaluated in various clinical trials. in the following sections, we will discuss briefly some of these agents and focus our primary attention on the newest of these agents, apixaban. dabigatran is a new oral direct thrombin inhibitor which has been approved by the united states food and drug administration (fda) for stroke prevention in nonvalvular af patients. dabigatran was extensively studied in the dabigatran with or without concomitant acetylsalicylic acid compared with warfarin alone in patients with nonvalvular atrial fibrillation (petro study) and the randomized evaluation of long- term anticoagulation therapy (re - ly study).2629 the re - ly study compared dabigatran etexilate with dose - adjusted warfarin in 18,113 patients in a multicenter, randomized, open - label study. results of this study showed that dabigatran 150 mg twice daily was superior to warfarin and dabigatran 110 mg twice daily was noninferior to warfarin in primary outcomes of stroke and systemic embolism. overall, there was a lower risk of bleeding (major, life - threatening, intracranial, and major or minor bleeding) but a higher incidence of dyspepsia compared to warfarin.29 based on the safety and efficacy results from the re - ly study, the fda approved dabigatran for stroke prevention in nonvalvular af patients (150 mg twice daily for patients with normal renal function and 75 mg twice daily for patients with renal impairment) in october 2010. dabigatran was added as a first - line option for anticoagulation in high - risk af patients in the 2011 updated of the acc / aha practice guideline for atrial fibrililation.30 dabigatran has been extensively used by clinicians around the world since its approval and there has been concern regarding an increased risk of gastrointestinal bleeding (especially in the elderly). in addition, the manufacturer has reported 260 fatal bleeding events after reviewing the postmarketing database. these findings have prompted several actions to be taken by different countries, including labeling updates in europe and the united states, safety advisories issued in japan and australia, and an fda investigation to determine whether the reports of bleeding in patients taking dabigatran are occurring more commonly than would be expected.31 an extension of the re - ly study, the rely - able study (rely - able long term multi - center extension of dabigatran treatment in patients with atrial fibrillation who completed re - ly trial nct00808067), is actively ongoing to evaluate the long - term safety of dabigatran. its primary role is to convert prothombin into thrombin which then participates in the thrombus formation. direct fxa inhibitors inactivate both prothrombinase - bound fxa and free fxa.14,32,33 several new agents have been developed as director fxa inhibitors, and these include rivaroxaban, apixaban, and edoxaban. rivaroxaban is the first direct fxa inhibitor that has been evaluated and recently approved for clinical use for stroke prophylaxis in atrial fibrillation. it has a half - life of 59 hours in the healthy individual and 913 hours in elderly patients. it is metabolized by the cyp3a4 enzyme pathyway.33 its use in atrial fibrillation was investigated in the rivaroxaban once - daily oral direct factor xa inhibition compared with the vitamin k antagonism for prevention of stroke and embolism trial in atrial fibrillation (rocket - af) trial as stroke prophylactic agent for af patients. the result of this study showed that rivaroxaban was noninferior to warfarin in preventing stroke and systemic embolism. the rivaroxaban group had fewer intracranial bleeding but more gastrointestinal bleeding events when compared to the warfarin group. these data demonstrated that rivaroxaban is a good alternative to warfarin in af patients who are at moderate to high risk of developing embolic strokes.34 although concern was raised during the approval process for its use as once - daily administration (due to its short half - life), it has been approved by the fda for prevention of stroke in patients with nonvalvular af in november 2011. there has not yet been sufficient substantive clinical exposure with rivaroxaban to evaluate its efficacy and safety in clinical practice. apixaban is the newest selective, reversible, oral, direct fxa inhibitor that has been evaluated for the prevention of strokes in patients with af in multiple trials. it has a rapid onset of action (3 hours) and a mean half - life of 12 hours. it is metabolized by the kidney, liver (primarily by cyp3a4, cyp3a5, and sulfotransferase 1a1), and the intestine. it is excreted 25% renally and 75% through the hepatobiliary system which is different from rivaroxaban (66% renal excretion). its absorption is not affected by food and it needs to be taken in a twice - daily dosing format. it is essential for clinicians to be cautious about concomitant use with cyp3a4 inhibitors and inducers, as the pharmacokinetics of the drug will be altered in such settings.32 a recent crossover study evaluated the pharmacokinetics, pharmacodynamics, and safety of coadministration of enoxaparin and apixaban in 20 healthy subjects. concomitant usage of apixaban and enoxaparin resulted in a 42% increase in peak anti - xa activity. similarly, the peak anti - xa activity was 15% higher when apixaban was coadministrated with enoxaparin compared to apixaban alone. overall, the study showed that the pharmacokinetics of apixaban were not affected by enoxaparin even though there was a modest increase in anti - xa activity when enoxaparin was used concurrently with apixaban. furthermore, the finding also suggested that co - administration of apixaban and enoxaparin was safe and well tolerated in healthy volunteers.35 apixaban has been evaluated in two recent phase iii randomized trials for stroke prevention in af patients. the first study was the apixaban versus acetylsalicylic acid to prevent stroke (averroes trial). it was an international (36 countries), multicenter (522 centers), double - blinded, and double - dummy trial which enrolled 5599 af patients over a 2-year period (september 2007december 2009). the patients enrolled in this trial were those who were not candidates for vitamin k antagonist therapy because of previous intolerance to or ineligibility for warfarin therapy.1,2 patients participating in the averroes trial had to be 50 years old and to have had atrial fibrillation documented in the 6 months before enrollment or by 12-lead electrocardiography on the day of screening. patients also had to have at least one of the risk factors for stroke which included prior stroke or tia, age 75 years, arterial hypertension (on treatment), diabetes mellitus (receiving treatment), heart failure (new york heart association class 2 at the time of enrollment), a left ventricular ejection fraction of 35%, or documented peripheral - arterial disease. patients were excluded if they needed long - term anticoagulation for reasons other than atrial fibrillation, if they had valvular disease requiring surgery, a history of a serious bleeding event in the past 6 months or if they were at high risk for bleeding, current alcohol or drug abuse or psychosocial issues, if they had a life expectancy 2.5 mg / dl or a calculated creatinine clearance of twice the upper limit of the normal range or a total bilirubin > 1.5 times the upper limit of the normal range), and allergy to acetylsalicylic acid.1,2 patients who met the inclusion and exclusion criteria were then randomly assigned through a 24-hour central, computerized, automated voice - response system to receive either acetylsalicylic acid (81324 mg per day) or apixaban (5 mg twice daily or 2.5 mg twice daily in those patients who met two of the following criteria : age 80 years, weight 60 kg, or serum creatinine concentration 1.5 mg / dl). primary efficacy outcome of the study was the occurrence of stroke (ischemic or hemorrhagic) or systemic embolism. the primary safety outcome was the occurrence of major bleeding defined by clinically overt bleeding with one of more of the following : drop in hemoglobin level of 2 g / dl over 24-hour period, transfusion of 2 units of packed red cells, bleeding at critical site, or fatal bleeding. study also included other outcomes such as rates of myocardial infarction, death from vascular causes, death from any cause, and composites of major vascular events. the averroes trial was terminated early by the data and safety monitoring committee when the interim efficacy analysis showed that apixaban was better than acetylsalicylic acid for the primary outcome in february 2010.36 as shown in table 4, both study groups had similar baseline clinical characteristics. of the patients enrolled in the study, 63%64% of patients had a chads2 score 2. the result of the study revealed that patients treated with apixaban had a statistically significant reduction in stroke and systemic embolic rates when compared with the acetylsalicylic acid group (1.6% per year vs 3.7% per year, hazard ratio [hr ] with apixaban 0.45, p 165 mg a day or needed both acetylsalicylic acid and clopidogrel, and severe renal insufficiency (serum creatinine level > 2.5 mg / dl or calculated creatinine clearance of < 25 ml / minute).3,4 eligible patients were randomized to receive either warfarin (dose adjusted to achieve an international normalized ratio of 2.03.0) or apixaban (5 mg twice daily or 2.5 mg twice daily in those patients who met two criteria : age 80 years, weight 60 kg, or serum creatinine concentration 1.5 mg / dl). international normalized ratios were monitored with the use of a blinded, encrypted, point - of - care device.3,4 the primary efficacy outcome was stroke (ischemic, hemorrhagic, or uncertain type) or systemic embolism. the key secondary efficacy outcome was death from any cause and rate of myocardial infarction. the primary safety outcome was major bleeding as defined by the international society on thrombosis and haemostasis (isth) criteria : clinically overt bleeding accompanied by a decrease in the hemoglobin level of 2 g / dl or transfusion of at least 2 units of packed red cells, occurring at a critical site, or resulting in death. the secondary safety outcome was a composite of major bleeding and clinically relevant nonmajor bleeding. other safety outcomes included any bleeding, other adverse events, and liver - function abnormalities.3,4 this study enrolled 18,201 patients from december 2006 to april 2010. as shown (table 6), both study groups had similar baseline clinical characteristics. of the patients enrolled in the study, 66% of patients had a chads2 score 2. the result of this study showed (table 7) that after the median follow - up of 1.8 years, patients in the apixaban group had a lower incidence of the primary outcome than the warfarin group (1.27% per year vs 1.60% per year, hr in the apixaban group 0.79, p < 0.001 for noninferiority and p = 0.01 for superiority). the apixaban group also had fewer hemorrhagic strokes (49% less) and fewer ischemic and uncertain types of stroke (8% less). patients who were treated with apixaban also had a reduced death rate from any cause, (3.52% per year vs 3.94% per year, hr 0.89, p = 0.047), from cardiovascular causes (1.80% per year vs 2.02% per year, hr 0.89), and from noncardiovascular cause (1.14% per year vs 1.22% per year, hr 0.93).3,4 the reduction in mortality observed with apixaban has not been shown in previous trials with other fxa inhibitors. as for safety outcomes, patients in the apixaban group had a lower incidence of major bleeding (as defined by the isth) as compared to warfarin (2.13% per year vs 3.09% per year, hr 0.69, p < 0.001). the apixaban group also had a reduced rate of intracranial bleeding (0.33% per year vs 0.80% per year, hr 0.42, p < 0.001). there was a 27% reduction in the rate of major bleeding in the apixaban group when analyzed by the intention - to - treat principle. moreover, there was no difference in the incidence of adverse events or liver - function testing.3,4 based on the results of the averroes and aristotle trials, apixaban clearly seems to offer better overall outcomes when compared with therapeutic anticoagulation with warfarin. it not only had a reduced incidence of primary outcome of stroke and systemic embolism, it also had fewer incidences of bleeding (including major bleeding and gastrointestinal bleeding) in all age groups. additionally, apixaban group also had a lower incidence of myocardial infarction. these findings demonstrate that apixaban is a suitable alternative to warfarin as a stroke prophylactic agent in patients with nonvalvular af. presently, dabigatran (direct thrombin inhibitor) and rivaroxaban (direct fxa inhibitor) are approved by the fda for stroke prophylaxis for patients with nonvalvular af and apixaban is currently undergoing review for same indication. results from phase iii trials have demonstrated that all three agents have the benefit of reducing the primary endpoint of stroke and systemic embolism ; however, they have different pharmacokinetic properties and metabolic pathways. both dabigatran and rivaroxaban are primarily renally excreted, and their dosages need to be adjusted in patients with renal impairment (eg, 75 mg twice daily in patients with renal impairment). some have even suggested avoidance of the use of these agents together in patients with severe renal dysfunction. in contrast, apixaban is mostly excreted in gastrointestinal tract (75%) with only 25% excretion through the kidneys.32,33 therefore, apixaban may be safer in patients with renal insufficiency ; however, its use in patients with hepatobiliary disease will require close monitoring. rivaroxaban and apixaban are both metabolized by the cytochrome cyp3a4 system and as such are subjected to drug - to - drug interactions with medications that are inducers or inhibitors of the cyp3a4 system. all of the newer oral anticoagulants are also subjected to interactions with p - glycoprotein inducers and inhibitors.32,33 comparison of the risk / benefit profiles of these new oral anticoagulants with warfarin reveals that all three resulted in lower rates of the primary outcome of stroke and systemic embolism as well as lower risks of bleeding (life - threatening bleeding, intracranial bleeding, and major and minor bleeding). of the three agents, only treatment with apixaban was found to be associated with a lower risk of overall mortality as well as cardiovascular mortality as compared to warfarin in the aristotle trial. although head - to - head comparison of the three newer anticoagulants is lacking, the available data do indicate that treatment with apixaban in the aristotle trial was associated with a lower rate of gastrointestinal bleeding. this is particularly important because, as stated earlier, clinical use of dabigatran has been reported to be associated with a significant risk of life - threatening gastrointestinal bleeding, such as a case report of fatal rectal bleeding.37 additionally, a recent pooled analysis of phase 3 trials compared the use of the three new oral anticoagulants (dabigatran, rivaroxaban, and apixaban) with enoxaparin for thromboprophylaxis after total hip or total knee replacement. the result showed that these new oral anticoagulants were more efficacious than enoxaparin in preventing venous thromboembolism after total hip or total knee replacement with similar bleeding risk. in addition, apixaban had the lowest clinically relevant bleeding risk (relative risk 0.81) among the three new oral anticoagulants.38 if apixaban use is indeed associated with a lower rate of gastrointestinal bleeding in clinical practice, it is likely to become the preferred agent. atrial fibrillation is the most common ar rhythmia encountered in clinical practice and it is associated with significant adverse clinical outcomes, especially cardioembolic strokes. although therapeutic anticoagulation with warfarin has been the standard therapy for stroke prophylaxis, it has been underutilized because of its narrow therapeutic window, significant drug - to - drug and drug - to - food interactions, need for frequent monitoring, and bleeding complications. there has been an ongoing search for newer and better oral anticoagulants for stroke prophylaxis in af. the research during the last decades has led to the recent introduction of two new oral anticoagulants (dabigatran and rivaroxaban) in clinical practice. apixaban is the newest oral direct fxa inhibitor which has been extensively studied for its use in stroke prophylaxis in the recently completed averroes and aristotle trials. the results of these trials have shown that treatment with apixaban is noninferior to warfarin in preventing stroke and systemic embolic events. compared with dabigatran and rivaroxaban, apixaban showed a reduced risk of gastrointestinal bleeding. the net clinical benefit observed during treatment with apixaban was also statistically superior to treatment with warfarin. if apixaban is approved by the fda (currently under review) for clinical use for stroke prophylaxis, it will certainly add to the therapeutic armory clinicians have in fighting against the cardioembolic strokes secondary to nonvalvular af in clinical practice.	atrial fibrillation (af) is a common cardiac arrhythmia, especially in the elderly population. it is associated with cardioembolic complications, particularly strokes, resulting in severe functional deficit or death. af patients are first stratified into low, intermediate, and high risk for thromboembolic events using the chads2 and cha2ds2-vasc score systems. depending on their risks, patients are treated with either therapeutic anticoagulation with warfarin or acetylsalicylic acid for stroke prevention. although warfarin is the recommended therapy, it is underutilized clinically due to concern for narrow therapeutic window, drug - to - drug and drug - to - food interactions, and hemorrhagic complications. newer anticoagulant agents such as dabigatran (a direct thrombin inhibitor) and rivaroxaban (a direct factor xa inhibitor) have already been approved by us food and drug administration for stroke prevention in patients with nonvalvular atrial fibrillation. apixaban is the newest oral direct factor xa inhibitor and it has been extensively studied in the averroes and aristotle trials. apixaban demonstrated reduced incidence of primary outcome of stroke and bleeding events when compared with warfarin. apixaban is currently being reviewed by the food and drug administration as a stroke prophylactic agent. in addition, there are several other indirect factor xa inhibitors and vitamin k antagonists under study presently. results from these studies will provide us with information about possible alternatives to warfarin.
the impact of heat on nerve conduction was initially described by hodgkin and katz in 19491. changes in sodium and potassium channel activation are primarily responsible for heat - related nerve conduction changes1, 2. with increasing temperature, voltage - gated sodium channel activation and deactivation becomes increasingly rapid, and channels remain open for a shorter duration. depending on the decreasing ion passage, the action potential amplitude diminishes and conduction velocity (cv) accelerates2. these differences that were defined at a single - fiber level were found to be similar in an in vivo environment3. in healthy nerve fibers, heating has been reported to increase both the sensory and motor nerve cv4, 5. in demyelinating diseases, nerve conduction blocks are observed with increasing body temperature6, 7. with respect to focal demyelinating conditions, superficial superficial heating modalities primarily increase the temperature of the skin and superficial subcutaneous tissues. in contrast, deep heating modalities change the temperature of deeper tissues to a depth of approximately 5 cm11. therapeutic ultrasound (us) is a deep - heating agent used to treat various musculoskeletal disorders12,13,14. the biophysical effects of us on tissues occur through 2 mechanisms : (1) thermal effects acquired with continuous application, and (2) non - thermal effects acquired from pulse application15. studies have reported contradicting results concerning the effects of deep heating produced by us on healthy nerve conduction parameters. with respect to motor nerve cvs, continuous zankel and farmer reported decreased ulnar motor nerve cvs with sonation intensities of 1 to 2 w / cm, but increased velocities at other intensities16, 17. madsen reported decreased cvs after sonation at 0.88 and 1.28 w / cm, but increased cvs with sonation at 1.92 w/ cm18. on the other hand, kramer19 observed increased motor cvs with all 5 tested sonation intensities in a range of 0.52.5 w/ cm. regarding sensory conduction velocities, us application is generally associated with decreased sensory latencies and increased cvs in healthy nerves20, 21. recently, burnham. reported no significant change in healthy nerve conduction parameters after us application5. carpal tunnel syndrome (cts), the most common neuropathy, is caused by entrapment of the median nerve under the flexor retinaculum. patients usually present with paresthesias, pain, and numbness or a tingling sensation in the fingers innervated by the median nerve. initial treatment of this condition is conservative, and includes splinting, exercises, medication, and physical therapy modalities. in a previous study, continuous us therapy at different intensities the researchers observed a decreased motor nerve conduction velocity and increased motor distal latency in median nerve conduction. the authors therefore suggested that continuous us should not be recommended for the treatment of carpal tunnel syndrome because of possible adverse effects on nerve conduction parameters due to overheating22. this negative effect of us on demyelinating nerve conduction must be considered, and therefore the biophysical effects of us should be assessed to confirm this result. previous reports do not appear to have described the instantaneous effects of deep heating on demyelinating nerves. however, such information would be important to the clarification of the effects of deep - heating modalities such as us on diseased nerves. therefore, the authors aimed to analyze and compare instantaneous changes after deep heating in demyelinating and healthy nerves in the present study. the effects of deep heating on motor and sensory nerve conduction studies were analyzed immediately before, at the midpoint, and immediately after the application of therapeutic us. the study was conducted in compliance with the principles of the declaration of helsinki. the study protocol was approved by the local ethical committee (dated 20.06.2012, no. 69), and patients provided written informed consent to participate. before all applications, the room temperature was recorded. patients were left to rest to allow their skin temperature to adapt to room temperature. the skin surface temperature on the palm was measured before, at the midpoint, and at the end of the us application using an infrared skin thermometer (medisana ag, neuss, germany). the infrared skin thermometer was reported to be highly reliable and valid for the purposes of an electrodiagnostic laboratory23. the probe head of the thermometer was placed on the skin surface of the palm, and the temperature was recorded in c. a total of 32 hands of 25 subjects (mean age : 51.6 9.5 years) were recruited for the study. eligible subjects were identified among patients referred to our electrodiagnostic laboratory with symptoms and diagnoses of cts24. a sample size of 19 would achieve 92% power for the detection of a mean difference in pre - treatment and post - treatment sensory velocities of 1.9 m / s with an estimated standard deviation of 2.4 m / s, and a significance level (alpha) of 0.05 using a 2-sided paired t - test25. patients were excluded if they had diseases that would affect nerve conduction, such as polyneuropathy, cervical radiculopathy, rheumatic diseases, or traumatic nerve injury. patients were also excluded if they had contraindications to us application and/or were administered a corticosteroid injection in the last 6 months. nerve conduction studies were conducted using a nihon kohden neuropack - s1 electromyogram device (tokyo, japan). motor conduction studies were conducted using low- and high - frequency amplifier settings at 5 and 10 khz, respectively. the amplifier settings for sensory conduction studies were 2 khz for the low and 20 khz for the high frequency. the sensitivity and sweep velocity were respectively set at 20 microvolts (v)/division and 2 msec / division for sensory studies and 5 millivolts / division and 2 msec / division for motor conduction studies. median motor nerve conduction parameters were recorded from the abductor pollicis brevis muscle, and ulnar motor nerve conduction parameters were recorded from the adductor digiti minimi muscle using superficial electrodes (orthodromic). the nerve was first stimulated from 7 cm proximal to the recorded muscle. a second stimulation was applied from the antecubital fossa for the median nerve, and from the cubital tunnel for the ulnar nerve. median sensory nerve conduction studies were recorded from the 2nd finger, and ulnar sensory nerve conduction studies were recorded from the 5th finger using a ring electrode (antidromic). median versus ulnar sensory conduction comparison studies were recorded from the 4th finger, and the peak latency of the sensory response was recorded. electrodiagnostic studies were repeated from the same marked points at the midpoint (4th min) and end (8th min) of us application. us applications were performed using an intellect mobile combo us unit (chattanooga, tn, usa). each subject was exposed to continuous - wave us treatment with a sonation intensity of 1.0 w / cm and frequency of 3.3 mhz. a 0.5-cm us head was used. a circular application technique with a soundhead movement speed of approximately 3 cm / second was used. the treatment area was 4 cm 2.5 cm, expanding from 1-cm distal to 2.5-cm proximal of the wrist crease, including the median and ulnar nerve traces. the size of the radiated area with respect to the diameter of the us head was within the recommended limits18. the kolmogorov - smirnov test was used to assess the normality of numeric variables. for numeric variables that were normally distributed, comparisons between 2 groups were made using the independent sample t - test, and results are expressed as means standard deviations. for the scores and numeric variables that were non - normally distributed, comparisons between 2 groups were made using the wilcoxon t - test or friedman test, and results are expressed as medians (25th75th percentiles). there were no statistically significant differences between skin surface temperature measurements before us application and at the 4th and 8th minutes (p > 0.05 ; table 1table 1.changes in skin surface temperaturebefore application4th minute8th minutetemperature (c)36.8 (36.537.0)36.8 (36.537.1)36.7 (36.537.2)absolute change values are presented as medians (2575 percentiles)). absolute change values are presented as medians (2575 percentiles) table 2table 2.summary of changes in nerve conduction parameters with deep heatinginitial4th minute8th minutemedian nervemotorlatency (msec)4.61 (4.264.95)4.58 (4.325.19)4.66 (4.095.14)cv (m / s)53.95 (50.6056.55)52.15 (50.1554.75)52.60 (50.8554.75)sensorylatency (msec)4.03 (3.334.61)4.03 (3.394.5)4.05 (3.424.64)cv (m / s)37.0 (33.545.0)36.0 (33.543.1)36.9 (33.341.3)ulnar nervemotorlatency (msec)2.60 (2.462.88)2.64 (2.542.95)2.77 (2.543.03)cv (m / s)62.65 (59.555.60)60.60 (58.1563.85)61.40 (57.6564.10)sensorylatency (msec)2.40 (2.342.67)2.43 (2.312.58)2.48 (2.312.59)cv (m / s)56.35 (51.5559.20)56.50 (52.0059.10)56.45 (51.9059.55)med - uln 4papld (msec)1.20 (0.922.30)1.18 (1.002.34)1.13 (0.982.26)absolute change values are median (25th75th percentiles). cv : conduction velocity ; med - uln 4p : median and ulnar nerve sensory conduction study to the fourth digit ; apld : absolute peak latency difference. the first measurement differs significantly from the 4th and 8th minute measurements (p < 0.05). the first measurement differs significantly from the 8th minute measurement (p < 0.05) summarizes the median values of changes in motor and sensory latencies and nerve cvs recorded from median and ulnar nerves before and at the 4th and 8th minutes of us application. regarding motor studies, the cvs of demyelinating median nerves exhibited significant decreases at the 4th and 8th minutes. in addition, a significant prolongation in the healthy ulnar nerve onset latency was identified at the 8th minute when compared to the initial value. there were no significant differences in the before- and after - us values for median motor latency, median sensory latency and cv, ulnar motor cv, ulnar sensory latency and cv, and median - ulnar 4th finger peak latency. cv : conduction velocity ; med - uln 4p : median and ulnar nerve sensory conduction study to the fourth digit ; apld : absolute peak latency difference. the first measurement differs significantly from the 4th and 8th minute measurements (p < 0.05). the first measurement differs significantly from the 8th minute measurement (p < 0.05) relative changes in electrophysiological parameters obtained from demyelinating and normal nerves at the 4th and 8th minutes of us application are compared in table 3table 3.comparison of changes in demyelinated and normal nerves with deep heatingmedian nerveulnar nerveml1-ml20.05 (0.28)0.06 (0.21)ml1-ml30.01 (0.230.21)0.08 (0.310.02)mcv1-mcv21.25 (0.004.77)1.30 (1.222.60)mcv1-mcv30.91 (3.77)1.57 (3.55)sl1-sl20.03 (0.100.08)0.01 (0.070.07)sl1-sl30.00 (0.100.10)0.01 (0.050.11)scv1-scv20.14 (1.99)0.30 (4.67)scv1-scv30.55 (0.601.72)0.25 (4.373.20)absolute change values are shown as means (standard deviations) or medians (25th75th percentiles) as indicated. 1 : initial value ; 2 : value at 4th minute ; 3 : value at 8th minute ; ml : motor latency ; mcv : motor conduction velocity ; sl : sensory peak latency ; scv : sensory conduction velocity. there were no significant differences between demyelinating and normal nerves in terms of velocity and latency changes due to deep - heat application. absolute change values are shown as means (standard deviations) or medians (25th75th percentiles) as indicated. 1 : initial value ; 2 : value at 4th minute ; 3 : value at 8th minute ; ml : motor latency ; mcv : motor conduction velocity ; sl : sensory peak latency ; scv : sensory conduction velocity the main question addressed in this study was whether the application of deep heating would differently affect demyelinating and normal nerves. some of these reports16,17,18 suggested that the cvs of healthy motor nerves decreased at sonation intensities of approximately 1 w / cm, except for 1 study25 that observed an increased cv. in addition, previous research indicated an association between sonation and increased sensory nerve cvs in healthy nerves19, 21, 25. in the present study, a statistically significant deceleration was observed with deep heating in both in demyelinating and healthy nerve motor cvs, but not in sensory cvs. previously, reduced healthy nerve motor conduction speed following us radiation has been attributed to micromassage action16 or a mechanical effect17 of us. this non - linear relationship between body temperature and nerve cvs might also explain the results of the present study. the authors of that earlier study also surmised that a decreased motor cv might be related to the cooling effect of the transmission gel. according to a report by kramer., intensities above 1.5 w / cm were necessary to exceed the cooling effect of the transmission gel25. in the present study, the sonation intensity was 1.0 w / cm, or below the lower limit determined by kramer. however, previous work has demonstrated temperature elevation in deeper tissue layers at a continuous us application of 1 mhz27. additionally, the effective radiating area and output power of us devices from different manufactures may differ, resulting in different degrees of tissue heating28, 29. consequently, these variables differed between studies and increased the difficulty of drawing a precise conclusion. the median and ulnar nerves, which have similar structures and anatomical locations, respond similarly to temperature changes30, 31. in the present study, healthy ulnar nerves in the arms affected by cts comprised the control group and were compared with demyelinating median nerves. use of the ipsilateral ulnar nerves as the control group enabled the elimination of potential biases such as deep tissue temperature differences or the amount of us radiation. as nerve conduction studies can be affected by many other parameters, such as height, age, sex, tissue temperature, and room temperature, the simultaneous comparison of healthy control and demyelinated nerves in the same arm is a strength of this study. several studies have evaluated the cumulative effect of us treatment in patients with cts18, 32, 33. in the first study, us pulsed mode 1:4 us was applied at 1 mhz and 1.0 w / cm for 15 minutes and 20 sessions, and significant improvements were observed in motor distal latency and sensory nerve conduction32. in another study, improvements in motor latencies and amplitudes of were observed in patients with cts after 15 sessions of us33. in contrast, another study found no significant difference in nerve conduction after 10 sessions of us with changing dosages, but identified a mild increase in motor latencies and deceleration of motor cvs with us applications at 1.5 w / cm and 0.8 w / cm22. in contrast, the present study focused on the immediate biophysiological results of deep heating from us. subcutaneous temperature measurement was not performed in this study, and this omission could be perceived as a limitation. however, previous reports described a significant correlation between intramuscular and skin surface temperatures, and skin surface temperature measurement is more reliable and reflective of the subcutaneous tissue temperature close to the nerve23, 30. therefore, in the present study used skin surface temperatures measured with an infrared thermometer, rather than subcutaneous tissue temperatures. the present study revealed a significant reduction in the demyelinating motor nerve cv, but not the sensory nerve cv, after deep heating. this finding could raise questions about the effectiveness of deep heating for diseased peripheral nerves. however, the present study only obtained results using continuous us at 3.3 mhz and 1w / cm, with application times of 4 and 8 minutes. in the future, similar studies involving different us parameters will enable researchers to obtain a wider variety of results.	[purpose ] physiotherapeutic heating agents are classified into two groups : superficial - heating agents and deep - heating agents. therapeutic ultrasound is a deep - heating agent used to treat various musculosketal disorders. numerous studies have attempted to determine the impact of ultrasound on healthy nerve conduction parameters. however, the instantaneous effects of deep heating via ultrasound on demyelinating nerves do not appear to have been described previously. the present study aimed to assess and compare the impact of ultrasound on demyelinating nerve and healthy nerve conduction parameters. [subjects and methods ] carpal tunnel syndrome was used as a focal demyelination model. thirty - two hands of 25 participants with carpal tunnel syndrome were enrolled in the study. ultrasound parameters were 3.3 mhz, 1.0 w / cm2, 8 minutes, and continuous wave. electrodiagnostic studies were performed initially, at the midpoint (4th min), and immediately after (8th min) ultrasound application. [results ] reduced motor conduction velocity was found in demyelinating nerves at the 4th and 8th minutes. ulnar nerve onset latency was significantly prolonged in the 8th minute recording, compared to the initial value. there were no significant differences in relative velocity and latency changes between demyelinating and normal nerves. [conclusion ] deep heating via ultrasound may inversely affect conduction velocity in demyelinating nerves.
attempted or accidental superwarfarin exposure has become an important public problem in western countries (1), as intoxication can cause prolonged bleeding diathesis, and sometimes fatal results. over 10,000 cases of superwarfarin intoxication were reported in 2008 to the poison control centers toxic exposure surveillance system in the united states (2). in korea, only sporadic reports of superwarfarin intoxication exist without any systemic and detailed investigations to the issue (3 - 5). we conducted an observational study of 10 cases of superwarfarin intoxication in korea, most of them broke out within a year. case report forms (crf) for patients with superwarfarin intoxication were requested to 8 physicians from october 2009 to april 2010 by electronic mails. a crf contained filling up section for basic information of a patient : age, sex, residence, occupation, past history of disease, and medication history of anti - coagulants or anti - platelet agents, etc. manifestations of bleeding were surveyed and initial laboratory data including prothrombin time (pt) and activated partial thromboplastin time (aptt) were collected. results of plasma mixing test, activity of coagulation factors and serum brodifacoum level test were also recorded. flow - sheet of treatment and change of laboratory data including pt and aptt of each patient were gathered. the crfs were analyzed by hong j and bang s - m and the results were reviewed by the other authors. this study was performed for the public good and had neither risk nor disadvantage for subjects. therefore, the institutional review board of seoul national hospital bundang hospital permitted this study without acquisition of informed consent (approval number : b-1007 - 106 - 105). this study was performed for the public good and had neither risk nor disadvantage for subjects. therefore, the institutional review board of seoul national hospital bundang hospital permitted this study without acquisition of informed consent (approval number : b-1007 - 106 - 105). only one patient (patient 6) lived in a metropolitan city, and outbreaks were nationwide. most of the cases occurred since 2009, except for a male patient who diagnosed in 2007 (4). two of them have been previously published as a case report (4, 5). reported bleeding events were oral mucosal bleeding (7 patients), hematuria (4 patients), easy bruising (5 patients), hematoma formation (5 patients), and epistaxis (3 patients). patients uniformly showed prolonged pt and aptt with decreased activity of vitamin k dependent coagulation factors. plasma mixing test and activities of coagulation factors were evaluated in all of the patients and the same result were showed : correction of pt and aptt and decreased activities of factors ii, vii, ix, and x. serum brodifacoum test were requested in 5 of 10 patients by physicians and 4 of them with a positive result were definitely diagnosed as superwarfarin intoxication. one patient (patient 2) repeatedly denied any contact with rodenticides, but she confessed to trying to kill herself by taking rodenticides after an interview with a psychiatrist. one patient (patient 3) was initially witnessed ingestion of rodenticides by her neighbors so serum brodifacoum test was not performed. environmental exposure was a possible cause in 3 patients (patients 4, 7, and 10) as they worked as a horticulturist, farmer, and ragman, respectively. four patients (patient 1, 3, 4, and 6) failed to achieve sufficient correction of pt and aptt initially so they had to increase doses of vitamin k1 administration. repeated prolongation of pt and aptt due to hasty discontinuation was observed in 6 patients (patient 1, 2, 3, 4, 6, and 8). ffp was initially transfused to all of the patients, but repeated ffp transfusion with low doses of vitamin k1 failed to maintain normalization of pt and aptt in 4 patients (patient 1, 3, 4, and 6). three patients (patient 1, 4, and 6) were completely discontinued vitamin k1 treatment, and their duration of vitamin k1 administration was 7.3, 6.2, and 3.3 months, respectively. all of the patients recovered or are on recovering from pt and aptt prolongation without sequalae and there was no mortality. only a patient (he confessed that he ingested excessive amount of fluconazole, an anti fungal agent, but repeatedly denied superwarfarin ingestion even positive result of brodifacoum level test was reported. it seems to be that he needed operation because of potentiated action of brodifacoum by drug interactions with the fluconazole. only one patient (patient 6) lived in a metropolitan city, and outbreaks were nationwide. most of the cases occurred since 2009, except for a male patient who diagnosed in 2007 (4). two of them have been previously published as a case report (4, 5). reported bleeding events were oral mucosal bleeding (7 patients), hematuria (4 patients), easy bruising (5 patients), hematoma formation (5 patients), and epistaxis (3 patients). patients uniformly showed prolonged pt and aptt with decreased activity of vitamin k dependent coagulation factors. plasma mixing test and activities of coagulation factors were evaluated in all of the patients and the same result were showed : correction of pt and aptt and decreased activities of factors ii, vii, ix, and x. serum brodifacoum test were requested in 5 of 10 patients by physicians and 4 of them with a positive result were definitely diagnosed as superwarfarin intoxication. one patient (patient 2) repeatedly denied any contact with rodenticides, but she confessed to trying to kill herself by taking rodenticides after an interview with a psychiatrist. one patient (patient 3) was initially witnessed ingestion of rodenticides by her neighbors so serum brodifacoum test was not performed. environmental exposure was a possible cause in 3 patients (patients 4, 7, and 10) as they worked as a horticulturist, farmer, and ragman, respectively. four patients (patient 1, 3, 4, and 6) failed to achieve sufficient correction of pt and aptt initially so they had to increase doses of vitamin k1 administration. repeated prolongation of pt and aptt due to hasty discontinuation was observed in 6 patients (patient 1, 2, 3, 4, 6, and 8). ffp was initially transfused to all of the patients, but repeated ffp transfusion with low doses of vitamin k1 failed to maintain normalization of pt and aptt in 4 patients (patient 1, 3, 4, and 6). three patients (patient 1, 4, and 6) were completely discontinued vitamin k1 treatment, and their duration of vitamin k1 administration was 7.3, 6.2, and 3.3 months, respectively. all of the patients recovered or are on recovering from pt and aptt prolongation without sequalae and there was no mortality. only a patient (patient 5) received major surgical procedure, evacuation of hemoperitoneum. he confessed that he ingested excessive amount of fluconazole, an anti fungal agent, but repeatedly denied superwarfarin ingestion even positive result of brodifacoum level test was reported. it seems to be that he needed operation because of potentiated action of brodifacoum by drug interactions with the fluconazole. the discovery of warfarin dates back to outbreaks of cattle death in the northern united states and canada in the 1920s (6). after isolation of the hemorrhagic compound in moldy silage which led to the death of the cattle, first generation anticoagulants including warfarin were introduced. warfarin is now widely used as a therapeutic anticoagulant, but its use as a rodenticide has declined because many rat populations have developed resistance to it (6). to overcome this resistance, second generation anticoagulants, they are highly lipid soluble and about 100 times more potent than warfarin because of the phenyl groups replacing the terminal methyl group in the structure (7). in a field test, 1 - 2 days feeding of brodifacoum was sufficient to achieve complete mortality in most of rodent species, compared to 21 days of warfarin (8). the reported half life of brodifacoum in humans is much longer than that of warfarin (16 - 36 days vs 17 - 37 hr) (9). the most common clinical feature of superwarfarin intoxication is bleeding, and this can occur from any mucosal site or organ. various symptoms of bleeding were reported in the literatures (1, 3, 4, 9 - 17) as in our study. superwarfarin intoxication should be suspected in any patient who presents with a suspicious history and marked prolongation of both pt and aptt without advanced liver disease or congenital coagulation factor deficiency. mixing studies result in complete correction of pt and aptt due to lack of inhibitors. measuring the activity of vitamin k dependent coagulation factors (factor ii, vii, ix, and x), plasma vitamin k levels, or pivka - ii can be helpful in narrowing down the differential diagnosis. the definitive diagnosis is made by superwarfarin blood level testing with high performance liquid chromatography (hplc) an accurate and effective way of determining the presence and concentration of superwarfarin. recent advances in hplc technology has made considerably accurate and rapid diagnosis possible (18), but such technology is not yet easily accessible in korea. in our study, although one patient (patient 2) was highly suspicious for superwarfarin intoxication and she even confessed ingestion, a negative result was reported. (9) evaluated half - life of brodifacoum in 3 patients with superwarfarin intoxication by measuring serial serum brodifacoum concentrations. they reported the half - life ranged from 16 to 36 days in 3 patients and serum brodifacoum had been no longer detected after 2 months from the time of initial diagnosis, although bleeding tendency continued more than 100 days. considering of their result, serum brodifacoum level in the patient of our study might be decreased to undetectable range at the time of sample request, as superwarfarin level test had been requested about 100 days after initial symptom presentation by her physician. oral vitamin k1 is preferred to intramuscular or intravenous injection, as it avoids hematoma formation and hypersensitivity reactions. an optimal dose and duration of vitamin k1 therapy has yet to be established (19). however, most previous studies support multiple, prolonged, and high dose vitamin k1 supplementation (1, 14 - 16, 19). as the half - life of vitamin k1 is 6 hr (7), multiple divided doses of vitamin k1 has the advantage of maintaining therapeutic effect. prolonged use of high dose vitamin k1 is necessary as the half - life of superwarfarins is substantially long (9). early tapering or discontinuation will result in repeat prolongation of pt and aptt even after normal levels have been achieved (12). this repeat prolongation due to hasty discontinuation was observed in 6 of our patients (patient 1, 2, 3, 4, 6, and 8) and in previous reports (10, 14). a phased and prudent tapering of vitamin k1 with regular follow up is thus mandatory (15). in a case series of 9 patients in taiwan (16), treatment duration ranged from 72 to 185 days. table 2 shows the doses and durations of vitamin k1 therapy performed in our study and several previous reports. in a situation of emergency due to bleeding, transfusion of coagulation factors is effective, although the effect of this will be transient because of long half - life of superwarfarin. urgent treatment with vitamin k1 and transfusion of coagulation factors based on clinical suspicion alone is justified because most detailed laboratory tests require several days to weeks. identifying the source of exposure is sometimes challenging in patients who deny ingestion of superwarfarin. after stabilization of symptoms, psychiatric interview should be recommended for patients in whom intentional administration is highly suspected as patient 2 of this study. we were unable to definitively identify the route of intoxication in 8 of the patients in this study. environmental exposure was a possible cause in 3 patients (patients 4, 7, and 10) considering their occupations. as these patients had no symptoms or history of mental illness, and taking into account the potency, lipid solubility and long - half life of superwarfarin, we can cautiously consider transdermal absorption and inhalation as potential routes of their superwarfarin exposure. such possibilities were highlighted in a previous report on 2 patients who worked in superwarfarin manufacture (3). the incidence of superwarfarin intoxication should be surveyed nationwide in light of these recent cases. supervision for rodenticide use and education of people who deal with rodenticides in their workplace are also necessary, along with further scientific research to achieve more accurate diagnosis and treatment of superwarfarin intoxication. in conclusion, early suspicion with a prompt request of serum brodifacoum test and sufficient dose and duration of vitamin k1 therapy is necessary for effective treatment of superwarfarin intoxication. further detailed investigation on the possibility and the actual condition of environmental exposure, and improving availability of the blood level test is mandatory.	this observational study aimed at evaluating recent superwarfarin intoxication of korean patients. ten patients were diagnosed as or highly suspicious for superwarfarin intoxication. case report forms described by attending hematologists of the patients were collected and analyzed. bleeding symptoms were varied among the patients. patients uniformly showed prolonged prothrombin time (pt) and activated thromboplastin time (aptt) with decreased activity of vitamin k dependent coagulation factors. positive serum brodifacoum test results in 4 of 5 requested patients contributed to confirmatory diagnosis. psychiatric interview revealed an attempted ingestion in one patient. high dose vitamin k1 therapy promptly corrected prolonged pt and aptt, but hasty discontinuation caused repeated bleeding diathesis in 6 patients. route of intoxication was unknown or not definite among 8 of 10 patients. three patients had a possibility of environmental exposure considering their occupations : there might be intoxication by transdermal absorption or inhalation. therefore, high dose and prolonged use of vitamin k1 therapy is necessary for effective detoxification. further detailed investigation on environmental exposure and efforts to improve availability of the blood level test in clinic are requested.
squamous cell carcinoma (scc) of the buccal mucosa accounts for 23% to 37% of all intraoral cancers. the highest incidence rate is observed in india, australia, brazil, france and south africa. a male to female ratio is about 2:1 with the largest number of oral sccs developing in the fourth and fifth decades of life. because of the high recurrence rate and invasive behavior of this tumor, the prognosis is generally poor. diaz 's showed that buccal squamous cell carcinoma (scc) is an aggressive cancer, with a tendency to recur locoregionally. strome 's revealed that 80% of patients had evidence of recurrence by 5 years, while lin cs 's found that recurance rate was 34% by 5 years. according to our search in pubmed medical data bases there was no similar reported case about the locoregional invasion and recuurence of buccal scc in iran. in march 2012,,a 32-year - old admitted in alzahra hospital medical center of isfahan, iran due to the swelling in the right cheek region that initiated from 6 months ago but rapidly grow during one month ago. he had a history of right cheek surgery with cervical lymph node dissection 2 years ago that was histopathologicaly diagnosed as squamous cell carcinoma (scc) at stage ii (t2n0m0) that justified no lymph node involvement in examination. extra oral examination, revealed a swelling of 3 2 cm in the right cheek region with a firm consistency. there were no palpable lymph node in the submandibular and cervical region and facial muscle paralysis was not existed. head and neck ct with soft tissue windowingin 2010, february showed a lesion in the right buccal region with dimensions of z 3.1 2.7 [figure 1 ]. that was surgically excised with all the involved surrounding tissues under the general anesthesia at that time. ct image, axial view, soft tissue windowing, showe a soft tissue mass lesion with dimension of 3.1 2.7 in the right buccal mucosa. (january 2010) in march 2012, ct images showed a soft tissue mass lesion with dimension of 2.5 3.64 in the right cheek region with erosive involvement of right zygomatic bone(body and anterior portion of the arc), palatal bone, ramus of the mandible and involvement of infra temporal fossa and pterygoid muscles [figure 2 ]. ct image, axial view, soft tissue windowing, showe a soft tissue mass lesion with dimension of 2.53.64 in the right cheek region with erosive involvement of right zygomatic bone (body and anterior portion of the arc), palatal bone, ramus of the mandible and involvement of infra temporal fossa and pterygoid muscles. (march 2012) magnetic resonance imaging (mri) of face showed a lobulated heterogeneous non enhancing mass lesion that was 2.5 cm in diameter in the right buccal mucosa with extension to theright masticatory muscles, right soft palate, uvula and also involving the right palatine tonsil, which may represent a neoplastic recurrence. there was a large non enhancing mass with diameters of 32 31 mm with low signal intensity in t1 images in the buccal mucosa representing the scar tissue in the previous surgical site. there is a fatty degeneration of the medial and lateral pterygoid muscles and right sided tongue muscles which may represent muscle denervation due to the peripheral spread of the neoplastic process, involving the maxillary and mandibular branches of the trigeminal nerve [figure 3 ]. mri, a lobulated heterogeneous non enhancing mass lesion in the right buccal mucosa with extension to the right masticatory muscles, right soft palate, uvula and also involving the right palatine tonsil, which may represent a neoplastic recurrence, also note to the non enhancing mass with diameters of 32 31 mm in the buccal mucosa representing the scar tissue in the previous surgical site (arrow). (march 2012) pathological examination revealed a neoplastic proliferation of squamous epithelial cells with cellular nests. the histopathological examination revealed a diagnosis of a well differentiated scc in the all affected tissues [figure 4 ]. histopathological views finally the complete excision of the mass in the right buccal mucosa and all affected tissues including right masticatory muscles, partial resection of the right maxillary and palatal bones and upper affected portion of mandibular ramus and zygomatic bone was done. due to the local invasion to the surrounding structures including adjascent bones and masticatory muscles and absence of lymph node involvement and distant metastasis, the stage of lesion was finally 4a (t4an0m0) after surgery. unfourthunetly, the patient did not refer to follow up after completion of the first chemotherapic duration. and in march 2012,,a 32-year - old admitted in alzahra hospital medical center of isfahan, iran due to the swelling in the right cheek region that initiated from 6 months ago but rapidly grow during one month ago. he had a history of right cheek surgery with cervical lymph node dissection 2 years ago that was histopathologicaly diagnosed as squamous cell carcinoma (scc) at stage ii (t2n0m0) that justified no lymph node involvement in examination. extra oral examination, revealed a swelling of 3 2 cm in the right cheek region with a firm consistency. there were no palpable lymph node in the submandibular and cervical region and facial muscle paralysis was not existed. head and neck ct with soft tissue windowingin 2010, february showed a lesion in the right buccal region with dimensions of z 3.1 2.7 [figure 1 ]. that was surgically excised with all the involved surrounding tissues under the general anesthesia at that time. ct image, axial view, soft tissue windowing, showe a soft tissue mass lesion with dimension of 3.1 2.7 in the right buccal mucosa. (january 2010) in march 2012, ct images showed a soft tissue mass lesion with dimension of 2.5 3.64 in the right cheek region with erosive involvement of right zygomatic bone(body and anterior portion of the arc), palatal bone, ramus of the mandible and involvement of infra temporal fossa and pterygoid muscles [figure 2 ]. ct image, axial view, soft tissue windowing, showe a soft tissue mass lesion with dimension of 2.53.64 in the right cheek region with erosive involvement of right zygomatic bone (body and anterior portion of the arc), palatal bone, ramus of the mandible and involvement of infra temporal fossa and pterygoid muscles. (march 2012) magnetic resonance imaging (mri) of face showed a lobulated heterogeneous non enhancing mass lesion that was 2.5 cm in diameter in the right buccal mucosa with extension to theright masticatory muscles, right soft palate, uvula and also involving the right palatine tonsil, which may represent a neoplastic recurrence. there was a large non enhancing mass with diameters of 32 31 mm with low signal intensity in t1 images in the buccal mucosa representing the scar tissue in the previous surgical site. there is a fatty degeneration of the medial and lateral pterygoid muscles and right sided tongue muscles which may represent muscle denervation due to the peripheral spread of the neoplastic process, involving the maxillary and mandibular branches of the trigeminal nerve [figure 3 ]. mri, a lobulated heterogeneous non enhancing mass lesion in the right buccal mucosa with extension to the right masticatory muscles, right soft palate, uvula and also involving the right palatine tonsil, which may represent a neoplastic recurrence, also note to the non enhancing mass with diameters of 32 31 mm in the buccal mucosa representing the scar tissue in the previous surgical site (arrow). (march 2012) pathological examination revealed a neoplastic proliferation of squamous epithelial cells with cellular nests. these cells had a high nucleus to cytoplasm ratio with atypism and pleomorphism. also keratin the histopathological examination revealed a diagnosis of a well differentiated scc in the all affected tissues [figure 4 ]. histopathological views finally the complete excision of the mass in the right buccal mucosa and all affected tissues including right masticatory muscles, partial resection of the right maxillary and palatal bones and upper affected portion of mandibular ramus and zygomatic bone was done. due to the local invasion to the surrounding structures including adjascent bones and masticatory muscles and absence of lymph node involvement and distant metastasis, the stage of lesion was finally 4a (t4an0m0) after surgery. unfourthunetly, the patient did not refer to follow up after completion of the first chemotherapic duration. and buccal scc is an important cause of morbidity and mortality worldwide with an incidence rate that varies widely by geographic location, sex, age and habit. buccal scc is an aggressive cancer with a high tendency to recur.. the incidence of buccal carcinoma is much higher in asia. in india, sharma revealed a male to female ratio of 2.2:1 with the largest number of sccs developing in the fourth and fifth decades of life. most of the reported cases of scc have a history of alcoholism and/or nicotine addiction, while our case did nt have any history of nicotine and alcohol consumption. squamous cell carcinoma (scc) of the buccal mucosa is a rare, but especially aggressive form of oral cavity cancer, associated with a high rate of recurrence. in contrast other oral cancer, buccal scc has a worse stage that affects the survival and become poor prognosis. scc of buccal mucosa has a failure rate even in patient with t1, 2 n0 stage that can be due to inadequate therapy and aggressive nature. there was a 100% overall incidence of local disease recurrence in patients with stage i and ii tumors treated with wide local excision alone and followed up for more than 2 years. patients with t1- or t2-sized tumors had only a 78% and 66% 5-year survival, respectively. postoperative radiotherapy was effective in decreasing locoregional failure in patients with close surgical margins, tumor thicker than 10 mm, high - grade tumors, positive node, and bone invasion. intra - arterial chemotherapy followed by radiotherapy is to be considered in advanced cases. similar to the presented case. the treatment of buccal carcinoma requires a multidisciplinary team approach because most of the patients are elderly and present with an advanced stage. nearly one - third of patients have localized disease, that is, t1 or t2 (stage i or stage ii) lesions without detectable lymph node involvement or distant metastases, these lesions are treated with curative intent by either surgery or radiation therapy. iin cs showed that scc of the buccal mucosa is an aggressive cancer with a high locoregional failure rate even in patients with t1 - 2n0 disease, so postoperative radiotherapy could led to a better locoregional control rate for patients and should be recommended for patients with t1 - 2n0 disease. the 5-year actuarial survival rates were 80% after surgery and 82% after surgery and postoperative radiation therapy. pop, showed a local recurrence rate of 45%, while schiza found the frequency as 56% in their research. postoperative radiotherapy has resulted in a better locoregional control rate for patients and should also be considered for patients with the t1 - 2n0 disease for whom adjuvant therapy after radical surgery currently is not recommended by most guidelines. in the head and neck cancer, the most important prognostic factor is the presence or absence of neck metastasis. deconde found that performance of neck dissection may decrease the risk of recurrence in primary scc of the buccal mucosa. finally the search in a procedure that diminishes recurrence may open the window of knowledge in treatment and increase survival. because of low survival and high recurrence, more research needs to perform to understanding the pathogenesis of the disease that led to new therapeutic strategies.	squamous cell carcinoma (scc) of the buccal mucosa accounts for 23% to 37% of all intraoral cancers, the prognosis is generally poor. we reported a case of local invasion of buccal squamous cell carcinoma. a 32-year - old man referred to the clinic with a chief complaint of swelling in the right cheek region that initiated from 6 months ago and rapidly grow from one month ago. history of the patient revealed that he was undergoing a surgery for buccal squamous cell carcinoma (scc) lesion 2 years ago. computed tomography (ct) and magnetic resonance(mri) images showed a heterogenous mass in the right maxillary, palate and mandibular regions that was histopathologically diagnosed as recurrence with locoregional invasion of scc.
although targeted endoscopic ultrasound - guided fine - needle aspiration (eus - fna) of the cyst wall following fluid aspiration has demonstrated improved overall diagnostic sensitivity for potentially malignant mucinous cysts 1, there are reports concerning a dissemination risk from eus - fna. the enhanced diagnostic capability of eus - fna must be balanced against the risk of tumor seeding. a 75-year - old man was admitted to our hospital due to a pancreatic cystic lesion accompanying a solid mass, and with liver metastasis. eus observation (uc240p - al5, olympus co, tokyo, japan) demonstrated a pancreatic cyst of 30-mm maximum diameter, and a solid mass beside the cystic lesion. using a 25-gauge needle (expect, boston scientific japan, tokyo) with a 20-ml syringe suction, we performed eus - fna on the solid mass, which also had a 30-mm maximum diameter through the posterior gastric wall. during the procedure, no early adverse events occurred. the pathological findings showed the lesion to be an adenocarcinoma that was diagnosed as intraductal papillary mucinous carcinoma (ipmc) (fig. 1). eus - fna on a mass beside cystic lesions through the posterior gastric wall using a 25-gauge needle (expect, boston scientific japan, tokyo). because the patient s condition was inoperable, gemcitabine - based chemotherapy was started at a dose of 1600 mg on days 1, 8, and 15 on a 4-week cycle. approximately 3 months after eus - fna, follow - up computed tomography scan showed a cystic mass in the posterior gastric wall (fig. 2). the cystic mass was visualized by esophagogastroduodenoscopy (egd) as a subepithelial lesion (sel) (fig. 3), and eus suggested that this sel was primarily localized in the third layer of the gastric wall. needle tract seeding of eus - fna for intraductal papillary mucinous carcinoma (ipmc) was suspected as the cause of this new lesion. eus - fna was thus performed on the sel and a definite diagnosis of ipmc that was similar to previous pathological findings was obtained (fig. 4). because the cystic mass was located where the eus - fna had been performed, the sel was therefore compatible with needle tract seeding from the initial eus - fna. as a second - line treatment, the patient underwent combined chemotherapy with one course comprising intravenous infusion of gemcitabine 1600 mg on day 1 and s-1 (tegafur + gimeracil + oteracil) 500 mg twice a day for 14 days with a 1-week rest period in between courses. despite chemotherapy, he died from the cancer 29 months after the initial eus - fna. a follow - up ct scan 3 months after eus - fna showed a new cystic lesion in the posterior gastric wall (arrow). eus showed an echoic cystic mass measuring 24 mm in diameter, primarily located in the third layer of the gastric wall. eus - fna for the gastric cystic lesion revealed a differentiated tubular adenocarcinoma that was consistent with the primary tumor in the pancreas. eus - fna for pancreatic cystic lesions is increasingly being used to differentiate benign cysts from cysts that have malignant potential or those with frank malignancy that may have otherwise been misdiagnosed as benign 2 3. 3 reported that the sensitivity, specificity and accuracy of eus - fna for the diagnosis of malignancy in ipmc were 75 %, 91 %, and 86 %, respectively. although a retrospective study reported no associated increased rate of gastric or peritoneal cancer recurrence following eus - fna 4, levy. 5 demonstrated the potential for tumor cell displacement during eus - fna. in the study by levy., the authors assessed the prevalence of luminal fluid - positive cytology among patients with luminal, extraluminal, and benign disease, and positive cytology was detected within post eus - fna luminal fluid in 3 of 26 patients (11.5 %) with pancreatic cancer. cytological examination of luminal fluid aspirates did not demonstrate malignant cells in patients with nonmalignant disease, suggesting that the process of eus - fna may withdraw malignant cells from a pancreatic cancer into the gastrointestinal luminal tract. this is likely an analogous method by which needle tract seeding occurs, and is concordant with reports on a dissemination risk from eus - fna. with respect to ipmc, 6 reported a case of ipmc dissemination to the peritoneum that was caused by eus - fna. due to copious mucus production in ipmc, mucus production after needle tract seeding may lead to widespread dissemination. in cases where eus - fna is performed for a cystic solid mass, therefore eus - fna should be performed only after the risks and benefits have been thoroughly evaluated.	background and study aims : we report on a 75-year - old man who was admitted due to pancreatic cystic lesion accompanied by a solid mass with liver metastasis. endoscopic ultrasound - guided fine - needle aspiration (eus - fna) was performed on the solid mass, and pathological findings revealed the lesion to be an adenocarcinoma diagnosed as intraductal papillary mucinous carcinoma (ipmc). approximately 3 months after, a cystic subepithelial lesion appeared in the posterior gastric wall where the eus - fna had been performed. we performed eus - fna again, which revealed that the cystic mass was ipmc with pathology similar to the original lesion. this is a rare case demonstrating needle tract seeding of eus - fna for ipmc.
a single - page questionnaire (technical appendix) was sent to the lead cf physician in all uk pediatric and adult cf centers identified by the principal uk cf charity, the cystic fibrosis trust, in 2009. results were tabulated and basic statistical analysis performed by using microsoft excel 2007 (microsoft, redmond, wa, usa). because m. chelonae and m. abscessus have common phenotypic characteristics and not all samples were fully sequenced by a reference laboratory, we combined these for reporting as mabsc. twenty - three adult and 29 pediatric uk cf centers, with 8,513 cf patients, were sent questionnaires. responses were received from 19 (83%) and 24 (83%) of these centers respectively, comprising 7,122 patients (3,805 adults, 3,317 children), or 84% of the uk cf population. as expected, the prevalence of any ntm isolate was higher in the adult (5.0%) than in the pediatric (3.3%) population (table). fifteen adult and 17 pediatric centers cultured samples for ntm yearly ; most remaining centers tested for ntm only if there was a specific clinical indication. the ntm pathogens most frequently cultured were the rapidly growing mabsc (62% of adults, 68% of children who were ntm - positive), followed by mac (28% of adults, 27% of children). other species (m. gordonae, kansasii, xenopi, fortuitum, simiae, malmoense, mucogenicum, perigrinum) together made up 8% of the total. wide geographic variation was noted, increasing from northwest to southeast, manifesting lowest prevalence in northern ireland (1.9%) and highest in south east england (7.5%) (figure). regional variation in percentage of cystic fibrosis patients in whom nontuberculous mycobacteria were isolated, united kingdom, 2009. nineteen of 23 adult and 24 of 29 pediatric cf centers, accounting for 7,122 of 8,513 (84%) cf patients, participated in the survey. most centers required clear clinical or radiologic deterioration in addition to positive cultures, but a significantly higher proportion of adult centers (42%) than pediatric centers (21%) were prepared to commence treatment on the basis of repeated culture or smear positivity alone. despite this, similar proportions of ntm - positive adults and children had received specific treatment for ntm in the 2 years preceding the survey : 44% of adults and 47% of children with any ntm isolate were treated, and 62% of adults and 70% of children in whom > 2 isolates were found were treated. six adults and 2 children who had cf had been refused a lung transplant on the basis of persistent ntm infection. our results differ from a 2003 multicenter us survey (9) in 2 crucial ways. first, the overall prevalence of ntm of 4.2% during the study period in the united kingdom was much lower than the 13% reported for the study done in the united states. this could be caused in part by lower sampling frequency, occurring annually in most uk clinics, compared with 3 times annually as reported in the us study. the proportion of patients having > 2 positive cultures was similar : 3.9% for the us study and 3.0% for our study. furthermore, the denominator used for prevalence estimation may have been overestimated in this study : it may have included some patients missed for annual culture ; and particularly for pediatric clinics, the denominator would include non sputum producers (cough swabs are normally not cultured for ntm). the questionnaire we used did not specify which source documents respondents used ; therefore, recall bias may be an issue. however, all uk centers extract and report ntm data to a national database annually. the second striking difference between this study and the us study was that rapid - growing mycobacteria (especially mabsc) predominated in this survey while mac predominated in the us study. our results are more in keeping with subsequent european and middle east prevalence studies (7,10,12). rapid - growing mycobacteria such as mabsc are now recognized to be of greater clinical importance in cf than mac (1), and a recent us single - center survey (13) has shown m. abscessus to be the current dominant ntm and to be associated with evidence of clinical decline. as in previous studies, we found marked geographic variation in prevalence : olivier. found higher rates in coastal centers in the united states (9), and roux. found the highest prevalence in west and southwest france (10). the higher prevalence in the south of the united kingdom could be caused by different sampling rates, but we found no systematic difference in screening policy between different uk regions. climatic differences are relatively small across a small temperate island such as the united kingdom. geology could be another factor ; ntm acquisition has been linked with water from aquifers (12,14) which are present mainly in the younger rocks in southeastern united kingdom. the varied threshold for instigating ntm treatment is perhaps not surprising in view of the lack of evidence to guide this. although there is no clear evidence to justify treatment on positive cultures alone, some centers may have lowered their threshold in the hope that (by analogy with pseudomonas aeruginosa) aggressive early treatment might lead to eradication. refusal of lung transplantation is clearly an issue and appears to affect the threshold for treatment, though the rationale for refusal by transplant centers remains controversial (15). this survey highlights the growing importance of ntm as a cf pathogen, the importance of routine, standardized surveillance, and the need to find out how best to manage ntm in cf. this will require further research into environmental, microbial, and host factors influencing acquisition and disease progression of ntm in the cf population.	incidence of pulmonary infection with nontuberculous mycobacteria (ntm) is increasing among persons with cystic fibrosis (cf). we assessed prevalence and management in cf centers in the united kingdom and found 5.0% of 3,805 adults and 3.3% of 3,317 children had recently been diagnosed with ntm. of those, 44% of adults and 47% of children received treatment.
descrever aspectos relacionados ao diagnstico e tratamento de pacientes com doena pulmonar por metal duro (dpmd) e realizar uma reviso da literatura. estudo retrospectivo dos pronturios mdicos de pacientes atendidos no servio de doenas respiratrias ocupacionais do instituto do corao, localizado na cidade de so paulo, entre 2010 e 2013. entre 320 pacientes atendidos no perodo do estudo, 5 (1,56%) foram diagnosticados com dpmd. todos os pacientes eram do sexo masculino, com mdia de idade de 42,0 13,6 anos e mdia de tempo de exposio a metal duro de 11,4 8,0 anos. os pacientes foram submetidos a avaliao clinica, histria ocupacional, tcar de trax, prova de funo pulmonar, broncoscopia com lba e bipsia pulmonar. todos apresentaram distrbio ventilatrio restritivo. o achado de imagem tcar de trax mais frequente foi de opacidades em vidro fosco (em 80%). houve o diagnstico de pneumonia intersticial descamativa associada bronquiolite celular em 1 paciente e de pneumonite de hipersensibilidade em 1. apesar de ser uma entidade rara, a dpmd deve ser sempre considerada em trabalhadores com risco ocupacional elevado de exposio a metais duros. a histria clnica e ocupacional associada a achados em tcar de trax e lba sugestivos da doena podem ser suficientes para o diagnstico. hard metal lung disease (hmld) is a rare disease caused by exposure to particles of hard metal alloys, whose major components are tungsten carbide (approximately 90%) and cobalt (approximately 10%) or cobalt and diamond. other components, such as tantalum, titanium, nickel, niobium, and chrome, are found in small amounts. because they are extremely hard and maintain their physical properties even at high temperatures, hard metals are used in tools for cutting and sharpening metals, drilling wells, polishing diamonds and dental prostheses, etc. workers are exposed to particles rich in cobalt (in ionized form) and tungsten carbide, which are absorbed by the lungs and gastrointestinal tract, both in cobalt powder production and when using cobalt alloy tools. the proposed mechanism for the pathogenesis of the interstitial disease involves a hypersensitivity reaction to cobalt. in addition, genetic susceptibility may play a role in the pathogenesis of the disease, although this role has yet to be fully understood. exposure to cobalt can cause several forms of lung disease, from asthma to various interstitial patterns in the lungs. the most widely known and typical histopathological presentation is giant cell interstitial pneumonia (gip), described by liebow in 1968. gip is characterized by the presence of multinucleated giant cells, which can be " macrophagic " in nature, in alveolar spaces. in addition to gip, other patterns described include usual interstitial pneumonia, hypersensitivity pneumonitis (hp), and desquamative interstitial pneumonia. in the present study, we describe diagnostic and treatment aspects of hmld and review the current literature on the topic. the study subjects were treated at the occupational respiratory diseases clinic of the pulmonology division of the instituto do corao of the university of so paulo school of medicine hospital das clnicas, in the city of so paulo, brazil, between 2010 and 2013. occupational histories were taken, after which all subjects underwent clinical evaluation, chest hrct, pulmonary function tests (elite dx series, medical graphics corporation, saint paul, mn, usa), bronchoscopy, bal, and lung biopsy. when the lung tissue obtained was considered insufficient for diagnosis, surgical biopsies were performed. we performed elemental analysis of lyophilized lung tissue specimens by energy - dispersive x - ray fluorescence spectrometry in 2 patients (edx 700-hs, shimadzu corporation, analytical instruments division, kyoto, japan). during the study period, 320 patients were treated at the facility. of those, 5 (1.56%) all of those 5 patients were male and were working at the time of the initial evaluation. one patient reported working as an industrial tool maintenance technician, 2 reported being industrial tool sharpeners, and 2 reported being grinder operators (table 1). table 1characteristics of the patients with hard metal lung disease.characteristic case 1case 2case 3case 4case 5age, years4430304363smokingnononoformer smokernooccupationgrinder operatorgrinder operatorindustrial tool sharpenerindustrial tool sharpenerindustrial tool maintenance technicianduration of exposure, years2568612chest hrct findingsextensive ground - glass opacities and poorly defined centrilobular micronodules, predominantly in the middle and upper lung fields. pattern suggestive of subacute hp fine reticulation, ground - glass opacities, and traction bronchiolectasis, distributed symmetrically and mainly peripherally, predominantly in the lung basesinterstitial micronodular infiltrates distributed in a perilymphatic pattern, predominantly in the upper lung fieldsground - glass opacities, peripheral reticulation, and traction bronchiectasis and bronchiolectasis, predominantly in the lower lung fieldsfine reticulation, ground - glass opacities, traction bronchiectasis and bronchiolectasis, distributed peripherally, and an area of air trapping in the left lower lobebal patternmultinucleated giant cells multinucleated giant cells multinucleated giant cells multinucleated giant cells lymphocytictype of biopsysurgicalsurgicaltransbronchialsurgicalsurgicalhistological pattern of the biopsy specimendip and cellular bronchiolitisgipgipgiphpeds resultscobalt + tungsten + cobalt - tungsten + nananatreatmentcorticosteroid therapy, azathioprinecorticosteroid therapycorticosteroid therapycorticosteroid therapy, azathioprine, placement on a lung transplant listcorticosteroid therapypatient courseworseningimprovementimprovementdeathworseningdip : desquamative interstitial pneumonia ; gip : giant cell interstitial pneumonia ; hp : hypersensitivity pneumonitis ; eds : energy - dispersive x - ray fluorescence spectrometry ; and na : not assessed. dip : desquamative interstitial pneumonia ; gip : giant cell interstitial pneumonia ; hp : hypersensitivity pneumonitis ; eds : energy - dispersive x - ray fluorescence spectrometry ; and na : not assessed. the most common chest hrct finding was ground glass opacities, in 4 patients (80% ; figure 1). other findings included peripheral reticular opacities with traction bronchiectasis and bronchiolectasis, in 3 (60% ; figure 2) ; and bronchial wall thickening, in 2 (40% ; table 1). restrictive lung disease, in varying degrees, was found in all subjects, and decreased dlco was found in 3 (table 2). case 2, before treatment initiation (figures a and b) : ground - glass opacities and parenchymal consolidations with some air bronchograms in both lungs, predominantly in the middle and upper lung fields, in a pattern similar to that of organizing pneumonia. case 2, after treatment (figures c and d) : the pulmonary changes were significantly reduced, there now being subtle foci of ground - glass opacification, mild, poorly defined centrilobular nodules, and scattered linear opacities, bilaterally. figure 2hrct scans. case 3 (figures a and b) : micronodular opacities distributed in a perilymphatic pattern, predominantly in the middle and upper lung fields. case 4 (figures c and d) : ground - glass opacities and foci of parenchymal consolidation, predominantly in the middle and upper lung fields, as well as mild peripheral reticulation with traction bronchiectasis and bronchiolectasis. variablecase 1 case 2 case 3 case 4 case 5 baselineafter 14mbaselineafter 60mbaselineafter 56mbaselineafter 30mbaselineafter 24mfvc, l4.17 (79%)3.74 (67%)2.85 (63%)2.88 (59%)3.47 (63%)4.12 (76%)2.22 (49%)1.76 (39%)2.81 (83%)2.33 (61%)fev1, l3.54 (83%)3.20 (73%)2.45 (65%)2.38 (60%)2.73 (60%)3.08 (68%)2.09 (56%)1.70 (46%)2.32 (86%)1.94 (69%)fev1/fvc0.850.860.860.830.810.750.940.940.820.83tlc, lna5.57 (75%)4.31 (68%)5.11 (83%)5.19 (66%)5.89 (75%)3.80 (56%)na4.07 (65%)4.06 (65%)rv, lna1.84 (91%)1.56 (96%)2.09 (142%)1.47 (79%)1.83 (98%)1.54 (77%)na1.29 (54%)1.67 (76%)dlco, ml / min / mmhgnana22.76 (72%)28.89 (106%)32.44 (93%)43.25 (107%)6.15 (20%)na19.39 (67%)11.29 (39%)m : months ; and na : not assessed. all patients underwent bronchoscopy, bal, and lung biopsy, with the diagnosis being established by examination of a bronchoscopy specimen in 1 patient and by examination of a surgical biopsy specimen in 4. large numbers of multinucleated giant cells were found in the bal fluid of 4 of the 5 patients (figure 3) and in the biopsy tissue of 3 (figure 4). desquamative interstitial pneumonia associated with cellular bronchiolitis was diagnosed in 1 patient ; a histological pattern suggestive of chronic hp, with no evidence of other occupational or environmental exposures that could be associated with hp, was identified in 1 ; the presence of tungsten was identified in 2, whose tissue samples were analyzed by energy - dispersive x - ray fluorescence spectrometry ; and the presence of cobalt was identified in 1 of those 2 (table 1). figure 3bal specimen containing macrophages, neutrophils, and lymphocytes, as well as multinucleated giant cells (papanicolaou stain ; magnification, 400). respiratory bronchiole stripped of its lining epithelium, filled with multinucleated giant cells (arrow), also observed in the adjacent alveoli, which are covered by metaplastic columnar epithelium (h&e ; magnification, 200). following diagnosis, all patients were withdrawn from their occupational exposure and received systemic corticosteroid therapy. two patients showed clinical, radiological, and functional improvement, although their lung function did not normalize (tables 1 and 2). three patients experienced disease progression despite corticosteroid therapy, 2 of whom additionally received azathioprine. one patient was placed on a lung transplant list, but he died before undergoing transplantation. in the case series presented here, ground - glass opacities were the most common chest hrct changes. multinucleated giant cells were detected in the bal fluid of most patients, and this finding was correlated with the typical histological pattern of gip. withdrawal from exposure combined with corticosteroid therapy was successful in 2 patients, and disease progression occurred in 3 patients. the functional, cytological, histopathological, and imaging findings are similar to those found in other studies. the published literature on cases of hmld in brazil is scarce and consists of the reporting of the disease in 4 patients. in the literature, data on the prevalence and incidence of hmld among individuals exposed to hard metals vary.. found no cases of hmld in cross - sectional studies involving 425 and 319 workers, respectively, with a mean duration of exposure to hard metals of 9 and 14 years, respectively.. found only 1 patient with hmld among 290 hard metal production workers evaluated. the diagnostic imaging methods available at the time might have contributed to the low hmld detection results observed in those studies. the paucity of cases and the varying latency period suggest that a hypersensitivity mechanism may be involved in the pathophysiology of hmld. found that 9% of the workers who had contact with hard metals had lung disease, and that the observed prevalence was not correlated with cobalt, tungsten, or tantalum levels, nor was it correlated with age, gender, or duration of occupational exposure, which, in the authors ' view, reinforced the hypersensitivity hypothesis. recurrence of hmld in unilateral or bilateral lung transplant recipients, even after cessation of exposure to hard metals, suggests the hypothesis that immune - mediated mechanisms are involved in the pathophysiology of the disease. in those 3 reported cases of recurrence, the histological pattern of gip was found, and no traces of tungsten or cobalt were detected in the transplanted lungs, which, in the authors ' view, reinforced this immune - mediated hypothesis.. demonstrated that hla - dp-glu69 was more prevalent among hard - metal - exposed workers with hmld than in those without. in that study, 19 (95%) of 20 hard - metal - exposed workers with hmld exhibited this polymorphism, compared with 17 (48.6%) of 35 hard - metal - exposed workers without the disease. the individual contribution of the major components of hard metal has also been investigated. in animal models, instillation of tungsten alone did not produce pulmonary inflammation, whereas cobalt alone was able to produce tissue injury. when combined, cobalt and tungsten had a synergistic effect and caused increased tissue inflammation. performed electron microprobe analysis of elemental levels in 17 patients with hmld and concluded that cobalt and tungsten accumulate in centrilobular fibrotic lesions. in that study, immunohistochemistry revealed the presence of tungsten particles within lesions surrounded by cd163 + macrophages and t cd8 + lymphocytes. the authors suggest that such macrophages play an important role in the development of fibrotic lesions. patients who have hmld develop restrictive lung disease and have a reduction in dlco, of varying severity. although chest x - ray and hrct findings are nonspecific, they are important in diagnosis, together with a history of exposure and histopathological changes. the major chest hrct changes found by choi. were ground - glass opacities and irregular linear opacities, predominantly in the lower lung fields ; these changes were also commonly seen in the 5 cases described here. unlike in other pneumoconioses, in which, in most cases, relevant occupational history and suggestive chest x - ray findings are sufficient to establish the diagnosis, in hmld, chest hrct and a specimen for cytological or histological analysis are required. bal, which is a minimally invasive method, can confirm the diagnosis without the need for lung biopsy. forni, in reviewing bal cytopathological findings described in the literature, concluded that there are two patterns that suggest the diagnosis of hmld. the first is characterized by significantly increased cellularity, resulting from characteristic multinucleated giant cells, lymphocytosis with a decreased cd4/cd8 ratio, and eosinophilia. to forni, these findings mirror the histopathological findings of gip, and, in such cases, a lung tissue biopsy would not be required. the second bal pattern is that of predominant lymphomononuclear infiltrates, with a decreased cd4/cd8 ratio, rare eosinophils, and giant cells, being similar to the pattern found in hp. in such cases, biopsy is essential to establish the diagnosis. in the present case series, the bal patterns coincided with those described by forni and reinforce the relationship between the presence of multinucleated giant cells in the bal and the histological diagnosis of gip. in a review of 100 patients who had undergone lung biopsy and had a diagnosis of hmld, reported that 59 of those patients had the pattern of gip, defined as the presence of typical multinucleated giant cells in the alveolar space. moriyama. highlighted the importance of the presence of centrilobular fibrotic lesions in the histological specimen for the diagnosis of hmld, even when there are no multinucleated giant cells. hard - metal - exposed workers often also use cutting oil, which may be contaminated with mycobacterium immunogenum, whose antigens can cause hp. when the observed histological patterns are inconsistent with gip, elemental analysis, by a combination of electron microscopy and energy - dispersive x - ray fluorescence spectrometry, is important in establishing the diagnosis. tungsten is usually found at high levels, whereas cobalt is usually found at moderate or low levels, since cobalt is highly soluble and is eliminated rapidly. although some authors have suggested that gip is a pathognomonic finding of hmld, there have been several published reports of cases with a histological pattern of gip without occupational exposure to hard metals and without tungsten or cobalt being detected in lung tissue. other reported cases of gip have been associated with exposure to titanium and to nitrofurantoin. treatment of hmld consists of withdrawal from the occupational exposure, which can stabilize or even improve lung function, and corticosteroid therapy. because of the scarcity of cases in the literature, there have been no studies comparing corticosteroid therapy and placebo treatment ; however, clinical experience is that corticosteroid therapy results in improvement. the use of immunosuppressive therapy also is not well established, although immunosuppressants are occasionally used. lung transplantation is a treatment option in patients with advanced lung disease that progresses despite treatment, despite the possibility of gip pattern recurrence in the transplanted lung. although hmld is rare, it should always be included in the differential diagnosis of respiratory dysfunction in hard - metal - exposed workers - such as tool sharpeners, toolmakers, operators of machines (such as milling cutters, grinders, and lathes), operators of diamond - blade cutting discs, and diamond polishers (using polishing discs made of hard metals)-who present with clinical complaints, functional changes, or imaging changes. data in the literature and our experience with the cases reported here suggest that adequate occupational history taking accompanied by chest hrct changes and bal findings (lymphocytosis with a decreased cd4/cd8 ratio and, especially, the presence of large numbers of giant cells) consistent with the disease might be sufficient for the diagnosis, there being no need for an open lung biopsy. treatment is based on permanent withdrawal from exposure and corticosteroid or immunosuppressive therapy, with varying results.	abstractobjective : to describe diagnostic and treatment aspects of hard metal lung disease (hmld) and to review the current literature on the topic. methods : this was a retrospective study based on the medical records of patients treated at the occupational respiratory diseases clinic of the instituto do corao, in the city of so paulo, brazil, between 2010 and 2013. results : of 320 patients treated during the study period, 5 (1.56%) were diagnosed with hmld. all of those 5 patients were male (mean age, 42.0 13.6 years ; mean duration of exposure to hard metals, 11.4 8.0 years). occupational histories were taken, after which the patients underwent clinical evaluation, chest hrct, pulmonary function tests, bronchoscopy, bal, and lung biopsy. restrictive lung disease was found in all subjects. the most common chest hrct finding was ground glass opacities (in 80%). in 4 patients, balf revealed multinucleated giant cells. in 3 patients, lung biopsy revealed giant cell interstitial pneumonia. one patient was diagnosed with desquamative interstitial pneumonia associated with cellular bronchiolitis, and another was diagnosed with a hypersensitivity pneumonitis pattern. all patients were withdrawn from exposure and treated with corticosteroid. clinical improvement occurred in 2 patients, whereas the disease progressed in 3. conclusions : although hmld is a rare entity, it should always be included in the differential diagnosis of respiratory dysfunction in workers with a high occupational risk of exposure to hard metal particles. a relevant history (clinical and occupational) accompanied by chest hrct and bal findings suggestive of the disease might be sufficient for the diagnosis.
acute megakaryoblastic leukemia (aml m7) is a rare hematological malignancy accounting for less than 5% of all cases of acute myeloid leukemia. aml m7 accounts for 7 to 10% of all cases of childhood aml but less than 1% of aml cases in adults. diagnosis is primarily based on bone marrow examination including both bone marrow aspiration and biopsy. there are only occasional case reports discussing the morphological features suggestive of megakaryocytic leukemia in extramedullary location on cytological smears. we report here a case of aml m7 presenting with cervical lymphadenopathy, emphasizing the cytological clues which may help in suspecting such a case on fine needle aspiration cytology (fnac) and cerebrospinal fluid. this is also the first report describing the morphology of aml m7 in cerebrospinal fluid. a 25-year - old male patient was treated with surgical excision for a mediastinal mixed germ cell tumor, the components of which included teratoma, embryonal carcinoma, and choriocarcinoma. subsequently he was administered four cycles of bep (bleomycin, etoposide, and carboplatin) and two cycles of etoposide and carboplatin in 2009. six months after completing the treatment, he presented with complaints of easy fatigability and generalized weakness. on examination, a clinical diagnosis of recurrent metastatic germ cell tumor was considered. a routine hemogram revealed hemoglobin of 6 g / dl. there was pancytopenia with a total leukocyte count of 3000/l and a platelet count of 20,000/l. the touch preparation showed few poorly preserved blasts, morphology of which was not clear. the decalcified sections of the biopsy were initially stained with hematoxylin and eosin stain and showed partial replacement of normal marrow by blasts. the blasts had scant to moderate cytoplasm with one to three prominent nucleoli, a few of them were binucleate and a few megakaryocytes showing multinucleation were also seen. on immunohistochemistry, the blasts were negative for myeloperoxidase (1:150 dilution, 59a5 clone, novocastra, new castle, united kingdom) and leukocyte common antigen (1:100 dilution, rp2/18 and rp2/22 clone, novocastra, new castle, united kingdom), but positive for cd 61 (1:100 dilution, 2f2 clone, novocastra, new castle, united kingdom) [figures 1a d ]. based on these findings, a diagnosis of acute megakaryoblastic leukemia was made and the patient was treated for the same with daunorubicin and cytarabin. however, six months later, the patient developed a single large cervical lymph node measuring 44 cm. a fine needle aspiration (fna) the smears were fixed in 95% ethyl alcohol for papanicolaou stain and immunocytochemistry, and air dried for may - grunewald giemsa (mgg) staining. there is partial replacement of normal marrow with blasts (h and e, 100) ; (b) the blasts have scant to moderate cytoplasm with prominent nucleoli (h and e, 400) ; (c) occasional multinucleated dysplastic megakaryocyte was also noted (arrow) (h and e, 400) ; (d) blasts showing strong cytoplasmic positivity cd 61, ihc (400) : microscopic examination of the aspiration smears revealed numerous blasts which varied in size from two to three times the size of mature lymphocytes. they had scant to moderate granular cytoplasm, fine chromatin, and single or multiple prominent nucleoli. some of the blasts had a notch in the nuclear membrane ; many had nuclear indentations and folds [figure 2a ]. interestingly at places, they were present in small clumps which on low power examination mimicked marrow particles as seen in smears prepared from bone marrow aspirate. within this population, there were cells which had single nucleus, but with the nucleus showing lobations, indentations and folds, some being horseshoe shaped [figures 3a c ]. bi to multinucleated cells reminiscent of megakaryocytes were also noted [figures 3d f ]. in addition, many anucleate pale blue staining masses were seen in the background, possibly representing shredded and clumped cytoplasmic fragments or giant platelets [figure 2b ]. immunocytochemistry showed the blasts to be negative for mpo, but positive for cd61 and cd34 (1:100 dilution, qbend 10 clone, dako, glostrup, denmark), thus confirming the diagnosis of extramedullary leukemic deposit of aml m7 [figure 2c ]. (a) blasts varying in size from medium to large, having scant to moderate granular cytoplasm, fine nuclear chromatin and single to multiple prominent nucleoli. occasional larger cells, having abundant cytoplasm and lobated nuclei (arrow) papanicolaou (200) ; (b) anucleate pale blue shredded cytoplasmic fragments in the background demonstrated better in low cellularity areas mgg (400) ; (c) blasts immunopositive for cd 61 icc (400) ; (d) bi and multinucleate forms on cytocentrifuge preparation of the csf specimen ; (inset) binucleate cell mimicking megakaryocyte mgg (400) (a) large cell with moderate amount of granular cytoplasm and a single nucleus showing multiple lobations papanicolaou (400) ; (b) cell having horse shoe shaped nucleus and dusky cytoplasm mgg (400) ; (c) another cell showing nuclear indentation and early lobulation mgg (400) ; (d, e, f) multinucleated cells with abundant cytoplasm and nuclear lobations resembling immature megakaryocytes mgg (400) a cytocentrifuge preparation (cytospin 4, thermo scientific, san diego, usa) of the csf specimen stained with mgg showed many blasts with scant to moderate granular cytoplasm, irregular nuclear margins, fine granular chromatin, and prominent nucleoli. there were binucleate forms (micromegakaryocytes) and multinucleate forms (immature megakaryocytes) [figure 2d ]. microscopic examination of the aspiration smears revealed numerous blasts which varied in size from two to three times the size of mature lymphocytes. they had scant to moderate granular cytoplasm, fine chromatin, and single or multiple prominent nucleoli. some of the blasts had a notch in the nuclear membrane ; many had nuclear indentations and folds [figure 2a ]. interestingly at places, they were present in small clumps which on low power examination mimicked marrow particles as seen in smears prepared from bone marrow aspirate. within this population, there were cells which had single nucleus, but with the nucleus showing lobations, indentations and folds, some being horseshoe shaped [figures 3a c ]. bi to multinucleated cells reminiscent of megakaryocytes were also noted [figures 3d f ]. in addition, many anucleate pale blue staining masses were seen in the background, possibly representing shredded and clumped cytoplasmic fragments or giant platelets [figure 2b ]. immunocytochemistry showed the blasts to be negative for mpo, but positive for cd61 and cd34 (1:100 dilution, qbend 10 clone, dako, glostrup, denmark), thus confirming the diagnosis of extramedullary leukemic deposit of aml m7 [figure 2c ]. (a) blasts varying in size from medium to large, having scant to moderate granular cytoplasm, fine nuclear chromatin and single to multiple prominent nucleoli. occasional larger cells, having abundant cytoplasm and lobated nuclei (arrow) papanicolaou (200) ; (b) anucleate pale blue shredded cytoplasmic fragments in the background demonstrated better in low cellularity areas mgg (400) ; (c) blasts immunopositive for cd 61 icc (400) ; (d) bi and multinucleate forms on cytocentrifuge preparation of the csf specimen ; (inset) binucleate cell mimicking megakaryocyte mgg (400) (a) large cell with moderate amount of granular cytoplasm and a single nucleus showing multiple lobations papanicolaou (400) ; (b) cell having horse shoe shaped nucleus and dusky cytoplasm mgg (400) ; (c) another cell showing nuclear indentation and early lobulation mgg (400) ; (d, e, f) multinucleated cells with abundant cytoplasm and nuclear lobations resembling immature megakaryocytes mgg (400) a cytocentrifuge preparation (cytospin 4, thermo scientific, san diego, usa) of the csf specimen stained with mgg showed many blasts with scant to moderate granular cytoplasm, irregular nuclear margins, fine granular chromatin, and prominent nucleoli. there were binucleate forms (micromegakaryocytes) and multinucleate forms (immature megakaryocytes) [figure 2d ]. extramedullary leukemic deposits often have to be differentiated clinically from infections and carcinomas for which fnac is an important diagnostic tool. there are only a few studies describing the morphological features of acute leukemia on fna smears, most of them being case reports.[521 ] however, of these, only one report discusses in detail the cytological features indicative of megakaryoblastic differentiation. we report the morphological features in a case of aml m7 with involvement of cervical lymphnode and cerebrospinal fluid. on fnac, granulocytic sarcomas can be classified into blastic, immature, and maturing forms of leukemias, based on the proportion of blasts to the more differentiating granulocytic cells. blasts from aml m7 typically show more abundant cytoplasm with a relatively lower nuclear to cytoplasmic ratio than what is seen in other subtypes of leukemia. presence of blasts of varying sizes including both small and large blasts, focal clumping of blasts forming small groups may also indicate the possibility of megakaryocytic differentiation. micromegakaryocytes in the form of large cells with single but multilobated nuclei, and/or bi or multinucleated cells having dusky cytoplasm or megakaryocytes along with dispersed giant platelets in the background may also be seen. although monoblasts also show nuclear membrane folds and indentations along with scant to moderate cytoplasm similar to megakaryocytes, they lack other features like multinucleation, multilobation, cytoplasmic shredding, micromegakaryocytes, and giant platelets. promyelocytes and promyeloblasts also show nuclear convolutions and low nucleo - cytoplasmic ratio, although some might have auer rods. kumar pv described many faggot cells (promyelocytes showing many auer rods) in a case of granulocytic sarcoma diagnosed by fnac. auer rods are however not seen in cases of aml m7. in the study by suh, auer rods were not seen in any of 27 cases of granulocytic sarcomas included in their study. another common differential is extramedullary hematopoiesis, especially when a deposit is characterized by presence of more maturing myeloid forms. a clinical history of myeloproliferative disorder or myelophthisic anemia, the presence of cells of erythroid lineage and also myeloid precursors besides megakaryoblasts can aid in making a correct diagnosis. metastatic carcinoma, when poorly differentiated, is an important differential diagnosis for aml m7 due to the presence of grouping of blasts, more abundant cytoplasm and large size of the blasts. poorly differentiated carcinoma may, however, have a suggestive clinical history. additionally, the tumor cells in carcinoma have coarser chromatin are more monomorphic and are cytokeratin positive on immunocytochemistry. multinucleated giant cells, if present, will have more bizarre hyperchromatic nuclei with or without prominent nucleoli in contrast to megakaryoblasts. in the case under discussion since there was a history of a mediastinal germ cell tumor, metastasis from the same may also be considered as a differential both clinically as well as on morphological assessment. of the germ cell tumors, seminoma especially shares some morphological features with leukemia / lymphoma infiltration as it is characterized by a hypercellular cytologic preparation with cells dispersed singly as well as in loosely dyscohesive clusters. nuclei are round to oval with thin nuclear membrane, vesicular chromatin, and prominent one central nucleolus or two to three smaller nucleoli. in contrast to megakaryoblastic leukemia which shows a more polymorphic population of cells of variable sizes with multinucleation and multilobation, multinucleated cells are usually absent in seminoma except for occasional syncytiotrophoblast - like cells. also background in the latter may have necrotic debris along with variable numbers of lymphocytes, plasma cells, and epithelioid histiocytes. a characteristic tigroid background may be noted on romanowsky stains. in comparison, however, some cases may be difficult to further characterize, in which case immunocytochemistry may be used. seminoma cells are immunopositive for cd117, placental - like alkaline phosphatase (plap), and sometimes for cytokeratin, all of which are negative in a case of megakaryoblastic leukemia except for cd117 which may be focally positive. smears in the former show mitotically active, markedly atypical cells arranged as glandular and papillary 3-dimensional structures. aspirates from choriocarcinoma show an admixture of multinucleated syncytiotrophoblasts and the mononuclear cytotrophoblasts in a background of hemorrhage and necrosis. although syncytiotrophoblasts may be misinterpreted for megakaryoblasts, they are much larger with more abundant cytoplasm, several pleomorphic nuclei and lack nuclear lobations. the tumor cells are mostly epithelioid - cell type, dispersed singly, having abundant cytoplasm ; marked anisokaryosis, bi- and multinucleation are common. cytoplasmic melanin pigment, present only in a minority, is beyond doubt the most diagnostic. confirmation in a case of amelanotic melanoma may be done with the aid of monoclonal antibodies for s-100 and hmb-45. in morphologically ambiguous cases, a panel of antibodies including lca, mpo, cd33, cd34, cd61, cd43, tdt, factor viii, cd117, plap, s-100, hmb-45, and cytokeratin should help in arriving at the diagnosis. suh and coauthors have used flow cytometry on their fna material to characterize their cases of granulocytic sarcomas. farray. also have described a case of megakaryoblastic leukemia involving pleural cavity ; they did not give detailed morphological description of the blasts though they performed a flow cytometry on pleural effusion fluid. in the cerebrospinal fluid sample evaluated in the present case, blasts, micromegakaryocytes and cells resembling megakaryocytes could all be identified as seen in the aspiration smears. this is the first report describing the finer cytomorphological features suggestive of megakaryoblastic differentiation on both aspiration and effusion smears. the features highlighted here may be of help in diagnosis and subtyping of extramedullary leukemic deposits of aml m7, especially in the event of a dry bone marrow tap, which is a common problem. all authors of this article declare that we qualify for authorship as defined by icmje. all authors are responsible for the conception of this study, participated in its design and coordination, and helped to draft the manuscript. as this is a case report without patient identifiers, approval from institutional review board (irb) is not required at our institution. to ensure 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 reviewers are blinded for authors)through automatic online system.	extramedullary deposits may be the presenting feature of acute myeloid leukemia. an early and accurate diagnosis on cytology will aid in correct patient management. this is especially true for patients with acute megakaryoblastic leukemia (aml m7), where bone marrow aspiration may yield only a dry tap. while cytomorphological features of myeloid sarcoma of other types are well recognized due to its rarity, there are only two case reports discussing the morphological details of megakaryoblastic differentiation on aspiration cytology. we present the case of a 25-year - old patient with extramedullary involvement of lymph node and cerebrospinal fluid by aml m7, describing in detail, the morphological features on aspiration as well as exfoliative cytology.
rubinstein taybi syndrome (rts) is characterized by mental retardation, postnatal growth deficiency, microcephaly, specific facial characteristics, broad thumbs, and big toes. a majority of patients have mutations in the gene that encodes the transcriptional coactivator, cyclic amp - responsive element - binding (creb) protein on chromosome 16p13, and about 3% of the patients have mutations in the e1a binding protein p300 (ep300) gene on chromosome 22q13. the behavioral aspects include a variable degree of mental retardation, impulsivity, distractibility, instability of mood, atypical depression, stereotypes, poor coordination, and overweight. there is one case report on recurrent manic episodes associated with rts and no reports on the association of rts with schizophrenia or non - affective psychosis. a 31-year - old, unmarried male presented with worsening behavioral problems for two months. he was the first of the three siblings, born of a second - degree consanguineous marriage among parents. his predominant symptoms included suspiciousness, delusions of reference and persecution, second person auditory hallucination giving derogatory comments on the patient, and occasional irritability. his symptoms were waxing and waning in nature, without full remission in between the exacerbation of symptoms. the patient was earlier treated by various psychiatrists with trifluoperazine, haloperidol, quetiapine, and valproate. the patient had partial improvement with the above - mentioned medications, but due to the side effects the patient had discontinued them. on physical examination the patient was pale, head circumference was 54 cms, height was 160 cms, weight was 60 kg, and vitals were stable. his physical anomalies included spiky hair, bushy eyebrows, synophrys, prominent supraorbital ridge, low set ears, long eyelashes, prominent nose, open mouth, carries in teeth, talon cusp (in the right upper canine), broad thumbs and toes, camptodactaly of little finger, cervical hyperkyphosis, and scoliosis. his intelligence quotient (iq) an x - ray of the lateral skull showed prominent occipital protuberance, an x - ray of the bilateral hands and feet revealed broad terminal phalanges, and echocardiography was within normal limits. his hemoglobin was 8.8 gm%, red blood cell (rbc) count was 2.38 millions / cmm, and peripheral smear was suggestive of dimorphic anemia. the patient was started on tab risperidone 2 mg / day and gradually the dose was built up to 6 mg / day, in divided doses, over two weeks. the patient had extrapyramidal symptoms and was managed with tab trihexyphenidyl 4 mg / day. he is under regular follow - up for the last six months and he is asymptomatic. a 20-year - old male presented with altered behavior for the last six months. he is the third of the three siblings born of a non - consanguineous marriage among the parents. the family members noticed that the patient had decreased social interaction, was suspicious that others may harm him, had occasional irritability, was not going to work, and was with poor personal care for the last six months. the patient had broad thumbs and toes, parrot beak shaped nose, long eyelashes, widows peak, synophrys, low set ears, deep palate, and malformed dentition, with talon cusp (upper lateral incisor). the patient was assessed using the mini scale and diagnosed as having a lifetime psychotic disorder. he was also diagnosed as having rts in view of the presence of physical anomalies. he was started on tab amisulpride 100 mg / day, which was later increased to 200 mg / day in divided doses. he is on regular follow - up for last four months and has reached a pre - morbid state. a 31-year - old, unmarried male presented with worsening behavioral problems for two months. he was the first of the three siblings, born of a second - degree consanguineous marriage among parents. his predominant symptoms included suspiciousness, delusions of reference and persecution, second person auditory hallucination giving derogatory comments on the patient, and occasional irritability. his symptoms were waxing and waning in nature, without full remission in between the exacerbation of symptoms. the patient was earlier treated by various psychiatrists with trifluoperazine, haloperidol, quetiapine, and valproate. the patient had partial improvement with the above - mentioned medications, but due to the side effects the patient had discontinued them. on physical examination the patient was pale, head circumference was 54 cms, height was 160 cms, weight was 60 kg, and vitals were stable. his physical anomalies included spiky hair, bushy eyebrows, synophrys, prominent supraorbital ridge, low set ears, long eyelashes, prominent nose, open mouth, carries in teeth, talon cusp (in the right upper canine), broad thumbs and toes, camptodactaly of little finger, cervical hyperkyphosis, and scoliosis. his intelligence quotient (iq) an x - ray of the lateral skull showed prominent occipital protuberance, an x - ray of the bilateral hands and feet revealed broad terminal phalanges, and echocardiography was within normal limits. his hemoglobin was 8.8 gm%, red blood cell (rbc) count was 2.38 millions / cmm, and peripheral smear was suggestive of dimorphic anemia. the patient was started on tab risperidone 2 mg / day and gradually the dose was built up to 6 mg / day, in divided doses, over two weeks. the patient had extrapyramidal symptoms and was managed with tab trihexyphenidyl 4 mg / day. he is under regular follow - up for the last six months and he is asymptomatic. he is the third of the three siblings born of a non - consanguineous marriage among the parents. the family members noticed that the patient had decreased social interaction, was suspicious that others may harm him, had occasional irritability, was not going to work, and was with poor personal care for the last six months. the patient had broad thumbs and toes, parrot beak shaped nose, long eyelashes, widows peak, synophrys, low set ears, deep palate, and malformed dentition, with talon cusp (upper lateral incisor). the patient was assessed using the mini scale and diagnosed as having a lifetime psychotic disorder. he was also diagnosed as having rts in view of the presence of physical anomalies. he was started on tab amisulpride 100 mg / day, which was later increased to 200 mg / day in divided doses. he is on regular follow - up for last four months and has reached a pre - morbid state. the two cases presented are the first cases described in literature about the association of schizophrenia / non - affective psychosis with rts. the analysis of genes potentially associated with schizophrenia is based on the observation that hypoxia prevails in the embryonic and fetal brain, and that interactions between the neuronal genes, molecular regulators of hypoxia, such as, hypoxia - inducible factor 1 alpha (hif1a), and intrinsic hypoxia, occur in the developing brain and may create conditions for complex changes in neurodevelopment., have evaluated the pc2 glutamine / q - rich - associated protein (pcqap) gene, which maps within the digeorge / velocardiofacial syndrome (dgs / vcfs) interval, as a potential candidate for schizophrenia susceptibility. the pcqap encodes for a subunit of the large multiprotein complex pc2, which exhibits a coactivator function in rna polymerase ii mediated transcription. it is a case control study involving schizophrenia patients (n = 378) and controls (n=444). the results indicate a possible involvement of the multiprotein complex pc2 in schizophrenia susceptibility. the camp signal cascade is implicated in the intracellular events mediated by various neurotransmitters and studies the creb protein is one of the messenger molecules involved in intracellular signal transduction pathways, used by most dopamine and serotonin receptor subtypes. in addition, creb stimulates the expression of a number of genes, alterations in the expression of which may be associated with schizophrenia. petrij., showed that the breakpoints at 16p13.3, demonstrated in patients with rsts, are all restricted to a region that contains the gene for the human creb protein, a nuclear protein participating as a coactivator in camp - regulated gene expression. the genes responsible for both the creb binding protein and ep300 are considered to be key regulators of rna polymerase ii - mediated transcription, acting as transcriptional coactivators in the regulation of gene expression through various signal transduction pathways. the crrb and ep 300 have also been identified as co - activators of hif1a. the factors discussed herewith, such as, regulators of rna polymerase ii and hif1a may be some common etiological factors for the association of schizophrenia or non - affective psychosis and rts. schizophrenia / non - affective psychosis can be a comorbid psychiatric condition with rts. in future the genetic analysis of such cases would help in the clear understanding of a complex disease like schizophrenia.	rubinstein taybi syndrome (rts) is a rare genetic disorder with characteristic physical anomalies. it is characterized by mental retardation, postnatal growth deficiency, microcephaly, specific facial characteristics, broad thumbs, and big toes. behavioral problems are common with rts ; they include mental retardation, impulsivity, distractibility, instability of mood, stereotypes, poor coordination, atypical depression, and mania. to date, there is lack of literature on the presence of schizophrenia or non - affective psychosis with rts. here, we describe two cases where there is co - morbid psychosis with rts. one case is diagnosed as paranoid schizophrenia and the other as psychosis possibly schizophrenia. genetic analysis was not done due to unavailability. the possible etiological factors for the association of psychosis with rts are discussed. factors such as regulators of rna polymerase ii and hypoxia - inducible factor 1 alpha (hif1a) may be some common etiological factors for the association of schizophrenia or non - affective psychosis and rts. schizophrenia / non - affective psychosis can be a comorbid psychiatric condition with rts.
eph receptors were identified in the late 1980 's and are known as largest family of receptor tyrosine kinases. they consist of a glycosylated extracellular domain with the immunoglobulin - like ligand - binding site, followed by a cysteine - rich region and two fibronectin type iii repeats (figure 1). connected via a single transmembrane spanning domain, the intracellular region contains a juxtamembrane domain, a tyrosine kinase domain, a sterile alpha motif, and a pdz-(postsynaptic density 95-discs large - zonula occludentes-1) binding motif [1, 2 ]. eph receptors bind membrane bound ligands, the ephrins, and both, receptors and ligands, are divided into two subclasses a or b based on binding properties and structural homologies. class a ephrins are membrane - bound via a glycosylphosphatidylinositol anchor and class b ephrins contain a transmembrane domain and a short cytoplasmic region with conserved tyrosine residues and a pdz - binding motif. class a eph receptors preferentially bind all a - type ephrins and class b eph receptors bind all b - type ligands. however, there are some exceptions, as epha1 primarily binds ephrina1, epha4 binds both, a- and b - type ligands, and ephrina5 binds epha receptors as well as ephb2 (figure 2) [36 ]. until today in contrast to other receptor tyrosine kinases, eph receptors / ephrins show unique properties in their activation and signaling. for the activation of the receptors not only dimerization as in most receptor tyrosine kinases multimer - induced signaling seems to be different from signals of normal dimers in so far as the degree of multimerization of the ephrins accounts for the kind and strength of biological effects. as eph receptors bind ligands which are also membrane bound, cell - cell contact is needed for eph receptor activation. on the other hand, recent work demonstrated that at least a - type ephrins can be released from the cell surface [9, 10 ]. these soluble proteins were shown to be functionally active and possibly represent an additional signaling mechanism without mandatory cell contact. forward and reverse [1113 ]. forward signaling involves binding of ephrins by the appropriate eph receptor. this leads to autophosphorylation of intracellular tyrosine residues of the eph receptor and further to activation of different downstream signal transduction cascades [14, 15 ]. in the case of b - type ephrins, if the cytoplasmic tail of the ephrin is phosphorylated which also results in activation of different signaling cascades. moreover, it should be noted that ephrina ligands might also have the potency to reverse signaling (overview in). many studies of the last decade indicate a complex cross - talk between eph receptors / ephrins and other signaling pathways which is necessary for consistent biological functions. the interactions between eph receptors / ephrins and different cell surface receptors, adhesion molecules, channels, pores, and cell surface proteases are reviewed in. taken together, eph receptors and their appropriate ephrin ligands represent an essential communication system that directs cell motility, repulsion and adhesion, cell - cell and cell - matrix contacts in a number of biological processes. due to the focused topic of this article, only two of them, angiogenesis and tumor angiogenesis, should be elucidated in detail, while other processes will be outlined in brief. eph receptor / ephrin signaling plays a crucial role in embryonic development. as an example, it has been shown that altered expression of epha3 and ephrina5 leads to defects in gastrulation and somite development. furthermore, together with integrin-5 and fibronectin, eph receptors / ephrins are discussed to mediate mesenchymal - to - epithelial transition and, hence, formation of somite boundaries. a further role of eph receptor / ephrin signaling is suggested in the developing and adult vertebrate brain. due to their complementary expression pattern, epha4, ephb2, ephb3, and their b - type ligands thereby, bidirectional signaling seems to be required for the restriction of cell intermingling between neighboring rhombomeres [20, 21 ]. furthermore, many studies analyzed the involvement of eph receptor / ephrin signaling in neuronal growth cone collapse, leading to axon guidance by inhibition. for instance, studies on epha4- and ephrinb3-null mice indicated that both proteins are required for normal formation of the corticospinal tract fibres, whereby eph receptor forward signaling is mandatory [22, 23 ]. the importance of proper ephrin ligand expression for correct outgrowth of retinal ganglion cell axons was analyzed by hornberger and colleagues. they demonstrated that unscheduled overexpression of ephrina2 in temporal axons leads to insensitivity of guiding outgrowing axons of the caudate tectum by repulsion. in the development of the visual system it was shown that ephb2 and ephb3 receptors and b - type ephrins are involved in axon pathfinding of retinal ganglion cells to the optic disc and that deletion of both ephb2 and ephb3 leads to increased frequency of axon guidance errors in this model. furthermore, the ephb2 receptor is also involved in synaptic functions (synaptic plasticity) in the adult mammalian central nervous system [26, 27 ]. in this regard, henderson and colleagues found that mice lacking the ephb2 receptor show reduced synaptic n - methyl - d - aspartate - mediated current and reduced long - term potentiation in hippocampal and dentate gyrus synapses. eph receptor / ephrin signaling plays not only a role in physiological processes, but also in pathophysiological processes such as tumorigenesis [7, 29 ]. thus, many ephrins and eph receptors were found to be upregulated in several human carcinomas such as breast, colon, liver, prostate, and melanoma and are often associated with tumor progression and metastasis (for overview see [7, 2931 ]). on the other hand, also downregulation of eph receptors can lead to increased metastasis and carcinogenesis as shown for epha1 in colorectal cancer, epha7 in prostate carcinomas, and ephb6 in melanoma [3234 ]. thereby, eph receptors do not operate like classical oncogenic growth factor receptors, because their activation does only in exceptional cases influence proliferation of the tumor cells [35, 36 ]. rather dysregulation of eph receptor activity seems to effect cell - matrix attachment, cell - cell attachment, organization of the cytoskeleton, and modification of tumor cell survival, which could result in increased cellular motility, tumor cell invasion, and metastasis. cell - matrix attachment can be influenced by eph receptors via modulating the integrin activity. for instance, epha2 stimulation with ephrina1 leads to decreased focal adhesion kinase (fak) phosphorylation which further results in inactive conformation of integrins and, finally, inhibition of integrin - mediated adhesion, cell spreading, and migration. it is assumed that also small gtpases of the ras and rho family could be linked to decreased integrin activation and cellular adhesion. however, the modification of cell attachment is probably dependent on the eph receptor / ephrin ligand ratio. a high expression of eph receptor and low expression of ephrin ligand could represent an advantage for tumor growth and metastasis. a possible cause for imbalanced eph receptor / ephrin ratio was recently analyzed by winter and colleagues who identified binding sites of multiple mrna - stabilizing and destabilizing factors at the 3'utr sequences of eph / ephrin transcripts. they found that binding of hur protein (a member of the embryonic lethal abnormal vision family of rna - binding proteins) to these regions destabilized eph / ephrin transcripts in tumor cell lines. the interaction of eph receptors and ephrins with other adhesion molecules such as e - cadherin could influence cell - cell attachment. thereby, it is assumed that e - cadherin can influence the expression and cellular localization of eph receptors and vice versa [4143 ]. the modification of the cytoskeleton is another important prerequisite for enhanced cellular motility and invasion, respectively, and there exists evidence of involvement of eph receptor / ephrin signaling. for instance, epha3/ephrina5 signaling induces growth cone collapse in retinal ganglion cells and cell rounding, blebbing, and detachment in epha3-expressing human kidney epithelial cells and melanoma cell lines [44, 45 ]. in both studies this was further confirmed by clifford and colleagues, who demonstrated that epha3 receptor suppresses motility through regulation of rho gtpases in rhabdomyosarcoma cell lines. moreover, eph / ephrin signaling can influence cell survival as shown recently by feng and colleagues. they demonstrated that overexpression of ephrina2 in hepatocellular carcinoma cells leads to enhanced tumor cell survival and proved that this is caused by resistance to tumor necrosis factor--(tnf--) induced apoptosis. in this regard, holen. demonstrated in jurkat tag cells that signaling through ephrina induced activation of scr and akt kinases, resulting in inhibition of antigen receptor - induced apoptosis. finally, it should be noted that some reports describe functionally relevant eph receptor mutations in some tumor entities. for instance, mutations have been identified in epha3 in melanoma and glioblastoma, and epha3, epha4, epha7, and ephb6 in colorectal cancers [49, 50 ]. angiogenesis is defined as growth of new blood vessels by sprouting from existing vessels [51, 52 ]. the lumen of blood vessels is faced by a single - layer squamous epithelium consisting of endothelial cells (ecs) which is separated from the circumjacent outer layers by the basal membrane. in small vessels (e.g., venules) ecs are enclosed by pericytes, in larger vessels by elastin fibres, smooth muscle cells and connective tissue. on one hand, ecs participate in the generation of blood vessels during embryonic development ; on the other hand, they retain their ability to proliferation and migration in adult organisms, where they renew the inner wall of existing blood vessels and rebuild new vessels, for instance, in uterus mucosa during menstruation and wound healing. at the beginning of the formation of a new capillary ecs form lateral pseudopodia which develop to a hollow tube. this new capillary sprout expands until it meets another capillary sprout for fusion, resulting in blood flow. this process is regulated by different expression of surface molecules on arterial and venous capillaries. the reaction of the ecs spans four periods : secretion of proteases to cleave the basal membrane of the parental capillary, migration of ecs towards the signal, proliferation of ecs, and, finally, formation of tubes and differentiation of the ecs. the most important factor is vascular endothelial growth factor (vegf) and its regulator hypoxia - inducing factor (hif-1), which stimulates transcription of the vegf gene [51, 53 ]. other important growth factors, like acidic and basic fibroblast growth factor (afgf, bfgf), can also initiate angiogenesis, whereby they affect not only ecs but also other cell types. additional vascular ecs - specific growth factors involve four members of the angiopoetin family and at least one member of the ephrin family, whereby those factors have to operate highly coordinated to form functional vessels. finally, factors not specific for ecs are required such as platelet - derived growth factor (pdgf) and tumor growth factor- (tgf-). generally, it is assumed that vegf functions as initiator of angiogenesis in development and adult organisms (with formation of immature vessels), followed by angiopoetin-1 and ephrinb2 function, necessary for maturation and stabilization of the vessel. moreover, angiogenesis is regulated not only by activating signals but also by inhibitors, for instance, thrombospondin-1, interferon-, platelet factor-4, and angiostatin. to date, more than 20 inducers or inhibitors of angiogenesis have been identified. concerning eph / ephrin signaling in angiogenesis, the pair of ephb4/ephrinb2 seems to play a key role. they are assumed to define vascular borders due to their reciprocal distribution : ephrinb2 on arteries and ephb4 on veins already in early developmental stages [5557 ]. the expression of ephrinb2 persists until late embryogenesis and adulthood, with distribution expanding from arterial ecs to surrounding smooth muscle cells and pericytes [54, 58 ]. generally, interplay between ecs and perivascular supporting cells mediated by ephrinb2/eph signaling is critical for vascular development as shown in several studies. for instance, foo and colleagues demonstrated that vascular smooth muscle cells require ephrinb2 for normal association with small - diameter blood vessels. in this context, oike. showed that unscheduled ubiquitous ephrinb2 expression in mice development leads to sudden death in embryonic stages due to defective recruitment of vascular smooth muscle cells to the ascending aorta. simultaneously, the authors suggest that bidirectional signaling is mandatory and that cell - to - cell repellent effects are important comparable to their role in the development of the central nervous system. in this regard, fller. hypothesized that distinct propulsive and repulsive effector functions of endothelial ephrinb2 and ephb4 prevent intermingling of cells and mediate spatial position signals during angiogenesis and vessel assembly. the importance of reverse signaling through ephrinb2 for vascular development is outlined by adams. and analyzed in detail by salvucci., who found that phosphorylation at the intracellular domain of ephrinb is dependent of src kinases and is assumed to play a role in pericyte - to - ecs assembly into vascular structures [62, 63 ]., who found that stimulation of ephb4 receptors with ephrinb2-fc fragments leads to phosphorylation of akt kinase and, furthermore, to increased proliferation and migration of the ecs. the authors show that this is mediated by the phosphatidylinositol 3-kinase / akt / endothelial nitric - oxide synthase / protein kinase g / mitogen - activated protein kinase axis. beside ephb4/ephrinb2 other b - class eph and ephrins play a role in vascularization and angiogenesis. in this regard, ephrinsb1, b3, and ephb2, b3, b4 are required for the regulation of the formation of the vascular network during cardiovascular development and for vascularization processes in the female reproductive system [6567 ]. furthermore, ephrinb1 is assumed to mediate ecs attachment on extracellular matrix by activation of integrins. in the case of a - class eph / ephrins, mainly epha2 and ephrina1 seem to be important for angiogenic processes. for instance ephrina1 is expressed in vascular development during embryogenesis in murine endocardium, dorsal aorta and primary head veins and later in intersomitic vessels and the limb bud vasculature. this implicates that ephrina1 expression corresponds to regions of vasculogenesis and/or angiogenesis, and presumably enhances angiogenesis [55, 69 ]. additional studies illuminated the role of involved pathways. referring to this, the role of vegf was analyzed by cheng., who demonstrated that soluble epha2-fc receptors inhibited vegf - induced survival, migration, sprouting of ecs and corneal angiogenesis. this was found to be mediated by jnk and p38mapk signaling pathways, leading to ecs migration and blood vessel assembly. another study showed that interaction of ephrina1 with epha2 induced activation of pi3 kinase and rac1 gtpase leading to ecs aggregation and migration. the role of epha2/ephrina1 in adult angiogenesis was further analyzed by different in vitro studies. for instance it was demonstrated that ephrina1 enhanced assembly of human umbilical venous endothelial cells (huvec) in matrigel and that soluble epha2-fc receptors inhibited microvessel formation in a rat aortic ring assay [73, 74 ]. angiogenesis can occur not only in physiological conditions but also in abnormal processes such as tumorigenesis. it is an early- to midstage event in many human cancers and a crucial step for the transition of a small, harmless cluster of mutated tumor cells into a large, malignant growth, capable of spreading to other organs throughout the body. without angiogenesis tumor size is restricted due to lack of nutrients, growth factors, and oxygen, resulting in counterbalance of dying and proliferating cells. hypoxia in solid tumors occurs at a distance of 70 m from functional blood vessels and it is generally accepted that tumors do not exceed a volume of 1 - 2 mm without the induction of angiogenesis [51, 76 ]. such proteins are produced by tumor cells themselves or by infiltrating immune cells, such as macrophages. alternatively, angiogenic proteins can be mobilized by tumor cells from the nearby tissue. once the process is initiated it can not be controlled or even stopped by the malignant cells. instead, newly dividing ecs release different proteins that can stimulate the proliferation or motility of tumor cells, leading to support of metastasis. generally, tumor cells produce two types of protein : one kind stimulates angiogenesis the other inhibits it, which lead to the hypothesis of an angiogenic switch in tumor angiogenesis [51, 76 ]. the most prominent angiogenic inducers are bfgf, afgf, and vegf with their corresponding receptors on ecs and among inhibitors are -interferon, platelet factor-4, and thrombospondin-1. fgf and other angiogenic factors can be sequestered in the extracellular matrix of many cell types, for instance ecs, and is believed to be released by proteolytic degradation of the extracellular matrix [51, 78 ]. for inhibitors alternative storage mechanisms are described : they are assumed to be stored as cryptic parts of larger molecules that are not per se inhibitors. among them are a 29 kda fragment of fibronectin, a 16 kda fragment of prolactin [80, 81 ], angiostatin as fragment of plasminogen, a small fragment of platelet factor-4, a propeptide of type 1 collagen, and a peptide fragment of endothelial growth factor. the balance between angiogenic inducers and inhibitors determines whether the tumor can switch on angiogenesis, whereby tumor angiogenesis is preferentially induced by a loss or decrease in the production of inhibitors. nevertheless, the underlying mechanisms are still poorly understood and dysregulation of transcription or the activation of different proteases are under discussion. an alternative way to facilitate tumor perfusion independent of tumor angiogenesis is the concept of vasculogenic mimicry [86, 87 ]. thereby it is assumed that tumor cells re - express endothelial and mesenchymal markers, normally appearing on embryonic cells. this is accompanied by induction of vascular structures mimicking blood vessels and thus promoting tumor growth. for instance, metastatic melanoma cells are able to constitute channels filled with blood cells. this tubules exhibit a basal lamina but no ecs and the formation seems not to be dependent of bfgf, tgf-, vegf, pdgf, tnf-, hypoxia, or integrins [87, 88 ]. in consequence, the formation of tubular networks on one hand results in better supply with nutrients and oxygen, on the other hand it can facilitate the invasion of tumor cells into the blood flow, thus, promoting metastasis. although the underlying mechanisms are not fully understood, the involvement of receptor tyrosine kinases, especially eph receptors, is strongly suggested. in an in vitro study hess and colleagues showed that transient knockout of epha2 expression in aggressive uveal melanoma tumor cells resulted in inhibition of tubular network formation. further the authors found that phosphorylation of epha2 by ephrina1 leads to activation of downstream signaling kinases such as fak and pi3 kinase and, furthermore, to the formation of vessel - like networks. the first reports concerning a direct connection between eph receptor / ephrin signaling and tumor angiogenesis appeared approximately 10 years ago. nikolova and colleagues investigated the b - class eph receptors and ephrins and found a spatially, temporarily, and hormonally coordinated expression of ephb4 and ephrinb2 during normal mouse mammary morphogenesis. the receptor was predominantly localized in the myoepithelial cells surrounding the ducts and alveoli whereas ligand expression was limited to the luminal epithelial cells. the disruption of the balanced expression lead to onset of carcinogenesis with loss of ligand expression and shift of receptor expression from myoepithelial cells surrounding the ducts to ecs with progressive malignancy. the importance of ephb4/ephrinb2 in tumor angiogenesis and tumor growth was also demonstrated in recent work on mouse models. in this regard, kimura and colleagues found that soluble ephrinb2-fc molecules suppressed growth of head and neck squamous cell carcinoma xenografts by inducing maturation of vessels in the tumor. other studies investigating the effects of ephb4/ephrinb2 on tumor microvasculature, tumor growth, and survival of tumor cells indicated that ephb4 could act as a survival advantage in head and neck squamous cell carcinoma and in breast cancer, respectively [93, 94 ]. they found both ephrina1 and epha2 expressed in tumor cells and endothelial cells in these xenografts, and also in vasculature and tumor cells of surgically removed human cancers. a further study revealed epha2, in combination with vegf, to be overexpressed in squamous cell carcinoma of oral tongue and, therefore, implicated in malignancy. today it is known that eph receptors and ephrins are expressed in both tumor cells and tumor vasculature of many types of cancer, often at higher levels than in endogenous tissue. thereby, eph receptor activation (forward signaling) is important as demonstrated by different studies using soluble receptors. blocking epha receptor signaling using soluble epha2-fc and epha3-fc receptors decreased tumor vascular density, tumor volume and cell proliferation in vivo, suggesting that the soluble receptors inhibited blood vessel recruitment by the tumor [74, 97, 98 ]. furthermore, epha2 kinase function in the tumor microenvironment seems necessary not only for tumor angiogenesis but also for metastatic progression [99, 100 ]. expression of truncated, soluble ephb4 receptor in breast cancer cells in a mouse xenograft model (with ephrinb2 ligand primarily expressed in the vasculature) increased tumor angiogenesis, suggesting that soluble ephb4 promotes tumor growth by stimulating angiogenesis through ephrinb2 signaling. another study showed that ephb4 and ephrinb2 are expressed by ecs of human malignant brain tumors and overexpression of different ephb4 variants in blood vessels in tumor xenografts leads to the assumption that ephb4 acts as negative regulator of blood vessel branching and vascular network formation. the involvement of additional eph receptors in the switch of dormant tumors to the fast - growing angiogenic phenotype was analyzed recently by almog and colleagues, who found increased epha5 plasma levels in mice and, furthermore, that mrna levels in tumor specimens of glioma patients correlated with disease stage. hence, among other investigated molecules, epha5 receptor possibly could represent a novel early cancer biomarker. an important question remains unanswered, concerning the initiation of the altered eph receptor / ephrin expression in tumor cells and tumor vasculature. until now it is not fully understood which mechanisms lead to this dysregulation, but it is hypothesized that hypoxia could play a role in this context. for instance in a mouse skin flap model of hypoxia vihanto and colleagues showed that hypoxia upregulates not only hif-1 and vegf but also ephb4, ephrinb2, epha2 and ephrina1 both on mrna and protein levels up to 48 hours after induction of hypoxia [30, 104 ]. furthermore, transcriptional profiles of umbilical cord blood and bone marrow - derived stem and progenitor cells showed that epha3 gene (among many other genes) is upregulated after hypoxia. another study, using hif-2 knockdown mice showed that also hif-2 interacts in hypoxia - induced tumor vascularization through activation of at least ephrina1. in contrast, in neonatal rats exposed to chronic hypoxia, among others, expression of hif-2 and ephrina1 was downregulated. however, it remains an important field and the identification of regulating mechanisms could provide novel targets for anti - angiogenic cancer therapies. in contrast to many other therapeutic approaches, anti - angiogenic therapy does not aim to destroy tumor cells directly. instead, it prevents tumor growth by its insufficient supply with nutrients and oxygen as a result of omitted blood vessel formation. numerous small molecule inhibitors and neutralizing antibodies targeting regulators of angiogenesis such as vegf / vegf receptors are recently under development and in clinical evaluation. for instance, recently the food and drug administration of the u.s.a. approved the anti - vegf - a - neutralizing antibody bevacizumab for treatment of stage iii - iv colorectal cancer in combination with chemotherapy and for treatment of nonsquamous non - small cell lung cancers, as well as small molecule tyrosine kinase inhibitors for treatment of renal cell cancer (sorafenib, sunitinib) and hepatocellular carcinoma (sorafenib). as eph receptors and ephrins are also significantly involved in angiogenesis and tumor angiogenesis and, therewith, in tumor progression and metastasis, they represent important targets for cancer therapy [19, 30 ]. to date, there are different approaches to target eph receptors and/or ephrins, either extracellularly by preventing receptor - ligand interactions or intracellularly through inhibition of tyrosine kinases or modification of gene transcription or translation (figure 3). one of them is the application of monoclonal antibodies, which show high specificity and are already well established tools in tumor therapy. the first ones were directed against epha2 and showed a significant inhibition of tumor growth in vitro [110, 111 ]. furthermore, effective targeting and internalizing into antigen - positive tumors in different mouse xenograft models have been reported for epha3 and ephb2 monoclonal antibodies [112, 113 ]. although the specificity for a particular binding partner is probably limited, another approach with great potential represents blocking of the eph receptor / ephrin signaling between tumor cells and ecs by the introduction of soluble eph receptors. in this regard, it was demonstrated that soluble monomeric ephb4 receptor resulted in dramatically reduced tumor growth in mouse models [114, 115 ]. furthermore, scehnet and colleagues fused the extracellular domain of ephb4 with human serum albumin for blocking ephrinb2 which results in inhibited migration and invasion of kaposi sarcoma cells in response to various growth factors. in addition, the role of a - class eph receptors was analyzed and inhibition of tumor angiogenesis and suppressed tumor growth in vivo was demonstrated for soluble epha2-fc and epha3-fc receptors [74, 97, 98 ]. not only eph receptors but also ephrins show therapeutic potency as truncated soluble forms. in this regard, soluble, monomeric ephrina1 is a functional ligand for epha2 in glioblastoma multiforme and modulates processes relevant to the progression of malignancy. beyond tumor pathology, soluble ephrinb2-fc or ephb4-fc chimeras, respectively, and soluble ephrinb2 were shown to reduce pathologic neovascularization in the retina [117, 118 ]. moreover, a possible therapeutic strategy represents conjugation of ephrins to gold - coated silica nanoshells, which was used to selectively target prostate tumor cells. an alternative strategy for targeting eph receptor / ephrin signaling is the application of mimetic or antagonist peptides, which were generated so far for a - class as well as for b - class eph receptors [120123 ]. finally, an alternative extracellular strategy is described by yamaguchi and colleagues who investigated peptide - pulsed dendritic cell vaccines and found that immunization with dendritic cells pulsed with epha2-derived peptides inhibited tumor growth in vivo in epha2-positive murine colorectal adenocarcinomas. therapeutical strategies focusing on intracellular structures involve inhibitors, selective for a single or for multiple tyrosine kinases. in this regard, several 2,5-dimethylpyrrolyl benzoic acid derivatives have been generated as selective small molecule inhibitors for epha4 receptors, as well as 2,4-bis - anilinopyrimidines for the inhibition of ephb4 receptors [125127 ]. in addition, various n - substituted 3-amino-4-methylbenzamide based type ii kinase inhibitors were analyzed concerning their potency to inhibit ephb2 receptor. it is a dual src / abl kinase inhibitor, whereby fak, crk - associated substrate, and epha2 receptor are assumed as additional targets. the inhibitor shows potent anti - proliferative activity against hematologic malignancies and has recently been approved for treatment of all stages of chronic myelogeneous leukemia. beneath its therapeutic effects in leukemias it was shown that dasatinib blocks migration and invasion of human melanoma cells without affecting proliferation and survival. furthermore, it was demonstrated that dasatinib blocks growth, migration and invasion of breast cancer cells, induced apoptosis and inhibited proliferation and invasion in different ovarian cancer cell lines. of importance an additional conceivable approach for therapies directed against intracellular targets is the regulation of the gene expression using small interfering rna or antisense oligodeoxynucleotides. in this regard, kumar. demonstrated that knockdown of ephb4 expression leads to anti - tumoral effects in breast cancer in vitro and in vivo. furthermore, it was demonstrated that knockdown of epha2 suppressed ephrina1- and vegf - induced endothelial cell migration and inhibited cell proliferation and induced apoptosis in human glioma cells [70, 134 ]. in part the pharmacological approaches against eph receptor-/ephrin - mediated tumor angiogenesis discussed above also provide the possibility to develop strategies for imaging of tumor vascularization, for instance, by means of fluorescent- or radiolabeled - small molecule kinase inhibitors or peptide ligands. overall, difficulties targeting eph receptor / ephrin signaling in cancer therapy should be kept in mind. heterogenous expression patterns of various eph receptors / ephrins in tumor and normal tissue complicate the discrimination of malignant cells from nonmalignant cells. furthermore, the effects of eph receptor - targeting agents on normal epithelial cells are insufficiently analyzed until today. another limitation in targeting eph receptors represents the occasional opposing effects of one eph receptor as tumor suppressor and tumor promoter. in this regard, signaling of ephrina1 and tumor cell - specific epha2 suppresses processes like growth and migration, whereas interaction of ephrina1 with ecs - specific epha2 seems to stimulate these same effects. furthermore, the efficacy of epha2 antibody - based therapy may depend on tumor type as no suppressive effect on tumor growth was observed in a colorectal tumor model, whereas mice harboring erbb2 in mammary epithelium were sensitive to therapeutic inhibition of epha2. when targeting the eph kinase activity, it should be noted that inhibition is useful in tumors where kinase activity promotes tumorigenesis (melanoma) but may instead be ineffective or even detrimental for the treatment of other types of cancer where eph receptor signaling suppresses tumorigenesis. in addition, the binding promiscuity of eph receptors and ephrin ligands as well as their capability to bidirectional signaling will further complicate targeting strategies and increase the potential for adverse side effects. therapies designed to either activate or block an eph receptor may also alter the signaling function of the ligand in adjacent cells [136, 140 ]. after all, possible interactions of eph receptor / ephrin therapeutic agents with other agents should be considered. it is assumed, that the kinase inhibitor imatinib can counteract the anti - oncogenic effects of ephb4 agonists in breast cancer. on the other hand, chemotherapeutic agents that target erbb receptors may enhance the effects of ephb4-targeted therapies. despite and due to the mentioned limitations it is necessary to understand the complex functions of eph receptors / ephrins in homeostasis and tumor progression to avoid undesirable side effects or unintentional exacerbation of disease functions. in this regard, targeting eph receptor / ephrin signaling to inhibit tumor angiogenesis and, therewith, tumor growth represents a promising approach in fighting cancer. eph receptors and their ligands, the ephrins, form a complex cellular communication system. its complexity is based on the large number of different receptor and ligand molecules, their promiscuous binding properties, the ability to bidirectional signaling, formation of multimers, and crosstalk with other signaling pathways and molecules. eph receptors and ephrins are involved in embryonic development, development of the nervous system, angiogenesis and also in tumorigenesis and tumor angiogenesis, respectively. they mediate cell - cell repellent effects, cell - cell and cell - matrix attachment, they influence cell survival and cytoskeleton dynamics, affecting cell motility, which could further result in tumor progression, invasion and metastasis. in the last decade eph receptors and ephrin ligands were put in perspective to anti - tumoral and anti - angiogenic therapy. to date, many different therapeutic strategies targeting eph receptors or ephrins are pursued and hopefully result in improvement of cancer treatment in the near future.	eph receptors and their ephrin ligands were identified in the late 1980 's. subsequently, they were linked to different physiological and pathophysiological processes like embryonic development, angiogenesis, and tumorigenesis. in this regard, recent work focused on the distribution and effects of eph receptors and ephrins on tumor cells and tumor microenvironment. the purpose of this review is to outline the role of these molecules in physiological angiogenesis and pathophysiological tumor angiogenesis. furthermore, novel therapeutical approaches are discussed as eph receptors and ephrins represent attractive targets for antiangiogenic therapy.
giant anal condyloma is a rare sexually transmitted disease involving the perianal and external genital tract area in the form of a large exophytic, cauliflower - like mass. complete surgical excision is the treatment of choice and often wide wounds are necessary to reach clear margins and prevent recurrence. various rotation or advancement flaps can be utilized as alternatives methods for covering wide wounds, decrease the length of recovery and minimize risk of anal stricture. we present two cases treated with an s - plasty rotation and a bilateral house advancement flap respectively. a 60-year - old man, heterosexual, presented in our department in june 2009,with a typical cauliflower - like tumor mass involving the perianal region. the patient was treated with daily wound cleansing with a good final result. a 64-year - old heterosexual man presented in our department in may 2012 with a typical cauliflower - like tumor mass involving the perianal region, and multiple condylomata involving the penis and prepuce. the patient was reluctant to undergo to medical treatment as symptoms had been present for 20 years. the patient was treated with a wide surgical excision and reconstruction with a bilateral house advancement flap (fig. the lesion on the penis and prepuce were excised by electrocautery scissors and a circumcision respectively. in this case dehiscence of the muco - cutaneus anastomosis developed during the second postoperative week maybe due to straining during defecation with consequent increased tension. at one month both patients were given a bowel preparation with polietilenglicole 4 l. intravenous antibiotic therapy with cephalosporine and metronidazole was administered for 7 days. a liquid diet and codeine were administered to all patients for three days after surgery to prevent evacuation and early contamination of the wound. a 60-year - old man, heterosexual, presented in our department in june 2009,with a typical cauliflower - like tumor mass involving the perianal region. a 64-year - old heterosexual man presented in our department in may 2012 with a typical cauliflower - like tumor mass involving the perianal region, and multiple condylomata involving the penis and prepuce. the patient was reluctant to undergo to medical treatment as symptoms had been present for 20 years. the patient was treated with a wide surgical excision and reconstruction with a bilateral house advancement flap (fig. the lesion on the penis and prepuce were excised by electrocautery scissors and a circumcision respectively. in this case dehiscence of the muco - cutaneus anastomosis developed during the second postoperative week maybe due to straining during defecation with consequent increased tension. at one month both patients were given a bowel preparation with polietilenglicole 4 l. intravenous antibiotic therapy with cephalosporine and metronidazole was administered for 7 days. a liquid diet and codeine were administered to all patients for three days after surgery to prevent evacuation and early contamination of the wound. giant anal condyloma also called buschke lwenstein (gcbl) tumor is a large exophytic, cauliflower - like mass that is characterized by local aggressive behavior. despite a benign histology in most cases, malignant degeneration into invasive squamous carcinoma (scc) is possible especially in immunocompromised patients. foci of invasive carcinoma are noted in 50% of the reports and carcinoma in situ in 8%. histologically gcbl presents with massive epidermal hyperplasia, hyperkeratosis and parakeratosis and is markedly exophytic. blunt - shaped masses of tumor project deeply into the dermis but tumor cells have little evidence of atypia and are not found inside blood vessels or lymphatics. lwenstein tumor is a sexually transmitted disease and like genital tract condylomata, bowen 's disease, anal squamous intraepithelial lesions and anal cancer are strongly associated with hpv infection. for this reason patient 's sexual habit should be investigated and if possible the patient 's sexual partners should also be examined. immunosuppression favors rapid growth of the condylomas and increases the risk of their malignant transformation maybe favoring the oncogenetic mechanisms caused by hpv infection. the patient 's immunological status must be checked including screening test serology for stds [human immunodeficiency virus (hiv), syphilis, hepatitis b virus (hbv) ] and hepatitis c virus (hcv). it allows histologically examination of the entire specimen and to evaluate for foci of scc. the cure rate with radical surgery reportedly is 61% and recurrences of giant condyloma acuminatum can be successfully treated with radical surgery. in limited lesions primary excision can be safely performed leaving wounds open to granulate without major primary flap reconstructions. in more extensive lesions flap or skin graft coverage is preferable to decrease the length of recovery and minimize risk of severe anal stricture. circumferential sleeve rectal advancement, house advancement, s - plasty rotations, v y advancement flaps are all effective alternatives methods for covering wide wounds. most authors do not perform colostomy for fecal diversion with acceptable postoperative complication rate. a combination of bowel cleansing, low fiber diet and loperamide can be administered to reduce early contamination with feces of the wound. in cases of anal canal involvement when mucosectomy extends beyond the dentate line colostomy helps to prevent potentially serious pelviperineal sepsis due to the high rate of suture dehiscence at this level. when performing anoplasty it is important to construct large flaps with a good blood supply and to obtain complete hemostasis of the raw surface to prevent hematomas as in our case 1. avoidance of tension on the muco - cutaneus anastomosis is important but not always technically simple. partial or complete dehiscence is not uncommon as in our experience but with a good follow up anal stenosis is rare. abdominoperineal resection should be performed for more extensive lesions with deep invasion, malignant transformation or tumor recurrence. no sufficient data are available to recommend any medical treatment such as interferon, radiotherapy or chemotherapy although sporadic cases of deeply infiltrating or recurrent tumors have been treated with apparent good results. postoperative complications such as hematoma or suture dehiscence are possible and should be avoided with accurate haemostasis and a good mobilization of the flap. local wound cleansing and an adequate follow up can avoid anal stenosis even without performing colostomy. the authors declare that written informed consent was obtained from the patient for publication of this case report and accompanying images.	introductiongiant anal condyloma also called buschke lwenstein tumor is a rare sexually transmitted disease involving anogenital region with potential malignant degeneration into invasive squamous carcinoma. complete surgical excision is the treatment of choice and often wide wounds are necessary to reach clear margins and prevent recurrence.presentation of casethe authors present two cases treated with an s - plasty rotating and a bilateral house advancement flap respectively with good functional result.discussiongiant anal condyloma also called buschke lwenstein tumor is a large exophytic, cauliflower - like mass that is characterized by local aggressive behavior. immunosuppression favors rapid growth of the condylomas and increases the risk of their malignant transformation. in limited lesions primary excision can be safely performed leaving wounds open to granulate while in more extensive lesions flap or skin graft coverage is preferable to decrease the length of recovery and minimize risk of severe anal stricture. abdominoperineal resection should be performed for more extensive lesions with deep invasion, malignant transformation or tumor recurrence.conclusiongiant anal condyloma also called buschke lwenstein is a rare pathology with mainly sporadic single center experience reported in literature. surgical complete excision remains the best treatment although elevate should be eventual recurrence. no sufficient data are available to recommend any medical treatment such as interferon, radiotherapy or chemotherapy.
due to a very extensive territory of yakutia (3,083,000 km), it is hardly possible to estimate permafrost temperatures at all of its 200 cattle burial grounds, especially because most grounds are situated in very remote and hard - to - reach places. we used the data of weather stations located in the same administrative districts as the burial grounds to estimate the trends in local air temperatures. the geographical coordinates (latitude, longitude, and altitude) and international identification indexes of all yakutia weather stations are listed at the website of russian meteorological service (www.meteo.ru) ; their locations are available on the interactive map at http://www.3planeta.com/googlemaps/karty-google-maps.html. we retrieved the daily temperature data for these weather stations from the us national climatic data centre website and selected years 19612010 as the study period. ncdc website maintains the most comprehensive archive of meteorological data collected all over the world up - to - date. the analysis of available archive data showed that only 17 out of 26 selected district weather stations reported their daily temperature data for all years between 1961 and 2010, whereas the data for the remaining 9 administrative districts were either incomplete or absent. regional models of climate change in yakutia have been developed only at a very broad geographic scale. there are only two such models : one covers all territories to the north of 65n and the other covers all territories to the south of 65n. the first model reports the following changes in annual average temperatures relative to the baseline : 0.2c for 19711980 ; 0.8c for 19811990 ; and 0.1c for 19912000. the second model reports the following temperature increments : 0.12c for 19711980 ; 1.6c for 19811990 ; and 0.3c for 19912000 [24. such aggregated information was not sufficient to estimate the temperature trends in individual administrative districts. for this purpose, we used the records of daily temperatures for those 17 districts where such data were available. even these datasets contained several periods of missing data and these were dealt in the following way. if, for any given year, more than 15% of daily data were missing, we excluded this year from the analysis. if 1 month of daily data were missing, we used daily temperatures during the same month of the preceding year and calculated the annual average temperature using these proxy data. then, the time series of annual average temperatures between 1961 and 2010 were tested for a linear trend using least squares method. the territory of yakutia can be subdivided into four climate - geographic zones : west part from laptev sea to the south boundaries of the republic ; central plains part ; northeast part that includes the arctic tundra and novosibirsk islands ; and south highlands part (www.atlas-yakutia.ru). the survey of cattle burial sites showed that most of them were located in the western part (112 sites) and in the central plains part (112 sites), whereas the smallest numbers (43 sites) were located in the east part, which is mostly occupied by mountains (fig. 1). geographic distribution of burial grounds of cattle that died (+) from anthrax in yakutia. the observed trends in the average annual temperatures in different parts of yakutia in 19612010 are summarized in table 1. the lowest air temperatures are typical for the east part, where the number of cattle burial grounds is the smallest. statistically significant linear temperature trends were obtained for the eight administrative districts with the highest annual average temperatures. the smallest change was observed in viluisky district, t=0.6c (thistoric=17.9c and tcurrent=18.5c). the greatest change was observed in churapchinsky district, t=1.0c (thistoric=17.9c and tcurrent=18.9c). in yakutsky district, it was interesting to observe that the annual warming rate in viluisky district was also minimal among all studied districts (0.02c / year), while the annual warming rate in churapchinsky district was maximal (0.04c / year). descriptive statistics of annual and july average temperatures in 19612010 and linear trends in annual average for selected administrative districts of yakutia with the greatest numbers of anthrax cattle burial grounds another characteristic of climate warming was the relative increase in the number of very hot days defined as the days with average daily temperature above the respective long - term average for this date, calculated over the past 50 years. the proportions of such days (n) were calculated for the historic and the current periods : nhistoric= 34.5% and ncurrent= 52% in viluisky district ; nhistoric= 39.1% and ncurrent= 56% in yakutsky district ; nhistoric= 38.9% and ncurrent= 52% in churapchinsky district. average daily temperatures in july 2010 were greater than the long - term average temperature of july during all 31 days of this month. the ranges of annual average temperatures observed in different parts of yakutia are shown in fig. the most pronounced temperature trend was observed in the administrative districts around yakutsk city (fig. statistical analysis confirmed a significant 0.1% level positive trend in the annual average temperatures in yakutsk. similar results have been obtained earlier (24) : climate warming is more pronounced in central and south yakutia than in north yakutia. the districts with the greatest increments of annual average temperatures also reported the highest numbers of outbreaks of anthrax : between 4 and 11 cases during the last 80 years. according to veterinary experts, these districts present the greatest risks of anthrax (14). from epidemiology standpoint, thorough monitoring of anthrax cattle burial grounds is recommended for the entire territory of yakutia, not just for several selected parts. this monitoring should include regular surveys of cattle burial sites, checks of fences around them, inspections of land - use permits, and other documentation, detailed measurements of permafrost parameters around such sites. the trends in permafrost temperatures may vary greatly even within a single administrative district (24). unfortunately, the authors could not find any information about the actual condition of siberian anthrax cattle burial sites, cropping out the remains or soil erosion. the authors recommend thorough monitoring of activity of airborne anthrax both in the northern and southern parts of yakutia, in the conditions of climate change, especially in light of the findings obtained in central asia (kazakhstan), where geographic distribution of b. anthracis was studied (25). statistically significant positive trends in the annual average temperatures were established in 8 administrative districts out of 17 districts of yakutia for which sufficient meteorological data were available. these eight districts should be carefully monitored in the first place, but all regions where the outbreaks of siberian anthrax took place in the past should probably deserve equal attention of epidemiologists. gradual phaseout of these burial grounds should use modern technologies of utilization of cattle remains. it is quite important, therefore, to estimate the threshold temperatures above which depreservation of the frozen remains becomes significant. temperature thresholds of permafrost degradation are estimated on the basis of geomorphological indicators (segregated frost heave mound detailed field surveys on the state of cattle burial grounds and the measurements of air and permafrost temperatures are needed for objective assessment of epidemiologic situation. besides massive vaccination of domestic animals has been proven to be effective to reduce the rates of this disease among both domestic animals and people living in the russian arctic. the authors have not received any funding or benefits from industry or elsewhere to conduct this study.	climate warming in the arctic may increase the risk of zoonoses due to expansion of vector habitats, improved chances of vector survival during winter, and permafrost degradation. monitoring of soil temperatures at siberian cryology control stations since 1970 showed correlations between air temperatures and the depth of permafrost layer that thawed during summer season. between 1900s and 1980s, the temperature of surface layer of permafrost increased by 24c ; and a further increase of 3c is expected. frequent outbreaks of anthrax caused death of 1.5 million deer in russian north between 1897 and 1925. anthrax among people or cattle has been reported in 29,000 settlements of the russian north, including more than 200 yakutia settlements, which are located near the burial grounds of cattle that died from anthrax. statistically significant positive trends in annual average temperatures were established in 8 out of 17 administrative districts of yakutia for which sufficient meteorological data were available. at present, it is not known whether further warming of the permafrost will lead to the release of viable anthrax organisms. nevertheless, we suggest that it would be prudent to undertake careful monitoring of permafrost conditions in all areas where an anthrax outbreak had occurred in the past.
histoplasmosis, also known as darling 's disease, is a systemic fungal infection caused by the dimorphic fungus, histoplasma capsulatum. infection is acquired by inhalation of conidia from soil contaminated by bird or bat excreta. histoplasmosis can manifest in a bewildering array of clinical types and variation and has been called the syphilis of fungal world. we report a case of chronic disseminated cutaneous histoplasmosis with varied skin lesions in an immunocompetent host. presence of cutaneous ulcers, linear erythematous plaques, skin coloured atrophic plaques and recurrent self - limiting oral ulcers in a single patient has not been documented in literature so far. a 74-year - old male, woodcutter by occupation, residing in a non - endemic area in south india, presented with multiple painful ulcers over face and oral mucosa and asymptomatic reddish raised lesions over neck and trunk of 5 days duration. there was history of recurrent episodes of self - healing ulcers over lower lip and soft palate for the past 10 years, and multiple asymptomatic skin coloured lesions over forearm and trunk for these years, which were not preceded by any other type of skin lesions. on examination, multiple ulcers of varying size with well - defined, irregular margin were seen on forehead, periorbital area, chin and left anterior axillary fold. multiple skin coloured atrophic plaques were present over forearm, trunk and thighs [figure 1 ]. leishman stained tissue smears from ulcers and erythematous crusted plaques revealed budding organism with peripheral halo [figure 2 ]. biopsies from these lesions and the skin coloured atrophic plaques showed parasitized macrophages, containing small round yeast - like organisms with a surrounding halo suggestive of histoplasma capsulatum, which was confirmed by gomori methenamine silver (gms) stain [figure 3 ]. patient was diagnosed with chronic disseminated cutaneous histoplasmosis and started on itraconazole 200 mg twice in a day. varied morphological lesions : (a) ulcerated plaques of varying size over forehead, periorbital area and cheeks ; (b) linear erythematous plaques over upper chest and crusted lesions over anterior axillary fold ; (c) atrophic plaques over thigh ; (d) linear atrophic plaque forearm tissue smears from ulcers and erythematous crusted plaques showing budding organism with peripheral halo. (leishman stain, 400) histology showing (a) heavy dermal inflammatory infiltrate containing poorly formed granulomas and many giant cells, hematoxylin and eosin stain, original magnification 40 ; (b) intracellular and extracellular yeast - like organisms with a surrounding halo, gms stain, original magnification 100 healed lesions after 6 weeks of antifungal therapy there are 2 varieties of histoplasma capsulatum that are pathogenic to man : h. capsulatum var. capsulatum occurs worldwide with endemic foci in central and north america, whereas h. capsulatum var. panja and sen reported the first case of disseminated histoplasmosis from india in 1954. among the forms of histoplasmosis reported from india, disseminated infection is acquired by inhalation of conidia from soil contaminated by bird or bat excreta. the risk factors for acquiring the infection include occupational and recreational activities in old buildings, bridges and caves. being a woodcutter, our patient had occupational exposure to multitude of trees inhabited by birds. though any person can acquire infection through inhalation, disseminated infection occurs in patients with defective cellular immunity. a myriad of lesions such as macules, papules, plaques, pustules, ulcers, abscesses and purpuric lesions have been described in histoplasmosis. molluscum contagiosum - like, erysipelas - like, pyoderma gangrenosum - like, ecthyma - like, rosacea - like and exfoliative forms are the rare manifestations reported. our patient, who was an immunocompetent elderly male, presented with diverse skin lesions including well defined irregular ulcers, linear erythematous crusted plaques, and skin coloured atrophic plaques. verma s.b. reported the first case of chronic disseminated cutaneous histoplasmosis presenting as linear erythematous plaques with central ulceration over the neck and upper chest in an immunocompetent host. we suspect this could be a specific manifestation of chronic disseminated cutaneous histoplasmosis in immunocompetent host. another unique feature in our patient was the presence of skin coloured atrophic plaques, from which we demonstrated the organism. our patient gave history of recurrent self - healing oral ulcers for about 10 years. most skin lesions of histoplasmosis arise following dissemination from a pulmonary focus and the only evidence of lung involvement could be calcification, which is indistinguishable on x - ray from tuberculosis. in our patient, history of chronic cough and chest x - ray finding of pulmonary calcification could have been due to pulmonary histoplasmosis, which might have been erroneously treated as tuberculosis. diagnosis of histoplasmosis was established by identifying small intracellular yeast - like cells of histoplasma in tissue smear and skin biopsy. in our case, the choice of therapy depends on the severity of illness. for patients with localized or few disseminated lesions, oral itraconazole of 200 - 400 mg daily intravenous amphotericin b up to 1 mg / kg daily is recommended for patients with widespread and severe infections. presence of cutaneous ulcers, linear erythematous plaques, skin coloured atrophic plaques and recurrent self - limiting oral ulcers in a single patient is reported. skin coloured atrophic plaques, from which we demonstrated the organism in our patient is a type of lesion that has not so far been documented in literature.	a case of chronic disseminated cutaneous histoplasmosis with unusual skin manifestations in an immunocompetent host is reported. presence of cutaneous ulcers, linear erythematous plaques, skin coloured atrophic plaques and recurrent self - limiting oral ulcers in a single patient has not been documented in literature so far. diagnosis was established by identifying small intracellular yeast - like cells of histoplasma in tissue smear and skin biopsy. leishman stained tissue smear proves to be an easy and simple procedure for diagnosis of histoplasmosis.
indonesia shows an amazing diversity of plants species that have been associated with the human health from time immemorial. many of them were reported to have various desirable activities ; however, only 2022% were cultivated. research of indonesian medicinal plants using modern laboratory facilities has been started since 1970, but only about 200 plants were studied. this figure shows a very small portion of the overall number of medicinal plant species that were reported. therefore, the analysis of chemical constituents would help in determining various biological activities of plants. the most important of these bioactive constituents of plant are alkaloids, flavonoids, terpenoids, steroids, tannins, and saponins. flavonoids are the most common and widely distributed group of plant phenolic compounds, occurring virtually in all plant parts, particularly the photosynthesising plant cells. flavonoids have been reported to exert multiple biological effects, including antioxidant, free radical scavenging abilities, anti - inflammatory, and anticarcinogen. several analytical techniques have been developed for determining total flavonoids concentration such as gas chromatographic (gc), mass spectrometry, thin layer chromatography, uv spectrometry, and high performance liquid chromatography (hplc). these methods are precise but all time - consuming, requiring many reagents, and costly. infrared spectroscopy is a technique based on the vibrations of the atoms of a molecule. the advantage of the infrared technique is that it can be nondestructive, requires a relatively small amount of sample, is fast, and is accurate [11, 12 ]. it has been proved to be a powerful analytical tool used in many fields. in recent years, nir spectroscopy also shows promising ability for discrimination of similar biological materials, such as pea, fruits, and wine. some papers have been published regarding nir quantitative analysis of active compound concentration in herbal products. the big advantage of multivariate statistical methods is their capability to extract the information of ir spectra and explore this spectral information for qualitative or quantitative applications. the most frequently used of multivariate statistical methods (often called chemometric methods) are linear discriminant analysis (lda) and partial least squares (pls) regression. the objective of this research is to develop a simple, rapid, and validated model of ir spectra for the determination of the flavonoid content. furthermore, ir spectroscopy and chemometric methods were applied for determining flavonoid content in commercial samples. in this study, samples used were leaves samples collected from materia medica botanical garden, malang, indonesia (table 1). methanol, ethanol, folin - ciocalteu, potassium acetate (e. merck, darmstadt, germany), and quercetin (sigma - aldrich) were of analytical grade reagent. commercial extract capsules, stimuno and daun salam, were purchased from a local pharmacy in jember, east java, indonesia (october 2015). 80.0 g of powdered sample was extracted with 800 ml of methanol in an ultrasonicator for an hour and continued being extracted by maceration for 24 hours. the extract was filtered through whatman filter paper and then the solvent was evaporated using a rotavapour at 60c. samples were scanned with a brimrose, luminar 3070 (brimrose corp, baltimore, md), with an integrating sphere. the monochromator entrance slit was set on 500 pm, the amplifier was set on 200. the response time is smooth (1 ms), and light intensity was set on 14 volts. the wavelength range of spectra is from 85002000 nm and the data were measured in 5 nm intervals, which resulted in 120 points reflection. ftir spectrometer (alpha ftir spectrometer from bruker optic), equipped with a deuterated triglycine sulphate (dtgs) as a detector and a germanium as beam splitter, interfaced to computer operating under windows - based system, and connected to software of opus operating system (version 7.0 bruker optic), was used during ftir spectra acquisition. a few drops of each sample were positioned in contact with attenuated total reflectance (atr) plate. ftir spectra were collected at frequency regions of 4000650 cm by coadding 32 scans and at resolution of 4 cm. all spectra were substracted against a background of air spectra. after every scan, a new reference of air background spectra was taken. the atr plate was carefully cleaned by scrubbing with isopropyl 70% twice followed by drying with soft tissue before being filled in with the next sample, making it possible to dry the atr plate. these spectra were recorded as absorbance values at each data point in replicate two times. the flavonoids content was determined by aluminum chloride method using quercetin as a reference compound. sample was prepared by mixing 0.5 ml of 4 mg / ml sample extract in ethanol with 3 ml of ethanol, 0.2 ml of 10% aluminum chloride, and 0.2 ml of 1 m potassium acetate and then diluted to 25 ml with distilled water. after incubation at room temperature for 30 min, the absorbance of the mixture solution was measured at 432 nm using spectrophotometer (uv - vis hitachi u 1800). various standard solutions of quercetin (2.0 up to 15.0 g / ml) were prepared from two stock solutions by dilution with ethanol. calibration and classification model for determination of flavonoids content were formed by training set samples that consists of fifteen medicinal plant extracts, quercetin, aquadest, and aerosil. the training set samples of medicinal plant extracts have been traced having varied flavonoid content which is expected to represent variations of flavonoid content of all plants. the linear discriminant analysis (lda), soft independent modelling of class analogies (simca), and support vector machines (svm) were used to develop classification model. matrix category was sample without flavonoids content (aquadest and aerosil) and flavonoid category was sample with flavonoid content (leaves extracts and quercetin). partial least square (pls) was used to develop calibration model for total flavonoids content. the pls model was then validated with leave - one - out cross - validation (loocv) and 2-fold cross - validation (five test set samples). the training set and test set samples were shown in table 2. the total flavonoids measurements were distributed around 4.03 up to 51.49 mg quercetin equivalence (qe)/g extract. figure 1 showed nir spectra of quercetin, dry extract, aquadest, and aerosil. those spectra have a different intensity and typical characteristic of absorption bands. in the pls calibration models, the evaluation of the linearity method was carried out in order to show a proportional relationship between the absorbance of nir spectra versus the concentrations of flavonoid. the correlation data of pls model in figure 2 showed good performance of pls model, indicated by coefficient of determination (r) higher than 0.99 and the low value of rmsec. r and the root mean square error of calibration (rmsec) were 0.9916499 and 2.1521897, respectively. therefore the calibration model can be used as a tool to predict the concentration of flavonoid content in medicinal plant. in order to validate the developed model, leave - one - out cross - validation (loocv) and 2-fold cross - validation were used. loocv was performed as follows : one sample was left out from the calibration set, a model was built with the remaining samples in the calibration set, then the left - out sample was predicted by this model, and the procedure was repeated by leaving out each sample in the calibration set. r and the root mean square error of prediction (rmsep) of loocv were 0.9986664 and 0.9136531, respectively (figure 3). twofold cross - validation was used to validate the developed model using independent samples (test set). the ability of nir model (lda, simca, and svm) to classify samples in flavonoid and matrix category can be seen through the accuracy of classification models. table 4 shows 100% of accuracy, which means that the model could classify fifteen training set samples in a correct category. figure 5 showed ftir spectra of quercetin, dry extract, aquadest, and aerosil. those spectra have similar intensity and characteristic of absorption bands in some segment of wavenumber. the results obtained from the pls in terms of r, rmsec for normal spectra, and segmentation were presented in table 5. pls calibrations in segment c (16501400 cm) revealed the highest of r and the lowest of rmsec compared with other segments (table 5). r and rmsec were 0.8653689 and 8.8958149, respectively. however, this result was not good due to the fact that r was less than 0.99 and rmsec was high. the ability of ftir model (lda, simca, and svm) was less than 100%, which means that the model could not classify fifteen training set samples in a correct category (table 6). pls and lda developed models of nir spectra were further used to predict flavonoid in commercial samples. the results of flavonoids content in samples measured by nir and uv - vis spectrophotometry method are presented in table 7. the paired samples t - test shows that flavonoid content that has been measured with both methods gave no significant difference (p > 0.05). furthermore, all of the commercial samples were in the flavonoid category in lda model. the nir spectroscopy combined with multivariate calibrations methods can be used to determine flavonoid in medicinal plant extract.	infrared (ir) spectroscopy combined with chemometrics has been developed for simple analysis of flavonoid in the medicinal plant extract. flavonoid was extracted from medicinal plant leaves by ultrasonication and maceration. ir spectra of selected medicinal plant extract were correlated with flavonoid content using chemometrics. the chemometric method used for calibration analysis was partial last square (pls) and the methods used for classification analysis were linear discriminant analysis (lda), soft independent modelling of class analogies (simca), and support vector machines (svm). in this study, the calibration of nir model that showed best calibration with r2 and rmsec value was 0.9916499 and 2.1521897, respectively, while the accuracy of all classification models (lda, simca, and svm) was 100%. r2 and rmsec of calibration of ftir model were 0.8653689 and 8.8958149, respectively, while the accuracy of lda, simca, and svm was 86.0%, 91.2%, and 77.3%, respectively. pls and lda of nir models were further used to predict unknown flavonoid content in commercial samples. using these models, the significance of flavonoid content that has been measured by nir and uv - vis spectrophotometry was evaluated with paired samples t - test. the flavonoid content that has been measured with both methods gave no significant difference.
osteopontin (opn) is a bone associated, extracellular matrix glycosylated phosphoprotein which is produced by several cell types, including osteoblasts, osteoclasts, immune cells, endothelial cells, epithelial cells and extra - osseous cells (skin, kidney and lung) (13). due to differences in post - translational modification (ptm) (phosphorylation, glycosylation, sulfation and proteolysis) from different cellular sources, opn has a molecular weight ranging from 41 to 75 kda, which may have a cell type - specific structure and function (47). opn plays a major role in various normal physiological processes, including bone remodelling, immune - regulation, inflammation and vascularisation (8,9). in addition, opn has also been shown to be involved in carcinogenesis with multi - functional activities (1012). opn is involved in a series of biological functions through interactions with different integrins and cd44. therefore, opn is classified as a member of the ' small integrin - binding ligand n - linked glycoproteins ' (siblings) together with other molecules, including bone sialoprotein (bsp), dentin matrix protein 1 (dmp1), dentin sialophosphoprotein (dspp) and matrix extracellular phosphoglycoprotein (mepe) (13). two critical integrin binding sequences of opn have been identified : arginine - glycine - aspartic acid (rgd) and serine - valine - valine - tyrosine - glutamate - leucine - arginine (svvyglr). opn interacts mainly with various v (particularly v1, v3, v5) integrin receptors via the classical rgd sequence, and interacts with 91, 41, 47 via svvyglr (1416). in addition, it also interacts with the cd44 splice variants, cd44v3, cd44v6 and cd44v7, via the c - terminal fragment calcium binding site (1720). these properties of opn induce the activation of signal transduction pathways, leading to cell proliferation, adhesion, invasion and migration, which have been demonstrated by both in vitro and in vivo models (2123). the binding of opn to integrins and cd44 initiates a downstream signalling cascade via the pi3k / akt signalling pathway leading to nf-b mediated cell proliferation and survival (2426). in additon, through the ras / raf / mek / erk signalling pathway, an opn - integrin complex and subsequent induction of ap-1-dependent gene expression, urokinase - type plasminogen activator (upa) and matrix metalloproteinases (mmps) confer a metastatic phenotype on some cancer cell types (2729). induced by vascular endothelial growth factor (vegf), opn and certain integrins are able to promote angiogenesis through enhanced endothelial cell migration, proliferation and the subsequent formation of capillaries, which are all essential requirements for the process of angiogenesis (30,31). opn has been shown to correlate with tumourigenesis, as well as with the progression and metastasis of different malignancies in both experimental and clinical studies (table i). the upregulation of opn expression has been identified in a variety of human cancers, including breast, prostate, lung, stomach, pancreatic and colorectal cancer, glioma and melanoma (table i). in these studies, in vitro experiments have also established a causal link between opn expression and cell functions. in vitro transfection experiments have demonstrated that downregulating opn gene expression suppressed the progression of breast cancer cells, which have invasive potential and metastatic competence (21). experimental evidence has further suggested that enhanced opn expression renders a highly metastatic phenotype of cancer cells. for example, the enforced expression of opn in non - metastatic rat mammary tumour cells has been shown to result in lung metastasis occurring in half of the animals that developed primary tumours (23). in another study, in glioma cells, elevated levels of opn and vegf synergistically enhanced the angiogenic properties, via integrin v3, on the endothelial cell surface (25). another study demonstrated that the knockdown of opn in prostate cancer cell lines enhanced the cytotoxicity of the chemotherapeutic drug, daunomycin (dun), through integrin - mediated fak / p - gp signalling (34). the ectopic expression of opn in dld1 colon cancer cells has been shown to stimulate emt activation and subsequent migration (41). in clinical investigations, elevated opn levels have been shown to correlate with increased tumour burden (stage, grade and tumour size), a poor prognosis and reduced survival, although discrepancies remain. for example, the increased expression of opn in plasma and tumour tissues has been identified in breast cancer patients and has been shown to be associated with metastatic disease and decreased survival (35). similarly, in advanced gastric cancer, patients with opn - positive cancer (as determined by immunohistochemistry) have a decrease in their 5-year survival, when compared with those with opn - negative cancer (40). in colorectal cancer (crc) patients, increased opn mrna levels have been shown to significantly correlate with stage, lymph node metastasis and lymphatic or venous invasion, as well as with lower disease - free and overall survival rates (41,42). a study on patients with pancreatic ductal adenocarcinoma (pdac) demonstrated that serum levels of opn are elevated with advanced tumour grades (44). elevated opn levels have also been strongly associated with increased stage, grade and tumour size in melanoma patients (52). in early stage non - small cell lung cancer (nsclc) however, in those patients which showed recurrence after surgery opn plasma levels re - elevated, indicating that opn is not only a potential diagnostic marker in nsclc, but also has potential as a tool for detecting tumour recurrence after treatment (53). thus, opn may be a useful biomarker to monitor cancer progression and a significant predictor of poor prognosis and survival rates. the alternative splice - generation of multiple mrna products from a single gene is a critical mechanism for generating proteomic diversity. the opn precusor - mrna (pre - mrna) is subject to alternative splicing, which generates three splice variants, opn - a (consists of all exons), opn - b (which lacks exon 5) and opn - c (which lacks exon 4) (fig. recent studies have demonstrated that the expression of opn splice variants in malignancies is cell - type / tissue specific and may have functional heterogeneity (table ii). for example, pang reported that opn - c is selectively expressed in breast cancer tissue (57). in their study on 170 breast cancer patients, opn - c protein staining was positive in 70% of all of the samples and was significantly higher in tumour tissues compared to normal tissues. the study indicated an inverse correlation between opn - c and e - cadherin, an established tumour suppressor. however, the observations by the same authors that opn - c also inversely correlated with -catenin and that e - cadherin positively correlated -catenin are intriguing. despite -catenin working with e - cadherin to form cell - cell adhesion complexes, -catenin has been classically regarded as an oncogenic protein. thus, the true link between opn - c and the cell adhesion complex requires further study and one has to delineate the cellular location of -catenin (namely membrane - associated, cytoplasmic and nucleus) in this context. however, the study by pang has clearly demonstrated that high levels of opn - c staining in breast tumours are associated with tnm staging, nodal involvement, recurrence and metastasis, and interestingly with the triple negativity of the tumours and, thus is an independent prognostic indicator for these patients (57). another study by sun demonstrated that each of the three opn splice variants was able to increase the growth of breast tumours, in vivo (56). it is noteworthy that opn - a appears more effective in promoting tumour growth than the other two splice forms. conflicting evidence also exists regarding the function of opn isoforms in hepatocellular carcinoma (hcc). it has been previously demonstrated that tumour tissue predominantly expressed opn - a and opn - b, and the ratio of opn - a and opn - b to opn - c increased substantially as the tumours progressed in sk - hep1 cells and hep3b cells (67). thus, it is possible that opn - a and opn - b may be associated with a poor prognosis, and opn - c may prevent both cell migration and invasion in more migratory and invasive cells. by contrast, in another study, takafuji (68) reported that the increased expression of opn - c in hep3b cells appeared to enhance cellular invasion and metastatic potential due to the formation of opn - c fragment by mmp-9. sun (59) found that, compared with normal lung tissue, the majority of nsclc samples predominantly expressed opn - a. a similar observation was made by blasberg (61), who reported that opn - a was the dominant isoform, whereas little opn - b and no opn - c expression was detected in lung cancer cell lines that endogenously expressed opn (a549, h460, h157, h1299 and calu-3). functionally, opn - a promotes angiogenesis in lung cancer by stimulating endothelial cells, likely by binding to the v3 integrin and increasing vegf expression and secretion. in their study, opn - b appeared to increase tubule formation merely by activating endothelial cells, and did so without a concomitant enhancement of vegf secretion or expression. however, zhao (60) suggested that opn - b strongly affected cell proliferation, while opn - c was closely related to the invasive behaviour of the nsclc cell line, a549. these studies emphasise that opn splice variants may have diverse expression patterns and different functional roles, which may be cancer type - specific. opn has mainly been studied as a secreted protein, but recent studies have shown that, in some cases, opn is not secreted and instead will be found in the cytoplasm and nucleus. mouse opn mrna has the canonical aug translation initiation site and an alternative translation initiation site. when translation initiates from the canonical site, the peptide produced is targeted to secretory vesicles. on the other hand, when translation starts from the alternative translation initiation site, the peptide produced is accompanied by deletion of a signal sequence and localises in the cytoplasm (76,77). although sopn and iopn are generated from the same opn mrna species, the biological outcomes mediated by the two isoforms may differ (78). the role of human sopn in cell physiology has been extensively studied in different cellular contexts. compared with sopn, the biological roles of iopn have only begun to emerge over the past several years. thus far, iopn has been found in calvarial cells, dendritic cells, macrophages and nerve cells (77,79,80). it is involved in cell motility, cytoskeletal rearrangement and mitosis by physical association with polo - like kinase-1 (plk-1) (8183). indeed, in patients with various solid tumours, sopn has been proposed as a diagnostic marker to distinguish either resectable cases or predict survival rates (43,84,85). however, cytoplasmic opn expression has appeared not to correlate with average tumour size, tumour stage and nodal status (10,86). for example, in a study on expression patterns of opn variants and its functions on cell apoptosis and invasion in glioma cells, yan presumed that the secretary nature of opn splice variants may be induced by the absence of exon 5 or exon 4. as a consequence, opn - b without out exon 5 may aggregate within the cytoplasm and exert a significant anti - apoptotic effect, while opn - c without exon 4 may be easily secreted to culture supernatants and has a remarkable effect on cellular invasion (74). therefore, it would be necessary to delineate which isoform of opn is responsible for pathophysiological events, and furthermore, sopn and iopn should be independently targeted in any potential therapies. the tumour microenvironment includes a variety of non - tumour cell types, such as fibroblasts, immune cells, vascular and smooth muscle cells. the host 's reaction to neoplastic cells and the ability of environmental modification by tumour cells themselves are both involved in tumour formation. a number of studies have demonstrated that opn can be synthesised by tumour and other cells types in the tumour microenvironment, such as macrophages and stromal cells (87,88). however, whether tumour - derived opn differs from host - derived opn, either structurally or functionally, is largely unknown. there is evidence indicating that the role of opn in metastasis is dependent on the site of production. for example, historically, stroma - derived opn has been considered to be intrinsically involved in the defensive mechanism against tumour development by acting as a macrophage chemoattractant, yet stroma - derived opn can effectively regulate melanoma growth, angiogenesis and metastasis with the host opn - tumour interaction (8991). in addition, tumour - derived opn may promote the metastatic process by inhibiting macrophage cytotoxicity against tumours (92). by contrast, some evidence suggests that at least in some instances, the presence of opn in macrophages promote the development and activity of type i natural killer (nk) t cells (nk t cells) and that opn may inhibit tumour activity via these nk cells (93). thus, host - derived opn and tumour - derived opn may mutually affect each other and the interaction between the tumour and tumour microenvironment may determine whether the overall effect of opn will be tumour promoting or tumour inhibiting. the differential effects of the three splice variants may be due to the distinctive functions of the spliced exons that individual opn isoforms contain. for example, the sequences encoded by exon 5, absent in opn - b, contain one of several clusters of phosphorylated serine / threonine residues. exon 4 however encodes two glutamine residues essential for transglutaminase cross - linking. as exon 4 is absent in opn - c, but present in opn - a and opn - b, opn - c has no specific functions exerted by exon 4 (94). in breast cancer cells, it has been reported that opn - a, not opn - c may have a tendency to aggregate in response to physical concentrations of calcium, and thus have a greater capacity to enhance cell adhesion (95). by contrast, opn - c may be more soluble and able to support anchorage - independent growth of breast cancer cells (96,97). opn - c possesses a more exposed arg - ser - lys (rsk) motif compared to opn - a (98). using the rsk motif as a cleavage site, systemically circulating thrombin can cleave opn - c easily into two fragments, the n - terminal and c - terminal, of approximately equivalent size (98). the n - terminal grgds - containing fragment produced by thrombin cleavage has the potential to enhance tumour cell migration (98). the thrombin - cleaved c - terminal fragment of opn has also been reported to influence breast cancer cell migration and invasion in vitro (99). the absence of the transglutaminase acting site in opn - c may explain why opn - c can not be cross - linked with the extracellular matrix (94). on the other hand, some investigations suggest that the possible reasons for opn isoforms cell - type specific patterns and their roles are closely related to these differing ptms of the three opn splice variants. gimba demonstrated that the potential of opn - a, opn - b and opn - c for specific phosphorylation patterns, and the deletion of exon 4 or 5 altered the pattern of ptms, ultimately resulting in functional modifications (46). opn has mainly been studied as a secreted protein, but recent studies have shown that, in some cases, opn is not secreted and instead will be found in the cytoplasm and nucleus. mouse opn mrna has the canonical aug translation initiation site and an alternative translation initiation site. when translation initiates from the canonical site, the peptide produced is targeted to secretory vesicles. on the other hand, when translation starts from the alternative translation initiation site, the peptide produced is accompanied by deletion of a signal sequence and localises in the cytoplasm (76,77). although sopn and iopn are generated from the same opn mrna species, the biological outcomes mediated by the two isoforms may differ (78). the role of human sopn in cell physiology has been extensively studied in different cellular contexts. compared with sopn, the biological roles of iopn have only begun to emerge over the past several years. thus far, iopn has been found in calvarial cells, dendritic cells, macrophages and nerve cells (77,79,80). it is involved in cell motility, cytoskeletal rearrangement and mitosis by physical association with polo - like kinase-1 (plk-1) (8183). indeed, in patients with various solid tumours, sopn has been proposed as a diagnostic marker to distinguish either resectable cases or predict survival rates (43,84,85). however, cytoplasmic opn expression has appeared not to correlate with average tumour size, tumour stage and nodal status (10,86). for example, in a study on expression patterns of opn variants and its functions on cell apoptosis and invasion in glioma cells, yan presumed that the secretary nature of opn splice variants may be induced by the absence of exon 5 or exon 4. as a consequence, opn - b without out exon 5 may aggregate within the cytoplasm and exert a significant anti - apoptotic effect, while opn - c without exon 4 may be easily secreted to culture supernatants and has a remarkable effect on cellular invasion (74). therefore, it would be necessary to delineate which isoform of opn is responsible for pathophysiological events, and furthermore, sopn and iopn should be independently targeted in any potential therapies. the tumour microenvironment includes a variety of non - tumour cell types, such as fibroblasts, immune cells, vascular and smooth muscle cells. the host 's reaction to neoplastic cells and the ability of environmental modification by tumour cells themselves are both involved in tumour formation. a number of studies have demonstrated that opn can be synthesised by tumour and other cells types in the tumour microenvironment, such as macrophages and stromal cells (87,88). however, whether tumour - derived opn differs from host - derived opn, either structurally or functionally, is largely unknown. there is evidence indicating that the role of opn in metastasis is dependent on the site of production. for example, historically, stroma - derived opn has been considered to be intrinsically involved in the defensive mechanism against tumour development by acting as a macrophage chemoattractant, yet stroma - derived opn can effectively regulate melanoma growth, angiogenesis and metastasis with the host opn - tumour interaction (8991). in addition, tumour - derived opn may promote the metastatic process by inhibiting macrophage cytotoxicity against tumours (92). by contrast, some evidence suggests that at least in some instances, the presence of opn in macrophages promote the development and activity of type i natural killer (nk) t cells (nk t cells) and that opn may inhibit tumour activity via these nk cells (93). thus, host - derived opn and tumour - derived opn may mutually affect each other and the interaction between the tumour and tumour microenvironment may determine whether the overall effect of opn will be tumour promoting or tumour inhibiting. the differential effects of the three splice variants may be due to the distinctive functions of the spliced exons that individual opn isoforms contain. for example, the sequences encoded by exon 5, absent in opn - b, contain one of several clusters of phosphorylated serine / threonine residues. exon 4 however encodes two glutamine residues essential for transglutaminase cross - linking. as exon 4 is absent in opn - c, but present in opn - a and opn - b, opn - c has no specific functions exerted by exon 4 (94). in breast cancer cells, it has been reported that opn - a, not opn - c may have a tendency to aggregate in response to physical concentrations of calcium, and thus have a greater capacity to enhance cell adhesion (95). by contrast, opn - c may be more soluble and able to support anchorage - independent growth of breast cancer cells (96,97). opn - c possesses a more exposed arg - ser - lys (rsk) motif compared to opn - a (98). using the rsk motif as a cleavage site, systemically circulating thrombin can cleave opn - c easily into two fragments, the n - terminal and c - terminal, of approximately equivalent size (98). the n - terminal grgds - containing fragment produced by thrombin cleavage has the potential to enhance tumour cell migration (98). the thrombin - cleaved c - terminal fragment of opn has also been reported to influence breast cancer cell migration and invasion in vitro (99). the absence of the transglutaminase acting site in opn - c may explain why opn - c can not be cross - linked with the extracellular matrix (94). on the other hand, some investigations suggest that the possible reasons for opn isoforms cell - type specific patterns and their roles are closely related to these differing ptms of the three opn splice variants. gimba demonstrated that the potential of opn - a, opn - b and opn - c for specific phosphorylation patterns, and the deletion of exon 4 or 5 altered the pattern of ptms, ultimately resulting in functional modifications (46). opn was initially identified in bone and later characterised based on its splice variants and their structures and functions. it is now well established that the isoforms of opn are differentially expressed in many tumour types and play critical roles at different stages of cancer development and progression. opn may be a useful biomarker to monitor cancer progression and one of the significant predictors of poor prognosis and survival rates, in certain cancers for example in breast cancer and lung cancer. thus, opn may have several potential clinical implications : firstly, as a convenient tool for clinical test. as a secreted protein, opn can easily detected in body fluids, such as blood, urine and body cavity fluids (namely pleural and peritoneal ascites). this gives rise to convenient sampling, taking advantages of sample quantity, safe detection, procedure speed and minimal invasiveness, collectively providing a convenient approach for clinical testing. secondly, the power of combining opn with other traditional biomarkers, such as vegf, mmps and e - cadherin, for example, provide more accurate predictions for prognosis and potential response to therapies (43,50,100). yet, changes remain, especially given the complexity of the distribution patterns and presence of the variant forms for opn. traditional protein detection methods such as elisa have shown their limit due to a lack of sensitivity and the nature of samples required for the testings. several studies have focused on the cost - effectiveness and ease of implementing immunosensors for opn detection demonstrating better sensitivity than elisa assays, and providing greater potential to develop simple test kits for opn combined with other protein biomarkers (101104). due to the pre- and post - translational regulation it has been suggested that this provides the possibility to develop cancer therapy strategies to target those opn isoforms which are specific to tumour cells or play a key role in tumour progression. but the pre- and post - translational regulation are complex and ubiquitous, developing drugs that target only cancer cells with minimal impact on healthy cells is extremely difficult. thus the expression patterns and activities of tumour - specific opn isoforms should be further defined with multidisciplinary and large scale clinical study. the development of specific opn - based approaches for individual cancer types is equally important as well. collectively, opn and its variants have been shown to play an important role in regulating the aggressive nature of cancer cells and promote the growth of tumours. although there is more to learn with regards to the mechanisms of action of the specific opn variants, it is clear that there is clinical prospect(s) for this protein and its variants. opn appears to have good clinical value in evaluating disease progression and cancer patient outcome. there is a good prospect for developing a opn based clinical test for cancer patients in order to evaluate their prognosis and response to therapies. wht is most exciting is the prospect of developing tools to target the protein and its variants in novel ways. it is anticipated that in the coming years we will see some significant progress along these fronts.	human osteopontin (opn) is a glycosylated phosphoprotein which is expressed in a variety of tissues in the body. in recent years, accumulating evidence has indicated that the aberrant expression of opn is closely associated with tumourigensis, progression and most prominently with metastasis in several tumour types. in this review, we present the current knowledge on the expression profiles of opn and its main splice variants in human cancers, as well as the potential implications in patient outcome. we also discuss its putative clinical application as a cancer biomarker and as a therapeutic target.
malignant peripheral nerve sheath tumor (mpnst) accounts for about 5 - 10% of all soft tissue sarcomas. the fact that the presence of this tumor in the buttock region is extremely rare has prompted the authors to report this case. this is an interesting tumor not encountered very much in the neuro - oncology literature. it can occur in sporadic form or over a setting of neurofibromatosis-1 (nf-1). when mtt develops over nf-1, the diagnosis can be confirmed based on morphologic grounds supported by an immunostain such as s-100 protein. a 40-year - old male presented with a large mass in the gluteal region gradually increasing in size over 6 months. ten years back, operation of a nodule in the same area had revealed histopathological diagnosis of neurofibroma. on further questioning, the patient revealed having two small painful nodules over his back since the presence of the operated nodule. his family history suggested that his father was a diagnosed case of von recklinghausen disease (nf-1). the resected mass consisted of a large globular mass measuring 10 7 6 cm. the cut section showed solid grey - white and hemorrhagic areas (fig. 1). multiple sections studied revealed alternate hypocellular and hypercellular areas composed of spindle cells with wavy nucleus and nuclear palisading. thick - walled blood vessels were seen with perivascular accentuation of tumor cells in the hypocellular areas (fig. mitotic figures were brisk in the cellular areas, 11/10 high power fields with few abnormal mitoses. the tumor also showed a good number of scattered large cells both rounded and elongated with abundant deep eosinophilic cytoplasm and clearly visible cross striations (fig. the patient was subjected to radiotherapy on being referred to the oncology department. during a follow - up period of 8 months, slightly more than half of the cases of mtt have been reported in conjunction with nf-1. in our case, when it occurs in sporadic form, other spindle cell sarcomas like fibrosarcoma, malignant fibrous histiocytoma and rhabdomyosarcoma can come as differential diagnosis. the tumor develops after a long latent period of 10 - 20 years. in our case there is an agreement that the diagnosis of mpnst can be on morphologic grounds supported by positivity for s-100 protein. the morphologic features are (a) alternating hypocellular and hypercellular regions, (b) the appearance of thin wavy comma - shaped / bullet - shaped nucleus in the hypocellular areas, (c) presence of nuclear palisading, (d) presence of nerve whorls or tactoid bodies resembling wagner - meissner corpuscles, (e) prominent thick - walled vasculature, and (f) presence of heterologous elements like rhabdomyoblasts, cartilage and bone. such tumors show focal positivity for s-100 protein in 50 - 90% of cases, suggesting a nerve sheath origin.. the 5-year survival rate of mtts is only 5 - 15% in contrast to mpnsts where it is 50 - 60%. integrated positron emission tomography and computed tomography have been used to assess remission and response to therapy. the prognosis of mtt depends on the location, grade and completeness of surgical margins. it is good for the head, neck, extremities and worse for other sites including the buttock. in a study it was observed that mtt in association with nf-1 has a poor prognosis compared to sporadic forms.	malignant peripheral nerve sheath tumor with rhabdomyoblastic differentiation, malignant triton tumor, has a rare incidence. we report such a case in a 40-year - old male who presented with a mass over the buttock. he was a previously diagnosed case of neurofibroma in the same area. histomorphology supported by immunostaining with s-100 protein confirmed the diagnosis. malignant triton tumor has a poor prognosis owing to its aggressive biological behavior. the fact that the presence of this tumor in the buttock region is extremely rare has prompted the authors to report this case.
according to the international agency for research on cancer (iarc), leather tanning and processing is not classifiable as to its carcinogenicity to humans, although the production process involves exposure to numerous chemicals, for some of which there is evidence of carcinogenicity in humans. however, in 1981, iarc had reported an increased risk for bladder cancer in the only study available at the time. tannery workers have been known from previous studies to have the potential for exposure to numerous known or suspected occupational carcinogens including hexavalent chromium salts, arsenic, organic solvents (benzene, formaldehyde, butyl acetate, ethanol, acetaacetate, toluene and acetone). the two major sources of chromium particulates in the tannery work environment are chromium chemicals used in the tannery work environment are chromium chemicals used in the tanning process in the form of baychrom and cr (oh)so4. chromium, which is a basic tanning agent, is available in trivalent form as chromium sulphate and in inorganic form and in the protein bound form that is known as leather dust. the leather dust produced by mechanical operations the exposures within the leather tanning industry have been suggested by some investigators[35 ] to result in the development of a variety of specific cancers including lung, bladder, kidney, pancreatic oral cavity, nasal and soft tissue sarcoma and skin along with dermatitis, ulcers, perforation of the nasal septum, respiratory illnesses. lung cancer : epidemiologic studies of tanners have shown an increased risk for cancer of the lung.[612 ] a review of cancer risk among tanners in italy was published and indicated an excess of lung cancer. increased mortality from lung cancer has also been reported earlier. an excess of lung cancer was observed in a case control study in the us, but these results were not supported by other studies. chromium and arsenicals were mentioned as possible contributors to the lung cancer excess. on the contrary, opined that the excess of lung cancer among tannery workers may partially be explained by cigarette smoking and not alone occupational exposure in the tannery industry as smoking is strongly correlated with cigarette smoking.[1617 ] pancreatic cancer : a three - fold statistically significant excess in pancreatic cancer mortality was noted in a swedish case referent study ; a 50% increase in pancreatic cancer was also noted in another study of three swedish tannery industries[910 ] and in an italian tannery. despite the excess risk of pancreatic cancer, no specific environmental agent was identified and dietary factors were considered as a possibility. these women were employed in the dyeing, stuffing and decorative process where they were basically exposed to organic solvents, formaldehyde, aniline and azo dyes. similar findings have also been reported who reported that the majority of the workers who died of pancreatic cancer had high exposure to formaldehyde and high dye and solvent exposure. high mortality form of pancreatic cancer is also shown in earlier studies showed the strong ability of proteins of leather dust to bind inorganic cr iii compound and facilitate their entrance into gastro intestinal tract. skin cancer and melanoma : one study in ussr showed skin carcinogenic effect of some synthetic fats used under leather stuffing. the workers exposed to leather dyes and fats may initiate skin cancer by direct impact. the females working in the dyeing, stuffing and decorating processes suffered from melanoma of skin and nonmelanoma skin cancer. high mortality of melanoma and skin cancers in females occupied in dyeing, stuffing and decorative workshops was observed in an recent study. kidney cancer : an excess of kidney cancer among tannery workers has been suggested by an earlier report. the dyes and pigments were mentioned as possible causative factors. buccal cavity and pharynx cancers : the workers in the tanning and liming workshops where exposure of leather dust was not high suffered from buccal cavity and pharynx cancers. pancreatic cancer : majority of workers who died with pancreatic cancer were either driers (high formaldehyde exposure) or painters (high dye - direct black 3 azo dye and solvents). in these conditions they presumed that azo dyes and/or formaldehyde could initiate dna mutation and chromium iii promote pancreatic cancer growth. bladder cancer : the analysis of recent literature data mainly based on case control studies showed the prevalence of bladder cancer in the leather industry. recent reports show one case of bladder cancer in man who was occupied in tanning and liming workshop. they pointed out gender difference in the prevalence of bladder cancer as women had much lower risk to develop bladder cancer than males. a study of the chinese leather tanning industry showed a statistical significance excess morbidity from bladder cancer among tanners were exposed to benzidine - based dyes, which increased with duration of exposure. a similar result was reported in her study of tuscan tannery industry and in her review of other studies among workers in leather finishing operation. however, earlier report suggest that in his mortality study bladder cancer risk was not elevated. sinonasal cancer : an excess risk of sinonasal cancer among leather tannery workers was observed in a case control study in italy. one case of nasal cancer was also reported earlier. however, iarc reported that there was no evidence of an increased risk of nasal cancer in the leather tanning and processing industry. testicular cancer : an excess risk of testicular cancer incidence was observed among leather tanners from the finishing department of the tannery and were exposed to dimethylformamide (dmf), a substance known to cause testicular damage. a statistically significant odds ratio of 7.2 for testicular cancer was observed among leather tanners. soft tissue sarcoma : two separate tannery studies indicated a significantly increased risk of this rare cancer. both investigators suggested that chlorophenols used in the pretanning operation and in the tanning process which have produced these tumors.	work in leather tanning involves exposure to a wide range of chemicals. some of these are carcinogens or suspected carcinogens. increased risks for a number of cancers have been reported among the tannery workers. in the present review, a detailed account of lung cancer, testicular cancer, soft tissue sarcoma, pancreatic cancer, bladder cancer among tannery workers is mentioned.
although twin and familial aggregation studies have revealed a significant genetic component involved in the etiology of stroke, not all of the genetic risk factors responsible for this heritability have been determined. classical linkage analysis approaches have reported an association of the pde4d and alox5ap genes with ischemic stroke, but replication of these findings has been inconsistent. in addition, candidate gene association studies have permitted the identification of several other candidate genes that may also be associated with ischemic stroke, such as apoe, il6, mthfr, and tnf. however, these studies have proven difficult to replicate for a number of reasons : it is because they may be false positive associations ; it is because of the fact that each gene may only contribute partially to overall heritability ; or it is because the studies did not study the ischemic stroke subtypes separately [4, 5 ]. genome - wide association study (gwas) approaches have highlighted the relationship of unsuspected genes with ischemic stroke subtypes. cardioembolic stroke is an ischemic stroke category that includes patients with arterial occlusion processes presumably due to an embolus arising in the heart mainly due to the presence of atrial fibrillation. atherothrombotic stroke or large vessel atherosclerosis is another ischemic stroke subtype that comprises strokes with > 50% stenosis or occlusion of a major brain artery or branch cortical artery, presumably due to atherosclerosis. a meta - analysis of gwas on ischemic stroke identified the zfhx3 gene on chromosome 16q22 as a locus specifically associated with atrial fibrillation and cardioembolic stroke [6, 7 ]. additionally, another ischemic stroke - related gwas showed intriguing results, including the identification of risk variants for atrial fibrillation and cardioembolic stroke on chromosome 4q25 near the pitx2 gene and for atherothrombotic stroke in the 9p21 locus. furthermore, an association between large vessel stroke and the hdac9 gene on chromosome 7p21.1 was recently identified. notably, the association of ischemic stroke with zfhx3, pitx2, the 9p21 locus, and hdac9 was verified in the collaborative metastroke study in which these loci were found to be specific to particular stroke subtypes. the changes to the atheromatous plaques caused by inflammation mediators in atherothrombotic stroke are associated with vessel occlusion processes and the subsequent development of this subtype of ischemic stroke. intuitively, it seems that inflammation mediators might be more involved in atherothrombotic stroke compared to cardioembolic or lacunar stroke. however, transcriptomic studies have revealed that inflammation could also have an important role in cardioembolic stroke. it has, therefore, become clear that genetic factors contribute to the development of ischemic stroke and that different genes are involved in the different subtypes of ischemic stroke and they only contribute partially to overall heritability. we hypothesize that most of the genetic studies failed to demonstrate true associations as they did not test ischemic stroke subtypes separately and as they did not take into account the fact that the risk polymorphisms only explain a small part of the ischemic stroke risk. our aim is to separately evaluate the genes associated with inflammation processes in the three main ischemic stroke subtypes and to combine the risk polymorphisms into a genetic scoring system in order to evaluate the value of inflammation mediator gene polymorphisms in diagnostic prediction. our discovery cohort and our replication cohort consisted of consecutive caucasian patients with an ischemic stroke that were admitted to the emergency room of a university hospital. healthy controls for both cohorts were recruited at the same hospital and were free of ischemic stroke episodes, myocardial infarction, or other cardiovascular diseases. the discovery cohort (n = 1,612 ; 1,456 ischemic stroke cases and 156 controls) was recruited between july 2005 and may 2009. the replication cohort (n = 1,073 ; 531 ischemic stroke cases and 542 controls) was recruited between november 2000 and may 2005 in a university hospital and the full clinical protocol etiological stroke subgroups were determined according to the trial of org 10172 in acute stroke treatment (toast) criteria. in the discovery cohort, 374 ischemic strokes were cardioembolic, 301 were atherothrombotic (large vessel stroke), 226 were lacunar, and 555 were undetermined ischemic stroke subtypes. in the replication cohort, 242 ischemic strokes were cardioembolic, 113 were atherothrombotic, and 176 were lacunar or undetermined ischemic stroke subtypes. patient information regarding established risk factors, including male gender, smoking, hypertension, diabetes mellitus, and dyslipidemia, was collected. also, written informed consent was obtained from all subjects, who were all of white european ancestry. we identified 68 single nucleotide polymorphisms (snps) in 30 genes selected from the literature related to inflammatory pathways associated with stroke (table 1). the most relevant candidate genes were selected by manual searching in pubmed using the keywords stroke and inflammation or inflammation for phenotypes and the keywords polymorphism, snp, mutation, variant for polymorphisms. snp selection was performed depending on previous literature (the most studied snps) and their functional effect, including those with an already known modification in transcription, translation, or protein activity or a hypothetical modification based on an amino acid substitution. whenever an interesting polymorphism involved more than a single nucleotide change a snp in perfect linkage disequilibrium was chosen for genotyping. in addition, we analyzed the whole gene region of the il6, mmp9, and nos3 genes with tag snps using data from the hapmap project (http://hapmap.ncbi.nlm.nih.gov/). we selected the snps using the tagger computer program in pairwise mode, maf > 0.1 (minor allele frequency), and linkage disequilibrium r > 0.8 and with central european population settings. genotyping was carried out at cegen (barcelona, spain), using snplex technology (applied biosystems, foster city, california) and genemapper 3.5 as the allele - calling algorithm. for quality control, two hapmap samples (na10860 and na10861) sample size calculation was performed using ene 2.0 software (servei d'estadstica aplicada, uab, barcelona, http://sct.uab.cat/estadistica/content/programari-d%27interes). a total of 110 subjects were needed in order to detect snp frequencies < 0.30 in the experimental group (ischemic stroke patients) and < 0.15 in the reference group (healthy controls) with a statistical power of 80% and p value = 0.05. statistical analysis was performed using spss software, v.15 (ibm, chicago, illinois). statistical significance for each snp in the discovery cohort continuous variables were compared by analysis of variance (anova) or mann - whitney or kruskal - wallis tests. we generated a predictive score based on logistic regression (lr) and odds ratio (or) coefficients, using forward stepwise procedure, with a p value of 0.05 as the threshold for entry as previously described. afterwards, to establish clinically relevant cut - off values, we automatically categorized this score into risk groups with the mathematic algorithm chi - squared automatic interaction detector (chaid), included in spss. the discovery cohort included 1,456 ischemic stroke cases and 156 controls and the replication cohort included 531 ischemic stroke cases and 542 controls. in terms of the toast etiology, in the discovery cohort, 374 ischemic strokes were cardioembolic, 301 were atherothrombotic (large vessel stroke), 226 were lacunar, and 555 were undetermined ischemic stroke subtypes. in the replication cohort, 242 ischemic strokes were cardioembolic, 113 were atherothrombotic, and 176 were lacunar or undetermined ischemic stroke subtypes. no snps were found to be associated with atherothrombotic stroke or lacunar stroke after statistical analyses on the discovery cohort. three snps were associated with cardioembolic stroke following a dominant / recessive genetic model (table 3). the snp rs1205 was located in the untranslated region (utr) of the c - reactive protein (crp) gene region and snps rs1800779 and rs2257073 in the nitric oxide synthase-3 (nos3) gene region. when we selected the cardioembolic stroke patients with atrial fibrillation before stroke onset (46.4%), the three snps remained associated with cardioembolic stroke with the exception of rs1205 (cc - cases : 40.1%, cc - controls : 51.3% ; p value = 0.052), rs1800779 (gg - cases : 22.7%, gg - controls : 11.6% ; p value = 0.033), and rs2257073 (tt - cases : 4.3%, tt - controls : 10.5% ; p value = 0.042). using the or of the risk factors ' genotypes (table 3) we generated a genetic risk score for cardioembolic stroke:(1)genetic risk score:1.5rs1205ct / tt+2.2rs1800779gg+2.1rs2257073ct / cc. this score was associated with the risk of cardioembolic stroke in the discovery cohort (cases : 3.4 points ; controls : 2.9 points ; p value = 0.001). the genetic score was validated in the replication cohort with the cardioembolic stroke group (cases : 3.5 points ; controls : 3.08 points ; p value = 0.017). however, this score was not associated with the risk of atherothrombotic stroke (p value = 0.24) or lacunar stroke (p value = 0.7). interestingly, we observed similar results in the undetermined stroke group compared to the cardioembolic stroke cases (cases : 3.3 points ; controls : 3.08 points ; p value = 0.07), although the results were not significant. hypertension and sex were the only clinical risk factors associated with cardioembolic stroke (table 2). when we included these variables in the score, the association with cardioembolic stroke was even more significant (cases : 7.6 points ; controls : 6.6 points ; p value = 8.3 10) and this was replicated in the replication cohort (cases : 5.1 points ; controls : 4.2 points ; p value = 0.002). however, for these two clinical variables the score was also significantly associated with atherothrombotic stroke (cases : 6.1 points ; controls : 4.2 points ; p value = 0.003) and undetermined stroke (cases : 5.1 points ; controls : 4.2 points ; p value = 0.025), although it was not associated with lacunar stroke (p value = 0.31). using the chaid method implemented by spss software we obtained three different risk groups for cardioembolic stroke and controls depending on the genetic risk score that we generated : a low risk group (score from 0 to 1.5 points), a medium risk group (from 1.6 to 4.3 points), and a high risk group (from 4.4 to 5.8 points). when we classified patients and controls using this classification, 92% of the high risk group presented a cardioembolic stroke (low risk : 50% of subjects with cardioembolic stroke, medium risk : 74.1% of subjects with cardioembolic stroke, and high risk : 92% of subjects with cardioembolic stroke ; p value = 0.002) (figure 1). we aimed to combine risk polymorphisms into a genetic scoring system in order to evaluate the diagnostic prediction of polymorphisms of inflammatory mediator genes for ischemic stroke subtypes. in addition, we aimed to study the role of inflammatory genes in ischemic stroke subtypes in order to establish which ischemic stroke subtype is most greatly influenced by the genetic background of the inflammatory mediator genes. the genetic risk score was weighted by the or of every genotype and was validated in an independent replication cohort. the genetic risk score was not associated with atherothrombotic stroke or with lacunar stroke, the other subtypes of ischemic stroke. we hypothesize that the trend of association between the score and undetermined stroke is due to a high percentage of undetermined strokes that are in fact cardioembolic strokes that have not been correctly diagnosed. when we generated a new score combining clinical risk factors (sex and hypertension) with genetic risk factors, we observed that this new clinical - genetic score was associated with the cardioembolic stroke subtype in both the discovery and replication cohorts. ischemic stroke subtypes have different genetic risk factors, and we observed that the inclusion of sex and hypertension introduces a confusing factor that reduces the accuracy of the genetic score. the use of these genetic scores could be very useful in the clinical practice to categorize the patients with atrial fibrillation. the genetic scores could detect those patients with the highest risk of suffering a future cardioembolic stroke and consequently initiate a treatment with anticoagulants. the polymorphisms and inflammatory mediator genes analyzed in this study were only associated with the cardioembolic stroke subtype. this is very interesting as inflammation has been classically more closely linked to atherosclerotic processes than to cardioembolism. during the last few years evidence has been found supporting the hypothesis that inflammation plays a key role in cardioembolic stroke and atrial fibrillation, the main risk factor for cardioembolic stroke. interestingly when we selected the cardioembolic stroke patients with atrial fibrillation before the stroke, the three snps of crp and nos3 were still associated with cardioembolic stroke, with very similar results compared to the whole group of cardioembolic stroke patients, indicating an association of these genetic markers with cardioembolic stroke patients with atrial fibrillation and without atrial fibrillation. moreover, previous transcriptomic studies using blood samples found inflammatory mediator genes played an important role in cardioembolic stroke. our study found that, in terms of genetics, inflammation also plays a key role in cardioembolic stroke. the nos3 gene codes for nitric oxide synthase (nos), an enzyme that synthesizes nitric oxide (no) from l - arginine. no is a reactive free radical that acts as a biological mediator in several processes, including the migration or proliferation of endothelial cells, platelet aggregation, and leukocyte adhesion. nos can regulate blood pressure by the synthesis of no, and it can also regulate metalloproteinase-2 (mmp2) and metalloproteinase-9 (mmp9), which have been associated with angiogenesis and inflammatory processes. our study found that the rs1800779 gg genotype and the c carriers of the rs2257073 snp were associated with a higher risk of cardioembolic stroke. the rs2257073 snp was a tag snp ; however, the rs1800779 snp of nos3 has been associated with an inhibition of the enzyme 's activity, causing a decrease in no production, and it has also been associated with the presence of leukoaraiosis by a previous paper. the gg genotype was associated with a higher risk of leukoaraiosis (a rarefaction of the brain white matter). interestingly, leukoaraiosis has been associated with inflammation, hypertension, and blood brain - barrier disruption. c - reactive protein (crp) is a marker of systemic inflammation that is significantly associated with an increased risk of cardiovascular disease in the general population. crp has previously been associated with cardioembolic ischemic stroke. studied 648 stroke patients with a first documented cerebral infarction and they measured crp within the first 6 hours after onset and the crp levels were then stratified in quartiles. the results showed that crp quartiles were mostly increased in cardioembolic strokes ; this suggests that, in the acute phase of the cerebral infarction, crp might be a marker of cardioembolism. another prospective study evaluated a cohort of 2,084 japanese ischemic stroke patients admitted in the first 7 days of onset ; the authors of the study showed that crp is an independent risk factor in the recurrence of cardioembolic ischemic stroke during the first year after symptom onset. in summary, we observed that polymorphisms of inflammatory mediator genes were more closely associated with cardioembolic stroke than with other subtypes of ischemic stroke including atherothrombotic stroke. in addition, we generated a genetic risk scoring system to predict the risk of cardioembolic stroke, which we also validated in an independent population. the implementation of genetic scoring systems can be useful in clinical practice to facilitate the prediction of the risk of stroke in healthy people ; however, further studies are needed to confirm these results.	inflammation has been associated with atherothrombotic stroke and recently with cardioembolic stroke. different genetic risk factors have been specifically associated with the subtypes of ischemic stroke (cardioembolic, atherothrombotic, and lacunar). however, there are no studies that have generated genetic risk scores for the different subtypes of ischemic stroke using polymorphisms associated with inflammation. methods. we have analyzed 68 polymorphisms of 30 inflammatory mediator genes in 2,685 subjects : 1,987 stroke cases and 698 controls. we generated a genetic scoring system with the most significant polymorphisms weighted by the odds ratio of every polymorphism and taken into consideration the stroke subtype. results. three polymorphisms, rs1205 (crp gene), rs1800779, and rs2257073 (nos3 gene), were associated with cardioembolic stroke (p value < 0.05). the score generated was only associated with the cardioembolic stroke subtype (p value : 0.001) and was replicated in an independent cohort (p value : 0.017). the subjects with the highest score presented a cardioembolic stroke in 92.2% of the cases (p value : 0.002). conclusion. the genetics of inflammatory markers is more closely associated with cardioembolic strokes than with atherothrombotic or lacunar strokes. the genetic risk scoring system could be useful in the prediction and differentiation of ischemic stroke ; however, it might be specific to particular ischemic stroke subtypes.
when estimating genetic parameters, the influence of environmental effects on milk yield is generally accounted for by using previously calculated correction factors or by including these effects directly in the model. these procedures decrease environmental variation and allow more reliable comparisons among animals raised in different environments (miller, 1973). according to bhat and batro (1978), a large portion of the variability in milk yield in milking buffaloes is explained by lactation length. buffalo lactation lasts around 250 days (tonhati., 2004), but shorter lactations are common. the criteria for deciding when to discard or include a short lactation in breeding value estimations are still unclear. the lack of information on the reasons or circumstances leading to the interruption of lactation is the major obstacle in including short lactation length records in genetic evaluations (bajwa., 2002). several studies have shown that 56 test days are required to extend the lactation period during genetic evaluations in dairy cattle (keown and van vleck, 1971 ; wilmink, 1987 ; pander., 1992 ; pander and hill, 1993 ; ribas., 1994). (2002) stated that short lactation length records can be dealt with in three ways : 1) by deleting them from the analysis, 2) by using them regardless of the lactation length, and 3) by adjusting them for days in milk and including this effect in the model. (2002) combining the average daily milk yield and the last test day milk yield. (2000) used quadratic logarithmic functions in their study. in brazil, tonhati. (2004) developed multiplicative correction factors for different classes of days in milk when working with milking buffaloes. (1992) found that deleting short lactation length records from the analyses or adjusting them for days in milk tended to reduce the additive genetic variance estimates of milk yield in crossbreed dairy cattle in brazil. the authors attributed this reduction to the high correlation between lactation length and milk production. however, more recently, khan. (2000) and bajwa. (2002), who studied buffaloes and dairy cattle, respectively, indicated an increase in heritability estimates when total milk yield was adjusted for days in milk. in brazil, the genetic evaluation for milking buffaloes is based on total milk yield, truncated at 305 days, with lactation length records < 120 days being discarded. there is no consensus on whether or not to adjust buffalo milk yield for days in milk, or how to best correct for this effect. consequently, there is an urgent need to develop alternative models that can be applied to genetic evaluation programs for milking buffaloes in brazil. the objectives of this study were to estimate the genetic parameters for unadjusted and adjusted milk yield by using multiplicative correction factors or including lactation length as a covariable in the model, and to examine the influence of adjusting for days in milk on sire rank. buffalo milk yields were obtained from the monthly test day records of a database maintained by the departamento de zootecnia at fcav, unesp, jaboticabal. the data were from 12 herds in the state of so paulo and were recorded from 1987 to 2004. all animals were from the murrah breed raised on pastures, with feed supplementation during the dry season (april to september). the data consisted of 4,408 complete lactation records from 1,879 buffaloes with an overall mean (se) of 1,617 14.4 kg per lactation. complete lactations from 90 or 150 days of lactation to 270 or 350 days of lactation were considered in the analyses. records of abnormal lactations or records for cows older than 144 months at calving were excluded. total milk yields were adjusted for days in milk by multiplicative correction factors (for 270 or 305 days) developed by tonhati. (2004) or by including lactation length as a covariable in the model (linear effect). the following traits were studied : 1) milk yield unadjusted for days in milk, considering complete lactations from 150 to 270 (um150_270), 150 to 305 (um150_305), 90 to 270 (um90_270) and 90 to 305 (um90_305) days in milk, 2) milk yield adjusted for days in milk using multiplicative correction factors, considering complete lactation from 150 to 270 (fm150_270), 150 to 305 (fm150_305), 90 to 270 (fm90_270) and 90 to 305 (fm90_305) days in milk, and 3) milk yield adjusted for days in milk including this effect as a covariable in the model, considering complete lactations from 150 to 270 (cm150_270), 150 to 305 (cm150_305), 90 to 270 (cm90_270) and 90 to 305 (cm90_305) days in milk. the model adopted, represented in matrix notation, was : y = x+za+wc+ewhere y is a vector of observed traits, x is the incidence matrix of fixed effects, is a vector of fixed effects, z is the incidence matrix of additive genetic random effects, a is a vector of additive genetic random effects, w is the incidence matrix of permanent environmental random effect, c is a vector of permanent environmental random effects, and e is a vector of random error effects. the assumptions about expectation and variances for multi - trait analyses were : e[y ] = xb, var(a) = a sa, var(c) = i sc and var(e) = i se, where sa is the additive genetic effect covariance matrix, sc, is the permanent environmental effect covariance matrix, se is the residual covariance matrix, a is the relationship matrix between animals, i is the identity matrix, and is the kroenecker product between matrices. the model of analysis included the fixed effects of a contemporary group (cg) and the covariable the covariable (linear and quadratic effects) days in milk was included in the model for cm150_270, cm150_305, cm90_270 and cm90_305. the cg was defined as cows that calved in the same herd, year and season (season 1 = from april to september, and season 2 = from october to march). cgs with less than five lactations and cows with a milk yield 3.0 standard deviations above or below the cg average were deleted from the analyses. variance and covariance components were estimated by the restricted maximum likelihood method (reml), using wombat (meyer, 2007). spearman correlations between the breeding values of sires and the percentage of common sires chosen for mating for milk yield unadjusted and adjusted for days in milk were calculated based on two selection intensities (5% and 10%) for the chosen sires. eighty - six percent, 70% and 11% of complete lactations had a length equal to or lower than 305, 270 and 150 days, respectively. permanent environmental, residual and phenotypic variances decreased when milk yield was adjusted for days in milk, independently of the method used, i.e. this adjustment decreased environmental variation, as expected. the highest additive genetic variances were obtained with multiplicative correction factors and the lowest when days in milk was included as a covariable in the model. days in milk as a covariable in the model adjusted the milk yield records for the average lactation length of the population. however, when milk yield was adjusted by multiplicative correction factors, the milk yield for 270 or 305 days in milk was affected and resulted in an increase in the average milk yield of the population. the heritability estimates for milk yield adjusted for days in milk were generally higher than the unadjusted values (table 2). 1992) observed a decrease in genetic variability when milk yield was adjusted for days in milk. the highest heritability estimates were obtained when the records were adjusted by multiplicative correction factors, probably as a consequence of the larger additive genetic variance estimates. (2002) also observed differences in milk yield heritability estimates when adjusted for days in milk by linear regression or by a method combining the average daily milk yield and the last test day milk yield. the differences between heritability estimates for unadjusted and adjusted milk yields were higher when short lactation length records (from 90 days) were included in the analyses (table 2). (2000) for milk yield in dairy cows were similar to those described here when they used projected lactations from 91, 151, 211 and 240 days to 305 days in milk. for nonadjusted records, the inclusion of lactations shorter than 150 days lead to a slight decrease in the genetic variance estimates. (1992) who reported an increase in genetic variability when short lactation length records (< 120 days) were included in the analyses without adjusting the records for days in milk. these authors worked with crossbred populations and did not exclude any lactations, regardless of their length. (2002) stated that the major obstacle to the inclusion of short lactation length records in genetic evaluations was the lack of information about the reasons or circumstances for the interruption of lactation, i.e., whether the interruption had an environmental or genetic origin. the heritability estimates obtained here for milk yield unadjusted and adjusted for days in milk were low to moderate. our results agree with those described of kuralkar and raheja (1997) in india who used milk yields adjusted for 305 days, and tonhati. (2004) in brazil who used milk yields unadjusted and adjusted for 305 days ; these two studies reported heritability estimates of 0.22 and 0.140.19, respectively, in milking buffaloes. in contrast, rosati and van vleck (2002) in italy and khan. (2000) in pakistan reported lower heritability estimates for milk yield (0.14 and 0.090.12, respectively) than those observed here. the genetic correlation estimates between adjusted and unadjusted milk yields were greater than 0.82, regardless of the method used to adjust milk yield for days in milk or lactation length. (2000) also reported high genetic correlations (close to 1.0) between extended and unextended milk yields for 305 days in holstein cows. table 3 provides a statistical summary of the breeding value estimates for the traits studied in this work. breeding value estimates this finding suggests that genetic variability increased when milk yield was adjusted for days in milk, in agreement with the higher heritability estimates obtained for adjusted milk yield (table 2). the highest mean breeding value estimate was obtained for fm90_270, which agreed with the highest heritability estimate obtained for this trait. table 4 provides a statistical summary of the accuracy of the breeding value estimates for the traits examined in this study. the accuracy of the breeding value estimates was greater when milk yield was adjusted for days in milk since higher heritability estimates were obtained for adjusted milk yields compared to unadjusted yields. the differences between the accuracy estimates for unadjusted and adjusted milk yields were greater when short length lactation records (from 90 days) were included in the analyses. the use of multiplicative factors to adjust milk yield resulted in more reliable breeding value estimates than those obtained for milk yields adjusted by including the accuracy of breeding value estimates were almost the same for lactation records from 90 or 150 days in milk. the inclusion of short length lactation records was desirable since this resulted in almost equal or more reliable breeding value estimates for milk yield. although the inclusion of short length lactation records decreased the heritability estimates, more animals and lactation records were considered in the analyses and this increased the accuracy of the breeding value estimates. for lactation records longer than 150 days, most of the sires would be the same if 5% of the sires were selected, regardless of whether adjustment was made by multiplicative factors or by including days in milk as a covariable in the model (table 5). in general, the percentage of sires selected for unadjusted milk yield that would also be selected for adjusted milk yield decreased when short length lactation records were included in the analyses. the inclusion of short length lactation records decreased the rank correlations between breeding value estimates for unadjusted and adjusted milk yields, particularly when multiplicative correction factors were applied. high selection intensity would change the profile of chosen sires, particularly for short lactations (90 days) and after adjustment by multiplicative factors. in conclusion, adjusting for days in milk affected the parameter estimates. milk yield must be adjusted for days in milk in order to decrease environmental variances and increase heritability estimates. more reliable breeding value estimates can be obtained by including short length lactation records in the analyses and adjusting milk yield for days in milk, regardless of the adjustment procedure used. with high selection intensity, changes in the sire rank and in the sires chosen can be expected if the records are adjusted by multiplicative factors and short length lactations are included in the data set. the results of this study should help to improve the genetic assessment of brazilian milking buffaloes through the use of procedures to adjust or correct the data for environmental effects.	the objectives of this study were to estimate the genetic parameters for milk yield unadjusted and adjusted for days in milk and, subsequently, to assess the influence of adjusting for days in milk on sire rank. complete lactations from 90 or 150 days of lactation to 270 or 350 days in milk were considered in these analyses. milk yield was adjusted for days in milk by multiplicative correction factors, or by including lactation length as a covariable in the model. milk yields adjusted by different procedures were considered as different traits. heritability estimates varied from 0.17 to 0.28. genetic correlation estimates between milk yields unadjusted and adjusted for days in milk were greater than 0.82. adjusting for days in milk affected the parameter estimates. multiplicative correction factors produced the highest heritability estimates. more reliable breeding value estimates can be expected by including short length lactation records in the analyses and adjusting the milk yields for days in milk, regardless of the method used for the adjustment. high selection intensity coupled to the inclusion of short length lactations and adjustment with multiplicative factors can change the sire rank..
the study of cancer in humans is impeded by several reasons, such as inaccessibility to the tumor sites, difficulty in the assessment of tumor biology, and ethical concerns. immunodeficient mice that can not reject xenotransplanted cells have been shown to be the best living recipients for developing xenograft models of human cancer.1 immunodeficient mice have also been important for investigating carcinogenesis, cancer therapy, and imaging of tumor growth and metastasis.2 to date, the most extensively used immunodeficient mouse strains for developing lung cancer xenograft models include nude,3 scid,4 and non - obese diabetic (nod)-scid57 mice. the interleukin-2 receptor (il-2r) -chain is known as the common cytokine receptor -chain. the il-2r -chain is a crucial component of the high - affinity receptors for il-2, il-4, il-7, il-9, and il-15, and is required for signaling through these receptors.8,9 absence of the il-2r -chain in mice leads to severe impairments in t- and b - cell development and function, and completely prevents natural killer (nk) cell development.911 the immunodeficient strains of il2rg mice include nod cg - prkdc il2rg / szj mice (abbreviated as nod / ltsz - scid il2rg and often referred to as nsg mice), nod cg - prkdc il2rg mice (abbreviated as nod / shi - scid il2rg and often referred to as nog mice), c.cg-rag2 il2rg mice (abbreviated as balb / c - rag2 il2rg mice) and stock (h2)-rag2 il2rg mice (referred to as h2-rag2 il2rg mice).2 nog mice have shown superiority in cancer xenoplantation systems compared with nude, scid, and nod - scid mice, when human cervical cancer,12 pancreatic cancer,13 and multiple myeloma14 cell lines were used. similarly, higher tumorigenicity in nsg mice has been reported with neuroblastoma1 and melanoma15 cells. acute leukemia cells injected in nsg mice generated faster and more efficient leukemic characteristics compared with other nod - scid - related strains.16 however, no lung cancer xenograft model using nod - scid il2rg mice (neither nsg nor nog) has been reported. to select a suitable mouse strain as a xenogenic host for testing tumorigenicity of lung cancer, we directly compared the susceptibility of nude, scid, nod - scid, and nsg mice for tumor formation from xenotransplanted lung cancer cell lines. nsg mice were originally generated at the jackson laboratory (bar harbor, me, usa). balb / c - nu, c.b-17 scid, nod - scid, and nsg mouse strains (50 female mice in each strain) were purchased from charles river laboratories japan, inc. (yokohama, japan) and maintained in the division of animal experiments, life science research center, kagawa university (kagawa, japan), according to the institutional regulations for animal experiments. the regulations included the best considerations on animal welfare and good practice of animal handling contributing to the replacement, refinement, and reduction of animal testing (3rs). animals were given free access to drinking water, and a basal diet, oriental mf (oriental yeast co, ltd, tokyo, japan), under controlled conditions of humidity (60%10%), lighting (12-hour light / dark cycle), and temperature (24c2c). two human lung cancer cell lines (ebc1, squamous cell carcinoma, and a549, adenocarcinoma) were obtained from the japan cancer research bank (tokyo, japan). cells were cultured in rpmi-1640 supplemented with 10% fetal bovine serum and passaged on reaching 80% confluence as reported previously.17 the protocols of the animal experiments were approved by the animal care and use committee for kagawa university. for direct comparison of susceptibility to cancer cell engraftment, various numbers of ebc1 or a549 cells (1010 cells / head, suspended in 0.1 ml of serum - free medium) were subcutaneously inoculated into five mice of each condition in each strain when mice were aged 6 weeks. the mice were monitored daily and tumor sizes were measured every week with calipers. the tumor volume (tv) was calculated using the formula tv = 1/2ab (where a= length in millimeters and b= width in millimeters) according to the previous study.12 the criteria for successive engraftment were as follows : progressive nodule growth at the site of injection and tv values exceeding 10 mm.12 mice were monitored up to 12 weeks after inoculation, and mice that developed a successive engraftment were euthanized. the engrafted tumors were fixed with 10% phosphate - buffered formalin, and paraffin - embedded sections were stained using hematoxylin and eosin. anti - human vimentin (clone v9 ; # m0725 ; dako, glostrup, denmark) and anti - human pancytokeratin (ae1/ae3 ; # m3515 ; dako) antibodies were used for the confirmation of human cell - derived tumors. a staining without primary antibodies secondary antibodies (i - view dab universal kit, # 518100032) were purchased from roche diagnostics kk (tokyo, japan). the differences in incidence of each engraftment were tested by fisher s direct probability method with p<0.05 as the cutoff for significance. all statistical analyses were performed using excel statistic 2012 (social survey research information co, ltd, tokyo, japan). nsg mice were originally generated at the jackson laboratory (bar harbor, me, usa). balb / c - nu, c.b-17 scid, nod - scid, and nsg mouse strains (50 female mice in each strain) were purchased from charles river laboratories japan, inc. (yokohama, japan) and maintained in the division of animal experiments, life science research center, kagawa university (kagawa, japan), according to the institutional regulations for animal experiments. the regulations included the best considerations on animal welfare and good practice of animal handling contributing to the replacement, refinement, and reduction of animal testing (3rs). animals were given free access to drinking water, and a basal diet, oriental mf (oriental yeast co, ltd, tokyo, japan), under controlled conditions of humidity (60%10%), lighting (12-hour light / dark cycle), and temperature (24c2c). two human lung cancer cell lines (ebc1, squamous cell carcinoma, and a549, adenocarcinoma) were obtained from the japan cancer research bank (tokyo, japan). cells were cultured in rpmi-1640 supplemented with 10% fetal bovine serum and passaged on reaching 80% confluence as reported previously.17 the protocols of the animal experiments were approved by the animal care and use committee for kagawa university. for direct comparison of susceptibility to cancer cell engraftment, various numbers of ebc1 or a549 cells (1010 cells / head, suspended in 0.1 ml of serum - free medium) were subcutaneously inoculated into five mice of each condition in each strain when mice were aged 6 weeks. the mice were monitored daily and tumor sizes were measured every week with calipers. the tumor volume (tv) was calculated using the formula tv = 1/2ab (where a= length in millimeters and b= width in millimeters) according to the previous study.12 the criteria for successive engraftment were as follows : progressive nodule growth at the site of injection and tv values exceeding 10 mm.12 mice were monitored up to 12 weeks after inoculation, and mice that developed a successive engraftment were euthanized. the engrafted tumors were fixed with 10% phosphate - buffered formalin, and paraffin - embedded sections were stained using hematoxylin and eosin. anti - human vimentin (clone v9 ; # m0725 ; dako, glostrup, denmark) and anti - human pancytokeratin (ae1/ae3 ; # m3515 ; dako) antibodies were used for the confirmation of human cell - derived tumors. a staining without primary antibodies secondary antibodies (i - view dab universal kit, # 518100032) were purchased from roche diagnostics kk (tokyo, japan). the differences in incidence of each engraftment were tested by fisher s direct probability method with p<0.05 as the cutoff for significance. all statistical analyses were performed using excel statistic 2012 (social survey research information co, ltd, tokyo, japan). we directly compared the susceptibility of four immunodeficient mouse strains, such as nude (balb / c - nu), scid (c.b-17 scid), nod - scid, and nsg, to xenotransplanted human lung cancer cell lines. when 10 ebc1 cells were inoculated subcutaneously, tumors formed in all mice tested within 9 weeks after inoculation (figure 1a). in nsg mice, all tumor formations had been completed in 3 weeks. when 10 ebc1 cells were inoculated, no tumor formation was observed in nude or scid mice (figure 1b). tumors developed in two of the five nod - scid mice (40%), while tumors formed in all the five nsg mice (100%). the superiority of nsg mice to xenotransplantation was clearer after 10 cells were inoculated (figure 1c, p=0.0079). no tumors developed in any strain other than nsg mice, in which 100% tumorigenesis was still observed in 9 weeks. all tumors in nsg mice grew progressively (figure 1d) and formed a large, hard, spheroid mass at 12 weeks (figure 1e). tumor formation developed in one of the five mice after inoculation of 10 ebc1 cells (figure 1c). similar to ebc1 cells, a higher xenotransplantability of a549 cells was observed in nsg mice when compared with other strains. although there was no statistical significance in final tumor number after inoculation of 10 and 10 a549 cells (figure 2a and b, respectively), tumorigenesis developed the fastest in nsg mice. after inoculation of 10 or fewer a549 cells, no tumor formation was observed in nude, scid, and nod - scid mice. in nsg mice, however, tumors developed in 6 of 15 mice (40%) in conditions of less than 10 cells (figure 2c), which is significantly higher when compared with other strains (p=0.0169). unexpectedly, only ten a549 cells formed a tumor in one of the five nsg mice. the pathologic findings of the developed tumor in the nsg mouse after inoculation of ten a549 cells showed, as expected, adenocarcinoma including glandular formation (figure 3a). the tumor was positive for anti - human cytokeratin and anti - human vimentin (figure 3b and c), suggesting that the developed tumor originated from a human source, that is, a549 cells. tumors showed negative staining with an isotype control antibody, confirming accuracy of immunohistochemistry (figure 3d). no metastatic tumor formation was observed in other organs including lungs and livers in all mice examined. we directly compared the susceptibility of four immunodeficient mouse strains, such as nude (balb / c - nu), scid (c.b-17 scid), nod - scid, and nsg, to xenotransplanted human lung cancer cell lines. when 10 ebc1 cells were inoculated subcutaneously, tumors formed in all mice tested within 9 weeks after inoculation (figure 1a). in nsg mice, all tumor formations had been completed in 3 weeks. when 10 ebc1 cells were inoculated, no tumor formation was observed in nude or scid mice (figure 1b). tumors developed in two of the five nod - scid mice (40%), while tumors formed in all the five nsg mice (100%). the superiority of nsg mice to xenotransplantation was clearer after 10 cells were inoculated (figure 1c, p=0.0079). no tumors developed in any strain other than nsg mice, in which 100% tumorigenesis was still observed in 9 weeks. all tumors in nsg mice grew progressively (figure 1d) and formed a large, hard, spheroid mass at 12 weeks (figure 1e). tumor formation developed in one of the five mice after inoculation of 10 ebc1 cells (figure 1c). similar to ebc1 cells, a higher xenotransplantability of a549 cells was observed in nsg mice when compared with other strains. although there was no statistical significance in final tumor number after inoculation of 10 and 10 a549 cells (figure 2a and b, respectively), tumorigenesis developed the fastest in nsg mice. after inoculation of 10 or fewer a549 cells, no tumor formation was observed in nude, scid, and nod - scid mice. in nsg mice, however, tumors developed in 6 of 15 mice (40%) in conditions of less than 10 cells (figure 2c), which is significantly higher when compared with other strains (p=0.0169). unexpectedly, only ten a549 cells formed a tumor in one of the five nsg mice. the pathologic findings of the developed tumor in the nsg mouse after inoculation of ten a549 cells showed, as expected, adenocarcinoma including glandular formation (figure 3a). the tumor was positive for anti - human cytokeratin and anti - human vimentin (figure 3b and c), suggesting that the developed tumor originated from a human source, that is, a549 cells. tumors showed negative staining with an isotype control antibody, confirming accuracy of immunohistochemistry (figure 3d). no metastatic tumor formation was observed in other organs including lungs and livers in all mice examined. this study demonstrates that the highest susceptibility to xenotransplantation of two lung cancer cell lines was observed in nsg mice. in nsg mice, the first immunodeficient mouse model of cancer was developed in nude mice, which allowed the growth of human solid tumors, after inoculation of 120 million cells.18 thereafter, several kinds of immunodeficient mice have been generated with more efficient xenotransplantability. among them, nod - scid mice showed a greater susceptibility to lung cancer cell lines and primary cells.57 the non - small cell lung carcinoma (nsclc) xenografts from patients undergoing surgery were implanted into nod - scid mice within 24 hours of surgery and they were successfully engrafted in 40% of cases.5 in this study, we demonstrated a dramatic improvement of xenotransplantation of fewer lung cancer cell lines in nsg compared with nod - scid mice. when 10 or fewer cells were inoculated, tumors developed only in nsg mice and grew progressively, suggesting higher susceptibility of nsg than nod - scid mice to xenotransplanted lung cancer cells. in addition, a subcutaneous tumor developed in one mouse that had been inoculated with only ten a549 cells. the most commonly used procedure for diagnosing nsclc is bronchoscopic examination,19 and only 20%25% of patients have resectable disease because of the diagnosis in an advanced stage.20 however, the number of cancer cells obtained by a transbronchial biopsy (tbb) is usually quite few. primary cell culture from patients with lung cancer has not fully been established, in particular, as the cell number is low, although many improvements in media used and culture technique have been reported for establishment of primary cell culture.21 if humanized mice can be available by successful xenotransplantation of lung cancer cells obtained by tbb, it might be useful for establishment of the best therapy for individual tumor. in this regard, nsg mice could be the best candidate. in fact, human breast cancers were propagated in scid / beige and nsg mice models.22 these models serve as a renewable, quality - controlled tissue resource for preclinical studies investigating metastasis and response to treatment.22 development of xenografts platform from patients with nsclc for drug testing in nsg mice models is also explored.23 in this study, tumorigenesis appeared faster after inoculation of lung cancer cells in nsg mice, which might also be attractive for rapid assessment of individual tumor biology. compared with nod - scid mice, nsg mice lack host nk cell activity and cytokines signaling are impaired. the contribution of il-2 is well known to induce nk cell cytotoxicity against various types of malignancy.2426 nk cell cytotoxicity for lung cancer cells was markedly augmented by stimulation with il-2.27,28 compared with normal subjects, patients with lung cancer consistently had higher levels of il-2 in their bronchoalveolar lavage fluid and this titer correlated with an increase in nk cell activity.29,30 a differential composition of the immune cell infiltration was also assessed in malignant and non - malignant lung tissue areas associated with nsclc.31 interestingly, nk cells were almost absent in the malignant areas, while non - malignant counterparts were selectively populated by nk cells and those nk cells showed strong cytotoxic activity ex vivo.31 the higher tumorigenicity in nsg mice observed in this study might be due to, in part, lack of il-2-dependent nk cell cytotoxicity, although cytokines other than il-2 might also have a role. the relevance of mouse models would further increase when assessed in an orthotopic organ site, ie, the lungs for lung cancer models, rather than assessment of subcutaneous growth. orthotopic models would have some advantages compared to subcutaneous models. in the previous reports, some lung cancer cells inoculated intravenously into immunodeficient mice induced multiple lung nodules and malignant pleural effusion32 and murine lung cancer cells injected into the pleural space of mice developed malignant pleural effusion.33 these models could make it possible to assess the therapeutic efficacy of cancer progression in a situation close to the actual target patients. on the other hand, in many previous studies, subcutaneous models have been used for assessment of lung cancer, because of some advantages in subcutaneous models. second, it is easier to confirm the course of tumor progression, without use of radiological techniques or euthanasia. third, subcutaneous inoculation of cells is an easier experimental procedure than intravenous inoculation or injection into the pleural space. in summary, we directly compared the ability of four immunodeficient mice to successfully engraft lung cancer cells. nsg mice were more susceptible to tumor formation than other strains including nod - scid mice. nsg mice could be a good choice in cases of limited cell numbers such as samples obtained by tbb or identification of a weakly tumorigenic phenotype.	purposeno lung cancer xenograft model using non - obese diabetic (nod)-scid il2rg/ mice has been reported. the purpose of this study is to select a suitable mouse strain as a xenogenic host for testing tumorigenicity of lung cancer.materials and methodswe directly compared the susceptibility of four immunodeficient mouse strains, c - nu, c.b-17 scid, nod - scid, and nod / ltsz - scid il2rg/ (nsg) mice, for tumor formation from xenotransplanted lung cancer cell lines. various numbers (101105 cells / head) of two lung cancer cell lines, a549 and ebc1, were subcutaneously inoculated and tumor sizes were measured every week up to 12 weeks.resultswhen 104 ebc1 cells were inoculated, no tumor formation was observed in balb / c - nu or c.b-17 scid mice. tumors developed in two of the five nod - scid mice (40%) and in all the five nsg mice (100%). when 103 ebc1 cells were injected, no tumors developed in any strain other than nsg mice, while tumorigenesis was achieved in all the five nsg mice (100%, p=0.0079) within 9 weeks. nsg mice similarly showed higher susceptibility to xenotransplantation of a549 cells. tumor formation was observed only in nsg mice after inoculation of 103 or fewer a549 cells (40% vs 0% in 15 nsg mice compared with others, respectively, p=0.0169). we confirmed that the engrafted tumors originated from inoculated human lung cancer cells by immunohistochemical staining with human cytokeratin and vimentin.conclusionnsg mice may be the most suitable strain for testing tumorigenicity of lung cancer, especially if only a few cells are available.
controlled induced hypotension is a common procedure during anaesthesia applied to patients undergoing, among others, endoscopic sinus interventions. decreased blood pressure allows reduction of bleeding in the surgical field, minimization of blood loss, better visibility and therefore, it increases the surgeon s comfort, reduces the surgery time and prevents complications emerging from blurred vision caused by coverage of the camera lens with blood. studies have shown a variety of methods reducing bleeding in the surgical field through : lowering the mean arterial pressure (map), lowering hr, local anemization with adrenaline, preoperative use of steroids (which reduce the inflammatory reaction, damage to the blood vessels, edema, and adrenergic receptors activation), and reversing the trendelenburg position, which reduces blood supply in the surgical field. magnesium, nitroglycerin, acei (angiotensin converting enzyme inhibitors), calcium channel blockers, beta - blockers, sodium nitroprusside, clonidine, and large doses of inhalational anaesthetics have all been used to achieve the target map. drug - induced vasodilatation is only beneficial if the cardiac output (co) is reduced. it can be achieved through the administration of labetalol, a short - acting beta - blocker, which stabilizes the hr at 60 bpm and simultaneously reduces the map. taking into consideration the co values as an index of both perfusion and bleeding, it is reasonable to stabilize it within lower normal range values [26 ]. additionally, remifentanil (rfn), an ultrashort - acting opioid and receptor agonist, also plays an important part in endoscopic surgery of paranasal sinuses. it enables easy adjustment of the depth of anaesthesia and reduction of the map and hr through cardiodepressive action. earlier studies also indicated the positive effect of propofol as an anaesthetic in reducing map through its effect on precapillary arterioles. moreover, when compared with the combination of sevoflurane and rfn, no significant differences in intraoperative bleeding were observed. additionally, this combination enables continuous monitoring of the volatile anaesthetic concentration as well as easy adjustment of map during the procedure. some studies have also shown that the return of cognitive functions to the initial level within the first hour seems to be faster in patients on sevoflurane than in those on propofol. although the positive effects of controlled hypotension (including reduced blood loss, time of intervention, and increased comfort of the surgeon) are well known, the method is still limited by concerns regarding the impact of severe hypotension. permanent brain damage, emboli in cerebral circulation, difficult awakening of the patient, and even death all these complications might occur when hypotension is too deep. the cerebral blood flow (cbf) is controlled by different metabolic, chemical (po2, pco2), and neural mechanisms and autoregulation. under physiological conditions, cbf remains constant despite map changes from 50 to 150 mmhg. brain ischemia, injuries, arterial hypertension, and other pathological and pharmacological factors may disturb the mechanisms of autoregulation. volatile anaesthetics used in anaesthesia may limit or even abolish autoregulation of cbf, thus increasing the vulnerability of brain tissues to map alterations during surgery. although brain tissues are susceptible to hypoxemic damage, research has shown that lowering the map to 2/3 of the initial value does not cause any damage. on the other hand, there is no evidence that these conditions influence the epidemiology of functional disorders, especially those affecting cognitive functions. earlier studies confirmed the impact of the patient s initial general condition, age, accompanying diseases, drugs, and addiction to psychoactive substances on cognitive functions. the influence of iatrogenic factors, including the duration of hospitalization, time of surgical procedure, and drugs administered, has also been noted. one of the main factors influencing patients condition is the quality of anaesthetic management, including hemodynamic stability. maintaining homeostasis of body functions is the golden rule of proper anaesthetic management and it is crucial to minimize complications. the objective of this study was to evaluate the effects of controlled induced hypotension on the cognitive functions of patients undergoing functional endoscopic sinus surgery (fess). the study was approved by the bioethics committee of the pozna university of medical sciences (decision number 298/13) and was conducted in accordance with the declaration of helsinki. the study was conducted on patients undergoing functional endoscopic sinus surgery in heliodor wicicki university hospital in pozna between 16 april and 18 june 2013 and between 15 october and 20 december 2014. the inclusion criteria were as follows : age 1875, good mental status confirmed with the mini - mental state examination (mmse) minimum score of 27 points, no drugs taken that influence cognitive functions, no vision impairment or vision corrected with glasses, and no severe hearing disorders influencing the quality of communication. patients with poorly controlled hypertension, ischemic disease, and autonomic system impairment were excluded from the study. all the subjects in the study were able to communicate in polish and had no manual dysfunctions. during the trial period, 84 patients were qualified, 76 of whom agreed to take part : 3 of them were disqualified due to pre - existent disorders of cognitive functions, with the mmse score under 27 points, 1 person due to daltonism (which makes it impossible to complete the stroop test), and 3 people due to psychiatric drugs taken. moreover, 21 patients withdrew on the day of the surgery, as they did not feel well enough to complete the psychometric tests. in 1 case, the surgery (mfess) was too short to achieve the target map and this patient was excluded from the study. eventually, 47 patients (american society of anesthesiologists asa score 13) were qualified for the study : 30 men (64%) and 17 women (36%), with a mean age of 46 years (sd 16). during the surgery, the anesthesiologists set the target map according to the patient s general condition as well as their own experience and preferences. the patients were allocated to 3 groups postoperatively, according to the mean map during the surgery as a fraction of the preoperative map (mapm%map0) : group i mild hypotension (mapm%map0>75%), group ii intermediate hypotension (65% < mapm%map0 75%), and group 3 severe hypotension (mapm% map0 65% with the minimum value of 53%). the groups were homogenous in terms of age, gender, bmi, initial results of basic blood tests, and the mmse result. one day before the surgery, all the patients qualified for the study completed psychometric tests, which evaluated cognitive functions : the stroop test, trail making test (tmt), and verbal fluency test (vft) in the stroop test variant a, the patient s task is to say aloud the names of colors printed on a sheet of paper. variant b evaluates the speed of naming colors in which the names are printed, but none of the names are printed in the proper color. the stroop test evaluates the interference of ambiguous stimuli describing colors, which prolongs the reaction time when the patient has to react to a new criterion. the larger the difference between variants b and a is and the number of mistakes made, the more impaired the cognitive functions are. the prefrontal cortex, cingulate cortex, cerebellum, and basal nuclei are all activated while the patient is performing this test [1619 ]. the stroop test was also used in the largest multicentre trials ispocd 1 and 2, evaluating patients undergoing non - cardiosurgical interventions. in the tmt, the patient has to connect dots : in variant a they represent numbers (1, 2, 3, 25), in variant b numbers and letters, alternately (1, a, 2, b, 3, c, 12, k). it allows assessment of scanning, processing speed, and mental elasticity [17,18,2027 ]. the vft evaluates both semantic (categorical) and phonemic (such as words that begin with the letter...) verbal fluency. for each of 6 categories (semantic : animals, vegetables, fruits ; phonemic : s, l, n) the number of unique words given by the patient within 1 minute was registered. the vft needs access to long - term memory (ltm), processing functions, executive functions, and memory processes [20,3033 ]. on the day of the surgery, 1 hour before induction, all the patients were administered midazolam (7.511.25 mg). an invasive blood pressure (ibp) cannula after 3-minute oxygenation with 100% o2 anaesthesia was induced with propofol (1.53 mg / kg), rfn (0.4 g / kg / h), rocuronium (5 mg) and suxamethonium (0.51 mg / kg). anaesthesia was maintained with rfn (0.050.5 g / kg / h) and sevoflurane (0.60.9 mac), according to the patient s individual needs. during the procedure, ecg, spo2, hr, map, etco2, fio2, and peak airway pressure (pap) were controlled continuously and the values were noted every 5 minutes. the flow of sevoflurane and rfn was stopped when the surgery terminated. in 18 cases (38%), the anesthesiologist decided to administer ephedrine (5 mg) to raise the map after lowering it below the target value. there were 37 patients who underwent the tests 3 times on the day before the surgery (day 1), on the day of the surgery (day 2), and on the day following the surgery (day 3). ten patients were discharged in the morning on the day following the surgery due to good general condition, which made it impossible to complete the tests for the third time. the tests were conducted every day between 6 pm and 9 pm. on the day of the surgery, the basic demographic data and the results of the screening tests were presented as the mean and standard deviation. the distribution of the demographic data, stroop test results, tmt results, vft results were analyzed with the kolmogorov - smirnov test. the differences in the test results between the groups were analyzed with anova for dependent variables. friedman s anova was used to investigate the number of mistakes made, due to their non - gaussian distribution. the time of completion of each test, the number of mistakes, and difference between variant b and a (b - a best represented cognitive functions in this test) were analyzed, separately for the whole group (with 2 attempts) and for those who completed 3 attempts. no statistically significant differences between the groups were observed. in each group, on day 2 the results of variant a were worse than on day 1. the results are shown in tables 2, 3, and figure 1a, 1b (showing the b - a result). the time of completion of each test, the number of mistakes, and difference between variant b and a (b - a best represented cognitive functions in this test) were analyzed, separately for the whole group (with 2 attempts) and for those who completed 3 attempts. a, the time of completion on day 2 was shorter in group a and longer in group b and c. however, the difference was not statistically significant. the comparison with the 3-day results and the comparison of the b - a results did not confirm this observation. the results are shown in tables 4, 5, and figure 1c, 1d (showing the b - a result). the number of words given and mistakes made were registered for each attempt and then analyzed. no statistically significant differences were shown. in a number of categories (fruits, vegetables, and animals, when 3 attempts were considered) an increase in the number of words given was noted in groups 1 and 2, while in group 3 the result remained constant or decreased. the time of completion of each test, the number of mistakes, and difference between variant b and a (b - a best represented cognitive functions in this test) were analyzed, separately for the whole group (with 2 attempts) and for those who completed 3 attempts. no statistically significant differences between the groups were observed. in each group, on day 2 the results of variant a were worse than on day 1. the results are shown in tables 2, 3, and figure 1a, 1b (showing the b - a result). the time of completion of each test, the number of mistakes, and difference between variant b and a (b - a best represented cognitive functions in this test) were analyzed, separately for the whole group (with 2 attempts) and for those who completed 3 attempts. a, the time of completion on day 2 was shorter in group a and longer in group b and c. however, the difference was not statistically significant. the comparison with the 3-day results and the comparison of the b - a results did not confirm this observation. the results are shown in tables 4, 5, and figure 1c, 1d (showing the b - a result). the number of words given and mistakes made were registered for each attempt and then analyzed. no statistically significant differences were shown. in a number of categories (fruits, vegetables, and animals, when 3 attempts were considered) an increase in the number of words given was noted in groups 1 and 2, while in group 3 the result remained constant or decreased. anaesthesia has both short - term (reversible) and long - term impacts on the central nervous system. these changes result from the direct and indirect effects of anaesthetic drugs, but they are also related with the patient s initial condition, other drugs administered, and environmental factors (e.g., stress and elevated sympathetic activity), which are still under investigation. there are 3 types of cognitive deterioration after surgery : delirium, short - term cognitive disturbance, and true pocd, which is a subtle deterioration in the cognitive function lasting weeks, months, or longer. recent studies show that applying peep does not influence the quality of the surgery field as long as the peak inspiratory pressure (pip) is maintained below 15 cmh2o. in our study all the patients were maintained with peep 5 cmh2o and pip below 15 cmh2o whenever possible. the results of the stroop test confirm that hypotension lasting about an hour does not influence the processing of interfering stimuli, even as soon as 34 hours postoperatively. the prolongation of the completion time of variant a on day 2 and 3 was similar in each group and it was probably caused by the patients discomfort due to the nasal pack inserted at the last phase of the surgery. however, variant b was performed better every day, additionally reducing the b - a index. this shows that the learning effect, characteristic of the stroop test at all ages, was also observed in all the groups after the surgery in controlled hypotension. this observation corresponds with the results of a study by davidson, who showed a correlation between practice and stroop test results. the results of the tmt are difficult to interpret due to significant differences between the patients results within each group, represented by high values of sd. although the results suggested that the patients from groups 2 and 3 needed more time to complete the test on day 2 and day 3, the statistical value of this observation was limited. another study proved the influence of age and education on the tmt results in a population of 911 individuals. the groups in this study were too small to achieve statistical significance when they were divided into subgroups. conducting the tmt in a larger population managed in controlled hypotension might provide new data regarding scanning, processing speed, and mental elasticity. no complications in the functions of kidneys, lungs, nervous system, and cardiovascular system were observed. the time of anaesthesia and post - operative pain in the numeral rating scale, which could have affected cognitive functions, did not differ significantly between the groups. the study was limited by lack of feedback from the surgeons regarding visibility in the surgical field. simultaneous evaluation of the vision quality (ideally assessed by the same surgeon) and cognitive function would be an interesting subject for further research. the results of psychometric tests conducted on the patients undergoing fess in controlled hypotension did not differ significantly between the groups. controlled hypotension seems to be equally safe for the patient as anaesthetic management in normotension, simultaneously decreasing the complication rate.	backgroundcontrolled induced hypotension guarantees less blood loss and better visibility of the surgical site. the impact of hypotension on post - operative cognitive functions is still being discussed. the objective of this study was to evaluate the effects of controlled induced hypotension on the cognitive functions of patients undergoing functional endoscopic sinus surgery (fess).material / methodswe allocated 47 patients with a good grade of preoperative cognitive functions evaluated with the mini - mental state examination to 3 groups (1 mild hypotension, 2 intermediate hypotension, 3 severe hypotension) according to the degree of mean intraoperative arterial pressure compared with preoperative blood pressure. cognitive functions were evaluated preoperatively, 6 h, and 30 h postoperatively with standardized tests : the stroop test, trail making test (tmt), and verbal fluency test (vft). a decrease in the test results and increase in the number of mistakes made were considered an impairment of cognitive functions.resultsa total of 47 patients (group 1 mild hypotension 15, group 2 intermediate hypotension 19, group 3 severe hypotension 13) were included in the study. a significant decrease was observed in all the 3 groups after stroop a test 6h postoperatively but it improved 30h postoperatively, without differences between the groups. neither a significant decrease in the test results nor an increase in the number of mistakes was noted for stroop b tests, tmt a&b tests and vft.conclusionsthe degree of controlled intraoperative hypotension during fess did not influence the results of psychometric tests.
this 2 year - old hispanic male has a past medical history of hypoplastic left heart syndrome (hlhs) that required multiple cardiothoracic surgeries including a norwood procedure with a sano shunt after birth, a bidirectional glenn procedure at 4 months of age, and two subsequent tricuspid valve repairs. he had a right middle cerebral artery (mca) stroke at 18 months of age manifesting as left face and arm weakness. at that time, a heterozygous prothrombin g20210a mutation was diagnosed causing thrombophilia. he ultimately made a full neurologic recovery and was discharged home with warfarin for the thrombophilia. he was most recently admitted for acute left hemiplegia. at 1430 on the day of presentation, his mother noted that he was demonstrating some left - sided weakness that progressed to complete hemiplegia. at 1800 in the emergency department, his mentation and speech were appropriate, but the left hemiplegia persisted and his modified rankin scale (mrs) was 4. his anticoagulation was subtherapeutic with an inr of 1.6. suspecting a stroke, a head ct scan was obtained demonstrating an area of hypodensity within the inferior right frontal lobe suggestive of a recent infarct. to obtain more information, the patient was electively intubated and an emergent mri was obtained (fig. an area of restricted diffusion was noted deep to the insular cortex involving the right lentiform nucleus and posterior limb of the internal capsule. patchy areas of restricted diffusion were demonstrated in right frontal region and posterior temporo - occipital junction without corresponding hypointensity on the adc map indicating subacute areas of infarction. given the findings of the mri, the patient was urgently transferred to the endovascular suite for intervention. he remained intubated and under general anesthesia while access was obtained in the right common femoral artery with a 4f sheath. a 65 cm - length, 4f angled glidecath (terumo medical corporation, somerset, nj), was navigated into the right internal carotid artery (ica) over a 0.035 inch glidewire (terumo medical corporation, somerset, nj) without difficulty given the history of aortic arch reconstruction. 2a) confirmed the occluded proximal m1 segment. a trevo pro 14 microcatheter (stryker, kalamazoo, mi) was navigated through the guide catheter into the right ica over a synchro 14 micro - guidewire (stryker, kalamazoo, mi). the microguidewire was then carefully advanced through the m1 segment thrombus followed by the microcatheter over the guidewire and an angiogram was obtained through the microcatheter after the guidewire was withdrawn (fig. 2b), confirming position of the catheter. a 3 20 mm trevo xp provue retriever stent (stryker, kalamazoo, mi) was then deployed into the occluded m1 segment (fig., the stent retriever and microcatheter were withdrawn together as a unit through the 4f guide catheter in the ica. an angiogram obtained after the pass (fig. 3a and 3b) revealed that the m1 segment was still occluded and a thrombus fragment had migrated into the right anterior cerebral artery (aca). after the failed first pass of the stent retriever, a larger 4 20 mm trevo xp provue retriever stent was deployed into the right m1 segment using a trevo pro 18 microcatheter that was first passed through the m1 thrombus over a synchro 14 microwire (fig. the microcatheter was carefully withdrawn out of the groin over the stent pusher wire, leaving only the stent retriever and 4f guide catheter in place. gentle aspiration was then applied to the guide catheter after 3 minutes while the stent retriever was carefully withdrawn from the m1 segment into the 4f catheter in the ica. a 4 20 mm stent retriever was once again deployed into the m1 clot and the microcatheter was withdrawn. aspiration was applied to the 4f guide catheter as the stent was withdrawn, resulting in a tici 2b recanalization of the right mca territory (fig. a 4 20 mm stent retriever was deployed through the aca thrombus after a microcatheter was passed through the clot over a microwire (fig. the microcatheter was then withdrawn, leaving only the guide catheter and stent retriever in place. the stent was allowed to incorporate into the thrombus over a period of approximately 3 minutes, and then aspiration was applied to the 4f guide catheter in the ica as the stent retriever was withdrawn. the resulting cerebral angiogram showed a partial reperfusion in the right mca territory and complete reperfusion in the right aca territory (fig. the puncture to recanalization time for the procedure was 55 minutes and time from symptom onset to full recanalization was 7 hours. the guide catheter and sheath were removed and pressure was held to the groin for 15 minutes. the patient was extubated at the end of the procedure and admitted to the pediatric intensive care unit. at that time, he was moving all of his extremities, but was weaker on the left side. an mri obtained on the following day did show an increased area of restricted diffusion deep to the insular cortex. he was placed on a heparin infusion as a bridge while warfarin was restarted with a goal inr of 2.0 - 2.5. the strength in his left arm and leg continued to improve over the course of his hospitalization and was discharged home after a week. at a 30 day return outpatient clinical visit, he was walking normally with only a mild motor deficit in his left hand and a slight facial droop. this 2 year - old hispanic male has a past medical history of hypoplastic left heart syndrome (hlhs) that required multiple cardiothoracic surgeries including a norwood procedure with a sano shunt after birth, a bidirectional glenn procedure at 4 months of age, and two subsequent tricuspid valve repairs. he had a right middle cerebral artery (mca) stroke at 18 months of age manifesting as left face and arm weakness. at that time, a heterozygous prothrombin g20210a mutation was diagnosed causing thrombophilia. he ultimately made a full neurologic recovery and was discharged home with warfarin for the thrombophilia. he was most recently admitted for acute left hemiplegia. at 1430 on the day of presentation, his mother noted that he was demonstrating some left - sided weakness that progressed to complete hemiplegia. at 1800 in the emergency department, his mentation and speech were appropriate, but the left hemiplegia persisted and his modified rankin scale (mrs) was 4. his anticoagulation was subtherapeutic with an inr of 1.6. suspecting a stroke, a head ct scan was obtained demonstrating an area of hypodensity within the inferior right frontal lobe suggestive of a recent infarct. to obtain more information, the patient was electively intubated and an emergent mri was obtained (fig. an area of restricted diffusion was noted deep to the insular cortex involving the right lentiform nucleus and posterior limb of the internal capsule. patchy areas of restricted diffusion were demonstrated in right frontal region and posterior temporo - occipital junction without corresponding hypointensity on the adc map indicating subacute areas of infarction. given the findings of the mri, the patient was urgently transferred to the endovascular suite for intervention. he remained intubated and under general anesthesia while access was obtained in the right common femoral artery with a 4f sheath. a 65 cm - length, 4f angled glidecath (terumo medical corporation, somerset, nj), was navigated into the right internal carotid artery (ica) over a 0.035 inch glidewire (terumo medical corporation, somerset, nj) without difficulty given the history of aortic arch reconstruction. 2a) confirmed the occluded proximal m1 segment. a trevo pro 14 microcatheter (stryker, kalamazoo, mi) was navigated through the guide catheter into the right ica over a synchro 14 micro - guidewire (stryker, kalamazoo, mi). the microguidewire was then carefully advanced through the m1 segment thrombus followed by the microcatheter over the guidewire and an angiogram was obtained through the microcatheter after the guidewire was withdrawn (fig. 2b), confirming position of the catheter. a 3 20 mm trevo xp provue retriever stent (stryker, kalamazoo, mi) 2c). after approximately 3 minutes, the stent retriever and microcatheter were withdrawn together as a unit through the 4f guide catheter in the ica. an angiogram obtained after the pass (fig. 3a and 3b) revealed that the m1 segment was still occluded and a thrombus fragment had migrated into the right anterior cerebral artery (aca). after the failed first pass of the stent retriever, a larger 4 20 mm trevo xp provue retriever stent was deployed into the right m1 segment using a trevo pro 18 microcatheter that was first passed through the m1 thrombus over a synchro 14 microwire (fig. the microcatheter was carefully withdrawn out of the groin over the stent pusher wire, leaving only the stent retriever and 4f guide catheter in place. gentle aspiration was then applied to the guide catheter after 3 minutes while the stent retriever was carefully withdrawn from the m1 segment into the 4f catheter in the ica. a 4 20 mm stent retriever was once again deployed into the m1 clot and the microcatheter was withdrawn. aspiration was applied to the 4f guide catheter as the stent was withdrawn, resulting in a tici 2b recanalization of the right mca territory (fig. a 4 20 mm stent retriever was deployed through the aca thrombus after a microcatheter was passed through the clot over a microwire (fig. the microcatheter was then withdrawn, leaving only the guide catheter and stent retriever in place. the stent was allowed to incorporate into the thrombus over a period of approximately 3 minutes, and then aspiration was applied to the 4f guide catheter in the ica as the stent retriever was withdrawn. the resulting cerebral angiogram showed a partial reperfusion in the right mca territory and complete reperfusion in the right aca territory (fig. the puncture to recanalization time for the procedure was 55 minutes and time from symptom onset to full recanalization was 7 hours. the guide catheter and sheath were removed and pressure was held to the groin for 15 minutes. the patient was extubated at the end of the procedure and admitted to the pediatric intensive care unit. at that time, he was moving all of his extremities, but was weaker on the left side. an mri obtained on the following day did show an increased area of restricted diffusion deep to the insular cortex. he was placed on a heparin infusion as a bridge while warfarin was restarted with a goal inr of 2.0 - 2.5. the strength in his left arm and leg continued to improve over the course of his hospitalization and was discharged home after a week. at a 30 day return outpatient clinical visit, he was walking normally with only a mild motor deficit in his left hand and a slight facial droop. ais in childhood is rare with an estimated incidence of 2.5 - 13 per 100,000 per year. while the mortality rate of pediatric ais is only 3 - 6%, 70% of cases will have lifelong morbidity, burdening society for decades after the event. this morbidity is higher than the estimated 50% morbidity of adult ais and may be attributed to delayed diagnosis. signs and symptoms of ais in the pediatric population can mimic other disease processes, contributing to the median 25 hour delay from clinical onset to radiologic confirmation of pediatric ais. treatment for pediatric ais poses unique challenges. with the exclusion of patients less than 18-year - old from major stroke treatment trials, recommendations of pediatric ais at this time are limited to supportive management and anticoagulation using aspirin or heparin [6, 7 ]. thrombolysis with intravenous tissue plasminogen activator (tpa) at the present is only recommended in the setting of clinic research protocols. the maturation of the hemostatic system that occurs throughout childhood illustrates physiologic differences between pediatric and adult populations, manifesting as different dose - related responses and pharmacokinetics of thrombolytic therapy [1, 9 ]. formal recommendations for intraarterial (ia) tpa or mechanical thrombectomy for pediatric ais are also lacking and evidence for these therapies are limited to published case reports. mechanical thrombectomy may serve as an important primary treatment of pediatric ais given that diagnosis is often delayed and appropriate thrombolysis dosing is still uncertain. a review of ais trials over the past 20 years showed that the recanalization rates have significantly improved, attributable to evolving mechanical thrombectomy technique and technology. though these trials have excluded pediatric cases, a total of 18 mechanical thrombectomy procedures performed in pediatric cases have been published and are summarized in table 1 [1, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 ]. all of these cases with the exception of one report have had favorable results, indicating that a mechanical thrombectomy can be safely performed for a pediatric patient. the average time to treatment after symptom onset of the reported cases was 12.9 hours, well beyond the recommended 8 hour window for treatment. anticoagulation was only reported in two cases [13, 17 ] and either iv or ia thrombolysis infusion was reported in 7 cases [1, 12, 13, 14, 16, 18 ]. in two reports where complete thrombus retrieval was not possible, one thrombus was transferred from an m1 branch to an ipsilateral a1 branch with a favorable clinical result, and a stent was used in the other case to recannulate an internal carotid artery with a favorable clinical result. other techniques of ia therapy including angioplasty with or without ia thrombolysis infusion are reported. the current case is an account of the youngest patient where a mechanical thrombectomy was performed for ais. as with other pediatric cases of ais, the acute onset of left hemiplegia was not immediately recognized as an ischemic event and presentation to the emergency department was delayed. in this case, once the diagnosis was definitively made, the patient was immediately transferred to the endovascular suite. the anatomy of the 2-year - old patient limited the available access in the right common femoral artery to a 4f sheath, which in turn limited the guide to a 4f catheter. due to concerns regarding the size of the occluded m1 branch, a 3 20 mm stent retriever was initially and unsuccessfully used without aspiration. ultimately, complete retrieval of the thrombus required a larger 4 20 mm stent retriever and aspiration through the 4f guide catheter after the microcatheter was first withdrawn. it was initially thought that the size of the m1 segment in the 2-year - old patient would not accommodate the 4 20 mm stent, but the larger stent retriever was used without hemorrhagic complication and the patient made a favorable recovery. the size of a stent retriever should be tailored to the specific vascular anatomy of pediatric patients presenting with ais. aspiration through the guide catheter during withdrawal of the stent retriever is also important to prevent thrombus migration. for pediatric patients that can only accommodate a 4f guide catheter, the delivery microcatheter must first be withdrawn after the stent retriever is deployed to provide enough space in the guide catheter to apply aspiration. pediatric ais is a rare event that is associated with a high incidence of poor neurologic outcomes because of delayed diagnosis and treatment. evidence for treatment of pediatric ais is sparse and based on expert opinion extrapolated from adult studies. as demonstrated in a growing number of reported cases, mechanical thrombectomy can be safely performed in the pediatric population. a review of the limited reported cases suggest that the therapeutic window for performing a mechanical thrombectomy in pediatric patient population extends beyond the 8 hour time period established for the adult population.	a 2-year - old boy with hypoplastic left heart syndrome that required multiple cardiovascular surgeries and a heterozygous prothrombin g20210a mutation with resulting thrombophilia maintained on warfarin presented with acute right middle cerebral artery (mca) infarction manifesting as a left hemiplegia. an mri revealed a complete occlusion of the right m1 segment with an area of restricted diffusion in the right basal ganglia representing only a small area of acute infarction. patchy areas of subacute infarction were also present in the right mca territory. he underwent endovascular mechanical thrombectomy with a stent retriever. this is an account of a successful mechanical thrombectomy performed in the youngest patient reported in the english literature to date.
multiple myeloma is a monoclonal malignant neoplasm of plasma cell origin which occurs in the bone marrow and may result in extensive destruction of skeletal structures. it most commonly affects the skull, vertebrae, and pelvis, with pain, fatigue, swelling, and anemia being some of the most common clinical presentations. an 83-year - old african american female presented to the emergency department with mild pain and swelling over the posterior left mandibular alveolar ridge [figure 1 ]. the lesion was approximately 2 cm 2 cm 1.5 cm in the area of the missing first molar. on palpation, it was firm with no fluctuance. radiographically, an ill - defined radiolucency of the alveolar bone was identified [figure 2 ]. initial clinical presentation of the patient with gingival swelling between teeth 18 and 20 radiograph showing an ill - defined radiolucent area in the bone between teeth 18 and 20 an incisional biopsy revealed a malignant neoplasm of hematopoietic origin with sheets of malignant plasma cells ranging from mature to immature forms as well as pleomorphic - cells and occasional binucleated cells [figure 3 ]. the neoplastic plasma cells were strongly positive with kappa light chain antibody and with cd138 [figure 4 ], both supporting the histological diagnosis of plasmacytoma. (a) low magnification shows stratified squamous mucosa on right upper end with cellular infiltrate in left lower portion of the image ; (b) magnification of h and e histology shows sheets of plasma cells, most are large and atypical with large nuclei and prominent nucleoli immunohistochemistry stain for cd138 antibody. note uniform positive staining with cd138, a marker for plasma cells a bone marrow aspirate biopsy was performed on the left posterior iliac crest where sheets of neoplastic plasma cells were identified. a random urine collection showed elevated bence - jones protein (0.09 g) and a subsequent 24 h urine collection demonstrated a level of 0.13 g (normal 0.0500.080). the disease is usually found in the sixth and seventh decades of life with a median age of 62, 66, 71. it is a multicentric and generalized bone marrow disease that affects multiple bones and can include the jaws. in a review of 193 patients with diagnosed multiple myeloma, epstein. reported that out of 783 multiple myeloma patients, 14.1% had oral manifestations in the form of jaw pain, severe periodontitis, tooth mobility, bone destruction, pathologic fracture, paresthesia, and soft tissue swelling. usually, if the jaws are involved, it is an indication of an advanced stage of the disease. in the case we report, no other lesions were identified on the osseous skeletal survey performed the same month as the biopsy. this patient had a systemic manifestation of multiple myeloma despite the jaw being the only bone affected ; she had mild anemia, elevated serum calcium, and bence - jones protein in her urine. she also had sheets of neoplastic plasma cells in her bone marrow biopsy performed on the left posterior iliac crest. the neoplastic plasma cells were positive with cd138 antibody confirming the presence of the disease in an area away from the jaw. we present a case of multiple myeloma first manifesting in the mouth as a mildly painful gingival swelling with underlying irregular bone destruction. we recommended that all patients receive a routine oral examination by their dentist and primary care physician to insure early recognition of malignant neoplasms of the mouth.	it is rare that multiple myeloma (mm) occurs as a primary lesion in the jaws ; we report such a case in an elderly patient involving the gingiva of the left posterior mandible. multiple myeloma is a monoclonal malignant neoplasm of plasma cell origin which occurs in the bone marrow and may result in extensive destruction of skeletal structures. if the jaws are involved, it usually indicates an advanced stage of the disease.thi s makes our case very unique due to the fact no other osteolytic lesions were identified at the time of the diagnosis of multiple myeloma. we report a rare case of multiple myeloma which was diagnosed from an intraoral gingival lesion on the lower left mandible.
dental implantation and fixation of screws for any reason are performed by drilling the bone and the success of these operations depend on many factors. through these factors, thermal injuries occurring due to temperature raise during drilling, the threshold level for thermal injuries on the bone is the 47c for a minute and the temperature can raise that level easily during drilling by rotational burs 1,3. as a consequence of thermal injury, bone is not only resorbed but also replaced with fat cells 4. as a result, the mechanical structure of the bone is weaken 5. to prevent the bone from the temperature raise during drilling, various irrigation systems are used and mostly, sterile saline solutions are the material of choice 6,7. although, to our knowledge, there is not a scientific data in the literature, there is a belief among the surgeons that cooled saline irrigation is more effective than the uncooled saline irrigation to protect the bone from the thermal injuries. the purpose of the present study was to investigate the effect of the irrigation temperature on the bone healing. for this aim, standardized drilling and miniscrew placement was performed in the tibias of 18 sprague dawley rats with rotating bur uncooled, cooled with 25c and 4c saline irrigation. experiments were designed to determine the effect of irrigation temperature on the bone after osteotomy and miniscrew placement. for this aim 3 months old weighing approximately 350 to 450 g 18 sprague - dawley rats were maintained at 22 0.5 c on a 12-h light/12-h dark cycle with free access to water and standartized food in pathogen free separate cages. all of the experiments were performed at the istanbul university, institutes of experimental science laboratories (istanbul, turkey). maintenance and all experimental procedures performed were fully in accordance with principles established by the existing governmental acts and approved by the university institutional animal welfare committee. animals were divided randomly in to 3 groups of 6 animals and 3 experiments were performed. anesthesia was induced with ketamine hcl (100 mg / kg i.p., ketalar, parke davis) by the injection through the peritonium of the rats and maintained with xylozin hcl (23.32 mg / ml i.p., rompun, bayer). after the induction of anesthesia, operation sites were shaved and the rats were placed and stabilized on the operation table. skin incision was performed parallel to the long axis of the right femurs and the bone was exposed by the dissection of hamstring and quadriceps femoris muscles. the drill speed was set to be 20,000 rpm, which is similar to that used during osteotomies by rotational systems and saline irrigation speed was standartized by using saline - dispencer. all of the holes were drilled with standard burs manufactured for 2 mm miniscrews and standart 2 mm wide 5 mm length titanium miniscrews (bone screw kit, biohorisons, usa) were inserted. in the group one, holes were drilled without irrigation (figure 1). in the group two, holes were drilled with 25c (2c) saline irrigation and in the group three, holes were drilled with 4c (2c) saline irrigation (figure 2). after the drilling and the miniscrew placement (figure 3), muscle and fascia layers were closed with simple resorbable 4.0 sutures (vicryl 4 - 0, ethicon) and finally the skin incisions were closed with continious 3.0 sutures (silk 3.0, dogsan). postoperative antibiotics (gentamicin, 0.5 mg / kg) and analgesics (xylocaine 0,5 mg / kg) were given subcutaneously. the positions of the miniscrews were controlled by cone beam ct scanning. during post - operative period, all of the animals were controlled day by day and none of them were lost. at the end of three weeks, animals were killed and right femurs of them were harvested. then, the specimens were sent to the pathology laboratory (istanbul university, institute of oncology, department of pathology, istanbul, turkey) for the histopathological evaluation. the cutting line was started parallel to the transverse axis of femurs at about 4 mm apart from femoral heads, where the first hole preparation was made. the specimens were fixed in 10% formalin for one week and decalcified in 10% formic acid solution (merck, darmstadt, germany) for 25 days. the decalcified specimens were embedded in paraffin and cut into 3 m thick sections on charged slides using a microtome (leica microsystemic rm 2125, germany), and routine hematoxylin and eosin (h&e) staining was performed. the sections were examined with a light microscope (olympus bx60 microscope) attached to a digital camera (olympus e-330) which connected to a computer. a histomorphological review was performed by a single blinded oral pathologist to evaluate the presence of infection, necrosis, fibrosis and new bone formation. all screw hole surroundings were evaluated by histopathologically with light microscope under 20, 40, 100 and 200x magnifications. the score was made up of 0 - 5% as (-), 5 - 30% as (+), 30 - 60% as (+ +) and 60% and over as (+ + +) to evaluation of new bone formation and fibrosis according to their surface covering at 40x magnification. the scores for infection and necrosis were determined by occurring in a standardized area at 20x magnification. the infection was determined if inflammatory cells were observed in the healing area the (+) score and if there were no inflammatory cells the (-) score were given. for necrosis the (-) score was given, if there was no necrosis whereas the (+) score was given if there was. the statistical differences between the control and test groups were compared by ki - square and fisher tests. in this research, all of the statistical evaluations were performed by using the ncss software (ncss inc., 2007, usa). ki - square and fisher tests were performed for the evaluation of the definitive statistical methods and also qualitative datas. the distribution difference of the new bone formation between 4c, 25c and control groups were observed as statistically significant (p=0,031)(table 1). the new bone formation of the control group was evaluated significantly lower than the 4c and 25c groups (p=0,020, p=0,049), however, there was no statistically significant difference evaluated between the 4c and 25c groups statistically(p=0,637)(table 2). statistically significant difference was observed between 4c, 25c and control groups regarding the distribution of the infection presence (p=0,0001)(table 3). the presence of the infection in the control group 9 (% 75), was observed as significantly higher than the 4c and 25c groups (p=0,0001), but no statistically significant difference was seen between the 4c and 25c groups (p=1)(table 4). the distribution of the bone necrosis existence between 4c, 25c and control groups were observed significantly different (p=0,0001) (table 5). the existence of the bone necrosis in the control 11 (% 91,7), group was evaluated significantly higher than the 4c and 25c groups (p=0,0001), but, no statistically difference was observed between the 4c and 25c groups (p=1) (table 6). no statistically significant difference was observed between 4c, 25c and control groups regarding the distribution of the fibrosis existence (p=0.355)(table 7). the fibrosis distribution of the control group was not significantly different than the 4c and 25c groups (p=0,334, p=0,164), and also, no statistically significant difference was observed between the 4c and 25c groups (p=0,232) (table 8). the temperature rise on the bone during osteotomies performed by rotational systems is affected by various factors 8 - 10. some of these factors are bone density, and the location of the osteotomy, in particular the amount of cortical versus cancellous bone, which may be influential. other important factors pertain to the drilling, including the speed at which the drill rotates, the sharpness of the margins on the drill, the thickness of the drill, and the force with which the drills applied to the bone 11. regardless of the reason for, this temperature rise can cause damage or impaired healing on the bone 1,3. as previously mentioned, the accepted threshold level that is required for the thermal injury on the bone is 47c for a period of 1 minute 1,3. for the protection of the bone from the thermal damage during osteotomy, saline solution is routinely applied to the drill and the osteotomy site in surgical practice. and also, most of the surgeons prefer cool saline solutions and they believe that it is more effective than the normal solutions for the reduction of the temperature. in the literature, there are some studies indicating the effect of the saline application on temperature rise 11, on the contrary, others indicate that application of saline solution to the rotational system to bone interface during osteotomy do not reduce the temperature during the osteotomy to any significant degree 12,13. in the authors ' knowledge, this is the first study focuses on the effect of the irrigation temperature to reduce thermal rise. at the same time, it is evaluated in this study that application of saline solution is effective or not for the reduction of the thermal damage. in the present study we evaluated the specimens in terms of new bone formation, presence of infection, necrosis and fibrosis our study demonstrated significant difference regarding the necrosis values between control group (without irrigation) and the group irrigated with 25c and 4c saline (figures 4 - 6). however copious saline irrigation was useful for cleaning the operation area from any remnants of the drill. although there was no statistically significant difference between the group irrigated with 25c and 4c saline for new bone formation, latter group had numerous osteoblasts and more prominent osteoblastic rim around the trabeculae of new bone (figures 4,5). these results indicate that there is no disadvantage to use 25c saline irrigation, but it may be better to use 4c saline irrigation for rapid healing. in this study, it is verified that the use of saline irrigation is not only useful to reduce temperature rise on the bone during osteotomy, but also effective for cleaning the osteotomy site from any bony remnants.	objective : osteotomies, performed by rotational instruments, can cause temperature rise on the bone and elevated temperature can disrupt the bone healing. when the osteotomies are performed for the insertion of miniscrews, the bone healing disruption may cause stability loosening or failures. saline irrigation is mostly used for the prevention of the heat generation during osteotomy.purpose : the purpose of this study was to evaluate the effect of the saline irrigation temperature on bone healing.material and method : standardized drilling and miniscrew placement was performed in the tibias of 18 sprague dawley rats with rotating bur uncooled, cooled with 25c and 4c saline irrigations. after the 21 days, the difference in healing was observed between the uncooled and cooled groups.results : although there was no statistically significant difference between the group irrigated with 25c and 4c saline for newly bone formation, osteoblasts were seen more active and bone marrow was more dynamic in group 4c than group 25c. there is no disadvantage to use 25c, but it may be better to use 4c for rapid healing.
in 2001, the human genome project (hgp) consortium and celera genomics reported the first drafts of sequences of the human genome [1, 2 ]. the hgp consortium used the hierarchical sequencing or clone - by - clone approach, whereas celera genomics used the whole genome shotgun (wgs) approach, which had been successfully used in 1995 to sequence the h. influenzae genome. in the hierarchical sequencing approach, a tiling of large dna sequences, such as bacterial artificial chromosome (bac) or yeast artificial chromosome (yac), are constructed for a genome, and each of the sequences is determined. the hgp consortium used bac as the large sequence, followed by shotgun sequencing of each bac. in sequencing the genome, owing to physical limitations of shotgun sequencing methods, the genome must be broken down into smaller portions, shotgun reads sized in the range of 600 bps (base - pairs) to 800 bps, and as the sequence data for each of these shotgun reads is produced, it must be connecting them with those adjacent and overlapping reads that have been previously sequenced, that is, to achieve an assembly of these smaller sequences into larger contiguous regions or contigs. in most cases, the sequences of shotgun reads are obtained by sequencing both ends of a dna fragment whose approximate size is known. such pair information, referred to as mate - pair information, constrains the placement of the reads within an assembly. in an ideal assembly, all read pairs are placed in such a manner as to satisfy the orientation and distance constraints imposed by the pairing. mate - pair information can be used to determine the quality of an assembly, because most types of misassemblies lead to violations of these constraints. in contrast to hierarchical sequencing, wgs breaks a whole genome into small pieces randomly, without shearing into large dna pieces of intermediate size. wgs is faster and cheaper than hierarchical sequencing because of the simplicity of the processing steps. the success of wgs [4, 5 ] has increased its usage and the size of the genome to be sequenced has increased. although contig assembly programs are well established, less is known about scaffold analysis. while some of its features have been implemented to sequence specific genomes [68 ], the features needed for general scaffold analysis and visualization consed, a graphical tool for contig assembly, provides good visualization and helps to finish sequencing by connecting with autofinish ; however, it does not have many features related to scaffold analysis. it has been suggested that the contig scaffolding problem can be solved by greedy - path merging algorithm. moreover, gigassembler can orient the contigs based on mrna, paired plasmid ends, est, and bac end pairs. this paper introduces a novel scaffold analysis tool, conpath, which calculates the longest scaffolds. due to the abundance of repeats in genomic dna sequences, a purely overlap - based approach for wgs assembly is not feasible, but the use of mate - pair information is crucial. the conpath program uses end read pairs of fixed - sized dna libraries as mate - pairs to calculate orientations, orders, and gap sizes. it reads a phrap output file (.out) and an ace format file, which contain contig structures and mate - pair information. the most important characteristic of conpath is its ability to exploit the mate - pair information of large dna fragments such as fosmids or cosmids, which are about 40 kbps(kilo base - pairs) in size, or bacs, which are about 100300 kbps in size, rather than plasmids, which are about 210 kbps in size. a mate - pair is composed of two end reads that always face each other. each end read, b or g, has an orientation relative to the contig containing it. if the direction of an end read is the same as the direction of the contig, the former has direction u, otherwise, it has direction c. in figure 1, b has direction u because the cl contig and b read are in the same direction, whereas g has direction c because the c2 contig and g read are in opposite directions. the size of the mate - pair helps to estimate the gap size between contigs c1 and c2. when one contig contains one end of a mate - pair and a second contig contains the other end of the mate - pair, the two contigs are said to be linked by the mate - pair. a scaffold is a series of contigs that can be linked by mate - pairs. the connection relationship of all the contigs can be represented as a graph in which each contig is represented as a vertex. an edge is created between two contig vertices when they are linked by at least one mate - pair, and the number of linking mate - pairs between two contigs is defined as the edge weight. to construct scaffolds using mate - pair information,, cn } a mate - pair set m = { m1, m2, m3,, ml } and a set of reads r = { r1, r2, r3,, rs } let g denote the scaffold graph using c and m : (1)g=(c, e). when a mate - pair mk = (ri, rj) exists, in which contig cs contains ri and contig ct contains rj there is an edge between contigs cs and ct. edge set e is expressed as (2)e={ecsct iff mk=(ri, rj) exists for rics, rjct, cs c, and ctc}. in constructing a scaffold graph, the linking level (l), the threshold value for the edge weights, was used as a filtering value in constructing and showing scaffolds on output. when an edge has a weight value smaller than the linking level (l), the edge is discarded from the graph. considering the errors that occur in base calling and contig assembly, the optimal construction of a scaffold graph is an np - complete problem. to practically solve this problem, conpath uses a simple greedy algorithm. whenever a new edge is added to the graph, graph it is worthwhile noting that conpath determines the relative orientations of all contigs using the orientations of the end reads. figure 2 shows the determination of the order and orientations of three contigs using two mate - pairs. in figure 2(a), b1 and g1 reads determine the relative orientation of contigs c1 and c2, and, in the same way, b2 and g2 reads determine the relative orientations of contigs c2 and c3 (see figure 2(b)). the relative orientations of contigs c1, c2, and c3 are determined by rotating the scaffold in figure 2(b), as shown in figure 2(c). assuming all mate - pairs have a fixed size, the size of the gap between two adjacent contigs is determined by the sizes of the two contigs and the positions of the end reads of contigs. suppose that contig c1 contains b read and contig c2 contains g read. let gap (c1, c2) be the gap size between c1 and c2. let ps(b) and pe(b) be the start and end positions of b read in c1, respectively, and let ps(g) and pe(g) be the start and end positions of g read in c2, respectively. considering all the possible directions of a mate - pair of two end reads, conpath estimates the gap size as b - u and g - u : gap (c1, c2) = matepair size { (c1 length ps(b)) + (c2 length ps(g)) } b - u and g - c : gap (c1, c2) = matepair size { (c1 length ps(b)) + pe(g)) } b - c and g - u : gap (c1, c2) = matepair size { (pe(b) + (c2 length ps(g))}b - c and g - c : gap (c1, c2) = matepair size { pe(b) + pe(g) } gap (c1, c2) = matepair size { (c1 length ps(b)) + (c2 length ps(g)) } gap (c1, c2) = matepair size { (c1 length ps(b)) + pe(g)) } gap (c1, c2) = matepair size { (pe(b) + (c2 length ps(g)) } gap (c1, c2) = matepair size { pe(b) + pe(g) } figure 3 shows the procedure for estimating the gap size between contigs when b and g have u and c directions, respectively. the orientations of contigs c1 and c2 are set in the same direction. the length of part of the mate - pair library in contig c1(c1 length ps(b)) and the length of part of the mate - pair library in contig c2(pe(g)) are calculated. finally, the gap size is calculated as (3)matepair size {(c1lengthps(b))+pe(g)}. one important feature of conpath is the verification of a contig assembly by identifying erroneous contigs. we have defined 4 types of contig assembly errors to check the quality of a contig assembly. self - collision errorwhen the number of mate - pairs connecting two adjacent contigs is more than 2, and there is an inconsistency in determining the orientation of contigs with mate - pairs, the error is defined as a self - collision error, the most serious error type. if this error occurs, the contigs should be inspected manually one by one. when the number of mate - pairs connecting two adjacent contigs is more than 2, and there is an inconsistency in determining the orientation of contigs with mate - pairs, the error is defined as a self - collision error, the most serious error type. if this error occurs, the contigs should be inspected manually one by one. when a mate - pair of an end read is contained in a contig, the real size of this mate - pair can be calculated. if the difference between the calculated and predefined sizes is larger than a threshold value, the error is defined as a mate - pair size error. if the gap size between two contigs is a negative value, it indicates that the two contigs should be merged in the contig assembly process ; this is defined as a gap size error. after calculating the distances of all adjacent contigs, any two nonadjacent contigs can be overlapped due to the accumulation of errors in gap size estimations. this type of error is defined as an overlap error, which happens rarely and is not so critical. identifying error types is useful in verifying and correcting the final result of a contig assembly. if a contig has more than two types of errors, it is highly probable that a misassembled contig is present. conpath assigns different colors to contigs by the number of error types, with nonerroneous contigs colored blue. when one contig has more than one error, conpath assigns this contig a reddish color, with the intensity proportional to the number of error types. therefore, we can check the quality of the final result of a contig assembly by simply inspecting the color information in the scaffold visualization window of conpath. it provides a user - friendly interface and shows visual and color - informative outputs, which can help analyze scaffolds both intuitively and informatively. conpath provides dialogue windows for mate - pair information, edge information, contig path, and invalid contigs by automatically checking for the 4 types of errors. scaffolds are displayed graphically in proportion to the real sizes of vertices and edges after aligning vertices and edges to avoid graphical collision, and the detailed information for each vertex and edge is shown on a pop - up window. conpath can produce a large picture for all scaffolds by assembling separately printed module pictures. artificial data were generated in two different versions : r (randomly) and u (uniformly). the r version consisted of contigs of random sizes, whereas the u version consisted of contigs of uniform size. in these artificial data experiments, conpath showed very successful scaffold constructions using mate - pair information. from experiments with artificial data, conpath worked very successfully and efficiently on real data sets, in sequencing the mannheimia succiniciproducens and vibro vulnificus genomes. four datasets were tested in sequencing the m. succiniciproducens genome, whereas one dataset was tested in sequencing the v. vulnificus genome, to verify the results of contig assembly. mh1, mh2, mh3 and mh4 are the contig assembly results of the m. succiniciproducens genome and vv is the contig assembly result for the v. vulnificus genome. for the m. succiniciproducens genome, going from mh1 to mh4 increased the reliability of the contig assembly results. conpath was most successful at linking level 2 by minimizing the loss of edges. table 3 shows the detected errors in scaffold construction for the 5 datasets. among the m. succiniciproducens datasets, mh1 had the most errors, whereas mh4 had no erroneous contigs. these results show that identifying the 4 types of errors for contigs is effective in verifying the result of contig assembly. figure 6 shows the constructed scaffolds at linking levels 2 and 3 for the mh1 dataset. contig 93 is suspected of being erroneous because it has several erroneous contigs on both sides. table 4 shows a comparison of features of several scaffold analysis tools, including conpath, consed, autofinish, and bambus. most importantly, conpath helps users to intuitively verify the contig assembly by providing many visualization features and additional information to detect erroneous contigs. a scaffold analyzer is a very important tool in genome sequencing, in that it can verify the results of contig assembly and to identify misassembled contigs. we have developed conpath, a scaffold analyzer that exploits mate - pair information to construct scaffolds by ordering and orienting separate sequence contigs. conpath provides various useful viewers and dialogue boxes for intuitive understanding. using end read pairs of a fixed - sized mate - pair library, conpath can determine the relative orientations of all contigs successfully, and estimate the gap size of each adjacent contig pair. conpath was used successfully in sequencing several microbial genomes, including the m. succiniciproducens genome. conpath is, therefore, a useful scaffold analyzer to verify contig assembly by detecting erroneous contigs. conpath will doubtless improve as its algorithm becomes more correct and efficient, as well as through the development of additional features, such as primer design for the finishing step and a sequence read viewer.	we have developed a windows - based program, conpath, as a scaffold analyzer. conpath constructs scaffolds by ordering and orienting separate sequence contigs by exploiting the mate - pair information between contig - pairs. our algorithm builds directed graphs from link information and traverses them to find the longest acyclic graphs. using end read pairs of fixed - sized mate - pair libraries, conpath determines relative orientations of all contigs, estimates the gap size of each adjacent contig pair, and reports wrong assembly information by validating orientations and gap sizes. we have utilized conpath in more than 10 microbial genome projects, including mannheimia succiniciproducens and vibro vulnificus, where we verified contig assembly and identified several erroneous contigs using the four types of error defined in conpath. also, conpath supports some convenient features and viewers that permit investigation of each contig in detail ; these include contig viewer, scaffold viewer, edge information list, mate - pair list, and the printing of complex scaffold structures.
very recently, the journal of medicinal chemistry embraced the qhnmr concept by including absolute qhnmr as an acceptable and established scientific method for purity analysis. as a quantitative method, qhnmr continues to gain popularity in the biopharmaceutical industry and acceptance by the international conference on harmonization (ich). notably, qnmr is a (relative) primary analytical method and is part of the general chapter of the united states pharmacopeia (usp) as well as an official method in the japanese pharmacopoeia (jp17). with respect to the development of quantitative methodology for botanical standardization, the application potential of qhnmr for purity analysis, residual complexity studies, metabolomics, multimarker standardization, and orthogonal quantitation has been demonstrated. due to the presence of very many components, possible excipients, and the resulting intense signal overlap, thus, while established qhnmr methods work well for a single component and relatively simple mixtures, analysis of such complex samples demands an expanded qhnmr tool box. in general, the choice of appropriate quantitative measures in qhnmr depends on the type of sample and the required accuracy. in classical qnmr, both relative and absolute quantitation methods rely on signal integration (int). as int - qhnmr is by far the most practiced form of qnmr, both accuracy and precision of most contemporary qnmr methods depend on the means of the integral measurement. presently, three mechanistically different quantitative measures exist apart from classical int - qhnmr (figure 1). one entails peak height measurements, but due to limitations related to resolution of resonances and signal multiplicity, it was rendered impractical for this, given the complex peak overlap in the rce qhnmr spectrum. before delving into the mechanistic differences of the two spectral deconvolution (sd)-based approaches used in this study, it is important to differentiate the often interchangeably used terms line, peak, and resonance. multiple lines constitute an nmr resonance (or a signal that is the digital visual output of a resonance) due to the presence of scalar couplings (giving rise to signal multiplicities). the number of observable lines depends on the digital resolution as well as the natural line width of the signals. peaks refer to only the visual top of each line or resonance. it involves sd of select lines or entire spectra via mathematical peak fitting (pf) algorithms. pf - qhnmr methods take a one - by - one approach to the fitting of visible peaks (not spectral lines) and do not involve quantum mechanical (qm) calculations of the underlying spin systems. the third alternative to integration, builds on the method of h iterative full spin analysis (hifsa) and consists of the qm - driven generation of a simulated qhnmr spectrum. this spectrum accumulates the subspectra of all individual components, and the underlying qm simulation includes their individual line shape characteristics as well as the relative intensities as present in the mixture. intrinsically, the qm simulations yield precise intensities for all resonances, including those belonging to non - first - order (i.e., higher order) spin systems. the presence of non - first - order situations is nearly inevitable in h nmr, even at very high magnetic field strengths (equivalent to 700 mhz h), and its impact on qhnmr quantitation continues to be underrated despite the availability of well - established spin simulation tools. in qhnmr, aside from peak height quantiation (see text), three principal methods with different characteristics (yes / no) exist for the extraction of quantitative measures (numbers) that reflect the molarity of the analytical response to be used for the quantitative calculation. (i) classical integration (int - qhnmr) calculates the areas - under - the - curve of discrete regions of the spectra. (ii) spectral deconvolution (sd) that employs peak fitting methods (pf - qhnmr), such as global sd (gsd), are limited by line and peak assignment, especially in the case of non - first - order effects. qm - qhnmr derives exact measures from the spin populations of all target analytes. it has the ability to account only for relevant individual line intensities, thereby excluding impurities from the quantification. during the initial stage of this study, it became evident that both sd methods (qm - based and non - qm / pf) outperform the classical region - based integration (int - qhnmr) in terms of achievable accuracy and precision, especially with complex samples such as the rce or similar natural products. with deconvolution processing being the common denominator in sd, one major aim of the study was a systematic comparison and exploration of the capabilities of the two methods for covering a wide dynamic range of analytes. additional study goals related to the multidisciplinary study context, supporting the continued biological studies of the rce, are the establishment of an orthogonal analytical means of chemical standardization, as a means of advancing the accuracy of standardization, and the need to (re)assess the stability of the extract, using these new methods. the recently developed hifsa fingerprinting and hifsa - qhnmr methodologies were enabling technologies for these aims : they could be applied to the relatively complex rce sample and results could be compared with those from previously published hplc - uv and lc - ms standardization. moreover, to generate an independent data set that is mechanistically congruent with the studies performed some 10 years ago, a fast 10 min uhplc - uv method was developed that allowed both chromatographic fingerprinting and quantitation. lc - uv / ms are the most commonly used analytical methods worldwide, partly as a result of the widely abundant instrumentation and the relatively high sensitivity of uv and ms detection. while often categorized as a relatively insensitive method, qnmr analysis typically requires only 1530 min of instrument time, especially when samples are not mass limited and/or when the mass sensitivity of the instrumentation is high (cryoprobes, microprobes). importantly, the identical and costly reference materials required for any lc - based quantitation are entirely dispensable in qnmr. another intrinsic advantage is that multitargeted analysis can be performed on a single nmr sample, using standard qhnmr conditions, and can be repeated as needed. furthermore, qhnmr provides valuable spin parameter information simultaneously with quantitation, thereby enabling a thorough structural characterization of the constituents. the use of well - established quantitative conditions assures that signal intensities in qhnmr spectra are directly proportional to the molarity of hydrogen atoms giving rise to them. notably, qhnmr is unaffected by response factors, which are inevitable in lc - based quantitation. considering these main advantages of qhnmr, including its time efficiency, simplicity of sample preparation, linearity of signal response, independence of sample intrinsic factors, and even cost considerations, qhnmr offers a unique set of attractive properties, making it a standardization method that deserves broader consideration in the botanical research community and industry. furthermore, qhnmr can recognize dynamic properties such as chemical exchange (e.g., keto enol tautomerism) or rotational / conformational isomerism, along with the identification of uv - transparent and/or poorly ionizable molecules. while such phenomena often evade lc / ms - based analysis, they can be important for the explanation of biological outcomes. in light of the natural variation of phytoconstituents in source plants and the potential overlap with (chemo-)taxonomically related and unrelated species (both authentic species and adulterants), the specificity of botanical standardization increases with the number of markers used and their individual chemotaxonomic (sub)species specificity. compared to the quantitation of a single marker, multimarker schemes enhance the significance of the standardization method by capturing a wider chemical window of the botanical metabolome. accordingly, over the years, the development of new standardization protocols at the uic botanical center has progressed from oligo- to multimarker / metabolomic schemes, in order to better capture the metabolic complexity of botanical preparations. the underlying hypothesis of this approach is that metabolomic standardization enhances the measure of botanical integrity and is the key to better reproducibility in botanical research. as the number of targeted markers increases, the experimental effort for lc - based methods increases overproportionally due to the requirement of having high - quality (purity) reference materials available for each of the target compounds. as the chemotaxonomic significance and abundance (% content) are often mutually exclusive properties, this leads to substantial isolation efforts associated with the purification of multi - milligram amounts of minor constituents. this is often rendered impractical and the main contributing factor why multimarker standardization schemes are not used more widely despite their acknowledged significant advantage. while there might be a need for the use of such compounds in the initial establishment of a qhnmr method (e.g., for the confirmation of peak assignments and assessment of method specificity, see discussion below), all subsequent qhnmr analyses can be performed without these reference materials. in addition, qhnmr is fully capable of multitargeted quantitation, as shown recently for preparations from ginkgo biloba and glycyrrhiza species. achieving a targeted multimarker standardization of the rce using qhnmr was one important methodological aim of this study. the first step toward a qhnmr - driven multimarker standardization is the identification and unambiguous assignment of the signals of the target components in the qhnmr spectrum of a mixture. isoflavones 19 (chart 1) and the flavonols 10 and 11 were identified in the rce spectrum. the isoflavone signals were divided into four key regions (figure 3) : region 1 consisting of the methoxy proton signals from the b - ring (3.54.0 ppm) ; region 2 with the signals of the a - ring protons h-6/h-8 (6.06.7 ppm) ; region 3 with the signals of the b - ring aaxx or amx spin systems (6.78.0 ppm) ; and region 4 containing the c - ring h-2 singlets (8.28.5 ppm). signal assignment in region 3 was the most challenging due to a combination of intense overlaps and higher order effects. the characteristic h-2 singlets in region 4 enabled the determination of the total isoflavone content of the extract. the h-2 resonances maintained their singlet property even after the application of very strong lorentzian gaussian resolution enhancement window functions, proving their pure singlet characteristics. this was in line with the predictable lack of observable long - range couplings for these protons : the only potential coupling partners are located in the b - ring, giving rise to j and j couplings that evidently are well below the natural line width of the h-2 signal (1 hz) and even lower than the achievable signal splitting (0.4 hz). non - qm deconvolution of region 4 of the rce qhnmr spectrum using the global spectral deconvolution approach. the deconvoluted signals of the internal calibrant (ic : 3,5-dinitrobenzoic acid), the isoflavones 17, and flavonoid 10 are labeled. the colored lines represent the sum (blue), the fitted peaks (green), and the residual (red) of the deconvolution process. stacked qhnmr spectra (dmso - d6 ; 600 mhz) of the red clover extract and the isoflavones were divided into four key regions representing different spin systems present in the isoflavones. 6, prunetin ; 5, calycosin ; 4, daidzein ; 3, genistein ; 2, formononetin ; 1, biochanin a ; rce, red clover extract. the nmr spin parameters for ps, pb, and ir were obtained from metidb. depending on the type of the spin system, the a - ring protons fall into either region 2 (aaxx) or 3 (amx). the signals of biochanin a (1), formononetin (2), irilone (7), and quercetin (10) were assigned unambiguously by stacking of reference spectra and, in the case of 7, based on the data in the literature. to demonstrate the specificity of close resonances, the assignments for genistein (3), daidzein (2), calycosin (5), and prunetin (6) were confirmed using spiking experiments. the resonances of the isoflavones pratensein (8) and pseudobaptigenin (9) were initially assigned based on previously published results. except for the methoxy protons, this naturally gave rise to severe overlap in the spectrum of an extract sample. for example, the 7.36 to 7.40 ppm portion of region 3 of the rce spectrum contained a set of two protons from an aaxx system belonging to four minor isoflavones, which altogether represented eight individual protons (figure 4b, blue region). such a high level of complexity precluded the use of classical integration and posed a significant challenge for an exact assignment. moreover, field - dependence resulting from the involvement of non - first - order spin particles intrinsically limits the transmission of classical integration and non - qm linear sd outcomes between laboratories. even when considering a region with relatively simple resonances, such as region 4, the observed signal resolution still does not allow sufficiently broad or even uniform integral ranges. for example, the h-2 singlet of genistein (3) at 8.3083 ppm overlapped with the corresponding h-2 singlet of pseudobaptigenin (9) at 8.3132 ppm, and the signals of both the minor components were riding on the feet of the h-2 singlet of the major isoflavone formononetin (2) (figure 4b, green region). another close overlap occurred between the h-2 singlets of daidzein (1) and calycosin (5) at 8.2743 and 8.2770 ppm, respectively. in this case, spiking experiments combined with hifsa were necessary to ensure unambiguous assignment of the minor constituents. regions 3 and 4 of the qhnmr spectrum of rce were the most relevant for the qhnmr analyses. panel a shows the signal assignments of the two major isoflavones 1 and 2. magnified expansions of the colored regions in panel b show the assignments of the seven minor isoflavones 39 and the flavonols 10 and 11 to demonstrate the importance of quantitative measures derived from qm - based deconvolution in strongly overlapping spectra (ic, internal calibrant ; 3,5-dinitrobenzoic acid). whereas signal overlap can be resolved for the purpose of analyte assignment, the complexity of the situation also shows that resolution of the underlying individual peak areas is not straightforward and can not be achieved by simple definition of (integral) ranges. this underscores again that classical integration is not a viable quantitative measure for most complex mixtures. some of the signals belonging to pratensein (8), pseudobaptigenin (9), and kaempferol (11) were completely masked by major isoflavone signals, thus lowering the confidence with which the minor markers can be quantified compared to other constituents. for instance, five out of the eight nmr signals for 9 were masked partially or completely by those of the major constituents. despite the exclusion of 8, 9, and 11 from full quantitative analysis, their identification and assignment in the rce nmr spectrum helped improve the overall match between the observed and the calculated spectrum when using the qm - based approach (hifsa), especially for the 6.807.05 ppm portion of region 3 (figure 4, red region). the spin parameter set (pms) (perch parameter file) files for 8 and 9 were built based on data obtained from the metidb. as per the literature, the h-2 singlets for both 8 and 9 in dmso - d6 appeared at 8.32 ppm. insufficient reporting of (relative) nmr chemical shifts in the literature precludes the successful dereplication and accurate assignment in a more complex sample, especially with regard to the frequently relevant non - first - order effects. this once more reinforces the importance of reporting h nmr chemical shifts with at least four decimal points (100 ppb level), as shown earlier. while concentration and sample matrix effects in complex samples such as extracts typically necessitate adjustments of absolute values (accuracy), the relative frequencies of the various resonances () of a given analyte are much more stable than generally considered. in fact, reporting or values with 100 ppb precision is a valuable means of identifying analytes and improving the specificity of a qhnmr method. this can be exemplified by pratensein (8) : the literature assignments of the chemical shifts of the two highly coupled protons h-5 and h-6 in 8 as 6.95 and 6.96 ppm, respectively, resulted in a higher order effect that transformed the h-2 signal into a pseudotriplet instead of a doublet. a side aspect of this observation is that it serves as a reminder of the often forgotten fact that peak separation in h nmr spectra is not identical to coupling constants. the first - order doublet property of the h-2 signal without higher order effect in the rce spectrum was contradictory and indicated that the between h-5 and h-6 must indeed be larger than their coupling constant (> 8 hz). thus, the signal assignments for 8 were refined through iterative optimization (figure 5a / b). however, we speculate that there might be two reasons behind this observation. one is the plausible matrix effect of the extract, and the second could be misreporting (incorrect or inadequate) of the nmr spectroscopic parameters in the literature, as indicated above. chemical shifts and/or line shapes of the signals of pure vs target compounds in the extract can be affected by the temperature, sample matrix, ph, and/or water content. notably, the h nmr spectrum of rce without 3,5-dinitrobenzoic acid (3,5-dnb) added also showed the signal for h-2 in 8 as a doublet, ruling out the possibility of a drastic chemical shift perturbation due to the addition of an internal calibrant (ic). hifsa was capable of resolving the higher order effects observed in the case of calycosin (5, figure 5c / d), as well as achieved assignments of the signals of 5 and 8 and in the spectral region with the most intense overlap. using an algorithm that sets strong probability distribution (prior) on metabolite resonance patterns, the recently developed bayesian sd approach allows automated peak (re)assignment under conditions of sample - dependent resonance shifts (), but by default can not overcome the intrinsic limitations of non - qm - based methods. representative portions of the qm - qhnmr spectra of rce showing the high congruence of the experimental (red) and qm - simulated (blue) spectra. panels a and b show the assignments of the b - ring amx system of the minor isoflavone pratensein (8) before and after the qm - based optimization (iteration), respectively. panels c and d demonstrate the higher order effect observed in the b - ring amx system of calycosin (5) in the pure isoflavone (c) and the signal assignments in the rce (d) (indicates the assignment of h-6 of 5 in the rce). along with the quantitation of individual components, qhnmr also enabled the close approximation of the total isoflavone content of the extract. the presence of a deshielded c - ring h-2 singlet resonance in the 8.20 to 8.55 ppm region is highly characteristic of all isoflavones and leads to the formation of chromatogram - like nine of the ten isoflavone singlets observed in rce could be identified, and seven could be quantified using the qm - based hifsa - qhnmr method. in this way, integration of h-2 resonances in the entire region 4 against the calibrant signals was used to estimate the tifco of the rce. the inter - isoflavone signal overlap was not critical, as region 4 was well isolated from the signals of the other regions in the spectrum (figure 3). if interfering signals were present, a more elaborate sd approach would be necessary. linear sd performed with the total line shape (tls) module of the perch software was used as a second means of estimating the tifco. quantitation was done with reference to the ic signals, and a weighted average of the individual molecular weights of the isoflavones was used to determine the tifco as a total average isoflavone content. using the integration and the tls methods, the tifco in the rce was estimated to be 34.5% and 36.5% w / w, respectively (table 2). given preference to the deconvolution method, this outcome showed that it is feasible to standardize an rce to its tifco, instead of, or in addition to, the individual isoflavone percentages, in a single - step qhnmr analysis. peak picking and deconvolution used the pac and tls modules of the perch software tool, respectively. the weight percentages of isoflavones obtained from the qm - qhnmr analyses were used to calculate the weighted average of the molecular weight of the isoflavones as a whole. the 100% normalization, internal calibrant (ic), and external calibrant (ec) methods are the three established modes of quantitation in qhnmr, differing in the way calibration is achieved. additionally, an ec can be used to calibrate the (residual) solvent signal, which can then be used as an ic for an extended series of qhnmr analyses (ecic method), provided the same batch of solvent is used throughout and the exact volume is known. traditionally, these quantitation modes have employed manual integration of the nmr signals as a quantitative measure. classical integration is most commonly used for the purity evaluation of single chemical entities such as isolated or synthesized compounds and can be used successfully for slightly complex mixtures. however, the classical integration approaches are limited or even inadequate for the analysis of more complex samples such as plant extracts, foods, or biological fluids. this is a result of the discrepancy between the widths of resonances and the chemical shift dispersion required for clean, nonoverlapped signals for integration. the former depends on the natural spectral line width (; 1 hz or slightly below in well - shimmed spectra) and the total width of the individual resonances, which is the sum of all j values plus 2, but notably only as long as first - order assumptions apply. moreover, reliable integration requires relatively wide integration ranges (510) in order to capture the full resonance, depending on the accuracy requirements. another confining factor is the presence of c satellite signals, which limits the width of integral ranges and produces an additional level of signal overlap with minor constituents. collectively, these challenges explain why advancement of quantitative measures is required to make 1d qhnmr fit for the purpose of metabolomic standardization.whereas chemometric approaches employing multivariate analysis can be used for untargeted nmr metabolomics, some form of deconvolution (peak fitting) is needed to achieve a targeted quantitative analysis of known metabolites, but also of unknowns when assuming molecular structures and weights. moreover, generic software tools exist for highly flexible curve fitting, such as fityk (http://fityk.nieto.pl/) and general commercial statistics tools. one of the recent developments in this area geared toward nmr spectra is the bayesian deconvolution and quantitation of metabolites using the automated batman software. this r package enables automated peak assignment and quantitation based on a list of chemical shifts of the metabolites entered by the user. the present study entailed a targeted qhnmr multimarker standardization using two methods : a qm - based approach, which utilized the previously developed h full spin analysis to deconvolute the entire qhnmr spectrum, and a non - qm sd method that used the gsd function of the mestrenova nmr software tool. the qm - based method had previously been termed hifsa - qhnmr, in reference to the use of the hifsa process for sd, and emphasizes the iterative nature of the qm deconvolution process for h nmr spectra. using the same approach for deriving quantitation measures in qhnmr has also been termed quantitative, quantum - mechanics - based spectral analysis (qqmsa). in this serum metabolomics study, qm - based stoichiometry of the target analytes required prior knowledge about the sample via the use of multiterm baseline functions to address extensive background, as well as optimizable and adjustable lines, regular multiplets, or constructs composed of spectral lines to account for unknown signals. however, in order to consolidate the nomenclature of the shared qm aspects, the present study used qm - qhnmr (quantum mechanical quantitative h nmr) to highlight the benefit of using quantum mechanical calculations to derive the quantitative measures, while continuing to refer to hifsa for the actual sd process, involving full spin analysis through iterative calculations. even under very careful experimental conditions, the analysis of qhnmr spectra is limited by a variety of factors such as incomplete signal assignments, extensive signal overlap, and imperfect baselines. these confounding factors are encountered commonly in the spectra of complex natural product samples such as rce. collectively, they influence the choice of the spectral window that is amenable to iteration or integration and, thereby, impact the achievable quantitative results. however, when compared to non - qm deconvolution, qm - based hifsa sd yields a much enhanced resolution of signal overlap by virtue of the qm - based calculation of individual spectral lines. this includes all interdependencies of the individual lines, especially in non - first - order situations, which are very common. for instance, the h-2/6 signals of irilone (7) and prunetin (8) almost perfectly coincided with each other, as well as overlapped with those of the aaxx spin systems of genistein (3) and daidzein (4) (figure 4b). the ability to assess the contribution of each component in such highly overlapped resonances and correctly assign peaks to individual components has to take into consideration the system of all spin particles (i.e., atoms) involved. thus, the resolution of signals where multiple degenerated lines from both the individual and one or several other species overlap inevitably requires a qm approach (figure 1). in contrast, non - qm, linear sd based approaches fail at this level of complexity, leaving the selection of the cleanest region with the least overlap as the only and predictably error - prone option for quantitation. collectively, this explains the intrinsic limitation of non - qm - based deconvolution as a quantitative measure in qhnmr. despite its advantage over classical integration, sd is flawed systematically and, thus, less rigorous for building comprehensive analyses of multiple components in complex mixtures. analogous to chromophores in uv- and molecular / fragment masses in ms - detected quantitation, qm is key to the definition of the spin parameters of the target compounds and ultimately guides the prerequisites of qm - qhnmr analyses. additionally, non - qm, peak - fitted lines of unknown components can be added to improve the overall fit, their quantitation requires the assignment of partial structures and amu values. while any assumptions made in this process become part of the systematic error of the initial quantitation, additional knowledge gained afterward can be used to eliminate such errors due to the inherent calibration characteristics of qhnmr. qm - qhnmr does not face this limitation for the development of multimarker standardization approaches. using the hifsa deconvolution mechanism, in contrast, other non - qm sd methods are nmr - blind and limited to modeling certain peak shape assumptions such as lorentzian or gaussian line shapes. as couplings and higher order effects involve multiple individual spins, non - first - order effects encode spectral information into the nmr resonances of coupled spin particles in other regions of the spectrum that might be subject to less overlap. as qm assures the integrity of these interdependencies, the degree of freedom and the number of iterative solutions that are consistent with the qm rules are reduced considerably, because only chemical shifts and coupling constants need to be optimized rather than frequencies and intensities for each single line. thus, an important thing to note is that application of any qm method does require at least partial characterization of an analyte of interest in order to enable the use of accurately predicted and simulated spin parameters. while fully defined spin systems of target compounds with known molecular weight are desirable, the qm definition of partial spin systems (spin cages ; e.g., sugars, amino acids) still enables targeted quantification of full or partial structures. qm - qhnmr utilizes the quantum mechanically simulated spectra, which represent the most accurate achievable replicas of the experimental spectra of the target analytes and can be derived from predicted initial spin parameters via an iterative qm calculation. importantly, because it employs the same mechanisms as hifsa profiling does for pure compounds, qm - qnmr performs a qm - based sd of all spectral lines, not just of the (visible) peaks, of a given species in a mixture. considering that individual peaks often consist of more than one line and that multiple peaks constitute the composite h signals (i.e., resonances) of a given proton, this has far reaching consequences for both the qualitative and quantitative interpretation of hnmr spectra : the distinction of lines and consideration of their intensities is fundamental to the accurate interpretation of all hnmr signals. the quantum mechanical description of a spin system correlates frequencies and intensities of the corresponding lines not only for each spin particle but via spin these constraints dramatically reduce the number of variables and allow the complete sd of even completely overlapping lines also in the presence of higher order effects. in the relatively rare case of a pure first - order spin system these constraints degenerate to the simple first - order rules. in practice, significant proportions of the intensity of hnmr signals can reside outside the peak range assigned to the respective proton (regardless of its assigned multiplicity), explaining at least in part the widely experienced relative weakness of int - qhnmr. taking into account the observed and/or achievable natural line width and signal - to - noise of the experimental spectra, it becomes evident that these effects can add considerable error to the accuracy and precision of sd methods in pf - qhnmr and, even more so, to classical int - qhnmr. in contrast, qm - based sd is less limited when analyzing the same experimental data. due to the ability to correlate spin particles through the underlying qm rules, the intensity information on individual lines is encoded in the intensity of other lines as well. as a result, qm - qhnmr has the ability to automatically exclude line(s) (intensity) that can not be explained by the given spin system(s) of the target analyte(s) from the quantification. in other words, qm - based sd methods have superior accuracy by virtue of their inherent capability to account only for the intensity of those lines (consequently also peaks and signals) that truly belong to the analyte. in addition to its higher intrinsic accuracy, the hifsa deconvolution process imparted in qm - qhnmr enabled a thorough qualitative analysis of the spectra and validated the identity of the target compounds simultaneously with their quantitation. once a library of hifsa spin parameter sets has been created for the individual compounds, a multitarget quantitation can be performed for any mixture containing these components. hifsa has been shown to be fit for the purpose of obtaining the exact chemical shifts up to ppb level accuracy, as well as coupling constants with 10 mhz precision. this level of detail is particularly critical in the case of regio-/stereoisomers, higher order spectra, and when determining small coupling constants that can not readily be detected visually. especially when considered as precise relative values (and), this helps in structural dereplication, confirmation, and differentiation of closely related analogues. practical considerations of hifsa processing involved the use of the integral optimization module (d - mode, coarse adjustment) of the perch iterator, which helped with the optimization of the chemical shifts, coupling constants, and signal intensities, thereby optimizing the relative ratios of the target components. the total line shape module (t - mode, fine adjustment) was used subsequently to refine the simulation of signal overlaps, deconvolute fine line shapes, and achieve the best overall match of the line characteristics of the spectrum. in this study, the iterations were performed until the observed and calculated spectrum were visually identical as indicated by a rms value of 0.1. as the j coupling constants are largely unaffected by extrinsic interactions in the sample, they were kept fixed during the hifsa iterative processes, typically after performing an initial optimization. also, a fit and lock method was utilized for certain spin parameters of the more problematic regions of the spectrum. alternatively, in principle it is possible to mask and, thereby, exclude a certain region that still can not be assigned reliably due to excessive overlap or unknown impurities, in order to achieve a converging iteration for the rest of the spectrum. in this scenario, hifsa sd retains its qm advantage via conservation of the underlying spin parameters, as long as the spin particles belonging to the molecule present in the overlapped region are also present elsewhere in the spectrum. additionally, overlap with unknown components can be mimicked by adding simulated (non - qm) lines that represent these unknowns, improving the overall fit and giving lower global rms values. a local rms value of 0.030 for the most relevant region of interest (7.6 to 9.0 ppm) indicated an excellent match between the calculated and experimental spectrum (figure 6). the percent w / w content of the isoflavones biochanin a (1), formononetin (2), genistein (3), daidzein (4), calycosin (5), prunetin (6), irilone (7), and the flavonol quercetin (10) in the rce was determined as 15.2, 16.0, 0.64, 0.40, 0.53, 0.71, 2.21, and 1.43, respectively, using the qm - qhnmr method (table 1). observed (top / blue), residual (middle / green), and calculated (bottom / red) qhnmr spectra of rce resulting from the qm - qhnmr analysis. the residual represents an rms value of 0.031 for the 7.44 to 9.00 ppm region. the more complex part of region 3 (7.05 to 6.79 ppm the final iteration was performed on the marked region, followed by simulation of the entire spectrum. the alternative quantitative measure for multitarget qhnmr explored in this study involved the non - qm sd of the relevant portions of the spectrum. the global spectral deconvolution (gsd) module of the mestrenova software tool was used for both automated peak picking and sd. gsd was primarily developed to improve the quality of an nmr spectrum by deconvoluting and performing deliberate subtraction of solvent and/or impurity peaks from the spectrum. unlike the hifsa - based qm - qhnmr approach, the initial time investment for gsd spectral processing is small, due to automation and the yield of acceptable deconvolution results. however, it must be kept in mind that gsd does not establish linkages between structural and spectroscopic features, nor can it verify any assignment and/or the recognition of (hidden) lines. this also brings up the need to distinguish qm - based lines (resonance frequencies) from experimentally distinguishable peaks (including peak shoulders). in the case of higher (non - first) order effects or excessive overlap, and due to its non - qm nature, gsd can not assign peaks and/or achieve resolution of peak overlap, leaving the choice of a simpler, cleaner region of the spectrum as the only option for accurate analysis. based on these considerations, a region inclusive of the ic signals and the c - ring h-2 singlets was used for gsd - assisted quantitation. after deconvolution, the resulting spectrum consisted of the original signal, the deconvoluted lines, the sum / fit peaks, and a residual (figure 2). the resulting percent w / w contents of each isoflavone, thus determined, were 14.5, 15.2, 0.64, 0.35, 0.53, 0.71, 2.13, and 1.40 for biochanin a, formononetin, genistein, daidzein, calycosin, prunetin, irilone, and quercetin, respectively (table 1 ; table s4). these values are relatively close to those obtained with hifsa - qhnmr, but show more substantial differences when compared to the published hplc quantitation results, despite the similar tifco. table 1 compares the quantitation results obtained by qm - qhnmr, non - qm qhnmr using gsd, and the newly developed uhplc - uv analysis with the 2009 hplc results. the goal of achieving a more comprehensive, metabolomic description of the sample calls for the most inclusive quantitative measure, which currently is offered by qm - qhnmr. although the initial data analysis and preparation of the hifsa profile (including all the relevant nmr parameters) can require significant effort for complex samples, the established profile can be readily transferred within and between laboratories. the generated spin parameter files of the individual compounds can serve as libraries for future analysis of mixtures containing any or all of these compounds. as the qm basis of nmr ensures their validity across the various magnetic field strengths, moreover, it can potentially be adopted for industrial quality control applications, e.g., to monitor batch - to - batch variations of known samples. more broadly, the present study showed that qm - qhnmr can be a valuable tool for the analyses of complex natural product matrices, including but not limited to botanical extracts. one of the continuing challenges that are inherent to qnmr and affect qm - qhnmr as well is baseline imperfections. while they can represent measurement artifacts, they can also be real and due to signal overlapping of a myriad of minor constituents that fall well below the limit of detection (lod), particularly in metabolomic samples. while masking of problematic regions from the iterations can improve the iteration and pf of resonances assigned to components that are identified by other means, these approaches may not be feasible with every sample and/or may require major additional analytical effort, at least for an exemplary sample. furthermore, as iterators typically give more statistical weight to major signals, optimization of the line characteristics of signals from minor components is often limited when samples exhibit a very broad dynamic range. certain weighting functions such as the intensity weight parameter may be used to give higher significance to minor components during iterations. in the experience of the authors, careful selection of processing and quantitation measure in sd - based qnmr the fundamental qm theory that underlies the observation of nmr infers that qm - based spectral deconvolution and quantitation like qm - qhnmr has a greater intrinsic accuracy compared to non - qm methods such as gsd (figure 1). the importance and achievable level of accuracy of a method depends on the intended application and the type of sample, respectively. for example, in the case of a pharmaceutical formulation that contains a specific active ingredient, inert excipients, and a well - defined pharmacological target, high levels of quantitative accuracy and precision are desired and justified, as both are intertwined with the efficacy and potency of the material. this situation differs for complex plant extracts : large numbers and dynamic range of the phytochemicals, including multiple unknowns, are natural limits of achievable accuracy. under such circumstances, the simultaneous identification and quantitation of as many components as possible, with a reasonable overall accuracy, becomes the primary goal. accuracy of both the qm - qhnmr and gsd methods was determined by spiking the ic - containing rce sample with a known amount of genistein (3). the spectra thus acquired were subjected to quantitative processing, and the amount of 3 before and after spiking was determined using these methods as described further in the experimental section. the accuracy of the qm - qhnmr and gsd methods for the multimarker quantitation of rce was determined to be associated with 2.3% and 6.5% relative errors, respectively (s5 and table s7). this outcome demonstrated that the qm - qhnmr approach holds its theoretical promise of higher accuracy in 1d qhnmr analysis. a 97% recovery associated with the quantitation accuracy for the spiked ge a comprehensive qhnmr study had reported previously that an s / n ratio of 150 leads to more accurate qhnmr results. this suggests that all components with abundance levels of > 0.9% w / w (s / n = 206 ; see also the supporting information) in the rce were quantified with high accuracy. a new, 10 min, uhplc method was developed for the quantitation of the four bioactive isoflavones biochanin a (1), formononetin (2), genistein (3), and daidzein (4). their percent w / w content was determined to be 13.0, 16.0, 0.43, and 0.25, respectively. compared to the qhnmr results, the differences between the quantities of the major isoflavones 1 and 2 could result from the observed different solubilities of the reference materials that likely affected the standard solutions for lc calibration and led to a slight underestimation of the quantities. the exploration of polymorphism and other batch - to - batch variations of reference standards was beyond the scope of the present qnmr study. as 1 exhibited lower solubility in meoh compared to 2, extended sonication with mild heating was required to solubilize 1. however, the uhplc - uv showed a general congruence with the lc - uv / ms methods previously established for this sample and served as an orthogonal approach to the current study. over a period of 10 years, the clinical rce had been stored in a sealed container at 20 c. the orthogonal analyses used in the present study confirmed its stability over the prolonged storage. a uhplc - uv profile (figure 7) and quantitation showed that 1 and 2 were found consistently to be the major constituents, whereas 3 and 4 were present as minor components. this was confirmed by the two qhnmr methods, and the present outcome was consistent with the original hplc standardization results. despite the 10-year consistency of the findings, it is important to realize that repetition of any lc - based analysis requires revalidation due to its dependence on numerous instrument - related factors, particularly those related to (non)linearity of the detectors (uv, ms), the injector system, and the characteristics of the columns. this does not apply to qhnmr methods : once established, they can be rerun anytime later, provided the nmr instrument is validated per se, and the qhnmr acquisition parameters are identical or demonstrated to be congruent between the different hardware and magnetic fields used. another unique advantage of qhnmr is that additional analytes can be included even years later when peak assignments have become available, to study the same sample or the original fid of the qhnmr measurement. the data can be used indefinitely not only for qualitative comparison but also for full - fledged quantitative measurements, provided ic is performed or ec / ecic calibration data are archived concurrently. in the present study, both qhnmr methods enabled the parallel quantitation of eight marker compounds : seven isoflavones, as well as quercetin. from a practical perspective, qhnmr proved to be the more time- and labor - efficient method in the long term and for large sample sets. the observed differences in the percentages of the markers determined using the three different methods are tolerable and can be attributed to the different intrinsic properties of the orthogonal analytical methods. as observed differences were within the combined error parameters of the methods, chemical degradation or interconversion being the cause of these variances seems unlikely. accordingly, the clinical rce was concluded to be phytochemically stable over the 10-year period and can be used confidently for future biological evaluations. the study identified quantum - mechanics - driven quantitative h nmr (qm - qhnmr) as currently the most capable and flexible method to achieve the targeted multimarker standardization of a complex botanical extract. using a 10-year - old clinical rce as study material, qhnmr allowed not only the quantitation of a total of nine biologically relevant and two additional markers but also the determination of the total isoflavone content. the tifco represents a unique reference point for botanical standardization, as it captures the entire group of bioactive isoflavones and their clinical relevance. the ability of the hifsa sd process to account for peak overlap and higher order effects of the target markers led to what can be considered the most accurate signal assignment and quantitative measurement currently achievable in qhnmr by available established methods. the calculated recovery rate following spiking with genistein (ge) (figure s5 and table s6) in rce was found to be 97.0%, demonstrating that the developed qm - qhnmr method is reasonably accurate and fit for the intended purpose of multimarker quantitation. additionally, considering the complexity of the rce h nmr spectrum, the extensive signal overlap, the large dynamic range of the major / minor components, the unavoidable presence of multiple unknowns, and the general limitations of reasonable experimental effort, the 2.3% relative error in quantitation observed for the qm - qhnmr method can be considered highly acceptable for the intended purpose of botanical standardization. comparing the two studied sd methods used to derive the quantitation measures, qm - based hifsa vs non - qm - based gsd, the qm - qhnmr method exhibited a significantly lower percent relative quantitation error. this is in line with the theoretically predictable higher accuracy of qm - based approaches to h nmr analysis. a particularly notable strength of qm - qhnmr lies in the wealth of qualitative information that can be obtained in conjunction with the quantitative data. due to its qm nature therefore, qm - qhnmr is a bivalent standardization method, which combines the identification of multiple marker compounds with their simultaneous quantitation (i.e., normalization), all in a single analysis. on a general note, (q)nmr analysis enables a highly efficient use of the information produced when made available in the scientific literature in its original form. accordingly, the fids underlying the present work are made available for future use. finally, the orthogonal uhplc - uv and qhnmr analyses confirmed the stability of rce over a period of 10 years. good overall consistency with previous standardization data was observed, and the extract was hence deemed fit for further biological studies. deuterated dimethyl sulfoxide (dmso - d6, 99.9% d) was obtained from cambridge isotope laboratories inc. biochanin a, daidzein, formononetin, and genistein were purchased from indofine chemical company inc. the internal calibrant, 3,5-dinitrobenzoic acid, was purchased from fluka analytical (buchs, switzerland). a gastight (1 ml) syringe, purchased from pressure - lok, precision sampling (baton rouge, la, usa), was used for volumetric nmr sample preparation. the qhnmr experiments were performed on a bruker (karlsruhe, germany) avance 600 nmr spectrometer equipped with a 5 mm txi cryoprobe. nmr data were analyzed and processed with mnova 10.0.2 software from mestrelab research s. l. (santiago de compostela, spain). (kuopio, finland) was used for all qm - based nmr spectroscopic analysis including iteration, simulation, and hifsa, as described previously. a shimadzu (kyoto, japan) nexera uhplc system equipped with a dad detector was used for the uhplc analysis of the extract. quantitation was performed on a kinetex 1.7 m xb - c18 100 column (50 mm 2.1 mm, s / n : 619546 - 18). the red clover extract prepared previously for clinical trials (as documented previously) was used in this study. the qhnmr spectrum of the rce was acquired using standard qhnmr conditions, which included a relaxation delay (d1) of 60 s, a calibrated 90 pulse (p1), and tuning and matching of the probe immediately preceding acquisition. for sample preparation, rce and the ic (3,5-dnb) were weighed accurately as 8.05 mg and 1.39 mg, respectively, into one glass vial and dissolved in a total of 200 l of dmso - d6. the solution containing the extract and the calibrant was transferred into a 3 mm nmr tube using 200 l drummond calibrated pipets. the qhnmr experiment consisted of 16 scans acquired with an rg (receiver gain) value of 64 and an acquisition time of 5.98 s. postacquisition processing included zero - filling of the 256k fid to 512k real data, a mild lorentzian gaussian window function (exponential factor 0.3, gaussian factor 0.05 in gf mode), and baseline correction (fifth - order polynomial). the residual dmso - d5 signal at 2.500 ppm was used for chemical shift referencing. the nmr spectrum was exported as a jdx (jcamp) file for qm - qhnmr processing. spectra of the pure isoflavones either had been acquired previously or were obtained from the metidb database. spiking experiments were performed for the minor components, genistein (3), daidzein (4), calycosin (5), and prunetin (6), in order to assign their nmr signals unambiguously. first, h iterative full spin analysis using the perch nmr software tool was performed for all isoflavones (s1, supporting information) identified in the extract, except for the isoflavones 79. the analysis involved initial prediction and subsequent iterative refinement of the spin parameters until the simulated spectrum matched the experimental spectrum. as a result, all chemical shifts, coupling constants, and individual line widths (, j, and w1/2) were determined for each compound, compiled into a spin parameter set (pms) file, one for each individual compound. once hifsa profiles for all the target components had been generated, the parameter sets for the individual compounds were copied and pasted to produce a combined pms file that will be utilized for the analysis of the rce (s2, supporting information). in this combined pms file the coupling constants previously determined for each individual compound were kept fixed (unalterable) before starting the iteration process against the original h nmr spectrum of the rce. a simulated spectrum of the combined pms file was then iterated against the experimental rce to produce a fully matched calculated qhnmr spectrum. the iterations were performed until a close visual spectral match, also determined by an rms value 0.1, was observed between the experimental and the calculated spectrum. along with the refined spin parameters for the mixture, the process generated relative populations (as % mol / mol) of the individual species in the extract. considering the exact weights of the extract and the ic, the absolute quantities of each individual component could be calculated using these populations (scheme 1). the resulting pms file contains all spin parameters (, j, and) and the individual populations of the target compounds (highlighted in red) ; t, target analyte, ic, internal calibrant ; w, weight ; m, molecular weight ; p, purity ; po, population. after postacquisition processing of the qhnmr spectrum, sd was performed for region 4 (7.69.00 ppm) containing the signals of the ic, h-2 singlets of all isoflavones, and the b - ring h-2 doublet of quercetin (10) (figure 2). the sd process was initiated by performing a peak picking of the entire spectrum using the global sd function of the mestrenova software tool with five fitting cycles. subsequently, the deconvoluted lines and their peak areas were assigned to the h-2 singlet resonances of the individual isoflavones. similarly, for composite resonances (doublets, triplets, etc.), the total area of their individual deconvoluted peaks additively constituted the area of the resonance, e.g., by adding the two individual peak areas of a doublet. alternatively, or in addition to the automated gsd - driven method, peak picking can be performed manually, followed by individual peak fitting and using the edit fit this generates a peak table, which contains the areas of the deconvoluted signals as qhnmr quantitation measures (s4, supporting information). the absolute weights of the target analytes can then be calculated using the ic method. accuracy was determined using the spike - recovery method. for the spiking solution, 5.10 mg of genistein (3) was accurately weighed into a glass vial and dissolved in 100.0 l of dmso - d6. three rce samples containing a known amount of 3,5-dnb (internal calibrant) were quantified by both the qm - qhnmr and gsd methods as presented above, both before and after spiking the samples with a 1.00 l aliquot of the genistein (3) standard solution amounting to 0.051 mg of 3. a hamilton gastight gc manual syringe (1% accuracy) was used for measuring the spiking solution. the hifsa profiles of the three samples used for accuracy determination are presented in s5 (supporting information) in the perch.pms text file format. from the percent w / w ge content of the three spiked samples, the percent relative error was calculated as follows. first, the amount of ge in each unspiked sample was determined by the qm (hifsa)-qhnmr method (table 1). on the basis of the known amount of ge in the spiked sample, the true value for each sample was determined as the sum of percent w / w ge in the unspiked sample and the spiked amount, 0.0510 mg (table s6, supporting information). the percent recovery was calculated using the following formula : the lod and loq thresholds were determined using the signal - to - noise ratio (s / n) method (table s7, supporting information). in order to keep the matrix effect consistent, another calibrant, caffeine, was added to both the rce and the 3,5-dnb - containing samples at seven concentrations ranging from 0.030% to 1.90% w / w. according to the ich guidelines, the lod at an s / n of 3:1 and loq at the s / n of 10:1 is commonly used in lc - based applications. a ratio of loq = 3.3 lod was used for the present study according to a previous qhnmr validation report. the s / n ratio calculator function in mestrenova was used to determine the s / n of individual signals. the methine proton singlet of caffeine at 7.98 ppm was used for the s / n determination, as it shares the h relative integral value (1.000) with those of the target signals and falls into a relatively flat baseline region compared to the range of the upfield methyl singlets. the mobile phase gradient used was as follows : 2026% b in 0.5 min, 2631.3% b at 6.5 min, held isocratic at 31.3% b up to 6.7 min ; re - equilibration from 31.320% b at 7 min ; and reconditioning at 20% b up to 10 min. the flow rate was 0.6 ml / min, and the injection volume was 1.0 l. the column oven, detector cell, and autosampler temperatures were maintained at 40, 40, and 4 c, respectively, throughout the analysis. uhplc - uv quantitation targeted the following four markers : biochanin a (1), formononetin (2), genistein (3), and daidzein (4). calibration curves with nine concentrations ranging from 1.00 to 500 g / ml and 0.25 to 125 g / ml were generated for the major (1 and 2) and minor (3 and 4) isoflavones, respectively (table s8, supporting information). all samples including the calibrants and the extract were run in triplicate with a blank injection in between each triplicate set. stock solutions of 2 (in meoh), 1, 3, and 4 (in etoh) were prepared at 0.50, 0.50, 0.125, and 0.125 mg / ml, respectively. uhplc - uv chromatogram (254 nm) of the red clover extract, showing the quantified major bioactive isoflavones 14.	chemical standardization, along with morphological and dna analysis ensures the authenticity and advances the integrity evaluation of botanical preparations. achievement of a more comprehensive, metabolomic standardization requires simultaneous quantitation of multiple marker compounds. employing quantitative 1h nmr (qhnmr), this study determined the total isoflavone content (tifco ; 34.536.5% w / w) via multimarker standardization and assessed the stability of a 10-year - old isoflavone - enriched red clover extract (rce). eleven markers (nine isoflavones, two flavonols) were targeted simultaneously, and outcomes were compared with lc - based standardization. two advanced quantitative measures in qhnmr were applied to derive quantities from complex and/or overlapping resonances : a quantum mechanical (qm) method (qm - qhnmr) that employs 1h iterative full spin analysis, and a non - qm method that uses linear peak fitting algorithms (pf - qhnmr). a 10 min uhplc - uv method provided auxiliary orthogonal quantitation. this is the first systematic evaluation of qm and non - qm deconvolution as qhnmr quantitation measures. it demonstrates that qm - qhnmr can account successfully for the complexity of 1h nmr spectra of individual analytes and how qm - qhnmr can be built for mixtures such as botanical extracts. the contents of the main bioactive markers were in good agreement with earlier hplc - uv results, demonstrating the chemical stability of the rce. qm - qhnmr advances chemical standardization by its inherent qm accuracy and the use of universal calibrants, avoiding the impractical need for identical reference materials.
natural orifice transluminal endoscopic surgery (notes) has recently generated significant interest amongst surgeons and gastroenterologists. pushing minimally invasive surgery a step forward, the concept of incisionless surgery is appealing to patients and physicians alike. accessing the abdominal cavity and its organs via natural orifices, such as the mouth, anus, vagina, and urethra, may enable surgeons in the future to approach operations they traditionally performed in open and laparoscopic fashions. the potential benefits of such techniques include less physiologic stress and trauma ; faster recovery ; less pain ; fewer complications, such as intestinal adhesions and hernia ; better cosmesis ; and decreased healthcare cost by decreasing the rate of hospitalization. several operations including cholecystectomy, splenectomy, appendectomy, gastrojejunostomy, and oophorectomy have been performed. however, little data are available in humans. a 61-year - old woman with ulcerative colitis refractory to medical therapy presented to our department following numerous admissions for anemia requiring blood transfusions. she had a history of deep venous thrombosis and pulmonary embolism status post placement of an inferior vena cava filter, cerebrovascular disease with left hemiplegia, and coronary artery disease. she underwent an uneventful proctocolectomy with end ileostomy. on postoperative day 1, the patient developed phlegmasia cerulea dolens, and an mri revealed thrombosis of her inferior vena cava and iliac veins. transanal drainage through the anal cuff was performed at the bedside with irrigation of the lower pelvis with a mushroom catheter, and antibiotic therapy was instituted. the patient remained septic and 2 days later underwent placement of a percutaneous drain by interventional radiology. the drain was ineffective due to the organized nature of the infected hematoma with multiple septations and phlegmonous reaction. re - exploration and drainage of the abdomen was entertained, but due to the frailty of the patient, a transanal endoscopic drainage was performed on postoperative day 17 in the endoscopy suite. room air under pressure was used to insufflate the abdominopelvic cavity for visualization. a large, multiseptated and organized intraperitoneal hematoma was encountered with the pigtail drain embedded in a fibrinous collection (figure 2). following mechanical and hydrogen peroxide fragmentation, the hematoma was retrieved with suction, forceps, and baskets, and the pelvis and lower abdomen were cleared (figure 3). ct scan images were used to guide the depth of intervention to minimize any injury to small bowel. two 19 french round blake drains were introduced via the anal opening and guided into the abdomen with the use of an endoscopic snare. the patient 's clinical status rapidly improved, and her white count normalized within 2 days. the evolution of minimally invasive surgery over the last 2 decades has redefined the practice of surgery. driven by technological advances and supporting clinical data, the implementation of these new techniques has been remarkable and has stimulated inquiry into future directions. undoubtedly, most of the initial human data will come from case reports and short series attesting to the feasibility, safety, and limitations of notes. rao and colleagues from india have already demonstrated that appendectomy via a transgastric route is feasible in humans. although transanal drainage of pelvic sepsis (the bedside drainage we performed in our patient as initial intervention) is a known procedure in the surgical armamentarium, this report illustrates the utilization of flexible endoscopy to reach a higher location in the abdominopelvic cavity and to perform more extensive drainage of a loculated infected hematoma. none of these interventions were successful due to the location, extent, and nature of the organized hematoma, and the overall debilitated state of the patient. although commonly used, percutaneous drainage is not always successful at controlling abdominal sepsis. in our case, re - exploratory laparotomy was the only remaining option to evacuate the patient 's infected hematoma. considering her clinical status, such an intervention would have carried significant morbidity. with the patient 's full consent, obviously in this case, the rectum was surgically missing so access to the peritoneal cavity was unhindered, and it was not necessary to close the anorectal stump. had the rectum been present, a transanal proctotomy would have been necessary to gain access to the abdomen. but this case illustrates that it is technically feasible to endoscopically tackle the postoperative abdomen, fragment, retrieve, and drain infected hematoma with current equipment. the equipment and endoscopic expertise to perform such a task in a routine, reliable, and safe fashion are currently limited but growing. in addition, several endoluminal methods and devices are being developed to gain entry access into the peritoneal cavity through the digestive tract. notes is a field in its infancy, and whether it will gain widespread acceptance and application is yet to be determined. although most advances in this field will be driven by animal experimentation, some may result from challenging situations such as our case that may provide opportunities to push the boundaries of our current surgical practices.	natural orifice transluminal endoscopic surgery (notes) is an evolving experimental field exploring the technical feasibility and outcome of therapeutic interventions performed through the natural orifices of the body. the knowledge accumulating in notes is the result of animal experimentation and ongoing early clinical experience in humans. in this report we describe a patient treated with transanal endoscopic drainage of postoperative abdominopelvic sepsis.
multiple randomized trials over the last four decades have established the equivalence of mastectomy and breast - conserving therapy (bct) in early stage breast cancer. more recently, accelerated partial breast irradiation (apbi) has become an acceptable means of delivering adjuvant radiation in early stage breast cancer patients who meet certain favorable criteria [2, 3 ]. the benefits of apbi include shorter treatment times, sparing of a larger amount of normal tissue, and focal irradiation of the area highest at risk of recurrence around the tumor bed. initially, apbi consisted of interstitial catheter placement either via a template based approach or ultrasound guided free - hand approach but these both required extensive expertise in their placement. after approval of mammosite (hologic inc, bedford, ma, usa) in 2002 and creation of newer multi - lumen devices such as contura (hologic inc, bedford, ma, usa) and savi (cianna medical, aliso viejo, ca, usa), the rates of utilization of brachytherapy - based apbi (b - apbi) have continued to rise and now approach 11% in patients older than 50 years old who have undergone bct. multiple consensus guidelines have been developed to specify the appropriate candidates for b - apbi [7, 8, 9, 10 ]. however, none of these address the role or feasibility of b - apbi in patients with existing breast implants who wish to undergo bct. given that now nearly 286,000 women undergo breast augmentation annually, an increase of 35% from 2000 to 2012, the rates of patients with pre - existing implants who will be diagnosed with breast cancer will continue to rise. therefore, it is imperative to understand the clinical implications of b - apbi in this patient population. the reported experience with b - apbi in the setting of breast augmentation is limited [12, 13 ], with no known specific dose - constraints for breast implants to date. we had also previously reported the long - term outcome and dosimetric considerations in a patient with pre - existing breast implants who was treated with b - apbi. the present study expands on our work by examining the outcome and dosimetric evaluation of all the patients with breast augmentation who have been treated in our institution with b - apbi. to our knowledge, this is the first such reported series with early clinical outcomes in this patient population and detailed examination of the dosimetric parameters of breast implants in b - apbi. after obtaining appropriate institutional review board approval, we examined the database of patients who had undergone b - apbi at our institution from 2007 to 2013 and identified seven patients with existing breast implants at the time of surgery and throughout treatment. all patients had undergone lumpectomy prior to radiation treatment. due to the complexity of device placement adjacent to the breast implants, the breast surgeons placed the devices. each patient was treated using either a contura or savi device, and all of the devices were placed via a closed cavity approach. the planning computed tomography (ct) simulation was performed 48 - 72 hours following device placement in our department (brilliance big bore, philips healthcare, andover, ma). all patients were simulated in the supine position, with the arm on the affected side raised over their head. a small amount of contrast (0.5 cc) was added to the saline mixture filling the balloon for contura patients to improve visualization of the balloon on ct. given the variations in multi - lumen or catheter based brachytherapy, the integrity, orientation and size of the device, as well as distance to skin and rib were all verified prior to treatment planning before each treatment. treatment planning was completed using brachyvision treatment planning system (varian, palo alto, ca, usa). the planning target volume for evaluation (ptv_eval) consisted of 1 cm of tissue surrounding the device but limited to a distance of 5 mm from the skin for the contura patients, 3 mm for the savi patients, and excluding the chest wall. we also attempted to minimize the v150 of normal breast tissue (volume receiving 150% of prescribed dose) to < 50 cc and v200 < 10 cc. the volume of air and seroma within the ptv_eval was less than 10% and this was also verified prior to each treatment. at the time of treatment planning, since no dose constraints were known for breast implants, no special considerations were made to exclude dose or ptv_eval from the implants. a total dose of 34 gy was prescribed to ptv_eval in 3.4 gy twice daily treatments using a high - dose - rate ir source. prior to each treatment, ct - based image guidance was used to verify the device size, position, and distance to skin. the volume of the breast implants was contoured retrospectively and dosimetric analysis were conducted based on the initial treatment plan on brachyvision. at the time of follow - up, harvard breast cosmesis scale was used to evaluate cosmetic outcome, which rates the outcome from excellent to poor depending on the difference compared to the untreated breast. follow - up pictures were obtained for visual comparison to the contralateral breast and the shape of the treated breast prior to treatment. all of the patients had undergone lumpectomy with the final pathology revealing invasive ductal carcinoma (idc), ductal carcinoma in situ (dcis), or both. the clinical stages ranged from tisn0m0 to t1cn0m0 and all patients with invasive disease had undergone sentinel lymph node biopsy. clinical characteristics and outcomes of the seven patients treated with brachytherapy - based accelerated partial breast irradiation (b - apbi) with existing breast implants idc invasive ductal carcinoma, dcis ductal carcinoma in situ, ajcc american joint committee on cancer, 7 edition. cosmesis : based on the harvard / nsabp / rtog cosmesis grading scale ; late toxicity based on rtog late morbidity scoring schema ; tac docetaxel, doxorubicin, cyclophosphamide at the time of last follow - up, all patients remained free of recurrence. figure 1 shows an axial image of each patient 's isodose lines along with their corresponding cosmetic outcome. five patients had a harvard breast cosmesis score of 1 or excellent, one patient had a score of 2 or good for difference in shape or size, and one had a score of 3 or fair for obvious difference in the size and shape of the treated breast. based on the rtog late radiation morbidity schema, three patients had a score of 0, three had a score of 1 for presence of telangiectasia, hyperpigmentation or erythema, and one had a score of 2 for tender and bright erythema with moderate edema. left column shows a representative cross - section of their respective isodose lines along with the device location. red line represents 340 cgy (100%), white line 323 cgy (95%), green line 306 cgy (90%), blue line 272 cgy (80%). images do not necessarily correlate with the last follow - up date but represent the latest available images from each patient one of the patients had to undergo replacement of bilateral breast implants due to age - related leakage in bilateral breasts. another patient with cosmetic outcome of 3 experienced intra and extracapsular rupture of the treated implant almost 1.5 years after completion of apbi as evidenced by breast mri. an ultrasound of the breasts about 6 months after completion of her treatment had previously shown intact implants, therefore ruling out implant rupture due to apbi device placement. she also developed a cystic fluid collection close to her implant, which was aspirated and found to be benign. the remainder of the implants remained intact through the duration of the study. at last follow - up, all patients expressed satisfaction with their cosmetic outcomes and graded the overall appearance of the treated breast as excellent or good. the average device to breast implant surface in our population was 1.2 mm and the average device to skin surface was 4.4 mm (range 2 - 7.6 mm). the mean volume of the ptv_eval amongst our patients was 55.9 cc and an air / seroma volume of 2.9 cc. the mean percentage of the ptv_eval volume receiving 90%, 95%, and 100% of the prescribed dose (v90, v95, v100) was 97.7%, 95.9%, and 93.1%, respectively. the maximum skin dose ranged from 95.6% to 119% and the maximum rib dose 14.2% to 113.4% of the prescribed dose. the average volume of the normal breast tissue receiving 150% of the prescribed dose (v150) and v200 was 20.1 cc and 9.4 cc, respectively. the average total breast implant volume was 279.3 cc and received an average mean dose of 12.1 gy, minimum dose of 1.9 gy, and a maximum dose of 234.1 gy. the percentage of breast implant volumes receiving 50%, 75%, 100%, 150%, and 200% of the prescribed dose (implant v50, v75, v100, v150, v200) was 15.6%, 7.03%, 4.6%, 1.58%, and 0.46%, respectively. dosimetric characteristics of the seven patients treated with brachytherapy - based accelerated partial breast irradiation (b - apbi) with existing breast implants v50, v75, v100, v150, v200 represent the percentage of breast implant volumes receiving 50%, 75%, 100%, 150%, and 200% of the prescribed dose. all maximum doses were point doses to less than 1 cc of breast implant volume table 3 shows the relative dose to the breast implant along with absolute volume of the implant receiving the same dose represented as v50, etc. the two patients who did not have an excellent cosmetic outcome (# 2 and # 7) also had the highest absolute volume of their implants receiving 50% of the prescribed dose as seen in figure 2 (v50 to 72.9 and 111.9 cc, respectively). the higher treated volume was also apparent across the increasing dose ranges, although not as pronounced. patient # 7, who has now experienced implant rupture, also had the highest maximum dose to the implant (535.1 gy) and the highest mean dose (23.1 gy). however, patient # 2 's mean and maximum dose to the implant was closer to the average of the population (15.4 gy and 72.8 gy, respectively). outcomes based on the harvard scale, 1 excellent, 2 good, 3 v50 (cc) represents the absolute volume of breast implant that received 50% of the prescribed dose. all patients with excellent cosmetic outcome had v50 (cc) of less than 50 correlation of absolute and relative dose to existing breast implants and correlation with cosmetic outcome v50, v75, v100, v150, v200 represent the percentage of breast implant volumes receiving 50%, 75%, 100%, 150%, and 200% of the prescribed dose. v50, v75, v100, v150, and v200 represent the absolute volume of the breast implant receiving 50%, 75%, 100%, 150%, and 200% of the prescribed dose, respectively with the advent of b - apbi and its shorter treatment duration, an increasing number of women with breast cancer have access to adjuvant radiation therapy. per multiple national and international guidelines, b - apbi is now considered a standard of care option in women with early stage breast cancer [2, 3, 7, 8, 9, 10 ]. although there are an increasing number of reports about the safety and efficacy of b - apbi, the appropriate patient population remains a contentious issue since only one randomized study has compared whole breast irradiation to b - apbi [16, 17 ]. furthermore, data regarding treatment of patients with existing breast implants using b - apbi is limited. were the first to report on utilization of b - apbi in the setting of breast augmentation in the abstract form. even though they had a median follow - up of 36 months and observed an excellent or good cosmetic outcome in 97% of the patients, the majority were treated with interstitial implants. also recently published his interstitial technique in the presence of breast implants but no dosimetric data were reported. were the first to report on dosimetric parameters in a case report of a patient who had undergone treatment with b - apbi with existing breast implants but their follow - up was only 6 months post - treatment. comparison of studies reporting treatment with accelerated partial breast irradiation (apbi) in the setting of breast implants we had previously reported the longest follow - up to our knowledge at nearly 5 years post - treatment and suggested several dosimetric parameters relating to breast implants that could be of significance. we have now expanded on our original report by including all of our treated patients, with the longest reported follow - up in this patient population, and moreover adds several dosimetric parameters that can be useful while treating apbi with pre - existing breast implants. all of our patients have been free of disease to this date, even patients # 1 and # 4 who had focally positive margins. it is important to note that their margin status could be somewhat misleading since it was adjacent to the breast implant and a negative margin would have been nearly impossible to obtain, since there were no remaining tissue except the implant itself. b - apbi in those cases allowed those patients to maintain their existing breast implants with excellent cosmetic outcomes. the majority of the other patients also had excellent or good cosmetic outcome, except patient # 7, who had a sizeable difference between her breasts even prior to start of her b - apbi and also has now experienced rupture of her implant on the treated side. importantly, all other women were able to maintain their breast implants except patient # 5 who had to undergo replacement of her implants bilaterally due to age - related leakage. there were no grade 3 or 4 late toxicities and all patients tolerated the treatments well. in terms of dose received by the breast implants, our values of mean dose of 12.1 gy, v50 of 15.6%, v75 of 7%, v100 of 4.6%, v150 of 1.58%, and v200 of 0.46% may represent benchmarks to be used for future treatment planning in patients with breast implants. overall, the lower the dose to the implant and the smaller the absolute implant volume treated, the more beneficial this appears to be in terms of late cosmetic outcome. as a result, b - apbi might be a better option compared to external beam radiation as well in patients who wish to maintain their implants since a smaller overall volume is treated. the correlation between volume of implant treated and cosmesis was apparent in our two patients who had the highest v50 along with the worst cosmetic outcomes. additionally, patient # 7 who had the highest mean, maximum, and absolute and relative dose to the implant went on to experience intra and extracapsular rupture of her implant, raising the possibility that there might exist an absolute implant dose tolerance. all of the patients with v50 of less than 50 cc had an excellent cosmetic outcome (figure 2), although this could potentially be a marker for overall lower dose to the implant. our patient with the worst cosmetic outcome also had the highest maximum and mean dose to the breast implant. furthermore, the distance from the device to the implant did not appear to have a significant impact on the cosmetic outcome as most devices were within 1 - 2 mm of the breast implants. we acknowledge that a larger population with longer follow - up is needed to confirm these findings. although the small number of patients in our series along with our limited follow - up limits our findings, we have nevertheless shown that b - apbi in patients with adjacent pre - existing breast implants is both safe and efficacious with good to excellent cosmetic outcomes in early clinical follow - up. ideally, the dose to the breast implants should be lowered as much as possible during the treatment planning process. based on the above results, unnecessary dose raises the possibility of future poorer cosmetic outcome and other complications like implant rupture. therefore, we believe that there is no need to expand the ptv_eval uniformly into the breast implant, since it contains no actual breast tissue and that ptv_eval can be pulled back as long as all of the necessary breast tissue is treated. additionally, the v50 can be used as a marker of appropriate amount of dose to the implant, with 50 cc or less receiving 50% of prescribed dose representing a reasonable marker. our other reported values of mean dose and the v50, v75, v100, v150, and v200 may be used as benchmarks to decrease the dose and any risks of possible complications. these parameters will need to be confirmed in a larger series with long - term follow - up. in the meantime, our dosimetric parameters have the added benefit of theoretically reducing the chances of capsular contracture, which is the most feared cosmetic complication in women with augmented breasts who undergo external beam whole breast irradiation. additionally, we have shown much better cosmetic outcomes compared to previously reported cases of whole breast irradiation in patients with existing breast implants, in which all twenty one patients were reported to have fair or poor cosmetic results. therefore, we conclude that with careful consideration during treatment planning and attention to tolerance of breast implants, b - apbi can be an excellent option for early stage breast cancer patients with augmented breasts who need to undergo adjuvant radiation therapy.	purposeaccelerated partial breast irradiation (apbi) is an accepted treatment option in breast - conserving therapy for early stage breast cancer. however, data regarding outcomes of patients treated with multi - lumen catheter systems who have existing breast implants is limited. the purpose of this study was to report treatment parameters, outcomes, and possible dosimetric correlation with cosmetic outcome for this population of patients at our institution.material and methodswe report the treatment and outcome of seven consecutive patients with existing breast implants and early stage breast cancer who were treated between 2009 and 2013 using apbi following lumpectomy. all patients were treated twice per day for five days to a total dose of 34 gy using a high - dose - rate 192ir source. cosmetic outcomes were evaluated using the harvard breast cosmesis scale, and late toxicities were reported using the radiation therapy oncology group (rtog) late radiation morbidity schema.resultsafter a mean follow - up of 32 months, all patients have remained cancer free. six out of seven patients had an excellent or good cosmetic outcome. there were no grade 3 or 4 late toxicities. the average total breast implant volume was 279.3 cc, received an average mean dose of 12.1 gy, and a maximum dose of 234.1 gy. the average percentage of breast implant volume receiving 50%, 75%, 100%, 150%, and 200% of the prescribed dose was 15.6%, 7.03%, 4.6%, 1.58%, and 0.46%, respectively. absolute volume of breast implants receiving more than 50% of prescribed dose correlated with worse cosmetic outcomes.conclusionsaccelerated partial breast irradiation using a multi - lumen applicator in patients with existing breast implants can safely be performed with promising early clinical results. the presence of the implant did not compromise the ability to achieve dosimetric criteria ; however, dose to the implant and the irradiated implant volume may be related with worse cosmetic outcomes.
le programme krescent (kidney research scientist core education and national training) a t lanc en 2005 pour augmenter la capacit de la recherche sur les maladies du rein travers le canada, et pour encourager la transmission des connaissances au sein des quatre axes de recherche en sant. cette tude avait pour but dvaluer les rpercussions du programme krescent sur ses principaux objectifs ainsi que des retombes sur la carrire des stagiaires participants, dix ans aprs sa cration. un sondage en ligne a t men en 2015 auprs des stagiaires (n = 53) ayant t admis ou ayant complt le programme krescent. des renseignements ont galement t obtenus par la consultation de curriculum vitae (cv). une analyse bibliomtrique a valu la productivit scientifique et la collaboration des participants ainsi que les rpercussions de leur participation krescent sur leur carrire. les donnes de cette analyse ont t compares celles des candidats nayant pas t retenus au cours de la mme priode. lanalyse comprenait galement une comparaison des donnes canadiennes avec celles obtenues en recherche sur les maladies du rein ailleurs dans le monde. trente - neuf stagiaires du krescent ont complt le sondage en ligne, soit 74% des personnes contactes, et quarante - quatre ont soumis leur cv. de manire gnrale, les stagiaires du krescent ont obtenu plus facilement des subventions des instituts de recherche en sant du canada (irsc) avec un taux de succs de 79%. de plus, 76% des dtenteurs dune bourse au niveau postdoctoral ont obtenu des charges professorales titre de professeur adjoint dans les 8 mois suivant leur formation. la trs grande majorit des stagiaires (90%) a indiqu que le krescent avait grandement contribu au fait quils aient obtenu les fonds des irsc, de mme qu la cration de nouveaux savoirs (93% des rpondants) et au dveloppement de nouvelles mthodes (50% des rpondants). lanalyse bibliomtrique a rvl un lger, quoique rgulier, dclin de la quantit de rsultats en recherche sur les maladies du rein dans le monde entre 2000 et 2014, lorsque converti en pourcentage des rsultats totaux en recherche sur la sant. et ce, bien que lincidence gnrale de la recherche sur les maladies du rein ait augment au canada de 2000 2005 ainsi quentre 2009 et 2014 en comparaison des autres pays. de manire gnrale, la suite de leur formation, les stagiaires du krescent ont dmontr une plus grande productivit, ont plus souvent particip la rdaction de publications collectives ou des collaborations internationales que les demandeurs nayant pas reu de financement. le programme krescent a favoris le perfectionnement professionnel en recherche sur les maladies du rein et a contribu augmenter la capacit de recherche, la productivit et la collaboration des participants. ainsi, pour poursuivre la cration de nouveaux savoirs en recherche sur les maladies du rein et faciliter leur transmission auprs des chercheurs canadiens, nous sommes davis que les programmes de formation tels que le krescent devraient continuer dtre financs sur le long terme par lentremise de partenariats. the kidney research scientist core education and national training (krescent) program was launched in 2005 to increase kidney research capacity in canada and enhance collaborations and knowledge translation. a preliminary review of the program in 2010 revealed enhanced training positions and high success rates for trainees in obtaining grants and academic appointments. the 10-year anniversary in 2015 provided an opportunity for a formal evaluation of krescent, using qualitative and quantitative data. survey data and a bibliometric analysis reveal that krescent has led to increased numbers and quality of research publications, and promoted the development of a collaborative community of leading kidney research investigators. krescent has significant potential to improve the lives of people affected by kidney disease in canada and should be sustained via long - term funding partnerships. the kidney research scientist core education and national training (krescent) program was established in canada in 2004, with enrollment of the first trainees in july 2005. krescent originated with recognition by the canadian research community of the rising prevalence of kidney disease, coupled with declining interest and engagement of trainees in kidney research. at its outset, the major objectives of the program were to enhance kidney research capacity in canada by training the next generation of leading investigators and to improve collaborations and knowledge translation (kt) across the 4 themes of health research : biomedical, clinical, health systems and services, and social, cultural, and environmental factors that affect the health of populations. since 2004, krescent has been sustained by a unique partnership funding model that includes the kidney foundation of canada (kfoc), the canadian society of nephrology (csn), and the canadian institutes of health research (cihr) institute of nutrition, metabolism, and diabetes. salary support is provided for up to 3 years to post - doctoral fellows (md and/or phd), new investigators (md and/or phd, within the first 3 years of academic appointment at a canadian institution), and allied health scholars (enrolled in graduate phd programs in canada). since 2005, the funding model has been supported by contributions (> $ 3.5 m) from the private sector and other granting agencies (additional file 1). seventy - two training positions have been funded in krescent since 2005 with representation in 7 provinces. fifteen post - doctoral fellows have received krescent salary support for training at academic centers outside canada. allied health scholars have been relatively underrepresented within the program (6 trainees since 2005), although changes have been incorporated into the peer review selection process in recent years to increase participation by this group of health professionals. a unique feature of krescent is its core curriculum, which is delivered at 2 workshops annually. the curriculum focuses yearly on selected major topics in kidney research and consists of core lectures from content experts spanning biomedical science to topics in health service delivery and population health, group discussions around pertinent journal articles, and oral presentations by trainees. career development sessions and grant- and manuscript - writing exercises are also built into the curriculum. krescent also provides mentorship support to trainees, particularly at the new investigator level, via biannual meetings with senior investigators involved in the program, and review of a checklist of topics relevant to career development. a preliminary review of the program in 2010 found that numbers of training positions in kidney research had increased due to krescent and that trainees had demonstrated a high success rate in obtaining peer review grant support and academic appointments. now that 10 years have passed, we conducted a formal evaluation of krescent to determine whether the program is meeting its objectives. the evaluation included both qualitative and quantitative components, using a survey and bibliometric analysis. furthermore, we assessed the overall state of kidney research in canada since 2000, in comparison with other countries, to frame the impact of krescent in this context. all recipients of krescent awards between 2005 and 2014 inclusive were contacted by e - mail in the summer of 2015 and asked to complete an online survey questionnaire. the survey consisted of 14 questions that covered a variety of areas, including success rate at obtaining cihr or other peer review operating grants, and numbers of publications or peer review grants with other krescent trainees (see additional file 2). electronic reminders were sent out to those who did not complete the survey, and the survey was closed after 6 weeks. survey results are reported either as categorical values, or in some cases, narrative responses were grouped into common themes identified by the authors, and arbitrarily selected responses depicted. in addition to the survey, krescent trainees were asked to submit their updated curriculum vitae (cvs) to kfoc for review. the cvs were reviewed, and data were collected regarding demographics, research theme and area of research, faculty appointment status, and grants / publications. all trainees consented to the use of anonymized submitted data and survey responses for publication for this formal evaluation. a bibliometric analysis of krescent trainees, before and after their training, in comparison with unsuccessful applicants to the krescent program in the same period this analysis compared, for the 2 groups, the average number of papers, average number of authors per paper, international collaborations (as determined by presence of international addresses in addition to canadian addresses on the manuscript), average of relative citations (arc), and average of relative impact factor (arif). arc measures the frequency in which the article is cited by other researchers, whereas arif indicates the overall prestige of the journal in which the manuscript was published. both indicators control for the disciplinary differences in papers citation rates. only articles and reviews were included in the analysis : abstracts, posters, presentations, and conference proceedings were excluded. to contextualize the results, data were compiled on the field of kidney research at the world level, providing insight into the impact of all canadian kidney researchers (includes krescent trainees and other kidney researchers) compared with kidney researchers from the top 20 countries. papers considered as belonging to the area of kidney research were those that had either one of the specific words in their title or keywords attributed, or that were published in one of the specialized journals (additional file 3). two time periods were examined (2000 - 2005 and 2009 - 2014) to assess impact before and after krescent. finally, the overall impact of kidney research relative to research in other health sectors was assessed over time. from 2005 to 2014 inclusive, there were 66 krescent training awards allocated (37 post - doctoral fellowships, 23 new investigators, and 6 allied health scholarships) to 53 individual recipients (some individuals received both post - doctoral fellowships and subsequent new investigator awards). thirty - eight awards were allocated to researchers with md degrees, whereas 22 awards went to non - md, phd scientists. of the awards to md researchers, 30 (78.9%) were allocated to adult nephrologists, whereas 8 (21.1%) were granted to pediatric nephrologists. the 6 allied health scholarships were held by awardees in clinical psychology (2), health services (2), rehabilitation sciences (1), and systems design engineering (1). in all, 45 individuals responded to at least 1 question on the online survey (85%), and 39 trainees completed the entire survey (74%). the 14 awardees who did not respond to the survey represented a mix of individuals in each of the 3 program categories (9 post - doctoral fellows, 3 new investigators, and 2 allied health scholars). forty - four (83%) trainees submitted cvs. of the respondents, 79% had secured operating grant funding from cihr as a principal investigator (pi) or co - investigator since completion of their training (41% had funding from cihr as a pi or co - pi). ninety percent of respondents reported that the krescent program had contributed importantly to their ability to obtain cihr funding, whereas 75% noted that krescent contributed to some extent or to a great extent in their success at obtaining other peer review grant support. three - quarters (76%) of post - doctoral fellows reported that they had been appointed to academic positions at the assistant professor level, within an average of 7.8 months of completing training. the leading 5 diseases / conditions reported to be impacted by krescent research were chronic kidney disease (ckd), kidney biology, diabetes, acute kidney injury, and transplantation. bar graph depicts responses to the survey question please select the disease or research area impacted by your research. percentage of responses is on y - axis (n = 44 responses), and number of responses is indicated in parentheses above each column. other category included renal failure (4), self - reporting of obesity / nutrition (1), ethics (1), drug - induced nephrotoxicity (1), urology (1), patient engagement (1), medication adherence (1), and development of artificial kidneys (1). krescent = kidney research scientist core education and national training. more than 9 in 10 (93%) respondents noted that krescent training had contributed to the achievement of research outcomes, with the majority reporting knowledge creation (93% of respondents) and development of new research methods (50% of respondents) as the leading outcomes (40 trainees responded to this survey question, and thus, the calculation of proportions is based on a denominator of 40, instead of 39). the development of collaborative research teams to enhance kt is one of the goals of krescent. the cihr research cycle categorizes kt1 as the opportunity to define research questions and methodologies. kt2 involves conducting research (participatory role), and kt3 refers to publishing research findings in plain language and accessible formats. in response to the question to what extent are you involved in kt and specifically kt1-, kt2-, or kt3-level translational research ? the answer was to some extent or to a great extent for 45% of respondents for kt1, 43% for kt2, and 30% for kt3. more than 4 in 5 (82%) respondents reported that since completion of training, they conducted research as part of a multidisciplinary team. interestingly, respondents reported an average of 3.4 publications per trainee (range, 0 - 36) and an average of 1.2 peer review grants per trainee (range, 0 - 17) arising from collaboration with other krescent awardees. there were 39 responses to the question what aspect of the krescent program did you value the most ? (table 1). three major themes emerged from these responses : (1) the opportunity for scientific interaction and networking (29 responses) ; (2) the value of the workshop meetings, including grant - writing exercises (14 responses) ; and (3) mentorship support (10 responses). sample responses to what aspect of the krescent program did you value the most ? there were also 39 responses to the question what aspect of the krescent program did you value the least ? (table 2). the predominant theme that emerged related to assignments and workload associated with the curriculum (17 responses). sample responses to what aspect of the krescent program did you value the least ? kims and kams refer to knowledge integration modules and knowledge application modules, respectively, which were written take - home assignments in the early years of krescent. note. krescent = kidney research scientist core education and national training. the final question of the survey asked for any additional comments, and 25 individuals responded. having the award gave me a level of credibility and visibility in the canadian kidney research community that would have been difficult to achieve in any other way. the bibliometric analysis revealed that international kidney - related research productivity has experienced a small but steady decline relative to other health sectors from 2000 to 2014 (4.5%-3.9%), as determined by numbers of published manuscripts (figure 2). in 2000, canada ranked eighth in the world in terms of number of kidney research publications (n = 600), and in 2014, canada had almost doubled the number of kidney research publications (n = 1124), ranking seventh in the world behind the united states, china, japan, germany, the united kingdom, and italy. decline in total international kidney research manuscripts from 2000 to 2014. graph depicts gradual decrease in the percentage of kidney research publications from 2000 to 2014, as the percentage of all health research publications. below graph is table illustrating numbers of publications per year and the percentage of yearly publications devoted to kidney research. kidney research manuscripts decreased from 4.5% to 3.9% of total health research manuscripts from 2000 to 2014. in comparison with other major countries, the relative impact of canadian kidney research increased in the period of 2009 - 2014, compared with 2000 - 2005. figure 3 illustrates a positional analysis of the top 20 countries publishing in kidney research as a function of the mean impact of their papers and the level of specialization in the field of kidney research. the arc for canada between 2000 and 2005 was 1.32, and in 2009 - 2014, it increased to 1.90. arc measures the frequency in which the article is cited by other researchers, whereas the specialization index (si) is a measure of the degree of specialization in a scientific discipline of a given entity in terms of world science. when the si is greater than 1, it indicates that the country of interest is more specialized in its priority area, compared with the world average. similarly, when the arc is greater than 1, the papers are cited more frequently than the world average in its priority area. for the period between 2009 and 2014, note that the arc and si include data from all kidney researchers in canada, not just krescent awardees. positional analysis of kidney research in 20 countries from 2000 to 2005 (a) and 2009 to 2014 (b). the x - axis depicts the si : when the si is greater than 1.0, the country produces more kidney research manuscripts than expected, and the opposite is true when the index is less than 1.0. for canada, both arc and si for kidney research (ie, includes krescent trainees and all other kidney investigators) increased in 2009 to 2014, compared with 2000 - 2005. the findings for a bibliometric analysis conducted for krescent awardees before and after their training, and for applicants to krescent who were not successful in receiving funding, before and after their applications are depicted in table 3. data from allied health scholars in krescent were excluded from this analysis because of low numbers of trainees. compared with nonfunded krescent applicants, krescent trainees (post - doctoral fellows and new investigators) had increases in average numbers of publications, average numbers of authors per manuscript, average number of addresses linked to authors on manuscripts, and percentage of manuscripts involving international collaborations. for post - doctoral krescent awardees, both arc and arif increased after training, compared with nonfunded applicants. while the arc increased for krescent new investigators after training examples of high - impact first - author publications supported by krescent that have improved diagnosis and management of kidney disease include a large registry cohort study that demonstrated an increased risk of adverse renal outcomes (including end - stage renal disease) with even a single kidney stone episode, development and validation of a predictive model for progression of ckd (which has been adopted internationally), and identification of a novel gene mutation causing atypical hemolytic - uremic syndrome. summative data for new investigators (within 3 years of first academic appointment) and post - doctoral fellows at the time of application to krescent (before), and after that time, until 2015 (after). krescent = kidney research scientist core education and national training ; arc = average of relative citation ; arif = average of relative impact factor. from 2005 to 2014 inclusive, there were 66 krescent training awards allocated (37 post - doctoral fellowships, 23 new investigators, and 6 allied health scholarships) to 53 individual recipients (some individuals received both post - doctoral fellowships and subsequent new investigator awards). thirty - eight awards were allocated to researchers with md degrees, whereas 22 awards went to non - md, phd scientists. of the awards to md researchers, 30 (78.9%) were allocated to adult nephrologists, whereas 8 (21.1%) were granted to pediatric nephrologists. the 6 allied health scholarships were held by awardees in clinical psychology (2), health services (2), rehabilitation sciences (1), and systems design engineering (1). in all, 45 individuals responded to at least 1 question on the online survey (85%), and 39 trainees completed the entire survey (74%). the 14 awardees who did not respond to the survey represented a mix of individuals in each of the 3 program categories (9 post - doctoral fellows, 3 new investigators, and 2 allied health scholars). forty - four (83%) trainees submitted cvs. of the respondents, 79% had secured operating grant funding from cihr as a principal investigator (pi) or co - investigator since completion of their training (41% had funding from cihr as a pi or co - pi). ninety percent of respondents reported that the krescent program had contributed importantly to their ability to obtain cihr funding, whereas 75% noted that krescent contributed to some extent or to a great extent in their success at obtaining other peer review grant support. three - quarters (76%) of post - doctoral fellows reported that they had been appointed to academic positions at the assistant professor level, within an average of 7.8 months of completing training. the leading 5 diseases / conditions reported to be impacted by krescent research were chronic kidney disease (ckd), kidney biology, diabetes, acute kidney injury, and transplantation. bar graph depicts responses to the survey question please select the disease or research area impacted by your research. percentage of responses is on y - axis (n = 44 responses), and number of responses is indicated in parentheses above each column. other category included renal failure (4), self - reporting of obesity / nutrition (1), ethics (1), drug - induced nephrotoxicity (1), urology (1), patient engagement (1), medication adherence (1), and development of artificial kidneys (1). krescent = kidney research scientist core education and national training. more than 9 in 10 (93%) respondents noted that krescent training had contributed to the achievement of research outcomes, with the majority reporting knowledge creation (93% of respondents) and development of new research methods (50% of respondents) as the leading outcomes (40 trainees responded to this survey question, and thus, the calculation of proportions is based on a denominator of 40, instead of 39). the development of collaborative research teams to enhance kt is one of the goals of krescent. the cihr research cycle categorizes kt1 as the opportunity to define research questions and methodologies. kt2 involves conducting research (participatory role), and kt3 refers to publishing research findings in plain language and accessible formats. in response to the question to what extent are you involved in kt and specifically kt1-, kt2-, or kt3-level translational research ? the answer was to some extent or to a great extent for 45% of respondents for kt1, 43% for kt2, and 30% for kt3. more than 4 in 5 (82%) respondents reported that since completion of training, they conducted research as part of a multidisciplinary team. interestingly, respondents reported an average of 3.4 publications per trainee (range, 0 - 36) and an average of 1.2 peer review grants per trainee (range, 0 - 17) arising from collaboration with other krescent awardees. there were 39 responses to the question what aspect of the krescent program did you value the most ? (table 1). three major themes emerged from these responses : (1) the opportunity for scientific interaction and networking (29 responses) ; (2) the value of the workshop meetings, including grant - writing exercises (14 responses) ; and (3) mentorship support (10 responses). sample responses to what aspect of the krescent program did you value the most ? there were also 39 responses to the question what aspect of the krescent program did you value the least ? (table 2). the predominant theme that emerged related to assignments and workload associated with the curriculum (17 responses). sample responses to what aspect of the krescent program did you value the least ? kims and kams refer to knowledge integration modules and knowledge application modules, respectively, which were written take - home assignments in the early years of krescent. note. krescent = kidney research scientist core education and national training. the final question of the survey asked for any additional comments, and 25 individuals responded. having the award gave me a level of credibility and visibility in the canadian kidney research community that would have been difficult to achieve in any other way. the bibliometric analysis revealed that international kidney - related research productivity has experienced a small but steady decline relative to other health sectors from 2000 to 2014 (4.5%-3.9%), as determined by numbers of published manuscripts (figure 2). in 2000, canada ranked eighth in the world in terms of number of kidney research publications (n = 600), and in 2014, canada had almost doubled the number of kidney research publications (n = 1124), ranking seventh in the world behind the united states, china, japan, germany, the united kingdom, and italy. graph depicts gradual decrease in the percentage of kidney research publications from 2000 to 2014, as the percentage of all health research publications. below graph is table illustrating numbers of publications per year and the percentage of yearly publications devoted to kidney research. kidney research manuscripts decreased from 4.5% to 3.9% of total health research manuscripts from 2000 to 2014. in comparison with other major countries, the relative impact of canadian kidney research increased in the period of 2009 - 2014, compared with 2000 - 2005. figure 3 illustrates a positional analysis of the top 20 countries publishing in kidney research as a function of the mean impact of their papers and the level of specialization in the field of kidney research. the arc for canada between 2000 and 2005 was 1.32, and in 2009 - 2014, it increased to 1.90. arc measures the frequency in which the article is cited by other researchers, whereas the specialization index (si) is a measure of the degree of specialization in a scientific discipline of a given entity in terms of world science. when the si is greater than 1, it indicates that the country of interest is more specialized in its priority area, compared with the world average. similarly, when the arc is greater than 1, the papers are cited more frequently than the world average in its priority area. for the period between 2009 and 2014, note that the arc and si include data from all kidney researchers in canada, not just krescent awardees. positional analysis of kidney research in 20 countries from 2000 to 2005 (a) and 2009 to 2014 (b). the x - axis depicts the si : when the si is greater than 1.0, the country produces more kidney research manuscripts than expected, and the opposite is true when the index is less than 1.0. for canada, both arc and si for kidney research (ie, includes krescent trainees and all other kidney investigators) increased in 2009 to 2014, compared with 2000 - 2005. the findings for a bibliometric analysis conducted for krescent awardees before and after their training, and for applicants to krescent who were not successful in receiving funding, before and after their applications are depicted in table 3. data from allied health scholars in krescent were excluded from this analysis because of low numbers of trainees. compared with nonfunded krescent applicants, krescent trainees (post - doctoral fellows and new investigators) had increases in average numbers of publications, average numbers of authors per manuscript, average number of addresses linked to authors on manuscripts, and percentage of manuscripts involving international collaborations. for post - doctoral krescent awardees, both arc and arif increased after training, compared with nonfunded applicants. while the arc increased for krescent new investigators after training examples of high - impact first - author publications supported by krescent that have improved diagnosis and management of kidney disease include a large registry cohort study that demonstrated an increased risk of adverse renal outcomes (including end - stage renal disease) with even a single kidney stone episode, development and validation of a predictive model for progression of ckd (which has been adopted internationally), and identification of a novel gene mutation causing atypical hemolytic - uremic syndrome. summative data for new investigators (within 3 years of first academic appointment) and post - doctoral fellows at the time of application to krescent (before), and after that time, until 2015 (after). krescent = kidney research scientist core education and national training ; arc = average of relative citation ; arif = average of relative impact factor. in 1999, the kfoc organized a national conference with its stakeholders to map out a long - term strategy for kidney research in canada. this conference, horizons 2000, established as a priority the enhancement of kidney research capacity in canada, which led to the development of the krescent program. the results of this review reveal several positive impacts of the program, including high rates of success in obtaining subsequent peer review operating grants and academic appointments at the level of assistant professor, enhanced numbers and quality of publications, and evidence for team - building and international collaboration. a recent series of articles in the canadian journal of kidney health and disease has focused on the serious challenges currently faced by kidney investigators in canada in establishing independent research programs, obtaining peer review grant support, and engaging in kt. the results of our survey indicate that a high proportion of krescent trainees were successful in receiving peer review operating grant support from cihr as either a pi or co - investigator (79%), and the majority of trainees acknowledged the importance of krescent in achieving this degree of success. these data are particularly impressive in view of the decline in recent years in success rates for cihr operating grant funding. a high proportion of post - doctoral fellows who completed krescent training were successful in obtaining academic appointments (76%), with the vast majority finding positions at academic institutions in canada. this extent of success is notable, especially when one considers recent calls for reductions in training positions for phds and post - doctoral fellows in the united states, due to a bottleneck in available academic appointments. however, a recent csn workforce survey reported that nephrology division heads across canada were seeking 14 full - time academic nephrologists immediately (defined as individuals with at least 75% of time devoted to teaching / research - related activities), and a total of 73 to 75 academic nephrologists are sought over the next 5 years. thus, young kidney research trainees (mds and/or phds) in canada have a unique opportunity for career development as independent investigators, and krescent appears poised to serve this need. interdisciplinary research and team work have been recently emphasized as critical to scientific progress, especially in the health sciences. our survey data indicate that since completion of training, the majority of krescent awardees conduct research as part of a multidisciplinary team (82%), and significant numbers are involved in kt1-, kt2-, or kt3-level translational research. the bibliometric data revealed that krescent trainees have increased numbers of authors on manuscripts after completion of training, and increased international collaborations, compared with unsuccessful applicants. furthermore, as shown for team - building and collaboration within the program, survey data indicated that trainees collaborate in publishing manuscripts and obtaining peer review grants together after completion of krescent. the data therefore support an increased likelihood for krescent - supported research findings to be widely disseminated. the bibliometric analysis demonstrated that krescent trainees at the new investigator or post - doctoral fellow level had substantial increases in research productivity upon completion of their training (as determined by the average annual number of manuscripts), compared with nonfunded applicants to the program. similarly, arc analysis showed that articles published by krescent trainees received more citations than those published by nonfunded applicants, suggesting a higher level of scientific impact. in this regard, after krescent training, the overall arc for new investigators was 1.88, and the arc for post - doctoral fellows was 2.26, values which compare favorably with the arc for program - supported articles arising from trainees in the canadian association of gastroenterology (cag)/cihr / partners training program between 2003 and 2011 (1.73). the results for arif reveal that krescent post - doctoral fellows published in journals of higher scientific impact after completion of training, compared with nonfunded applicants, and the average normalized impact factors were comparable with those observed for the cag / cihr / partners training program. however, the arif for krescent new investigators did not increase compared with values before krescent training or compared with nonfunded applicants, nor was the increase in arc higher than that observed with nonfunded applicants (0.20 vs. 0.21, respectively). although reasons for this observation are unclear, it is notable that the average normalized arif prior to krescent training for new investigators was relatively high (1.59), as was the arc (1.68), supporting a high performance level of krescent awardees at baseline. perhaps the most revealing data in our analysis derive from the narrative comments obtained from the survey questionnaire. the responses support the importance of face - to - face workshop meetings in research training, which foster networking and team - building. unfavorable comments regarding krescent were few and largely restricted to the workload burden arising from the core curriculum. in this regard, work assignments in krescent have been modified extensively since 2005, with reduction in time commitments and introduction of exercises with distinct potential benefits (including publication of review manuscripts, and mock grant- and manuscript - review panels). our analysis of krescent has certain limitations. although the survey questionnaire completion rate was significant (74%), those trainees who did not respond may have differing views on the strengths and weaknesses of the program. data collected from trainees cvs are subject to potential error due to self - reporting, and a verification process was not conducted. the subjective nature of some of the survey responses can also be viewed as a limitation. with regard to the bibliometric data, we did not perform statistical comparisons, because use of tests of statistical significance is considered problematic and potentially detrimental in bibliometric analyses, as the data do not meet most assumptions for performance of these tests. finally, the use of data from nonfunded applicants to krescent as a comparator group may be viewed as a weakness, because it might be expected that krescent trainees should outperform this group on a number of research metrics. nonetheless, our analysis is strengthened by the relatively high response rate to the questionnaire, and the robust bibliometric analysis, which allowed for comparison of research metrics before and after krescent training. the bibliometric data indicate that canada s position within the international kidney research community has strengthened in the period from 2009 to 2014, compared with 2000 - 2005, in terms of both impact and numbers of publications. although the increase in the number of canadian kidney - related publications and the arc correspond temporally to the period when the krescent program was launched and implemented, the evaluation methodology does not permit direct attribution. besides the potential contribution of krescent, several factors likely played important roles in enhancing canada s position in the period between 2000 and 2014, including an increase in the overall number of funded canadian kidney researchers. from this evaluation, we conclude that the krescent program has enhanced kidney research capacity in canada and fostered collaboration and kt. the program has resulted in increased numbers and quality of research publications, and promoted the development of a collaborative community of leading kidney research investigators. our data suggest that to meet the needs of the kidney research community and stakeholders in canada, krescent should be sustained via long - term funding partnerships.	background : the kidney research scientist core education and national training (krescent) program was launched in 2005 to enhance kidney research capacity in canada and foster knowledge translation across the 4 themes of health research.objective:to evaluate the impact of krescent on its major objectives and on the careers of trainees after its first 10 years.methods:an online survey of trainees (n = 53) who had completed or were enrolled in krescent was conducted in 2015. information was also obtained from curriculum vitae (cvs). a bibliometric analysis assessed scientific productivity, collaboration, and impact in comparison with unsuccessful applicants to krescent over the same period. the analysis included a comparison of canadian with international kidney research metrics from 2000 to 2014.results:thirty-nine krescent trainees completed the survey (74%), and 44 trainees (83%) submitted cvs. krescent trainees had a high success rate at obtaining grant funding from the canadian institutes of health research (cihr ; 79%), and 76% of post - doctoral fellows received academic appointments at the assistant professor level within 8 months of completing training. the majority of trainees reported that krescent had contributed significantly to their success in securing cihr funding (90%), and to the creation of knowledge (93%) and development of new methodologies (50%). bibliometric analysis revealed a small but steady decline in total international kidney research output from 2000 to 2014, as a percentage of all health research, although overall impact of kidney research in canada increased from 2000 - 2005 to 2009 - 2014 compared with other countries. krescent trainees demonstrated increased productivity, multiauthored papers, impact, and international collaborations after their training, compared with nonfunded applicants.conclusions:the krescent program has fostered kidney research career development and contributed to increased capacity, productivity, and collaboration. to further enhance knowledge creation and translation in kidney research in canada, programs such as krescent should be sustained via long - term funding partnerships.
as an international megacity with a population of over 20 million, beijing has been suffering from frequent smog events in recent years. since the official daily monitoring data became available in 1999, particulate matter has been shown to be a major air pollutant in beijing, and its impact to the public health may be profound. categorized by pm2.5 and pm10 (particulate matter with nominal mean aerodynamic diameters of 2.5 and 10 m, respectively), pm pollutants of different sizes deposit and affect different regions of the respiratory tract : when inhaled, coarse particles (pm2.510) deposit primarily in the head airways, while fine particles (pm2.5) are more likely to penetrate and deposit deeper in the tracheobronchial and alveolar regions. historical data suggest that exposure to such atmospheric particulate matter is linked to increases in morbidity and mortality, and decreases in life expectancy. during the period of january 1014, 2013, the city of beijing, along with the rest of the mideastern region of china experienced a massive, severe smog event. the highest daily average pm2.5 concentration in beijing measured greater than 500 g / m at times (20-fold higher than the who guideline value), raising serious public health concerns. beijing cough) and significant increases of outpatient cases related to respiratory diseases have been reported. here we asked the question of what microorganisms, particularly inhalable allergens and pathogens, are in beijing s pm2.5 and pm10 pollutants and what potential effects they may have on the public health during severe smog events like this. the public health effects of pm, particularly those of pm2.5, have been well documented in the literature. while the physical and chemical properties of pm2.5 and pm10 pollutants have been extensively studied, relatively less is known about inhalable biological particles such as bacteria, fungi, viruses, pollens, and cell debris in the micrometer to submicrometer size range. it has been suggested that materials of biological origin may contribute as much as 25% to the atmospheric aerosol, and they are responsible for various diseases and allergies. the abundance of airborne bacteria measured from 10 to 10 cells per m, depending on the environment. while culture - based methods have been used to detect airborne microorganisms, they are constrained to the identification of a limited number of cultivatable species. although the use of amplicon - based (e.g., 16s or 18s ribosomal rna (rrna) gene) sequencing and related techniques have allowed us to detect both cultivatable and noncultivatable microorganisms (although dna from cell debris may also be detected) and categorize the microbial populations in airborne particles, it has been challenging to sequence the fine, inhalable pm2.5 samples (which are more relevant to human health) due to the low dna yield, unless with amplification of the extracted dna. yet amplicon - based sequencing methods often result in biases, and most importantly, they are generally limited to categorizing bacteria or fungi at the family or genus level (without the use of marker genes). because microbial species within the same family or genus may differ significantly in pathogenic potential, the discovery of microbial allergens and pathogens requires the identification of bacteria, fungi, and viruses at the species or even strain level. thus, microbial metagenomic sequencing represents a powerful alternative for studying complex microbial communities, particularly for its ability to discover clinically relevant microbes at the species level. pm2.5 and pm10 samples were collected from the roof top of the environmental science building (40017n, 1161934e, 10 m above the ground, 20 m and 690 m from the nearest river and hospital, respectively) at tsinghua university, an area without major pollution sources nearby. sampling was conducted by three high volume air samplers (thermo electron corp., ma, u.s.), two of which were equipped with pm2.5 fractionating inlets, the third one being equipped with a pm10 fractionating inlet. ambient air was drawn at an average flow rate of 1.13 m / min for 23 h (10:00 am to 9:00 am the next day) per sampling day from january 8 to january 14 (including january 8 as a nonsmog control, according to the chinese class ii standard), resulting in approximately 1559 m of air flow - through per sampling day. particulate matter with aerodynamic diameters of 2.5 and 10 m were collected on 20.32 25.4 cm tissuquartz filters (pall, ny, u.s.) with 99.9% typical aerosol retention. all the filters were sterilized by baking in a muffle furnace at 500 c for 5 h prior to sampling. each sterilized filter was packaged in sterilized aluminum foil and stored in a sealed bag until being loaded into the filter cartridge. the filter holder and all the tools used for changing new filters were cleaned with 75% ethanol or autoclaved every day to avoid contamination. the net weight of each filter was recorded at mg accuracy before and after sampling. the concentrations of pm2.5 and pm10 at our sampling site were estimated by the net weight of each sample (average weight of the two pm2.5 samples) divided by the 23 h flow - through volume per sampling day (to avoid microbial contamination, samples were not kept under 45% relative humidity at 20 c as typically required for pm measurements). a 47 mm diameter filter punch was taken from the pm10 sample and one of the pm2.5 samples each day for chemical component and elemental analyses. the filter punches were kept in size adaptive chambers and stored at 20 c. all other samples were stored at 80 c until downstream analyses were performed. to overcome the issue of low yield during genomic dna extraction, several technical improvements were made to optimize the extraction of high - quality dna from pm samples. considering the different dna yield of pm2.5 and pm10 samples, 1/4 of pm10 filter (a total of 103.04 cm) and 1 and 3/4 of the pm2.5 filters (a total of 721.28 cm) from each sampling day were used for dna extraction. the filters were cut into 8.96 11.5 cm pieces and were placed in 50 ml centrifuge tubes filled with sterilized 1x pbs buffer. the pm samples were then pelleted at 4 c by centrifugation at 200 g for 2 h. after gentle vortexing, the resuspension was filtered with a 0.2 m supor 200 pes membrane disc filter (pall, ny, u.s.), which was then cut into small pieces and used for dna extraction using the mo - bio powersoil dna isolation kit (carlsbad, ca, u.s.). the samples were then heated to 65 c in powerbead tubes for 10 min followed by vortexing for 2 min. the remaining steps of the extraction were performed according to the standard mo - bio powersoil dna isolation protocol except for the column purification step, which was replaced with magnetic bead purification (agencourt ampure xp, beckman, ca, u.s.) for improved yield. genomic dna quality and concentration were analyzed by gel electrophoresis and a fluorescent dsdna - binding dye assay (qubit fluorometer, life technologies, ca, u.s.). blank control samples were collected by placing a sterilized filter inside of the sampler without operation for 23 h, and treated similarly as above. dna extraction of blank control samples resulted in dna concentrations below the detection limit of our instruments, and library generation efforts failed to generate useable sequencing libraries. the illumina miseq (for library validation) and hiseq 2000 sequencing systems (illumina, ca, u.s.) were used for sequencing, and the library preparation kits were purchased from new england biolabs (ma, u.s.). sequencing library construction and template preparation were performed according to the neb library preparation protocols. we constructed a paired - end library with insert size of 500 bp for each sample. an aliquot of 5 ng dna from each sample was used as the starting amount (except for 3 samples, the total quantities of dna of which were less than 5 ng, supporting information (si), table s1) for library preparation in order to ensure sample consistency. in order to minimize possible bias introduced by pcr, each sample was barcoded and equal quantities of barcoded libraries were used for sequencing (for index sequences, see si table s1). adaptor contamination and low - quality reads were discarded from the raw data. in total, 98 gb sequence with a uniform read length of 90 bp was obtained and an average of 7 gb high - quality hiseq sequences were generated from each sample (si table s1). the rarefaction curve (analyzed by the metagenomics rast server (mg - rast, release 3.3)) suggested that the sequencing depth of the hiseq data was sufficient to capture most of the microorganisms but not the miseq data (average data set of 683 mb) (si figure s1). metaphlan (metagenomic phylogenetic analysis) was used to estimate the relative abundance of bacteria and archaea with unique clade - specific genes at the species level (si figures s2 and s3). the illumina hiseq reads were aligned to a cohort of nonredundant ncbi complete genomes (2637 complete genomes, including bacteria, fungi, archaea, and viruses) using the short oligonucleotide analysis package (soap) alignment tool (release 2.21 t) to profile the common core species. we used a 90% identity threshold for bacteria, archaea, and fungi, and 100% identity for viruses due to their smaller genome sizes. bacterial, fungal, and viral species with coverage of 5%, 0.5% (average alignment of all chromosomes), and 1%, respectively, in either pm2.5 or pm10 samples of 7 consecutive sampling days were listed in si table s2. the genome - size - normalized relative abundance of these species was calculated based on the number of aligned reads normalized by the species genome size (si figure s4 and table s3). the variations of the hit abundance of species across 7 sampling days were estimated based on the hit numbers normalized by number of total aligned reads (si figure s5). the greengenes 16s rrna gene database was used for 16s rrna phylogenetic analysis with the following alignment parameters : > 97% identity, minimal alignment 40 bp. dna sequence data have been deposited in mg - rast (http://metagenomics.anl.gov/) at the following url : http://metagenomics.anl.gov/linkin.cgi?project=3756. the greengenes 16s rrna gene database was used for assigning the hiseq reads at > 97% identity threshold (only uniquely aligned reads were used for following calculations). all of the greengenes database sequences with available information of bacteria habitats were classified into the four categories without overlaps. in addition, the 16s sequences of previous studies on high - altitude and urban airborne bacteria of milan and new york were assigned to the same habitat categories and compared to our results (si figure s6). we sought to sequence the metagenome of inhalable airborne microorganisms in beijing s pm2.5 and pm10 pollutants, after having overcome the technical issues involved in high - volume pm2.5 and pm10 sample collection, dna extraction, and library generation (for details of the dna extraction methods, see materials and methods). pm2.5 and pm10 samples collected at a beijing tsinghua pm monitoring site (40017n, 1161934e) from january 814, 2013, during which period beijing s pm2.5 and pm10 pollution indexes rapidly deteriorated from healthy to record - high hazardous levels, were used for sequencing (figure 1a, si figures s7s9 and table s4 ; air temperatures are typically low in january in beijing, creating a unique high pm, low temperature environment). the illumina hiseq data from a total of 7 daily pm2.5 and 7 pm10 samples is more than 1000-fold larger than those of three previous studies on airborne bacteria combined, from which original sequence data were publicly available (si table s1). by aligning to the greengenes 16s rrna gene database at a 97% identity threshold, we discovered 255 more bacteria genera than those identified by the three previous studies (si figure s10 and table s5). overall, the pm2.5 samples contained 86.1% bacterial, 13.0% eukaryotic, 0.8% archaeal, and 0.1% viral reads, while the pm10 samples contained 80.8% bacterial, 18.3% eukaryotic, 0.8% archaeal, and 0.1% viral reads (figure 1b). the higher relative abundance of eukaryotic reads (which included those from fungi, plants, algae, and animal debris), as well as the higher alpha diversity (a measurement of species diversity) found in pm10 compared with those of pm2.5 samples (figure 1c), may in part be attributed to the fact that the aerodynamic diameters of many fungal spore agglomerates were between 2.5 and 10 m. principal component analysis (pca) of the microbial relative abundance (figure 1d and si table s6) and dinucleotide frequency (si figure s11) suggested that the metagenomes of airborne microbes were distinct from those of other environments, though relatively more related to the soil metagenomes. (a) daily average pm2.5 and pm10 concentrations estimated from the collected samples during january 814, 2013. (b) relative abundance of the mg - rast taxonomic hits at the domain level in pm2.5 and pm10 samples. (c) estimated average alpha diversity of the pm2.5 and pm10 samples (error bars represent sd of the 7 daily pm2.5 and 7 pm10 samples, respectively). (d) principal component analysis of the relative abundance of microorganisms at the phylum level of the 14 sequenced pm metagenomes (red) compared to those of other environments (other colors). bacteria appeared to be the most abundant prokaryotic microorganisms in pm2.5 and pm10 pollutants. to identify the prokaryotic species and to estimate their relative abundance, we used the metagenomic phylogenetic analysis (metaphlan) toolbox to reveal a picture of complex bacteria and archaea community (figure 2a and si figure s2). we show that the most abundant phyla were actinobacteria, proteobacteria, chloroflexi, firmicutes, bacteroidetes, and euryarchaeota (relative abundance 1%). at the species level, 1315 distinct bacterial and archaeal species were identified from the 14 samples. an unclassified bacterium in the nitrogen fixing, filamentous bacteria genus frankia appeared to be the most abundant (figure 2a and si figure s3). the most abundant classified bacterial species appeared to be geodermatophilus obscurus, a bacterium commonly found in dry soil environments (si table s7). by aligning to the greengenes 16s database, categorized by terrestrial, fecal, freshwater, and marine - associated bacteria (see materials and methods), we show that the majority (> 85%) of the categorized bacteria in the collected pm2.5 and pm10 samples were related to fecal and terrestrial sources (figure 2b and si figure s6). the proportion of bacteria from terrestrial - related sources appeared to be higher than those identified from the three previous studies (figure 2c and si figure s6). this may in part be attributed to the lack of vegetation coverage and abundance of dry, exposed soil, and construction sites in the city of beijing and its surrounding areas, especially during the winter seasons. in addition, while the proportion of freshwater and marine - associated bacteria remained relatively constant, the fraction of fecal - associated bacteria appeared to have increased (from 4.5% to as high as 11.4% in pm2.5 samples) (figure 2b) with progressively increased concentrations of pm pollution. (a) phylogenetic tree of the bacteria and archaea identified from pm2.5 samples, analyzed by metaphlan. the sizes of the nodes correspond to the relative abundance at the corresponding levels in the cohort. the family, genus, and species levels of the most abundant order actinomycetales are plotted. only nodes with 1% relative abundance are labeled. (b) original habitats of the identified bacteria in daily pm2.5 and pm10 samples, categorized by terrestrial, fecal, freshwater, and marine sources. (c) bacterial and archaeal species in beijing 's pm samples were pooled and compared with those identified from the grip high - altitude, milan urban, and new york subway studies. since not only bacteria, but also fungi and viruses are responsible for various human allergies and diseases, we sought to identify the microbial species including fungi and viruses (which are currently not supported by the metaphlan toolbox) in pm2.5 and pm10 pollutants. we employed the short oligonucleotide analysis package (soap) alignment tool to align the hiseq reads from each sample to a cohort of 2637 nonredundant species of ncbi complete genomes, including bacteria, archaea, fungi, and viruses. at a 90% identity threshold and 5% coverage of the complete bacterial genomes (for a typical bacterial genome of 4 mb, it corresponds to a minimal alignment length of 200 kb, > 100-fold longer than the 16s rrna gene and thus provides more confidence) or 0.5% coverage for fungal genomes, the 48 most abundant bacterial and 2 fungal species were identified (si table s2). because of the smaller genome size of viruses, we used a more stringent alignment strategy (i.e., 100% identity and 1% coverage), and 3 most abundant viral species were identified. we next estimated the genome - size - normalized relative abundance (defined as the number of unique hit reads normalized by genome size) of each species within the most common ones (si table s3) and analyzed the daily variations of their relative abundance during the 7 sampling days (figure 3). consistent with the metaphlan results, the soil - associated bacteria g. obscurus appeared to be the most abundant classified bacterial species (with an average genome coverage of 42.7% and relative abundance of 14.6% in the pm2.5 samples), followed by modestobacter marinus, blastococcus saxobsidens, kocuria rhizophila, and micrococcus luteus, all of which are commonly found in soil habitats and some with the abilities to survive under tough (e.g., uv radiation) conditions (si table s7). although the relative abundance of most of the bacterial species remained stable during the 7 sampling days, as was found in previous studies, some showed considerable variations. for example, the relative abundance of thermobifida fusca, an important bacterial degrader of plant cell walls and commonly found in decaying organic matter (si table s7), increased 5-fold from an average of 0.7% during the first 2 less polluted days to an average of 3.7 2.5% in the 5 heavily polluted days in pm2.5 samples (si figures s4 and s12). box plot of the daily variations of the relative abundance of 48 most common bacterial, 2 fungal, and 3 viral species in pm samples. boxes correspond to the interquartile range between the 25th and 75th percentiles, and the central lines represent the 50th percentile. whiskers correspond to the lowest and highest values no more than 1.5 times the interquartile range from the box, while dots are the outliers beyond the whiskers. pm2.5 samples are labeled pink and pm10 are black. among the identified microbial species, several are known to cause human allergies and respiratory diseases, including streptococcus pneumoniae, aspergillus fumigatus, and human adenovirus c (with average genome coverage of 2.0%, 14.5%, and 6.5%, respectively). among them, s. pneumoniae is the most common cause for community - acquired pneumonia (cap), having been isolated from nearly 50% of cap cases. its representation within the entire bacteria community (analyzed by metaphlan) was 0.012% in pm2.5 samples and 0.017% in pm10 samples, and the normalized number of hit reads (hit abundance) appeared to have increased by 2 fold from an average of 0.024% during the first 2 less polluted days to an average of 0.05 0.02% in the 5 heavily polluted days in pm2.5 samples (figure 4a). a. fumigatus, likely collected in the form of spores, is known as a major fungal allergen and opportunistic pathogen that causes airway or lung invasion in immunodeficient patients. its average hit abundance was found to be higher in pm10 than in pm2.5 samples (4.5% vs 1.7%), most likely because the aerodynamic diameters of the fungal spore agglomerates are between 2.5 and 10 m. the hit abundance of a. fumigates also appeared to be correlated with the increase of pm pollution levels, increasing 4-fold from an average of 1.5% during the first 2 less polluted days to an average of 5.8 1.8% in the 5 heavily polluted days in pm10 samples (figure 4b). to confirm the existence of a. fumigatus in our samples, we cultured the fungus and validated its existence by sequencing the 18s rrna gene and a species - specific gene (glii) (si table s8), as well as sem imaging (si figure s13). human adenovirus, a dsdna virus that accounts for 510% of upper and lower respiratory tract infections in children, was also found (with 100% sequence matched to human adenovirus c in all 14 samples). the hit abundance of adenovirus in our samples also appeared to have increased during the heavily polluted days, though with more daily variations than those of s. pneumonia and a. fumigates (si figure s5). daily variations of the normalized hit abundance of microbial pathogens and allergens in the collected pm2.5 and pm10 samples. to put our findings in the context of aerosol chemistry, we analyzed the organic and elemental carbon, water - soluble ions, and elemental composition of the collected samples (si tables s9s11). we found that sulfate, ammonium, nitrate, and organic matter were among the most abundant, and altogether their relative abundance in pm2.5 and pm10 samples were 80% and 71% (w / w), respectively. these results, as well as the high weight ratio of pm2.5 to pm10 (0.7), suggested that secondary formation of fine particles likely led to the high pm concentrations during this smog event. additionally, the high relative humidity (si figure s8) during the period may have contributed to particle growth through water uptake and promoting aqueous redox chemistry (e.g., the oxidation of sulfur dioxide to sulfate). this also suggested that most of the particles were rich in water content during the polluted days and thus would favor the survival of microbes. pm pollution has been studied extensively in the context of aerosol chemistry and physics, and statistical correlations between pm pollution and decreased life expectancy have been made. so far, no specific components of pm have been conclusively shown to be harmless. in particular, previous studies have shown that bioaerosols containing pathogens are responsible for the spread of respiratory diseases, and thus it is crucial to understand the composition of airborne microbes at the species level and to identify the potential microbial allergens and pathogens. most of the clinically relevant studies on inhalable pathogens were conducted in hospital environments, yet in beijing, a significant increase of outpatient cases related to respiratory diseases during the same severe pm pollution period studied here has been reported. our results have provided sequence - based evidence for the existence of inhalable microbial allergen and pathogen species in an open environment, and suggested that high pm pollution may pose health threats to the susceptible population (e.g., the elderlies and the immunodeficient). besides, information on the original habitats of airborne bacteria provides important insights for understanding the source of the biological particles, and may be used as a reference for future urban planning efforts to reduce pm pollution and the spread of airborne microbial allergens and pathogens. in future studies, clinical samples (e.g., sputum samples from respiratory disease patients) during severely polluted and unpolluted days can be obtained, and the sequence information can be compared to those from collected pm samples for comparison. furthermore, pm exposure studies on animal models can be performed to characterize the effects of pm - associated allergens and pathogens, leading to better understandings of their pathogenicity. using the current methods, we were able to identify bacterial, archaeal, fungal, and dsdna viral species in the collected pm samples. we also attempted to culture other bacteria and fungi species, but not all were successfully cultured (data not shown) since some species were slow - growing or difficult to culture, and the samples were stored at 80 c before use. rna viruses such as rhinovirus and influenza virus are undoubtedly important viral agents that affect the public health. yet in our experience, it appeared to be technically challenging to extract sufficient quantities of rna for reverse transcription and sequencing from pm samples containing various rna - degrading containments such as divalent cations. low - bias preamplification techniques may be used to generate sufficient libraries for the sequencing of rna viruses in pm samples in future studies. as for human dsdna viruses, human adenovirus c appeared to be the most abundant in our pm samples based on our sequencing results. more importantly, the current study was limited by the daily sampling capacity and availability of sampling sites, as well as the trade - off between obtaining high - depth sequence data for species - level characterization vs more sampling days (98 gb data from 14 samples). in particular, though pm pollution levels are typically high in the winter of beijing, low temperatures are often associated with lower overall microbial abundance compared to warmer seasons. thus, future longitudinal and multiple location studies to identify airborne microorganisms should be performed to compare with our current results and to provide better insights on the increased incidences of respiratory diseases during urban smog events, and to correlate with meteorological data, chemical components, and clinically obtained pathogen samples. additionally, the establishment of a monitoring network for airborne microbes can be invaluable during outbreaks of deadly respiratory diseases. information on the abundance of particular airborne pathogens and their regional and seasonal variations will be of particular importance for the prevention of respiratory diseases at a public scale, in areas such as vaccine design and distribution, as well as for understanding the spread of drug resistant respiratory pathogens.	particulate matter (pm) air pollution poses a formidable public health threat to the city of beijing. among the various hazards of pm pollutants, microorganisms in pm2.5 and pm10 are thought to be responsible for various allergies and for the spread of respiratory diseases. while the physical and chemical properties of pm pollutants have been extensively studied, much less is known about the inhalable microorganisms. most existing data on airborne microbial communities using 16s or 18s rrna gene sequencing to categorize bacteria or fungi into the family or genus levels do not provide information on their allergenic and pathogenic potentials. here we employed metagenomic methods to analyze the microbial composition of beijing s pm pollutants during a severe january smog event. we show that with sufficient sequencing depth, airborne microbes including bacteria, archaea, fungi, and dsdna viruses can be identified at the species level. our results suggested that the majority of the inhalable microorganisms were soil - associated and nonpathogenic to human. nevertheless, the sequences of several respiratory microbial allergens and pathogens were identified and their relative abundance appeared to have increased with increased concentrations of pm pollution. our findings may serve as an important reference for environmental scientists, health workers, and city planners.
five thoroughbred horses (two females and three geldings), aged 4 to 7 years old with body weight of 493.8 26.6 (mean sd) kg, belonging to the equine research institute, japan racing association, were assigned to the experiment. these horses had been routinely grazed for 2 months and specially trained for treadmill exercise prior to this study. hydrogen - rich water (hw) was made by special equipment (high density hydrogen water, melodian co., tokyo, japan) for the present study. the hw consisted of filtrated water (ph=6.82) with a concentration of molecular hydrogen (h2) higher than 1 ppm. fresh hw that was made about 3 hr before the treadmill exercise was stored in an aluminum bag (10 l in volume) and tightly sealed in order to prevent hydrogen gas escape. two liters of hw were administered into the esophagus via a nasogastric catheter 30 min before the treadmill exercise. for the placebo trial, the same volume of normal water without hydrogen was administered by the same method. horses were exercised with increment of load every 2 min at speeds of 1.6, 3.6, 7, 10, 12 and 13 m / s until they were exhausted. the first treadmill exercise was performed after placebo administration and the second treadmill exercise was performed after hydrogen - rich water (hw) administration, after one - week interval. two litters of placebo water or hw were administered to the esophagus by a nasogastric catheter in each horse at 30 min before the treadmill exercise. blood samples (serum) were collected 4 times : 30 min before the exercise, immediately before the exercise, post - exercise, and at 30 min after the exercise.. treadmill exercise on a 6% incline was performed by each horse. each horse was exercised on the treadmill with stepwise increases of exercise intensity every 2 min, at treadmill running speed of 1.6, 3.6, 7, 10, 12, 13 and 14 m / sec, until the horses became thoroughly exhausted. by thoroughly exhausted, we mean that the horses were too exhausted to maintain their position at the front of the treadmill with humane encouragement. horses were exercised with increment of load every 2 min at speeds of 1.6, 3.6, 7, 10, 12 and 13 m / s until they were exhausted. the first treadmill exercise was performed after placebo administration and the second treadmill exercise was performed after hydrogen - rich water (hw) administration, after one - week interval. two litters of placebo water or hw were administered to the esophagus by a nasogastric catheter in each horse at 30 min before the treadmill exercise. blood samples (serum) were collected 4 times : 30 min before the exercise, immediately before the exercise, post - exercise, and at 30 min after the exercise. the first treadmill exercise was performed after the placebo administration, and the second treadmill exercise was performed after the hw administration, after one week interval. in order to measure serum d - roms and bap, venous blood samples of 10 ml the blood samples were collected from the jugular vein immediately before the administration of placebo water (distilled water) or hw, 30 min before the treadmill exercise. blood samples were also collected at pre - exercise, immediately before the onset of treadmill exercise, at post - exercise, immediately after treadmill exercise, and at 30 min after the end of treadmill exercise. in addition to the samples, blood was collected at 10:00 and 13:00 on a day without treadmill exercise from all five horses to determine their background levels of d - roms and bap. the measurement of d - roms was performed using a colorimetric method of final derivatives, i.e., hydroperoxide produced by free radicals, in which hydroperoxide in the serum reacts with n, n - diethyl - p - phenylenediamine to form [a - nh2 ] using a free radical analyzer (free carpe diem, wismerll, tokyo, japan). this d - roms test was invented and developed by carratelli, m. and the validity of this method has been demonstrated by comparisons with the results of the electron spin resonance (esr) method, which serves as a direct measurement of unpaired electrons [2, 39, 40 ]. bap was determined by color reaction of thiocyanate which reflects reduction potency from fe to fe due to electrons (e) in the blood using the same free radical analyzer. the d - roms, bap and bap / d - roms values were statistically evaluated using two - way repeated - measures analysis of variance (two - way repeated - measures anova). in addition, the statistical significance of differences was tested by two - factor anova with only one observation in each cell at pre - exercise, post - exercise and 30 min after exercise for each of the placebo and hw groups, and the dunnet test was used to compare post - exercise and 30 min after exercise data with that at pre - exercise. wilcoxon s signed - rank test was also used to examine the data of the blood samples at 30 min before treadmill exercise, before the placebo or hw administration, in order to examine if the basal conditions were the same. for all the data, differences were considered significant if the p value was less than 0.05. five thoroughbred horses (two females and three geldings), aged 4 to 7 years old with body weight of 493.8 26.6 (mean sd) kg, belonging to the equine research institute, japan racing association, were assigned to the experiment. these horses had been routinely grazed for 2 months and specially trained for treadmill exercise prior to this study. hydrogen - rich water (hw) was made by special equipment (high density hydrogen water, melodian co., tokyo, japan) for the present study. the hw consisted of filtrated water (ph=6.82) with a concentration of molecular hydrogen (h2) higher than 1 ppm. fresh hw that was made about 3 hr before the treadmill exercise was stored in an aluminum bag (10 l in volume) and tightly sealed in order to prevent hydrogen gas escape. two liters of hw were administered into the esophagus via a nasogastric catheter 30 min before the treadmill exercise. for the placebo trial, the same volume of normal water without hydrogen was administered by the same method. horses were exercised with increment of load every 2 min at speeds of 1.6, 3.6, 7, 10, 12 and 13 m / s until they were exhausted. the first treadmill exercise was performed after placebo administration and the second treadmill exercise was performed after hydrogen - rich water (hw) administration, after one - week interval. two litters of placebo water or hw were administered to the esophagus by a nasogastric catheter in each horse at 30 min before the treadmill exercise. blood samples (serum) were collected 4 times : 30 min before the exercise, immediately before the exercise, post - exercise, and at 30 min after the exercise.. treadmill exercise on a 6% incline was performed by each horse. each horse was exercised on the treadmill with stepwise increases of exercise intensity every 2 min, at treadmill running speed of 1.6, 3.6, 7, 10, 12, 13 and 14 m / sec, until the horses became thoroughly exhausted. by thoroughly exhausted, we mean that the horses were too exhausted to maintain their position at the front of the treadmill with humane encouragement. horses were exercised with increment of load every 2 min at speeds of 1.6, 3.6, 7, 10, 12 and 13 m / s until they were exhausted. the first treadmill exercise was performed after placebo administration and the second treadmill exercise was performed after hydrogen - rich water (hw) administration, after one - week interval. two litters of placebo water or hw were administered to the esophagus by a nasogastric catheter in each horse at 30 min before the treadmill exercise. blood samples (serum) were collected 4 times : 30 min before the exercise, immediately before the exercise, post - exercise, and at 30 min after the exercise. the first treadmill exercise was performed after the placebo administration, and the second treadmill exercise was performed after the hw administration, after one week interval. in order to measure serum d - roms and bap, venous blood samples of 10 ml the blood samples were collected from the jugular vein immediately before the administration of placebo water (distilled water) or hw, 30 min before the treadmill exercise. blood samples were also collected at pre - exercise, immediately before the onset of treadmill exercise, at post - exercise, immediately after treadmill exercise, and at 30 min after the end of treadmill exercise. in addition to the samples, blood was collected at 10:00 and 13:00 on a day without treadmill exercise from all five horses to determine their background levels of d - roms and bap. the measurement of d - roms was performed using a colorimetric method of final derivatives, i.e., hydroperoxide produced by free radicals, in which hydroperoxide in the serum reacts with n, n - diethyl - p - phenylenediamine to form [a - nh2 ] using a free radical analyzer (free carpe diem, wismerll, tokyo, japan). this d - roms test was invented and developed by carratelli, m. and the validity of this method has been demonstrated by comparisons with the results of the electron spin resonance (esr) method, which serves as a direct measurement of unpaired electrons [2, 39, 40 ]. bap was determined by color reaction of thiocyanate which reflects reduction potency from fe to fe due to electrons (e) in the blood using the same free radical analyzer. the d - roms, bap and bap / d - roms values were statistically evaluated using two - way repeated - measures analysis of variance (two - way repeated - measures anova). in addition, the statistical significance of differences was tested by two - factor anova with only one observation in each cell at pre - exercise, post - exercise and 30 min after exercise for each of the placebo and hw groups, and the dunnet test was used to compare post - exercise and 30 min after exercise data with that at pre - exercise. wilcoxon s signed - rank test was also used to examine the data of the blood samples at 30 min before treadmill exercise, before the placebo or hw administration, in order to examine if the basal conditions were the same. for all the data, differences were considered significant if the p value was less than 0.05. on the days of placebo and hw administration, the mean values of d - roms immediately before administration of placebo or hw at 30 min preceding the treadmill exercise were 148.3 15.3 (placebo) and 152.8 9.4 (hw), respectively. likewise, the mean values of bap at 30 min before treadmill exercise were 2,555.9 92.5 (placebo) and 2,774.1 32.7 (hw), respectively. there were no significant d - roms or bap between the placebo and hw treatment trials. d - roms values of each horse at pre - exercise, post - exercise and 30 min after the treadmill exercise are shown in fig. 2fig. 2.d - roms values at pre - exercise, post - exercise and 30 min after exercise of each horse after administration of placebo and hw.. in all horses, d - roms tended to elevate at post - exercise, i.e., immediately after the treadmill exercise. the average changes in d - roms induced by the treadmill exercise in the placebo and hw trials are shown in fig. 3fig. 3.average changes of d - roms of all horses after administration of placebo and hw.30 min : at 30 min after the treadmill exercise. data are shown as means se.. a significant difference in d - roms was found among the three measurement times of pre - exercise, post - exercise and 30 min after exercise in the placebo (p<0.005) and hw (p<0.005) trials. the d - roms value significantly increased at post - exercise, compared to pre - exercise, in the placebo (p<0.005) and hw (p<0.001) trials. no significant changes in d - roms were observed at 30 min after exercise in both the placebo and hw trials. the d - roms value at pre - exercise was lower for the hw trial (148 14.3 u.carr ; mean se) than for the placebo trial (179 12.1 u.carr ; mean se), but the difference was not significant. d - roms values at pre - exercise, post - exercise and 30 min after exercise of each horse after administration of placebo and hw. average changes of d - roms of all horses after administration of placebo and hw. the bap values of each horse at pre - exercise, post - exercise and 30 min after the treadmill exercise are shown in fig. 4.bap values at pre - exercise, post - exercise and 30 min after exercise of each horse after administration of placebo and hw.. the bap value increased in all horses at post - exercise. the average change in bap values of the placebo and hw trials are shown in fig. 5.average changes of bap of all horses after administration of placebo and hw.30 min : at 30 min after the treadmill exercise. data are shown as means se.. there were significant differences among the three measurement times in the placebo (p<0.0001) and hw (p<0.0001) trials ; the bap values at post - exercise increased to 150% and 153% of the pre - exercise value in the placebo and hw trials, respectively. significant differences from pre - exercise were found at post - exercise (p<0.0001) and 30 min after exercise (p<0.005) in the placebo trial, whereas a significant difference was present only at post - exercise (p<0.0001) in the hw trial. bap values at pre - exercise, post - exercise and 30 min after exercise of each horse after administration of placebo and hw. the value of the bap / d - roms ratio is shown in fig. 6.average changes of bap / d - roms of all horses after administration of placebo and hw.30 min : at 30 min after the treadmill exercise. data are shown as means se.. significant differences from pre - exercise were found in the placebo (p<0.005) and hw (p<0.05) trials ; the differences were greatest at post - exercise in the placebo (p<0.001) and hw (p<0.01) trials, and they were also significant at 30 min after the exercise in the placebo (p<0.001) and hw (p<0.05) trials. as for comparisons between the placebo and hw trials on d - roms, bap and bap / d - roms ratio, a significant difference (p=0.04) was observed only for bap / d - roms (two - way repeated - measures anova) without interaction between substance (placebo or hw) and measurement time points (p=0.87). average changes of bap / d - roms of all horses after administration of placebo and hw. the excessive production of intramuscular or extra - muscular ros induced by intensive exercise such as supra - maximum exercise might play an important role in enhancing inflammation of the muscle. however, concurrent action of protective factors, represented by various antioxidative substances such as super oxide dismutase (sod), catalase, peroxidase, glutathione (gsh), homocysteine, ascorbic acid (vitamin c), -tocopherol (vitamin e) and some minerals, might be enhanced or recruited against the oxidative stress. therefore, the balance of oxidative and antioxidative functions in tissues and blood are thought to be more important than the production of oxidative substance alone. in the present study, significant increases in d - roms and bap were induced by the intensive treadmill exercise (figs. 3 and 5), and were accompanied by elevation of the bap / d - roms ratio (fig. 6). there are several reports on the association of exercise with oxidative stress in equine. it has been suggested that biochemical parameters such as lipid hydroperoxides, which are indicative of oxidative stress, are changed by exercise, and that the changes are exacerbated during exercise at high temperature and humidity. also, the exercise - induced increase in plasma lipid peroxide was reduced by allopurinol - induced inhibition of xanthine oxidase, which resulted in the formation of ros during exercise. changes in blood contents of malondialdehyde (mda) and gsh were evident in race horses (ten 3 year - old stallions) subjected to physical exercise with a progressive strength of training, where the plasma mda and gsh significantly increased after the ride and the increase in mda was still present at 18 hr after the exercise. the present study demonstrated that the oxidative parameter (d - roms) and antioxidative parameter (bap) in the blood were clearly and simultaneously elevated by intensive treadmill exercise of thoroughbred horses. the finding of increase in d - roms may be supportive of the finding of increased lipid hydroperoxidase in horses after exercise [25, 26 ]. moreover, in the present study the marked increase in bap observed at post - exercise and rapid return to the pre - exercise level at 30 min after the exercise is worthy of note. this evidence implies that antagonistic action to strong oxidative stress is designed to onset in rapid time - course and bap recovery to the pre - exercise level occurs within a short period after the exercise. this antioxidative function may be reinforced by exercise training of thoroughbred horses because the antioxidative capacity has been suggested to be improved by exercise training. some evidence of the efficacy of dietary supplementation with antioxidants has previously been presented. the antioxidative influence of vitamin e and selenium supplementation in 3-year - old stallions has been reported. the antioxidant capacity, total antioxidant activities and thiobarbiturate reactive substances in thoroughbred race horses were elevated after a race, and the intravenous administration of ascorbate reduced the oxidative stress, although the creatine kinase activities were not influenced by the administration. in recent years, possible therapeutic effects of hw, which contains molecular hydrogen (h2), have been noticed in experimental studies with animals. in rats with periodontitis, the intake of hw for 4 weeks lowered serum levels of ros and oxidised low - density lipoprotein - cholesterol. in mice subjected to physical restraint stress for 10 hr per day for 6 days each week, in addition, the inhalation of h2 gas protected the brain from ischemia and reperfusion - induced damage in the rat. brain slices derived from mice administered hydrogen - rich pure water for 33 days showed significantly less superoxide formation than the control. in a rat model of sepsis, the intraperitoneal administration of hydrogen - rich saline inhibited the increase of oxidative responses, such as ros and malondialdehyde, in the hippocampus in a dose - dependent manner. also, drinking hw significantly suppressed intimal hyperplasia of the inferior vena cava in the rat. it is of interest to understand the biochemical mechanism of the antioxidative action of hw. the antioxidative action of molecular hydrogen (h2) has been described in detail. h2 dissolved in culture medium of pc12 cells significantly decreased the level of hydroxyl radicals (oh) without decreasing the level of in super oxide anion radicals (o2) or hydrogen peroxide (h2o2) derived from mitochondria. the absorption of hw from the digestive organs and its time - course change in the body was described in a rat study. hydrogen in the blood was detected at 3 min after the direct instillation of saturated hw into the stomach, and the half - life of hydrogen in the muscle was estimated to be approximately 20 min after the instillation. therefore, it is assumed that the molecular hydrogen is rapidly absorbed by the digestive organs and most of it can be metabolized within one hour in the living body. in the present study, the d - roms value showed a tendency to decrease already at pre - exercise after the intake of hw, compared to the placebo intake. in addition, there were significant increases in the bap / d - roms ration in the hw trial, compared with the placebo trial, at pre - exercise, post - exercises and 30 min after exercise (two - way repeated - measures anova), while no significant difference was found in the same horses at 30 min before the treadmill exercise. this finding may indicate that the hw administered into the digestive tract of the horse is rapidly distributed throughout the body after administration and acts to reduce a part of free radicals in the blood. in addition, the earlier recovery of the bap / d - roms ratio in the hw trial, compared to the placebo trial, may reflect lesser amounts of free radicals (d - roms) during and immediately after the exercise, as compared to the placebo trial. in conclusion, the present study demonstrated that oxidative and antioxidative changes in the blood are significantly induced by treadmill exercise of thoroughbred horses, and that the recent developed measurements, i.e. d - roms and bap tests, are useful and convenient methods for determining exercise - related physiological changes in horses. furthermore, we suggested a possibility that the supplementation of hydrogen - rich water has efficacy in lowering oxidative stress in horses.	the present study aimed to clarify changes of oxidative stress and antioxidative functions in treadmill - exercised thoroughbred horses (n=5, 3 to 7 years old), using recently developed techniques for measurement of serum d - roms for oxidative stress, and bap for antioxidative markers. also, the effect of nasogastric administration of hydrogen - rich water (hw) or placebo water preceding the treadmill exercise on these parameters was examined. each horse was subjected to a maximum level of treadmill exercise in which the horses were exhausted at an average speed of 13.2 0.84 m / sec. blood samples were taken 4 times, immediately before the intake of hw or placebo water at 30 min preceding the treadmill exercise, immediately before the exercise (pre - exercise), immediately after the exercise (post - exercise) and at 30 min following the exercise. in all horses, both d - roms and bap values significantly increased at post - exercise. the increase in d - roms tended to be lower in the hw trial, as compared to the placebo trial at pre - exercise. the increase in bap was considerable at approximately 150% of the pre - exercise values in both the hw and placebo treatment trials. the bap / d - roms ratio was significantly elevated at post - exercise in both treatment trials, while a significant elevation was also observed at pre - exercise in the hw trial. bap, d - rom, and the bap / d - rom ratio tended to decline at 30 min after the exercise, except bap and bap / d - roms in the placebo trial. these results demonstrate that the marked elevation of oxidative stress and anitioxidative functions occurred simultaneously in the intensively exercised horses, and suggest a possibility that hw has some antioxidative efficacy.
hyperthyroidism (ht) due to thyroiditis is rarely observed in children treated for neoplastic diseases. one of the causes of ht may be an infectious process in the thyroid gland. fungi, most commonly candida and aspergillus species, belong to infectious factors of ht [13 ]. literature gives only few reports on thyroid involvement in the history of mucormycosis, even though a constant increase in invasive fungal infections (ifis) due to members of mucorales has been observed in recent years, especially in children treated because of proliferative diseases of the hematopoietic system [4, 5 ]. no hyperthyroidism in a child with thyroiditis due to mucormycosis has been so far reported in the literature. a 12-year - old girl was hospitalized due to acute lymphoblastic leukemia (allic 2002 protocol, intermediate risk group). routine imaging examinations before treatment introduction (brain magnetic resonance imaging, abdominal ultrasound scan as well as neck and thyroid ultrasound scan) showed no abnormalities. agranulocytosis with neutrophils count below 200 per l was observed since the introduction of antineoplastic therapy (prednisone 60 mg / m, vincristine, daunorubicin, l - asparaginase). on the 21st day of therapy, computed tomography of the chest showed diffuse inflammatory infiltration in the majority of the right lung and free fluid in the right pleural cavity. the child required mechanical ventilation for 7 days. since the etiological factor of the pulmonary infiltration remained unknown and because there was no clinical improvement in spite of the introduced antibacterial (cefepime 100 mg / kg daily replaced with meropenem 60 mg / kg daily and vancomycin 40 mg / kg daily) and antifungal treatment (voriconazole 12 mg / kg daily), we decided to perform an exploratory pleural puncture. pleural fluid was incubated at 37 c the first 24 h and then at room temperature. the outgrowing colonies on sabouraud agar and columbia agar with 5 % blood were fast growing, floccose and white in color (becoming darker after several days). microscopic examination in lactophenol blue stain revealed wide non - septate hyphae and sporangiophores with short branches bearing spherical sporangia with columellae. the strain was susceptible to amphotericin b (mic 0.5) and resistance to caspofungin (mic 32) and voriconazole (mic 32). amphotericin b lipid complex (10 mg / kg daily) and posaconazole (600 mg daily). after extubation attempt, we observed a change in the voice timbre, which was related to intratracheal intubation and mechanical ventilation. three weeks after the combined antifungal treatment, the patient demonstrated alarming clinical symptoms such as increasing anxiety, psychomotor agitation, psychotic symptoms and sleeplessness. all microbiologic examinations of the blood, urine and stool as well as viral examination of the stool turned out to be negative. on examination, we observed tachycardia (heart rate > 180 per min) and arterial hypertension with a high systolic and diastolic amplitude. the thyroid gland was enlarged and tender on examination, with no flare or increased temperature of the skin over the gland. hormonal evaluation confirmed the tentative diagnosis of hyperthyroidism (tsh0.088 u / ml, ft426.56 pmol / l, ft33.95 pmol / l). the level of antithyroid antibodies was unremarkable, including the level of tsh receptor antibodies. an ultrasound scan of the thyroid gland was also performed, and it revealed signs of an intense inflammatory process with an increased flow (fig. 1). an uneventful ultrasound - guided fine - needle aspiration biopsy of the thyroid gland was carried out under general anesthesia. the direct gomori methenamine silver - stained preparation revealed broad irregular, rarely septated fungal hyphae typical for mucorales ; the hyphae were rarely branching with wide angle and bulbous dilatations (fig. 2). intravenous infusions with thiamazole derivative and propranolol were introduced in the treatment and gave a rapid regression of hyperthyroidism, including the psychotic symptoms. control hormonal evaluation proved to be normal 18 days after the introduction of antithyroid treatment (tsh4.59 u / ml, ft412.96 pmol / l, ft32.28 pmol / l). despite the complex antifungal therapy, further progression of mucormycosis was observed. even though a pulmonectomy was carried out, further spread of the infection to the right kidney and the contralateral lung was seen. the child died because of multiorgan failure due to general fungal infection 49 days after the invasive fungal infection was diagnosed. 1right thyroid lobe : a longitudinal scan diffused hypoechoic area with hyperechoic linear fibrous septa and focal isoechoic areas ; b transverse scan decreased echo of thyroid gland with isoechoic areas in the central part of right lobe and isthmusfig. gomori methenamine silver stain right thyroid lobe : a longitudinal scan diffused hypoechoic area with hyperechoic linear fibrous septa and focal isoechoic areas ; b transverse scan decreased echo of thyroid gland with isoechoic areas in the central part of right lobe and isthmus fungal hyphae with wide angle branching in thyroid tissue. gomori methenamine silver stain after 2 years, we tried to amplify the fungi dna from tissue in reference mycology center. formalin - fixed, paraffin - embedded (ffpe) tissue was sent to the mycology laboratory at the new york state department of health, albany, new york, for molecular identification of etiologic agent identified by histopathology. the tissue was processed for dna extraction using qiaamp dna ffpe tissue (qiagen, valencia, ca) kit as per the manufacturers instructions, with minor modifications. after tissue lysis step, glass beads were added ; and the mixture was disrupted in a cell disrupter for 30 min, followed by dna extraction using reagents provided in the kit. the conventional pcr was carried out to amplify internal transcribed spacer 1 (its1) and its2 regions and the 5.8s ribosomal dna (rdna) region by using universal primers its1 and its4. these results indicated that either there was complete lack of fungal dna in the extracted tissue dna or the amount of fungal dna was not sufficient to be amplified by conventional its - pcr. it is also important to note that paraffin - embedded tissue has historically been viewed as an insensitive source for pcr assay. in the available literature, there is no case report of a child with ht due to mucormycosis. the diagnosis of ht is based on the presence of clinical signs such as fever, tachycardia and heart palpitations, nausea, diarrhea, muscle weakness, fatigue and insomnia. however, in children who undergo antineoplastic therapy, such symptoms most commonly suggest the onset of general bacterial or fungal infection. therefore, the diagnosis of ht in the described patient was not easy for oncohematologists. it is also important that the child presented no previous signs of hyperthyroidism, which would make the diagnosis easier. the ultrasound scan of the thyroid gland performed during the initial diagnosis of leukemia showed no abnormalities. it should also be stressed that psychotic symptoms came long before the other symptoms of ht. that is why it is extremely important for pediatric hematologists to include ht in the differential diagnosis of those symptoms. involvement of the thyroid gland in fungal infections is rarely diagnosed ; such diagnosis is often reached postmortem [7, 8 ]. infections due to members of the mucorales have been an increasing clinical problem in recent years, and the prognosis in generalized infections due to those fungi is usually very poor [911 ]. the most common manifestations of mucormycosis in europe were pulmonary (30 %), rhinocerebral (27 %), soft tissue (26 %) and disseminated disease (15 %). the involvement of the thyroid gland in the history of mucormycosis is described in the literature extremely seldom (table 1). the intra vitam diagnosis of thyroiditis due to a member of the mucorales in our patient was based on the clinical signs, hormonal evaluation, ultrasound scan and histopathological examination. we supposed thyroiditis due to mucor sp. in the result of generalized fungal infection with the beginning in the right lung (positive culture from the pleural fluid). the introduction of the appropriate therapeutic procedure in our patient led to regression of ht symptoms. unfortunately, despite the complex antifungal therapy and pulmonectomy, further spread of the infection to other organs was observed. such an unfavorable course of generalized mucormycosis is reported by numerous authors [5, 12 ]. however, due to the serious general condition of the patient and the severity of the elective pulmonectomy after consulting anesthesiologists the patient was disqualified from thyroidectomy.table 1thyroid gland involvement in mucormycosis [3, 7, 1318]authorpublication yearpatient s age (years)underlying conditionpathogendiagnosisoutcomechiba. 201142acute myelogenous leukemia (aml)cunninghamella bertholletiaeautopsydiedpresent case201112acute lymphoblastic leukemia (all)mucormycosisbiopsydiedpathogen original description, the currently accepted name of absidia corymbifera is lichtheimia corymbifera thyroid gland involvement in mucormycosis [3, 7, 1318 ] pathogen original description, the currently accepted name of absidia corymbifera is lichtheimia corymbifera the child died due to general fungal infection because of multiorgan failure.	thyroiditis due to fungal infection is an extremely rare cause of hyperthyroidism. the most common etiological factor of thyroiditis is aspergillus. infections due to members of the mucorales have been an increasing clinical problem in recent years, and the prognosis in generalized infections due to those fungi is usually very poor. no hyperthyroidism in a child with thyroiditis due to mucormycosis has been reported in the literature so far. we describe a clinical course of generalized mucormycosis with thyroid involvement in a 12-year - old girl treated for acute lymphoblastic leukemia. the child underwent a hyperthyroidism connected with thyroid involvement due to a fungal process. the diagnosis was based on the clinical signs, laboratory findings and typical ultrasound scan ; however, later attempt to amplify the fungi dna from the tissue block has failed. the child died because of multiorgan failure due to general fungal infection 49 days after the invasive fungal infection was diagnosed. the generalized mucormycosis is always connected with poor prognosis and the mortality is high.
although renal embolization is reported to be a safe and effective therapeutic procedure for embolization of small branches of renal artery, it is mainly used for urological purposes, i.e. vascular malformations, angiomyolipomas, and renal tumors that are not amenable to surgical resection [1, 2 ]. a search of the literature for the past 20 years reveals that only a few cases of renal amyloidosis [3, 4, 5, 6, 7 ] and severe nephrotic syndrome [8, 9, 10, 11 ] have resulted in bilateral renal artery embolization (rae) for severe proteinuria. the limited use of bilateral rae for nephrological purposes may be partly related to its tendency to destroy renal function, which results in anuria and subsequent regular dialysis. regular dialysis is usually stressful for patients, so some patients may be reluctant to receive bilateral rae for severe proteinuria, which can induce hypoalbuminemia and increase the risk of morbidity and mortality [13, 14 ]. therefore, doctors and patients with renal amyloidosis and proteinuria face a difficult dilemma in deciding whether to use bilateral rae or supportive treatments. this study reports a 66-year - old patient with renal amyloidosis and severe proteinuria, who received delayed bilateral rae, until a life - threatening pulmonary edema occurred. finally, bilateral rae, followed by regular hemodialysis (hd), successfully cured the severe proteinuria and its related symptoms. a 66-year - old man diagnosed with chronic lymphocytic leukemia had been treated with prednisolone 5 mg per day and cyclophosphamide 25 mg per day since 1995. he was referred to our clinic because he had been suffering from renal amyloidosis - related heavy proteinuria (21.77 g / day), hypoalbuminemia (1.4 g / dl), and anasarca edema for 6 months. because bilateral rae was not performed, progressive anasarca edema, increased proteinuria (31.2 g / day), reduced serum albumin (0.9 g / dl), and dyspnea developed after further conservative treatment with bed rest, salt and water restriction, diuretics such as furosemide 80 mg and hydrochlorothiazide 50 mg trice daily, ramipril 1.25 mg once daily, indomethacin 25 mg trice daily and albumin infusion, for 1 month. the serum bun (normal range 820 mg / dl) and creatinine (normal range 0.81.5 mg / dl) levels were increased to 43 and 4.8 mg / dl from 36 and 3.0 mg / dl, respectively. on physical examination, body weight was 55 kg, body height 162 cm, blood pressure 7080/5060 mm hg, pulse rate 84 beats per minute, respiratory rate 20 breaths per minute, and body temperature 37c. laboratory investigation revealed white blood cells (wbc), 4.3 10/l (normal range 4.511 10/l) ; hemoglobin, 92 g / l), and platelets, 212 10/l (normal range 150350 10/l). unfortunately, further breathing difficulty developed in hospital, despite aggressive treatment with intravenous furosemide and albumin infusion. therefore, the patient underwent bilateral rae ; pure alcohol mixed with lipidol was injected via the orifices of the right and left renal arteries to obliterate the arteries and their branches (fig. 1). after bilateral rae, no proteinuria was noted, due to anuria. there was only mild nausea, flank pain, and mild fever for 1 day after embolization. the serum albumin level was increased from 0.9 to 3.5 g / dl within 3 months (fig. an increase in muscle mass (the arm girth increased from 18 to 23 cm, thigh girth from 25 to 35 cm, waistline from 60 to 71 cm, and dry weight from 46 to 57 kg) was noted during the follow - up period. hospitalization and protein substitution were no longer needed in the following 2 years (fig., blood pressure was increased from about 7080/5060 to 100120/6070 mm hg in the first year, and 120135/7080 mm hg in the second year after bilateral rae. although our case had developed severe proteinuria (> 20 g / day), hypoalbuminemia (1.4 g / dl), and anasarca edema under aggressive diuretic treatment, bed rest, fluid control, angiotensin converting enzymes, and nonsteroid anti - inflammatory drugs and albumin infusion, the patient and family agreed to bilateral rae when the conditions worsened (urine protein 31.2 g / day and serum albumin 0.9 g / dl) and a pulmonary edema occurred. after bilateral rae and following regular hd, the patient 's refractory proteinuria, leg edema, and respiratory distress subsided (fig. because of the successful outcome in this case and previous reports [3, 4, 5, 6, 7, 8, 9, 10, 11 ], it is suggested that treatment with bilateral rae as early as possible may be worthwhile for patients with severe proteinuria resistant to medical therapy. in our case, concerns about the subsequent regular hd meant that the patient and family refused early bilateral rae. the patient and family were anxious and wary of further loss of renal function and the stress of regular hd as a result of treatment with rae. however, prolonged severe proteinuria resistant to medical therapy had caused the patient to reach a life - threatening cachexic state. in contrast, bilateral rae and subsequent regular hd not only rapidly alleviated proteinuria, anasarca edema, and pulmonary edema, but also resulted in an increase in serum albumin level, with an obvious improvement in nutritional status, muscle mass and body weight, and clinical condition, with an obvious improvement in quality of life. the patient maintained a steady status for at least the next 2 years. in the meantime, no specific complications have occurred, except for the mild, self - limiting symptoms of post - infarction syndrome, which presented as mild flank pain, fever, and nausea for 1 day after the bilateral rae. this case and selected previous reports [3, 4, 5, 6, 7, 8, 9, 10, 11 ] reveal important information for physicians and patients in discussing prognoses and considering the pros and cons of bilateral rae. in conclusion, bilateral rae is an alternative, effective, rapid, and safe procedure for the treatment of heavy proteinuria with nephrotic syndrome. bilateral rae with subsequent hd is useful in breaking a vicious circle and can improve nutritional status and alleviate the life - threatening complications associated with hypoalbuminemia.	in the case reported here, after prolonged medical therapy resistance, severe proteinuria subsided following bilateral renal artery embolization (rae). thereafter, respiratory distress, anasarca edema, muscle mass, and serum albumin level improved after regular hemodialysis. although rae is reported to be a safe and effective therapeutic procedure, it is rarely used for severe proteinuria with prolonged medical therapy resistance. the limited use of bilateral rae for nephrological purposes may be partly related to its tendency to destroy renal function, which results in anuria and subsequent regular dialysis. however, delayed rae could cause the patient to reach a life - threatening cachexic state and could increase the risk of morbidity and mortality due to severe proteinuria - induced hypoalbuminemia. our case and selected previous reports reveal important information for physicians and patients while discussing prognoses and considering the pros and cons of bilateral rae.
in 1902, albert moll published his comprehensive, 650-page book rztliche ethik : die pflichten des arztes in allen beziehungen seiner thtigkeit [medical ethics : the doctor s duties in all relations of his work ]. at this time, moll was already well known as a medical author : his 1889 book on hypnotism and his 1891 monograph on homosexuality had gone through several editions and translations, and his studies of the libido sexualis had come out in 1897/8. with his work on medical ethics he contributed to a literary genre that had been flourishing at the end of the nineteenth century. books on medical deontology that is, on doctors professional duties had been authored in the 1890s by the berlin physician and historian of medicine julius pagel (18511912), the berlin medical practitioner jacob wolff, the bremen psychiatrist friedrich scholz (18311907) and others. the common context for all of them was the so - called overcrowding of the medical profession in those years. doctors not only competed for lucrative private patients, but also for contracts with the health insurance organisations [krankenkassen ], which gave them access to the insured working class and lower - middle - class patients. books on the doctors duties were seen as one means to counteract overly competitive behaviour among medical practitioners, and to emphasise a need for collegiality and solidarity. other means to safeguard professional conduct were the disciplinary tribunals of the medical societies and doctors chambers [rztekammern ]. in prussia, such medical courts of honour [rztliche ehrengerichte ], attached to the regional doctors chambers, had been established by law in 1899. moreover, many medical societies had adopted professional codes of conduct that served as guidance for their disciplinary committees. accordingly, writing about medical ethics predominantly meant discussing the requirements of fair conduct among practitioners and how to display behaviour that would enhance the reputation of the profession in the eyes of the public. moll s medical ethics contained much of this type of professional ethics, yet he took a distinctive approach to medical ethics an approach that was fuelled by his outrage about abuses in clinical experimentation on hospital patients. one of the most notorious cases, which had recently been discussed in the daily press, was that of dr alexander strubell, who had locked up a patient with diabetes insipidus in an attic of the jena university clinics without any access to water in order to try to break the patient s allegedly, the patient became so desperately thirsty that he ended up drinking his own urine. another widely debated case was that of the breslau professor of dermatology albert neisser (18551916), who had injected, without consent, eight female hospital patients, some of them minors and some of them prostitutes, with blood serum from syphilis patients in the hope of developing a vaccination for the disease. none of these patients suffered from syphilis at the time of the experiment (1892) ; they had been hospitalised because of other skin or venereal diseases. four of the subjects, all of them prostitutes, developed syphilis some years later, which raised the question of whether the infection had been caused by the experimental injections or through their occupation. moll was particularly upset about the many bacteriological experiments that were carried out on hospital patients around this time, including inoculation of dying patients with the germs of gonorrhoea and other infectious diseases. against this background, moll advocated a medical ethics that focused on the doctor s relationship and duties to his patients, not just his professional obligations to medical colleagues. in this article, i will first outline moll s patient - centred type of ethics and illustrate it with some moral issues that he regarded as more important than professional etiquette : truth - telling and the justifiability of deceiving patients ; the question of euthanasia ; and the so - called perforation of the fetus ie. late abortion when a natural birth turned out to be impossible. i will complement these in the second part of the paper with two cases of moll s personal involvement in publicly debated ethical issues : the above - mentioned issue of human experimentation in hospitals ; and the so - called patient trade scandal of 1908/9, in which four professors of the berlin university clinics were accused of paying middlemen to bring them lucrative private patients. in the final sections, i will assess the impact of moll s ethics and his contemporary image as a guardian of medical ethics. in writing medical ethics, moll had received advice from two philosophers : his friend max dessoir (18671947), with whom he also collaborated in the berlin society for experimental psychology on questions of hypnotism and psychical research ; and georg simmel (18581918), who had been appointed to an extraordinary professorship at berlin university in 1901. their input may, to some extent, explain why moll started his discussion of medical ethics by delineating its relationship to moral philosophy, an aspect that one does not usually find in other works of this genre at that time. moll made it clear that none of the current systems of moral philosophy could provide a basis for medical ethics because one system of thought could be used to argue for entirely contrary positions, thus failing to give reliable guidance for the doctor. average morality [durchschnittsmoral ] of the people, a kind of intuitive common - sense ethics in contemporary society, that the doctor shared with the layperson. this did not mean, however, as moll cautioned, that the doctor s ethical decisions were determined by public opinion. with his critical stance towards moral theories, moll echoed positions of simmel, who in his introduction to ethics (1892) had argued that ethics had to be developed into an inductive science in order to progress, and that this task was separate from the normative role of ethics. moreover, as moll stressed, moral systems such as evolutionary ethics and utilitarianism could lead to conclusions that negated the role of the doctor as a healer. for instance, from an evolutionary standpoint, it might be argued that patients with hereditary illnesses or disabilities should not be treated in order not to be helped to pass on their condition to the next generation, that is, a social darwinist and eugenic position might be supported. or from a utilitarian perspective, experimentation on a dying patient might be justified in the interest of developing treatment for future patients. none of these conclusions appeared acceptable to moll, who asserted that the essential characteristic of the doctor was that of the healer who is committed to the well - being of the individual patient. while this principle of salus aegroti suprema lex was common among medical authors writing on ethics, moll gave it a characteristic, quasi - legal shape. patient relationship was a (tacit) contract, with duties and rights for both parties. this contract relationship implied full commitment of the doctor to his patient once he had agreed to take on the treatment, though it also gave a right to the doctor to refuse treatment in the first place except in emergencies. it also gave room for the self - determination of the patient in questions of his or her health, but simultaneously implied an expectation of compliance by the patient with the agreed treatment, at least as far as the patient s personal circumstances permitted it. this basic concept ran through the whole of moll s work on medical ethics, giving it some coherence, despite a strong tendency towards long - winded and overly detailed discussion and excessive casuistry. these latter problems were also observed by reviewers and probably limited the book s success. while two russian editions of medical ethics appeared in 1903 and 1904, there was no further german edition a marked contrast to the success of moll s books on hypnosis and on sexology. perhaps over - ambitious in its comprehensiveness, moll s medical ethics not only covered the doctor patient relationship, or as moll called it, the relations between doctor and client, but discussed in detail the various forms of practice, as a house doctor, specialist, panel doctor, hospital doctor, country doctor, etc. ; dangerous or morally problematic actions of the doctor, such as abortion or risky surgical interventions ; economic aspects of medical practice, including the health insurance system ; the doctor s professional and private conduct ; the role of public and personal hygiene ; the doctor as an expert for the authorities, courts and insurance organisations ; the ethics of medical science and research on animal and human subjects ; and finally, questions of medical education. this scope was not dissimilar to pagel s book on doctors duties ; the latter, however, had managed to limit his ein kleiner katechismus fr angehende praktiker [a brief catechism for incoming practitioners ], as he subtitled it, to a mere ninety - seven pages. pagel covered, in his text, the setting up and running of a medical practice ; the doctor s conduct in society, towards patients and colleagues, including behaviour during consultations ; medical societies and professional discipline ; the relationships to pharmacists, midwives and other healers ; town and country practice, as well as the roles of poor law physician, panel doctor and officer of health ; and the doctor s fees and book - keeping. unlike moll s approach to the doctor patient relationship, pagel s was overtly paternalistic : the doctor had to be, or at least appear to be, this view was in line with that of many other medical practitioners of his generation. in the following sections, i will illustrate moll s particular ethical argumentation with the examples of deceiving patients, euthanasia and abortion. moll was aware of philosophical positions such as those of immanuel kant (17241804) and johann gottlieb fichte (17621814), who had both argued that truthfulness was an unconditional duty in any situation, but he rejected this point of view as unsuitable for the demands of medical practice. according to moll, the doctor had to distinguish whether he had only been asked for his expert opinion on a medical question, or whether he was also in charge of the patient s care. in the former case, an exception could only be made if the prognosis was so dire that suicide of the patient had to be feared. in the second scenario, when the doctor had taken on a mandate for the patient s care, the decision about truthfulness or deception depended on the best interests of the patient. here, deception could be permissible ; for example, a patient with hysteric paralysis who had put hope in a course of magneto - therapy should be encouraged by the doctor in order to make use of the suggestive effect of such a harmless treatment. in dangerous diseases, this might also extend to misleading the relatives of the patient, who might otherwise reveal the seriousness of the condition through their words or behaviour. in this context, moll addressed particularly the question of truth - telling in incurably ill patients. tactful disclosure of the dire prognosis seemed justified in the patient s interest, if the patient had to sort out his or her personal affairs eg. by drawing up a last will or if the patient was religious (catholic) and would want to be given the last rites. if the doctor had merely been asked for his expert opinion, disclosure of the prognosis was rather unproblematic in these two situations. if, however, the doctor was also responsible for the patient s treatment, and perhaps, as the house doctor, for the health of the patient s family, he carefully had to weigh the potential damage caused by telling the truth against the potential benefits. moll advised the doctor in this case to involve a third person to communicate the bad prognosis, which would affect the patient s psychological condition less and would keep the doctor in his role as a source of hope and support. moll s discussion of the problem of truth - telling thus gave careful attention to what the specific contract with the patient and his / her family implied. the demands of the contract eventually determined the ethical decision. with his application of his contract theory, it was not, however, radically different from the traditional medical view in this matter, which likewise permitted restrictions on truthfulness vis - - vis the terminally ill. prominent nineteenth - century medical authors, such as christoph wilhelm hufeland (17621836) and karl friedrich heinrich marx (17961877), had warned that telling the truth in this situation might seriously harm, even this silence of the doctors could, as karen nolte has recently shown, conflict with the intentions of nurses belonging to religious orders, who wished to inform incurably ill patients about their imminent end, so that they could be spiritually prepared for death. typical of the attitude of doctors was the advice given by the viennese surgeon robert gersuny (18441924) in his deontological booklet arzt und patient : winke fr beide [doctor and patient : hints for both ] (1884) to be very restrictive in giving information to patients with serious illnesses. diagnoses such as tuberculosis and cancer should, in his view, not be mentioned because they would deprive the patient of hope. the doctor should even exercise caution in speaking with the relatives in such cases, because they might pass the information on to the patient or become unable to be carers as a result of their own sense of hopelessness. similarly, the munich surgeon albert krecke (18631932) claimed that a cancer patient gains nothing by knowing the nature of his malady. a doctor acts in obedience to the highest dictates of humanity if he conceals the true nature of the complaint from his patient, whilst at the same time endeavouring to effect a radical cure of the gruesome disease. in his memoirs, published in 1936, moll portrayed his position as one of absolute truthfulness towards his patients a claim that clearly contradicted the advice that he had given in his medical ethics. within his discussion of the doctor patient relationship, moll also addressed the then controversially discussed question of euthanasia. from the perspective of social darwinism, racial hygiene and eugenics, the berlin physician alfred ploetz (18601940) had propagated, in 1895, his vision of a society that would practise active euthanasia with morphine of weak and disabled newborns, and that would refrain from caring for the sick, the blind and the deaf - mute in order to avoid counteracting natural selection. a few years earlier, the philosopher friedrich nietzsche (18441900), in his gtzendmmerung [twilight of the idols ] written in 1888 had denounced the sick as parasites on society, who from a certain stage of illness that made them entirely dependent on doctors and other practitioners should be treated with social contempt. doctors, he suggested, should then be the agents of this contempt not offering prescriptions, but instead a daily dose of disgust at their patients. also in 1895, the notorious booklet das recht auf den tod [the right to death ] by adolf jost had been published. jost, a student of philosophy, mathematics and physics at the university of gttingen, argued here that in some cases of incurable physical or mental illness, death was desirable both from the patient s and from society s perspective. taking a utilitarian approach, he claimed that the intensity of the individual s suffering and the amount of harm caused to society by the patient s sickness could result in a negative value of the human life. since killing on demand was punishable with not less than three years imprisonment under the german penal code of 1871 (section 216), jost called for legal reform. a new law would permit, in such cases, voluntary active euthanasia performed by physicians. once this had been established in society, a further step of legal reform might extend also to euthanasia, without consent, of incurably ill mental patients. moll firmly rejected such ideas in his medical ethics. for him, any measures that deliberately shortened a patient s life were inadmissible from the point of view of criminal law as well as of morality. moll used here what we now call a slippery slope argument : if one once admitted such a right to kill the terminally ill, it might also be applied to shorten a patient s life by months and even years, if a painful and socially unproductive remaining lifespan was predicted. life should be the doctor s highest good, and death should be seen by him as the worst evil. moll s argument here was a classical piece of medical ethics, virtually the same as hufeland had used at the beginning of the nineteenth century. as michael stolberg has recently shown, however, there were already individual cases of german medical practitioners performing active life - shortening measures on terminally ill patients around 1800. although moll very rarely made reference to hippocratic ethics in his work, he pointed to the hippocratic oath s prohibition of giving a deadly poison to a patient, even on demand. moll s position on the question of euthanasia was in line with the prevailing view of the medical profession at his time at least on the level of normative writing and debate. moll rejected traditional techniques of euthanasia, such as pulling away the pillow from underneath the dying person s head or turning the patient on their face. he did, however, concede a generous use of narcotics to mitigate pain, even if they led to unconsciousness. the objection that inducing unconsciousness was incompatible with the dying person s dignity was, in his view, invalid. moll was aware of the fact that high doses of painkillers, such as morphine, could shorten the lifespan of the dying patient, but he regarded this as a case of applying risky treatments, which were permissible according to his contract theory with the patient s consent. there was no clear sense in moll s discussion of this matter of the problematic nature of so - called indirect or double - effect euthanasia. his understanding of the concept of euthanasia was the traditional one, as part of palliative care. in fact, he referred to the berlin professor of clinical medicine johann christian reil (17591813), who had described in detail how good nursing care could ease a patient s final hours. still, moll s contract theory also influenced his advice in this area. heroic treatment efforts, such as strong electric stimulation, which would prolong the dying patient s life for just a short time and would only increase his or her pain, should not be undertaken, even if the patient s family urgently asked for them. the contract relationship, moll emphasised, was still with the dying patient, not with the relatives, so the presumed interest of the patient in not suffering even more than necessary in their last moments was paramount. similarly, from his contract - perspective, moll condemned medical experimentation on dying patients as ethically entirely unacceptable and as a shameful act of brutality. the self - determination of the patient or, if this could no longer be exercised, the patient s presumed interest, likewise guided moll s advice in another morally contested issue, the practice of craniotomy or perforation of the fetus when a natural delivery was impossible eg. because of too narrow a pelvis. the alternative, a caesarean section, at that time still carried a relatively high risk of mortality about ten per cent for the mother. craniotomy of the fetus, which inevitably killed it, was seen as a desperate measure for saving the mother s life, although around 1900 it had a similar mortality risk for the mother as a caesarean section. jurists had argued, for example, that the killing of the fetus in this situation could be seen as self - defence or as an act in a state of emergency. moll, however, examined the issue from an ethical point of view. if the woman requested the perforation, the doctor had to follow her wish and carry out the procedure. also, if the woman was unable to express her will due to her condition, or left the decision to the doctor, the craniotomy should be performed. for moll, the fetus was not an independent human being, which meant that its life could not be balanced against the life of the mother. if, however, the mother deliberately wanted to take the risk of a caesarean section, the doctor should also follow her wishes, because self - sacrifice for a high purpose was regarded as ethically permissible. if the mother was dying and unconscious, the caesarean section could also be performed, because the mother s consent to an attempt to save the baby s life could be presumed. moll s emphasis on the wishes of the mother contrasted with that of leading gynaecologists and obstetricians of the time, such as bernhard krnig (18631917) in freiburg, who wanted to make the decision only on medical grounds, which could mean that a caesarean section was performed against the mother s will. moll s position on the question of the perforation of the fetus also matched his rather liberal views on the morality of earlier abortions. the german penal code, which at the time punished abortion with imprisonment or penal servitude for up to five years (section 218), was, in his opinion, out of tune with public sentiment. the public did not invariably regard abortion as something unethical, as moll pointed out, especially not if carried out in an early stage of pregnancy. in fact, it has been estimated that in late nineteenth - century germany, between three hundred thousand and five hundred thousand abortions were performed every year. only a small proportion of these cases, less than one thousand per year around the turn of the century (18821912), ended with a criminal conviction. moll himself was sympathetic to women who wanted to terminate a pregnancy after rape or if they already had too many children to support. he was also not alone in his view that a doctor should not denounce the woman to the police, if he was called to attend to complications after a botched abortion. patient relationship through a breach of medical confidentiality in this situation, and the serious damage to the woman s reputation, had to be weighed against the value of reporting a crime. in 1911, moll commented on a case in which a doctor had reported the woman concerned, with her consent, in order to initiate prosecution of the abortionist. both the abortionist and the woman had subsequently been convicted, but the latter had been able to submit a plea for clemency which was supported by the public prosecutor. in moll s view, a case like this was a matter of personal, conscientious decision - making for the doctor. many doctors, it seems, did not report abortions. moll s views on medical confidentiality were differentiated. while he leaned towards protecting patients confidence on the issue of abortion, in 1905, he successfully supported the defence of a berlin doctor who was accused of a breach of professional secrecy under section 300 of the german penal code for having warned the relatives of a syphilitic patient of the danger of infection. we can conclude from moll s argumentation in the three examined ethical issues of truth - telling, euthanasia and abortion that the self - determination of the patient was a central factor. it competed, to some extent, with the best interest of the patient especially in the case of truth - telling but unlike other writers on medical ethics at the time, such as pagel and scholz, moll was not a paternalist. patient relationship committed him to the well - being and interests of the individual patient, and this included respect for the patient s wishes. in this sense, moll s theory of medical ethics might be seen as a forerunner of the modern concept of patient autonomy, although his practical conclusions were rather moderate and often in line with medical traditions. the patient s consent to treatment, after adequate information and advice, was a crucial element in moll s ethics. this goes some way to explain his position and actions on the issue of clinical experimentation on hospital patients, to which i will turn in the following sections. in his medical ethics, moll provided a summary of about 600 research papers that explicitly or implicitly reported non - therapeutic experimental interventions on human subjects. he had collected these publications from the international literature, as he emphasised : from germany, austria, switzerland, france, italy, england, russia, norway, sweden, denmark, romania, the united states, chile and egypt. many of the examples that moll cited indicated that patients had been harmed, or at least molested or exposed to risks, through experimentation. moreover, in many cases, the human subjects did not seem to have been informed about the nature and implications of the experiment, nor been asked for their consent to being a subject in a trial. typically, the experiments had been carried out on hospital patients or other institutionalised persons, such as orphans or prisoners. while moll s tone in his medical ethics was generally neutral and considered, his outrage about abuses in human experimentation became clearly recognisable at this point : i have observed with increasing surprise that some medics, obsessed by a kind of research mania, have ignored the areas of law and morality in a most problematic manner. for them, the borderline between human beings and animals is blurred for them. the unfortunate sick person who has entrusted herself to their treatment is shamefully betrayed by them, her trust is betrayed, and the human being is degraded to a guinea pig. some of these cases have occurred in clinics whose directors can not talk enough about i have observed with increasing surprise that some medics, obsessed by a kind of research mania, have ignored the areas of law and morality in a most problematic manner. for them, the borderline between human beings and animals is blurred for them. the unfortunate sick person who has entrusted herself to their treatment is shamefully betrayed by them, her trust is betrayed, and the human being is degraded to a guinea pig. some of these cases have occurred in clinics whose directors can not talk enough about there seem to be no national or political borders for this aberration. despite its international dimension, alerted by the scandal surrounding the syphilis experiments of albert neisser, which were discussed in the prussian parliament, the prussian minister for religious, educational and medical affairs [minister der geistlichen, unterrichts- und medizinal - angelegenheiten ] had commissioned, in march 1899, an expert report on human experimentation from the ministry s scientific committee for medicine [wissenschaftliche deputation fr das medizinalwesen ]. neisser himself, as a university professor, was punished with a reprimand and a fine by the royal disciplinary court for civil servants [kniglicher disziplinarhof fr nicht - richterliche beamte ] because he had failed to obtain consent from the parents or guardians of the minors on whom he had experimented. the syphilis serum injections given to those of his subjects who were prostitutes were, however, regarded by the court as legitimate therapeutic trials, because they might have protected the subjects against the risk of catching syphilis through their occupation. as a kind of immunisation therapy for prostitutes, who could legally be subjected to compulsory medical treatment, these trials did not require consent. this distinction between therapeutic and non - therapeutic trials was central in a directive that the prussian minister issued to the directors of hospitals and clinics on 29 december 1900. it required information about the risks and consent of the subjects in scientific, non - therapeutic trials, but did not make this a requirement for interventions that served therapeutic, diagnostic or immunisation purposes. moreover, the directive made the hospital or clinic directors personally responsible for the scientific trials, which had to be documented including the compliance with information and consent requirements in the patients files. non - therapeutic experiments on minors and other persons lacking full legal competence were forbidden. moll criticised these regulations in medical ethics, arguing that on the one hand they did not provide enough protection for the human subjects, and that on the other, they went too far in their requirements. they went too far in ruling out scientific experiments on minors ; for example, taking a blood sample from a twenty - year - old within a scientific study would not be allowed in prussia. but the regulations were not stringent enough, as they did not make clear whether the therapeutic, diagnostic or immunisation purposes referred to the individual patient or to all interventions of this type, regardless of whether they might be useful to the individual subject concerned. moll also doubted that simple recording in the patient s file was sufficient to ensure that full information had been given and valid consent obtained. he was aware of the authoritarian milieu in the hospitals and university clinics of the time, in which the mostly lower - class patients were expected to follow doctors orders. many patients would also lack the education to fully comprehend the implications of a proposed trial. moll therefore demanded a guarantor for the proper information and consent of the subjects, and written consent for serious interventions. beyond this critique in his book on medical ethics, moll became personally involved in the issue. with the exception of the cases that were well known through the press, such as those of neisser and strubell, moll deliberately omitted the names of the experimenters in his account of problematic human trials in medical ethics. in a preceding article on the issue of human experimentation, published in november 1899 in the critical political weekly die zukunft his aim was to draw attention to abuses in clinical trials as a general problem, not to individual researchers such as neisser. after publication of this article, however, moll was approached by friedrich althoff (18391908), the influential ministerial director in the prussian ministry for religious, educational and medical affairs, who requested to see moll s material on this topic. the request was sent on 30 december 1900, just one day after the minister s directive on scientific trials had been issued. obviously, the ministry planned investigations, and possibly disciplinary proceedings, against some of the researchers who were resident in prussia. in early january 1901, moll replied to althoff s letter, stating that he was willing to give access to his material under two conditions : that it was explicitly acknowledged by the ministry that he, moll, had supplied the information upon althoff s request ; and that he was given an assurance that his material was not going to be used in investigations of individual persons. rather, he wanted his material to provide background information for the drawing up of future regulations on human trials. as can be gathered from a memorandum of the ministerial official ludwig elster (18561935) at the end of february 1901, moll was subsequently told that the assurance mentioned in his second condition could not officially be granted. moll then continued to refuse permission for access to his material, but agreed that the ministerial official could meet with him privately to look into his documentation, provided he was given the requested assurance by this official. after elster had given moll the assurance, he was able to study the material, a total of ninety - five cases, and to take notes. as elster remarked, however, most cases were totally harmless. the ministry does not seem to have taken much further action on the issue of human experimentation in the following years. as barbara elkeles has found in her research on this matter, only six cases were investigated by the ministry between 1900 and 1913, but were not pursued any further due to when moll described the affair in his memoirs of 1936, he indicated that his reluctance to reveal the identity of the experimenters had led to dissonances with the ministry, and he associated these with his failure to obtain an allegedly planned titular professorship at berlin university. the incident had, in fact, drawn the ministry s attention to moll as a person and physician. after moll had published, in march 1901, another critical article on the issue of human experimentation, this time in the popular illustrated news magazine die woche, elster requested, on behalf of the ministry, a report on moll from the berlin chief of police, specifically on moll s personal circumstances and his standing in medical circles. the report, authored by a medical civil servant in the police department, richard wehmer (18541909), was highly ambivalent. good, that he lived unmarried in orderly and prosperous financial circumstances in his berlin flat, and that his recent scientific works had found recognition, but on the other hand, it pointed out that moll had specialised in hypnotism and had fallen out with leading experts over scientific questions ; that he had attacked the work of asylum doctors in the journal die zukunft ; and that he liked to study sexual perversions and had served as an expert in trials against sexual offenders. the report even contained a piece of innuendo, saying that detectives were often seen in his flat, that prostitutes were among his patients, and that there were rumours that something mysterious was going on at his home. the account ended with the observation that moll did not vote in the 1893 general elections, and that he was a member of the progressive party [fortschrittspartei ], but that he had never attracted the attention of the police through any political campaigning. the report was filed in the ministry and no further action seems to have been taken. the whole affair revealed a pattern in moll s behaviour : engagement in a public debate on the ethics of medicine ; confident and almost arrogant dealings with the authorities ; and anxious concern for his reputation as well as the reputation of the medical profession. moll was aware of the broader implications of the contemporary debate on human experiments, which included a general lay criticism of scientific medicine and of medical authority, especially as practised in hospital. to the extent that mostly working - class patients were the subjects of clinical experimentation, the issue had also a political dimension. the case of strubell, for example, had been made public by the social democratic newspaper vorwrts. friedrich scholz, in his widely read ethical reflections von aerzten und patienten [on doctors and patients ] (first published in 1899), noted complaints about the autocratic attitude of some hospital physicians who regarded the patients that were dependent on them as material and abused them for the sake of science. syphilis inoculations, made out of scientific curiosity and with the knowledge that they would do harm, were in his eyes criminal. moreover, the public attacks against neisser had anti - semitic undertones, something that moll, who had converted from judaism to protestantism in 1895, as the police report on him noted, is likely to have felt. julius pagel, who, like neisser, was jewish, defended the latter s controversial human trials on syphilis immunisation by explaining their scientific context and medical significance. to a degree, moll s manoeuvring between going public and protecting himself and the profession becomes understandable from a consideration of these various contexts. however, a similar behavioural pattern became apparent again a few years later when moll became involved in the so - called in october 1908, the medical society of berlin - schneberg [verein der schneberger aerzte ] began an investigation of medical practitioners referrals of foreign private patients to specialist consultants in berlin hospitals and university clinics. the main target of this investigation was the russian institute for medical consultations in berlin [russisches institut fr medizinische consultationen zu berlin ], run by doctors semjon lipliawsky (born c.1875) and siegfried weissbein (born c.1877), who arranged, for a fee, consultations with leading medical specialists for russian patients visiting the german capital. there were also other such institutes, as well as individual agents in berlin, who provided this service to foreign visitors. allegedly, these middlemen were not only paid by the patients but also by the clinicians consulted. for the medical practitioners of schneberg this would constitute unfair competition for lucrative private patients. through a series of appeals in the berliner rzte - correspondenz ie. the official newsletter of the berlin medical chamber and journal of the local medical professional societies the deputy chairman of the medical society of berlin - schneberg, julius friedemann (b. 1858), called for information on such practices, in the interest of the professional dignity [standeswrde ] of the german and especially the berlin medical profession. clearly, the intention was to initiate disciplinary proceedings against consultants who made underhand payments to middlemen for bringing them private patients. moll, as chairman of the so - called committee of fifteen [fnfzehner - ausschu ], a group representing the interests of the medical profession in the area of berlin, soon sided with friedemann in this campaign and himself collected the explosive nature of the issue became quickly apparent as ernst von leyden (18321910), the doyen of internal medicine and former director of the first medical clinic of the charit - hospital of berlin university, had agreed to be a consultant for a planned outpatient clinic of the russian institute for medical consultations. after friedemann and moll had personally spoken with him, von leyden publicly retracted from this agreement. soon, however, another prominent clinician, karl anton ewald (18451915), director of the department for internal medicine at the augusta - hospital in berlin and professor at berlin university, came under suspicion of being involved in the patient trade. such a suspicion was all the more delicate because ewald was, at the time, a candidate in the elections for the council of the prestigious berlin medical society [berliner medizinische gesellschaft ]. as in the case of von leyden, private conversations took place. on behalf of ewald, hermann senator (18341911), the chairman of the berlin medical society and director of the third medical clinic and outpatient clinic of the charit - hospital, followed moll s invitation to speak with him and friedemann about the matter at moll s home. tricked him on this occasion to implicate himself in the dubious practices surrounding russian private patients. the contents of the conversation became public in a libel trial in may 1909, after senator had been mentioned, in an article in the newspaper berliner zeitung am mittag in march 1909, as one of those making payments to the russian institute for sending patients to them. as moll later declared as a witness in disciplinary proceedings against ewald, he also had, in early january 1909, a private conversation about the issue with the latter. before the trial of senator against the editor of the newspaper, however, further revelations about the patient trade and the russian institute had been made in a libel trial of moll against dr albert levin (b. 1867), a member of the medical society of berlin - schneberg, in march 1909. levin belonged to the society s committee that had taken on the investigation of this matter. when levin asked moll to give him access to all his collected material on the patient trade issue, moll refused. incensed about this lack of collaboration, levin wrote a letter to moll, in which he accused him of cowardice or of holding back the material for improper reasons, or of actually having no further material at all. initially, moll had taken the view that one should deal discreetly with the patient trade issue, in order to stop abuses but not to expose individual colleagues. however, by the time of the trial against levin his tactics had clearly changed. moll now used the trial to make the conduct of the berlin university professors public, and friedemann became his ally in this enterprise. in his witness statement, friedemann reported that von leyden, senator, and the urologist carl posner (18541928), who was an extraordinary professor at berlin university, had admitted to having offered or made payments to middlemen from the russian institute for medical consultations or other agents that had brought them private patients. the judge, keen to limit proceedings to the specific issue of libel, concluded that the payments described had actually happened, that moll had made material on this matter available to friedemann, and that the tone of levin s letter to moll was libellous. the appeal proceedings, held in late may 1909, shortly after the libel trial of senator against the editor of the berliner zeitung am mittag in which moll appeared as a witness this time, moll s lawyer submitted an extensive motion to take evidence, in which he gave further details about the berlin medical professors relationships with the russian institute. dr weissbein, as one of the russian institute s directors, had a defensive letter read out at the trial, in which he suggested that moll and levin had stage - managed the whole libel case to obtain a public platform for their accusations against the professors and the institute. the trial ended with a settlement : levin acknowledged that moll had had honourable reasons not to release his substantial material, withdrew the accusations that he had made against moll in his letter, and took on the costs for the trial. the details of the trials were not only reported and commented upon in the medical press, such as the berliner rzte - correspondenz and the berliner klinische wochenschrift, but also in newspapers across the political spectrum. the allegations that prominent members of the berlin medical establishment had paid bribes to get private patients provided the right sort of material for a public scandal. as in the neisser case a few years earlier, anti - semitic comments were made. obviously being aware that moll, friedemann, levin and senator, as well as lipliawsky and weissbein, had a jewish background, the deutsche tageszeitung and das reich emphasised that jewish doctors were prominently involved in the affair. when the matter was also mentioned in the lower house of the prussian parliament [abgeordnetenhaus ], the ministry for religious, educational and medical affairs promised to investigate the allegations. at their own request, weissbein and friedemann initiated court of honour proceedings against themselves in order to have their conduct vindicated. in the disciplinary proceedings against the three professors, moll was heard four times as a witness by the university judge and government official, geheimer regierungsrat (senior executive officer) dr daude. the minister for religious, educational and medical affairs, to whom reports on the hearings were sent, asked daude to obtain more detailed evidence from moll because the latter initially refused to confirm that the accused professors had paid the middlemen specifically for bringing patients. only after daude threatened legal action against moll himself if he refused to give full evidence did moll provide further details that incriminated ewald and posner. in the end, however, none of the involved professors was found guilty. while it was noted that the medical professors concerned had occasionally given money to agents who had brought them patients, the ministry accepted their justification that these payments had just been small tips or small rewards for having acted as interpreters, or for having assisted when the russian patients were seen and examined by the professors. the disciplinary proceedings against senator and ewald were abandoned in november 1909, as were proceedings against the elderly and ill von leyden which had been initiated only in the previous month. posner, who had regularly paid one specific agent, the russian interpreter and health assistant bernhard rosenberg (born c.1860) had to face a full trial by the royal disciplinary court for civil servants in january 1910, but was acquitted. with his intervention in this issue of professional conduct moll expert in medical ethics, but clearly at a reputational cost. while his name was now all over the newspapers, and he had turned himself into a champion of the economic interests of berlin s medical practitioners, his relationship to university medicine had suffered further damage ; for example, in the widely reported libel trial of senator against the berliner zeitung am mittag, senator sneeringly called moll and friedemann... those two gentlemen, who make their appearance as guardians of medical ethics and of collegiality. the young doctor and medical journalist hans lungwitz (18811967), who had recently completed his medical training in ewald s department, published a series of polemical articles about the affair in his weekly therapeutische rundschau, in which he ridiculed moll as god s ethicist [ethiker von gottes gnaden ] and as a medical sherlock holmes who had interrogated the professors in his flat. lungwitz also shared weissbein s suspicion that the moll vs levin libel case had been staged by these two in order to go public with their allegations. as lungwitz claimed, moll s and friedemann s campaign against the berlin professors had damaged the international reputation of german medicine and was anti - german [deutschfeindlich ]. the latter remark may well have been understood by informed readers as an anti - semitic gesture towards the jewish descent of both friedemann and moll. in principle, moll had repeated his strategy to intervene in an ethical issue, but then to try to protect his reputation and that of the profession by withholding incriminating material until forced to release it to the authorities ; but, as in the issue of human experimentation, such manoeuvring put him in an unfavourable light. guardian of medical ethics [wchter der medizinischen ethik ] by his medical peers was rather derogatory, and, apparently, after the patient trade affair, moll s continuing membership as a representative in the berlin medical chamber was called into question by some of his colleagues. from my analysis of moll s medical ethics, there appears to be a discrepancy between the appreciation that they might deserve from a present - day, ethical point of view, and the historical impact they actually made. his focus on the contract relationship between doctors and patients gave space to the self - determination of the patient, though he moderated patients wishes with what he thought to be in the patient s best interest. moll can thus be seen, to some extent, as a pioneer of the concept of patient autonomy in medical ethics. carefully discussing conflicts of interest in many areas of medicine, his voluminous text on medical ethics was the most elaborate consideration of the topic at this time. his contemporaries, however, do not seem to have picked up his point about respect for patients wishes. a friendly but bland review in the mnchener medizinische wochenschrift by the hamburg gynaecologist karl jaff (18541917) asserted that moll s book was a true reflection of the current views on medical ethics and praised his impartial and objective point of view. incoming as well as experienced doctors might learn from it, suggested jaff, but he also expressed his general doubts about the practical usefulness of writings on medical ethics. a similar point was made by the reviewer for the deutsche medizinische wochenschrift, the berlin physician leopold henius, who thought that it was more helpful to seek the advice of an experienced colleague in doubtful cases than to plough through a voluminous handbook. many things in moll s book were not new, claimed henius, and other parts, such as that on the perforation of the fetus, too sophisticated to be convincing. both jaff and henius acknowledged moll s enormous industriousness in writing medical ethics, and, like jaff, henius acknowledged moll s calm, noble and objective tone. but in the end, henius assessment must have been frustrating for moll : on the whole () we have here an industrious work which deserves recognition, and we wish it a large readership, but, for the above - mentioned reasons, we do not expect that it will have it. some reviewers in less prominent medical journals more warmly recommended moll s book to their readership. generally appreciative were also the reviews in the viennese medical periodicals. however, henius prediction seems to have been correct if one considers that there was no further german edition of moll s medical ethics. the reception of the book in the legal profession was perhaps more positive ; for example, melchior stenglein (18251903), formerly an official at the german supreme court, praised, in a review in the legal journal der gerichtssaal, the similarity of moll s views to current legal opinion on the issues of euthanasia and consent to surgery. likewise, the berlin lawyer erich sello (18521912), a friend of moll s, emphasised, in his review, the many links to legal questions that the book provided. in fact, legal publications on aspects of medical practice, such as consent and confidentiality, regularly cited moll s work on ethics while often ignoring other medical literature. it would be an exaggeration, however, to say that moll s medical ethics was a success, despite its two translations into russian. the editor of the 1904 translation, the marxist medical doctor and writer vikentii veresaev (18671945), characterised moll as a cautious, moderate, and prudent philistine, who is devoid of noble purpose and saw parts of the book as illustrating the bourgeois outlook of the modern, ordinary german physician. russian reviewers of medical ethics criticised it for its lack of a foundation in a philosophical system and for its application of the term moll himself, in his memoirs of 1936, repeatedly claimed that his outrage about abuses in experimentation on hospital patients had made him feel compelled to write medical ethics. in view of his later active engagement in medical politics, as a member of the berlin medical chamber from december 1908, one may also assume that a more general interest in questions of professional conduct had been a motivating factor. his emphasis on self - determination of the patient might have been a reflection of the specific circumstances of his own practice, in which he saw private patients who paid for the consultations themselves. only in the 1980s and 1990s, when medical ethics and bioethics became increasingly important subjects in german academia, did historians of medicine engage with the contents of moll s book on medical ethics. situation ethics in moll s text, and to his subjective response to the rise of science in medicine, as exemplified by the issue of human experimentation. julius henri schultz, in a short monograph on moll s medical ethics in 1986, emphasised the latter s closeness to legal thought and philosophical positivism ; and for antonia eben, in her medical doctoral dissertation of 1998, the his collected materials on human experimentation and his criticism of the prussian ministerial directive of 1900 did not lead to revisions of the regulations, as he had hoped. his activities in the patient trade affair also failed to effect disciplinary punishment for the professors involved. on the other hand, the publicity that his interventions in this matter gained may well have put a temporary stop to the practice of clinicians payments to middlemen. however, a hundred years on, when in august and september 2009 a new patient trade affair went through the german media, nobody remembered that berlin had experienced all this before	in 1902, albert moll, who at that time ran a private practice for nervous diseases in berlin, published his comprehensive book on medical ethics, rztliche ethik. based on the concept of a contractual relationship between doctor and client, it gave more room to the self - determination of patients than the contemporary, usually rather paternalistic, works of this genre. in the first part of the present paper this is illustrated by examining moll s views and advice on matters such as truthfulness towards patients, euthanasia, and abortion. the second part of this article discusses how moll engaged with the then publicly debated issues of experimentation on hospital patients and the trade of foreign private patients between agents and medical consultants. in both matters moll collected evidence of unethical practices and tried to use it to bring about change without damaging his or the profession s reputation. however, with his tactical manoeuvres, moll made no friends for himself among his colleagues or the authorities ; his book on ethics also met with a generally cool response from the medical profession and seems to have been more appreciated by lawyers than by other doctors.
to react appropriately to danger, both animals and humans must rapidly marshal a complex set of behavioral responses. the locus ceruleus (lc), which is located in the dorsal pons, plays a crucial role in activating central and peripheral nervous system responses to threat. through its broad connections with cortical structures, the hippocampus, hypothalamus, amygdala, and spinal cord, the lc organizes affective, cognitive, and motor responses to acute stressors. activation of lc neurons leads to secretion of norepinephrine (ne), which recruits the multiple pathways involved in modulating behavioral responses to acute stressors. for example, upon receiving electrical stimulation to their locus ceruleus, restrained monkeys will immediately wake up and exhibit behaviors such as head and body turning, eye scanning, tongue movement, hair pulling, and escape struggling. these behavioral responses are similar to those elicited when they arc threatened in their natural environment. repeated exposure to a stressful stimulus leads to increased ne secretion and facilitates the process of behavioral stress sensitization, whereby the animal develops a heightened behavioral response to further presentations of the same stimulus. exposure to severe and repeated stress depletes brain ne concentrations and leads to behavioral changes such as decreased exploration in a plus - maze novelty task, decreased appetite, and deficits in previously well - learned behavioral tasks. such behavioral changes induced by chronic stress have been characterized by the term learned helplessness. these animal models differ from ptsd in that the development of stress sensitization and learned helplessness requires repeated exposure to stressful stimuli, while ptsd can develop after only a single exposure to traumatic stress. despite this important difference, stress sensitization and learned helplessness models are useful in explaining behavioral changes associated with ptsd, such as heightened reactions to traumarelated stimuli and decreased interest in usual - life activities. through its reciprocal connections with the amygdala, the lc / nf, axis also mediates classically conditioned fear responses in animals. in this model, the repeated pairing of a neutral stimulus such as a bright light with a noxious stimulus, such as an electrical shock, eventually results in a conditioned fear response to the previously neutral stimulus when it is presented alone. reactivation of the neuronal connections between the lc and amygdala that are established during acute stress exposure may explain the failure of animals to extinguish stress - related associations. conditioned fear patterns may underlie features of ptsd such as heightened arousal responses to ordinary noises and increased avoidance behaviors, while failure of extinction may subserve persistent alarm reactions to reminders of past trauma. research on noradrenergic function in ptsd includes hormone- and receptor - binding assays, assessment of autonomic reactivity, and pharmacological probes involving central 2-adrencrgic receptor agonists. one method to assess noradrenergic function in ptsd has been to measure plasma nf, levels or levels of ne metabolites in 24-hour urine collections. studies have found increased urinary concentrations of ne among hospitalized ptsd patients compared with hospitalized patients with other mental disorders. other investigators have noted decreases in the density of platelet cell 2-adrenergic receptors in combat veterans with ptsd and in traumatized children. reduction of these ne - binding receptors may indicate an adaptive downregulation in response to chronically elevated plasma ne levels. since the noradrenergic axil also modulates peripheral autonomic responses, investigators have also assessed noradrenergic function in ptsd by comparing autonomic sesponses in ptsd subjects and controls. automnomic measures in these studies have includes heart rate, systolic and diastolic blood pressure, and galvanic skin responses. while early studyies noted baseline autonomic differences between combat veterans with ptsd and non - ptsd controls, later studies did not control for the effects of anticipatory anxiety and study demand characteristics. studies that have compared autonomic responses in ptsd and non - ptsd subjects to stressful but nontraumatic stimuli such as having to perform arithmetic caclulations or watch unpleasant films have not identified autonomic differences between ptsd subjects and controls. thus, there is little evidence to suggest that ptsd involves changes in resting autonomic function or in autonomic responsivity to nontraumatic stimuli. in contrast to these negative findings, there is compelling evidence to indicate that individuals with ptsd exhibit an increased autonomic responsivity to trauma - related stimuli. compared with traumaexposed controls, ptsd subjects exhibit greater autonomic arousal to trauma - related stimuli such as audiotapes of combat sounds, videotapes of war zone scenes, and trauma - related smells. pitman noted increased autonomic arousal in ptsd subjects using a script - driven imagery technique in which trauma survivors listened to their own trauma narrative while viewing trauma - related slides. these findings prompted a multisite veterans affairs cooperative study to evaluate the diagnostic utility of psychophysiological assessments in vietnam combat veterans with ptsd. this study included three groups : veterans with current ptsd (n=778), veterans with lifetime but not current ptsd (n=181), and veterans who never had ptsd (n=369). using physiological variables alone, researchers correctly classified 67 % of the current ptsd group and a similar percentage of the non - ptsd group. collectively, these studies suggest that increased autonomic reactivity to traumatic stimuli is an important feature of manyindividuals with ptsd. investigators have also examined noradrenergic axis activity in ptsd using pharmacologic probes such as yohimbine, which activates noradrenergic neurons in the lc region by blocking inhibitory 2-adrenergic autoreceptors at the presynaptic terminal. southwick found that after receiving yohimbine, a subset of ptsd patients not only exhibited physiological arousal such as increased heart rate and blood pressure, but also developed severe anxiety symptoms including acute panic attacks and increased ptsd symptoms such as intrusive thoughts, flashbacks, and emotional numbing. morgan demonstrated that yohimbine infusion enhanced acoustic startle responses in combat veterans with ptsd, but did not affect startle responses in combat veterans without ptsd. consistent with psychophysiologic findings, these result further support the hypothesis that increased noradrenergic responsivity is a core biological feature of ptsd. in addition to activating the noradrenergic system, exposure to acute stress elicits important neuroendocrine changes that are modulated by the hpa axis. in response to acute stress, corticotropin - releasing hormone (crh) crh is a 41-amino - acid peptide that is transported to the anterior lobe of the pituitary gland where it stimulates pituitary secretion of adrenocorticotropic hormone (acth). acth enters the systemic circulation and binds to cells in the adrenal cortex, thereby stimulating the secretion of cortisol. cortisol binds to the type i and type ii glucocorticoid receptors that are present on cell membranes and activates a cascade of physiologic stress responses involving altered metabolism, increased cellular uptake of glucose, modulation of immune activity, and induction of hepatic enzymes. cortisol also blocks further secretion of crh and acth, thereby curtailing the acute stress response once the stress is over. this is a crucial function of cortisol, since uncontrolled activation of acute stress hormones can significantly harm host tissue. there is clear evidence from animal studies that persistent activation of the hpa axis by chronic and repetitive stress can have deleterious effects such as the acceleration of aging, disruption of reproductive function, immunosuppression, and reduced ability to fight cancers : these findings have been reviewed by johnson. noting that increased hpa axis activity is associated with chronic stress in preclinical studies, investigators initially predicted that individuals with ptsd would have elevated plasma cortisol levels and would fail to suppress cortisol levels after being administered dexamethasone. however, evidence indicates that hpa axis patterns in ptsd are quite different from patterns seen in studies of chronic nontraumatic stress. ' mason and colleagues first noted that veteran inpatients with ptsd had lower 24-hour urinary cortisol levels than other psychiatric inpatients. this finding has been replicated in studies involving both psychiatric and healthy controls and in other trauma - exposed populations such as holocaust survivors, rape victims, and adolescents exposed to a natural disaster. differences in study results may reflect differences in study settings, different assay techniques, and patient differences such as inpatient versus outpatient status, obesity, substance abuse, use of medications, and comorbid illnesses. two studies have found that cortisol release in ptsd patients is comparable to that of healthy subjects during the daytime (7 am to 7 pm), but significantly lower during the late evening and early morning, leading to wider diurnal fluctuations in the ptsd group. other studies have examined target receptor alterations in ptsd and have identified increased glucocorticoid receptors on lymphocyte cell membranes in ptsd subjects compared with controls. in addition to activating the noradrenergic system, exposure to acute stress elicits important neuroendocrine changes that are modulated by the hpa axis. in response to acute stress, corticotropin - releasing hormone (crh) crh is a 41-amino - acid peptide that is transported to the anterior lobe of the pituitary gland where it stimulates pituitary secretion of adrenocorticotropic hormone (acth). acth enters the systemic circulation and binds to cells in the adrenal cortex, thereby stimulating the secretion of cortisol. cortisol binds to the type i and type ii glucocorticoid receptors that are present on cell membranes and activates a cascade of physiologic stress responses involving altered metabolism, increased cellular uptake of glucose, modulation of immune activity, and induction of hepatic enzymes. cortisol also blocks further secretion of crh and acth, thereby curtailing the acute stress response once the stress is over. this is a crucial function of cortisol, since uncontrolled activation of acute stress hormones can significantly harm host tissue. there is clear evidence from animal studies that persistent activation of the hpa axis by chronic and repetitive stress can have deleterious effects such as the acceleration of aging, disruption of reproductive function, immunosuppression, and reduced ability to fight cancers : these findings have been reviewed by johnson. noting that increased hpa axis activity is associated with chronic stress in preclinical studies, investigators initially predicted that individuals with ptsd would have elevated plasma cortisol levels and would fail to suppress cortisol levels after being administered dexamethasone. however, evidence indicates that hpa axis patterns in ptsd are quite different from patterns seen in studies of chronic nontraumatic stress. ' mason and colleagues first noted that veteran inpatients with ptsd had lower 24-hour urinary cortisol levels than other psychiatric inpatients. this finding has been replicated in studies involving both psychiatric and healthy controls and in other trauma - exposed populations such as holocaust survivors, rape victims, and adolescents exposed to a natural disaster. differences in study results may reflect differences in study settings, different assay techniques, and patient differences such as inpatient versus outpatient status, obesity, substance abuse, use of medications, and comorbid illnesses. two studies have found that cortisol release in ptsd patients is comparable to that of healthy subjects during the daytime (7 am to 7 pm), but significantly lower during the late evening and early morning, leading to wider diurnal fluctuations in the ptsd group. other studies have examined target receptor alterations in ptsd and have identified increased glucocorticoid receptors on lymphocyte cell membranes in ptsd subjects compared with controls. to evaluate the dynamic responsivity of the hpa axis in ptsd, investigators have used exogenous hormones that stimulate or inhibit the hpa axis at a specific locus. a well - established paradigm involves measuring acth and cortisol levels after administering dexamethasone. dexamethasone is a synthetic glucocorticoid that mimics the negative feedback effects of cortisol on the hpa axis. it inhibits acth release from the pituitary gland, which subsequently leads to a decrease in scrum cortisol levels. four studies have reported that, in response to dexamethasone, individuals with ptsd demonstrate a more robust suppression of acth and cortisol release that normal controls. this contrasts with the nonsuppression of cortisol levels seen in almost half of all depressed patients after dexamethasone administration. other studies have measured acth and cortisol levels after infusing crh, which stimulates the pituitary gland to release acth. two studies found decreased acth responses to crh infusion in adult ptsd subjects compared with controls, while another study found no differences in acth response to crh infusion in sample of adolescent girls. finally, metyrapone, which blocks the synthesis of cortisol in the adrenal gland, has been used to examine hypothalamic and pituitary responses to decrease cortisol in ptsd. one study found that ptsd patients show larger increases in acth levels following metyrapone administration that do normal controls. these findings led yehuda and colleagues to propose that ptsd may involve an hpa axis that is characterized by enhanced sensitivity to feedback inhibition. according to this model, individuals with ptsd experience chronic and recurrent stress events that lead to increased secretion of crh. pituitary sensitivity to crh decreases the need to compensate for increased crh release, as reflected by blunted acth responses to crh infusion. to protect against the toxic effects of elevated cortisol, the hpa axis in ptsd becomes increasingly sensitized to feedback inhibition from cortisol through upregulation of glucocorticoid receptors and other mechanisms. this is evidenced by low baseline acth and cortisol levels and robust suppression of acth and cortisol release after dexamethasone administration. by tightly controlling cortisol secretion and responding aggressively to acute rises in cortisol levels, the neuroendocrine system may serve to buffer vulnerable neuronal structures such as the hippocampus from cellular toxicity induced by elevated scrum cortisol levels. while evidence that severe stress can affect noradrenergic and neuroendocrine function has been well - established, recent animal studies have identified important neurotic effects of stress - mediated increases in glucocorticoid levels. one neuroanatomical structure that appears to be particularly susceptible to stress - induced damage is the hippocampus, which is involved in learning and memory circuits. studies of monkeys exposed to the stressors of disrupted attachment found damage to cells in the hippocampal region ; similar patterns of cell damage could be induced by implanting glucocorticoids directly into the hippocampus. this suggests that elevated glucocorticoid levels, such as might occur acutely during exposure to traumatic stress, could lead to hippocampal damage. other studies examining stress - induced hippocampal damage in mice have identified important memory deficits that are correlated with the extent of hippocampal damage, suggesting that structural damage to the hippocampus may also be associated with functional memory deficits. these findings have led investigators to hypothesize that ptsd may be associated with hippocampal changes resulting from either the acute neurotoxic effects of elevated scrum cortisol during exposure to traumatic stress or the gradual deterioration resulting from glucocorticoid - mediated effects of chronic stress. using magnetic resonance imaging (mri) techniques to measure hippocampal volume, brcmner compared hippocampal size in 26 male vietnam combat veterans with ptsd and 22 healthy controls, and found a statistically significant 8 % reduction in right hippocampal volume in the ptsd group. however gurvits and colleagues compared hippocampal volumes in veterans with ptsd (n=7) and matched controls (n=7). the ptsd group showed bilateral reductions in hippocampal size (26 % on the left, 22 % on the right), which remained significant after controlling for age, brain volume, drinking history, and combat exposure. total hippocampal size was negatively correlated with combat exposure (r=0.72, p=0.003) and number of ptsd symptoms (r=0.78, p=0.0q1), but only weakly associated with memory performance. examining a different population, bremner compared hippocampal volumes in adult child abuse survivors with ptsd (n=17) versus healthy controls (n=17) and found a statistically significant 12 % reduction in left hippocampal size in the ptsd group after controlling for alcohol use, age, and educational status. however, hippocampal volume was not associated with memory deficits, number of ptsd symptoms, or exposure. finally, stein examined hippocampal volumes in 21 female survivors of childhood sexual abuse with ptsd and 21 nonvictimized controls, and noted a statistically significant 5 % reduction in left hippocampal size in the abused group. combining mri measurements with proton magnetic resonance spectroscopy (mrsi), schuff observed a 6 % decrease in right hippocampal volume which was associated with an 18 % decrease in hippocampal activity as measured by the ratio of jy - acetyl aspartate signal activity to that of choline and creatinine. their results suggest that utilizing mrsi measurement may enhance our ability to detect subtle hippocampal changes in ptsd. while the above studies included only adults, de bellis compared hippocampal size in 43 abused children with ptsd and 61 matched controls, and found no corresponding decrease in hippocampal volume in the ptsd group. collectively, these studies provide preliminary evidence that changes in hippocampal size and function may be an important feature of chronic ptsd. the findings reviewed in this paper provide tantalizing new insights into ptsd and offer the promise of a richer understanding of this complex disorder. however, for these findings to be truly meaningful, important empirical questions need to be addressed. most studies have employed a cross - sectional design and included ptsd subjects who suffer from comorbid disorders such as major depression or alcohol abuse. ' ittiis makes it difficult to identify whether a biological finding associated with ptsd represents a premorbid condition, reflects the impact of a comorbid disorder, or actually results from prsd there is a need for prospective longitudinal studies to measure biological variables prior to the onset of ptsd and track their change across time. furthermore, animal models of ptsd have primarily examined biological responses that develop over days to weeks : findings from such animal models may be less applicable to a disorder such as ptsd, which develops over a period of months to years. improved animal paradigms are needed to anchor future research in the biology of ptsd. another critical issue is to determine which biological responses in ptsd are similar to biological stress responses in other populations, and which biological patterns are uniquely associated with ptsd. exciting research lies ahead and promises to advance our scientific understanding of this major public health challenge.	since posttraumatic stress disorder (ptsd) was first recognized as a psychiatric disorder, it has generated a great deal of scientific interest. recent studies on the neurobiology of ptsd provide evidence that ptsd is biologically distinct from other types of traumatic and nontraumatic stress responses. this paper reviews three important directions of neurobiological research in ptsd : noradrenergic axis changes and associated alterations in autonomic responsivity neuroendocrine changes involving the hypothalamic - pituitary - adrenocortical (hpa) axis, and neuroanatomy changes involving the hippocampus. each section reviews the salient aspects of preclinical research on the biology of stress and their bearing on the understanding of ptsd, and summarizes prominent findings from clinical biological studies of ptsd, tentative models that integrate current findings from the clinical study of ptsd are reviewed. to conclude, the important methodological and empirical issues that need to be addressed by future studies are indicated.
a 28-year - old unmarried male from low socioeconomic status came to the hospital for consultation of psychiatric problems along with his caregivers. the presenting complaints were unprovoked aggressive outbursts, poor self - care, disorganized behavior and self - mutilating behaviors for last 4 years. physical examination revealed old burnt scar over the forehead, fused ear lobules bilaterally and polydactyly in right upper limb and crush injury of left thumb and index finger. on mental status examination, he was diagnosed as undifferentiated schizophrenia according to international classification of disease-10 version. patient was advised to take in - patient psychiatric care on the same day of consultation. he remained aggressive, disorganized and on two occasions, he tried to crush his left index finger and thumb by hammering it with a stone. he was started on electro convulsive therapy (ect) sessions considering aggressive outburst and self - mutilating behavior. during pre - ect evaluation computed tomography scan of the head revealed grade ii csp [refer figure 1 ]. along with ect, patient also received oral chlorpromazine 600 mg / day, which was hiked gradually over 2 weeks. plain computed tomography image of the patient and arrow head pointing towards cavum septum pellucidum patient started showing improvement in aggressive and self - mutilating behavior. on mental status examination, he reported that he became angry for no apparent reason, following which he would start hammering his fingers with stones. he received nine sessions of ect with minimal cognitive impairment and was continued on oral chlorpromazine 600 mg / day without any side effects. there was improvement in self - mutilating behavior, disorganized behavior and poor self - care, but occasional aggressive outbursts persisted. anatomically, the septum pellucidum consists of two laminae of tissue composed of white matter surrounded by gray matter. the septum is known to be a part of the limbic system and is connected via the medial forebrain bundle to the hypothalamus and via the fornix to the hippocampus and amygdala. csp is present in 100% of fetuses and premature infants, but the posterior half of the leaves normally fuse by 36 months of age. the presence of a csp later in life reflects neurodevelopmental abnormalities of structures bordering the septum pellucidum, such as the corpus callosum and hippocampus and may be considered as a marker of limbic system dysgenesis. despite being a normal variant, csp has been seen in various psychiatric disorders, most commonly in schizophrenia. in a recent meta - analysis, trzesniak. have mentioned that incidence of csp ranges between 1.1% and 89.7% in healthy volunteers, and from 10.0% to 89.5% in patients with schizophrenia spectrum disorder. they have also suggested that the clinical significance of csp may depend more on its size rather than presence or absence in schizophrenia. nevertheless, one recent longitudinal study has also found that csp may not be linked to the neurobiology of emerging psychotic disorders and may be related to the progression of the disorder per se. the exact function of the septum is not yet completely understood ; however it may appear to moderate behaviors such as rage and arousal. interestingly, csp is associated with aggressive features of antisocial personality disorder compared to nonaggressive ones, indicating particular relevance of septal disruption to aggression in human. researchers have also found that csp influences neighboring dense synaptic networks and cause irregularities in the distribution of serotonin (5-ht2a) receptors that may affect the septal regulatory role in the limbic system. in addition, improvement of severe self - mutilation has been reported after limbic leucotomy. based on these studies, we hypothesize that limbic dysgenesis may be the reason for aggressive and self - mutilating behavior in our patient. self - mutilation has an aggressive element, wherein the person, through self - mutilation directs anger inward which enables him or her to feel in control. though several line of management have been proposed including clozapine, lithium, and naltrexone and so on, we preferred ect and chlorpromazine as there have been previous successful treatment of self - mutilating behaviors with ect and we chose chlorpromazine considering its sedating property which also reduce psychotic aggression. in our patient, self - mutilating behavior was not secondary to any psychotic process or any affective disturbance ; neither it was explainable from mental status examination. it may be argued that csp, which has been found to be associated with aggression in a different patient population, also might play a role in self - mutilating behavior in our patient with schizophrenia. we conclude that the self - mutilating behavior in our patient may be an aggressive behavior directed towards self and might be neurobiologically linked to csp and its relationship with limbic system dysgenesis.	cavum septum pellucidum (csp) is a neurodevelopmental anomaly, which is commonly reported in schizophrenia patients. various symptoms of schizophrenia, including thought disturbances have been associated with csp. we present a rare case of undifferentiated schizophrenia with csp who presented with self - mutilating behaviors.
glucagon - like peptide 1 receptor agonists (glp1ras) have become an accepted part of the diabetes pharmaco - therapeutic landscape. the glp1ras are included in guidelines from various professional bodies such as the international diabetes federation (idf), the american diabetes association (ada), european association for study of diabetes (easd), and american association of clinical endocrinologists (aace). glp1ras can be used as second - line and third - line agents, in combination with metformin and insulin. there is also evidence to suggest that glp1ras should be used earlier on in the natural history of diabetes, so as to utilize their full potential. an increasing number of glp1ras are available for use, or are in advanced phases of development. while they have earlier been divided according to chemical structure, glp1ras can also be classified based on their duration of action (table 1). this approach makes it easier to compare and contrast the various pharmacokinetic and pharmacodynamic properties of these drugs. short - acting glp1ras are those with a half - life of 12 h, which allow once daily injection. long - acting glp1ras have half - lives of > 24 h [5, 6 ] and can be injected as once - weekly doses. the once - daily glp1ra, viz, liraglutide and lixisenatide, are also being developed as fixed ratio co - formulations with basal insulins, viz, degludec and glargine, respectively.table 1glucagon - like peptide 1 receptor agonists (glp1ra)short acting twice - daily dosage exenatide once - daily dosage lixisenatideintermediate acting (once - daily dosage) liraglutidelong acting (once - weekly dosage) exenatide lar albiglutide semaglutidefixed ratio combinations liraglutide + degludec lixisenatide + glargine glucagon - like peptide 1 receptor agonists (glp1ra) various glp1ras have been utilizing differing strategies to prolong or protract their duration of action. the shortest - acting glp1ra, viz exenatide and lixisenatide [3, 7 ], achieve this by exchanging amino acids at the n2 and n3 positions, thus making them resistant to breakdown by dipeptidyl peptidase 4 (dpp4). the renal elimination of the molecule, however, continues as per physiology, and so drug levels fall to baseline prior to the next injection. the short half - life of 24 h means that peak concentrations are maintained for just 6 h. liraglutide achieves a longer half - life (1213 h) and duration of action, by having a fatty acid side chain. this increases its size and slows down release of the glp 1 moiety from plasma albumin, thus prolonging its action. albiglutide uses a similar albumin - binding based approach, but binds in a covalent manner to the protein. the long half - life of the albumin glp1 conjugate allows for a long duration of action and once - weekly administration [5, 9 ]. it conjugates glp-1 with the fc fragment of igg and thus achieves a longer duration of action [10, 11 ]. exenatide long - acting release (lar) is a chemical modification of exenatide, which is coupled to microspheres that allow slow and steady release of the molecule from its subcutaneous site. in essence, a patient - centered approach implies crafting of a management strategy which involves the patient and is respectful of his or her opinion. viewed through a larger prism, patient - centered care assumes acceptance of the bio - psychosocial model of disease. it is understood that biomedical, psychological, and social factors impact not only the presentation of diabetes, but also its management. apart from biological characteristics, psychosocial reality should be taken into consideration while planning a therapeutic regime. the varieties of glp1ras available today and their unique pharmacological properties allow a patient - centered choice to be made in this class of drugs. various factors which influence the choice of a glp1ra can be categorized according to the bio - psychosocial model (table 2).table 2comparison of glp1rashort - acting glp1raintermediate- and long - acting glp1racontrol of fasting glycemiasuitablemore suitablecontrol of postprandial glycemiamore suitablesuitablecontrol of hba1ceffectiveeffectivefrequency of dosagedaily / twice dailyweekly / once dailycombination with basal insulincomplementary effectcomplementary effect for liraglutidecombination with premixed insulinmay show beneficial effectmay show complementary effectuse as directly observed therapy (dot)not feasiblefeasiblegauge of needlethin (31 g, 32g)thick (23 g) for exenatide larinjection techniquesimplerequires manual dexterity for exenatide lar, injection technique is also simple for liraglutide, titrated with the same peninjection site reactionsrarecommon, seldom with liraglutidegastrointestinal symptomsmore commonless commonincrease in pulse rateless commonmore commonweight losseffectiveeffectiveimprovement in lipid profileminimalminimalantibody formationrelatively high with exenatiderelatively high with exenatide lar ; low for liraglutideability to stop in case of adverse eventsretainedlost for once - weekly injections glp1ra glucagon - like peptide 1 receptor agonists, lar long - acting release glp1ra glucagon - like peptide 1 receptor agonists, lar long - acting release the biomedical factors which inform the appropriate choice of glp1ra include efficacy, safety, tolerability, and versatility in combination with insulin. the shorter - acting glp1ras have a greater effect on postprandial glycemia, while the longer - acting molecules lower fasting glucose to a greater extent. short - acting glp1ras delay gastric emptying, decrease the rate of glucose delivery to the duodenum, blunt postprandial glycemia excursions, and reduce insulin levels as well. however, their short duration of action means that optimal glucose control may not be achieved over the full 24 h. exenatide lowers breakfast and post - dinner glucose levels to a much greater degree than post - lunch excursions. this is expected with a twice - daily dose, keeping its pharmacokinetic profile in mind. the hba1c reduction varies from 0.8% to 1.5% with the recommended dose of 10 g twice daily [1316 ]. lixisenatide is associated with modest improvements in postprandial glucose after each meal, and in hba1c (0.71.0%), with a once - daily dose of 20 g [1719 ]. this drug has a lower risk of nausea, vomiting and hypoglycemia as compared to exenatide. the intermediate - acting liraglutide, used in a dose of 1.21.8 mg / day, improves both fasting and postprandial glucose values, leading to a 1.11.8% reduction of hba1c [1, 2023 ]. gastrointestinal effects and hypoglycemia are less common than with exenatide ; liraglutide also exhibits added benefits of low antibody formation, reduction in systolic blood pressure, and greater weight loss. the drug is associated with a slight increase in pulse rate, and the impact of this positive chronotropic effect on cardiovascular health is uncertain. intermediate- and long - acting glp1ras exhibit greater effect on fasting glucose levels, as they continue to act during the nighttime. they do not have a significant effect on gastric motility, as the initial slowing is abolished due to tachyphylaxis. the intermediate - acting and long - acting glp1ras cover all 24 h glucose control including fasting glucose. the short - acting glp1ras probably also achieve weight loss through central effects mediated via the hypothalamus. longer - acting drugs such as albiglutide have a lower hba1c reduction (0.78% vs. 0.99%) and lower body weight reduction (0.62 vs. 2.21 kg) probably because of the larger size which prevents entry into the central nervous system [25, 26 ]. the weekly dose of 30 mg, facilitated by a long half - life of 68 days, is associated with low rates of nausea and hypoglycemia, but significant local injection site reaction [27, 28 ]. dulaglutide, too, can be administered once weekly with its half - life of 90 h [11, 12 ]. significant hba1c reduction (1.35%) and weight reduction (2.43 kg) have been noted with doses of 1 mg once weekly for the first 4 weeks, followed by 2 mg weekly for 12 weeks. injection site reactions such as nodule formation are most often observed with exenatide lar, which also needs thicker gauge needle for administration [2932 ]. while diarrhea occurs more frequently with longer - acting compounds, nausea seems to be more common with short - acting glp1ra. gastroparesis may be an indication to choose intermediate- or longer - acting glp1ra, while being a relative contraindication for the shorter - acting glp1ra. the duration of action of glp-1 analogs determines the possible combination with a short - acting insulin or a basal insulin. the short - acting exenatide can be given with a basal insulin as it has a greater effect on postprandial glucose levels because of its shorter half - life. in contrast, the longer - acting glp-1 analogs exert greater effect on fasting glucose levels than shorter - acting glp-1 analogs. the possible combination of a long - acting glp-1 analog with a short - acting insulin would also offer the advantages of complementary pharmacologies and could theoretically reduce both fasting and postprandial blood glucose levels. a positive chronotropic effect has been noted with intermediate- and long - acting glp1ra, but not with the short - acting molecules. the short - acting drugs are also associated with greater improvements in postprandial lipid excursions : these factors may encourage their use in specific patients with cardiovascular morbidity. another biomedical factor which determines the choice of glp analog prescribed is the effect on autonomic functions. liraglutide has less effect on gastrointestinal motility than short - acting glp-1 analogs like exenatide and lixisenatide [34, 35 ]. thus, liraglutide is a better choice in diabetic patients with impaired autonomic functions. on the other hand, exenatide and lixisenatide thus, careful assessment of the expected efficacy (fasting vs. postprandial vs. overall glycemic control), anticipated risk of side effects (gastrointestinal symptoms, hypoglycemia, antibody formation, injection site reactions), effect on weight, cost and comorbid conditions (dyslipidaemia, hypertension) can help individualize the choice of glp1ra. the ability of the patient to self - inject, to adhere to pre - specified times of injection(30 min before meals for exenatide), manual dexterity, frequency of contact with health - care providers, meal pattern followed, and adherence to therapy are some of these issues. for a person who can self - inject, use of exenatide lar, however, requires some amount of manual dexterity, as it has to be self - mixed prior to injection. for those unable or unwilling to self - inject and dependent upon a caregiver for the same, a once - weekly injection of long - acting glp1ra represents a useful therapeutic strategy this once - weekly injection can be administered as directly observed therapy (dot), akin to that used in tuberculosis control programs. this approach has the added advantage of encouraging regular patient provider contact, which facilitates early detection of adverse events and complications, and encourages more efficient lifestyle modification. it is also helpful in patients who may not adhere to more frequently administered therapy. patients who consume heavy breakfast and dinner may benefit from exenatide twice daily, while those who take a light dinner may wish to take lixisenatide. patients with irregular meal habit and lifestyles, which put them at risk of hypoglycemia, will respond to once - weekly drugs without major safety concerns. the different glp-1 analogs have been compared with respect to the improvement in the quality of life and treatment satisfaction. the increase in patients treatment satisfaction from baseline is significantly higher with 1.8 mg liraglutide than with sitagliptin, but the increase with 1.2 mg liraglutide versus sitagliptin is not significant. no difference was perceived in the convenience of treatment between the oral versus injectable. compared to patients on traditional therapy which was insulin, liraglutide 1.8 mg versus glimepiride improves psychological and emotional well - being and health perceptions by reducing anxiety and worry associated with weight gain. the preceding discussion provides a reasonably robust evidence - based assessment upon which an appropriate glp1ra can be chosen or changed. further research and experience will help utilize glp1ra therapy in the best possible manner for the benefit of people with diabetes.	the individualized treatment of type 2 diabetes mellitus, using various glucagon - like peptide receptor agonists (glp1ras), has recently been described. as experience with existing glp1ras grows, and as newer molecules in the development pipeline continue to progress, interest related to these drugs continues to grow. this article describes a person - centered approach, using the bio - psychosocial model of health, to help individualized decision making related to choice of glp1ra. it utilizes an evidence - based approach to discuss various biomedical and psychosocial factors which may influence choice of glp1ra.
bony fractures are one of the most common reasons for physician visits worldwide. with greater public interest in physical fitness activities to maintain a healthy lifestyle, metatarsal stress fractures are commonly encountered by clinicians. metatarsal stress fractures, which account for nearly 25% of all stress fractures, often heal with nonoperative measures. immobilization of the foot and avoidance of strenuous activity often result in bone healing and return to full functional capacity within 68 weeks of the initial injury. impaired fracture healing in which a patient may fail to form a unionization of bone, or have delayed unionization, is frequently encountered in both inpatient and outpatient settings. patients at risk for impaired fracture healing include elderly patients, diabetics, smokers, patients with osteoporosis, and postmenopausal females due to estrogen deficiency. impaired fracture healing can have devastating consequences in this patient population, and may increase the fracture - related morbidity and mortality. teriparatide, a drug approved for the treatment of osteoporosis in postmenopausal women, has shown promise in the realm of fracture healing. in the initial stages of fracture healing, an increase in bone formation is required. by increasing bone formation through stimulation of osteoblasts this may be useful in treatment of patients who have impaired fracture healing, ultimately affecting quality of life. we present two cases of women with metatarsal fractures who had risk factors for impaired fracture healing, and report the time to fracture healing with the use of teriparatide. a 35-year - old premenopausal caucasian female presented to the emergency department with a complaint of pain in her right foot which occurred abruptly while running. she described the pain as sharp and unremitting, and denied any recent trauma to the affected foot. medical history was significant for a stress fracture of the left second metatarsal two years prior, which took approximately 14 weeks to obtain radiographic evidence of fracture healing with the use of an immobilizing boot. she denied the use of any medications, alcohol, or illicit drugs, but did have a 21 pack - year history of tobacco use. physical examination revealed a well developed female in mild distress. on examination of the right foot radiography of the right foot was consistent with a stress fracture at the base of the fifth metatarsal with associated soft tissue inflammation (fig. 1). the patient was instructed to wear an immobilizing boot on the right foot and to avoid strenuous activity. ibuprofen was suggested to the patient for analgesia. nearly 6 weeks after sustaining the fracture, the patient returned to the emergency department with continued pain in the right foot despite the use of the immobilizing boot and adequate analgesia. the patient refused further use of the immobilization boot. due to non - compliance with the immobilization boot, she was started on a daily dose of teriparatide, 50,000 iu of vitamin d3, and 2000 mg of calcium citrate. four weeks into therapy with teriparatide, repeat imaging revealed evidence of bony callus and new bone formation of the right fifth metatarsal, consistent with a healing fracture (fig. 2). the patient was pain free at this time, and shortly thereafter resumed all prior activities, including running. a 40-year - old caucasian female was seen in the orthopedic clinic with complaints of moderate pain in her left foot. she described the pain as sharp, and worsening with movement of her left foot. medical history was significant for hypothyroidism secondary to hashimoto s disease, celiac disease, and premature ovarian failure. she had no history of osteoporosis but had a previous fracture of the right distal radius, which took approximately 10 weeks to heal as per clinical findings and imaging studies. home medications included vitamin d3 50,000 iu weekly, levothyroxine 175 g daily, and glucosamine / chondroitin sulfate 1500 mg/1200 mg daily. physical examination was significant for tenderness to palpation of the left foot, with the point of maximal tenderness being over the first phalanx. radiography of the left foot confirmed the presence of a first left phalanx stress fracture (fig. 3), and the patient was instructed to wear an immobilizing boot for at least 6 weeks. because the patient appeared to be reluctant to wear an immobilizing boot for such a long duration, she was also started on teriparatide, 50,000 iu of vitamin d3 daily, and calcium citrate 2000 mg daily. she continued this medication regimen without any additions or changes for a total duration of 4 weeks. one week into therapy with teriparatide, the patient s pain and swelling had subsided and she was able to remove her immobilizing boot. at the end of 4 weeks, repeat imaging revealed a fracture that was well healed with callus formation seen (fig. a 35-year - old premenopausal caucasian female presented to the emergency department with a complaint of pain in her right foot which occurred abruptly while running. she described the pain as sharp and unremitting, and denied any recent trauma to the affected foot. medical history was significant for a stress fracture of the left second metatarsal two years prior, which took approximately 14 weeks to obtain radiographic evidence of fracture healing with the use of an immobilizing boot. she denied the use of any medications, alcohol, or illicit drugs, but did have a 21 pack - year history of tobacco use. physical examination revealed a well developed female in mild distress. on examination of the right foot radiography of the right foot was consistent with a stress fracture at the base of the fifth metatarsal with associated soft tissue inflammation (fig. 1). the patient was instructed to wear an immobilizing boot on the right foot and to avoid strenuous activity. ibuprofen was suggested to the patient for analgesia. nearly 6 weeks after sustaining the fracture, the patient returned to the emergency department with continued pain in the right foot despite the use of the immobilizing boot and adequate analgesia. the patient refused further use of the immobilization boot. due to non - compliance with the immobilization boot, she was started on a daily dose of teriparatide, 50,000 iu of vitamin d3, and 2000 mg of calcium citrate. four weeks into therapy with teriparatide, repeat imaging revealed evidence of bony callus and new bone formation of the right fifth metatarsal, consistent with a healing fracture (fig. 2). the patient was pain free at this time, and shortly thereafter resumed all prior activities, including running. a 40-year - old caucasian female was seen in the orthopedic clinic with complaints of moderate pain in her left foot. she described the pain as sharp, and worsening with movement of her left foot. medical history was significant for hypothyroidism secondary to hashimoto s disease, celiac disease, and premature ovarian failure. she had no history of osteoporosis but had a previous fracture of the right distal radius, which took approximately 10 weeks to heal as per clinical findings and imaging studies. home medications included vitamin d3 50,000 iu weekly, levothyroxine 175 g daily, and glucosamine / chondroitin sulfate 1500 mg/1200 mg daily. physical examination was significant for tenderness to palpation of the left foot, with the point of maximal tenderness being over the first phalanx. radiography of the left foot confirmed the presence of a first left phalanx stress fracture (fig. 3), and the patient was instructed to wear an immobilizing boot for at least 6 weeks. because the patient appeared to be reluctant to wear an immobilizing boot for such a long duration, she was also started on teriparatide, 50,000 iu of vitamin d3 daily, and calcium citrate 2000 mg daily. she continued this medication regimen without any additions or changes for a total duration of 4 weeks. one week into therapy with teriparatide, the patient s pain and swelling had subsided and she was able to remove her immobilizing boot. at the end of 4 weeks, repeat imaging revealed a fracture that was well healed with callus formation seen (fig. the incidence of fractures has been steadily increasing, with an estimated 15.3 million fractures occurring in the us annually.1,2 stress fractures of the metatarsal bones are common, and account for approximately 5% of fractures seen in the primary care setting annually.3 with the treatment of metatarsal stress fractures based primarily on conservative measures such as immobilization, the morbidity associated with such fractures depends largely on the rate of an individual s natural fracture healing time. the first phase involves inflammation and formation of a hematoma at the site of fracture due to disruption of the blood supply at the site of injury.4 in the second phase, chondrogenesis begins and a soft callus forms, which is then mineralized.4 the third phase marks the beginning of osteoblast cells forming woven bone, resulting in formation of a hard callus and union of the fracture.5 in the final phase of fracture healing, remodeling of bone occurs, resulting in the formation of lamellar bone, which is mechanically stronger bone.5 stress fractures of the metatarsal bones are often misdiagnosed initially as a soft tissue injury, because initial radiographs may not show evidence of fracture. 6 if clinical suspicion for a metatarsal stress fracture is high, or diagnosis is confirmed via radiographic imaging, conservative treatment modalities are first - line options. patients with metatarsal stress fractures should refrain from vigorous physical activity, and immobilization of the affected foot is often recommended. in most cases, nonweight - bearing activity and placement of an immobilizing boot leads to decreased pain associated with metatarsal fractures. under normal circumstances, it takes approximately 68 weeks for a metatarsal stress fracture to heal.7 a visible callus is typically seen on radiographs by 6 weeks after the initial injury.6 however, in approximately 10% of patients, impaired fracture healing is encountered, leading to delayed union or nonunion of metatarsal stress fractures. these fractures take longer to heal because of the poor blood supply in the region of the fifth metatarsal. they typically show radiographic evidence of union within 8 weeks with immobilization and use of a rigid boot.8 jones fractures involve the proximal diaphysis, and without surgery, take approximately 20 weeks to heal.8 avoiding weight - bearing is usually recommended for several months, and many patients find it difficult to comply with this. half of these fractures treated with immobilization alone tend to result in refracture or nonunionization of the fracture.9 thus many patients, including athletes, opt for surgical intervention to minimize the healing time. this involves placement of a screw into the canal of the fifth metatarsal, followed by immobilization for at least 2 weeks.10 with surgery, these fractures typically heal within 68 weeks. although the pathophysiology of impaired fracture healing is largely unknown, certain risk factors have been well reported. mechanical causes, such as insufficient immobilization of the fracture, distraction of fracture fragments by fixation devices, or repeated manipulations of a fracture, may lead to delayed or impaired union. nonmechanical risk factors, including patient comorbidities, have also been suggested to result in impaired fracture healing. diabetes, smoking, osteoporosis, and estrogen deficiency have all been identified as possible risk factors for impaired fracture healing.11 in such patient populations, impaired fracture healing may result in significant morbidity and negatively impact quality of life.12 with established risk factors for impaired fracture healing identified, there comes a greater need for therapy that can accelerate fracture healing in patients at high risk for delayed union or nonunion fractures. teriparatide, a recombinant human parathyroid hormone analog (134), was approved in 2002 by the food and drug administration for the treatment of osteoporosis in postmenopausal women who are at high risk for fractures.13 the only anabolic agent available for the treatment of osteoporosis, its mechanism of action is unique in comparison with other agents used currently. chronic exposure to endogenous human parathyroid hormone leads to an increase in bone resorption via stimulation of osteoclasts, further exacerbating the condition of osteoporosis.14 teriparatide, because of its ability to reach peak serum concentrations in 30 minutes and return to nondetectable serum levels in a few hours, has the paradoxical effect of stimulating new bone formation by favoring stimulation of osteoblasts over osteoclasts.14 in the natural process of fracture healing, a transient increase in bone formation at the site of fracture occurs. by increasing cortical thickness and bone formation on all bone surfaces, teriparatide may accelerate and augment this process of increased bone formation and fracture healing that happens naturally.4 andreassen were the first to report accelerated healing of fractures with teriparatide. the results demonstrated enhanced callus formation and mechanical strength of tibial fractures in rats treated with teriparatide.15,16 in 2006, manabe used teriparatide for fracture healing in cynomolgus monkeys, because their bone remodeling systems are closest to those of humans.17 in the study by manabe, the results suggested that by attenuating the degree of mineralization and shrinking the size of the callus, teriparatide may have accelerated the rate of fracture healing.17 although prior case studies regarding the use of teriparatide for accelerated fracture healing in humans exist, the only randomized, double - blind study in humans testing the hypothesis of acceleration of fracture repair was done by aspenberg in 2010. in this study, involving 102 postmenopausal women with fractures of the distal radius, it was concluded that there was a shortened time of fracture healing in a group of patients using 20 g of teriparatide compared with placebo.18 their results suggest that teriparatide may lead to acceleration of fracture healing in humans.18 our cases reported here suggest the possibility of accelerating metatarsal stress fracture healing with the use of teriparatide. 13 taking cost into account, it may be best used to accelerate fracture healing in those patients at high risk for delayed union or nonunion of fractures, or in patients who require a return to baseline activity at a faster rate than is possible with the normal healing process. this includes high - performance athletes, but also includes a large percentage of the workforce where jobs entail manual labor or constant walking throughout the day. for those who have occupations that involve walking throughout the day, patients with jobs entailing manual labor may suffer a greater loss of income than the cost of teriparatide for one month, due to inability to return to work until healing of the metatarsal fracture has occurred. in addition, many employers may not allow for a month away from work, and this could result in loss of employment for the patient. according to data from the centers for disease control published in 1990, the value of lost wages stemming from health conditions related to both physical and mental impairment as well as costs of medical care related to this reaches over a billion dollars in the us annually.19 therefore, in patients with jobs where the loss of income is greater than the cost of treatment with teriparatide, the drug may be useful in acceleration of fracture healing, allowing earlier return to work. although teriparatide is also indicated for the treatment of osteoporosis associated with use of long - term systemic glucocorticoids in men, there have been no data published on the use of teriparatide in the acceleration of fracture healing in men and premenopausal women. there are ongoing phase ii and phase ii clinical trials investigating the benefits of this medication in these specific patient populations. although more randomized, double - blind, placebo - controlled trials in humans will need to be pursued to investigate further the potential of this drug in fracture healing, clinicians should be aware that teriparatide may become useful in the clinical setting for acceleration of fracture healing.	bone fractures are one of the leading causes of emergency room visits worldwide, with approximately 8 million bony fractures occurring annually in the us alone. although the majority of fractures do not cause significant long - term morbidity and mortality, approximately 10% of these fractures result in impaired fracture healing, drastically affecting quality of life in affected patients. by increasing bone formation, teriparatide, an anabolic agent used in the treatment of postmenopausal osteoporosis, has shown promise in accelerating the rate of fracture healing. we present two patients with impaired healing of metatarsal fractures who were subsequently treated with teriparatide. both patients experienced successful bony union of the fracture after the use of teriparatide. these findings suggest that teriparatide may be useful in the clinical setting for the acceleration of fracture healing, especially in patients who are at risk for impaired fracture healing.
he was admitted for an elective left typanomastoidectomy and removal of the incus and malleus with tympanoplasty. his medical history included hypertension, dyslipidemia, gout, polycystic kidney disease and gastroesophageal reflux disease. his medications at the time of admission were rosuvastatin, domperidone, esomeprazole, allopurinol, irbesartan / hydrochlorothiazide and labetalol. on the first day postoperatively the patient experienced a sudden drop in his level of consciousness accompanied by marked agitation, and required intubation. his glasgow coma scale score was 9 (eyes 3, verbal 3, motor 3). there were no focal neurological signs, his pupils were symmetrical but sluggish to react and the fundi appeared normal. a computed tomography scan of the patient s head showed no structural abnormalities, no masses and no hematoma. lumbar puncture was performed and revealed cloudy cerebrospinal fluid (csf) with an elevated protein level (5.78 g / l), low glucose level (< 1.0 mmol / l) and a leukocyte count of 11,97410/l, with 95% neutrophils. based on these findings, the patient was treated empirically for bacterial meningitis with intravenous (iv) vancomycin, ceftriaxone and dexamethasone pending culture results and sensitivities. further blood work revealed a blood leukocyte count of 13.810/l, hemoglobin level of 123 g / l and platelet count of 15410/l. mmol / l, potassium level 3.6 mmol / l, chloride level 106 mmol / l, urea level 12.6 mmol / l and creatinine level 190 mol / l. initial gram stain of the csf using the cytospin technique revealed abundant polymorphonuclear leukocytes and no organisms. preliminary reports revealed growth of gram - negative coccobacilli, and at this point metronidazole was also added to the treatment regime until an anaerobic cause was ruled out. ultimately, the organism was identified from aerobic cultures as p multocida using the vitek 2 identification system (biomrieux, usa). interestingly, a swab of the left ear performed on postoperative day 2 grew the same organism as that cultured from the csf (growth on chocolate and blood agar ; no growth on macconkey s or inhibitory mold agar). the empirical antibiotics and dexamethasone were discontinued, and the patient was started on a 14-day course of iv penicillin g at a dose of 2,000,000 units every 4 h. the patient made a rapid recovery from his meningitis, and he was discharged on postoperative day 10 to continue treatment as an outpatient. on further questioning, it was revealed that the patient was the primary caregiver of several pet cats and a dog, although he reported no history of bites. the pets were allowed on the furniture, including his bed, and would occasionally lick his face. meningitis is an uncommon outcome of p multocida infection (3), making p multocida a rare cause of adult bacterial meningitis. two reviews spanning 1950 to 1999 report only 29 cases published in the english literature during that time period (4,5). animal contact was a major risk factor, present in 89% of cases, and a history of a bite was much less common, occurring only 15% of the time (4). previous cranial / facial surgery or skull fracture has been reported as a cause of p multocida meningitis (513). table 1 summarizes adult cases of p multocida meningitis published in the english literature after 1999 (1322). animal contact was present in all cases, while only two (20%) reported a history of a bite. the current report presents one of only a handful of cases of p multocida meningitis ever documented in the literature from a canadian site (5,6,9,21,23). the patient had the typical csf findings of bacterial meningitis (low glucose, high protein, high leukocytes). penicillin is the most commonly used antibiotic to treat p multocida meningitis (4,15), and our patient recovered fully with a course of iv penicillin g. many of the more recent cases describe treating with third generation cephalosporins (table 1). p multocida meningitis has been reported following mastoidectomy (11,12), and the pathogenesis of infection is hypothesized to involve contiguous spread of the organism from a colonized ear canal. supporting this theory, local spread from an adjacent infected site has been proposed as an etiology (4) because chronic otitis media and otorrhea have been found in association with p multocida meningitis (4,19,2427). our patient showed no signs of clinical meningitis preoperatively ; therefore, extension to the surgical site is the likely mechanism in this case. a preoperative ear swab has been proposed for patients having a mastoidectomy that have a history of exposure to animals (12), and may be supported by the present case.	pasteurella multocida is a gram - negative anaerobe that is known to colonize household pets ; in fact, it has been reported to be present in a majority of cats and dogs. p multocida can cause a variety of infections in humans, of which skin infections are the most common. this article describes a case involving a 56-year - old man who developed meningitis caused by p multocida following tympanomastoidectomy. the authors discuss the treatment course and most likely route of infection in this case, and summarize the cases of p multocida - associated meningitis presented in the literature.
the incidence of differentiated thyroid cancer (dtc) has increased globally over the last several decades, and iodine-131 is routinely used to treat dtc after total or near - total thyroidectomy to ablate the remnant thyroid tissue and enable physicians to detect tumor recurrence or distant metastasis more easily. iodine-131 is also used to treat benign thyroid conditions such as grave s disease and benign nodular goiter. few case reports have found that iodine-131 induced liver injury, including a case report involving a patient with grave s disease. here we present a case of drug - induced liver injury (dili) that occurred 10 days after iodine-131 ablative therapy and responded favorably to oral corticosteroid. a 47-year - old woman was referred to our hepatology unit in may 2015 for the evaluation of increased liver enzymes. the patient was previously diagnosed with thyroid cancer (t3n1am0) in march 2015 and had undergone total thyroidectomy with right neck lymph node dissection. at 8 weeks after surgery, she underwent iodine-131 therapy at 100 mci ; 10 days later, she was admitted with jaundice and elevated liver enzymes. the patient was taking only prescribed medications, including levothyroxine 150 g, calcitriol 0.25 g, and calcium carbonate 500 mg daily for 2 months after the total thyroidectomy. she denied having any alcohol or smoking history and taking any possible agents causing hepatitis such as health supplements or herbal medications. on admission, her vital signs were normal, and a physical examination showed no remarkable findings except icteric sclerae and skin. mild hepatomegaly was observed ; however, there was no evidence of splenomegaly or ascites. her blood count revealed a white blood cell count of 3,550/l (neutrophils, 35.9% ; lymphocytes, 49.9% ; and eosinophils 3.2%), and hemoglobin level, hematocrit level, and platelet count were 13.4 g / dl, 38.9%, and 148,000/l, respectively. serum biochemical assay gave the following results : blood urea nitrogen (bun) level, 8.0 mg / dl ; creatinine level, 0.85 mg / dl ; sodium level, 142 meq / l ; potassium level, 3.9 meq / l ; albumin level, 4.3 g / dl ; aspartate aminotransferase (ast) level, 1,136 iu / l ; alanine transaminase (alt) level, 1,632 iu / l ; total bilirubin level, 2.0 mg / dl ; direct bilirubin level, 1.3 mg / dl ; -glutamyl transpeptidase (-gt) level, 303 iu / l ; alkaline phosphatase (alp) level, 713 iu / l ; and lactate dehydrogenase (ldh) level, 502 iu / l. serological tests for immunoglobulin m (igm) anti - hepatitis a virus, hepatitis b surface antigen, igm anti - hepatitis b core antibody, anti - hepatitis c virus (hcv) antibody, hcv real - time polymerase chain reaction, anti - hepatitis e virus antibody, human immunodeficiency virus (hiv) antigen, anti - hiv antibody, igm anti - cytomegalovirus, igm anti - epstein - barr virus viral - capsid antigen, and igm anti - herpes simplex virus were negative, as were results for autoimmune markers, including anti - nuclear antibody, anti - mitochondrial ab, anti - smooth muscle ab, and anti - liver kidney microsome antibody-1. miu / l (0.175.65 miu / l), serum free t4 level was 1.46 ng / dl (0.801.90 ng / dl), and serum t3 level was 124.9 ng / dl (78.0182.0 ng / dl). 1). a liver biopsy was performed to confirm the diagnosis, and a histological examination revealed that the lesion was composed of moderate lobular inflammation and mild portal inflammation without fibrosis (fig. the roussel uclaf causality assessment method (rucam) score was 6, which was consistent with probable dili (table 1). in the analysis of r value, our case was 2.3, suggestive of mixed - type dili. a diagnosis of dili caused by iodine-131 was made as the administration of levothyroxine, calcitriol, and calcium carbonate for 2 months did not raise any special issues. a blood test conducted on the third day of hospitalization showed that ast / alt was 1,308/1,920 iu / l, iu / l, indicating the aggravation of jaundice. since her symptoms of nausea, vomiting, and abdominal pain persisted, subsequently, her clinical indicators improved and her ast / alt, total bilirubin, and alp levels started to decrease. after 10 days on prednisolone, the patient was discharged with considerably improved ast / alt and total bilirubin levels (180/174 iu / l and 0.9 mg / dl ; fig. iodine-131 ablation therapy after the surgical procedure of total or near - total thyroidectomy is standard management for thyroid cancer. iodine-131 is generally considered able to ablate thyroid cells without affecting healthy tissues in the body because of their levothyroxine (t4) and triiodothyronine (t3) contents. however, iodine-131 can accumulate in other body tissues and destroy normal tissues because iodine is distributed through the bloodstream and can be accumulated in any other organs associated with thyroid hormone metabolism. according to previous published reports, diffuse iodine-131 uptake in the liver was revealed frequently in the post - therapy scan, and the uptake amount increases in the status of total thyroidectomy [7 - 9 ]. therefore, the status of thyroidectomy might be associated with an increased risk of iodine-131-related liver injury ; however, the detailed mechanism involved is not well understood. lin. reported hepatotoxicity after iodine-131 therapy in a patient with thyroid cancer who subsequently responded to methylprednisolone. another report described two cases of liver toxicity after iodine-131 ablation therapy in two patients with grave s disease, both of whom were treated with prednisolone. in our case, several factors are able to potentiate the diagnosis of iodine-131-induced hepatotoxicity, including female sex, timing of drug intake and withdrawal, clinical course of liver injury, and exclusion of other causes of liver injury. when the rucam scale was applied to our patient, the assessed score was consistent with probable dili (table 1). thus, we concluded that liver injury in this case was probably caused by iodine-131 ablative therapy. since most cases of dili follow a benign course, the main treatment for dili is withdrawal of the causative agent with careful observation for risk of acute liver failure. however, 1317% of acute liver failure cases have been attributed to idiosyncratic drug reactions, with a high mortality rate (80%) without liver transplantation. elevated liver enzyme (alt > 3 times, bilirubin > 2 times the upper limit of normal) is considered a predictive factor of severe dili. in such cases s clinical course, the liver enzymes were markedly increased (ast / alt was 1,308/1,920 iu / l, total bilirubin was 2.9 mg / dl) and gastrointestinal symptoms (abdominal pain, nausea, and vomiting) were aggravated after hospitalization despite the provision of supportive treatment. a skin rash also developed on her trunk and limbs. these findings indicated rapid progression of dili, for which we decided to start oral corticosteroid (prednisolone 30 mg per day) to relieve the symptoms and achieve the expected therapeutic effect. steroid therapy is generally used to recover the features of autoimmune hepatitis (aih) in dili and reduce the allergic symptoms in other types of dili. since the mechanism of dili after iodine-131 ablation was not established, nevertheless we could give consideration that it might be partly induced by a hypersensitivity reaction to the iodine-131 in the same way as aih. in fact, it is difficult to distinguish aih from dili, because overall histological and serological features of these diseases are not far different from each other. and the frequent appearance of autoimmune hepatitis at the second episode of dili, named drug - induced autoimmune hepatitis (di - aih), makes it more challenging. the immune - mediated reactions is the leading mechanism of di - aih, but it has no pathognomonic features revealed and specific diagnostic criteria confirmed until now. previous studies reported that patients with dili after iodine-131 ablation responded dramatically to steroid therapy. these results can be supported by our finding that our patient also responded favorably to steroid therapy ; however, further studies are needed to support the actual therapeutic efficacy of this agent. the incidence of thyroid cancer continues to increase, and iodine-131 ablative therapy is routinely considered after total thyroidectomy. however, it is important to consider the possibility of dili by monitoring the liver enzymes. more evidence is required to determine whether iodine-131 is related to the incidence of hepatitis and whether some patients are more vulnerable than others.	iodine-131 is a radioisotope that is routinely used for the treatment of differentiated thyroid cancer after total or near - total thyroidectomy. however, there is some evidence that iodine-131 can induce liver injury. here we report a rare case of drug - induced liver injury (dili) caused by iodine-131 in a patient with regional lymph node metastasis after total thyroidectomy. a 47-year - old woman was admitted with elevated liver enzymes and symptoms of general weakness and nausea. ten weeks earlier she had undergone a total thyroidectomy for papillary thyroid carcinoma and had subsequently been prescribed levothyroxine to reduce the level of thyroid - stimulating hormone. eight weeks after surgery she underwent iodine-131 ablative therapy at a dose of 100 millicuries, and subsequently presented with acute hepatitis after 10 days. to rule out all possible causative factors, abdominal ultrasonography, endoscopic ultrasonography (on the biliary tree and gall bladder), and a liver biopsy were performed. dili caused by iodine-131 was suspected. oral prednisolone was started at 30 mg / day, to which the patient responded well.
the postoperative care has changed from admission in the hospital for several days to total ambulatory care. european and north american studies have examined the safety and early recovery of patients and have justified this change [13 ]. the less developed countries including india have also adopted this change in technique and patient care. a nationwide or large single - center study on the safety and complications related to these changes is sparse in india. we believe these studies will help planning the management policies of cataract surgery and finally formulate a uniform health care planning in india. there were three objectives of this study : (1) to estimate the rate of acute endophthalmitis in a large tertiary care eye hospital in south india, (2) to correlate the events of endophthalmitis with change in surgical technique from extracapsular cataract extraction (ecce) to phacoemulsification and from the inpatient to ambulatory patient care, and (3) to evaluate the differential infection rate in the higher and lower socioeconomic strata of the society. the study was done in a large tertiary care referral eye center in south india. comprehensive and total eye care is provided to patients of lower socioeconomic group (nonpaying patients) at no cost to the family. the materials for this study were obtained from the patient records of the institute after due clearance from the institutional review board. all adult cataract surgeries (excluding 2114 complicated cataracts following uveitis and trauma) performed between january 1993 and december 1998 were included. the study did not include the referred patients of postcataract surgery acute endophthalmitis operated outside the institute facilities. all patients were operated either by institute full - time faculty or ophthalmology fellows after a sufficient period of training (certified by the faculty). apart from specific technique related to the ecce and phacoemulsification, all processes related to pre-, intra-, and postoperative care were uniform as per the institute protocol earlier published by us. the inpatient care patients stayed overnight in the institute, and the ambulatory care patients were discharged usually within one hour of surgery. the eye patch was removed in all patients on the first postoperative day and replaced with a plastic eye shield or a pair of protective goggles. the first day evaluation included uncorrected and pinhole snellen acuity under standard conditions, applanation tonometry, a detailed slit lamp examination of the anterior segment, and fundus biomicroscopy using a + 78/90 d lens. the postoperative medications included topical fluoroquinolones four times daily for two weeks and topical 1% prednisolone acetate six times daily for four weeks and tapered thereafter. all patients were routinely instructed to report immediately should they notice / experience increased redness, pain, unusual discharge from the eye, and reduction in vision. all patients who returned with these symptoms and were suspected to have inflammatory or infective endophthalmitis were examined in the retina - vitreous service (td, sj, abm) for further management as per the standards we have earlier published. prior to december 1995 (publication of evs results), all patients received immediate vitrectomy ; beginning from january 1996, the evs recommendations for postcataract surgery endophthalmitis were followed, though surgeon - specific variations were allowed. in the pretransition period (19931995), all patients also received intravenous antibiotics, and this was discontinued after publication of the evs results. the collected undiluted vitreous fluid was evaluated in the microbiology laboratory for microscopy and culture (aerobic and anaerobic bacteria and fungi) as per the institutional protocol. all cultures were kept at least for a period of 4 days (14 days when fungus was suspected) before declaring them negative. a positive culture was defined as confluent growth of organism(s) at the site of inoculation on one solid medium and nonconfluent growth in one solid medium along with growth in one or more liquid media ; growth of the same microorganism in one liquid medium which was also identified in microscopy. patients were kept admitted to the institute for a period of 3 to 5 days (five days when patients received intravenous antibiotics). during this period, they were treated with intensive topical antibiotics, topical cycloplegic, intensive topical, and oral corticosteroids (except in cases of suspected or confirmed fungal endophthalmitis). case control analysis was done for the purpose of identifying all factors presumably associated with acute clinical endophthalmitis. five controls per case were selected from the surgical registry amongst all cataract surgeries done in the same time period. the control for each case was identified using the systematic random sampling strategy from the entire time period of the study. they were systematically chosen from the chronologically sorted list of cataract surgeries done in this period. this method of ascertaining the controls was adopted so that the effect of factors such as change in surgical technique and patient care was not lost. the analyzed factors included the type of patient care (inpatient and ambulatory), economic status (higher and lower), type of surgery (ecce and phacoemulsification ; iol and no iol implantation), systemic conditions (diabetes and hypertension), habitat (city limit and outstation), and surgeon factor (faculty and fellow - in - training). the years 19931995 were termed pretransition period, and the years 19961998 were termed posttransition period. the transition was from the ecce and inpatient postoperative care in 19931995 to phacoemulsification and ambulatory postoperative care in 19961998. the 19931995 period also served as evs prepublication time, and the 19961998 served as evs postpublication time. the incidence of acute endophthalmitis in the entire sample was compared between patient groups using fisher 's exact test. the risk of endophthalmitis was compared between patient groups using risk ratios and their 95% confidence intervals (cis). independent factors in the case - control study were tested for significance in univariate level using fisher 's exact test, t - tests, unadjusted odds ratios, and adjusted odds ratios using logistic regression in the multivariate level. significant factors in the univariate analysis at p < 0.15 were used for further multivariate testing. stata-7 intercooled stata for windows 7.0 (texas, 2001) was used for all statistical analyses. in the study period 19931998, a total of 46,095 cases of uncomplicated adult cataract extraction with iol implantation surgeries were performed23,727 (51.48%) paying and 22,368 (48.52%) nonpaying patients. the transition time marked shift in technology of surgery and techniques of patient care and also simultaneously coincided with the publication of the first evs report. in pretransition period, 20,039 patients (paying : 10,560 ; nonpaying : 9,479) and in posttransition period, 26,056 (paying : 13,167 ; nonpaying : 12,889) were operated for cataract. in the former period, all patients received ecce (with / without iol) and were treated as inpatients. in the later period, 80% (20,848 of 26,056) of patients received phacoemulsification, and in 95% of instances (24,752 of 26,056), the patients were provided ambulatory care. based on the clinical examination, acute endophthalmitis was suspected in 62 patients, with an incidence of 0.13% (62 of 46,095). thirty six vitreous samples were culture positive, a rate of 58.06% (36 of 62), and 37 microorganisms were isolated (one sample had polymicrobial infection). the interval between cataract surgery and presentation with symptoms and signs of endophthalmitis was 15 12 days. the patient 's age ranged from 42 to 81 years (mean 52 + 11 years ; median 60 years). primary surgery was ecce in 4 (6.5%) eyes, ecce and iol implantation in 42 (67.7%) eyes, and phacoemulsification and iol implantation in 16 (25.8%) eyes. primary pars plana vitrectomy was done in 41 (66.1%) eyes, and primary vitreous biopsy in the remaining 21 (33.9%) eyes. three patients in the vitreous biopsy group (3 of 21 ; 14.3%) needed vitrectomy. all patients received two intraocular antibiotics (cefazoline / vancomycin + amikacin / ceftazidime), and the antibiotics (culture adjusted) were repeated in 3 patients who had received deferred vitrectomy. sixteen of 37 microorganisms (43.2%) were staphylococcus epidermidis, and five (13.5%) were pseudomonas aeruginosa (table 1). gram - positive cocci (gpc) grew in 64.9% instances (24 of 37 growths) ; gram - negative bacilli (gnb) grew in 24.3% instances (9 of 37 growths). eleven of 62 patients (17.7%) in the entire series and 8 of 36 (22.2%) culture - positive patients regained a final visual acuity of 20/40 or better. thirty five of 62 (56.5%) patients in the entire series and 21 of 36 (58.3%) culture - proven infected eyes obtained final acuity of 20/100 or better. the incidence of endophthalmitis was higher in the ambulatory patient care group compared to the inpatient care group, and this difference was statistically significant (p = 0.054). this difference in the incidence of endophthalmitis rates was not observed in the higher socioeconomic group of patients (p = 0.351). the age and gender distribution of cases and controls was not significant (table 3 ; univariate analysis). ambulatory patient care (p = 0.025) and patients residing within the city limit (p = 0.04) the low socioeconomic group of patients was a significant factor at the 10% level (p = 0.157). the significance of patient residence in the multivariate analyses may be related to sampling variations of the case control itself. the interaction of socioeconomic status with type of patient care was a significant factor (p = 0.038) (tables 5 and 6). the risk of infection was higher in ambulatory patient care of nonpaying patients (lower socioeconomic status) compared to inpatient care (p = 0.001) and compared to all paying patients (p = 0.001). all paying patients (higher socioeconomic patients) and all inpatient care were not associated with a higher risk of infection. type of cataract surgery (ece or phacoemulsification) systemic disease (diabetes and hypertension) and surgeon factor (faculty and fellow) did not have statistical significance in univariate analysis. the current tertiary eye care study center in south india caters to both higher and lower socioeconomic group without change in quality of care. the nonpaying category of patients are those who possess bpl (below poverty line) card issued by the state government. being a retrospective case control study, it also ensured that any special efforts other than specified by the institute were not administered to affect a reduction of infection incidence. nationwide surveys and large case series of postcataract endophthalmitis in different countries suggest endophthalmitis incidence from 0.06% to 0.31% [2, 3, 723 ]. briefly, they include ecce with iol to phacoemulsification with iol and ambulatory patient care. this has obviously saved the overall expenses both for the patient and the hospitals, without compromising the surgical outcome and the quality of care [2, 3, 710 ]. while the reports of safety and efficacy of the new technology and patient care are available from developed countries, similar reports are sparsely available from less developed countries. this study has documented a higher risk of developing endophthalmitis in the ambulatory care lower socioeconomic group of patients. important factors associated with this higher incidence may include the residential environment and health education. poor residential environment and suboptimal health education could have a strong association with higher risk of endophthalmitis. a tertiary - care - hospital - based study may not actually reflect the true incidence of postcataract surgery acute endophthalmitis in india, particularly when mass cataract surgery is actively advocated to reduce the back log of cataract blindness. it is also possible that all patients of endophthalmitis, particularly from distant and rural locations, may not have returned for examination. but this possibility is unlikely since most of the noninstitutionalized eye care facilities in india will normally refer these patients to a higher eye care center. we excluded the endophthalmitis patients referred after cataract surgery done outside the institute facilities, so as to obtain uniform pre- and post - operative information. the uniform system adopted in the institute also allowed us to divide the patients into higher and lower socioeconomic groups nearly accurately. we also believe that such a large case control study involving over 46,000 patients and spanning six years probably overcomes some of the deficiencies of the study. this study suggests that when deciding on to whom to offer ambulatory care cataract surgery and when developing policy related to such surgery, the increased incidence of endophthalmitis in lower socioeconomic class patients compared to those in higher economic categories should be considered. since a long - term economic benefit lies in one hundred percent ambulatory care, improvement in housing, sanitation, and health education together is likely to improve the surgical outcome. cataract is the major cause of reversible blindness, and several efforts are made to reduce the cataract blindness. cataract surgery in itself does not decrease blindness without qualitative effort to improve quality of surgery and postoperative care. cataract - surgery - related blindness varies from 17% to 43% in india, china and africa [24, 2632 ]. while the efforts of the governmental and nongovernmental organizations to combat reversible blindness in developing countries are commendable, education on good eye health and care of the operated eye should yield superior outcomes after cataract surgery. an effective and assured ambulatory care will reduce the burden of housing the patients for longer period of time in a hospital. this will also reduce employing health care personnel excess of requirement, thus providing much needed flexibility to the available resources. the modern tools of surgery and management yield better results only when combined with healthy management strategy. in the absence of the later, the technological advancements can never be adequately exploited to advantage in developing countries.	purpose. we investigated acute endophthalmitis incidence following cataract surgery vis - a - vis the current technological and postoperative care changes in higher and lower socioeconomic categories of patients in south india. methods. in a retrospective case control study, we analyzed 62 cases of acute endophthalmitis and 5 controls for each endophthalmitis case from 46,095 cataract surgeries done between years 1993 and 1998. the time period covered the transition of surgical technique and after care. in addition, we analyzed systemic diseases, surgeon factor, habitat, and socioeconomic status. results. clinical and culture positive endophthalmitis incidence were 0.13% and 0.07%, respectively. differential incidence of 0.10% and 0.17% for in- and ambulatory care surgeries, respectively, was close to statistical significance (p = 0.054). lower economy category ambulatory patients had higher risk of infection. conclusion. ambulatory cataract surgery carried additional risk for post - operative infection in lower socioeconomic group. improved health education could ensure greater safety.
ageing is one of the most complex biological processes of growing older and defined as the intrinsic, inevitable, and irreversible age - related process of loss of viability and increase in vulnerability or a progressive functional decline, or a gradual deterioration of physiological function with age, including a decrease in fecundity. some workers refer to it as senescence. recently, ageing is defined as the age - dependant fractal process consisting in increasing of quantity of homeostasis disturbances at molecular, subcellular, cell - tissue, and system levels. the life cycle of human cells are determined by strings of dna called telomeres, present at the ends of chromosomes. the telomere shortens each time a cell divides, leading to ageing and eventually the death of the cell, once the telomere becomes too short to sustain life. genes regulate the ageing speed by indirection and controlling organism resistance to damages by exogenous and endogenous stresses. silencing of genomic dna was first observed by repression of genes near certain translocation breakpoints in drosophila. factors, such as proteins encoded by the sirtuin genes, have been identified in drosophila and yeast that act in trans to mediate silencing. sirtuins (sir, silent information regulator) are a group of nad - dependent deacetylases, hypothesized to play a key role in an organism 's response to stresses (such as heat or starvation) and to be responsible for the lifespan - extending effects of calorie restriction and/or a mutation in one or two genes, such as, ras2 and sch9. sir2 is required for silencing in the rdna, but sir2, sir3, and sir4 are all required for silencing at mating - type loci and telomeres. limited overexpression of the sir2 has been observed to result in a lifespan extension of about 30% and the deletion of sir2 results in a 50% reduction in lifespan. the sir2 homolog in mammals is known as sirt1 or sir2. it has been proposed that sir2 and sirt1 may block the organism from entering an extreme survival mode characterized by the absence of reproduction, improved dna repair, and increased protection against cell damage. mice that overexpress sirt1 show properties of calorie restriction, including low cholesterol, low blood glucose, and low insulin levels and also show increased numbers of mitochondria in their neurons. the human sirt1 protein has been demonstrated to deacetylate several downstream effector proteins including ku70, nbs1, the foxo transcription factor family, and p53, several of which are in response to dna damage events occurring within the cell. sirt1 may also stimulate autophagy by preventing acetylation of proteins required for autophagy in cultured cells and embryonic and neonatal tissues which shows a link between sirtuin expression and the cellular response to limited nutrients due to caloric restriction. nicotinamide riboside, a new nadprecursor, regulates sir2 deacetylase activity and life span in yeast and the ability of nicotinamide riboside to enhance lifespan does not depend on calorie restriction. it has been observed that the dual specificity tyrosine phosphorylation - regulated kinase 1a (dyrk1a) and 3 (dyrk3) promote cell survival through phosphorylation and activation of sirt1 and single copy loss - of - function of dyrk1a leads to increased apoptosis. klotho gene(named after a greek goddess who spins life 's thread) to high blood pressure. boosting the activity of the klotho gene appears to extend the natural lives of male mice as well as it also seems to delay many of the effects of old age, like the weakening of bones, clogging of the arteries, and loss of muscle fitness. increased p53 activity has also been implicated in ageing. it has been revealed that tap63 (a p53 family member) is a critical gene in preventing organismal ageing by controlling the maintenance of dermal and epidermal precursor and stem cells. gene expression is imperfectly controlled, and it is possible that random fluctuations in the expression levels of many genes contribute to the ageing process. individual cells, which are genetically identical but can have substantially different responses to outside stimuli, and markedly different life spans, indicate that the epigenetic factors also play a role in gene expression and ageing. increasing evidences have shown that autophagy, a highly conserved lysosome - mediated catabolic process, plays important roles in maintenance of energy homeostasis and the quality control of proteins and small organelles. autophagy is involved in a number of pathophysiologies, including ageing and age - related diseases ; however, its roles in these processes are far from straightforward. it was shown that this replicative exhaustion is essentially telomere shortening, which activates a persistent dna damage response. based on the intensity of the stress and acuteness of the process, rs and oncogene - induced senescence (ois) may reflect natural ageing and age - related disease, respectively. autophagy in lower eukaryotes has been shown to be critical for the anti - ageing effects of dietary restriction and negative modulation of insulin - signaling. in rs, there is a gradual shift from the proteasome pathway to autophagy within polyubiquitinated protein degradation systems which is mediated through at least two members of the bag (bcl-2-associated athanogene) protein family, which can bind to chaperones of the hsc / hsp70 family and thereby modulate protein quality control. the increase of bag3/bag1 ratio and activation of autophagy is also found in tissue ageing ; however, autophagy capacity declines with age in vivo. interleukin 6 (il6) and interleukin 8 (il8) have recently been shown to reinforce the senescence phenotype. the timing of their induction has been correlated with autophagy activation during the transition phase. strikingly, rnai - mediated repression of atg5 or atg7, which are essential genes for autophagy, suppresses il6/8 production, indicating a functional relevance of autophagy in senescence. components of the pi3k pathway, including mtor, a negative regulator of autophagy, are attenuated after their acute activation following ras expression during the transition phase of ois. cellular energy generation in the mitochondria is both a key source and key target of oxidative stress in the cell. cytochrome c oxidase, nadh dehydrogenase, and succinate dehydrogenase that regulate both oxidation and cellular respiration in mitochondria are believed to play a role in age - related increases in oxidation. an increased production of free radicals has been proposed to compromise mitochondrial efficiency, and eventually energy output, in a detrimental feedback loop. these energy - deficient cells lack the minimum metabolic capacity necessary to carry out an orchestrated cell - removal program known as apoptosis. another pathway to ageing involves the accumulation of proteins with toxic carbonyl groups in cells. carbonylation results from protein oxidation and reactions of proteins with sugars, aldehydes, and lipid peroxidation products. protein carbonylation increases with age, damaging about one - third of the body 's proteins later in life. these dysfunctional proteins accumulate in vital organs including the skin and eye, clogging the cellular machinery. with growing age, our neuroendocrine and immune systems decline functionally and this may cause these systems to send inflammatory chemical signals contributing to cell senescence or cell death. also, with growing age, there is a decline in the levels of essential polypeptide hormone insulin - like growth factor 1 (igf-1, somatomedin c). igf-1 coordinates cellular function and regulate cell growth and division and is responsible for many of the age - defying effects attributed to hgh. igf-1 plays an important role in preventing apoptosis in the early development of the embryo, as well as in the progressive regulation of organ development. igf-1 research shows that igf-1 can reverse many of the physical signs of ageing, such as loss of muscle strength, mass, and endurance, sagging skin, wrinkles, etc. shortages or imbalances of igf-1 are now thought to be a key factor in the ageing process and also in the development of ageing - related health disorders such as alzheimer 's disease, diabetes, and atherosclerosis. a recent study suggested that higher growth hormone / igf-1 levels in adulthood play a determinant role not only for regressive manifestations, but also for longer lifespan. marked changes in the expression levels of the neurotrophin receptors, trka and p75, occur with ageing of the brain as trka expression predominates in younger animals which switches to p75 predominating in older animals. the igf-1 receptor (igf1-r), the common regulator of lifespan and age - related events in many different organisms, has been shown responsible for the trka - to - p75 switch in both human neuroblastoma cell lines and primary neurons from mouse brain. the signaling pathway that controls the level of trka and p75 downstream of the igf1-r requires irs2, pip3/akt, and is under the control of pten and p44, the short isoform of p53. the hyperactivation of igf1-r signaling in p44 transgenic animals, which show an accelerated form of ageing, is characterized by early trka - to - p75 switch and increased production of a in the brain. recently, scientists have shown that yeast cells age through succeeding cell divisions and chromosomal instability dramatically increases when a threshold is crossed, thereby increasing genetic defects and age - related degeneration. telomeres are also considered important indicators of cellular senescence and vascular ageing and found to be shortened in senescent cells as a consequence of the increased production of mitochondrial reactive oxygen species (ros). the phosphorylation of the telomerase reverse transcriptase (tert) by tyrosine kinase src results into its export from the nucleus. the ageing - induced lack of nuclear tert activity finally leads to cellular senescence due to telomere shortening and consequent chromosome instability. p66shc protein has been investigated extensively as part of a critical pathway for loss of endothelial integrity associated with ageing and ros generation. a recent report has proposed that mediators other than p66shc, like o2-, are actively involved in determining the injurious effects of both ageing and hypertension. mutations causing a reduction in protein kinase a signaling have been shown to extend lifespan in yeast. it also delayed the incidence and severity of age - related disease and promoted leanness and longevity in mice. persons who are genetically predisposed to produce high levels of il-6 have a reduced capacity to reach the extreme limits of human life, whereas the high il-10-producer genotype is increased among centenarians. recently, two genes for which rnai knockdown delayed age - associated locomotory decline, conferring a high performance in advanced age phenotype (hpa), hpa-1 and hpa-2, have been identified as novel negative regulators of egf signaling, and egf signaling has been revealed as a major pathway for healthy ageing, acting through downstream phospholipase c - gammaplc-3 and inositol-3-phosphate receptor itr-1. inflammaging, an increased inflammation state with ageing, has been proposed mainly on the basis of peripheral levels of inflammatory cytokines and acute phase reaction proteins. it is linked to immunosenescence, an age - related decline in the functionality of the immune system. although the signaling pathways and mechanisms whereby inflammation inhibits muscle protein synthesis are not fully understood, chronic inflammation is an important player in sarcopenia and muscle weakness. adipocytes from old c57bl mice have significantly higher mrna expression of the proinflammatory cytokines il-1, il-6, tnf-, and lipid inflammatory mediator cox-2 and lower expression of anti - inflammatory nuclear receptor ppar- than those of young mice. an age - associated upregulation in nuclear factor (nf)-b binding activity has been shown to be present in various types of mouse tissues, and this elevated nf-b activity was diminished by a ppar agonist, suggesting that activation of nf-b in adipocytes is upregulated with ageing, resulting in increased expression of its target genes and, consequently, secretion of their products. highly significant differences between cytokine generation by t cells and monocytes of old and young subjects with gender specificity has also been observed. patients with mutations in the nalp3 gene secrete more il-1 and il-18 and suffer from systemic inflammatory diseases. they also have high circulating levels of il-6 and c - reactive protein (crp) which decrease rapidly upon blockade of the il-1 receptor, suggesting that il-1 contributes to the elevation of these markers of the inflammatory mechanisms of ageing. in a recent study of sfas, which is a known inhibitor of apoptosis, sfasl, a known stimulator of apoptosis, and total cytochrome c, which is released from cells during apoptosis, serum levels of sfas were significantly higher while sfasl and cytochrome c levels were lower in men compared with women. with increasing age, there was a decrease in apoptotic markers (cytochrome c) and pro - apoptotic factors (sfasl) and an increase in anti - apoptotic factors (sfas) in circulation. this shift toward less global apoptosis with increasing age in normal subjects is consistent with increased incidence of diseases whose pathophysiology involves apoptosis dysregulation. the understanding of pathophysiology of ageing over the past few years has posed tremendous challenges for the development of anti - ageing medicine for targeted therapy. in order to extend lifespan, an anti - ageing drug must delay ageing process or at least age - related diseases. inhibition of the signal transduction pathway for il6-mediated inflammation is suggested as the key to the prevention and treatment of ageing and age - related disorders, which may be achieved either indirectly through regulation of endogenous cholesterol synthesis and isoprenoid depletion or by direct inhibition of the il-6 signal transduction pathway. statins and bisphosphonates inhibit the mevalonate to cholesterol conversion pathway and cause isoprenoid depletion with inhibition of il-6 inflammation. statins inhibit the enzyme hmg - coa reductase and bisphosphonates inhibit the enzyme fpp synthase. polyphenolic compounds inhibit multiple pathways of signal transduction for il-6-mediated inflammation including inhibition of tyrosine kinase activity, inhibition of activation of nf-b, and inhibition of activation of ikk complex. statins, bisphosphonates, and polyphenolic compounds inhibit the jak / stat3 signaling pathway for il-6-mediated inflammation. resveratrol and rapamycin target conserved longevity pathways and may mimic some aspects of dietary restriction. they have also been reported to slow ageing in yeast and invertebrate species. in addition, both compounds also show beneficiary effects in rodent models of age - associated diseases. these compounds hold great promise as therapies to target multiple age - related diseases by modulating the molecular causes of ageing. resveratrol also increases the activity of sir2, which is the postulated reason for its beneficial effects, although with debated validity. free radical damage causes cellular energy depletion and further generation of more toxic free radicals. these cells may not go through normal apoptosis, produce inflammatory cytokines further damaging healthy cells, and also become chromosomally instable. antioxidant drugs could protect against the ageing effects of free radicals and slow down ageing considerably. plant sterols (ps) and stanols consumption is known to decrease low - density lipoprotein cholesterol (ldl - c) levels. furthermore, ps have recently been investigated for the prevention of other age - related diseases. ps may also have other potential beneficial effects including anti - inflammatory, antioxidant and anti - cancer activities. zingerone, found in ginger root, has been shown to have antioxidative and anti - inflammatory activities. zingerone has not only the antioxidant effect by constitutive suppression of ros, but also anti - inflammatory effects by suppression of nf - kappab activation in aged rat. in addition, zingerone treatment suppresses gene activation of proinflammatory enzymes, cox-2 and inos, which were upregulated with ageing through nf - kappab activation and ikk / mapk signaling pathway. experiments strongly indicate that zingerone treatment exerts a beneficial efficacy by suppressing both oxidative stress and age - related inflammation through the modulation of several key proinflammatory genes and transcription factors. madeo., hypothesized that increased autophagic turnover of cytoplasmic organelles or long - lived proteins is involved in most if not all lifespan - prolonging therapies. exogenous supply of spermidine, a polyamine, prolongs the lifespan of several model organisms and significantly reduces age - related oxidative protein damage in mice. spermidine induces autophagy in cultured yeast, nematodes, and flies as well as in mammalian cells.. many therapists also believe that the human growth hormones, melatonin and testosterone, can also prevent ageing. dehydroepiandrosterone (dhea) and its sulfate dheas are the most abundant sex steroids in women, the levels of which decline with age. it has been proposed that restoring the circulating levels of these steroids to those found in young women may have anti - ageing effects and improve sexual function and wellbeing in postmenopausal women. it is postulated that morin 's anti - nf - kappab activation depends on its ability to scavenge excessive reactive species (rs). anthocyanidins and related compounds extracted from the fruits and seeds of shrubs belonging to genus vaccinium have been reported to possess antioxidant and anti - inflammatory properties. extracts of the fruits have been applied for the inhibition of nonenzymatic glycosylation in anti - ageing preparations. histometric evaluations have shown that the dimethylaminoethanol (dmae)-supplemented anti - ageing formulation led to increased dermal thickness and also increase in collagen fiber thickness. dmae also enhanced the stratum corneum water content in the forearm skin without significantly modifying the mechanical properties. peptides, such as palmitoyl - kttks pentapeptide, applied topically in moisturizer - type cosmetic products are known to improve the appearance of fine lines, wrinkles, and other signs of facial skin ageing. a dipeptide, pal - kt, increases expression of skin structural biomarkers in human skin equivalents. gene microarray analysis indicated that pal - kt does not affect just the dermis and basement membrane in vitro, but can also affect biomarkers associated with epidermal differentiation, wound healing, and longevity, and has anti - ageing effects on skin. n - acetyl glucosamine (nag), a precursor to hyaluronic acid, serves important structural and hydration roles in extracellular matrix in both the epidermis and the dermis, and improves the appearance of uneven coloration and hyperpigmented spots in ageing facial skin. human skin equivalent cultures treated topically with nag show a dose - dependent increase in hyaluronic acid in parallel with an increase in procollagen-1 expression. a number of molecular mechanisms that cause ageing and ageing - associated disease have been identified and found to have interconnected pathways. to date, most studies have focused on interconnectedness of inflammation and its mediators and age or age - related diseases. future research areas must include targeted role of systemic inflammatory markers such as crp and il-6 and other biochemical and genetic studies including gene signaling pathways, gene microarray analysis, gene modulation, gene therapy, and development of animal / human models for potential therapeutic measures and evaluations.	ageing, also called as senescence, is one of the most complex, intrinsic, biological processes of growing older and resulting into reduced functional ability of the organism. telomerase, environment, low calorie diets, free radicals, etc., are all believed to affect this ageing process. a number of genetic components of ageing have been identified using model organisms. genes, mainly the sirtuins, regulate the ageing speed by indirection and controlling organism resistance to damages by exogenous and endogenous stresses. in higher organisms, ageing is likely to be regulated, in part, through the insulin / insulin - like growth factor 1 pathway. besides this, the induction of apoptosis in stem and progenitor cells, increased p53 activity, and autophagy is also thought to trigger premature organismal ageing. ageing has also been shown to upregulate expression of inflammatory mediators in mouse adipose tissue. the understanding of pathophysiology of ageing over the past few years has posed tremendous challenges for the development of anti - ageing medicine for targeted therapy. future research areas must include targeted role of systemic inflammatory markers such as c - reactive protein and interleukin 6 and other biochemical and genetic studies including gene signaling pathways, gene microarray analysis, gene modulation, gene therapy, and development of animal / human models for potential therapeutic measures and evaluations.
we aim at presenting a simple way to prioritize reservoir species in relation to their capacity to carry multiple agents of potential diseases. we illustrate this simple method with rodent - borne diseases in thailand, a country that showed major outbreaks of several rodent - borne diseases in the past years (1). we focused on rodents as carriers, vectors or reservoirs of numerous zoonotic diseases, notably microparasites (2). thailand presents the advantage to harbour a rich biodiversity, although at threat (3), and to have been quite intensively surveyed for rodent - borne diseases. using published surveys investigating rodent - borne diseases in rodents in this country we aim at identifying the major rodent species reservoirs, at least in term of their capacity to host multiple pathogens. for this, we first investigate the relationship between the pathogen species richness observed among rodent species and the screening effort and, second, we use the residual variations of this relationship to prioritize rodents and evaluate the potential risky habitats. we compiled surveys of microparasites investigated in rodents trapped in thailand (from 27 references given in supplementary appendix). the data comprise a total of 17,358 rodents from 18 species of murine rodents that have been investigated for a total of 10 microparasites (table 1). viruses were : hantaviruses, lymphocytic choriomeningitis virus (family arenaviridae, genus arenavirus), rabies virus and hepatitis e virus. these viruses are directly transmitted between rodent hosts and are all major pathogens of humans. are indirectly transmitted via contact with water or soils contaminated by urine of infected rodents. from the three protozoans, altogether 1,716 rodents (10%) have been found positive for at least one pathogen. microparasite richness was defined as the number of pathogen species for which each rodent species was found positive. survey of infection by microparasites (viruses, bacteria, protozoans) of rodent species in thailand, with number of positive individuals and number of investigated individuals between brackets (see references in supplementary materials) we used information on main habitats of rodents following jittapalapong. (4, 5), ivanova. (6) and suntsov. (7) : forests, dry lands near forests, non - flooded lands, paddy fields and irrigated / flooded agricultural lands, houses. total host sample size varies widely among rodent species, from 5,683 rattus tanezumi to 9 mus musculus. this last species was removed from the analysis due to its low sample size. this great variability can be explained by the variability in abundance of each species but also by unequal sampling among habitats. all rodent species have not been screened for all selected microparasites, with number of pathogens investigated varying according among rodent species (table 1). the detection of microparasites then depends on both the pathogens screening effort (number of pathogens tested) and the host sampling size (number of individual hosts trapped and tested for a given microparasite). using multiple regression analysis between microparasite richness and host sample size and pathogens screening effort as independent variables, we found that microparasite species richness was positively related to both independent variables (p < 0.0001, host sample size being log transformed). by investigating the residual variations among rodent hosts (fig. 1b), we show that several rodent species harboured more pathogens than that was expected by the regression model (i.e. positive residual values), particularly rattus adamanensis, bandicota savilei, r. argentiventer and r. norvegicus. two species appeared to harbour less pathogen species than expected by the regression model (i.e. negative residuals values) : mus cervicolor and m. caroli. residual values in microparasite richness, corrected for rodent sample size and pathogens screening effort, positively sorted according (a) to rodent species and (b) to habitats. high positive values of residuals in microparasite richness indicate higher species diversity of microparasites than expected by the regression modelling and can help at identifying good rodent carriers of pathogens or risky habitats, at least in term of high diversity of pathogens than can be encountered herein. similar reasoning on habitats suggests that higher pathogen richness than expected from correlation with sampling effort (i.e. positive residual values) are found in non - flooded lands, forests and paddy fields. we have developed here a simple method for prioritizing / targeting rodents that are best carriers of rodent - borne diseases, in a sense that they harbour more pathogen species that expected on the basis of the relationship between pathogen richness and sampling effort. controlling sampling effort in comparative analysis is usual as pathogen / parasite richness is highly correlated with the efforts done to sample their hosts. however, host sample size is also often positively correlated with some host features such as host density or host geographical range (8). a host living in high density and a wide geographical range is more sampled than a host living in low density and on restricted range. as parasite / pathogen richness is found correlated with host density and/or host geographical range (9, 10), there are potential confounding effects between these host features and the sampling effort to detect these parasites / pathogens. here, rather to determine the potential determinants of pathogen richness in rodents, which is a difficult task due to the lack of knowledge on many life traits of the species living in southeast asia, we used the residual variations of the pathogen richness / sampling effort relationship. interestingly, our results suggest some rodent species that are not commonly investigated to target for pathogen screening or surveillance such as r. adamanensis or b. savilei. the second result suggest that non - flooded lands and forests should be more taken into caution, whereas much surveys focused on paddy rice fields and households, although for obvious reasons. there is growing empirical evidence that some ecosystems are prone to alter or improve parasite transmissions (1113). (14) emphasizes that ecosystems with high productivity (and then high host densities) could select hosts for being more resistant to infections to limit epidemics and parasite transmission but also less fecund (due to trade - offs between reproduction and immunity) compare to host living in ecosystems with low productivity. assuming the reality of differences in pathogen richness between rodent species and the various habitats, our results call for future studies in asian ecosystems to improve the processes prone to explain such patterns. finally the simple method developed here based on known research effort in pathogens screening of wildlife can present some interest in surveillance prioritization (15) by allocating wildlife surveillance effort to specific rodent species in specific habitats. this study is supported by the french anr biodiversity anr 07 bdiv 012, project ceropath, community ecology of rodents and their pathogens in a changing environment.	backgroundcomparative analysis, which aims at investigating ecological and evolutionary patterns among species, may help at targeting reservoirs of zoonotic diseases particularly in countries presenting high biodiversity. here, we developed a simple method to target rodent reservoirs using published studies screening microparasite infections.methodswe compiled surveys of microparasites investigated in rodents trapped in thailand. the data comprise a total of 17,358 rodents from 18 species that have been investigated for a total of 10 microparasites (viruses, bacteria and protozoans). we used residual variation of microparasite richness controlled for both rodent sample size and pathogens screening effort to identify major rodent reservoirs and potential risky habitats.resultsmicroparasite species richness was positively related to rodent sample size and pathogens screening effort. the investigation of the residual variations of microparasite species richness showed that several rodent species harboured more pathogens than expected by the regression model. similarly, higher pathogen richness than expected was observed in rodents living in non - flooded lands, forests and paddy fields.conclusionour results suggest to target some rodent species that are not commonly investigated for pathogen screening or surveillance such as r. adamanensis or b. savilei, and that non - flooded lands and forests should be more taken into caution, whereas much surveys focused on paddy rice fields and households.
epithelioid hemangioendothelioma (ehe) is a rare tumor of the vascular endothelium that is considered to be intermediate between hemangioma and angiosarcoma. typically, the soft tissue is involved and, less frequently, the skin, bone, liver and lung are involved. in this report, we describe a case of cutaneous ehe that was present since birth involving the whole of the right lower limb. a 16-year - old female presented with painful progressive swelling and erythematous skin lesions involving the whole of her right lower limb. she had lesions since birth and also had restricted movements but with no other systemic complaints. an examination revealed diffuse swelling of the right lower limb, including the vulva and the lower abdomen. there were multiple erythematous papules, nodules and plaques of varying size that were firm in consistency [figure 1 ]. diffuse swelling of the whole of the right limb with multiple erythematous papules, nodules and plaques on subjecting the patient for investigations, her bleeding time, clotting time, prothrombin time and complete blood count were normal. x - ray [figure 2 ] and computed tomography scan revealed soft tissue swelling with diffuse osteolysis of the femur, tibia and fibula. the other differential diagnosis was klippel trenaunay syndrome, but due to the presence of osteoporotic changes in the bone, this was excluded. x - ray showing diffuse osteolysis of the right lower limb bones compared with the normal left limb biopsy of the erythematous nodule showed nodular tumor in the dermis, which was composed of oval- to spindle - shaped epithelioid cells. the nuclei showed a coarse chromatin pattern, and occasional mitotic figures were also noted. the cells were strongly immunoreactive for cd34 [figure 4 ] and factor viii - related antigen. finally, a diagnosis of ehe was made and the patient was referred to the vascular surgery department for further management. hematoxylin and eosin showing multiple vascular spaces with red blood cells (40) cd 34 antigen positivity (40) ehe has also been reported in the liver, bone, gingiva, mediastinum and lung. ehe is most commonly located on the extremities, and tumor is evenly distributed through all the adult age groups. the neoplasm usually presents as solitary, rarely multiple, slightly painful erythematous papules, nodules, plaque and nonhealing ulcer. microscopic examination reveals aggregates of oval to polygonal cells with abundant eosinophilic cytoplasm and round nucleus. the nuclear chromatin pattern is vesicular and frequent prominent nucleoli are present. the more commonly used antisera are factor viii - related antigen, cd 31, cd34 and ulex europeus lectin. our case is unusual because of its presence since birth and because of its location in the dermis and involving the underlying bone with diffuse osteolysis. a case of cutaneous ehe with underlying bone involvement was described by malane, sau, benson in 1992. the prognosis of ehe is uncertain as the mortality rate for ehe of the liver is 35% and lung is 65%. treatment should be limited to simple surgical excision, provided the possibility of an underlying bone lesion has been excluded.	epithelioid hemangioendothelioma is an intermediate - grade vascular tumor arising from the vascular endothelium, which usually arises in soft tissue, and skin involvement is extremely rare. we report a case that presented with primary cutaneous tumor involving the whole limb and was present since birth.
moreover, leadership is increasingly considered an important driver in terms of organisational performance, in particular in relation to the implementation of policy designed to solve the wicked issues of society. yet, despite leadership being viewed as an essential component of integrative public sector performance, there is relatively little thoughtful work analysing the relationship between the two sets of ideas. leadership in collaborative settings is simultaneously represented as being both the same, and yet different, to these roles in more traditional settings. what this means in practice is that much of the literature appears somewhat at best platitudinous and at worst confused posing practical difficulties for leaders and managers of collaborations who are looking for evidence or guidance on how to enact leadership. this paper examines the literature and asks how different leadership in inter - agency settings is from more traditional settings, before going on to map out lessons which may be useful for leaders and managers to draw upon in more effectively navigating this difficult terrain. this research is based on an extensive review of the literature surrounding leadership, collaboration and broader theories of networks. the paper finds that this distinction is overstated ; there are also significant overlaps in the types of tasks and challenges that both sets of leaders and managers will face and these should not be underestimated. this has clear implications for training and development of these individuals where understanding of the contexts for and nature of partnerships and thus the sensemaking and performance that may be most effective may be as important as the skills and attributes themselves.	introductionhealth and social care collaboration is currently a key feature of improvement efforts internationally. moreover, leadership is increasingly considered an important driver in terms of organisational performance, in particular in relation to the implementation of policy designed to solve the wicked issues of society. yet, despite leadership being viewed as an essential component of integrative public sector performance, there is relatively little thoughtful work analysing the relationship between the two sets of ideas. leadership in collaborative settings is simultaneously represented as being both the same, and yet different, to these roles in more traditional settings. what this means in practice is that much of the literature appears somewhat at best platitudinous and at worst confused posing practical difficulties for leaders and managers of collaborations who are looking for evidence or guidance on how to enact leadership.aims and objectivesthis paper examines the literature and asks how different leadership in inter - agency settings is from more traditional settings, before going on to map out lessons which may be useful for leaders and managers to draw upon in more effectively navigating this difficult terrain.methodsthis research is based on an extensive review of the literature surrounding leadership, collaboration and broader theories of networks.resultsthe paper finds that this distinction is overstated ; there are also significant overlaps in the types of tasks and challenges that both sets of leaders and managers will face and these should not be underestimated.conclusionsthis has clear implications for training and development of these individuals where understanding of the contexts for and nature of partnerships and thus the sensemaking and performance that may be most effective may be as important as the skills and attributes themselves.
necropsies of 95 harbor seals (p. vitulina) collected from july to december 2002 showed a moderate - to - severe pulmonary alveolar and interstitial emphysema and alveolar edema as the predominant findings. histologic lesions consisted of interstitial pneumonia with multinucleated syncytial cells and a moderate - to - severe lymphocytic depletion in the lymphoid tissues. cytoplasmic and nuclear acidophilic inclusion bodies were detected in respiratory epithelial cells, gastric surface mucous and chief cells, intestinal crypt epithelial cells, and hepatic and pancreatic duct epithelial cells. in the urogenital tract, inclusion bodies were observed in endometrial, vaginal, and epididymal epithelial cells as well as epithelial cells of the renal pelvis and urinary bladder. occasionally, inclusion bodies were present in neuronal and glial cells of the central nervous system. tissue lesions from a harbor seal (phoca vitulina) with phocine distemper virus infection. (b) immunohistochemical labeling of morbilliviral antigen in glandular epithelial cells of the lung. immunohistochemical analyses were performed by using a cross - reacting murine monoclonal antibody specific for the morbillivirus nucleoprotein. morbillivirus antigen was detected in lung, trachea, stomach, intestine, liver, pancreas, kidneys, urinary bladder, female genital mucosa, and epididymal tubules (figure 1b). in the lymphoid tissues, variable numbers of lymphocytes and macrophages of the follicular and parafollicular areas screening for morbillivirus - specific nucleic acid in tissue samples from lung, spleen, and lymph nodes as well as in blood samples from 85 seals was performed by reverse transcription polymerase chain reaction (rt - pcr). for this procedure, universal morbillivirus primers based on the conserved sequence of a 457-bp fragment of the phosphoprotein gene (8,6) were used. pdv - specific rna was detected in 46 (54%) of the 85 seals from german waters affected from july onward. both pdv - specific rna and morbillivirus antigen were detected in 33 (43%) of 77 animals. seals with no detectable morbillivirus antigen or nucleic acid had pneumonia and endoparasitosis of varying degrees of severity or died of undetermined causes. sequence analysis of the rt - pcr product showed an identity of 97% compared to the dutch isolate of 1988. the german isolate was 100% identical with the pdv isolate from the netherlands and differed in 1 nt from the danish isolate (6) (not shown). phylogenetic analysis showed that the phocine isolates from the two epidemics in european waters formed a discrete cluster, separated from the cdv isolates, including those from lion and siberan seal (figure 2). unrooted neighbor - joining phylogenetic tree constructed by using 369 nt from the gene coding for the morbillivirus p protein. treeview (version 1.6.5) was used for the graphic display of the tree. the canine distemper virus (cdv) sequences included were from vaccine strains rockborn (af181446) and onderstepoort (af378705), siberian seal (af259551), lion (u76708) ; cdv isolates originating from dogs 5804/89 (aj582384), a129/98 (aj582385), a77/98 (aj582386), 1489/98 (aj582387), and 1259/95 (aj582388) ; and 207/97 (aj582389), which was isolated from a marten. phocine distemper virus (pdv) isolates were pdv-1/nl/88 (af525289) ; pdv-1/dk/2002 (af525287) ; and pdv-1/nl/2002 (af525288). the german pdv isolates 1435 and 1419 are in a discrete cluster with a dutch and with a danish seal pdv isolate from 1988 and 2002, respectively. neutralization assays using the cdv strain onderstepoort were performed to determine the titers of serum samples from 187 harbor seals from german waters, collected from 1996 until the outbreak of the epidemic in 2002 (9,10). because of the cytotoxicity of some serum samples, only titers of > 10 were considered positive. titers from 22 (12%) of the 187 animals ranged from 14 to 240 (mean 50.5, 52.6 standard deviation). the morphologic and immunohistochemical findings in harbor seals from german waters during the recent morbillivirus epidemic in northwestern europe closely resembled those observed in 1988 (1113,5) and confirmed the epithelio-, lympho- and neurotropism of the pdv. the distribution of the viral antigen indicates that the respiratory tract was the primary route of morbillivirus infection. in contrast to reports about european harbor seals from 1988, no demyelination was detected in seals from german waters in 2002 (5). whether this finding represents a distinct feature of the 2002 epidemic or is a result of the small number of investigated animals remains unclear. seals that died during the morbillivirus epidemic with no detectable viral antigen or nucleic acid may have cleared the virus but still have virus - induced immunosuppression, which could result in fatal secondary bacterial or parasitic infections. the rna sequences of the recent virus isolates showed a virus population along the german coast during this epidemic that was almost identical to the isolates from the netherlands and denmark in 2002 and that had a high identity to the isolate from 1988 (6). protective morbillivirus - specific antibody titers were detectable in only a few seals from german waters before the outbreak in 2002, suggesting a high susceptibility for morbillivirus infection in this naive population. during the morbillivirus epidemic in 1988, approximately 65% of the dutch, danish, and german wadden sea seal population died (7). the death rate in 2002 is estimated at approximately 51% on the basis of the number of dead seals and the count of the wadden sea seal population in 2003 (14). the lower death rate in 2002 may have been influenced by different factors, such as decreased social contacts at the beginning of the epidemic during the late breeding season. in addition, genetic selection of a less susceptible population originating from the survivors of the 1988 outbreak might have resulted in a lower number of deaths during the second epidemic. it remains unclear why both outbreaks started at the danish kattegat isle of anholt. in the past, migrating arctic seal species, such as harp seals from greenland, have been suspected as carriers that introduced a morbillivirus into an immunologically naive population (15). several epizootics of infectious diseases in marine mammals with increases in air temperature were observed, indicating that environmental influences may have also resulted in the emergence of new epidemics (16). further studies are needed to determine whether alterations in migration patterns of arctic seal species caused by changes in climatic conditions are responsible for the two pdv epidemics in northwestern europe.	approximately 21,700 seals died during a morbillivirus epidemic in northwestern europe in 2002. phocine distemper virus 1 was isolated from seals in german waters. the sequence of the p gene showed 97% identity with the dutch virus isolated in 1988. there was 100% identity with the dutch isolate from 2002 and a single nucleotide mismatch with the danish isolate.
the transient receptor potential family is widely involved in many sensory processes, with a subset of channels implicated in the reception of temperatures. they are named thermotrp and are responsible for thermosensation (patapoutian 2005). in mammals, there are six thermotrps : two activated by cold (trpm8 and trpa1) and four activated by heat (trpv1 - 4) (dhaka. 2006). the first known receptor in mammals activated by noxious heat was vanilloid receptor subtype 1 (trpv1) (caterina. 1997). in humans, trpv1 is widely expressed in neuronal (dorsal root ganglion, trigeminal ganglion and various brain regions including hypothalamus or hippocampus) and nonneuronal tissues (among others, urinary bladder, liver or macrophages) (vennekens. 2008). vanilloid receptor subtype 1 is a non - selective cation channel with substantial calcium permeability, activated by various chemical and physical stimuli. chemical substances that activate the channel include various vanilloids (such as capsaicin) and endogenous lipid metabolites, while physical stimuli are acidic ph and high temperature > 43c (oneil and brown 2003 ; benham. 2003). other mammalian vanilloid receptors (trpv2 - 4) also respond to warmth : trpv2 is activated by noxious heat (~52c), trpv3 opens at 3439c and trpv4 at 2534c (caterina 2007). trp receptors are also implicated in insects thermosensation, especially the channels of the trpa subfamily (mckemy 2007). drospohila trpa channels, on the contrary to mammalian trpa, respond to warm temperatures (dillon. vanilloid receptors have also been discovered in insects (for details see : olszewska 2010). these include two receptors found in drosophila melanogaster : nanchung and inactive calcium - permeable cation channels activated by mechanical and osmotic stimuli. both receptors form one interdependent complex in the antennal chordotonal organ and are required for the insect s hearing (gong. 2004 ; liedtke and kim 2005). in our research, we tried to assess the effect of trpv1 agonist capsaicin and antagonist capsaicin is a natural alkaloid derived from pepper (clapham 1997). through activation of mammalian trpv1, this substance causes a feeling of burning pain (szallasi and blumberg 1999). kobayashi. (1998) showed that this substance induces hypothermia in rats, which is afterward followed by hyperthermia. preliminary studies at our laboratory revealed that capsaicin affects behavioral thermoregulation of the american cockroach periplaneta americana application of this vanilloid in submicromolar concentrations induced selection of lower temperatures in the thermal gradient system compared to cockroaches, which were not intoxicated (adamkiewicz. capsazepine, on the other hand, is a competitive antagonist of mammalian trpv1 and it inhibits the organism s response to vanilloids (walpole. it has been shown that administration of different trpv1 antagonists induces hyperthermia in rats (gavva. the aim of our study was to assess the effect of capsaicin and capsazepine on insect thermoregulation. based on our preliminary results on american cockroach, we hypothesized that capsaicin affects insect thermoregulation through receptors functionally similar to mammalian trpv1 channel. to evaluate that, we examined behavioral thermoregulation of mealworms intoxicated with capsaicin and capsazepine in a thermal gradient system. we hypothesized that the examined insects would respond to the tested substances similarly to mammals, which means that capsaicin would induce seeking lower ambient temperatures by the insects and that capsazepine s action would be opposite. the mealworm larvae (tenebrio molitor) were reared in room temperature and under 12:12 ld photoperiod in box containers filled with flour and oat flakes for hiding and as food. slices of apples were provided at regular time intervals (every 24 h) as a source of food and humidity. the individuals were chosen for the experiments according to body weight ; we selected insects with mean body weight 0.14 g per individual. to avoid the possibility of significant differences between body weight and actual larval stage, only the mealworms reared under the same conditions were used for the experiments. we tested two concentrations of both substances : 10 and 10 m. one group of insects was intoxicated with alcohol only (1%the same concentration as in the tested substance solutions) to evaluate the effect of the solvent alone., the mealworms were given 10 l of the tested substances on the ventral part of the prothorax. the system consists of a long and narrow aluminum trough (60 5 cm) cooled down by a cryostat on the one end and heated up by a thermostat (fisherbrand fbh 612) on the other. in this way the thermal gradient system was divided into 20 compartments of equal length and the temperature in each compartment was measured with a thermocouple prior to each experiment. behavioral thermoregulation of the mealworms after intoxication was recorded for 3 days with a camera (sony hdr - xr 200ve) and saved on a computer disc. the image from the camera was recorded every 3 min with biovid program (ferro software). we recorded thermal preferences of the animals in the 12-h cycles of light and dark. the mealworms were put into the thermal gradient system immediately after intoxication with the tested substances. in each experimental series, we examined six insects placed together into one thermal gradient system. the mealworms movements were not restricted in the thermal gradient system and the slices of apples were placed as a source of food and humidity. slices of apples were placed in different parts of the thermal gradient system with different ambient temperatures (15, 22, and 30c) in order not to affect the insects distribution. food was exchanged every 24 h. the data recorded from the thermal gradient system served for estimating the temperatures preferred by the intoxicated insect. for assessing the effect of the tested substances on thermal preferences of mealworms, one - way analysis of variance (one - way anova) and post hoc tukey hsd test were performed in pasw statisitcs 18 (spss inc.) all values are presented as mean sd and the results were considered statistically significant at p 0.05) (fig. 1temperature preferred (c) (mean se) by the mealworms intoxicated with alcohol (1%), capsaicin and capsazepine in a concentration of 10 m temperature preferred (c) (mean se) by the mealworms intoxicated with alcohol (1%), capsaicin and capsazepine in a concentration of 10 m after intoxication with capsaicin in a concentration of 10 m, an interesting change in behavioral thermoregulation of mealworms was observed. in the first 24 h, the insects preferred lower temperatures than the control animals (18.40 0.53c ; p = 0.02) (fig. 3). in the next 2 days, the examined mealworms chose temperatures similar to the control group (fig. 1). capsaicin in a concentration of 10 m lowered slightly the temperature preferred by the insects during 3 days of experiment (19.57 0.60c), but the changes were not significant (p > 0.05) (fig. 2).fig. 2temperature preferred (c) (mean se) by the mealworms intoxicated with alcohol (1%), capsaicin and capsazepine in a concentration of 10 m temperature preferred (c) (mean se) by the mealworms intoxicated with alcohol (1%), capsaicin and capsazepine in a concentration of 10 m capsazepine in low concentration (10 m) affected the insects thermoregulation in an opposite way to capsaicin in low concentration. (22.47 1.27c) (fig. 1), especially in the first 24 h of the experiment (23.55 0.78c ; p = 0.001) (fig. the insects intoxicated with capsazepine in high concentration (10 m) stayed at the lowest temperatures (18.25 0.77c) (fig. 2).fig. 3mean temperature (c) preferred in the first 24 h of experiment by the mealworms intoxicated with alcohol (1%), capsaicin (ca) and capsazepine (cpz) in two concentrations : 10 and 10 m. lines show statistical significance between series (one - way anova ; post hoc tukey hsd test) : p < 0.001 ; p < 0.01 ; p < 0.05 mean temperature (c) preferred in the first 24 h of experiment by the mealworms intoxicated with alcohol (1%), capsaicin (ca) and capsazepine (cpz) in two concentrations : 10 and 10 m. lines show statistical significance between series (one - way anova ; post hoc tukey hsd test) : p < 0.001 ; since insects are ectothermic animals, ambient temperature affects profoundly all their physiological processes. for that reason, we do not have complete knowledge about the structures involved in the reception of temperatures in insects. the sensilla that respond to warm or cold stimuli are mostly located on insects antennae. cold receptors are usually combined with two hygroreceptor cells (moist and dry receptor), as reported for example in apis, triatoma, antherea, carausius, aedes and gryllus. in some insect species, such as periplaneta, locust and leucophaea, there is an additional type of cold receptor combined with olfactory receptors in the antennal olfactory and thermoreceptive sensillum. two kinds of thermoreceptors (warm and cold) associated with mechanoreceptive hairs were reported in speophyes (steinbrecht and mller 1991 ; nishikawa. 1992 ; gingl and tichy 2001). 2011) revealed that in drosophila, two antennal structures (arista and sacculus) contain hot and cold receptors. these thermoreceptors project onto anatomically and functionally distinct glomeruli in the proximal antennal protocerebrum of the fly s brain. the preferred temperature is suggested to be set by independent action of hot and cold receptor systems. the authors demonstrated that members of the trp family belonging to trpp (polycistin) subfamily brivido trp (brv 1, 2 and 3) might function as antennal cold receptors. also, other transient receptor potential family members involved in insects temperature sensation are known. the first known receptor involved in nociception of noxious temperatures in drosophila was painless, a member of the trpa subfamily (tracey. other trpa channels involved in drosophila reception of warm temperatures are pyrexia, activated by temperatures above 40c (lee. 2005), and dtrpa1, important for thermotaxis (rosenzweig. 2005). there are also members of trp family that form a different subfamily (trpc) : trp and trpl, which are also implicated in phototransduction. it is notable that although trp receptors take part in thermosensation both in insects and mammals, the sensation of different ambient temperatures is mediated by different channels in those groups of animals. in insects, the members of the trpa subfamily are warm activated, while the mammalian trpa channel is responsible for reception of low temperatures. mammalian trpv channels were implicated in sensing warm temperatures, while insect trpv channels, nanchung and inactive, expressed in chinese hamster ovary tissue culture cells, were not activated by temperature (liedtke and kim 2005). recent research showed, however, that in drosophila larvae inactive is required for the selection of preferred (17.5c) over cooler temperatures (1416c), implying it takes part in insects thermoregulation (kwon., we do not have any direct evidence for capsaicin sensitivity of trpv channels in insects. kim. (2003) reported that in heterologous expression systems, chinese hamster ovary cells expressing nanchung channels were unresponsive to capsaicin. a similar situation occurred with inactive, the second insect trpv (gong. it is suggested that capsaicin sensitivity of trpv1 is a recent evolutionary acquisition in mammals, as avian vanilloid receptor shows only residual sensitivity to this substance (jordt and julius 2002). although insect vanilloid receptors do not respond to capsaicin, there are reports that this substance may repel some insect species. capsaicin is used as a repellent against cotton pests and one species of stored - products beetle (sitophilus zeamais) (spurr and mcgregor 2003). this shows that at least some insect species are sensitive to capsaicin, although it does not clearly imply that they possess receptors sensitive to capsaicin. the studies conducted at our laboratory on cockroaches revealed that insects respond to mammalian trpv1 agonist, capsaicin, which affects their behavioral thermoregulation. however, we did not investigate insects reaction to capsazepine then. in the presented research, we hypothesized that capsaicin and capsazepine affect insects thermoregulation through receptors functionally similar to the mammalian trpv1 channel. the experiments performed on mealworms in the thermal gradient system seem to confirm our assumptions. the larvae intoxicated with capsaicin in low concentration (10 m) in the first 24 h of the experiments preferred temperatures lower by about 23c than the control group. subcutaneous injection of capsaicin (5 mg / kg) to rats induced cutaneous vasodilation, increase of skin temperature and decrease of body temperature leading to hypothermia that lasted for about 2 h. simultaneously, capsaicin increased rats oxygen consumption and heat production, which in turn resulted in hyperthermia that occurred after hypothermia (kobayashi. capsazepine (in low concentration), a trpv1 antagonist, elicited an opposite reaction in the examined insects. the mealworms stayed at warmer parts of the thermal gradient system than the control animals for almost 45 h from intoxication, and then moved to the temperatures similar to the ones chosen by the larvae which were not intoxicated. in mammals, the application of various trpv1 antagonists (except capsazepine) induces an increase in body temperature (gavva. it is suggested that hyperthermia appears because trpv1 receptors are tonically active in vivo, keeping body temperature at normal level, and when antagonists block trpv1 channel, the body temperature increases. although capsazepine blocks mammalian trpv1 and was used in thermoregulation studies in rats and mice, it was not shown that this substance causes hyperthermia. it was reported that capsazepine does not block activation of vanilloid receptor subtype 1 by low ph, which presumably may be the reason for it not causing hyperthermia (romanovsky. hemolymph ph may change in a broader range than in mammals (the reported hemolymph phs in insects range from 6.4 to 7.5 ; harrison 2001), insects vanilloid receptor may vary in reaction to protons. here, we report that capsazepine in a concentration of 10 m affects insect behavioral thermoregulation, inducing selection of warm temperatures. the observed opposite effect of capsaicin and capsazepine in the same concentration on the mealworms thermoregulation may suggest that the tested substances act as agonist we suppose that this may be a receptor functionally similar to mammalian trpv1. as far as higher concentration (10 m) of the tested substances is concerned, the observed changes in the mealworms behavioral thermoregulation are similar for capsaicin and capsazepine. the insects stayed at cooler parts of the thermal gradient system than the group of insects not intoxicated. (2005) showed that capsaicin acts through trpv1 only at submicromolar concentrations, whereas at micro- to millimolar concentrations this substance alters the physical properties of biological membranes. capsaicin adsorbs to lipid bilayers and affects inter alia membrane fluidity, causing a decrease in its stiffness. this process leads to changes in functioning of many membrane proteins, and similar action is observed after capsazepine in micro- to millimolar concentrations. our previous studies revealed that in the thermal gradient system the colorado potato beetle (leptinotarsa decemlineata say) larvae fed on red pepper chose ambient temperatures higher by 10c than the larvae fed on potatoes (tgowska. 2005). it is very interesting to observe a distinct reaction of insects exposed to pure capsaicin (mealworms) or red pepper (colorado potato beetles). the observed changes in insects behavioral thermoregulation may stem from the increased membrane fluidity caused by the examined substances (at higher concentration, 10 m). when the lipid bilayer stiffness is decreased, the insect may choose cooler ambient temperatures to restore regular fluidity of the membrane. since we did not examine the changes in the insects lipid bilayer fluidity after capsaicin or capsazepine application, we can not be sure whether the observed behavior of the mealworms resulted from these or other reasons. it is remarkable that capsaicin affects not only mammalian, but also insect, thermoregulation. the observed opposite effect of trpv1 agonist and antagonist on insect behavioral thermoregulation, which at the same time remains similar to the effect of these substances on thermoregulation in mammals, indicates indirectly that capsaicin may act on receptors in insects that are functionally similar to trpv1. however, it can not be excluded that the observed results reflect non - specific actions unrelated to trp channels. further investigations are required to explain through what way trpv1 agonist and antagonist influence behavioral thermoregulation of insects.	transient receptor potential channels are implicated in thermosensation both in mammals and insects. the aim of our study was to assess the effect of mammalian vanilloid receptor subtype 1 (trpv1) agonist (capsaicin) and antagonist (capsazepine) on insect behavioral thermoregulation. we tested behavioral thermoregulation of mealworms larvae intoxicated with capsaicin and capsazepine in two concentrations (107 and 104 m) in a thermal gradient system for 3 days. our results revealed that in low concentration, capsaicin induces seeking lower temperatures than the ones selected by the insects that were not intoxicated. after application of capsazepine in the same concentration, the mealworms prefer higher temperatures than the control group. the observed opposite effect of trpv1 agonist and antagonist on insect behavioral thermoregulation, which is similar to the effect of these substances on thermoregulation in mammals, indicates indirectly that capsaicin may act on receptors in insects that are functionally similar to trpv1.
enterococcus faecalis is the cause of 85 - 90% of enterococcal, and third cause of nosocomial infections, especially bacteremia, sepsis in children, endocarditis, urinary tract infection (uti), and wound infections. it plays a significant role in treatment of the disease, knowledge of bacterial resistance pattern to antimicrobial agents is important for the successful management of diseases. there are five resistance genes whose products are responsible for resistance to glycopeptides antibiotics in vancomycin - resistant enterococci strains (vre). two of such genes (van a and van b) are most common than others, especially in e. faecalis and e. faecium. strains with van a gene are resistant to vancomycin and tycoplanin, and strains with van b are resistant to vancomycin but sensitive to tycoplanin. resistant enterococcal infections are usually treated by synergistic action of a glycopeptide and an aminoglycoside. vancomycin - resistant enterococci strains are usually transferred via the hands of health care workers, who are fecal carrier and are in close contact with patients. those patients who have long - time hospital stay and long - time antibiotic therapy, as well as children and the elderly with a critical situation, such as those who are hospitalized in intensive care units (icu), are more prone to take the disease. culture is the most - used way for detecting enterococci in the blood, however, for effective treatment of enterococcal bacteremia, characterization of the bacteria and their pattern of resistance to antibiotics is necessary. this requires some diagnostic biological tests as well as determination of its antibiogram pattern and mic, which usually takes about five days. some rapid methods such as e test for mic, api 20 and api 32 for characterization, and selective - differential specific media and choromogenic agars for direct detection of vre such as eva, can - vga, and beaa with 6 g / ml vancomycin have been introduced in recent years. these methods have shown different sensitivity and specificity in different studies, and need 2 - 3 days for final confirmation of their results. the aim of this study was to evaluate the feasibility of a pcr assay (four - pair primers) as an alternative for a routine method, which is time - consuming and expensive, to characterize e. faecalis in the blood and to reveal its resistance type to vancomycin. we used a standard strain vre (ptcc 1447, and ptcc 1237) prepared by the division of bacteria and fungi collections, iranian institute of industrial and scientific researches, tehran, iran). a suspension 10 cfu / ml was made in normal saline by adding some single colonies, which were grown on tsa by adjusting its optical density to half mcfarland solution and checking their absorbance in 700 nm with spectrophotometer. then, diluted solutions with different bacterial contents (10 cfu / ml, 10 cfu / ml and 10 cfu / ml) were made by diluting it in normal saline. they are used for inoculating to blood. by adding certain amount of each bacterial solution to certain amount of defibrinated sheep blood, some blood samples with different bacterial content (10 cfu / ml, 10 cfu / ml, 10 cfu / ml, 10 cfu / ml, 5 cfu / ml and zero as control) were prepared. ten - milliliter - samples of each dilution were prepared to be used in ten experiments of each of the pcr and routine assays. for routine assay, we used initial enrichment procedure for each specimen by inoculating to tsb and incubation at 37c for 24 hours, passage to tsa and incubation in 37c for more 24 hours, identifying by catalase test, pyr test, growth on tsa with 6.5% nacl, and hydrolysis esculin in the presence of bile on bea. differentiation of e. faecalis from e. faecium was done by three tests including ability to use pyruvate, fermentation of sorbitol, and reduction of tellurite. for screening vre transferring 100 l of each blood sample to a 2 ml ependorf vial contain 400 l sterile double distilled h2o and incubation in 37c for 30 minutes, adding 500 l red cell lysis buffer (nahco3 10 mm, nh4cl 0.155 m, ph=7) and incubation at 37c for one hour, centrifugation at 10,000 rpm for 15 minutes, discarding the supernatant, adding 200 l lysis buffer for bacteria (tris 10 mm, sucrose 0.3 m, mgcl2 5 mm) to the pellet with 10 l lysozyme (0.1 mg / ml, sinagen, lot : mr7735) and incubation at 37c for one hour, adding 4 l proteinase k (900 u / ml, fermentaze, lot : 00022411) and incubation at 65c for one hour, extraction of dna by standard phenol - chloroform method and precipitation of dna by cold isopropanol. pcr mix was prepared as 3 l of 10x pcr buffer, 2 l of mgcl2 (25 mm), 0.5 l of dntp 10 mm, 100 pm of each primer, 0.2 l dna pol (5 u/l), 2 l dna, and double distilled h20 to final volume of 25 l. special features of primers that were used in this study are shown in table 1. special features of the used primers the gene of each primer ; name of primers ; the reference for the primer design pcr programs was adjusted as one cycle at 94c for 7 minutes, 34 cycle at 94c for 40 seconds, 46c for 40 seconds, 72c for 50 seconds, and final extension at 72c for 10 minutes. electrophoresis of pcr product was done on 1.5% agarose gel including etidium bromide by 100 volts for one hour. all blood samples with different bacterial content of 5 cfu / ml were positive in the routine assay and pcr (figure 1). gel electrophoresis of pcr with genus specific primers c1 and c2) to enterococcus on blood samples with different contents of enterococcus faecalis (ptcc 1447). l : ladder 100 bp ; 1 : negative control (blood without bacteria) ; 2 : blood with 5 cfu / ml ; 3 : blood with 10 cfu / ml ; 4 : blood with 102 cfu / ml ; 5 : blood with 103 cfu / ml ; 6 : blood with 104 cfu / ml ; size of all bands is 320 bp pcr with species specific primers (d1 and d2) on two blood samples with different bacterial contents of e. faecalis (ptcc 1447) is shown in figure 2. gel electrophoresis of pcr with species specific primers (d1 and d2) on a blood samples. l : ladder 100 bp ; 1 : blood with 103 cfu / ml enterococcus faecalis (ptcc 1237) ; 2 : negative control (blood without bacteria) ; 3 : blood with 103 cfu / ml enterococcus faecalis (ptcc 1447) ; size of both specific bands is 940 bp pcr with species specific primers van a (a1, a2) and van b (b1, b2) on two blood samples with the same number but different strains of e. faecalis (ptcc 1447) and e. faecalis (ptcc 1237) is shown in figure 3. gel electrophoresis of pcr with specific primers for van a (a1, a2) and van b (b1, b2) on two blood samples. l : ladder 100 bp ; 1 : blood with 103 cfu / ml enterococcus faecalis (ptcc 1447). two specific bands are obvious : 940 bp for ddi gene and 732 bp for van a ; 2 : blood with 103 cfu / ml enterococcus faecalis (ptcc 1237). two specific bands are obvious : 940 bp for ddi gene and 635 bp for van b ; 3 : negative control (blood without bacteria for enterococcus faecalis ptcc 1447) ; 4 : negative control (blood without bacteria for enterococcus faecalis ptcc 1237) routine assay is time - consuming and expensive, and commercial automatic screening tests and disc diffusion agar are not efficient for highly resistant bacteria that make most of the hospital isolates. the most - studied selective - differential medias are eva, can - vga, and beaa with 60 g / ml vancomycin. although is more specific than can - vga, eva is slower (24 vs 48 hours). dutka - malen used six primer pairs todetect a number of standard strains and clinical isolates. the multiplex - pcr assay that with 4 primer pairs that stake, used for screening many clinical isolates, had a sensitivity of 85.0% and a specificity of 100% specificity, but the one that with three primers that jayartne used for screening of 657 isolates had a sensitivity of 95.4% and a specificity of 99.8%. ke, use primers designed from tuf enterococcal gene to diagnose 14 of 20 enterococcal species. angelleti, used four pair of primers to detecting 279 isolates, and found that it was more rapid than routine assay. paul, used two pairs of specific primers for van a and van b to screen clinical isolates, and found it was more sensitive than culture methods. in our study, the sensitivity of pcr was the same as that of routine test. it seems that the quality and quantity of dna, which is related to dna extraction method, is critical to the higher sensitivity. kariyama, used multiplex - pcr with seven pairs of primers to screen many clinical isolates and standard strains, and found that it was simpler and more efficient than the routine method. false - positive cases is mainly the result of amplifying dna of dead bacteria in the sample and amplifying resistance genes like van a and van b that are present in some other bacteria. this is critical for fecal samples that contain different bacteria, but not for blood ones. regulating the concentrations of several primers in pcr mix is a technical problem for multiplex - pcr. kariyama found that inhibition of van a primers can be neutralized by increasing their concentrations to two - folds. angeletti, and stake, performed the multiplex - pcr in two steps, and in one step they only used van a primers. we used one step and the same concentrations of primers, which seems to be the cause of weak view of bands (figure 3). primers designed by ke, from tuf gene could amplify two species of abiotrophia and four species of lysteria. however, they are not the usual causes of bacteremia and this problem is important for fecal screening. it has been recommended to use molecular typing methods such as rflp on pcr product, or very specific primers for e. faecalis, for characterization. it has been recommended to use genus specific or universal primers as the internal control for detecting false - negative cases. however, we used species specific primers to diagnose e. faecalis and as an internal control (figure 2). one of the main technical problems in the diagnosis of bacteremia by pcr is the obtainment of high quality and quantity bacterial dna from the whole blood. zang, used quiagen kit, and detected five cfu / ml bacteria in the blood. newcomb, used boom method and klausegger, used dna zol buffer for lysis bacteria in blood. however, anthony, used double distilled h20 for lysis blood cells and boiling for extraction bacterial dna. we used sterile double distilled h2o and red cell lysis buffer for lysis blood cell, proteinase k for eliminating pcr inhibitors, phenol - cholroform and alcohol precipitation to extract dna of bacteria in blood, however, the quality of extracted dnas was not very good. it seems that the poor quality of dna was the main cause of weak bands of pcr products in our study (figure 1). we recommend a very efficient method or a commercial kit to increase the accuracy of pcr assays in such a case. we also recommend further studies to overcome other technical problems of pcr assay to make it more simple and reliable relative to new and very improved culture based methods such as vre - bmx chromogenic commercial media. such kits has several advantages such as differentiation at the species level, inhibition of growth of sensitive enterococci to vancomycin, promoting growth of resistant strains, and shorter duration than the traditional methods (24 vs 48 hours). pcr is a more rapid and a sensitive method for simultaneous detection and characterization of e. faecalis, and determination of its antimicrobial pattern to vancomycin. it can be considered as an alternative assay, but a more efficient dna extraction method to increase the sensitivity of the assay is suggested.	background : bacteremia due to enterococcus faecalis is usually caused by strains resistant to most antibiotics. effective management of the disease is dependent on rapid detection and characterization of the bacteria, and determination its sensitivity pattern to antimicrobial drugs. the aim of this study was to investigate a more rapid and reliable assay for simultaneous diagnosis of enterococcal bacteremia and its sensitivity pattern to antimicrobial drugs. methods : several bacterial suspensions with different content of two standard strains of enterococcus faecalis resistant to vancomycin were used for inoculation to defibrinated sheep blood samples. pcr and routine assay was performed on all blood samples with different bacterial content. results : routine assay and pcr for all inoculated blood samples with 5 cfu / ml was positive. mean time for pcr and routine assays was 10 hours and 5 days, respectively. conclusion : pcr is a more rapid and sensitive assay for simultaneous detection and characterization for enterococcus faecalis, and determination of its sensitivity pattern to vancomycin.
atraumatic splenic rupture (asr) is an uncommon pathologic condition in which bleeding from the spleen occurs for a variety of reasons [13 ]. while the current trend in traumatic splenic rupture is nonoperative management including transcatheter arterial embolization [4, 5 ], splenectomy is recommended in most patients with asr as a consequence of underlying splenic pathology such as bacterial infection, malarial infection, hematological disorders, drugs or amyloidosis. two patients with asr, concomitant hemorrhagic shock and anticoagulation therapy, presented to us for definitive treatment. our successful treatment algorithm included a three - step strategy : (i) rapid transient hemostasis by splenic artery occlusion using a microballoon catheter, (ii) damage control resuscitation in the intensive care unit (icu) and (iii) splenectomy as a definitive hemostatic treatment. a 66-year - old man presented after suffering sudden hypotension and abdominal distension, followed by a rapid and progressive anemia. after arriving at hospital, he collapsed and went into cardiac arrest. return of spontaneous circulation occurred after 36 min of cardiopulmonary resuscitation and rapid blood transfusion. abdominal contrast - enhanced computed tomography (cect) revealed a large hematoma around the spleen and liver, in paracolic gutters and the pelvis with active arterial extravasation and apparent capsular disruption (fig. 1). his systolic blood pressure (bp) was 90 mmhg ; heart rate (hr), 80 bpm ; hemoglobin (hb) level, 6.7 g / dl ; hematocrit (ht), 19.0% ; prothrombin time - international normalized ratio (pt - inr), 2.16 and activated partial thromboplastin time (aptt), 63.9 s (under massive transfusion). he was currently on warfarin therapy for anticoagulation following a total aortic arch replacement including aortic valve replacement at the age of 60. he presented with no history of trauma, and moraxella catarrhalis had been detected in a blood culture taken on initial presentation. however, his transfusion requirement was massive and the risk of significantly worsened hemorrhage during any operative procedure was increased by his iatrogenic coagulopathy. after discussion with surgery team and interventional radiology (ir) team, transient occlusion of the splenic artery using a microballoon catheter was performed (logos, piolax, inc., kanagawa, japan ; arrival to occlusion, 90 min ; arrival to angiography suite, 60 min ; procedure time to occlusion, 15 min ; fig. 2). soon after balloon occlusion, the patient 's hemodynamic state improved and his anemia stabilized. perioperatively, 6 units of red cell concentrate (rcc) and 10 units of fresh frozen plasma (ffp) were transfused. the patient 's coagulopathy improved on hospital day 2 (ht, 25% ; pt - inr, 1.35 and aptt, 33.5 s) and was taken to the operating theater for open splenectomy. the spleen was swollen and capsular rupture was indeed identified, but there was no finding of a solid tumor or abscess formation. he was extubated on day 4, and was discharged from the icu on day 6. the patient was transferred to another hospital on day 25 without residual deficits or complications from his cardiac arrest. figure 1:there was a massive hematoma in the peritoneum surrounding the spleen and the liver, and extending into the paracolic gutter and the pelvic cavity. the capsule of the spleen was disrupted, and active arterial extravasation can be seen around the spleen (arrow). there was a massive hematoma in the peritoneum surrounding the spleen and the liver, and extending into the paracolic gutter and the pelvic cavity. the capsule of the spleen was disrupted, and active arterial extravasation can be seen around the spleen (arrow). the right gastroepiploic artery with platinum coils after embolization. a 52-year - old woman was admitted to the emergency department with altered mental status. on admission, her mental status improved to e3v4m6 on the glasgow coma scale with a bp of 95/72 mmhg and hr of 92 bpm. she was found to be anemic and coagulopathic (hb, 9.6 g / dl ; ht, 31.0% ; pt - inr, 2.68 and aptt, 38.4 s). she had a history of mitral valve replacement 3 months ago and was taking warfarin and sarpogrelate. cect showed a giant splenic artery aneurysm, splenic laceration and hematoma (fig. a diagnosis of hemorrhagic shock secondary to ruptured splenic aneurysm was made and ir was consulted. the source of hemorrhage could not be identified because of the giant aneurysm and therefore hemorrhagic shock persisted, despite aggressive resuscitation. localization of the hemorrhagic source was abandoned and transient occlusion of the splenic artery using a microballoon catheter was performed (arrival to occlusion, 140 min ; arrival to angiography suite, 110 min and procedure time to occlusion, 17 min). the hemorrhage and patient 's hemodynamics subsequently improved after a total of 4 units of rcc and 6 units of ffp being transfused. a splenectomy was performed on day 2 after correction of the patient 's coagulopathy (ht, 27% ; pt - inr, 1.02 and aptt, 33.8 s). on examination, the giant splenic aneurysm was found to be a pseudoaneurysm complicated by an abscess at the hilum of the spleen, yielding a final diagnosis of asr due to infection. the patient was moved to the general ward on day 3, and discharged from the hospital on day 12. a giant splenic aneurysm (40 50 mm) and splenic laceration are seen (arrow). a giant splenic aneurysm (40 50 mm) and splenic laceration are seen (arrow). a 66-year - old man presented after suffering sudden hypotension and abdominal distension, followed by a rapid and progressive anemia. after arriving at hospital, he collapsed and went into cardiac arrest. return of spontaneous circulation occurred after 36 min of cardiopulmonary resuscitation and rapid blood transfusion. abdominal contrast - enhanced computed tomography (cect) revealed a large hematoma around the spleen and liver, in paracolic gutters and the pelvis with active arterial extravasation and apparent capsular disruption (fig. 1). his systolic blood pressure (bp) was 90 mmhg ; heart rate (hr), 80 bpm ; hemoglobin (hb) level, 6.7 g / dl ; hematocrit (ht), 19.0% ; prothrombin time - international normalized ratio (pt - inr), 2.16 and activated partial thromboplastin time (aptt), 63.9 s (under massive transfusion). he was currently on warfarin therapy for anticoagulation following a total aortic arch replacement including aortic valve replacement at the age of 60. he presented with no history of trauma, and moraxella catarrhalis had been detected in a blood culture taken on initial presentation. however, his transfusion requirement was massive and the risk of significantly worsened hemorrhage during any operative procedure was increased by his iatrogenic coagulopathy. after discussion with surgery team and interventional radiology (ir) team, transient occlusion of the splenic artery using a microballoon catheter was performed (logos, piolax, inc., kanagawa, japan ; arrival to occlusion, 90 min ; arrival to angiography suite, 60 min ; procedure time to occlusion, 15 min ; fig. 2). soon after balloon occlusion, the patient 's hemodynamic state improved and his anemia stabilized. perioperatively, 6 units of red cell concentrate (rcc) and 10 units of fresh frozen plasma (ffp) were transfused. the patient 's coagulopathy improved on hospital day 2 (ht, 25% ; pt - inr, 1.35 and aptt, 33.5 s) and was taken to the operating theater for open splenectomy. the spleen was swollen and capsular rupture was indeed identified, but there was no finding of a solid tumor or abscess formation. he was extubated on day 4, and was discharged from the icu on day 6. the patient was transferred to another hospital on day 25 without residual deficits or complications from his cardiac arrest. figure 1:there was a massive hematoma in the peritoneum surrounding the spleen and the liver, and extending into the paracolic gutter and the pelvic cavity. the capsule of the spleen was disrupted, and active arterial extravasation can be seen around the spleen (arrow). there was a massive hematoma in the peritoneum surrounding the spleen and the liver, and extending into the paracolic gutter and the pelvic cavity. the capsule of the spleen was disrupted, and active arterial extravasation can be seen around the spleen (arrow). the right gastroepiploic artery with platinum coils after embolization. a 52-year - old woman was admitted to the emergency department with altered mental status. on admission, her mental status improved to e3v4m6 on the glasgow coma scale with a bp of 95/72 mmhg and hr of 92 bpm. she was found to be anemic and coagulopathic (hb, 9.6 g / dl ; ht, 31.0% ; pt - inr, 2.68 and aptt, 38.4 s). she had a history of mitral valve replacement 3 months ago and was taking warfarin and sarpogrelate. cect showed a giant splenic artery aneurysm, splenic laceration and hematoma (fig. 3). a diagnosis of hemorrhagic shock secondary to ruptured splenic aneurysm was made and ir was consulted. the source of hemorrhage could not be identified because of the giant aneurysm and therefore hemorrhagic shock persisted, despite aggressive resuscitation. localization of the hemorrhagic source was abandoned and transient occlusion of the splenic artery using a microballoon catheter was performed (arrival to occlusion, 140 min ; arrival to angiography suite, 110 min and procedure time to occlusion, 17 min). the hemorrhage and patient 's hemodynamics subsequently improved after a total of 4 units of rcc and 6 units of ffp being transfused. a splenectomy was performed on day 2 after correction of the patient 's coagulopathy (ht, 27% ; pt - inr, 1.02 and aptt, 33.8 s). on examination, the giant splenic aneurysm was found to be a pseudoaneurysm complicated by an abscess at the hilum of the spleen, yielding a final diagnosis of asr due to infection. the patient was moved to the general ward on day 3, and discharged from the hospital on day 12. a giant splenic aneurysm (40 50 mm) and splenic laceration are seen (arrow). a giant splenic aneurysm (40 50 mm) and splenic laceration are seen (arrow). splenectomy is still considered the treatment of choice for asr. however, ir hemostasis in emergency or trauma patients is gaining acceptance. our three - step strategy using temporary balloon occlusion was chosen over the more widely used coils, because balloon occlusion occludes the vessel lumen completely and is therefore effective in the setting of coagulopathy. combined with critical care resuscitation and operative intervention, ir may allow a more rapid and safer hemostatic strategy than conventional ir or surgery alone. there have been few reports on the effectiveness of balloon catheters used in damage control interventional radiology for trauma or critical hemorrhage. however, we believe they represent a useful hemostatic device for transient, proximal bleeding control, especially in hemorrhagic shock with coagulopathy conventional balloon catheters (e.g. selecon mp, terumo clinical supply, gifu, japan, or moiyan, tokai medical products, aichi, japan) could be used as well. however, anatomical and/or technical problems might make it difficult to select and advance the catheter in similar cases. as described in the presented cases, the microballoon catheter (e.g. logos, piolax, inc. or attendant, terumo clinical supply) the presented cases demonstrate that splenic artery occlusion using a microballoon catheter before splenectomy can be a useful hemostatic strategy in asr patients with hemorrhagic shock and coagulopathy.	atraumatic splenic rupture (asr) is an uncommon pathologic condition in which bleeding from the spleen occurs for a variety of nontraumatic reasons. while the current trend in traumatic splenic rupture is nonoperative management including transcatheter arterial embolization, the current recommendation for the treatment of most patients with asr is splenectomy. in this report, we describe two cases of asr presenting with hemorrhagic shock and complicated by anticoagulation therapy. in patients with severe hemorrhagic shock and coagulopathy, a damage control strategy is recommended. our successful treatment of these patients included a three - step strategy as a damage control : (i) rapid transient hemostasis by splenic artery occlusion using a microballoon catheter, (ii) damage control resuscitation and (iii) splenectomy as a definitive hemostatic treatment.
they are often caused by high - energy trauma and are associated with a high mortality rate. however, such severe injuries with complex patterns are seen more commonly nowadays due to high - speed trauma caused in road traffic accidents (rta) on motorways. high mortality rates (50%77%) have been reported for femoral shaft fractures associated with pelvic fractures and injuries to other systems. these injuries have a great impact not only on the social and economic life of individual, but also on the society. we present a rare case of bilateral unstable pelvic fractures (acetabular and sacral fractures), bilateral segmental femoral shaft fracture with multiple right rib fractures and associated haemothorax. to the best of our knowledge, a 21-year - old female sustained multiple injuries in a major rta 6 months ago. she was travelling with family and friends in a four wheeler jeep when the driver of her vehicle tried to overtake a truck in high speed and had a head - on collision with the truck coming from opposite side. only 2 young ladies, sitting in the rear seat of the jeep, survived this fatal accident (fig. 1). they were admitted in a nearby hospital where they had primary resuscitative treatment and were then referred to our tertiary care center 5 days after the accident. on admission, this patient was in a state of hemorrhagic shock and was found to have multiple injuries, including multiple right rib fractures with haemothorax, bilateral segmental femur fractures (fig. her medical condition was stabilized by multiple blood transfusions, intravenous broad - spectrum antibiotics, splinting of fractures by skeletal traction, right chest tube drainage and other supportive measures. she was then operated on in 2 sittings at the gap of 2 days to fix all the major fractures. firstly, the segmental fractures of both femora were fixed under general anaesthesia using intramedullary interlocking nails, taking the adequate precaution of padding the perineal and sacral areas. right side segmental femur fracture was fixed with an antegrade femoral nail ; whereas left comminuted segmental fracture was more proximal (subtrochanteric) therefore was fixed with synthes ' shaft fracture nailing system. she responded very well to the first surgery and hence after two days the sacral fractures were fixed using a sacral bar in a prone position. and then the patient was turned supine and anterior column fractures of both the acetabulum were fixed with pre - contoured reconstruction plates through the bilateral ilioinguinal approach. the postoperative courses after both surgeries were uneventful. she was allowed sitting in bed from the 2nd postoperative day and active and passive exercises were started. physiotherapy and wheelchair mobilization were advised for 6 weeks. at a follow - up of 3 months, she had shown neurological recovery and the ankle dorsiflexors were improved from the medical research council (mrc) grade 0 to grade 3 and at 6 months to grade 4 +. follow - up radiographs showed satisfactory healing of all the fractures in good alignment at 12 months (fig. 4, fig. 5, fig. 6) she was allowed partial weight bearing mobilization with crutches after 2 months of the surgery. she not only survived this major accident but escaped from any major complication related to her fracture and treatment. there is paucity in the literature about the mechanism, priority and optimal treatment of the concomitant unstable pelvic and segmental femoral shaft fractures. due to non - availability of suitable guidelines to treat such complex injuries we consider that combined femoral shaft and unstable pelvic fractures are severe injuries and necessitate timely and aggressive management. the femoral fractures need precedence over pelvic fracture fixation. understanding the mechanism of such fracture pattern remains a challenge. malgaigne 's fracture is an unstable pelvic fracture through both pubic rami and the ilium or sacroiliac joint with vertical displacement. these fractures result from application of axial force through the femur, through extended knees when a motor vehicle occupant is bracing during frontal collision, or from dashboard injury to the knee, vertical shear forces are involved. it is an uncommon injury occurring from the axial force through the femur resulting in a posterior acetabular fracture and femoral shaft fracture. the mechanism of injury, in this case, could have been due to an axial force through the extended knee resulting in vertical shear force in the pelvis leading to bilateral acetabulum fracture and bilateral sacral fracture. the segmental comminuted fracture of bilateral shaft femur might have been due to crushing of both the bones between some heavy objects of the jeep body parts. it is, however, difficult to conceive and explain these injuries with any single mode of injury ; as the 4 wheeler had tumbled many times after the initial strike and, therefore, a multi - hit theory only can explain such an injury pattern. bilateral femoral fractures associated with unstable pelvic fractures and visceral damage have been reported with a very high mortality and morbidity rate. these patients should be initially managed with the advanced trauma life support (atls) protocol5, 6 and once stable, surgical intervention should be considered. if the patient remains unstable the principles of damage control orthopaedics (dco) should be followed. this patient presented to us in a state of shock, thus priority for us was to stabilize the patient hemodynamically and once the patient was stabilized we proceeded for the definitive fixation of the fractures in staged manner, giving priority to femoral shaft fractures. in this case, we fixed both the femora in the first sitting with an interlocking nail, with minimal reaming. the patient was moved gently to the fracture table for the fixation of the femur and gentle traction was applied intraoperatively to prevent secondary injury to the pelvic fractures or to prevent re - bleeding from the pelvic cavity. subsequently, in the second sitting an open reduction and internal fixation (orif) of bilateral acetabulum fracture and bilateral sacral fractures was done. tscherne suggested that in patients with multiple closed fractures, femur fractures must be given priority over pelvis. an early and timely surgical intervention to fix all the major fractures helps the patient to overcome the medical complications and provide better rehabilitation. the data suggest that aggressive resuscitation for multiply - injured patients and early or delayed orif without application of external fixation will lead to overall low mortality rate.	the management of multiple complicated injured patients remains a great challenge despite advancements in modern medical care. we present a rare case of bilateral unstable pelvic fractures associated with bilateral segmental femoral shaft fractures. we have proposed a mechanism of such complex injury pattern and discussed the plan of management. we believe that a timely and aggressive surgical intervention to fix all the major fractures soon after medically stabilizing the patient helped our patient to overcome these serious and lethal injuries. it is necessary to establish an optimal protocol for management of such complex fractures by conducting prospective and multicentric studies in the future.
many patients with non - small cell lung cancer or gastrointestinal tract cancer have pleural effusion or ascites at diagnosis. generally, cytological examination is the most common method to detect malignant cells in pleural fluid or ascites ; however, the technique is known to miss up to more than 50% of tumors. it is crucial to detect malignant cells in pleural fluid or ascites for staging work - up and for decision of treatment strategy, especially in non - small cell lung cancer. pleural or peritoneal biopsy, which is the gold standard for diagnosis, is invasive for routine examination. hence, it is important to develop a sensitive and less invasive diagnostic method to improve the detection of malignant cells in pleural fluid or ascites. cd44 is a cell surface adhesion molecule implicated in diverse biologic processes, such as lymphocyte activation and homing, extracellular matrix adhesion and cellular migration. cd44 is composed of at least 20 exons, of which 10 exons can be alternatively spliced into different combinations to make up various isoforms. it has been reported that various human malignant tumors have been associated with over - expression of cd44 variant isoforms, and widespread attention has been focused on cd44 variant isoforms a candidate marker for detection of cancer. however, the variant exons that have been associated with cancer have been detected in corresponding normal tissues in other studies. recently, a novel molecular approach, cd44v8 - 10/cd44v10 competitive rt - pcr, has been developed for the detection of cancer cells over - expressing cd44v8 - 10. this method is based on the concept that the relative expression of cd44v8 - 10 transcripts to cd44v10 transcripts can be quantified by using the endogenous cd44v10 transcripts as a source of internal standard competitor rna. we analyzed from benign and malignant pleural effusion and ascites by cd44 competitive rt - pcr for the detection of cancer cells over - expressing cd44v8 - 10. ags, a human gastric adenocarcinoma cell line(korean cell line bank, seoul, south korea) was maintained in rpmi supplemented with 10% heat - inactivated fetal calf serum(fcs), 100 unit / ml penicillin and 0.1mg / ml streptomycin at 37c in an atmosphere containing 5% co2. the pleural fluid or ascites were obtained from 11 patients with malignant disease (5 non - small cell lung cancer, 5 gastric cancer, 1 pancreatic cancer) and 24 patients with benign disease (14 tbc pleurisy, 10 liver cirrhosis), who were treated at hanyang medical center (table 1, 2, 3). cellular components from pleural fluid or ascites were examined by both cytological and competitive rt - pcr analysis. for competitive rt - pcr analysis, 25 ml of pleural fluid or ascites samples obtained from patients were centrifuged at 3,000 rpm for 10 min and the precipitated cells were washed with phosphate- buffered saline (pbs) and stored at 70c until use. total rna was spectrophotometrically quantified at 260/280 nm and its quality was tested in 1% agarose gel to find intact 28s, and 18s rnas. cdna was synthesized from 5 g of total rna in a 25 l reaction mixture containing 5 l of 5 reverse transcriptase reaction buffer, 200 m dntp, 100 m of random hexamer, 50 units of rnasin, 2 l of 0.1 m dithiothreitol, and 200 units of moloney leukemia virus reverse transcriptase. the mixture was incubated at 37 c for 60 min, heated to 95 c for 10 min, and then chilled on ice. the competitive rt - pcr was performed by using the sense primer, s2 (5-gacagaatccctgctaccaata-3) and the antisense primer, as2 (5-atgtgtcttggtctcctgataa-3) that were designed to amplify both cd44v8 - 10 and cd44v10 cdna, simultaneously, by utilizing sequence identity(17). five microliters of cdna were transferred into an eppendorf tube containing 200 m dntp, 1.5 mm mgcl2, 2.5 u taq polymerase and 20 pmoles of primer s2 and as2. thirty - five cycles of amplification were performed in a thermocycler (robocycler 40 : stratagene, la jolla, ca) at 94 c for 1 min, 56 c for 1 min, and 72 c for 1 min, with a final extension step performed for 10 min at 72 c. to ensure the rna was of sufficient purity for rt - pcr, a pcr assay with primers specific for the gene -actin cdna was carried out in each case. the primer sequences for -actin primers were as follows : 5-tgacggggtcacccacactgtgcccatcta-3 and 5-ctagaagcattgcggtggacgatggaggg-3. each series of rt - pcr reactions included mononuclear cell samples from peripheral blood as a negative control and ags cell samples as a positive control, respectively. the pcr products were electrophoresed on a 1.5% agarose gel and visualized by ethidium bromide staining followed by uv transillumination. the resulting images were density scanned and peak intensity of each band corresponding to the amplimers of cd44v8 - 10 and cd44v10 were then quantified by use of image analyzer, and cd44v8 - 10/cd44v10 (v8- 10/v10) ratio was calculated (biorad, fluor - s image analyzer, bms, usa). all the samples were assessed in 2 independent pcr reactions, and the reported v8- 10/v10 ratios represent the mean values from these duplicate determinations. ags, a human gastric adenocarcinoma cell line(korean cell line bank, seoul, south korea) was maintained in rpmi supplemented with 10% heat - inactivated fetal calf serum(fcs), 100 unit / ml penicillin and 0.1mg / ml streptomycin at 37c in an atmosphere containing 5% co2. the pleural fluid or ascites were obtained from 11 patients with malignant disease (5 non - small cell lung cancer, 5 gastric cancer, 1 pancreatic cancer) and 24 patients with benign disease (14 tbc pleurisy, 10 liver cirrhosis), who were treated at hanyang medical center (table 1, 2, 3). cellular components from pleural fluid or ascites were examined by both cytological and competitive rt - pcr analysis. for competitive rt - pcr analysis, 25 ml of pleural fluid or ascites samples obtained from patients were centrifuged at 3,000 rpm for 10 min and the precipitated cells were washed with phosphate- buffered saline (pbs) and stored at 70c until use. total rna was spectrophotometrically quantified at 260/280 nm and its quality was tested in 1% agarose gel to find intact 28s, and 18s rnas. cdna was synthesized from 5 g of total rna in a 25 l reaction mixture containing 5 l of 5 reverse transcriptase reaction buffer, 200 m dntp, 100 m of random hexamer, 50 units of rnasin, 2 l of 0.1 m dithiothreitol, and 200 units of moloney leukemia virus reverse transcriptase. the mixture was incubated at 37 c for 60 min, heated to 95 c for 10 min, and then chilled on ice. the competitive rt - pcr was performed by using the sense primer, s2 (5-gacagaatccctgctaccaata-3) and the antisense primer, as2 (5-atgtgtcttggtctcctgataa-3) that were designed to amplify both cd44v8 - 10 and cd44v10 cdna, simultaneously, by utilizing sequence identity(17). five microliters of cdna were transferred into an eppendorf tube containing 200 m dntp, 1.5 mm mgcl2, 2.5 u taq polymerase and 20 pmoles of primer s2 and as2. thirty - five cycles of amplification were performed in a thermocycler (robocycler 40 : stratagene, la jolla, ca) at 94 c for 1 min, 56 c for 1 min, and 72 c for 1 min, with a final extension step performed for 10 min at 72 c. to ensure the rna was of sufficient purity for rt - pcr, a pcr assay with primers specific for the gene -actin cdna was carried out in each case. the primer sequences for -actin primers were as follows : 5-tgacggggtcacccacactgtgcccatcta-3 and 5-ctagaagcattgcggtggacgatggaggg-3. each series of rt - pcr reactions included mononuclear cell samples from peripheral blood as a negative control and ags cell samples as a positive control, respectively. the pcr products were electrophoresed on a 1.5% agarose gel and visualized by ethidium bromide staining followed by uv transillumination. the resulting images were density scanned and peak intensity of each band corresponding to the amplimers of cd44v8 - 10 and cd44v10 were then quantified by use of image analyzer, and cd44v8 - 10/cd44v10 (v8- 10/v10) ratio was calculated (biorad, fluor - s image analyzer, bms, usa). all the samples were assessed in 2 independent pcr reactions, and the reported v8- 10/v10 ratios represent the mean values from these duplicate determinations. to examine the validity of the rt - pcr method as a quantitative assay, 210 normal mononuclear cells were mixed in separate tests with an increasing number of ags gastric cancer cells expressing cd44v8 - 10. the resultant mixtures were subjected to competitive rt - pcr. as shown in fig. 1 when the v8- 10/v10 ratios were plotted against the number of added ags cells, linear relationships were observed. twenty one samples, 14 from patients with benign pleural fluid and 7 from patients with malignant effusion were analyzed by competitive rt - pcr by using the set of primers, s2 and as2, that can co - amplify cd44v8 - 10 and cd44v10. the cd44v10 transcript was predominantly expressed in comparison with the cd44v8 - 10 transcript in all 14 of the pleural effusion samples from patients with benign disease (v8 - 10/v10 ratio : 0.2190.948), whereas 6 of 7 samples from patients with malignant cancers (5 non - small cell lung cancer, 2 gastric cancer) presented a predominant expression of cd44v8 - 10 transcript (table 1,3). the representative data are shown in figure 2 and 3. when we place the diagnostic cutoff value for the v8 - 10/v10 ratio at 1.0, the sensitivity and specificity of competitive rt - pcr were 85.7% and 100%, respectively. fourteen samples, 10 from patients with benign ascites (10 liver cirrhosis) and 4 from patients with malignant ascites (3 gastric cancer, 1 pancreatic cancer) were analyzed as previously described. 1.0 (table 2,3). to examine the validity of the rt - pcr method as a quantitative assay, 210 normal mononuclear cells were mixed in separate tests with an increasing number of ags gastric cancer cells expressing cd44v8 - 10. the resultant mixtures were subjected to competitive rt - pcr. as shown in fig. 1 when the v8- 10/v10 ratios were plotted against the number of added ags cells, linear relationships were observed. twenty one samples, 14 from patients with benign pleural fluid and 7 from patients with malignant effusion were analyzed by competitive rt - pcr by using the set of primers, s2 and as2, that can co - amplify cd44v8 - 10 and cd44v10. the cd44v10 transcript was predominantly expressed in comparison with the cd44v8 - 10 transcript in all 14 of the pleural effusion samples from patients with benign disease (v8 - 10/v10 ratio : 0.2190.948), whereas 6 of 7 samples from patients with malignant cancers (5 non - small cell lung cancer, 2 gastric cancer) presented a predominant expression of cd44v8 - 10 transcript (table 1,3). when we place the diagnostic cutoff value for the v8 - 10/v10 ratio at 1.0, the sensitivity and specificity of competitive rt - pcr were 85.7% and 100%, respectively. fourteen samples, 10 from patients with benign ascites (10 liver cirrhosis) and 4 from patients with malignant ascites (3 gastric cancer, 1 pancreatic cancer) were analyzed as previously described. 1.0 (table 2,3). it has been known that conventional cytological analysis does not have high sensitivity to detect malignant cells in pleural fluid or ascites. at present, there is no reliable biological marker that is used for routine clinical examinations. recently, molecular biological techniques make it possible to detect cancer cells in those samples. since it has been reported that a cd44 splice variant containing variant exon 6 could confer metastatic potential on rat pancreatic adenocarcinoma cells, a great deal of interest many investigators reported that the expression of specific cd44 variant exons was detected in human carcinomas, such as bladder cancer, non - small cell lung cancer and breast cancer, indicating that molecular approaches of cd44 splice variant might be a promising tool for cancer diagnosis. other investigators demonstrated that among the cd44 splice variants, cd44v8 - 10 was predominantly expressed in human colon cancer and non - small cell lung cancer by rt - pcr using the set of primers that are able to amplify all cd44 variant isoforms and dna sequence analysis. however, cd44v10 was also expressed in normal leukocytes, so the discrimination method should be needed. recently, okamoto developed noble competitive rt - pcr with which to quantify the relative expression of cd44v8 - 10 transcripts to cd44v10 transcripts. the endogenous cd44v10 transcripts, which were identified as the predominant cd44 splice variants in leukocytes, were used as internal standard, competitor rnas for measuring the expression level of cd44v8 - 10 transcripts. when we applied the competitive rt - pcr assay to pleural fluid and ascites, our results showed that the v8 - 10/v10 ratios in all the samples associated with benign disease were less than < 1.0, whereas malignant diseases revealed significantly higher than those associated with benign diseases. although the v8 - 10/v10 ratio displayed large variation between the benign diseases, the ratios were consistently low in various benign diseases suggesting that the v8- 10/v10 ratio might be a useful molecular marker for cancer diagnosis. these findings indicate that the cd44v8 - 10/v10 ratio may be an important molecular marker for cancer diagnosis, especially in exfoliated cells from pleural fluid. cd44 competitive rt - pcr assay is a useful and adjunct to cytological examination in cancer diagnosis, especially in detecting exfoliated cancer cells in pleural effusion. to determine the exact role of cd44 variant in the diagnosis of exfoliated cells from pleural	background : cd44 is a cell surface adhesion molecule which has been implicated in various biologic functions as lymphocyte homing and activation, cellular migration and extracellular matrix adhesion. over - expression of cd44v8 - 10 has been found in several cancers and is considered to be associated with tumor progression and metastasis. recently, a novel molecular method, cd44v8 - 10/cd44v10 competitive reverse transcription - polymerase chain reaction (rt - pcr) has been developed for detecting cancer cells over - expressing cd44v8-10.methods : we analyzed from benign and malignant pleural effusion and ascites by cd44 competitive rt - pcr and compared to the conventional cytology.results : the cd44 competitive rt - pcr analysis showed that all the 24 samples associated with benign disease presented a predominant expression of the cd44v10 transcript (v8 - 10/v10 ratio : 0.1260.948), whereas 6 of 7 malignant pleural samples associated with cytology positive cancer expressed the cd44v8 - 10 transcript (v8 - 10/v10 ratio > 1.00).conclusion : these results indicate that cd44 competitive rt - pcr assay is a useful and adjunct to cytological examination in cancer diagnosis, especially in detecting exfoliated cancer cells in pleural effusion.
recent studies demonstrate roles for regulatory noncoding rna molecules (ncrnas) in normal cns development and function and in the onset and progression of various neurodegenerative diseases. ncrnas are functional rna molecules expressed specifically in the central nervous system that do not encode proteins. they are classified as small ncrnas comprising fewer than 400 nucleotides and long ncrnas comprising more than 400 nucleotides. small ncrnas include ribosomal rnas (rrnas), transfer rnas (trnas), small nucleolar rnas (snrnas), micrornas (mirnas), short small interference rnas (sirnas), and piwi - interacting rnas. long noncoding rnas (lncrnas) include heterogeneous regulatory molecules such as long intergenic noncoding rnas (linc rnas) and natural antisense transcripts (nats) [13 ]. ncrnas play critical roles in neuronal processes such as transcription of neuronal genes, brain morphogenesis, neuronal cell specification, and formation of memory. micrornas (mirnas) are small rna molecules (2123 nucleotides) involved in the regulation of gene expression that bind posttranscriptionally to the 3-untranslated region of target mrnas and either inhibit translation or degrade the target mrna. a single - stranded rna, mirna, is derived from a 70100-nucleotide hairpin precursor called pre - mirna that plays a key role in posttranscriptional regulation of target gene expression. due to their small size, mirnas are potentially a valuable tool for therapy of cancer, neurodegenerative, and cardiovascular diseases [5, 6 ]. the mirnas are transcribed by either rna polymerase ii or iii in the nucleus to yield primary transcripts (pri - mirnas) of different lengths that are processed by the drosha / dgcr8 enzyme complex into a ~70 base pair precursor mirna. the precursor mirnas are transported into the cytoplasm where cytoplasmic rnase iii enzymes, dicer and loquacious, process them into ~22-nucleotide mirna duplexes with guide and passenger strands. the guide strand functions as a mature mirna and is incorporated into an rna - induced silencing complex (risc). this complex contains an argonaute (ago) protein as a primary component that binds to the target mrna and degrades the passenger strand. mature mirna guides risc to recognize target sequences located in the 3utr of mrnas, which leads to the inhibition of translation or degradation of mrna [6, 7 ]. neurodegeneration refers to the death of neurons during the normal course of brain development, and many studies demonstrate the involvement of mirnas. the mirnas are key regulators of cns development involving brain plasticity, neuronal cell maturation, neuronal cell differentiation, and synaptogenesis [8, 9 ]. dysregulation of mirnas leads to the development of neurodegenerative diseases such as alzheimer 's disease, parkinson 's disease, and huntington 's disease. alzheimer 's disease (ad) is the most common form of dementia, among elderly adults, and causes memory impairment as well as problems with thinking, decision - making, and behavior. ad is characterized by dysregulated processing of amyloid precursor protein (app) and the accumulation of amyloid beta (a) peptide in brain hippocampus. -secretase, also known as -site app - cleaving enzyme 1 (bace 1), cleaves app to generate a peptides. several studies show that increased expression of app, an integral membrane protein, is associated with ad.. showed that mir-101 negatively regulates the expression of app and a accumulation in cultured hippocampal neurons, thus suggesting an essential role for mirna in the development and progression of ad. postmortem studies of human brain hippocampus showed increased levels of bace1, upregulation of mir-9, mir-125b, and mir-125b, mir-146a, and downregulation of mir-15a, mir-107, and mir-29a / b-1, which suggests a link between mirnas and protease in the progression of the disease [3, 9 ]. decrease in the expression of mir-107 was observed in patients who exhibit early stages of ad. affymetrix exon array microarrays demonstrated a sharp increase in bace1 mrna expression and a decrease in mir-107 expression. in addition, the mir-29a / b-1 cluster is down - regulated in ad patient 's brain while upregulation of bace1 is observed, which suggests a role for the mir-29a / b-1 cluster in negative regulation of bace1 expression that leads to the accumulation of a peptide. certain patterns of mirna expression in the grey matter of cortex also lead to ad pathogenesis. mir-211 is downregulated in alzheimer 's disease brain, whereas there is an upregulation of mir-424. a murine model of ad showed that there was no correlation between bace1 mrna and levels of proteins in the hippocampus of appswe / ps1 mice.. supports the concept that there is an association between dysfunctional mirna regulation of bace1 expression and ad by further analysis of mir-298 and mir-328 in the hippocampus of appswe / ps1 mice. in addition, a novel noncoding rna 17a expressed in human brain is upregulated in cerebral tissues of ad patients and leads to increased secretion of a peptides, gpr51 alternative splicing, and ultimately, impaired gaba b function. a similar study showed that neuroblastoma differentiation marker 29 (ndm29), a rna polymerase iii - dependent noncoding rna, enhances the synthesis of amyloid precursor protein, eventually resulting in an increase of a secretion that promotes the formation of a peptides as they may occur in alzheimer 's disease. nf-b signaling regulates mir-146a, which is upregulated in ad brains, and downregulation of cfh protein is observed in ad brains, which suggests that mir-146a targets cfh protein, a brain inflammatory response repressor. the death of dopamine generating cells in the brain substantia nigra and accumulation of alpha - synuclein leads to the development of parkinson 's disease, which is characterized by motor abnormalities and sensory, mood, and cognitive changes. a study suggests that -synuclein is regulated posttranscriptionally by mir-7 and mir-153, which bind directly to the 3-untranslated region of synuclein and suppress its expression. in addition, downregulation of synuclein due to mir-7 and mir-153 protects cells from oxidative stress. in primary neurons, increased translation of a luciferase construct that bears synuclein 3-untranslated region was reportedly caused by inhibition of micro - rnas mir-7 and mir-153. elevated levels of fibroblast growth factor 20 (fgf20) genes also lead to overexpression of synuclein and eventually to the death of dopaminergic neurons. furthermore, this demonstrates that binding of mir-433 to a single nucleotide polymorphism in the promoter region of fgf20 disrupts the target site for mir-433, which eventually results in overexpression of synuclein. downregulation of mir-7 in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-(mptp-) induced neurotoxin pd mouse model suggests that it may protect cells against oxidative stress. the most vital gene in parkinson 's disease, lrrk2 that functions in the dopamine generating cells negatively regulates let-7 and mir-184, which causes overexpression of their targets e2f1 and dp eventually, leads to defects in cell division and cell death. expression of mir-133b is detected in the midbrain dopaminergic neurons but not detected in parkinson 's disease midbrain tissue. a negative feedback loop with the transcription factor pitx3, found in midbrain dopamine generating neurons, is formed by the downregulation of mir-133b, which is a dopaminergic neuron - specific mirna. furthermore, a recent study suggests that overexpression of synuclein by mir-433 leads to increased risk of ad. also, activation of two neuroprotective genes pten - induced putative kinase (pink1) and parkinson disease 7 (park7) normally involved in protecting cells from oxidative stress leads to development of parkinson 's disease through pten signaling. huntington 's disease is an autosomal dominant neurodegenerative disease characterized by cognitive decline and impaired movement. it is caused by trinucleotide expansion in the gene that encodes a polyglutamine stretch near the n - terminus of huntingtin. multiple studies of mirnas in mouse and human brain have shown mir-22, mir-29c, and mir-128 are down - regulated in mice and mir-9/9, mir-29b, and mir-124a are down - regulated in humans [25, 26 ]. showed that mir-221 and mir-222 are both down - regulated in hd. also, downregulation of mirnas let-7a, let-7c, let-7d, and let 7e was observed in hd - fc (frontal cortex) and hd - st (striatum) whereas there was upregulation of mir-30b, mir-30c, and mir-30e in hd - fc and hd - st. the mir-30a, that is, upregulated in hd - fc and hd - st has been shown to target bdnf, which is a rest - modulated neuronal gene. loss of bdnf leads to loss of medium spiny neurons [28, 29 ] rest / corest regulates many mirnas. rest repressor complex regulates mir-9 and mir-9 that target rest / corest expression and are considered to be a part of a double - negative feedback loop. expression of mir-9/9, mir-124a, and mir-132 is regulated by the rest repressor complex including msin3, rest corepressor 1 (corest), and mecp2. rest silencing complex and mir-9/9 give rise to a negative feedback loop during the development of huntington 's disease due to the effects of mir-9 and mir-9 on rest and corest. several mirnas such as mir-9/9, mir-129a, mir-132, mir-29b, mir-29a, mir-330, mir-17, mir-196, mir-222, mir-485, and mir-486 are affected in hd due to the disrupted mirna transcriptome [27, 30 ]. moreover, reports suggest that mir-34b is elevated in plasma of huntington 's disease patients even before the onset of symptoms, which implies that mir-34b may be useful as a potential diagnostic biomarker for hd [32, 33 ]. clearly, there is evidence for mirnas as potential biomarkers to aid in the diagnosis of neurodegenerative diseases. for instance, rna interference (rnai) knocks down the translation of certain disease - associated molecules due to the presence of short hairpin rnas, synthetic double - stranded rnas, and the precursors of artificial mirnas that are expressed by viral vectors. these drugs are synthetic rna molecules that mimic a mechanism of action similar to that of endogenous ncrnas sirnas or mirnas. thus, rna interference technology has emerged as a potential therapeutic tool for several neurodegenerative diseases. wang. suggest the following two different potential roles of mir-146 : (1) as a useful biomarker in the early diagnosis of ad since it can be detected in human blood monocytes, which aids in easy collection, and (2) as a therapeutic tool. they also suggest that anti - mir-146a can be used with therapeutic potential to suppress the up - regulatory effects of mir-146. another study conducted to examine the pattern of a driven neurogenesis in a transgenic mouse model of ad - tg-19959 showed that the use of lna - modified sirnas led to a marked decrease in the levels of insoluble a peptides and altered the pattern of aggregation of soluble a in the tg-19959 mouse brain by targeting bace1 and bace - as. identified two diagnostic biomarkers : dysregulation of mirna in ad patients ' brains and changes in mirna specific to ad in cerebrospinal fluid. several mirnas such as mir-133b, mir-7, mir-153, let-7, mir184, and mir-433 may be involved in the pathophysiology of pd and offer a novel approach for therapeutic targets. in addition, the mirna pathway is known to play a crucial role in translational regulation in the pathophysiology of pd and hence is considered one of the most important translation regulation pathways. (2005) showed notable signs of improvement in the behavioral abnormalities and neuropathology among hd patients with the use of an adenoassociated virus serotype 1 (aav1) vector that targets the mutant human htt in the brain of mouse giving rise to u6 promoter - driven shrna effector system. in addition, noncoding small rna - based technology plays a pivotal role in the treatment of huntington 's disease. antisense oligonucleotides (aos) help to prevent mrnas bearing mutations by redirecting pre - mrna splicing. overall, data suggest that various noncoding rnas could serve as useful diagnostic biomarkers and therapeutic targets for neurodegenerative diseases. ncrnas, especially mirnas, are known for their roles in normal development and function of the central nervous system. they are involved in targeted gene expression and act as key regulators in several neuroprotective mechanisms. it is a well - established fact that dysregulation of mirnas eventually leads to the development of various neurodegenerative diseases. alterations in the patterns of mirna expression will probably serve as diagnostic biomarkers of brain function, and the initiation and progression of neurological disorders and neurodegenerative diseases. further research in mirna expression patterns and profiling will result in the discovery of many more novel biomarkers. in - depth study to determine the functions of long noncoding rnas in rna - mediated gene regulation is likely to remain an area of intense research interest.	noncoding rnas are widely known for their various essential roles in the development of central nervous system. it involves neurogenesis, neural stem cells generation, maintenance and maturation, neurotransmission, neural network plasticity, formation of synapses, and even brain aging and dna damage responses. in this review, we will discuss the biogenesis of microrna, various functions of noncoding rna 's specifically micrornas (mirnas) that act as the chief regulators of gene expression, and focus in particular on misregulation of mirnas which leads to several neurodegenerative diseases as well as its therapeutic outcome. recent evidences has shown that mirnas expression levels are changed in patients with neurodegenerative diseases ; hence, mirna can be used as a potential diagnostic biomarker and serve as an effective therapeutic tool in overcoming various neurodegenerative disease processes.
stress urinary incontinence (sui), which is a complaint of involuntary urine leakage on effort, exertion, sneezing, or coughing, is a common functional disease in women. understanding of the physiopathological concepts of female sui has steadily developed and the application of this improved understanding has led to improved surgical procedures aimed at correcting this disorder. since the tension - free vaginal tape (tvt) procedure was first reported by ulmsten. in 1996, midurethral sling procedures have been considered safe and effective for treating female sui. in 2002, delorme. described the transobturator technique for a midurethral sling in which they passed the tape through the obturator foramen from outside to inside. de leval demonstrated a transobturator midurethral sling procedure in which the sling passes the obturator foramen from inside to outside. in long - term follow - up studies, the transobturator technique has similar efficacy and safety compared with that of tvt. despite the popularity of these procedures, prognostic factors for the surgical outcome of correcting sui are still debated. valsalva leak point pressure (vlpp) is defined by the international continence society (ics) as the intravesical pressure generated by the valsalva maneuver at which urine leakage occurs owing to increased abdominal pressure in the absence of detrusor contraction. clinical application of vlpp remains controversial, however, and the effect of vlpp on treatment outcome of distal polypropylene sling procedures or pubovaginal sling procedures is undetermined. only a few studies have examined outcomes of transobturator midurethral slings stratified by the preoperative severity of urinary incontinence. additionally, few studies have assessed the relationship between incontinence severity and validated disease - specific quality of life. thus, we designed this study to assess the influence of preoperative vlpp and stamey grade on quality of life and surgical outcome from transobturator midurethral tape implantation for treating sui in women. a total of 204 female patients who underwent transobturator midurethral sling placement for the treatment of urodynamic sui from march 2008 to february 2012 were included in this retrospective study. the preoperative evaluation consisted of a detailed history, urologic physical examination, and a urodynamic study (uds). the physical examination included a pelvic organ prolapse quantification system classification, q - tip test, and a stress test in the lithotomy position at physiological maximum bladder capacity. all patients underwent a preoperative uds, including uroflowmetry, provocative cystometry, and urethral profilometry. the stamey incontinence score (grade 0, continent ; grade 1, loss of urine with sudden increase in abdominal pressure such as from coughing, sneezing, or laughing ; grade 2, leaks with lesser degrees of physical stress such as walking, standing erect from a sitting position, or sitting up in bed ; grade 3, total incontinence, urine is lost without any relation to physical activity or position) was checked to represent sui severity in all enrolled patients. in cases of grade 3, we executed cystoscopy or a radiological evaluation to rule out the possibility of a fistula. patients were then stratified by stamey incontinence score into three groups (grades 1, 2, 3). all surgery was performed under spinal or general anesthesia according to the decision of the anesthesiologist. suburethral tape was inserted via an inside - out transobturator midurethral sling (tvt - o ; gynecare, chaska, mn, usa) by the same experienced surgeon in all patients (s.o.k.). the follow - up examination at 6 months included a physical examination with a stress test, an interview for signs and symptoms, and assessment of patient satisfaction with the procedure. the procedure for this study complied with the guidelines provided by the declaration of helsinki. patients were excluded from this study according to the following criteria : patients who had undergone previous spine or brain surgery, patients with concomitant high - grade pelvic organ prolapse (> grade iii), patients with a history of urinary retention with residual urine > 200 ml, and patients with active urinary tract infection (confirmed by nlaton catheterization and urinalysis) and other urologic disease. patients using drugs that could have an impact on bladder and urethral function, such as alpha - adrenergic receptor agonists or antagonists, were also excluded. the incontinence quality of life questionnaire (i - qol), a self - reported quality of life measure specific to urinary incontinence, was completed, and the objective cure rate of incontinence was measured by a stress test before and 6 months after surgery. cure was defined as no leakage of urine postoperatively both subjectively and objectively, and improvement was defined as no urine leakage on a subjective exam with subjective urine leakage sometimes but with overall satisfaction, whereas failure was defined as objective loss of urine during the stress test. we compared the pre- and postoperative i - qol scores and cure rates according to preoperative vlpp and stamey grade. the bladder was filled at a constant rate of 50 ml / min by using normal saline solution at room temperature for standard uds. vlpp was obtained with the subject seated when the total infused volume of sterile water reached 200 ml by asking the patient to perform a valsalva maneuver until urine loss was directly observed. the vlpp, a measurement of the lowest abdominal pressure required to produce urine leakage, was recorded and was repeated to verify the initial finding. if a urine leak was not noted with the valsalva maneuver, the patient was asked to cough. patients were categorized into three groups on the basis of preoperative vlpp : group 1 (vlpp60 cm h2o), group 2 (6090 cm h2o). 17 (spss inc., chicago, il, usa) was used for the statistical analyses. data were analyzed by one - way analysis of variance and bonferroni 's post hoc test. a total of 204 female patients who underwent transobturator midurethral sling placement for the treatment of urodynamic sui from march 2008 to february 2012 were included in this retrospective study. the preoperative evaluation consisted of a detailed history, urologic physical examination, and a urodynamic study (uds). the physical examination included a pelvic organ prolapse quantification system classification, q - tip test, and a stress test in the lithotomy position at physiological maximum bladder capacity. all patients underwent a preoperative uds, including uroflowmetry, provocative cystometry, and urethral profilometry. the stamey incontinence score (grade 0, continent ; grade 1, loss of urine with sudden increase in abdominal pressure such as from coughing, sneezing, or laughing ; grade 2, leaks with lesser degrees of physical stress such as walking, standing erect from a sitting position, or sitting up in bed ; grade 3, total incontinence, urine is lost without any relation to physical activity or position) was checked to represent sui severity in all enrolled patients. in cases of grade 3, we executed cystoscopy or a radiological evaluation to rule out the possibility of a fistula. patients were then stratified by stamey incontinence score into three groups (grades 1, 2, 3). all surgery was performed under spinal or general anesthesia according to the decision of the anesthesiologist. suburethral tape was inserted via an inside - out transobturator midurethral sling (tvt - o ; gynecare, chaska, mn, usa) by the same experienced surgeon in all patients (s.o.k.). the follow - up examination at 6 months included a physical examination with a stress test, an interview for signs and symptoms, and assessment of patient satisfaction with the procedure. the procedure for this study complied with the guidelines provided by the declaration of helsinki. patients were excluded from this study according to the following criteria : patients who had undergone previous spine or brain surgery, patients with concomitant high - grade pelvic organ prolapse (> grade iii), patients with a history of urinary retention with residual urine > 200 ml, and patients with active urinary tract infection (confirmed by nlaton catheterization and urinalysis) and other urologic disease. patients using drugs that could have an impact on bladder and urethral function, such as alpha - adrenergic receptor agonists or antagonists, were also excluded. the incontinence quality of life questionnaire (i - qol), a self - reported quality of life measure specific to urinary incontinence, was completed, and the objective cure rate of incontinence was measured by a stress test before and 6 months after surgery. cure was defined as no leakage of urine postoperatively both subjectively and objectively, and improvement was defined as no urine leakage on a subjective exam with subjective urine leakage sometimes but with overall satisfaction, whereas failure was defined as objective loss of urine during the stress test. we compared the pre- and postoperative i - qol scores and cure rates according to preoperative vlpp and stamey grade. the bladder was filled at a constant rate of 50 ml / min by using normal saline solution at room temperature for standard uds. vlpp was obtained with the subject seated when the total infused volume of sterile water reached 200 ml by asking the patient to perform a valsalva maneuver until urine loss was directly observed. the vlpp, a measurement of the lowest abdominal pressure required to produce urine leakage, was recorded and was repeated to verify the initial finding. if a urine leak was not noted with the valsalva maneuver, the patient was asked to cough. patients were categorized into three groups on the basis of preoperative vlpp : group 1 (vlpp60 cm h2o), group 2 (6090 cm h2o). spss ver. 17 (spss inc., chicago, il, usa) was used for the statistical analyses. data were analyzed by one - way analysis of variance and bonferroni 's post hoc test. the mean age of the patients was 52.89.6 years, and the duration of disease was 61.458.0 months. three patients had a hormonal therapy history, and no patient had undergone radiation therapy. incontinence was the pure sui type in 171 patients (83.8%), whereas 33 patients (16.2%) showed a mixed type of stress and urgency urinary incontinence. the numbers of patients with stamey grades i, ii, and iii were 99 (48.5%), 84 (41.2%), and 21 (10.3%), respectively, and 30 (14.7%), 87 (42.6%), and 87 patients (42.6%) showed vlpp60 cm h2o, 6090 cm h2o, respectively. table 2 shows the objective cure rate and changes in i - qol scores according to preoperative vlpp. no significant difference was observed in objective cure rate (p=0.860), preoperative i - qol (p=0.327), postoperative i - qol (p=0.560), or changes in i - qol (p=0.924) between the groups when we compared outcome by preoperative vlpp. measuring outcome by preoperative stamey grade also did not make a significant difference in objective cure rate (p=0.985) or postoperative i - qol (p=0.944). however, preoperative i - qol scores were significantly different (p=0.001) between the groups. a significantly lower i - qol score was found and the score improved remarkably after surgery (p=0.001) when patients had a high preoperative stamey grade (table 3). we found that vlpp was not related to the cure rate or quality of life after the midurethral sling operation for treating sui. however, preoperative quality of life was significantly different between the groups, with a significantly lower i - qol score and pronounced improvement after surgery when patients had a high preoperative stamey grade. these results suggest that stamey grade rather than vlpp is important for predicting subjective satisfaction and improved incontinence - related quality of life after midurethral sling surgery in women. vlpp has been used as an objective determination of sui severity in women to distinguish between intrinsic sphincter deficiency and urethral hypermobility as a cause of sui. women with low vlpp on uds have been considered to be at increased risk for surgical failure after anti - incontinence surgery. vlpp has been used to predict the surgical outcome of midurethral sling surgery for correcting sui and to choose an optimal surgical treatment in each woman. these findings were confirmed by o'connor., who reported that 77% of patients with a preoperative vlpp>60 cm h2o were cured compared with 25% of patients with vlpp60 cm h2o. similarly, romancik. specifically investigated vlpp as an outcome - predicting factor in 65 patients who underwent tot and found cure and success rates to be significantly higher in patients with vlpp>60 cm h2o (78% and 100%, respectively) than in patients with vlpp60 cm h2o (25% and 78% ; p80 cm h2o ; group 3, vlpp 30 - 80 cm h2o ; group 4, vlpp60 cm h2o. when cure rates were compared with preoperative vlpp, no significant difference emerged at a mean follow - up of 21.6 months (89.7% vs. 88.9%, respectively). furthermore, a multicenter well - powered study confirmed that vlpp does not predict outcome success after burch or an autologous fascia lata sling procedure in women with pure or predominant sui at a 24-month follow - up. thus, consensus is lacking on whether preoperative vlpp predicts outcome from anti - incontinence surgery, but the current trend on this issue is that surgical outcome is not associated with preoperative vlpp. reports suggesting that all types of incontinence can be treated by a similar surgical method and that a distal urethral sling cures incontinence without correcting urethral hypermobility support the results of the present study. however, our results showed that stamey grade rather than vlpp was important for predicting baseline subjective score of quality of life and improvement in incontinence - related quality of life after midurethral anti - incontinence surgery in women with sui. the necessity and usage of preoperative uds is still questioned, and related studies have been conducted. nager. recently reported in their multicenter randomized study that a preoperative office evaluation alone is not inferior to a uds for surgical outcome in women with uncomplicated, demonstrable sui. the national institutes for health and clinical excellence advised against routine uds in women with a clearly defined clinical diagnosis of pure sui, although some organizations recommend routine uds before surgery for sui. moreover, it is not easy to use vlpp to assist in choosing the surgical method or surgical decision because the methodology is not standardized. many related factors such as catheter size, bladder volume, patient position, and valsalva method or cough could influence the vlpp measurement, and all clinicians must recognize this fully when they analyze the results. we could not address the role of vlpp in women with more challenging issues including urgency - predominant incontinence, incontinence combined with neurologic disease, previous surgery, or sui concomitant with pelvic organ prolapse. we included some patients with mixed urinary incontinence, and the urgency component of incontinence was not completely excluded when we analyzed the data, which possibly affected the results. stamey grade rather than vlpp was related with preoperative baseline quality of life and improvement of quality of life after surgery.	purposewe investigated whether the valsalva leak point pressure (vlpp) is valuable for predicting postoperative outcome measurement after transobturator suburethral tape (tvt - o) implantation for treating stress urinary incontinence (sui) in women.materials and methodsa total of 204 female patients who underwent tvt - o placement for treatment of sui from march 2008 to february 2012 were enrolled in this retrospective study. all patients completed the incontinence quality of life questionnaire (i - qol), a self - reported quality of life measure specific to urinary incontinence, and the cure rate of incontinence was measured before and 6 months after surgery. cure was defined as no leakage of urine postoperatively both subjectively and objectively. we compared pre- and postoperative i - qol scores according to preoperative vlpp and stamey grade.resultsthe numbers of patients with stamey grades i, ii, and iii were 99 (48.5%), 84 (41.2%), and 21 (10.3%), respectively. a total of 30 (14.7%), 87 (42.6%), and 87 patients (42.6%) showed vlpp60, 6090 cm h2o, respectively. preoperative vlpp was not significantly different according to preoperative i - qol or change in i - qol after surgery. however, i - qol after surgery improved in patients with a high preoperative stamey grade (p=0.001).conclusionsvlpp was not a factor related to surgical outcome from the midurethral sling procedure. stamey grade rather than vlpp was important for predicting subjective quality of life and improved incontinence - related quality of life after surgery.
they have a theoretical advantage over metal analogues in their secure initial fixation and subsequent degradation with eventual replacement by host tissues. the rigidfix anterior cruciate ligament (acl) cross pin (mitek, westwood, ma, usa) utilises polylactic (pla) pins to give the advantage of 360 bone - to - graft contact at the graft tunnel, with clinically acceptable pull - out strength and proven patient outcomes [2, 3 ]. this fixation method does, however, have the disadvantages of local tissue response, such as inflammation and swelling [4, 5 ]. we report the case of implant migration and the loose body presenting as a subcutaneous collection. a 25-year - old fit and well male sustained a left complete acl rupture in august 2001 due to a rotational soccer injury. this was confirmed at subsequent arthroscopy, and after a course of physiotherapy and a period of nonoperative treatment he elected for a reconstruction. this procedure was performed 3 years after the initial injury in august 2004 using bone patellar tendon bone (btb) without complication. 9 and 10 mm bone plugs were harvested and fixed in the femoral canal with two rigidfix cross pins placed from the lateral side of the femur as per the standard operative technique. the tibia was fixed with a 20 mm 9 mm metal interference screw. a concomitant posterior 1/3 tear of the medial meniscus was repaired at the time of operation with 2 mitek bioabsorbable sutures. postoperatively, the patient made a full recovery and returned to sporting activity at 6 months. whilst on holiday in february 2008, the patient developed an acute collection on the medial side of his knee overlying the medial femoral condyle. this was incised and drained, and a 22 mm fragment consistent with the rigidfix implant was retrieved. the wound healed and was treated with antibiotics, although no evidence of infection was documented. the patient s inflammatory markers were normal and at last follow - up there was no evidence of superficial or deep infection. the range of movement in the knee was 0120 and the acl was stable based on the normal anterior drawer and lachman tests.fig. 1retrieved rigidfix cross pin retrieved rigidfix cross pin magnetic resonance imaging (mri) undertaken in october 2008 demonstrated an intact reconstructed acl with a well - healed medial meniscus (figs. 2, 3).fig. 2post operative mri scan showing intact acl and healed medial meniscus (coronal view)fig. 3post operative mri scan showing intact acl (sagittal view) post operative mri scan showing intact acl and healed medial meniscus (coronal view) post operative mri scan showing intact acl (sagittal view) the patient gave informed consent prior to being included in the study. we believe this to be the second reported case of loosening of the rigidfix bioabsorbable pin for btb acl reconstruction. unlike in the previous case, where the entire pin was found in two sections within the joint, we describe a subcutaneous pla implant fragment that caused local inflammation within the tissues on the medial aspect of the knee. cross - pin transverse femoral fixation in acl reconstruction principally leads to increased pull - out strength of the graft. this, however, can lead to backing out of the pin if not positioned correctly, which is not the case in this patient, as the operation was done according to the standard rigidfix surgical technique. the timing of the backing out of the pin is unclear. what was apparent, however, was that the graft appeared to incorporate well on mri and give a clinically acceptable outcome, even after losing one pin [3, 4 ]. the pla implant remained unabsorbed 42 months postoperatively (fig. 1). this is in keeping with previous studies demonstrating the presence of bioabsorbable implant remnants up to 5.7 years after implantation [1, 4 ]. while the backing out of these smooth pins did not cause any loss of acl function in this case, it did result in the implant acting as a loose body. this loose body was in contact with the joint for an undefined period of time before it became embedded in the medial soft tissues. this intraarticular contact theoretically placed the joint at risk of both mechanical and chemical damage. this is thought to promote inflammation, and has been directly linked to a reduction in cultured cell growth and possible chondral damage. local clearance by macrophages and polymophonuclear leukocytes is thought to be overloaded by excessive breakdown of bioabsorbable implants, which in theory could be increased by the presence of a loose pla pin in the intra - articular space. it is unclear as to the stage at which the pin backed out, and therefore how long the loose body was indeed loose. the fragment was also discovered in the subcutaneous tissues on the contralateral side of the knee to its insertion point, so it can only be postulated that it must have travelled through the joint itself. while bioabsorbable implants do have the benefits of good initial fixation and a larger surface area for integration than interference screws, they do carry a risk of backing out and the subsequent loose body reaction. we have not been able to determine the cause of the pins backing out in this case. it is, however, a rare occurrence for what is a well - tolerated operation with good functional outcome. it is worth considering a loose body from this graft in the event of knee pain or failure to progress in the postoperative period.	we report a case of loosening of a bioabsorbable cross - pin fixation device for anterior cruciate ligament reconstruction. forty - two months following a bone tendon bone reconstruction of the anterior cruciate ligament, the patient presented with a subcutaneous collection in the medial side of the knee. at subsequent surgery, a rigidfix cross - pin fixator (mitek, westwood, ma, usa) was retrieved, intact, from the sterile fluctuant mass around the superomedial aspect of the knee. the graft was stable both radiologically and clinically, and the patient remains symptom free. this case raises concern about the use of this smooth cross - pin fixator and the consequences of backing out and the resultant intraarticular loose body. we suggest consideration of a loose body if the patient becomes symptomatic postoperatively, and early intervention to prevent chondral damage is recommended.
data were drawn from project eat (eating and activity in teens and young adults), a 10-year longitudinal study of eating - related, anthropometric, and psychosocial factors among young people. paul, minnesota assessed in early / middle adolescence [eati ; time 1 (t1) ; age 11 - 18y ] from 1998 - 1999. follow - up data were collected 5- and 10-years later [eat - ii : time 2 (t2)=middle adolescence / early young adulthood ; age 16 - 23y ; eat - iii : time 3 (t3)=early / middle young adulthood ; age 21 - 29y ] to investigate changes in weight - related outcomes and their correlates. the eat survey assesses cognitions, behaviors, and attitudes related to eating and psychological functioning. test - retest data for the eat - i survey were collected on 161 adolescents completing identical versions of the survey approximately two weeks apart. self - reported height and weight were used to determine body mass index (bmi ; kg / m). overweight referred to a bmi85 percentile at t1 and t2, and a bmi25 at t3. self - report of height and weight was validated in 125 eat - iii participants, representing a range of demographic and anthropometric features, who were consecutively invited to have height and weight measured at the university of minnesota by trained research staff. five levels of socioeconomic status (ses) were based on participant report of the highest educational attainment by either parent at t1. eligibility for public assistance, eligibility for free / reduced - price school meals, and parental employment status were used to approximate missing ses information. kandel and davies scale for adolescents assessed the frequency of depression symptoms during the past year. binge - eating was ascertained as follows : in the past year, have you ever eaten so much food in a short period of time that you would be embarrassed if others saw you ? ; during the times when you ate this way, did you feel you could n't stop eating or control what or how much you were eating ? these items have good concurrent validity and test - retest reliability (present study test - retest agreement=92% for overeating, 84% for loss of control). fruit and vegetable intake (f / v), assessed by the youth form of the willett semi - quantitative food frequency questionnaire, served as a proxy for dietary quality. fruit intake (excluding juice) was estimated by summing the reported consumption of 9 varieties, including berries, melons, citrus, and stone fruits. vegetable intake (excluding french fries) was estimated by summing the reported consumption of 19 varieties, including legumes and root, starchy, and cruciferous vegetables. a modified leisure time exercise questionnaire assessed moderate - to - vigorous physical activity (mvpa). participants report how many hours per week they engage in vigorous (your heart beats rapidly), moderate (not exhausting), and mild (require little effort) activities (range=.05). this study expands upon the previously - identified longitudinal relationship between depression and overweight by identifying behavioral mediators explaining this relationship. among females, higher early / middle adolescent depression symptoms predicted binge eating frequency during middle adolescence / early young adulthood, which in turn predicted overweight in early / middle young adulthood. the mediation effect for binge eating frequency was small, and f / v and mvpa were not significant mediators. thus, the relationship between adolescent depression and adult overweight may also operate through other behavioral (or non - behavioral) variables. binge eating affects 10 - 30% of obese individuals, and our results indicate that it may represent one pathway to overweight among females with depression symptoms in adolescence. binge eating may develop as a method of alleviating depression symptoms, ultimately contributing to weight gain and overweight status. females presenting with depression symptoms should be screened and treated for eating pathology to prevent the later onset of overweight ; this is especially important given that binge eating prevalence increased over time in our sample (although this may reflect improved understanding of the term interventions should aim to resolve current depression symptoms, and help individuals develop healthier affect regulation strategies. limitations include the use of brief, self - report measures to assess anthropometric, behavioral, and psychosocial variables. assessments occurred at 5-year intervals, which may have precluded the detection of more nuanced changes in depression symptoms, eating and activity behaviors, and weight status occurring between assessments. finally, treatment - seeking (for either overweight or depression) was not assessed, which may have impacted our findings, especially since antidepressant usage can impact weight control. a clearer understanding of mechanisms explaining the relation between depression and overweight is needed. binge eating may represent one pathway among females ; however, modest effects and absence of other mediation effects may indicate that environmental variables and/or their interaction with behavioral factors contribute more heavily to overweight risk than behavioral factors alone. future studies should further explore the relationship between depression and overweight to prevent adverse health outcomes throughout development.	depression may be a risk factor for overweight status, but mechanisms involved in this relationship are unclear. this study explored behavioral factors involved in the relationship between adolescent depression symptoms and adult overweight status. a population - based cohort of female participants in project eat (n=1,035) was followed over 10 years and reported on psychological functioning, weight status, and eating and activity patterns in early / middle adolescence (1999=time 1 ; t1), middle adolescence / early young adulthood (2004=time 2 ; t2), and early / middle young adulthood (2009=time 3 ; t3). structural equation models were fit which included t1 depression scores predicting overweight status at t3, with t2 fruit and vegetable consumption, moderate - to - vigorous physical activity, and binge eating examined as mediators. there were small but significant effects of t1 depression scores predicting an increased likelihood of t3 overweight status (standardized estimate=0.038 ; p=.007), and of t2 binge eating mediating the relation between t1 depression and t3 overweight status (standardized indirect effect estimate=.036 ; p=.009). binge eating may be one pathway to overweight among depressed females, suggesting that recognition and treatment of eating pathology in individuals with depression may help prevent overweight. examination of other behavioral (and non - behavioral) factors explaining the relationship between depression and overweight is warranted.
airway remodeling, including alterations in the thickness of the basement membrane, an increase in the number of mucus producing cells, an increase in the number of blood vessels (angiogenesis), and a change in the extracellular matrix (ecm) protein profile and hypertrophy / hyperplasia resulting in an increase in the bulk of the airway smooth muscle (asm), is now recognized as a hallmark feature of asthma. little is known about the effectiveness of current asthma therapies upon these structural changes in the airways, particularly in the vicinity of the asm. we have previously reported that neither corticosteroids nor long - acting 2-agonists (labas) alone are effective at preventing or reversing in vitro parameters of asm - driven airway remodeling. the critical question that remained was whether the combination of these two drug classes would be more effective. whilst the combination of inhaled corticosteroids and labas improves asthma control and lung function and decreases the frequency of asthma exacerbations compared to placebo or high doses of inhaled corticosteroids alone [26 ], one exception is the study by orsida and colleagues who reported that the combination of labas and inhaled corticosteroids reduces blood vessel number. given our previous finding of the lack of effectiveness of these drugs singly in reducing parameters of airway remodeling, it was vital to assess their efficacy in combination. several studies have examined the in vitro effectiveness of combined corticosteroids and labas in fibroblasts with conflicting results. goulet. found that corticosteroids and labas had opposing effects on matrix protein deposition in the presence of serum and their combination counteracted each other. in contrast, also in fibroblasts, descalzi. reported corticosteroids had significant anti - proliferative effects and that combination with labas strengthened these effects. reported that corticosteroids reduced and the combination with labas abolished proteoglycan production induced by serum. in the absence of serum, regardless of whether transforming growth factor (tgf) was present or not, fluticasone increased fibronectin at both the mrna and protein levels ; however, it decreased tenascin - c at both levels. salmeterol did not affect fibronectin or tenascin - c nor did it alter the effect of fluticasone when the drugs were applied in combination. whilst we, and others, have begun elucidating the molecular mechanism underlying the synergistic effect of the combination of corticosteroids and labas in asm cells [12, 13 ], the effect of the combined drugs on the release of ecm proteins from asm cells remains to be investigated. in this study, we hypothesized that the combination of corticosteroids and labas would be ineffective at inhibiting the production of ecm proteins in vitro. to investigate this hypothesis, we examined the effect of the combination of corticosteroids and labas in a well - characterized model of in vitro airway remodeling, namely, tgf-induced fibronectin in human asthmatic and nonasthmatic asm cells in vitro and in nonasthmatic bronchial rings ex vivo. approval for all experiments with human lung was provided by the human ethics committees of the university of sydney and the sydney south west area health service. asthmatic asm was obtained from 7 patients (mean age 32.7 11.5 years sd) either undergoing resection for lung transplantation or deep endobronchial biopsies. nonasthmatic asm was obtained from bronchial airways of 9 patients (mean age 58.6 11.6 years sd) undergoing resection for either lung transplantation or carcinoma. pure asm bundles were dissected free and grown as explants as previously described [1315 ]. asm cell characteristics were determined by light microscopy and immunofluorescence for the detection of -smooth muscle actin and calponin. asm cells from 6 asthmatic and 8 nonasthmatic patients were seeded for 24 hours in 5% fetal bovine serum (fbs) (jrh biosciences, melbourne, australia) dulbecco 's modified eagle 's medium (dmem) (safc biosciences, lenexa, ks) in the presence of 20 u / ml penicillin, 20 g / ml streptomycin, and 2.5 g / ml amphotericin b (invitrogen, heidelberg, australia) at a density of 1 10 cells per cm. medium was then changed to 0.1% insulin transferrin selenium (its) (invitrogen, heidelberg, australia) dmem for 24 hours before addition of formoterol (0.1 and 10 nmol / l) and budesonide (0.1 and 10 nmol / l) alone or in combination as indicated 30 minutes prior to stimulation with tgf1 (1 ng / ml) for the time periods described below. the effect of the drugs in unstimulated cells was assessed by omission of the tgf stimulation in cells maintained in 0.1% its. asm cells from 6 asthmatic and 6 nonasthmatic patients were seeded in 96 well plates and treated as described above for 48 hours. ecm free of cells was prepared by treatment with sterile hypotonic ammonium hydroxide [1719 ]. fibronectin was measured by elisa as previously described using an antibody to fibronectin (mouse antihuman plasma fibronectin 2 g / ml, clone 868a11, chemicon, temecula ca) and a purified mouse igg1 isotype control 2 g / ml, clone mopc-31c, (becton and dickinson pharmingen, san jose, ca). asm cells from 6 asthmatic and 8 nonasthmatic patients were seeded in 24 well plates and treated as described above for 48 hours. il-6 release was detected using an il-6 elisa kit according to the manufacturer 's instructions (duoset, becton and dickinson, san jose, ca). supernatants collected as described above were also assayed for soluble fibronectin release using a quantimatrix human fibronectin elisa kit according to the manufacturer 's instructions (chemicon international, temecula, ca). bronchial rings (25 mm diameter and 3 mm in length) were dissected free from surrounding parenchymal tissue., tissues were frozen in optimal cutting temperature (oct) embedding medium (fronine laboratory supplies, riverstone, australia), sectioned on a cryostat and immunohistochemistry performed using mouse anti - fibronectin (1 g / ml chemicon international, temecula, ca) coupled with a horseradish peroxidase labeled polymer. to help identify the morphology of the tissue, hematoxylin and eosin (h&e) staining results from duplicate wells from each individual subject were averaged and the absorbance from media alone subtracted before an overall mean and standard error of the mean (sem) were obtained from asthmatic and nonasthmatic cells. analysis of variance (anova) repeated measures with bonferonni posttests or student 's paired t - tests were performed on the results for ecm elisas where appropriate. in all cases a p value of less than or equal to 0.05 was considered significant. budesonide alone (10 m) induced fibronectin deposition in both asthmatic and nonasthmatic asm cells (figure 1), in agreement with our previous study. the addition of formoterol (10 m but not 10 m) abolished the induction of fibronectin by budesonide 10 m and 10 m (figure 1(a) and table 2). tgf induced the deposition of fibronectin from both asthmatic and nonasthmatic asm cells, in agreement with our previous reports [1, 20, 21 ] (figure 1). the addition of formoterol (10 m and 10 m) or budesonide (10 m or 10 m), alone or in combination, did not significantly alter fibronectin deposition in the presence of tgf in either cell type. the release of soluble fibronectin from asthmatic and nonasthmatic asm cells was increased by tgf but the presence of the drugs, in any combination, did not alter the release of fibronectin (data not shown). in nonasthmatic asm cells, budesonide (10 m) alone the addition of formoterol (10 or 10 m) did not reverse this reduction (figure 2(a) and table 3). the release of il-6 from asthmatic asm cells was more variable but followed the same pattern. budesonide reduced the release of il-6 even in the presence of tgf in both cell types (figure 2(b) and table 3). once again, formoterol (10 and 10 m) did not reverse the inhibitory effect of budesonide (asthmatic 13.66 4.0 and 21.08 5.5, nonasthmatic 17.34 2.9 and 17.55 2.9% of tgf) (figure 2(b)). to examine the effectiveness of the combination of corticosteroids and labas on ecm deposition in the whole airway bronchial rings from two nonasthmatic individuals stimulated with tgf showed increased deposition of fibronectin, in agreement with our previous findings [1, 20 ]. neither formoterol nor budesonide alone, or in combination, reduced the tgf-induced fibronectin deposition (figure 3). our previous work demonstrated that neither long - acting beta agonists nor corticosteroids reduced the release of ecm proteins from asm cells. the question remained as to whether the combination of these two therapeutic drug classes might be more effective. the results of the current study with formoterol and budesonide demonstrate that this is not the case, regardless of whether the cells were derived from asthmatic or nonasthmatic subjects. moreover, in bronchial rings stimulated with tgf, fibronectin deposition was not reduced by formoterol, budesonide, nor their combination. there are many examples of the efficacy of combined labas and corticosteroids in both in vivo [5, 22 ] and in vitro [13, 2325 ] studies. there are very few reports, however, of the modulation of remodeling parameters by these drugs, although the combination of labas and inhaled corticosteroids does reduce angiogenesis one of the features of remodeling. in addition, we have reported a synergistic inhibition of asm proliferation when these drugs are studied in combination. however, budesonide and salbutamol, alone or in combination, had no effect on collagen fiber tractional remodeling as asm cells migrated through collagen gels.. found that the combination of beclomethasone dipropionate (bdp) with either a short or long - acting beta agonist decreased fibronectin production induced by basic fibroblast growth factor, and that this effect was greater than with bdp alone. their study was carried out in fibroblasts stimulated with basic fibroblast growth factor, as opposed to smooth muscle cells stimulated with tgf in the current study, and this may be the basis for the differences observed. again, in fibroblasts, corticosteroids in the presence of serum increased ecm deposition, which we also found in asm cells, but labas decreased ecm deposition and the net result of the combination was simply additive. in contrast, degen. found, in fibroblasts, that fluticasone increased fibronectin but decreased tenascin - c mrna and protein induced by fbs, tgf, or in the absence of stimulation. under these experimental conditions we also examined the effect of corticosteroids and labas alone and in combination on tgf-induced soluble, as opposed to matrix - associated, fibronectin release but again these interventions were without effect in either asthmatic or nonasthmatic cells. to our knowledge, there are no previous reports examining the effect of combination therapy in asthmatic asm. a consistent finding from our laboratory has been the increase in release of ecm proteins from asm in response to corticosteroids. beclomethasone increased release of fibronectin from asm and this effect has also been reported by goulet. and degen. this is consistent with the fact that budesonide increased fibronectin release from asm cells derived from both asthmatic and nonasthmatic subjects in our current study, and furthermore this occurred whether or not cells were stimulated with tgf. interestingly, formoterol was able to attenuate budesonide - induced ecm fibronectin deposition even though alone it was without effect. the differential response of fibroblasts to fluticasone in relation to the production of fibronectin and tenascin - c observed by degen. therefore, caution should be taken in interpreting the results of this in vitro study as a global representation of the effectiveness of current therapies on altering parameters of airway remodeling. although we found in the present study that the combination of labas and corticosteroids did not decrease fibronectin release, corticosteroids, as previously reported [1, 27 ], inhibited il-6 release from the asm cells. reported that in (myo) fibroblasts, salmeterol inhibited il-6 release, and this was amplified by the addition of low concentrations of fluticasone dipropionate. others have found, also in fibroblasts, that corticosteroids inhibited and labas had no effect on il-6 release and the effect of the combination was that of corticosteroids alone. in contrast, il-6 release from asm is increased by 2-agonists in both asthmatic and nonasthmatic cells [1, 29 ], and our findings in the current study confirm this. the study of cells in culture is associated with limitations, and this is where we find the bronchial ring preparation a useful model. it enables us to observe, in an intact airway, changes in ecm proteins [1, 17 ] and cytokine deposition [15, 30 ] in response to profibrotic stimuli such as tgf and, in addition, to investigate the effects of intervention with relevant therapeutic agents such as labas and corticosteroids. here we confirmed our previous findings that neither labas nor corticosteroids alone decreased fibronectin deposition in response to tgf and extended them to include the combination of the two drug classes which were without effect. in contrast, we have previously reported that the phosphodiesterase inhibitor roflumilast abolished tgf-induced fibronectin deposition. in summary, in our cell and tissue models of ecm protein deposition, we investigated whether the combination of a laba and a corticosteroid would be more effective in inhibiting or reversing tgf-induced fibronectin release. this was not the case either in cells derived from asthmatic or nonasthmatic volunteers, or in intact bronchial rings. airway remodeling is detrimental in the pathophysiology of asthma, and ecm protein deposition is a major component of said remodeling ; therefore, these results highlight the need for further development of agents to reverse or prevent parameters of airway remodeling.	background. persistent asthma is characterized by airway remodeling. whereas we have previously shown that neither 2-agonists nor corticosteroids inhibit extracellular matrix (ecm) protein release from airway smooth muscle (asm) cells, the effect of their combination is unknown and this forms the rationale for the present study. methods. asm cells from people with and without asthma were stimulated with tgf1 (1 ng / ml) with or without budesonide (108 m) and formoterol (1010 and 108 m), and fibronectin expression and il-6 release were measured by elisa. bronchial rings from nonasthmatic individuals were incubated with tgf1 (1 ng / ml) with or without the drugs, and fibronectin expression was measured using immunohistochemistry. results. budesonide stimulated fibronectin deposition, in the presence or absence of tgf1, and this was partially reversed by formoterol (108 m) in both asthmatic and nonasthmatic cells. budesonide and formoterol in combination failed to inhibit tgf-induced fibronectin in either cell type. a similar pattern of expression of fibronectin was seen in bronchial rings. tgf1-induced il-6 release was inhibited by the combination of drugs. conclusion. current combination asthma therapies are unable to prevent or reverse remodeling events regulated by asm cells.

Subsets and Splits

No community queries yet

The top public SQL queries from the community will appear here once available.