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The treatment of chronic hepatitis c virus (hcv) infection has seen tremendous advances over just a quarter of a century, since 1989 when the virus was discovered (1), to the present day, when the infection can be cured, which is something quite unique in viral infections and even in modern medicine (2). The interferon (ifn) monotherapy used in the late 1980s (3) was followed by dual therapy (recombinant ifn alpha + ribavirin) at the end of the 1990s (4), then, in 2001, by pegylated interferons (peg - ifn) + ribavirin (5), and the use of 1st generation direct acting antivirals (daas) in 2011 (6, 7), culminating with a new wave of daas in 2013 (8), administered in different combinations without interferon . These therapeutic improvements were presented in the hepatogastroenterology literature as milestones, a new era, and nowadays as revolutionary . Dual therapy with pegifn + ribavirin is currently the only treatment for chronic hcv infection in romania as well as in other eastern european countries, where genotype 1 is present in over 95% of infected patients, and the rate of sustained virological response (svr) is around 50% or even less (9). Triple therapy (pegifn + ribavirin + telaprevir or boceprevir) was approved in 2011 by the food and drug administration (fda) and the european medicines agency (ema), yet it was not made available in romania and some neighboring countries . However, triple therapy had only a short lifetime from its launching to the moment when it was not anymore recommended both by the european association for the study of the liver (easl) and the american association for the study of the liver disease (aasld) (2014), mainly because of the arrival of the new revolutionary highly effective and safe daas on the market . The year 2013 announced an unprecedented therapeutic victory with several new daas having different viral targets, including ns3 protease inhibitors, nucleoside / nucleotide analogues and non - nucleoside inhibitors of the rna - dependent polymerase and ns5a inhibitors, some already approved by the ema and/or fda in 2013 (sofosbuvir and simeprevir) and 2014 (daclatasvir; abbvie s viekira pak (ombitasvir and ritonavir boosted paritaprevir plus dasabuvir); harvoni (ledipasvir + sofosbuvir)), with more to expect in 2015 and in the next two to three years . All these new daas were proved to be amazingly efficient (90% - 100% svr), safe, and well tolerated, while all oral, ifn - free combinations cure and even eradicate hcv infection (10, 11). A contagious enthusiasm spread among the authors of hundreds of articles; almost all with opening statements such as the advent of the new daas has revolutionized the treatment of hcv infection, and some with closing remarks as hcv could be cured and even eradicated . According to the dictionary, to revolutionize means to completely change the way something is done, thought about, or made (macmillan english dictionary). Does this mean that the standard care of dual - and triple - therapy should be dropped? Logically and ethically, once agreed that hcv infection can be cured and even eradicated with the new daas treatment, the answer is yes and it is strongly sustained by professional societies guidelines (aasld and easl). What does this therapeutic revolution mean for us and other eastern european countries? For sofosbuvir, the initial cost was 84000 usd per 12-week course, and 1000 usd per pill, making it one of the most (if not the most) expensive drug product ever marketed . At such prices, our country, other eastern european countries and many other low- and middle - income countries from all over the world, where the hcv pandemic is mainly concentrated (85% of the patients) cannot afford the new treatments . In europe, there have always been significant socioeconomic disparities between some western rich countries (e.g. Germany, france, uk etc .) And eastern lower middle - income countries (e.g. Romania, bulgaria etc . ). All of us, physicians and patients, still remember the grim times of the berlin wall, symbol of the differentiation made, among other things, in terms of health care facilities, including the access to new treatments, between eastern and western european countries . Paradoxically, 25 years after the political revolution that led to the fall of the berlin wall, the therapeutic revolution has now raised a new wall for eastern european patients, restricting their access to therapy, while in some western countries, such as germany or france, with large health budgets even with a very strong will from the part of the governments, most of the eastern european countries could not afford to purchase the new treatments at the current price for all who need it, simply because this would mean spending their entire annual health budget . Consequently, the gap in the therapeutic approach will widen between the rich countries in western and northern europe and those in the east . No one will understand and accept the high sale price of the new daas, despite producers attempts to justify it (costly research programs etc . ), considering that manufacturing costs are very low (between 68 and 136 usd for sofosbuvir, 10 and 30 usd for daclatasvir, and 130 and 270 usd for simeprevir). Unless companies producing daas, make their drugs widely affordable, hcv infected patients from eastern europe will never benefit from equitable access to treatment . Unfortunately, gilead s voluntary licensing with indian - based generic companies, allowing the production and sale of their new daas, sofosbuvir and ledipasvir, in 91 countries (mostly low - income and only few middle - income countries), excluded all eastern european countries from receiving affordable treatment (12). A common attitude together with concerted efforts of eastern european governments, non - governmental organizations, public health officials, clinical care providers, patient advocacy groups, media representatives and the community at large are needed to negotiate agreements with pharmaceutical companies to make these new life - saving treatments accessible and affordable (13). The situation of hcv therapy today is similar to the debates in 2000 on human immunodeficiency virus infection and acquired immune deficiency syndrome (hiv / aids) treatment, when the initial price of triple antiretroviral therapy was very high (unaffordable for millions of infected people). Over the last 15 years, generic competition has driven prices down by 99%, and this should be a lesson for today s battle against the exorbitant prices of the new hcv treatment (14). Egypt has shown that commitment from the part of the government and tough negotiations with gilead brought them a 99% discount on the us price of sofosbuvir (15). Hepatitis c virus infection can be cured and even eradicated by new treatments and this undeniable truth has become more contagious than hcv infection itself, to such a degree that nobody can stay in its way . Rarely, if ever, has mankind seen a curative therapy for a chronic viral infection as for hepatitis c. it would be unimaginable and unforgivable for the present generations if this medical triumph would be wasted and hcv patients, as citizens of low- and middle - income countries, are left excluded from new treatments on grounds related to high prices and financial constraints . We, physicians and patients, from both eastern and western europe, share the same dreams and goals: to cure and eradicate hepatitis c. in other words, we should all be treated like in a real family (gens una sumus) and have equal access to new therapy throughout europe and ideally, worldwide.
A 22-year - old male presented with decreased vision in his right eye since 1 month . Best - corrected visual acuity (bcva) was hand motions close to face with accurate projection of rays in his right eye, and 20/60 in his left eye . On fundus examination, he was found to have xlr with typical foveal schisis bilaterally [fig . The right eye had subtotal rd with inferotemporal retinoschisis and retinal breaks and grade c-2 posterior proliferative vitreoretinopathy [fig . Spectral domain optical coherence tomography (sd - oct) confirmed the presence of foveal schisis with a split in the outer plexiform layer in both eyes [fig . (a) fundus photograph of the right eye at presentation showing subtotal retinal detachment with proliferative vitreoretinopathy inferotemporally . (b) the left eye fundus showed typical foveal schisis with peripheral retinoschisis in the temporal and inferotemporal quadrants . (c) spectral - domain optical coherence tomography of the right eye demonstrating retinoschisis with a large intraretinal cyst and retinal detachment . (d) spectral - domain optical coherence tomography scan through the left macula showing schisis at the level of the outer plexiform layer (a) preoperative fundus photograph of the right eye showing subtotal retinal detachment with a large foveal cyst . (b) spectraldomain optical coherence tomography confirmed the presence of foveal schisis, with a split in the outer plexiform layer, and showed neurosensory detachment . (c) postoperative fundus photograph of the right eye with silicone oil in situ showing an attached retina . (d) spectral - domain optical coherence tomography demonstrated macular thinning with collapse of the schitic cavities noted earlier . (e) fundus photograph of the right eye at 3 weeks following silicone oil removal showing an attached retina . (f) spectral - domain optical coherence tomography showed reappearance of the schitic cavities the patient underwent 23 gauge pars plana vitrectomy with induction of posterior vitreous detachment, subretinal fluid drainage, endolaser, and silicone oil tamponade in the right eye . Postoperatively, bcva improved to 20/120 with an attached retina [fig . 2c]. Sd - oct showed macular thinning with the collapse of the schitic cavities in the presence of silicone oil [fig . While the retina remained attached postoperatively with a bcva of 20/80, the foveal schitic cavities reappeared with a split in the outer plexiform layer at 3 weeks following silicone oil removal [fig . In xlr, accumulation of defective retinoschisin protein within and around muller cells results in the formation of cystic spaces in multiple retinal layers, which coalesce to form larger schisis cavities . Sd - oct has demonstrated that the foveal schisis cavities are commonly located at the inner nuclear layer, outer plexiform layer or the outer nuclear layer . This is contrary to previous histopathologic reports that showed the involvement of the superficial retina (retinal nerve fiber layer and ganglion cell layer). Over time, with the structural loss of muller cells, defective retinoschisin might no longer be released but would likely be degraded and as a consequence cystic spaces would no longer be sustained . It has been observed that in adulthood, some patients may have a collapse of the retinoschisis and development of macular atrophy . The removal of vitreous and the internal limiting membrane (ilm) could be helpful for resolution of the foveal schisis and restoration of the retinal structure . It has been shown that following the complete removal of the vitreous cortex, retinoschisis resolves, suggesting that vitreous traction may play a role in the development of the foveal schisis . The rs1 gene encodes a secretory protein, retinoschisin that is secreted from the bipolar cells and rod and cone photoreceptors and functions as a cellular adhesion protein . The absence of retinoschisin as in xlr may cause intraretinal cell - to - cell dehiscence . Thus, elimination of vitreous traction and gas tamponade may promote structural restoration of the retina . Temporary tamponade with silicone oil has also been shown to stabilize the retinoschisis, persisting even after oil removal . It has been noted that ilm peeling may be a risk factor for a macular hole or retinal breaks in xlr due to the weakened retinal structure in the schitic area . This case adds to the limited literature elaborating on the course and outcomes of foveoschisis in xlr following vitrectomy . The unusual sequence of events that occurred in the above case have not been described earlier.
In 1990, the direct gene transfer of plasmid dna into mouse muscle in vivo without the need for a special delivery system was demonstrated . Furthermore, intramuscular inoculation with plasmid dna encoding reporter genes induced protein expression within the muscle cells . This study provided evidence for the idea that naked dna could be delivered in vivo to direct protein expression . Subsequently, a further study reported the gene expression a year or more after intramuscular injection of plasmid dna . Since these initial studies, many more experiments have been carried out to evaluate different factors that determine the efficiency of gene transfer and immunogenicity of plasmid dna . Furthermore, plasmid dna has been used to immunise against a variety of diseases (known as dna vaccination). Alternatively, plasmid dna has been used to treat genetic diseases and similar factors may affect the efficacy of this gene therapy . Dna vaccines usually consist of plasmid vectors (derived from bacteria) that contain heterologous genes (transgenes) inserted under the control of a eukaryotic promoter, allowing protein expression in mammalian cells . An important consideration when optimising the efficacy of dna vaccines is the appropriate choice of plasmid vector . The basic requirements for the backbone of a plasmid dna vector are a eukaryotic promoter, a cloning site, a polyadenylation sequence, a selectable marker and a bacterial origin of replication . A strong promoter may be required for optimal expression in mammalian cells . For this, some promoters derived from viruses such as cytomegalovirus (cmv) or simian virus 40 (sv40) have been used . A cloning site downstream of the promoter should be provided for insertion of heterologous genes, and inclusion of a polyadenylation (polya) sequence such as the bovine growth hormone (bgh) or sv40 polyadenylation sequence provides stabilisation of mrna transcripts . The most commonly used selectable markers are bacterial antibiotic resistance genes, such as the ampicillin resistance gene . However, since the ampicillin resistance gene is precluded for use in humans, a kanamycin resistance gene is often used . Finally, the escherichia coli cole1 origin of replication, which is found in plasmids such as those in the puc series, is most often used in dna vaccines because it provides high plasmid copy numbers in bacteria enabling high yields of plasmid dna on purification . Various reports have described the strength of promoter / enhancers or other transcriptional elements in dna vaccines (see table 1) [5 - 20]. In general, virally - derived promoters have provided greater gene expression in vivo than other eukaryotic promoters . In particular, the cmv immediate early enhancer - promoter (known as the cmv promoter) has often been shown to direct the highest level of transgene expression in eukaryotic tissues when compared with other promoters . For example, in one study a plasmid expressing human immunodeficiency virus type 1 (hiv-1) gag / env under the regulation of the cmv promoter / enhancer was compared to a comparable plasmid utilising the endogenous akv murine leukemia long terminal repeat . Analysis of the immune responses in macaques injected with the plasmids showed that the cmv - containing plasmid elicited higher gag- and env - specific humoral and t - cell proliferative responses, reflecting the greater transcriptional activity of the cmv promoter . Furthermore, it has been demonstrated that inclusion of the cmv intron a improved the level of expression of transgenes expressed by the cmv promoter or other promoter / enhancers . It is thought that the beneficial effect of introns on expression is primarily due to an enhanced rate of polyadenylation and/or nuclear transport associated with rna splicing . However, some widely used virally - derived promoters, such as the cmv promoter, may not be suitable for some gene therapy applications since treatment with interferon- or tumour necrosis factor- may inhibit transgene expression from dna vaccines containing these promoters . For example, the desmin promoter / enhancer, which controls expression of the muscle - specific cytoskeletal protein desmin, was used effectively to drive expression of the hepatitis b surface antigen priming both humoral and cellular immunity against the antigen . These responses were shown to be of a comparable magnitude to those in mice immunised with comparable dna vaccines containing the cmv promoter . Other tissue - specific promoters that have been studied include the creatine kinase promoter, also specific to muscle cells, and the metallothionein and 1,24-vitamind(3)(oh)(2) dehydroxylase promoters, both of which are specific to keratinocytes . Comparison of promoters used in dna expression studies in vitro and in vivo since the rate of transcriptional initiation is generally increased by the use of strong promoter / enhancers, the rate of transcriptional termination may become rate - limiting . In addition, the efficiency of primary rna transcript processing and polyadenylation is known to vary between the polyadenylation sequences of different genes . Thus, the polyadenylation sequence used within a dna vaccine may also have significant effects on transgene expression . For example, it was demonstrated that the commonly used sv40 polyadenylation sequence was less efficient than the minimal rabbit -globin and bovine growth hormone polyadenylation sequences in mouse liver, although addition of a second sv40 enhancer downstream of the sv40 polyadenylation signal did increase expression to a level comparable to the other signals . Therefore, it is possible that the strategy of inserting a second sv40 enhancer downstream of a sv40 polyadenylation sequence may be utilised in the construction of more efficient vectors . Sequences flanking the aug initiator codon within mrna influence its recognition by eukaryotic ribosomes . As a result of studying the conditions required for optimal translational efficiency of expressed mammalian genes, it has been proposed that this defined translational inititiating sequence (gcca / gccaugg) should be included in vertebrate mrnas located around the initiator codon . It has also been suggested that efficient translation is obtained when the -3 position contains a purine base or, in the absence of a purine base, a guanine is positioned at + 4 . Therefore, the expression level of these genes might be increased by the insertion of a kozak sequence . Codon bias is observed in all species, and the use of selective codons in genes often correlates with gene expression efficiency . In general, taxonomically - close organisms, such as e. coli and salmonella enterica serovar typhimurium, for example, use similar codons for their protein synthesis whereas taxonomically - distant organisms, such as e. coli and saccharomyces cerevisiae, utilise very different codons . Nagata et al . Studied the effect of codon optimisation for mammalian cells of cytotoxic t - lymphocyte (ctl) epitopes derived from the intracellular bacterium, listeria monocytogenes, and the parasite plasmodium yoelii, and reported that the codon optimisation level of the genes correlated well with translational efficiency in mammalian cells . The greatest deviation from random codon usage in an organism occurs in the most highly expressed genes as a result of selection for codons that maximise translational efficiency . Thus, differences between codon usage in a heterologous gene and the host organism may affect expression . To improve expression of human immunodeficiency virus type 1 gp120 from a dna vaccine vector, andr et al . Generated a synthetic gp120 sequence in which most of the wild - type codons were replaced with codons from the resulting construct showed increased in vitro expression of gp120 compared to the wild - type sequence . In addition, significantly increased antibody titres and ctl reactivity were observed following administration of the vector containing the synthetic sequence . Similarly, a dna vaccine vector encoding a synthetic epitope of listeriolysin o with mammalian codon usage showed higher translation efficiency than a vector containing the wild - type sequence in murine cells . Furthermore, the first dna vaccine was capable of inducing specific cd8 t cells able to confer partial protection against challenge with l. monocytogenes where the second dna vaccine could not . A number of other studies have reported that increased immune responses may be obtained by dna vaccination with a transgene sequence with optimised codon usage . In this review the methodologies by which antigen expression has been optimised to date, i.e. Optimisation of vector and transgene sequences, have been discussed . It is clear that transgene expression may be increased through the use of optimised promoters and polya sequences . However, in some circumstances it may be necessary to optimise dna vaccines to produce reduced transgene expression . For example, the weaker sv40 promoter has been used rather than the cmv promoter to drive expression of antigens that induce cell death upon overexpression . Such tissue - specific expression systems may be able to produce stable expression by reducing the probability of inducing an immune response to the transgene . It may be possible to design vectors for gene therapeutic purposes that avoid inducing unwanted immune responses against the encoded antigen by using tissue - specific promoters . Restricting the site of expression of genes should minimise the risks related to aberrant expression of a gene product . Furthermore, it should be possible to develop expression systems where gene products are only expressed in the critical cell types for dna vaccination or gene therapy, for example, dendritic cells (dcs). As a better understanding of the proteins whose expression is limited to dcs is obtained, novel expression systems will be generated . Finally, through increased knowledge of the regulation of expression of antigens, it is now possible to produce multivalent systems whereby multiple antigens may be expressed from a single dna vaccine vector . It is clear that the optimisation of antigen expression is an important consideration in dna vaccine vector design . However, it is important to recognise that other aspects of vector design may influence the efficacy of the vaccine / gene therapy . A rational approach to improve the efficacy of dna vaccination or gene therapy would optimise the: (i) vector backbone dna sequence; (ii) transgene sequence; (iii) co - expression of stimulatory sequences; (iv) delivery system used for the vector; (v) targeting of the vector for appropriate immune stimulation . The backbone of a dna vaccine vector could be further modified to enhance immunogenicity via the manipulation of the dna to include certain sequences, so that the dna itself will have an adjuvantising effect . Dna vaccine vectors contain many cpg motifs (consisting of unmethylated cpg dinucleotides flanked by two 5' purines and two 3' pyrimidines) that, overall, induce a th1-like pattern of cytokine production, and are thought to account for strong ctl responses frequently seen following dna vaccination . It is possible to augment responses to dna vaccine vectors by incorporating cpg motifs into the dna backbone of the plasmid . Alternatively, immune responses may be modulated or enhanced by the co - expression of stimulatory molecules or cytokines or through the use of localisation or secretory signals [47 - 49], or ligand fusions [50 - 54] to direct antigens to sites appropriate for immune modulation . Finally, a variety of routes of administration of dna vaccines have been studied, including intramuscular, intradermal, subcutaneous, intravenous, intraperitoneal, oral, vaginal, intranasal and, more recently, non - invasive delivery to the skin (reviewed by gurunathan et al . ). The approaches outlined above will together allow for the rational and optimised design for dna vaccines and gene therapy vectors . The ability to improve antigen expression through the use of optimisation of regulatory elements, kozak sequences and codon usage is highlighted in this review, as part of this rational approach.
It acts as a primary restraint against posterior displacement and, in concert with the anterior cruciate ligament (acl), to regulate external rotation of the knee during extension . The most commonly reported mechanism of injury to the pcl is a posteriorly directed blow to the anterior aspect of the proximal tibia with the knee flexed at 90 . Another mechanism is sudden hyperextension in conjunction with associated varus or valgus force or hyperflexion of the knee . Clinical examination shows a posterior sag and a positive posterior drawer test if the pcl is ruptured or avulsed . In case of avulsion, radiological examination avulsion of the tibial insertion of the pcl is believed to be an uncommon injury . A non - displaced bony avulsion can be treated conservatively with a plaster cast, holding the knee flexed and the tibia pulled forward to diminish tension on the pcl . There is general consensus that a displaced bony pcl avulsion should be surgically reduced and fixed immediately to stabilize the knee joint and prevent non - union . All publications on the repair of acute bony avulsion of the tibial attachment of the pcl report excellent clinical results [1, 2, 4, 6, 912, 14]. Trickey states that surgical repair of an avulsion the tibial attachment of the pcl after 3 weeks is not successful . There is one report in the literature of a reduction and screw fixation combined with posterolateral stabilization of a 4.5-year - old bony avulsion of the pcl with good clinical result . To the authors knowledge, there is no report of pseudo - arthrosis repair of a pcl avulsion fracture . We report the case of a pseudo - arthrosis repair of a 4-year - old bony avulsion fracture of the pcl using a minimally invasive technique, screw fixation, and bone grafting . Four years prior, she suffered from distorsion trauma of her left knee during a skiing accident . At the time, the knee was examined both physically and radiologically but the diagnosis of an avulsion fracture of the tibial eminence was not made . In the years following the initial trauma, the left knee had a full range of motion without any apparent instability but did show a slight posterior sag . Standard lateral and posterior - anterior radiographs revealed an evident pseudo - arthrosis with a radiolucent and sclerotic line under the posterior tibial eminence . The avulsed fragment measured 32 mm 24 mm 12 mm (fig . 1standard radiographs showing an evident pseudo - arthrosis of the posterior tibial eminence standard radiographs showing an evident pseudo - arthrosis of the posterior tibial eminence magnetic resonance imaging and computed tomography showed pseudo - arthrosis of a large bony avulsion of the tibial attachment of the pcl extending to both the medial and lateral tibial plateau the magnetic resonance imaging also showed an intact pcl and did not reveal any signs of concomitant intra - articular injuries of the knee (fig . 2). Bone scintigraphy demonstrated increased activity at the proximal left tibia.fig . 2computed tomography revealing a large bony avulsion of the tibial attachment of the pcl . Magnetic resonance imaging shows an intact pcl computed tomography revealing a large bony avulsion of the tibial attachment of the pcl . Magnetic resonance imaging shows an intact pcl it was concluded that the patient s complaints could be caused by the pseudo - arthrosis of a bony avulsion of the tibial attachment of the pcl; this was deemed an appropriate indication for pseudo - arthrosis repair . A single 2-g dose of intravenous ceftazidime was administered . Under fluoroscopic control, an entrance point for a 10-mm cannulated drill along the medial aspect of the proximal tibia was located . A stab - incision was made, and the cannulated drill was inserted and directed cranially and posteriorly towards the centre of the pseudo - arthrosis until the tip of the drill was located just subchondral at the lateral tibial plateau . 3).fig . 3positioning of the cannulated drill and its fluoroscopic verification . Removed pseudo - arthrosis shows the fibrous tissue and healthy cancellous bone positioning of the cannulated drill and its fluoroscopic verification . Removed pseudo - arthrosis shows the fibrous tissue and healthy cancellous bone the drill and the collected tissue, consisting of normal cancellous bone and fibrous tissue, were removed . A similar manoeuvre was performed, only now the 10 mm cannulated drill was directed more posteriorly in order to access more of the pseudo - arthrosis . The fibrous tissue was separated from the healthy cancellous bone for microscopic evaluation (fig . A combination of the removed cancellous bone mixed with homologous bone was used to fill the defect created by the cannulated drill . A tibial acl guide was used to guide kirschner wires for two 4.5-mm cannulated partially threaded screws . 4removal of the remaining pseudo - arthrosis using a curette and bone grafting of the defect created by the cannulated drill removal of the remaining pseudo - arthrosis using a curette and bone grafting of the defect created by the cannulated drill there was a good fixation and compression of the avulsed fragment . Radiographic evaluation demonstrated good positioning of the screws with all screw threads in the avulsed bony fragment (fig . 5standard radiographs showing positioning of the lag screws and complete disappearance of the pseudo - arthrosis of the bony avulsion of the tibial insertion of the pcl standard radiographs showing positioning of the lag screws and complete disappearance of the pseudo - arthrosis of the bony avulsion of the tibial insertion of the pcl postoperatively, the patient was mobilized immediately with a full leg cylinder plaster cast . There was no weight bearing for the first 6 weeks followed by full weight bearing without the plaster cast . Three months after surgery, the patient was satisfied with the result of the surgical intervention . Radiographs showed a complete disappearance of the pseudo - arthrosis of the bony avulsion of the tibial insertion of the pcl and good incorporation of the bone graft (fig . In this case, there was a good functional result following minimally invasive pseudo - arthrosis repair of a posterior cruciate ligament avulsion fracture . In displaced bony pcl avulsion injuries, there is general consensus that surgical reduction and fixation in the acute phase is indicated to stabilize the joint and prevent non - union . Trickey states that surgical repair of an avulsion the tibial attachment of the pcl after 3 weeks is not successful . Kim et al . Reported two patients with non - union tibial avulsion fractures who underwent surgical reduction and fixation 19 and 20 months after the causal trauma . Both were described to have grade c (abnormal) function postoperatively according to the assessment with the ikdc form . In contrast, torisu reported good results in three patients with delayed repair of bony pcl avulsion fractures; he stated that late repair is recommended . Meyers reported two patients with delayed treatment of displaced pcl avulsion fractures operated three and 6 months after the initial trauma with good functional results . Reported reduction and screw fixation of a 4.5-year - old displaced pcl avulsion fracture . Using a posterior approach and single screw fixation, good stability and function of the knee the case presented here seems to be rather unique due to the fragment size and the approach for pseudo - arthrosis repair . In this case of a 4-year - old pseudo - arthrosis, it was hypothesized that it would be necessary to remove the pseudo - arthrosis tissue to achieve a solid bone union . The removal of the pseudo - arthrosis tissue and the filling with homologous bone was deemed easier and safer using a medio - anterior rather than a posterior approach . A minimally invasive technique was used to reduce soft tissue damage and reduce the risk of injury to the popliteal neurovascular branch . Furthermore, a medio - anterior approach was preferred over a posterior one for optimal screw fixation in the centre of the avulsed bony fragment . Partially threaded screws were chosen to achieve a rigid and stable fixation with compression between the bony avulsion fragment and the proximal tibia . There are no previous reports of similar pseudo - arthrosis repairs, but good results of delayed refixation of pcl avulsion fractures have been described . Therefore, refixation and pseudo - arthrosis repair should be considered as a viable treatment.
Primary hepatic lymphoma (phl) is a rare malignancy usually presenting with abdominal pain, anorexia, hepatomegaly and jaundice . Diffuse hepatic involvement is exceedingly rare with only few cases of fulminant hepatic failure (fhf) due to phl reported in the literature.1- 5 here, we present a case with prolonged fever who underwent several investigations and, in the meantime, developed hepatic failure before the diagnosis could be reached . A 47-year old man was referred with a history of fever and jaundice for two months . The fever (oral temperature = 38.5c without chills) and jaundice persisted despite the fact that two weeks ago, he had undergone cholecystectomy in another center for cholelithiasis . The patient was admitted to our center and a full work - up for sepsis was performed, including blood and urine cultures and chest radiography . Laboratory studies revealed abnormal liver enzymes (alt=80; ast=60; alp=1,100) and abnormal liver function tests (pt=18; aptt=40; hb=11; mcv=90; wbc=12,000; plt=121,000). Serologic evaluations for brucellosis, leishmaniasis, borreliosis, and bone marrow aspiration and biopsy all returned normal . Chest and abdominal ct scans were normal (no mass lesion or lymphadenopathy), except for mild hepatomegaly (figure 1). Magnetic resonance cholangiopancreatography was also performed, yielding a normal result . A liver biopsy, performed after correction of prothrombin time with fresh frozen plasma, revealed diffuse large b - cell lymphoma (figure 2). Immunohistochemical assessments indicated the expression of cd20 and bcl2 on blast, while cd10 was negative . Nodular infiltration of the liver by a diffuse large b - cell lymphoma (centroblastic subtype in the meantime, the patient expired as a result of fulminant hepatic failure . Phl is associated with a poor prognosis, with a median survival of 8 to 16 months and low rate of complete remission (<20%). Very rare cases of phl presenting with fhf have almost always been fatal because of the ambiguous features and rapid progression, with most cases diagnosed on autopsy with an average survival of 10.7 days from diagnosis . Few cases have been reported to survive fhf caused by hematologic malignancies involving the liver . Cameron et al . Reported a patient with fhf from phl, who underwent urgent orthotopic liver transplantation . Reported the case of a woman who developed fhf due to infiltration by a non - hodgkin s lymphoma . In this case, rapid administration of chemotherapy resulted in complete recovery . Finally, shehab reported a patient with fhf due to richter s transformation of cll who was successfully treated with a chop regimen . The key to successful treatment in all these cases was early diagnosis by prompt liver biopsy . The importance of liver biopsy in these patients is highlighted by another issue . While liver transplantation can be lifesaving in patients with fhf, it is imperative to perform a thorough evaluation to screen for potential contraindications . Reported a case of fhf in whom liver biopsy revealed natural killer (nk)-like t cell leukemia/ lymphoma to be the cause, thus obliterating liver transplantation once this diagnosis was made . Patients with phl have prominent b symptoms, including fever and weight loss, that mimic infectious and inflammatory disorders and therefore delay the diagnosis . In fact, we believe that the delay in appropriate diagnostic work - up led to the unfavorable outcome in our patient . Although the patient had mentioned no history of biliary pain, he was subjected to an unnecessary cholecystectomy for cholelithiasis that could not explain the initial clinical presentation . The resulting delay allowed the aggressive nature of the disease to ultimately progress to fhf, even though the initial presentation did not include hepatic failure . Metastasis from gastrointestinal tract (mostly colon) and hepatoma present similarly and are much more common . The standard approach to prolonged fever or fever of unknown origin in a patient with jaundice requires a liver biopsy following minimum diagnostic evaluations, including blood tests for infectious and inflammatory disorders and ct scans of the abdomen and chest . As in our case, the most frequent pathology for phl is diffuse large b - cell followed by small lymphocytic, follicular and marginal b - cell lymphoma . Phl is a rare disease that may be neglected in patients with fever and jaundice or fever and hepatomegaly . Adherence to standard approaches to prolonged fever or fever of unknown origin and timely diagnostic workup without a need for a high index of suspicion for this rare condition may contribute to the correct diagnosis.
Asthma is characterized by irreversible obstruction and chronic inflammation of the airways, and is traditionally considered a t helper 2 (th2-)cell driven inflammatory disorder . However, an important role for the innate immune system in addition to the adaptive immune system is increasingly being recognized in asthma . Macrophages are key cells in innate immune responses in the lung: they are among the most abundant cells and one of the first to encounter allergens and other threats to homeostasis . They also have the plasticity to quickly deal with those without endangering normal gas exchange . Depending on the signals received, macrophages can be pro- or anti - inflammatory, immunogenic or tolerogenic, and destroying or repairing tissue . Each characteristic may belong to a different macrophage phenotype with distinct functions [3, 4]. Tumor necrosis factor (tnf) and interferon (ifn) induce, under the influence of the transcription factor interferon - regulatory factor 5 (irf5), a phenotype of m1 macrophages with increased microbicidal and/or tumoricidal activities [4, 5]. Exposure to il-4 or il-13 results in a population of m2 macrophages that is involved in anti - parasite and tissue repair responses [6, 7]. In mice, these cells are recognized by high production of chitinase and chitinase - like molecules such as ym1 [7, 8]. These macrophages can be induced by a variety of stimuli including exposure to a tlr - ligand in the presence of il-10 or many more compounds . The main characteristic of the subtly different m2-like population is the production of il-10 . Since il-10 is a potent anti - inflammatory cytokine, these m2-like macrophages are effective inhibitors of inflammation . Despite the broad the spectrum of macrophage activation, the role of macrophages in asthma has scarcely been studied . From what is known, all three macrophage phenotypes have been implicated in the development of murine and human asthma [1012]. In mice, depending on the protocol used, asthma phenotypes can greatly differ [13, 14]. We aimed to investigate the distribution of the three main macrophages phenotypes in three different models of hdm - induced asthma and also included the effects of sex on asthma development . First, we show the general differences in airway inflammation in the three hdm models and next we study the distribution of the macrophage phenotypes with regard to severity of allergic airway inflammation . Male and female balb / c mice (aged 68 weeks) were obtained from harlan (horst, the netherlands). The mice were fed ad libitum with standard food and water and were held under specific pathogen - free conditions in groups of 4 mice per cage . The animal procedures, approved by the institutional animal care and use committee of the university of groningen (application number 5318), were performed under strict governmental and international guidelines . Male (n = 4 per model) and female mice (n = 4 per model) were anaesthetized with isoflurane and exposed intranasally to whole body hdm extract (dermatophagoides pteronyssinus, greer laboratories, lenoir, usa) in 40 l phosphate - buffered saline (pbs) according to three different protocols . Control animals (n = 8) were exposed to 40 l pbs according the 21-day protocol described . Mice of the first model (n = 8) received 100 g hdm extract intranasally on day 0, were subsequently exposed to 10 g hdm on day 711 according to the protocol of hammad et al . And were sacrificed on day 14 (abbreviated as 14-day hdm). In the second model, according to gregory et al ., mice (n = 8) were exposed to 25 g hdm extract three times a week during three weeks and were sacrificed on day 21 (abbreviated as 21-day hdm). For the last model (n = 7, due to illness one female was excluded from the study), mice were intranasally exposed to 100 g hdm on days 0, 7, 14 and 21 according to the protocol of arora et al . And were sacrificed on day 24 (abbreviated as 24-day hdm). During sacrifice lungs were lavaged three times with 1 ml cold pbs to determine the number of eosinophils and ym1 levels . Then, the left lung lobe was collected to isolate lung cells from digested lung for flow cytometry and the right lung was inflated with 0.5 ml 50% tissue - tek o.c.t . Zoeterwoude, the netherlands) in pbs and snap frozen / formalin - fixed for histological analyses . Arbitrary elisa units of hdm - specific ige titers were calculated as relative values to the optical density of pooled sera from hdm - exposed mice . Balf was collected and total numbers of cells were determined using a casy cell counter (roche innovatis ag, reutlingen, germany). After centrifugation at 300 gfor 10 minutes, balf supernatants were stored at 80c for further analysis (ym1 elisa) and the cells were resuspended in rpmi medium (biowhittaker europe, verviers, belgium) for preparation of cytospots . Approximately 50,000 cells were spotted onto slides using a cytospin 3 (thermo shandon, waltham, ma, usa) at 450 rpm for 5 minutes . To determine the percentage of eosinophils in the cytospots, a giemsa staining (sigma - aldrich, zwijndrecht, the netherlands) was performed and the number of eosinophils was counted in a total of 300 cells . The level of mecf - l (ym1) in balf supernatants was determined by an elisa kit according to the manufacturer's instructions (r&d systems, oxon, uk). After bronchoalveolar lavage, the left lung was minced and incubated in rpmi medium supplemented with 10% fetal calf serum (both lonza, verviers, belgium), 10 g / ml dnase i (grade ii from bovine pancreas, roche applied science, almere, netherlands), and 0.7 mg / ml collagenase a (sigma - aldrich) for 45 minutes at 37c in a shaking water bath . The digested lung tissue was passed through a 70 m nylon strainer (bd biosciences, breda, netherlands) to obtain single cell suspensions . Incubation with 10 times diluted pharmlyse (bd biosciences, breda, the netherlands) was performed to lyze contaminating erythrocytes . Cells were centrifuged through 70 m strainer caps and counted using a casy cell counter (roche innovatis ag). The single lung cell suspensions were stained for t - cell subsets using antibodies for flow cytometry . Frequencies of effector t cells (cd3+cd4+cd25+foxp3) and regulatory t cells (cd3+cd4+cd25+foxp3 +) were examined using cd3-apc / cy7 (biolegend, fell, germany), cd4-pe / cy7 (biolegend), cd25-pe (biolegend), and foxp3-apc (ebioscience, vienna, austria). An appropriate isotype control was used for the foxp3 staining (rat igg2ak - apc, ebioscience). Approximately 10 cells were incubated with the appropriate antibody mix including 1% normal mouse serum for 30 minutes on ice, protected from light . After washing the cells with pbs supplemented with 2% fcs and 5 mm edta, the cells were fixed and permeabilized for 30 minutes using a fixation and permeabilization buffer (ebioscience). Then cells were washed with permeabilization buffer and incubated with anti - foxp3 including 1% normal mouse serum for 30 minutes . Subsequently, the cells were washed with permeabilization buffer, resuspended in facs lysing solution (bd biosciences) and kept in the dark on ice until flow cytometric analysis . The fluorescent staining of the cells was measured on a lsr - ii flow cytometer (bd biosciences) and data were analyzed using flowjo software (tree star, ashland, usa). Sections of 4 m were cut from the frozen part of the right lung and stained for all macrophages (rat cd68, serotec, oxford, uk). The numbers of m2 macrophages were determined in frozen sections by staining for ym1 (goat -mecf - l, r&d systems, oxon, uk) using standard immunohistochemical procedures . Cd68 and ym1 were visualized with 3-amino-9-ethylcarbazole (aec, sigma aldrich, zwijndrecht, the netherlands). The formalin - fixed part of the right lung was embedded in paraffinthen sections of 3 m were cut . To identify the m1 macrophages in tissue sections, antigen retrieval was performed by overnight incubation in tris - hcl buffer ph 9.0 at 80c and then sections were stained for irf5 (rabbit -irf5, proteintech europe, manchester uk) using standard immunohistochemical procedures . To determine the number of il-10 producing cells, antigen retrieval was performed by boiling the sections in citrate buffer ph 6.0 for 10 minutes . The sections were pretreated with 1% bovine serum albumin (sigma aldrich) and 5% milk powder in pbs for 30 minutes and incubated with rabbit -il-10 overnight (hycult biotech, uden, the netherlands). Irf5 and il-10 were both visualized with immpact novared kit (vector, burlingame, ca, usa). Positive cells were quantified by manual counting in parenchymal lung tissue (thus excluding large airways, vessels, and infiltrates, magnification 200400x) and the total tissue area was quantified by morphometric analysis using imagescope analysis software (aperio, vista, ca, usa). The numbers of cells were expressed per mm of tissue . To determine if the data were normally distributed a kolmogorov - smirnov test was used . The differences between the models were tested using one - way analysis of variance (anova) with tukey's post - hoc test for multiple comparisons and sex differences were tested with the student's t test . Pearson correlation coefficients were calculated to analyze the correlation between the inflammation parameters and macrophages phenotypes, and correlations within macrophage phenotypes (graphpad software, la jolla, ca, usa). Differences were considered significant when p <0.05, and p <0.10 was considered a statistical trend . To test whether exposure to hdm, according to three different protocols, induced allergic airway inflammation differently, we studied a number of general inflammation parameters . Higher percentages of eosinophils in balf were found in all three hdm - exposed groups as compared to control mice (figure 2(a)). No differences in percentage of eosinophils in balf were observed between the three hdm protocols . Hdm - specific ige levels in serum were not affected by the different hdm exposures, only a trend of higher levels was found in the group that was exposed to hdm in the 21-day protocol . In all protocols of hdm exposure, hdm - specific ige levels were very low measuring just above the limit of detection in the calibration curve (figure 2(b)). The higher airway inflammation in the three hdm - exposed groups was accompanied by higher percentages of effector t cells in lung tissue as compared to the control group (figure 3(a)). The 24-day protocol showed a higher percentage of effector t cells in lungs than the 14- and 21-day protocol . After hdm exposure the percentage of regulatory t cells was also higher in all three protocols as compared to control mice (figure 3(b)). The 24-day hdm protocol induced higher percentages of regulatory t cells in lungs compared to the 14-day protocol . The ratio of effector t cells to regulatory t cells was higher in the 24-day hdm protocol as compared to the control group and the other two hdm protocols (figure 3(c)). In females, hdm exposure induced more eosinophilia (p <0.01), effector t cells (p <0.05), regulatory t cells (p <0.05), and higher levels of hdm - specific ige (p <0.05) than in males . To study the presence of macrophage phenotypes after hdm exposure according to the three different protocols, we stained lung tissue for markers of total macrophages (cd68), m1 macrophages (irf5), m2 macrophages (ym1), and m2-like macrophages (il-10) and counted positive cells in parenchymal tissue . Hdm - exposed mice had more cd68-positive cells in lung tissue as compared to control mice (figure 4(a)). No differences in cd68-positive numbers were observed between the hdm protocols . Compared to control mice, irf5-positive cell numbers were higher in 14- and 21-day protocol, but not in mice exposed to hdm according to the 24-day protocol (figure 4(b)). Between the hdm models, lower irf5-positive numbers were found in lungs of mice that were exposed to hdm according to the 24-day protocol as compared to the mice of the 14-day hdm protocol . For ym1, all hdm protocols induced more ym1-positive cells as compared to control (figure 4(c)). However, mice that were exposed in the 24-day hdm protocol had higher numbers of ym1-positive cells in lung tissue than the mice of the 14-day hdm protocol . Ym1 levels in balf were elevated in all hdm models as compared to control, but no differences were found between the models (figure 5). Interestingly, hdm exposure resulted in significantly lower numbers of il-10-positive cells in all three protocols compared to the control - treated group (figure 4(d)). There were no differences observed in il-10-positive cell numbers between the three hdm protocols . Hdm - exposed females had more cd68-positive cells (p <0.05), ym1-positive cells (p <0.01), and higher levels of balf ym1 (p <0.05) than males, whereas no differences were found in irf5- and il-10-positve cells numbers between the two sexes . To assess how severity of airway inflammation is reflected by the presence of the three main macrophage phenotypes, we correlated parameters of allergic airway inflammation with the different macrophage phenotypes in hdm - exposed mice (table 1). Numbers of cd68-positive cells correlated positively with the percentage of eosinophils in balf (r = 0.58), effector t cells (trend, r = 0.37) and regulatory t cells (r = 0.42) in lungs of hdm - exposed mice, indicating that more severe disease was accompanied by more macrophages . Most of these macrophages appear to be ym1-positive as only ym1-positive cell numbers correlated significantly with the percentage of eosinophils in balf (r = 0.48, figure 6) and the percentage of regulatory t cells (r = 0.51) in lung tissue . No differences were found between males and females . To study the relationship between the different macrophage phenotypes in allergic airway inflammation, correlations were made between ym1-postive, irf5-positive, il-10-positive, and cd68-positive cells in lung tissue of all hdm - exposed mice (table 2). Numbers of irf5-positive cells negatively correlated with cells positive for cd68 (trend, r = 0.40) and ym1 in lung tissue (r = 0.70, figure 7(a)). Ym1-positive cell numbers correlated negatively with numbers of il-10-positve cells (r = 0.48, figure 7(b)) and positively with numbers of cd68-positive cells in lung tissue (r = 0.66). Our study has shown that the balance between macrophage phenotype changes as the severity of allergic inflammation increases . Higher numbers of m2 macrophages in hdm - exposed mice correlated with higher percentages of eosinophils in balf . At the same time lower numbers of m1 macrophages and m2-like macrophages were found in the mice with more severe inflammation and these therefore correlated negatively with m2 macrophages . In addition, we have confirmed again that females have more pronounced airway inflammation with higher numbers of m2 macrophages as compared to males . The models we used for our study were short - term exposure to hdm and they give us much information about the distribution of the different macrophage phenotypes during induction of asthma . In these models we found that longer exposure to hdm did not induce more severe eosinophilic inflammation but it did lead to higher numbers of m2 macrophages and higher percentages of effector and regulatory t cells in lungs of mice . The fact that we found no differences in eosinophils between the models is probably due to the large variation within the groups . However, when analyzing all hdm - exposed mice separately, eosinophils correlated positively with total macrophages and m2 macrophages, confirming our previous findings in humans that m2 macrophages increase with increasing asthma severity . Another important parameter of allergic airway inflammation, serum hdm - specific ige, could barely be detected probably because the duration of the models was too short . It takes around 3 weeks for naive b cells to mature to plasma cells and switch from igm production to ige after first contact with an antigen . Our models lasted 24 days at the most and we therefore sacrificed our animals before a full - blown ige response could develop . Also, studies from other groups using these models did not show hdm - specific ige in serum [1517]. To investigate how macrophage phenotypes are distributed during and contribute to more chronic disease, longer models of hdm - exposure need to be used . We sought to phenotype the distinct macrophage subsets in lung tissue using markers that could distinguish each phenotype . We identified m2 macrophages using expression of ym1, which is unique for this phenotype in the lung . Previous studies found that only macrophages express ym1 and the staining is not complicated by other cells staining positive [2224]. A unique marker for identifying m1 macrophages in lung tissue is, however, more difficult to find . To our knowledge selective surface markers for tissue are not available and therefore the production of il-12 and oxygen radicals have been used in several mouse studies [25, 26]. In asthma, oxygen radicals cannot be used to stain for m1 macrophages because these are also copiously produced by eosinophils that are present in great numbers . Our study was the first to use irf5 as an m1 marker on lung tissue and it is a fairly selective marker . Bronchial epithelial cells and incoming leukocytes in infiltrates also stain positive for irf5 but these could be excluded from the quantification of parenchymal tissue based on localization . A double staining with cd68 to make sure only macrophages are included in the quantification is unfortunately at present not possible due to technical incompatibilities . In previous studies, m2-like macrophages are often not distinguished from m2 macrophages because they share many markers, including mannose receptors . The production of the immunosuppressive cytokine il-10 is the most important and reliable characteristic of m2-like macrophages and can be used to identify these cells with . Similar to irf5, bronchial epithelial cells and some cells in infiltrates are also expressing il-10 but these can easily be excluded from the quantification of parenchymal tissue . To exclude other il-10-producing cells from the analysis a double staining with cd68 these limitations in the stainings for irf5 and il-10 may also explain why the total number of ym1-, irf5- and il-10-positive cells is higher than the number of cd68-positive cells . Higher numbers of m2 macrophages were found in lungs of hdm - exposed mice, which correlates with previous studies [19, 20, 28]. Interestingly, numbers of m2 macrophages and the levels of ym1 in balf correlated strongly with eosinophils in balf . Ym1 is also known as eosinophil chemotactic factor (ecf - l), but it was suggested that ym1 only has weak chemotactic properties for eosinophils [24, 30]. The chemotactic strength of ym1 is still debated and our findings and those of others do suggest otherwise [23, 31]. The number of m2 macrophages also showed a positive correlation with regulatory t cells . In an interesting study by tiemessen et al ., regulatory t cells were shown to promote the induction of m2 macrophages to help with trying to maintain tissue homeostasis and preventing too much tissue damage of the inflammatory response . Our data could be explained along these lines with incoming regulatory t cells inducing m2 macrophages in an attempt to restrict inflammation and tissue damage induced by hdm . The m2 macrophages would then be the result of inflammation and be beneficial instead of contributing to allergic airway inflammation, which is still an ongoing debate [19, 33, 34]. Asthma is dominated by a th2-driven inflammation, but evidence shows that m1 macrophages are also present in this disease . In two interesting studies, ifn-stimulated macrophages were shown to prevent the onset of allergic airway inflammation [35, 36]. Our findings with higher numbers of m1 macrophages in the shorter protocols and in less severe diseases suggest that these macrophages are induced as a counterregulatory mechanism to dampen inflammation . However, higher numbers of m1 macrophages in asthma have also been shown in severe asthma and markers of m1 macrophages correlate with asthma severity, suggesting that m1 macrophages play a role in severe asthma as well [3739]. This appears not to be the case for our results because we find higher numbers of m1 macrophages at the onset of allergic airway inflammation when numbers of effector t cells are lower, and lower numbers of m1 macrophages in the 24-day hdm protocol when effector t - cell numbers are higher . M1 macrophages appear to have a beneficial role in preventing allergic sensitization, but in already established disease they promote the development of a severe phenotype . A similar double role has been reported for the m1 cytokine il-12 in allergic airway inflammation . Since our hdm models are short and focus on the onset of the disease, we do not have information on the presence of m1 macrophages in established, more severe disease . Since m2-like macrophages have anti - inflammatory functions, we were interested in the presence of this macrophage phenotype in hdm - induced asthma . Others have reported that interstitial macrophages are the il-10-producing macrophages and treatment of sensitized mice with these macrophages prevented the development of allergic airway inflammation . Our observation of lower numbers of m2-like macrophages in asthma as compared to control is in line with these and previous findings in human asthmatics [42, 43]. Interestingly, it was shown that il-10 production in severe asthmatics is even lower as compared to moderate asthmatics and that treatment with corticosteroids induces il-10 production by macrophages [43, 44]. This suggests that specific stimulation of macrophages to polarize to m2-like macrophages that produce il-10 may reduce asthma symptoms . Women suffer from more severe asthma than men and this phenomenon was also found in mouse models of asthma [45, 46]. A role for sex hormones has been suggested, but the underlying mechanisms are unknown . In accordance with the previous findings, we show that hdm - exposed female mice had more pronounced airway inflammation than male mice . In macrophage phenotypes, we only found a difference in the m2 macrophages . Since we and others found a correlation between m2 macrophages and asthma severity, it may suggest that this phenotype could play a role in the increased airway inflammation in females . Taken together, our data suggest that during the development of allergic airway inflammation m1 and m2 macrophages are induced or recruited, whereas m2-like macrophages are prevented from moving in or inhibited . As hdm - induced inflammation progresses, m1 macrophages are diminished in favor of m2 macrophages possibly under the influence of regulatory t cells that try to restrict inflammation . Their work may be hampered by the fact that il-10-producing m2-like macrophages do not develop in hdm - induced inflammation and the inflammation can progress . Influencing this imbalanced relationship by therapeutic macrophage targeting could be a novel way to treat asthma.
Depression is an extremely common pathological complex with psychological, neuroendocrine, and pathological symptoms . It is a leading cause of disability worldwide and has a very significant impact on morbidity, mortality, and health care cost [24]. Disconcertion in monoaminergic neurotransmission especially serotonin and noradrenaline neurotransmission is considered the major cause of the observed symptoms of depression . Unfortunately the efficacy of these medications is unsatisfactory and multiple side effects are common . It is estimated that about 40% of patients have conditions refractory to current medications . Furthermore, these drugs require at least 24 weeks of administration before producing clinically meaningful improvement in the symptoms . These reasons underpin the need for novel therapeutic agents with less side effects and faster onset of action [7, 8]. Also both preclinical and clinical studies support the role of nmda receptor antagonists as possible therapeutic agents for depression [5, 9, 10]. For instance ketamine and memantine have demonstrated rapid and profound antidepressant effects clinically [11, 12]. Numerous behavioural studies have further demonstrated that antagonists and partial agonists at the glycine coagonist site of the nmda receptor have antidepressant potentials with less severe side effects . While the search of newer antidepressants continues, renewed interest in medicinal plants, for example, mallotus oppositifolius, for the treatment of many cns disorders has been on the ascendancy [1416]. Despite the use of mallotus oppositifolius in treating psychiatric and affective disorders in ghana, there is no scientific data on its antidepressant effect . Thus the present study investigated the effect of the hydroalcoholic leaf extract of the plant in acute antidepressant models the forced swim (fst) and tail suspension tests (tst). The effects of the extract on the monoaminergic system (serotonin and noradrenaline) as well as the glycine / nmda receptor complex were investigated in order to elucidate the possible mechanism(s) of action of the extract . Leaves of the plant mallotus oppositifolius were collected from the wild around the kwame nkrumah university of science and technology (knust), kumasi, ghana (6416.4n, 13342.8w) and authenticated at the department of herbal medicine of the faculty of pharmacy and pharmaceutical sciences, knust, kumasi, where a voucher specimen (knust / fp/035/09) has been deposited . After air - drying for 7 days, the leaves were pulverized with a hammer - mill and the powder extracted by cold maceration using 70% (v / v) ethanol in water over a period of 72 h. the resulting extract was concentrated under moderate temperature (60c) and pressure to a syrupy mass on a rotary evaporator . The syrupy mass was then dried to a dark brown semisolid mass using water bath and kept in a desiccator till it was ready to be used . This is subsequently referred to as mallotus oppositifolius extract (moe) or extract . Male icr mice were obtained from and housed at the animal facility of the department of pharmacology, knust, kumasi, ghana . The animals were housed in groups of five in stainless steel cages (34 47 18 cm) with soft wood shavings as bedding, fed with normal commercial pellet diet (gafco, tema), given water ad libitum, and maintained under laboratory conditions . All animals used in these studies were treated in accordance with the guide for the care and use of laboratory animals and experiments were approved by the college ethics committee . Fluoxetine hydrochloride (prozac) was from eli lilly and co., basingstoke, england . Desipramine hydrochloride, d - serine, d - cycloserine, -methyldopa, reserpine, and para - chlorophenylalanine were purchased from sigma - aldrich inc ., st . Louis, mo, usa . The fst was based on that described by porsolt et al . . Mice were divided into ten groups of five animals each and received the vehicle (water), extract (10, 30, or 100 mg kg, p.o . ), or the standard reference drugs fluoxetine (3, 10, or 30 mg kg, p.o . ), imipramine (3, 10, or 30 mg kg, p.o . ). One hour after oral administration of the test compounds, mice were gently placed individually into transparent cylindrical polyethylene tanks (25 cm high, 10 cm internal diameter) containing water (25 to 28c) up to a level of 20 cm and left there for 5 min . Each session was recorded by a video camera suspended approximately 100 cm above the cylinders . An observer scored the duration of immobility (when mouse floated upright in the water and made only small movements to keep its head above water), during the last 5 min test, from the videotapes with the aid of the public domain software jwatcher version 1.0 (university of california, los angeles, usa, and macquarie university, sydney, australia, available at http://www.jwatcher.ucla.edu/). Mice were allowed to acclimatize to the room for 3.54 h before the test . Groups of ten mice were treated with moe (10, 30, or 100 mg kg, p.o . ), fluoxetine (3, 10, or 30 mg kg, p.o . ), and imipramine (3, 10, or 30 mg kg, p.o .) Or vehicle . One hour after oral administration of the test compounds, mice were individually suspended by the tail from a horizontal bar (distance from floor = 30 cm) using adhesive tape (distance from tip of tail = 1 cm). Duration of immobility, defined as the absence of all movement except for those required for respiration, was recorded by an observer for 5 min from video recordings of the test as described above for forced swimming test . Mice that climbed up on their tails during the test session were gently pulled down and testing continued . Mice were pretreated with reserpine and/or -methyldopa (-md) in order to investigate the possible role of noradrenergic system in the actions of moe . The doses of -md and reserpine were chosen on the basis of work done by others [20, 21]. To deplete newly synthesized pools of noradrenaline (ne) and dopamine (da), mice were treated with a single dose of -md (400 mg kg, i.p .) 3.5 hours before behavioural testing . To deplete vesicular pools of ne and da, mice were treated with a single dose of reserpine (1 mg kg, s.c .) 24 h before behavioral testing . In an effort to deplete both the vesicular and cytoplasmic pools of ne and da, mice were pretreated with a combination of reserpine (1 mg kg, s.c ., 24 h before behavioral testing) and -md (200 mg kg, i.p ., 3.5 hours before behavioral testing), respectively . Mice were grouped into groups of five (5) forming ten (20) groups . Pcpa (200 mg kg, i.p .) Was administered once daily for 3 consecutive days to some of the animals . On the fourth day, group 1 received saline without pretreatment; group 2 received pcpa after pretreatment; groups 3 to 5 received moe (10, 30, and 100 mg kg) without pretreatment; groups 6 to 8 received moe (10, 30, and 100 mg kg, p.o .) After pretreatment; groups 9 to 11 received fluoxetine (3, 10, and 30 mg kg, p.o) alone; groups 12 to 14 received fluoxetine (3, 10, and 30 mg kg, p.o .) After pretreatment; groups 1517 received imipramine (3, 10, and 30 mg kg, p.o .) Alone; and finally groups 1820 received imipramine (3, 10, and 30 mg kg, p.o .) After pretreatment . After the tail suspension sessions, mice were taken through the modified forced swimming test . The modified forced swimming test followed the same procedure described above except that the depth of water was changed to 20 cm . For tail suspension, immobility period was scored, whilst for the modified swimming test mean immobility counts, mean swimming counts and mean climbing counts were scored . Mice were divided into 2 groups, a and b. groups a and b were further subdivided into 6 groups each (n = 8). Briefly five groups of mice from group a received d - cycloserine (2.5 mg kg, i.p .) And 30 min after the first three groups received an oral dose of the extract (10100 mg kg) with the last two groups receiving either fluoxetine (10 mg kg) or desipramine (10 mg kg, i.p . ). Five groups of mice from group b received d - serine (600 mg kg) and 30 min after the first three groups received an oral dose of the extract (10100 mg kg) with the last two groups receiving either fluoxetine (10 mg kg, p.o .) Or desipramine (10 mg kg, i.p . ). The forced swim and tail suspension tests were used as described above to investigate the antidepressant mechanism . Pedalling behaviour was defined as the continuous movement of the paw of the mice without moving the body while curling was defined as the raising of the head of the mouse towards its hind paws . Graphpad prism for windows version 4.03 (graphpad software, san diego, ca, usa) was used for all data and statistical analyses . P <0.05 was considered statistically significant . In all the tests, a sample size of ten animals the time - course curves were subjected to two - way (treatment time) repeated measures analysis of variance (anova) with bonferroni's post hoc test . Total immobility time, distance travelled, and time taken to find the hidden platform and change in weight for each treatment were calculated in arbitrary unit as the area under the curve (auc). Differences in aucs were analysed by anova followed by newman keuls' post hoc test . Moe (10100 mg kg, p.o . ), administered 60 min before the test period, significantly decreased the frequency of immobility (f3,19 = 21.47, p <0.001) (figure 1(a)) and immobility periods of mice (f3,19 = 143.4, p <0.001) (figure 1(d)) in a dose dependent manner in the fst . In the tst both frequency (f6,54 = 0.486, p = 0.8159) (figures 2(a), 2(b), and 2(c)) and duration (f6,52 = 25.57, p <0.001) (figures 2(d), 2(e), and 2(f)) of immobility decreased, indicating significant antidepressant activity . Pretreatment with reserpine (1 mg kg, s.c .) Alone, -methyldopa (400 mg kg, p.o .) Alone, or a concomitant administration of reserpine (1 mg kg, s.c .) And -methyldopa (200 mg kg, p.o .) Did not reverse the decline in immobility caused by the extract, moe (10100 mg kg, p.o . ), in the forced swim test (fst) (figures 3(a), 3(d), and 3(g)). Results obtained for fluoxetine - treated groups (flx) (330 mg kg, p.o .) Were similar to that of the extract - treated groups (figures 3(b), 3(e), and 3(h)). In contrast, the antidepressant effect of imipramine (imi) was reversed by either reserpine alone, -methyldopa alone, or a concomitant administration of reserpine and -methyldopa (figures 3(c), 3(f), and 3(i)). Pretreatment of mice with pcpa (200 mg kg) abolished the antidepressant effect of moe (10100 mg kg, p.o . ), and fluoxetine, flx (330 mg kg, p.o . ), but not imipramine, imi (330 mg kg, p.o . ), in the fst . The mean counts for immobility (f7,32 = 14.63; p <0.0001) (figure 4(a)), swimming (f7,32 = 44.74; p <0.0001) (figure 4(d)), and climbing (f7,32 = 1.121; p = 0.3742) (figure 4(g)) in the extract - treated group after pcpa pretreatment did not show any difference when compared with the control . Similar results as above were observed for flx - treated groups but not imipramine (figures 4(b)-4(c), 4(e)-4(f), and 4(h)-4(i)). In an attempt to investigate the possible involvement of 5-ht2a receptor activation in the antidepressant action of the extract, mice were given 5-hydroxytryptophan after extract pretreatment to induced head twitch responses . It was observed from the time course curve that the extract as well as fluoxetine increased the head twitch responses significantly for the period of 30 minutes (figures 5(a) and 5(c)). One - way anova followed by newman keuls' test of the areas under the curve (aucs) showed a dose dependent increase in the head twitch response for both extract and fluoxetine (figures 5(b) and 5(d)). In the tst, moe (100 mg kg, p.o . ), fluoxetine, flx (10 mg kg, p.o . ), and desipramine, dsp (10 mg kg, i.p . ), exhibited significant antidepressant effect by decreasing mean immobility score which was reversed by d - serine, ds (600 mg kg, i.p . ), pretreatment (figure 6(a)). Pretreatment with d - cycloserine, dcs (2.5 mg kg, i.p . ), potentiated the effect of moe and flx (but not dsp) by further decreasing mean immobility score (figure 6(b)). Moe alone did not affect curling score and this was not changed by ds pretreatment (figure 6(e)). Flx and dsp alone caused slight increase in the curling score which was reversed by ds . Pretreatment with dcs significantly increased curling score of moe but caused only a modest increase in both flx and dsp treated groups (figure 6(f)). Moe, ds, dcs, flx, and dsp alone increased swinging score . Ds pretreatment partially inhibited swinging behaviour by moe but totally in flx and dsp treated groups (figure 6(c)). Dcs pretreatment also inhibited swinging behaviour by moe and dsp but not flx (figure 6(d)). In the forced swim test, moe, flx, and dsp decreased immobility score and pretreatment with dcs potentiated the effect of moe and flx (but not dsp) by further decreasing immobility score (figure 7(b)). Moe and flx, unlike dsp, increased swimming behaviour which was inhibited by ds but increased by dcs pretreatment (figures 7(c) and 7(d)). Climbing scores were decreased by both moe and flx and this was not affected by both ds and dcs pretreatment (figures 7(e) and 7(f)). Dsp, on the contrary, increased climbing score which was unaffected by dcs pretreatment but decreased by ds . Results of the study demonstrated that moe has significant antidepressant effect in both the forced swimming and the tail suspension tests . In both animal models, percentage immobility and frequency of immobility were decreased by extract treatment . Reduction in immobility has been used as the primary index for antidepressant effect of test substances in these models almost all antidepressants in clinical use induce a decrease in immobility in rodents whilst other drugs fail to give the same response [22, 23]. Preclinical and clinical studies suggest that depletion of a monoamine implicated in depression pathophysiology may abolish the antidepressant effect of a substance if the substance depends on that particular monoamine for its antidepressant effect [20, 24]. Hence when 5-ht was depleted by pretreating mice for 3 days with the tryptophan hydroxylase inhibitor para - chlorophenylalanine (pcpa), the effects of drugs that act by enhancing 5-ht neurotransmission were abolished [25, 26] while those that act on noradrenergic pathways were not affected [2628]. The inhibition of the antidepressant effect of moe by pcpa in both tst and fst suggests that its antidepressant effect is dependent on enhancement of serotoninergic neurotransmission . The lack of antidepressant effect of fluoxetine in pcpa - treated mice is consistent with the hypothesis that fluoxetine elicits its acute behavioural effects by increasing extracellular 5-ht after blockade of the serotonin transporter . The extract and fluoxetine increased swimming score which was reversed with pcpa pretreatment, further supporting their action on the serotoninergic system [27, 30]. Both moe and fluoxetine did not affect mean climbing score, suggesting that their behavioural effect may not depend on noradrenergic pathways . Further evidence suggesting that the extract enhances 5-ht neurotransmission was derived from its ability to increase head twitch responses induced by 5-htp . The head twitch response (htr) in rodents induced by 5-hydroxytryptophan (5-htp), a precursor of 5-ht, is considered as a specific behavioural model for the activation of serotoninergic neuron specifically 5-ht2a receptors . Thus it can be inferred that moe may be acting via direct or indirect activation of 5-ht2a receptors . This result is consistent with a number of studies where fluoxetine elicited similar responses [33, 34]. The present experiments also examined the role of noradrenaline and dopamine in the acute behavioural effects of the extract in the modified fst by using drugs that interfere with their neurotransmitter synthesis or release . Depletion with -methyldopa, an l - aromatic amino acid decarboxylase inhibitor that inhibits the biosynthesis of catecholamines and 5-ht, failed to attenuate the behavioural effects of moe and fluoxetine while that of imipramine was abolished . This suggests that moe may not be affected by the biosynthesis of noradrenaline or dopamine . Moreover, when vesicular pools were depleted by reserpine, the decrease in immobility elicited by moe or fluoxetine was not affected . Here reserpine is an irreversible inhibitor of the vesicular monoamine transporter 2 (vmat-2) which is located primarily within the cns and is responsible for transporting monoamines from the cytoplasm into secretory vesicles [36, 37]. Treatment with reserpine therefore leads to depletion of vesicular monoamine stores both serotonin and noradrenaline suggesting both serotonin and noradrenaline might be important in the antidepressant effects of imipramine . The inability of reserpine pretreatment to reverse the antidepressant effects of the extract and fluoxetine, however, seems to suggest that reserpine does not affect vesicular storage of 5-ht to the same extent as that of noradrenaline . In fact this assertion is consistent with the results obtained by o'leary et al . And woode et al . [20, 39]the former demonstrating that reserpine at the dose used produced a 93 and 95% depletion of cortical noradrenaline and dopamine content, respectively, and a 78% depletion of 5-ht . To inhibit synthesis as well as deplete vesicular pools of noradrenaline and dopamine the work published by o'leary et al . Indicated that, when reserpine was combined with -methyl para - tyrosine ampt (ne and da biosynthetic inhibitor), a depletion of cortical da (95%), ne (97%), and 5-ht (78%) was observed . The combination had only a modest effect on ne and da but failed to affect 5-ht . Though -methyldopa was used instead of the ampt used by o'leary and colleagues, the results from the combined effect of reserpine and -methyldopa did not differ significantly from when reserpine was used alone . A more recent publication by woode et al . Used -methyldopa and similar results was observed . These results demonstrate that the antidepressant effect of moe may not be dependent on noradrenergic neurotransmission . Both clinical and preclinical studies support the antidepressant activity of antagonists on functional glycine / nmda receptor complex . These compounds are thought to have lower side effect profiles compared to the competitive and noncompetitive nmda antagonists . The effect of moe on glutamatergic neurotransmission was assessed by pretreating mice with d - serine (ds), a full agonist on glycine / nmda receptors, or d - cycloserine (dcs), a partial agonist on these receptors . In both the tst and fst, ds reversed the decline in immobility by moe and fluoxetine but dcs pretreatment potentiated the decline, demonstrating that moe may be acting as an antagonist on the glycine / nmda receptor complex . In contrast, the decrease in immobility of desipramine was reversed by ds but dcs had no effect on it . This suggests that the antidepressant effect of both serotonin and noradrenaline - based compounds depends on the inhibition of the glycine / nmda receptor complex but the enhancement of antidepressant activity depends on the serotoninergic pathway and not the noradrenaline pathway . This explains why the antidepressant effect of the extract, fluoxetine, and desipramine was abolished by d - serine but only the effect of the extract and fluoxetine (which act via serotoninergic pathway) was potentiated by d - cycloserine . Moreover, moe increased curling score in the tst slightly (suggestive of opioidergic activity) though not significant . Pretreatment with dcs significantly increased curling score of moe but did not affect both flx and dsp treated groups . Ds pretreatment partially inhibited pedaling behaviour by moe but totally in flx and dsp treated groups . Dcs pretreatment also inhibited pedaling behaviour by moe and dsp but not flx . According to berrocoso et al . This suggests that the extract on its own may have little effect on opioidergic activity but may interact with opioid receptors when combined with dcs . In the fst, moe and flx unlike dsp increased swimming behaviour which is sensitive to selective serotonin reuptake inhibitors (ssris) and 5ht agonists [22, 42]. This behavior exhibited by moe and fluoxetine - treated mice was inhibited by ds but increased by dcs pretreatment further supporting the theory that antidepressant effect of serotonin based drugs depends on the inhibition of the glycine / nmda receptor complex and the enhancement of antidepressant activity depends on the serotoninergic pathway . Climbing scores were decreased by both moe and flx and this was not affected by both ds and dcs pretreatment . Dsp, a selective noradrenergic reuptake inhibitor, on the contrary, increased climbing score which was unaffected by dcs pretreatment but decreased by ds . It is worth mentioning that the extract, fluoxetine, desipramine alone, or their combination with either d - serine or d - cycloserine did not impair motor coordination . The present study shows that mallotus oppositifolius has antidepressant - like effect in mice and this may be mediated via enhancement of serotoninergic neurotransmission and inhibition of glycine / nmda receptor complex activation.
Without much risk of controversy, one can safely assume that we all want more people in our society to learn, develop expertise, and innovate; fewer people to fail to thrive or to become dependent on damaging substances or behaviors; and for more people to experience a greater sense of well being . But we would also agree that some people in our society learn more, create more, become more emotionally and physically resilient, and have more fun . The question is: why? For answers to this question we can achieve very little consensus . Is it because people are constitutionally endowed with different personal assets native intelligence, motivation, optimism, capacity for pleasure? . They can be skills (such as calculation or literacy), levels of expertise, knowledge, creativity, emotional or social biases, likes and dislikes; they are, in short, relatively persistent traits and habits individuality, if you like . Questions about the origins of differences among people are of intrinsic interest to many, but from a biomedical perspective, the incentive to study them comes from the well - documented observation that many seriously adverse outcomes evolve disproportionately in the context of specific behavioral phenotypes . Risk phenotypes have been linked to many behavioral disorders, including substance use disorders (volkow, 2005; clark et al ., 2013), affective disorders (klein et al ., 2011), psychosis (brent et al ., 2014), adhd (castellanos and tannock, 2002), dyslexia (peterson and pennington, 2012), and dyscalculia (butterworth et al ., 2011). One could say that from conception to the onset of these disorders, evolving risk phenotypes are simply forms of individuality, but a deeper understanding of the divergence of high - risk from low - risk phenotypes eludes us . This article will describe some lines of evidence relevant to these issues, including work from behavioral genetics, neurogenetics, and cognitive neuroscience, and will pose important questions that we must answer in future research . Finally, impediments to progress in the field, and some recommendations for how to reduce them, are outlined . For example, scores from intelligence tests, performance on cognitive tasks, and self - report scales measuring personality have consistently exhibited moderate heritability in twin studies (bartels et al ., 2002; rettew et al ., 2006; kremen et al ., 2007), as have measures of academic skills such as reading and calculation (luo et al ., 2003). These observations highlight the influence of genetic factors on developing behaviors, but do not identify them . A few behavioral phenotypes have been examined in large, genome wide association studies (gwas), and surprisingly, given reported high estimates of heritability, these studies initially revealed few replicable associations with specific genetic variants, and those variants identified accounted for only very small proportions of the phenotypic variability (butcher et al ., 2008; calboli et al ., 2010; rietveld et al ., 2013; rietveld et al ., however, more recent studies have applied novel methods that exploit gwas results to estimate heritability attributable to common genetic variants in aggregate (so called chip heritability), and these suggest that substantial variability in behavioral phenotypes may indeed be explained by common variants, but the genetic architecture of these traits is likely to be highly polygenic, and extremely complex . So, in summary, genetic differences probably do play a large role in engendering behavioral individual differences, but it appears that thousands of variants may in some way influence these pheno - types, each with a very small additive effect (purcell et al . A number of studies have examined the heritability of brain imaging phenotypes, again mostly using twin designs . Global measures, such as brain volume (baar et al ., 2001), gray matter volume (baar et al ., 2001), and both surface area and thickness of the cortex (panizzon et al ., 2009; eyler et al ., 2011) exhibit even higher levels of heritability, sometimes over .9, with heritability estimates of regional measures significant but more modest . Interestingly, cortical surface area and mean thickness of the cortex, both with estimated heritability in the .8.9 range, nevertheless exhibited no evidence for genetic correlation in a study of middle - aged male twins (panizzon et al ., 2009), suggesting that the genetic factors influencing variability of these brain phenotypes are essentially nonoverlapping . A series of subsequent analyses of imaging data from this study examined more closely the genetic architecture of individual difference variability in cortical regionalization, that is, individual differences in the relative sizes of different cortical regions . Work with rodent models has shown that arealization of the developing cortex is linked to gradients of secreted morphogens and expression of transcription factors in the neocortical proliferative zone during the embryonic period (mallamaci and stoykova, 2006; o'leary et al ., 2007). Transgenic models have shown that alterations of these gradients are associated with different relative sizes of sensorimotor regions in the later developing cortex . Now that surface - based methods are available for examining the relative sizes of human cortical regions more precisely, one might ask whether genetic variation gives rise to individual differences in human cortical regionalization . Using data from 400 middle - aged male veterans from the vietnam era twin study cohort, chen et al . (2012) applied conventional twin methodology to compute the vertex - to - vertex genetic correlations among measures of cortical surface expansion at 2500 vertices (after smoothing). This large matrix of genetic correlations was then further analyzed using a data - driven, fuzzy clustering method to identify 12 sets (or clusters) of vertices with relatively higher genetic inter - correlations and relatively lower genetic correlations with other sets (or clusters). These clusters are color - coded in fig . 1 and they represent regions of the cortex where the twin data suggest that individual differences in relative surface area expansion are related to distinct sets of underlying genetic factors . Interestingly, this analysis yields a novel method for defining cortical parcels, one that is driven by analysis and simplification of the genetic architecture . After this analysis of cortical surface area variability was published, a similar analysis of genetic correlations for vertex - wise measures of cortical thickness was performed . The results of this analysis (chen et al ., 2013) also revealed regional clustering of genetic inter - correlations, and the 12-cluster solution resembled the regional pattern for surface area expansion . However, the dominant patterns of genetic inter - correlation for thickness measures (as expressed in the 24 cluster solutions) differed substantially from those for surface area, and as previously reported for total surface area and mean cortical thickness (panizzon et al ., 2009), the genetic correlations between areal expansion and thickness within comparable genetic clusters were also very low . These results suggest that genetic factors strongly influence both global area and thickness of the cortex as well as regional differences in these biomarkers; however, the overlap in genetic factors contributing to the high heritability of area and thickness of the cortex is negligible . Attempts to identify specific genetic variants that may contribute to the heritability of imaging phenotypes have been slow to appear because of the paucity of large studies with comparable brain imaging data . In recent years, however, larger studies with imaging and genotyping have been conducted . Early attempts to identify variants with effects strong enough to survive gwas significance criteria either failed or led to associations with low replicability, however a few studies have now reported findings in discovery and replication samples . Bakken et al . (2012) performed gwas to identify specific variants associated with the proportional surface area of the visual cortex . This variant was in a regulatory region of a gene that is highly expressed in the occipital cortex (gpcpd1). (2013) identified a variant related to lenticular nucleus size that was also replicated in an independent sample . Even these important gwas studies, however, examined fewer individuals than have been studied in most successful gwas studies of other complex human phenotypes, including several behavioral phenotypes . Recently, the important enhancing neuro imaging genetics through meta - analysis (enigma) project has begun to aggregate imaging genomics data across many studies worldwide to facilitate the search for specific variants related to imaging phenotypes . In an early enigma report on gwas of volumes of subcortical structures in over 30 thousand individuals, hibar et al . (2015) report that several variants contributing to these volumes survived stringent gwas criteria and replicated across samples . So far, however, these early reports suggest that, like heritable behavioral phenotypes, neuroimaging phenotypes may have highly complex genetic architecture, with few variants exerting large effects on the phenotypes, but perhaps many variants contributing strongly in aggregate . Together these results suggest that substantial individual differences in the relative sizes of and average thickness in different functional regions of the cortex are genetically mediated to a significant degree, that across regions they are influenced by different sets of genetic factors, and that genetic factors influencing cortical regionalization show little overlap with those influencing thickness variability across the cortex . In other words, the patterns suggest that the genetic architecture of the cortex is exceedingly complex, and each phenotype is likely to be mediated by a different large set of (probably small additive) genetic effects . An important question, of course, is whether these differences are associated with individual differences in behavioral phenotypes . Whatever the factors that give rise to them, genetic or otherwise, of one thing we can be certain: most behavioral phenotypes do not emerge at a particular point in time, but gradually, through the cumulative effects of factors influencing them over the life of the individual . As an example, the left - hand graph in fig . 2 shows the long time course of developing calculation skills in children between ages 5 and 16 . Such a protracted increase in calculation skills is consistent with the expected influence of instruction and practice in formal and informal learning environments . Perhaps more surprising is that even the basic capacity to inhibit a primed motor response (shown in the figure's right panel) exhibits a similarly protracted course of development . Why, for example, do some children from this study at age 14 exhibit calculation skills more typical of the average 9 year old, and some children at 9 exhibit motor inhibitory functions more typical of 15 year olds? Again, there is evidence for some degree of genetic mediation of variability on both of these kinds of skills, but developing calculation skills are clearly modified by the instruction and learning experiences of children, and by other cultural factors, and even performance on a simple motor inhibitory task like this improves with practice . For simplicity's sake, we might begin with this basic conceptual model (fig . 3) for an emerging behavioral phenotype in a developing child: that it represents some function of the effects exerted by (1) the hypothetical domain - relevant neural genotype; and sometimes by (2) environmental effects on the neural apparatus relevant to the skill or domain (e.g., in cases in which damage or toxic exposure to the brain occurs); but, more generally, also by (3) cumulative experiences throughout development encountering and manipulating material in the domain; and finally by (4) interactions between these factors . Much ongoing research in human developmental neuroscience is aimed at improving models like this one . However if we are ever to achieve our translational aims that is, to prevent adverse outcomes and lift the trajectories of well - being in at - risk individuals in our society then we must strive to answer some big questions that strike at the heart of the matter . We might ask: to what degree, and via which biological mechanisms, does common genetic variation constrain or bias functions of maturing circuits in the human brain? And to what degree, and under which circumstances, are there consequences (of these gene effects) for experience - dependent developmental processes, or for responses of the developing brain to neuroactive environmental factors (such as trauma, toxicity, drug exposure, etc . )? Only 2 decades ago it seemed unlikely to many that more knowledge about the biological development of the brain across the postnatal period would provide further insight into the nature of individual differences in part because the time course of the evolution of these complex behavioral phenotypes seemed much more protracted than what was thought of as a foreshortened period of the brain's biological development . It was assumed by many that although early events might fix the range of outcomes to some extent, everything changing in the school age years and beyond was necessarily due to learning in a biologically mature brain . This view began to change with the advent of noninvasive brain imaging . On this occasion, when we honor peter huttenlocher, we revisit one of the first papers published in this area 23 years ago (jernigan et al ., 1991); not, certainly, because it is a particularly interesting paper by today's standards, on the contrary, it will serve to illustrate how far we have come in those 2 decades; but it is one of the earliest imaging papers to cite peter huttenlocher's remarkable observations . In those days, jernigan was applying early semi - automated morphometry methods developed in her lab to analyze mr imaging data . The regions of interest (rois) for examining the cortex were essentially stereotactically - defined quadrants of the cerebrum, and are a long way back from the sophisticated surface - based methods we apply in our work today . By performing tissue segmentation based on dual echo mr images, jernigan et al . Measured cortical gray matter volumes in these large rois; and in a modest sample of 39 young people ranging in age from 7 to 35, observed highly significant, and linear, decreases in the cortical gray matter volumes (adjusted for volumes of the supratentorial cranial vault) in the two dorsal regions, with no real evidence of change in the ventral areas . Obviously, we know a lot more about the age functions and anatomical pattern of these apparent changes in gray matter volume now, but at the time they were quite surprising to many people; and the biological explanation for them was far from clear . Since the one largely postnatal phenomenon of which we were all aware was the protracted course of myelination and oligodendrocyte maturation (yakovlev and lecours, 1967), we, and others, speculated that signal changes associated with the newly generated myelin sheaths of axons coursing beneath the cortex, or even intracortically, might contribute to these apparent volume reductions in cortical gray . In other words, the apparent cortical gray matter reductions might represent the shifting signal characteristics of immature tissue comprised of more lightly myelinated axons (i.e., resembling gray matter) to the signal characteristics of tissue comprised of more fully myelinated axons . But we also had just become aware of huttenlocher's observations suggesting synaptic pruning in what seemed to be an anatomically heterochronous pattern across the cortex (huttenlocher and de courten, 1987; huttenlocher, 1990; huttenlocher and dabholkar, 1997). We therefore speculated that our observations and those reported by huttenlocher might in both cases reflect some later process of maturation in the dorsal cortical regions during adolescence . Before we move into the modern era of neuroimaging, we would emphasize one other result from this early paper, rarely mentioned in the subsequent literature, that seemed to suggest that the mechanisms underlying these apparent cortical volume changes probably involved both loss of tissue volume as well as changing signal contrast on mri . That was the observation that csf volume increased in the adjacent subarachnoid space in a very similar anatomical pattern to the cortical volume reductions, as though nearby tissue loss resulted in expansion of this space, ex vacuo . These early observations demonstrated that the neural architecture itself, like the behavioral phenotypes, continued to show dynamic age - related change over a similarly protracted period of development . Furthermore, imaging highlighted the considerable individual difference variability in brain morphology present across the entire age range, for example, the apparent differences in regionalization of the cortex, sometimes reflected in sulcal and gyral patterns, that were later the subject of the bakken et al . And however, little was known about whether the differences in brain morphology might be mirrored in any way in the behavioral variability, particularly in typically developing children . Since these early observations, much elegant imaging work has been done revealing robust indices of ongoing biological development of the brain that can be monitored noninvasively in children . Many of these neurodevelopmental biomarkers and functional imaging phenotypes show very protracted trajectories of change with age and exhibit regional variation . Though a number of studies have now examined age - effects on measures of cortical architecture during the postnatal years, and a few have included longitudinal data, details about the pattern of change have been inconsistent, probably in part because of modest sample sizes, different age - ranges examined, and variable imaging protocols and analysis methods . Recently, investigators throughout the country collaborated on the large, multisite pediatric imaging, neurocognition, and genetics (ping) project in which well over 1000 children were studied . This imaging genetics study of children between the ages of 3 and 20 enrolled participants at 10 sites throughout the us . The design was cross - sectional and involved only a limited number of developmental and cognitive phenotypes, but the dataset is now shared freely with the research community and has been accessed by people all around the world, through a web - based tool called the ping portal (pingstudy.ucsd.edu). Users can apply for access by filing a data use request and a data use agreement on the portal . Approved users can download the dataset for offline analysis and/or explore the data using advanced interactive statistical and visualization utilities in the data exploration module (brown et al ., 2012; this dataset provides several advantages for defining postnatal changes on imaging phenotypes, including: the large number of participants studied with harmonized and standardized methods; the wide age range of the participants (and therefore the long developmental trajectories that can be estimated); and the availability of genome - wide genotyping, which among other things made it possible to compute sensitive measures of genetic ancestry, in the form of 6 genetic ancestry factors (alexander et al ., 2009; jernigan et al ., thus in this dataset it has been possible to estimate age - differences and extrapolated trajectories while holding constant the scanner used, socioeconomic status of the family, and genetic ancestry, variables that could otherwise introduce cohort effects in a cross - sectional study . Application of extended freesurfer methods for computational morphometry produced a set of cortical biomarkers that, for example, isolated variability in surface area from variability in apparent cortical thickness . 4 shows plots produced with the data exploration module of the ping portal of age - differences (and smooth functions of age) for two global cortical phenotypes, total surface area and mean thickness (across the entire cortical surface). The effect of age on surface area is nonmonotonic; surface area expands during early childhood years, and expansion decelerates during middle childhood giving way to gradual contraction during adolescence and thereafter . In contrast, the apparent thickness of the cortex exhibits (mostly linear) monotonic decrease across the entire range, from 3 to 21 years . With other features of the portal one can estimate these effects at each vertex on the (tessellated) surface of the cortex, and create visualizations that code, for each vertex, an age - specific estimate of annualized rate of change . These estimates of rate of change were calculated from the ping data using a generalized additive model (gam) implemented with the r - package mgcv . For each vertex a gam estimated a smooth function of age (like the one shown above for total surface area), controlling for scanner, gender, ses (family income and highest parental education), and a set of six genetic ancestry factors . The predicted values obtained from the age model were sampled at regular intervals (100 equally spaced intervals across the age range). Instantaneous rates of change were calculated from the sampled values using the method of finite differences and normalized to produce estimates for annualized rates of change in surface area and thickness at each vertex . Rate of change maps for surface area are shown in fig . 5 and confirm that the global pattern is observed across the entire cortical surface, i.e., early expansion followed by contraction during adolescence . Note that the maps of change at ages 4 and 6 are coded differently than those at ages 8 and above to better visualize regional variability in the generally higher rates of expansion at these earlier ages . To further highlight regional differences we computed the smooth age functions from the gam models for 3 larger rois generated by the 12-cluster genetic parcellation of surface area (shown in fig . 1 above adapted from chen et al ., 2012). 6 are the trajectories for (covariate - adjusted) mean expansion coefficients for parcels in the dorsolateral prefrontal cortex (blue), dorsome - dial frontal cortex (red), and occipital cortex (green); labeled as parcels 2, 3, and 12, respectively . Comparing the models visually suggests that early to middle childhood expansion is more rapid in the dorsolateral prefrontal than in the occipital parcel . Thinning of the cortex appears to be present across the entire cortex and across the entire age range from 3 to 21, contrasting with some previous reports of early childhood cortical thickening . Again, the maps suggest some degree of regional heterochronicity in apparent thinning of the cortex, as highlighted by comparing the smooth age functions from the gams for the genetically - derived parcels (note that mean thickness measures in rois derived from the surface area genetic parcels are used for consistency with fig . 6). 8 are the trajectories for covariate - adjusted mean cortical thickness measures for the parcels in the dorsolateral prefrontal cortex (blue), dorsomedial frontal cortex (red), and occipital cortex (green); labeled as parcels 2, 3, and 12, respectively, in fig . 1 . The age functions vary slightly, suggesting that unlike in the frontal parcels, where the rate of thinning appears to be fairly constant, thinning may decelerate slightly after age 10 in the occipital parcel . In summary, these results from ping illustrate the advantages of a more integrative, data - sharing approach to research that, in this case, yields more definitive estimates of parameters that relate imaging biomarkers to brain development . They highlight the distinct trajectories that relate age to cortical surface area and apparent cortical thickness, and they suggest that both the non - monotonic surface area function and the monotonic function for apparent thickness may exhibit some regional heterochronicity . However, while apparent differences among the regional trajectories are intriguing, the statistical reliability of these regional patterns is more difficult to assess and may require even larger samples, and certainly longitudinal data, for confirmation . These models describe the modal developmental course of surface area expansion (and contraction) and of apparent cortical thinning, but one might ask: what do we know about individual differences in these processes of cortical development and how might they relate to behavioral differences? In recent years a number of observations in children have associated individual differences in the cortical architecture with individual differences in behavioral phenotypes, and a few suggest that trajectories of biological maturation in specific neural systems may themselves map onto emerging phenotypes . (2001) using early global regional measures, and more recently by investigators using surface - based methods (tamnes et al ., 2010; porter et al ., 2011; squeglia et al ., 2013), regressive changes (volume loss or thinning) of the cortex has been associated with better performance on memory, cognitive, and executive functions in developing children and adolescents . These results would seem to suggest that more mature cortical phenotypes are mirrored in more mature performance profiles during development, since thinner cortex is rarely associated with performance improvements in other contexts . In other words, they suggest that phase advance of apparent cortical thinning might be associated with precocious functional development . Cortical surface area phenotypes have only rarely been correlated with behavioral measures in developing children, but in two reports from the ping study, such associations have been found . (2012) observed, in the 512 year old children, an association between greater (relative) cortical expansion in the anterior cingulate region and better performance on a flanker task, the latter measured as reduced effects of incongruent cues on reaction time . This association was independent of age . Since this region continues to expand in surface area over the 512 year age range (figs . 5 and 6), one explanation is that earlier anterior cingulate surface area expansion is associated with greater functional maturity of circuitry involved in response conflict resolution on this task . An alternative explanation is that individuals with relatively larger anterior cingulate, e.g., through regionalization effects, regardless of their developmental status, are more adept at such tasks . That the associations seemed to be absent in older individuals in the study is more consistent with the former than the latter interpretation, but these null effects in older participants could also be due to differences in measurement sensitivity or other factors . A second ping study (newman et al ., 2015) revealed a surface area phenotype related to high self - reported levels of generalized anxiety in 287 ping participants aged 720 years . Independent of age, gender, and genetic ancestry factors, anxiety was negatively associated with relative cortical surface area of the ventrome - dial prefrontal cortex, as well as with global cortical thickness, and these associations significantly diminished with age . These findings suggest that higher levels of anxiety, even in typically - developing children, may be characterized by both delayed expansion of the ventromedial prefrontal cortex and an altered trajectory of global cortical thinning . Though these early clues about neural correlates of individual differences in children are intriguing, unfortunately, many important questions remain about what they mean and why they occur . For example, for most of the associations reported here there is no direct evidence for or against a role of genetic factors in mediating them, though such effects are plausible given the findings in twin studies . Even when genetic factors can be implicated, it is not clear whether this represents variable functional constraint within the circuitry of different individuals, or effects of functional biases rather than constraints per se . In either case, it is unclear when such constraining or biasing effects were exerted during the functional development of the relevant neural systems . Thus, we still have little understanding of the meaning of the associations, and therefore they provide little guidance toward promising routes for intervention . An important question, with clear translational implications, is whether the associations are mediated to a greater degree by direct effects of genetic variation on developing neural systems, by direct effects of experience through neuroadaptive, or activity - dependent, processes, or by indirect effects of genetic factors, mediated by experience, through gene - experience correlations? Note that to the degree that the answer is the latter, even phenotypes with high heritability are likely to exhibit robust effects of intelligent behavioral (or other environmental) interventions . Information about possible gene - experience correlations is lacking, at least in part, because of the absence of large - scale, genetically - informed studies of behavioral phenotypes with sufficiently comprehensive assessments of putative experiential and environmental mediators . Almost certainly, the most definitive answers to these questions will require longitudinal observations . To answer these and a significant part of the problem stems from the fact that we do not yet know what the imaging biomarkers represent at a cellular level . Focusing on the developmental signals we can monitor in maturing human cortex, we could ask: what differences in the architecture of the cortex attend early cortical surface expansion and later contraction? Could these changes reflect widening or narrowing of cortical columns as might attend expanding and contracting dendritic mass? Although investigators have often hypothesized that synaptic pruning might be related to cortical thinning during development, recent studies of human postmortem material suggest a different pattern of nonmonotonic change in synaptic density and dendritic extent, i.e., protracted increases into childhood with more modest but similarly protracted decreases through adolescence and adulthood (glantz et al ., 2007; petanjek et al . The time courses reported in these studies are more similar to the age differences in surface area than thickness . Other possible contributors to surface area are late maturation of neuronal or glial morphological phenotypes, or even ongoing proliferation and differentiation of glial or microglial populations . Similar questions remain about the biological alterations that we measure as apparent cortical thinning . Although a role for intracortical and subcortical myelination is often suggested, direct evidence is lacking . In summary, a critical priority for advancing human developmental science should be establishing which cellular changes in the neural architecture attend the changes in imaging biomarkers that allow us to monitor biological development of the brain noninvasively . More information about changing levels and regional distribution of gene expression in the human brain across the developmental age range could provide important clues about the underlying biology . However, longitudinal studies that include careful assessment of plausible experiential moderators of neurodevelopmental processes, and, ideally, prospective behavioral intervention studies, are also needed to examine the role of activity - dependent processes . Further study is needed to determine which among the genetically mediated associations are due to effects of early patterning (e.g., that may be present throughout the lifespan), and which to genetic effects on the time course of brain development; and of the latter, which exert temporary effects on brain and behavioral development and which exert persisting effects . Among the demonstrated experiential effects (i.e., of practice, training, or emotion), it remains to determine how robust these effects are, how persistent they are, and which specific neural circuits they affect . We would argue that in order to answer these questions we need to take the research to a different level, and that a significant impediment to that aim is an unfortunate degree of fragmentation in developmental science generally, and in the science of the developing human mind and brain in particular . Human developmental neuroscience, developmental psychology, child psychiatry, pediatric neurology, education research, and other relevant branches of developmental science are siloed, with few unifying or integrative structures . Support for the research is provided through many small programs distributed across multiple institutes, agencies, and foundations . And unfortunately the behavioral disciplines are even further isolated from basic developmental neurobiology, in spite of the fact that both have much to gain from closer integration . Above we posed the following as examples of big questions at the heart of our understanding of behavioral phenotypes: to what degree, and via which biological mechanisms, does common genetic variation constrain or bias functions of maturing circuits in the human brain? And to what degree, and under which circumstances, are there consequences (of these gene effects) for experience - dependent developmental processes, or for responses of the developing brain to neuroactive environmental factors (such as trauma, toxicity, drug exposure, etc . )? So how do we achieve the integration needed to answer big questions with this degree of complexity? First, by beginning to accumulate large, informative, high - dimensional datasets for sharing, populated with harmonized assessments of developing children obtained repeatedly over time as they mature . These studies must attempt to capture genetic and epigenetic variability, and include noninvasive imaging and other neurophysiological measures sensitive to differences in the neural architecture and to the status of the brain's biological development . They should also include measures of the activity within maturing neural systems as well as behavioral and experiential assessments . These assessments must go beyond conventional forms of formal testing, observation, and parent and self - report methods to include innovative methods for sampling behavior and the environment with mobile devices and quantified life apps . It cannot be emphasized enough that we must collect richer data detailing the behaviors and experiences of children, and the physical, social, and cultural contexts in which those experiences occur . Data repositories populated with observations like these would yield high scientific dividends for decades, as new methods and conceptual approaches were applied to them over time . Whenever opportunities exist we should embed translational arms within these large - scale, longitudinal studies . Translation can mean implementation of models derived from observational (or theoretical) work in applications that would intervene in the observed process, as well as transfer from basic to clinical, or from clinical to basic, research; and for human developmental neuro - science all of these forms of translation are needed . First, we should consider implementing the prototyping, testing, and modification of novel interventions for mitigating risks in struggling or other high - risk groups, as well as interventions for enhancing learning and growth more generally, in high - information cohorts of longitudinal studies . Thus the additional behavioral, developmental, neurobiological, and genetic information already available for these participants could be leveraged in models of the effects of the interventions . The availability of elective interventions, delivered, for example, in after - school or summer programs held in the same venues as ongoing longitudinal studies, or via web - based interactive applications, could also help to consolidate longer - term relationships with families, and would serve as a catalyst for more interdisciplinary work on translation, e.g., involving teams of developmental scientists, teachers, and clinicians . At a scientific level, these studies would not only accelerate progress toward more effective prevention and mitigation of adverse developmental outcomes, they would address critical remaining questions about the role of experience - dependent processes, and their interactions with other factors, during development . Finally, a more integrative science would involve nested basic science programs explicitly designed to bridge gaps between the results from noninvasive studies in children and those from parallel studies of relevant cellular and molecular phenomena in animals, and in human postmortem and in vitro tissue . Interdisciplinary teams should propose studies designed collaboratively by scientists studying human development with others studying animal models . The aims of these studies would be to create new bridging behavioral paradigms for cross - species comparisons and to develop animal models of human developmental biomarkers by applying noninvasive methods parallel to those used in children . Developmental neurobiologists, working more directly and closely with human developmental neuroscientists, should be encouraged to generate novel hypotheses about the neurobiological sources of many robust developmental signals that we now monitor with noninvasive assessments . More comprehensive data from human pediatric postmortem material is a critical need, and should be given high priority; but, again, this work would have larger impact if linked directly to noninvasive biomarkers, for example by retrieving relevant in vivo imaging or neurophysiological data or applying postmortem imaging protocols for bridging the gap . Modeling of effects within these new cross - level, multimodality datasets will present significant challenges, and computational scientists with both the biostatistical expertise to implement appropriate mixed - models and sufficient domain knowledge to contribute to the interpretation of the effects must be trained and recruited to a new, more integrative developmental science . In order to achieve these goals, our sponsors also have a role to play, by: creating structures for integrating and leveraging resources within nih and through collaboration with nsf, department of education, and private sponsors.offering new mechanisms for funding of multi - pi, multidisciplinary collaborations (cross - level, cross species, observations + interventions).providing support for infrastructure and informatics specifically designed for developmental data.revising review criteria to incentivize: method harmonization across studies early data release and sharing integrative elements within programs creating structures for integrating and leveraging resources within nih and through collaboration with nsf, department of education, and private sponsors . Offering new mechanisms for funding of multi - pi, multidisciplinary collaborations (cross - level, cross species, observations + interventions). Providing support for infrastructure and informatics specifically designed for developmental data . Revising review criteria to incentivize: method harmonization across studies early data release and sharing integrative elements within programs method harmonization across studies early data release and sharing integrative elements within programs in conclusion, the remarkable progress made in developmental cognitive neuroscience in the last two decades is undeniable and very exciting but progress toward answering our big questions will languish unless we demand and create a more integrative science of the developing mind and brain . Certainly the most daunting challenge we face is the enormous complexity of the subject, but this challenge is all the greater when we are hamstrung by an unnecessary degree of fragmentation in our field . No societal campaign is more universally embraced than the pledge to ensure that each child can reach his or her own potential, but there is no consensus about how to accomplish this, and this situation reflects a sobering fact: insufficient evidence exists to reconcile contradictory assumptions and predictions . The answers to our questions are not beyond our reach, but they may be coming more slowly because we are not deploying our scientific assets in the most powerful way to answer the most important questions.
Yoga is an ancient indian science as well as the way of life, which includes the practice of specific posture (asana) and regulated breathing (pranayama). Different types of pranayama were reported to produce different cardiovascular and autonomic responses in healthy individuals . Bhramari pranayama (humming bee breath) is one of the common pranayama practice, which involves inhaling through both nostrils and while exhaling produce sound of humming bee . A previous study stated that the practice of bhramari pranayama influences the parasympathetic dominance on cardiovascular system due to its effect in reducing systolic blood pressure (sbp), diastolic bp (dbp), and mean arterial pressure (map). There are several methods available to measure cardiac autonomic nervous system, of which heart rate variability (hrv) has been established as a noninvasive tool . Many studies have reported the hrv of pranayama practices before and after the practice but only very few studies have reported the hrv during the pranayama practices such as alternate nostril breathing and kapalbhati . Since, bhramari pranayama was reported to produce parasympathetic activity without assessing autonomic measures, we had a research question as does bhramari pranayama produce parasympathetic activity? . Based on the previous study observations and reports, we hypothesized that bhramari pranayama may produce parasympathetic activity and hence, this present study aims at evaluating hrv (a noninvasive tool used to find sympathetic and parasympathetic activity) changes during and after the practice of bhramari pranayama . Sixteen (9 males, 7 females) healthy volunteers with the mean standard deviation age of 23.50 3.01 years were recruited from a residential yoga university, south india . Subjects aged 18 years and above experienced in practicing yoga for more than 1 year and willingness to participate in the study were included . Subjects with the history of any systemic and mental illness, regular use of medication for any diseases, chronic smoking, and alcoholism were excluded from this study . The study protocol was approved by the institutional ethics committee and a written informed consent was obtained from each subject . Of 40 subjects assessed for eligibility, 22 subjects did not fulfill the inclusion criteria and hence, did not include in the study . Demographic variables of the study group (n=16) this is a single group repeated measures study, in which all the subjects were asked to perform bhramari pranayama for the duration of 5 min . Hr and hrv: hr and hrv were assessed before, during, and after the intervention using a four - channel polygraph (polyrite d, recorders and medicare systems, chandigarh, india). The ag / agcl pregelled electrodes were placed according to the standard limb lead ii configuration for recording electrocardiogram (ecg). Frequency domain and time domain analysis of the hrv data were carried out at baseline, during and post - intervention (5 min recordings for each). The data recorded were visually inspected off - line, and only noise - free data were included for the analysis . The data were analyzed with an hrv analysis software (kubios hrv version 2.0, biomedical signal analysis group, department of physics, university of kuopio, finland). The energy in the hrv series in the following specific frequency bands was studied: low frequency (lf) band (0.040.15 hz), and high - frequency (hf) band (0.150.4 hz). The following components of the time domain hrv were analyzed: (1) the mean of the intervals between adjacent qrs complexes or the instantaneous hr (rr intervals), (2) standard deviation of rr intervals (sdnn) (3) hr, (4) the square root of the mean of the sum of the squares of differences between adjacent normal - to - normal (nn) intervals (rmssd), (5) the number of interval differences of successive nn intervals> 50 ms (nn50), and (6) the proportion derived by dividing nn50 by the total number of nn intervals (pnn50). Assessments such as pulse pressure (pp), map were derived using following formulas . Pp was calculated as (sbp dbp); map as (dbp + pp). All the subjects were asked to perform bhramari pranayama by inhaling through both nostrils and while exhaling produced the sound of humming bee for the duration of 5 min at the rate of 6 breath / min . Of 18 subjects, 2 female subjects rrinterval could not be extracted from the ecg due to rests of the data were checked for the normality using shapiro wilk test . Statistical analysis for bp was performed using student's paired samples t - test (data that were normally distributed) and wilcoxon signed - ranks test (data that were not normally distributed) and hrv data were analyzed using repeated measures of analysis of variance and post - hoc analysis with bonferroni adjustment for multiple comparisons with the use of statistical package for the social sciences (spss) for windows, version 16.0 . Sixteen (9 males, 7 females) healthy volunteers with the mean standard deviation age of 23.50 3.01 years were recruited from a residential yoga university, south india . Subjects aged 18 years and above experienced in practicing yoga for more than 1 year and willingness to participate in the study were included . Subjects with the history of any systemic and mental illness, regular use of medication for any diseases, chronic smoking, and alcoholism were excluded from this study . The study protocol was approved by the institutional ethics committee and a written informed consent was obtained from each subject . Of 40 subjects assessed for eligibility, 22 subjects did not fulfill the inclusion criteria and hence, did not include in the study . This is a single group repeated measures study, in which all the subjects were asked to perform bhramari pranayama for the duration of 5 min . Hr and hrv: hr and hrv were assessed before, during, and after the intervention using a four - channel polygraph (polyrite d, recorders and medicare systems, chandigarh, india). The ag / agcl pregelled electrodes were placed according to the standard limb lead ii configuration for recording electrocardiogram (ecg). Frequency domain and time domain analysis of the hrv data were carried out at baseline, during and post - intervention (5 min recordings for each). The data recorded were visually inspected off - line, and only noise - free data were included for the analysis . The data were analyzed with an hrv analysis software (kubios hrv version 2.0, biomedical signal analysis group, department of physics, university of kuopio, finland). The energy in the hrv series in the following specific frequency bands was studied: low frequency (lf) band (0.040.15 hz), and high - frequency (hf) band (0.150.4 hz). The following components of the time domain hrv were analyzed: (1) the mean of the intervals between adjacent qrs complexes or the instantaneous hr (rr intervals), (2) standard deviation of rr intervals (sdnn) (3) hr, (4) the square root of the mean of the sum of the squares of differences between adjacent normal - to - normal (nn) intervals (rmssd), (5) the number of interval differences of successive nn intervals> 50 ms (nn50), and (6) the proportion derived by dividing nn50 by the total number of nn intervals (pnn50). Assessments such as pulse pressure (pp), map were derived using following formulas . Pp was calculated as (sbp dbp); map as (dbp + pp). All the subjects were asked to perform bhramari pranayama by inhaling through both nostrils and while exhaling produced the sound of humming bee for the duration of 5 min at the rate of 6 breath / min . Of 18 subjects, 2 female subjects rrinterval could not be extracted from the ecg due to t wave elevation and hence, these data were not included in the statistical analysis . Rests of the data were checked for the normality using shapiro wilk test . Statistical analysis for bp was performed using student's paired samples t - test (data that were normally distributed) and wilcoxon signed - ranks test (data that were not normally distributed) and hrv data were analyzed using repeated measures of analysis of variance and post - hoc analysis with bonferroni adjustment for multiple comparisons with the use of statistical package for the social sciences (spss) for windows, version 16.0 . Results of this present showed a significant increase in hr and lf spectrum of hrv and a significant reduction in hf spectrum of hrv during the practice of bhramari, and it reverts back to normal during the recovery period after the practice . It also showed a significant reduction in sbp, dbp, and map after the practice and such significant changes were observed in rest of the variables [table 2]. Physiological oscillations that lead to variable beat - to - beat fluctuations in hr are known as hrv . Hence, hr and hrv are the most sensitive and easily accessible indicators of sympathetic and parasympathetic activity and autonomic regulation . The time domain analysis of hrv mainly reflects parasympathetic outflow and frequency domain analysis reflects overall autonomic balance and is the most widely used tool to investigate hrv and involves decomposition of sequential rrintervals into sinusoidal components of different amplitude and frequency . Just as hf band power is related to parasympathetic activity, lf band power is often related to sympathetic activity, yet the interpretation and clinical significance of hrv in the lf band have aroused intense controversy . The relationship between the lf band and sympathetic activity has been disputed because lf band power has been shown to be partly under parasympathetic control . Results of this present showed a significant increase in hr and lf spectrum of hrv and a significant reduction in hf spectrum of hrv during the practice of bhramari compared to its baseline, and it reverts back to normal during the recovery period after the practice . This effect might be possibly through its slow breathing techniques because slow yoga breathing practices were reported to increase hr fluctuations in the lf band with simultaneous increases in hr; or breathing at 4.5 and 6.5/min frequency or other rhythmical stimulation at this frequency such as rhythmical skeletal muscle contraction were reported to reflect in large increases in the lf band and simultaneous decreases in the hf band . Such resonance effects were also reported with yoga slow breathing practices, mantra chanting, and some meditative practices . A comprehensive review reported as the large amplitude hr oscillations occurring in the lf range resulting from breathing at an optimal frequency may reflect resonance, also known as coherence occurring due to entrainment between hr, bp, and the relaxation response rather than sympathetic tone . And since, the time domain variables of hrv and the hf band power of frequency domain are mainly the indicative of the parasympathetic activities, the insignificant reduction in these variables such as rri, sdnn, rmssd, nn50, pnn50 (time domain), and significant reduction in hf band power (frequency domain) along with significant increase in hr during the practice of bhramari pranayama, we believe that there might be a parasympathetic withdrawal while practicing bhramari pranayama . Due to the reduction in the bp level after the practice of bhramari pranayama bhramari pranayama induces parasympathetic dominance on cardiovascular system whereas, findings of this present study is contradicting the statement of the previous studies . At the same time, the previous study's findings such as significant reduction in sbp, dbp, map, and mild fall in hr after the bhramari pranayama were similar to this present study findings . Hence, reduction in the bp might not attribute to the increased level of parasympathetic activity, but it might attribute to other mechanisms which are unclear and need to be evaluated in the future studies . The first study evaluating the hrv during the practice of bhramari pranayama . Although the bp was assessed using sphygmomanometer, the assessment was performed by the intern who was not in the part of the study . The study was conducted in the healthy volunteers, hence limiting the application of its findings to pathological conditions . Additional assessments, such as continuous bp monitoring, baroreceptor sensitivity, photoplethysmography, and galvanic skin resistance would have given a better understanding of the state of the autonomic nervous system . The present study assessed only the hrv and bp changes in one group and did not have the control group and also did not assess its underlying mechanisms . Hence, further studies are required (i.e., randomized controlled trials) engaging a larger sample size, using advanced techniques, and taking place over a greater period, to evaluate its precise physiological effects and underlying mechanisms . Although the bp was assessed using sphygmomanometer, the assessment was performed by the intern who was not in the part of the study . The study was conducted in the healthy volunteers, hence limiting the application of its findings to pathological conditions . Additional assessments, such as continuous bp monitoring, baroreceptor sensitivity, photoplethysmography, and galvanic skin resistance would have given a better understanding of the state of the autonomic nervous system . The present study assessed only the hrv and bp changes in one group and did not have the control group and also did not assess its underlying mechanisms . Hence, further studies are required (i.e., randomized controlled trials) engaging a larger sample size, using advanced techniques, and taking place over a greater period, to evaluate its precise physiological effects and underlying mechanisms . Results of this study suggest that there might be a parasympathetic withdrawal during the practice of bhramari that revert back to normal after the practice.
Pupil dilatation unresponsive to light stimuli and decreased constriction response to accommodation, with denervation supersensitivity to dilute cholinergic agents, is known as tonic pupil . Most tonic pupil cases are idiopathic, but among all known causes, trauma is the most frequent . It has been described following ocular surgery; there are two classic reports of tonic pupil following retinal detachment (rd) repair, one using an encircling band (eb) and another after an old electrocoagulation [2, 3], and one recent report after pars plana vitrectomy (ppv). We describe the onset of a tonic pupil in a child after ppv and eb for a rd . In this case, an 11-year - old boy attended our emergency department for decreased visual acuity (va) in his right eye (re) for the last week . The patient had been a premature newborn, with 26 weeks and 800 g at birth, and he received a bilateral 360 argon photocoagulation for active retinopathy of prematurity (rop). Va, at the emergency room, was hand movement in his re, with normal anterior segment . Fundoscopy showed a total rd with macula off, and an 180 inferior giant tear at the posterior edge of the 360 intense laser scar . The left eye did not show any significant alteration, with an attached retina and a 360 peripheral laser scar . He underwent a ppv and placement of a 2.5-mm eb and heavy silicone oil (densiron) endotamponade in his re . A mild infrared laser (810 nm), he developed a painless persistent corneal epithelial defect (4 4 mm) in his re . One month after the ppv, the patient was started on intense autologous serum treatment, achieving complete corneal epithelisation within five weeks . When postoperative dilating drops were discontinued a moderate mydriasis in his re, unresponsive to light, was observed (fig . Four months later, pupil diameter of the re decreased and a slight anisocoria for right miosis was present (fig . 2c). One year after the ppv, corneal sensibility remains severely decreased and a moderate residual corneal scarring is still present . Pupil and accommodation defects have been described following extensive retinal laser treatment . The main explanation for this complication is thermal lesion of the short ciliary nerves, containing parasympathetic motor fibers, travelling through the choroid and suprachoroidal space . Infrared diode laser (810 nm) produces a much deeper coagulation when compared to argon laser and affects the retinal pigment epithelium and choriocapillaris, and thus easily damages the nerves running in the suprachoroidal area . There are few reports published in the literature, though, on tonic pupil following retinal surgery [2, 3, 4]. Report a case of a tonic pupil after eb with lamellar scleral resection and diathermy . They attribute their clinical findings to a compromise of the short ciliary nerves as they pass around the globe. Ebrahim et al . Recently published a case after ppv, argon endolaser and silicone endotamponade, performed under retrobulbar anesthesia . They speculate that endolaser damaged the short ciliary nerves and that it is responsible for the tonic pupil . It is surprising, though, that such a small amount of argon laser as described in their case can cause such a serious injury to the short ciliary nerves as to induce a tonic pupil . Nevertheless, they did not rule out a possible causative effect of the retrobulbar anesthesia or even an idiopathic etiology of the case . Direct injury to the ciliary ganglion by the anesthetic needle can be another cause of tonic pupil onset . There are, to our knowledge, no reports in the literature on this etiology . This is not applicable to our case because the patient underwent surgery under general anesthesia . On the other hand, direct compression of the ciliary ganglion exerted by the eb could be another plausible explanation in our case . Though we believe eb played a role in the development of the tonic pupil, this could be more related to short ciliary nerve damage . Eb is usually placed under the rectus muscles, in a much anterior position . In our case, the two long posterior ciliary nerves, branches of the nasociliary nerves, are responsible for the sensitivity of the iris, cornea and ciliary muscle . Experimental studies support that parasympathetic fibers travel with the long posterior ciliary nerves proximally, but arborice farther posteriorly near the posterior pole, where the choroid is thickest . Intraocular connections of the long and short ciliary nerves exist within or on the outer surface of the choroid . This suggests that all innervation types can be damaged with an intense laser photocoagulation and that respecting horizontal retinal meridians during the treatment may not always help to preserve these nerves . We hypothesize that there might also be a vascular etiology in the ciliary nerves lesion of our patient . Eb has been reported to induce relative ischemia to anterior ocular structures by means of venous obstruction or kinking of the long posterior ciliary arteries . Nevertheless, we cannot rule out that the tonic pupil and corneal anesthesia may arise from the prematurity state and treatments the patient had previously received, especially after observing a slight hypoesthesia of the contralateral cornea . In conclusion, we believe that a combination of different pathogenic factors have been involved in our case . Thermal injury of the ciliary nerves, initially by the old argon rop laser and recently by the infrared diode laser for rd repair, combined to the hypothetical blood support deficiency induced by the eb may have resulted in a long ciliary nerves and parasympathetic denervation causing the permanent corneal anesthesia and the tonic pupil.
Despite the improvement in the diagnostic and therapeutic strategies, oral squamous cell carcinoma (oscc) remains a major public health problem; it is the eighth most common cancer worldwide . Oral leukoplakia (olk) and oral submucous fibrosis (osf) are potentially malignant disorders with a high risk of transitioning to oscc . The potential for malignant transformation of olk and osf lesions with evidence of epithelial dysplasia ranges from 5%-15% and 3%-19%, respectively . Therefore, attempts are being made to identify specific molecular biomarkers to predict which lesions have the potential to be malignant . Activation of the canonical wnt pathway occurs by binding extracellular ligands to the frizzled receptor and the low - density lipoprotein (lrp5/6) co - receptors, which ultimately leads to stabilization of -catenin . -catenin translocates to the nucleus, interacts with tcf / lef family, and activates transcription by replacing repressor complexes like the groucho repressor complex . The human dickkopf family comprises four members (dkk1, 2, 3, and 4), which function as inhibitors of the canonical wnt pathway . Dkk3 is a potential tumor suppressor gene (tsg) that is localized on 11p15, a locus often deleted in cancerous cells . Dkk3 interacts with the krm receptors intracellularly, which exerts its inhibitory effect on wnt signaling either by binding to btrcp, thus blocking -catenin from entering the nucleus, or by increasing the localization of -catenin in the cell membrane . Dkk3 has been shown to be downregulated in various cancer tissues, suggesting that this gene may be a tsg, while hypermethylation of its promoter correlates with the occurrence of cancers . Since the expression of dkk3 in potentially premalignant disorders has not been examined, in this study we aim to assess and compare immunohistochemical expression of dkk3 in olk and osf as well as correlation with clinicopathological features . Seventy - five formalin - fixed paraffin - embedded blocks with histologically proven nom, osf, olk, and oscc were collected retrospectively . The samples consisted of 5 nom, 14 osf without dysplasia, 16 osf with dysplasia, 18 olk with mild dysplasia, 12 olk with moderate / severe dysplasia, and 10 oscc . All patients in this study provided informed consent, and the study was approved by the school of medicine, central south university . Oscc was diagnosed according to who criteria of 2005 . According to pindborg s criteria, osf cases were further categorized as early stage (n=5), moderately advanced (n=16), or advanced (n=9). Nom samples were obtained from the buccal mucosa during the surgical removal of third molars . The biopsy samples were fixed in 10% neutral buffered formalin, left 24 h at room temperature, routinely processed for histology and embedded in paraffin wax . Four - micrometer - thick sections were cut, dewaxed in xylene, and dehydrated in a graded alcohol . After the slides were rinsed, endogenous peroxidase activity was blocked by treatment with 3% h2o2 for 15 min at room temperature . The slides were pretreated with citrate buffer (10% citrate buffer stock in distilled water, ph 6.0) and microwaved for 20 min to retrieve the antigen . Non - reactive staining was blocked by 1.0% goat serum in tris - buffered saline (ph 6.0) applied for 3 min . Primary rabbit polyclonal antibodies against human dkk3 (ap1523a, abgent, san diego, ca, usa; 1:50) were diluted in phosphate - buffered saline (pbs, ph 7.4) and then incubated for 1 h at 37c in a humidified chamber . After the slides were rinsed with pbs, they were incubated with a secondary antibody (biotinylated polylink; dako, hamburg, germany) for 30 min at 37c in a humidified chamber and then rinsed with pbs . Dkk3 expression and epithelial thickness was measured in all tissue samples using imagej, java - based image processing software (national institute of mental health, bethesda, md, usa). The mean values for each group of measurements were compared using the student s t - test . The chi - square test was used to determine the relationship between dkk3 expression and clinicopathological parameters . Dkk3 expression was compared between different olk and osf groups using the mann - whitney u test . Differences with a probability value of less than 0.05 were considered to be statistically significant . Seventy - five formalin - fixed paraffin - embedded blocks with histologically proven nom, osf, olk, and oscc were collected retrospectively . The samples consisted of 5 nom, 14 osf without dysplasia, 16 osf with dysplasia, 18 olk with mild dysplasia, 12 olk with moderate / severe dysplasia, and 10 oscc . All patients in this study provided informed consent, and the study was approved by the school of medicine, central south university . Oscc was diagnosed according to who criteria of 2005 . According to pindborg s criteria, osf cases were further categorized as early stage (n=5), moderately advanced (n=16), or advanced (n=9). Nom samples were obtained from the buccal mucosa during the surgical removal of third molars . The biopsy samples were fixed in 10% neutral buffered formalin, left 24 h at room temperature, routinely processed for histology and embedded in paraffin wax . Four - micrometer - thick sections were cut, dewaxed in xylene, and dehydrated in a graded alcohol . After the slides were rinsed, endogenous peroxidase activity was blocked by treatment with 3% h2o2 for 15 min at room temperature . The slides were pretreated with citrate buffer (10% citrate buffer stock in distilled water, ph 6.0) and microwaved for 20 min to retrieve the antigen . Non - reactive staining was blocked by 1.0% goat serum in tris - buffered saline (ph 6.0) applied for 3 min . Primary rabbit polyclonal antibodies against human dkk3 (ap1523a, abgent, san diego, ca, usa; 1:50) were diluted in phosphate - buffered saline (pbs, ph 7.4) and then incubated for 1 h at 37c in a humidified chamber . After the slides were rinsed with pbs, they were incubated with a secondary antibody (biotinylated polylink; dako, hamburg, germany) for 30 min at 37c in a humidified chamber and then rinsed with pbs . Dkk3 expression and epithelial thickness was measured in all tissue samples using imagej, java - based image processing software (national institute of mental health, bethesda, md, usa). The mean values for each group of measurements were compared using the student s t - test . Chicago, il, usa). The chi - square test was used to determine the relationship between dkk3 expression and clinicopathological parameters . Dkk3 expression was compared between different olk and osf groups using the mann - whitney u test . Differences with a probability value of less than 0.05 were considered to be statistically significant . Dkk3 staining was observed in 60 of the samples and was lost in 15 . In nom, dkk3 was localized only in the cell membrane of suprabasal cell layers, whereas the basal cell layer was negatively stained except for some scattered spots of weak dkk3 expression . It was localized in both the cell membrane and cytoplasm . However, the percentage of cytoplasmic expression in cases of olk with severe dysplasia was higher compared to olk with mild dysplasia . In contrast, the membranous expression of the suprabasal cells was higher in olk with mild dysplasia compared to olk with severe dysplasia (figure 1 c - f). Dkk3 expression was detected in 15 of 30 tissue samples of patients with osf (table 2). Dkk3 expression was detected in 13 cases of osf without dysplasia and was lost in 1, whereas dkk3 was detected in 2 cases of osf with dysplasia and was lost in 14 . A significant difference was observed when comparing dkk3 staining among cases of osf with and without dysplasia (figure 2 a - f). Dkk3 expression was observed in 4 out of 5 patients in the early stage, 9 out of 16 in the moderately advanced stage, and 2 out of 9 in the advanced stage . There was a gradual decline in dkk3 expression while epithelial atrophy progressed, but this was not statistically significant . No dkk3 expression was detected in the cell membrane of any osf case . In the early stage of osf, dkk3 expression was localized in the cytoplasm of the basal and suprabasal layers, but it was only localized in the cytoplasm of the basal layer in the moderately advanced and advanced stages (figure 2 a - f). Cytoplasmic dkk3 expression was detected in all oscc cases (figure 3 a, b). The relationship between dkk3 expression and the various clinicopathological characteristics of olk and osf was studied . Only the dysplastic status of osf, which shows significant correlation with dkk3 expression, other shows no significance (tables 1 and 2). When dkk3 expression was compared between olk groups and osf groups, an important aim of this study was to assess and compare dkk3 expression in olk and osf patients . The current study is the first to investigate dkk3 expression in both olk and osf . In nom, dkk3 was expressed on the cell membrane at suprabasal layers and was absent at the basal layer . In olk, the percentage of dkk3-positive cytoplasmic expression in mild dysplasia was less compared with severe dysplasia . Further, the staining was localized in the cytoplasm of the basal layer in both mild and severe dysplasia . Our finding was in line with the discovery that dkk3 expression gradually increases from nom to olk and from olk to oscc . Findings regarding the location of the expression either in the cell membrane of the nom or in the cytoplasm of the olk and osf . The precise mechanism by which dkk3 expression shifts from cell membrane to the cytoplasm is not fully clear . However, this does not appear to be through inhibition of the canonical wnt/-catenin signaling pathway . The most striking finding in the present study was the loss of dkk3 expression in 50% of osf cases . In contrast to olk cases, the percentage of positive cytoplasmic expression was higher in cases of osf without dysplasia than in cases of osf with dysplasia (p=0.0001). It is suggested that dkk3 is involved in scc carcinogenesis in the head, neck, and oral regions . Showed that by performing a genome - wide loss of heterozygosity (loh) analysis . An allele at the dkk3 locus is frequently deleted in head and neck scc suggesting that dkk3 functions as a tsg . Dkk3 knockdown in oscc - derived cells resulted in reduced cell migration and invasion and may have an oncogenic function that is independent of wnt signaling . An intriguing question was raised: why was dkk3 expressed in all olk cases, but loss of protein expression was reported in 50% of osf cases? To answer this question, we should first understand the cause of osf development, which is still not clear . Therefore, it is tempting to hypothesize that there may be underlying factors responsible for loss of dkk3 expression in osf with dysplasia . The possible explanation for aberrant dkk3 expression may be the chemicals in areca nuts: arecoline, arecaidine, guvacine, and guvacoline . These compounds have been proposed to be highly cytotoxic and genotoxic to cultured human buccal epithelial cells . Teh et al . Demonstrated that genomic instability is due to loh in several chromosomal loci containing known oncogenes and tsgs associated with head and neck carcinogenesis in osf tissues . Whether this reflects that dkk3 is instable or hypermethylation occurs in the promoter remains to be confirmed by molecular biology techniques . Surprisingly, a gradual loss / reduced expression of dkk3 in osf cases, from overexpression in the early stage to partial loss in the moderately advanced stage, and then to severe loss in the advanced stage was observed . In other words, dkk3 expression progressively decreased with increased severity of epithelial atrophy from the early to advanced stages of osf . However, the results were not significant (p=0.547). In their investigation in colorectal cancer, zitt et al . Reported that dkk3 positive samples showed a higher mean microvessel count than did dkk3 negative samples . Suggesting that dkk3 can be considered a putative pro - angiogenic protein in colorectal cancer . Since osf occurs because of the stromal changes, which undergo progressive hyalinization, decrease in vascularity and cellularity . We hypothesized that dkk3 expression in osf samples may decrease with the decrease in vascularity of the stromal layer . Recently, dkk3 is the clinical target of gene therapy in prostate, gastric scirrhous and renal fibrosis . . Demonstrated that dkk3 might serve as a diagnostic marker to identify the degree of atrophy and fibrosis in human patients with different types of chronic kidney diseases . Findings as well as our study findings, dkk3 may serve as a diagnostic marker to identify different stages of osf based on the severity of epithelial atrophy . Further, it may also help to assess the osf carcinogenesis based on the dysplastic status of osf, as most predictions about the onset of malignancy are currently based on the severity of dysplasia . Therefore, further well - designed study with large sample size and multiple markers are needed.
He also had a history of sexual dysfunction, for which he was prescribed udenafil . He took his first dose of 100 mg udenafil three days before his visit to our clinic . The next day he found that his eye was mildly irritated, and he had a mild headache . He discussed these symptoms with his urologist, and his symptoms subsided on their own after several hours . One day before his visit to our clinic, he took his second dose of udenafil . The following morning, approximately 12 hours later, he reported blurred vision and a decrease in the visual field of his right eye . This patient did have a 20-year history of smoking but no history of either hypertension or hypotension, diabetes mellitus, hyperlipidemia, or cardiovascular disease . Upon examination, his best - corrected visual acuity (bcva) was 20 / 25 in the right eye and 20 / 20 in the left eye . A dilated fundus examination of the right eye revealed prominent swelling of the disc with a disc rim hemorrhage (fig . Fundoscopy of the left eye revealed a healthy but crowded disc with a cup - to - disc ratio of 0.2 (fig . 1). Fundus fluorescein angiography showed both an inferior choroidal filling delay and an inferior sector filling delay of the optic disc in the arteriovenous phase and diffuse leakage of the optic disc in the late phase (fig . Automated visual fields (humphrey 30 - 2) revealed a generalized constriction of the visual field in the right eye and a normal visual field in the left eye (fig . 3). Optical coherence tomography revealed a prominent thickening of the inferior retinal nerve fiber layer (fig . Laboratory testing revealed a normal blood count, an erythrocyte sedimentation rate of 10 and a normal crp titer . A systemic evaluation was performed by a physician, and there was no evidence of cardiovascular disease, including hypertension, hypotension, diabetes, and hyperlipidemia . A neurologic examination was performed by a neurologist and was normal, and a magnetic resonance image (mri) scan of the brain with gadolinium enhancement was able to demonstrate that the optic nerves appeared normal, and that there were no white matter lesions, which would have been suggestive of demyelinating disease . The patient was counseled to discontinue using udenafil and to regularly monitor his blood pressure . The patient's bcva was improved to 20 / 20, and fundoscopy showed a slightly pale disc one month after he had discontinued the use of udenafil . Aion is the most common acute optic neuropathy in people over the age of 50 . It is generally accepted that the site of the infarction is located in the retrolaminar portion of the optic nerve head, which is supplied by the short posterior ciliary arteries (spca), as this has been demonstrated histopathologically . Doppler studies have also corroborated these findings by showing reduced blood flow in the spca . The location of the impaired blood flow has been further refined by fluorescein and indocyanine green angiographic studies . These studies have demonstrated that delayed optic disc filling occurs without impairment of the choroidal circulation, which suggests that the vasculopathy is located in the para - optic branches of the spca after their branching from the choroidal branches rather than in the short ciliary arteries . Have suggested that nocturnal hypotension may be an important precipitating factor in the pathophysiology of aion . This concept has been demonstrated by monitoring the ambulatory blood pressure over the course of 24 hours, which has shown that nocturnal hypotension may act as the final insult that leads to ischemia and aion in optic nerves that have been previously rendered vulnerable to ischemia by predisposing factors . Beck et al . Have also noted that some discs have certain anatomic features that seem to predispose them to aion . Burde has coined the term " disc at risk " to describe these structurally crowded discs, which are characterized by a small nerve head with a small or absent physiologic cup, abnormal branching of the central vessels, and full nerve fiber bundles that obscure the disc margin . During sexual stimulation, the cavernous nerves release nitric oxide, which induces cyclic guanosine monophosphate (cgmp) formation and leads to smooth muscle relaxation and increased blood flow to the corpus cavernosum . Pde5 is a naturally occurring enzyme that is found in high concentrations in the corporis cavernosum and that breaks down cgmp . Sildenafil citrate selectively inhibits pde5, thus blocking the breakdown of cgmp and facilitating the erectile process . It is hypothesized that the mild hypotensive effect of pde5 inhibitors accentuate physiological nocturnal hypotension, which may result in ischemia of the optic nerve head and compartment syndrome in a susceptible disc . An alternative explanation is that these medications reduce optic nerve head perfusion or disrupt the ability to autoregulate due to the potentiation of nitric oxide . There have been several case reports that have described aion in users of pde5 inhibitors . Egan and pomeranz have reported on sildenafil - associated aion, and cunningham and smith have also reported a case of anterior ischemic optic neuropathy associated with sildenafil (viagra; pfizer, new york, ny, usa). Bollinger and lee have reported a recurrent visual field defect and ischemic optic neuropathy associated with tadalafil rechallenge . There have been no published case reports that have linked an episode of aion to udenafil . The commonly associated systemic diseases include arterial hypertension, diabetes mellitus, ischemic heart disease, hyperlipidemia, cerebrovascular accidents, and arteriosclerosis . Various associated systemic diseases may interfere with the autoregulation of blood flow in the optic nerve head and make the optic nerve head vulnerable to aion . Have reported that hypertension, high cholesterol and decreased cup - to - disc ratio were observed in more than 60% of korean patients with aion and thereby defined these as major risk factors . Have reported that, of the 137 patients identified with aion in their study, the 28 smokers were statistically younger, with a mean age of 51 years, than the 83 nonsmokers, who had a mean age of 64 years . They concluded that cigarette smoking is an important risk factor in the development of aion, and that the cessation of smoking appears to reduce the risk of aion to that of the nonsmoking population . Have also reported that, of the 48 patients with aion in their study, the mean age of the nine smokers, was less than the 50.5 years of the nonsmokers . Of the nine smokers, four had smoking history as the only risk factor for aion . The authors also suggested that smoking is an important risk factor of aion and may be the only risk factor in younger patients . The differential diagnosis of aion included functional visual loss, retinopathy (hypertensive retinopathy, central serous retinopathy, congenital maculopathy, pigmentary reitnaopathy, amelanotic melanoma, and cellophane maculopathy), amblyopia, glaucoma, keratoconus, pseudopapilledema, miscorrected refractive error, leber's congenital amaurosis, and cortical visual loss . Although the funduscopic image and fundus fluorescein angiography were most consistent with aion, the possibility of inflammatory optic neuropathy (optic neuritis) was also considered . The neurologic examination was also normal, and the mri scan of the brain with gadolinium enhancement demonstrated that the optic nerves appeared normal, and the lack of white matter lesions were not suggestive of demyelinating disease . Patients are told to take udenafil approximately 90 to 120 minutes prior to sexual intercourse . Since the patient in this case study took the udenafil at night and then fell asleep after sexual intercourse, he noticed the visual field defect the following morning . We believe that udenafil may have contributed to the episode of aion in this patient . The risk factors of this patient were a decreased cup - to - disc ratio and a smoking history . The udenafil may have sufficiently accentuated his physiologic nocturnal hypotension to decrease the perfusion pressure in the posterior ciliary arteries . This may have resulted in ischemia of the disc that was already predisposed to aion due to the anatomic disc - at - risk configuration . The temporal relationship between the dose of udenafil and the onset of visual field defect make it difficult to accept the notion that these were unrelated coincidental events . During the evaluation of patients with signs and symptoms of aion, patients may be reluctant to inform their doctors about their use of udenafil as a result of the stigma that is associated with erectile dysfunction.
Type 1 diabetes mellitus (t1 dm) is one of the most prevalent endocrine disorders among children worldwide; annually an estimated of 65,000 children develop the disease and its incidence is increasing 3% each year (1). Between 10 to 70% of these children present in diabetic ketoacidosis (dka) as their first presentation of the disease (1). In addition to its life threatening aspects, dka is associated with a heavy financial burden; it accounts for more than 500,000 hospital days per year with an estimated annual cost of 2.4 billion usd (2). Some studies have shown a higher prevalence of autoimmune disorders among patients with diabetes mellitus which might increase their chance of developing dka as the first manifestation of their disease (3). Hypothyroidism is prevalent among pediatric patients with t1 dm; while the reported prevalence of hypothyroidism among general pediatric population is 0.1 to 2% (4, 5), the prevalence of hypothyroidism in patients with t1 dm is much higher ranging from 3 to 30% (6, 7). This could be due to the shared autoimmune disposition for both t1 dm and hypothyroidism; recent studies have identified some shared genes involved in the susceptibility for both conditions (7, 8). Additionally hypothyroidism and metabolic derangement in patients with t1 dm might form a vicious cycle aggravating disease severity; hypothyroidism have been shown to increase insulin resistance and impair glucose metabolism (8, 9). While metabolic derangement have been shown to depress pituitary thyroid axis impairing thyroid function (9). Given the high prevalence of hypothyroidism among pediatric patients with t1 dm and the increased insulin resistance caused by hypothyroidism state and the increased risk of developing dka as the first manifestation of diabetes in individuals who have other autoimmune disorders (3, 6), we postulate that children with t1 dm who have simultaneous hypothyroidism might have a more aggressive form of the disease requiring tighter control and also might have higher chance of developing dka at initial diagnosis . Searching the literature we found a considerable gap in this area of research; we could not find enough studies searching for the relationship between hypothyroidism and the severity of t1 dm, the hemoglobin a1c (hba1c) levels, and the required insulin doses to control the disease . Whether pediatric patients with both t1 dm and hypothyroidism have more aggressive onset of the disease (dka) is not clearly identified . Furthermore it needs to be identified whether the family history of diabetes or hypothyroidism could affect the presence of both conditions in their affected children . We therefore conducted this study to assess the prevalence of hypothyroidism among pediatric patients with t1 dm in a developing country where the rate of consanguineous marriage is very high and to evaluate whether the presence of hypothyroidism has any associations with the severity of t1 dm as reflected in the required insulin dose, age at initial diagnosis, hba1c levels, and the occurrence of dka as the first manifestations of the disease . We studied the pediatric patients with t1 dm who were referred to the diabetes clinic of our institute, from january 2013 through january 2015 in a hospital based retrospective cohort study . The patients were considered eligible if they had less than 18 yr of age, had a documented t1 dm diagnosis based on the american diabetes association (ada) criteria and had not been on any thyroid medication . A total of 357 patients were eligible to participate in the study, and 27 were excluded due to the following criteria: age more than 18 yr, missing the medical records including the records on the first presentation of the disease and missing an informed consent . A total of 330 children aged from 2 to 18 yr completed this study which was approved by the research deputy and the ethics committee of our institute . After explaining the whole process an informed written consent was obtained from all the parents and when applicable from the patients who accepted to participate in the study . The patients were visited in a separate appointment and a questionnaire was completed for them through medical interviews and investigating their medical records . Then a blood sample was obtained from patients to measure their thyroid stimulating hormone (tsh), free thyroxin (t4), triiodothyronine (t3), anti - thyroid peroxidase (anti - tpo) antibodies, fasting blood glucose (fbs), and glycated hemoglobin (hba1c) levels . The following information was retrieved from the patients medical records: age at diagnosis of t1 dm, occurrence of dka as the first clinical manifestation of the disease, the duration of t1 dm, the required insulin dose, and family history of autoimmune diseases in the first degree relatives . Dka was diagnosed based on the following criteria: hyperglycemia over 250 mg / dl, plasma ph less than 7.3, plasma hco3 less than 15 meq / l and the presence of ketonuria . Clinical hypothyroidism was diagnosed based on the increased tsh level and decreased free t4 and/or t3 levels according to the normal range in different pediatric age groups (table1table 1.reference range for the thyroid function tests in different pediatric age groups) (10). Patients were divided into two groups, based on thyroid function tests; patients with t1 dm and hypothyroidism and patients with t1 dm and normal thyroid function . The hba1c levels, required insulin dose, presence of thyroid antibodies, age at initial diagnosis of t1 dm, the occurrence of dka at initial diagnosis and demographics were compared between the two study groups . All statistical analyses were performed using spss statistical software (version 18.0.0: pasw, chicago, il). Chi - squared analysis, fisher s exact test, independent - samples t test and multivariate logistic regression were used to for statistical analysis of the study . Sample size was calculated for a power of 80% and an alpha error of 0.05 where at least 28 persons would be required in each group to detect a difference in presenting with dka at initial diagnosis of t1 dm . Estimated odds ratios (ors) with 95% confidence intervals (95% cis) and p value were used to evaluate the statistical significant . We studied the pediatric patients with t1 dm who were referred to the diabetes clinic of our institute, from january 2013 through january 2015 in a hospital based retrospective cohort study . The patients were considered eligible if they had less than 18 yr of age, had a documented t1 dm diagnosis based on the american diabetes association (ada) criteria and had not been on any thyroid medication . A total of 357 patients were eligible to participate in the study, and 27 were excluded due to the following criteria: age more than 18 yr, missing the medical records including the records on the first presentation of the disease and missing an informed consent . A total of 330 children aged from 2 to 18 yr completed this study which was approved by the research deputy and the ethics committee of our institute . After explaining the whole process an informed written consent was obtained from all the parents and when applicable from the patients who accepted to participate in the study . The patients were visited in a separate appointment and a questionnaire was completed for them through medical interviews and investigating their medical records . Then a blood sample was obtained from patients to measure their thyroid stimulating hormone (tsh), free thyroxin (t4), triiodothyronine (t3), anti - thyroid peroxidase (anti - tpo) antibodies, fasting blood glucose (fbs), and glycated hemoglobin (hba1c) levels . The following information was retrieved from the patients medical records: age at diagnosis of t1 dm, occurrence of dka as the first clinical manifestation of the disease, the duration of t1 dm, the required insulin dose, and family history of autoimmune diseases in the first degree relatives . Dka was diagnosed based on the following criteria: hyperglycemia over 250 mg / dl, plasma ph less than 7.3, plasma hco3 less than 15 meq / l and the presence of ketonuria . Clinical hypothyroidism was diagnosed based on the increased tsh level and decreased free t4 and/or t3 levels according to the normal range in different pediatric age groups (table1table 1.reference range for the thyroid function tests in different pediatric age groups) (10). Patients were divided into two groups, based on thyroid function tests; patients with t1 dm and hypothyroidism and patients with t1 dm and normal thyroid function . The hba1c levels, required insulin dose, presence of thyroid antibodies, age at initial diagnosis of t1 dm, the occurrence of dka at initial diagnosis and demographics were compared between the two study groups . All statistical analyses were performed using spss statistical software (version 18.0.0: pasw, chicago, il). Chi - squared analysis, fisher s exact test, independent - samples t test and multivariate logistic regression were used to for statistical analysis of the study . Sample size was calculated for a power of 80% and an alpha error of 0.05 where at least 28 persons would be required in each group to detect a difference in presenting with dka at initial diagnosis of t1 dm . Estimated odds ratios (ors) with 95% confidence intervals (95% cis) and this study evaluated 330 children with t1 dm who were referred to our diabetes clinic between 2013 and 2015 . The mean standard deviation (sd) for the patients age was 11.6 2.4 yr; the duration of t1 dm was 2.2 1.2 ys; the age at diagnosing t1 dm was 9.1 2.3 yr; the required insulin dose was 0.83 0.23 international unit (iu)/kg / d; the hba1c level was 8.8 1.9 . 193 patients (58.4%) were female with a male: female ratio of 1:1.4; 32 patients (9.6%) had hypothyroidism; 63 patient (19%) had positive anti - tpo antibodies; in 123 children (37.2%) dka was the first clinical presentation of t1 dm; parents consanguinity was observed in 86 patients (26%); 55 children (16.6%) had a family history of diabetes mellitus in their first degree relatives; 39 children (11.8%) had a family history of hypothyroidism in their first degree relatives . Children with t1 dm and hypothyroidism tended to have a higher rate of consanguinity in their parents (43.7 vs. 24.1%, p = 0.01), and a higher rate of diabetes mellitus in their first degree relatives (31.2 vs. 15.1%, p = 0.02) compared to children with t1 dm who had normal thyroid function, while the family history of hypothyroidism was not significantly different between the two groups (18.7 vs. 11%, p = 0.2) (table 2table 2.comparison of the patients demographics between diabetic patients with and without hypothyroidism). Patients with t1 dm and hypothyroidism had significantly higher rates of dka at initial diagnosis (62.5 vs. 34.5%, p = 0.002), younger age at initial diagnosis (p = 0.04), higher rates of positive anti - tpo antibodies (75 vs. 13%, p <0.001), and higher levels of hba1c upon enrolment in the study (p = 0.02) compared to patients with t1 dm who had normal thyroid function . Patients with hypothyroidism also required higher doses of insulin to control their disease (p = 0.03) (table 3table 3.comparison of the disease severity between diabetic patients with and without hypothyroidism). We used logistic regression model to adjust the results for patients sex, age at diagnosis, positive anti - tpo antibodies, parents consanguinity and family history of diabetes mellitus; after these adjustments hypothyroidism remained significantly associated with the occurrence of dka as the initial manifestation of the disease in patients with t1 dm (b = 1.1, p = 0.004, or = 3.03, 95%ci = 1.416.48) (table 4table 4.logistic regression analysis of the factors associated with the occurrence of dka at the initial diagnosis of t1 dm). In this study hypothyroidism was detected in 9.6% of children who suffer from t1 dm and was associated with a more aggressive disease; in comparison to children with t1 dm who had normal thyroid function, children who had both t1 dm and hypothyroidism had significantly higher hba1c levels at enrolment, required higher insulin doses to control their disease, their disease tended to present at younger ages and most of them experienced dka as the first presentation of their disease . Additionally children with t1 dm and hypothyroidism had significantly higher rates of anti - tpo antibodies, consanguinity among their parents and higher rates of diabetes in their first - degree relatives . In the current study hypothyroidism was a prevalent problem among children with t1 dm, complicating 9.6% of these children . This was in accordance with other studies; based on a 2010 review 38% of pediatric patients with t1 dm have been reported to develop autoimmune hypothyroidism (6). While a recently published review and a meta - analysis reported a much higher rate of 730% for the prevalence of hypothyroidism in patients with t1 dm (7, 11). The heterogeneity in the reported prevalence of hypothyroidism in these studies could be due to the variable population characteristics including age and ethnicity of patients, the differences in study design including the cut - off levels and classification of the disease with different definitions including autoimmune, subclinical, and clinical hypothyroidism (6, 7, 11). In our study more than 70% of the patients with t1 dm and hypothyroidism had positive anti - tpo antibodies, in addition parental consanguinity and the presence of diabetes mellitus in the first - degree relatives were significantly higher among patients with both t1 dm and hypothyroidism, compared to patients with t1 dm but without hypothyroidism . These results strongly support the role of autoimmunity and the shared genetic susceptibility in the pathogenesis of hypothyroidism in patients with t1 dm . Recent studies have revealed some genes that might be responsible for the joint susceptibility to t1 dm and autoimmune thyroid dysfunction (8); hla class ii loci, ctla4, ins (12, 13, 15), ptpn22 (14, 15), and foxp3 (15), are among the identified genes . These genes have been recognized as the key role players in the regulation of the immune response (15,16,17,18); these genes are involved in the differentiation, regulation, activation and function of regulatory t - cells (15,16,17,18), and their polymorphism have been linked to a number of autoimmune diseases including t1 dm and autoimmune thyroid dysfunction (12,13,14,15,16,17,18). In this study hypothyroidism was associated with a more aggressive disease in pediatric patients with t1 dm; patients with t1 dm and hypothyroidism had higher rates of dka at presentation, were younger at the onset of the disease, had higher hba1c levels and required higher insulin doses for the control of the disease . Documented the association of severe metabolic derangement and impaired thyroid function in patients with newly diagnosed t1 dm (9). Lin et al . Also showed a significantly lower levels of t3, t4 and free t4 among children newly diagnosed t1 dm who presented with dka compared to patients without dka at initial diagnosis (19). The association of thyroid dysfunction with the disease severity in t1 dm patients could be explained through several mechanisms; our results supported by the aforementioned studies (12,13,14,15,16,17,18), document that the occurrence of both t1 dm and hypothyroidism might show the presence of polymorphism in some of the immune response regulatory genes (12,13,14,15,16,17,18), causing a more severe disease with stronger features of autoimmunity (15,16,17,18). Another mechanism might be through the effect of hypothyroidism on increasing insulin resistance and glucose metabolism(8, 19,20,21,22); thyroid hormones stimulate glucose uptake into peripheral tissues and enhance an additive effect on insulin action on glucose transport into cells (19,20,21,22). Therefore in hypothyroid patients insulin - dependent glucose clearance could get defected (19,20,21,22), putting diabetic patients with hypothyroidism at significantly higher risk of experiencing acute and possibly chronic complications of diabetes (8). Furthermore metabolic derangement and poor diabetic control for a long time might cause complete depression of hypothalamus - pituitary - thyroid axis causing clinical hypothyroidism (9), which in turn aggravates insulin resistance, thus might form a vicious cycle resulting in a more aggressive disease . This study is among the rare studies that evaluate the clinical severity, insulin requirement and hba1c levels in pediatric patients with t1 dm who have hypothyroidism . However our study has some limitations that should be considered when interpreting the results; we do not have information regarding thyroid function at the onset of t1 dm therefore it is not clear whether thyroid dysfunction developed though the course of the disease or it was present at the initial presentation of t1 dm . Another limitation was that our patients were not followed to evaluate whether treating hypothyroidism could improve the severity of diabetes and lower the required insulin dose . Additionally in our study thyroid scan and anti - thyroglobulin antibodies were not evaluated, this might have caused an underestimation of the presence of autoimmune thyroiditis among the patients with t1 dm . Prospective and interventional studies are required to evaluate whether treating hypothyroidism could alleviate the severity of diabetes and lower the required insulin dose in patients with concurrent t1 dm and hypothyroidism, and to answer whether controlling diabetes could result in improvement of thyroid function in these patients . Hypothyroidism is prevalent among pediatric patients with t1 dm and is associated with a more aggressive form of the disease . Patients with t1 dm and hypothyroidism have higher rates of dka, develop the disease at younger ages, and require higher insulin doses . Therefore all patients with t1 dm especially those with poorly controlled disease and those with positive family history for diabetes should be screened for thyroid dysfunction . Interventional studies are required to evaluate the efficacy of treating thyroid dysfunction in these patients and to assess if hormone therapy in these patients could help in a better management of diabetes and reduce its complications.
Antiphospholipid syndrome (aps) in pregnancy may trigger the life - threatening catastrophic antiphospholipid syndrome (caps). Complement activation is implicated in the pathogenesis, and inhibition of complement factor c5 is suggested as an additional treatment option . We present a pregnant patient treated with the c5-inhibitor eculizumab due to high risk of developing devastating aps - related complications . The complement inhibitory effects of the treatment were examined both in the patient and the premature infant . Complement activity in the mother recovered considerably faster than anticipated; however, no new thrombosis or caps developed during the last week of pregnancy or postpartum . Blood sampling from the umbilical vein and artery, and from the infant after delivery showed low complement activity; however, only 0.3% of the eculizumab concentration detected in the mother, consistent with low placental passage of eculizumab . The data underscore the importance of close monitoring of complement inhibition and individualizing dosage regimens in pregnant patients receiving eculizumab . We document how traditional functional complement activity tests cannot assess the effect of eculizumab in premature infants due to the very low levels of complement factors detected in this infant born in gestational week 33 . Complement c5 inhibition might be a safe candidate treatment option for aps during pregnancy and delivery, and additionally, enables prolongation of pregnancy with important weeks . Antiphospholipid syndrome (aps) is characterized by arterial, venous, or small - vessel thrombosis and/or pregnancy morbidity in the presence of persistent antiphospholipid antibodies (anticardiolipin antibodies, antibeta2 glycoprotein 1 antibodies, and lupus anticoagulant). Although the pathogenesis is not fully understood, the binding of antiphospholipid antibodies to 2 beta2 glycoprotein 1 promotes endothelial cell activation determined by upregulation of adhesion molecules, tissue factor, and production and secretion of proinflammatory cytokines, which enhance the risk of thrombosis formation . Complement appears to play a significant role in the pathophysiology based on both in vitro and in vivo studies . Catastrophic aps (caps), although rare, is a devastating and life - threatening syndrome featured by multiorgan thrombosis . Current treatment options in addition to anticoagulation are glucocorticoids, plasma exchange, or intravenous immunoglobulins; however, case reports have reported that inhibition of complement may be lifesaving . A 22-year - old primigravida was admitted to hospital in the 2nd trimester with painful ulcerations of ischemic origin in her right leg . Barely 14 years old, she developed her 1st episode of lower limb arterial thrombosis which was treated with bypass grafting and digital amputations . No arteriosclerosis or vasculitis was detected and she was diagnosed with aps, fulfilling the sydney criteria with persistent triple positive antiphospholipid antibodies: anticardiolipin immunoglobulin g (igg) 205gpl - u / l (ref <10 gpl - u / l), antibeta 2 glycoprotein 1 igg 125 u / ml (ref <10 u / ml), and positive lupus anticoagulant 2.41 (ref <1.3 silica clotting time). A recurrent episode of thrombosis was treated with percutaneous transluminal angioplasty, and an episode of microemboli resolved with intensified anticoagulant treatment . In conjunction with pregnancy, warfarin was substituted with low molecular weight heparin adjusted up to 10,000 iu twice daily (antifactor xa levels of 0.91.1 iu / ml) and low dose aspirin (75 mg daily). Ischemia was treated conservatively with analgesia in addition to anticoagulation therapy, and pregnancy was monitored by regular ultrasounds following fetal growth and placental function . Based on her multiple previous arterial thromboses and ongoing ischemia during pregnancy, the risk of developing caps in relation to pregnancy, delivery, and puerperium was considered significant . Ruffatti et al published data suggesting that addition of 2nd - line therapy increases live - birth rates in high risk pregnant patients with aps, although no guidelines are currently available on the ideal treatment strategy . Previous experience with the efficacy of the complement c5 inhibitor eculizumab in treatment of caps and described safety in pregnancy prompted the choice of eculizumab . Thus, 600 mg of eculizumab was administered 8 days before delivery (day 0) in addition to prophylactic antibiotics . Serum (prepared by drawing whole blood into empty tubes, left for clotting 60 minutes followed by centrifugation 15 minutes, 3500 g, 4 c) and ethylenediaminetetraacetic acid (edta) plasma (prepared by drawing blood into k2edta tubes, followed by immediate centrifugation 15 minutes, 3500 g, 4 c) samples were obtained from the patient before and at several time points after eculizumab administration and analyzed directly or stored at 70 c . Complement activity in plasma (total complement system screen, wieslab, malmo, sweden) decreased to zero after the 1st eculizumab infusion and remained low at day 2, however had returned to normal levels already by day 7 (fig . Eculizumab - c5 (e - c5) complexes in serum (enzyme immunoassay as described in ref) increased from zero to 67 after the 1st eculizumab dose (fig . Interestingly, the patient reported decreased ischemic pain following the 1st dose of eculizumab, and opioid analgesia was successfully reduced . Complement activity and e - c5 complexes in a pregnant patient with aps and the newborn infant . (a) the patient received eculizumab 600 mg day 0 and 7 and a caesarean section was performed on day 8 . Effect of eculizumab on complement functional activity was measured as a common readout (c5b-9 formation) for the cp, lp, and ap, by elisa in patient serum obtained before and repeatedly after the administration of eculizumab . E - c5 complexes were measured by elisa at day 0, 2, and 8 in the patient serum before and after administration of eculizumab . Complement activity was completely abolished by eculizumab 600 mg, however normalized within 7 days . Consistently, e - c5 complexes showed an inverse pattern with high levels following eculizumab administration . (b) the infant's e - c5 complexes were measured by elisa in infant serum (left column) and subsequently after in vitro challenge with purified complement protein c5 (50 g / ml) (middle column) and eculizumab (100 g / ml) (right column). The increased e - c5 complex formation following challenge with eculizumab, but not c5, is consistent with the presence of free c5 in infant serum and negligible levels of eculizumab . Ap = alternative pathway, aps = antiphospholipid syndrome, caps = catastrophic antiphospholipid syndrome, cp = classical pathway, e - c5 = eculizumab - c5, edta = ethylenediaminetetraacetic acid, elisa = enzyme - linked immunosorbent assay, lp = lectin pathway . A 2nd dose of eculizumab 600 mg was infused on day 7, and a caesarean section was performed the following day (day 8) in gestational week 32 + 4, resulting in delivery of a healthy infant (apgar 8/9/9) with a normal weight (1875 g) for gestational age . Serum and edta plasma samples were obtained from the umbilical cord artery and vein by careful needle puncture to avoid contamination from mothers blood and wharton jelly, directly after cord clamping during caesarean section . In addition, edta - plasma was obtained from the infant 2 hours after delivery . The infant's total complement activity was <1%, however consistent with the measured low complement protein concentrations (standard routine immunochemical methods, department of clinical immunology and transfusion medicine, lund, sweden) as expected at gestational week 32 (table 1). Umbilical and infant e - c5 complexes were 0.3 g / ml . Concomitant patient e - c5 complexes were 98 g / ml consistent with a 0.3% placental passage of eculizumab (table 1). To further support the findings of low eculizumab passage, we added eculizumab (final concentration of 100 g / ml) or purified c5 (50 g / ml), respectively, to the infant serum in vitro and subsequently analyzed for e - c5 complexes . Eculizumab, but not c5, increased the e - c5 complex formation in infant serum substantially (fig . 1b), consistent with the presence of free c5 (table 1) and negligible amounts of eculizumab . Complement protein levels, complement activity, and eculizumab - c5 complexes in samples from a pregnant eculizumab - treated patient with antiphospholipid syndrome before delivery and from the patient and newborn infant immediately after delivery . Close monitoring of the patient's complement activity after the 2nd dose revealed increased activity already after 3 days and again normal activity within 1 week (fig . The patient did not develop new thrombosis or caps during the last week of pregnancy or postpartum . The intervention given (eculizumab) was part of normal health care and thus ethical approval was neither obliged nor sought . However, written consent was obtained from the patient in order to publish the case report . A history of thrombosis and triple positive antiphospholipid antibodies, as seen in the present patient, is associated with a higher risk of maternal and fetal complications in pregnant women with aps . Our patient demonstrated multiple risk factors and showed clinical signs of thrombosis despite intensive anticoagulant treatment . Additionally, the risk of peri- and postoperative hemorrhage due to intense anticoagulant treatment was high, while on the other hand, surgery and cessation of anticoagulant treatment would increase the risk of triggering caps . Eculizumab has been suggested in several recent reports as a 2nd - line treatment option in aps and caps . Hence, a short term course of eculizumab was chosen before cesarean section to reduce the risk of caps triggered by delivery and puerperium . Complement activity surprizingly increased to normal levels within 7 days after both doses of eculizumab despite complete inhibition of complement activity after each infusion . Others have observed a similar requirement of an increased eculizumab dosage regimen as pregnancy proceeds . However, pregnancy - induced changes of the normal physiology may influence the pharmacokinetics and pharmacodynamics of eculizumab, underscoring the importance of individual treatment monitoring . Data from pregnancies in patients with paroxysmal nocturnal hemoglobinuria treated with eculizumab suggest that the treatment is safe . We examined both arterial and venous blood samples from the umbilical cord, as well as a blood sample from the infant 2 hours after delivery . All 3 samples showed similar results, suggesting that virtually no eculizumab (approximately 0.3%) had passed the placenta . To our knowledge the amount of eculizumab has previously not been examined in preterm newborns at this stage of gestation . Collectively, these data indicate that the igg subclass 2/4 chimeric structure of eculizumab largely prevents the molecule from passing the placental barrier . The observed complement activity of less than 1% in the infant could have been misinterpreted as an eculizumab effect . Eculizumab blocks cleavage of c5 and thus prevents assembly of the terminal c5b-9 complement complex detected in the complement function enzyme - linked immunosorbent assay (elisa). However, a normal complement activity test requires adequate levels of complement proteins to produce sufficient amounts of tcc for assay detection . Premature infants have low levels of most complement proteins, as did the infant in this report . The detection of merely trace levels of e - c5 complexes in the infant samples and the results from the in vitro assay demonstrating that only the addition of eculizumab, but not purified c5, could increase the e - c5 complex formation in infant serum, indicate that the low complement activity resulted from the low levels of most of the complement proteins (eg, c4, c3, c5, c6, and c8), and was not due to the presence of eculizumab . Consequently, abnormal results in complement activity assays, such as in premature infants, must be carefully interpreted to avoid incorrect conclusions . In conclusion, these results add up to the increasing body of literature stating that eculizumab treatment can be considered safe in pregnancy as negligible amounts pass over the placental barrier . Our data suggest complement inhibition as a treatment option to safely prolong pregnancy and reduce the risk of caps triggered by pregnancy, surgery, and puerperium without affecting the infant . In addition, we stress the importance of monitoring treatment effects of eculizumab using reliable methods to ensure individual adequate complement inhibition.
Uveitis is inflammation of the middle - lining layer of the eye, comprising the iris, ciliary body, and choroid . It may involve other adjacent tissues, such as the retina, optic nerve, and vitreous humor . It accounts for up to 10% of legal blindness in united states and about 25% in the developing countries [2, 3]. This disease is considered to affect young patients with median age at presentation in the third and fourth decade . This impression is based on epidemiology studies published in the 1960s, which demonstrated that uveitis occurs mainly in young adults at 20 to 50 years of age . On the other hand, more recent reports from the united states showed significant increase in number of uveitis cases among elderly patients [4, 5]. According to world population aging report (2009), elderly or aged people are persons of 60 years of age or more . Globally the population of older persons is growing considerably faster than the population as a whole, and it is expected to continue growing more rapidly than other age groups at least till 2050 . Taking that into consideration, our knowledge of the prevalence of uveitis and its common types among elderly patients is essential to formulate and evaluate goals and programs and to enhance our understanding of this disease and prevent its long term complications . Furthermore, we aim to discuss the pattern of presentation and diagnosis among elderly patients with uveitis and analyze any change over the past 5 decades . We also aim to delineate a thorough differential diagnosis of de novo uveitis in the elderly . We performed an extensive literature search using the medline database, from 1964 to 2014 . The search subject included uveitis: epidemiology, uveitis among elderly, etiology, classification, and diagnosis . The search was limited to the literature pertaining to humans, with no language limitation . An arbitrary method was used to select the studies to be included in our review, the emphasis being placed on obtaining studies that were representative of each region in different time era . Data pertaining to age, gender, location of uveitis, chronicity, and diagnosis were extracted and analyzed in accordance with the international uveitis study group (iusg) recommendations . Data from studies including adequate patient numbers spanning 5 decades to date was evaluated and analyzed . Uveitis diagnosis among elderly patients older than 60 years of age was considered uncommon . The previous impression about the common age of presentation in uveitis darrell et al . In 1962 demonstrated that only 14% of uveitis patients were considered elderly (> 60 years of age). However, this trend is changing especially in the developed countries in the last two decades [4, 5]. Reviewing the published uveitis epidemiology studies in the last 50 years, we find that the number of published reports has almost doubled after the year 2000 (15 reports published between 1960 and 1999 [1, 820] versus 26 reports published after 2000 [4, 5, 16, 2043]). Specifically, much more reports have been published from developing countries after the year 2000 (3 reports were published prior to 2000 [10, 15, 44] compared to 12 reports after 2000 [3, 21, 22, 2426, 30, 32, 34, 35, 39, 41]). Studying the total data from studies in the past 5 decades revealed that mean age at presentation is 38.0 5.0 (range: 29.046.5) (table 1). Comparing the mean age at presentation between developed versus developing countries shows that mean age at presentation in developed countries is more than in developing countries: 40.6 4.7 (range: 33.846.1) versus 34.4 2.7 (range: 29.039.9), respectively . If we compare data published before year 2000 to that published after, we find that there is no difference in the mean age at presentation (38.3 versus 37.8) (table 1). Also, comparing the mean proportion of elderly patients among total uveitis patients between developed versus developing countries revealed that this group of patients occupies a large number of patients in most of the epidemiology studies in developed countries with mean percentile of 18.6% 5.7 (range: 13.629.9%). However, the mean percentile of elderly patients among uveitis patients in developing countries is limited to 7.2% 2.2 (range: 3.010.2%) only . The percentile of elderly patients increased in reports from developing countries from 5.7 prior to year 2000 to 8.4% after . This discrepancy in the mean percentile of elderly patients among total uveitis patients between developed and developing countries may have two potential explanations . First, most of the developed countries are considered according to the latest who report as aging countries with an increasing proportion of elderly population . Second, with an increasing awareness of the recommended screening tests and an improvement in the health care systems in the developed countries, more patients are expected to be examined and to be followed up in these countries including elderly patients . Most of the major uveitis epidemiology studies demonstrated no gender preference or slight preference toward females . However, reviewing the data of eight epidemiology studies reporting the gender preference among elderly patients revealed that uveitis among elderly females is significantly more than elderly males (f: m = 2.0) (p = 0.021) [11, 12, 14, 27, 28, 38, 42, 45]. Generally, the most common location of uveitis worldwide is anterior uveitis . Similarly, most of the uveitis epidemiology studies in the elderly reported anterior uveitis as the most common location of this disease at presentation (507 out of 823 cases), followed by panuveitis (129 out of 823 cases) and posterior uveitis (112 out of 823 cases) (table 3). Also, the most common diagnosis of uveitis among elderly was reported as idiopathic uveitis in five out of ten reports . However, acute anterior uveitis was found to be the most common diagnosis in three studies . In addition, herpetic induced uveitis was reported as the second or the third most common cause of uveitis in elderly in seven out of ten studies . Most of the reviewed epidemiology studies based their diagnosis of hsv, vzv, and cmv anterior uveitis on careful ocular and medical history in combination with positive antibody titers or dna detection in the intraocular fluid using pcr method [17, 21, 25, 34, 42, 43]. However, two studies based their diagnosis of herpetic and cmv uveitis on the clinical findings only [26, 39]. Ocular tuberculosis, toxoplasmosis, birdshot, and lymphoma were all reported in the elderly population after the year 2000 . That might be due to the increased reporting from developing countries after the turn of the century or to increased awareness and advances in diagnostic modalities of some conditions such as ocular tuberculosis and lymphoma (table 4). Interestingly, none of the available epidemiology studies found that masquerades or neoplasm was a common cause of uveitis among elderly patients . In addition, only 4 of the reviewed papers reported neoplasms as a cause of uveitis in this age group (12 out of 261 cases) [10, 15, 18, 20]. Primary or metastatic ocular lymphoma can present with wide spectrum of age distribution, including young patients [4649]. Actually, our current epidemiology studies demonstrated that ocular lymphoma was one of the common etiologies of ocular inflammation in only one study . As the disease progresses, it can mimic the inflammation of uveitis and is often inappropriately treated with corticosteroids . In primacy intraocular lymphoma, definitive diagnosis requires identification of malignant lymphoid cells from ocular tissue or csf . Several techniques exist to obtain the required tissue, including aqueous aspiration, diagnostic vitrectomy, and diagnostic retinal or choroidal biopsy [4649]. Only one study reported the role of diagnostic pars plana vitrectomy (ppv) in our currently reviewed epidemiology reports . . Showed that, of 19 cases that underwent diagnostic ppv, 2 cases had ocular lymphoma and 3 cases were diagnosed with intraocular infection . In addition, many reports previously demonstrated the importance of diagnostic ppv in cases of uveitis with unknown etiology . It was shown that ppv is a helpful tool with diagnostic yield ranging from 14.3% to 61.5% of uveitis with unknown causes [5058]. Similarly to the younger patients, autoimmune diseases like sarcoidosis, inflammatory bowel diseases, and insulin dependent diabetes mellitus were reported as common comorbidities among the elderly patients with uveitis . Noninsulin dependent diabetes mellitus was listed by some reports as a common comorbidity among elderly patients with uveitis [17, 20]. However, further data analysis by chatzistefanou et al . Failed to reproduce any specific correlation between diabetes type ii and uveitis among the elderly in their study . Uveitis is an inflammatory process affecting one or more of the eye globe layers . Understanding the mechanism of work of the immune system and the changes associated with age is key to explaining the differences observed in uveitis demographics among the elderly patients . It is known that the elements of the innate and the acquired immune system undergo changes with age . This process is labeled immunosenescence . T - cells among other elements of the immune system are believed to play a major role in ocular inflammatory processes . In particular, th1 mediated response by t - cells gets altered with age secondary to irregular cell - cell interactions that take place at many levels including the antigen presenting cells (apc) [2, 59]. This explains the increased rate of infection by certain pathogens in the elderly, like influenza, herpes, and tuberculosis [60, 61]. As for the paradoxical increase in the number of antibodies produced by b - cells, the weaker immune system among the elderly renders them more prone to develop uveitis secondary to infectious causes . This was obviously translated in our paper by an increase in the proportion of patients diagnosed with herpetic uveitis . It should be noted also that a weak immune reaction in the elderly alters the classical clinical presentation of a disease . Similarly, endophthalmitis in elderly might not develop a severe inflammatory reaction except late in the course of the disease . In our study, we have found that the proportion of elderly (aging 60 years or more) among the uveitis patients is more in the developed countries (18.6%) compared to the developing countries (7.2%). This observation can be mainly explained by the increasingly aging populations of the developed countries compared to the younger societies in the developing countries . Even with the increased reporting from the developing countries after the year of 2000, the proportion of the elderly among the uveitis patients in these areas did not exceed 8.5% . This suggests that the fewer number of reports published in the developing countries is not responsible for the difference mentioned above between the developing countries and the developed countries . Extrapolating from the bigger proportion of elderly among the uveitis patients in the developed countries, it is expected that the mean age at presentation of patients with uveitis should be more in those countries compared to the developing countries . In our paper, we found a mean age of presentation of 40.6 in the developed countries, compared to 34.4 in the developing countries . In our paper, we report that de novo uveitis attacks among the elderly have a female predominance . However female predominance was not always reproducible in all the reports included in our review . It should be noted however that masquerades occur more frequently among the elderly population and in case they are suspected, an extensive workup is sometimes required in order to rule them out including ppv and tissue analysis . Uveitis affects all age groups, but the differential diagnosis steers toward specific entities with each age category . Seronegative spondyloarthropathies very rarely manifest as uveitis de novo in elderly patients . On the other hand, masquerades are more common among the elderly and high level of suspicion should be kept in mind while ruling them out . Location of uveitis is the main subcategory used to differentiate the major entities of uveitis . Clinicopathologic picture, onset and course of the disease, signs and symptoms, and review of system are other useful categories that are employed to narrow down the differential diagnosis . Segregation was based on the onset of the disease, presence or absence of granulomatous reactions, and some distinguishing feature . Special attention should be given to ischemic syndrome secondary to carotid disease in the elderly . The altered immunologic status of the elderly, generally characterized by a relative cellular immune deficiency, may mask some of the clinical findings warranting definitive diagnostic workup in suspected cases of hsv, vzv, and cmv uveitis using viral pcr of the aqueous humor tap . It should be noted that pars planitis and multiple sclerosis are not part of the differential listed because these entities are rarely reported in patients older than 60 years of age . It is also worth noting that lens / iol - induced uveitis is much more common in the elderly cataractous lens . Fuch's heterochromic iridocyclitis and lens - induced uveitis may present as intermediate or as panuveitis . Figure 3 delineates the entities that present as de novo posterior uveitis in the elderly . Many of them are characterized by an extensive inflammation and may present as panuveitis like sympathetic ophthalmia and birdshot chorioretinopathy . Of particular interest, one must keep in mind that immunocompromised elderly have a weaker immune system and hence a milder form of inflammation should be anticipated . Sarcoidosis, tuberculosis, and syphilis can mimic any form of uveitis, including panuveitis (figures 13). Furthermore, in elderly patients with altered immunologic status which may mask some of the clinical findings and with the availability of the less invasive 25-gauge vitrectomy, diagnostic ppv may be very helpful in determining the unknown etiology of uveitis in the elderly particularly ruling out infections or cancers . As in uveitis of any age, treatment is directed to the specific etiology . In case of an infectious etiology, specific anti - infectious medications are given according to the specific organism . While in cases of inflammatory / autoimmune uveitis, treatment with corticosteroids and immune suppressive therapy is instituted . On the other hand, malignant conditions such as lymphoma are treated with local / systemic chemotherapy . However, care should be taken in this elderly population to monitor for drug side effects especially hepatic and renal toxicity and bone marrow suppression, as this age group tends to have other comorbidities and chronic diseases such as osteoporosis, diabetes, and hypertension that puts them at added risks of drug induced complications . Furthermore, drug interactions should be taken into account given the higher likelihood in this age group that the patient will be on medications for other diseases . It is more commonly reported in the developing world representing 18.6% of the uveitis population . The percentage of elderly uveitis patients is increasing with recent reports after the year 2000 . Moreover, there is a female preponderance in the elderly uveitis group; however, this ratio is decreasing with recent reports . Anterior uveitis is the most common presentation worldwide and infections such as tuberculosis and toxoplasmosis are increasingly being reported in recent years . Furthermore, neoplasms do not constitute a sizeable proportion of uveitis in the elderly but need to be ruled out as a masquerade syndrome in this age group . The socioeconomic burden of this disease relies on the fact that it affects mainly the population of productive ages (aging 20 to 60). Little is mentioned about uveitis as being a cause of blindness among the elderly . But more recently, it was reported that it is underestimated and that such a rare disease among the elderly has a lot of socioeconomic impact especially with the continuously aging societies in the developed countries.
Following the discovery of the lysosome by the nobel prize winner christian de duve in 1955 (de duve et al . 1955), research into the biochemistry, molecular biology and pathology of the lysosomal system unravelled many of its functions . Nowadays, genetic lysosomal storage disorders (lsds) represent a group of about 50 disorders (scriver et al . All of them are characterized by intralysosomal accumulation of compound(s), e.g., enzyme substrates or products, mishandled bioconjugates, or segregated cytoplasmic components, in a variety of cell types and tissues . Most lsds result from a deficient enzyme activity caused by a mutation in the gene encoding either the relevant enzyme, protecting protein, protein activators, or the endoplasmic reticulum however, some are caused by dysfunction of the lysosomal system itself due to mutations in genes encoding lysosomal membrane components or components closely associated with the lysosomal system (saftig 2005; jalanko and braulke 2009). The number of lysosomal enzymes and non - catalytic proteins reported in the literature has steadily increased and, therefore, more defects in the lysosomal system are expected to be discovered . The progress in the research into limp-2 deficiency serves as a recent example (balreira et al . Overall frequencies of occurrence of lsds have been reported for several countries (meikle et al . 2004), while other reports focused on lsd groups (e.g., mucopolysaccharidoses, (mps): nelson et al . 2005; malm et al . 2008; lipidoses: ozkara and topcu 2004; neuronal ceroid lipofuscinoses, ncl: claussen et al . 1992), or on particular lsds (e.g., mps i: moore et al . The frequency of lsds as a group varies among populations from 7.6 to 25 per 100,000 (meikle et al . The aim of this study was to calculate the birth prevalence and carrier frequencies of lsds in the czech republic population, and to compare our results with reported epidemiologic data from other populations . We analyzed data from individuals who had been diagnosed with a lsd at the institute of inherited metabolic disorders in prague between 1975 and 2008 . Our institute is the only laboratory providing diagnostic testing for lsds, including prenatal testing, in the czech republic . The patients had been referred to, or their blood samples had been sent to, the institute by departments of paediatrics, neurology, haematology, cardiology, nephrology, dermatology, gastroenterology, ophthalmology, pathology, and clinical genetics from hospitals all over the country . The initial diagnosis of most lsds was based on demonstration of accumulated substrates (e.g., sphingolipids, oligosaccharides, or glycosaminoglycans) in tissues and/or body fluids using chromatographic or electrophoretic methods, or on histological proof of storage . The definite diagnosis was made by demonstrating the deficiency of the relevant enzyme and/or presence of pathogenic mutation, and/or by detection of undegraded substrate by loading tests in cell cultures . Mucolipidosis ii was confirmed by detecting elevated activities of multiple lysosomal enzymes in serum and by molecular analysis . Patients with unspecified types of mps or ncl, who had died before the availability of confirmatory tests, were also included in the birth prevalence calculation . Their diagnosis was based on biochemical analyses of storage compound, evaluation of clinical data, and histochemical and/or ultrastructural examinations of samples obtained at biopsy or autopsy . For the calculation of the prevalence the prevalence was calculated as the total number of patients diagnosed with a particular lsd, divided by the total number of live births in the same period, as was the birth period of the diagnosed cases (i.e., the birth period is the interval between the year of the birth of the oldest patient and the year of the birth of the youngest patient) the total number of live births in particular years was calculated using birth rates taken from the czech statistical office web page . To avoid underestimation, some patients were not included into the prevalence calculation (e.g., if there was only one patient diagnosed with the disease, the prevalence was calculated using the number of live births (i.e., 4,261,897) in the examined period (i.e., between 1975 and 2008), as used previously by pinto et al . Familial cases and 11 affected fetuses [fabry disease (1), niemann - pick disease type c (1), mps i (3), mps iii a (3) and mps iv a (3)] were also included in the calculation of birth prevalence . For some specific lsds, the patient group consisted of subgroups with distinct clinical phenotypes (e.g., mld, gaucher disease). Using the method mentioned above, we calculated the prevalence of the subgroups separately, and the overall prevalence of the specific lsd was estimated as a sum of the prevalences in the subgroups (poorthuis et al . Calculation of the frequency of fabry disease was done using two approaches, i.e., excluding and including female heterozygotes . Exclusion of heterozygotes (n = 78) enabled us to compare the prevalence to other reported male - specific prevalence data (meikle et al . 1999; poorthuis et al . 1999; ozkara and topcu 2004; pinto et al . Fabry heterozygotes, who can be as severely affected as male patients, are nowadays classified as patients instead of carriers of the defective gene (dobrovolny et al . 2005; wang et al . 2007; gibas et al . Confidence intervals (95%) for the birth prevalences were estimated from the poisson distribution using sisa (simple interactive statistical analysis) software (uitenbroek 1997). Carrier frequency was calculated using the hardy weinberg equation (strachan and read 2004). In the past three decades, a total of 478 patients have been diagnosed with one of 34 specific types of lsds in the czech republic (fig . 1). The number of diagnosed patients gradually increased during the study period, from less than 10 cases per year in the 1970s to 2030 new cases diagnosed annually in recent years (fig . 2). More than half of all lsd patients had a lipidosis, about one - quarter a mucopolysaccharidosis, and the remaining quarter suffered from ncl, gsd ii, mucolipidoses, or glycoproteinoses (fig . 1). The combined birth prevalence for all lsds is 12.25 (95% ci 6.2524.1) per 100,000 live births . Data on patients with lipidoses, mucopolysaccharidoses, and other lsds detected during the study period, including carrier frequencies in the czech population, are summarized in tables 1, 2 and 3, respectively . Detailed data on the birth prevalence of specific disorders and their subtypes can be found in tables 4, 5 and 6 . Mps mucopolysaccharidosis, ncl neuronal ceroidlipofuscinosis, gsd ii glycogenosis type ii, ml mucolipidosis, gp glycoproteinosis, npa / b / c niemann - pick a / b / c, mld metachromatic leukodystrophy, cesd cholesterol ester storage disease, msd multiple sulphatase deficiency, gm1 gm1 gangliosidosis, gm2 gm2 gangliosidosis (tay - sachs and sandhoff diseases), -man -mannosidosis, -man -mannosidosis, issd infantile sialic acid storage diseasefig . 2number of live births () and number of lsd diagnoses () in the czech population from 1975 to 2008 . In 1985, electrophoretic analysis of glycosaminoglycans (elfo gag) and thin layer chromatography of oligosaccharides (tlc ols) in urine were introduced . From 1990 laboratory diagnostic methods for 45 lsds are available (enzyme and dna analyses, loading assays)table 1lipidoses: comparison of data in different populationsdiseaseczech republicother countriesno . Of patients 10nlportugalaustraliaturkeyprevalence per 100,000 live birthsgaucher (all types)491.130.611.956.711.161.351.750.45niemann - pick a / b230.330.160.613.650.530.60.4niemann - pick c540.910.671.216.020.352.20.47fabry male490.52 (1.0)0.380.690.010.210.120.860.015fabry female780.770.600.98krabbe150.400.210.684.01.351.210.711mld (all types)250.690.291.385.261.421.851.091.43cesd, wolman180.270.150.453.310.19gm1 gangliosidosis130.260.140.463.250.410.620.260.54gm2 tay - sachs100.300.140.553.450.413.130.50.23gm2 sandhoff30.190.040.552.740.341.490.260.95lipidoses all types259/3375.0/5.772.79 (3.39)8.53 (9.51)6.212.66.64.6nl netherlands, mld metachromatic leukodystrophy, cesd cholesteryl ester storage diseasetotal number of patients diagnosed between 1975 and 2008data for prevalence calculations are in table 4carrier per 1 live birthmale live births onlyfabry females not includedfabry females includedtable 2mucopolysaccharidoses: comparison of data in different populationsdiseaseczech republicother countriesno . Of patients 10nlportugalaustraliagermanyprevalence per 100,000 live birthsmps i200.720.441.115.381.191.331.140.69mps ii220.43 (0.83)0.260.650.00850.671.090.740.64mps iii a180.470.270.754.311.1600.88mps iii b10.020.00.130.970.420.720.47mps iii c50.420.140.994.120.210.120.07mps iii d000.10.10mps iii (all types)240.910.411.871.890.841.421.57mps iv a140.710.381.225.350.220.60.59mps iv b10.020.00.130.970.1400mps iv (a + b)150.730.381.350.38mps vi20.050.010.181.400.150.420.430.23mps vii10.020.00.130.970.2400.05mps unspecified320.600.390.87msd30.260.050.773.240.050.480.07mps all types1193.721.946.934.54.84.443.53nl netherlands, mps mucopolysaccharidosis, msd multiple sulphatase deficiencytotal number of patients diagnosed between 1975 and 2008data for prevalence calculations are in table 5carrier per 1 live birthmale live births onlyunspecified cases of mps were diagnosed on the basis of analysis of glycosaminoglycan excreted in urine combined with the evaluation of clinical data . No material was available for enzyme and dna analysistable 3glycoproteinoses, mucolipidoses, glycogenosis type ii and neuronal ceroid lipofuscinoses: comparison of data in different populationsdiseaseczech republicother countriesno . Of patients 19752008prevalence per 100,000poisson 95% confidence intervalcarrier frequency ii / iii30.220.050.652.980.240.810.31mucolipidosis iv10.020.00.130.97glycogenosis type ii (all)120.370.130.813.8620.170.69ncl 120.190.020.702.790.17ncl 2280.360.240.523.790.07ncl 320.270.030.963.270.48ncl 410.020.00.130.97ncl 520.210.030.772.92ncl 610.020.00.130.971.43ncl 7180.850.491.365.81ncl unspecified200.370.220.58ncl (all types)742.291.035.152.14nl netherlands, issd infantile sialic acid storage disease, ncl neuronal ceroid lipofuscinosistotal number of patients diagnosed between 1975 and 2008data for prevalence calculations are in table 6carrier per 1 live birthunspecified ncl were diagnosed on the basis of histochemical and ultrastructural examination of biopsy samples combined with the evaluation of clinical data . No material was available for enzyme and dna analysistable 4lipidoses: data for calculation of birth prevalencediseaseno . Of patients 19752008years of birthno . Of live birthsno . Of patientsprevalence (1 per numbers of live births)prevalence per 100,000carrier frequencygaucher type i, early12 (10)197419963 090 90212257 5750.39254gaucher type i, late29 (28)194519805 956 02824248 1680.40249gaucher type ii and iii8 (8)198019982 352 4928294 0620.34271gaucher (all types)494488 3971.13149niemann - pick a10 (8)196320025 480 88510548 0890.18370niemann - pick b + atyp13 (11)194819967 362 14611669 2860.15409niemann - pick a / b2321301 3270.33274niemann - pick c54 (45)196520035 271 31048109 8190.91166fabry49 (24)194520028 518 60144193 6050.5296 803male4 382 0684499 5921.00fabry female78 (24)194520058 812 16168129 5910.77krabbe15 (15)197720023 249 15013249 9350.40250mld infantile13 (11)197019994 195 56413322 7360.31284 juvenile5 (4)197020044 661 3245932 2650.11483 adult7 (7)196819802 188 0046364 6670.27302mld (all types)2524144 6470.69190cesd, wolman18 (15)196320025 480 88515365 3920.27302gm1 gangliosidosis13 (13)197020034 563 66012380 3050.26308gm2 tay - sachs10 (10)197119923 362 88910336 2890.30290gm2 sandhoff3 (3)199220071 594 9493531 6500.19365lipidoses all types259/337234/30220 0005.0/5.7771mld metachromatic leukodystrophy, cesd cholesteryl ester storage diseasetotal number of patients diagnosed between 1975 and 2008number of families with particular disorder in parenthesesnumber of patients taken for the calculation of birth prevalence . To avoid underestimation, some patients were not included into the prevalence calculation (e.g., patients born before 1945 see patients and methodsexpressed as 1 carrier per number of shown live birthsone stillborn casenp type b in 6 slowly progressive visceral cases and atypical protracted neurovisceral phenotype in 7 cases [pavl - pereira h, et al . J inherit metab dis 2005;28:203227]including one case of np type c2 [elleder m. et al . Virchows arch 2001;439:206211]classical phenotype in 43 male patients and cardiac variant in 6 male patients (two families) [elleder m, et al . Cas lek cesk 1990;129:368372]infantile form in 12 patients, late infantile in 2 patients, and adult form in one patient [kostalova e, et al . Ceska slov neurol neurochir 2006;69/102:200210]one infantile patient (wolman disease) and 17 juvenile and adult cases [elleder m, et al . Cas lek cesk 1999;138:719724]including two adult patientsfabry females not includedfabry females includedmale live birthscarrier frequency of lipidoses except for fabry diseasetable 5mucopolysaccharidoses: data for calculation of birth prevalencediseaseno . Of patients 19752008years of birthno . Of live birthsno . Of patientsprevalence (1 per number of live births)prevalence per 100,000carrier frequencymps i20 (17)198420082,772,55920138,6280.72186mps ii22 (19)196920054,906,70021233,6520.43116,826male2,520,37321120,0180.83mps iii a18 (14)197720063,648,54117214,6200.47232mps iii b1197520084,261,89714,261,8970.021,032mps iii c5 (4)198419921,179,7455235,9490.42243mps iii (all types)2423109,5020.91165mps iv a14 (9)198720031,826,47313140,4980.71187mps iv b1197520084,261,89714,261,8970.021,032mps iv (a + b)1514136,9860.73185mps vi2 (2)196819954,115,05722,057,5290.05717mps vii1197520084,261,89714,261,8970.021,032mps i vii848134,9092.8693mps - unspecified32 (27)196019894,533,49627167,9070.60205msd3 (1)196219691,144,4673381,4890.26309mps all types11911126,8653.7282mps mucopolysaccharidosis, msd multiple sulphatase deficiencytotal number of patients diagnosed between 1975 and 2008number of families with particular disorder in parenthesesnumber of patients taken for the calculation of birth prevalence . To avoid underestimation, some patients were not included into the prevalence calculation (e.g., patients born before 1945 were excluded). For prevalence definition, see patients and methodsexpressed as 1 carrier per number of shown live birthshurler syndrom in 16 patients, hurler / scheie in 2 patients and scheie syndrom in 2 patientsmale live birthscarrier frequency of mps excluding mps iitable 6glycoproteinoses, mucolipidoses, glycogenosis type ii and neuronal ceroidlipofuscinoses: data for calculation of birth prevalencediseaseno . Of patients 19752008years of birthno . (1 per numbers of live births)prevalence per 100,000carrier frequency-mannosidosis4 (3)198519921,042,8044260,7010.38255-mannosidosis2 (1)195019561,223,3862611,6930.16391issd1197520084,261,89714,261,8970.021,032mucolipidosis i3 (2)196919994,338,72931,446,2430.07601mucolipidosis ii / iii3 (3)199420071,352,2193450,7400.22336mucolipidosis iv1197520084,261,89714,261,8970.021,032gp + ml (all types)1414112,9950.87170glycogenosis type ii infantile6 (5)197919992,614,0756435,6790.23330glycogenosis type ii juvenile6 (5)197220024,167,9306694,6550.14417glycogenosis type ii (all)1212267,7500.37259ncl 12 (2)197019751,033,4472516,7240.19359ncl 228 (25)194920037,803,06828278,6810.36264ncl 32 (2)19791983749,5202374,7600.27306ncl 41197520084,261,89714,261,8970.021,032ncl 52 (1)19831989935,5532467,7770.21342ncl 61197520084,261,89714,261,8970.021,032ncl 718 (16)198520022,002,02517117,7660.85172ncl - unspecified20 (17)196519994,903,21418272,4010.37261ncl (all types)747143,5512.29104issd infantile sialic acid storage disease, gp glycoproteinosis, ml mucolipidosis, ncl neuronal ceroid lipofuscinosistotal number of patients diagnosed between 1975 and 2008number of families with particular disorder in parenthesesnumber of patients taken for the calculation of birth prevalence . To avoid underestimation, some patients were not included into the prevalence calculation (e.g. Patients born before 1945 were excluded). For prevalence definition see patients and methodsexpressed as 1 carrier per number of shown live birthsone infantile patient and two juvenile siblings with cherry - red spot myoclonus syndrome [ledvinova j, et al . J inherit metab dis 1994;17:118119]atypical case of ml iv with ocular restricted phenotype [dobrovolny r, et al . Am j ophthalmol 2007; 143:663671] relative rate of lysosomal storage disorders in the czech republic . Mps mucopolysaccharidosis, ncl neuronal ceroidlipofuscinosis, gsd ii glycogenosis type ii, ml mucolipidosis, gp glycoproteinosis, npa / b / c niemann - pick a / b / c, mld metachromatic leukodystrophy, cesd cholesterol ester storage disease, msd multiple sulphatase deficiency, gm1 gm1 gangliosidosis, gm2 gm2 gangliosidosis (tay - sachs and sandhoff diseases), -man -mannosidosis, -man -mannosidosis, issd infantile sialic acid storage disease number of live births () and number of lsd diagnoses () in the czech population from 1975 to 2008 . In 1985, electrophoretic analysis of glycosaminoglycans (elfo gag) and thin layer chromatography of oligosaccharides (tlc ols) in urine were introduced . From 1990 nowadays, laboratory diagnostic methods for 45 lsds are available (enzyme and dna analyses, loading assays) lipidoses: comparison of data in different populations nl netherlands, mld metachromatic leukodystrophy, cesd cholesteryl ester storage disease total number of patients diagnosed between 1975 and 2008 data for prevalence calculations are in table 4 carrier per 1 live birth male live births only fabry females not included fabry females included mucopolysaccharidoses: comparison of data in different populations nl netherlands, mps mucopolysaccharidosis, msd multiple sulphatase deficiency total number of patients diagnosed between 1975 and 2008 data for prevalence calculations are in table 5 carrier per 1 live birth male live births only unspecified cases of mps were diagnosed on the basis of analysis of glycosaminoglycan excreted in urine combined with the evaluation of clinical data . No material was available for enzyme and dna analysis glycoproteinoses, mucolipidoses, glycogenosis type ii and neuronal ceroid lipofuscinoses: comparison of data in different populations nl netherlands, issd infantile sialic acid storage disease, ncl neuronal ceroid lipofuscinosis total number of patients diagnosed between 1975 and 2008 data for prevalence calculations are in table 6 carrier per 1 live birth unspecified ncl were diagnosed on the basis of histochemical and ultrastructural examination of biopsy samples combined with the evaluation of clinical data . No material was available for enzyme and dna analysis lipidoses: data for calculation of birth prevalence mld metachromatic leukodystrophy, cesd cholesteryl ester storage disease total number of patients diagnosed between 1975 and 2008 number of families with particular disorder in parentheses number of patients taken for the calculation of birth prevalence . To avoid underestimation, some patients were not included into the prevalence calculation (e.g., patients born before 1945 were excluded). For prevalence definition, see patients and methods expressed as 1 carrier per number of shown live births np type b in 6 slowly progressive visceral cases and atypical protracted neurovisceral phenotype in 7 cases [pavl - pereira h, et al . J inherit metab dis 2005;28:203227] including one case of np type c2 [elleder m. et al . Virchows arch 2001;439:206211] classical phenotype in 43 male patients and cardiac variant in 6 male patients (two families) [elleder m, et al . Cas lek cesk 1990;129:368372] infantile form in 12 patients, late infantile in 2 patients, and adult form in one patient [kostalova e, et al . Ceska slov neurol neurochir 2006;69/102:200210] one infantile patient (wolman disease) and 17 juvenile and adult cases [elleder m, et al . Cas lek cesk 1999;138:719724] including two adult patients fabry females not included fabry females included carrier frequency of lipidoses except for fabry disease mucopolysaccharidoses: data for calculation of birth prevalence mps mucopolysaccharidosis, msd multiple sulphatase deficiency total number of patients diagnosed between 1975 and 2008 number of families with particular disorder in parentheses number of patients taken for the calculation of birth prevalence . To avoid underestimation, some patients were not included into the prevalence calculation (e.g., patients born before 1945 were excluded). For prevalence definition, see patients and methods expressed as 1 carrier per number of shown live births hurler syndrom in 16 patients, hurler / scheie in 2 patients and scheie syndrom in 2 patients carrier frequency of mps excluding mps ii glycoproteinoses, mucolipidoses, glycogenosis type ii and neuronal ceroidlipofuscinoses: data for calculation of birth prevalence issd infantile sialic acid storage disease, gp glycoproteinosis, ml mucolipidosis, ncl neuronal ceroid lipofuscinosis total number of patients diagnosed between 1975 and 2008 number of families with particular disorder in parentheses number of patients taken for the calculation of birth prevalence . To avoid underestimation, some patients were not included into the prevalence calculation (e.g. Patients born before 1945 were excluded). For prevalence definition see patients and methods expressed as 1 carrier per number of shown live births one infantile patient and two juvenile siblings with cherry - red spot myoclonus syndrome [ledvinova j, et al . J inherit metab dis 1994;17:118119] atypical case of ml iv with ocular restricted phenotype [dobrovolny r, et al . Am j ophthalmol 2007; 143:663671] to date, no individuals in the czech population have been conclusively diagnosed with farber disease, schindler disease, danon disease, sanfilippo d disease, galactosialidosis, fucosidosis, aspartylglucosaminuria, or prosaposin, saposins, or gm2 activator protein deficiencies . The overall prevalence of lsds in the czech population (12.25 per 100,000), which represents a typical central european population, is comparable to prevalences reported for australia (12.9 per 100,000), the netherlands (14 per 100,000) and italy (12.1 per 100,000) (meikle et al . . The higher prevalence of lsds reported for the northern portuguese population (25 per 100,000) might reflect the relative geographic isolation and more extensive studying of that particular population of this region (pinto et al . The most frequent single lsd is gaucher disease in the czech republic, italy and australia, gm2 gangliosidosis in portugal, and pompe disease in the netherlands . In the czech population, lipidoses were most frequently diagnosed (5.0 per 100,000 live births), followed by mps (3.72) and ncl (2.29). In addition to the lsd diagnoses in czech patients, 96 cases from slovakia were confirmed at our institute during the studied period; 41% with lipidoses, 39% with mps, and 20% with gsd ii or ncls . These slovakian patients have not been included in our prevalence calculations as former czechoslovakia was divided into two independent states, the czech republic and the slovak republic, in 1993 . Interestingly, the four cases of prosaposin deficiency so far diagnosed at our institute came from neighboring populations (slovak, german) (hulkova et al . 2001; elleder et al . The birth prevalence of the most frequent lsd in the czech republic, gaucher disease, is 1.13 per 100,000 live births, followed by niemann - pick c (0.91), mps iii (0.91), mps iv (0.73), and mps i (0.72). Comparison of these prevalences to those in selected foreign populations (tables 1, 2 and 3) is complicated by the differences in methods used for calculation . When we compared the calculations of prevalence in the czech population based on the method of poorthuis et al . (1999) used in this study (see patients and methods) to the method reported by meikle et al . (1999) (the prevalence calculated as the number of patients divided by the total number of live births during the study period), the calculations resulted in underestimation or overestimations of some individual disorders . This is the case for disorders with delayed onset (e.g., frequencies of fabry disease were 0.52 and 1.14 per 100,000, and frequencies of cholesterol ester storage disease were 0.27 and 0.42, if calculated using the methods of poorthuis et al . Also, unavailability of some diagnostic methods during the study period (some methods were introduced as late as the 1990s) may explain some discrepancies found in our calculations using the two methods . Although prevalence values for some disorders calculated using either of the methods were within the confidence intervals (data not shown), for other disorders prevalence values fell outside this interval (e.g., the frequencies differed for mps iii c (0.42 and 0.11 per 100,000), mps iva (0.71 and 0.32), ncl 2 (0.36 and 0.65) and ncl 7 (0.85 and 0.42), if calculated according to the methods used by poorthuis et al . Thus, establishment of standard rules for calculation of birth prevalence - a defined term in clinical epidemiology - is highly recommended . Based on our experience, the calculation of the ratio of the number of patients to the number of live births during the patients births period (poorthuis et al . Underestimation of the birth prevalence may result from lack of recognition of early clinical signs and symptoms or phenotypic diversity, including adult forms of the disease (e.g., fabry disease, adult krabbe disease, adult mld, cholesteryl ester storage disease, and adult gsd ii). Thus, the calculated birth prevalence is probably the lowest estimate as the diagnosis is commonly delayed for years . Screening studies have shown that certain groups of patients, for example individuals with renal failure on haemodialysis, may harbor missed fabry patients (nakao et al . The nationwide screening study among 3,370 chronic haemodialysis patients in the czech republic disclosed four previously unrecognized fabry males and one female from five unrelated families . Subsequent family screening ascertained fabry disease in another 12 individuals, including 8 females (merta et al . Patients with unexplained cardiomyopathy represent another group at risk of having fabry disease where screening is justified (monserrat et al . 2007). Such lsd screening initiatives for selected lsds will contribute to the identification of the true prevalence of these disorders (spada et al . Fabry heterozygotes have not been included in the calculation of birth prevalence in previous reports (meikle et al . However, they commonly develop clinical symptoms and severe complications, generally a few years later, as in affected men (deegan et al . 2006; wilcox et al . 2008). When we included fabry heterozygotes in the calculations, the prevalence of fabry disease increased by 2.5 times to 1.29 per 100,000 live births, that of lipidoses from 5.0 to 5.77 per 100,000 and the overall prevalence of lsds reached 13.02 per 100,000 (table 4) therefore, we propose that fabry heterozygotes ought to be included in the calculation of the birth prevalence of fabry disease . Reliable calculation of the birth prevalence is dependant on a correctly defined pathogenic cause underlying the condition . Recent molecular studies have provided evidence for at least ten separate genetic loci (cln1cln10) for ncl (peltonen et al . These new findings necessitated reclassification of our 37 cases that had formerly been provisionally classified as ncl6 into ncl5, ncl6, and ncl7 subgroups . The latter turned out to be of high prevalence in the roma gypsy population (kousi et al . 2009). About a quarter of the ncl cases remain unspecified due to the lack of material suitable for mutation analysis . Athough specific lsds are generally reckoned to be rare disorders, as a group of inherited metabolic disorders they are quite common, as seen from published and our data . The information on birth prevalencies of lsds is available from countries with a long tradition of diagnosis of lsds; however, for most populations, these date are missing . Epidemiological data on these disorders are important in genetic counselling for calculation of the risk for the disorders in the other members of affected families and subsequently for the public health care systems . Importantly, awareness of the clinical aspects of these life - threatening and progressively disabling genetic diseases among the medical community should increase, allowing earlier diagnosis . Dependant on the diagnosed disorder, such intervention may include enzyme replacement therapy, bone marrow transplantation, or other current or future therapies . Last but not least, it should be borne in mind that genetic counselling and prenatal diagnosis in families affected by a genetic metabolic disorder still form the basic strategy for lowering the number of patients with these severe disorders.
Dna sequences were designed using program sequin.23 dna strands, including iodinated derivatives were synthesized by standard phosphoramidite techniques on an applied biosystems 394 dna synthesizer . Strands were doubly purified by reverse - phase hplc using a c-18 column (waters). Crystals were grown from 80 l sitting drops in a thermally - controlled incubator containing 0.25 g/l dna, 30 mm sodium cacodylate, 50 mm magnesium acetate, 50 mm ammonium sulfate, 5 mm magnesium chloride, 25 mm tris (ph 8.5), equilibrated against a 1.5 ml reservoir of 1.7 m ammonium sulfate . Rhombohedral - shaped crystals with dimensions as large as 250250250 m were obtained by slow annealing, in which the temperature was decreased from 60 c to room temperature (~20 c) with a cooling rate of 0.2 c per hour over a period of 7 days, during which the volume of the drop diminished by about 90% . Crystals were obtained at the end of the cooling step, and appeared full - sized within a day . Crystals were transferred to a cryosolvent of 30% glycerol, 100 mm ammonium sulfate, 10 mm mgcl2, and 50 mm tris and were frozen by immersion into liquid nitrogen . X - ray data diffraction data were collected from crystals of iodinated derivatives (12 iodine atoms per triangle--on the fourth and thirteenth nucleotide of each green strand and on the sixth and eleventh nucleotide of each red strand) at 1.7 on beamlines x6a and x25 at the national synchrotron light source (brookhaven national laboratory, upton, new york, usa). A complete sphere of native x - ray data were collected at the aps beamline 19id.24 complete crystallographic details and associated references are available in the supplementary information in the online version of the paper at www.nature.com/nature . Dna sequences were designed using program sequin.23 dna strands, including iodinated derivatives were synthesized by standard phosphoramidite techniques on an applied biosystems 394 dna synthesizer . Strands were doubly purified by reverse - phase hplc using a c-18 column (waters). Crystals were grown from 80 l sitting drops in a thermally - controlled incubator containing 0.25 g/l dna, 30 mm sodium cacodylate, 50 mm magnesium acetate, 50 mm ammonium sulfate, 5 mm magnesium chloride, 25 mm tris (ph 8.5), equilibrated against a 1.5 ml reservoir of 1.7 m ammonium sulfate . Rhombohedral - shaped crystals with dimensions as large as 250250250 m were obtained by slow annealing, in which the temperature was decreased from 60 c to room temperature (~20 c) with a cooling rate of 0.2 c per hour over a period of 7 days, during which the volume of the drop diminished by about 90% . Crystals were obtained at the end of the cooling step, and appeared full - sized within a day . Crystals were transferred to a cryosolvent of 30% glycerol, 100 mm ammonium sulfate, 10 mm mgcl2, and 50 mm tris and were frozen by immersion into liquid nitrogen . X - ray data diffraction data were collected from crystals of iodinated derivatives (12 iodine atoms per triangle--on the fourth and thirteenth nucleotide of each green strand and on the sixth and eleventh nucleotide of each red strand) at 1.7 on beamlines x6a and x25 at the national synchrotron light source (brookhaven national laboratory, upton, new york, usa). A complete sphere of native x - ray data were collected at the aps beamline 19id.24 complete crystallographic details and associated references are available in the supplementary information in the online version of the paper at www.nature.com/nature.
As indicated in the millennium development goals, decreasing maternal deaths is one of the most important global health objectives . With improved access to maternal health services, particularly in rural areas, maternal mortality ratios (mmr) however, although access to maternal health services has improved, their use has often not grown as expected . It is commonly observed that women do not recognize their health needs or that they are unable to make the right decisions about seeking health care . To advance women s health, it is important not only to provide them with greater access to health services (supply side), but also to empower them so that they choose to employ such services (demand side). Mmr in egypt has been decreased dramatically during the last two decades . According to national surveys, such as the egypt national maternal mortality study, mmr was 174 per 100,000 live births in 1992, 84 in 2000, and 55 in 2008 . The greater availability of public health services has contributed to this decrease, particularly in unprivileged areas such as southern governorates . Access to basic health services is guaranteed to 99% of the egyptian population, which is concentrated in a narrow area along the nile river . Despite this achievement, mmr in egypt continue to be higher than those of many other middle income countries, including arab nations such as jordan . In addition, about one - third of the maternal deaths are caused by delays in recognizing problems or seeking care . Furthermore, such delays are closely related to the low socio - economic status of women in egypt and several other eastern mediterranean region countries . This research aims to investigate the association between the household status of women and the use of maternal health services in rural egypt . A cross - sectional survey was carried out in a village in the giza governorate of the arab republic of egypt in november 2007 . The village, which has a population of around 20,000, is located around 14 km south of cairo . Although the village was formerly composed of traditional extended families engaging in agriculture, it expanded rapidly by adding migrant nuclear families from other governorates because of its vicinity to the capital . We assumed that women had good access to health services, since the village had a public health centre that was staffed with a gynaecologist and nurses and that actively provided maternal and child health care at nominal fees . A total of 201 married women, younger than 50 years of age, were selected using a stratified sampling technique . Subsequently, we randomly visited about 40 households in each section and chose one woman from each household . Excluding the women who did not respond to necessary questions, we obtained a sample of 189 women between 20 and 50 years of age . Face - to - face interviews with a structured questionnaire were carried out in arabic by trained junior faculty members and students of faculty of nursing, cairo university . The questionnaire was developed by referring to various survey questionnaires, such as egypt demographic and health survey, and revised after pretesting in the target field . It consisted of questions on women s health service utilization, such as frequency of antenatal care (anc) attendance during pregnancies, places of last deliveries, and persons who assisted last deliveries . Questions regarding women s socio - economic status, such as levels of education of both wives and husbands; ages of marriage; cash incomes; availability of assistance with household chores; and women s status within households, including household decision - making; experiences of physical assaults by husbands, and awareness of and participation in community activities . The three variables for women s health service utilization were transformed into dichotomous values as follows: (1) whether a participant had attended anc at least four times during her last pregnancy, a frequency regarded as regular by the world health organization (who); (2) whether she had been attended in her last delivery by skilled health professionals; and (3) whether she had given birth to her last child at a health facility . Variables for participation in decision - making were transformed into scores, by aggregating the answers of relevant sub - questions . In each sub - question, women who could make a decision alone were given a full score of two points, those who could only partly decide one point, and those who did not participate in the decision - making process zero points . The variable of decision - making on household issues was scored from zero to six and that on household expenditure was scored from zero to ten . Associations between each of the outcome variables and socio - economic variables were tested by the chi - square test, and p <0.05 was considered statistically significant . Crude odds ratios of variables for the use of maternal health services were calculated for each variable proxy to women s socio - economic status . Ethical clearances for the study were obtained from the ethics review committee of nagoya university school of medicine in nagoya, japan, and faculty of nursing, cairo university in cairo, egypt . Written informed consent over 60% married when they were less than 18 years old, and 25% married before they turned 16, the legally approved age of marriage . More than half of them had never attended school, while most of their husbands had attended at least primary schools and about 38% of them had studied at secondary or higher level schools . About 20% had a job with cash income, and most of them stated that they could use their earnings without the permission of their husbands or other family members . Several community - based activities, such as vocational training for women and literacy classes, had been implemented in the village, by both the government and non - governmental organizations; however, only 34% of the women were aware of these activities, and very few had actively participated in them . No less than 48% had attended regular anc, while about 30% stated that they never attended anc during their last pregnancies . At the same time, 79% had been attended by skilled health professionals at their last deliveries . Sample characteristics (n = 189) the total score of decision - making on household issues of three sub - questions (02 points each): (1)what to cook (2)children s schooling (3)family planning the total score of decision - making on household expenditure of five sub - questions (02 points each): (1)furniture and household items, such as electric appliance, bed (2)daily use items and groceries, such as fruit, vegetable (3)your personal items, such as clothes, shoes (4)husband s personal items, such as clothes, shoes (5)children s items, such as clothes, toys, stationery table 2 shows the crude odds ratios of the variables of maternal health service utilization for each proxy variable of socio - economic status . The age of the first marriage and level of women s education are significantly associated with the three variables for use of maternal health services . Association between women s health service utilization and other variables women who had never been physically assaulted by their husbands and those who were aware of community activities were more likely to attend anc regularly . However, these variables did not show a significant association with deliveries attended by skilled health professionals and deliveries at health facilities . Participation in household decision - making such as purchases and availability of assistance with household chores had no significant association with the three variables . Marriage to husbands with secondary or higher levels of education was significantly associated with the increase of regular anc attendance, deliveries attended by skilled health professionals, and deliveries at health facilities, as was residence in extended families . Although access to maternal health services was guaranteed in the study village, the rate of utilization was still low, as regular anc attendance, deliveries attended by skilled health professionals, and deliveries at health facilities equalled only 48%, 79%, and 61% of the total sample, respectively . This result was similar to or a little better than an earlier nationwide survey in 2005 . To improve women s health in developing countries, every woman must have access to appropriate health services, particularly during pregnancy and childbirth, when women s health is most likely to be at risk . However, as mentioned previously, even though such services are affordable and accessible, women do not always use them because of various social constraints . Our study seeks to identify the social factors that influence the use of maternal health services in rural egypt . Our findings suggest that women who married before the age of 18 and who received insufficient formal education were less likely to use maternal health services than the women who married later . Similar results have been observed in nepal, where the use of maternal health services increased along with the amelioration of women s educational and income levels . Women who married early without formal education were likely to be subordinated to their husbands and parents - in - law and were unable to participate in household decision - making, including their own health needs . Formal education and marriage at a mature age contributed to the improvement of the familial status of women by providing them with higher self - esteem and confidence . Thus, they were able to seek health services according to their own needs; in other words, their demands for such services increased . Similarly, the experience of physical assaults by husbands may reflect the subordinate status of women in their families . Our findings show that physical assaults by husbands were negatively associated with regular anc attendance but not with the other two outcome variables deliveries attended by skilled health professionals and deliveries at heath facilities . However, since childbirths were major family events, and the outcome could be easily recognized by anyone in the family, household decision - makers might opt for seeking proper delivery care, regardless of the status of women . However, in the case of attending anc, pregnant women might need to make decisions by themselves or to convince household decision - makers to permit such attendance, even though they had no serious symptoms . This fact indicates that regular attendance at anc required women to understand the importance of preventive care, enjoy a good status in the household, and be allowed to go out freely . This could explain another finding why women who were aware of community activities were more likely to attend regular anc than those who were not . Women with a good familial status could easily obtain information outside the household and participate freely in community activities . Participating in them allowed women to attain greater self - esteem and self - confidence through social achievement and the enhancement of their decision - making abilities . Consequently, such women were likely to understand the importance of preventive maternal health care . Women with educated husbands were also more likely to seek maternal health services, because these men were likely to understand their wives health needs and offer proper advice . We expected that women who lived with parents - in - law would have difficulty seeking health services, as observed in previous studies in nepal and india . However, our results show that women who lived with extended families were more likely to avail themselves of such facilities than those living with only husbands and children . In addition, the availability of assistance with household chores was not significantly associated with a reliance on maternal health services . Our results suggest that members of extended families were likely to encourage women to seek health care, rather than to share household work . Therefore, two factors, the educational levels of husbands and extended family households pointed to the existence of moral and not just physical or economic support for women . Perhaps we arrived at this finding because we selected maternal health services as a variable of women s access to health services . Childbirth would be an important family event; thus, all family members might be willing to give moral support to pregnant women seeking medical supervision . The attitude of family members might be different in case the women suffered from other illnesses . We expected that the use of maternal health services would increase, if women could make various household decisions or had cash income that they could freely spend . Previous studies show that women with authority and economic autonomy in the family could decide and act according to their own needs, including medical ones . However, our findings do not show the linkage between familial decision - making authority and economic autonomy and a rise in the employment of maternal health services . In addition, the fees of maternal health services in rural egypt might be too low to be influenced by household income . As our findings indicate, currently younger generation made greater use of maternal health services, perhaps because they had fewer childbirth experiences than older women . The egypt demographic and health survey reveals that lower birth order is associated with more involvement with maternal health services . Another possibility is that the supply for maternal health services might have been different among age groups . However, as our sample size is not big enough to stratify the results by age group, further studies are needed to exclude the influence of age . Through this study, we sought to show that women s utilization of or demand for health services would increase with the improvement of their household status . Our results suggested that improved status of women in the household, as well as moral support from family members, contribute to the greater reliance of women on maternal health services and particularly on regular anc attendance . The authors wish to thank faculty members and students of faculty of nursing, cairo university for data collection . This study was in part supported by a grant - in - aid for scientific research (b, 19406024) to a.a . From the japan society for the promotion of sciences and the international cooperation research grant (17 - 3) to a.a . From the japanese ministry of health, labour and welfare.
Advanced chronic renal failure is a progressive and irreversible disorder in which the kidneys ability to excrete metabolic waste products and to maintain fluid and electrolytes is lost and can lead to uremia . Chronic renal failure and end - stage renal diseases (esrds) are progressive and irreversible disorders and one of the major problems around the world . The incidence of ckd in the usa, taiwan, and some regions in mexico is approaching 400 cases per million . In iran, the number of patients beginning maintenance renal replacement therapy (rrt) increased by 130% from 2000 to 2006 . The incidence of esrd linearly increased from 13.82 per million population in 1997 to 49.9 per million population in 2000 and to 63.8 per million population in 2006 . Widespread and easy access of patients requiring hemodialysis treatment has resulted in increase in longevity of thousands of patients with esrds . Hemodialysis is the most common treatment for the patients suffering from the esrds or irreversible progressive renal failure . Changes in living patterns and its limitations impose complex and changed lives on the patients and their families and, finally, reduce their quality of life . In such situations, optimizing their quality of life will be one of the most important objectives of health care . Nowadays, hemodialysis patients, in addition to increasing the lifetime thanks to hemodialysis, want to improve their quality of life . The results of studies on the health - related quality of life in the patients requiring hemodialysis have shown that related diseases have undesirable effects on patients physical, mental, and social functions, and measuring the quality of life in such patients has special importance . Despite advances in the treatment of hemodialysis patients, their quality of life is affected by various factors that may make their physical and mental performances difficult . One of the ways of improving the patients quality of life is through self - care education . Developing and using theories and models in nursing is one of the available training methods . Development of strategic plans for hemodialysis patients for them to achieve efficient care is essential . Those patients who have received self - care education, in comparison with those who have never received such training, have higher quality of life . Hemodialysis patients in their own self - care do not have high self - efficacy . Therefore, in order to improve their quality of life, training them in self - care programs has been taken into consideration as a strategy . Several factors have effects on increasing the hemodialysis patients quality of life, one of which is self - care education . Self - care is the patients ongoing efforts to promote their health and welfare and to make their lives better . The results of some studies have shown that there are positive and significant relationships between self - care ability and different aspects of quality of life . The results of a study conducted in taiwan in 2002 indicated that the quality of life in hemodialysis patients was lower than that in kidney transplant patients, and breast and colon cancer patients . Several studies have been conducted in iran to assess the quality of life in hemodialysis patients . Concluded in their study that more than half of hemodialysis patients had undesirable quality of life . 's study showed that self - care educational program had effects on decreasing the hemodialysis patients problems and improving their quality of life . 's study, the health - related quality of life in hemodialysis patients in iran was lower than that in european and asian countries due to the differences in lifestyle, socioeconomic status, the general level of education of the patients, as well as physician patient communication . In another study conducted by moattari et al . In iran, the results showed a positive effect of empowerment on hemodialysis patients self - efficacy and quality of life . Controlling the problems and complications and improving quality of life requires patients participation in treatment and care . Patients treated with hemodialysis need special and ongoing training as they have multiple drug treatments and a special diet, as well as for acquiring the required skills to cope with physical and mental disabilities . There are several models and approaches for educating and training patients . Considering the chronic problems of these patients, hemodialysis inefficiency and inadequacy in addressing these problems, as well as the need for continuous care, education and training should provide active and informed participation of patients in their own self - care . According to the problems mentioned above, including the type, extent, and frequency of such patients physical and psychological problems and, on the other hand, the need for providing education to the patients so that they are qualified for their continuous self - care, this study aimed to determine how self - care education in these patients can be effective in reducing their problems and improving their quality of life, in order to provide more evidences in this area . The use of a nursing model helps to evaluate patients health status, establish good communications between patients and nurses, set goals of care, and improve the quality of care . The main goal of the self - care models is to improve the patients quality of life . The partnership care model focuses on the participation of the patient, his / her family, nurses, physicians, and other healthcare providers in the treatment processes and, based on the partnership approach, provides programs to ensure the continuity of this participation in order to promote and maintain the health of the patient . This model, in addition to explaining the collaborative approach, examines and explains the relationship between care and this participation, and in this regard, the assumptions, structure, basic concepts, and objectives are defined . This model pays special attention to the care and emphasizes two major dimensions including: skills, techniques, and special carethe caring relationship developed among patients, physicians, and nurses to promote and maintain patients health . Skills, techniques, and special care the caring relationship developed among patients, physicians, and nurses to promote and maintain patients health . However, the second one is a software dimension, does not have a fixed and defined nature, and has a dynamic status . Although certain skills and activities are necessary in the care processes, caring relationship plays an important role in the quality of care and promoting and maintaining the health . This model has several objectives, the most important of which is paying attention to the patients quality of life . In other words, this model provides an overall strategy to utilize all the capabilities of those involved in the care and treatment, including physicians, nurses, and the patients, and based on this model, providing care is only possible when the relationship among physicians, nurses, patients, and their families is continued and all these people have the same common objectives and understanding of patient care activities . This model includes four stages: (1) motivating, (2) preparing, (3) involving, and (4) evaluating . The results of some studies have shown that using partnership care model in patients with cardiovascular disease and hypertension has had positive effects on the patients quality of life, and given that using this model is not limited to a particular disease, implementing it for patients with diseases which reduce their quality of life has been considered as a necessity by researchers . Therefore, this study aimed to determine and evaluate the effects of using this model on the quality of life in patients treated with hemodialysis . It was assumed that the implementation of the partnership care model has a positive impact on improving the quality of life in hemodialysis patients . In iran, the researchers of this study had used this model for the first time for hemodialysis patients in a military hospital to determine how self - care education based on partnership care model could be effective in reducing problems and improving the quality of life of these patients . This was a quasi - experimental study conducted in 2012 on patients who were referred to a military hospital in tehran, iran to be treated with hemodialysis . Inclusion criteria were: willingness to participate in this study, loss of function of both kidneys based on clinical evidence, laboratory tests, and expert opinions, glomerular filtration rate (gfr) <10%, having passed at least 6 months from starting hemodialysis, performing hemodialysis two times per week, and not suffering from any underlying acute or chronic diseases . The first section was related to the demographic data, including age, sex, marital status, employment status, education level, and duration of hemodialysis . The second section included data on weight, blood pressure, sleep, itching, and doing exercise by patients . The second questionnaire was the short form-36 (sf-36) standardized questionnaire consisting of eight dimensions, including general health, vitality, physical function, physical role, bodily pain, mental health, social function, and emotional role . Generally, this questionnaire has two components, including physical and mental components . Its scales are scored from 0 to 100 in which 0 represents the worst quality of life and 100 represent the best quality of life . The content validity of the first questionnaire was approved using the opinions of 15 faculty members of nursing and midwifery school of tehran university of medical sciences, and its reliability was confirmed using test retest reliability coefficient (r = 0.85). Validity and reliability of sf-36 questionnaire have been confirmed in previous studies . The model used in this study as one of the self - care models was partnership care model . This model consists of four stages: (1) motivating, (2) preparing, (3) involving, and (4) evaluating . During the motivating stage, patients became aware of their disease and its outcomes and consequences by attending a training session . At the preparing stage, the objectives, schedules, and time tables of partnership education programs, as well as how to track them were determined . During the involving stage, the educational meetings were held and, finally, at the evaluating stage, achieving the objectives of the model was evaluated . Individuals should attend such a system based on their desire and motivation . On the other hand, care is also a voluntary relationship initiated and formed by choosing a physician, a nurse, or even the type of care and treatment by the patient . Because all care and treatments are considered as activities invading patients privacy, giving informed choices to the patients is the prerequisite for providing care and treatment . Explanation of what should be done, how, why, who, where, and when is essential . The second stage of the model is developing an appropriate plan to engage all members of the partnership team, including patients, nurses, and physicians, because the best ideas and programs, even with the strongest and most well - intentioned people and with the best working facilities, cannot be implemented without members involvement or they will lead to very poor results . To create appropriate opportunities for involving and engaging all members, because care and treatments involve a series of specialized activities, it is necessary to define the required activities and their relationship with the members duties and responsibilities according to their abilities and skills . Therefore, at this stage, it is required to do proper planning before any other action . One other important step at the preparing stage is planning for educational partnership visits, as well as following the achievement of this model's objective, i.e., ensuring the participation and compliance . At the next stage, time and content planning of collaborative visits will be done as follows: these visits are conducted according to prior planning . Therefore, the objectives of the educational content and the teaching methods and tools required are determined . This plan can be classically developed according to the patients problems and diagnoses, and in collaboration with the physician, nurse, and the patients . However, it should be noted that it is not essential to implement the education plan classically and formallythe implementation of the education plan should be based on the philosophy and approach of participation and partnership, so that both patients and other participants are active . In other words, only the nurse and the physician are not responsible for trainingthe methods of undertaking the visits and providing education can be according to the patients conditions and by applying appropriate methods of explaining, question and answer methods, or based on problem solving and problem - based learning modelcollaborative visits are undertaken with the presence of those patients who have similar conditions . After the planning stage, care and treatments are provided according to the defined objectives . If the partnership plan is implemented without applying an effective management, the concept and principles of participation will be forgotten over time or will be filed and archived in the patients charts as a few slips of useless instructions . Since a manager or coordinator should be selected for implementing each plan in the groups, the following steps should also be taken in the partnership plan: determining the head and manager of the partnership teamaccording to the nurses capabilities, it is better to give the responsibility of the team to its nurse based on members consensusteam members consensus on delegating the responsibility of the team to its head and manager, as well as cooperating with him / herimplementing the plan and undertaking the educational visits and required follow - ups . Therefore, the objectives of the educational content and the teaching methods and tools required are determined . This plan can be classically developed according to the patients problems and diagnoses, and in collaboration with the physician, nurse, and the patients . However, it should be noted that it is not essential to implement the education plan classically and formally the implementation of the education plan should be based on the philosophy and approach of participation and partnership, so that both patients and other participants are active . In other words, only the nurse and the physician are not responsible for training the methods of undertaking the visits and providing education can be according to the patients conditions and by applying appropriate methods of explaining, question and answer methods, or based on problem solving and problem - based learning model collaborative visits are undertaken with the presence of those patients who have similar conditions . After the planning stage, care and treatments are provided according to the defined objectives . If the partnership plan is implemented without applying an effective management, the concept and principles of participation will be forgotten over time or will be filed and archived in the patients charts as a few slips of useless instructions . Since a manager or coordinator should be selected for implementing each plan in the groups, the following steps should also be taken in the partnership plan: determining the head and manager of the partnership teamaccording to the nurses capabilities, it is better to give the responsibility of the team to its nurse based on members consensusteam members consensus on delegating the responsibility of the team to its head and manager, as well as cooperating with him / herimplementing the plan and undertaking the educational visits and required follow - ups . Determining the head and manager of the partnership team according to the nurses capabilities, it is better to give the responsibility of the team to its nurse based on members consensus team members consensus on delegating the responsibility of the team to its head and manager, as well as cooperating with him / her implementing the plan and undertaking the educational visits and required follow - ups . To identify patients problems, the measurement of biochemical compositions of the blood, weight gain between two hemodialysis sessions, edema, and hypertension was used . The patients were weighed before and after hemodialysis with predetermined dress and with a scale that was calibrated and was constant during the intervention . For measuring edema, the patients edema in the wrist, leg, and around the eyes the edema in the hip and abdominal circumference was also measured by the meter . To gain more confidence in the data collected, blood biochemical compositions were measured within 2 months before training and other variables were measured two to four times with 2-week intervals before training and their mean values were considered as the data before training . Also, after training, the blood biochemical compositions were measured within 2 months and other variables were measured for two to four times with 2-week intervals and their mean values were considered as the mean after training . Quality of life was measured after training for two times, at 6 and 8 weeks after training, and their mean was calculated as the data after training . The study patients blood pressure was measured after 5 min of rest, in sitting mode, from the arm which did not have any fistula, in a position at heart level, and in a position in which the patient had an appropriate support and backrest . The education was provided to the patients through a training manual which had been prepared based on valid and scientific articles about exercise, sleep, nutrition, stress, hemodialysis equipment, hemodialysis methods, and psychology . Patients companions were also trained for 2 h in a class, and then, practical and additional training was provided for them during hemodialysis . The study patients were trained in all educational topics including the topics of nutrition, exercise, sleep, ways to prevent itching, etc . For example, for training the patients in the topic of exercise, a physiotherapist and the researcher gave theoretical and practical training to all the patients of a work shift (eight patients) for 2 h. afterward, in the next session, the researcher evaluated those patients, responded to their related questions, and explained that topic again to them (if needed) so that they learned the topic completely . Then, the researcher started training the other topic . Between the 1 and 2 weeks, the topic that was dealt with in the 1 week was evaluated . In all stages of the research,, chicago, il, usa) and some statistical tests including paired samples t - test, wilcoxon and mcnemar tests . Paired samples t - test was used for analyzing and comparing the data collected on weight and blood pressure before and after the intervention . In order to assess insomnia, edema, and the itching status before and after the intervention, required data were collected through another part of the questionnaire with yes / no items and analyzed using mcnemar test . Individuals should attend such a system based on their desire and motivation . On the other hand, care is also a voluntary relationship initiated and formed by choosing a physician, a nurse, or even the type of care and treatment by the patient . Because all care and treatments are considered as activities invading patients privacy, giving informed choices to the patients is the prerequisite for providing care and treatment . Explanation of what should be done, how, why, who, where, and when is essential . The second stage of the model is developing an appropriate plan to engage all members of the partnership team, including patients, nurses, and physicians, because the best ideas and programs, even with the strongest and most well - intentioned people and with the best working facilities, cannot be implemented without members involvement or they will lead to very poor results . To create appropriate opportunities for involving and engaging all members, because care and treatments involve a series of specialized activities, it is necessary to define the required activities and their relationship with the members duties and responsibilities according to their abilities and skills . Therefore, at this stage, it is required to do proper planning before any other action . One other important step at the preparing stage is planning for educational partnership visits, as well as following the achievement of this model's objective, i.e., ensuring the participation and compliance . At the next stage, time and content planning of collaborative visits will be done as follows: these visits are conducted according to prior planning . Therefore, the objectives of the educational content and the teaching methods and tools required are determined . This plan can be classically developed according to the patients problems and diagnoses, and in collaboration with the physician, nurse, and the patients . However, it should be noted that it is not essential to implement the education plan classically and formallythe implementation of the education plan should be based on the philosophy and approach of participation and partnership, so that both patients and other participants are active . In other words, only the nurse and the physician are not responsible for trainingthe methods of undertaking the visits and providing education can be according to the patients conditions and by applying appropriate methods of explaining, question and answer methods, or based on problem solving and problem - based learning modelcollaborative visits are undertaken with the presence of those patients who have similar conditions . After the planning stage, care and treatments are provided according to the defined objectives . If the partnership plan is implemented without applying an effective management, the concept and principles of participation will be forgotten over time or will be filed and archived in the patients charts as a few slips of useless instructions . Since a manager or coordinator should be selected for implementing each plan in the groups, the following steps should also be taken in the partnership plan: determining the head and manager of the partnership teamaccording to the nurses capabilities, it is better to give the responsibility of the team to its nurse based on members consensusteam members consensus on delegating the responsibility of the team to its head and manager, as well as cooperating with him / herimplementing the plan and undertaking the educational visits and required follow - ups . Therefore, the objectives of the educational content and the teaching methods and tools required are determined . This plan can be classically developed according to the patients problems and diagnoses, and in collaboration with the physician, nurse, and the patients . However, it should be noted that it is not essential to implement the education plan classically and formally the implementation of the education plan should be based on the philosophy and approach of participation and partnership, so that both patients and other participants are active . In other words, only the nurse and the physician are not responsible for training the methods of undertaking the visits and providing education can be according to the patients conditions and by applying appropriate methods of explaining, question and answer methods, or based on problem solving and problem - based learning model collaborative visits are undertaken with the presence of those patients who have similar conditions . After the planning stage, care and treatments are provided according to the defined objectives . If the partnership plan is implemented without applying an effective management, the concept and principles of participation will be forgotten over time or will be filed and archived in the patients charts as a few slips of useless instructions . Since a manager or coordinator should be selected for implementing each plan in the groups, the following steps should also be taken in the partnership plan: determining the head and manager of the partnership teamaccording to the nurses capabilities, it is better to give the responsibility of the team to its nurse based on members consensusteam members consensus on delegating the responsibility of the team to its head and manager, as well as cooperating with him / herimplementing the plan and undertaking the educational visits and required follow - ups . Determining the head and manager of the partnership team according to the nurses capabilities, it is better to give the responsibility of the team to its nurse based on members consensus team members consensus on delegating the responsibility of the team to its head and manager, as well as cooperating with him / her implementing the plan and undertaking the educational visits and required follow - ups . To identify patients problems, the measurement of biochemical compositions of the blood, weight gain between two hemodialysis sessions, edema, and hypertension was used . The patients were weighed before and after hemodialysis with predetermined dress and with a scale that was calibrated and was constant during the intervention . For measuring edema, the patients edema in the wrist, leg, and around the eyes was measured using the pitting edema grading scale . The edema in the hip and abdominal circumference was also measured by the meter . To gain more confidence in the data collected, blood biochemical compositions were measured within 2 months before training and other variables were measured two to four times with 2-week intervals before training and their mean values were considered as the data before training . Also, after training, the blood biochemical compositions were measured within 2 months and other variables were measured for two to four times with 2-week intervals and their mean values were considered as the mean after training . Quality of life was measured after training for two times, at 6 and 8 weeks after training, and their mean was calculated as the data after training . The study patients blood pressure was measured after 5 min of rest, in sitting mode, from the arm which did not have any fistula, in a position at heart level, and in a position in which the patient had an appropriate support and backrest . The education was provided to the patients through a training manual which had been prepared based on valid and scientific articles about exercise, sleep, nutrition, stress, hemodialysis equipment, hemodialysis methods, and psychology . Educating patients patients companions were also trained for 2 h in a class, and then, practical and additional training was provided for them during hemodialysis . The study patients were trained in all educational topics including the topics of nutrition, exercise, sleep, ways to prevent itching, etc ., for training the patients in the topic of exercise, a physiotherapist and the researcher gave theoretical and practical training to all the patients of a work shift (eight patients) for 2 h. afterward, in the next session, the researcher evaluated those patients, responded to their related questions, and explained that topic again to them (if needed) so that they learned the topic completely . Then, the researcher started training the other topic . Between the 1 and 2 weeks, the topic that was dealt with in the 1 week was evaluated . In all stages of the research, chicago, il, usa) and some statistical tests including paired samples t - test, wilcoxon and mcnemar tests . Paired samples t - test was used for analyzing and comparing the data collected on weight and blood pressure before and after the intervention . In order to assess insomnia, edema, and the itching status before and after the intervention, required data were collected through another part of the questionnaire with yes / no items and analyzed using mcnemar test . The results showed that most of the study patients were men (n = 17, 53.1%), in the 56 - 65 years age group, illiterate (n = 9, 28.1%), married (n = 23, 71.9%), employees (n = 18, 56.3%), and being treated with hemodialysis for 1 - 3 years (n = 13, 40.6%) [table 1]. Also, the results showed that the mean and standard deviation (sd) of patients parameters including weight and blood pressure improved significantly after the educational intervention compared to before the intervention (p <0.001). In addition, the mean of systolic and diastolic blood pressure decreased after the intervention (p <0.001) [table 2]. Demographic characteristics of the patients referred to the hospital for hemodialysis mean and standard deviation of the parameters considered (weight and blood pressure) in the study patients before and after the intervention while 46.8% of the study patients did exercise rarely before the intervention, 59.4% of them started doing exercise after the educational intervention . Before the intervention, all study patients were complaining of insomnia . However, after the educational intervention, only 59.4% patients had this problem (p <0.001). Also, the number of study patients with edema and itching before and after the educational intervention had significant differences (p <0.001) [table 3]. Comparison of the percentages of the study patients based on changes in their exercise, sleep, edema, and itching before and after the educational intervention table 4 shows that the differences between the mean values of the study patients laboratory tests before and after the intervention were statistically significant (p <0.001). Mean and standard deviation of the study patients laboratory test results before and after the intervention the mean and sd of the quality of life dimensions (including general health, vitality, physical function, role physical, bodily pain, mental health, social function, and role emotional) had been improved after the educational intervention . These differences were statistically significant before and after the intervention (p <0.001) [table 5]. Mean and standard deviation of the dimensions of the study patients quality of life before and after the educational intervention due to the low quality of life in hemodialysis patients, taking some measures to improve the quality of life of these patients is essential . The results of studies show that the quality of life indicates the quality of health care provided and is a part of hemodialysis patient care program . Measuring these patients quality of life will provide more information for the health team . In the field of nursing, conducting studies on the patients quality of life and forming interventions that would result in improved quality of life are growing rapidly, which leads to improvements in the lives of people with such chronic diseases . In the present study, in baraz et al . 's study, it was 46.69 . In another study which was conducted by pakpour et al . In their study, the performance of mental component was more than that of physical component . According to the results of rostami et al . 's study, the mean of hemodialysis patients quality of life was 44.29 . Therefore, the results of the present study and other studies conducted in iran show that the quality of life in hemodialysis patients in iran is not at a desirable level . In other words, it can be concluded that the psychological and physical quality of life in the patients treated by hemodialysis for a long period has been poor . 's study showed that the mean of all eight dimensions of the quality of life was higher than 50% and the overall mean of quality of life was 68.38 . In diaz - buxom et al . 's study, the mean of hemodialysis patients quality of life was more than that in the present study and other similar studies conducted in iran . Overall, the mean of quality of life in iranian hemodialysis patients compared to that in other countries is at a lower level, and the difference may be related to the factors such as study patients lifestyle, socioeconomic status, low level of awareness, and lack of appropriate self - care . Despite the lower quality of life of the patients in the present study, the mean of quality of life after implementing the educational plan significantly increased and indicated that the provided education was effective . In baraz's study, the mean of quality of life had increased from 46.69 to 54.64 after education, indicating its lower effectiveness compared to that in the present study results . Although these two studies have been conducted with the same methodology, their results are slightly different because of the differences in the factors among the study patients, such as sex, education level, and socioeconomic status . If they do not pay careful attention to their daily fluid intake, they are faced with fluid retention and problems such as general body swelling, shortness of breath, cardiac and pulmonary disorders, weight gain, etc . The present study results show that using the partnership care model had positive effects on some parameters in the study patients, such as their sleep, exercise, weight, and blood pressure . For instance, all these patients had sleep problem before the educational intervention; however, this problem decreased significantly and only 40.6% of them had it after the intervention . . 's study results showed that insomnia was a common problem in hemodialysis patients which had significant relationship with their low quality of life . Results of another study showed that implementing continuous care model had a positive effect on the quality of sleep in the hemodialysis patients . Braz found in his study that self - care education decreased the amount of edema significantly in hemodialysis patients . In the present study, the results of lugon's study showed that itching is an annoying sensation which is the common problem in most of the hemodialysis patients and can affect their quality of life and cause depression . In the current study, while the study patients were not exercising before the intervention, a large number of them (59.4%) started exercising after the intervention . The results of jennen and uhlenbruck's study showed that exercising increased the sense of satisfaction and the scores of quality of life dimensions . The mean of blood pressure and weight loss had significant difference before and after the intervention, as they improved after the intervention . The results of salehi's study showed that education had a positive effect on weight loss between two hemodialysis sessions . In the present study, that the mean of serum urea decreased significantly after training, which is similar to the present study results . Uremia causes irritability, loss of appetite, insomnia, drowsiness, fatigue, memory loss, confusion, making mistakes in judgments, and lack of focus, each of which has effects on the patients quality of life . The results of salehi's study showed that unlike the present study results, the mean of potassium levels did not show any statistically significant decrease after training . Advanced chronic renal failure is the main cause of increased blood potassium because the kidneys are unable to excrete excess potassium in the blood . However, in the present study, the blood potassium levels of the study patients decreased because of following correct nutrition and diet patterns . The results of the current study showed a statistically significant decrease in the mean of serum phosphorus levels after training . The study results of shichiri et al . Also showed that the serum phosphorus levels of the study patients decreased significantly after 5 weeks of training . One of the major complications of renal failure is renal osteodystrophy which occurs as a result of decrease in blood calcium and increase in blood phosphorus levels . Since hemodialysis cannot remove excess blood phosphorus, choosing the proper diet is important in reducing this type of disorder . In the present study,'s study showed that following a low - protein diet reduced the serum uric acid levels significantly . In the current study, systolic and the results of sarafi's study also indicated the positive effects of the training program on decreasing the blood pressures in hemodialysis patients . In the present study, the weight gain between two hemodialysis sessions increased the patients blood pressures by 3 mm hg / kg at the same time . The results of oka et al . 's study showed that the control of feeding behavior had significant negative correlations with blood urea nitrogen, potassium, phosphorus, as well as the weight gain between two hemodialysis sessions . 's study indicated that training hemodialysis patients in nutrition and diet, as well as in fluid intake can result in fluid intake restrictions and, subsequently, weight loss . The present study results show that the use of self - care model had a positive and significant effect on the study patients, as all quality of life dimensions improved after the educational intervention . Rahimi's study results showed that using continuous care model had positive effects on several parameters and indicators of the hemodialysis patients, such as their quality of life . Found in their study that after a 12-week exercise program, the psychological status, quality of life, and work capacity in hemodialysis patients significantly improved, which confirms the present study results . The results of tsay and lee's study showed that implementing an adaptation training program for patients with esrd decreased their stress and increased the physical and mental dimensions of their quality of life significantly, which is similar to the results of the current study . Learning rate of the study patients was different due to their individual and motivational differences and could not be controlled . Use of care models, especially those models which are compatible with the culture of our society, can increase the quality of life in the hemodialysis patients . Assisting the hemodialysis patients the results of the present study showed that using self - care model had significant effects on all quality of life dimensions, including social and physical function, general health, etc ., therefore, matrons and nursing managers in their planning for applying different education methods can use the self - care model in their units to improve the inpatients overall health status . In order to show the positive effects of using the self - care model and develop strategies for better implementation of this model, it is recommended to compare the effects of using this model with those of providing traditional physician visits and also conduct similar studies on hemodialysis patients in other hospitals with larger sample sizes . Also, conducting a study on the problems of and barriers to using this model is suggested . It is also suggested conducting similar studies using different educational methods and models to determine and compare their effectiveness.
Pioglitazone is the only thiazolidinedione (tzd) that is currently available in europe . During the last few years, the focus has been on the side - effect profile of this group of medications . In 2007, following a meta - analysis by nissen et al . On the cardiovascular safety of rosiglitazone, it was shown that rosiglitazone was associated with cardiovascular risk in terms of increased risk of myocardial infarction (mi). Further to this, the us food and drug administration (fda) have given strict criteria for new prescriptions and additional warning labeling for rosiglitazone . The european medicine agency (ema) has withdrawn the marketing authorization for rosiglitazone . Of late, concerns have also been raised regarding a possible link between pioglitazone use and bladder cancer [24]. Here, the authors review the risk benefit profile of pioglitazone, with a particular focus on the putative link between pioglitazone and bladder cancer . English language articles were included in the literature search, including the key terms pioglitazone, bladder cancer, and type 2 diabetes . The literature search included clinical trials, preclinical trials, and epidemiology and evidence reviews . Following concerns regarding the cardiovascular safety of rosiglitazone, the effect of pioglitazone on cardiovascular outcomes has been closely evaluated in recent years . Even though they belong to the same class, unlike rosiglitazone, pioglitazone has a favorable cardiovascular outcome . A meta - analysis of 19 trials comparing pioglitazone with placebo or active comparator in 16,390 patients with diabetes has shown that pioglitazone was associated with a significantly lower risk of death, mi, or stroke . Death, mi, or stroke occurred in 375 out of 8,554 patients (4.4%) on pioglitazone therapy as compared to 450 of 7,836 patients (5.7%) receiving controlled therapy [hazard ratio (hr) = 0.82; 95% ci, 0.720.94; p = 0.005]. In the prospective pioglitazone clinical trial in macrovascular events (proactive) study, the primary composite endpoint (mortality, nonfatal mi, silent mi, stroke, acute coronary syndrome, endovascular or surgical intervention on the coronary or leg arteries, major leg amputation) analysis failed to show a beneficial effect for pioglitazone . The hr for primary composite endpoint was 0.904 in favor of pioglitazone (95% ci, 0.801.08; p = 0.095). For the primary endpoint, the 3-year placebo event rate was 23.5% and the pioglitazone event rate was 21% (relative risk reduction of 10%); the number needed to treat (nnt) to prevent one cardiovascular event was 120 per year [69]. Analysis of the main secondary endpoint has shown a significant benefit for pioglitazone (hr = 0.84; 95% ci, 0.7220.981; p = 0.027). For the main secondary endpoint (placebo event rate 13.6% and pioglitazone event rate 11.6%), furthermore, the statistical significance is lost only when revascularization procedures are included within the primary composite endpoint . In other words, when commonly used cardiovascular outcomes (death, mi, or stroke) are evaluated, the statistically significant beneficial effect of pioglitazone is revealed . A further subgroup analysis of the proactive study (proactive 05) looked at the effect of pioglitazone on recurrent mi in 2,445 patients with diabetes and previous mi . This showed that patients treated with pioglitazone had a statistically significant beneficial effect on the prespecified endpoint of fatal and nonfatal mi (p = 0.045; 28% relative risk reduction) which translates into an estimated nnt of approximately 149 per year and acute coronary syndrome (p = 0.035; 37% risk reduction) compared with those treated with placebo . Two other studies which looked at the effect of pioglitazone on the progression of atherosclerosis have also shown a favorable outcome . Carotid artery intima media thickness (cimt) is a surrogate marker for cardiovascular disease . A study comparing the effect of pioglitazone versus glimepiride on changes in cimt in type 2 diabetes patients has shown that over an 18-month period, pioglitazone slowed progression of cimt compared with glimepiride . In the prospective evaluation of a risk score for postoperative pulmonary complications in europe (periscope) study, coronary intravascular ultrasonography (ivus) was used to assess the change in percent atheroma volume in patients with type 2 diabetes and coronary artery disease, and the study compared pioglitazone with glimepiride . In total, 360 patients completed the final ivus assessment at 18 months, which showed that pioglitazone - treated patients had a significantly lower rate of progression of coronary atherosclerosis, compared with patients treated with glimepiride . Both these studies have used surrogate markers to assess the effect of pioglitazone on atherosclerosis, which has shown that the pioglitazone slows the progression of atherosclerosis compared to an active comparator (sulfonylurea). In summary, pioglitazone appears to be associated with potential cardiovascular outcome benefits (table 1) [510].table 1evidence of cardiovascular outcome benefits from pioglitazonestudystudy designnumber of patientsendpointshazard ratiop valuenumber needed to treatpioglitazone meta - analysismeta - analysis on pioglitazone: 19 trials16,390death, mi, or stroke0.820.005proactive studypioglitazone vs. placebo5,238primary composite endpoint0.900.095120proactive studypioglitazone vs. placebo5,238secondary endpoint of death, mi, and stroke0.840.02143proactive 05 studypioglitazone vs. placebo but with history of mi2,445fatal and nonfatal mi0.720.045149pioglitazone on cimt studypioglitazone vs. glimepiride on changes in cimt462absolute change in mean posterior wall cimt0.02periscope studyivus assessment of progression of coronary atherosclerosis in patients treated with pioglitazone vs. glimepiride360 (study completion group)change in percent atheroma volume0.002cimt carotid artery intima media thickness, ivus intravascular ultrasonography, mi myocardial infarction, periscope prospective evaluation of a risk score for postoperative pulmonary complications in europe, proactive prospective pioglitazone clinical trial in macrovascular events primary composite endpoint of death, nonfatal mi, silent mi, acute coronary syndrome, stroke, revascularization, and major leg amputation evidence of cardiovascular outcome benefits from pioglitazone cimt carotid artery intima media thickness, ivus intravascular ultrasonography, mi myocardial infarction, periscope prospective evaluation of a risk score for postoperative pulmonary complications in europe, proactive prospective pioglitazone clinical trial in macrovascular events * primary composite endpoint of death, nonfatal mi, silent mi, acute coronary syndrome, stroke, revascularization, and major leg amputation tzds cause fluid retention and peripheral edema, predisposing the susceptible patients to heart failure . The pioglitazone meta - analysis revealed that serious heart failure was reported in 200 (2.3%) of pioglitazone - treated patients, compared with 139 (1.8%) of the control patients (hr = 1.41; 95% ci, 1.141.76; p = 0.002), but the composite endpoint of death and serious heart failure was not statistically different in the two groups . The data from the proactive study specifically looking at pioglitazone use and heart failure (proactive 08) also showed a significant increase in heart failure in pioglitazone - treated patients, but subsequent analysis of patients who developed serious heart failure has shown that the all - cause mortality was proportionately lower with the pioglitazone group (40/149; 26.8%) compared with the placebo group (37/108; 34.3%) (p = 0.1338). The composite endpoint of death, nonfatal mi, or stroke was much less in pioglitazone patients with serious heart failure (34.9%) compared with placebo (47.2%), which was statistically significant (p = 0.025). Experimental studies have reported that peroxisome proliferator - activated receptor (ppar) agonists like pioglitazone and dual ppar gamma / ppar alpha agonists like muraglitazar induce urothelial bladder tumors . Urothilium has plenty of ppar gamma receptors, and direct interaction of the agonist with the receptors ultimately leading to induction of cancer was one possible mechanism suggested . But it has been shown that even though the ppar gamma expression was at similar levels in rat and mouse urothelium, the tzd produced bladder tumors only in the rat and not in the mouse . In - vitro studies using human urothelial cell lines have shown that ppar gamma agonists inhibit cell proliferation, hence a direct carcinogenic effect to the urothelium by the ppar gamma agonist is less likely . Also, as ppar agonists are highly lipophilic, only a small percentage of administered drugs get excreted in the urine, reducing the exposure of the drug to the urothelium . The second postulated mechanism involves alteration to the urinary composition leading to cytotoxicity, necrosis, and urothelial proliferation . Studies have shown that both ppar gamma agonists and ppar gamma / ppar alpha agonists alter urinary composition, resulting in calcium containing urinary solids to varying degrees . The amount of urinary solids detected in rats treated with pioglitazone was less than that observed for rats treated with muraglitazar . As expected, muraglitazar induced a significantly higher incidence of bladder tumors than the small number induced by pioglitazone . The evidence from experimental studies supports the hypothesis that potential cytotoxicity may be related to urinary solids, namely calcium containing crystals and calculi . An interim report of a longitudinal cohort study conducted between 1997 and 2002, that assessed the risk of bladder cancer among diabetic patients treated with pioglitazone (including 193,099 patients in the kaiser permanente northern california diabetes registry,> 40 years of age) showed that after adjusting for age, sex, tobacco use, and use of other categories of diabetes medications, there was no significant increase in the risk of bladder cancer in patients ever exposed to pioglitazone, compared with patients never exposed to pioglitazone (hr = 1.2; 95% ci, 0.91.5), with similar results in men and women . However, the risk of bladder cancer rose with increasing duration of pioglitazone use . Compared to never being used, pioglitazone treatment for more than 24 months was associated with a 40% increase in relative risk of developing bladder cancer (hr = 1.4; 95% ci, 0.92.1). Based on these data, the fda calculated that duration of therapy longer than 12 months was associated with 27.5 excess cases of bladder cancer per 100,000 person - years follow - up, compared with no use of pioglitazone . More recently, piccinni et al . Have reported further evidence for the association of pioglitazone use and bladder cancer after analyzing the data from the fda s drug adverse event reporting system . The association between antidiabetic drugs and bladder cancer was analyzed by case / non - case methodology using reporting odds ratio (ror) as a measure of disproportionality . Overall, 93 reports of bladder cancer were retrieved, corresponding to 138 drug reaction pairs (obtained by splitting co - medications and multiple reactions reported for each case). The ror for bladder cancer was 4.3 for pioglitazone (95% ci, 2.826.52; p <0.001). The ror for gliclazide was 3.56 (95% ci, 1.428.39) and for acarbose it was 5.12 (95% ci, 1.014.33). With the number of bladder cancer cases reported with gliclazide and acarbose being small, it is too susceptible to reporting biases, and hence not clinically relevant . But this report concluded that there was a definite signal for bladder cancer associated with pioglitazone use . A retrospective cohort study using the data from the french national health insurance system also showed an increased risk of bladder cancer with pioglitazone use . This study was from a cohort of 1,491,060 diabetes patients on drug therapy, aged 4079, followed - up for up to 4 years (20062009). The results showed that after adjusting for age, sex, and use of other antidiabetic medications, there was a statistically significant increase in the risk of bladder cancer in patients exposed to pioglitazone, compared with patients exposed to other antidiabetic agents (hr = 1.22; 95% ci, 1.031.43). The results also showed a dose effect related to cumulative dose> 28,000 mg (hr = 1.75; 95% ci, 1.222.5) and for exposure longer than 1 year (hr = 1.34; 95% ci, 1.021.75). A significant increase in risk was observed in males (hr = 1.28; 95% ci, 1.091.51). Mesenchymal stem cells in the bone act as the common progenitor of both adipocytes and osteoblasts . This affects the bone density and osteoblast functioning, which leads to reduced osteoblast activity, shifting the balance of bone homeostasis to favor bone loss . Clinical studies have shown an increased risk of fractures, especially in females . In the periscope study, fractures occurred in 3% of patients treated with pioglitazone, compared with none in the glimepiride group . An analysis of the clinical trial database of the pioglitazone manufacturer with a special focus on fractures has shown an excess risk in female patients . The majority of fractures observed in female patients who received pioglitazone were in the distal upper limb (forearm, hand, and wrist) or distal lower limb (foot, ankle, fibula, and tibia). The calculated fracture incidence was 1.9 per 100 patient - years in the pioglitazone - treated group and 1.1 fractures per 100 patient - years in the comparator treated group, resulting in an excess fracture risk of 0.8 fractures per 100 patient - years of pioglitazone use . Following the retrospective study conducted by the national health insurance agency, the french agency for drug regulations (afssaps) have suspended the use of pioglitazone in france since june 2011 . The fda safety communication in june 2011 announced that the use of pioglitazone for more than 1 year may be associated with an increased risk of bladder cancer . It recommended not to use pioglitazone in patients with active bladder cancer, and to evaluate risks and benefits before initiating pioglitazone . The fda encourages physicians to counsel patients regarding the symptoms of bladder cancer . In july 2011, the ema concluded that the evidence from different sources showed a small increased risk of bladder cancer with pioglitazone, in particular in patients treated for the longest durations and with the highest cumulative doses . The ema advised the clinicians not to use pioglitazone in patients with a past or current history of bladder cancer or in patients with uninvestigated macroscopic hematuria . Various risk factors for bladder cancer (elderly people, smoking, previous exposure to occupational risk factors, or drugs related to a risk of bladder cancer) should be assessed before initiating pioglitazone . The balance of risk and benefits should be assessed before as well as during treatment with pioglitazone, to ensure that only patients who are deriving sufficient benefit continue to take it . Pioglitazone remains a useful blood glucose - lowering therapy and may produce significant clinical benefits for some patients, particularly from the perspective of potential cardiovascular outcomes . A number of safety concerns have been identified with pioglitazone, including an increased risk of cardiac failure, bone fracture, and a small but definite increase in the risk of bladder cancer . Thus, when considering the use of pioglitazone, an individualized patient risk benefit evaluation is essential with the use of pioglitazone being inappropriate in people considered at risk of bone fracture, cardiac failure, or bladder cancer.
The clinical history and investigation could raise the possibility of malignant disease in differential diagnoses . Young adult known case of essential hypertension on medication started to complain of bilateral flank pain which was intermittent not severe localized to his flank with no other symptoms . His laboratory investigation came pack normal he underwent abdominal ultrasound initially, which showed bilateral cystic renal masses images in fig . 1, ct scan done for further evaluation, which shows the same finding of non - enhancing cystic renal masses images in fig . The diagnosis being discussed with the patient himself after explaining the rarity of disease and management options available in the clinic and ease of follow up he decided for not persue any active surgical intervention with regular follow up in our out patient clinics no active surgical intervention were offered to the patient based on his request except analgesic medications as needed . Renal lymphoangiomatosis, rare benign congenital anomaly due to failure of lymphatic system around the kidney to drain to retroperitoneal lymphatic system, which will cause dilation of lymphatic duct and formation multiple cystic lesion in perinephric space and renal sinus . It might be familial, . This rare anomaly can be asymptomatic or can present as flank pain, hypertension, hematuria and prtienuria . The diagnosis of renal lymphangimatosis can be confirmed with needle aspiration of cystic fluide but the radiological feature can make the clinician make the diagnosis confidently, . The reported complication of renal lymphohangiomatosis are obstructive uropathy secondary hypertension due to obstruction and mass effect of the lesion . Pregnancy can be an exacerbating factor for this disease by increasing the cyst size and fluid collection . Percutaneous drainage has proved it's effectiveness in treating symptomatic patient and pregnant women, . 1.renal lymphangiomatsis is extremely rare disease with no sexual difference or age group tendency.2.can be asymptomatic or symptomatic due to the cyst it self or it's mass effect.3.radiological finding can help in making the diagnosis with no need for invasive procedure.4.usually require no treatment unless in specific condition . Can be asymptomatic or symptomatic due to the cyst it self or it's mass effect . Alqahtani, raed primary author alkhateeb, sultan secondary author who supervise the review almutaiti, bader the radiologist who report the image and made the diagnoses.
Histopathologic classification of pancreatic endocrine neoplasms is complex and the recent world health organization (who) classification of tumors of the digestive system divides pancreatic endocrine tumors into three grades based on mitotic rate and proliferation index (pi) as determined by ki-67 immunohistochemical staining . The who classification is formulated for the evaluation of histologic specimens which are paraffin embedded and formalin fixed . The papanicolaou society of cytopathology (psc) has developed a set of guidelines for the investigation and diagnosis of pancreatic and biliary tract lesions . The neoplastic category is further sub - divided into neoplasm, benign, and pancreatic endocrine neoplasms are placed in either the neoplasm, other or malignant categories depending on cytologic features . Several authors have applied the who recommendations for classification to formalin fixed paraffin - embedded cell block specimens obtained by fine needle aspiration (fna). These studies have shown good correlation between results obtained from cell block specimens and subsequent surgical specimens . Endoscopic ultrasound (eus) guided fna biopsies of pancreatic lesions do not invariably result in cell block material and may produce only smear preparations . Such preparations may be stained by either the diff - quik or the papanicolaou methods, and if rapid on - site evaluation is utilized, a significant proportion of the resultant smears are diff - quik stained . This may result in the only material available for evaluation being air - dried diff - quik stained smears . Little information exists as to criteria for separating cytologic specimens of pancreatic endocrine neoplasms into low-, intermediate-, and high - grade lesions corresponding to the who system . The psc guidelines recommend categorizing pancreatic endocrine neoplasms as high - grade lesions (malignant, pancreatic endocrine carcinoma) and low- or intermediate - grade lesions (neoplasm, other, pancreatic endocrine tumor). While the high - grade malignant lesions are invariably aggressive neoplasms and require an aggressive therapeutic approach, neoplasms cytologically characterized as neoplasm, other (pancreatic endocrine tumor) have a variable biological behavior with some showing aggressive features including metastases while others have a more indolent behavior . Malignant neuroendocrine carcinomas in the psc classification are histologically small cell and large cell neuroendocrine carcinomas with aggressive behavior . Neoplasm, other into those likely to behave aggressively and those with a more indolent clinical course . Prior studies have demonstrated the utility of cell block material for such separation but criteria for the analysis of smear preparations have not been published . We utilized a series of 36 pancreatic endocrine tumors including low-, intermediate-, and high - grade examples to develop a set of cytomorphologic criteria and a related scoring system to identify pancreatic endocrine neoplasms with a poor outcome in smear preparations . The present study was reviewed by the institutional review board and determined to be exempt . The case files of the department of pathology and anatomical sciences at the university of missouri and the department of pathology and laboratory medicine at the university of utah / arup laboratories were searched for all fna biopsies of pancreatic endocrine neoplasms performed between january 2008 and january 2014 . Neoplasm, other, pancreatic endocrine neoplasm and four classified as malignant, pancreatic endocrine carcinoma were identified and the corresponding slides obtained . Cases with biopsy or resection specimen were graded using the who recommendations and classified as neuroendocrine neoplasm grade 1, neuroendocrine neoplasm; grade 2 or neuroendocrine neoplasm; grade 3 . The histologic grading was based on ki-67 index, mitotic counts, the presence or absence of necrosis, and the presence or absence of angioinvasion . Histology, in some cases, was performed on only biopsy specimens while others were resection specimens . All cases had diff - quik stained smears, and these smears formed the basis of the present study . For each case, twenty morphologic features [table 1] were independently evaluated by two raters . Evaluations were made only on the basis of morphology as seen in diff - quik stained slide material . Diff - quik material was used as this is the preparation type used for rapid on - site evaluation . Raters were blinded to the original diagnoses and did not have access to clinical information . Both raters were board - certified cytopathologists with 8 and 30 years of experience, respectively . Chromatin stringing is defined as artifactual pulling of nuclear chromatinic material into long chords due to the smearing process . For cytologic evaluation, mitotic activity was graded as absent (no mitotic figures seen) or present (one or more mitotic figures seen in the smear). Poor outcomes included metastases, death, or high - grade lesions (pancreatic endocrine carcinoma) as determined by histologic evaluation of surgical specimens . Inter - rater agreement statistics for morphologic features associations between morphologic features (absent vs. present) and outcomes (poor vs. not poor) were evaluated using hierarchical logistic regression . Inter - rater agreement was assessed with absolute agreement and chance - corrected agreement (kappa). Discriminatory power was assessed using the area under the receiver operating characteristic (auroc) curve . Statistical calculations (kappa, logistic regression, roc analysis) were performed using the stata 13 (stata corporation, college station, tx, usa). Recursive partitioning was conducted using the jpm 11.0 (sas corporation, cary, nc, usa). Absolute inter - rater agreement ranged from 51.4% to 97.1% across the 20 morphologic features [table 1]. Table 2 shows the corresponding histologic diagnoses and clinical outcome with the cytologic category as poor or not poor outcome . Raters achieved kappa scores of at least 0.3 for seven morphologic features (chromatin stringing, prominent nucleoli, irregular nuclear membranes, nuclear grooves, mitotic figures, necrosis, and 3-fold variation in nuclear size). Tumor histologic grade, clinical follow - up, and cytologic risk for a poor outcome sensitivity and specificity both range from 4% to 100% [table 3]. Twelve of the twenty morphologic features showed statistically significant associations with poor outcomes [table 4]. Overall discriminatory power was highest for irregular nuclear membranes (auroc = 0.76), mitoses (auroc = 0.71), 3-fold variation in nuclear size (auroc = 0.67), and necrosis (auroc = 0.65). Accuracy statistics for morphologic features, area under the receiver operating characteristic curve strength of association between morphologic features and poor outcomes many of the morphologic features had statistically significant associations [table 5]. For example, chromatin stringing had a statistically significant correlation with mitotic figures (r = 0.50, p <0.001). Irregular nuclear membranes had statistically significant correlations with nuclear grooves, mitotic figures, 3-fold variation in nuclear size, and the presence of necrosis . Pairwise correlation matrix for selected morphologic features recursive partitioning analysis showed that mitotic figures had the highest discriminatory power [figure 1]. All cases with mitotic figures were associated with poor outcomes (specificity of mitoses = 100%). Cases without mitoses could be further classified on the basis of irregular nuclear membranes and 3-fold variation in the nuclear size . There were no poor outcomes in cases, which lacked mitoses, irregular nuclear membranes, and a 3-fold or greater variation in nuclear size . We developed two different scoring systems based on odds ratios [table 6] and recursive partitioning [table 7 and figure 1]. For the odds ratio method, we use the relative odds ratio of three factors: classification and regression tree for morphological features . The tree splits the cases based on the features with the highest discriminatory power (mitotic figures). Then for each branch, the feature with the highest discriminatory power is selected scoring based on odds ratios scoring based on recursive portioning score = 4l (mitoses) + 4l (irregular nuclear membranes) + l (3-fold variation in nuclear size) where l (x) is the indicator function and l (x) = 1 if feature x is present and l (x) = 0 if the feature x is absent [table 5]. For the partitioning method, roc analysis showed that there was no statistically significant difference between the two scoring systems . An roc curve for the score based on odds ratios is presented in figure 2 . Using the regression tree classification shown in figure 1, cytologic features stratified malignancy risk between 0% and 100% [table 1]. Using a risk of malignancy of 60% for assigning a specimen to the poor risk category, cytology correctly assigned specimens to the poor outcome category in 9 of 12 cases (75%) and when high - grade lesions were excluded, correct assignment to the poor outcome category occurred in 5 of 8 cases (62.5%). The scoring system correctly assigned a case to the favorable category (risk 20% or below) in 20 of 24 cases (83%). Histologic grading using the who system correctly predicted aggressive behavior in 14 of 13 cases (37.7%) and when high - grade lesions were excluded histologic grading failed to predict aggressive behavior in any case . Figures 35 illustrate grade 1 (risk of poor outcome 20% or less), grade 2 (risk of poor outcome> 20 and 60%), and grade 3 (risk of poor outcome 100%). Receiver operating characteristic curved for scoring scheme based on odds ratio photomicrograph of low - grade neuroendocrine tumor . No mitotic figures were present (diff - quik, 1000) photomicrograph of intermediate - grade neuroendocrine tumor . Cells have smooth nuclear contours, but greater than a 3-fold variation in nuclear size (diff - quik, 400) photomicrograph of high - grade (small cell carcinoma) neuroendocrine tumor . In 2010, the who published a classification for pancreatic endocrine neoplasms addressing both staging and grading issues . They proposed a three - tiered categorization system based on mitotic rate and pi as determined by ki-67 staining . The who grading scheme is based on histopathologic material, and specific guidance was not given for the classification's application to cytologic material . The psc developed guidelines for pancreatic and biliary tract cytology which include a categorization scheme with diagnostic criteria . In the psc guidelines, grade 1 and 2 neoplasms of the who recommendations are placed in the neoplasm, other category and further categorized as pancreatic endocrine neoplasms . Who high - grade lesions are placed in the malignant category with further categorization as pancreatic neuroendocrine carcinoma, either small or large cell variants . The who grading system for pancreatic endocrine neoplasms is most appropriately performed on resection specimens and not small biopsies as the small biopsy may not be representative of the final resection specimen grade . This limitation on histologic grading may be a reason for the superior predictive value of the cytologic grade over the histologic grade obtained from small biopsy specimens in the present study . Assignment of who low- and intermediate - grade pancreatic endocrine tumors to the neoplasm, other category of the psc guidelines is based on the desire to maximize therapeutic options for treating clinicians and their patients . Thus, patients with histologically low- and intermediate - grade pancreatic neuroendocrine tumors could be followed if the patient's clinical status made operative intervention undesirable . These lower grade neoplasms were not assigned to the malignant category because it was believed such an assignment would make it difficult to clinically follow patients with low- and intermediate - grade pancreatic endocrine neoplasms if such neoplasms were designated cytologically malignant . The psc guidelines made no attempt to distinguish between low- and intermediate - grade pancreatic endocrine tumors of the who classification and assigned both to a single category . A number of authors have recommended performing ki-67 immunohistochemical staining on formalin fixed paraffin - embedded cell block preparations to prognostically stratify pancreatic endocrine neoplasms and have shown good correlation between such cytologic stratification and subsequent histologic grading of surgical resections . This approach does require cell block preparations which may not always be obtained during eus - fna sampling . In some cases, only smear preparations are available for analysis and little data exists as to the possibility of and the methodology for stratifying pancreatic endocrine tumors in smear preparations . We analyzed a series of 36 pancreatic endocrine neoplasms of which 32 were designated as neoplasm, other (pancreatic endocrine tumor) and four were diagnosed as malignant: pancreatic endocrine carcinoma . We rated the diff - quik stained smear preparations for the presence or absence of 20 morphologic features and correlated these features with poor outcome (development of metastatic disease, death or high - grade history morphology on resection). While several of the features analyzed are nuclear features and might be better evaluated on papanicolaou - stained preparations, the staining preparation generally used for rapid on - site assessment is air - dried diff - quik stained material . This preparation appears suitable for assessment of the features utilized in this study . Using these correlations poor outcomes were unlikely (<20%) if mitoses were not present and the cells demonstrated no nuclear membrane irregularity or 3-fold variation in nuclear size . Cases that lacked mitotic figures on smear preparations but demonstrated nuclear membrane irregularity and a 3-fold variation in nuclear size had a 60% chance of a poor outcome . Morphologic features in our study were highly correlated [table 3], so that it is difficult to build a predictive model . Once a feature is selected, correlating other features adds relatively little information to the model . The scoring system developed by the odds ratio method utilized three morphologic factors as shown below: score = 4l (mitoses) + 4l (irregular nuclear membranes) + l (3-fold variation in nuclear size) where l (x) is the indicator function and l (x) = 1 if the feature is present and 0 if the feature is absent [table 6]. For the partitioning method, scores were based on the five categories shown in figure 1 [table 7]. Using recursive partitioning analysis, mitotic figures were shown to have the highest discriminatory power [figure 1]. All cases which had mitotic figures were associated with a poor outcome and specificity for mitoses was 100% . Cases lacking mitoses were further classified on the basis of irregular nuclear membranes and 3-fold variation in nuclear size . When mitotic figures, irregular nuclear membranes and 3-fold variation in nuclear size were absent, no poor outcomes were present . The evaluation of specimens for these three markers is a sensitive measure for poor outcome . Roc analysis demonstrated that there was no statistically significant difference between the two scoring systems . The present study demonstrated that a simple scoring system utilizing the presence of mitotic figures, irregular nuclear membranes, and 3-fold or greater variation in nuclear size can subdivide pancreatic endocrine neoplasms into those with poor and those with a more favorable outcome . Given the prominence of mitotic rate and pi (ki-67) in the who classification, it is not surprising that the presence of any mitotic figures is a strong predictor of poor outcome in smear preparations . The proposed scoring system is applicable to diff - quik stained smear preparations and is useful for evaluating both of the psc categories neoplasm, other (pancreatic endocrine neoplasm) and malignant (pancreatic endocrine carcinoma). The cytologic system had superior predictive value for poor outcome than did the who grading system which had an accuracy of classification of 69% (24 of 36 cases). The cytologic system is most useful when cell block preparations are not available for analysis . From the previously published data, cell block immunohistochemical staining for ki-67 appears to represent the optimal technique to evaluate grade in cytologic specimens obtained from pancreatic endocrine neoplasms . Because our proposed cytologic scoring system yielded higher accuracy than histologic evaluation of small biopsy specimens, it might be considered as superior to evaluation by the who system for small samples . However, several studies have demonstrated the high reliability of cell blocks for grading, so we recommend their use when such material is available . When only smears are available for analysis, our scoring system appears to be a helpful alternative . Lester j. layfield, m.d . Developed the study concept and design, evaluated the specimens for data collection and wrote the draft article . Robert l. schmidt m.d ., ph.d . Performed the data analysis and helped prepare the draft article . Jack campbell, ba collected the cases for analysis and prepared the data for analysis . The present study was reviewed by the institutional review board and determined to be exempt . Eus - endoscopic ultrasound fna - fine needle aspiration pi - proliferation index psc - papanicolaou society of cytopathology who - world health organization to ensure the integrity and highest quality of cytojournal publications, the review process of this manuscript was conducted under a double - blind model (authors are blinded for reviewers and vice versa) through automatic online system.
The first line therapeutic modality for non - surgical patients with large and multifocal hepatocellular carcinoma (hcc) who do not have vascular invasion or extrahepatic spread is transcatheter arterial chemoembolization (tace). However, transcatheter arterial radioembolization (tare) with yttrium-90 (y)-labeled microspheres has an emerging role in treatment of unresectable hcc patients with or without portal vein thrombosis, or as a bridge to transplantation.1,2 radioembolization refers to injection of embolic beads loaded with radioisotopes using transarterial technique . Microsphere radioembolization allows the delivery of radioactive beads directly to the site of malignancy, thereby minimizing toxicity to adjacent organs . Despite direct delivery to tumor site, complications still occur when the microembolic effect or radiation injury occurs in organs other than liver . Reported side effects are predominantly constitutional symptoms such as fever while others are gastrointestinal ulceration or bleeding, cholecystitis, pancreatitis, radiation pneumonitis and hepatic decompensation.3 herein, we report a case of severe gastric ulceration with bleeding that eventually required surgery due to aberrant deposition of microspheres . A 67-year - old male, inactive hepatitis b virus (hbv) carrier (hbeag negative with undetectable serum hbv dna level), was admitted for further evaluation and treatment of hepatic masses . Laboratory tests showed serum albumin level of 3.7 g / dl, bilirubin level of 1.5 mg / dl, and prothrombin time international normalized ratio of 1.40 . Neither ascites nor hepatic encephalopathy was found, which was compatible with child - pugh class a. serologic marker for hepatitis c was negative . Dynamic enhanced computed tomography (ct) revealed underlying liver cirrhosis with a hepatic mass measuring 2.7 cm in segment 4 and another lesion measuring 0.6 cm in segment 7 with typical patterns of arterial enhancement and delayed washout . Gadoxetic acid - enhanced liver magnetic resonance imaging showed slightly high signal intensity in the t2 and diffusion - weighted image with a defect in 20-minute delayed image for these two lesions (fig . 1). These findings were compatible with hcc (t3n0m0), stage iii . Radiofrequency ablation (rfa) or tace can be considered as locoregional treatments for these unresectable hcc nodules in this cirrhotic patient who had no living donor for liver transplantation . However the lesion in segment 4 located adjacent to small bowel and gallbladder, the interventional radiologist concerned about the risk of ablation - related complications and recommended tare . Angiographic studies to detect the variation of arterial vessels that distribute to the liver and other vessels that give access to extrahepatic organs as well as the review of the portal vein patency were done . After angiographic study, technetium (tc)-99 m macroaggregated albumin (maa) was injected to determine the degree of shunt to the lung as well as gastrointestinal tract . The calculated extrahepatic shunt was 10.71% . In the pre - evaluation study, prior to the embolization, empirical coil embolization for the gastroduodenal artery was done to prevent complication of non - target lesion . A dose of 0.85 gbq of y microspheres was delivered via left hepatic artery and right posterior segmental artery, through 2.7f microcatheter (fig . Immediately after the procedure, patient complained severe abdominal pain but post - tare scan without injection of tc-99 m maa demonstrated tumoral uptake of y but no gastrointestinal uptake was observed . Pain was controlled with tramadol and the patient was discharged one week after tare . However, the patient was readmitted due to severe abdominal pain four days after discharge . Upper endoscopy revealed geographic active ulcer at the lesser curvature of gastric antrum and mid - body . Histopathologic findings of endoscopic biopsy showed several deposited microspheres without evidence of helicobacter pylori infection . Gastric ulcer was treated with full ulcer medications including an intravenous bolus followed by continuous - infusion of proton pump inhibitor . Follow - up enhanced computed tomography which was performed one month after tare showed diffuse gastric wall thickening of antrum and lower body while hcc nodules in segment 4 and 7 were completely treated without recurrence . As abdominal pain failed to resolve with continuous medications, follow - up upper endoscopy was done two weeks later . Worsening of ulcerative mucosal lesions with exudative bases accompanied by diffuse mucosal friability and easy - touch bleeding were noted . Three additional endoscopic examinations were done to assess the lesions, but the aforementioned ulcers as well as the territory of the lesion were not grossly changed (fig . Surgical intervention was recommended due to persisting excruciating abdominal pain and refractory endoscopic findings, and finally, subtotal gastrectomy was performed three months after tare . Gross pathologic specimen revealed diffuse ulceration with multiple scattered microspheres deposited in the whole gastric layers, vessels and perigastric lymph nodes (fig . The patient's symptom resolved after surgery and he was discharged two weeks later without any complication . Patients treated with chemoembolization for the treatment of hcc are reported to show similar survival time to those treated with y labeled microspheres radioembolization . One of the advantages of tare is decreased embolic effect compared to tace allowing treatment for those with main portal vein thrombosis . Furthermore, tare resulted in longer time - to - progression and less toxicity than tace.4,5 although complication rate may be less than tace, gastrointestinal ulceration following tare can lead to fatal bleeding, small bowel obstruction and perforation.6 these complications occur due to non - target spheres distributed to aberrant vessels supplying gastrointestinal tract as well as changes in flow dynamics during infusion . Early studies showed high incidence (25%) of gastrointestinal ulceration, but the incidence dropped to less than 5% after more extensive angiographic search to assess the hepatic vasculature and its collateral vessels that supply gastrointestinal tract as well as lung shunting.6,7,8 one of the studies reported that median ulceration rate was 8%, ranging from 0 to 20%, with 6% (0.4% of the entire cohort) requiring surgery.6 prior to treatment, angiographic study is performed to minimize any complication . According to study by song et al . Based on 250 patients,9 the most common aberrant artery was the right gastric artery (rga) 78.4% and left gastric artery 17.2% while hepatic falciform artery, posterior superior pancreaticoduodenal artery and left inferior phrenic artery comprising the rest of the aberrancy . In our case, celiac and superior mesenteric arteries were evaluated, especially the right, and left gastric arteries which supply gastrointestinal tract with high aberrancy rate but no aberrant vessel was observed . Microcoil embolization is the most widely adopted method for preventing extraheptic arterial embolization, while balloon occlusion technique is another choice to occlude hepatopetal flow in the common hepatic artery which is less commonly used.10,11,12 finally, tc-99 m maa is injected into hepatic artery followed by single photon emission computed tomography to determine the degree of pulmonary and extrahepatic shunting.7,8 all these planning studies were performed in this patient and coiling was done for gastroduodenal artery but not for rga before tare . Despite aggressive pre - evaluation, 3 - 5% incidence of gastrointestinal complication occurs due to failure in detecting smaller vessels or collaterals developing in several weeks after empirical coil embolization.13 another possibility of gastrointestinal injury is due to dynamic changes which may occur from embolic effect of microspheres due to slowing of antegrade flow that increases the risk of particle reflux.14 besides this, catheter stimulus may cause vessel spasm, infrequently . In this case, complication could have been prevented if the rga was coil embolized prior to radioembolization considering the high incidence of gastric ulceration due to reflux of particles into rga . Small collateral vessels and hypertrophy of other vessels might develop after coil embolizations which were unnoticed during the procedure . Our case emphasizes the importance of finding collateral vessels meticulously even after coil embolization to ensure that there is no other vessels that might lead to reflux of particle into vessels feeding gastrointestinal tract . Time to diagnosis varies from less than 1 month to over 9 months and mean time to diagnosis is reported to be 3.2 months.8 therefore gastric ulceration due to tare should be suspected if severe abdominal pain persists and clinicians must not hesitate to perform endoscopy . Antral and pyloric channel ulcerations with extension to duodenum are common.13 endoscopic findings including erosions, erythema, exudation, friability and bleeding are not specific findings, so biopsy is required to definitely distinguish radioembolization - induced ulceration from other causes of ulcer . The characteristic finding that allows diagnosis of radioembolization - induced ulceration is microsphere particles lodged in vessels of hematoxylin and eosin stained specimen . Other supporting findings of radioembolization - induced ulceration are apoptosis, epithelial flattening, and glandular cystic dilatation to nuclear atypia, capillary ectasia, and prominent endothelial cells.15 since radioembolization associated ulceration is diagnosed pathologically, repeated endoscopic examinations and multiple biopsies are needed if initial biopsy reveals negative result . Treatment of radioembolization - induced gastric ulcer is challenging because there is no consensus for management . Currently, the treatment is no different from medications that are prescribed for peptic ulcer disease, namely acid suppression . Ulceration result from ischemic injury due to occlusion of arteries supplying gastrointestinal tract, direct toxic effect of radiation or both . Radiation destroys parietal cells leading to decreased gastric secretion, thus unlike peptic ulcer disease that originates from acid - induced mucosa, injury, acid suppression treatment may not be effective.16 additionally radioembolization induced ulcers occur at the serosal surface not mucosal surface causing lack of response of acid suppression therapy which acts primarily at the mucosa.3 radiation enteritis is known to be caused by excessive stimulation of transforming growth factor (tgf)-1 which leads to fibrosis and organ failure . Interferon- inhibits the effect of tgf-1.17 therefore, pentoxyfylline, a phosphodiesterase inhibitor that reduces inflammation mediated by cytokines including tgf- can be effective in radioembolization associated gastric ulcer.7 besides this, the effectiveness of anti - inflammatory agent such as sulfasalazine and antioxidant that reduces radiation induced free radical formation such as tocopherol can be considered as treatment options.8 since there is no one validated effective treatment, outcome can be fatal if appropriate time for surgical approach is missed . Early surgical intervention should be considered when abdominal pain persists and mucosal ulceration aggravates in endoscopic findings . In summary, radioembolization - induced ulcer can be irreversible and can be life - threatening and therefore, aggressive pre - evaluation angiography and preventive coiling of aberrant vessels should be done before performing the procedure . This is the first reported, preceptive case of radioembolization - induced severe gastric ulcer in korea which emphasizes the operator perception of the possibility of reflux into gastrointestinal artery and the need of surgery when gastric ulcer is refractory to medical treatment.
Parkinson's disease (pd) is associated with a selective loss of the neurons in the midbrain area called the substantia nigra pars compacta . These neurons contain the neurotransmitter dopamine (da), and their projecting nerve fibers reside in the striatum . Because these neurons control voluntary movements, the degeneration leads to four cardinal, debilitating symptoms: resting tremor, muscular rigidity, bradykinesia, and postural imbalance . Occupational uses of herbicides or pesticides, exposure to organic solvents, carbon monoxide, and carbon disulfide, and more generally, industrialization, rural environment, well water, plant - derived toxins, and bacterial and viral infection are all thought to play roles . Aging is an obvious factor associated with the onset of pd, and failure of normal cellular processes that occurs with aging is believed to cause increased vulnerability of daergic neurons . Familial forms of pd involving mutations in a number of genes have also been described . The mechanism by which mutation of these genes lead to degeneration of the nigral neurons have shed light to understanding of the pathophysiology of pd . In both idiopathic and genetic cases of pd, oxidative stress is thought to be the common underlying mechanism that leads to cellular dysfunction and demise . As such, the substantia nigra of pd patients exhibit increased levels of oxidized lipids, proteins and dna and decreased levels of reduced glutathione (gsh). Oxidative stress occurs when an imbalance is formed between production of reactive oxygen species (ros) and cellular antioxidant activity . Because of the presence of ros - generating enzymes such as tyrosine hydroxylase and monoamine oxidase, the daergic neurons are particularly prone to oxidative stress . In addition, the nigral daergic neurons contain iron, which catalyzes the fenton reaction, in which superoxide radicals and hydrogen peroxide can contribute to further oxidative stress . Because of this intrinsic sensitivity to reactive species, a moderate oxidative stress can trigger a cascade of events that lead to cell demise . The major sources of such oxidative stress generated for the nigral daergic neurons are thought to be the ros produced during da metabolism, mitochondrial dysfunction, and neuroinflammation, as discussed below in more detail . . Lines of evidence suggest oxidation of da and consequent quinone modification and oxidative stress as major factors contributing to the vulnerability of daergic cells . Although da is normally stored in vesicles, excess cytosolic da is easily oxidized both spontaneously and enzymatically to produce da quinone . The da quinone species are capable of covalently modifying cellular nucleophiles, including low molecular weight sulfhydryls such as gsh and protein cysteinyl residues, whose normal functions are important for cell survival . Notably, da quinone has been shown to modify a number of proteins whose dysfunctions have been linked to pd pathophysiology, such as -synuclein, parkin, dj-1, and uch - l1 . Da quinone covalently modifies -synuclein monomer and promotes the conversion of -synuclein to the cytotoxic protofibril form . The da quinone - modified -synuclein is not only poorly degraded but also inhibits the normal degradation of other proteins by chaperone - mediated autophagy . Conversely, -synuclein can bind to and permeabilize the vesicle membrane, causing leakage of da into the cytosol and this would in turn induce da quinone generation . Parkin is also covalently modified by da and becomes insoluble, which leads to inactivation of its e2 ubiquitin ligase activity . Catechol - modified parkin has been detected in the substantia nigra but not other regions of the human brain, and parkin insolubility is observed in pd brain . In addition, da quinone modification of uch - l1 and dj-1 has also been observed both in brain mitochondrial preparations and daergic cells . Since both uch - l1 and dj-1 contain a cysteine residue that is important for their activity [12, 13] and their oxidative modification at cysteine has been observed in pd [14, 15], the da quinone modification is likely the cause of inactivation of these enzymes . Da quinone has also been shown to cause inactivation of the da transporter and tyrosine hydroxyalse . Accordingly, the subunits of complex i and complex iii of the electron transport chain, whose dysfunction will deter mitochondrial respiration and cause ros production, were also shown to be targets of da quinone modification . In addition, er-60/grp58/erp57 and protein disulfide isomerase-5, the proteins that assist in protein folding in the endoplasmic reticulum, are also modified by da quinine . Da metabolites have also been shown to induce proteasomal inhibition, which can lead the cells to undergo apoptosis . Furthermore, da quinone can cyclize to become the highly reactive aminochrome, whose redox - cycling leads to generation of superoxide and depletion of cellular nadph, and which ultimately polymerizes to form neuromelanin . Moreover, hydrogen peroxide is generated during da metabolism by monoamine oxidase and is subsequently converted to the highly reactive hydroxyl radical in the presence of transition metal ions, contributing to oxidative stress . Evidence of the existence of in vivo da oxidation and its toxicity is also available . Neuromelanin, the final product of da oxidation, is accumulated in the nigral region of the human brain . Higher levels of cysteinyl - catechol derivatives are found in postmortem nigral tissues of pd patients compared to age - matched controls, suggesting cytotoxic nature of da oxidation . In animals, da directly injected into the striatum caused selective toxicity to daergic terminals that was proportional to the levels of da oxidation and quinone - modified proteins . Mice expressing a low level of ventricular monoamine transporter-2, presumably with increased cytosolic da level, showed evidence of da oxidation and age - dependent loss of nigral da neurons . Neurons depend heavily on aerobic respiration for atp, and hydrogen peroxide and superoxide radicals are normally produced during oxidative phosphorylation as byproducts in the mitochondria . Any pathological situation leading to mitochondrial dysfunction can cause a dramatic increase in ros and overwhelm the cellular antioxidant mechanisms . Oxidative stress causes peroxidation of the mitochondria - specific lipid cardiolipin, which results in release of cytochrome c to the cytosol, triggering apoptosis . Because daergic neurons are intrinsically more ros - generating and vulnerable as described above, any event that triggers further oxidative stress can be harmful to the cell . Damage to mitochondrial complex i in the electron transport chain causes leakage of electrons, which in turn causes ros generation . As such, the complex i inhibitors rotenone and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (mptp), when injected intraperitoneally, exert preferential cytotoxicity to the daergic neurons . Indeed, reduced complex i activity has been found in tissues from subjects with pd . Higher numbers of respiratory chain deficient da neurons have been found in pd patients than in age - matched controls . A line of evidence for mitochondrial dysfunction related to oxidative stress and daergic cell damage comes from the findings that mutations in genes of mitochondrial proteins parkin, dj-1, and pink are linked to familial forms of pd . Mice deficient in parkin gene have shown reduced striatal respiratory chain activity along with oxidative damage . Dj-1 is a mitochondrially enriched, redox - sensitive protein and an atypical peroxiredoxin - like peroxidase that scavenges h2o2, and dj-1 ko mice accumulate more ros and exhibit fragmented mitochondrial phenotype . In addition, -synuclein, although mostly cytosolic, seems to interact with mitochondrial membranes and to inhibit complex i . Neuronal loss in pd is associated with chronic neuroinflammation, which is controlled primarily by microglia, the resident innate immune cells and the main immune responsive cells in the central nervous system . Microglial reaction has been found in the sn of sporadic pd patients as well as familial pd patients and in the sn and/or striatum of pd animal models elicited by mptp . Microglia are activated in response to injury or toxic insult as a self - defensive mechanism to remove cell debris and pathogens . When activated, they release free radicals such as nitric oxide and superoxide, which can in turn contribute to oxidative stress in the microenvironment . Overactivated and/or chronically activated state of microglia causes excessive and uncontrolled neuroinflammatory responses, leading to a self - perpetuating vicious cycle of neurodegeneration . This is thought to be exacerbated by inflammatory signals generated by molecules released from damaged neurons, leading to induction of reactive microgliosis . The oxidized or ros - induced molecules that are released from damaged nigral daergic neurons and trigger microglial activation include neuromelanin, -synuclein, and active form of mmp-3, as described below . Neuromelanin is the dark insoluble polymer produced from da oxidation and confers the dark pigmentation to the substantia nigra . Insoluble extraneuronal neuromelanin granules have been observed in patients of juvenile pd and idiopathic pd, as well as those with mptp - induced parkinsonism . Addition of neuromelanin extracted from pd brain to microglia culture caused increases in and nitric oxide . Intracerebral injection of neuromelanin caused strong microglia activation and a loss of daergic neurons in the substantia nigra . Neuromelanin appears to remain for a very long time in the extracellular space and thus thought to be one of the molecules responsible for inducing chronic neuroinflammation in pd . Although mostly intracellular, a fraction of -synuclein fibril is released from neurons, and -synuclein is found in the cerebrospinal fluid from pd patients and normal subjects, and in human plasma . The addition of aggregated human -synuclein to a primary mesencephalic neuron - glia culture causes activation of microglia and daergic neurodegeneration, and this cytotoxicity does not occur in the absence of microglia . In addition, neuron - derived -synuclein stimulates astrocytes to produce inflammatory modulators that augment microglial chemotaxis, activation and proliferation . Nitration of -synuclein, presumably due to increased nitric oxide, facilitates the neuroinflammatory responses . More recently, it has been shown that transgenic mice expressing mutant -synuclein developed persistent neuroinflammation and chronic progressive degeneration of the nigrostriatal da pathway when inflammation was triggered by a low level of lipopolysaccharide . The active form of mmp-3 is increased in response to oxidative stress in daergic cells, and mmp-3 causes activation of microglia, which in turn would generate reactive nitrogen species and ros [48 - 51]. In mmp-3 knockout mice, the microglial activation following exposure to mptp is abrogated, and this is accompanied by a lower level of superoxide production compared to their wild type . Mmp-3 causes cleavage of protease activated receptor-1 (par-1), whose removal of n - terminal extracellular domain renders the remaining domain acting as a tethered ligand, subsequently triggering generation of intracellular signals and activation of microglia . In addition, mmp-3 participates in expression of inflammatory cytokines in activated microglia, and conversely, mmp-3 is induced by free radicals and the cytokines in these cells . Therefore, a vicious cycle may exist, where mmp-3, whose expression is induced by oxidative stress, is released from daergic neurons and leads to production of free radicals and cytokines in the microglia . Mmp-3 can also cause degradation of blood brain barrier and infiltration of neutrophils, which can further contribute to neuroinflammation . Currently, there is no therapy clinically available that delays the neurodegenerative process itself, and therefore modification of the disease course via neuroprotective therapy is an important unmet clinical need . Thus, understanding of the pathophysiology and etiology of the disease at cellular and molecular levels and finding molecular targets for neuroprotective / disease - modifying therapy is the crucial issue in the field of basic pd research . As described above, oxidative stress originating from da metabolism, neuroinflammation and mitochondrial dysfunction is thought to be the hallmark of pd pathogenesis, and antioxidant mechanism should prove to be an effective neuroprotective therapy for pd . However, no direct antioxidant, such as vitamin c, vitamin e, and coenzyme q10, has provided disease modification in pd patients . Doxycycline, a tetracycline derivative that penetrates the blood brain barrier, suppresses the increase in mmp-3 gene expression as well as nitric oxide and inflammatory cytokines and provides protection of the nigral daergic neurons in the mptp - induced mouse model of pd . A novel synthetic compound 7-hydroxy-6-methoxy-2-propionyl-1,2,3,4-tetrahydroisoquinoline, which downregulated expression of mmp-3 along with il-1, tnf- and cyclooxygenase-2, provided neuroprotection in both cell culture and animal models of pd . The enzyme nad(p)h: quinone reductase (dt - diaphorase; nad(p)h-(quinone acceptor) oxidoreductase; ec 1.6.99.2; nqo1) catalyzes two - electron reduction of quinone to the redox - stable hydroquinone . Since da and its metabolites have been implicated in the pathogenesis of pd, nqo1 may exert a protective effect against such conditions . Indeed, nqo1 protected against damaging effects of cyclized quinones and oxidative stress induced during their redox cycling . Induction of nqo1 by sulforaphane protected against neurocytotoxicity associated with da quinone in vitro and against mptp - elicited toxicity in vivo . In addition, nqo1 is known to maintain both -tocopherol and coenzyme q10 in their reduced, antioxidant state . While nqo1 is abundant in the liver where it participates in the phase ii detoxification, the enzyme is also expressed in the brain . In addition to its predominant expression in astrocytes, nqo1 is also expressed, albeit to a lesser degree, in dargic neurons in the substantia nigra . Moreover, a marked increase in the neuronal expression of nqo1 was consistently observed in the parkinsonian substantia nigra . A polymorphism (c609 t) of nqo1 that results in a decrease or total loss of its expression is reported to be associated with pd, although another group reported no such association . Cellular induction of nqo1 is achieved by the transcription factor nrf-2 binding to a cis - acting enhancer sequence termed antioxidant response element (are). Nrf-2 is normally present in the cytosol bound by the cytosolic protein keap1, but is released and translocated into the nucleus in response to a variety of cellular or exogenous signals . Ways to induce nqo1 expression and nrf2 activation should therefore serve as viable approaches to develop neuroprotective therapy for pd . Pd pathogenesis seems to be closely related to oxidative stress due to ros generated by da metabolism, mitochondrial dysfunction and neuroiniflammation . Because there is no current therapy available that delays the neurodegenerative process, development of drugs that will modify the course of pd is crucial . Intensive studies are being carried out worldwide toward understanding the molecular mechanism of cell demise in pd, and the results are actively being utilized in attempts to design disease - modifying drugs for this devastating disease.
Female patient aged 75 years who had no history of chronic disease apart from depression presented at outpatient clinic with complaints of lassitude, loss of appetite, exertional dyspnea for 4 months, and dry coughing for 2 months . Her examination revealed marked decrease in respiratory sounds at basal segments of the right lung . Hematological values, sedimentation, glucose, urea, creatinine, sodium, potassium, total protein, and albumin levels were within normal limits . Levels of alanine aminotransferase (189 u / l), aspartate aminotransferase (113 u / l), cholesterol (251 mg/ dl), and lactate dehydrogenase (ldh) (193 u / l) were also measured . X - ray graphy revealed a dark shadow, consistent with pleural fluid masking the demarcation line between mediastinum and pleura at the inferior zone of the right lung (figure 1). Thoracic computed tomography (ct) revealed multiple sites of lymphadenopathy, the largest containing dense calcification 22 mm in size, and marked pleural effusion on the right side (figure 2). Milky fluid with high density sampled from pleural effusion did not precipitate after centrifugation, and contained tg (1072 mg / dl), cholesterol (173 mg / dl), ldh (148 u / l), and albumin (2.7 g/ dl) at indicated concentrations . Sputum arb -negativity was detected 3 times, and bronchoscopy of the patient did not reveal any endobronchial pathology . Positron emission tomography - ct was obtained to rule out mediastinal tumor . During lymphatic scanning performed to detect etiology of chylothorax, images taken at second, fourth, and sixth hours demonstrated passage of radiographic contrast substance through bilateral inguinal, parailiac, paracaval, and paraaortic lymph nodes, and accumulation of radioactivity in these lymph nodes at popliteal, femoral, and pelvic regions, respectively . On delayed thoracic images, no uptake of radioactivity apart from background activity was observed . Mediastinal lymph node biopsy performed at external center revealed presence of granulomas demonstrating caseous necrosis . Symptoms regressed with treatment, and level of liver enzymes decreased . At sixth month, marked regression in pleural effusion was observed (figure 3). A dark shadow can be seen, consistent with pleural fluid masking the demarcation line between mediastinum and pleura at the inferior zone of the right lung . Chylothorax was first defined by bartolet in 1633, and the first case was presented by quinke in 1875 . Trauma is the most frequent cause of chylothorax; however, etiology may also be tumor or other cause may be responsible . Chylothorax may occur secondary to mobilization of subclavian vein during cardiovascular, pulmonary, or esophageal surgery . It can also be seen following trauma such as fall from height, motor vehicle accidents, and abdominal and thoracic crush injury . The second most frequently detected cause is malignancy; 75% are due to lymphoma, but bronchogenic carcinoma, and rarely leukemias may also be seen . Many idiopathic cases are thought to occur secondary to minor trauma, such as coughing or hiccupping, or eating fatty foods, or it may be congenital . The fourth category includes vena cava superior or subclavian vein thrombosis, cirrhosis, lymphangioleiomyomatosis (a rarely seen interstitial parenchymal disease), gorham s syndrome, kaposi s sarcoma, castleman s disease, filariasis, and familial lymphedema, sarcoidosis, radiation - related mediastinal fibrosis, and hypothyroidism [47]. Kim et al ., incubated tuberculosis bacilli in sputum and pleural fluid of a 17-year - old female patient with diffuse nodular opacities in her pulmonary parenchyma multiple necrotic abdominal lymphadenopathies, chylothorax, and chylous ascites . They reported improvement of the patient s health with antituberculosis treatment, dietary regimen rich in protein but low in fat, and supportive treatment . Hammoumi et al ., also reported that they achieved cure with antituberculosis and supportive medical treatment in a patient with chylothorax secondary to mediastinal and abdominal tuberculous lymphadenitis [2, 5]. In our country, buyuksirin et al ., detected growth of m.tuberculosis in chylous pleural aspiration fluid of a 22-year - old female patient who presented with pleural effusion, and achieved favorable response with antituberculosis treatment . We also achieved cure with antituberculosis treatment and further medical care in present case with chylothorax secondary to mediastinal tuberculous lymphadenitis . Though pathogenesis of chylothorax is not known for sure, it is thought to be associated with compression of cisterna chyli, and thoracic duct by enlarged mediastinal and hilar lymph nodes, and opening of collateral anastomoses between thoracic duct system, azygos, and intercostal veins . With compression exerted by lymph nodes, pressure inside lymphatic system increases with resultant seeping of chyle into pleural cavity [14]. The most characteristic finding in diagnosis of chylothorax is level of tg in pleural fluid . Pleural fluid tg level> 110 mg / dl, pleural fluid / serum tg ratio> 1, but pleural fluid / serum cholesterol ratio <1 establish diagnosis of chylothorax . If pleural fluid tg level is 50100 mg / dl, then presence of chylomicrons in pleural fluid may establish diagnosis [2, 46]. Milky pleural fluid should be discriminated from pseudochylothorax and drainage of empyema or parenteral fluid through subclavian vein into thoracic cavity . Pseudochylothorax is associated with high cholesterol level and induces chronic pleuritis, which is characterized by increase in cholesterol or lecithin - globulin complexes in pleural fluid . It has milky appearance, as in chylothorax; however, it leads chronic clinical course . If, after centrifuging, turbidity of the fluid persists and addition of ethyl ether eliminates turbidity, then diagnosis favors psedochylothorax . Important for this discrimination is that pleural fluid / serum cholesterol ratio should be> 1 . Fluid may have milky appearance and there may be acute onset, as seen in chylothorax; however, it has strong scent, unlike odorless fluid seen in chylothorax . If parenteral fluid drains into pleural space through subclavian vein, only tg level in fluid increases, while in chylothorax, chylomicrons, tg, and lymphocytes are present . Fluid accumulates at acute onset and precipitation does not occur after centrifugation [4, 6]. In treatment of chylothorax associated with tuberculous lymphadenitis, when necessary, antituberculosis treatment in combination with therapeutic thoracentesis is performed . In case of serious dyspnea, pleuroperitoneal shunt, or drainage with chest tube may be required . Chyle contains high concentrations of protein, lipid, electrolyte, and lymphocytes . In some case reports defect of the thoracic duct generally closes spontaneously; however, if chylothorax persists for longer than 4 weeks, ligation of the thoracic duct during surgical exploration may be considered [1, 2, 4, 7]. Typical appearance of the pleural fluid of our patient, high tg concentration in the sample obtained from pleural fluid, high pleural fluid / serum tg ratio, and relatively low pleural fluid / serum cholesterol established diagnosis of chylothorax . Our patient responded to antituberculosis treatment favorably . While analyzing pleural fluid to make diagnosis of chylothorax, tuberculosis should also be considered in differential diagnosis.
A major blunt abdominal trauma is the leading cause of hepatic injury and significant damage to the biliary tract causing intrahepatic biloma, intraperitoneal bile leak and hemobilia . Partial liver resection has been widely used as an effective treatment for severely injured liver . Bile leak is a serious complication that may lead to sepsis and peritonitis and, therefore, has high mortality rate if left untreated . This incidence has remained the same over the last decade despite a significant improvement in the results of liver surgery . Endoscopic therapy has become the predominant modality of choice used in both the diagnosis and treatment of biliary tract injuries . In this paper, we describe a novel endoscopic treatment used in a patient who underwent right hepatectomy for severe liver injury complicated by a major bile leak who was not a candidate for further surgery . A 21-year - old man was the driver of a motor vehicle and sustained a blunt abdominal trauma in a high - speed collision leading to major liver laceration . The patient was initially managed at a secondary care hospital where he underwent liver packing . He received 20 units of packed red blood cells (prbc) with fresh frozen plasma (ffp) and was transferred to the armed forces hospital (afh) for further management . At afh however, he continued to bleed from the site of the liver laceration and underwent emergency right hepatectomy . He remained intubated with mechanical ventilation in the intensive care unit (icu) for three months . His prolonged icu course was complicated by pneumothorax, sepsis, bed sores, renal failure, coagulopathy, and cardiac arrest on day 15 of his icu admission . He had deranged liver enzymes and abnormal liver function associated with abdominal distension and drain of bile from the abdominal drainage . Endoscopic retrograde cholangiopancreatography (ercp) was performed which confirmed a major bile leak from one of the main branches of the left main bile duct on the resected edge (fig . There was an improvement in the abdominal distension, bile drainage and liver function for about three weeks . He started to have abdominal distension some days later with deranged liver chemistry, mainly high bilirubin and alkaline phosphate . Blocked or migrated stent was anticipated and he underwent a second ercp for stent replacement . However, the previous stent was in place with good drainage . Injection of contrast into the biliary tree beside the stent revealed a major leak from the right hepatic duct stump (fig . The plastic stent was removed and the leaking bile branch was selectively cannulated using a 0.035 mm boston scientific guide wire . A metallic endovascular coil (3/30 mm), (boston scientific) was deployed in the terminal end of the leaking bile duct followed by an injection of 1.5 ml of a mixture of nbca and lipidol at a ratio of 2:1 (fig . 4). The endovascular coil remained in place on ct abdomen done three months postendoscopic intervention (fig . Ct abdomen revealed mildly dilated cbd with a radio - opaque object most likely migrated endovascular coil at the distal cbd causing biliary obstruction (fig . However, the cbd was found to be patent; most likely he passed the endovascular coil . Bile leak still represents one of the most common complications after liver resection and its incidence has not decreased during the past decade . Bile leak can be managed by various therapeutic techniques, aiming at decreasing the intrabiliary duct pressure, to allow bile to flow through the path of least resistance and, therefore, passive closure of bile leak . Multiple studies, supporting early intervention, have shown an improved outcome with repair by experienced hepatobiliary specialists . In selected cases, ercp is preferable to surgery as the first step in the management of non - complicated biliary duct injury . Different modalities of endoscopic management of persistent bile leak such as sphincterotomy, plastic biliary stents, nasobiliary drainage, embolization with micropledgets, obliteration with n - butyl cyanoacrylate (nbca) and coil embolization have been described ., can be managed by sphincterotomy . However, in high grade bile leak, a biliary stent is a must ., a 10 fr 5 cm plastic stent was inserted . However, after a few weeks of improvement, he started to have more bile coming out of the abdominal drain . Stent migration or blockage was anticipated . However, a second ercp revealed another major leak coming out from a biliary branch, different from the first one . Since the patient had a major bile leak, despite the presence of a functioning stent and his condition did not permit further major surgery, the decision was made to deploy an endovascular coil within the terminal end of the leaking bile duct followed by an injection of cyanacrylate . There are few reports in the literature describing embolization of biliary leaks using metallic endovascular coils . The percutaneous use of hydrocoil to manage cystic duct stump leak is described by doshi et al . Hydrocoils have the advantage of swelling or expansion when the hydrophilic polymer comes into contact with fluids . Endoscopic approach is less traumatic and associated with less complications compared to the percutaneous approach . Reported the first successful ercp management of persistent cystic duct leak using embolization of fibred platinum coils into the cystic stump . The presence of coil within the leaking duct and the high viscosity of the bile will most likely lead to mechanical occlusion . Nbca is a low viscosity tissue glue monomer that polymerizes and become solid once in contact with body fluids at neutral ph . Reported the successful endoscopic application of nbca in seven out of nine cases with no complication . Ganguly et al . Reported the first case of the endoscopic combination of coils and nbca . A 0.5 ml mixture of nbca and lipidol at a ratio of 3:1 was used . Due to the high output biliary leak, despite the present of functioning plastic stent, we decided to use a combination of metallic coil embolization followed by obliteration with nbca . The short stump of a leaking bile duct was a challenge in our case due to the high risk of common bile duct occlusion . One short metallic coil (3/30 mm) was deployed to act as a matrix for the nbca . This was followed by an injection of 1.5 ml of a mixture of nbca and lipidol at a ratio of 2:1 . Subsequent ct abdomen a month after the procedure showed the coil to be in place (fig . The short and long term complications of such a procedure is difficult to assess due to the small number of patients treated with such a technique . This is the third description of coil migration to the common bile duct from its site of insertion . The combined use of coil and nbca as in our case did not prevent coil migration . To our knowledge this is the second reported case in english speaking literature describing the endoscopic combination of metallic biliary coils and nbca for the management of biliary leak . Written informed consent was obtained from the patient for publication of this case report and accompanying images . A copy of the written consent is available for review by the editor - in - chief of this journal on request . Khalid alnaamani contributed to the study design, data collections, data analysis, writing and review all the case.
Peripheral nerve injuries produce acute pain and often induce neuropathic pain, a severe clinical problem and chronic debilitating condition that affects the nervous system . Neuropathic pain is relatively common and impairs the quality of life of sufferers . In the past few decades, neuropathic pain animal models have been used to study pain mechanisms and analgesia effects . Chronic constrictive injury (cci) has been the common neuropathic pain model since 1988 [1, 2]. The hippocampus, a part of the limbic system, has the function of learning, memory, emotion, and affect and also has relationships with chronic and acute pain . It has been reported that hippocampal formation plays an important role in pain information processing, including anatomical features, behavioral experiments, electrophysiology, functional imaging, and other molecular research . Hippocampal n - acetylaspartate / creatine decreased in elderly patients suffering from acute pain [4, 5]. Neuropathic pain induced hippocampal interleukin-1 beta (il-1) mrna levels upregulation, and the changes of il-1 mrna expression correlated with the injured side of the hippocampus . Xiao et al ., yang et al ., and li et al . Reported in a series of studies that acetylcholine (ach) influences the pain - induced discharge frequency and the electric activities of pain - related neurons in the hippocampus of rats . Acupuncture has been widely applied in china and other asian countries for thousands of years to ameliorate a number of diseases, including acute and chronic pain . Acupuncture analgesia has been gradually accepted by people due to its advantages, but its mechanism has not yet been clarified in detail . Mounting evidence from many laboratories over the past years suggests that acupuncture has effects on the hippocampus . Traditional acupuncture treatments significantly decreased functional magnetic resonance imaging (fmri) signals and the metabolism in the hippocampus; 2 hz electrical acupoint stimulation - induced analgesia has negative correlations with the averaged fmri activation levels of the bilateral hippocampus . Electroacupuncture (ea) could modulate the function of interneurons in the hippocampus, significantly enhancing long - term potentiation (ltp). In some of our previous studies, chronic pain had an effect on the hippocampal cholinergic system, and ea treatment relieved pain by regulating hippocampal cholinergic neurons . Ea had an obvious analgesic effect and in cci rats significantly diminished the injury - induced increase in synaptic cleft width and thinning of the postsynaptic density [15, 16]. It also activated the extracellular regulated protein kinases (erk) signaling pathway in the hippocampus . Previous studies suggested that erk / mitogen - activated protein kinase (mek) play an essential role in neuropathic pain . In the spinal cord, cci - induced neuropathic pain activated the erk- and camp - response element binding protein (creb) signaling pathway . However, it remains largely unknown how the erk is involved in pain modulation in the hippocampus . On the basis of these studies, the objective of this research is to investigate the effects of ea and the erk signaling pathway on the acute pain - induced responses of pain - related neurons in the hippocampus of wistar rats under both control and neuropathic pain conditions . Adult male wistar rats, weighing from 220 g to 280 g (n = 43), purchased from beijing union medical college, were acclimatized to standard laboratory conditions (12-hour light and dark cycle) at the beijing acupuncture and moxibustion institute for a week and given free access to standard chow pellet diet and water . The rats were randomly divided into three groups: (1) sham / control group: n = 15 (sham - operated rats); (2) cci group, n = 13; (3) u0126 (erk1/2 inhibitor) group, n = 15; u0126 (2.26 l, 10 g) was injected to the rats . With the automatic injector, all surgical interventions and postoperative animal care were performed in accordance with the guidelines for declaration of the national institutes of health guide for the care and use of laboratory animals (publication number 80 - 23, revised 1996). The rats were anesthetized by a mixture of a solution of urethane (28 mg/100 g) plus chloralose (3.3 mg/100 g). Rectal body temperature was monitored throughout the experiment, and a heating pad was used to maintain the temperature of the animals at 37.0c 0.5c . The rats were fixed on the back and the hair on the neck was shaved . The trachea was exposed by blunt dissection through neck muscles after cutting off the neck skin above the manubrium . A t - shaped incision was sheared at the bottom of the thyroid isthmus, and then tracheal intubation was performed using special intubation designed by the laboratory of the institute of acupuncture and moxibustion . In the prone position, the left sciatic nerve was isolated (the chronic constrictive injured nerve in the cci group) and covered with liquid paraffin . As described by bennett and xie, cci was used as the neuropathic pain model . Local application of antibiotics (sodium penicillin, 900010000 u / rat) was used to avoid postoperative infection . The rats were anesthetized by a mixture of urethane (28 mg/100 g) and chloralose (3.3 mg/100 g). The head of the rat was fixed on the stereotaxic apparatus (sr-6r, nihon kohden, tokyo, japan). With the aid of the stereotaxic atlas of the rat brain, two small skull windows were opened and covered with warm liquid paraffin at the positions for inserting recording electrodes and microsyringe (kds-310-plus, kd scientific, holliston, ma, usa). A glass microelectrode (5 m, filled with 3 mol / l kcl) was inserted into the right hippocampal ca1 area (ap: 3.23.6 mm; ml: 2.53.0 mm; dv: 2.53.2 mm) by a micromanipulator (sm-21, nihon kohden, tokyo, japan) as recording electrode . Another micromanipulator was used to insert the microsyringe into the lateral ventricle (ap: 1.0 mm; ml: 1.32.0 mm; dv: 3.0 mm) to inject the experimental drug (mek1/2 inhibitor). The neuronal discharges were monitored by an oscilloscope (vc-10, nihon kohden, tokyo, japan) at the same time . After recording spontaneous neural discharge for 5 min as control, the sciatic nerve was stimulated by a double stainless electrode (delay 0, interval 50 msec, duration 0.3 msec, and current intensity 5 ma) for 10 s as noxious stimulation . If there was no change in the neural discharge after noxious stimulation, the neural discharges were not recorded anymore . When the discharge frequency returned to control level (about 10 min after giving the noxious stimulation), the sciatic nerve was given another noxious stimulation for 10 s. at the end of noxious stimulation, bilateral acupoints were punctured with stainless - steel acupuncture needles (gauge 28, 0.20 mm in diameter) to a depth of about 4 mm, respectively, and stimulated electrically using a hans ea stimulator for 1 min, and u0126 was administered intracerebroventricularly for 2 min at the same time . Zusanli (st36) and yanglingquan (gb34) are the main points for treating sciatica and other types of leg pain according to the theory of traditional chinese medicine . (st36) and yanglingquan (gb34) has cumulative analgesic effects for neuropathic pain [1417]. The response of the neurons to noxious stimuli can have three forms: excitement, inhibition, and no response . Neurons that respond to nociceptive stimulation are defined as pain - related neurons, neurons excited after noxious stimuli are called pain - excited neurons (pens), and the inhibited neurons are defined as pain - inhibited neurons (pins). The discharge frequencies before the noxious stimulus served as control (corresponding to 100%) to observe the changes of discharge frequencies of pain - related neurons . We analyzed the electrical activities of pain - related neurons whose discharge frequencies increased or decreased by more than 20% after noxious stimuli . The experimental data were scanned on a computer with spike ii (ced instruments, cambridge, united kingdom) after management and analyzed with spike ii software . All data were expressed as mean sem (standard error of the mean). 29 pain - related neurons were recorded in the control group (n = 15). The electric activities of 17 pens (58.6%) were increased while those of 12 pins (41.4%) were decreased after noxious stimuli . In the cci group (n = 13), we recorded 18 pain - related neurons, 14 of which were pens (78%) and 4 were pins (22%), so the number of recorded pins in the control group was larger than in the cci group . The u0126 group (n = 15) showed 15 pain - related neurons, among which there were 9 pens (60%) and 6 pins (40%). In sham rats, brief sciatic nerve stimulation significantly increased the electrical activities of 17 hippocampal pens in the control group (134.53 18.50%) and ea group (126.1 8.97%), and there was no difference between the two groups (p> 0.05, figure 1(c)). Ea reduced the excitatory effects of brief sciatic nerve stimulation on the firing rates of 17 pens . At 2 min after the noxious stimuli, the discharge frequency changes of hippocampal pens in the ea group (121.18 7.45%) were markedly lower than those in the control group (168.68 10.64%, p <0.05). At 4 min after the noxious stimuli, although the firing rates of hippocampal pens in the control group (145.08 7.22%) were still increased, the firing rates of hippocampal pens in the ea group (108.65 4.48%) had almost returned to normal level . No significant difference was found between the control group (116.86 8.21%, 103.22 2.13%) and the ea group (102.14 6.03%, 97.65 4.12%) at 8 and 10 min after the noxious stimuli . The electric activities of pens in the control group almost returned to normal level at 8 min after the noxious stimuli . It is suggested that ea played an inhibiting role in regulating excitatory effects of the acute noxious stimulus on the electrical activity of pens in sham rats . The electric activities of 12 hippocampal pins were decreased by the brief sciatic nerve stimulation in sham rats (figure 1(d)), and the frequency changes in the control group were larger than those in the ea group . At 0 minutes (immediately) after brief sciatic nerve stimulation, the firing rates in the control group (67.88% 6.00) were lower than those in the ea group (93.35% 6.46, p <0.05). The frequencies of pins in the control group were still decreased at 2 min (54.35% 4.97), 4 min (53.01% 6.12), and 6 min (67.93% 7.64) and returned to normal level at 10 min (94.96% 6.11) after the stimulation . The frequencies of pins in the ea group were still at a low level at 2 min (74.36% 6.28) and 4 min (87.37% 3.78) and returned to normal level at 6 min (100.59% 8.80), earlier than in the control group . In comparison with the control group, the frequency changes of pins in the ea group were higher (p <0.05) at 2, 4, and 6 min after giving the noxious stimuli . Those findings suggested that ea played an exciting role in regulating inhibitory effects of the acute noxious stimulus on the electrical activity of pins in sham rats . The electric activities of 14 hippocampal pens reached 216.46 25.40% in the cci group and 219.57 44.15% in the cci + ea group after the noxious stimulus . There was no significant difference between the two groups (p> 0.05, figure 2(c)) of cci rats . Similar to the sham rats, the discharge frequencies of 14 pens were decreased after ea treatment . The frequency changes of hippocampal pens in the cci group (193.54 21.50%) were markedly higher than those in the cci + ea group (139.40% 12.78, p <0.05) at 2 min after the noxious stimuli . The firing rates of pens in the cci + ea group (116.99% 20.90) returned to normal level and were observably lower than those in the cci group (188.82% 16.16, p <0.05) at 6 min . The frequency changes of pens in the cci + ea group were 105.87% 13.26 and 99.58% 9.06 at 8 min and 10 min after the brief sciatic nerve stimulation, which are lower than those in the cci group (167.27% 14.86, 148.82% 20.71, p <0.05). It is suggested that ea treatment also reduced the excitatory effects of brief nerve stimulation on the firing rate of pens in cci rats . The discharge frequencies of 4 hippocampal pins were decreased significantly in both the cci (56.44% 8.68) and the cci + ea (68.27% 7.96) group after brief sciatic nerve stimulation (figure 2(d)). There was no significant difference between the two groups at 0, 2, and 4 min . At 6 min after giving the noxious stimuli, the frequencies of pins in the cci group (59.82% 9.57) were still inhibited, and the frequency of pins in the cci + ea group (81.40% 16.29) had almost returned to normal level . At 8 and 10 min after giving the noxious stimuli, the frequency changes of hippocampal pins reached 70.65 10.31% and 77.94 2.67%, respectively, in the cci group; however, the frequencies in the cci + ea group were still at the normal level (100.30% 15.36, 96.69% 5.66). The discharge frequency changes of the cci and the cci + ea group showed no significant differences at 10 min . Although the number of recorded pins was small, it is also suggested that ea treatment reduced the effect of brief sciatic nerve stimulation on the frequency of pins in cci rats . The discharges of 17 pens and 12 pins were recorded in the hippocampus of 15 sham rats, and those of 14 pens and 4 pins were recorded in the cci rats . At 2 min, there was no difference between sham and cci rats in the frequency changes (p> 0.05, figure 3(a)); however, brief sciatic nerve stimulation induced bigger frequency changes of pens in the cci rats than in the sham rats (p <0.05) from 4 min to 8 min . The discharge frequencies of pens in the cci rats were still increased at 10 min, while the discharge frequencies of pens in the sham rats had almost returned to normal level at 8 min after the acute noxious stimuli . The discharge frequency changes of pins in the cci group were slightly higher than in the control group at 2 and 10 min after the noxious stimuli (p> 0.05, figure 3(b)). This might be associated with the small number of pins recorded in the cci group . Compared with the ea group, the discharge frequency changes of pens in the cci + ea group were slightly increased (p> 0.05, figure 3(c)), and those of pins in the cci + ea group were mildly decreased (p> 0.05, figure 3(d)) from 2 to 10 min after the noxious stimuli . Ea suppressed the excitatory and inhibitory effects of the acute noxious stimulus on the electric activities of pens and pins in both sham and cci rats . Compared with the ea group, the discharge frequency changes of 9 hippocampal pens in the u0126 group were not significant (p> 0.05, figure 4(b)) at 0 min . Compared with the ea group, the discharge frequency changes of hippocampal pens in the u0126 group (195.20 22.98%) increased significantly (p <0.05) at 2 min after the noxious stimuli (after injection of u0126). From 4 to 8 min after giving the noxious stimuli, the discharge frequency changes of pens in the u0126 group (165.80 39.32%, 188.32 25.35%, and 181.14 43.41%) were still higher than those in the ea group (p <0.05). At 10 min after giving the noxious stimuli, no significant differences were found between the u0126 group (118.34% 20.80) and the ea group . There was no significant difference between the u0126 group and the ea group in the discharge frequency changes of 6 hippocampal pins (p> 0.05) at 0 min . The discharge frequency changes of hippocampal pins in the u0126 group (55.6% 13.27) were bigger compared to those in the ea group at 2 min after the noxious stimuli (and after injection of u0126). During a period of 6 to 10 min, the discharge frequency changes of pins in the u0126 group exhibited obvious differences when compared with the same period in the ea group (p <0.05). The electrical activities of pins gradually returned to baseline levels at 6 min after the noxious stimuli in the ea group, while those in the u0126 group did not . Combination of u0126 + ea in sham rats produced effects markedly greater than those observed when brief sciatic nerve stimulation was performed alone (without drugs), so it may be assumed that u0126 increased the effect of noxious stimulation through a pathway other than that of the ea pathway, thus masking the effect of ea . It is suggested that the mek1/2 inhibitor u0126 blocked the ea effect on acute noxious stimulation . Pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage . Acute pain is very common in clinical practice, including pain in the perioperative setting, pain in patients with severe or concurrent medical illnesses (such as arthritis), pain related to cancer or cancer treatment, and labor pain . Tens of thousands of people are affected by acute pain, and the treatment costs amount to billions of dollars every year . To enhance the quality of life and maintain the patient's function ability, clinical medicine is used to treat acute pain, but these medications have substantial side effects . Acupuncture analgesia has its advantages, such as an obvious analgesic effect and little side effects, but its mechanism of action is not clarified at the moment . As a part of the limbic system, the hippocampus is associated with memory, but also with pain and acupuncture analgesia . The dorsal hippocampal dopamine receptors exert an analgesic effect during the orofacial pain test; microinjections of nonsteroidal anti - inflammatory drugs or 2-ag into the hippocampus induce antinociception . A recent study indicates that persistent peripheral nociception induced by subcutaneous injection of bee venom upregulated mtor target p70 s6 kinase signaling and facilitated long - term potentiation which could be reversed by mtor inhibitor in the hippocampus . In our experiment, we observed that the discharge frequencies of hippocampal ca1 pain - related neurons (pens or pins) were changed after brief electrical impulses applied to the sciatic nerve . The electric activities of pens and pins returned to normal level at 8 and 10 min after the noxious stimuli, respectively . Focused on electric activities of pain - related neurons in the hippocampus after noxious stimuli for many years . In the hippocampal ca1 and ca3 area and dentate gyrus, cholinergic neurons and muscarinic receptors have effects on the electric activities of pens and pins, so that they are involved in pain modulation [79, 32]. Glutamate and its receptors, noradrenaline (ne), phentolamine, and alpha - adrenoceptors also have effects on pain modulation by regulating electric activities of pens and pins in the hippocampal ca3 region [33, 34]. These studies reported the effects of pain - induced discharges of hippocampal neurons, but few research papers pay attention to electric activities of hippocampal neurons in a chronic pain state . Hains et al . Reported that after spinal cord contusion injury the changes of the spontaneous discharge and afterdischarge of extracellular neurons in the thalamus are related to an upregulated sodium channel expression . In cci rats, the acute noxious stimulation evoked greater changes of electric activities of pens and pins, and the recorded number of hippocampal pens was obviously bigger and that of pins smaller compared with sham rats . It took more time for the pain - related neurons to return to normal level in the cci rats than in sham rats . This might be because the effect of brief sciatic nerve stimulation on firing rates of pens and pins was enhanced under the advanced cci situation . The cci rats may have experienced more pain than the sham rats after the short noxious stimulation . The cci rats were in a state of hyperalgesia, an increased response to noxious stimuli, which means the same intensity of electrical stimulation causes more pain intensity and finally a prolonged time of recovery to normal . Shi et al . Reported that pain - related neurons were involved in the modulation of ea analgesia, and ea stimulation resulted in an inhibiting effect on the electrical activity of pens and an activating effect on the electrical activity of pins . After ea at the acupoint hegu, or dolantin given intravenously, gao et al . Stimulated the head of the caudate nucleus, eliciting an inhibitory effect on pens and a reduction of inhibition or release on pins . Ea has been shown to suppress pens and excite pins, which can be taken as an electrophysiological index for ea analgesia [38, 39]. Yang et al . Also reported that ea treatment could play an inhibiting role in mediating the evoked discharge of pens and an activating role in that of pins, and cholecystokinin-8's (cck-8) b receptor antagonist could be antagonistic to the effect of cck on ea analgesia . Figure 1) showed that, in the hippocampal ca1 area, ea inhibited the excitatory effect of brief sciatic nerve stimulation on the electric activities of pens and activated the inhibitory effect of brief sciatic nerve stimulation on electric activities of pins in sham rats, which caused the firing rates of pens and pins to return to normal level at 6 min after the noxious stimuli, earlier than in the control group . These findings suggest that the analgesic effect of ea is related to the electric activities of pain - related neurons in the ca1 area, which were affected by noxious stimulation . Ea reduced the effect of acute noxious stimulation on the electric activities of pens and pins . The electric activities of pens and pins play an important role in mediating ea analgesia, as reported before . In cci rats, ea also had an effect on the firing rate of pens and pins similar to that evoked by acute noxious stimuli, but the electric activities returned to normal level at 6 or 8 min after administration of the noxious stimulus . It is suggested that the effect of ea treatment is closely related to the severity of pain . Intrathecal administration of the inhibitor of the mapk family members mek1/2, such as u0126, pd198306, and pd98059, had analgesic effects and significantly potentiated the effectiveness of opioids in neuropathy in the spinal cord [4143]. U0126 downregulated the increased late responses and afterdischarge induced by melittin (a pain - related peptidergic component) in wide - dynamic - range neurons of the spinal cord . Accumulating evidence showed that erk expression increased at the peripheral nerve and spinal cord horn [4547]. Phospho - erk (perk) in the spinal cord is activated immediately in neurons (<6 h), then in microglia on days 1 and 3 and both in astrocytes and in microglia on day 10, and finally in satellite cells on days 10 and 21 after spinal nerve ligation, and this activation contributes to mechanical allodynia . Pd 98059 may modulate the nociceptive factors and antinociceptive factors that are released by glial cells, which have a close relationship with reduced symptoms of neuropathy [43, 46]. These reports suggested that mek1/2 inhibitors have an analgesic effect on neuropathic models in the primary injury and that neuropathic pain is associated with the activity of the mapk / erk signal pathway within the spinal cord . Few studies focused on the relationship between the expression of erk and the hippocampus in chronic neuropathic pain, and an agreement has not been reached [48, 49]. The phosphorylation of the erk signal transduction pathway was enhanced by ea in depression rat tissue; however, there was no report on the study of ea analgesia on the hippocampus erk signal pathway . Our previous results showed that in the hippocampus the erk signal pathway was inhibited in chronic neuropathic rats, and after acupuncture treatment the erk signaling pathway was activated . Our experimental results showed that, compared with the ea group, brief sciatic nerve stimulation produced a greater excitatory and inhibitory effect on the firing rate of pens and pins in u0126 + ea group, suggesting that u0126 suppressed the effect of ea on the analgesic pathway . It is suggested that involvement of the activation of erk signaling pathway in the hippocampal ca1 region in ea treatment induced pain relief . In cci rats, acute noxious stimulation required a longer time for the firing rate of pain - related neurons to return to normal level; ea treatment could suppress the effect of the noxious stimulus on pens and pins in both sham and cci rats, which suggests a close relationship with the ea analgesic effect; and the erk signal pathway is probably involved in pain and ea analgesia.
Respiratory muscle weakness is commonplace in critically ill patients, impairing the ability of those patients to breath, prolonging the need for ventilatory support, and increasing the likelihood of respiratory failure when that support is removed . Infections and endotoxemia reduce respiratory muscle strength, probably acting through several mechanisms - including increased oxidative stress, caspase activation leading to protein breakdown, and activation of the proteasome and calpain proteolytic systems resulting in protein loss . Supinski and colleagues report that the omega-3 fatty acid eicosapentaenoic acid (epa) attenuates the loss in diaphragm specific force generation (that is, diaphragm strength) induced by bacterial endotoxin treatment in rats . Epa is found in fish oils, along with its derivative docosahexaenoic acid . In the present study, pure epa was administered orally on two occasions: the first at the same time as endotoxin, and the second 24 hours later (g. supinski, personal communication). Epa was given at a dose of 1 g / kg body weight / day (that is, 2 g / kg). For a 250 g rat, the epa dose equates to 0.25 g / day (0.5 g in total). This is similar to the amount of epa that would be consumed by rats fed on a diet containing 5 to 10% by weight as fish oil, as is commonly used in experimental studies . On the contrary, the amount of epa provided here could not be translated directly to humans (70 g / day in a 70 kg individual) and is greatly in excess of amounts provided to patients receiving artificial nutrition either parenterally or enterally . In this new study, endotoxin decreased diaphragm specific force generation by about 50%, while epa almost totally prevented this reduction . Epa might attenuate the loss of muscle strength through a variety of actions: epa has been shown to act as a weak antioxidant, to inhibit proteasomes, to inhibit caspase activation and to reduce inflammation . In the study of supinksi and colleagues, epa did not prevent the caspase activation or oxidative stress pathways in the diaphragm but it did reduce calpain activation, suggesting a specific effect on this proteolytic pathway . Supinski and colleagues thus report a highly novel effect of epa (attenuation of endotoxin - induced loss of respiratory muscle strength) and a novel mechanism of action (reduced calpain activation). The implication of this work is that administration of epa may be able to decrease the respiratory, and perhaps other, muscle weakness that accompanies critical illness and sepsis . Oral fish oil provided for a period of time prior to endotoxin administration has been shown to decrease post - endotoxin metabolic perturbations and inflammation, to improve heart and lung function, and to reduce mortality . These studies provide epa (and docosahexaenoic acid) in advance of endotoxin treatment, which is not likely to be the best model for the clinical situation, and may favour an effect of epa since it will be incorporated into cells and tissues in advance of the endotoxin stimulus . In supinski and colleagues' study, epa was administered at the same time as, and following, endotoxin administration, which is closer to the clinical situation . In critically ill patients in the intensive care unit, parenteral fish oil improved lung function and decreased the length of hospital stay while an enteral formula that included fish oil improved lung function, reduced requirement for ventilatory support, reduced risk of new organ failures [10 - 12], decreased the intensive care unit stay and reduced mortality in patients with acute respiratory distress syndrome, with acute lung injury or with sepsis . This latest study, however, suggests that there may be an alternative mechanism: improved (or maintained) diaphragm function . The findings of supinski and colleagues indicate a rapid effect of epa on the diaphragm that is consistent with the relatively short period of time required to improve lung function and to reduce the need for ventilatory support seen in these studies in critically ill patients . . It will be important, however, to identify whether epa administration at a time after the initial insult (in this case, endotoxin) is also protective, since this would be most relevant to the clinical situation, to identify whether doses relevant to the human clinical situation are effective, to examine whether this effect occurs in patients receiving epa, and to understand more about the mechanism that underpins this effect.
Enhancing the binding potency of carbohydrate inhibitors of protein carbohydrate interactions is an important step towards improved medicinal applications.1 this is mostly the case because of the relatively weak interactions between proteins and carbohydrate ligands . Ensuring multivalency of ligands is a proven method to achieve this.2 leca is an important virulence factor of pseudomonas aeruginosa and is involved in bacterial adhesion and biofilm formation.3 the protein has recently become a popular target for the design of multivalent inhibitors.4 the tetrameric protein promotes the adhesion of the bacteria to tissue cell surfaces, thus facilitating subsequent steps such as cell invasion and biofilm formation . Inhibiting bacterial adhesion proteins has the potential to become a mild and less resistance - prone method to treat and prevent bacterial infections.5 two of the four binding sites, with specificity for galactosides, are relatively close together with a separation of about 26 .6 this arrangement has led us to design and synthesize divalent galactoside ligands with well - defined and rigid spacers that should allow a chelation - type divalent binding mode.7 flexible spacers are commonly used as they are forgiving of imperfect design and usually yield sizeable potency enhancements in multivalent systems.7 they are, however, not optimal as there will be a significant entropy loss upon binding and, moreover, achieving selectivity will be less likely . In our search for an optimal spacer we found compound 1 (figure 1 a) to be a highly potent divalent ligand with nanomolar inhibitory potency.8a the spacer of this structure contains direct linkages between the glucose moieties and the connected triazoles.8 this arrangement leads to a relatively rigid structure, in which rotations of the components can take place, but the overall geometry remains mostly linear . A) structure of potent divalent leca inhibitor 1 with the relatively rigid glucose - triazole - based spacer; b) schematic divalent binding mode of a divalent ligand to two leca subunits; c) x - ray structure of leca with bound galactose moieties . The two asp 47 carboxylates in the spacer path are shown explicitly.6 most importantly, good solubility in water was observed . We found that three glucose - triazole units was the optimal length for leca inhibition, while divalent ligands with two and four units showed far inferior inhibition . All data were consistent with a chelating binding mode; especially convincing was the stoichiometry derived from isothermal titration calorimetry (itc) binding experiments . Furthermore, the short linkage of just a single carbon between the galactoside ligand and the triazole proved to be a major contributing factor to the success of this compound . In order to further optimize the potency of the compound and to explore the principle of protein spacer interactions, we now report on our functionalization of the spacer of 1 with various functional groups . The presence of the two carboxylates (asp 47 in each subunit) is apparent when looking at the path between the two bound galactosides, which the spacer is likely to span on the leca protein surface when chelating bivalent binding occurs (figure 1 c). These two carboxylates are likely to be in close proximity to the 6-oh group on the terminal spacer glucoside units, depending on the rotational state of the molecule . Molecular modeling was used in order to gauge whether positively charged functional groups at the c-6 position on the terminal spacer glucoside units would be able to interact with the asp 47 residues . Firstly, ammonium groups were used (as derived from 12, see below). Creating a stabilized conformation with the positive charges in close proximity to the asp 47 carboxylates was possible, and this orientation was used as the starting point of additional simulations . When running an unrestrained nanosecond molecular dynamics (md) simulation with explicit water molecules, the stabilized geometry persisted throughout the simulation (see supporting information). Especially, the two hydrogen - bonded salt bridges, between the asp 47 side chains and the ammonium groups of the protonated form of 12, remained within 3 and were compatible with a geometry that included two fully bound galactoside ligand units . Such a bound geometry was also possible for a compound that included a pyridinium group (as in 13), but resulted in longer distances between the carboxylate oxygens and the pyridinium nitrogens, due to bigger steric requirements of the pyridinium units . Furthermore, while performing a similar md simulation, this structure was not maintained, indicating indeed the larger steric requirements of the pyridinium group when facing the protein surface . These experiments lead to the conclusion that the introduction of ammonium groups would be most promising . We chose to prepare a series of derivatives with various new substituents in the spacer in hopes of further enhancing the leca inhibitory potency of the molecules . Positively charged substituents were included, as reasoned above . Also, negatively charged and more lipophilic groups were included as these could possibly benefit from interacting with other proximate parts of the protein that would not have been previously obvious . The synthesis of the modified spacers started with two previously prepared building blocks 2 and 3.8 these building blocks were coupled by a double copper(i)-catalyzed azide alkyne cycloaddition (cuaac) reaction . Next, the two galactosyl axial 4-oh groups of the resulting product 4 were turned into triflates, thus enabling the subsequent substitution by azide with inversion to give 5 . The terminal azides were subsequently linked to the protected galactosyl ligand 6 a by cuaac yielding 7 . Selective removal of the tert - butyldimethylsilyl (tbdms) groups then gave 8 which is ready for further functionalization . Reagents and conditions: a) cuso45 h2o, naasc, dmf with 10% h2o, 80 c, 30 min, 85%; b) 1) tf2o, 10% pyridine in ch2cl2, 0 c, 3 h, 2) nan3, acetone / h2o (4:1), rt, o / n, 98%; c) 6 a, cuso45 h2o, naasc, dmf with 10% h2o, 80 c, 30 min, 64%; d) p - tsoh, ch3cn / h2o (7:1), rt, 6 h, 83% . The two hydroxymethylene groups of compound 8 were oxidized to carboxylic acids using 2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl (tempo) (scheme 2). Reaction of 8 with triflic anhydride in the presence of pyridine followed by zempln deprotection gave the bis pyridinium compound 10 . Tosylation of the primary hydroxy groups of 8 followed by reaction with sodium azide gave the intermediate 11 . Zempln deprotection of 11, followed by the hydrogenation of the azido groups gave the diamine 12 . Cuaac coupling of phenylacetylene to 11 and subsequent zempln deprotection gave the bis - triazole 13 . All final products were purified by preparative high - performance liquid chromatography (hplc). Reagents and conditions: a) 1) tempo, naocl, nabr, bu4nbr, nahco3/na2co3 ph 9.5, 0 c, 2 h, 2) naome, meoh, rt, o / n, 38%; b) 1) tf2o, 10% pyridine in ch2cl2, 0crt, 3 h, 2) naome, meoh, rt, o / n, 44%, c) 1) tscl, dabco, ch2cl2, rt, o / n, 2) nan3, dmf, 95 c, o / n, 34%, d) 1) naome, meoh, rt, o / n, 2) h2, pd / c, rt, 60%, e) 1) phenylacetylene, cuso45 h2o, naasc, dmf with 10% h2o, 80 c, 30 min, 2) naome, meoh, rt, o / n, 24% . The compounds were tested on an array chip in an assay similar to an enzyme - linked immunosorbent assay (elisa), as previously reported.8 fluorescein - labeled leca was incubated with the inhibitors and exposed to a galactoside - functionalized chip surface . Monovalent ligand 6 b was previously determined to have an ic50 in this assay of 22 m.8a clearly all divalent compounds were far more potent and showed major multivalency effects (table 1). The previous best inhibitor, 1, still remained the most potent compound in the present series with an ic50 in the 2 - 3 nm range, as before . The pyridinium - functionalized 10 and the phenylacetylene - derived 13 showed only a minor drop in potency, with ic50 values in the 5 nm range . Larger potency drops of about an order of magnitude were observed for both the negatively charged bis - carboxylate 9 and the positively charged bis - amine 12 . Subsequently, itc experiments were conducted, which confirmed the divalent binding mode in all cases, with the stoichiometry n values being close to 0.5 . As before8a the dissociation constants (kd) were somewhat higher than the ic50 values from the chip - based elisa - like assay . Furthermore the small potency differences of the chip - based assay were not seen in the itc assay . Interestingly, when looking at the enthalpic and entropic components of the binding event, the lipophilic and noncharged compound 13, showed enthalpy entropy compensation, with a lower beneficial binding enthalpy and, at the same time, a lowered entropy loss upon binding . The latter is understandable as an example of the hydrophobic effect where water molecules in an ice - like structure are liberated from the lipophilic surfaces . Inhibitory potencies (ic50) of the divalent inhibitors on leca binding[a] and dissociation constants (kd), stoichiometry, and thermodynamic binding parameters[b] [a] chip - based elisa - like assay: fitc - labeled leca (5 g ml) binding to a galactoside functionalized chip surface . [b] obtained from isothermal titration microcalorimetry (itc): [leca]=2040 m . It proved possible to use selectively protected 6-oh groups to build up the ligand in its protected form . Subsequently, the selectively deprotectable silyl groups were removed, and the resulting primary hydroxy groups were converted to carboxylate groups by oxidation, to tosylates and subsequently to azides by substitution, and to pyridiniums via their corresponding triflates . The azides allowed cuaac coupling and also further reduction to the corresponding amino groups . While it was anticipated that some of these groups would be able to take advantage of additional beneficial interactions with the nearby protein surface, we did not observe this through enhanced inhibitory or binding potencies . This was true even for the positively charged groups that could possibly take advantage of the nearby positioned carboxylate groups of asp 47 . However, a possible alternative scenario, where the newly added groups point into the solution and thus away from the protein surface, is apparently more favorable in this case . In future designs nevertheless, the functionalization of the spacers will also be important in order to fine - tune other properties of this type of ligand, such as toxicity and absorption, distribution, metabolism, and excretion (adme), which are important for drug development . General: chemicals were obtained from commercial sources and were used without further purification unless noted otherwise . Compounds 2, 3, and 6 were synthesized following literature procedures.7 solvents were purchased from biosolve (valkenswaard, the netherlands). Anhydrous solvents were dried over molecular sieves of 4 or 3 . Thin - layer chromatography (tlc) was performed on merck precoated silica 60 plates . Spots were visualized by uv light and also by 10% h2so4 in meoh . Microwave reactions were carried out in a microwave initiator system (biotage, uppsala, sweden). Analytical hplc runs were performed on an automated hplc system (shimadzu, kyoto, japan) with a reversed - phase column (reprospher 100, c4, 5 m, 2504.6 mm, dr . Maisch gmbh, ammerbuch - entringen, germany) which was equipped with an evaporative light scattering detector (plels 1000, polymer laboratories, amherst, usa) and a uv / vis detector operating at 220 nm and 250 nm . Preparative hplc runs were performed on an applied biosystems (waltham, usa) workstation . Elution was effected by using a linear gradient of 5% ch3cn/0.1% trifluoroacetic acid (tfa) in h2o to 5% h2o/0.1% tfa in ch3cn or by a gradient of h2o to 30% ch3cn in h2o . H and c nmr spectroscopy was carried on an 400-mr spectrometer (agilent, santa clara, usa) operating at 400 mhz for h and 100 mhz for c. heteronuclear single quantum coherence (hsqc) and total correlated spectroscopy (tocsy) nmr (500 mhz) were performed with an inova 500 instrument (varian, palo alto, usa). Electrospray ionization mass spectrometry (esi - ms) experiments were performed with a shimadzu lcms qp-8000 . High - resolution mass spectrometry (hrms) analysis was recorded using an esi - q - tof ii spectrometer (bruker, billerica, usa). The proton numbering scheme of all compounds can be found in the supporting information and is used in the assignments of the signals for the nmr spectra below . Isothermal titration microcalorimetry (itc): the lectin leca was obtained from sigma aldrich and was dissolved in buffer (0.1 m tris - hcl, 6 mm cacl2, ph 7.5) and degassed . Protein concentration (between 20 and 40 m depending on the ligand affinity) was checked by measurement of optical density by using a theoretical molar extinction coefficient of 28 000 . Carbohydrate ligands were dissolved directly into the same buffer, degassed, and placed in the injection syringe (concentration range: 0.10.2 mm). Itc was performed using a microcal auto itc200 (malvern, worcestershire, uk). Leca (0.020.04 mm) was placed into the 200 l sample cell at 25 c . Titration was performed with injections of carbohydrate ligands (2.5 l) every 120 s. data were fitted using the one - site model using microcal origin 7 software according to standard procedures . Fitted data yielded the stoichiometry (n), the association constant (ka), the enthalpy (h) and the entropy of binding . The kd value was calculated as 1/ka, and t is 298 k. each ligand test was performed in duplicate . Leca inhibiton assay: lectin leca was labeled with fluorescein isothiocyanate (fitc) according to a literature procedure.9 microarray experiments were performed by using a pamchip array run on a pamstation 12 instrument (pam - gene, s - hertogenbosch, the netherlands). Data were obtained by real - time imaging of the fluorescence signal by a ccd camera . The fluorescence intensities were expressed in arbitrary units, and the relative intensities were the average of the two duplicate spots . Aliquots of a solution of fitc - labeled leca (5 g ml for all tested compounds) in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid / bovine serum albumin (hepes / bsa) buffer (10 mm hepes, 100 mm nacl, 0.1% bsa . Ph 7.4), containing different concentrations of the inhibitors were incubated for 1 h at rt and subsequently added to the galactoside - functionalized chip . The binding process was monitored for 2 h, and the end values of the fluorescence detection were taken for the determination of the ic50 by using prism 5 (graphpad software, inc ., la jolla, usa). Molecular modeling: all molecular modeling studies were performed using the molecular graphics, modeling, and simulation program yasara version 13.9.8 (yasara biosciences, vienna, austria). The bivalent ligands were first constructed in yasara as isolated molecules . Subsequently, the complex with leca was built by superposition of one of the galactose units of the ligand with a bound galactose of one of the subunits of the leca crystal structure (pdb i d: 1oko).3a the other galactose unit of the divalent ligand was then pulled into the adjacent galactose binding site of leca by restraint md using distance restraints based on the position of the bound galactose present in the x - ray structure with respect to a number of leca residues . Possible electrostatic interactions between two positively charged amino and pyridinium substitutions in the linker region with two asp 47s of leca were investigated in more detail . In order to induce these interactions, the subunits comprising the substituted moieties were first rotated, and subsequently, the nitrogen atoms of respectively the amino and the pyridinium group were restrained to bring them in close proximity to the carbon atoms of the carboxylic acid moieties of asp 47 . After restrained md, the molecules were subjected to a 1000 ps free md simulation in water . General procedure of microwave - assisted click reaction: to a solution of the azide and alkyne compounds in dimethylformamide (dmf) with 10% h2o, was added cuso45 the mixture was then heated by microwave irradiation at 80 c for 30 min . When the mixture cooled to rt,0 the copper salts were removed by a resin (cuprisorb), and the solvents were removed under reduced pressure . Compound 4: a click reaction of a mixture of compound 2 (0.253 g, 0.71 mmol), compound 3 (0.83 g, 1.63 mmol), cuso45 h2o (0.06 g, 0.24 mmol), and naasc (0.096 g, 0.48 mmol) in dmf (5 ml) containing 10% h2o was performed following the general procedure described above to afford compound 4 (0.84 g, 85%); h nmr (400 mhz, cdcl3): = 8.037.89 (m, 5 h, 4ch benzoyl, h-1), 7.867.76 (m, 5 h, 4ch benzoyl, h-1), 7.527.26 (m, 12 h, 12ch benzoyl), 6.08 (dd, j4,3=j4,5=10 hz, 1 h, h-4), 6.025.90 (m, 2 h, h-4, h-3), 5.81 (dd, j5,4=j5,6=9 hz, 1 h, h-5), 5.60 (dd, j4,3=j4,5=9 hz, 1 h, h-4), 5.515.41 (m, 2 h, 2h-5), 5.024.92 (m, 2 h, 2h-3), 4.81 (dd, j6,5=j6,7=10 hz, 1 h, h-6), 4.674.54 (m, 3 h, h-7, 2 h-6), 4.164.07 (m, 1 h h-8a), 4.073.93 (m, 2 h, 2h-8), 3.913.82 (m, 2h-7), 3.703.60 (m, 1 h, h-8b), 3.573.51 (m, 1 h, 6-oh), 3.353.29 (1 h, 6-oh), 2.02, 1.65, 1.58 (s, 9 h, 3ch3, acetyl), 0.90 (s, 18 h, 2sic(ch3)3), 0.07 ppm (2 s, 12 h, 2si(ch3)2); c nmr (100 mhz, cdcl3): =69.97, 169.10, 168.95 (3c = o acetyl), 166.14, 166.05, 165.63, 165.55 (4c = o benzoyl), 145.61, 145.58 (2c-2), 133.44133.21 (ch benzoyl), 129.99128.41 (ch benzoyl), 129.39129.20 (c benzoyl) 123.43, 122.13 (2c-1), 85.55 (c-3), 77.97 (2c-7), 75.75 (c-5), 75.48 (c-5), 74.84 (c-7), 74.03, 73.77 (2c-3), 72.41 (c-5), 70.85 (c-4), 69.94, 69.59 (2c-4), 68.92, 68.53 (2c-6), 63.65, 63.13 (2c-8), 61.61 (c-8), 59.84 (c-6), 25.99 (sic(ch3)3), 20.72, 19.98, 19.89 (3ch3 acetyl), 18.44 (sic(ch3)3), 5.31 ppm (si(ch3)2); ms (esi) m / z [m+h] calcd for c68h84n6o21si2: 1378.59, found 1378.00; hrms (q - tof) m / z [m+h] calcd for c68h84n6o21si2: 1377.5228, found 1377.5326 . Compound 5: compound 4 (0.84 g, 0.61 mmol) in anhydrous ch2cl2 (30 ml) containing pyridine (4.22 ml) was treated with triflic anhydride (12.2 ml of a 1 m solution in ch2cl2, 12.2 mmol). The mixture was stirred at 0 c for 3 h, after which cold 1 n khso4 (20 ml) was added . The organic layer was washed with cold h2o (220 ml) and, once with cold brine (20 ml), dried over na2so4, filtered, and concentrated . The residue was dissolved in an acetone / h2o mixture (15 ml, 4:1), and nan3 (0.397 g, 6.1 mmol) was added . The mixture was stirred at rt overnight and diluted with a cold h2o / ch2cl2 mixture (50 ml, 4:1). The water layer was separated and extracted once with ch2cl2 (10 ml). The combined organic layers were dried on na2so4, filtered, and concentrated to afford compound 5 as a yellowish solid (0.86 g, 0.60 mmol, 98%); h nmr (400 mhz, cdcl3): =8.007.93 (m, 4 h, 4ch benzoyl), 7.857.72 (m, 5 h, 4ch benzoyl, h-1), 7.65 (s, 1 h, h-1), 7.587.26 (m, 12 h, 12ch benzoyl), 5.90 (d, j3,4=9 hz, 1 h, h-3), 5.865.65 (m, 3 h, h-4, 2h-5), 5.57 (m, 2 h, h-4, h-5), 5.42 (dd, j4,3=j45=10 hz, 1 h, h-4), 4.974.88 (m, 2 h, 2h-3), 4.81 (dd, j6,5=j6,7=10 hz, 1 h, h-6), 4.614.51 (m, 1 h, h-7), 4.174.03 (m, 3 h, h-8a, 2h-6), 4.033.93 (m, 2 h, 2h-8), 3.683.55 (m, 3 h, h-8b, 2h-7), 2.01, 1.69, 1.62 (s, 9 h, 3ch3, acetyl), 0.95 (s, 18 h, 2sic(ch3)3), 0.180.04 ppm (m, 12 h, 2si(ch3)2); c nmr (100 mhz, cdcl3): =169.85, 169.01, 168.96 (3c = o acetyl), 165.85, 165.78, 165.56, 165.47 (4c = o benzoyl), 145.18 (2c-2), 133.57133.39 (ch benzoyl), 129.96128.44 (ch benzoyl), 129.39129.20 (c benzoyl) 123.08, 121.61 (2c-1), 85.64 (c-3), 79.88, 79.86 (2c-7), 75.01, 74.96 (2c-5), 74.69 (c-7), 73.60, 73.35 (2c-3), 72.60 (c-4), 72.17, 72.12 (2c-4), 70.73 (c-5), 62.50, 62.40 (2c-8), 61.50 (c-8), 60.43 (2c-6), 59.84 (c-6), 26.04 (sic(ch3)3), 20.67, 20.01, 19.94 (3ch3 acetyl), 18.57 (sic(ch3)3), 4.97(5.26) ppm (si(ch3)2); ms (esi) m / z [m+h] calcd for c68h82n12o19si2: 1427.54, found 1427.75; hrms (q - tof) m / z [m+h] calcd for c68h82n12o19si2: 1427.5358, found 1427.5389 . Compound 7: a click reaction of a mixture of compound 5 (0.88 g, 0.62 mmol) and compound 6 a (0.570 g, 1.48 mmol) with cuso45 h2o (28 mg, 0.11 mmol) and naasc (0.0443 g, 0.22 mmol) in dmf (15 ml) containing 10% h2o was performed following the general procedure described above to afford compound 7 (0.87 g, 64%); h nmr (400 mhz, cdcl3): =7.88 (s, 1 h, h-1), 7.827.68 (m, 9 h, 8ch benzoyl, h-1), 7.687.59 (2 s, 2 h, 2h-1), 7.517.39 (m, 4 h, 4ch benzoyl), 7.367.26 (m, 9 h, 8ch benzoyl), 6.356.22 (m, 2 h, 2h-5), 5.94 (d, j3,4=9 hz, 1 h, h-3), 5.885.73 (m, 2 h, 2h-4), 5.675.52 (m, 2 h, h-5, h-4), 5.435.33 (m, 2 h, 2h-13), 5.275.07 (m, 6 h, 2h-3, 2h-11, 2h-6), 5.034.71 (m, 7 h, 2 h-12, 2h-9a, h-6, 2h-9b), 4.634.53 (m, 1 h, h-7), 4.484.37 (m, 4 h, 2h-7, 2h-10), 4.294.04 (m, 5 h, 2h-15a, h-8a, 2h-15b), 3.943.85 (m, 2 h, 2h-14), 3.80 (d, j8a,8b=12 hz, 2 h, 2h-8a), 3.65 (dd, j8b,8a=12 hz, j8b,7=3 hz, 1 h, h-8b), 3.453.32 (m, 2 h, 2 h-8b), 2.201.88 (m, 21 h, 7ch3, acetyl), 1.771.63 (m, 12 h, 4ch3, acetyl), 0.940.84 (m, 18 h, 2sic(ch3)3), 0.05(0.07) ppm (m, 12 h, 2si(ch3)2); c nmr (100 mhz, cdcl3): =170.83168.96 (c = o acetyl), 165.51 (c = o benzoyl), 165.39 (c = o benzoyl), 165.07 (c = o benzoyl), 144.90 (2c-2), 143.54 (2c-2), 133.64, 129.81128.53 (ch benzoyl), 128.81 (c benzoyl) 123.66, 123.57, 123.14, 121.67 (4c-1), 99.29, 99.26 (2c-10), 85.70 (c-3), 79.40, 79.33 (2c-7), 75.02 (c-7), 74.20, 73.85 (2c-5), 73.67, 73.42 (2c-3), 72.70 (c-5), 72.22, 72.15 (2c-4), 70.99 (2c-12), 70.78 (2c-14), 70.73 (c-4), 68.74 (2c-11), 67.25 (2c-13), 61.84 (2c-9, 2c-8), 61.43 (2c-15, c-8), 60.17, 60.12 (2c-6), 59.81 (c-6), 25.96 (sic(ch3)3), 20.9719.92 (ch3 acetyl), 18.52 (sic(ch3)3), 5.11(5.34) ppm (si(ch3)2); ms (esi) m / z [m+2 h]calcd for c102h126n12o39si2: 1100.89, found 1100.60; hrms (q - tof) m / z [m+2 h] calcd for c102h126n12o39si2: 1100.3892, found 1100.8980 . Compound 8: compound 7 (0.87 g, 0.40 mmol) was dissolved in ch3cn / h2o (24 ml, 7:1), and p - tsoh (0.114 g, 0.60 mmol) was added . The mixture was stirred at rt for 6 h and was then diluted with ch2cl2 (50 ml), which was followed by the addition of 10% nahco3 (20 ml). The organic layer was washed once with brine (20 ml) and dried over na2so4, filtered, and concentrated . The residue was purified by silica gel chromatography to afford compound 8 (0.65 g, 0.33 mmol, 83%); h nmr (400 mhz, cdcl3): =8.22 (s, 1 h, h-1), 8.12 (s, 1 h, h-1), 7.92 (s, 1 h, h-1), 7.87 (s, 1 h, h-1), 7.81 (d, j=8 hz, 4 h, 4ch benzoyl), 7.767.69 (m, 4 h, 4ch benzoyl), 7.477.35 (m, 4 h, 4ch benzoyl), 7.317.16 (m, 8 h, 8ch benzoyl), 6.456.33 (m, 2 h, 2h-5), 6.30 (d, j3,4=8 hz, 1 h, h-3), 6.015.88 (m, 2 h, 2h-4), 5.81 (dd, j5,4=j5,6=10 hz, 1 h, h-5), 5.68 (dd, j4,3=j4,5=10 hz 1 h, h-4), 5.535.37 (m, 4 h, 2 h-13, 2h-6), 5.32 (d, j3,4=12 hz, 2 h, 2h-3), 5.225.09 (m, 3 h, 2h-11, h-6), 5.044.80 (m, 6 h, 2h-12, 2h-9a, 2h-9b), 4.734.65 (m, 1 h, h-7), 4.514.40 (m, 4 h, 2h-7, 2h-10), 4.334.23 (m, 2 h, 2h-15a), 4.174.07 (m, 2 h, 2h-15b), 4.043.93 (m, 3 h, 2h-14, h-8a), 3.80 (d, j8a,8b=12 hz, 2 h, 2h-8a), 3.56 (dd, j8b,8a=12 hz, j8b,7=4 hz, 1 h, h-8b), 3.29 (d, j8b,8a=12 hz, 2 h, 2h-8b), 2.201.93 (m, 21 h, 7ch3, acetyl), 1.861.48 ppm (m, 12 h, 4ch3, acetyl); c nmr (100 mhz, cdcl3): =170.88169.10 (c = o acetyl), 165.51 (c = o benzoyl), 165.26 (c = o benzoyl), 165.11 (c = o benzoyl), 144.59, 144.40, 143.75, 143.71 (4c-2), 133.60, 129.91128.44 (ch benzoyl), 128.77, 128.37 (c benzoyl) 123.79 (2c-1), 123.63 (2c-1), 99.20, 99.10 (2c-10), 85.29 (c-3), 79.35, 79.15 (2c-7), 74.85 (c-7), 73.99 (2c-5), 73.68, 73.33 (2c-3), 72.72 (c-5), 72.12 (2c-4), 70.94 (2c-12), 70.83 (2c-14), 70.81 (c-4), 68.84 (2c-11), 67.27 (2c-13), 61.64 (2c-9), 61.45 (2c-15), 61.31 (c-8), 60.64, 60.56 (2c-8), 59.92, 59.88 (2c-6), 59.84 (c-6), 20.9619.83 ppm (ch3 acetyl); hrms (q - tof) m / z [m+2 h] calcd for c90h98n12o39: 986.3027, found 986.8078 . Compound 9: a solution of compound 8 (51.6 mg, 0.026 mmol) and tempo (0.164 mg, 1.05 mol) in ch2cl2 (1 ml) was added to a solution of nabr (1.1 mg, 10.5 mol) and bu4nbr (3.4 mg, 10.5 mol) in nahco3/na2co3 buffer (1 ml, ph was adjusted to 9.5 with saturated na2co3). The mixture was cooled to 0 c, and naocl (614%, 105 l) was added dropwise . The mixture was stirred vigorously at 0 c for 2 h and then quenched by the addition of na2s2o3 (sat ., 0.5 ml), after which h2o (1.5 ml) and ch2cl2 (4 ml) were added, and the ph was adjusted to ph 3 with aqueous hcl (6 n). The organic phase was washed once with brine (1 ml), dried over na2so4, and concentrated in vacuo . The resulting oxidized compound was exposed to zmpln conditions, followed by h resin, and was concentrated . The residue was subjected to preparative hplc purification, which gave compound 9 (11.1 mg, 0.010 mmol, 38%); h nmr (400 mhz, d2o): =8.56 (s, 1 h, h-1), 8.43 (s, 1 h, h-1), 8.26 (s, 2 h, 2h-1), 6.10 (d, j3,4=12 hz, 1 h, h-3), 5.06 (d, j9a,9b=12 hz, 2 h, 2h-9a), 5.014.91 (m, 5 h, 2h-9b, h-6, 2h-3), 4.894.72 (m, 4 h, 2h-7, 2h-6), 4.584.41 (m, 6 h, h-7, 2h-10, h-5, 2h-5), 4.30 (dd, j4,3=j4,5=9.2 hz, 1 h, h-4), 4.08 (dd, j4,3=j4,5=9.2 hz, 2 h, 2h-4), 3.973.92 (m, 2 h, 2h-13), 3.883.70 (m, 6 h, 2h-15ab, 2h-14), 3.693.61 (m, 3 h, 2h-12, h-8a), 3.56 (dd, j11,10=j11,12=8 hz, 2 h, 2h-11), 3.40 ppm (dd, j8b,8a=12 hz, j8b,7=4 hz, 1 h, h-8b); c nmr (100 mhz, d2o): =172.26 (2c-8), 145.12 (c-2), 145.01 (c-2), 144.38 (2c-2), 126.65 (2c-1), 126.51 (c-1), 125.47 (c-1), 102.56 (2c-10), 88.28 (c-3), 78.82 (2c-7), 77.71 (c-7), 75.95 (2c-14), 75.04 (c-5), 75.00 (c-5), 74.40 (c-3), 74.38 (c-3), 74.28 (c-5), 73.64 (c-4), 73.59 (c-4), 73.42 (2c-12), 73.30 (c-4), 71.39 (2c-11), 69.33 (2c-13), 64.60 (2 c-6), 62.41 (2c-9), 62.33 (c-6), 61.68 (2c-15), 60.32 ppm (c-8); ms (esi) m / z [m+h] calcd for c40h56n12o26: 1121.34, found 1121.05; hrms (q - tof) m / z [m+h] calcd for c40h56n12o26: 1121.3429, found 1121.3465, [m+na] 1143.3282 . Compound 10: compound 8 (49.2 mg, 0.025 mmol) was dissolved in dry ch2cl2 (2 ml) with pyridine (200 l). The mixture was cooled down to 0 c, after which triflic anhydride (203 l, 0.203 mmol) was added dropwise to the above solution . The reaction was allowed to warm up to rt and stirred for 3 h after which 1 n khso4 (2 ml) and ch2cl2 (15 ml) were added . The organic layer was washed once with h2o (5 ml) and once with brine (5 ml), dried on na2so4, filtered, and concentrated . The resulting material was then treated with 0.5 m naome in meoh (5 ml), stirred at rt overnight, after which 1 n hcl was added to adjust to ph6, and the solvents were evaporated in vacuo . The residue was purified by preparative hplc, which gave compound 10 (13.3 mg, 0.011 mmol, 44%); h nmr (400 mhz, d2o): =8.69 (d, j16,17=8 hz, 4 h, 4h-16), 8.56 (dd, j18,17=j18,17=8 hz, 2 h, 2h-18), 8.42 (s, 1 h, h-1), 8.38 (s, 2 h, 2h-1), 8.28 (s, 2 h, h-1), 8.057.98 (m, 4 h, 4h-17), 6.06 (d, j3,4=8 hz, 1 h, h-3), 5.12 (d, j9a,9b=12 hz, 2 h, 2h-9a), 5.004.92 (m, 3 h, 2h-9b, h-6), 4.924.73 (m, 8 h, 2h-7, 2h-6, 2h-3, 2h-8a), 4.624.42 (m, 6 h, 2h-10, 2h-8b, h-7, h-5), 4.344.23 (m, 3 h, 2h-5, h-4), 4.073.98 (m, 2 h, 2h-4), 3.983.93 (m, 2 h, 2h-13), 3.883.73 (m, 6 h, 2h-15ab, 2h-14), 3.713.63 (m, 3 h, 2h-12, h-8a), 3.633.54 (m, 2 h, 2h-11), 3.35 ppm (dd, j8b,8a=12 hz, j8b,7=4 hz, 1 h, h-8b); c nmr (100 mhz, d2o): =146.77 (2 c-18), 145.32 (4c-16) 144.63 (2c-2), 144.37 (c-2), 144.23 (c-2), 128.21 (4c-17), 125.96 (2c-1), 125.56 (c-1), 124.75 (c-1), 102.42 (2c-10), 87.61 (c-3), 77.08 (c-7), 75.87 (2c-7), 75.49 (2c-14), 74.68(2c-5), 73.84 (2c-3), 73.73 (c-5), 72.96(2c-4), 72.89 (2c-12), 72.72 (c-4), 70.84 (2c-11), 68.76 (2c-13), 63.61 (2c-6), 62.07 (2c-9), 61.71 (c-6), 61.43 (2c-8), 61.20 (2c-15), 59.76 ppm (c-8); ms (esi) m / z [m] calcd for c50h68n14o22: 608.23, found 608.30; hrms (q - tof) m / z [m] calcd for c50h68n14o22: 608.2311, found 608.2356 . Compound 11: compound 8 (246 mg, 0.125 mmol) was dissolved in dry ch2cl2 (5 ml), and tosyl chloride (238.4 mg, 1.25 mmol) and 1,4-diazabicyclo[2.2.2]octane (dabco) (38 mg, 0.34 mmol) were added . The residue was redissolved in ch2cl2 (15 ml) and washed once with h2o (5 ml) and once with brine (5 ml), dried on na2so4, filtered, and concentrated . The resulting material was purified by silica gel chromatography to give the tosylated compound (220 mg, 77.2%). The residue was then dissolved in dry dmf (8 ml) to which nan3 (33.6 mg, 0.52 mmol) was added . The mixture was stirred at 95 c overnight after which the solvent was removed . The residue was dissolved in ch2cl2 (15 ml), washed once with 1 n khso4 (5 ml) and once with h2o (5 ml), dried on na2so4, filtered, and concentrated . The residue was purified by silica gel chromatography to afford compound 11 (86.8 mg, 0.043 mmol, 34%); h nmr (400 mhz, cdcl3): =7.95 (s, 1 h, h-1), 7.827.765 (m, 11 h, 4ch benzoyl, 3h-1), 7.517.39 (m, 4 h, 4ch benzoyl), 7.367.22 (m, 8 h, 8ch benzoyl), 6.366.23 (m, 2 h, 2h-5), 5.98 (d, j3,4=8 hz, 1 h, h-3), 5.925.76 (m, 2 h, 2h-4), 5.65 (t, j5,4=j5,6=8 hz, 1 h, h-5), 5.57 (t, j4,3=j4,5=8 hz, 1 h, h-4), 5.435.35 (m, 2 h, 2h-13), 5.29 (d, j3,4=8 hz, 2 h, 2h-3), 5.225.09 (m, 4 h, 2h-11, 2h-6), 5.024.83 (m, 5 h, 2h-12, 2h-9a, h-6), 4.77 (d, j9b,9a=12 hz, 2 h, 2h-9b), 4.714.56 (m, 3 h, 2h-7, h-7), 4.44 (d, j10,11=8 hz, 2 h, 2h-10), 4.284.18 (m, 2 h, 2h-15a), 4.184.05 (m, 3 h, 2h-15b, h-8a), 3.943.86 (m, 2 h, 2h-14), 3.723.62 (m, 1 h, h-8b), 3.56 (t, j8a,8b=12 hz, 2 h, 2 h-8a), 2.95, 2.88 (2dd, j8b,8a=12 hz, j8b,7=4 hz, 2 h, 2h-8b), 2.211.92 (21 h, 7ch3, acetyl), 1.831.59 ppm (12 h, 4ch3, acetyl); c nmr (100 mhz, cdcl3): =170.88169.06 (c = o acetyl), 165.36 (c = o benzoyl), 165.28 (c = o benzoyl), 164.99 (c = o benzoyl), 144.72, 144.65, 144.25, 144.24 (4c-2), 133.75133.54, 129.85128.49 (ch benzoyl), 128.72, 128.29, 128.26 (c benzoyl) 123.49, 123.40, 123.30 (3c-1), 121.72 (2c-1), 99.66, 99.60 (2c-10), 85.68 (c-3), 77.93, 77.72 (2c-7), 75.00 (c-7), 73.76, 73.71 (2c-5), 73.50, 73.28 (2c-3), 72.66 (c-5), 72.07, 72.03 (2c-4), 70.91 (2 c-12, c-4), 70.85 (2c-14), 70.81 (c-4), 68.78 (2c-11), 67.23 (2 c-13), 62.17, 62.13 (2c-9), 61.43 (2c-15, c-8), 60.81 (2c-6), 59.72 (c-6), 50.58, 50.37 (2c-8), 20.9419.92 ppm (ch3 acetyl); ms (esi) m / z calcd for c90h96n18o37 (m+2 h) 1011.91, found 1012.35; hrms (q - tof) m / z [m+2 h] calcd for c90h96n18o37: 1011.3092, found 1011.3091, [m+h+na] 1022.8011, [m+2na] 1033.2923 . Compound 12: compound 11 (22.6 mg, 0.020 mmol) was treated with 0.5 m naome in meoh (2.5 ml) at rt overnight and briefly with h resin . The residue was concentrated and dissolved in h2o (2 ml), and pd / c (15 mg, 10% pd) was added . The ph was adjusted to ph 1 by 6 n hcl, and the mixture was stirred at rt under an h2 atmosphere until the hydrogenation was complete . The filtrate was concentrated under reduced pressure and subjected to preparative hplc purification, which gave compound 12 (12.9 mg, 0.012 mmol, 60%); h nmr (400 mhz, d2o): =8.56 (s, 1 h, h-1), 8.43 (s, 1 h, h-1), 8.33 (s, 2 h, 2h-1), 6.13 (d, j3,4=8 hz, 1 h, h-3), 5.09 (d, j9a,9b=10 hz, 2 h, 2h-9a), 5.034.89 (m, 5 h, 2h-3, h-6, 2h-9b), 4.894.70 (m, 2 h, 2h-6), 4.664.47 (m, 6 h, 2h-7, 2h-10, h-7, h-5), 4.414.28 (m, 3 h, 2h-5, h-4), 4.083.99 (m, 2 h, 2h-4), 3.993.93 (m, 2 h, 2h-13), 3.893.73 (m, 6 h, 2h-15ab, 2h-14), 3.713.64 (m, 3 h, 2h-12, h-8a), 3.57 (dd, j11,10=j11,12=8 hz, 2 h, 2h-11), 3.40 (dd, j8b,8a=12 hz, j8b,7=4 hz, 1 h, h-8b), 3.243.11 (m, 2 h, 2h-8a), 2.86 ppm (d, j8b,8a=12 hz, 2 h, 2h-8b); c nmr (100 mhz, d2o): =144.94, 144.82 (2c-2), 144.69 (2c-2), 126.18 (2c-1), 126.13 (c-1), 125.07 (c-1), 102.63 (2c-10), 87.97 (c-3), 77.46 (c-7), 75.76 (2c-14), 74.90 (2c-5), 74.51 (2c-7), 74.33 (2c-3), 74.07 (c-5), 73.45 (2c-4), 73.18 (2c-12), 73.13 (c-4), 71.14 (2c-11), 69.09 (2c-13), 64.27 (2c-6), 62.39 (2c-9), 62.08 (c-6), 61.48 (2c-15), 60.12 (c-8), 40.59 ppm (2c-8); ms (esi) m / z [m+2 h] calcd for c40h62n14o22: 546.21, found 546.90; hrms (q - tof) m / z [m+h] calcd for c40h62n14o22: 1091.4163, found 1091.4282, [m+na] 1113.4132 . Compound 13: a click reaction of a mixture of compound 11 (61.8 mg, 0.031 mmol), phenylacetylene (11.1 mg, 0.109 mmol), naasc (6.5 mg, 0.033 mmol), and cuso45 h2o (4.1 mg, 0.016 mmol) was performed following the general procedure described above to afford the coupling product compound (54 mg, 80%), which was then treated with naome in meoh (0.5 m, 5 ml). The mixture was stirred at rt overnight, treated with h resin, concentrated, and subjected to preparative hplc purification, which gave compound 13 (9.8 mg, 7.3 mol, 24%); h nmr (400 mhz, d2o with 30% cd3cn): =8.62 (s, 1 h, h-1), 8.50, 8.49 (2 s, 2 h, 2h-1), 8.46 (s, 1 h, h-1), 8.42, 8.41 (2 s, 2 h, 2h-1), 8.057.98 (m, 4 h, 4c-17), 7.75 (t, j18,17=j18,19=8 hz, 4 h, 4c-18), 7.717.62 (m, 2 h, 2c-19), 6.21 (d, j34=9 hz, 1 h, h-3), 5.27 (d, j9a,9b=12 hz, 2 h, 2h-9a), 5.164.99 (m, 7 h, 2h-9b, 2h-3, h-6, 2h-7), 4.924.83 (m, 2 h, 2h-6), 4.834.70 (m, 4 h, 2h-8a, 2h-10), 4.704.58 (m, 4 h, 2h-8b, h-5, h-7), 4.574.48 (m, 2 h, 2h-5), 4.43 (t, j4,3=j4,5=8 hz, 1 h, h-4), 4.214.11 (m, 4 h, 2h-4, 2h-13), 4.083.90 (m, 6 h, 2h-15ab, 2h-14), 3.893.82 (m, 2 h, 2h-12), 3.823.73 (m, 3 h, 2h-11, h-8a), 3.46 ppm (dd, j8b,8a=13 hz, j8b,7=4 hz, 1 h, h-8b); c nmr (100 mhz, d2o with 30% cd3cn): =147.90 (4c-2), 145.44 (2c-2), 130.82129.48 (4c-18, 2c-19), 130.00 (2c-16), 126.40 (2c-1), 126.21 (4c-17), 125.78 (c-1), 124.76 (c-1), 123.66 (2c-1), 102.73 (2c-10), 88.11 (c-3), 77.554 (c-7), 76.27 (2c-7), 75.81 (2c-14), 75.31 (2c-5), 74.50 (2c-3), 74.20 (c-5), 73.62 (2c-4), 73.38 (2c-12), 73.25 (c-4), 71.29 (2c-11), 69.18 (2 c-13), 64.29 (2c-6), 62.37 (2c-9), 62.07 (c-6), 61.55 (2c-15), 60.22 (c-8), 51.44 ppm (2c-8); ms (esi) m / z [m+2 h] calcd for c56h70n18o22: 674.63, found 674.95; hrms (q - tof) as a service to our authors and readers, this journal provides supporting information supplied by the authors . 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Cardiopulmonary resuscitation (cpr) is a potentially life - saving intervention following cardiac arrest . However, it may be associated with traumatic injury to the upper abdominal organs, namely the liver, stomach and spleen [24]. We here describe a rare case of occult splenic rupture complicating resuscitation efforts in post - operative cardiac arrest occurring immediately after left pneumonectomy . A 63-year - old male smoker was admitted for elective surgical resection of a left upper lobe squamous cell carcinoma, diagnosed 2 months previously . His medical history included psoriasis, lumbar disc prolapse and previous laparoscopic cholecystectomy without concomitant haematological or neoplastic disease . Mediastinoscopy demonstrated only reactive changes in the sampled lymph nodes; therefore, informed consent was obtained for left upper lobectomy . At bronchoscopy, tumour was seen to involve the orifice of the left upper lobe bronchus . A left upper lobe sleeve resection was initially performed via left posterolateral thoracotomy, with routine stapling and division of the left superior pulmonary vein and upper lobe pulmonary artery branches . Following this however, lung reinflation was not possible and left pneumonectomy was completed uneventfully with surgical haemostasis and placement of a single basal chest drain . With the patient in a right lateral position, external cpr alongside emergent re - thoracotomy were performed . Active bleeding from a disrupted left inferior pulmonary vein staple line was controlled with sutures . Once cardiac output was regained, the patient was stabilized with minimal inotropic support, and left ventricular pacing wires were sited . Unfortunately, haemodynamic instability recurred after chest closure, this time without large - volume chest drainage, and the patient could not be resuscitated after a second cardiac arrest . Examination demonstrated a distended peritoneal cavity containing at least 1 200 ml of blood . In addition, a subcapsular haematoma measuring 9 5 cm was noted on the upper pole of the spleen in association with a 3 2 cm laceration; a second 3.5 4 cm laceration was also present on the inferior pole . The left 2nd7th ribs, right 2nd5th ribs and the right 8th rib were fractured in the mid - axillary line . The coroner concluded that the intra - abdominal bleeding had originated from the spleen, although there was no pathological evidence attributing this to the recent surgical intervention . The acute splenic rupture was considered to be consequent to the first episode of cpr . External cardiac massage in combination with ventilatory support and timely defibrillation has been demonstrated to enhance outcomes following cardiac arrest . Nevertheless, recognized complications associated with the technique include rib and sternal fractures, and injuries to the pleura, pericardium, heart and great vessels . Gastric mucosal tears have been found in 912% of patients following cpr, while liver lacerations are reported in 2% . Splenic rupture is a rarer occurrence, present in 0.3% of 705 patients undergoing post - mortem examination after cpr . We believe that the present case is the only report of splenic rupture subsequent to cpr, occurring immediately post - operatively . The prompt and accurate identification of occult intra - abdominal bleeding after cpr, particularly so soon after major thoracic surgery, represents a diagnostic challenge . We recognized the onset of hypovolaemic cardiac arrest following left pneumonectomy, instituted cpr accordingly and were able to control active bleeding from a disrupted staple line via emergency thoracotomy . The patient s rapid deterioration following the initial successful resuscitation, however, was not accompanied by profuse chest drainage and was attributed to a primary myocardial event rather than intrathoracic haemorrhage . A second re - thoracotomy at this stage would likely have yielded no additional diagnostic benefit, since the culprit splenic lesion would not have been visualized . Basic radiological studies, such as an erect chest radiograph to reveal free subdiaphragmatic air, or abdominal ultrasound, could have been performed had the patient not destabilized after the first cardiac arrest . Furthermore, exploratory laparotomy in this already precarious situation would be very hazardous and rapid arrest of bleeding might not have been achieved within the confines of a cardiac arrest scenario . In the present case, pre - existing infective or neoplastic diseases causing splenomegaly might have predisposed this enlarged organ to rupture during cpr, but none were present in our patient . While left - sided rib fractures were sustained during resuscitation, the spontaneous rupture of an apparently normal spleen following left - sided lung resection has been described, but splenic rupture in the context of cpr immediately subsequent to left pneumonectomy has not been reported before . It is known that major left lung resections may be accompanied by varying degrees of left diaphragmatic elevation . Thus, we propose that elevation of the left hemidiaphragm to occupy part of the cavity resulting from left pneumonectomy may have caused the spleen to reside in a slightly higher anatomical position than normal, especially in a supine patient . This, perhaps combined with chest compressions over the lower border of the sternum, may have led to significant compressive forces being transmitted over the spleen, precipitating laceration and rupture . This mechanism of injury may also happen following left - sided lobectomy, especially if the lower lobe is resected . An alternative explanation would be upper abdominal adhesions after previous cholecystectomy, which might have anchored the spleen to an abnormally high subcostal position . In conclusion, concealed intra - abdominal visceral injury may occur during cpr and complicate resuscitation efforts by contributing to persistent hypotension . Clinicians should be aware that intrathoracic volume changes following major procedures, such as pneumonectomy or lobectomy, may lift the spleen into a higher anatomical location, rendering it at risk of injury during cpr . Finally, special attention should be paid to the desired position of chest compressions to prevent excessive force being directed over the upper abdominal viscera.
Nocturnal sleep of patients suffering from various forms of dementia is often impaired by nocturnal agitation or nocturnal wandering . Anticonvulsives such as carbamazepine or valproate are reported to have some therapeutic efficacy, but there is little information about other drugs suitable for treatment of this condition . Our patient, a 77-year - old czech woman with incipient vascular dementia, received gabapentin 400 mg at bedtime for 6 months and showed convincing improvement . Excessive motor activity during the night impairs the sleep of patients with various forms of dementia . Lack of sleep due to nocturnal agitation or nocturnal wandering is a significant burden on families of elderly demented patients and is the primary cause of institutionalization [14]. Nocturnal agitation may result from discomfort, pain or environmental factors; hence, identification of these potential sources is crucial . For insomnia management, eliminating alcohol and restricting caffeine intake in the early morning may help in alleviating nocturnal agitation . However, if necessary, atypical neuroleptics, anticonvulsives (carbamazepine, valproate) and benzodiazepines, as well as trazodone and chloral hydrate are recommended . We present the case of a patient with incipient vascular dementia accompanied by nocturnal agitation, which was successfuly treated with gabapentin . To our best knowledge, no controlled study has yet been conducted to prove its efficacy in this condition . We present the case of a czech woman, now 77 years old, who was treated for ischemic heart disease, arterial hypertension, chronic obstructive pulmonary disease and migrainous cephalalgia . The patient was referred to our center for sleep disorders for the first time in 2006 due to a history of 2 nocturnal episodes of confusion with abnormal behavior and retrograde amnesia . The patient herself could not recall these episodes, and it was not possible to retrieve further objective history . Eeg showed a mild slowing of the background activity, but no epileptiform discharges were found . As partial complex epileptic seizure was suspected, gabapentin (300 mg 3 times daily) was administered and subsequently found to have brought the patient considerable relief . Gabapentin is generally not the first - choice drug in the cases of temporal lobe epilepsy; nevertheless, we considered it appropriate because of diagnostic uncertainty and only sporadic occurrence of the episodes . Next, the patient consulted our department in may 2008 because of 3 episodes of syncope . Cardiologic examination excluded cardiac disease as the cause, which appeared to be of cerebrovascular etiology . Since our previous diagnosis of temporal lobe epilepsy was uncertain and with respect to the 4-year seizure - free period, gabapentin was discontinued in june 2008 . Two weeks after its withdrawal the nocturnal episodes of confusion with abnormal behavior reappeared . According to a room - mate, the patient was agitated in the night, shuffled about in her apartment, did not know where she was, talked aloud and was sometimes verbally aggressive . In the morning the patient could not recall the episodes, though these recurred several times during the night . These nocturnal states of confusion were the reason for her admission and video - eeg monitoring at our department . During the monitoring, performed on 2 consecutive nights, the patient repeatedly sat up and attempted to get out of bed, but could not due to being attached to the monitor . Though confused, she reacted to the technician s statements appropriately, but with a delay . She complained of discomfort caused by the electrodes and the recurrent urge to void . The technician repeatedly told her where she was, but she continued to ask the same question . During both nights we recorded the above - described episodes during the first night every 20 minutes, and during the second night every 40 minutes . The quality of the sleep was considerably impaired by fragmentation, by a higher percentage of stage 1nrem, and by a worse configuration of k - complexes and sleep spindles . No epileptiform activity or respiratory disturbances or periodic leg movements were recorded . During follow - up clinical examination after the video - eeg monitoring, the patient complained of memory failures for the first time, and for this reason a psychological examination was performed, which revealed low - grade organic deterioration of cognitive functions with excursive thinking, impairment of encoding and retrieval of information and normal spatial orientation . The mini - mental state examination (mmse) score was 26 and the cohen - mansfield agitation inventory (cmai - long form) score was 39 . With regard to the fact that the nocturnal episodes of confusion, sometimes accompanied by verbal aggression, were evoked by environmental influences and discomfort and by concomitant incipient vascular dementia, we concluded the case was nocturnal agitation (nocturnal wandering). We recommended elimination of the evoking factors, and following the recommended regimen with the goal of reducing the insomnia . In spite of the regimen precautions the episodes repeated several times during most nights and we decided to begin pharmacological treatment . Since gabapentin had proved effective and well - tolerated before, we prescribed it again, with very satisfactory results . To date, the patient has been taking 400 mg of gabapentin at bedtime for 9 months, with no recurrence of her former problems despite the relatively low dosage . Thus, we consider the effectiveness of gabapentin in this condition to be clearly demonstrated . We confirm that the patient was able to provide informed consent . The use of gabapentin has thus far been only sporadically reported in this condition [710]. Therefore, we should like to call attention to our satisfactory experience of gabapentin in cases like ours . In particular, we found it suitable in geriatric patients for its minimal adverse effects and absence of pharmacokinetic interactions with respect to geriatric polymorbidity . It is noted for its anti - anxiety and hypnotic effects in psychiatric patients . Its positive effect on the structure / quality of sleep is well - known . While its mechanism of action is not yet fully understood, it would appear to be via the modulation of glutamate - mediated excitatory synaptic transmission and gamma - aminobutyric acid (gaba)-mediated inhibitory synaptic transmission . Unlike carbamazepine, it does not interfere with bone metabolism, nor does it have any adverse cardiac effects . Nevertheless, there are still limited data on its efficacy, and no controlled studies have been published to date on this topic . As we mentioned above, in most of the reviewed cases gabapentin is reported to be a well - tolerated and effective treatment for dementia - associated agitation . However, several case reports in which gabapentin was used for agitation in dementia with lewy bodies question its appropriateness for all types of dementia - related agitation [1416]. Indeed, paradoxical gabapentin - induced exacerbation of psychosis in a patient with schizophrenia has already been described . The relevant literature recommends drugs such as benzodiazepines; however, these can aggravate the cognitive deficit, a fact of importance in our patient . We present the case of a patient with incipient vascular dementia accompanied by nocturnal agitation, which was successfully treated with gabapentin.
Moreover, secondary syphilis should be considered in the differential diagnosis of any dermatosis which is atypical . We are reporting a case of secondary syphilis, without any history of a primary lesion, that resembled borderline lepromatous (bl) leprosy, and in whom the diagnosis was considered on clinical grounds . A 57 year old middle class male patient presented with asymptomatic, generalized, annular plaques present for 2 months, and without constitutional symptoms . The plaques were situated on the front and back of trunk [figure 1], on the upper extremities and thighs, and on the face, with confluent nodules on the forehead . The larger plaques were 4 - 5 cm in size, and had succulent papulonodules at the margin [figure 1, site of biopsy 1]. Other plaques had scaly margins, which were rolled out [figure 1]. On the right forearm there were eroded, annular plaques, with sharply raised overhanging, and scaly margins [figure 2]. However, on noting that the plaques showed increased sensitivity to pain (buschke - ollendorf sign), secondary syphilis was also considered . Moreover, a generalized and epitrochlear lymphadenopathy was also noted, but there were no mucous patches or condyloma lata . The plaque with a scaly margin is seen on the left scapular area (biopsy 2) the eroded, annular plaques with scaly margins, on the right forearm, before treatment subsequent investigations confirmed the diagnosis of secondary syphilis, as his rapid plasma reagin (rpr) test was reactive in 1:128 dilution, his skin smears were negative for lepra bacilli, and his treponema pallidum haemagglutination test was also positive . On direct questioning, he also had concomitant hiv infection by the elisa test, and his cd4 count was 269 cells / cmm . His chest x - ray was normal, and his eye, cardiovascular, and neurological examination did not reveal any abnormality . His cerebrospinal fluid (csf) examination revealed asymptomatic neurosyphilis, with 10 cells / cmm and 50 mg protein / dl, but with a negative csf venereal disease research laboratory (vdrl) test . His skin biopsy performed from two different plaques showed essentially similar features, with the presence of a florid subepidermal granulomatous infiltrate without a free grenz zone, in biopsy 2 . A few langhans and multinucleate giant cells were also seen, apart from a periappendageal and perineural granulomatous infiltrate rich in plasma cells [figure 3, biopsy 1] that was breaking up the arrectores pilorum muscle . In addition, endothelial swelling, with luminal narrowing, and a perivascular infiltrate of lymphocytes and plasma cells was also present . A periappendageal and perineural infiltrate (h and e 200, biopsy 1) the patient received three weekly injections of 2.4 million units of intramuscular benzathine penicillin, along with 2 g of daily intravenous ceftriaxone for 15 days . He did not develop any fever after any of his injections (negative jarisch - herxheimer reaction). The plaques on the back had flattened 50% within one week of the penicillin injection [figure 4] and the eroded plaques on the forearm had regressed 70% by then . Within 20 days, the plaques had almost totally flattened, and 1 months later, only hyperpigmented macules were remaining . After 4 months his repeat rpr had fallen to 1:64 dilution, and his cd4 count had risen to 547 cells / cmm . When last seen one year later, his rpr had further fallen to 1:16 dilution, and there was no recurrence of his skin lesions . Hence, it is suggested that an rpr / vdrl test should be routinely performed in any atypical dermatosis . In our patient, there was both a clinical and a histological resemblance to leprosy, as well as atypical features . However, on careful evaluation, a diagnosis of granulomatous secondary syphilis could have been considered, as there were numerous plasma cells, associated with changes in the blood vessels . There have been similar reports of secondary syphilis resembling bl or ll leprosy, with, . And without associated hiv . Syphilis, when associated with hiv, could be more severe and unusual, with an increased risk of neurological involvement, hastening of the late stage, and treatment failure . Hence, treatment as for late syphilis has been advocated in cases of secondary syphilis, with hiv, along with a mandatory csf test . We treated his asymptomatic neurosyphilis with the alternative recommended dose of ceftriaxone, . And also decided to complete the course of benzathine penicillin, as it had already been started . Syphilis facilitates the transmission of hiv, by increasing the viral load, and reduces the cd4 count, which increases after the syphilis is successfully treated . In conclusion, it may be stated that this case has demonstrated the primacy of the time - tested method of a thorough clinical examination and history, followed by the relevant and interpretative investigations, and a good, clinico - pathological correlation for making the correct diagnosis in any unusual dermatosis.
Bone marrow was isolated from adult male new zealand white rabbit femurs via aspiration with an 18-gauge needle . The cells in the bone marrow were expanded in monolayer culture and split and subcultured according to standard protocols developed for rat models.16 17 approximately 5 10 cells were placed in each treatment well of a 24-well plate and exposed to one of three treatments: (1) minimum essential medium (mem); (2) 40 ng / ml dexamethasone and mem; or (3) 50 ng / ml bmp-2, 40 ng / ml dexamethasone, and mem . The noncontrol treatments were given additional mineralizing agents of 2.8 10 mol / l concentration of l - ascorbic acid and 10 mmol / l concentration of -glycerophosphate starting on day 7 and every media change thereafter.18 19 the media was changed every 3 days . Louis, mo) to determine the activity of the alkaline phosphatase enzyme, an early indicator of osteogenesis,20 and von kossa,21 which indicates mineralization, a late stage of osteogenesis . The amount of staining was quantified as follows: digital images of the stained wells were obtained at low power such that the entire well could be accommodated in one picture frame (as can be seen in figs . 1 2 3 4). The images were imported into image analysis software (image j: http://rsbweb.nih.gov/ij/). Using a custom written macro the measured area depended on the threshold value used for the image (portions of the image with staining intensity below the threshold would be ignored; thus the higher the threshold value, the lower the stained area). The appropriate threshold value was determined from the control samples; in the control samples, the threshold value was adjusted until the stained area became 0 . (a) rabbit bone marrow derived cells were cultured in monolayer under different conditions and tested for alkaline phosphatase activity (proportional to red color) at different times . (b) alkaline phosphatase enzyme activity increased through day 21 and declined by day 28 . Analysis of variance showed that some groups were significantly different from others (f = 0.0021 at p = 0.05 level) in terms of alkaline phosphatase activity . Mem, minimum essential medium; dexa, dexamethasone; bmp, bone morphogenic protein . (a) rabbit bone marrow derived cells were cultured in monolayer and tested for calcium deposition by von kossa staining (black) at different time points . In the negative controls, a chelator of calcium was added before von kossa staining . The clear wells of the negative control indicated that the stain seen was due to deposited calcium and was not an artifact . (b) von kossa staining was not observed through day 21 but appeared at day 28 . Treatment groups showed significantly higher mineralization than controls on day 28 (p = 0.001). Mem, minimum essential medium; dexa, dexamethasone; bmp, bone morphogenic protein . Rabbit bone marrow derived cells were cultured in monolayer under different conditions and tested for alkaline phosphatase activity at different time points . Alkaline phosphatase enzyme activity was significantly enhanced by nicotine at 100-g / ml dose over the control at p <0.05 . Rabbit bone marrow derived cells were cultured in monolayer and tested for calcium deposition by von kossa staining at different time points . Both 100 and 250 g / ml treatments showed significantly higher mineralization than control on day 28 (p <0.05). Approximately 3 10 cells were placed in each treatment well of a 24-well plate and culture medium containing mem and 40 ng / ml dexamethasone was added . All treatments were given 2.8 10 mol / l concentration of l - ascorbic acid and 10 mmol / l concentration of -glycerophosphate after day 7 and with every media change after that . Cells were exposed to one of six concentrations of nicotine: 20, 40, 80 ng / ml and 10, 100, 250 g / ml . Control cells were not exposed to nicotine . Wells were stained with an alkaline phosphatase staining kit (sigma) to determine the activity of the alkaline phosphatase enzyme . Bone marrow was isolated from adult male new zealand white rabbit femurs via aspiration with an 18-gauge needle . The cells in the bone marrow were expanded in monolayer culture and split and subcultured according to standard protocols developed for rat models.16 17 approximately 5 10 cells were placed in each treatment well of a 24-well plate and exposed to one of three treatments: (1) minimum essential medium (mem); (2) 40 ng / ml dexamethasone and mem; or (3) 50 ng / ml bmp-2, 40 ng / ml dexamethasone, and mem . The noncontrol treatments were given additional mineralizing agents of 2.8 10 mol / l concentration of l - ascorbic acid and 10 mmol / l concentration of -glycerophosphate starting on day 7 and every media change thereafter.18 19 the media was changed every 3 days . Louis, mo) to determine the activity of the alkaline phosphatase enzyme, an early indicator of osteogenesis,20 and von kossa,21 which indicates mineralization, a late stage of osteogenesis . The amount of staining was quantified as follows: digital images of the stained wells were obtained at low power such that the entire well could be accommodated in one picture frame (as can be seen in figs . 1 2 3 4). The images were imported into image analysis software (image j: http://rsbweb.nih.gov/ij/). Using a custom written macro the measured area depended on the threshold value used for the image (portions of the image with staining intensity below the threshold would be ignored; thus the higher the threshold value, the lower the stained area). The appropriate threshold value was determined from the control samples; in the control samples, the threshold value was adjusted until the stained area became 0 . (a) rabbit bone marrow derived cells were cultured in monolayer under different conditions and tested for alkaline phosphatase activity (proportional to red color) at different times . (b) alkaline phosphatase enzyme activity increased through day 21 and declined by day 28 . Analysis of variance showed that some groups were significantly different from others (f = 0.0021 at p = 0.05 level) in terms of alkaline phosphatase activity . Mem, minimum essential medium; dexa, dexamethasone; bmp, bone morphogenic protein . (a) rabbit bone marrow derived cells were cultured in monolayer and tested for calcium deposition by von kossa staining (black) at different time points . In the negative controls, a chelator of calcium was added before von kossa staining . The clear wells of the negative control indicated that the stain seen was due to deposited calcium and was not an artifact . (b) von kossa staining was not observed through day 21 but appeared at day 28 . Treatment groups showed significantly higher mineralization than controls on day 28 (p = 0.001). Mem, minimum essential medium; dexa, dexamethasone; bmp, bone morphogenic protein . Rabbit bone marrow derived cells were cultured in monolayer under different conditions and tested for alkaline phosphatase activity at different time points . Alkaline phosphatase enzyme activity was significantly enhanced by nicotine at 100-g / ml dose over the control at p <0.05 . Rabbit bone marrow derived cells were cultured in monolayer and tested for calcium deposition by von kossa staining at different time points . Both 100 and 250 g / ml treatments showed significantly higher mineralization than control on day 28 (p <0.05). Approximately 3 10 cells were placed in each treatment well of a 24-well plate and culture medium containing mem and 40 ng / ml dexamethasone was added . All treatments were given 2.8 10 mol / l concentration of l - ascorbic acid and 10 mmol / l concentration of -glycerophosphate after day 7 and with every media change after that . Cells were exposed to one of six concentrations of nicotine: 20, 40, 80 ng / ml and 10, 100, 250 g / ml . Control cells were not exposed to nicotine . Wells were stained with an alkaline phosphatase staining kit (sigma) to determine the activity of the alkaline phosphatase enzyme . The trend in the data showed that alkaline phosphatase activity increased over a 3-week period with a decline in activity seen at week 4 (figs . 2a, 2b) with the mem + bmp-2 + dexamethasone treatment having the largest percent of area coverage (p = 0.001). This mimicked the behavior of other preosteoblastic cells that have been induced to undergo osteogenesis.17 22 control cells behaved as expected, with increasing alkaline phosphatase activity up to 3 weeks and dropping off slightly at 4 weeks . As expected, there was no appreciable von kossa staining up to 3 weeks but after 4 weeks, the control cells stained positively for von kossa . A two - way analysis of variance (anova) using dose and time as variables showed significant differences among groups (f = 0.0021). A post hoc analysis (least squares means differences student t test) among groups showed that the 100-g / ml dose of nicotine significantly enhanced alkaline phosphatase activity over controls (fig . 3). Von kossa staining was not observed at sampling times of weeks 1 through 3 . A one - way anova using dose as the variable was performed, and the 100- and 250-g / ml dose had significantly greater mineralization than controls . The dose - response analysis revealed a statistically significant (p = 0.001) effect of nicotine dose on alkaline phosphatase activity and von kossa activity at 4 weeks (figs . 5a, 5b). The alkaline phosphatase activity at 1, 2, and 3 weeks showed the same trends but the results were not statistically significant . A dose - response analysis showed a statistically significant effect of dose on osteoblastic activity markers (a) alkaline phosphatase (alpase) activity (p = 0.0139) and (b) von kossa (p = 0.0352) staining after 4 weeks of culture . The doses are shown in ng / ml concentrations . The peak effect was seen for the 100-g / ml dose and dropped off for higher doses . The trend in the data showed that alkaline phosphatase activity increased over a 3-week period with a decline in activity seen at week 4 (figs . 2a, 2b) with the mem + bmp-2 + dexamethasone treatment having the largest percent of area coverage (p = 0.001). This mimicked the behavior of other preosteoblastic cells that have been induced to undergo osteogenesis.17 22 control cells behaved as expected, with increasing alkaline phosphatase activity up to 3 weeks and dropping off slightly at 4 weeks . As expected, there was no appreciable von kossa staining up to 3 weeks but after 4 weeks, the control cells stained positively for von kossa . A two - way analysis of variance (anova) using dose and time as variables showed significant differences among groups (f = 0.0021). A post hoc analysis (least squares means differences student t test) among groups showed that the 100-g / ml dose of nicotine significantly enhanced alkaline phosphatase activity over controls (fig . 3). Von kossa staining was not observed at sampling times of weeks 1 through 3 . 4). A one - way anova using dose as the variable was performed, and the 100- and 250-g / ml dose had significantly greater mineralization than controls . The dose - response analysis revealed a statistically significant (p = 0.001) effect of nicotine dose on alkaline phosphatase activity and von kossa activity at 4 weeks (figs . 5a, 5b). The alkaline phosphatase activity at 1, 2, and 3 weeks showed the same trends but the results were not statistically significant . A dose - response analysis showed a statistically significant effect of dose on osteoblastic activity markers (a) alkaline phosphatase (alpase) activity (p = 0.0139) and (b) von kossa (p = 0.0352) staining after 4 weeks of culture . The doses are shown in ng / ml concentrations . The peak effect was seen for the 100-g / ml dose and dropped off for higher doses . Our study demonstrated a statistically significant effect of nicotine dose on osteoblast activity in vitro . Bone marrow derived osteoblastic cells increase alkaline phosphatase activity in the presence of nicotine, although statistically significant effects were only seen for high doses in the g / ml range . Mineralization was enhanced when nicotine was administered with higher dosages, resulting in more mineralization (100- and 250-g / ml dosages). In a similar study, nicotine has been shown to have a significant dose - dependent effect on the activity of osteoblastic cell lines with increased alkaline phosphatase activity and deposition of calcium.23 fang et al24 showed that nicotine suppressed proliferation and increased alkaline phosphatase activity in umr 601 osteoblast - like cells . Walker et al.25 showed that nicotine had a direct effect on osteoblasts that includes increased expression of osteopontin . In that study, cells expressed the nicotine receptor 4 unit and the effects of nicotine were blocked by receptor antagonists . Rothem et al reported a dose - dependent effect of nicotine on bone metabolism in osteoblastic cells, although low - dose nicotine (equivalent to a light smoker) up - regulated expression of osteocalcin, type i collagen, and alkaline phosphatase, and a high concentration inhibited expression of these genes.26 kim et al27 showed a bimodal effect of nicotine on the proliferation and osteoblastic differentiation of mesenchymal stem cells derived from alveolar bone marrow . These studies support the fact that nicotine can have a positive role in mesenchymal stem cells and osteoblast function and may act in a dose - dependent manner . Thus, the increased fusion rates we previously observed in rabbits exposed to nicotine in vivo might be explained partially by enhanced osteoblastic activity due to nicotine . The dose of nicotine exposure in the current study, however, is substantially higher than that administered during our prior in vivo study.15 this disparity in doses that causes responses in the in vivo and in vitro systems continues to be a concern and demands further investigation . One potential explanation could be that the bone marrow aspirate we used contains many cell types, only a small number of which are osteoblast progenitors, and thus a higher dose is required to elicit a statistically significant difference . However, the trend is consistent with dosage, although very high doses were required for statistical significance . The fact that our in vivo and in vitro data trend in the same direction despite dose differences is encouraging . Gullihorn et al,36 working with an osteoblastic cell line, showed statistically significant responses of nicotine for as low as 12.5 ng / ml, which is well within the circulating levels of nicotine in smokers . Kim et al27 showed bimodal responses that changed as the concentration of nicotine shifted between the 1 to 2 mmol / l range . Thus the specific cell - culture model seems to heavily influence the dosages at which the effects of nicotine are seen . Interestingly, in many models of posterior spinal fusion, in vivo administration of nicotine has been shown to decrease fusion rates . Decreased angiogenesis measured by decreased vascular ingrowth into autogenous cancellous bone grafts9 and inhibition of the expression of cytokines associated with neovascularization10 have been observed in these models . However, in other settings, such as in tumor or ischemia models, nicotine is a potent enhancer of angiogenesis.13 the overall relationship between bony fusion rates and nicotine may depend on many factors including both angiogenesis and osteoblastic activity . Nicotine seems to positively affect osteoblasts and negatively affect vascularization in bone and increase angiogenesis in other models . High doses of nicotine administered via mini osmotic pumps (1.5 to 4 times as much as heavy smokers) administered for 12 weeks caused no difference in bone mass and strength in female rats,28 although bone mineral density was lowered in male rats given nicotine via drinking water in a different study.29 in growing female rats, nicotine had no effect on bone mineral content, bone strength, or markers of bone metabolism such as vitamin d, calcitonin, and parathyroid hormone.30 in adult female rats, a limited effect was noticed for high concentrations of nicotine.31 gotfredsen et al reported that long - term (6 months) exposure to nicotine did not have a detrimental effect on osseointegration of titanium implants in a rabbit osteotomy model.32 numerous clinical studies have documented deleterious effects of smoking on bone healing and spinal fusion.2 33 34 35 one must be careful, however, to differentiate smoking exposure from nicotine exposure . Gullihorn et al reported that although exposure of osteoblast - like cells to nicotine elicited a dose - dependent increase in metabolic activity, preparations composed of cigarette smoke condensate with equivalent levels of nicotine showed reduced alkaline phosphatase activity and decreased total protein and collagen synthesis.36 skott et al reported the results of exposure to nicotine, tobacco extract, and both in a rat femur fracture model.37 when mechanically testing the fracture healing, they found that ultimate torque and torque at yield point in rats receiving tobacco extract alone were decreased 21% and 23%, respectively, compared with the control (saline infusion) group and 20% and 26%, respectively, compared with the nicotine - only group . The combined group (tobacco extract plus nicotine) demonstrated an 18% torque reduction compared with the nicotine only group, and no difference was found between the tobacco - only group and the combined group.37 this suggests that smoking, rather than just nicotine, may be the prime culprit . Some authors have even hypothesized that low - dose nicotine exposure may be utilized to reduce the incidence of osteoporosis.38 in conclusion, the results of this study suggest that the effects of nicotine on osteoblast activity are complex and dose - dependent and may not always be detrimental . More research needs to be performed before the effects of nicotine on healing bone can be determined.
Three - dimensional (3d) computed tomography (ct) provides accurate and detailed information for diagnosis and treatment planning of dentofacial deformities . Although volume - rendered and multiplanar reconstructed images are mostly used, 3d surface models are more useful in some circumstances, such as evaluation of facial asymmetry1,2 and computer - assisted surgical simulation.3,4,5,6 despite the many advantages of 3d ct, detailed occlusal and accurate interocclusal data cannot be obtained . Moreover, image quality is affected by artifacts induced by various factors such as beam hardening, extinction, scatter, noise, exponential edge gradient effect, aliasing, partial volume effect, and object motion.7,8,9,10 in particular, the quality is worsened by the existence of metals such as orthodontic brackets and dental restorations.7,11,12 to overcome this limitation, attempts have been made to combine maxillofacial ct images with digital dental models.13,14,15,16,17,18 in this method, the dental part of a ct image is replaced with a 3d dental surface model created by optical14 or laser scanning.13,15,16,17,18 nevertheless, artifacts influence image accuracy when surface registration is used for the fusion.14,18 the purpose of this study was to assess artifacts induced by metallic restorations in 3d dental surface models derived by cone - beam computed tomography (cbct). For this study, extracted human premolars and molars were prepared by removing soft tissue, residual bone, and calculus and embedded in plaster blocks (15 specimens with four different teeth per specimen) such that their crowns were aligned as in the natural dentition . In each specimen, cavities of increasing size were prepared in the second premolar according to the standard methods19 and the tooth was restored with dental amalgam, as follows: no restoration (control), small occlusal restoration, large occlusal restoration, disto - occlusal restoration, and mesio - occluso - distal restoration (figure 1). Five sequential scans were obtained with a cbct scanner (alphard vega; asahi roentgen ind . Co., kyoto, japan) before and after the restorations under the following conditions: 80 kv, 5 ma, 0.39 0.39 0.39 mm voxel size, and 200 179 mm field of view (fov). The images were saved in digital imaging and communication in medicine (dicom) format and imported into imaging software (invivodental 5.0; anatomage, san jose, ca, usa). Then, 3d surface models were constructed by using segmentation threshold values ranging from 600 to 3,071 according to the program's default function and converted to stereolithographic format . To reveal artifacts, each restored model was registered with the control model by using the iterative closest point algorithm20 in 3d reverse engineering software (rapidform 2006; inus, seoul, korea). Regional registration was used to calculate rotation and translation from surface information of the two data sets . Corresponding points and shapes were searched and their distance was minimized after rotation and translation . Discrepancies between the models were shown by color mapping . For quantitative assessment, maximum linear discrepancy, area, and intensity of artifacts were measured . Maximum linear discrepancy was measured as the distance between shells at the most protruded point of the graphic (figure 2a and 2b). The shell / shell deviation function of the reverse engineering software was used to neglect discrepancies smaller than 0.5 mm . The graphics were exported to image analysis software (image - pro plus 4.1; media cybernetics, bethesda, md, usa), and artifact area in the buccal, lingual, and occlusal views was measured by using the area function of the software (figure 2c and 2d). Artifact intensity was determined from cross - sectional views at the level of the maximum linear discrepancy and 0.5 and 1.0 mm above and below . The cross - sectional graphics were exported to the image analysis software, and the area of discrepancy at each level was measured . The sum of the five areas was defined as artifact intensity (figure 2e and 2f). One - way analysis of variance (anova) was used to analyze parametric differences according to increasing restoration size, and the tukey test was used for post - hoc comparisons . All analyses were carried out by the spss software program (version 18.0; spss inc . One - way analysis of variance (anova) was used to analyze parametric differences according to increasing restoration size, and the tukey test was used for post - hoc comparisons . All analyses were carried out by the spss software program (version 18.0; spss inc ., chicago, il, usa). Color mapping revealed artifacts in all cases . They were present not only on the second premolar but also on the adjacent teeth and mostly on the buccal and lingual surfaces . On these surfaces, the maximum linear discrepancy was the least for the small occlusal restoration and the greatest for the mesiooccluso - distal restoration (table 1), with significant differences among the models . Similarly, artifact area and intensity significantly increased with increasing restoration size (tables 2 and 3, respectively). A 3d surface model is influenced by several factors such as scan field and segmentation threshold value.21 this study used the specimen which consisted of extracted teeth . We wanted to simulate cbct scans of actual patient as much as possible while we used the specimen . The segmentation threshold value also followed the default value of the program which is used as a routine in clinical practice . Measurements of 3d surface models reconstructed from cbct scan data are reportedly larger than physical measurements.22,23,24 ye et al.24 showed that volumetric measurements from cbct scans are larger than those from laser scans of extracted human teeth . Laser - scanned images might be the gold standard for experiments with 3d surface models . However, cbct scans without restoration were used as the control in this study because the aim was to assess artifacts from metallic restorations in cbct scans . Comparison of the accuracy of 3d surface models derived from cbct with those from other imaging methods was not the focus . After registering each restored model with the control model of a specimen, discrepancies between the shells were evaluated both graphically and numerically . They were present not only on the second premolar but also on the adjacent teeth . It causes beam hardening artifacts which are the most prominent artifacts induced by high - density objects in the beam's path.11,12,25 in beam hardening, a polychromatic x - ray beam gradually becomes harder when passing through high - density objects, by which lesser - energy photons are absorbed and only higher - energy photons pass through . While the artifact appears as streaks in 2d images, it " bulges " in 3d surface models . In the case of the larger restorations in this study, the finding of artifacts mostly on the buccal and lingual surfaces compared with the occlusal surface indicates that artifacts appear in the horizontal direction, not vertically, on 3d surface models . The difference is likely attributable to the direction of the x - ray beam of the scanner, because the emitter of a cbct scanner rotates around an object in the horizontal direction . In this study, the maximum linear discrepancy was the least and greatest in models of the small occlusal and mesio - occluso - distal restorations, respectively . Positive relationships between the extent of artifact and the restoration size were also observed in the artifact area and intensity measurements . While artifact reduction can be attempted by reconstruction algorithms,26 a simple alternative is to replace the highly absorbing material with a less dense one, such as composite resin, before cbct scanning . During orthodontic treatment, the resulting changes in artifacts can be evaluated quantitatively by the method used in this study . Another way to avoid artifacts in 3d surface models of the head is to replace the artifact - laden dental part with optical or laser - scanned dental images . For fabricating appliances such as surgical splint and indirect bonding tray, the dental part should be replaced with images containing detailed occlusal and accurate interocclusal data . However, these procedures are complicated for practitioners, particularly when fiducial markers are used for registering models derived from two imaging modalities.13,15,27 a simpler method is to integrate the digital data of a plaster cast with ct data by surface registration13,16,17,18 using an iterative closest point algorithm,20 without fiducial markers . However, it should be noted that artifacts affect registration accuracy in case of using surface registration in the implementation of digital dental model into ct scan data.18,28 lin et al.18 reported greater registration errors in models with artifacts than in those without artifacts although all the measurements exhibited acceptable interoperator and intraoperator reproducibility . In this study, cbct images were obtained with a large fov, so a large voxel size was used . As artifacts differ according to voxel size and fov, it needs to be evaluated under other scan conditions, such as smaller voxel size . Further, comparative studies with other dental materials, such as composite resin, are needed, considering that beam hardening artifacts are influenced by object density . In particular, the 3d surface models created in this study do not represent actual patients . Metallic restorations induce considerable artifacts in 3d dental surface models . Artifact reduction should be taken into consideration for a proper diagnosis and treatment planning when using 3d surface model derived by cbct in dentofacial deformity patients . On the other hand, the present study would be useful for assessing various artifact - inducing factors, such as scan field, voxel size, segmentation threshold value, and dental material type.
Growing teratoma syndrome (gts) is a metastatic form of a mature teratoma and involves the clinical and radiological enlargement of metastases during or after chemotherapy for malignant germ cell tumors . In many reported cases, gts occurred at the site of the primary tumor, and the peritoneum of the pelvis and abdomen and the retroperitoneum were the most frequent sites of metastasis . Here, we report a 19-year - old woman with gts showing three patterns of metastasis: dissemination, lymphogenous metastasis, and hematogenous metastasis . A 14-year - old girl presented to a local hospital in june 2006 with abdominal fullness . Computed tomography (ct) scans revealed a 15-cm mass and a small amount of ascites, but no sign of lymph node enlargement (fig . Serum tumor markers were slightly elevated: ca19 - 9 = 113.8 u / ml, ca125 = 60 u / ml, scc = 2.5 ng / ml, and -fetoprotein (afp) = 297.4 iu / ml . Liver and renal function tests, electrolytes, and a complete blood count were all normal . The initial laparotomy revealed that the left ovary was larger than the size of a child's head and had already ruptured with slightly bloody fluid in the pelvic cavity . Histological examination revealed the tumor to be a mixed germ cell tumor which included a grade 3 immature teratoma (75%), mature teratoma (10%), embryonal carcinoma (10%), and yolk sac tumor (5%) (fig . The patient was therefore diagnosed with a left ovarian mixed germ cell tumor, clinical stage 1c [pt1c(2) nxmx]. She underwent 6 cycles of chemotherapy postoperatively with cisplatin, etoposide, and bleomycin (bep). Five years after the completion of chemotherapy, routine ct scans revealed dissemination in the pelvic region, para - aortic lymph node metastasis, and lung metastasis (fig . 3). However, the interval since the last examination had been 18 months based on the patient's wishes . No tumor marker was elevated . The patient was admitted to nagoya university hospital for resection of the three types of metastatic mass . Secondary laparotomy showed a 3-cm dissemination on the mesentery of the sigmoid colon and two 1-cm disseminations on the posterior uterus . Next, we removed an 8-cm metastatic tumor in the para - aortic lymph node . One month after the secondary laparotomy, video - assisted thoracoscopic surgery was performed for a right lung metastasis . Pathologic analysis of the resected specimens revealed that all metastatic tumors were mature teratomas (fig . The patient is now undergoing regular follow - up and remains disease free 11 months after the second surgery . Logothetis et al . Described gts as an increase in tumor size in a patient with a germ cell tumor either during or after chemotherapy, while the histology showed only mature teratoma and the initial tumor markers were normal . The first is that chemotherapy induces a selective exclusion of immature components, whereas mature components, resistant to chemotherapy, persist and grow alone as gts . The second is that chemotherapy influences the differentiation potency and induces the malignant cell differentiation of an immature into a mature teratoma . Recently, amsalem et al . Concluded that gts and chemotherapeutic retroconversion were probably the same phenomenon . In many cases, only one group reported an ovarian gts patient with gts nodules in the neck lymph nodes and lungs . In our case, there were three metastases in the peritoneum, para - aortic lymph node, and lung . Para - aortic lymph node and lung metastases were not detected during the initial treatment . In our case, considering the above progress, the second hypothesis is reasonable; however, the possible existence of micrometastases cannot be denied . The time of gts onset was within 5 years in most cases, in some cases exceeding 5 years . In our case, the time of gts onset was 67 months after the adjuvant chemotherapy . Therefore, long - term follow - up is necessary to detect gts . Regarding sites of recurrence, the peritoneum, lymph nodes, liver, and lungs have been reported [2, 5, 6]. In our case, the patient had three metastases in the peritoneum, para - aortic lymph node, and lung . The case is rare as she showed three patterns (dissemination, lymphogenous metastasis, and hematogenous metastasis), and this is the first report of gts showing three simultaneous patterns of metastasis . Surgical resection is recommended for initial treatment because growing tumors apply pressure on surrounding organs and possibly include malignant parts, caused by malignant transformation . Malignant transformation could be to a sarcoma, adenocarcinoma, or a primitive neuroectodermal tumor [8, 9] and carcinoid . Considering the surgical procedure, laparoscopic surgery is an option in the case of a small tumor . However, in many cases, surgery was selected based on the tumor size and spread . In our case, the interval between examinations was 18 months . For early detection, examination of the whole body using magnetic resonance imaging and ct is needed . Previous studies revealed 1 patient with gts who showed a positive fluorodeoxyglucose positron emission tomography (fdg - pet) uptake [5, 12]. In our case,, we report the first case of ovarian gts with three patterns of metastasis: dissemination, lymphogenous metastasis, and hematogenous metastasis . Our case highlights the importance of long - term follow - up and a whole - body search.
Early life events may play a critical role in determining the susceptibility to chronic diseases (gluckman et al ., 2005). Epidemiological evidence suggests a close relationship between the exposure to a suboptimal in utero environment, whose consequence is intrauterine growth retardation (iugr), and the development of insulin resistance, type 2 diabetes, hypertension, hyperlipidemia, and cardiovascular disease in adult life (barker et al ., 1989; robinson et al ., 1992; ravelli et al ., introduced the concept of programming, based on the observation that early diet influenced brain development and growth in preterm babies . This concept was subsequently expanded to include the long - term effects of in utero programming induced by maternal cues on metabolic and endocrine functions of the fetus . The programming process occurs during critical periods of embryo fetal life characterized by high cell proliferation rate in the developing tissues and may involve structural and functional changes in genes, cells, tissues, and even whole organs . This ability of the organism to change structure and function in response to environmental signals is named developmental plasticity such plasticity permits a range of phenotypes to develop from a single genotype and is finalized to allow the organism to match its environment (gluckman et al ., 2009). Acts through epigenetic changes in gene transcription, alterations in tissue differentiation, and changes in homeostatic processes (gluckman and hanson, 2004). Epigenetic changes are established in early life and modulate gene expression during development, thus mediating the adaptation of the organism to the environment (gluckman and hanson, 2004; gluckman et al ., 2008). However, when environmental conditions change a mismatch may occur, rendering the organism less adapt to cope to the new environmental conditions (such as postnatal overfeeding), eventually leading to disease (gluckman et al ., 2009). Epigenetic mechanisms are commonly associated to gene silencing, genomic imprinting and transcriptional regulation of tissue - specific genes during cellular differentiation (schbeler et al ., 2000). The epigenetic control of gene expression is based on modulation of chromatin structure and accessibility to transcription factors (figure 1). Demethylation of cytidine guanosine (cpg) sequences in the promoter regions, acetylation deacetylation of lysine residues of core histones in the nucleosome and presence of microrna molecules which bind to complementary sequences in the 3 end of mrna and reduce the rate of protein synthesis (goldberg et al ., 2007). Cpg - rich regions of dna reside in 60% of promoters utilized by human rna polymerase ii and are often found in association with housekeeping and tissue - specific genes . Transcriptionally active chromatin is characterized by unmethylation of cpg sequences, which permits an open structure of the chromatin, thus allowing access to transcription factors . The same effect is given by acetylation of lysine residues of histones, which decreases their binding to dna (wolffe and matzke, 1999). Transcriptionally active chromatin is characterized by the presence of acetyl groups (ac) on specific lysine residues of histones in the nucleosome, which decreases their binding to dna, eventually leading to an open chromatin structure that permits access to transcription factors (tf). In addition, demethylation of cytidine guanosine (cpg) sequences in the promoter region (p) of actively transcribed genes allows for the binding of transcription factors (tf). Transcriptionally inactive chromatin is characterized by histone deacetylation, promoter cpg methylation (as indicated by the presence of methyl groups, me), and decreased binding of transcription factors . A further level of epigenetic control is provided by microrna molecules (1922 nucleotides in length) which bind to mrna thus reducing the rate of translation . Major epigenetic programming, involving the removal of epigenetic marks in the nucleus, followed by establishment of a different set of marks occurs physiologically upon fertilization when many gametic marks are erased and replaced with embryonic marks important for early embryonic development and toti- or pluripotency . Major programming also takes place in primordial germ cells in which parental imprints are erased and totipotency is restored (morgan et al ., 2005). One of the mechanisms that trigger programming and, hence, epigenetic changes, is intrauterine malnutrition (cianfarani et al ., 1999), which can be induced by several causes affecting the placental transfer of nutrients from mother to fetus (fowden et al ., 2006). To explain the relationship between prenatal undernourishment and postnatal risk of metabolic disease, hales and barker proposed the thrifty phenotype hypothesis (hales and barker, 1992). According to the thrifty phenotype model, the growing fetus exposed to nutritional deprivation adopts at least two strategies to aid survival . First, it diverts nutrients to the brain to preserve brain growth at the expense of body growth and the development of other organs such as pancreas, liver, and muscle . Second, metabolic programming occurs in a manner that is beneficial to survival under conditions of poor postnatal nutrition . However, if the organism is born into conditions of adequate or overnutrition, then this may conflict with the earlier programming and insulin resistance, and, later on, type 2 diabetes, may result (geremia and cianfarani, 2004). Several animal models have been created to reproduce a poor uterine environment leading to fetal undernourishment and, consequently, developmental programming . Most animal models of iugr are based on uteroplacental insufficiency (simmons et al ., 2001), which limits the supply of substrates to the fetus, or suboptimal maternal nutrition (armitage et al ., 2004). Altered intrauterine milieu associated with uteroplacental (placental) insufficiency affects dna methylation and histone h3 acetylation . In liver, increased levels of s - adenosylhomocysteine together with dna hypomethylation and histone hyperacetylation of histone h3 on lysine 9 (h3k9), lysine 14 (h3k14), and lysine 18 (h3k18) are present at birth (maclennan et al ., 2004). These features persist up to day 21 of postnatal life, suggesting a permanent change in hepatic gene expression . (2004) showed that the hyperacetylation on histone h3 in the liver of iugr rats occurs in association with decreased nuclear protein levels of histone deacetylase 1 (hdac1) and hdac activity . These site - specific changes in histone h3 acetylation alter the histone association with the promoter regions of ppar - gamma coactivator (pgc-1) and carnitine palmitoyl - transferase i (cpti), two genes that we have previously demonstrated to be persistently altered in the iugr rat . Pgc-1 expression is increased whereas cpti expression is reduced in iugr rats who will develop diabetes (lane et al ., 2001, 2002). Pgc-1 is a transcriptional coactivator that mediates hepatic glucose production by controlling mrna levels of key gluconeogenic enzymes, such as glucose-6-phosphatase, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase (yoon et al ., 2001). Cpti is a part of the carnitine shuttle and is considered to be a rate - limiting transporter in mitochondrial fatty acid -oxidation (mcgarry and brown, 1997). Altered mrna levels of these genes characterize the iugr liver at birth, and these changes persist postnatally . Finally, these epigenetic modifications may be gender specific as at day 21, the neonatal pattern of h3 hyperacetylation persist only in male iugr rats (fu et al . . The major limitations of these studies are the lack of information on the effects of the epigenetic changes on gene expression and the short postnatal follow - up of the study animals . A key developmental gene whose epigenetic modulation has been studied to explain the intrauterine metabolic programming predisposing to type 2 diabetes is pdx1 (pancreatic and duodenal homeobox 1). Reductions in pdx1 expression in human and animal models have been shown to cause type 2 diabetes, -cell dysfunction, and impaired islet compensation in the presence of insulin resistance (holland et al ., 2005; stoffers et al ., uteroplacental insufficiency causes multiple epigenetic changes of pdx1 involving histone modifications, dna methylation, and chromatin remodeling in rats (park et al ., 2008). In this animal model, -cell mass is normal at birth, whilst pdx1 expression is reduced . In adult animals, the epigenetic mechanisms underlying these events are characterized by the progressive histone h3 and h4 deacetylation, lysine 4 on histone h3 (h3k4) demethylation, and lysine 9 on histone h3 (h3k9) methylation in pdx1 proximal promoter . All these changes lead to a silenced chromatin, with decreased usf-1 (a key transcription factor) binding and increased recruitment of histone deacetylase 1 (hdac1) and its corepressor sin3a . During the neonatal period, these epigenetic changes and the reduction in pdx1 expression could be reversed by hdac1 inhibition (islets cultured in the presence of trichostatin). However, as h3k9me2 accumulates, dnmt3a (a dna methyltransferase) is recruited to the promoter and initiates de novo dna methylation, which locks in the silenced state the pancreas, eventually resulting in diabetes (park et al ., 2008). Exendin-4 (ex-4), a pancreatic -cell trophic factor, has been shown to reverse the silencing of pdx-1 . Administration of ex-4 during the prediabetic neonatal period dramatically prevents the development of diabetes in iugr rats by restoring expression of pdx1 to normal levels and normalizing islet -cell proliferation rate (stoffers et al ., 2003). (2008) explored epigenetic mechanisms underlying diminished skeletal muscle glut4 mrna in a rodent model of iugr obtained by nutrient restriction . Glut4 is the major insulin - responsive isoform in the family of membrane - spanning glycoproteins with the function of glucose transporters . At 450 days of life this gender - specific difference may originate from early developmental perturbations in pancreatic -islet cell insulin synthesis and secretion (chamson - reig et al ., 2006), young females demonstrating persistent postnatal hypoinsulinemia that regulates skeletal muscle glut4 transcription . No significant increase in the methylation of cpg regions within glut4 promoter was observed in skeletal muscle of iugr rats . On the contrary, hypomethylation of most cpg islands was found . In this iugr model deacetylation and di - methylation of specific amino acid residues in the n - tail of histone 3 taken together these findings indicate that epigenetic changes of histone code may inhibit skeletal muscle glut4 transcription in adult female iugr rats . 2010) have recently studied dna methylation of the whole genome in pancreatic islets of iugr rats at 7 weeks of age . Using the hpaii tiny fragment enrichment by ligation - mediated pcr (help) assay, they generated a dna methylation map at almost 1 million unique sites throughout the rat genome in normal pancreatic islet cells . Male iugr animals showed a different cytosine methylation pattern in approximately 1400 loci at 7 weeks of age . Epigenetic dysregulation occurred preferentially in conserved intergenic sequences, frequently near genes regulating processes involved in vascularization, -cell proliferation, insulin secretion, and cell death . This epigenomic dysregulation precedes the development of diabetes and probably represents a link between intrauterine growth restriction and development of type 2 diabetes in adulthood . Candidate dysregulated loci were investigated with quantitative assay of cytosine methylation and gene expression was tested by rt - pcr . Gtp cyclohydrolase 1 (cgh-1) gene (a gene with a role in the endothelial dysfunction and in -cell development) showed a three - fold reduction in mrna expression, associated with hypermethylation at a conserved intergenic site . Hypomethylation of fibroblast growth factor receptor 1 (fgfr1) gene (whose signaling is modulated by -cell microenvironment) was associated with increased mrna expression . Proprotein convertase subtilisin / ketin type 5 (pcsk5) gene (a gene that impairs -cell activity and regulates -cell adhesion to extracellular matrix) showed significantly reduced mrna expression associated with hypermethylation . These findings strongly suggest that early epigenetic modifications in pancreatic islets may mediate the long - term metabolic consequences of exposure to suboptimal intrauterine environment (thompson et al ., the different genes whose function is linked to the development of type 2 diabetes (and its complications). References to the relative studies, subjects of the studies and type of epigenetic modifications are also indicated . Iugr, intrauterine growth - retarded; aga, adequate for gestational age; sga, small for gestational age . In humans, evidence that epigenetic changes predispose the organism to type 2 diabetes stems from the studies on the individuals who were prenatally exposed to famine during the dutch hunger winter in 194445 . This period of famine was the consequence of a german - imposed food embargo in the western part of the netherlands toward the end of world war ii during the winter of 194445 . As official food rations were documented, the reported average daily rations were 667 kcal and there was little variation in the percentage of calories from proteins (12%, of which 4% of animal origin), fat (19%), and carbohydrates (69%). It was demonstrated that individuals exposed to famine prenatally were at higher risk of developing cardiovascular and metabolic diseases in adulthood (lumey et al ., 2007). (2008) selected 60 individuals whose mothers were exposed to famine during the periconceptional period and 62 individuals exposed late in gestation for at least 10 weeks . Igf - ii gene is maternally imprinted and strongly epigenetically regulated (hypomethylation leads to bi - allelic expression). Among the 60 individuals exposed to famine periconceptionally, all cytosine guanine (cpg) sites of the igf - ii gene but one were significantly less methylated (in comparison with the same - sex siblings). Among the 62 individuals exposed to famine late in gestation, no difference in igf - ii methylation was found between the exposed individuals and their unexposed siblings . They concluded that periconceptional exposure to famine is associated with lower methylation of the igf - ii gene 6 decades later (heijmans et al ., 2008). The reduced methylation of igf - ii may represent the consequence of intrauterine exposure to deficient methyl donors supply, such as the aminoacid methionine, although additional contribution of other stressors such as cold and emotional stress cannot be ruled out . Consistent with the potential role of methyl donors in determining igf - ii gene methylation status is the recent observation that periconceptional folic acid use of the mother is related to an increased methylation of the igf - ii gene of the offspring (steegers - theunissen et al ., 2009). However, we point out that remains to be determined whether the changes in igf - ii gene methylation are associated with changes in gene expression . The transcriptional coactivator peroxisome proliferator activated receptor coactivator-1 (protein pgc-1; gene ppar--c1-) is an important factor regulating the expression of genes for oxidative phosphorylation and atp production in target tissues through coactivation of nuclear receptors (lin et al ., 2005). Ppar--c1- shows an epigenetically regulated decrease of expression in muscle and pancreatic islets from patients with type 2 diabetes . Insulin secretion in pancreatic islets is dependent upon mitochondrial function and this transcriptional coactivator is a master regulator of mitochondrial genes: this protein can interact with and regulate the activities of camp response element binding protein and nuclear respiratory factors . It provides a direct link between external physiological stimuli and the regulation of mitochondrial biogenesis . The epigenetic modulation results in increased dna methylation on ppar--c1- gene promoter (ling et al ., 2008). The highest proportion of cytosine methylation within ppar--c1- is found within non - cpg nucleotides . As alterations in the extracellular milieu, including hyperglycemia, hyperinsulinemia, elevated free fatty acids (ffa), and elevated cytokines can cause peripheral insulin resistance, barrs et al . (2009) incubated human skeletal muscle from normal glucose - tolerant subjects with tumor necrosis factor- (tnf-), ffa, insulin and glucose . In these experimental conditions non - cpg methylation selective silencing of the dna methyltransferase 3b (dnmt3b) prevented palmitate - induced non - cpg methylation of ppar--c1- and decreased mitochondrial dna and pgc-1 mrna . These data suggest that dnmt3b is linked to the acute fatty acid - induced non - cpg methylation of ppar--c1- promoter . Dna methylation and gene expression of ppar--c1- in human muscle is influenced by high - fat diet in a birth weight dependent manner (brns et al ., 2010). Studied 26 young, healthy, lean men with low birth weight (lbw, <10th percentile) and 20 subjects with normal birth weight (nbw, 5090th percentile). All subjects were born at term, and the groups were matched according to age and body mass index (bmi). Subjects were examined twice with a hyperinsulinemic euglycemic clamp after intake of a 3-days control diet including 30% fat and after a 5-days high - fat diet containing 50% extra calories and 60% fat . When challenged with high - fat diet, lbw subjects developed peripheral insulin resistance and reduced ppar--c1- and oxphos (the cluster of genes from both the nuclear and mitochondrial genomes controlling the mitochondrial oxidative phosphorylation) gene expression in the muscle . Ppar--c1- methylation was significantly higher in lbw subjects during the control diet . When exposed to overfeeding, methylation of ppar--c1- increased in the nbw group only . Lbw subjects showed similar ppar--c1- methylation during the two diets . When shifted to the control diet after overfeeding, reversibility of ppar--c1- methylation epigenetic alterations observed in lbw subjects at increased risk of developing type 2 diabetes could reflect dna methylation remnants established during fetal life, possibly affecting tissue development in organs relevant to type 2 diabetes pathophysiology during phases of active cell divisions . This study shows that the same ppar--c1- cpg sites reported to be more highly methylated in pancreatic cells from type 2 diabetes subjects are methylated to a higher extent in young and lean lbw compared with nbw subjects when studied during an isocaloric control diet (brns et al ., 2010). The increased methylation of ppar--c1- observed after overfeeding in nbw only may reflect a more widespread genome - wide response, probably part of a normal physiological response involved in the day - to - day regulation of mechanisms influenced by the diet . The majority of known environmentally induced epigenetic effects will involve direct exposures and action on somatic tissues . However, experimental evidence suggests that programming induced by suboptimal in utero environment can be transmitted to the following generations (jablonka and raz, 2009). The daughters of women exposed to nutrient restriction and environmental stress during pregnancy as a result of the dutch hunger winter, showed a decreased birth weight and an increased risk of insulin resistance, and their daughters were born with a lower birth weight (stein and lumey, 2000; painter et al ., 2005). In rats, feeding a protein - restricted diet to the f0 generation during pregnancy resulted in alterations of glucose and insulin metabolism in male and female f2 offspring (zambrano et al ., the adverse effects on glucose homeostasis of feeding a protein - restricted diet during pregnancy in the f0 generation have been found in the offspring up to f3 generation (benyshek et al ., 2006). The administration of dexamethasone to dams in late pregnancy induced an increased expression of the glucocorticoid receptor (gr) and its target gene phosphoenolpyruvate carboxykinase (pepck) in the liver of the f1 and f2 offspring (drake et al ., 2005). Hepatic peroxisome proliferated activated receptor (ppar) and gr promoter methylation has been reported to be significantly reduced in rats prenatally exposed to protein - restricted diet in both f1 and f2 generation (burdge et al ., 2007). Intrinsic epigenetic transgenerational processes will require the involvement of the germline to allow the transmission of an epigenetic abnormality between multiple generations . It is known that postfertilization deletion of epigenetic marks is incomplete for imprinted genes and, probably, some non - imprinted genes (chong and whitelaw, 2004). The process by which early cues at critical stages of development lead to permanent changes in tissue structure and function is known as intrauterine programming and is finalized to allow the organism to match its environment . Programming induces epigenetic changes that may permanently affect the expression of specific genes in selected target tissues . Epigenetic changes permit a range of phenotypes to develop from a single genotype, and may be transmitted across generations (figure 2). However, when the organism is exposed to a different environment, the epigenetic pattern induced in utero may become detrimental and may predispose the individual to the development of chronic diseases . Therefore, the identification of the cues and the target genes involved in programming would permit early interventions to prevent severe adult illnesses such as type 2 diabetes and cardiovascular disease . Epidemiological and experimental evidence shows the importance of the periconceptional period, when maternal suboptimal nutrition can have long - lasting effects on the offspring, and suggests the potential importance of designing specific nutritional supplementation programs during the pre - pregnancy period . In addition, the identification of the epigenetic markers associated with increased risk of metabolic disease may allow to identify the individuals at metabolic risk and to propose a prevention strategy based on a healthy life - style as well as nutritional and pharmacological interventions . In utero epigenetic programming . Epigenetic programming permits a range of phenotypes to develop from a single genotype and is finalized to allow the organism to match its environment . When environmental conditions change a mismatch may occur, rendering the organism less adapt to cope to the new environment, eventually leading to disease . A. liguori, a. puglianiello, d. germani, a. deodati, and e. peschiaroli have nothing to declare . S. cianfarani received lecture fees from ipsen, eli lilly, novo nordisk, and pfizer, and consulting fees from ipsen, eli lilly, and pfizer.
The root of ginseng (panax ginseng meyer) has been traditionally used for medicine and food . The primary physiologically - active substances of ginseng are ginsenosides, polyacetylenes, ginseng proteins, polysaccharides, and phenolic compounds . Ginsenosides in particular have been identified as the principal component of ginseng, displaying various biochemical and pharmacological properties . A number of researchers have studied the components of ginseng since the late 1960s, starting with the research of shibata et al, whose research group identified the chemical structures of ginsenosides . Notably, the amount of ginsenoside re in the berries was four to six times more than that in the roots . Research in the area has shown that ginsenoside re exhibits multiple pharmacological activities via different mechanisms both in vivo and in vitro [38]. However, the pharmacological effects of ginsenoside re on gastritis or gastric ulcer have not yet been studied . A gastric ulcer is one of the most common diseases in the world, which affects approximately 510% of people during their lives . The therapy used to treat gastric ulcers includes control of acid secretion as well as the inflammation reversal to the mucosa . Korea red ginseng can assist in the eradication of helicobacter pylori and alleviate h. pylori - induced halitosis . A recent pharmacological investigation reports the antihistamine and anticytokine releasing effects of ginsenoside re isolated from the berries of panax ginseng . For the common treatment of mild gastritis, antacids in liquid or tablet form are typically used . When antacids do not provide sufficient relief, h2 blocking medications, such as cimetidine, ranitidine, nizatidine, and famotidine, which help reduce the amount of acid famotidine, the most potent h2 receptor antagonist, was used as a positive control . The present study examined the protective effect of ginsenoside re on acute gastric mucosal lesion progression in rats treated with compound 48/80 (c48/80). C48/80 causes degranulation of mast cells in connective tissue with the release of histamine from the cells, and causes the development of acute gastric mucosal lesions with neutrophils infiltrating into the gastric mucosal tissue . Injecting c48/80 is consequently suggested as a good model for elucidating the mechanisms of clinical acute gastric lesions . Ginsenoside re was prepared according to a previously reported method . In brief, dried ginseng berries (5 kg) were ground to powder and extracted twice with 1 l of 95% ethyl alcohol for 2 h in a water bath (60c). The extracts were concentrated by a vacuum evaporator (eyela co., tokyo, japan). The lyophilized extract was dissolved in distilled water, and was rinsed 10 times with diethyl ether to remove unnecessary compounds . The water fraction was suspended in distilled water and was adsorbed in a diaion hp-20 (mitsubishi chemical corporation, tokyo, japan) ion exchange resin column . A 30% meoh fraction, 50% meoh fraction, 70% meoh fraction, and 100% meoh fraction were eluted in the order named . The 30% meoh fraction was then subjected to an octadecylsilyl (ods) gel column by gradient elution with 30100% meoh, and resulted in four subfractions (f1f4). The f3 subfraction was rechromatographed on a silica gel column with a mixture of the solvents (chcl3:meoh: h2o = 70:30:4 v / v), and ginsenoside re was isolated and identified . The authenticity of ginsenoside re was tested by spectroscopic methods including h - nmr, c - nmr, and fast atom bombardment - mass spectrometry (fab - ms). Male wistar rats of 6 wk of age were purchased from samtako (osan, korea) and housed in controlled temperature (23 2c), relative humidity (60 5%), and 12 h light / dark cycle (7:00 am7:00 pm) with free access to water . The experiment was reviewed and approved by the animal care and research ethics committee of the semyung university, jecheon, south korea (smecae 08 - 12 - 03). Rats were divided into five groups (n = 8, respectively): normal (no gastric lesion and administered with distilled water), gastric lesion control (administered with distilled water), gastric lesion positive control (administered with famotidine 4 mg / kg; nelson korea co., seoul, korea), and gastric lesion administered with two levels of ginsenoside re (20 mg / kg and 100 mg / kg). The dosage of 20 mg / kg of ginsenoside re was chosen from previous published data . The 100 mg / kg dosage was determined to discover the maximum effects of ginsenoside re . The animals were maintained with free access to rat chow, and famotidine and ginsenoside re were orally administered with a stomach tube . After 5 d of sample administration, c48/80 (0.75 mg / kg; sigma - aldrich inc ., ny, usa), dissolved in saline, was intraperitoneally injected into the rats fasted for 24 h. the normal group received a saline injection . The animals were sacrificed by decapitation under ether anesthesia 3 h after the c48/80 injection, and blood samples were obtained from the cervical wound . The stomachs were removed, inflated with 10 ml of 0.9% nacl, and put into 10% formalin for 10 min . The isolated stomachs were cut open along the greater curvature and washed in ice - cold saline . The parts of the mucosa were immediately fixed with 10% formalin solution, and routinely processed for embedding in paraffin wax . The sections were cut 5 m thick and stained using the periodic acid schiff (pas) method to observe mucus secretion . The measurement of gastric mucosal adherent mucus was assayed using alcian blue staining . In brief, the parts of the stomach mucosa were rinsed with ice - cold 0.25 m sucrose . A 50 mm (approximately 8 mm - diameter) portion of the glandular region of the stomach was excised with a scalpel, and soaked in 0.1% alcian blue dissolved in 0.16 m of sucrose buffered with 0.05 m sodium acetate (ph 5.8) for 2 h. the unbound dye was removed using two successive washes with 0.25 m sucrose . The dye complex with mucus was extracted using 30% docusate sodium salt (sigma - aldrich inc ., ny, usa) for 2 h. after centrifugation at 2,060 g for 10 min, the optimal density of the alcian blue solution was measured at 620 nm, and calculated using the calibration curve . The adherent gastric mucosal mucus was expressed as the percentage of the alcian blue adhering to the gastric mucosal surface of the gastric lesion control group . The measurement of gastric mucosal hexosamine has been used as another indicator of gastric mucus secretion, and was assayed by the method of neuhaus and letzring . In brief, the parts of the gastric mucosal tissue were homogenized and centrifuged for 10 min at 9,000 g and the supernatant was used for malondialdehyde (mda), myeloperoxidase (mpo), and xanthine oxidase (xo) analyses . Mda levels of gastric mucosa were determined by the thiobarbituric acid reactive substance (tbars) colorimetric assay (synergy2; biotek co., usa). Gastric mucosal mpo activity was used to examine the degree of neutrophil infiltration and inflammation . Mpo activity was assayed by the method of suzuki et al, measuring the h2o2-dependent oxidation of tetramethylbenzidine at 37c . Gastric mucosal xo was assayed according to the method of hashimoto by measuring the increase in absorbance at 292 nm following the formation of uric acid at 30c . The immunofluorescence analysis was performed with mouse monoclonal anti - bax antibody and rabbit monoclonal anti - bcl2 antibody (santa cruz biotechnology, inc ., dallas, tx, usa) and fluorescein isothiocyanate (fitc)-conjugated antimouse and antirabbit igg antibodies, respectively (sigma chemical co., st louis, mo, usa). The nuclei were counterstained with 1 g / ml propidium iodide (pi; sigma chemical co.). The fluorescence images were taken with a laser confocal microscope (fluoview fv1000; olympus, tokyo, japan). The optical density was measured using bio1d software (vilber lourmat, marne - la - valle cedex, france). For laser microdissection (lmd), a 10-m thick section prepared from the same tissue block was attached onto provided slides (jungwoo f&b co., bucheon, republic of korea). Sixteen fragments of gastric tissues were collected in a 0.5-ml tube cap using an ion lmd (jungwoo f&b co.). Briefly, the tissue fragments were deparaffinized and boiled at 100c in tris hcl buffer solutions of ph 8 containing 2% sodium dodecyl sulfate (sds). Protein concentrations were measured using a dc protein assay kit (bio - rad, hercules, ca, usa). Five l of standards and protein samples were transferred to a 96-well plate and 25 l of alkaline copper tartrate solution containing reagent s was added to each well . Then 200 l of dilute folin reagent was added to each well and the 96-well plate was incubated at room temperature . After 15 min, the protein concentrations were measured at 750 nm using an enzyme - linked immunosorbent assay (elisa) reader (synergy2; biotek, winooski, vt, usa). Each protein was then fractionated by electrophoresis through a 10% sds polyacrylamide gel at 100 v for 2 h, and the proteins were transferred onto polyvinylidene fluoride (pvdf) membranes at 100 v for 60 min . Hcl, ph 7.4, 150 mm nacl, 0.1% tween-20) containing 5% bovine serum albumin (bsa) for 1 h and then incubated with primary antibodies (mouse anti - bax and rabbit anti - bcl2 antibodies) in tbst buffer containing 1% bsa at 4c overnight . The membranes were washed three times with tbst buffer and further incubated with antimouse and anti - rabbit immunoglobulin g (igg) secondary antibodies conjugated with horseradish peroxidase for 2 h, respectively . Each membrane was filmed with a chemiluminescent imaging system (fusion sl2; vilber lourmat), and analyzed using bio1d software (vilber lourmat). Statistical analysis was performed using one - way analysis of variance (anova) followed by duncan's multiple range tests . A p value <0.05 was considered to indicate statistical significance . For all analyses, a commercially available statistical package software was used (spss version 19; spss inc ., . The apical surface of the mucous cells in normal rats was strongly stained with pas (arrows in fig . However, pas reaction was significantly reduced in surface cells of the control group (arrows in fig . 1d)-treated rats compared with the control group, suggesting an increase in mucus secretion and alleviation of the erosion in the gastric mucosal cell layer in these groups . A significant decrease in adherent gastric mucus content was seen in c48/80-induced gastric lesion control rats compared with normal rats (table 1). Pre - administration with famotidine and ginsenoside re significantly attenuated the decrease in adherent gastric mucus content . Gastric mucosal hexosamine is the best indicator of mucin production, which is the first line of gastric mucosal defense . A significant decrease in mucosal hexosamine content, like in the adherent gastric mucus, was seen in c48/80-induced gastric lesion control rats compared with normal rats (table 1). Pre - administration with famotidine and ginsenoside re significantly attenuated the decrease in mucosal hexosamine content . Gastric mucosal mda content, mpo, and xo activities significantly increased in c48/80-treated control rats compared to those of the normal group (table 2). The mda content, mpo, and xo activities in the c48/80-treated control group were 3.6, 2.3, and 1.4 times higher, respectively, than those in the normal group . Immunofluorescence staining clearly showed that bax was expressed and limited to the cytosol of the gastric mucosal cells (fig . 2). Bax positive cells were found predominantly in part of the gastric gland (arrow in fig . The bax staining in submucosa and muscularis externa was very strong (arrowhead in fig . 2d)-treated rats compared with the control group suggesting the alleviation of apoptotic damage in the gastric mucosal cell layer in these groups . By contrast, bcl2 positive cells were found predominantly in part of the normal gastric gland (arrow in fig . Bcl2 staining in submucosa and muscularis externa was extremely strong in the normal group (arrowhead in fig . Bcl2 staining became weak in both gastric mucosa and submucosa in the control group (arrow and arrowhead in fig . Famotidine and ginsenoside re attenuated the diminishment of the bcl2 staining in both gastric mucosa and submucosa (arrow and arrowhead in fig . Parts of the gastric gland, submucosa, and muscularis externa were microdissected (fig . Bax protein increased in the c48/80-treated control and decreased in the famotidine- and ginsenoside re - treated groups . By contrast, bcl2 protein decreased in the c48/80-treated control and increased in the famotidine- and ginsenoside re - treated groups (fig . The ratio of bax and bcl2 significantly increased in the c48/80-treated control group compared with the normal group (fig . The famotidine- and ginsenoside re - treated groups showed significantly decreased bax / bcl2 ratios compared with the c48/80-treated control (p <0.05). Ginsenoside re showed multiple pharmacological activities including antidiabetic, antiobese, antioxidant, anticancer, memory - enhancing, and anti - inflammatory effects, and inhibitory activities on histamine release . Histamine is an organic nitrogen compound involved in regulating physiological function in the digestive system and local immune responses ., the h2 receptor antagonists are used in the treatment of peptic ulcer disease, gastroesophageal reflux disease, and dyspepsia, as well as in the prevention of stress ulcers . Famotidine, an h2 receptor antagonist with a thiazole nucleus, is approximately 7.5 times more potent than ranitidine and 20 times more potent than cimetidine on an equimolar basis . Famotidine was, therefore, used as a positive control in the present study . Among the variety of biological activities of ginsenoside re reported in vitro and in animal models, we have noticed the antihistamine and anti - inflammatory activities . In this study, we attempted to examine the effect of ginsenoside re on acute gastric lesion progression induced by c48/80 . The c48/80 promotes histamine release and causes acute gastric mucosal lesions . The model of acute gastric mucosal lesions in rats treated once with c48/80 has been thought to be important for clarifying the roles of oxidative stress and inflammation in the pathogenesis of gastritis in humans . The results of the present study have clearly shown that ginsenoside re administered orally to c48/80-treated rats protects gastric mucosal lesion progression, and its potency is similar to famotidine . Pre - administration of ginsenoside re ameliorated gastric mucosal damage, mucus secretion, mda content, mpo, and xo activities . Mucus secretion is a crucial factor in the protection of gastric mucosa from gastric lesions and has been regarded as an important defensive factor in the gastric mucus barrier . A decrease in the synthesis of mucus has been implicated in the etiology of gastric ulcers . The mucus layer protects the newly formed cells against the damage caused by acidic ph and the proteolytic potential of gastric secretions . The wide distribution of adherent mucus content in the gastrointestinal tract plays a pivotal role in cytoprotection and repair of the gastric mucosa . The results showed that severity of erosion induced by c48/80 treatment was alleviated by ginsenoside re administration, and gastric mucosal damage and mucus secretion assessed by alcian blue staining and gastric mucosal hexosamine were dose - dependently improved by ginsenoside re administration . Ohta et al suggested that neutrophil infiltration plays a critical role in c48/80-induced acute gastric mucosal lesion formation and progression . In the present study, ginsenoside re normalized the increased gastric mucosal neutrophil infiltration assessed by mpo activity the level of mpo activity is directly proportional to numbers of neutrophils, and krawisz et al suggested that mpo activity can be used to quantitate inflammation . Therefore, the results of the present study suggest the suppressive effect on neutrophil infiltration and anti - inflammatory action of ginsenoside re . It has been shown that changes in gastric mucosal reactive oxygen species (ros) and neutrophil infiltration into gastric mucosal tissues are closely related to the development of gastric mucosal lesions in rats with a single c48/80 treatment . Xo generates ros during the oxidation of hypoxanthine or xanthine, and ohta et al suggested that the xanthine xo system in the gastric mucosal tissue participates in the progression of gastric mucosal lesion . In the present study, increased mpo activity an index of neutrophil infiltration of the gastric lesion control group was reduced, and ros - related parameters such as mda content and xo activity were normalized by ginsenoside re administration . From the present study, it seems likely that administration of ginsenoside re exerts a preventive effect on the progression of c48/80-induced acute gastric mucosal lesions by protecting the gastric mucosal barrier and tissue against the attack of ros derived from infiltrated neutrophils and the xanthine xo system through preservation of gastric mucus . The protein encoded by the bcl2 gene is a regulator of programmed cell death and apoptosis . The cell survival - promoting activity of this protein is contrary to the cell death - promoting activity of bax, a homologous protein that forms heterodimers with bcl2 and accelerates rates of cell death . Bax protein significantly increased 3 h after hypoxic ischemic brain injury in neonatal brain tissue and it increased in gastric mucosa after ischemia reperfusion damage . In the present results, we have observed that the increased bax expression by c48/80 treatment was attenuated when ginsenoside re was administered . In contrast to bax, bcl2 expression decreased after c48/80 induced acute gastritis and ginsenoside re attenuated the diminution . In western blotting analysis, the bax / bcl2 ratio result also confirmed the protective effects of ginsenoside re on c48/80-induced acute gastritis . In conclusion, the results of the present study indicate that ginsenoside re exerts a preventive effect on the progression of c48/80-induced acute gastric mucosal lesion in rats, possibly by inducing mucus secretion and attenuating enhanced neutrophil infiltration, inflammation, and oxidative stress in gastric mucosa.
There is no single topical antiacne medication that acts against all four major pathophysiologic acne features . Retinoids, such as adapalene, are well known for targeting comedones, including microcomedos and precursors of both inflammatory and non - inflammatory lesions, and exerting anti - inflammatory activity on the innate immunity.57 they are more and more often applied in combination with topical antibacterials, including antibiotics and benzoyl peroxide (bpo).8,9 the latter is known to cause irritation, while the use of topical antibiotics is less and less recommended due to the increased development of antimicrobial - resistant bacteria . Laboratoire bioderma, france, developed a new dermocosmetic cream containing a triple complex (hereafter bgm complex, sbium global cream). This complex mimics the effect of vitamin e, allowing protection from potentially harmful effects of squalene oxidation products, such as hyperkeratinization and inflammation.10 the product contains a triple complex consisting of bakuchiol, ginkgo biloba extract, and mannitol.1114 bakuchiol regulates seborrhea, is antibacterial, and has antiacne properties.1519 in addition to its excipients, the active formulation contains enoxolone (18-glycyrrhetinic acid), salicylic acid, alpha hydroxy acid esters, and zinc gluconate, all of which are all well - recognized pharmacological active agents used in the treatment of acne.2023 the aim of the present trial was to assess the potential of bgm complex to improve the established clinical efficacy of adapalene 0.1% gel . This multicenter and comparative double - blind clinical trial was conducted according to the declaration of helsinki of 1973 and all its amendments and its successive updates, to good clinical practices for the conduct of clinical trials and to local regulatory requirements . Subjects had to be 1225 years of age, present with mild - to - moderate acne (grades 23 on the investigator global assessment [iga] scale), and had to be prescribed adapalene 0.1% gel for at least 2 months . Subjects were randomized at investigational sites in a 1:1 ratio to either an association of bgm complex cream and adapalene 0.1% gel, hereafter active association, or to an association of vehicle cream and adapalene (hereafter vehicle association). Adapalene was prescribed by the investigators on the day of inclusion and had to be used as per the instructions . Bgm complex or the vehicle, containing water (aqua), glycerin, hydroxyethyl acrylate / sodium acryloyldimethyl taurate copolymer, isohexadecane, titanium dioxide, phenoxyethanol, chlorphenesin, polysorbate 60, alumina, and stearic acid, was to be applied on the entire face in the morning, after the face had been cleaned with a gentle cleanser . Make - up, except foundation cream, was permitted, while no other topical skin care product or treatment was authorized on the face during the trial . Oral contraception for women of childbearing potential had to remain unchanged during the course of the trial . Clinical evaluations of inflammatory and non - inflammatory lesions count, seborrhea intensity on a visual scale (from 0: nil to 3: high), global acne severity using the iga score (from 0: none to 4: severe), and global efficacy assessed on a scale (from 0 [lowest score] to 10 [highest possible score]) were performed on d0, d28, and d56 (d standing for day). Quality of life using the cardiff acne disability index24 was assessed on d0 and d56 . Safety was assessed through adverse event reporting; local tolerance signs and symptoms comprising erythema, desquamation, pruritus, burning, prickling, tightness, vesicles, papules, and edema were assessed on d28 and d56 by the investigator on a scale from 0 to 3 (0= none, 1= light, 2= average, 3= high). The student s t - test on paired data or paired wilcoxon test was applied to compare the number of non - inflammatory and inflammatory lesions, iga and intensity of seborrhea between d0 and d28, and d0 and d56 in each group . Differences between groups for lesion count, intensity of seborrhea and iga were calculated using the student s t - test or mann whitney test . This multicenter and comparative double - blind clinical trial was conducted according to the declaration of helsinki of 1973 and all its amendments and its successive updates, to good clinical practices for the conduct of clinical trials and to local regulatory requirements . Subjects had to be 1225 years of age, present with mild - to - moderate acne (grades 23 on the investigator global assessment [iga] scale), and had to be prescribed adapalene 0.1% gel for at least 2 months . Subjects were randomized at investigational sites in a 1:1 ratio to either an association of bgm complex cream and adapalene 0.1% gel, hereafter active association, or to an association of vehicle cream and adapalene (hereafter vehicle association). Adapalene was prescribed by the investigators on the day of inclusion and had to be used as per the instructions . Bgm complex or the vehicle, containing water (aqua), glycerin, hydroxyethyl acrylate / sodium acryloyldimethyl taurate copolymer, isohexadecane, titanium dioxide, phenoxyethanol, chlorphenesin, polysorbate 60, alumina, and stearic acid, was to be applied on the entire face in the morning, after the face had been cleaned with a gentle cleanser . Make - up, except foundation cream, was permitted, while no other topical skin care product or treatment was authorized on the face during the trial . Oral contraception for women of childbearing potential had to remain unchanged during the course of the trial . Clinical evaluations of inflammatory and non - inflammatory lesions count, seborrhea intensity on a visual scale (from 0: nil to 3: high), global acne severity using the iga score (from 0: none to 4: severe), and global efficacy assessed on a scale (from 0 [lowest score] to 10 [highest possible score]) were performed on d0, d28, and d56 (d standing for day). Quality of life using the cardiff acne disability index24 was assessed on d0 and d56 . Safety was assessed through adverse event reporting; local tolerance signs and symptoms comprising erythema, desquamation, pruritus, burning, prickling, tightness, vesicles, papules, and edema were assessed on d28 and d56 by the investigator on a scale from 0 to 3 (0= none, 1= light, 2= average, 3= high). The student s t - test on paired data or paired wilcoxon test was applied to compare the number of non - inflammatory and inflammatory lesions, iga and intensity of seborrhea between d0 and d28, and d0 and d56 in each group . Differences between groups for lesion count, intensity of seborrhea and iga were calculated using the student s t - test or mann whitney test . A total of 111 subjects participated in this trial, 55 in the active association and 56 in the vehicle association group (table 1). Three patients presented with an acne severity of less than mild (iga grade 1) at inclusion . The investigator decided to maintain these subjects in the trial . After 56 days of treatment, the mean number of non - inflammatory lesions had decreased from 34.423.5 to 15.213.5 lesions in the active association group and from 29.320.3 to 14.68.5 lesions in the vehicle association group . The percent decrease at d56 from d0 reached 56% and 50.2% in the active association and the vehicle association groups, respectively (figure 1a). These reductions were statistically significant (p<0.005) for each group and sustained from d28 . At d56 compared to d0, the mean number of inflammatory lesions had decreased in both groups: from 18.410.9 to 6.96.6 and from 17.311.5 to 10.110.4 lesions in the active association and vehicle association groups, respectively . At the end of the trial, the percent decrease from d0 was 62.7% for the active association group and 41.5% for the vehicle association group (figure 1b). The difference between both groups was statistically significant (p<0.05) and in favor of the active association group . Table 2 shows results for seborrhea intensity, iga, global efficacy evaluation, and quality of life at d0 and d56 . In the active association group, 23 subjects reported at least one local adverse event (burning sensation, erythema, desquamation, and pruritus). In 15 subjects, they were considered related to adapalene and in four subjects, related to bgm complex . A total of 12 subjects had to stop adapalene and four had to stop bgm complex . The interruption of applications was temporary in all subjects except for one subject who experienced an allergy considered to be related to adapalene . In the vehicle association group, 17 subjects reported at least one local adverse event (erythema, burning sensation, pruritus, and desquamation). Of those, 12 were related to adapalene . The majority of all local adverse events were mild in intensity . In eleven subjects, adapalene had to be interrupted temporarily; a total of 111 subjects participated in this trial, 55 in the active association and 56 in the vehicle association group (table 1). Three patients presented with an acne severity of less than mild (iga grade 1) at inclusion . The investigator decided to maintain these subjects in the trial . After 56 days of treatment, the mean number of non - inflammatory lesions had decreased from 34.423.5 to 15.213.5 lesions in the active association group and from 29.320.3 to 14.68.5 lesions in the vehicle association group . The percent decrease at d56 from d0 reached 56% and 50.2% in the active association and the vehicle association groups, respectively (figure 1a). These reductions were statistically significant (p<0.005) for each group and sustained from d28 . At d56 compared to d0, the mean number of inflammatory lesions had decreased in both groups: from 18.410.9 to 6.96.6 and from 17.311.5 to 10.110.4 lesions in the active association and vehicle association groups, respectively . At the end of the trial, the percent decrease from d0 was 62.7% for the active association group and 41.5% for the vehicle association group (figure 1b). The difference between both groups was statistically significant (p<0.05) and in favor of the active association group . Table 2 shows results for seborrhea intensity, iga, global efficacy evaluation, and quality of life at d0 and d56 . After 56 days of treatment, the mean number of non - inflammatory lesions had decreased from 34.423.5 to 15.213.5 lesions in the active association group and from 29.320.3 to 14.68.5 lesions in the vehicle association group . The percent decrease at d56 from d0 reached 56% and 50.2% in the active association and the vehicle association groups, respectively (figure 1a). These reductions were statistically significant (p<0.005) for each group and sustained from d28 . At d56 compared to d0, the mean number of inflammatory lesions had decreased in both groups: from 18.410.9 to 6.96.6 and from 17.311.5 to 10.110.4 lesions in the active association and vehicle association groups, respectively . At the end of the trial, the percent decrease from d0 was 62.7% for the active association group and 41.5% for the vehicle association group (figure 1b). The difference between both groups was statistically significant (p<0.05) and in favor of the active association group . Table 2 shows results for seborrhea intensity, iga, global efficacy evaluation, and quality of life at d0 and d56 . In the active association group, 23 subjects reported at least one local adverse event (burning sensation, erythema, desquamation, and pruritus). In 15 subjects, they were considered related to adapalene and in four subjects, related to bgm complex . The majority of all local adverse events were mild in intensity . A total of 12 subjects had to stop adapalene and four had to stop bgm complex . The interruption of applications was temporary in all subjects except for one subject who experienced an allergy considered to be related to adapalene . In the vehicle association group, 17 subjects reported at least one local adverse event (erythema, burning sensation, pruritus, and desquamation). Of those, 12 were related to adapalene . The majority of all local adverse events were mild in intensity . In eleven subjects, adapalene had to be interrupted temporarily; after 2 months of combined use with adapalene, bgm complex improved significantly the clinical outcome for inflammatory lesions when compared to the vehicle . The fact that bgm complex enhances the anti - inflammatory potential of adapalene may be due to its antibacterial properties on p. acnes as well as its anti - irritation potential . Results observed for the non - inflammatory and inflammatory lesion counts were confirmed by the iga, the seborrhea intensity, and the global efficacy rated by the investigator and the subject, which at the end of the trial were all significantly in favor of the bgm complex / adapalene regimen; quality of life of subjects had significantly improved . Even though results are not shown, the excellent subjects perceptions of bgm complex may have contributed to making them adhere to their treatment, especially in the light of acne being a chronic disease.25 one may argue that subjective evaluation is secondary to objective clinical assessments . However, disfigurement from inflamed lesions, post - inflammatory hyper - pigmentation, and scars often causes embarrassment and frequently undermines confidence and lowers self - esteem . Hence, adherence to treatment, reducing sequels from acne is crucial for a positive treatment outcome . The fact that bakuchiol, one of bgm complex agents, has antibacterial properties may open new perspectives in the issue of p. acnes strains, becoming more and more resistant to antibiotics . Indeed, to limit the use of topical antibiotics, retinoids such as adapalene are more and more combined with bpo . However, bpo may cause irritation and is hence contraindicated in a certain number of patients . As for bpo, bakuchiol does not lead to antibiotic resistance . Hence, in such patients, bgm complex may be considered an alternative to bpo . To confirm this, future clinical trials may be conducted to evaluate the efficacy of bgm complex combined to a topical retinoid compared to a combination of topical retinoids and topical antibacterials . A similar number of subjects reported mild and transient local adverse events with bgm complex and with the vehicle cream; the majority of local adverse events were caused by adapalene, even though known to be the less - irritating topical retinoid.26,27 a missing treatment arm with adapalene in monotherapy and the short treatment duration, treatment of adapalene should last 3 months, may raise concerns for limitations . However, we feel that the results obtained from the active and the vehicle groups confirmed the expected treatment outcome of adapalene . Therefore, we consider that the trial is robust enough to show that bgm complex improves the treatment outcome of adapalene in patients with acne vulgaris . In conclusion, the dermocosmetic containing bgm complex improves the treatment outcome of adapalene in patients with acne vulgaris and is well tolerated.
From 1992 to 2000, we collected serum samples from 317 (65 breeds, 146 male, 171 female) dogs seen at a veterinary clinic in arendal, in southern norway . The laboratory received 436 serum specimens . In case of multiple specimens from one dog, collected during several months or years, we controlled the results for possible changes in antibody levels . The presence and level of igg antibodies to tbev were tested by an enzyme immunoassay for the detection of igg antibodies to tbev (enzygnost anti - tbe virus igg, dade behring marburg gmbh, marburg, germany). Igg to tbev was detected by a specific sheep, anti - dog, heavy and light chain igg antibody (a40 - 105p-7, bethyl laboratories, montgomery, tx) in a dilution of 1:20,000 . Positive specimens were confirmed by a second elisa (baxter - immuno, orth, austria), as previously described (7). In this assay, titers> 100 were considered to be positive . A total of 52 (16.4%) of 317 dogs had igg antibodies to tbev; 40 (12.6%) had igg antibody titers to tbev> 450, while 12 dogs (3.8%) had moderate levels (> 100<450) (table 1). Positive serum specimens, including samples with 11 to <100 u in the enzymeimmunoassay (eia)-e test, were confirmed with the baxter - immuno (b - i) test (table 2). The confirmatory test included five extra serum samples in instances where such blood samples were drawn; thus the number of positive specimens to be confirmed was 57 . Positive by enzyme immunoassay e - test (eia - e) and confirmed by a second test (enzyme immunoassay baxter - immuno [eia - bi]). In five cases, we had two or more serum specimens with high positive results . 287) with 116 u in the enzygnost (eia - e) by the immuno elisa . Of the low - positive specimens in the enzygnost (<100 u), only four specimens had low - positive results in the b - i elisa; four low - positive eia - e specimens gave positive results in the b - i test . On the other hand, 9 low - positive specimens in the eia - e (2037 u) were negative by the b - i test . Thus serum specimens sampled and coded at different times were in some cases collected from a single dog . Nevertheless, high positive antibody levels were reproducible even after several years . In five instances, we had two or more serum specimens from one dog with high positive results at our disposal . All these samples were tested by both elisa techniques . Only results of> 450 u in the enzygnost test could be registered, which in two instances gave lower results in the new specimens . The immuno elisa was in agreement with the enzygnost in case a, and it showed stable titers in case b. in cases c and d, one could observe an increase in titers by the b - i test . The average age of the dogs at the time of blood sampling was 6.6 years (0.515). The 52 dogs with 100 u were 8.02 years versus dogs with <100 u, which were younger, 6.29 years . The distribution of antibodies according to the size of the dogs is shown in table 3 . A total of 34 (21.8%) of 151 large dogs had antibodies to tbev 100 u versus 18 (10.8%) of 166 small and medium - sized dogs . This difference is statistically significant: with odds ratio = 2.39, = 7.03, p = 0.008 with yates correction . Among dogs with> 450 u, 25 (62.5%) of 40 were large . Antibodies to tbev were detected in 16.4% of dogs in aust - agder county of southern norway . Although the first human cases prove the existence of tbev in southern norway, the levels of seropositivity in dogs were still unanticipated in a region where tbe has previously not been seen . Tbe in dogs has been reported from several european countries (7), and the number of cases is growing . Searching for antibodies to tbev in our canine population would be useful since dogs are suitable serologic indicators of tbev in a geographic area, and canine serum has been used to reveal natural epidemic foci . Our data support the recent findings of human tbe cases in norway and the notion of an emerging disease, especially because the serum samples were collected from the same geographic area where the first human cases were described.
Xanthogranulomas are benign, usually asymptomatic, self - healing, red, yellow, or brown papules, nodules composed of histiocytic cells that predominantly occur in infancy and childhood . Adamson first reported juvenile xanthogranuloma (jxg) in 1905 . But during 1912, mcdonough reviewed the condition and renamed it as nevoxanthoendothelioma . In 1954, helwig and hackney re - termed it as jxg, reflecting its histopathological appearance . It belongs to a heterogeneous group of non - langerhans cell histiocytoses, which are characterized by benign dermal proliferations of histiocytic cells in the absence of any known stimuli . Approximately 35% of cases of jxg occur at birth, with as many as 71% of cases occurring in first year . Usually, xanthogranuloma is termed as jxg though around 10% of cases manifest in adulthood . Up to 81% of cutaneous jxg cases extracutaneous jxg is rare (3.9%) and most commonly involves eye . Histopathological examination of jxg demonstrates a variety of findings . A time - dependant progression exists in development of characteristic histological features which correlates with age of lesions . A mixed cellular infiltrate of neutrophils, lymphocytes, eosinophils and rarely mast cells may be noted . Anticipatory care, with patient reassurance, is appropriate because of self - limiting benign nature of disease . Ocular and systemic lesions may respond to steroids or radiotherapy . But diffuse and multiple cutaneous lesions also need some treatment . A 28-year - old healthy male patient presented in our department with one - month history of diffuse numerous papulonodular eruptions at extremities, ears, face, and trunk . Cutaneous examination revealed yellow - brown, relatively well - demarcated papulonodular lesions with variable sizes (15 mm in diameter). Lesions were shiny, soft to elastic in consistency consistency present almost all over the body and majority being over upper and lower extremities, ears, and chin [figure 1a c]. The mucous membranes, palms and soles were unaffected and ophthalmologic examination was normal . No other systemic involvement was noted . Yellow - brown relatively well - demarcated shiny elastic papulonodular eruptions the following differential diagnosis was made: lepromatous leprosy, tuberous xanthoma and xanthogranuloma . Laboratory investigations, including routine hematological examination, liver and renal function test, were within normal range . Histopathological examination revealed dense granulomatous dermal infiltrates consisting of foam cells, giant cells (mainly touton type), histiocytes, lymphocytes, and a few eosinophils and neutrophils mainly at upper dermis . The epidermis was thinned out without any grenz zone and inflammatory cells extended toward lower dermis to subcutaneous tissue [figure 2a c . Granulomatous dermal infiltrates of foam cells, giant cells (touton type), histiocytes, lymphocytes, neutrophils . [h & e stain; original magnifications: 2b 10; 2a and c 40] the prognosis of disease was discussed with patient . As there was a diffuse involvement with disfigurement, we planed to give some treatment to improve disease process rapidly . Patient was reviewed after one month and noted about 50% reduction of size of lesion [figure 3]. He continued treatment for another one month and noticed that most of the lesions had flattened with yellowish and hyperpigmented macules [figure 4]. Post - treatment photograph after one month of treatment post - treatment photograph after two month of treatment serum lipid profiles are normal in patients with both juvenile and adult form of xanthogranuloma . The number of lesions in adult xanthogranuloma is lesser than in juvenile form . Of 31 patients, 27 (87%) with adult onset xanthogranuloma in japanese literature had a solitary lesion . There seemed to be no definite sites of predilection for adult onset xanthogranuloma and lesions were usually asymptomatic . Spontaneous resolution does not occur in adult form, whereas juvenile form usually involutes spontaneously within a year . Earlier published report of nine cases of adult xanthogranuloma did not notice any spontaneous resolution . Extracutaneous involvements of eye, lung, testis and pericardium have been reported in patients with jxg . In contrast, no concomitant extracutaneous lesions have been found in adult form, although solitary extracutaneous xanthogranuloma without cutaneous lesions have been reported . These cells express phenotype of dermal dendrocytes, although a recent study has suggested that cell of its origin could be plasmacytoid monocytes . The appearance of giant cell and foamy lipid - laden histiocyte occur late and they are almost certainly secondary events, possibly in response to cytokine production by the lesion histiocyte . Jxg has been noted in association with different diseases like neurofibromatosis, niemann - pick disease, urticaria pigmentosa, juvenile chronic myelogenous leukemia . Ocular and systemic lesions may respond to steroids or radiotherapy, and severe systemic jxg have required single and mutagenic chemotherapeutic regimens . Commonly, no treatment is necessary for cutaneous xanthogranuloma, but severe cutaneous involvement of adult form like our case may require some treatment to hasten disease improvement . The growth of a number of tumor cell lines seems to be inhibited by retinoids, but response may be variable . It affects transformed cell surfaces and leads to anchorage - independent growth, cell adhesiveness and density - dependant growth . For these reasons, we started isotretinoin and noticed significant clinical improvement within two months . As spontaneous resolution does not occur in adult xanthogranuloma, we think that this type of early resolution may be due to isotretinoin . But further large study is required to see the effectiveness of isotretinoin in xanthogranuloma.
Allogeneic hematopoietic stem cell transplantation (allo - hsct) is an established treatment for hematologic malignancy, and more than 15,000 procedures are performed worldwide each year . After hsct, chronic graft - versus - host disease (cgvhd) is the most common cause of morbidity and mortality . Indeed, the incidence of cgvhd is reported to be 6080% during long - term follow - up, and the incidence has recently been increasing because of the extensive use of unrelated donor transplants, older donor age, increased use of donor leukocyte infusion, and peripheral blood stem cell transplantation (pbsct). Symptoms of cgvhd can vary depending on the site of involvement, which may include the skin, eyes, oropharynx, or respiratory and gastrointestinal tracts . However, renal involvement associated with gvhd, particularly glomerulopathy, is very rare . In general, renal injury after hsct occurs due to hemodynamic compromise, medications, radiation, or thrombotic microangiopathy, which manifests as tubulointerstitial nephropathy . Cases of nephrotic or nephritic syndrome after hsct have recently been reported, and these glomerulopathies are presumably related to cgvhd . Herein, we report a case of membranous nephropathy (mn) in a patient who underwent hsct 21 months before this unusual nephrotic syndrome (ns) developed . A 39-year - old female patient was admitted to our hospital due to generalized edema and fatigue . The patient was diagnosed with aplastic anemia 3 years previously, and had no history of diabetes or hypertension . Most importantly, she had undergone allogeneic pbsct from her human leukocyte antigen - identical brother 21 months before admission following myeloablative conditioning chemotherapy with cyclophosphamide and anti - thymoglobulin . Grade iv acute gastrointestinal gvhd accompanied by diarrhea developed 12 days after transplantation despite gvhd prophylaxis with cyclosporine, methotrexate, and steroids, for which a continuous maintenance regimen of cyclosporine and prednisolone resulted in resolution . The patient also suffered from cytomegalovirus colitis 4 months after transplantation, and recovered after a 2-week administration of gancyclovir while cyclosporine was discontinued and prednisolone was tapered to 5 mg / day . Eighteen months after cytomegalovirus infection, the patient suddenly developed generalized edema and gained 5 kg of body weight over a 2-week period . At this time initial laboratory tests showed the following values: hemoglobin, 10.5 g / dl; platelets, 32010/l; serum albumin, 2.2 g / dl; total cholesterol, 402 mg / dl; low - density lipoprotein cholesterol, 248 mg / dl; serum creatinine, 0.77 mg / dl; random urine protein - to - creatinine ratio (upcr), 7.85 g / g; and 24-hour urinary protein excretion, 5.03 g / day . Hepatitis b surface antigen, hepatitis c antibody and anti - nuclear antibody titers were undetectable, and serum concentrations of c3, c4, and immunoglobulins g, a, and m were within reference range . 1a), eight nonsclerotic glomeruli were normocellular without mesangial expansion . The glomerular basement membrane was not thickened and double contours or subepithelial spikes were not noted . The interstitium was moderately infiltrated by mononuclear inflammatory cells, which immunohistochemical staining confirmed as cd3 + t cells (fig . Electron microscopy demonstrated numerous nodular electron - dense deposits that were mainly located in the subepithelial space along with diffusely effaced epithelial foot processes (fig . As such, oral prednisolone at a dose of 1 mg / kg was immediately started, and an 8-week treatment resulted in partial remission with a upcr of 1.9 g / g . Because the patient was intolerant to the side effects of corticosteroid treatment, prednisolone was tapered . Two weeks later, however, the random upcr increased to 7.58 g / g; thus, cyclosporine at a dose of 5 mg / kg was added . Complete remission was achieved as demonstrated by a upcr of 0.23 g / g 4 months after the combined treatment with cyclosporine and 10 mg / day of low - dose prednisolone . Acute kidney injury (aki) is a common complication in patients with allo - hsct . In general, aki can be attributed to preexisting renal disease, underlying malignancy, previous chemotherapies, irradiation, and various nephrotoxic agents such as antimicrobials, antifungals, and antivirals . Accordingly, acute tubular necrosis, hemolytic uremic syndrome, thrombotic microangiopathy, and radiation nephritis are the most common forms of aki after hsct . However, glomerulopathy such as ns or nephritis rarely occurs as a manifestation of cgvhd . The notion that graft - versus - host reaction is directly related to glomerular injury has recently been gaining acceptance . One of these reports showed that nine of 889 patients who underwent allo - hsct from 1994 to 2002 developed ns, with an overall incidence of 1% . When only sibling transplant patients with or without cgvhd were taken into account, the incidence of ns was 1.8% and 0.6%, respectively, suggesting that it is not a common complication . Glomerular diseases tend to occur in allogeneic pbsct patients from unrelated donors who received nonmyeloablative therapy shortly after immunosuppression was decreased or stopped,, and the median time elapsed to the onset of ns from cessation of immunosuppression was 1.5 months . Regarding pathologic features, mn (61%) was the most common type of ns, followed by minimal change disease (22%). Approximately 70% of patients had concomitant cgvhd at the onset of ns and, in particular, 7593% of patients with posthsct mn had recent or current cgvhd, . In addition, development of ns appears to be a late event after hsct because ns was diagnosed 15.524 months after hsct . In line with these findings, our patient developed ns 21 months after allogeneic pbsct, whereby mn was confirmed by renal biopsy . Unlike previous observations, the patient did not show other features of cgvhd before the onset of ns, although she did experience acute gvhd 12 days after hsct . However, a prior episode of acute gvhd is unlikely to be related to the development of glomerular disease considering its late onset after hsct . Despite the high incidence of cgvhd, only a small portion of patients with cgvhd developed glomerular disease, and this low incidence is no different than that of the whole hsct population, . Furthermore, cgvhd was not evident in approximately 30% of patients who presented with ns, suggesting that ns can develop irrespective of gvhd . In this regard, it is not clear whether preexisting cgvhd can indeed contribute to the development of glomerular disease . Nevertheless, in this case, immunohistochemical studies clearly identified t cells in the interstitium . Because complete donor chimerism was confirmed, these t cells originated from the donor and were presumed to be associated with kidney injury . This finding may be nonspecific because kidney injury could be caused by other conditions such as drugs or infections . However, considering the fact that the patient had no evidence of kidney injury associated with such conditions before the onset of ns, this pathologic finding partly explains the presence of gvhd in the kidney . Previous observations showed that ns usually occurred shortly after immunosuppressive medications were stopped or their dose was reduced . However, it is uncertain whether cessation or reduction of immunosuppression may play a role in the development of ns . In fact, 40% of patients that have ns may develop glomerular disease while on immunosuppressive agents . Although controversy remains over the role of preexisting cgvhd in posthsct ns, it has been suggested that immune complex, which is formed as a result of gvhd, can deposit within the glomeruli and cause damage . In particular, b - cell dysregulation with a high prevalence of autoantibodies to cellular antigens and minor histocompatibility antigens has been demonstrated in patients with cgvhd,,, . Besides b - cell dysregulation in fact, cgvhd is considered an immune reaction between donor cd4+/th2 cells and the recipient's minor histocompatibility complex antigens . Furthermore, prehsct conditioning chemotherapy or irradiation can cause renal damage, which renders hiding autoantigen exposed or altered, ultimately leading to selective activation of alloreactive donor cd4 + cells and the production of immune complex . The pathogenesis of hsct - associated ns cannot be explained by a single factor, and is more likely to be attributed to the combination of b cell dysregulation, alloreactive t cells, preconditioning chemotherapy, etc . Treatment of hsct - associated ns has not yet been defined, but, similar to idiopathic ns, corticosteroids and cyclosporine have been most commonly used . Other immunosuppressive agents such as cyclophosphamide, mycofenolate mofetil, and rituximab have also been attempted to induce remission . Overall responsiveness to these drugs appears favorable: according to a previous report, 27% and 62% of patients with mn attained complete and partial remission, respectively, and 90% of patients with minimal change disease achieved complete remission . Likewise, in our case, cyclosporine in addition to corticosteroids resulted in complete resolution of proteinuria, and the patient has been in complete remission . In conclusion, we present a case of posthsct mn as a rare manifestation of gvhd . However, ns occurred without other features of cgvhd while the patient was on low - dose corticosteroids . Our report contradicts previous assumptions that concomitant gvhd is responsible for the development of ns, suggesting that ns can develop as a new, independent manifestation of gvhd.
It has numerous adverse health effects and is considered as one of the main predisposing factors for the emerging epidemic of noncommunicable diseases . Nowadays, overweight and obesity are growing in populations with different levels of economic situation . It is estimated that by continuing the actual trend, the global prevalence rate of 33.0% for overweight and obesity among adult population (1.3 billion people) in 2005 would reach up to 57.8% (3.3 billion people) by 2030 . The world health organization included excess weight, with a prevalence higher than undernutrition, as one of the top 10 health risks worldwide . The rise in the incidence in obesity matches the rise in the use and distribution of industrial chemicals that may have a role in development of obesity . In her interesting review in 2002, baillie - hamilton postulated a role for chemical toxins in the etiology of obesity by presenting the coincidence of the obesity epidemic with the noticeable increase of industrial chemicals in the environment over the past four decades . An accumulating body of evidence suggests that substances as endocrine - disrupting chemicals (edcs) may be linked to the obesity epidemic . Edcs are chemicals that alter the normal functioning of hormones and other signaling molecules in the body . Obesogens, molecules with adverse effects on lipid metabolism and adipogenesis, and in turn resulting in obesity [6, 7]. The environmental obesogen hypothesis suggests that prenatal or early - life exposure to certain substances as edcs may predispose exposed individuals to increased fat mass and excess weight . It is suggested that exposure to obesogens can modify the epigenome of multipotent stromal stem cells, biasing them to the adipocyte lineage at the expense of bone . Hence, humans exposed to obesogens during early life might have an altered stem cell compartment, already preprogrammed for an adipogenic outcome . The list of chemicals studied as possible obesogens continues to grow and includes diethylstilbestrol (des), bisphenol a (bpa), phthalates, organotins, polybrominated diphenyl ethers (pbdes), polyfluoroalkyl chemicals (pfcs), organochlorine (oc) pesticides, and polychlorinated biphenyls (pcbs) and some solvents caused weight gain, and it is proposed that these chemicals were interfering with weight homeostasis by changing weight - controlling hormones, modifying sensitivity to neurotransmitters, or altering the sympathetic nervous system activity . The purpose of this paper is to systematically review the experimental and human studies on obesogenic chemicals and their mechanisms of action to provide a comprehensive view on underlying mechanisms and the multifactorial aspects of obesity for clinicians and public health stakeholders . Relevant literature reporting the environmental obesogens was identified through electronic search of medline, pubmed, isi web of science, and scopus / embase with no time or language restrictions . We searched the databases using the following strategy: for scopus / embase we used the emtree thesaurus terms; for pubmed search, we considered medical subheading (mesh) words, and for other databases we used keywords (text words). For pubmed search, we used (endocrine disruptors [mesh] or endocrine disrupting chemicals or obesogen [mh] or polychlorinated biphenyls [mesh] or hydrocarbons, chlorinated [mesh] or dioxins [mesh] or polybrominated biphenyls [mesh] or carbon tetrachloride [mesh] or organothiophosphorus compounds [mesh] or phthalic acid [substance name] or phthalic acids [mesh] or organotin compounds [mesh] or bisphenol a [substance name] and ((obesity [mh] or fat deposition [mh]) and (publisher[sb] or in process [sb]). Duplicates were removed; the relevant papers were selected in three phases . In the first and second phases, titles and abstracts of papers were screened and irrelevant papers were excluded . In the last phase, the full text of recruited papers was explored intensely to select only relevant papers . For any additional pertinent studies, all these three screening phases were done by two independent reviewers (fj and pp). In the next step, the eligibility of relevant papers was checked . Identification of main findings of studies was conducted on a case - by - case basis and included consideration of any statistical analyses that might have been conducted, consistency of the general pattern across exposure groups . The required information that was extracted from all eligible papers was as follows: (i) general characteristics of the study (first author's name, publication year, study year, study design) (ii) characteristics of the chemical, (iii) reason for using the chemical, (iv) suggested obesogen mechanism, and (v) adverse effects on humans or animals . Two reviewers (fj and pp) extracted the data while another (rk) checked their extracted data . We found that actually many environmental obesogens are identified; they are mainly classified as chemical simulators of metabolic hormones or brain neurotransmitters [10, 11]. Bisphenol a [1215], tributyltin (tbt) [16, 17], nonylphenol [18, 19] and genistein [20, 21], phatalate, perfluoroalkyl compounds (pfcs), and perfluorooctanoic acid (pfoa) are some of the obesogen chemicals described by experimental studies . Diverse mechanisms are explained for obesogen chemicals; mainly they have disruptive effects on homeostasis of energy balance, glucose and lipid metabolism, and control of adipogenesis . The concentrations of many industrial obesogen chemicals are found to be high in general population . For instance, some studies examined the obesogenic effects of phthalates, which are esters mainly added to plastics to increase their flexibility, transparency, durability, and longevity . Cross - sectional data from the national health and nutrition examination survey (nhanes) in the usa found significant associations between several phthalates metabolites (monobenzyl phthalate (mbzp), mono-(2-ethyl-5-hydroxyhexyl) phthalate (mehhp), and mono-(2-ethyl-5-oxohexyl) phthalate (meohp)) and measures of abdominal obesity and insulin resistance in men but not in women . A cross - sectional study on 90 girls aged 68 years found slightly higher concentrations of some phthalate metabolites as monoethyl phthalate (mep), mono-(2-ethyl-5-hydroxyhexyl) phthalate (mehhp), and mono - n - butyl phthalate (mbp) among overweight girls than in their other counterparts some epidemiologic studies documented the obesogenic effects of some environmental chemicals, as pcb and bpa, whereas such effects are conflicting for some other chemicals as organochlorine pesticides [3032]. Phytoestrogens, notably soy products, have beneficial health effects and are added to several food and food supplements . Genistein is one the mostly used phytoestrogens in the human diet, and by its estrogenic activity, it has favorable effects for regulating the homeostasis of lipids and carbohydrates . Though its beneficial effects in inhibiting fat deposition in the adipose tissue are considered to be obtained at high pharmacological doses, its low doses in foods are found to increase adiposity and mild peripheral insulin resistance particularly in males . In this review, we summarized information regarding environmental chemicals that can be associated with obesity . Most evidence comes from experimental and laboratory studies; however a growing number of human studies also support the role of obesogen chemicals . Chemicals as heavy metals, some solvents, pesticides, bpa, organophosphates, phthalates, pcb, pbbs, and many other substances are documented to cause weight gain . These chemicals interfere with weight and lipid homeostasis by various mechanisms related to weight - controlling hormones, activity of the sympathetic nervous system, and sensitivity to neurotransmitters . Exposure to these chemicals varies in different age groups; their effects during fetal and infancy periods may be irreversible and long - lasting for adulthood . Even exposure to low doses of edcs during critical times of differentiation can change the developmental programming and may result in obesity . Barker's hypothesis on the effects of intrauterine growth on fetal programming and fetal origins of adult diseases is well documented [36, 37]; however, other characteristics as later growth spurt and environmental factors are considered to influence this programming . Exposure to environmental chemicals with endocrine - disrupting activities in early life may result in everlasting adverse health effects . Such health consequences may become apparent not only in childhood, but also in adulthood, and even in succeeding generations . Transgenerational effects may be because of mutations as well as because of factors regulating gene expression . Our findings support the role of obesogens, as chemicals with disruptive effects on fat homeostasis and various weight controlling mechanisms, in programming the development of excess weight from early life . Although all obesogen chemicals are not yet identified, and their detailed mechanisms of action remain to be explored, generally it is assumed that exposure to different doses of these environmental chemicals in various periods of life from fetal to adult period interacts with some endocrine signaling mechanisms and in turn leads to obesity . Edcs act by diverse mechanisms; accumulating body of evidence supports that these chemicals disrupt some epigenetic, structural, and functional mechanisms, which control energy homeostasis, lipid metabolism, appetite regulation, and adipogenesis [4044]. Chemical obesogens are considered to function through various factors as leptin, ghrelin, melanocyte - stimulating hormones, neuropeptide y, amphetamine - regulated transcript, agouti - related protein, and cocaine, as well as through inhibiting aromatases as the p450 family members (cyp19 and cyp3a1) [4244] or through modifying the expression of various receptors for steroid hormones, retinoic x, peroxisome proliferator - activated, and glucocorticoids . The exposure to obesogen chemicals may influence the steroid hormone receptors or may change serum levels of metabolic hormones or may influence nuclear receptor signaling pathways in preadipocytes, which would result in adipocyte differentiation and a tendency to excess weight [44, 45]. The systemic reactions to exposure to environmental chemical factors can potentially increase the risk for obesity - related health effects, as metabolic syndrome, insulin resistance, prediabetes, diabetes, oxidative stress, prehypertension, hypertension, and nonalcoholic fatty liver diseases even in the pediatric age group . Even in the pediatric age group, environmental chemicals can influence oxidative stress and proinflammatory cytokines [46, 47, 50], which in turn would initiate the second hit suggested in the two - hit hypothesis [51, 52] for the progression of fatty liver to metabolic syndrome and diabetes . The other aspect of the influences of environmental factors on obesity and its health consequences can be the impact of these chemicals on intrauterine growth retardation, low birth weight, and prematurity [5355], which are documented as predisposing factors for obesity and adult chronic diseases . Whether the results of laboratory models can be generalized to health hazards in humans remain to be determined, but a growing number of epidemiologic studies also suggest a link between exposure to environmental chemicals with obesity . However, it should be considered that in many human studies, weight gain has not been an endpoint in the original proposal, and excess weight has been reported as an adverse effect . Environmental factors have diverse health effects [4750, 5658]. Although rapid changes in lifestyle habits, along with increased energy intake and decreased energy expenditure, are considered as the main causes of excess weight, but by considering the rapid escalating trend of obesity in various age groups and in populations with different lifestyle habits and diverse socioeconomic levels, it is obvious that it is simple - minded to consider only these two factors responsible for this expanding global problem; the role of other environmental determinants as obesogen chemicals is being proposed in this regard . Large - scale longitudinal studies with long - term followup are necessary to document the clinical importance of exposure to environmental chemicals . The current evidence proposes that the systemic responses to exposure to environmental factors, notably during developmental phases of life, could potentially increase the risk of excess weight . By taking into account the current knowledge on the adverse transgenerational effects of obesogen chemicals on human health, the global obesity epidemic should be considered as a multifactorial complex disorder necessitating the emphasis of public health interventions for environmental protection.
Tuberomamillary nucleus (tmn) neurons expressing the histidine decarboxylase (hdc) gene are the sole neuronal source of histamine [1315]. The hdc gene shows haploinsufficiency: a 2-fold decrease in hdc mrna levels halves the brain content of histamine in mice [16, 17], and in humans, having only one functional hdc allele produces a type of tourette syndrome . Thus, modest changes in hdc transcript levels in tmn neurons can change the amount of histamine released and influence behavior . Hdc mrna levels in human hypothalamus are 1.6-fold higher for daytime deaths, and hdc enzyme activity and histamine levels in rat brain vary with time of day [1921]. In agreement with these data, immunocytochemical staining for hdc protein in mouse tmn neurons was stronger at zeitgeber time (zt)18 (night, mid - lights off, the period when the animals are more active) than at zt6 (day, mid - lights on) (3.5 0.19 versus 1 0.09 arbitrary units [aus]; unpaired two - tailed t test, p <0.001) (figures 1a and 1b). In control mice there was also a 1.5-fold variation in hdc transcript levels over 24 hr (unpaired two - tailed, t test, p <0.05): hdc mrna was highest at the start of the night (zt12), declined during the night, and increased during the day (figure 1c). N - methyltransferase (hnmt), did not show daily variation in the tmn area (figure 1c). This daily variation in hdc expression could indicate a clock - like mechanism in histaminergic neurons . Indeed, histaminergic neurons express the core clock protein bmal1 (figure 1d). (bmal1 antisera specificity was confirmed by the absence of staining in suprachiasmatic nucleus [scn] sections from bmal1 global knockout brains [figure s1a available online].) We crossed hdc - cre mice with animals containing a floxed bmal1 gene (figure s1b). The resulting hdc-bmal1 mice were similar to littermate controls in weight (control weight, 26.6 0.6 g, n = 5; hdc-bmal1 weight, 27 0.7 g, n = 5; unpaired two - tailed t test, p = 0.34) and seemed healthy . All the hdc - positive cells lost bmal1 (figure 1d). In our characterization of the hdc - cre mice, we found that transient developmental expression of the hdc gene produced recombination in several additional places, in particular the dorsal lateral geniculate (dlg) thalamic nucleus, the ventral medial (vm) hypothalamic nucleus, and purkinje neurons . By immunostaining, there was no indication of bmal1 loss from the dlg, vmh, and cerebellum of hdc-bmal1 brains (figures s1c s1e); bmal1 and per1 transcript levels in these regions were also unchanged (two - way anova and post hoc bonferroni, p> 0.05) (figure s1f). The failure to delete the bmal1 gene in these areas likely reflects that the particular floxed allele is relatively cre insensitive, requiring sustained doses of cre to produce recombination . Bmal1 could serve housekeeping functions unrelated to its clock role . To see whether removing bmal1 from histaminergic neurons disrupted the local clock, we examined the expression of core clockwork - associated genes in the tmn of control and hdc-bmal1 mice . In littermate control mice, per1, cry1, and rev - erb mrna levels peaked around the beginning of the night (figure s1 g); in hdc-bmal1 mice, the expression rhythms of these three genes across the light - dark cycle were flattened; per1 and cry1 mrna levels were, on average, higher, whereas rev - erb levels were significantly lower (figure s1 g) (two - way anova and post hoc bonferroni, p <0.05, p <0.01; cosinor analysis [cosinor.exe, version 2.3; http://www.circadian.org/softwar.html]; per1: control: amplitude, 0.63, p <0.05; hdc-bmal1: p = 0.27; cry1: control: amplitude, 0.25, p <0.05; hdc-bmal1: p = 0.25; rev - erb: control: amplitude, 0.9, p = 0.01; hdc-bmal1: amplitude, 0.29, p = 0.05; cosinor p values are related to comparisons of goodness of cosine fit). Furthermore, the rhythmic expression of per2 protein was abolished in histaminergic neurons in hdc-bmal1 mice (figure s1h; the specificity of the per2 antiserum was confirmed in per2 knockout mice). These results indicate that bmal1 deletion from histaminergic neurons has likely disrupted their local clock mechanism . In the hdc-bmal1 mice, hdc gene expression showed a disrupted 24 hr pattern (two - way anova and post hoc bonferroni, p <0.01, p <0.001), and hdc transcript levels and protein were overall higher in the day and the late night . This produced increased brain histamine levels in the day (figure 1f; two - way anova or one - way anova and post hoc bonferroni, p <0.05). To test the behavioral consequence of upregulated hdc gene expression in tmn neurons, we examined locomotor activity in an open field . Mice traveled farther and at higher speeds (figures 1 g and 1h) than littermate controls (unpaired two - tailed t test, p <0.05, p <0.01). Bmal1-clock dose - dependently increased hdc promoter - luciferase gene expression (figure s2a) (one - way anova and post hoc bonferroni, p <0.001), but not when the e box was mutated (figure s2b). This was the opposite of the in vivo situation, when hdc transcript levels increased after bmal1 deletion . Thus, in histaminergic neurons, bmal1 could recruit a repressor complex onto the hdc promoter . Alternatively, rore sequences in the hdc gene could bind the repressor and core clock protein rev - erb [28, 29]. Diminished rev - erb levels in the tmn of hdc-bmal1 mice (figure s1 scn neurons show cell - intrinsic circadian regulation of their electrophysiological parameters, partly determining when these neurons fire [3032]. We made whole - cell current - clamp recordings of histaminergic neurons from littermate and hdc-bmal1 mice during night and day (figure s3c). Resting membrane potential, input conductance, current injection to threshold of action potential firing, capacitance, and membrane time constant were unaffected by time of day or the absence of bmal1 (figure s3c). We expect that hdc-bmal1 histaminergic neurons will fire action potentials normally but release more histamine . Hdc-bmal1 knockout mice had an unchanged behavioral circadian rhythm and phase, compared with littermate controls, as assessed by wheel running in free - running conditions of constant darkness (dd) (unpaired two - tailed t test, p> 0.05) (figures 2a and 2b). In free - running constant light (ll), both genotypes were more variable in period length than in ld or dd (figure 2a). However, the amplitude of the peak period was lower and more variable in ll than in ld and dd, indicating the mice were equally less active in ll than in ld or dd, regardless of genotype (figures 2a and 2b). Within the scn, the circadian variation in bmal1 and per2 proteins was unchanged between hdc-bmal1 knockout mice and littermate controls (figures 2c and 2d); there was little variation in bmal1 staining intensity in the scn between zt6 and zt18 (figure 2c), highlighting that although bmal1 is the core component of the clock, its levels change little during the circadian cycle . The critical rhythm for bmal1-clock activity arises from per - cry, which arrives to inhibit, and then dissociates from, the bmal1-clock complex . Per2 staining in the scn of both groups of mice increased at zt18 compared with zt6 (figure 2d). Thus, the hdc-bmal mice had an unaffected scn molecular clock and circadian pace making . Mice unable to synthesize histamine (hdc knockouts) show normal sleep - wake behavior throughout most of the 24 hr cycle, except they are significantly less awake just before, and for the first few hours after, the start of the night . It is intriguing that hdc knockout mice have a selective deficit in anticipating lights off, further suggesting a circadian involvement of histaminergic neurons . In contrast to hdc knockout mice, the hdc-bmal1 mice have a gain of function in the histaminergic system . We looked at the consequences for the sleep - wake cycle (figure 3; figure s4). Sleep experiments and nontethered electroencephalogram (eeg) analysis were performed using neurologger2 devices [22, 34]. For the first part of the night, hdc-bmal1 mice were more awake, as assessed by electromyogram (emg) and the ratio of: power in the eeg, than littermate controls (figures s4a s4c), namely the opposite of hdc knockout mice . As the night progressed, the eeg: emg ratios of the hdc-bmal1 mice became similar to littermate controls (figure s4a). Some the hdc-bmal1 mice had long (up to 40 min) periods of uninterrupted waking (figure 3b). The total wake time, hr was unchanged (693 21 min versus 693 12 min, unpaired two - tailed t test, p> 0.05), but over the night they spent more time awake than littermate control mice and less time awake during the day (night: 420 16 min versus 461 10 min, unpaired two - tailed t test, p <0.05; day: 273 9 min versus 231 7 min, unpaired two - tailed t test, p <0.05) (figure s4a). Throughout the 24 hr, during the wake periods, the hdc-bmal1 mice had higher frequencies in the eeg than littermate controls (two - way anova and post hoc bonferroni, p <0.05) (figure s4d). The amount of nonrapid eye movement (nrem) sleep was similar between hdc-bmal1 and control mice (figure s4b) (488 11 min versus 427 20 min, unpaired two - tailed t test, p> 0.05), but nrem power was lower (figure s4e; see next section) (two - way anova and post hoc bonferroni, p <0.05). During the day, hdc-bmal1 mice had more nrem episodes than controls (figure 3c), but these episodes were shorter (figure 3d) (3.5 0.3 min versus 2.4 0.3 min, unpaired two - tailed t test, p <0.01). The amount of rem sleep in hdc-bmal1 mice compared with littermate controls was higher in the day (figure s4c): there were more episodes (figure 3e), although episode duration was unchanged (figure 3f) (1.7 0.04 min versus 1.8 0.04 min, unpaired two - tailed t test, p> 0.05); however, rem episode duration was shorter in the hdc-bmal1 mice (figure 3f) during the night (1.6 0.04 min versus 1.4 0.03 min, unpaired two - tailed t test, p <0.01). The daytime sleep architecture of hdc-bmal1 mice differed from littermate control mice (figure 3 g). Nrem - to - rem (39 2 versus 68 2, unpaired two - tailed t test, p <0.001) and rem - to - nrem (29 1 versus 58 2, unpaired two - tailed t test, p <0.001) transitions during the day (figure 3 g). During the night, there was no difference between genotypes in nrem - rem transitions (22 3 versus 28 2, unpaired two - tailed t test, p> 0.05) (figure 3 g); however, hdc-bmal1 mice had fewer wake - to - nrem transitions (29 3 versus 17 1, unpaired two - tailed t test, p <0.001) and vice versa (21 3 versus 11 1, unpaired two - tailed t test, p <0.001) (figure 3 g), reflecting that they were awake more (figure 3b). Hdc-bmal1 mice and littermate controls were sleep deprived for 5 hr during the start of the day . Mice were placed into a novel cage, and objects (plastic tubes, pieces of paper) were introduced that were exchanged each hour . Consistent with the raised brain histamine levels in hdc-bmal1 mice, the eeg profiles between the genotypes differed during sleep deprivation: littermate control mice had frequencies distributed in the -to- range (210 hz), with two peaks at 2 and 8 hz, but the hdc-bmal1 mice had a single broad peak of enhanced power relative to controls, centered in the range (figures s4 g and s4h). Littermate control mice had a recovery sleep lasting about 1012 hr (figure 4a), with sustained nrem periods remaining 6 they reaccumulated their nrem sleep at a rate of approximately 30 min extra nrem sleep per hour (figure 4b). The power in the eeg of littermate control mice remained elevated, compared with presleep deprivation levels, for some 12 hr after sleep deprivation (figure 4c). By contrast, hdc-bmal1 mice did not sustain their recovery sleep: it was about 6 hr shorter than sleep - deprived control littermates (figure 4a), and their enhanced power of recovery sleep, already lower compared with littermate controls before sleep deprivation, remained lower as it declined to baseline (figure 4c; figure s4h). Hdc-bmal1 mice reaccumulated their nrem sleep at a slower rate than control mice: 12.5 min extra nrem sleep per hour (figure 4b). Because hdc-bmal1 mice had more rem baseline sleep than littermate controls during the day (figure 3 g), they had more rem loss during sleep deprivation, namely they had more rem sleep to lose by sleep deprivation (figure s4j). Hdc-bmal1 mice also had a quicker reaccumulation of rem sleep during recovery sleep (figure s4j). In the recovery stage after sleep deprivation, hdc-bmal1 mice had more transitions from nrem to rem, which caused more rem gain but less nrem gain . The reason could be that hdc expression was stronger in the hdc-bmal1 mice during recovery sleep (see next section). We examined hdc expression in tmn neurons at the end of the sleep deprivation period (zt5; 5 hr of sleep deprivation). Zt5 is when hdc and histamine levels are normally lower (figure 1). At the end of the deprivation period, however, hdc expression was at the higher nighttime levels in both littermate controls and hdc-bmal1 mice (figure 4d), suggesting that sleep deprivation increases hdc gene expression . Consistently, sleep deprivation raises histamine levels in cerebrospinal fluid . In control mice, 4 hr into recovery sleep, hdc protein expression had decreased (roughly halved) to typical zt6 levels (1% 0.05% versus 0.36% 0.03%, aus, one - way anova and post hoc bonferroni, p <hdc protein expression remained elevated (figure 4e). Presumably, in wild - type mice, hdc expression increases to combat the effects of sleep deprivation, and bmal1 represses hdc gene expression back to baseline levels, ensuring good recovery sleep . Without bmal1 in histaminergic neurons, the hdc gene expression level stayed flat and higher because it was already high before sleep deprivation and we investigated whether the diminished recovery sleep after sleep deprivation of hdc-bmal1 mice affected their ability at novel object recognition (figure 4f). In mice, control littermates and hdc-bmal1 mice were tested either during the night phase of their normal sleep - wake cycle or after 5 hr of sleep deprivation followed by 17 hr of recovery sleep . Mice were trained for 10 min in the open field with the same objects; control littermates and hdc-bmal1 mice spent equal time exploring the two objects . In normal sleep - wake cycle conditions, control littermates and hdc-bmal1 mice performed the same (72% 2% versus 65% 3%, one - way anova and post hoc bonferroni, p> 0.05) (figure 4f). For both genotypes, sleep deprivation impaired performance in recognizing the novel object, even after 17 hr of recovery sleep (figure 4f); however, hdc-bmal1 mice performed worse (56% 3% versus 39% 3%, one - way anova and post hoc bonferroni, p <0.01) (figure 4f). Thus, the reduced recovery of nrem sleep in hdc-bmal1 mice, compared to littermate controls, impaired cognitive function (figures 4a and 4b). Circadian transcription factors regulate arousal and sleep [3, 12, 38, 39]. Our work reveals a specified function for local clock factors in histaminergic circuitry controlling arousal . Bmal1 in histaminergic neurons promotes a daily 1.5-fold fluctuation in hdc gene expression, with lower mrna levels during the day . We propose that the local bmal1-dependent clock mechanism suppresses daytime histaminergic tone and thereby facilitates appropriately timed intervals of sleep and wake synchronized to the animal s overall circadian behavior.
A popular option for the surgical management of gastroesophageal reflux disease is minimally invasive nissen fundoplication . Similar to that of other nonamputative procedures, the successful outcome of a nissen fundoplication is dependent on a technically precise operation in a well - selected patient . Floppy fundoplication) to the nissen procedure is in common use, although precise statistics are not available . The floppy nissen fundoplication is favored for the treatment of gastroesophageal re - flux disease because this procedure maximizes reflux control while minimizing dysphagia . Subsequently, the indications, preoperative evaluation, technical aspects, and outcome for minimally invasive nissen fundoplication have been well described . One observation we have made in our own series of minimally invasive nissen procedures is the routine occurrence of temporary postoperative gastrointestinal complaints (this observation also has been made by others). We also have observed that many patients are treated by their referring physicians for these temporary postoperative gastrointestinal symptoms, perhaps too aggressively, as noted by others . We therefore wanted to identify the incidence, severity, and duration of these symptoms in our patient population to provide prospective patients with quantitative data on this issue . We found that the vast majority of postoperative gastrointestinal complaints after minimally invasive nissen fundoplication are temporary, and do not require prolonged (if any) medical treatment . Over a 10-year period, 628 patients underwent laparoscopic nissen fundoplication under the supervision of the first author (ctf). The typical indication for fundoplication in this series was gastroesophageal reflux disease; this diagnosis required objective evidence that usually was in the form of esophagitis on endoscopy or a positive ambulatory ph study, or both . The components of this procedure that we believe to be critical include circumferential esophageal mobilization resulting in an adequate length (3 cm to 5 cm) of intraabdominal esophagus; closure of the diaphragmatic crura with posterior (and occasionally anterior) cruroplasty over a large (56 f to 60 f) bougie; fundal mobilization with division of the short gastric vessels; a short (about 2 cm), floppy 360 wrap that utilizes the fundus only (not the gastric body); and fixation of the wrap to the anterior crural arch (but not to the esophagus itself). In addition, we used prosthetic reinforcement of the cruroplasty when faced with a large hiatal defect; this practice reduced hiatal hernia recurrence in a randomized trial . Postoperative follow - up was performed in the office of the supervising surgeon at 1 week, then at 1, 3, and 6 months, and then yearly . Postfundoplication dietary restrictions included avoidance of carbonated beverages and gas - producing food for 3 months, and eliminating meat for 1 month after surgery . Each follow - up visit, each patient was queried regarding early satiety, dysphagia, odynophagia, hiccups, diarrhea, nausea, bloating / flatulence, and constipation (table 1). The incidence of each symptom during different postoperative periods was compared with the fischer exact or chi square test; the level of significance was p<0.05 . If so, does the difficulty seem to be in the upper or lower chest? Is the difficulty with solids or liquids or both? Do you have bloating, or flatulence (gas from below), or both? Twelve - month follow - up data were available in 615/628 patients (98%). Recurrent reflux (ie, a failed procedure) occurred in 16 patients (2.5%); in 14 of these, the failure of the procedure was evident by the 6-month follow - up visit . Exclusion of the above 16 patients (ie, those with reflux recurrent at 3 months postoperatively) left 599 patients for analysis . During the first 3 postoperative months, 100% of these patients had at least 1 gastrointestinal complaint; early satiety (88%), bloating / flatulence (64%), and dysphagia (34%) were the most common symptoms (table 2). The vast majority of these symptoms were resolved within the first 4 weeks to 6 weeks after the procedure (data on the precise time of symptom resolution are not available). In the 3-month to 12-month postoperative interval, the incidence of gastrointestinal complaints in the 599 patients decreased markedly; specifically, 94% of these patients did not report symptoms during this period (table 2). Gastrointestinal symptoms 0 to 3 and 3 to 12 months after minimally invasive nissen fundoplication * comparison of the incidence between the 2 periods was performed with either the fisher exact or chi square test . The symptoms of bloating and flatulence (n = 21), dysphagia, and diarrhea were persistent in 33 patients beyond the third postoperative month (table 2). Of note, 5 patients with persistent flatulence or diarrhea carried a preoperative diagnosis of irritable bowel syndrome . Upper endoscopy and barium radiography were performed in the 5 patients with persistent postoperative dysphagia; the associated causes included a slipped fundoplication (n=2), and a tight cruroplasty, a tight wrap, and no identifiable cause (n=1 each). Eleven patients with bloating and flatulence and 4 patients with diarrhea were evaluated with upper endoscopy, colonoscopy, and clostridium difficile testing; none of this evaluation identified causative pathology . Up to 62% of patients who have undergone an antireflux procedure may be treated chronically with antireflux medication in the postoperative period; such postoperative antireflux treatment is given in some cases without documentation of reflux . We have documented a high incidence (100% of our series) of temporary postoperative gastrointestinal complaints after laparoscopic nissen fundoplication . It has been our experience that these postoperative gastrointestinal complaints resolve by 3 months in over 90% of the patients . Interestingly, it has been shown that the accuracy of postoperative symptoms in predicting objective reflux (as measured by 24-hr ph monitoring) is poor, and that some asymptomatic patients actually may have subclinical reflux after a successful we would argue that the more relevant observation is that laparoscopic antireflux surgery, when performed by experienced surgeons, results in a long - term clinical success rate of 90% . Postoperative symptoms like bloating, flatulence, and dysphagia can be minimized with the avoidance of carbonated beverages, gas - producing foods (eg, beans), and coarse substances (eg, red meat). This is not necessarily indicative of recurrent reflux, but possibly due to irritation of the esophageal mucosa secondary to drinking a relatively caustic beverage, such as citrus juice or alcohol . These beverages also should be avoided for several months so that the patient's esophagitis has a chance to resolve . If gastrointestinal symptoms occur during the first 3 months after fundoplication, our evidence suggests that the symptoms resolve in the vast majority of patients . The only treatment needed at this point is patient education and reassurance . If a patient has symptoms that persist beyond the 3 months postoperatively, however, an endoscopic and contrast fluoroscopic evaluation should be undertaken . Other than recurrent reflux, the most common persistent symptom requiring intervention in our series was dysphagia, which happened in 1% of our patients . The cause of postoperative dysphagia is multifactorial, including factors such as an undiagnosed motility disorder (eg, achalasia), a wrap failure (eg, slippage, overly tight wrap or cruroplasty, or corpus wrap), retroperitoneal hematoma, or peptic stricture . Treatment for dysphagia is specific to its cause, such as revisional surgery for wrap failure . Persistent bloating / flatulence may be treated with simethicone and dietary modification; further treatment can consist of speech therapy to retrain / control swallowing frequency . The cause of persistent diarrhea often is not clear; vagal injury often is implicated . The treatment of persistent diarrhea includes antidiarrheals; further evaluation of the lower gastrointestinal tract (for irritable bowel syndrome, pseudomembranous colitis, etc) might be considered . Since 1995 laparoscopic nissen fundoplication has been shown to be safe and effective therapy for gastroesophageal reflux disease . Although many patients will have gastrointestinal complaints during the first 3 months after laparoscopic nissen fundoplication, the vast majority of these symptoms will resolve and do not require chronic treatment . We suggest patient education and reassurance for the complaints that come up during this initial period, and reserve further evaluation for symptoms that persist beyond 3 months.
Affective disorders are a leading cause of disabilities worldwide, and the etiology of these many affective disorders such as depression and posttraumatic stress disorder (ptsd) is due to hormone changes, which includes hypothalamus - pituitary - adrenal (hpa) axis in the peripheral nervous system and neuromodulators in the central nervous system . The hypothalamic - pituitary - adrenal (hpa) axis plays a pivotal role in stress induced physiological changes, so that the levels of hpa axis, such as cortisol, are a good biomarker for certain mental disorders . In the central nervous system, neuromodulators, such as norepinephrine (ne), are regarded as a critical part of the central stress circuitry, which is consistent with pharmacological studies indicating that medical treatment acts by increasing the concentration of catecholamine . Hpa axis and ne both can interact and interfere with each other; for example, ne release in the central nervous system facilitates activation of the hypothalamic - pituitary - adrenal axis in response to acute stress . The major function of lc / ne system is to induce fight or flight hormonal hypothalamus - pituitary - adrenal (hpa) stress axis and the peripheral sympathoadrenal autonomic response system are possibly very good biomarkers for the physiological stresses, while the activities of the locus coeruleus (lc) or ne levels in the central nervous system are very good biomarkers for the mental stress . Stress can be defined as any threat, either real or perceived, to the homeostasis and wellbeing of an organism . Stress can be induced by two broad and qualitatively differentiated categories of stressors: physiological stress and psychological stressors . Physiologic stress is a physical threat to the wellbeing, and the psychogenic stress is the cognitive processing or interpretation of the stimulus as stressful . The first category is a real and imminent physiological threat to health and wellbeing, and the second psychogenic stressor is cognitive interpretation of the stimulus as a stressful event . The locus coeruleus (lc) neurons can be activated by both stressful events, which means that information from both the external and the internal environment can activate lc / ne system . Many electrophysiological and neurochemical studies have shown that the brain ne system is physically and robustly activated by a diverse array of acutely stressful stimuli . The locus coeruleus (lc) is regarded as a part of the central stress circuitry [6, 7], because robust activation of the lc was reported after stressful stimuli . The lc is the largest norepinephrine (ne) in the brain and projects axons to almost all brain regions . It is a pure ne nucleus in rodents, comprising approximately 1500 cells on each side of the brain stem in rats . The hpa axis is regulated by a number of neural and hormonal inputs to the hypothalamus, and noradrenergic system has been implicated as one of the important systems for the hpa stress axis, mainly through its action upon the corticotrophin - releasing factor neurons in the paraventricular nucleus (pvn) of the hypothalamus . The ne afferents to the pvn originate mainly from the medullary ne nuclei and reach the pvn via the ventral ne bundle . The crf neurons in the pvn play the most important role in handling neuroendocrine stress response . The pvn contain neurons producing vasopressin and oxytocin, also regarded as hormones to cope with stressors, as well as regulating the water and electrolytes balance and parturition, respectively . The functional significance of this pathway in mediating stress responses was demonstrated in many studies, such as lesion of na pathway . Microinjection of ne directly into the pvn of conscious rats increases crf gene expression in the pvn in parallel with acth release from the pituitary . The ne influence upon the pvn crf neurons stress induced activation of ne neurotransmission facilitates behavioral responses evoked by acute stress [9, 11], whether these responses represented inhibition of ongoing behavior, consistent with the behavioral function of lc / ne to say no, or activation of behavior that would not otherwise occur in the absence of acute stress . An inability to appropriately initiate or regulate aspects of the stress response has been proposed as a potentially critical factor in the pathophysiology of various stress - related disorders . The lc has been implicated in a variety of physiological functions, such as fear and anxiety . Multiple brain regions have been implicated in emotional processing including anxiety and fear, the amygdala, the lc, the septohippocampal system, and the raphe nuclei . Consistently, the noradrenergic system in the brain is considered to play an important role in emotion, and central responses to stress, and has also been implicated in affective disorders . A considerable amount of evidence has suggested the relationship between the central ne and fear / anxiety . Emotions are one possible means by which stress responses may be tailored to specific stress . Ne is especially important to conditioned fear because fear learning occurs during times of threat and stress . Dysregulation of lc / ne systems has been implicated in stress and its related psychiatric diseases such as depression, posttraumatic stress disorder (ptsd), and other anxiety disorders . This notion is supported by the knowledge of the pharmacological actions of many psychotherapeutic drugs, including tricyclic antidepressants and noradrenaline reuptake inhibitors . The central noradrenergic system is also involved in panic disorders, especially posttraumatic stress disorders, because an alpha-2-adrenergic agonist, clonidine, is effective in alleviating the symptoms of patients with panic disorders . It was more than 30 years ago that the functional relationship of the lc with fear or anger was first suggested in monkeys by redmond et al . : electrical stimulation of the lc resulted in particular behaviors that were observed in fearful or threatening situations in the wild . Ne is especially important to conditioned fear because fear learning occurs during times of threat and stress . Thereafter, considerable effort was made to examine whether the ablation of the lc of rodents elicits the behaviors consistent with those in monkeys . However, the rats with 6-ohda induced lc ablation were more reluctant to leave a familiar place and took longer to consume the food pellets in an unfamiliar place, suggesting an increase in fear following the lesion . This is exactly opposite to the predictions of the fear hypothesis derived from monkey studies . Similarly, increase in neophobia (i.e., fear in response to novelty) was also observed in subsequent studies . The reason for this discrepancy between monkey and rat studies is not clear . It might be possible that ne has different controls over behaviors in different animals, for example, in prey and predators (anger in prey, fear in predator). For example, when a deer meets a lion, ne is released in the brains of both animals, but the reaction of the deer is flight (fear), while the reaction of a lion is fight (anger). So fear and anger are twin emotions coming from the same neuromodulator ne, but with different reaction depending upon the inner state of the animals . (master yoda (george lucas)) fear is the path to the dark side . (master yoda (george lucas)) yoda was so wise and insightful that he said this . It is so true that you can test it in every situation when you are fearful; you will see it quickly turns into anger . Everything in the world, whether it is positive or negative, occurs in ways expected or unexpected by us . Our ancestors negotiating rich environments were faced with a set of hugely complex inference and learning problems, involving many forms of variability . Therefore, our ancestors evolved an adaptive mechanism to first have a safety check for everything in the world with available information, to see whether they are as expected or unanticipated . If something happens as anticipated, people feel calm; instead if it happens surprisingly, the first reaction of people would be to get scared and angry . This is an early evolutionary adaptation to allow better coping with dangerous and unexpected situations . Therefore, fear and anger are due to unexpected ways in which these things happen . Let us take one example from izard's paper; when rafe was hit from the back by a wheelchair, the first reaction of him was to get scared and angry and he showed angry expression and clenched fist . But after he turned back to see rebecca, a person with hemiplegia whose wheelchair had gone out of control and caused her to crash into rafe, rafe's understanding changed his anger to sadness and sympathy . So when something happens, people will first evaluate whether it is dangerous (fear / anger) or not (calm) and next evaluate whether it fits into their need (happy / sad) (figure 1). Similar emotional flow happens all the time in our everyday life: life is normally calm; everything is as expected; then something unexpected happens; people first feel unsafe and scared (fear) and then blame (anger) the reasons for the unexpectancy after fear is gone; then people feel happy after successfully coping with the unsafe stimulation and/or feel sad if the individual failed to cope successfully . Finally, the stressful events go away, and people calm down . This kind of emotional wave, big or small, long or short, constitutes our everyday stressful emotional flow . So fear - anger - happiness - sadness - calm constitutes the rainbow of emotions or emotional flow in our everyday life (figure 1). Even though fear and anger are the most frequently experienced emotions we have, there is no clear definition about them, let alone the relationship reports about them . From above, we can find that fear and anger are twin emotions that always come together at stressful events, usually fear first and anger next in tandem . For example, when an aggressive driver cuts in front of you, your first reaction is being scared (fear), and next you will feel angry after and only if fear is gone . Similarly, lazarus proposed a relational concept for stimulus and emotion: emotions are due to a relationship with the environment that the person regards as significant for his wellbeing . Lazarus borrowed the concept of appraisal from arnold and elaborated the concept as a key factor for emotions: emotional processes depend on the predictability of the stressful events . He distinguishes two basic forms of appraisal: primary and secondary appraisal, and he proposed that the primary appraisal and its induced emotions are faster activating, automatic processing, which is similar to the safety need . Indeed, lazarus distinguished three types of stressful events: harm, threat, and challenge, which are related to primary appraisal . The secondary appraisal concerns coping options, which include blame or credit, coping potential, and future expectations . It seems that the primary appraisal is related to fear, and the secondary one is related to anger (figure 1). Therefore, these two kinds of appraisals are related to safety needs: for personal safety, which are related to the unexpected ways of stimulus occurring . Evolutionarily, fear is related to threats, or aversive unconditional stimuli, such as pain and height . Due to our rational ability to think in the future and fantasize about what might happen, fear is getting broader in humans, such as being afraid of losing the needed things, in addition to getting the threat, because of the uncertainty / unexpectancy . Actually, the major reason is the uncertainty, because no matter what the hedonic value is, it has two sides of uncertainty, get it and lose it, which make us worry . Anger is a nature passion aroused by a real or fancied injury or insult and involves a desire for retaliation . Anger always comes after fear at stressful events, and anger is due to fear, or more accurately, anger is due to the unexpected things, which induced fear . Therefore, it seems that fear and anger are twin emotions that always come together, but fear and anger exclude each other in that they never occur at the same time in the same person . We give the equation below for the relationship between anger and fear, which fits in both duration and tension: (1)the total amount of stress = fear (duration, tension)+anger (duration, tension). Therefore, for a certain amount of stress, if fear is stronger, anger would be relatively less . For example, there is no time left for anger while riding a rollercoaster . On the contrary, when the fear is less, anger should dominate, such as betrayal by a lover . At most of the time, fear and anger come in tandem with fear first and anger next . For example, after long time preparation for the test, nancy was told to have failed in the test . She was scared at first, and then she was angry after she found out that it was not her fault, and her fear was gone for it was the teacher's mistake . Fear and anger are basic emotions, which are built into us to allow us to react to dangerous situations, just like in other animals . Yet the problems come because we have the rational ability to think in the future and past and fantasize about so we might be angry, sometimes unperceived, because fear, most of which is based on subjective thinking, can quickly overload causing us to react in anger . Anger is the secondary reaction to fear; if we are fearful, we will also have the tendency to be angry . Therefore, we have to keep our fears checked in order to control our anger . Anger plays a significant role in everyday life, but there was no clear scientific definition about it before . If there is any, it would be very confusing; for example, anger is an internal, mental, subjective feeling state with associated cognitions and physiological arousal patterns . Barries gave a simple definition: anger is the emotion humans experience when they do not get what they should or must get; therefore, anger is due to unexpectancy . It would be very useful to break down anger types, because anger takes many forms, such as bitterness, malice, clamor, envy, resentment, intolerance, criticism, revenge, wrath, hatred, sedition, jealousy, attack, gossip, sarcasm, and unforgiveness . Hughes divided anger into three types: the first form of anger, named hasty and sudden anger by joseph butler, is connected to the impulse for self - preservation . The second type of anger is named settled and deliberate anger and is a reaction to perceived deliberate harm of unfair treatment by others . The third type of anger is dispositional and is related more to the character traits than to instincts or cognition . Irritability, sullenness, and churlishness postures are examples of the last form of anger . One of the most common forms of breaking down anger into anger traits is habitual anger responses such as those determined by the state - trait anger expression inventory . This test addresses four different categories: trait anger, anger - out, anger - in, and anger control . Anger - in itself refers more to the affective experience and is not to be confused with hostility or aggression . Anger - out implies that a person is more likely to deal with anger through outward expression such as verbal or physical behavior . The last factor, anger control, refers to the tendency to engage in behavior that is intended to reduce overt anger expression . Anger is experienced more often than other emotions, and it is very intense and results in adrenaline rush . There are many people that have anger (fear) problems, and their problems are due to wrong beliefs . Our definition clearly states that anger is due to unexpectancy, so expectancy plays a key role in anger . So ellis proposed that anger is not caused by the things that happen to us but is the result of wrong belief in our minds: we like to put the things we would like to have, wish to have, hope to have, and prefer to having into things we believe we must have, should have, ought to have, are outght to have, and demand to have [sic]. So in one sentence, anger / fear is due to expectancy, or anticipation, which depends on your belief . Anger pushes others away for people are often fearful of being exposed to angry people . Even the angry people themselves tend to isolate themselves from the world, because the angrier they get and the more the ne release, the more fearful they will be next time, and this results in social phobia . In the emotional flow, for the phobia patients, their problems might be that they cannot successfully accomplish the emotional flow (fear - anger - happiness - sadness - calm). The best way to remove fear is anger; these patients have problems with expression of anger, so their emotions are checked at emotional flowing from fear to anger . Depression . Depression is characterized by unrelenting sadness accompanied by an inability to derive pleasure from positively hedonic situations . Therefore, depression might be related to the primary appraisal, the worrying about safety, instead of hedonic satisfaction . Indeed, excessive self - blame and feeling worthless are symptoms of major depression episodes across cultures, which is similar to lazarus's secondary appraisal . So depressed patients have problems with anger or with coping appraisal, or their problem is due to inability to cope with the unsafe stressful situation, and they showed inward anger . Recently, compelling evidence from genetic studies and pharmacological studies points to dysfunction of the central catecholaminergic network . Genetic analysis suggests that adhd is caused by multiple genes, including dopamine receptors, the adrenergic receptors, and possibly also other monoamine transporters . Reduced levels of ne and da have been demonstrated in rodent models of adhd and in adhd patients, consistent with pharmacological studies indicating that medical treatment acts by increasing the concentration of catecholamine . A growing number of studies suggest that the activation of brain ne neurotransmission by acute stress acts to facilitate an array of neuroendocrine, autonomic, behavioral components of integrated, organismic response to stress . Given such a widespread modulatory effect on a number of adaptive responses to stress, it is possible that dysregulation of the brain ne system may represent a potential substrate for the stress - related psychopathology, such as depression, ptsd, or other anxiety disorders . Therefore, the neuromodulator levels in the cerebral spinal fluid and blood flow might act as biomarkers for stress induced mental disorders.
According to who, all people aged more than 65 years are defined as old (1). The average annual growth rate of aged population over 65 is annually 2.5% . In iran, the growth rate of 4.3% in 1995 has increased to 5.2% in 2005, and it is estimated to reach more than 7.2% . According to the report of who, by 2050, the number of elderly population in iran will reach 26,393,000 people and at that time, elderly population will form 26% of population . Therefore, iran society will change to aged population and we will face common aging disorders (2). Thus, memory impairment and amnesia is the most common disease in the old age . According to the statistics reported by the iranian alzheimer association, about 450,000 patients suffer from this disease and the costs of medical treatments and care for them is very high . Hence, this trend in the coming years will cause a high financial cost and emotional burden on the health care system in iran . The amount of time spent providing care to these patients is significantly associated with the level of cognitive impairment and the patient s dysfunction . Psychological stress which caregivers incur, including the time spent away from community to care patient (an average of 11 hours a day), illness stigma, behavioral and psychological symptoms of dementia (bpsd), insomnia, memory impairment and cognitive dysfunction(4). Early diagnosis and treatment of the disease at early stages of the disease before can stop the course of destruction and consequently improve the quality of these patients lives and reduce the burden of future maintenance costs . Dementia has not been considered as a major problem in iran health system so diagnosis of dementia and create tools which are free of educational level and culture is very crucial in improving the national health care system . Most of available neuropsychological tests to recognize dementia are highly dependent to culture and education (years of schooling). Due to high rates of dementia in developing countries, illiteracy and low literacy levels in the countries such as iran, there is an unmet need to develop appropriate scales, which are independent to education and culture (5). This research was done to provide the psychometric properties of the persian version of csadl questionnaire for the patients with dementia and ad in iran . Used method in the present research was cross - sectional, descriptive and correlation one . The aim was to examine the convergent validity of the correlation between test performance evaluations with csadl as the evaluation of the correlation function, and cleveland were also calculated on the same number of sample . The study population included all patients diagnosed with dementia and were referred to memory clinic at roozbeh hospital and iaa in 2011 2012 and were selected randomly from these centers and observed . Samples were recruited through purposive sampling . The sample size according to number of questions of questionnaire that was 46 was estimated 235 patients . Among them 72 patients suffered ad, 137 people suffered other types of dementia and 26 people suffered mci . In total, main caregivers of 235 subjects completed the questionnaire, which 107 of the patients were male . An average year of schooling in men was 9 years and in women was 5 years . Instrument used in this study was cleveland scale of activities daily living (csadl) (6). Csadl has been designed to evaluate the dependence on others to perform activities of daily living (adl). The assessed people should have a normal developmental record and behavior of patients before suffering dementia should be like a normal adults . The scale has 48 questions, and two questions (number 32 and 48) are not used in scoring . Any questions should be rated with five following options: fully autonomous, rarely dependent, the time - dependent, dependent, not assessed . An informed person who respond the questionnaire should be aware of patient dependency rate and should take care of patient in a way that have direct relation with patient at least two or three days a week . Implementation time is 15 to 20 minutes . According to research main objective, in the psychometric properties of the cleveland questionnaire, instruments standard making operations, including reliability assessment practicality, validity and software troubleshooting according to exploratory factor analysis with promax method and using spss version 18 software . Questions whole validity was 0.95 . The correlation of each score with whole score showed that all questions have proper and high relation with scale so it is better not to make changes in questions and delete them . Final observation showed coefficient after two weeks with retest method . To perform main factor analysis value and prove the point that correlation matrix of the data is not zero in society, bartlett s test of sphericity is used so gained results was meaningful statistically (kmo=0.93, p<0.001). Therefore, based on the analysis of both criteria, correlation matrix of the observed sample group can be explained . To determine the observed assessment instrument (whole question) saturated by several factors, 1 eigen value 2 - eigen value rotated diagram 3 - the amount of variance explained by each factor . To extract the relevant factors, factor analysis has done multiple times with a variety of solutions, including solutions, 8.7, 6, 5, 4 factors . Eventually it became clear that the three factor solution is more adequate, and this solution was used . Scary chart has been shown in fig . 1, so we can find out that first factor share in total variance of the variables is significant and is different from the other factors share . The lowest communality for each question is 0.174 belongs to question number 34 (effectively takes into things) and 0.071 belong to question 46 (write complex expressions). The highest communality is 0.71 belongs to question 7 (at the end of urination or defecation) and 0.694 belong to question 3 (ability to enter and exit the bath) and 23 (use the drug in specified time and amount). To simplify factors extraction and factor analysis several results with different methods showed that the factors extracted by both methods are almost equal to each other . However, the results obtained from using the promax rotation that is more suitable and less number of questions were deleted . Factor matrix which created by promax rotation is shown in table 2 . In this matrix, questions 47, 35, 12 and 34 deleted in factor rotation, as they had no relation with factors and at last question numbers reached 42 . The factor scores for the three total, instrumental and basic respectively is 0.867 and 0.819 and 0.828, which is a high correlation and significant at the 0.01 level . To evaluate the sensitivity and specificity of the questionnaire for the diagnosis of dementia syndrome of the roc curve was used . Scores of 20 for the total score was chosen as cut of point . In this cut - off point test, sensitivity is 90% and specificity rate is 93% . If you select 26 for cut of point, sensitivity is 87% and specificity will be 100% . According to importance and sensitivity of test in related research, both of cut - off points can be chosen . The cut - off score bas, 3 was the score at 88% sensitivity and 98% specificity . The cut- off score ins, was 20 the test sensitivity is 91% and specificity is 100% . Compared to the average of the three groups of patients with ad and others dementia and x2 (2, n=235) = 30.95, p=0.001, (ad, n=72: dementia, n=137: mci, n=26). Experimental tests showed that the dementia group (md = 93.5) and alzheimer groups (md = 91) had higher median score than the (md = 19.5) mci . There was no significant difference in the scores of the two groups of others dementia and alzheimer s (p=0.640, r=0.045, u=4738, z=0.467). To control type i error, the alpha level was used for correction bon ferroni and significant alpha level of 0.017 was used as the criterion . To determine the relationship between two genders variable and the risk of dementia, alzheimer and mci chi - square test was used . Two chi - square test for independence (with yets correction) showed that no significant relationship exists between gender and dementia syndrome . This research was done with psychometric properties of the activities of csadl . Whereas the number of published studies concerning csadl measure are rare, but there are some studies about adl in old population for providing methods relevant to assessment of dementia . In a study the authors developed an 11 item scale to screen for dementia in illiterate population in india with good internal consistency (cronbach s =0.82). Another research was done in 1996 to develop the bristol adl scale specifically for use with people with dementia that assess 20 daily - living abilities (8). In our study, three factors extraction were followed by an additional equity and scary chart confirms that these three factors are self - care (21 items), language skills (14 items), planning (7 items). Four questions from the original questionnaire was eliminated by removing any of these questions, the cronbach s alpha was increased only 0.002 and explained percentage of variance increased to 0.004 so due to the very small changes you can keep all the questions in the questionnaire . Scale reliability was assessed using the test retest verification . (6). In their study to determine the ability of the csadl to distinguish between ad patients and healthy elderly individuals and patients with physically impaired they compared this three groups and concluded that the csadl is a reliable measure of functional deficits in individuals with ad . They developed csadl to evaluate a broad range of adl including basic and complex activities (6). In our study correlation between scale scores and performance level, evaluation scale showed that in terms of theory, scales, and measures the desired characteristics . These findings indicate persian version of csadl is a reliable scale of activities of daily living in iranian old population by using obtained roc curve and cut - off points . The sensitivity and specificity of this questionnaire is able to separate the healthy subjects from the patients with mci and dementia . It can be claimed that persian version of csadl questionnaire have appropriate indicators to serve as a useful tool for research in dementia and its early detection . It can also enable the implementation of scientific research in academic and medical centers about dementia syndrome and ad in iran . Considering the fact that ad is the most common cause of dementia, it is recommended to do more research to differentiate ad and other types of dementia such as vascular dementia, lewy body dementia, fronto temporal dementia, etc . The limitation of this study was the limited number of the caregivers who have enough time and energy to participate in our study, which shows the high burden of this devastating disease . This research is specifically done on dementia as a whole . With a view to increasing age of our population and the burden of ad as the most common cause of dementia syndrome - including direct and indirect costs of the disease- the results of the psychometric tool for early detection and treatment of early stage disease could be helpful significantly in reducing healthcare costs imposed by illness and caregivers suffer loss due to delay in the progress of the disease . Moreover, attention of health planning authorities and scientific and research centers into the disease is necessary and can reduce the burden of the disease in the coming years significantly . Social intervention to improve attitudes, knowledge and performance of individuals is possible by public education through the mass media to enables the professionals make dementia diagnosis as early as possible . Ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc) have been completely observed by the authors.
To study bacteria at the micrometer scale or single - cell resolution (kreft et al . This can be achieved by microscopy in conjunction with fluorescent markers, e.g. Dyes such as propidium iodide and syto9 allowing to determine the membrane integrity of bacterial cells (berney et al . 2007), fluorescence in situ hybridization (remus - emsermann et al . 2014), or by labeling bacteria using fluorescent proteins (fps) (ledermann et al . 2015). Often, fps were used under the control of inducible promoters as bioreporters, which allow the assessment of the availability of inducing agents, such as fructose (leveau and lindow 2001a), or under the control of constitutive promoters to identify bacteria in situ (bloemberg et al . The use of minitn7-transposon delivery plasmids is widespread in molecular and environmental microbiology, e.g. To chromosomally integrate promoter - reporter gene constructs (choi and schweizer 2006, mckenzie and craig 2006, lagendijk et al . 2010) or for the complementation of knockout mutants (crepin, harel and dozois 2012). The bacterial tn7 transposon provides an excellent non - destructive tool for several reasons: (i) it integrates with high affinity at the atttn7 site within a wide range of gram - negative bacteria; (ii) it does not disrupt genes, omitting potential pleiotropic effect; (iii) and antibiotics are not required to maintain the inserted transposons (choi and schweizer 2006, choi and kim 2009). Most tn7-transposon delivery systems have in common that they employ a suicide plasmid - based approach using vectors carrying a puc origin of replication (lambertsen, sternberg and molin 2004, choi and schweizer 2006, lagendijk et al . Plasmids featuring this origin of replication do not replicate in bacterial recipients and classically, the transposon machinery is provided on a helper plasmid . In enterobacteriaceae, however, the used puc origin of replication works efficiently, thereby excluding escherichia coli and salmonella spp . As possible recipients . Using a temperature - sensitive tn7-delivery system constructed by mckenzie and craig (2006), we provide a ready - to - use alternative for fluorescent labeling of enterobacteriaceae . We provide two different promoters, the synthetic, constitutive, laci - repressible tac promoter (ptac) (de boer, comstock and vasser 1983) and the constitutive nptii promoter (pnptii) (ledermann et al . 2015) in combination with fluorescent marker genes, encoding blue, cyan, yellow or red fps . In combination with two - parental mating employing e. coli s17 - 1, which provides the tra operon necessary for the mobilization of plasmids as a chromosomal insertion, we overcome the often tedious low efficiency of transposition and preparation of competent recipient cells . The here - proposed sets of plasmids offer a convenient alternative to generate multicolored sets of stably fluorescently labeled bacteria . The fps allow for multichannel fluorescent microscopy analysis with little to no overlap of fp emissions to study multistrain synthetic communities (fig . S2, supporting information). Employing the repressability of the ptac by laci, e.g. By coexpressing laci, it is possible to construct so - called reproductive success, or cusper, bioreporter strains (reproductive success backwards = cusper) (remus - emsermann and leveau 2010, remus - emsermann et al . Cusper bioreporters are equipped with a ptac - controlled fp and express laci, thereby the expression of the fp is repressed . When derepressing the expression, i.e. By adding lactose or isopropyl -d-1-thiogalactopyranoside to growing cells, cells can be loaded with fp (leveau and lindow 2001b). If the derepressor is subsequently removed, no de novo production of fp occurs and the preformed fp is then diluted from growing cells (remus - emsermann and leveau 2010). The fluorescence intensity of growing cells relative to their ancestors thereby becomes a proxy for the number of divisions individual cells underwent (remus - emsermann and leveau 2010). Cusper bioreporters were used to study the probability of successful colonization of bacterial cells on plant leaves, the heterogeneity of microhabitats on plant leaf surface, the probability of secondary bacterial colonization on precolonized leaves (remus - emsermann and leveau 2010, remus - emsermann et al . 2012, remus - emsermann, kowalchuk and leveau 2013), and to estimate reproductive success in a spatial context in mice spleens and plant leaves (helaine et al . 2010, tecon and leveau 2012). Using the model strain e. coli o157:h7 stx, we demonstrate the transposon delivery of the promoter fluorescence reporter constructs and resulting fluorescence at the population and single - cell resolution . Escherichia coli neb 5-alpha (new england biolabs, ipswich, ma, usa), e. coli s17 - 1 and e. coli o157:h7 stx (nctc 12900) were routinely grown on lysogeny broth (lb) or lb agar at 37c . To prevent plasmids loss, for counterselection of auxotroph e. coli s17 - 1 after mating, mm2 medium agar (4 g l l - asparagine, 2 g l k2hpo4, 0.2 g l mgso4, 3 g l nacl, 10 g l sorbitol, 15 g l agar) was used . Where necessary, media were supplemented with 100 g ml ampicillin, 50 g ml kanamycin or 20 g ml tetracycline . For microtiter plate reader experiments, cells were grown in m9 minimal medium (20 ml l 20% (w / v) casamino acids (amresco), 40 ml l 10% (w / v) carbon source, 2 ml l 1 m mgso4, 1 ml l 0.1 m cacl2, 100 ml l 10 m9 salts (85.1 g l na2hpo4 2 h2o, 30 g l kh2po4, 5 g l nacl, 10 g l nh4cl, ph 7)) containing either glucose or lactose as sole carbon source . All plasmids used in this study are given in table 1, a generic map of all produced plasmids is given in fig . 1 . All pcrs were conducted using phusion polymerase (new england biolabs, ipswich, ma, usa) following the manufacturer's recommendations and annealing temperatures were chosen based on the respective melting temperature of the primers (table 2). All plasmids that were used for pcr amplification were isolated using the nucleospin plasmid extraction kit (macherey - nagel, oensingen, switzerland) following the manufacturer's recommendations . The designated plasmid backbone of the herein constructed plasmids, pgrg36 (a gift from nancy craig (addgene plasmid #16666)), was isolated using the nucleobond xtra plus midiprep kit (macherey - nagel, oensingen, switzerland) following the manufacturer's recommendations . The map shows pgrg36 including the insertion site of the different promoter fp constructs and other relevant features . Arabad promoter = arabinose - inducible promoter, tnsa - d = transposase genes, ampr = ampicillin resistance conferring beta - lactamase, orit = origin of transfer, psc101 = origin of replication, tn7 left end = left border of tn7 transposon, mcs = multiple cloning site, tn7 right end = right border of tn7 transposon . Tm specific to the overlap to the specified targets, overlaps for isothermal assembly were designed to anneal at 50c; abr = antibiotic resistance: amp = ampicillin, kan = kanamycin, zeo = zeocin, tet = tetracycline . As source of ptac mcherry gene fragment, plasmid pmp7607 was used . The fragment was amplified and equipped with restriction sites xhoi and noti using primers xho_ptac_mche_for and noti_mche_rev . The resulting amplicon was noti / xhoi digested and ligated into equally digested pgrg36 to generate pmre100-ptac - mche . The ligation reaction was transformed into e. coli neb 5-alpha chemically competent cells following the manufacturer's recommendations and selected on lb agar containing ampicillin . To construct pmre101-ptac - ecfp and pmre102-ptac - eyfp, ecfp and eyfp were amplified from puc18t - minitn7-zeo - ecfp and puc18t - minitn7-zeo - eyfp, respectively, using primers ptac_c / yfp.for, which contains ptac, and ptac_c / yfp.rev . The resulting amplicons were amplified using primers gib_ptac+c / yfp.fwd and gib_ptac+c / yfp.rev to generate amplicons with overlaps to smai - digested pgrg36 . Each amplicon was fused into pgrg36 using isothermal assembly as described by gibson et al . Briefly, amplicons were mixed in equimolar ratios with 100 ng smai - linearized pgrg36 each in a total of 5 l before 15 l isothermal assembly reaction mix was added (for 1.2 ml isothermal assembly mix, combine 320 l isothermal assembly buffer (25% peg-8000 (amresco, cleveland, oh, usa), 500 mm tris - hcl ph 7.5 (sigma, saint louise, mo, usa), 50 mm mgcl2 (rockland, limerick, pa, usa), 50 mm dtt (amresco, cleveland, oh, usa), 1 mm each of the four dntps (amresco, cleveland, oh, usa) and 5 mm nad) with 1.2 l t5 exonuclease (new england biolabs, ipswich, ma, usa), 20 l phusion polymerase (new england biolabs, ipswich, ma, usa), 160 l taq dna ligase (new england biolabs, ipswich, ma, usa) and 700 l ddh2o). After incubation at 50c for 15 min, 10 l of the reactions were used to transform e. coli neb 5-alpha chemically competent cells . To construct pmre103-pnptii - mche, pnptii was amplified from pfru97 using primers gib_pnptii.fwd and gib_pnptii.rev, yielding an amplicon with 5 overlap to smai - digested pgrg36 and 3 overlap to the 5 end of gib_mche.fwd and gib_mche.rev amplified mcherry . The two amplicons and smai - digested pgrg36 were isothermal assembled as described above, yielding pmre103-pnptii - mche . Pmre104-pnptii - ecfp and pmre105-pnptii - eyfp were constructed by amplifying pnptii from pfru97 using primers gib_pnptii.for and gib_c / yfp_pnptii.rev and amplifying ecfp and eyfp from pmre101-ptac - ecfp and pmre102-ptac - eyfp, respectively, using primers gib_pnptii_c / yfp.for and gib_pnptii_c / yfp.rev . Plasmids pmre106-pnptii - ebfp2 and pmre107-pnptii - mtq2 were constructed by amplifying the respective fluorophore genes and pnptii using primers gib_pnptii_ngfps.for and gib_ngfps.rev from prjaph_ebfp2 or prjaph_mtq2 . All generated plasmids were purified from e. coli neb 5-alpha, verified by pcr sequencing and transformed into e. coli s17 - 1 to allow two - parental mating of the mobilizable pmre - series plasmids . Plasmid nucleotide sequences were deposited under genbank accession numbers: ku973693ku973700 . For two - parental mating, donor e. coli s17 - 1 containing minitn7-delivery plasmids were grown overnight as a lawn on lb agar containing ampicillin at 32c and recipient e. coli o157:h7 were grown overnight as a lawn on lb agar at 37c . Freshly grown lawns of donor and recipient were harvested using inoculation loops, resuspended in 1pbs (8 g l nacl, 0.24 g l kcl, 1.42 g l na2hpo4, 0.24 g l kh2po4), washed twice by centrifugation at 3500 g and resuspension in 1pbs . Donor and recipient were mixed to reach the same number of cells before they were harvested by centrifugation at 3500 g. the donor / recipient mixture was resuspended in 1pbs to reach a final od600 nm of 20 . 100 l of this suspension was spotted onto lb containing no antibiotics and left to dry in a laminar flow . Afterwards, spotted cell mixes were incubated at 32c overnight before they were harvested using an inoculation loop . Harvested cells were resuspended in 1 pbs and plated on mm2 containing ampicillin where they were incubated at 32c, preventing auxotrophic e. coli s17 - 1 to grow . Briefly, e. coli were cultivated in lb containing ampicillin at 220 rpm and 32c overnight to promote transposition (in e. coli leaky expression of the arabinose promoter - driven tnsabcd, transposase genes were sufficient for transposition into atttn7 locus, for other species and strains it might be advantageous to add 0.1% arabinose to the medium to induce the transposons promoter). Overnight cultures were plated onto lb agar containing no ampicillin and were cultivated at 42c to block replication of the heat - sensitive plasmid . Colonies growing on non - selective agar were screened for their ability to fluoresce on a fluorescence microscope . To verify that the ampicillin resistance conferring plasmid was lost, 10 individual colonies were streaked onto lb agar containing the antibiotic as well as onto lb agar plates without . Colonies that were not able to grow on ampicillin but grew on lb were screened once more for their ability to fluoresce . Bacterial strains containing chromosomally inserted fps under the control of ptac were cultivated in m9 containing lactose and casamino acids or in m9 containing glucose and casamino acids . Strains containing chromosomally inserted fps under the control of pnptii were cultivated in m9 containing glucose and casamino acids . Strains were cultivated in triplets of 300 l at 28c and 3 s of orbital shaking every 10 min in a polystyrene, 96-well, flat bottom microtiter plate (greiner bio - one, frickenhausen, germany) in an infinite m200 microtiter plate reader (tecan, mnnedorf, switzerland). The initial density of the cultures was between 0.001 and 0.005 od600 as measured by the plate reader . Fluorescence was determined by exciting the culture at 383 9 nm, 420 9 nm, 497 9 or 560 9 nm and measuring emission at 448 20 nm, 481 20 nm, 533 20 nm or 610 20 nm for ebfp2, cyan fps (ecfp and mturquoise2), eyfp or mcherry, respectively . Fluorescence and absorbance were determined every 10 min for a total of 48 h or 288 cycles, respectively . The determined fluorescence intensity was background subtracted using the respective t0 fluorescence intensity . Microscopy was performed on a zeiss axioimager.z2 microscope equipped with a zeiss axiocam mrm for image acquisition . Ebfp2 was visualized using zeiss filter set 49 (g 365 nm / ft 395 nm / bp 445/50 nm), ecfp and mturquoise2 were visualized using zeiss filter set 47 he (bp 436/25 nm / ft 455 nm / bp 480/40 nm), eyfp was visualized using zeiss filter set 46 he (bp 500/25 nm / ft 515 nm / bp 535/30 nm) and mcherry was visualized using zeiss filter set 43 he (bp 550/25 nm / ft 570 nm / bp 605/70 nm). To visualize fluorescence of individual bacterial cells, an ec plan - neofluar 100 objective (1.30 na, oil, ph3) or an ec plan - neofluar 40 objective (0.75 na, air, ph2) was used . Individual colonies were investigated using a plan - apochromat 5 objective (0.16 na). To measure fluorescence intensity of individual cells, freshly grown colonies were harvested using an inoculation loop and resuspended in 1 pbs . Serial dilutions of the suspensions were spotted onto gelatin coated 10-well microscope slides (thermo scientific, dreieich, germany) and dried for 15 min to bind cells to the surface of the slide . Dilutions of appropriated densities, e.g. Containing several well - separated individual cells per field of view at 100 magnification, were further investigated . Single - cell fluorescence intensity was determined of at least 100 cells per strain by acquiring multichannel images of respective fluorescence signals and phase contrast signals . Cells were separated from the background based on their phase contrast using the fiji thresholding command and standard settings . Cells were added to the fiji region of interest manager using the analyze particles command . The multimeasure command of the region of interest manager was then used to determine the average fluorescence of individual cells . When grown in media lacking lactose, escherichia coli o157:h7 cells harboring:: mre100,:: mre101 or:: mre102 exhibited weak fluorescence (fig . 2) due to the presence of the ptac repressor gene laci in e. coli o157:h7 . Addition of lactose to minimal medium led to the induction of fps in e. coli o157:h7 harboring:: mre - ptac constructs causing an accumulation of fp in cells compared to their repressed controls as shown by microscopy and microtiter plate reader (fig . 2). Chromosomally inserted mcherry in e. coli o157:h7::mre100 exhibited a significantly stronger signal after induction and the strongest observed signal of the here - introduced reporter set (fig . Escherichia coli o157:h7::mre101 expressing ecfp exhibited a slightly stronger, however not significant, fluorescence signal after induction, which could be shown only using microscopy . Possibly, ecfps short excitation wavelength led to a high autofluorescent signal of the medium and/or microtiter plate plastic in the plate reader which decreased the signal - to - noise ratio . Lastly, e. coli o157:h7::mre102 expressing eyfp exhibited the second strongest fluorescence, however not significantly increased, signal after induction . Analysis of chromosomally inserted ptac fluorescence protein gene labels using microscopy and microtiter plate reader . (a) phase contrast and fluorescence micrographs of e. coli o157:h7 carrying chromosomally integrated tn7-transposons containing mcherry, ecfp or eyfp encoding genes under the control of the lactose derepressible tac promoter . The left two columns show phase contrast and fluorescence images of non - induced cells, the right two columns show lactose - induced cells . For fair comparisons of fluorescence intensities and background, (exposure times for the respectively measured fluorophores are given in fluorescence micrographs, scale bar = 5 m .) (b) average single - cell fluorescence intensity per millisecond exposure after background subtraction . Non - induced cells are represented as white bars, and induced cells as black bars . Statistical differences in fluorescence intensity between treatments were assessed by performing a one - way anova . * * = p <0.01; * * * * = p <0.0001 . (c) background - subtracted fluorescence of e. coli o157:h7 carrying the tn7 insertions or wild - type cells cultivated under non - induced or induced conditions . Lines reflect floating averages of three replicates; stippled lines reflect the standard deviation of the mean . These results show that the repression of the ptac by the in e. coli endogenously present laci was not sufficient to tightly control the activity of ptac as also non - induced bacterial cells exhibited fluorescence, albeit at lower average intensities, which were, however, only significantly lower in the case of mcherry expressing e. coli o157:h7::mre100 (fig . To show that the activity of ptac can be controlled more tightly, the ptac repressor laci was expressed from the multicopy plasmid pcpp39 (leveau and lindow 2001b, remus - emsermann and leveau 2010) and transformed into e. coli o157:h7::mre100 . In e. coli o157:h7::mre100 (pcpp39), absolutely no fluorescence was detected in cells grown on minimal medium containing glucose as sole carbon, while cells grown on minimal medium containing lactose as sole carbon were fluorescent (fig . S1, supporting information). This tightly controlled gene expression system can be employed to generate cusper bioreporters (remus - emsermann and leveau 2010, remus - emsermann et al . Cells can then be loaded with a fluorophore by adding the derepressor and after its removal, the constitutive repression abolishes the de novo production of the fp . The now fp - loaded cells can be used as cusper bioreporters, i.e. While growing, they will evenly dilute the fp to daughter cells, which will contain less fp than the mother cell . All fps that were placed under the control of pnptii were functionally expressed in e. coli o157:h7 while present on plasmids (data not shown) or after chromosomal insertion (fig . The strongest discernable fluorescence, as assessed by microscopy and microtiter plate reader, was exhibited by mcherry expressing e. coli o157:h7::mre103, followed by cells e. coli o157:h7::mre107 expressing mturquoise2, e. coli o157:h7::mre105 expressing eyfp, e. coli o157:h7::mre104 expressing ecfp and finally e. coli o157:h7::mre106 expressing ebfp2 . Notably, the fluorescence of chromosomally inserted ebfp2 was very low using the available fluorescence microscope and plate reader, with signals barely above the limit of detection . Furthermore, the fluorescence of cells expressing ecfp was far inferior to cells expressing mturquoise2 . (a) phase contrast and fluorescence micrographs of e. coli o157:h7 carrying chromosomally integrated tn7 transposons containing mcherry, ecfp, eyfp, ebfp2 or mturquoise2 encoding genes under the control of the constitutive nptii promoter . The first column shows phase contrast images, the second corresponding fluorescence images (exposure times are given in the fluorescent images, scale bars = 5 m). For fair comparisons of fluorescence intensities and background, (b) fluorescence intensity of e. coli o157:h7 carrying the tn7 insertions or wild - type cells . Lines reflect the mean of three replicates; stippled lines reflect the standard deviation of the mean . (c) average single - cell fluorescence intensity per millisecond exposure after background subtraction . Statistical differences in fluorescence intensity between treatments were assessed by performing a one - way anova, and significant differences are indicated in the graph . The here - presented plasmids feature a narrow - host range origin of replication and should be functional in many relevant enterobacteriaceae including e. coli, salmonella spp . And shigella spp (mckenzie and craig 2006). The tn7-transposon delivery machinery and promoters are considered to be functional in a broad host range including all enterobacteriaceae (miller, leveau and lindow 2000, peters and craig 2001). Thereby the plasmids are of value for many different studies and allow to rapidly setup and perform experiments at single - cell resolution, for example, the investigation of multispecies biofilms and interactions within (sternberg et al . 2014) and bacterial behavior in microbe microbe or microbe host interactions (bloemberg et al . 2000, remus - emsermann, kowalchuk and leveau 2013, janissen et al . 2015, ledermann et al . 2015). As the promoter - reporter constructs are inserted chromosomally, furthermore, by avoiding the integration of antibiotic resistances it is possible to add additional genetic elements that require antibiotics pressure, such as plasmids or transposons, in the host bacteria . With non - optimized wide - field microscopy systems, it is possible to combine the following three fps to allow for overlap - free observations of subpopulations of bacteria: ecfp / mturquoise2, eyfp and mcherry (fig . 4; fig . S2, supporting information). To add a fourth population, i.e. An ebfp2-labeled strain, it is necessary to optimize the detection system by using optimized fluorescence emission and excitation filters or a filter - free confocal microscopy system . The observation of several subpopulations at the same time allows for the application of spatial statistics (daims, lcker and wagner 2006, remus - emsermann et al . 2014), which will give deep insights into the behavior of bacterial strains towards each other in a given environment, e.g. If they aggregate or segregate . Artificial mixtures of e. coli o157:h7 containing different tn7-transposon inserted promoter fluorescent - protein labels . (a) a mix of induced e. coli o157:h7::mre100, e. coli o157:h7::mre101 and e. coli o157:h7::mre102 . Left image, phase contrast image, right image, false color overlay of mcherry (b) a mix of e. coli o157:h7::mre103, e. coli o157:h7::mre105 and e. coli o157:h7::mre107 . Left image, phase contrast image; right image, false color overlay of mcherry (red), eyfp (green) and mturquoise2 (blue). With this novel set of fp labels harbored on minitn7-transposon delivery plasmids in combination with the conjugation strain e. coli s17 - 1, we provide a convenient ready - to - go tool to generate fluorescently labeled enterobacterial strains for microbe microbe and microbe mre and dd conceived the study, mre planned the experiments, mre and pg performed the experiments, mre analyzed the data and mre wrote the manuscript with critical input from dd . This work was financially supported by the agroscope research program reduction and dynamics of antibiotic - resistant and persistent microorganisms along food chains (redymo).
Diabetes is considered the fifth leading cause of death, and it is a leading cause of morbidity and mortality in the developed world, as well as in many developing countries . Diabetes prevalence (in adults) is reported to be 24% in saudi arabia,1 which is higher than that reported in the developed countries . According to the international diabetes federation, the diabetes rate in saudi arabia in 2015 was 17.6%.2 diabetic ketoacidosis (dka) is one of the life - threatening acute complications of diabetes mellitus (dm) that mainly occurs in type 1 diabetes patients, as well as in some patients with type 2 diabetes . It tends to present under stressful conditions or in association with illnesses that feature metabolic decompensation . Dka is characterized by hyperglycemia, ketoacidosis, and ketonuria.3 dka affects both children and adults and requires immediate attention . The true annual incidence rate for dka is difficult to establish, but population - based studies have reported ranges from 4.6 to 8 cases per 1,000 patients with diabetes.4,5 dka rates may be between 5% and 7% in individuals aged <18 years.6 the global incidence of dka is influenced by various factors and is reflective of the prevalence of diabetes in that population.7,8 mortality due to dka is <5% according to the american diabetes association (ada).8,9 most cases of dka arise due to missed insulin doses, either as a result of negligence or poor socioeconomic status.10 other precipitators of dka include infections, cerebrovascular accidents, alcohol / drug abuse, pancreatitis, myocardial infarction, and trauma . Simple lifestyle modifications, such as educating the patients about not missing any insulin doses especially during illness and providing the patients with an adequate insulin regimen, can greatly reduce dka occurrence . The mortality rate associated with dka depends on the experience of the treating hospital in dealing with this condition;11 thus, it is critical that patients detect dka and get medical help as soon as possible . The management of dka has improved over the years, as evident by the decrease in the death rate.12 it is recommended that ketoacidosis should be considered upon the first admission of diabetic children and high - risk patients; in this vein, blood glucose and ketones should be measured upon first admission to the emergency room . Despite the major health burden of dka, there have been a few studies conducted on its clinical and biochemical characteristics in the kingdom of saudi arabia (ksa) or the arab world . Diabetic education and the importance of correct medication should be taught from the beginning of diabetes diagnosis, especially for type 1 diabetes patients, in order to identify dka symptoms at the earliest possible time . Accordingly, we propose to perform a retrospective cohort study of the clinical and biochemical characteristics of dka in diabetic patients presenting to a tertiary center in riyadh, ksa . Our study will provide the critical information required for the ksa; it will allow the appropriate measures to be implemented to optimally address the prevention, diagnosis, and treatment of dka; and it will also provide intensive education to increase the awareness of dka, dm, and other risk factors . This study is a retrospective observational cohort study of 400 consecutive patients with dka who presented to the emergency room at the tertiary center from june 2014 to may 2015 . The sample size is calculated to be 400 patients (154 male and 246 female) at the level of significance of = 0.05, with an estimated prevalence rate of 30% and 50% for male and female diabetic patients, respectively, with a power of 94% . The large sample size of consecutive cases provides the necessary power to perform a detailed analysis in order to study dka . Patients under 12 years of age are seen by pediatric endocrinologists; as such, all type 1 and type 2 diabetes patients aged 12 years and older with dka are included . Cases of dka will be identified by the ada (2006) criteria for the definition of dka; metabolic acidosis is often the most prevalent finding, while the serum glucose concentration is generally below 800 mg / dl (44 mmol / l). However, serum glucose concentrations may exceed 900 mg / dl (50 mmol / l) in comatose patients with dka . We review all the cases and classify them according to these criteria, with the previously demonstrated validity for enrollment in this retrospective cohort study . Patients with hyperosmolar state and hyperglycemia without ketacidosis were excluded . The availability of clinical data regarding dka characteristics, glycemic data, the presence of other comorbidities, and documented hba1c levels were also prerequisites for inclusion in this study . For each patient, case report forms were completed, and the following variables were collected and recorded in an excel sheet: age, sex, type and duration of diabetes, missed insulin doses, presenting signs and symptoms, vitals, body mass index, comorbidities, complications, duration of hospital stay, altered consciousness, and mental obtundation . The following investigations were also performed, including electrolyte changes, such as plasma glucose (mg / dl), arterial ph, serum bicarbonate (meq / l), urine ketones, serum ketones, effective serum osmolality (mosm / kg), anion gap, hba1c results, vitamin d, thyroid - stimulating hormone (tsh), ft4, calcium, creatinine, sodium, potassium, and lactate . The data were analyzed using the statistical package for the social sciences for windows version 12 . This study included 400 dka admissions with a mean sd age of 21.4 10.1 years . The admissions included 154 (38.5%) male and 246 (61.5%) female patients . Of the 400 admissions, most (n = 395; 98.8%) of them were saudis with type 1 diabetes (n = 372; 93%). A previous history of dka was recorded in 241 (65%) patients, including both type 1 and type 2 diabetes patients . Intensive care unit (icu) admission was required in 77 (19.3%) patients . The factors that warranted icu admission were severe dka as per ada criteria and classification of dka . The patients other baseline characteristics are listed in table 1 . As documented in the patients case report forms, of the 400 admissions, most had vomiting (n = 319; 79.8%), nausea (n = 282; 70.5%), and abdominal pain (n = 303; 75.8%) as the presenting symptoms . Other symptoms included polyuria (n = 105; 26.3%), polydipsia (n = 113; 28.2%), impaired level of consciousness (n = 83; 20.8%), shortness of breath (n = 55; 13.8%), and fever (n = 56; 14.1%). The predominant precipitating cause of dka was noncompliance to the insulin regimen (n = 215; 54.2%). Symptoms and signs of a triggering illness were pursued with appropriate studies (eg, cultures, imaging studies). Tachycardia was the most common clinical sign noted among the patients on admission (n = 243; 61.8%). One intrauterine fetal death (suspected due to no fetal movement and confirmed through ultrasound) occurred due to dka complications in one patient . Bicarbonate treatment was administered to 18 patients (4.5%), while diabetic education was given to most patients (n = 384; 94%). Table 4 presents the average laboratory results of the patients blood tests (ph, blood glucose, hco3, anion gap, hba1c, tsh, ft4, and vitamin d). The mean sd recurrence rate of dka (number of dka admissions) was 1 8 . Recurrent dka admissions in type 1 diabetes patients were higher than in type 2 diabetes patients (n = 232 versus n = 9, respectively; p = 0.002). There were more female patients than male patients who required recurrent dka admissions (n = 167 versus n = 74, respectively; p = 0.002). A study conducted in riyadh over the course of 20 years found that 80.4% of dka episodes occurred in intermediate, secondary school, and university - level students.13 a total of 16.6% patients with no history of diabetes presented with dka as the first diabetes - related episode . The study found that precipitating factors for dka included missed insulin dose in 51.2% of patients and infections in 22.5% of patients.13 the mean duration of hospital stay was 6.56 3.4 days, and there were no dka - related deaths reported . Another study from jeddah found that both infection and poor compliance to treatment were the most common precipitating factors for dka, as these were responsible for 54.4% and 28% of cases, respectively . Dka occurrence had a male: female ratio of 1.4:1.10 a study from iran showed a higher prevalence of dka among girls than boys (53.1% and 46.9%, respectively).14 moreover, a recent study conducted on pediatric cases in riyadh showed that nonadherence to insulin accounted for 79.4% of dka admissions.15 female patients accounted for 56% of admissions . Our study found a similar pattern as those reported in these studies, in that nonadherence / discontinued insulin therapy accounted for 54.2% of dka admissions, which has been previously reported from various studies in the ksa10,13,1517 and in the neighboring countries.14,18 this is mostly due to socioeconomic reasons, where the patients try to stretch their medicine or they think they do not need it after they return to good health . Similarly, they may alter their medication throughout the course of the illness, or simply, they may not strictly adhere to their medication . There was a predominance of female cases in our study, which concurs with the findings of previous studies, as well as with universal findings . Females are mostly noncompliant with their treatment due to social, personal, and domestic factors . A lack of knowledge of the importance of their medication is also an important factor . The dka rates can be greatly reduced by providing good diabetic education on the importance of their insulin dosage;19 we offered this education to most patients (93%). With regard to presenting complaints, one study found that most patients had a history of vomiting for a duration of at least one day (74%), and 69% of the patients had abdominal pain.15 another study found vomiting to be a complaint in 64.7% of admissions.11 the 20-year study conducted in riyadh showed that vomiting occurred in 61.6% of patients, abdominal pain was present in 56.6% of admissions, polyuria was present in 57% cases, and polydipsia was present in 54.1% of admissions.13 these findings are in agreement with our results . Gastrointestinal manifestations, including abdominal pain, are common in patients with dka.20 the blood glucose level observed at presentation is close to what was stated by other studies,10,13 while blood ph and bicarbonate levels were found to be lower . These characteristics were noted in diabetics with poor overall control of diabetes, as evidenced by the high mean a1c levels, which were primarily due to the fact that most patients were adolescents who were nonadherent to their insulin regime . Moreover, about 19.3% of patients required icu admission; these findings are in agreement with the values associated with dka.21 the use of sugary drinks and chocolates by patients thinking they have hypoglycemia when they wrongly self - interpret their symptoms of nausea, vomiting, and abdominal pain complicates matters . Sick - day management should be taught to all patients; the patient should be advised to never discontinue insulin during their illness and that they should seek medical advice early in the course of their illness instead.19 the 20-year study also found a reoccurrence of dka in 54.5% of cases,9 while our study yielded a value of 65% . One study that was recently performed in damascus showed that the recurrence of dka was found in 75% of females and in 87% of type 1 diabetes cases.18 our study reported that there was a recurrence rate of 69.29% in females and 67% in type 1 diabetes patients . The zero mortality rate is highly outstanding compared with the rates found in other studies conducted in the ksa (values of 2.9%, 4.1%, and 3.5% were reported in three previous studies from the ksa13). There was only one intrauterine fetal death arising from dka complications in one patient in our study . This could be attributed to the immediate medical attention given at the center in diagnosing dka episodes . Continued diabetic education on the importance of adhering to the prescribed medical regime, addressing the social and cultural barriers that precipitate dka, and the provision of timely medical attention may greatly reduce dka episodes . Sick - day management should be taught to all patients; in fact, the patient should be advised to never discontinue insulin during the illness . Rather, he / she should seek medical advice early in the course of the illness . Continued diabetic education and counseling on the importance of adhering to medical regimes, addressing the social and cultural barriers that precipitate dka, and the provision of timely medical attention can greatly reduce dka episodes and their associated complications . It is our hope that a major reduction in morbidity and hospitalizations due to dka can be achieved following our study.
A stroke involves the rapid loss of brain function due to a disturbance in the blood vessels supplied to the brain1 . Common post - stroke motor disorders are spasticity (hypertonia) and muscle synergy that induce distinctive and complex movement patterns1, 2 . Muscle synergy is the mass contraction of multiple muscle groups and occurs in the arm due to various upper extremity movements . Spasticity (hypertonia) includes disabling and persistent symptoms that affect many stroke survivors and is a motor disorder characterized by a velocity - dependent resistance in muscle tone3,4,5,6 . Muscle tone is defined as the state of activity or tension of a muscle beyond its physical properties7 . Skeletal muscle tone reflects intrinsic viscoelastic muscle properties (passive tone, non - reflex, or emg silent) and includes neurogenic factors that are activated by stimuli; it is represented mainly as the stretch reflex and is also identified as active tone, reflex tone, or neurogenic tone8 . Increased muscle tone in the post - stroke hemiplegic limb often negatively interferes with functional motor recovery of the upper limb and with correct postural control3, 6 . In chronic stroke patients, flexor muscle tone often appears as a complex pattern with hypertonia, thus affecting upper - extremity joints2 . Therefore, appropriate management of flexor muscle tone has been an important topic in rehabilitation for stroke patients . Several management options help to decrease muscle tone and spasticity in stroke patients, such as neuromuscular electrical stimulation9, stretching devices3, vibratory simulation10, serial cast application11, and taping8, 12,13,14,15,16 . In a clinical setting, several types of tape are applied directly to the skin in a special manner in order to achieve various therapeutic effects, such as improved circulation, subcutaneous lymphatic drainage, muscle facilitation or inhibition, fascia correction, and mechanical correction4, 17 . Mcconnell taping was introduced in 1984 by jenny mcconnell18, this method normally involves preparation of the skin and application of a protective undertape followed by a non - elastic, rigid overtape that applies tension to the underlying soft tissues17 . Non - elastic taping enhances the ability to generate force14, improves joint realignment, and leads to decreased joint reaction forces17 . Non - elastic taping also immediately corrects faulty joint alignment or restricts joint range of motion (rom)19, 20 . Recent studies have reported significant effects on proprioception and neuromuscular control18, 21, and non - elastic taping appears to be the rehabilitation technique of choice in the clinic . Global synkinesis (gs), known as mirror movement, motor overflow, and contralateral irradiation, is common in hemiparetic subjects . When the homologous part of the opposite limb is active in hemiparetic subjects, involuntary pathological muscle activity and movements elicited at several or all of the joints can be observed22 . Brain images reveal that gs is identified by bilateral excitation of the motor cortex in which one hemisphere reduces adequate inhibitory influences on the opposite hemisphere23 . The level of gs intensity in the paretic arm is related to the functional outcome of patients with post - stroke hemiparesis and is especially dependent on contralateral elbow flexor muscle contractions22 . Even though gs is well recognized by clinicians, there is still no consensus as to whether attempts should be made to reduce the synkinetic movement . In the present study, we attempt to replicate the effect of elbow re - positioning tape by using non - elastic tape to modulate abnormal muscle tone in chronic hemiplegic stroke patients . We aimed to measure gs intensity using electromyography (emg) in the paretic upper limb during elbow flexion and extension movements of the contralateral arm . We used viscoelastic components (passive tone) and the modified ashworth scale (mas) for neurogenic components (active tone)7, 8, 22, 23 . The aim of the present pilot study was to determine the influence of non - elastic taping on abnormal elbow flexor tone in patients with strokes . Fourteen people with post - stroke were recruited from a pool of appropriate patients at the rehabilitation hospital in changwon city . The time ranged from seventeen months to fifty - eight months; mean ages and standard deviations were 49.52 9.2 . None of the subjects with a stroke were taking any antispastic medication or had any limitations in passive range of motion . The criterion for inclusion was a diagnosis of stroke with spasticity (hypertonia) in the elbow flexors, according to the modified ashworth scale24, 25; this scale quantitatively evaluates the degree of passive movement during muscle stretching and determines the degree of spasticity in stroke subjects . The scale ranges from 0 to 4: grade 0, grade 1, grade 1 +, grade 2, grade 3 and grade 46 . Those subjects who demonstrated deficits in language, attention, or cognition were excluded from the study because of their possible inability to follow experimental instructions22 . All subjects understood the purpose of this study and submitted written consent prior to their participation, in accordance with the ethical standards of the declaration of helsinki ., the subjects completed the target tasks using the joints in the unaffected extremity . The positions in performing the isometric muscle contractions were constant with the elbow flexed at 90, the shoulder in slight abduction (15), and the forearm in a neutral position23 . The relaxed (affected) upper extremity was suspended without support parallel to the trunk22 . For these positions, each subject was seated in a comfortable chair: the back was curved, the hips and knees were positioned at approximately 90 of flexion, and the ankles were flexed at 90 and reached on the floor . Before and immediately after application of the non - elastic tape, we measured the degree of spasticity in the elbow flexor muscles using the modified ashworth scale and measured emg activity in the affected arm . Global synkinesis was defined operationally as the emg activity of two elbow muscles on the affected arm during maximal isometric contralateral elbow contractions . The delsys triagno wireless emg system (delsys inc ., boston, ma, usa) was used to record gs activity of the bicep brachii and tricep brachii muscles; active surface electrodes were placed on the respective muscle belly23 . The elbow hypertonic position associated with post - stroke is typically present during elbow flexion, though forearm pronation appears to be more common25 . Forearm pronation is formed by the biceps, the brachioradialis, the brachialis muscles, and the pronator teres6 . Application of non - elastic tape has been reported to be a useful intervention for restricting joint range of motion (rom) or unwanted postures . We modified the placement of non - elastic tape as suggested by martin13 and elkhatib26 . To inhibit hypertonic positioning and to provide joint stability with neutral alignment, non - elastic tape applied in a serpentine fashion along the elbow when flexed slightly and to the forearm when in supination (fig . Non - elastic taping application a strip of hypoallergenic tape (endura fix tape, endura - tape pty . Ltd ., australia) was applied under the non - elastic tape to minimize the effects on the skin . The hypoallergenic tape was placed from the anterior surface of the upper third of the ulna and moved in an upward spiral past the posterior aspect of the elbow joint . The tape continued in the same manner, passing and ending at the distal lateral border of the humerus . A manual was placed below the elbow joint with the intention of maintaining the elbow joint extension and forearm supination positions while a strip of non - elastic tape (endura sports tape, endura - tape pty, ltd ., gs intensity (irradiated muscle activity, viscoelastic components, passive tone) was recorded for three seconds at affected (relaxed) arm22 during maximal voluntary isometric muscle contraction . When the mean maximal voluntary isometric contraction (mvic) level was reached, irradiated muscle activity was recorded . The sampling rate was 2,000 hz, with a bandwidth of 20500 hz, and the raw data were converted into the root mean square (rms) data . The activity of two muscles was recorded three times over a period of three seconds . Based on the research methods described by hwang22, the mean rms value was determined by averaging the rms values of the three trials for each task . The modified ashworth scale (mas) score was used to evaluate the neurogenic components (active tone) in the flexor muscles of the elbow joint . The mas uses a graded six - level scale with varying descriptors ranging from 0 (no increase in muscle tone) to 4 (affected parts rigid in flexion or extension)24 . The evaluation was performed by a proficient physician who was not blinded to the purpose . Categories 1 + to 4 of the mas were modified to 2 to 5 for statistical analysis . Differences in the gs intensity between the pre - taping and post - taping conditions were assessed using a paired t - test . Application of non - elastic tape at the elbow joint significantly changed the gs intensity by contralateral voluntary isometric flexion (t(1,13)=3.218, p=0.007), but no significant by extension was identified (t(1,13)=1.838, p=0.089) (table 1)table 1.gs intensity by contralateral maximum voluntary isometric elbow flexion / extension pre and post non elastic tapinggs intensityprepostelbow flexion1.0020.3410.4480.270elbow extension1.2790.3220.8260.304gs: global synkinesis*p<0.05 . Values are meansse . Compared to the mas, the average score of spasticity ranged from 2.29 to 2.14, which was not found to be statistically significant (z=1.414, asymp . Sig. (2-tailed)=0.157) (table 2)table 2.clinical assessment of spastic elbow flexor pre and post non - elastic taping using the masmas coresubjectabcdefghijklmnpre22133123333332post22132122332322 . Normal upper limb function depends on the ability to statically and dynamically position the limb in an optimal coordinated fashion20, but a stroke creates an imbalance between agonist and antagonist muscle pairs . A stoke can also contribute to impairments in passive and/or active elbow motion, resulting in the loss of inhibitory control . This reduction in motion creates functional losses, limits reachable workspace activities, and restricts daily activities11 . Weakness in the muscles is commonly seen after a stroke and often persists chronically, disrupting the stabilizers of the joint . The hemiplegia has a direct influence on the worsening of neurologic and articular patterns when posturing with a dominant flexor tone in the upper limbs9 . Therefore, preventing increased abnormal tone and spasticity in a timely manner is essential . To address this concern, this pattern of excessive elbow flexion can worsen with walking (e.g., increased flexor posturing with walking or running), emotional excitement, or with the use of the uninvolved extremity for activities such as getting dressed, washing one s hands, or writing11 . The management of abnormal flexor muscle tone of the elbow in stroke patients is considered an important factor for functional recovery2 . In a study described by castilho25, researchers applied neural mobilization of the upper limb contralateral to the hemiplegia and found a reduction in electromyographic activity in the biceps brachii but found an increase in electromyographic activity when the process of capturing the signals was performed with the arm in extension . They also report that the central and peripheral nervous systems are considered a single continuous system and that any limb movement has mechanical consequences in the neuro axis . The principal aim of this study was to evaluate the effect of non - elastic tape on an elbow flexor muscle in hemiplegic patients that was measured by gs intensity and mas . In our study, non - elastic tape was applied in a spiral manner across the muscle belly and along the elbow extension and forearm when in the supinated position . This was done to inhibit the flexor tone that is associated with gs . As a result, the gs intensity was significantly decreased in contralateral isometric flexion . According to a study completed by hwang22, gs activity was observed to spread through the muscles of the contralateral upper extremity in association with target movements . The level of gs intensity in the paretic arm related to the functional outcome of post - stroke patients23 and was directly linked to spastic resistance and angular velocity mirrored in their elbow flexor tone28; this was especially true when gs was triggered by contraction of the contralateral elbow flexors22 . Studies using electromyography (emg) in hemiparetic subjects show that emg levels were significantly higher during contralateral upper extremity tasks . The neurophysiological mechanisms involved demonstrated a decrease of cortical inhibition acting on the spinal cord22 . Taping techniques can be used as an adjunct during the rehabilitation process to enhance functional recovery by improving alignment, stimulating or inhibiting muscle function, and improving proprioceptive function of the joint structure12 . Proper taping is a useful adjunct to these processes and has the particular advantage of lasting well beyond patient - therapist contact, thus extending the duration of therapy20 . Following the applied direction, taping perpendicular to a muscle inhibits activity and taping parallel to a muscle promotes activity14, 27 . The inhibitory effects of this study could be explained on the basis of cutaneous effects being produced by laying the tape upon the skin . Cutaneous afferents have the ability to both facilitate and inhibit local motoneurone pool excitability14 . And they suggest that if the muscle is held by the tension of the tape, a reduction in tonic muscle spindle activity may result . This would reduce the spindle afferent input upon the motoneurone pool, which may lead to its inhibition . The mechanisms of cutaneous mechanoreceptors have been explained in several studies8, 17,18,19, 29, 30, and most studies consider taping the skin to be an effective way to stimulate cutaneous mechanoreceptors, thereby allowing more sensory signals to be carried to the central nervous system for information integration31, 32 . Recent studies have documented increasing proprioceptive function resulting from the cutaneous afferent stimulation of the skin17, 31 . Skin sensation plays an important role in detecting joint position and movement31; proprioception refers to mechanoreceptor detect information regarding joint position, movement, and the perception of these movements by the central nervous system32 . Afferent input from the joint capsule, ligaments, and muscle spindles to allow the central nervous system (cns) to build precise proprioceptive information and to induce changes in local muscle tone . Chou21 found that the application of the non - elastic tape provided proper body alignment and suggestss that this method contributes to improve stability, improved proprioception, and neuromuscular control33 . Taping is used to maintain the joint in an appropriate anatomic position to prevent or reduce positioning default27 . It also helps to decrease the stress on the joint and prevent subluxation by reducing the gravitational pull on the joint . Elkhatib26 suggests that the position of the upper extremity following a stroke affects not only the patients ability to reach, hold, and manipulate an object but also their ability to stand up and walk; therefore, taping can lead to improvements in the upper extremity after the stroke . Kneeshaw34 suggests that taping from the onset of stroke until restoration of muscle tone may prevent the onset of hemiplegic pain by enforcing proper positioning . Recent studies have shown that the pull involved in applying the second of the two tapes is critical to the electromyographic and mechanical positional changes observed during successful taping application20 . Likewise, someeh33 showed that repositioning non - elastic tape can significantly improve postural control in healthy subjects and can be applied immediately prior to increase joint awareness . In addition, the repetitive feedback formation of the cerebrum through the taping triggers a decrease in movement related cortical potential (mrcp), positively influencing functional movements35 . In patients with stroke, neuronal irradiation was manifested and mutually coupled with the flexor muscles of the affected upper limb22 . Although the specific pathophysiological mechanisms underlying the development of spasticity are not fully understood, evidence suggests that abnormalities in spinal pathways regulating the stretch reflex may contribute to the hypertonia and hyperreflexia that characterize spasticity9 . The technique of positioning a limb in a reflex - inhibiting pattern can help to prevent inefficient movement and maintain muscle tone27 . We supposed that proper joint position through non - elastic tape provides adequate joint stability and proprioceptive information feedback and can also contribute to changes in local muscle tone . The mas scale showed no significant mas grades associated with a significant decrease in passive range of motion (prom) at the wrist and elbow, particularly in those who had decreased extension due to the greater relative strength in the flexor muscles . Although the ashworth scale measures hypertonia by gauging the resistance to passive displacement of the limb, pizzi6 suggested that the mas does not provide a valid measure of spasticity at lower grades, especially between the 1 and 1 + grades of the scale; the mas score is quite low in the present study . Furthermore, we have concluded that changes in the active tone are not significant enough . We must consider the function of the shoulder, the elbow, and the wrist but our study focused only on the elbow joint . Although the non - elastic tape contributed to a decrease in abnormal passive flexor tone, we have a small sample size, and the mechanism is are not clear . Thus, there is a need for future studies dealing to a greater degree with abnormal muscle tone . These results may be used to guide the choice of appropriate interventions, such as those involving muscle tone and practices to prevent spastic elbow flexion.
We report a case of a 41-year old man presented with a solid mass located in the left temporo - occipital region . The 3d computed tomography showed a large solid mass with high vascularity, skull erosion and supra - infratentorial epidural mass effect . The patient underwent surgery where a soft mass with transverse sinus invasion was encountered; the tumour was successfully resected employing microsurgical techniques . Histological examination revealed a thyroid follicular neoplasm with positive staining for follicular carcinoma in immunohistochemical analysis . Treatment was planned for the thyroid gland, patient receiving 6 courses of chemotherapy including paclitaxel . The present case emphasizes that although they are uncommon, dural metastasis can be mistaken for meningiomas . The definitive diagnosis of a meningioma should be established only after the histopathological analysis . Thyroid follicular carcinoma should be included in the differential diagnosis in cases of extrinsic tumoral lesions . Metastasis in the skull associated with carcinoma of the thyroid accounts for only 2.55.8% of cases, but the initial presentation with distant metastasis is uncommon . Isolated forms have radiological features that strongly suggest a primary tumor, and furthermore, their macroscopic appearance during surgery may even be taken for a meningioma . In this paper, we described a patient who initially presented a tumor that invaded the scalp, dura mater, transverse sinus, supra and infratentorial space, mimicking a malignant meningioma . A 41-year - old man was referred to our institute with a 1-year history of persistent headache and a mass in the left temporo - occipital region, the mass had developed in the last year and rapidly grown within 6 months . The computed angiotomography showed a large extrinsic solid lesion with epidural mass effect and contrast enhancement causing bone destruction . Mri showed a supra - infratentorial tumour location with invasion of the scalp and left transverse dural sinus (fig . 1). Mri showing an extrinsic mass with scalp, bone and dura involvement . The mass causes bone erosion and has supra - infratentorial, and left transverse sinus extension . Intraoperatively, the tumour was found immediately in the scalp appearing as a soft, reddish, highly vascularizated mass with dural invasion . The inferior and posterior borders of the tumour encased the left transverse sinus; the tumour was successfully dissected employing microsurgical techniques . Histological examination revealed a thyroid differentiated neoplasm with positive staining for thyroglobulin and follicular carcinoma in immunohistochemical analysis (fig . A tissue removed from the tumour zone showing cubic cells resembling thyroid follicular cells (hemotoxylin and eosin, original magnification 9400). The incidence of thyroid carcinoma is about 1 per 25,000 populations, accounts for approximately 1% of all thyroid tumours . The mean age of presentation in a case series of 12 patients reported was 60 years and a female preponderance was seen . Batson demonstrated a vertebral venous plexus which consisted of a valveless vascular bed within the spinal canal and extended from the skull to the pelvis . Batson and eckenhoff showed that there were multiple anastomosis and free connections between this venous plexus and the dural sinuses . More recently arterial spread has also been suggested because of the association with secondary cutaneous locations in the territory of ipsilateral external carotid artery . Patients usually have a long clinical course before the diagnosis of skull lesion, and the principal clinical features are a palpable scalp tumour, disturbance of consciousness, hemiparesis, headache, cranial nerve dysfunction and exophthalmos have all been reported . In our case, the period until diagnosis of the definite metastatic focus was 1 year . Eighty percent of patients with thyroid follicular carcinoma are seen initially with a solitary thyroid nodule . Nevertheless, there are very few reports regarding the initial presentation of patients with distant metastasis leading to diagnosis of follicular carcinoma[1320]. Reported a rare initial manifestation of a giant mass on the right scapula of a female patient . The diagnosis in our case was difficult, because based on neuroimaging findings the most likely diagnosis was malignant meningioma . Anatomically, skull metastatic lesions are most frequently located over the occipital region, isolated papers report sellar region, posterior fossa, skull base[1416]. Skull metastatic lesions were found to be osteolytic on ct scan, and highly vascular on angiographic assessment, the same as occurred in our case . The differential diagnosis of sarcoma and metastasis should always be considered when a lytic skull lesion with irregular edges and absence of peripheral sclerosis is identified, even in the young patient . The primary focus of thyroid metastasis, which causes large bone defects, is difficult to define, metastatic tumours with unidentified primary tumour histology have been reported in patients with normal thyroid glands . The best treatment for skull metastasis remain to be determined, but the current literature supports the excision of the lesion of the skull, removal of the thyroid tissue and maintenance tsh - suppression . Radiotherapy and iodine internal radiation are other treatment options recommended for highly vascularised metastatic skull tumours . Only 17% of metastatic lesions to the brain take up iodine, so the effect of radioactive ablation on brain metastasis is very restricted . Intracranial metastasis have been treated by external beam radiation or radioactive ablation using iodine but the effect was very limited . The primary tumor is treated with radioactive iodine . In the absence of established treatment protocols to follow up in patients with intracranial metastasis from follicular carcinoma, this is a rare case of follicular thyroid carcinoma metastasized to the bone with supra - infratentorial extension.
Surgery is the major therapeutic method in soft tissue sarcomas of the extremity (e - sts). Treatment of large high - grade tumours, which resection cannot be performed with a wide safe margin, should include complementary radiation and/or chemo - therapy . Hopefully, the use of adjuvant brachytherapy will improve the prognosis of e - sts . After a long process of diagnosing a tumour in the medial compartment of the thigh, a 65-year - old woman with diagnosed synovial sarcoma underwent a surgery . Compartment resection was performed and the tumour was removed with a 10 mm safety margin of healthy tissue . Adjuvant brachytherapy was delivered with ir (microselectron, nucletron electa group, stockholm, sweden) with 10 ci of nominal activity to a dose of 55 gy in 16 days because of large tumour size (99 78 73 mm) and its proximity to the neurovascular bundle . The wound healed without any complications and the outpatient follow - up is being continued . Adjuvant brachytherapy is rarely used after surgical treatment due to its limited accessibility in hospitals with surgical and orthopaedic departments . There are numerous publications proving positive influence of brachytherapy on local control and decreased number of recurrences . The recurrence - free survival time also increased significantly, however no direct impact on the number of distant metastases was found . The most common adverse effects include: peripheral neuropathy, skin necrosis and osteonecrosis of the long bones . Treatment of large soft tissue sarcomas of the extremity (e - sts) should include combination of surgical intervention and external beam radiotherapy or brachytherapy . Adjuvant brachytherapy improves local control rate up to 78%, is well tolerated and rarely causes complications . The extremities are the most common anatomic site where soft tissue sarcomas develop i.e. Almost 50% of all cases [1, 2]. Symptoms of these malignancies are not characteristic . Usually, tumours don't cause any pain . Knowledge about this type of neoplasms presented by surgeons and orthopaedists is incomplete, which often results with delays and even errors concerning diagnostic and therapeutic interventions . One should be aware that every tumour, especially fast growing ones, located beneath the fascia requires special attention and is always an indication for a diagnostic biopsy prior final surgical intervention . Diagnostic procedures should include mri scans of the extremity with the tumour and ct scans of the lungs in order to exclude pulmonary metastases [37]. Surgical resection of the tumour with a safety margin of healthy tissue is the fundamental method of treatment of soft tissue sarcomas . At present, an amputation is avoided and the number of these procedures performed in soft tissue sarcomas has decreased by 20% . However, an amputation is recommended in sarcomas infiltrating the neurovascular bundle and tumours causing large changes extending outside compartments where limb salvage surgery cannot preserve extremity function . The application of brachytherapy and chemotherapy seems to be associated with improved local control and the lower rate of recurrences [35]. At present, brachytherapy is not a standard method used for complementary treatment of soft tissue sarcoma of the extremity (e - sts) despite reports about its effectiveness . The biggest problem seems to be its limited accessibility and technical limitations due to the fact that brachytherapy facilities are rarely located near surgical or oncologic orthopaedic departments with the possibility of shared surgical path . Currently three types of brachytherapy are used: neoadjuvant, adjuvant and a separate treatment method in tumours which cannot be removed surgically . The application of brachytherapy as a complementary therapy in soft tissue sarcomas has a history of 30-years . For many years, due to a small number of patients, published reports were fragmentary and unequivocal as far as recommendations were concerned . At present, publications include comparisons of different brachytherapy techniques i.e. Hdr (high - dose - rate), ldr (low - dose - rate), and a recent one ultra - low - does - rate meaning the application of radioactive seeds . Effectiveness of brachytherapy is a result of its conformality as well as imaging methods used in order to spare critical organs . The major advantage of brachytherapy is its clear influence on the increased recurrence - free five - year survival rate and better local control . These adverse effects concern peripheral nerves, less frequently skin, and very rarely radiation - induced muscle damage and bone loss . Few randomized trials comparing the effectiveness of brachytherapy and external beam radiation therapy didn't unambiguously indicate which method was better [9, 10]. A 65-year - old woman was diagnosed outside the oncology centre because of a fast growing tumour localised in the medial compartment of the left thigh . At the beginning the patient was treated in an outpatient surgical clinic where a puncture was performed as the surgeon suspected a synovial cyst . The tumour was growing between and infiltrating three muscles: adductor magnus, sartorius and gracilis . The large size of the malignancy (99 78 73 mm), its fast growth, increasing pain and numbness in the foot forced the need of faster diagnostics and treatment (fig . Mri images of patient's left thigh revealing a large soft tissue tumour located in the medial compartment . Sagittal plane through the largest dimension of the tumour (a) and a cross section (b) histopathological examination of tissue taken during the excision biopsy confirmed the suspected malignant change . The patient had a second mri scan and the decision about limb salvage surgery was made . Ct scans of the lungs and abdominal cavity didn't reveal any metastases (fig . 2). A picture of a soft tissue tumour of the left thigh before the surgery . A scar after the prior biopsy is visible on october 22, 2012, compartment resection with intraoperative histopathological assessment of safety margins was performed . 3). The pictures of a tumour bed after resection of the neoplasm and muscle compartment (a) and the tumour with a tunnel after the biopsy (b) the surgery was performed in the presence of brachytherapy specialists (radiation oncologists). After resection was finished applicators (nucletron elekta group) the distance between adjacent applicators was 1 cm whereas the proximal and distal margins vs. the tumour location in the longitudinal axis of the limb was 2 cm . 4). The pictures presents the tumour bed in the thigh (a) and secured applicators for brachytherapy (b) brachytherapy was performed using hdr technique with an ir radioactive source . Treatment planning (reconstruction) was based on ct images (ge bright speed 8) (figs . 5 and 6). The total dose was 55 gy (fraction dose of 2.5 gy) delivered twice a day with a minimum 6-hour inter - fraction interval . 3d reconstruction of target volume (green), bone structures (white), nerve structures (blue), muscles (red) with inserted applicators dose distribution for the target volume and organs at risk with corresponding dose volume histograms (dvh) no complications were reported during the surgery as well as postoperative period and brachytherapy . The patient was discharged from the hospital on the 28 day after the limb salvage surgery and is in outpatient follow - up care . We reviewed literature and found that doses delivered in similar localizations and application of hdr technique ranged between 32 and 55 gy regardless of treatment sequence i.e. Perioperative or postoperative . In our case fraction dose was limited while the total dose remained unchanged mainly due to the close proximity of femoral nerve . It seems that r0 resection is the optimal solution in small tumours graded as g1 and g2 . Large malignancies and those graded as g3 require resection with an adequate safety margin of soft tissue, which usually is impossible . Therefore, a complementary therapy should be delivered . Chemotherapy in soft tissue sarcoma is usually delivered within the frames of clinical trials rather than standard treatment protocols . Brachytherapy in sts is rarely administered as a therapy complementary to surgical interventions due to its technical requirements and limited accessibility . The advantage of brachytherapy is the fact that applicators are inserted under visual control therefore the process is very precise and decreases the number of complications . However, recent publications report that new techniques with j seeds were used to deliver the dose ranging from 40 to 70 gy . The complications rate is estimated to be around 10% and peripheral nerve damage is the major adverse effect comprising 5% [8, 10]. According to published reports, the 5-year recurrence free survival rate was 78% [1215]. Brachytherapy in sts significantly decreases the number of local recurrences, however it has no direct effect on the number of distant metastases . At present it is impossible to decide which type of radiation therapy is more effective, but shorter time of treatment seems to be relevant . Postoperative brachytherapy usually takes from 5 to 10 days whereas external beam radiation therapy starts from 4 to 6 weeks after the surgery . There are no clear reports indicating that teletherapy is necessary in extremity soft tissue sarcomas . Surgical treatment is the basic therapeutic method in soft tissue sarcomas of the extremity (e - sts), however delays in the diagnostic process often force the use of complementary oncologic therapy in order to maintain local control . Brachytherapy seems to be a good method of adjuvant therapy in e - sts, however its accessibility is limited . Further multicentre randomized studies should decide which method of adjuvant treatment (external beam radiation therapy vs. brachytherapy) is better and what is the optimal sequence of therapies improving local control.
With the increasing dependence on smartphones, computers, and other electronic products, myopia has become the most common vision problem . In the latest nation - wide student physical health monitoring report, the myopia percentage of 22% (nearly 400 million people) in china is 1.5 times of the world average level . In the high myopia group, china is experiencing the highest incidence of myopia in the world, and the incidence also rises year by year . Poor vision can directly affect students performance in school or at work and thus, will cause a negative influence on their future . Therefore, the prevention and control of adolescent myopia it is particularly important . Genetic and environmental factors, including high heritability, sex, distance from home to workplace, time devoted for the rest and physical exercise, and habits related to eye utilization contribute to the onset and progression of myopia . Through the recent surveys of the domestic and foreign population, results have indicated that the myopia prevalence of female was higher than that of male . Based on those epidemiological data, it can be concluded that myopia is more prevalent in females . Relevant research result which indicates that myopia prevalence of males is different from that of females in the early development stages of growth, and the difference becomes small in young adults, gives rise to the issue that gender is a risk factor for the high prevalence of juvenile myopia . After puberty, under the effect of growth hormone and estrogen, the growth and development of young females reach a peak . At this point, the organ dysfunction is prone to cause the change of the tissue structure, including those in eyes . Female myopia occurs earlier than male due to the fact that female puberty starts earlier than that of male and the degree also gradually increases with body growth and development . Wickham et al . Confirmed that estrogen receptor (er) mrna was expressed in the cornea, meibomian glands, retinal / choroidal and retinal pigment epithelial cells of rats, rabbits, and human beings . Estrogen had a certain influence on cornea thickness, which was positively correlated to myopic diopter . Therefore, this study is conducted aiming to evaluate the effect of estrogen on ocular parameters in adolescent females . The results may not only increase our understanding of the effects of reproductive hormones on myopia, but can also help elucidate that vision changes along with estrogen change in the menstrual cycle . The study was approved by the institutional review board of wenzhou medical university, and the research followed the tenets of the declaration of helsinki . The nature of the study was explained to the participants and written informed consent was obtained . A total of 120 girls (1516 years old) with myopia was included in the study . Eligibility criteria were: (1) without family hereditary myopia, hyperopia, or dyschromatopsia and visual acuity of 5.0 or higher (bare or corrected); (2) normal external eye and fundus examination; (3) non had use hormones or any drugs with a potential vision effect within the last 6 months; (4) good health and normal body mass 10% were maintained during the investigation; (5) regular menstrual cycle (average length of cycle = 28.52 days, standard deviation = 1.65) without severe premenstrual or menstrual syndrome; (6) intraocular pressure <21 mmhg; (7) normal anterior segment examination by slit lamp and no retinopathy or glaucoma . Subjects with a history of eye diseases, glaucoma excimer laser eye operation, diabetes, hypertension, and other systemic diseases were not included in the study . One hour later, each participant was administered with automatic optometry (nidek ark-710a, japan). Unaided or aided visual acuities were taken within the far and short distances before commencing refraction . Retinoscopy was then performed on the same eye, whose result was used as a starting point for subjective refraction, which was also performed on the same eye firstly . One diopter was added, and the power was reduced to give the best visual acuity . Monocular subjective refraction was performed based on subjective refinement of the autorefractor readings until the best - corrected visual acuity was achieved . Main outcomes of the eye examination included spherical lens, cylindrical power, axis, interpupillary distance (ipd), and vision . All participants provided the information of their menstrual cycle (the average cycle length, the length of menstrual period, and the prediction of the next menstruation date). We gave each subject an oral thermometer to measure basal body temperature every day before getting up in a month before the test . The rise of temperature to a high level (about 0.3c) indicated ovulation . After being exposed to the items (learning phase) fast blood samples were drawn from an antecubital vein for clinical routine laboratory diagnosed in the second affiliated hospital of wenzhou medical university on the 2 or 3 day, 14 and 28 day after menstruation, respectively according to participants menstrual cycles . In the same way, cylindrical lens, spherical lens, ipd, and visual acuity were detected by the outpatient department of ophthalmology based on automatic optometry method and comprehensive optometry detection . Blood specimens were drawn and immediately centrifuged at 2000 r / min for 10 min at 4c to obtain serum or ethylenediaminetetraacetic acid plasma, which were stored at 75c for further analysis . Serum was absorbed by capillary burette and then divided into two parts, which were marked as a and b, respectively . Serum estradiol (e2) levels were detected using the access automatic microparticle chemiluminescence immunoassay analyzer (beckman coulter, usa). If the detection data of specimen a were significantly abnormal, specimen b would be re - detected for revising the data . The visual parameter data did not follow a normal distribution and were paired, so the wilcoxon matched - pairs ranks test of sas proc univariate was used to determine the magnitude of the differences of related ocular parameters between two different time points in menstrual cycle . Friedman m test was used to compare visual acuity differences at different time points . For univariate significance levels, the study was approved by the institutional review board of wenzhou medical university, and the research followed the tenets of the declaration of helsinki . The nature of the study was explained to the participants and written informed consent was obtained . A total of 120 girls (1516 years old) with myopia was included in the study . Eligibility criteria were: (1) without family hereditary myopia, hyperopia, or dyschromatopsia and visual acuity of 5.0 or higher (bare or corrected); (2) normal external eye and fundus examination; (3) non had use hormones or any drugs with a potential vision effect within the last 6 months; (4) good health and normal body mass 10% were maintained during the investigation; (5) regular menstrual cycle (average length of cycle = 28.52 days, standard deviation = 1.65) without severe premenstrual or menstrual syndrome; (6) intraocular pressure <21 mmhg; (7) normal anterior segment examination by slit lamp and no retinopathy or glaucoma . Subjects with a history of eye diseases, glaucoma excimer laser eye operation, diabetes, hypertension, and other systemic diseases were not included in the study . One hour later, each participant was administered with automatic optometry (nidek ark-710a, japan). Unaided or aided visual acuities were taken within the far and short distances before commencing refraction . Retinoscopy was then performed on the same eye, whose result was used as a starting point for subjective refraction, which was also performed on the same eye firstly . One diopter was added, and the power was reduced to give the best visual acuity . Monocular subjective refraction was performed based on subjective refinement of the autorefractor readings until the best - corrected visual acuity was achieved . Main outcomes of the eye examination included spherical lens, cylindrical power, axis, interpupillary distance (ipd), and vision . All participants provided the information of their menstrual cycle (the average cycle length, the length of menstrual period, and the prediction of the next menstruation date). We gave each subject an oral thermometer to measure basal body temperature every day before getting up in a month before the test . The rise of temperature to a high level (about 0.3c) indicated ovulation . After being exposed to the items (learning phase), after an overnight, fast blood samples were drawn from an antecubital vein for clinical routine laboratory diagnosed in the second affiliated hospital of wenzhou medical university on the 2 or 3 day, 14 and 28 day after menstruation, respectively according to participants menstrual cycles . In the same way, cylindrical lens, spherical lens, ipd, and visual acuity were detected by the outpatient department of ophthalmology based on automatic optometry method and comprehensive optometry detection . Blood specimens were drawn and immediately centrifuged at 2000 r / min for 10 min at 4c to obtain serum or ethylenediaminetetraacetic acid plasma, which were stored at 75c for further analysis . Serum was absorbed by capillary burette and then divided into two parts, which were marked as a and b, respectively . Serum estradiol (e2) levels were detected using the access automatic microparticle chemiluminescence immunoassay analyzer (beckman coulter, usa). If the detection data of specimen a were significantly abnormal, specimen b would be re - detected for revising the data . The visual parameter data did not follow a normal distribution and were paired, so the wilcoxon matched - pairs ranks test of sas proc univariate was used to determine the magnitude of the differences of related ocular parameters between two different time points in menstrual cycle . Friedman m test was used to compare visual acuity differences at different time points . For univariate significance levels, a total of 120 participants with myopia had a mean age of 15.6 0.4 years and body mass index of 20.7 1.2 kg / m; the differences were nonsignificant (p> 0.05). There was the statistically significant difference of the spherical lens among all different times in menstrual cycle (p <0.0001), as shown in table 1 . Sphere correlation between two different time points in menstrual cycle of the same subject in young females with myopia, the cylindrical lens, axis, and ipd were changed significantly during the menstrual cycle (p <0.05), as shown in table 2 . Related ocular parameters of the same subject at different time points in the menstrual cycle (mean sd) sd: standard deviation . Friedman m test results of the vision changes during the menstrual cycle suggested a statistically significant difference of vision in three time points of the menstrual cycle (= 6.35, p = 0.042) [table 3]. Interestingly, the vision of the 2 or 3 day showed statistically significant difference of vision on the 14 day and the 28 day (p = 0.021). Vision change of the same subject at different time in menstrual cycle (median, q) table 4 presents the serum e2 levels during the menstrual cycle . The mean e2 levels were significantly different at different time points in the menstrual cycle (p <0.05). E2 levels in all subjects reached the maximum value on the 14 day while the minimum value appeared on the 2 or 3 day . We found a significant positive correlation between serum e2 levels and vision in the total group of participants, and the vision gradually became better with the increase of e2 level after menstruation [tables 3 and 4]. E2 level of the same subject at different time in menstrual cycle the mean e2 level is significantly different among all different time points in menstrual cycle (all p<0.05). Estrogen is a type of the female hormone mainly generated in the ovaries and placenta . After entering puberty stage, female teenagers ovary begins to secrete estrogen, promoting the uterus development and menses formation . Several epidemiologic observations suggest a potential participation of estrogen in the homeostasis of the eye, but the mechanisms involved remain unclear . Some scholars believed that the modulatory properties of estrogen may be mediated by the expression of the er in the ocular tissues through gene regulation or nongene regulation . Estrogen also has an effect on the expression of more than 600 genes in corneal epithelial cells . Estrogen treatments can change the transcriptional activities of many genes, thus affecting cell growth, cell communication, signal transmission and modification on corresponding eye tissues or organs . Juvenile myopia has been one of the hotspots in recent 150 years . At the current stage, the exact pathogenesis is not very clear while different pathogenesis opinions are available . Estrogen has been shown to upregulate matrix metalloproteinase-2 (mmp-2) and/or mmp-9 in human ocular cells, and mmp-2 upregulation was suggested as part of the scleral remodeling process in myopia development . In recent years, a large number of researchers have found that the myopia prevalence of female was higher than that of male and results indicated that myopia incidence between males and females showed the significant difference (p <0.005). Many factors are responsible for the higher myopia rate of girls than that of boys, including earlier growth conditions, more limited outdoor activity time than boys . However, certain internal factors, like hormone endocrine, are yet to be investigated . The present cross - sectional study investigated the relation of ocular parameters and serum e2 level as well as vision changes along with e2 change in menstrual cycle of a total group of healthy female adolescent without severe menstrual diseases and with regular menstrual cycle . As a first important finding, the spherical lens varies with e2 level in menstrual cycle of adolescent females . The significant difference of spherical lens was observed between the 2 or 3 day and the 14 day after menstruation when e2 level reached the maximum value on the 14 day and the minimum value on the 2 or 3 day . Our study showed that during the menstrual cycle, not only the spherical lens but also cylindrical lens, axis, and ipd were changed significantly . Nevertheless, our results were inconsistent with what zhao had previously reported . Moreover, the data obtained and investigated in this study showed a positive relationship between vision and e2, which corroborated the findings of previous research that showed diopter decreased with the increase of estrogen level during the menstrual cycle and was negatively correlated with estrogen level . Detected levels of various serum sex hormones (luteinizing hormone (lh), follicle - stimulating hormone (fsh), e2, and testosterone) of young students (1214 years old) in a large sample of different myopia degrees as well as nonmyopia controls . However, xie et al . Found a relationship between myopia and estrogen by detecting serum lh and fsh in adolescent females . In contrast, we further studied the vision changes of adolescent females along with the e2 level in menstrual cycle by directly detecting serum e2 levels . In that prior study, related ocular parameters, such as a spherical lens, cylindrical lens, axial, and ipd were further detected in our study . Based on the above data, it can be further confirmed that the serum level of e2 is closely related to the occurrence and development of juvenile myopia . The change of estrogen levels during the menstrual cycle can affect a person's vision for three reasons . First, metabolic disorders of estrogen in menses cause the hypothalamus and plant nerve function disorder . Therefore, the visual function of females may experience some temporary changes . Decreased vision, like refractive error second, the changes of sex hormone levels can cause retinal function change for some females . The cornea varies during the menstruation process, being thinnest at the end of the menstrual cycle while thickest in the period of ovulation . The corneal thickness in the low - degree myopia patients is much thicker than in the high - degree myopia patients . By the reasons above, conclusions achieved in this study are supported and proved objectively, with the discovery that vision in late menstrual cycle is significantly ameliorated compared to the premenstrual cycle . In our study, monocular vision is different from binocular vision in the results of computer automatic optometry and comprehensive optometry . In addition, anisometropia (especially mild anisometropia) interference with binocular vision is the second reason . A substantial number of individuals exhibit binocular gain for low contrast targets indicates that the monocular assessment would result in an underestimate of the individual's function as they go about daily life . For our study, participants were performed both binocularly and monocularly, making us better understand the vision function of the individual participant . The rather inhomogeneous and small group of female adolescents is an obvious limitation of our study . The difficult recruitment may at least in part be due to the fact that these participants, having to cope with an invasive procedure, may not easily agree to a three times repeated blood test and a time - consuming study . Second, in this study, the confounding factors produced by the rapid growth and development in the young stage were deliberately avoided . However, due to the special physiological features and condition limitations of the subjects involved in the study, we only studied one menstrual cycle . Finally, the estrogen level varies with each menstrual cycle, and may also be interfered by other hormones or presents periodic variation . All of these preliminary results should be confirmed in future studies, with greater sample size and longitudinal designs . In conclusion, we found that estrogen level fluctuations in the menstrual cycle simultaneously occurred to the related eye parameters and vision sensitivity of healthy female teenagers . It can be used to explain the changes in visual acuity at different time points during the menstrual cycle . Through this study, we also obtained that myopia development of adolescent females is variable peculiarly with periodic fluctuation . According to our current study and other related research results, the sensitivity of ers in human eyes varies with the estrogen levels during the menstrual cycle, thus inducing the change of vision . The wide distribution of ers in the ocular tissue helps us better comprehended the pathogenesis of several eye diseases and has opened up a new direction for the clinical prevention and treatment of myopia to us . Understanding the mechanism of the effect of estrogen on myopia may provide evidence of targeted drug therapy for individual treatment in the future . In addition, with hormone replacement therapy has become a research hotspot, the effects of hrp on eyes cannot be ignored . At present, the corresponding research literature indicating the relation between estrogen and myopia at home and abroad is rare . Therefore, we sincerely hope that some exploratory effort in theory and practice can be conducted by us at the following stage . We also believe that with the progress of basic studies, the development of new targeted drugs and new therapeutic approaches, a new platform for the clinical application of estrogen in adolescent myopia will be built and constructed.
Over the past two decades, the emergence of metal organic frameworks (mofs) and covalent organic frameworks (cofs) has reinvigorated the field of crystalline microporous materials, previously dominated by zeolites . However, with widespread applications in areas including, but not restricted to, heterogeneous catalysis, the adsorption of pollutants, and resistant coatings, zeolites are still regarded as the benchmark for functional porous materials . Although the porosity of zeolites and related materials has been exploited for centuries, real insights into the behavior of guest molecules in confined environments, and the role of the host in which they reside, have only recently become possible through advanced crystallographic techniques . Composed primarily of the naturally abundant elements o, si, and al, zeolites have remarkable thermal stability upon evacuation, regular pore sizes, and high acidity . However, tuning zeolite functionality is generally limited to controlling the ratio of si: al in the framework, while extensive structure modulation of these otherwise simple inorganic materials relies on the introduction of organic molecules, either as structure - directing agents (sdas) or as functional organic platforms incorporated within the zeolite scaffold itself . Mof materials, including hybrid zeolite analogues, are born of remarkably sophisticated design principles, and display a richly diverse range of properties . Synthetic control in mof chemistry is largely attributable to the modular nature of mof preparation, with secondary building units (sbus) utilized as geometrically well - defined components to access many of the great variety of framework topologies currently realized . Despite rapid progress made in this field and the widespread application of mofs, there remains scope to explore and extend the thermal and chemical stability range of the compounds due to the nature of the organic constituents . Therefore, the development of porous transition metal oxide - based molecular materials could be interesting due to the promise of flexibility and tunable redox, catalytic, and thermal properties . As such, pom - frameworks promise to combine the thermal stability and general applicability of zeolites, combined with the synthetic control and tunable properties of mofs . Polyoxometalates (poms) are a diverse class of early transition metal oxide clusters, typically comprised of multiple w, mo, or v centers connected through shared oxygen atoms, and commonly incorporating additional heteroelements such as si, p, or ge . Although by their definition poms are discrete (0-d) molecules, the introduction of transition metals (tms) and/or organic moieties to pom solutions may lead to connectivity between clusters, and their extension into coordinatively linked 1-d chains, 2-d sheets, or 3-d networks . By application of the topological node and linker designation given to the structural components of a mof, in multidimensional inorganic pom assemblies the pom units may be considered as inorganic nodes, which extend into infinite lattice arrangements through the coordination of tm linkers . However, in contrast to mofs, it is primarily the versatility of the node, and not the linker, which generates the diversity of structures found in tm - linked pom framework materials . Like mofs, these purely inorganic networks have been shown to exhibit tunable properties through rational synthetic strategies . Furthermore, the recent trend for the incorporation of pom guests inside porous frameworks recognizes the acidic, electronic, and catalytic properties that these clusters may embellish on the overall framework structure . It is therefore clear that the direct incorporation of poms into porous framework scaffolds is a valid strategy for the modular synthesis of robust functional materials, without the need for an organic component . Two main strategies are typically employed in the construction of modular frameworks with high porosity: (1) the selection of long, narrow linkers (which may lead to the interpenetration of multiple frameworks), and (2) the pre - fabrication of a pore within one of the components . The latter approach is most commonly adopted to incorporate poms within porous framework scaffolds, since these clusters often assume cyclic configurations . In particular, the crown - type heteropolyanion, [p8w48o184] (hereafter referred to as {p8w48}) is notable for several properties, including its high - negative charge and remarkable electrochemistry . Its intrinsic nanometer - sized cavity means {p8w48} is an ideal candidate to be utilized as a network synthon, to prepare open framework materials with microporosity (figure 1). Such structures have already been prepared by introducing first - row tms to aqueous solutions of {p8w48}; however, the desired control in preparing {p8w48}-based porous frameworks is yet to be realized . (a) nanoporous [p8w48o184] structure, with simplified ring representation showing both (b) endocyclic (red) and (c) exocyclic (green) coordination of transition metals . Dark green spheres, w; red spheres, o; light blue rings, [p8w48o184]. Detailed herein are the syntheses of three coordinatively linked framework structures, based on the minimal building block library of {p8w48} nodes and tm linkers . In addition, the tm coordination sites of {p8w48} are fully mapped, allowing a comprehensive analysis of ring connectivity within the known {p8w48}-based frameworks . The term pomzites is introduced to apply to this unified class of materials, alluding both to their pom - based constituents and to their zeolitic nature . Despite the various similarities between pomzites and zeolites in terms of their composition and properties, the two compound families are accessed via contrasting routes of assembly . As described here, pomzites are prepared in a modular fashion, whereas the synthesis of zeolites typically follows a one - pot methodology . The modular nature of pomzite preparation affords a valuable synthetic handle to exert greater control over the eventual framework topologies, which could enable the precise tuning of these porous inorganic materials toward tailored applications . K28li5h7[p8w48o184]92h2o and li17(nh4)21h2[p8w48o184]85h2o were prepared according to the published syntheses with minor modifications for optimization . Single - crystal xrd data sets were collected at 150 k on an oxford diffraction gemini ultra s instrument equipped with a graphite monochromator and atlas ccd detector, or a bruker apex ii quasar instrument with ccd detector (mo k = 0.71073). Inductively coupled plasma optical emission spectroscopy (icp - oes) was carried out on a tja - iris - advantage spectrometer, with echelle optics and a cid detector used to observe in the 170900 nm wavelength range . A minimum of 10 mg of each compound was submitted to the institut fr festkrperforschung in jlich for analysis . Where relevant, carbon, hydrogen, and nitrogen content were determined by the microanalysis services within the school of chemistry, university of glasgow, using an ea 1110 chns, ce-440 elemental analyzer . For fourier transform infrared (ft - ir) spectroscopy, the materials were prepared as kbr pellets, and ft - ir spectra were collected in transmission mode using a jasco ft / ir 4100 spectrometer . Wavenumbers () are given in cm; intensities are denoted as wk = weak, sh = sharp, m = medium, br = broad, s = strong . Thermogravimetric analysis (tga) was performed on a ta instruments q500 thermogravimetric analyzer . To 20 ml of 2 mol l lich3co2 buffer solution (ph 4.0) in a 50 ml round - bottomed flask was added mn(clo4)2xh2o (102 mg, 0.40 mmol), followed 5 min later by k28li5h7[p8w48o184]92h2o (100 mg, 6.75 mol). After a further 5 min of rapid stirring, the resulting pale yellow solution was then heated to 80 c overnight on a hot plate with a reflux condenser attached (approximately 20 h) upon which it gained a more vibrant golden color . After cooling slowly back to room temperature while still on the hot plate and connected to the condenser, the solution was transferred to a 50 ml conical flask and covered, before being placed in the refrigerator (4 c) for crystallization . Large, rectangular yellow plate crystals formed overnight and were collected after 5 days . Yield: 19.8 mg, 1.36 mol, 20.1% based on [p8w48o184]. Icp - oes analysis for the hydrated material, h182k8li17mn6.5o275.5p8w48.5, mw = 14543.82 g mol . Calculated values (found values in parentheses): k 2.14 (2.18), li 0.80 (0.79), mn 2.45 (2.54), p 1.70 (1.70) w 61.3 (63.1). Characteristic ftir bands: 1620 (s), 1133 (s, sh), 1086 (s, sh), 1022 (w), 933 (s), 798 (br), 698 (br). Tga water loss from 20 to 250 c, calculated (found): 11.3 (11.3)% . To a 20 ml buffer solution of 2 mol l lich3co2 at ph 4.0 in a 50 ml round - bottomed flask was first added mn(clo4)2xh2o (102 mg, 0.40 mmol), followed 5 min later by li17(nh4)21h2[p8w48o184]85h2o (93 mg, 6.75 mol). After a further 5 min of rapid stirring, the white suspension was then heated to 90 c on a hot plate with a reflux condenser attached, giving a lightly colored, clear, golden solution . After stirring at 90 c overnight (approximately 20 h), the reaction mixture was allowed to cool slowly back to room temperature while still on the hot plate and connected to the condenser, before being filtered into four narrow test tubes . The test tubes were plugged with cotton wool and placed in a container, which was roughly 10% filled with methanol, to facilitate crystal growth . Well - formed, pale yellow, hexagonal block crystals appeared in solution after 5 days . Yield: 22.0 mg, 1.49 mol, 22.1% based on [p8w48o184]. Icp - oes and chn analysis for the hydrated material, h250li18mn8n6o297p8w48, mw = 14725.04 g mol . Calculated values (found values in parentheses): li 0.86 (0.92), mn 2.98 (2.93), n 0.57 (0 . ), p 1.68 (1.71), w 59.9 (62.9). Characteristic ftir bands: 1620 (s), 1402 (s), 1134 (s, sh), 1084 (s, sh), 1022 (w), 932 (s), 797 (m), 673 (br). Tga water loss from 20 to 250 c, calculated (found): 13.8 (13.8)% . To 20 ml of 2 mol l lich3co2 buffer solution at ph 4.0 in a 50 ml round - bottomed flask was added nicl26h2o (88 mg, 0.37 mmol), followed 5 min later by li12(nh4)21h7[p8w48o184]85h2o (102 mg, 6.75 mol). After a further 5 min of rapid stirring, the resulting apple - green solution was heated to 90 c for 25 h on a hot plate with a reflux condenser attached . After cooling slowly back to room temperature while still on the hot plate and connected to the condenser, the solution was syringe - filtered and divided between four narrow test tubes, which were plugged with cotton wool . Each test tube was place in the same large container, which was roughly 10% filled with acetone . Pale green plate crystals were observed in the test tubes after 5 days and collected 1 week later . Yield: 11.5 mg, 0.80 mol, 11.9% based on [p8w48o184]. Icp - oes and chn analysis for the hydrated material, h200li22n5ni6o274.25p8w48.25, mw = 14283.57 g mol . Calculated values (found values in parentheses): li 1.08 (1.12), n 0.49 (0.49), ni 2.43 (2.45), p 1.73 (1.82), w 62.1 (59.4). Characteristic ftir bands: 1635 (br), 1399 (sh), 1132 (s, sh), 1083 (s, sh), 1019 (w), 934 (s, br), 704 (m), 663 (br). Tga water loss from 20 to 250 c, calculated (found): 11.4 (11.4)% . By utilizing the minimal building block library of {p8w48} nodes and tm linkers, three new coordinatively linked inorganic frameworks have been synthesized . Each compound has been prepared in a modular fashion, with the intrinsically porous pom building block formed prior to the assembly of the frameworks . The same general synthetic system was employed in each case, based on a lithium acetate buffer solution at ph 4.0, heated to just below boiling and stirred overnight . These compounds further expand the growing family of tm - linked {p8w48} structures . K8li17[mn6.5{w0.5o0.5}p8w48o184]91h2o (compound 1, figure 2) was crystallized from lithium acetate buffer solution following the reaction of mn(clo4)2 with k28li5h7[p8w48o184]92h2o . Each {p8w48} ring in the structure is coordinated to 8.5 mn centers (4 mn centers are shared between adjacent rings, hence there are only 6.5 mn atoms in the formula). Two mn atoms are positioned in the internal cavity of the ring, occupying hinge sites on opposite sides of the cluster, in octahedral coordination environments of two pom terminal oxygen ligands (w = ot), and four water molecules . Two mn atoms are coordinated on the outside of the remaining two hinge regions, also through two w = ot sites . These mn atoms, and another two mn atoms situated on the outside of the ring (coordinated to only one w = ot), are shared between adjacent {p8w48} rings . Mn atoms occupy a further two equivalent sites on the external surface of the ring in a disordered fashion across roughly one in every four clusters in the crystal . As has been observed previously, the four - fold symmetry of the parent cluster is distorted, and an additional 49th tungsten center is accommodated within the hinge region of the {p8w48} rings, occupationally disordered across half of the clusters throughout the crystal . Ball - and - stick representation of the {p8w48} sbu in compound 1 from (a) top - down and (b) side - on . Dark green spheres, w; red spheres, o; orange spheres, mn; dark blue spheres, p. the four shared mn atoms which are coordinated to the outer surface of {p8w48} connect the rings into a one - dimensional chain . Each ring is oriented in the same manner, but coordinated rings are displaced both horizontally and vertically, resulting in a diagonal, stepped, polymeric structure . In the crystal packing, potassium and lithium cations, as well as solvent water molecules, form a supramolecular bridge between chains in a disorder fashion . Similarly, these alkali metal cations and water molecules are also situated inside the {p8w48} ring cavities, and could potentially be exchanged by solid - state cation sorption experiments, as observed for similar structures . In accordance with compound 1, li18mn8(nh4)6[p8w48o184]113h2o (compound 2, figure 3) was formed in a lithium acetate buffered reaction mixture of mn(clo4)2 and {p8w48}. However, in contrast to the preparation of compound 1, the synthesis of compound 2 utilized the li17(nh4)21h2[p8w48o184]85h2o starting material instead of k28li5h7[p8w48o184]92h2o . The influence of specific alkali metal cations on the assembly of {p8w48}-based frameworks has previously been discussed; however, until the recent preparation of a potassium - deficient salt of {p8w48}, this effect could not be directly investigated . Ball - and - stick representation of the {p8w48} sbu in compound 2 from (a) top - down and (b) side - on . Connectivity of pomzite-13(mn) in three dimensions from (c) top - down and (d) side - on . Dark green spheres, w; red spheres, o; orange spheres, mn; dark blue spheres, p. compound 2 has eight mn centers situated on the outside of the {p8w48} hinge regions, each coordinated to two pom w = ot sites (these are all shared with adjacent rings, and so constitute only four mn atoms in the formula of the compound). Incidentally, this position is also occupied in compound 1, but only at two hinge sites instead of eight . The remaining coordinated mn centers in compound 2 reside in the center of the {p8w48} cavity, disordered across the inside of each hinge site . Additional mn content, identified by icp - oes but not observed crystallographically, is assumed not to be coordinated to {p8w48}, but to reside in the pores of the framework structure as charge - balancing, and potentially exchangeable, [mn(h2o)6] countercation . Each ring in compound 2 retains the approximate d4h symmetry of the parent {p8w48} structure, with no additional tungsten content in the hinge positions . {(w o)2mn(o w)2} bridges connect each ring to eight surrounding clusters . All rings are oriented in a uniform manner throughout the framework, creating a three - dimensional framework with layers stacked directly on top of each other in an abab fashion . This arrangement of {p8w48} into parallel columns creates cylindrical channels of roughly 1 nm diameter, in which reside a mixture of the crystallographically unaccounted - for [mn(h2o)6] cations, nh4 cations, li cations, and solvent water molecules . By recognizing the role played by nh4 cations in the synthesis of compound 2, the potassium - free starting material, li17(nh4)21h2[p8w48o184]85h2o, was investigated for its effect on the synthesis of further {p8w48}-based compounds . Similarly, the recent publication of the first ni - linked {p8w48} framework indicated that ni could be a suitable tm to link {p8w48} clusters and add further new compounds based on this system . Consequently, li22(nh4)5ni6[{w0.25o0.25}p8w48o184]90h2o (compound 3, figure 4), an ni - linked, potassium - free {p8w48}-based compound was formed under typical reaction conditions . In this compound, a total of six ni atoms have been identified by icp - oes; however, only 4.2 ni centers have been located crystallographically . As in compound 1, the inner hinge sites are filled in two positions, on opposite sides of the ring . The remaining ni atoms are located on the outer surface of the ring, with significant positional disorder preventing crystallographic resolution of their complete coordination environments . Again similarly to compound 1, the four - fold symmetry of the parent cluster is distorted, and an additional 49th tungsten center is accommodated within the hinge region of the rings . In compound 3, this supplementary position is occupationally disordered over a quarter of the clusters throughout the crystal . Ball - and - stick representation of the {p8w48} sbu in compound 3 from (a) top - down and (b) side - on . Dark green spheres, w; red spheres, o; light green spheres, mn; dark blue spheres, p. compound 3 has a planar one - dimensional chain - type structure, and as with the two previously described compounds, each {p8w48} cluster is oriented uniformly throughout the crystal . However, in contrast to the chains of compound 1, each of the connected rings resides in the same plane . Vacancies in the structure of compound 3 are occupied by a combination of nh4 and li cations, solvent water molecules, and the remaining [ni(h2o)6] cations which are not coordinated directly to the ring via dative bonds . During the course of this work, a number of additional frameworks based on {p8w48} and tms have been identified . However, many of the compounds currently suffer from a lack of reproducibility and are yet to be fully characterized . Among the strategies which have shown promise so far include the introduction of secondary cations to the reaction mixture, the results of which approach will be reported in due course . However, the rapid expansion of this class of materials has highlighted the need for a greater understanding of the nature of tm coordination to {p8w48}, and its subsequent extension into multidimensional frameworks . Further, the establishment of a classification system to cover all one-, two-, and three - dimensional {p8w48}-based structures is needed to facilitate both the identification and discussion of new compounds . Although the nanometer - sized cavity of {p8w48} makes it an ideal building block for the modular construction of porous frameworks, its tm - mediated extension into multidimensional architectures has so far been plagued by unpredictability . This is primarily because there are several sites around the {p8w48} ring to which tms may coordinate, causing rings which are connected through one or more common tm atoms to adopt a variety of different positions and orientations in relation to each other . Crucially, the specific nanoscale arrangement of neighboring rings determines the long - range organization of {p8w48} on the mesoscale, into coordinatively linked, one-, two-, or three - dimensional structures . Such topological diversity arises despite the same minimal library of building blocks being employed in each case . In order to control the extension of the effectively 0-d {p8w48} sbu into higher - dimensional structures, it is therefore crucial to gain a clearer insight into tm coordination to {p8w48} by fully mapping the binding sites of the heteropolyanion, and using the wealth of information available from the tm - linked {p8w48} structures which are currently known (a complete list of the compounds considered in this study is given in the supporting information). In general, poms act as nucleophilic o - donor ligands for the coordination of first - row tms . More specifically, the {p8w48} ring features a total of 64 reactive, coordinatively unsaturated terminal oxygen (w = ot) sites to which 3d tms may bind . Despite this apparent complexity, however, in its idealized d4h configuration there are only four inequivalent groups of w = ot, each made up of 16 equiv sites around the ring, and labeled either a, b, c, or d according to their environment (figure 5). (a) {p8w48} ring - shaped sbu node, with close - up of the smallest repeating [p0.5w3o11.5] subunit, highlighting the three inequivalent w atoms and four inequivalent w = ot (labeled a, b, c, and d). (b) octahedral {tmo6} linker (tm = mn, co, ni) which can be used to connect adjacent {p8w48} sbus . Although tm binding is a prerequisite for the extension of {p8w48} into a coordination polymer, it does not always result in ring connectivity . Pendant metal coordination, in cases where tm cations are bound to only one {p8w48} sbu, has been observed at each of the four binding sites around the ring (figure 6). In accordance with the labeling system for these sites, the binding modes of the four possible monodentate pendant positions can in turn be labeled a, b, c, or d according to the identity of the coordinated w = ot . Four types of monodentate pendant coordination modes, labeled a, b, c, and d according to the identity of the coordinated terminal oxygen . In addition to the simple monodentate nonbridging modes of tm coordination to {p8w48}, the two components may also be combined in bidentate fashion (figure 7). There are 10 potential bidentate modes of coordination based on the binary combination of recognized w = ot sites; however, only aa and dd combinations have been observed for the first - row tms . In addition to these sites at the hinge region of {p8w48}, a third mode is possible when {p8w48} is reacted with f - block metals . Labeled dd , these binding sites are on the inner face of {p8w48}, and are seemingly favored by bulkier cations, due to the typically larger spacing between the o - termini in comparison with the dd coordination mode . Moreover, the three observed bidentate pendant modes of tm binding to {p8w48} suggest that bidentate nonbridging coordination is forbidden for two different w = ot sites of the same {p8w48} ring . Three observed modes of bidentate pendant coordination, labeled aa, dd, and dd according to the identity of the coordinated terminal oxygens . Note that dd * coordination has only been observed for f - block transition metals . Transition metals which are bound to {p8w48} in pendant modes undoubtedly have a significant influence on the general properties of the compound . Pendant tms typically have at least four coordination sites occupied by aqua ligands, which may be exchangeable in nonaqueous solvents . In mof chemistry, open metal sites are commonly attributed with enhancing catalytic activity, improving uptake, and increasing the specificity of interactions . We have also demonstrated that pendant sites in {p8w48}-based structures may be activated to form linkers, and in turn entirely new frameworks, which are only accessible through an intermediary, preorganized structure with nonbridging tms . Although pendant sites must not be ignored in pomzite chemistry, the extension of {p8w48} into multidimensional structures requires the coordination of tm centers which are common to two or more {p8w48} sbus . As with the small number of observed bidentate pendant coordination modes, there are many potential combinations of pendant - type coordination modes which are not, or are only rarely, observed as linkers . Of the common linker modes, three may be considered as homotopic i.e., bridging between the same sites of two {p8w48} sbus (figure 8). These are labeled aaaa, bb, and cc, where the prime indicates the coordination of a site from a second ring . Further, there are four common heterotopic linker modes which bridge neighboring rings via two different binding sites (figure 9). Following the same convention as for the labeling of homotopic linkers, the heterotopic linkers are designated as aab, aac, bc, and ddc. As the seven common linker modes are crucial in determining the positional and orientational relationships between neighboring rings, they play a crucial structure - directing role in the extension of {p8w48} into coordinatively linked frameworks, and are responsible for the structural diversity seen for the pomzite family of materials . Three main homotopic linker coordination modes observed in pomzites: a - type linkage, green; b - type linkage, blue; c - type linkage, yellow . Four main heterotopic linker coordination modes observed in pomzites: a - type linkage, green; b - type linkage, blue; c - type linkage, yellow; d - type linkage, red . Four main structural types of pomzite have been observed so far, namely chain, column, herringbone, and cube (figure 10). The simplest structural type is the chain arrangement, seen for both compounds 1 and 3, in which {p8w48} rings are linked edge - to - edge in only one dimension . Each ring in the chain structure is orientated in a uniform fashion, and 2-coordinated, i.e., linked to two other rings . Similarly, each ring in the column structure has the same orientation, but the rings are connected in a face - to - face manner . The coordination number is also 2 if the rings are connected in only one dimension; however, column structures may also be linked into two or three dimensions by edge - to - edge connectivity . For two - dimensional structures, which may be considered as rows of columns, the coordination number is typically 6, while for three - dimensional structures rings are 8-connected and reside in a columnar matrix . In both the herringbone and cube structures, there are two different ways in which the rings are orientated . In the two - dimensional herringbone structure, rings are related by an approximate 45 rotation, with edge - to - face connectivity . Herringbone structures are typically 4-coordinated, but may also be linked edge - to - edge to become three - dimensional with higher coordination numbers . Finally, the cube architecture is a three - dimensional construct of 8-coordinated rings in a stacked - box it seems highly likely that further structure types will be identified in future . In particular, a sheet structure based on the chain arrangement with additional edge - to - edge connectivity in the second dimension is easy to visualize . Representative models for the four structural types of known pomzites: chain, column, cube, and herringbone . One of the major benefits of disentangling the complex connectivity of {p8w48} rings in pomzite structures by defining a limited number of linker modes, is that it allows simplified representations of the structures to be presented . This gives the reader a much greater insight into the three - dimensional arrangements of {p8w48}-based compounds than with conventional ball - and - stick models, and enables facile comparisons between pomzites to be made . In total, 14 unique pomzite architectures have been observed so far, and are numbered chronologically according to their date of publication (table 1). If multiple compounds were presented in a single publication, the order of numbering within the article has been retained in the current system . Additionally, if more than one compound has been identified with any given structure, it is the publication date of the first compound to be presented which determines its numbering here . For example, the first multidimensional {p8w48} compound to be presented was the co - linked structure, k15li5[co10(h2o)34(p8w48o184)]54h2o, labeled compound 1 in a communication published in 2009 which presented two new compounds . Pomzite-1(co) to indicate that this was the first pomzite discovered, and that it incorporated co linkers . Compound 1, presented herein, is a mn - linked analogue of pomzite-1(co), appropriately termed isostructural pomzites have the same tm substitution patterns and connectivity, although they may differ in the actual occupancy of each tm coordination site . Indeed, we have observed small variations in heterometal content between crystals from different batches of a number of pom framework materials, although the batches are still considered to be of the same compound . In the future, reports of new pomzites should indicate the range of tm incorporation with which a material can be prepared . As the chemical formulas of pomzites are typically complex, and often quite similar, this universal system of nomenclature should simplify discussions concerning all pomzites . For each pomzite the left - hand column details the pendant modes present and the right - hand column the linker modes (as described in the main text). The figures in parentheses are the tm occupancies of such sites per {p8w48} ring . For detailed composition of each pomzite architecture, it is apparent that a distinction has been made between several structures with similar substitution patterns and linker modes . In particular, pomzite-1, pomzite-4, and pomzite-9 all have chain structures with aab connectivity . In pomzite-1, the presence of an external tm coordinated in the b position sets it apart from the two other compounds for two reasons: (1) pendant functionality is important to the general properties of the framework and can be activated by single crystal to single crystal transformations, and (2) the steric effect of a tm positioned externally causes spatial displacement of adjacent chains in the solid state . As the aab linkers present in both compounds are heterotopic, they also have direction; i.e., they connect two inequivalent termini . In pomzite-4 and pomzite-9, the same positions are occupied in both compounds, but the direction of the aab linkers is reversed . Notably, the authors of the latter publication did not refer to the prior - published compound when presenting pomzite-9(mn), despite its close similarity to pomzite-4(co). Both the binding site analysis carried out here, and the structural model representations which this allowed, make compound comparisons much simpler . The differences between the three compounds discussed here can be much better understood using this system . Three new compounds based on a minimal building block library of the cyclic polyoxometalate {p8w48} and transition metal cations have been prepared . Each material consists of a purely inorganic metal oxide scaffold, with intrinsic porosity resulting from the use of a crown - type building block . Despite the apparent limitations of employing only two main structural components, this study expands the number of frameworks based on this system to 14 . Furthermore, a comprehensive {p8w48} binding site analysis of the literature has been performed to gain a greater understanding of how the various structure - types are accessed . As a result, a previously disparate group of compounds has been united and proposed to be named pomzite materials have tremendous potential to bridge the gap in porous framework materials between mof tunability and zeolite functionality . Previously, the structural complexity of these materials, and a lack of understanding regarding the connectivity within them, has held back their development . Due to their light nature, mofs hold a clear technological advantage over pom framework materials for such applications as gas storage . The most obvious advantage is in catalysis, where the benefit of poms is already realized by the dispersion of these clusters throughout mof architectures . Pomzites are self - supported porous materials with pom functionality inherent in their structure . In the future, the use of organic sdas, a well - known strategy in zeolite synthesis, will be employed to access novel pomzite archetypes . The potential for further expanding this library is great, with possibilities to introduce other tms and employ other sdas representing just a couple of examples of how this might be achieved . It is highly likely that many more structures will arise from this fruitful chemical system through careful control of reaction conditions . Work is currently underway in our research group to fully utilize the pore space within these materials to exploit their unique pore environments.
Radial nerve injury is the most common damage to the peripheral nervous system associated with shaft fractures of long bones . Radial nerve palsy (rnp) occurs in 2% to 17% of the cases in this group . Rnp may be either partial or complete, and complete motor loss occurs in approximately 50% of cases . Depending on the time of occurrence, radial nerve injuries can be divided into primary and secondary . In primary nerve palsy, loss of function occurs at the time of injury and is associated with closed fractures . In secondary nerve palsy, it occurs after conservative treatments, such as manipulation, impingement by or between fracture fragments, entrapment by a fracture callus, and scar tissue formation, as well as after surgery (iatrogenic nerve palsy). Iatrogenic radial nerve palsy may be a result of plate fixation or intramedullary nailing treatment . It is estimated that damage to the radial nerve occurs in 4% to 32% of patients who undergo surgical treatments to stabilize a fracture . The high risk of radial nerve damage is associated with the very complicated anatomy in the area of the nerve . Together with the accompanying vessels, it then proceeds from the medial side to the side of the posterior surface of the humerus in the groove of the radial nerve, and on the border of the middle and distal 1/3 of the humeral shaft turns to the side - arm front surface . However, studies have shown a very large variability in the course of the nerve . The distance between the acromion and the point of entry to the groove of the radial nerve is estimated to be from 10 cm to 19 cm, whereas the distance between the exit point of the groove of the radial nerve and the lateral epicondyle is from 6 cm to 16 cm . . Other studies have indicated a higher course for the radial nerve, from the proximal humerus (53% of the humeral length) at 12.02.3 cm (range, 7.416.6 cm) and from the olecranon fossa (36% of the humeral length) at 16.00.4 cm (range, 9.020.5 cm). The large anatomical variability in the course of the nerve, which increases the risk of damage to the radial nerve, affects both the surgical access and type of stabilization . This is why many different surgical approaches, including anterior, anterolateral, lateral, posterior, and modified approaches, have been used to expose the humerus and to complete fixation . The aim of the present study was to analyze the causes that lead to secondary damage of the radial nerve and to discuss the results of reconstructive treatments . The study group consisted of 33 patients treated for radial nerve palsy after humerus fractures during 20072013 . The ages of the patients ranged from 19 to 67 years (mean age 41). The study group included 12 (36%) women and 21 (64%) men . In 19 (57%) cases, surgery was performed on the right arm and in 14 (43%) cases on the left arm . Initially, the patients were treated outside our clinic and were sent to our clinic because it is a center for peripheral nerve surgery treatment . Of the patients with a humeral fracture with an associated rnp, 11 patients had an injury as a result of a traffic accident, 10 from a sports injury, and 12 from a fall onto the same level . The fractures were localized in the middle humerus, at the mid - third / distal - third junction, and at the proximal - third / mid - third junction . An analysis of x - ray images revealed spiral (12 a 1, b 1), oblique (12 a 2, b 2), transverse (12 a 3), and comminuted (12 c 3) fracture patterns according to ao classification . Inclusion criteria for our study were a closed humerus fracture, confirmed proper function of the radial nerve after the fracture, and a complete palsy of radial nerve function after conservative treatment or surgery . Patients were diagnosed based on clinical examinations, ultrasonography (us), electromyography (emg), or nerve conduction velocity (ncv). During each operation, the location and type of nerve damage were analyzed . We determined the location of the radial nerve based on where it leaves the spiral groove distally, which depends on the lateral and medial epicondyle . The following techniques were used as treatment: releasing the nerve (neurolysis), direct neurorrhaphy, and reconstruction using a nerve graft (sural nerve). The strength of the muscles (triceps, wrist and digit extensors, and supinator) was evaluated using the medical research council (mrc) scales, where grade 0 corresponds to no movement and grade 5 to normal muscle contraction against full resistance, and the quick dash score . The mean follow - up time was 4.6 years (range, 27 years) after surgery . The results were analyzed according to the type of damage to the radial nerve, time from injury to second surgery, type of reconstructive injury, localization of the radial nerve injury, type of fracture, and type of stabilization during the first operation . Based on surgical reports from other centers, the radial nerve was exposed and protected in 10 cases . The failure of the function of the radial nerve was observed for up to 6 h after the treatment . One superficial wound infection occurred, but it did not require any treatment . During the revision operation, a lacerated nerve or entrapment in a callus was found (figure 1a). In other cases, radial nerve neurotmesis with a gap of less than 1 cm (figure 2a) or more than 1 cm (figure 3a) was observed . Analysis of causes of damage to the radial nerve, type of injury, type of treatment, surgical approaches, visualized nerve, and stabilization methods during the first operation are presented in table 1 . During surgery, neurolysis (figure 1b), direct neurorrhaphy (figure 2b, 2c), or reconstruction with a nerve graft was performed (figure 3b, 3c). A full return of function was observed in 18 patients and 11 patients achieved partial return of function . A response from the radial nerve a clinical and radiological union occurred at a mean of 8 weeks (range, 712 weeks). In the 4 patients who did not achieve satisfactory improvement in functional recovery of the radial nerve, one of these patients was initially treated conservatively because the injury was in the middle third of the humerus . The 3 other patients had a mid - shaft spiral fracture with radial nerve neurotmesis treated by use of an intramedullary nail . When we analyzed the time between nerve injury and reconstruction surgery, the results observed for operations performed with less than 6 weeks between the injury and second surgery were significantly better than those observed for operations performed after 12 weeks (mrc: median 5 vs. 1, p<0.001; dash: median . 2.25 vs. 75.0, p=0.006) (table 2). The best results were reported in groups treated less than 6 weeks after the radial nerve injury . The outcome of the treatment depended on the type of damage to the radial nerve . The patients with entrapment of the radial nerve had significantly better results than those with radial nerve neurotmesis (mrc: median 5 vs. 2, p<0.001; dash: median 0 vs. 67.05, p<0.001), but not when we compared the results of the groups with a lesion of less than 1 cm with the results of groups with lesions greater than 1 cm (table 2). The results of the surgical treatment were significantly different in patients with neurolysis compared to reconstruction with a sural nerve graft (mrc: median 5 vs. 2, p<0.001; dash: median 0 vs. 34.1, p<0.001). However, the difference was not statistically significant (table 2). Regarding the type of fracture, there were no statistically significant differences among the spiral, oblique, transverse, and comminuted groups . However, the best results were observed in the groups with oblique and spiral fractures (table 2). The difference between the orif and crif fixation methods were statistically significant (mrc: median 4 vs. 2, p<0.025; dash: median 10.25 vs. 59.1, p<0.022) (table 2). The location at which the radial nerve leaves the spiral groove distally depends on the lateral epicondyle, which was 11.53.5 cm, and on the lateral and medial epicondyle diameters, which were 2.5 1 times greater . The mean time to initial radial nerve recovery after the revision operation was 8.3 weeks (range, 6 weeks to 6.6 months), and the mean time to recover full function was 6.1 months (range, 3.412 months). The problem of radial nerve palsy after the treatment of a humeral fracture is not uncommon . Nerve injury could exist at the level of the fracture, under and on a plate, as well as when a lateral or posterior approach is used . Newly formed callus, reduction techniques (e.g., use of clamps, forceps, or hooks), compression or nerve rupture by plate, or compression by fracture were the causes of this injuries . On the other hand, crif can occur at the level of the fracture or interlocking screw and may the cause of the newly formed callus, reaming of the medullary canal, compression by fracture, or insertion interlocking screw from lateral or anterior side . Several studies have compared the incidence rates of radial nerve palsy between plate fixation and intramedullary nailing [1418]. Because of the consistent results in the literature, a fixed - effects model was performed, which showed that the difference in radial nerve damage between these 2 groups was not significant . However, the above work did not refer to the severity of damage . In our study the outcomes in contrast to those studies, we found statistically significant differences . On the other hand, we examined already damaged nerves without studying the population that had been treated or how the damage occurred (although treatment was performed by qualified people from various centers, no data on the number of complications in these centers were available). In our opinion, better results for orif are associated with less damage (i.e., more frequent entrapment and minor nerve deficit) and a faster decision on the revision of the nerve, compared to crif . Some authors stressed that for cases using orif, surgeons should explore the nerve to avoid damage, while others emphasized that exposure and protection of the nerve does not guarantee avoidance of nerve injury and may cause fibrosis around the nerve in a small number of cases . In our opinion, visualizing the nerve without separating it from the surrounding tissue significantly reduces the risk of damage . In a retrospective analysis of operational protocols from the first surgery, this technique was not routinely used . In our opinion, this is useful especially because of the wide range of variability in anatomic relationships . As regards the type of damage to the radial nerve and surgery treatment, the best results were obtained when treated by entrapment neurolysis, in contrast to the damage of the gap> 1 cm treated using the sural nerve . One explanation might be the type of nerve injury and nerve regeneration process . At the beginning of the regeneration nerve process directly after injury, chromatolysis and swelling take place in the cell body and nucleus, after which, wallerian degeneration (axonal and myelin disintegration) proceeds both in a distal (antegrade) and proximal (retrograde) direction . Antegrade wallerian degeneration then continues with schwann cells and macrophage infiltration to remove cell debris, leaving only the basement membrane for about 36 weeks . In subsequent stages, schwann cells start to proliferate and guide the axonal sprouts between the basement membranes of the 2 nerve ends . The difference is that for entrapment, only the axon is affected and wallerian degeneration appears in the distal part of the nerve (axonotmesis). In case of interruption (neurotmesis), wallerian degeneration takes place in both antegrade and retrograde directions . To analyze the variable course of the distal radial nerve, we compared our results with the most prominent landmark bone points (the lateral and medial epicondyle). The results were comparable with anatomic studies of these points in which the distal extent of the radial nerve in the spiral groove was 12.61.1 cm proximal to the lateral epicondyle of the humerus, and along the posterior aspect of the humerus from 20.71.2 cm proximal to the medial epicondyle . In addition, we studied the position of the nerve in relation to the distance between the 2 epicondyles . Our results also emphasize considerable variability in nerve position, which may, in our opinion, cause damage . In the case of intramedullary nails, the risk of damage to the radial nerve occurs during repositioning, drilling, and distal locking . Some authors have emphasized the advantages of locking from the side, while others have encouraged locking from the front . In cases of proximal interlocking in the frontal and sagittal planes, the theoretically high risk notwithstanding, only a few cases of iatrogenic injury to these nerves in anterograde and 1 case in retrograde interlocking i m nailing have been described to date . Two cases of this type are described in the scientific literature . Because of the risk of damage during locking, we recommend exposing the bone surface and locking under direct vision . In the present study, the subsequent stages of the radial nerve treatment, from fracture to final results, were analyzed . It can be assumed that the type of fracture influences the type of fixation, the type of fixation influences the severity of the nerve lesion, the severity of the nerve injury influences the reconstruction technique, and the reconstruction technique influences the functional results . Although the results of the secondary radial nerve palsy treatment were analyzed, we also analyzed the type of fracture because choice of fracture treatment method depends on it . However, the final results were not statistically significant, depending on type of fracture . Neurophysiologic testing (electromyography and nerve conduction velocity) may be useful for characterizing both the level and the extent of nerve dysfunction . However, testing should be performed at a minimum of 4 weeks after an injury . The brachioradialis and extensor carpi radialis are the first muscles to be reinnervated, and the extensor indicis proprius is the last muscle to recover . Diagnostics of radial nerve damage can complete an ultrasound examination, although the effectiveness of this protocol is debated . Some studies reported success using high - resolution ultrasound to evaluate the injured radial nerve, but others reported that the role of ultrasound has yet to be properly determined and cannot be used as part of an exemplary algorithm study . Nerve function often spontaneously recovers and a lack of clear markers of nerve damage makes the decision to re - explore difficult . In the treatment of the radial nerve palsy there is no single algorithm for treatment no exploration can be generally applied in closed fractures, where most often there is no interruption of the nerve because spontaneous recovery after such injuries is reported to occur in more than 70% of patients . This has been confirmed by other studies that show radial nerve palsy is caused by a nerve contusion . Early exploration has been advocated due to concerns, especially of iatrogenic nerve entrapment . However, a review of published series demonstrated that the rate of spontaneous recovery is comparable to that of primary radial nerve palsy following humeral shaft fractures . Although limited, the literature supports nonsurgical management of a patient with a humeral shaft fracture and secondary radial nerve palsy . Early exploration may not be indicated in every case, but it allows for the assessment of the degree of damage apart from entrapment . Additionally, if the nerve is lacerated, quick repair after the injury allows tension reduction and promotes healing . Furthermore, if a nerve is ruptured with a large defect and reconstruction cannot be performed, nerve grafting or tendon transfer can be used at the beginning as a method of treatment . Some studies also suggest that functional nerve recovery is more complete and consistent with this approach . Entrapment during late exploration ranges from 6% to 25% and nerve laceration in 20% to 42% of cases is observed; however, late exploration can allow for spontaneous return of function, thus avoiding an unnecessary operation . In addition, delayed surgery may allow the neurilemmal sheath to thicken, which facilitates repair if a neurorrhaphy is needed . In contrast, delayed surgical intervention can include scarring, which can result in difficulty with nerve preparation . We believe that the risk of a bad result from the postponement of an operation justifies early exploration in cases of uncertain nerve damage . We realize that the main limitation of this study is in the analysis of results of the emg and ncv . The studies did not follow a set protocol, which did not allow us to carry out a statistical analysis . However, we believe that the most important is clinical examination; therefore, we have used quantitative (i.e., full return of function, partial improvement, no improvement), not qualitative, evaluation criteria of emg and ncv . Surgical techniques are associated with the risk of secondary radial nerve palsy, due in part to the large anatomical variability . The potential risk of radial nerve neurotmesis justifies an operational intervention in the treatment of neurological complications after a humeral fracture . Adequate surgical treatment in many of these cases allows for functional recovery of the radial nerve.
Genome - wide analyses of transcripts have revealed that a significant portion of the rna transcribed by rna polymerase ii is nonprotein - coding (noncoding) rna . In the fission yeast schizosaccharomyces pombe, 371 species of noncoding rna are predicted to be transcribed in vegetatively growing cells (wilhelm et al 2008), but most of them are not well characterized . Rna transcripts often form bodies inside the nucleus . Unlike protein - coding rna, which is transported to the cytoplasm and engaged by ribosomes for translation to protein, the most significant nuclear rna body is the nucleolus, which is formed around the ribosomal rna - coding genes . Several other examples of nuclear rna bodies formed by long noncoding rnas have been introduced in the literature (clark and mattrick 2011; mao et al . 2011; ip and nakagawa 2012). The best characterized long noncoding rna in s. pombe is meirna, a polyadenylated noncoding rna that forms a nuclear body in meiotic cells (yamashita et al ., we describe the properties of the meirna body in light of similar rna bodies in other species . It is essential for premeiotic dna synthesis and entry into meiosis (watanabe and yamamoto 1994; watanabe et al . Mei2 binds to meirna transcribed from the sme2 gene; meirna is essential for progression of meiosis (watanabe and yamamoto 1994). It was initially obtained as a multicopy suppressor of a mei2 mutant and was reported to be 508 nucleotides in length (watanabe and yamamoto 1994). However, the full length of meirna turned out to be 1,586 nucleotides (ding et al . The 508 and the full - length 1,586 nucleotide meirnas are designated meirna - s and meirna - l, respectively (fig . Mei2 binds to the meirna - s portion of meirna during entry into meiosis; meirna - l is necessary for homologous chromosome pairing . As described below, the different domains of meirna have different roles in meiosis entry, chromosomal retention, and homologous chromosome pairing (fig 1molecular dissection of meirna . A schematic diagrams of the sme2 gene, the positions of dsr and poly - a sites, and transcripts of meirna - s, meirna - l, and three kinds of deletion mutants . The 0.5-kb meirna - s was the transcript originally annotated (watanabe and yamamoto 1994). A longer transcript of 1.5 kb was later characterized and named as meirna - l (ding et al . B summary table of the phenotype of meirna - l and deletion mutants in relation to meiosis progression; meirna, mei2, and mmi1 localization in the meiotic prophase nucleus; and robust pairing . Results for 1 - 574, 1058 - 1586, and 1425 - 1586 are adapted from a previous report (ding et al . 2012) molecular dissection of meirna . A schematic diagrams of the sme2 gene, the positions of dsr and poly - a sites, and transcripts of meirna - s, meirna - l, and three kinds of deletion mutants . The 0.5-kb meirna - s was the transcript originally annotated (watanabe and yamamoto 1994). A longer transcript of 1.5 kb was later characterized and named as meirna - l (ding et al . B summary table of the phenotype of meirna - l and deletion mutants in relation to meiosis progression; meirna, mei2, and mmi1 localization in the meiotic prophase nucleus; and robust pairing . Results for 1 - 574, 1058 - 1586, and 1425 - 1586 are adapted from a previous report (ding et al . Mmi1 is involved in the selective elimination of meiotic gene transcripts in vegetatively growing cells to prevent untimely entry into meiosis (fig . In vegetatively growing s. pombe, transcripts from genes with meiosis - specific functions are quickly degraded by nuclear exosomes through several pathways (harigaya et al . 2012, 2013), and one of the major pathways is the mmi1-mediated pathway . Mmi1 is a rna - binding protein which recognizes hexanucleotide the determinant of selective removal (dsr) motifs on meiosis - specific rnas and induces their degradation (harigaya et al . Mmi1-mediated degradation of meiosis - specific rnas likely requires other proteins in addition to mmi1 . One of such factors is red1, which is present only in mitotic cells and plays a critical role in the degradation of dsr - containing rnas in vegetatively growing cells (sugiyama and sugioka - sugiyama 2011). Thus, a role for mmi1 is to recruit dsr - containing rnas to the red1-mediated degradation pathway in exosomes (fig . B sequestration of mmi1 by meirna to the sme2 locus in meiotic cells roles for mmi1 and meirna . A mmi1-mediated selective elimination of dsr - containing meiotic rnas in vegetative cells . B sequestration of mmi1 by meirna to the sme2 locus in meiotic cells meirna has 13 core dsr motifs, which are distributed along its entire sequence but are more concentrated in the region between 500 and 1,000 nucleotides (fig . Like other meiosis - specific gene products, meirna transcribed in vegetatively growing cells is completely degraded by the mmi1-mediated degradation pathway; meirna - l can be detected in vegetatively growing cells in mmi1-deficient mutants (yamashita et al . 2012). The meirna - s fragment originally identified (watanabe and yamamoto 1994) is probably a degradation product of meirna - l; the meirna - s fragment can be found in mmi1-hypomorphic conditions (yamashita et al . 2012). In seeming contrast to the function of mmi1 in the degradation of rna, on entering meiosis, mmi1 is sequestered from the rna degradation pathway by its binding to meirna (fig . Meirna and mmi1 form a nuclear rna body and are sequestered away from exosomes (harigaya et al . It is thought that meirna acts as a decoy for mmi1 with the meirna dsr motifs acting to bind and sequester mmi1 (harigaya et al . 2006; yamashita et al . 2012): meirna binds the mmi1 protein and forms a rna body in the meiotic prophase nucleus (ding et al . 2012). Because inactivation of mmi1 rescues the meiotic defects observed in sme2 deletion cells (harigaya et al . 2012), a role for meirna in entry into meiosis is subject to sequestration of mmi1 from the rna degradation pathway . As a consequence, dsr - containing meiotic rnas although meirna - s was first identified as an essential noncoding rna for entry into meiosis, it is interesting to point out that even 1 - 574 meirna - l, lacking this region, can promote normal progression of meiosis (fig . Thus, we speculate that any fragment of meirna - l that contains a sufficient number of dsr can act as a decoy for mmi1 and promote entry to meiosis . Observation of living cells demonstrated that the sme2 locus shows a significantly higher pairing frequency in the early stages of meiotic prophase and that this robust pairing requires transcription of meirna (ding et al . As mentioned above, meirna was first annotated as a 508-nucleotide rna (meirna - s) essential for the progression of meiosis . We therefore reexamined transcripts of the sme2 gene and found a 1.5-kb rna as the major transcript of the sme2 gene (designated meirna - l, as noted above) and concluded that meirna - l was required for robust pairing (ding et al . 1998), which is located at the sme2 locus on chromosome ii in the meiotic nucleus (shimada et al . 2003). Deletion of a 5 region of the sme2 gene (1 - 574) resulted in a loss of the chromosomal localization of mei2, but this 1 - 574 meirna - l still accumulated on the chromosome, and robust pairing of the sme2 gene locus was observed . These results indicate that the 5 region of the sme2 gene locus is necessary for recruiting mei2 protein to the locus, but that mei2 recruitment is not necessary for robust pairing at the sme2 locus . Instead, the 3 region of meirna - l is sufficient for robust pairing at the sme2 locus . These results suggest that the transcribed rna accumulated at the sme2 locus may play an active role in recognition and pairing of homologous chromosomes . A model has been previously proposed in the lily in which a group of meiosis - specific polyadenylated rna transcripts initiate the pairing process; these rna transcripts are collectively called zygrna for zygotene transcripts (hotta et al . 1985). Zygrna in the lily is homologous to zygrna in mouse spermatocytes, suggesting a conserved mechanism across the phylogenic spectrum (hotta et al . 1985). As discussed above, these meirna transcripts bind and sequester mmi1 and also colocalize with mei2 at the sme2 gene locus . Meirna and its associated proteins, such as mei2 and mmi1, remain on the chromosome throughout meiotic prophase . Deletion of the polyadenylation sites of the sme2 gene, yielding longer read - through transcripts expressed from the sme2 gene, eliminated the meirna body at the sme2 locus and did not promote robust paring of the sme2 loci (ding et al . Addition of an adh1 terminator to the 3 end of the polyadenylation site - deleted sme2 gene, yielding the 1425 - 1586 transcript shown in fig . 1a, resulted in the recovery of the formation of a single meirna body together with mmi1 (fig . 3c). A shorter adh1 terminator fragment (1058 - 1586 in fig . This suggests that properly terminated meirna transcripts are necessary for their chromosomal retention and that chromosomal retention is necessary for the robust pairing of homologous chromosomes . Although mechanisms for retention of rna transcripts at their respective genes are still largely unknown, these results suggest that chromosomal retention may be coupled with polyadenylation.fig . A, b time lapse imaging of meirna and gfp - mmi1 in living meiotic cells of 1058 - 1586-t (a) or 1425 - 1586-t (b); the phenotype of these cells is summarized in fig . The meirna transcript was visualized using a 4xu1a tag inserted at the 5 end of the sme2 gene and a u1a gfp fusion construct (ding et al ., the 3-end 1058 - 1586 dna fragment was replaced with an adh1 terminator and a marker gene . In b, the 3-end 1425 - 1586 dna fragment was replaced with an adh1 terminator and a marker gene . C pairing frequencies of the sme2 locus in the wild - type and 3 deletion mutants are indicated . Results for 1058 - 1586 and 1425 - 1586 are adapted from a previous report (ding et al . A, b time lapse imaging of meirna and gfp - mmi1 in living meiotic cells of 1058 - 1586-t (a) or 1425 - 1586-t (b); the phenotype of these cells is summarized in fig . The meirna transcript was visualized using a 4xu1a tag inserted at the 5 end of the sme2 gene and a u1a gfp fusion construct (ding et al ., the 3-end 1058 - 1586 dna fragment was replaced with an adh1 terminator and a marker gene . In b, the 3-end 1425 - 1586 dna fragment was replaced with an adh1 terminator and a marker gene . C pairing frequencies of the sme2 locus in the wild - type and 3 deletion mutants are indicated . Results for 1058 - 1586 and 1425 - 1586 are adapted from a previous report (ding et al . 2012) formation of a single meirna body in the meiotic nucleus is correlated with robust pairing of homologous chromosomes, but not with progression of meiosis (fig . 1b). In the 3-truncated mutants of sme2 (1058 - 1586 and 1425 - 1586 with or without adh1 terminator, t), progression of meiosis is normal, while meirna does not form a single dot except for 14251586-t (fig . (1 - 574), mmi1 but not mei2 was found in the meirna body . Thus, mei2 interacts with the 5 portion of meirna - l, and mmi1 is sequestered by meirna independently of mei2 (fig . Finally, the results presented here indicate that the meirna body is necessary for the robust pairing of homologous chromosomes at the sme2 locus.fig . 4a model for rna - mediated homologous chromosome recognition . A distinct functional domains of meirna . The meirna transcript can be divided into two distinct domains: the 5 portion of meirna - l corresponding to meirna - s and the 3-extended region specific to meirna - l . We speculate that as yet unknown proteins may bind to the 3 end of meirna - l and play a role in retention of rna on the chromosome . B complexes containing rna transcripts act as chromosome recognition sites along each of the chromosomes aligned by telomeres . C to find a homologous chromosome partner, recognition complexes (yellow, green, and orange bulbs) on homologous chromosome arms, as shown in a, are aligned by telomere - mediated chromosome movements . A distinct functional domains of meirna . The meirna transcript can be divided into two distinct domains: the 5 portion of meirna - l corresponding to meirna - s and the 3-extended region specific to meirna - l . We speculate that as yet unknown proteins may bind to the 3 end of meirna - l and play a role in retention of rna on the chromosome . B complexes containing rna transcripts act as chromosome recognition sites along each of the chromosomes aligned by telomeres . C to find a homologous chromosome partner, recognition complexes (yellow, green, and orange bulbs) on homologous chromosome arms, as shown in a, are aligned by telomere - mediated chromosome movements . It has been proposed that interactions between homologous dnas with double - strand break (dsb) are involved in homology searching in yeast saccharomyces cerevisiae (gerton and hawley 2005). On the other hand, there are many examples in which homologous pairing occurs independently of dsb formation (gerton and hawley 2005; zickler 2006). As a common phenomenon in many organisms, it is known that chromosomes are bundled at the telomere in meiotic prophase (reviewed in scherthan 2001; hiraoka and dernburg 2009). In nematode caenorhabditis elegans, special nontelomeric chromosomal regions play a role analogous to telomeres, acting as a pairing center (villeneuve 1994; macqueen et al . 2005); the pairing center is bound by one of the four zinc finger proteins him-8, zim-1, zim-2, and zim-3, which provide a mechanism for homologous recognition (phillips et al . 2005; phillips and dernburg 2006). Homologous pairing is promoted by clustering and movements of telomeres prior to dsb formation (chikashige et al . 2004, 2010; reviewed in chikashige et al . 2007; hiraoka and dernburg 2009). During this process, meirna - l directly or indirectly mediates robust pairing at the sme2 locus . This pairing is independent of dsb formation and, hence, independent of recombination (ding et al . This strongly suggests that chromosomes can recognize their homologous partners without direct interaction between dna sequences . Rather, homozygous transcription of the meirna - l sequence is essential for the robust pairing at the sme2 locus . These results suggest a model in which rna transcripts accumulate at their respective gene loci and act as recognition sites in homology searching . Rna may be directly involved in the recognition of homologous loci through rna rna or rna dna interactions . Dna interaction is less likely as homozygous transcription of meirna - l is required for robust pairing . Alternatively, meirna - l may play a role in recruiting specific rna - binding proteins essential for recognition . As discussed above, mei2 forms a distinct dot at the sme2 locus, but mei2 protein alone was not found to be necessary to confer robust pairing . In addition to mei2, at least three other proteins, mmi1, spo5, and dot2, also colocalize at the sme2 locus in meiotic prophase (harigaya et al . 2006). To date, components critical for robust pairing have not been identified among these proteins . It is possible that other unidentified critical factors may be contained in the meirna body . Alternatively rna transcripts can form nuclear bodies at their respective gene loci autonomously (mao et al . 2011; shevtsov and dundr 2011; carmo - fonseca and rino 2011), and a linear array of transcription factories formed along the chromosome may act as a bar code for recognition of homologous chromosomes as proposed previously (cook 1997; xu and cook 2008). 2004, 2010), such models provide a possible mechanism for how rna bodies result in recognition and pairing of homologous chromosomes when chromosomes are prealigned by telomere clustering (fig . It should be emphasized that the transcription itself or chromatin structural changes associated with transcription are not driving forces for the recognition of homologous chromosomes . Instead, rna bodies formed on the chromosome are important because robust pairing is not promoted when transcripts are not retained on the chromosome . A search for other chromosome loci which trigger chromosomal loci from which noncoding (or protein - coding) rna is transcribed in the early stages of meiosis may be candidates for such pairing sites in s. pombe . Arrays of rna bodies along chromosomes likely act as chromosome identifiers for the recognition of homologous chromosomes . Further studies will provide more insight into the role of rna nuclear bodies in the recognition of homologous loci during meiosis.
Sampled squirrels were from varying locations: private holdings with up to 45 animals per holding, zoological gardens, or roadkill (table 1). Locations represented 3 countries: germany (399 samples from 28 holdings), the netherlands (49 samples from 4 holdings), and the united kingdom (20 samples from roadkill). Most live squirrels were sampled at the request of the owners . At least 1 swab sample and, if possible, duplicates of oral swab samples were collected from living animals, and brain samples were tested from dead squirrels . Blood samples (edta / heparin) were also available from 164 squirrels (table 1). We obtained congruent results when we tested swabs and brain samples by 2 different vsbv-1specific quantitative reverse transcription pcr (qrt - pcr) assays (11). Blood samples were centrifuged, and the resulting plasma was tested for the presence of viral rna by using qrt - pcr and for bornavirus - specific igg by using an indirect immunofluorescence assay (11). * * swab samples were taken from the oral cavity of each investigated animal . Oral swab samples and, if available, brain samples were tested by rt - qpcr . Blood samples (in edta or heparin), wherever available, were tested by quantitative reverse transcription pcr as well as by indirect immunofluorescence assay . B, blood, bm, brain material; ss, swab sample; vsbv-1, variegated squirrel bornavirus 1 . We detected vsbv-1 rna and bornavirus - specific antibodies in 11 (2.6%) of the 468 animals . These 11 vsbv-1positive squirrels (7 male, 4 female) belonged to the family sciuridae, subfamilies sciurinae (variegated squirrels, s. variegatoides; 6 animals) or callosciurinae (prevost s squirrel, c. prevostii; 5 animals) (table 1). Of the 11 vsbv-1positive animals, 9 were from 4 squirrel breeders in germany (holdings i iv). The remaining 2 were from a private holding in the netherlands (holding v). Of the 6 variegated squirrels, 3 originated from holding i, where the first vsbv-1positive animal was identified in 2014 (11), and the remaining 3 were from holdings ii and iii . Of the 5 vsbv-1positive prevost s squirrels, 3 came from holding iv in germany, and 2 were from holding v. the positive squirrels in the netherlands had been transferred from a breeder in germany in 20112012, but we could establish no direct epidemiologic link to the holdings in germany where we found vsbv-1positive animals . All animals with detectable virus as well as 40 negative squirrels from the same holdings were euthanized and checked for macroscopic lesions . We applied hematoxylin and eosin staining to formalin - fixed, paraffin - embedded brain sections from 9 of 11 animals and performed immunohistochemistry as described previously (12). Histopathology showed that 5 of 9 vsbv-1positive animals, variegated squirrels as well as prevost s squirrels, had mild nonsuppurative meningitis or encephalitis . In 8 of 9 animals, we also detected intranuclear eosinophilic joest - degen inclusion bodies in scattered neurons of brain, spinal cord, and trigeminal as well as spinal ganglia (data not shown). We observed bornavirus - specific phosphoprotein and x protein throughout the brain in neurons, glial cells, and in a few ependymal cells in nuclei, cytoplasm, and cellular processes (figure 1). We tested a panel of organ samples from all euthanized animals by qrt - pcr . All animals for which swab samples were vsbv-1positive harbored considerable vsbv-1 genome loads, whereas animals for which swab samples were negative for viral rna were also negative for viral loads in all tested organs (table 2; data for control animals not shown). We found the highest viral rna loads in the cns and organs (kidney, nose, bladder, salivary gland, and sex organs), which could play a role in viral shedding and transmission (table 2). Skin sections were also positive, indicating that vsbv-1 has broad cell and organ tropism . We were able to cocultivate infected primary squirrel cells with a permanent cell line and to isolate infectious virus from these passaged cells . Immunohistochemical detection of bornavirus x protein (a) and phosphoprotein (b) in hippocampal neurons of a brain of a prevost s squirrel (callosciurus prevostii) collected in germany in 2015 . Inset shows intranuclear dot (inclusion body) in cells with and without cytoplasmic immunostaining (arrows). No staining was observed for bornavirus x protein (c) or phosphoprotein (d) in a bornavirus - negative variegated squirrel . Original magnification 400 * data are represented in vsbv-1 genome equivalent copies per milliliter of template: + + +,> 10; + +, 1010; +, 1010; 1010). The vsbv-1specific mix 10 quantitative real- time pcr assay was used for quantification (limit of detection 1 genome equivalent/l template). We sequenced the vsbv-1 genome (8,786 nt; missing only the 5 and 3 noncoding regions) from all 11 squirrels and compared the sequences with the published vsbv-1 prototype sequence from a variegated squirrel (genbank accession no . Accession numbers of the vsbv-1 sequences from this study are lt 594381lt5943919 (european nucleotide archive). Genomes of viruses detected in squirrels from holding i showed the highest similarity with 16 nt substitutions, resulting in 13 aa changes, whereas the other sequences exhibited more variability . The mutations were evenly distributed over the coding regions, but in viral genomes of squirrels from the same holding, they often occurred at the same position (figure 2, panel a). Analysis of 11 newly identified vsbv-1 genomes from squirrels collected in germany and the netherlands, 2015, in comparison with related bornaviruses . A) new sequences aligned with published squirrel - derived vsbv-1 genome (genbank accession no . G, glycoprotein; l, large structural protein; m, matrix; n, nucleoprotein; p, phosphoprotein; x, nonstructural protein . B) phylogenetic tree of vsbv-1 isolates from this study (labeled) and comparison sequences . Ger, germany; nl, the netherlands; vsbv-1, variegated squirrel bornavirus 1 . We aligned the 11 vsbv-1 sequences with those from other bornaviruses by using mafft (multiple alignment using fast fourier transform), and we used the best - fit model tim2+i+g4 to construct a phylogenetic tree (iq - tree version 1.3.10; http://iqtree.cibiv.univie.ac.at) with 1,000 bootstrap replicates . The novel vsbv-1 genomes clustered with the 2 human- and squirrel - derived prototype sequences ln 713680 and ln71368, forming a unique vsbv-1 clade separate from other mammal, avian, and snake bornaviruses (sequence identity <69%; figure 2, panel b) we screened 468 squirrels and identified 11 vsbv-1positive animals, including squirrels belonging to 2 subfamilies of the family sciuridae . Although the vsbv-1positive squirrels originated from different holdings and belonged to different subfamilies, the viral genome sequences formed a distinct vsbv-1 cluster . Whole - genome analyses provided no evidence for specific mutation patterns with regard to zoonotic potential or species - specific adaptations . Highest viral genome loads were found in the cns, followed by the oral cavity and skin, indicating the potential for transmission to humans through scratching or biting . Only those squirrels positive for vsbv-1 by qrt - pcr displayed bornavirus - specific antibodies . Although serologic analyses support the qrt - pcr results, collecting serum samples is difficult and often not feasible for private breeders . Our data suggest that screening of swab samples is a suitable and reliable tool for noninvasive monitoring of squirrels for vsbv-1 infection . The prevalence of 3.3% for s. variegatoides and 8.8% for c. prevostii squirrels indicates a considerable risk for transmission to humans handling those animals without taking precautionary measures . We therefore recommend routine testing of squirrels in contact with humans, such as those in breeding and holding facilities or zoological gardens, at least from the subfamilies sciurinae and callosciurinae.
Microwave imaging has recently emerged as one of the most promising non - invasive imaging modalities of the last two decades . Its low cost, non - ionising characteristics justify the considerable interest of the scientific community . Although breast cancer imaging has been the most widely explored applications [1, 2], other biomedical diagnostic areas are also proposed [3, 4]. The operating principle is based on the dielectric contrast between the targeted and surrounding tissues . The imaging formation procedure involves illuminating the tissue with an electromagnetic radiation and analysing the reflected / scattered (and sometimes transmitted) signals from dielectric boundaries within the tissue . These algorithms can be divided into two broad categories: those that seek to identify the presence and location of significant dielectric scatterers in the tissue (termed as radar approaches); and those whose aim is to reconstruct the entire dielectric profile of the tissue under examination (termed as tomographic approaches). Ultra - wideband (uwb) antennas have been proven suitable for microwave medical imaging under radar approaches due to their large operating bandwidth combined with stable radiation / coupling properties and compact designs [5, 6]. Limited field penetration or non - uniform frequency - dependent near - field distribution can generate artefacts and degrade or destroy the imaging process . In order to fully assess the near - field microwave detection system performance, the antenna presence must be taken into account . In [4, 7] microwave tomography the technique was proposed as non - invasive screening modality of osteoporosis bone cortex degradation . In this letter, we present a microwave radar technique to image bone profiles in a more complex multi - tissue phantom that mimics a large leg . The system is capable of detecting the dielectric boundaries of two bones with different cross - sections that are embedded within a non - uniform multi - layer structure of skin, adipose and muscle tissues . An uwb signal is transmitted and received in the frequency bandwidth 0.54 ghz by two vivaldi antennas in a radar monostatic fashion . The signal travels across different impedance conditions due to the heterogeneous scenario due to non - uniform multi - layer structure of skin, adipose, muscle and bone tissues . However, it also penetrates a highly attenuating muscle tissue that accounts for about 50% of the entire investigated volume . This work shows the viability of microwave bone radar imaging technology which is low cost, portable, non - ionising and does not require specially trained personnel . In fact, microwave technology allows using compact and moderately expensive devices compared to other conventional imaging technologies . Images are reconstructed by using non - coherent migration, which is a particular version of beamforming [8, 9]. In particular, using non - coherent migration to reconstruct bone profiles from measured reflection coefficients makes unnecessary any pre - scan antenna characterization . This feature both speeds up the scanning time and ease usability . After a brief description of its functional parts, results are shown in the following scenarios: a metal scatterer in homogeneous background totally filled with adipose tissue for delocalisation assessment.bones inserted in a multi - layer structure including skin, fat and muscle . A metal scatterer in homogeneous background totally filled with adipose tissue for delocalisation assessment . The 3d scanner proposed here extends the investigation carried out in for breast cancer detection in a more controlled environment and on a diverse target (fig . 1). The prototype is made of the following parts: two printed antipodal vivaldi antennas that can be manoeuvred to adjust their height and distance from the phantom.a turntable that rotates the phantom with 1 accuracy.a tank that contains the measuring setup immersed in a coupling medium with permittivity of 12.a vector network analyser to measure the s - parameters at the antennas' terminals.an acquisition unit to synchronise the antenna / phantom positioning with the data acquisition.a data processing unit to generate reconstructed images from the measured datasets . 13d microwave scanner and phantoma schematics of the scanning procedure with two antennas positioned on opposite sides of the phantom to half the scanning timeb detail of the scanner two printed antipodal vivaldi antennas that can be manoeuvred to adjust their height and distance from the phantom . A turntable that rotates the phantom with 1 accuracy . A tank that contains the measuring setup immersed in a coupling medium with permittivity of 12 . A vector network analyser to measure the s - parameters at the antennas' terminals . An acquisition unit to synchronise the antenna / phantom positioning with the data acquisition . A data processing unit to generate reconstructed images from the measured datasets . 3d microwave scanner and phantom a schematics of the scanning procedure with two antennas positioned on opposite sides of the phantom to half the scanning time b detail of the scanner the geometry of the antenna is shown in fig . 2 and consists of a printed element on 1.6-mm thick fr4 substrate . The dimensions and the taper of the two metallised parts were optimised to combine compactness with satisfactory field penetration inside the phantom . Each prototyped antenna was sprayed with 3m novec coating to prevent electro - mechanical change of the dielectric when immersed in the coupling medium . 2geometry of the printed antipodal vivaldi antenna geometry of the printed antipodal vivaldi antenna the system records s11 and s22 sampled across 801 equally spaced points in the frequency range 0.54 ghz, where satisfactory impedance matching is achieved (better than 8 db). In this configuration, instead of having one single antenna scanning the phantom across 360 as in, two antennas are positioned on opposite sides of the phantom to half the scanning time . The coupling medium used a mixture of 50% kerosene50% safflower oil solution and de - ionised water in the proportion of 80 and 20%, respectively . A realistic phantom that mimics a cow's leg was prepared by using a bovine tibia and fibula bone section with muscle tissue attached and embedded in pork fat and turkey skin for convenience (fig . 3). By assembling the phantom, more control on the ratio between adipose and non - adipose tissue volumes can be achieved . This phantom was manufactured with the bone + muscle accounting for more than 80% of the entire volume in order to challenge the imaging procedure with high percentage of electromagnetically dense tissue in the scanned volume . The dielectric properties of the corresponding human tissues in the phantom are listed in table 1 at the centre frequency of 2.75 ghz [1012]. The phantom has an overall approximate diameter of 100 mm and a length of 250 mm with the bone - section of diameter 28 mm . Three slices in the central region spaced 10 mm apart were considered for screening with 36 scans per slice (18 per antenna). 3realistic phantom that mimics a cow's leg was prepared by using a bovine tibia and fibula bone sectiona phantom's cross - sectionb sagittal x - ray scan of the phantom table 1dielectric properties of corresponding human tissues in phantom at 2.75 ghzconductivity, s / mrelative permittivitybone cortical0.4542911.207marrow0.108775.2644muscle1.951552.363fat0.118095.2492skin1.780242.442 realistic phantom that mimics a cow's leg was prepared by using a bovine tibia and fibula bone section a phantom's cross - section b sagittal x - ray scan of the phantom dielectric properties of corresponding human tissues in phantom at 2.75 ghz the dielectric contrast between the target (i.e. Bone) and surrounding tissues is about 4.6 which is significantly greater than that observed in microwave breast cancer imaging . However, this scenario presents different challenges as the target bone tissue is embedded in a thick and electromagnetically denser layer of muscle tissue, hence results in a weaker signal reaching the target tissue . The observation domain is a set of circular lines each located at different heights, zh . For a fixed height, the field scattered by the phantom is collected by a tx / rx antenna over n scanning positions (r01, r02,, the data of a complete scan can be arranged accordingly in the n nf scattering matrix \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $\underline {\underline{{\bi s}}} = \left[{{\underline{{\bi s}}} _ {1}\comma \; \ldots \comma \; \; {\underline{{\bi s}}} _ {n_{\rm f}}\comma \; \ldots \comma \; \; {\underline{{\bi s}}} _ {n_{\rm f}}} \right]$\end{document}s__=s_1,,s_nf,,s_nf where \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $\underline{{\bi s}} _ {n_{\rm f}}$\end{document}s_nf is the column vector of data collected over the observation positions at nfth frequency . These data include the information signal, but also strong clutter components generated from the antenna's internal reflection, skin and other non - targeted tissues . As the clutter tends to mask the bone - interface signal, it has to be reduced before the image construction procedure . Different clutter rejection methods exist in the literature such as the differential approach, the subspace projection method and the entropy - based time windowing algorithm . However, considering the cylindrical layout of the adopted phantom for this proof - of - concept, the average trace subtraction strategy is implemented . In particular, in frequency domain one obtains (1)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $$\underline {\underline {{\bi s}}} _ {{\rm av}} = \underline {\underline {{\bi s}}} - \underline {\underline {\check{{\bi s}}}} \; \eqno\lpar 1\rpar $$\end{document}s__av = s__s__where the subscript av indicates that the average trace subtraction algorithm is used . Each column of the matrix \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $\underline {\underline {\check{{\bi s}}}} = \left[\underline {\check{{\bi s}}}_{1}\comma \; \ldots \comma \; \; \underline{\check{{\bi s}}}_{n_{\rm f}}\comma \; \ldots \comma \; \; \underline{\check{{\bi s}}} _ {n_{\rm f}} \right]$\end{document}s__=s_1,,s_nf,,s_nf has values all equal to the average over the sensors' positions (fixed the frequency). To test the detection system, the non - coherent migration is adopted according to the expression in the following equation (2)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $$\; \phi _ {n - m}\left({{\underline{r}} _ {k}\comma \; z_{\rm h}} \right)= \mathop \sum \limits_{i = 1}^{n_{\rm f}} \eqno\lpar 2\rpar $$\end{document}nmr_k, zh=i=1nfwhere a(fi) is the steering vector calculated at the trial position rk within the spatial domain d. the positions of the target are identified where the pseudo - spectrum nm (.) Peaks, with mi[.] Being migration operator . Note that by employing non - coherent migration no information about the antenna is needed . In fact, as shown in this algorithm actually represents a no - windowing beamforming image method . Therefore, as the time window is not considered, the travel time within the antenna is not required . The data collected for each measurement position on the observation circle is used to generate a reconstruction of the corresponding 2d slice (i.e., in the x y - plane). More in detail, both the de - clutter procedure and the calculation of the pseudo - spectrum nm (.) Hence, the final 3d reconstruction is obtained by performing a collection of 2d single pseudo - spectra at different heights . Once the 2d slice reconstructions are obtained, the 3d image of the target is obtained by superimposing the 2d reconstructions (3)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $$\; i\left({{\underline{r}} _ k} \right)= \left({\matrix {{\phi _ {n - m}\left({{\underline{r}} _ {k}\comma \; z_1} \right)} \cr \cdots \cr {\phi _ {n - m}\left({{\underline{r}} _ {k}\comma \; z_{\rm h}} \right)} \cr}} \right)\eqno\lpar 3\rpar $$\end{document}ir_k=nmr_k, z1nmr_k, zhwhere zh (z1,, zh) are the different heights at which the slices are taken . Finally, the actual 3d reconstruction is obtained by interpolating in the z - direction . The scanner was assessed for a very simple homogeneous scenario with the investigation scene totally filled with the coupling medium and an 8 mm thick cylindrical metal bar used as target . This test was performed in order to estimate the delocalisation error due to the mismatch of the equivalent permittivity used by the algorithm to generate the image from the actual permittivity of the coupling medium . Thirty - six equally spaced measurements were taken across three sections spaced 10 mm from each other . In the top panel of fig . 4, the target located at (x = 20 mm; y = 5 mm) is imaged . Although the object appears slightly oblique, the 3d reconstruction is successfully performed . In each of the three 2d reconstructions (omitted here for brevity) the target is detected with a consistent spatial displacement (sd) of 2.78, 2.85 and 4.23 mm, respectively . The sd measures the error in the target localisation and accounts for the difference between the target centre position as a peak value in the reconstruction and as actual centre position in the scanned phantom . This is due to the mismatch between the adopted equivalent phantom permittivity in the reference green's function and the actual unknown permittivity mapping of the detection scene . 4 highlights once again the effect of the mismatch described above . Fig . 4target located at (x = 20 mm; y = 5 mm) is imageda 3d reconstruction of the investigation scene totally filled with adipose tissue and an 8-mm thick cylindrical metal bar used as targetb overhead view of 3d reconstruction target located at (x = 20 mm; y = 5 mm) is imaged a 3d reconstruction of the investigation scene totally filled with adipose tissue and an 8-mm thick cylindrical metal bar used as target b overhead view of 3d reconstruction the phantom 3d image is shown in fig . 5 when scanned across three sections spaced 10 mm apart . The presence of both tibia and fibula is correctly detected and their 3d profile is reconstructed . Although the different cross - sections of the bones are not distinguished by the imaging algorithm, the targets are localised in the scanned area with good accuracy . 5phantom 3d imagea preparation of the realistic phantom (tibia and fibula are marked in yellow circle)b 3d image reconstruction of the tibia and fibula in the phantom a preparation of the realistic phantom (tibia and fibula are marked in yellow circle) b 3d image reconstruction of the tibia and fibula in the phantom the microwave scanning system presented here shows promising results for bone imaging in a multi - layer complex limb - mimicking phantom . Without a priori antenna characterisation, the image formation procedure based on non - coherent migration successfully reconstructs the 3d profile of both bones in the phantom . Although the scenario under investigation presents several degrees of difficulty such as its multi - layer structure and the presence of more than one target (i.e. Tibia and fibula) with different cross - sections, the scanner is able to discriminate the bone - muscle interface and reconstruct its 3d profile . Future work will explore the integration of a multi - element antenna array to enable multi - view radar configurations . On a more engineering side, a more compact version of the prototyped scanner will be designed to deliver a portable bone scanning device that can be used in site of accidents . This work was supported by italian ministry of university and research through the firb initiative under the project micenea (rbfr12a7cd) and the cost actions td1301 mimed and bm1309 emf - med.
Peritoneal tuberculosis has common symptoms with advanced ovarian carcinoma including pelvic pain, mass, ascites, and elevated ca-125 levels . We present here three different cases of coexisting genital carcinoma along with tuberculosis diagnosed on histopathology . Tuberculosis complicating malignant disease may occur in regions with a high prevalence of disease; with a resurgence of tuberculosis worldwide, this association may not be uncommon . The diagnosis and treatment of tuberculosis in a patient with cancer assume importance as a high morbidity has been seen in patients with coexistent disease . Whether tuberculosis, a chronic inflammatory condition, facilitates carcinogenesis is yet to be determined . We present a case of a 45-year - old para 4 female who reported to the gynecological outpatient department with a complaint of lump abdomen since 1 year, which was gradually increasing in size and had reached up to umbilicus . There was associated dull aching pelvic pain and amenorrhea for last 7 months . On examination, she had a body mass index (bmi) of 22.5 with minimal pallor and no lymphadenopathy . On bimanual examination, a firm mass of size approximately 15 cm 12 cm 12 cm felt arising from left adnexa . Her hemoglobin was 9.7 gm%, total leukocyte count of 11,900/mm, and erythrocyte sedimentation rate (esr) of 30 mm in 1 h. ca-125 level was raised to 1000 units / ml . Magnetic resonance imaging (mri) pelvis was done which also showed a smoothly marginated complex multiseptated predominantly cystic lesion with solid component within the dependent portion, arising from the left ovary and was 10.6 cm 14.3 cm 16.8 cm in size . A provisional diagnosis of ovarian malignancy was made and a staging laparotomy followed by total abdominal hysterectomy and optimum debulking surgery was performed in this patient . Intraoperatively, there was a large right - sided ovarian sized 15 cm 15 cm 12 cm occupying almost whole of the pelvis . Thick cheesy material was expressed from the tumor and a solid area of size 6 cm 3 cm 3 cm was present inside the tumor . Histopathology report showed moderately differentiated papillary serous cystadenocarcinoma involving left ovary spreading to the ipsilateral fallopian tube . The tumor involved the capsule which showed focal dense chronic inflammation comprising caseating epithelioid granulomas indicative of tuberculosis [figure 1]. Right fallopian tube showed multiple epithelioid granulomas, few with caseation suggestive of tuberculosis [figure 2]. Ovarian tissue showing moderately differentiated papillary serous adenocarcinoma along with features of tuberculosis tubal mucosa showing infiltration of carcinoma along with tubercular changes finally, stage iia ovarian carcinoma was made . The who category 1 antitubercular therapy was started on postoperative day 10 and one cycle of chemotherapy comprising carboplatin and paclitaxel was given . She has taken antitubercular treatment for 6 months and received 6 cycles of chemotherapy after surgery and is doing fine . A 53-year - old female postmenopausal since 56 years presented with three episodes of postmenopausal bleeding over the last 6 months . Her hemoglobin was 9.0 gm%, total leukocyte count of 12,000/mm, and an esr of 35 mm in 1 h. her liver function test, kidney function test, and blood sugars were within normal limits . Transvaginal ultrasound showed an enlarged uterus of size 10 cm 5 cm 4 cm with disrupted endomyometrial junction . Six out of 13 lymph nodes dissected showed caseation along with epithelioid granulomas without any evidence of malignancy . A final diagnosis of stage ib grade iii endometrial mucinous adenocarcinoma with coexisting tuberculosis was made . The who category i antitubercular therapy was started on postoperative day 10, and she received adjuvant radiotherapy . She took antitubercular therapy for 6 months along with adjuvant treatment for carcinoma and is doing well on follow - up . A 50-year - old postmenopausal female presented with a complaint of postmenopausal bleeding since 3 months . Examination, growth was seen on the anterior lip of cervix of size 2 cm 2 cm . All the fornices, bilateral parametria, and rectal mucosa were free of tumor growth . Punch biopsy from the cervical growth revealed well - differentiated squamous cell carcinoma . A final diagnosis of stage her chest x - ray was unremarkable, and her liver function test, kidney function test, and blood sugar were within normal range . Intraoperatively, tumor was found limited to only cervix . Vaginal cuff, bilateral parametria, and adnexa were free of tumor . She took antitubercular therapy under the who category i for 6 months and is doing well on follow - up . We present a case of a 45-year - old para 4 female who reported to the gynecological outpatient department with a complaint of lump abdomen since 1 year, which was gradually increasing in size and had reached up to umbilicus . There was associated dull aching pelvic pain and amenorrhea for last 7 months . On examination, she had a body mass index (bmi) of 22.5 with minimal pallor and no lymphadenopathy . On bimanual examination, a firm mass of size approximately 15 cm 12 cm 12 cm felt arising from left adnexa . Her hemoglobin was 9.7 gm%, total leukocyte count of 11,900/mm, and erythrocyte sedimentation rate (esr) of 30 mm in 1 h. ca-125 level was raised to 1000 units / ml . Magnetic resonance imaging (mri) pelvis was done which also showed a smoothly marginated complex multiseptated predominantly cystic lesion with solid component within the dependent portion, arising from the left ovary and was 10.6 cm 14.3 cm 16.8 cm in size . A provisional diagnosis of ovarian malignancy was made and a staging laparotomy followed by total abdominal hysterectomy and optimum debulking surgery was performed in this patient . Intraoperatively, there was a large right - sided ovarian sized 15 cm 15 cm 12 cm occupying almost whole of the pelvis . Thick cheesy material was expressed from the tumor and a solid area of size 6 cm 3 cm 3 cm was present inside the tumor . Histopathology report showed moderately differentiated papillary serous cystadenocarcinoma involving left ovary spreading to the ipsilateral fallopian tube . The tumor involved the capsule which showed focal dense chronic inflammation comprising caseating epithelioid granulomas indicative of tuberculosis [figure 1]. Right fallopian tube showed multiple epithelioid granulomas, few with caseation suggestive of tuberculosis [figure 2]. Ovarian tissue showing moderately differentiated papillary serous adenocarcinoma along with features of tuberculosis tubal mucosa showing infiltration of carcinoma along with tubercular changes finally, stage iia ovarian carcinoma was made . The who category 1 antitubercular therapy was started on postoperative day 10 and one cycle of chemotherapy comprising carboplatin and paclitaxel was given . She has taken antitubercular treatment for 6 months and received 6 cycles of chemotherapy after surgery and is doing fine . A 53-year - old female postmenopausal since 56 years presented with three episodes of postmenopausal bleeding over the last 6 months . Her hemoglobin was 9.0 gm%, total leukocyte count of 12,000/mm, and an esr of 35 mm in 1 h. her liver function test, kidney function test, and blood sugars were within normal limits . Transvaginal ultrasound showed an enlarged uterus of size 10 cm 5 cm 4 cm with disrupted endomyometrial junction . Six out of 13 lymph nodes dissected showed caseation along with epithelioid granulomas without any evidence of malignancy . Ziehl - neelsen stain for acid - fast bacilli was, however, negative . A final diagnosis of stage the who category i antitubercular therapy was started on postoperative day 10, and she received adjuvant radiotherapy . She took antitubercular therapy for 6 months along with adjuvant treatment for carcinoma and is doing well on follow - up . A 50-year - old postmenopausal female presented with a complaint of postmenopausal bleeding since 3 months . Examination, growth was seen on the anterior lip of cervix of size 2 cm 2 cm . All the fornices, bilateral parametria, and rectal mucosa were free of tumor growth . Punch biopsy from the cervical growth revealed well - differentiated squamous cell carcinoma . A final diagnosis of stage ib carcinoma cervix was made . Her chest x - ray was unremarkable, and her liver function test, kidney function test, and blood sugar were within normal range . Intraoperatively, tumor was found limited to only cervix . Vaginal cuff, bilateral parametria, and adnexa were free of tumor . She took antitubercular therapy under the who category i for 6 months and is doing well on follow - up . Pelvic tuberculosis infection is usually caused by reactivation of organisms from systemic distribution of mycobacterium tuberculosis during primary infection . The most common organ involved in genital tuberculosis is fallopian tube (95%-100%) followed by endometrium (50%-60%), ovary (20%-30%), cervix (5%-15%), and vulva and vagina (1%). The symptoms of chronic pelvic pain, abdominal mass, and weight loss along with the formation of adnexal mass with or without ascites can be present in both pelvic tuberculosis and ovarian carcinoma . Various authors have reported pelvic tuberculosis mimicking an advanced ovarian carcinoma; however, none reported coexistence of ovarian carcinoma with tuberculosis . First case presented with a complex adnexal mass and elevated ca-125 levels which led us to the diagnosis of ovarian cancer preoperatively . Whether tuberculosis, a chronic inflammatory condition, facilitates carcinogenesis is yet to be determined . Similarly, hsu et al . Reported two cases from china in 1985, where both the cases presented with postmenopausal bleeding . Reported a case of carcinoma cervix with coexisting endometrial tuberculosis in 2004 . Even coexisting endometrioid adenocarcinoma and endometrial tuberculosis have been reported as an extremely rare condition . Due to rarity of the association, data regarding outcome of such patients, the influence of one condition over the other as well as survival it is controversial whether the coexistence of genital malignancy and tuberculosis is an incidental finding or whether cancerous cachexia offers a good nutritional basis for dormant tuberculosis bacilli to survive or whether carcinoma was superimposed on chronic progressive tuberculosis . Usually, the patients are cachectic on presentation as both the malignancy and tuberculosis cause cachexia . Cancer cervix has been included in the list of acquired immune deficiency syndrome - defining illnesses in patients infected with hiv and tuberculosis too has resurged with the hiv pandemic . The immune suppression caused by hiv infection is a risk factor for the development of cancer cervix as well as tuberculosis, thus cancer cervix occurring along with tuberculosis may increase in the hiv era . Data regarding outcome of such patients, the influence of one condition over the other as well as survival are unavailable due to the rarity of the association . Thus, a regular follow - up of such patients is necessary . Although we could not show an acid - fast microorganism by ziehl neelsen stain from the specimens obtained at laparotomy in any of the cases, histological findings confirmed tuberculosis along with carcinoma . Since genital tuberculosis is a paucibacillary form of the disease, smears and cultures are usually negative . Microscopic examination of acid - fast bacilli requires at least 10,000 organisms / ml in the sample . Although polymerase chain reaction dna is a rapid and sensitive molecular method dna for diagnosing m. tuberculosis in extrapulmonary samples, it was not done as it is not routinely performed at our institution . Further research is needed to determine if tubercular infection being a chronic inflammatory condition facilitates carcinogenesis . Since coexisting tuberculosis with carcinoma is a histopathological diagnosis, it is important to keep its diagnosis in mind while reporting genital cancer.
Cutaneous metastases are of diagnostic importance as they may be the first manifestation of an undiscovered internal malignancy or the first indication of metastasis from a supposedly adequately treated malignancy . As a rule, most regional metastases probably occur through the lymphatic system, while distant metastases are more likely to occur via the hematogenous route . In - transit metastases are cutaneous metastatic foci located between the tumor and the closest regional lymph nodes . A case of squamous cell carcinoma (scc) of the penis with numerous in - transit metastases in the pubic region and scrotum in may 2010, a 65-year - old male patient presented at our hospital with complaints of numerous papules and nodules in the pubic and suprapubic region and the scrotum . According to him the lesions had been present for the last 4 months . In october 2009, he had undergone partial amputation of the penis at a tertiary care hospital for an ulceroproliferative growth involving the glans penis, which was histologically proven to be a moderately differentiated scc . A fine needle aspiration cytology (fnac) of the left inguinal nodes at the same teritiary care hospital where patient underwent surgery, revealed well - differentiated metastatic scc on the left side and reactive lymphoid hyperplasia on the right side . Bilateral block resection of inguinal lymph nodes, wth skin grafting from a donor site from the thigh, was performed . However, 4 months later he developed edema of both lower limbs, scrotum, and the suprapubic region . He also developed multiple painless papules and nodules in the above areas, with some of the lesions discharging a clear fluid . A diagnosis of post - lymphadenectomy lymphangiectasia, was entertained at that time at the tertiary care hospital and the patient was treated conservatively and reassured . He presented at our hospital in may, 2010, with multiple papules and nodules (some of them ulcerated) on the scrotum and the pubic and suprapubic regions . Local examination revealed edema of the scrotal skin and suprapubic region, a short stump of the amputated penis, postsurgical scars in both inguinal regions, and postinflammatory pigmentary changes both hypo- and hyperpigmentation at the donor site on the thigh [figure 1]. There were multiple firm papules and nodules of about 23 mm in size, some of them ulcerated and discharging serous fluid, in the pubis, suprapubic region, and scrotum . [figure 2] roentgenogram of the chest and ultrasonogram of the abdomen did not reveal any metastatic deposits . Other than anemia (with hemoglobin of 8.1 gm%), the routine hematologic and biochemical investigations did not reveal any abnormality . Edema of the scrotal skin and suprapubic region, the short stump of the amputated penis, and postsurgical scars in both inguinal regions . Islands of metastatic squamous cell carcinoma with a clear zone between the deposit and the epidermis . Overlying stratified squamous epithelium is normal (h and e; a 200, b 400) a diagnosis of scc with in- transit cutaneous metastasis was made and the patient was referred back to the tertiary hospital for further management . Carcinomas arising from modified skin such as the glans penis and the vulva and from the oral mucosa have a rather high rate of metastasis unless they are recognized and adequately treated at an early stage . Carcinomas arising in sun - damaged skin have a very low propensity to metastasize, the incidence being only about 0.5% . The rate of metastases is higher in adenoid and mucin - producing scc of the skin than in the common type . However, in our patient, details about the histological type of the primary lesion were unavailable . Dissemination may take place via the lymphatics or the blood stream . In - transit metastases are cutaneous metastatic foci located between the tumor and the closest lymph node region . Analogous to in - transit metastasis found in melanoma, these represent metastatic spread along lymphatic vessels and/or nerves and their presence is an indicator of poor prognosis . In the present case, in view of the innumerable papulonodules and the previous history of regional (i.e., inguinal) lymph node involvement as proved by fnac study, lymphatic spread was probably the reason for these in transit metastases . A case of penile cancer has been reported earlier where the patient presented with primary high - grade penile squamous carcinoma and secondary skin metastases, in addition to metastases to liver, lungs, and other organs . However, in our patient there was no clinical or radiological evidence of involvement of other organs . Overall, in - transit metastases occur most frequently from scc risk stratified as high - risk lesions; however, all scc that occur in immunosuppressed patients have the potential for distant spread . Ninety percent of metastatic scc occurs within 3 years of diagnosis of the primary tumor . In our patient, however, the metastasis occurred within a much shorter period of 4 months . It is possible that the tumor emboli were already lodged in the lymphatic channels at the time the inguinal node resection was undertaken and hence this early recurrence . Clinically, in - transit metastasis are nondescript, subtle, waxy, gray - white or flesh - colored papules of about 26 mm diameter and are not contiguous with the primary lesion . Such a diagnosis was entertained in the present case also at the tertiary care hospital 3 months prior to his visit to our hospital . In a review of 21 cases with in - transit metastases, carucci et al . Reported that patients presented most commonly with discrete dermal papules distinct from, but in the vicinity of, the primary tumor site . In their series, local control of in - transit metastasis should be achieved with mohs surgical technique or some other surgical method, where the surgical margins are rigorously evaluated for residual tumor, perineural extension, or intravascular invasion (e.g., excision with intraoperative frozen section control or excision with postoperative margin assessment). The radiation field often involves the primary tumor site, the in - transit metastatic site, and the draining lymph node basin . Our patient was referred for further treatment to the same tertiary care hospital where he had earlier undergone surgery for his scc penis, but he was lost to follow - up . This case is presented because of the uncommon presentation of in - transit metastases after the primary tumor and the regional lymph nodes were resected.
Clear cell odontogenic carcinoma (ccoc) is an extremely uncommon tumor of odontogenic origin, first described by hansen in 1985 . Ccocs were formerly called clear cell ameloblastomas or clear cell odontogenic tumors and were classified under the category of benign tumors in the 1992 world health organization classification . However, owing to its high potential for regional spread and distant metastases, it was reclassified as a malignant tumor of odontogenic origin in 2005 . To date, of the reported cases, all have presented in a single jaw, with none having tmj involvement . We report, to the best of our knowledge, the first case of an aggressive ccoc involving the mandible and the temporomandibuar joint (tmj) with cervical lymph node metastasis at presentation . A 50-year - old female was referred our institution for evaluation of a swelling in the left mandible of 3 months duration . She gave a history of extraction of a mobile left lower third molar one month ago elsewhere ., there was an extra oral swelling overlying the left posterior mandible measuring 4 5 cm . Intraorally, the swelling extended from the region of the left first molar to the ascending ramus of the mandible, obliterating the left retromolar trigone . The patient was also found to have grade 1 trismus and numbness of the left tongue . Examination of the neck revealed a significant, firm, submandibular lymph node approximately 1.0 cm in diameter . Orthopantomogram and computed tomography scans revealed an expansile lytic lesion involving the posterior body and ramus of the left mandible [figure 1]. There was perforation of both the buccal and lingual cortical plates and involvement of the adjacent soft tissues . An intraoral trucut biopsy was performed, and a diagnosis of ccoc was established . A chest skiagram and ultrasound of the abdomen, done as a part of the metastatic work - up, (a) opg showing a radiolucent lesion in the posterior segment of the left hemi - mandible . (b) reconstructed axial ct scan showing the lytic lesion along the left angle and ascending ramus of the mandible and involvement of the temporomandibular joint the patient then underwent a composite resection which included an en bloc excision of the posterior segment of the left mandible, tmj, infratemporal fossa clearance, and a left modified radical neck dissection [figure 2]. (a) inraoperative clinical photograph following composite resection (b) postoperative clinical photograph of the operated specimen the gross pathology revealed a firm tumor measuring 5 3 4.5 cm involving the mandible, tmj, and the adjoining soft tissues . Microscopically, tumor cells were seen arranged in sheets, cords and islands, separated by thin and thick bands of fibrocollagenous stroma with hyalinization . The tumor cells were round to polygonal with well - defined cell outlines and abundant clear cytoplasm . The tumor cells were immunoreactive for keratin, epithelial membrane antigen, smooth muscle actin, and s-100 and negative for vimentin, and hmb-45 [figures 3 and 4]. H and e x10 (a) the tumor cells were round to polygonal with well - defined cell outlines and abundant clear cytoplasm . (b) tumor showing infiltration of the mandible . (c) tumor cell infiltrating the temporomandibular joint . (d) metastatic tumor deposits in the cervical lymph nodes ihc x100 (a) tumor cells showing strong immunopositivity to keratin . (b) tumor cells showing weak immunopositivity to ema . (c) tumor cells showing weak immunopositivity to sma . (d) tumor cells showing mild - to - moderate immunopositivity to s-100 adjuvant external beam radiotherapy was advised in view of the large erosive tumor with perineural infiltration and the presence of cervical lymph node metastasis . The patient subsequently received 40 grey of external beam radiotherapy to the tumor bed and neck . Insight into the behavior of this tumor is based on a limited number of case reports . The peak incidence is reported to occur in the fifth to seventh decades of life with a female preponderance . (1.62: 1) the mean age at presentation was 54.45 years (range 1789 years). The most common presenting sign was as a painless swelling of the jaw, often with mobility of the teeth in that region . Palpable lymph nodes on initial presentation were noted in about 10% of the reported cases . The etiology of this neoplasm remains poorly understood and the diagnosis of ccoc depends on the exclusion of other clear cell tumors . The biphasic pattern is the most common, in which clear cell nests are intermixed with polygonal cells having eosinophilic cytoplasms . The ameloblastomatous pattern is the least common and has palisaded ameloblastomatous cells at the periphery of clear cell nests . The case that we report was characteristic of the monophasic pattern . Ccoc must be distinguished histopathologically from clear cell carcinoma arising from salivary glands and other benign odontogenic tumors which may also have clear cells, like ameloblastoma and calcifying epithelial odontogenic tumor . Metastatic tumors, such as metastatic renal clear cell carcinoma must also be ruled out . The presence of perineural or vascular invasion is an indicator of malignancy and can be used to identify ccocs . The treatment of choice as agreed upon by most authors is wide excision of the tumor, including the adjoining soft tissue when involved . The recurrence rate following such treatments appears to be much lower than when treated by enucleation or curettage . Elective neck dissection has been performed by several authors and it is interesting to note that none of these patients had recurrences . Adjuvant radiation therapy is generally considered in cases with extensive soft tissue invasion, perineural spread, lymph node metastasis with extra nodal involvement or in those where tumor - free margins are not possible . Long - term follow - up of patients with ccoc is imperative in view of the aggressive biological potential.
Suicidal behavior is known to exist on a continuum ranging from occasional suicidal ideas to serious contemplation to planning to attempt and finally culminating in completed suicides . Understanding this continuum suggests that suicides can be prevented if appropriate steps are taken in identifying the persons at risk . One of the important risk factors for completed suicides is history of previous suicidal attempt . It is suggested that suicide attempters have about 10 times higher risk of future attempts and completed suicides . Although there is reasonable data from india on understanding the risk factors for completed suicides and attempted suicides in adult population, there is relative lack of data for children and adolescents . Studies suggest that about one - fourth to three - fourths of deaths in the young population in india could be due to suicides . The handful of studies which have evaluated the different levels of suicidal behavior in children and adolescents report that 6% and 21.7% of children and adolescents report current and life time suicidal ideations respectively and about one - tenth of the study population (11.7%) report suicidal ideations in last 1 year . In terms of suicide attempt 8% of children and adolescents studies from india which have evaluated the risk factors for suicidal behavior have reported that suicidal behavior is more commonly seen in those belonging to hindu religion, older adolescent, female gender and working for income after school / college . Other important risk factors which have been shown to predict suicidal behavior in children and adolescents include school and academic related problems, family pathology, actual or anticipated punishment, failure in love, social conflict, history of suicide by a friend, presence of physical illness, psychiatric morbidity in parents in the form of alcoholism, generalized anxiety disorder and depression . Death of one of the parents by suicide has also been shown to be a strong predictor of suicidal behaviour among offspring and this association persists despite controlling for comorbidity of parental disorders and for the presence of mental disorders among offspring . With regards to psychiatric morbidity studies suggest that only one third to half (30 - 52%) of the adolescents who present with attempted suicide or deliberate self - harm have psychiatric morbidity . However, it is important to recognize that most of these studies have included small study sample and only occasional study has focused on children and adolescents presenting with deliberate self - harm . Accordingly, the aim of this retrospective chart review was to study the socio - demographic and clinical profile of children and adolescents presenting with intentional self - harm and referred to psychiatry consultation liaison (cl) services for evaluation . This study was conducted in a tertiary care multicentric public sector hospital in north india . The department of psychiatry runs round the clock cl services to all the wards and emergency services of the hospital . Most of the cases who come to emergency or are admitted in various wards, who have history suggestive of self - harm / attempted suicide are usually referred to psychiatry cl services . Each of these patients is evaluated in detail initially by a trainee (junior) resident under the supervision of a senior resident (qualified psychiatrist). Then all the cases are evaluated by the consultant psychiatrists and final diagnosis is made and management is carried out . During the evaluation of the case, information is obtained from as many sources as possible including the family members and diagnosis is made as per the international classification of diseases-10 . The cases are followed - up by the cl psychiatry services until the patient is either discharged, has a fatal outcome or is transferred to the psychiatry ward for further management . All the cases seen by the cl psychiatry team are recorded in a registry and the cl psychiatry team maintains separate records of all the cases seen . The data included in the registry include age, gender, psychiatric diagnosis, physical diagnosis, treatment given and outcome of the patient at the time of last evaluation . Prior to 2010, only the cases seen in various wards were entered into the registry and from 2010 all the cases seen in various wards and emergency services are also entered into the registry . This chart review involved screening of the cl psychiatry registry to identify patients aged less than equal to 19 years who were diagnosed to have intentional self - harm by the cl psychiatry team during the period of 2000 - 2012 . Once the cases were identified the treatment records maintained by the cl psychiatry team of these patients were traced and the data was extracted in a structured performa designed for the study . Demographic information was extracted pertaining to the age, gender, marital status, educational qualifications, present occupation, religion, place of residence and the type of family . Clinical information relating to the method used, preceding life events, psychiatric morbidity and family history of psychiatric illness and any use of substances was also extracted . Descriptive analysis was conducted for demographic and clinical variables and included mean, standard deviations, frequencies and percentage . Comparisons were carried out by using chi - square test, fisher exact test and mann whitney u - test . This study was conducted in a tertiary care multicentric public sector hospital in north india . The department of psychiatry runs round the clock cl services to all the wards and emergency services of the hospital . Most of the cases who come to emergency or are admitted in various wards, who have history suggestive of self - harm / attempted suicide are usually referred to psychiatry cl services . Each of these patients is evaluated in detail initially by a trainee (junior) resident under the supervision of a senior resident (qualified psychiatrist). Then all the cases are evaluated by the consultant psychiatrists and final diagnosis is made and management is carried out . During the evaluation of the case, information is obtained from as many sources as possible including the family members and diagnosis is made as per the international classification of diseases-10 . The cases are followed - up by the cl psychiatry services until the patient is either discharged, has a fatal outcome or is transferred to the psychiatry ward for further management . All the cases seen by the cl psychiatry team are recorded in a registry and the cl psychiatry team maintains separate records of all the cases seen . The data included in the registry include age, gender, psychiatric diagnosis, physical diagnosis, treatment given and outcome of the patient at the time of last evaluation . Prior to 2010, only the cases seen in various wards were entered into the registry and from 2010 all the cases seen in various wards and emergency services are also entered into the registry . This chart review involved screening of the cl psychiatry registry to identify patients aged less than equal to 19 years who were diagnosed to have intentional self - harm by the cl psychiatry team during the period of 2000 - 2012 . Once the cases were identified the treatment records maintained by the cl psychiatry team of these patients were traced and the data was extracted in a structured performa designed for the study . Demographic information was extracted pertaining to the age, gender, marital status, educational qualifications, present occupation, religion, place of residence and the type of family . Clinical information relating to the method used, preceding life events, psychiatric morbidity and family history of psychiatric illness and any use of substances was also extracted . Descriptive analysis was conducted for demographic and clinical variables and included mean, standard deviations, frequencies and percentage . Comparisons were carried out by using chi - square test, fisher exact test and mann whitney u - test . During the period of 2000 - 2012, 9830 referrals were received by cl psychiatry team from various wards and about 2450 numbers of referrals were received from various emergency services during the period of 2010 - 2012 . Out of these, 101 cases (0.82%) were diagnosed with intentional self - harm in adolescents by the cl psychiatry team . The mean age of this sample was 17.0 years (standard deviation 1.58 years) with youngest patient being 12-year - old child . The interquartile range of the sample was 16 - 18 years and median was 17 years . The study sample comprised of 40 males (39.6%) and 61 females (60.4%). As you can see in table 1, the most common methods of intentional self - harm was consumption of insecticide (62.4%) followed by corrosives (10.9%). Violent methods (hanging, strangulation, jumping from height, self - stabbing and self - immolation) were used by 11 individuals (10.9%), 9 out them were males . It was seen that males had significantly increased likelihood of using violent methods than females (fischer's exact test p = 0.006). Self - harm methods used in the entire sample (n = 101) two of these patients had expired (2.0%) during the hospital stay and in 99 patients (98.0%) the intentional self - harm was not associated with fatal outcome . The use of violent method was related to poorer survival outcome (fischer's exact value = 0.011), but not related to the age (mann whitney u = 405, p = 0.310). However, detail treatment records were available for only 57 cases (56.4% of the sample). The demographic and clinical characteristics of the sample for which the detailed case records were available are shown in table 2 . Majority of the patients were students, hindus, belonged to nuclear families of middle socio - economic status . Demographic and clinical characteristics of patients whose case records available (n = 57) the patients for whom detailed records were available did not differ from those patients for which detailed records were not available in terms of gender (2 = 0.056, p = 0.814), age (mann whitney u = 1215, p = 0.782), marital status (fisher's exact test p = 1.000), violent methods being used (2 = 0.018, p = 0.893) and outcome (fisher's exact test value = 1.000). The precipitating events of attempting self - harm included inter - personal relationship (ipr) issues with family members in about half of the patients, followed by stress of failure in examinations . When we looked at the specific interpersonal familial issues, the events which precipitated self - harm included issues like altercation with one or either the parents or sibling mainly related to the demands of patients being not met . Having ipr issues with family members was not associated with type of family (nuclear / non - nuclear), the locality (2 = 0.338, p = 0.561) or the gender of the patient (2 = 0.011, p = 0.916). However, the presence of ipr issues as precipitating event for self - harm was associated with family history of psychiatric illness, most of which included alcohol use disorder (fisher's exact test value = 0.048). India has the largest population of adolescents in the world . According to the census of 2001, 45% of population of india is in the age range of 0 - 19 years, with one - fifth of the population (21.8%) in the age group of 10 - 19 years . Considering the fact that about one - fourth to three fourths of deaths in the young population in india could be due to suicides, it is very important to identify the risk factors associated with suicide so that preventive strategies can be planned . One of the important precursors of completed suicides is suicidal attempt or intentional self - harm and evidence suggest that there is certain level of overlap in the risk factors associated with completed suicides and suicide attempters . Hence, understanding the profile of suicide attempts can help in prevention of completed suicides . The present study attempted to study the profile of patients referred to cl psychiatry team after an attempt of self - harm . Present study suggests that majority of patients who attempt self - harm are females and this is in keeping with international literature and studies from india . However, this is in contrast to indian literature involving patients who are in the adult and elderly age group, which suggests preponderance of males . The present study also shows that the patients who attempted self - harm more frequently belonged to nuclear families . This possibly reflects the lack of emotional support available to the children, which possibly contributes to the self - harming behavior . Similar to previous report, self - harm behavior was more common in those belonging to hindu religion and older adolescents . In the present study too most of the adolescents who presented with self - harm were aged 16 - 18 years . In the present study, the most common method of self - harm was poisoning, by consumption of insecticides and that too organophosphates . This is similar to the method of self - harm reported in the previous studies from india, involving adolescents, as well as adults . This may be due to easy availability of insecticides as india is an agrarian based economy and people routinely use insecticides for use in the farms as well as for storage of grains in the houses . Violent methods of self - harm were used by about 10% of the sample, that to was more commonly by males . This is in keeping with the western literature which suggests that adolescent males usually use more lethal and irreversible methods to attempt self - harm and suicide and more frequently results in completed suicide . Studies from other parts of india have also reported interpersonal problems with parents, physical abuse by parents, feeling of being neglected by parents, conflicts with parents or siblings and parental disharmony to be associated with self - harm behavior . Findings of the present study too support this, as in half of the patients; act of self - harm was precipitated by ipr problems with family members . In the present study too like previous studies involving adolescents, academic issues and failure in love were associated with self - harm in few patients . However, in contrast to the previous studies, none of the patient in the present study had physical illnesses . Slightly more than one - fifth of the patients had diagnosable psychiatric illness in the present study, this is less that the reported range of 30 - 52% in previous studies from india which have studies children and adolescents presenting with attempted suicide or deliberate self - harm . In the present study, about one - tenth of patients had family history of alcohol dependence, which has been identified as a risk factor for self - harm in adolescents . Further present study suggests that interpersonal problems were more common in households where one of the parents had a psychiatric disorder, especially in the form of substance use disorder . Based on the above study it can be concluded that self - harm is more common in children and adolescents who are females, from nuclear families, middle socio - economic status and hindu by religion . The common method of self - harm in adolescents is by ingestion of insecticides and the self - harm behavior is often precipitated by interpersonal problems in the family context . About one - fifth of the patients have psychiatric morbidity . Taking the findings of the present study into consideration there is an urgent need to develop a clear policy for the sale and purchase of insecticides . Further, the families which use the same for occupational use should be advised to store the insecticides safely, so that access to the same can be minimized . Further considering the fact that in most cases self - harm behavior was precipitated by interpersonal issues, there is an urgent need for enhancing the parenting skills of the parents . With regards to the academic issues, there is a need to reduce the stress of performance and failure in exams should not be stigmatized . Schools should have adequate number of counselors who could guide the students and their parents based on the intelligence and aptitude of the adolescents . In terms of secondary prevention, the psychiatric morbidity at any level, i.e., in parents and children and adolescents present study was limited by its retrospective design and limited to the patients referred to cl psychiatry services . It may not be true reflection of entire population of treatment seekers presenting to the hospital . It is possible that for many of the cases who were too sick to be interviewed, a psychiatric consultation was not sought . Furthermore, it is possible that the patient or the family members did not disclose crucial information fearing the legal implications of suicidal attempts in india . Hence, the findings of the study should be interpreted in the light of the above mentioned limitations.
Hemoglobin is a remarkable molecular machine that uses motion and small structural changes to regulate its action . Once the first heme binds oxygen, it introduces small changes in the structure of the corresponding protein chain . The main function of red blood cell is transfer of o2 from lungs to tissue and transfer of co2 from tissue to lungs . Glassy carbon derives its name from exhibiting fracture behavior similar to glass, from having a disordered structure over large dimensions (although it contains a graphitic microcrystalline structure), and because it is a hard shiny material, capable of high polish [35]. Glassy carbon is particularly useful in electrochemical applications because of its low electrical resistivity, impermeability to gases, high chemical resistance, and because it has the widest potential range observed for carbon electrode . Glassy carbon was first prepared by yamada and sato in 1962 by the hight emperature pyrolysis of phenolic resin, and later by davison who used cellulose as the starting material [6, 7]. They concluded that glassy carbon consists of long microfibrils that twist, bend, and interlock to form interfibrillar bonds, and that these microfibrils are randomly oriented . Because of the desire to impart selectivity to electrochemical reactions and to control electron transfer kinetics, several investigators have utilized adsorption or covalent bonding of catalysts to the glassy carbon surface . The intent of these modifications is to control the interaction of molecules and ions with the electrode surface . Small fibers in the submicron range, in comparison with larger ones, are well known to provide better filter efficiency at the same pressure drop in the interception and inertial impaction regimes [10, 11]. While smaller fiber size leads to higher - pressure drop, interception and inertial impaction efficiencies will increase faster, more than compensating for the pressure drop increase . Thus, in the particle size of interest, that is, from submicron and up, better filter efficiency can be achieved at the same pressure drop, or conversely, the same filter efficiency at lower pressure drop can be achieved with smaller fiber sizes . Polymeric nanofibers can be made using the electrospinning process, which has been described in the literature and in patents . Electrospinning uses an electric field to draw a polymer solution from the tip of a capillary to a collector . A voltage is applied to the polymer solution, which causes a jet of the solution to be drawn toward a grounded collector . The fine jets dry to form polymeric fibers, which can be collected on a web . The electrospinning process has been documented using a variety of fiber - forming polymers [15, 16]. By choosing a suitable polymer and solvent system, nanofibers with diameters in the range of 402000 nm can be made . Pvp is a kind of polymers which is able to adsorb through many points of the molecule on the surface of steel, copper, and zinc and reduce the corrosion rate of the material [1719]. Pvps are considered as food additives and, hence, their addition will not raise health concerns for treating ro water . However, the effect of the molecular weight of polyvinylpyrrolidone, its concentration, and temperature on the corrosion properties of 316 l stainless steel in reverse osmosis water were not evaluated . Electrospinning is a process of applying a high - voltage electric field (several to tens of kilovolts) to generate electrically charged jets from polymer solutions or melts and further to produce polymer (nano) fibers . This technique is quite similar with the commercial process for drawing microscale fibers; however, it is more suitable for generating nanofibers, because the elongation can be accomplished by a contactless scheme through the application of an external electric field . Generally, generation of porous surface on a bulk electrospinning nanofiber can be realized through two different ways . The first one is based on the selective removal of a component from nanofibers made of a composite or blend material, while the other one involved the use of phase separation of different polymers during electrospinning under the application of proper sinning parameters . For instance, in pla / pvp electrospinning nanofibers, more porosity can be generated when the two materials are loaded in equal amounts comparing to the 11 corresponding products by different proportion of pla / pvp . It can be attributed to the rapid - phase separation and solidification in the spinning jet [1417]. Pla is contraction name of polylactic acid, and it is a thermoplastic polymer made from lactic acid and has mainly been used for biodegradable products, such as plastic bags and planting cups . It was found that the average diameter of the pla / pvp / nanofibers lead fibers became larger, and the morphology of the fibers became finer with the content of pla increasing . The formation of pores is also affected by the solvent vapor pressure and the humidity in atmosphere . The cooling effect that comes from rapid evaporation of a highly volatile solvent might induce the polymers to separate into different phases in liquid jet . Because of evaporative cooling and condensation, water droplets could also be formed within the fibers to promote the formation of porous nanofibers [2022]. The electrospun nanofibers exhibit several unique features, which enable the prevalent utilization of them . Because electrospinning is a continuous process without any contact force for elongation, the fibers can be as long as several kilometers and can be further assembled into a 3d mat with porous structure . At the same time, electrospun fibers can have a thinner diameter and surface - to - volume ratio, due to the presence of porous structure . In addition, due to the simple fabrication process and the diversity of suitable materials, the electrospinning technique and its resultant nanofiber product have attracted increasing attention . These properties potentiate the use of the electrospun nanofibers in various applications such as reinforced composites, nanofiber - based membranes, nanofiber - based support for enzyme, and catalyst [2325]. Manganese(ii) nitrate tetrahydrate, and poly(vinylpyrrolidone) (pvp, mw = 1,300,000) were purchased from sigma . 0.1 m phosphate buffer solutions with various ph values were prepared by mixing stock standard solutions of na2hpo4 and nah2po4 and adjusting the ph values with naoh and h3po4 solution . All solutions used in the experiments were prepared with deionized water generated by a barnstead water system . 44 wt% mn(no3)2, 11 wt% agno3, and 44 wt% pvp were dissolved in dmf . The solution was kept under magnetic stirring for 2 h and then loaded into a plastic syringe equipped with a 23-gauge needle made of stainless steel . Electrospinning process was conducted at an applied voltage of 20 kv with a feeding rate of 0.3 ml / h and a collection distance of 15 cm . The nanofibers were collected on aluminum foil and then calcined under air atmosphere at 500c for 3 h for the degradation of pvp and the decomposition of mn(no3)2 and agno3: (1)4mn(no3)2mn2o3 + 8no2+ o22agno32ag+2no2+ o2. Electrospinning is a process of applying a high - voltage electric field (several to tens of kilovolts) to generate electrically charged jets from polymer solutions or melts and further to produce polymer (nano) fibers . So, in this study, we used from this method to produce mn2o3-ag nanofibers . The most commonly used carbon - based electrode in the analytical laboratory is glassy carbon (gc). It is made by pyrolyzing a carbon polymer, under carefully controlled conditions, to a high temperature like 2000c . An intertwining ribbon - like material results with retention of high conductivity, hardness, and inertness . Glassy carbon electrode (gce, dia . 3 mm) was polished with 1 m and 0.05 m alumina slurries sequentially and then rinsed with di water . After that, the electrode was sonicated in deionized water and finally dried under ambient conditions . To prepare the modified gce, the mn2o3-ag nanofibers / glassy carbon electrode was placed into a fresh pbs including 5 mg ml hb (ph 7.0, 3 to 5c) for 8 h. at the end, the modified electrode was washed in deionized water and placed in pbs (ph 7.0) at a refrigerator (3 to 5c), before being employed in the electrochemical measurements as the working electrode . A jeol 6335f field - emission scanning electron microscope (sem) was used to examine the morphology and the size of the as - prepared nanofibers . More detailed morphology and selected area electron diffraction (saed) patterns of mn2o3-ag nanofibers were obtained with a tecnai t12 transmission electron microscope (tem) operated at 120 kv . Xrd pattern was obtained with oxford diffraction xcaliburtm px ultra with onyx detector to study the crystal structure of mn2o3-ag nanofibers measurements which were carried out with a potentiostat / galvanostat (model 263a, eg&g, usa) using a single compartment voltammetric cell, equipped with a platinum rod auxiliary electrode . All experiments were conducted using a three - electrode electrochemical cell (10 ml volume with a working volume of 5 ml), with a working electrode, an ag / agcl reference electrode, and a platinum wire counter electrode . Electrochemical measurements were carried out with a potentiostat / galvanostat (model 263a, eg&g, usa) using a single compartment voltammetric cell, equipped with a platinum rod auxiliary electrode . Figure 2(a) presents a typical sem image of electrospun precursory pvp - mn(no3)2-agno3 nanofibers . Pvp was used for characterization of mn2o3-ag nanofibers, because pvp binds to polar molecules exceptionally well, owing to its polarity . This has led to its application in coatings for increase of photo quality in microscopic studies . When dry, it is a light flaky powder, which readily absorbs up to 40% of its weight in atmospheric water . In solution pvp is a branched polymer, that is, its structure is more complicated than linear polymer, but it too is in a two - dimensional plane . Moreover, because mn2o3-ag nanofibers have polar groups too, a good bind was created between this nanofiber and pvp . After calcination, the as - prepared mn2o3-ag composite nanofibers (figure 2(b)) exhibit a porous network structure, and their surfaces are no longer as smooth as the precursory nanofibers . Such feature endows the nanofibers with high surface - to - volume ratio, which could provide not only a large surface area for hb loading but also a large interface for direct electron transfer of hb . As a comparison, the nanofibers prepared by single metal salt (mn(no3)2) with pvp and its calcined product (mn2o nanofibers) are presented in figures 2(c) and 2(d), respectively . The xrd spectrum of mn2o3-ag nanofibers matches the combination of the standard spectrum of jcpds 41 - 1442 (mn2o3) and jcpds 04 - 0783 (ag). The formation of face - centered cubic crystalline mn2o3 is revealed by the diffraction peaks at 2 values of 32.951, 38.234, 45.178, 49.347, 55.189, and 65.806, corresponding to (111), (200), (220), (311), (222), and (400) crystal planes, respectively, while the diffraction peaks at 2 values of 38.116, 44.277, and 64.426, which correspond to (111), (200), and (220) crystal planes, respectively, indicate the formation of cubic crystalline ag . The cyclic voltammograms (cvs) of different modified electrodes were obtained in 0.1 m pbs with ph 7.0 . Compared with bare gc electrode, the background current of mn2o3-ag - nanofibers - modified electrode is apparently larger, which indicates that the effective electrode surface area is significantly enhanced by use of mn2o3-ag nanofibers to modify the electrode . However, the cvs of hb / mn2o3-ag nanofibers / gc electrode give a pair of well - defined redox peaks at 75 mv at scan rate of 100 mv / s, characteristic of heme fe(iii)/fe(ii) redox couples of hb, suggesting that direct electron transfer has been achieved between hb and modified electrode . The electron transfer of the proteins, at the bare electrodes is very slow so that the redox peak of proteins can usually be observed . Figure 4(a) shows a cyclic voltammogram (cv) of the bare glassy carbon electrode . Figure 4(b) shows a cyclic voltammogram of hb / mn2o3-ag nanofibers / glassy carbon electrode in 0.1 m phosphate buffer at ph 7.0 . The hb showed quasireversible electrochemical behavior with a formal potential of 49 mv (versus ag / agcl); cathodic and anodic peaks were not observed using the bare graphite electrode . This shows that mn2o3-ag nanofibers act as a facilitator of electron transfer from the redox species of hb to the electrode surface and vice versa . These results are in line with the previous work that explains the behavior of nanoparticles as the facilitators of electron transfer . In figure 4(a), one sees cyclic voltammogram for bare and modified glassy carbon electrode in 50, 100, 200, 300, 400, and 500 scan rates . These data showed that the mn2o3-ag nanofibers were successfully assembled on the glassy carbon electrode surface . The redox peak currents increase linearly with the square root of the scan rate . With an increasing scan rate, the anodic peak potential of adsorbed hb shifted to a more positive value, while the cathodic peak current shifted in a negative direction . The redox peak currents were proportional to the scan rate (figure 5); thus, the electrode reaction was typical of the surface - controlled quasireversible process . One can see in figure 5(b) the dependence of the anodic and cathodic peak currents on the scan rates that the red line is cathodic and the blue line is anodic . The plot of cathodic peak potentials versus the logarithm of scan rates gave a charge transfer coefficient of 0.52 . For scan rates from 50 to 500 mv / s, the electron transfer rate constant k s was estimated to be (1.9) s. these statements discussed below indicate that hb is strongly adsorbed onto the surface of modified electrode . As could be seen in figure 5(c), in the range from 200 to 500 mv s, the catholic peak potential (e pc) changed linearly versus lnv with a linear regression equation of y = 0.2425x 0.4254, r = 0.945, according to the following equation [2628], (2)ep = e+rtnfrtnflnv, where is the cathodic electron transfer coefficient, n is the number of electrons, r, t, and f are gas, temperature, and faraday constant, respectively (r = 8.314 j molk, f = 96493 c / mol, t = 298 k), and n is calculated to be 0.52 . Given 0.3 <<0.7 in general, it could be concluded that n = 1 and = 0.52 . From the width of the peak at midheight and low scan rate, therefore, the redox reaction between hb and modified graphite electrode is a single electron transfer process . In order to calculate the value of apparent heterogeneous electron transfer rate constant (k s), the following equation was used [2628]: (3)logks=log(1)+(1)loglog(rtnfv) (1)nfep2.3rt . The k s was calculated to be 1.90 s. moreover, this k s indicates that transfer of electron is good and fast in hb - modified glassy carbon electrode . The designed biosensor is introduced as a hydrogen biosensor because it has oxidation and reduction reactions in hb; when fe changes to fe, the oxidation reaction occurred, and when fe changes to fe, the reduction reaction occurred . The electrocatalytic reactivity of hb / mn2o3-ag nanofibers / gc electrode toward h2o2 was investigated by cv . Figure 6 displays the cyclic voltammograms obtained for the hydrogen peroxide biosensor in pbs (ph 7.0) containing varied concentration of h2o2 in the absence of oxygen . The catalytic reduction of hydrogen peroxide at the biosensor can be seen clearly in figure 5 . With the addition of h2o2, the reduction peak current increases obviously, while the oxidation peak current decreases (figures 6(a) and 6(b)), indicating a typical electrocatalytic reduction process of h2o2 . However, no similar cathodic peak corresponding to the reduction of h2o2 can be observed at bare gc, mn2o3-ag nanofibers / gc electrode under the same condition, so it can be concluded that hb immobilized on mn2o3-ag nanofibers / gc electrode shows good catalytic activity toward hydrogen peroxide . The sensor was found to have sensitivity in the range of 20 to 700 m based upon the mean of the slope found from the points on the response curve . As can be observed, the sensor response shows good linearity in this range . The correlation factor, r, was found to be 0.9962 . In order to obtain an efficient biosensor for h2o2, the influence of ph and applied potential on the response of hb / mn2o3-ag nanofibers / gc electrode were investigated . The change of chronoamperometric current with the ph under constant hydrogen peroxide concentration (50.0 m) is shown in figure 7 . This designed biosensor possesses good stability and reproducibility and achieves 95% of the steady - state current in less than 5 s. a new biosensor for h2o2 was prepared based on hb / mn2o3-ag nanofibers / glassy carbon electrode . Hb retained well in mn2o3-ag nanofibers / gce, which combined the utilities of mn2o3-ag nanofibers facilitating the electron transfer . At hb / mn2o3-ag nanofibers / gce, the cyclic voltammogram exhibits a pair of redox peaks corresponding to a surface - controlled electrode process with a single - proton transfer.
An 87-year - old female presented to the prince charles hospital medical imaging service requiring insertion of a peripherally inserted central catheter (picc). The catheter was inserted by an operator who was a radiographer sonographer, credentialed locally in performing the procedure . The catheter's tip position was confirmed by a radiology registrar prior to discharge from medical imaging (fig.1). Two hours following the procedure, a second read of the fluoroscopic imaging was performed by an interventional radiology consultant who reported the picc tip position in the azygous vein . The patient was recalled and under fluoroscopic imaging the catheter was retracted to the distal superior vena cava (svc) (fig.2). Prior to commencing the picc insertion procedure, previous imaging was reviewed, a verbal discussion between the operator and patient occurred explaining the procedure, confirming it was the correct procedure and provided the patient with a final opportunity to ask questions . The patient was placed in a supinated position on a fluoroscopy table, with the left arm abducted laterally and placed supine on an arm board . A tourniquet was positioned as close to the axilla as possible and the deep veins of the arm were sonographically assessed with a high frequency linear transducer from cubital fossa to the axilla (tourniquet). A sterile pack including basic procedural ancillary equipment was used along with a sterile pre - packaged picc line kit . The operator employed personal and procedural protective equipment including lead gown, face mask, hair cap, 5 min aseptic hand wash, sterile gown and sterile gloves . Aseptic technique was employed for the procedure, with the patient's upper limb prepared using a solu-i.v.md maxi swabstick, containing 2% chlorhexidine gluconate in alcohol . A fenestrated sterile drape was then placed over the marked cannulation site to maintain the maximum barrier . The procedure required administration of 1 ml 1% lignocaine, which was infiltrated at the level of the cannulation site into the subcutaneous fat layer under ultrasound guidance . A modified seldinger technique was employed to insert a 5fr arrow (arrow international, reading, pa) double lumen pressure injectable picc line into the venous system under ultrasound guidance . The picc line was advanced blindly 45 cm and then fluoroscopy used to position the tip of the catheter in the svc . A radiology registrar confirmed the catheter's tip position in the svc and the procedure was completed by securing the picc with a catheter stabilization device called a statlock (bard, c. r. bard, inc ., covington, ga), applying sterile dressings to the puncture site region and flushing both lumens of the catheter with normal saline . They are indicated in patients requiring long - term venous access including antibiotics, chemotherapy, total parental nutrition or in patients with poor venous access.1 numerous complications encountered in picc insertions are reported in the literature, with the most common types being vascular in nature, such as haemorrhage and thrombus formation; and infection of both the catheter line and the skin puncture site . Intraluminal and extraluminal occlusion causing malfunction and catheter malposition are also reported complications.2 optimal catheter position described in the literature is with tip placement in the distal third of the svc.3 ideally and more precisely, it is at the location of the cavoatrial junction . It is practice at our institute that a picc line tip position is checked by performing either a chest radiograph, or with the use of fluoroscopy image, such as that produced by a dedicated room outlined above . In our practice once an image has been obtained by the radiographer sonographer, the operator would ascertain if they believed the catheter had been advanced successfully to the distal third of the svc . Medical officer confirmation is then obtained prior to the catheter's use as per local protocol . It is not unusual for the radiographer sonographer to manipulate a catheter that was initially advanced into a jugular, either subclavian, axillary or peripheral arm vein segment during a picc insertion procedure . In some circumstances a hand injection of intravenous contrast is required to map or describe vasculature to assist optimal tip position . Malposition of a picc line tip has been reported to cause serious central complication including cardiac tamponade, air embolism, pneumothorax, haemothorax, hydrothorax, thoracic duct injury and extraluminal occlusion.2 the azygous vein, meaning unpaired, is one of the seven veins of the thorax . It originates opposite the first or second lumbar vertebra, courses to the right of the vertebral column and arches ventrally over the superior aspect of the right main bronchus just distal to the level of the tracheal bifurcation . Many veins drain into the azygous including the hemiazygous vein.4,5 the inadvertent placement of a catheter into the azygous vein is reported as rare . Although an azygous catheter tip placement is recognized as an alternative in some patients with co - morbidity such as severe venous occlusion, the azygous is more susceptible to complications.6 it is proposed that due to the smaller calibre of the vessel (68 mm) there is a greater risk of thrombus formation, perforation of the vessel, stenosis and extravasation.5 reports in the literature describe a small number of risk factors that increase the likelihood of catheter preferential course into the azygous vein . These conditions can in turn significantly dilate the azygous vein calibre allowing a more easily accessed course.6 this risk factor is particularly relevant for our institute as it is a tertiary referral centre for cardiothoracic care . Reported that a picc is also more or less heading in the direction of the azygous vein when being inserted from the patient's left side due to human anatomy . They proposed a decreased probability of azygous placement if a right - sided approach is used.5 performing an anteroposterior chest image such as an x - ray or fluoroscopic image can indicate a high probability that the picc has entered the azygous vein . Pua states that tracheobronchial angle or the location that the trachea bifurcates to right main bronchus is the precise location of the azygous vein arching over the right main bronchus to enter the svc.6 therefore, a picc line would be seen tracking laterally at the level of the trachea bifurcation, with an acute angle back towards the midline . The course (of catheter) would then continue medially across the midline of the spinous process as it heads caudally . Catheter kink in a case of azygous catheter placement.6 our case study demonstrated that catheter foreshortening was not evident and that the initial lateral course of the catheter, followed by a medial and caudal course, was an indicating sign . In our practice it is commonplace to use a picc kit that has a limiting catheter guide wire / stylet length such as 70 cm . At this length it may be unable to initially trouble shoot for suspected azygous tip placement during an insertion procedure . We have found that with significant length of wire a brief test for projecting the course of catheter can be performed assessing the projected course . If the wire can be demonstrated coursing either below the diaphragm and right of midline (inferior vena cava) or into the cardiac shadow while remaining above the diaphragm, one can more confidently rule out a suspected azygous placement . Furthermore, a hand injection of radiopaque contrast media during the insertion procedure can be used to delineate vessel anatomy as an alternative to a wire - based trouble shooting approach . Highly accurate diagnostic information regarding picc line tip malposition report diagnosing a central venous catheter with the tip position in the azygous vein.5 however, as a frontline picc check modality, it is the opinion of the authors of this paper that this may not be cost - effective or as timely as a during procedure fluoroscopic imaging. Knowledge of venous anatomy beyond the major vessels of the deep system in the upper limb is invaluable . Azygous vein anatomy and image interpretation of suspected azygous catheter placement should be highlighted to all healthcare professionals with a stake in quality picc line placement.
Root - end filling materials are in contact with periradicular tissues, so they should have good sealing ability and be biocompatible to promote healing . Because many of these materials are not able to ensure a perfect seal, a microscopic space is likely to create at the root - end cavity / filling interface, through which micro organisms and their products can penetrate . Thus, besides good sealing ability and biocompatibility, root - end filling materials should ideally have some antibacterial activity . More over, these materials should have low solubility to avoid that components leaching from the endodontic space might exercise undesirable biologic effects on the surrounding tissues . Finally, radiopacity is a very important property for all root - end filling materials because the material has to be detected radiographically, and thus distinguished from surrounding anatomic structures . Intermediate restorative material (irm) has addressed some drawbacks including moisture sensitivity, irritation to vital tissues and difficulty in handling . Mta was developed by parirokh and torabinejad to address shortcomings of commonly used root - end filling materials . Several studies have recognized mta as a bioactive material, that is, hard tissue conductive, hard tissue inductive, and biocompatible . The sealing ability of mta in root - end fillings was found to be superior to that of amalgam, irm, and super - ethoxy benzoic acid (eba) with both dye and bacterial leakage methods . However, its handling characteristics are less than ideal as a result of long setting time and difficulty in maintaining consistency of mixture . Several new calcium silicate - based materials have recently been developed; aiming to improve some mta drawbacks such as its difficult handling property and long setting time . Biodentine is amongst these materials and is claimed to be used as a dentine restorative material in addition to endodontic indications similar to those of mta . The purpose of the present study is to evaluate and compare the biological (cytotoxicity and antibacterial efficacy) and chemical - physical (solubility, ph, and radiopacity) properties of four different root - end filling materials: biodentine (septodont, saint - maur - des - fosses, france), mta - angelus (angelus, londrina, pr, brazil), proroot mta (dentsply, johnson city, tn, usa), and irm (dentsply, johnson city, tn, usa). Transwell insert methodology (sigma - aldrich, st louis, mo, usa) was performed . Cytotoxicity was assessed with mdpc-23 cells grown in the lower chamber of a 24-mm diameter transwell plate with a 0.3 mm pore size polycarbonate membrane (sigma - aldrich, st . The transwell membrane of the inner chamber, filled with the paper disks, were placed into the lower chamber of the 24-well culture plate containing at the bottom 5 10 cells / well and incubated at 37c in 5% co2 atmosphere for 24, 48, and 72 h, respectively . Some wells were incubated with only tissue culture media (negative control) whereas others with a 10% dilution of 30% h2o2 (positive control). At the end of incubation time the results were presented as percentage of cell viability referred at cells incubated in absence of materials set at 100% . The vitality test to alamar blue reagent acts as an indicator of cell health, determining the reducing power in order to measure quantitatively the proliferative capacity . The reagent was added in a ratio of 1:10 to the cell culture and then the cells were kept in the incubator for 3 - 4 h at 37c . The degree of fluorescence and the relative values of absorbance were then acquired by using a spectrophotometer at a wavelength of 595 nm . For a further control, the percentage of vitality of murine odontoblasts, at 72 h, was also assessed with the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (mtt) assay (sigma - aldrich, st louis, mo, usa). The mtt test is a standard colorimetric assay for measuring the activity of enzymes that reduce the mtt to formazan (a salt blue) in the mitochondria, giving the substance a blue / purple color . This reaction was assessed and measured by the spectrophotometric reading of the sample, at a wavelength of 570 nm by a microplate reader (bio - rad laboratories, hercules, ca, usa). The streptococcal strains used in this study were provided from the culture collection of university of goteborg (ccug): streptococcus mutans (ccug 35176), s. salivarius (ccug 11878), and s. sanguis (ccug 17826). The cultures were grown and maintained in a brain heart infusion (bhi; difco lab ., detroit, mi, usa). S. mutans culture medium was supplemented with 10% (v / v) heat - inactivated horse blood serum (oxoid s.p.a ., the culture of all bacterial strains was statically incubated for 16 h at 37c under aerobic conditions . This overnight culture, used as source for the experiments, was reduced at a final density of 1 10 cells / ml as determined by comparing the optical density at 600 nm (od600) of the sample with a standard curve relating od600 to cell number . Sterile paper discs (diameter: 6 mm, thickness: 1 mm) (watman international ltd . Maidstone, uk) were impregnated with 10 l of each root - endfilling material . Then, bhi - agar plates were incubated with 1 10 cells / ml of an overnight culture of each streptococcal strain at 37c for 20 min . The excess of bacterial suspension was removed from the plates and incubated with the paper disks impregnated with the root - end filling materials at 37c for 24 h. the diameter of the halo formed around the paper disc (inhibition zone) was measured by the same operator in two perpendicular locations with a millimeter ruler with accuracy of 0.5 mm, after 24 and 48 h. the size of the inhibition zone was calculated as follows: size of inhibition zone = (diameter of halo diameter of specimen). All the assays were conducted in triplicate and the results were recorded in terms of the average diameter of inhibition zone . Stainless steel ring molds with an internal diameter of 20 0.1 mm and a height of 1.5 0.1 mm were used for sample preparation . All molds were weighed three times prior to use (accuracy 0.0001 g) on a mettler ae-163 balance (mettler - toledo s.p.a ., novate milanese, mi, italy), which was used throughout the experiment . After filling the molds, a glass plate covered with a mylar strip was placed on top of the molds, exerting a light pressure in order to remove any excess . All samples were left to set for 24 h on a grating in a cabinet at 37c and 100% relative humidity . The samples in their molds were then exposed to air for 15 min, weighed three times, and the average reading was recorded to three decimal places . The specimens of each material were individually placed in tarred bottles, containing 5 ml of distilled water . The bottles were then transferred to an oven at 37c where they remained for 24 h. they were removed from the oven and rinsed with distilled water, which was then collected in the same bottle . Bottles and residues were dried in an oven at 105c, cooled down in desiccators, and weighed . The differences found between this weight and the original bottle weight were divided by the initial dry weight of the specimens and multiplied by 100 . The solubility of the set root - end filling materials should not exceed 3% mass fraction (iso 6876 clause 4.3.6). The samples were stored at 37c, and ph measurement was performed after incubation of 3 and 24 h. the ph value was measured by a digital hi 2210 ph meter (hanna instruments us, woonsocket, ri, usa), previously calibrated . Biodentine, mta - angelus, proroot mta, and irm were mixed and placed into silicon molds measuring 1 mm in thickness and 4 mm in internal diameter . The specimens were covered with glass plates on each side to allow for the removal of excessive material . The specimens were kept in a chamber at 37c and 95% relative humidity for 24 h. ten arrays were set by collecting three samples for each material in order to scan them with prodigy dxa system (ge healthcare, madison, wi, usa). All materials were scanned on a ge healthcare lunar prodigy and idxa in routine clinical manner per manufacturer recommendations . For the prodigy, encore software (ge healthcare, madison, wi, usa) version 9.2 was used for acquisition and 11.4 for analysis; with idxa, encore software version 9.3 was used to acquire scans with version 11.0 used for analyses . The precision assessments were performed in routine clinical manner according to international society for clinical densitometry (iscd) recommendations . College station, tx: statacorp lp . ). To assess the normality of the data obtained from the cytotoxicity assays, the antibacterial tests, the solubility evaluations, and from the radiographic densities; the shapiro wilk test was applied (p <0.05). The number of vital cells was analyzed with tukey's test . Whitney test; the inhibition zones values after 48 h were used for the statistical analysis . Whitney test . To determine whether time influenced the solubility of the pulp capping materials, an analysis of longitudinal data was performed using t - test for paired data (p <0.05). T - test for paired data test was applied to determine whether significant differences existed in ph values after 3 and 24 h of incubation (p <0.05). Tukey's test was then applied to evaluate the differences in ph values among the materials (p <0.05). Radiographic densities obtained from dual - energy x - ray absorptiometry (dxa) measurements were analyzed using tukey's test . Transwell insert methodology (sigma - aldrich, st louis, mo, usa) was performed . Cytotoxicity was assessed with mdpc-23 cells grown in the lower chamber of a 24-mm diameter transwell plate with a 0.3 mm pore size polycarbonate membrane (sigma - aldrich, st . The transwell membrane of the inner chamber, filled with the paper disks, were placed into the lower chamber of the 24-well culture plate containing at the bottom 5 10 cells / well and incubated at 37c in 5% co2 atmosphere for 24, 48, and 72 h, respectively . Some wells were incubated with only tissue culture media (negative control) whereas others with a 10% dilution of 30% h2o2 (positive control). At the end of incubation time the results were presented as percentage of cell viability referred at cells incubated in absence of materials set at 100% . The vitality test to alamar blue reagent acts as an indicator of cell health, determining the reducing power in order to measure quantitatively the proliferative capacity . The reagent was added in a ratio of 1:10 to the cell culture and then the cells were kept in the incubator for 3 - 4 h at 37c . The degree of fluorescence and the relative values of absorbance were then acquired by using a spectrophotometer at a wavelength of 595 nm . For a further control, the percentage of vitality of murine odontoblasts, at 72 h, was also assessed with the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (mtt) assay (sigma - aldrich, st louis, mo, usa). The mtt test is a standard colorimetric assay for measuring the activity of enzymes that reduce the mtt to formazan (a salt blue) in the mitochondria, giving the substance a blue / purple color . This reaction was assessed and measured by the spectrophotometric reading of the sample, at a wavelength of 570 nm by a microplate reader (bio - rad laboratories, hercules, ca, usa). The streptococcal strains used in this study were provided from the culture collection of university of goteborg (ccug): streptococcus mutans (ccug 35176), s. salivarius (ccug 11878), and s. sanguis (ccug 17826). The cultures were grown and maintained in a brain heart infusion (bhi; difco lab ., detroit, mi, usa). S. mutans culture medium was supplemented with 10% (v / v) heat - inactivated horse blood serum (oxoid s.p.a ., rodano, mi, italy) to improve its growth . The culture of all bacterial strains was statically incubated for 16 h at 37c under aerobic conditions . This overnight culture, used as source for the experiments, was reduced at a final density of 1 10 cells / ml as determined by comparing the optical density at 600 nm (od600) of the sample with a standard curve relating od600 to cell number . Sterile paper discs (diameter: 6 mm, thickness: 1 mm) (watman international ltd . Maidstone, uk) were impregnated with 10 l of each root - endfilling material . Then, bhi - agar plates were incubated with 1 10 cells / ml of an overnight culture of each streptococcal strain at 37c for 20 min . The excess of bacterial suspension was removed from the plates and incubated with the paper disks impregnated with the root - end filling materials at 37c for 24 h. the diameter of the halo formed around the paper disc (inhibition zone) was measured by the same operator in two perpendicular locations with a millimeter ruler with accuracy of 0.5 mm, after 24 and 48 h. the size of the inhibition zone was calculated as follows: size of inhibition zone = (diameter of halo diameter of specimen). All the assays were conducted in triplicate and the results were recorded in terms of the average diameter of inhibition zone . Stainless steel ring molds with an internal diameter of 20 0.1 mm and a height of 1.5 0.1 mm were used for sample preparation . All molds were weighed three times prior to use (accuracy 0.0001 g) on a mettler ae-163 balance (mettler - toledo s.p.a ., novate milanese, mi, italy), which was used throughout the experiment . After filling the molds, a glass plate covered with a mylar strip was placed on top of the molds, exerting a light pressure in order to remove any excess . All samples were left to set for 24 h on a grating in a cabinet at 37c and 100% relative humidity . The samples in their molds were then exposed to air for 15 min, weighed three times, and the average reading was recorded to three decimal places . The specimens of each material were individually placed in tarred bottles, containing 5 ml of distilled water . The bottles were then transferred to an oven at 37c where they remained for 24 h. they were removed from the oven and rinsed with distilled water, which was then collected in the same bottle . Bottles and residues were dried in an oven at 105c, cooled down in desiccators, and weighed . The differences found between this weight and the original bottle weight were divided by the initial dry weight of the specimens and multiplied by 100 . The solubility of the set root - end filling materials should not exceed 3% mass fraction (iso 6876 clause 4.3.6). The samples were stored at 37c, and ph measurement was performed after incubation of 3 and 24 h. the ph value was measured by a digital hi 2210 ph meter (hanna instruments us, woonsocket, ri, usa), previously calibrated . Biodentine, mta - angelus, proroot mta, and irm were mixed and placed into silicon molds measuring 1 mm in thickness and 4 mm in internal diameter . The specimens were covered with glass plates on each side to allow for the removal of excessive material . The specimens were kept in a chamber at 37c and 95% relative humidity for 24 h. ten arrays were set by collecting three samples for each material in order to scan them with prodigy dxa system (ge healthcare, madison, wi, usa). All materials were scanned on a ge healthcare lunar prodigy and idxa in routine clinical manner per manufacturer recommendations . For the prodigy, encore software (ge healthcare, madison, wi, usa) version 9.2 was used for acquisition and 11.4 for analysis; with idxa, encore software version 9.3 was used to acquire scans with version 11.0 used for analyses . The precision assessments were performed in routine clinical manner according to international society for clinical densitometry (iscd) recommendations . Stata statistical software: release 12 . College station, tx: statacorp lp . ). To assess the normality of the data obtained from the cytotoxicity assays, the antibacterial tests, the solubility evaluations, and from the radiographic densities; the shapiro whitney test; the inhibition zones values after 48 h were used for the statistical analysis . Whitney test . To determine whether time influenced the solubility of the pulp capping materials, an analysis of longitudinal data was performed using t - test for paired data (p <0.05). T - test for paired data test was applied to determine whether significant differences existed in ph values after 3 and 24 h of incubation (p <0.05). Tukey's test was then applied to evaluate the differences in ph values among the materials (p <0.05). Radiographic densities obtained from dual - energy x - ray absorptiometry (dxa) measurements were analyzed using tukey's test . Descriptive statistics analysis for biocompatibility investigation is reported in table 1 . As regards alamar blue test [table 1], after 24 h there were no differences in the number of vital cells among proroot mta, mta - angelus, biodentine, and the negative control (p> 0.05). Irm showed a significant reduction in the number of vital cells (p <0.05). These results changed uniformly after 48 h and maintained no significant differences among the materials, except for irm . After 72 h all three materials varied from the negative control, in particular proroot mta and mta - angelus gained a similar and lower number of vital cells than the control, while biodentine showed no decrease, as confirmed by the analysis for paired data . When the mtt test [table 1] was applied, no significant differences were recorded among proroot mta, mta - angelus, biodentine, and the negative control (p> 0.05), while irm maintained lower percentages of vitality (p <0.05). Mean standard deviation of the number of vital cells for each material tested with alamar blue at different times table 2 shows the mean diameter of the inhibition zones after 48 h and the standard deviation . Except for biodentine when tested for s. mutans, the root - end filling materials caused zones of inhibition . However; mta - angelus, proroot mta, and irm showed the highest values for the inhibition zones when tested for s. mutans (p <0.05). No significant differences between mta - angelus, proroot mta, and irm (p> 0.05) were found when materials were tested for s. salivarius . Biodentine showed the largest inhibition zone when tested for s. sanguis (p <0.05), while mta - angelus and proroot mta produced similar smaller inhibition zones (p> 0.05). Mean diameter and standard deviation of inhibition zones (mm) formed by the root - endfilling materials after 48 h by the paper disc method the results of solubility test (after 24 h and 2 months) are listed in table 3 . All the materials fulfilled the requirements of the international standard 6876, demonstrating a weight loss of less than 3% . There was no statistical significance in solubility among the materials tested after 24 h (p <0.05). Similar results were obtained after 2 months, except for irm for which solubility significantly raised (p <0.05). For remnant materials the weight loss after 2 months was not significantly different from the weight loss after 24 h: all the materials tested provided a very alkaline ph after 3 h [table 3]; proroot mta showed the highest value among the materials tested . Biodentine and irm provided similar ph values after 3 h (p> 0.05). For all materials tested, a nonsignificant reduction in ph value was recorded after 24 h (p> 0.05). For each root - end filling material descriptive statistics for radiographic densities and the results obtained with the analyses of multiple comparisons were calculated and reported in table 4 . Proroot mta and mta - angelus showed the highest values of density when compared with the other materials (p <0.05). Mean percentage values of solubility and standard deviation (sd) for each material and for each immersion period radiographic densities: descriptive statistics for each material tested and multiple comparisons calculated with tukey test (p = 0.05) when comparing the cytotoxicity of the root - end materials tested in this study, irm demonstrated the lower rates of vitality and a strong cytotoxic capability . Irm has shown the lowest mean number of cells in the colorimetric assay performed with alamar blue, with assessments at 24, 48, and 72 h, and in the mtt assay at 72 h. these results are in agreement with previous studies . It has been demonstrated that cell response to irm was characterized by marked rounding of the cells and depletion of cell numbers, indicating that irm was not biocompatible . Very different results were obtained by mta - based materials . At 72 h, with alamar blue test and mtt assay, parirokh and torabinejad found that proroot mta was less toxic than amalgam, super eba, and irm . Biodentine, in measurements made at 24, 48, and 72 h; reported percentage of vitality above the negative control . Biodentine is new bioactive cement based on calcium silicate, whose high biocompatibility has been shown in recent studies . The agar diffusion test has been widely used to evaluate the antibacterial activity of dental materials . In our study, except for biodentine against s. mutans, all the materials demonstrate antibacterial activity, causing zones of inhibition against the three streptococcal strains tested small differences in the antibacterial effect on the different streptococcal strains were recorded ., in which proroot mta and irm proved to be strong inhibitors . While we have little data about the antibacterial capability of biodentine it has been shown that in aerobic conditions, mta could generate reactive oxygen species with antimicrobial activity . Parirokh and torabinejad found that mta had no antibacterial effect against any of the strictly anaerobic bacteria . However, as showed by our results, it is possible that mta's highly alkaline ph affords its antimicrobial activity even when in anaerobic condition . The test was repeated after 2 months, because schafer and zandbiglari stated that the 24-h period of the specification test is not sufficient . In our study all the materials tested showed minimal solubility, fulfilling the requirements of the iso 6876 and demonstrating a weight loss of less than 3% . These conclusions are in accordance with our previous study about solubility of proroot mta, irm, and super - eba . A very alkaline ph was recorded after 3 h; with the highest value registered for proroot mta . No significant reduction in ph value was recorded after 24 h. proroot mta showed ph values greater than biodentine . This may be probably caused by the higher presence of metallic oxides in mta that could promote ion dissociation . Finally, as concerned for radiopacity, in our study mta - based materials demonstrated higher values of density compared to irm and biodentine . Radiopacity of biodentine has been recently evaluated . Comparing the density of biodentine and irm, grech et al ., found higher values for irm; while the relatively lower radiopacity of biodentine, compared to mta, has been recently shown in a study by tanalp et al . Within the limitation of this in vitro study, the differences shown by the materials do not cover completely the ideal clinical requests for root - end filling . Irm could not be considered as the ideal material due to the high cytotoxicity; while mta - based cement and biodentine proved to be biocompatible . However, biodentine, comparing to mta, did not cause zone of inhibition for the entire microorganism tested and it showed lower values of radiopacity . Therefore, mta - angelus and proroot mta can be considered as the best material for root - end filling among those tested due to the fact that they presented at once biocompatibility, antibacterial properties, radiopacity, and low solubility.
In toxicogenomics, gene expression data has been used to understand the transcriptional changes in response to exposure to environmental stressors and toxicants . The transcriptome may be a more effective marker or predictor for drug - induced liver injury (dili) compared to other biomarkers (for a review see watkins, 2009). Several toxicogenomics studies have monitored the gene expression changes in the liver and/or the blood following exposure to hepatotoxicants (bushel et al . Figure 1a illustrates a few examples of descriptors used in toxicogenomics studies . For a given classical dose response curve, regions can be ill - defined as having no effect, an observable effect on the transcription of genes, or bracketed into a maximum limit where the highest dose produces an effect without appreciable toxicity (figure 1a left inset). In other cases, measurements of serum enzymes alanine transaminase (alt) and aspartate transaminase (ast) are typically used to monitor liver damage and separate samples into responders and non - responders of an exposure to a toxicant . However, increases in these transaminases are not good prognosticators of liver injury and as such, have limitations in their use as biomarkers (blei, 2005). There can be cases where the variation of the alt measure among samples sharing the same necrosis severity score is large (huang et al ., 2010). In a more practical approach, liver injury severity measures can be grouped into a composite score or according to the similarity of the biological processes of the samples to reflective a more intuitive phenotypic representation of the toxicant effect (figure 1a right inset). Furthermore, and to complicate matters even further, when time is a function of the exposure along with dose, biological and transcriptional responses take the form of those exhibited in an acute / short - term effect that may or may not be reversible vs. a chronic or repeated effect resulting in a long - term manifestation of an injury . Cleary these limited, albeit general, representations of the interplay between toxicology and genomics provide good examples of how the desired end result of a toxicogenomics study ultimately depend on how one structures the study design and categorizes the phenotypic response(s). Main inset: alt over time as a function of sample type . Left inset: classical dose response curve . Right inset: liver injury severity measures grouped according to similarity to gene ontology biological processes (go bps) (b, c) intersection of coi - specific predictive gene signature probes (b) and gene ontologies enriched in those predictive gene signatures (c). (d) network analysis of predictive gene signatures shows a common central gene relational network . Corresponding gene symbols of predictive gene signatures were submitted to string to generate a gene relational network (solid black lines; line width correlates to string combined score, which is the computed final confidence score for association between two proteins). A region of interest (roi) is commonly used in scientific research and analysis to focus on a particular area or subset of data identified for a particular purpose . In digital imaging for example, a roi might be the focal point of a digital representation of an object . The ability to focus on a specific subset of data is useful to investigators in order to have a broader understanding of the larger experimental system or problem . Similarly, in toxicogenomics the classification of interest (coi) is defined as the a priori partitioning of biological samples according to a common toxicological characteristic . Here, 2007; huang et al ., 2008, 2010), each of which used the same toxicant for exposure, but varied according to coi . (2007), investigated the utility of rat blood gene expression data to predict exposure to acetaminophen (apap). The samples were segregated based on a binary coi defined as either no or low dose exposure vs. high dose exposure . In another study, huang et al . (2008) targeted rat liver gene expression data to predict the extent of necrosis in the liver . Here the coi was defined as the severity of liver necrosis determined by visual histopathology . In the final study, huang et al . (2010) explored the use of rat blood gene expression data to predict liver necrosis . The coi for this latter study was no visual liver necrosis vs. some observable sign of necrosis in the treated samples . Although each study was similarly designed to identify predictive markers based on rat gene expression in response to apap exposure, the predictive gene signatures generated share very few microarray probes in common . Further, the predictive gene signatures from the three studies differ widely in enriched biological processes and thus biological interpretation . It is unclear how these subtle differences in the study coi contributed to the identification of predictor genes, biological interpretation of these gene signatures and therefore the ultimate conclusions . The three toxicogenomic studies examined each define similar predictive transcriptomic signatures based on related yet distinctive coi, i.e., blood transcriptomic signatures that predict dose of apap exposure, liver transcriptomic signatures that predict the severity of liver necrosis, and blood and liver transcriptomic signatures that predict dili (table 1). To give an overview of the studies, summary of predictive transcriptomics for apap / dili . Using a blood gene expression dataset in rat, bushel et al . (2007) reported a gene signature that predicted an endpoint of apap exposure and classified the endpoint by level (no dose / low dose vs. high dose). Here into called the dose - classification . Using the agilent 011868 rat oligo microarray g4130a, pooling of the highly accurate classifiers common to any two out of the four prediction algorithms (k - nn / dme - anova, pca / epig, fuzzy - artmap / dme - anova, fuzzy - artmap / dce - anova) resulted in 27 gene - probes . The classifiers were built to take advantage of dose as a main effect in the study with treatments levels divided into non\sub - toxic (0, 150 mg / kg) and toxic (1500, 2000 mg / kg) without considering the duration of exposure . Huang et al . (2008) used rat liver gene expression assayed on the same array platform as bushel et al . (2007) but from samples exposed to eight hepatotoxicants as a training set to predict the severity of liver injury . Here an independent test set comprised of liver gene expression from rats treated with apap, carbon tetrachloride and allyl alcohol . The union of the classifiers from the random forest and support vector machine prediction algorithms resulted in 22 gene - probes . The classifiers were trained using the partition of groups created by merging severity levels of a histopathological endpoint depending on the similarity of the biological processes of samples (group1: no necrosis; group2: minimal and mild necrosis; group3: moderate and marked necrosis) irrespective of the dose and/or time of exposure . Huang et al . (2010) used liver and blood gene expression data from the huang et al . (2008) study but predicted dili within and across tissues, i.e., used the same predictive gene signature trained on liver expression to predict dili based on both liver and blood gene expression data (and vice verse). Here into called the dili - classification . Dili was defined as a binary response (no necrosis vs. some observable sign of necrosis) without considering the dose and/or time of exposure . From the various, highly accurate predictors, 10 gene - probes occurred most often . A meta - analysis of the data from these studies illustrates the effect of the coi in the identification of predictor genes and the interpretation of results . Although each study was similarly designed to identify predictive markers related to samples treated with apap, there are very few probes common to all three gene signatures . While no gene - probes are common to each predictive gene signature, half of the dili - classification predictive gene signature probes (n = 10) overlap with either the dose - classification or severity - classification predictive gene signatures (n = 27 and n = 22, respectively), which have no gene - probes common amongst themselves (figure 1b). The pattern of overlap of the gene signatures from the three studies is only partially reinforced by similar patterns of intersection of functionally enriched ontologies in the predictive gene signatures (figure 1c) as the dose - classification signature is not functionally enriched for gene ontologies after multiple test correction . Yet, despite the presence of only a single gene - probe common to both predictive signatures (figure 1c), the severity - classification and dili - classification studies revealed a large amount of functional overlap (figure 1c; table 2). Although there are no gene - probes or enriched gene ontologies common to all three gene signatures, surprisingly, genes from the three signatures together comprise a string - generated gene relational network (figure 1d) that is enriched for inflammatory and immune response ontologies (table 2). Top eight bingo gene ontology terms enriched in severity and dili predictive gene signatures, and string gene relational network generated from the three predictive signatures . P, benjamini and hochberg false discovery rate correction; x, number of genes from list in go term; n, number of genes from background in go term . Common to gene signatures that predict liver injury based on both liver and blood gene expression (severity - classification and dili - classification) was chemokine (c c motif) ligand 2 (ccl2), a key chemokine that regulates migration and infiltration of monocytes and macrophages, and has a putative role in apap exposure and dili . The ccl2 protein measured by elisa was shown to be elevated in blood from patients following acute apap overdose (james et al ., 2005). The role of ccl2 in dili was further implicated in a study of ccl2-deficient mice which showed protection from necrosis when administered carbon tetrachloride (zamara et al ., 2007). In addition, when a mouse model genetically deficient in the c - c chemokine receptor and negatively mediates ccl2 expression is challenged with apap, an elisa revealed elevated liver necrosis and increased ccl2 protein (hogaboam et al ., 2000). The genes from the three cois together form a relational network dominated by inflammatory responsive genes . These inflammatory responsive genes appear to be central to the major biological pathways that are presumably part of the mechanism(s) leading to apap liver tissue damage . However, there is no reason why very few genes, even a single one would not have the predictive power to accurately classify the exposed samples . If more mechanistic information needs to be gleaned from the predictors, the number of genes in the classifiers can be enlarged . This will undoubtedly bring more biological context to the classifiers but definitely at the expense of losing prediction accuracy due to the addition of potentially noisy genes or genes as false positives . What is certain is that depending on a typical toxicogenomics study, if the gene expression data is analyzed using different cois, then several different predictors can be derived as potential biomarkers which may have a small but biologically relevant common theme, yet still captures the underpinnings of the data surveyor s interest . Possible solutions to this problem may be (1) include more descriptors for a study design, i.e., increase the size of the data used, (2) leverage a composite phenotype of many endpoint measurements, and/or (3) focus on a more informative classifier and improved training set . The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . Gene ontologies enriched in the assembled gene signatures for the three studies were created using bingo as described below . Biomolecular interaction networks of predictive gene signatures were generated using string (szklarczyk et al ., 2011). Gene symbols from the three assembled gene signatures were submitted to string using the default settings, the output was downloaded as a text file and then imported into cytoscape (smoot et al ., 2011) for graphical visualization . Interactions between genes and nodes representing a coi were manually added prior to import into cytoscape . Over - representation analysis of gene signatures and networks rendered in cytoscape was by the bingo v. 2.44 plug - in (maere et al ., 2005) using the default settings, go_full as the selected ontology file and rattus norvegicus as the selected annotation . For intersection comparison of gene ontologies enriched in gene signatures, all terms with benjamini and hochberg fdr correction p, terms were filtered to remove less significant ontologies with redundant gene lists; and, only the top eight filtered ontologies ranked by increasing p were reported.
Poxviruses are large, double - stranded dna viruses that infect a wide variety of hosts, ranging fromcaterpillars to humans . They are the largest animal virus, with a brick - shape, ranging in size from 200 to 400 nm long and 170 to 200 nm wide . Due to their large size, poxviruses can be visualized with light microscopy, although the fine details of the virus remain obscure . Poxvirus genomes encode the genes necessary for replication and immune modulation, with replication occurring in the host cell's cytoplasm . The genome is large compared to most viruses, containing 130300 kb, depending on the virus type . Poxviruses are classified into two categories: entomopox (insect infecting) and chordopox (vertebrate infecting). The chordopox viruses are further divided into eight categories, including orthopox, which includes variola major and vaccinia virus, and avipox, which includes fowlpox virus . Variola major, the virus that causes the contagious disease smallpox, has been considered one of the most destructive viruses in history, affecting 1/10th of all humankind and killing 300 million people in the 20th century alone . The disease was considered fully eradicated in 1980 . However, although variola major no longer exists in any known animal reservoirs and nature, stocks are still kept in the united states and russia . Smallpox has a 2030% lethality rate in unvaccinated individuals, although analysis of historical data suggests that 77.6% of cases are still protected against severe disease 70 years after vaccination . Vaccina virus, long considered the prototype of variola major, has been used as both a simulant and vaccine for variola major . They show remarkable sequence similarity; however, vaccinia virus does not cause disease in healthy individuals, possibly due to the fact that variola major virus contains complement inhibitors 100 times more potent than those of vaccinia virus . However, vaccinia can still cause serious infection in humans and is therefore not an ideal simulant . The purpose of this paper is to investigate whether fowlpox, also a member of the chordopox family, can be used as a simulant for variola major . Fowlpox virus naturally infects chicken and turkey; however, it is not infectious to humans . Fowlpox virus has a genome consisting of 260 genes . Of the 260 genes, 65 are conserved homologues of chordopox genes . When compared by hexamer and glimer computer programs, these genes encode for transcription factors, rna - polymerase subunits, dna repair, protein modification, structural proteins, and immune evasion proteins . Several genes encoding cellular function show homology with proteins of unknown function from yeast, bacteria, roundworm, plant, and human . Another gene shows similar homology to a protein found in yeast, human, tomato and fruit fly . Secondly, the fowlpox genome contains host range genes, limiting infection to members of the avian family . Thirdly, the virus is so large due to the presence of novel cellular homologues . Vaccinia strain wr (vr-119) and fowlpox strain fpv - mdvgbh (merck's disease vaccine strain) (vr-2330) were obtained from the american type culture collection (atcc, manassas, va). Bhk-21 (ccl-10), bsc-40 cells (crl-2761) and umnsah / df-1 cells (chicken embryo fibroblast cells) (crl-12203) were also obtained from atcc . Vaccinia virus was propagated in bhk-21 monolayer cultures in dulbecco's modified eagle's medium (dmem) containing 10% heat - inactivated fetal bovine serum (fbs) at 37c . Infected cells were harvested and centrifuged at 3,210 g at 4c for 10 minutes . Pellets were resuspended in dmem containing 10% fbs, freeze - thawed for three cycles, sonicated for four minutes on ice and then centrifuged at 3,210 g at 4c for 10 minutes . The supernatant was collected and titered in bsc-40 cells and served as the source of virus for all experiments described in this paper . Bsc-40 cells were plated at a density of 5.0 10 cells per well in six well plates for vaccinia plaque assays . Cells were allowed to reach confluence overnight at 37c . In dmem containing 10% fbs . Following absorption, medium was removed and replaced with minimal essential medium (mem) containing 5% fbs and 1% seaplaque agarose . Forty - eight hours after infection, plaques were visualized by staining with 0.01% crystal violet . The plaques were counted, and the results were reported in plaque forming units per milliliter (pfu / ml). Fowlpox virus was propagated in umnsah / df-1 monolayer cultures in dmem containing 10% fbs at 39c . Infected cells were harvested and centrifuged at 3,210 g at 4c for 10 minutes . Pellets were resuspended in dmem containing 10% fbs, freeze - thawed for three cycles, sonicated for four minutes on ice, and centrifuged at 3,210 g at 4c for 10 minutes . The supernatant was collected and titered in umnsah / df-1 cells and served as the source of virus for all experiments described in this paper . Umnsah / df-1 cells were plated at a density of 1.0 10 cells per well in six well plates for the fowlpox plaque assay . Cells were allowed to reach confluence overnight at 39c in dmem containing 10% fbs . Following absorption, media was removed and replaced with 50% mem, 50% dmem, 2.5% fbs, and 1% seaplaque agarose . The following disinfectants were tested for their efficacy against vaccinia and fowlpox viruses: ethanol (70%), isopropyl alcohol (50%), sodium hypochlorite (0.5%), formaldehyde (30%), benzalkonium chloride (10%), a mixture of cetyltrimethylammonium chloride (6.67%) and benzalkonium chloride (3.33%), and a mixture of iodine (1.75%) and polyethyleneglycol nonylphenyl ether (10%). All disinfectants were prepared in distilled, deionized water and then filtered through a 0.25 m filter . The following concentrations of disinfectants were used to determine the inactivation kinetics of vaccinia and fowlpox viruses: ethanol (40%), sodium hypochlorite (0.05% and 0.025%), a mixture of cetyltrimethylammonium chloride (0.1%) and benzalkonium chloride (0.05%), a mixture of iodine (0.1%) and polyethyleneglycol nonylphenyl ether (5.7%), and a mixture of iodine (0.05%) and polyethyleneglycol nonylphenyl ether (0.3%). All disinfectants were prepared in distilled, deionized water and then filtered through a 0.25 m filter . The plaque assays described above for vaccinia and fowlpox viruses were followed to determine the activity of the disinfectants, with minor changes . Vaccinia and fowlpox viruses were diluted 1: 10 in sterile water (control) or disinfectant . To test the infectivity of the treated viruses, the virus was treated with disinfectants for 1, 3, 5, and 10 minutes . To determine the inactivation kinetics of the individual disinfectants, the viruses were treated for 15, 30, 45, 60, 90, 120, 150, and 180 seconds . In order to determine the thermal stability of vaccinia and fowlpox viruses were diluted in 0.85% saline, ph 4.5, phosphate buffered saline (pbs), ph 7.4, 10% skim milk, and heart infusion broth as described by hahon and kozikowski for their work with variola major . Solutions were equilibrated to 40c, 45c, 50c, and 55c and then seeded with virus, resulting in a 1: 100 dilution . Samples were removed at 0, 15, 30, 45, and 60 minutes and cooled on wet ice . The plaque assays described above were then performed . Aliquots of vaccinia and fowlpox viruses were stored at 80c and 4c for one week . After the one week storage period, the viruses were diluted 1: 100 in sterile pbs heated to 56c as described by hahon and kozikowski . Samples were incubated at 56c for 0, 15, 30, 45, and 60 minutes . At the indicated timepoint, samples were removed and cooled on wet ice . The plaque assays described above were then performed . To determine the order of the reaction, the following graphs were produced . To determine a first - order reaction, graphs of the natural log of the virus concentration versus time were produced . A linear line with a negative slope indicated a first - order reaction . To determine a second - order reaction, graphs of 1/virus concentration versus time were produced . The rate of reaction for the first - order reactions was determined using the following equation: vt / v0 = e, where vt is the concentration of virus at time, t, and v0 is the concentration of virus at the zero timepoint, and k is the reaction rate . The rate of reaction for the second - order reactions was determined using the following equation: (1/vt) = (1/v0) + kt, where vt is the concentration of virus at time, t, v0 is the concentration of virus at the zero timepoint, and k is the reaction rate . The virus half - life (t1/2) for viruses showing a first - order reaction rate was determined using the following equation: t1/2 = ln 2/k, where k is the rate constant determined using the equations described above . The t1/2 for viruses showing a second - order reaction rate was determined using the following equation: t1/2 = 1/(v0 k), where v0 is the concentration of virus at the zero timepoint . The rate constant and virus half - life for the thermal inactivation studies were calculated as described above . The calculations of hahon and kozikowski were used to determine h and s . As shown in figure 3, h may be obtained by the arrhenius plot of the natural logarithm of the k values versus the reciprocal of the absolute temperature (1/t). A straight line with the slope h / r is obtained, where r is the universal gas constant, from which the h can be calculated from . The eyring equation is as follows: k = (kt / h) e(h / rt) e(s / r), where k is boltzmann's constant, t is the absolute temperature, h is plank's constant, r is the universal gas constant, and k is the rate constant calculated for the reaction . Chemical toxicity assays were performed by incubating bsc-40 and umnsah / df-1 cells with the highest dose of chemical for the viral absorption time (one hour for bsc-40 and two hours for umnsah / df-1). Following incubation, toxicity effects were noted to ensure that cells were healthy enough to produce viral protein, allowing the infection process to occur . Additionally, the determinations of toxicity effects were important to ensure that any cell death was due to viral infection and not the treatment chemical . The ethanol, isopropyl alcohol, and sodium hypochlorite had no observable effects on either cell line . The formaldehyde and the surface - active detergents (benzalkonium chloride, cetyltrimethylammonium chloride, and a mixture of iodine and polyethyleneglycol nonylphenyl ether) had some toxic effects on cells at the highest two dilutions, but cells that recovered and grew had normal morphology . All the chemicals tested disinfected both vaccinia virus and fowlpox virus (tables 1 and 2). . For each chemical tested, triplicate test plates of untreated virus (positive control) were also cultured on cells . In order for the results of the chemical decontamination timepoint to be considered valid, the average positive control titer was within 1-log unit of the known virus titer . These results were identical to the results reported by tanabe and hotta, using vaccinia and variola major . The four chemicals chosen for the inactivation kinetic studies were shown by tanabe and hotta to demonstrate high viracidal properties at minimum concentrations . As shown in figure 1, 0.05% and 0.025% sodium hypochlorite inactivated the vaccinia virus within 15 seconds of treatment . Within 30 and 45 seconds following treatment, 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride inactivated vaccinia virus, respectively . Two concentrations of chemicals were not high enough to result in complete viral inactivation . Following a three minute treatment with 40% ethanol, although treatment for three minutes with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether did not result in complete inactivation, viral titers dropped by 5-log pfu / ml units . The 40% ethanol inactivation kinetics in this study vary somewhat from the data reported by tanabe and hotta . Purified variola major virus is inactivated by 40% ethanol within 1 minute, while a nonpurified culture is inactivated within three minutes . Secondly, tanabe and hotta reported their data in percent reduction in plaque count . If the 40% ethanol inactivation kinetics described here were reported in percent reduction in plaque count, there would be a 95.4% reduction in plaques at the 60-second timepoint, which is the point at which tanabe and hotta describe a 100% reduction . Fowlpox virus had an inactivation profile similar to that of vaccinia virus . Like vaccinia virus, 0.05% and 0.025% sodium hypochlorite inactivated the fowlpox virus within 15 seconds of treatment, as shown in figure 2 . Within 15 and 45 seconds following treatment, 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride inactivated fowlpox virus, respectively . The inactivation kinetic curves for 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride treated fowlpox are similar to the inactivation kinetic curves of vaccinia virus treated with those same chemicals . Like vaccinia virus, treatment for 3 minutes with 40% ethanol and 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether did not result in complete inactivation of fowlpox virus following treatment of fowlpox virus with 40% ethanol for three minutes, the concentration of fowlpox virus dropped by approximately 2-log pfu / ml units . Fowlpox virus treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether had a decreased titer of approximately 1-log pfu / ml unit following a three - minute treatment . Inactivation rate constants were calculated for vaccinia and fowlpox viruses treated with ethanol (40%), sodium hypochlorite (0.05% and 0.025%), a mixture of cetyltrimethylammonium chloride (0.1%) and benzalkonium chloride (0.05%), a mixture of iodine (0.1%) and polyethyleneglycol nonylphenyl ether (5.7%), and a mixture of iodine (0.05%) and polyethyleneglycol nonylphenyl ether (0.3%). The calculated rate constants for vaccinia and fowlpox viruses are shown in table 3 . Overall, vaccinia virus exhibited greater inactivation rate constants than fowlpox virus treated with the same chemicals . For both viruses, the order of the inactivation reaction when treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether was a second - order reaction . Vaccinia virus exhibited a first - order reaction, while fowlpox virus exhibited a second - order reaction . A first - order reaction was observed in both vaccinia and fowlpox virus treated with 0.05% and 0.025% sodium hypochlorite, a mixture of 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride, and 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether . Vaccinia and fowlpox viruses treated with 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether, 0.05% sodium hypochlorite, 0.1% sodium hypochlorite, and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride had a calculated half - life of less than 8.41 seconds (table 3). The difference in virus half - life between the two viruses treated with the above chemicals was less than two seconds . When treated with 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether, 0.05% sodium hypochlorite, 0.1% sodium hypochlorite, and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride, fowlpox had a slightly greater half - life than vaccinia virus by 12 seconds . For two chemicals, fowlpox virus treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether had a half - life 2.4 10 times greater than the half - life of vaccinia virus treated with the same chemical . However, vaccinia virus treated with 40% ethanol had a virus half - life over 20 times greater than fowlpox virus treated with the same disinfectant . Vaccinia virus was completely inactivated in 0.85% saline when heated to 55c . Approximately two - log reductions of viral titer were observed in the vaccinia diluted in 0.85% saline and heated at 40c, 45c and 50c . According to the arrhenius plot, the h for vaccinia virus is 44,138 calories per mole in 0.85% saline (see table 4). Unlike vaccinia virus, after one hour of heating at 55c, fowlpox virus had a 2-log decrease in plaques . After heating for one hour at 40c, 45c, and 50c, fowlpox virus titers did not decrease by even 1-log unit . The h for fowlpox virus in 0.85% saline was 53,481 calories per mol in 0.85% saline (see table 4). The entropy of activation (s) for vaccinia virus in 0.85% saline ranged from 67.61 cal/c at 40c to 65.85 cal/c at 55c (see table 5). The entropy of activation (s) for fowlpox virus in 0.85% saline ranged from 71.70 cal/c at 40c to 63.93 cal/c at 55c (see table 6). Vaccinia and fowlpox viruses behaved oppositely in pbs, ph 7.4 as compared to their behavior in 0.85% saline, ph 4.5 . Unlike the vaccinia virus treated in 0.85% saline that was completely inactivated at 55c, vaccinia virus in pbs, ph 7.4 is stable, decreasing by only 2-log units over the 60 minute incubation period . Vaccinia virus heated for one hour at 40c, 45c, and 50c decreased by less than one - log unit . The calculated h for vaccinia virus in pbs, ph 7.4, is 34,239 calories per mole (see table 4). Unlike vaccinia virus, fowlpox virus was completely inactivated in pbs, ph 7.4 at 55c . However, at 40c, 45c, and 50c, fowlpox, like vaccinia, was stable, with titers decreasing by less than one - log unit over the hour long incubation period . The calculated h for fowlpox virus in pbs, ph 7.4, is 35,868 calories per mole (see table 4). The entropy of activation (s) for vaccinia virus in pbs, ph 7.4, ranged from 69.08 cal/c at 40c to 63.17 cal/c at 55c (see table 5). The entropy of activation (s) for fowlpox virus in pbs, ph 7.4 ranged from 68.34 cal/c at 40c to 62.45 cal/c at 55c (see table 6). Vaccinia and fowlpox viruses behaved similarly when heated in a solution of 10% skim milk . After a one hour treatment at 55c in 10% skim milk, vaccinia titers decreased by 4-log units and fowlpox virus decreased by 2-log units . After a one hour treatment in 10% skim milk at 50c, vaccinia virus titers decreased by 2-log units, while fowlpox virus titers decreased by one - log unit . At the lower temperatures (40c and 45c) titers only dropped by one - log unit for both vaccinia and fowlpox viruses the calculated h for vaccinia virus in 10% skim milk is 44,741 calories per mole; while the calculated h fowlpox virus in 10% skim milk is 22,378 calories per mole (see table 4). The entropy of activation (s) for vaccinia virus in skim milk ranged from 70.04 cal/c at 40c to 62.82 cal/c at 55c (see table 5). The entropy of activation (s) for fowlpox virus in skim milk ranged from 67.74 cal/c at 40c to 65.12 cal/c at 55c (see table 6). Vaccina and fowlpox viruses also behaved similarly in heart infusion broth, both in their inactivation curves, but also in the fact that both viruses showed decreased aggregation at lower temperatures in this test liquid . Both viruses, especially noted in vaccinia as the time course progressed, plaque size dramatically decreased and titers actually increased between the 30- and 45- minute timepoints . At the 30- and 45-minute timepoints, both vaccinia and fowlpox plaques were approximately 10 times smaller than those observed during plaque assay optimization procedures, during which the virus was not treated with chemicals or heated . However, by the 60-minute timepoint, titers had decreased to below or similar to the starting titer and plaque size returned to the normal sizes observed during plaque assay optimization . Neither vaccinia virus nor fowlpox virus was completely inactivated at 55c in brain heart infusion broth . Vaccinia titers decreased by 4-log units after a one hour incubation in heart infusion broth, while fowlpox virus titers decreased by 2-log units after a one hour incubation . At both 45c and 50c for both vaccinia and fowlpox viruses, the calculated h for vaccinia virus in heart infusion broth is 95,204 calories per mole; while the calculated h fowlpox virus in heart infusion broth is 65,229 calories per mole (see table 4). The entropy of activation (s) for vaccinia virus in heart infusion broth ranged from 63.12 cal/c at 40c to 64.86 cal/c at 55c (see table 5). The entropy of activation (s) for fowlpox virus in heart infusion broth ranged from 73.95 cal/c at 40c to 63.55 cal/c at 55c (see table 6). Vaccinia and fowlpox viruses were stored for one week at either 4c or 80c, then diluted in pbs, ph 7.4, and inactivated at 56c . Vaccinia virus was more stable than fowlpox virus under both storage conditions (see figure 3). Following 60 minutes of heating at 56c, vaccinia stored at 4c showed a 3-log decrease in plaque forming units . Although titers decreased, viable virus still remained after the 60-minute incubation period . The reaction orders for both the virus stored at 4c and 80c were both first - order reactions . Fowlpox varied significantly from vaccinia virus in its stability in pbs following different storage conditions (see figure 4). Initially, fowlpox virus was heated at 56c for 60 minutes; however, half - way through the incubation period, no viable fowlpox virus could be detected (data not shown). The experiment was repeated and instead of incubating the virus for 60 minutes at 56c, the fowlpox was only incubated for 15 minutes at 56c . When incubated for 15 minutes, inactivation kinetics could be determined . When the virus stored at 4c was inactivated at 56c, viral titers only dropped approximately one - log at the 15-minute timepoint, however, by the 30 minute timepoint, no active virus could be detected . When the virus stored at 80c was inactivated at 56c, viral titers declined ~0.2-log unit within the first 15 minutes, however like the sample stored at 4c, after 30 minutes no active virus could be detected . Like the vaccinia virus, the fowlpox inactivation curves were both first - order reactions . The viruses are so closely related that it has been hypothesized that vaccinia virus may have evolved from variola virus through continual passaging in cows and humans . Thus, vaccinia virus data common between this work and that of tanabe and hotta and hahon and kozikowski allow for an approximate comparison of the decontamination and thermal stability of fowlpox and variola major . Vaccinia virus is a well characterized virus that has been grown extensively in a variety of cell lines, from monkey and hamster kidney cells to chicken embryo fibroblasts . Fowlpox, due to the presence of host limiting genes in the viral genome, does not infect a wide variety of species like vaccinia . Based on literature searches, fowlpox virus is normally grown in primary chicken embryo fibroblast (cef) cells . Due to the more difficult nature of maintaining primary cell lines, it has been demonstrated in this work that fowlpox can be grown in an immortalized cef line, the umnsah / df-1 cell line . Although the concentration of fowlpox virus was on the order of 1 - 2 log units less than the concentration of vaccinia virus, the minimum concentration of fowlpox virus produced by these cells was approximately 7 10 pfu / ml . Vaccinia virus titers were in the 2 10 pfu / ml range . The virus used in this work was cell - associated virus that was lysed out of the cells . The virus was then centrifuged to remove any cell debris, which is similar to the manner in which tanabe and hotta prepared the variola major used in their work . The purities of the vaccinia and fowlpox viruses are comparable, as the virus harvesting methods were very similar to the methods described by tanabe and hotta . Data from this study suggest that fowlpox virus is a suitable decontamination simulant for variola major when using disinfectants at full strength (as they would typically be used). Fowlpox virus and vaccine virus are both inactivated within 1 minute when using the following disinfectants: 70% ethanol, 50% isopropyl alcohol, 0.5% sodium hypochlorite, 30% formaldehyde, 10% benzalkonium chloride, a mixture of 6.67% cetyltrimethylammonium chloride and 3.33% benzalkonium chloride, and a mixture of 1.75% iodine and 10% polyethyleneglycol nonylphenyl ether . Viral inactivation with minimal concentrations of the same chemicals was also examined in the present study, for the purpose of determining the inactivation kinetics of both vaccinia and fowlpox . To first principles, the inactivation curves for each virus and chemical appear similar; however mathematical analysis of the data reveals distinct differences for vaccinia and fowlpox viruses treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol . The reaction orders described in this study are both first- and second - order decay reactions . First - order reactions were observed with vaccinia and fowlpox viruses treated with 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether, 0.025% and 0.05% sodium hypochlorite, and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride . Vaccinia virus treated with 40% ethanol also showed a first - order reaction, while a second - order reaction was observed when the virus was treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether . Fowlpox virus exhibited second - order reactions when treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol . The first - order decay reaction is typically expected when observing viral kinetics . However, further analysis of the unique properties of poxviruses suggests possible reasons for the observed second - order reaction of fowlpox virus treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol and the vaccinia virus treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether . First, vertebrate poxviruses have the unique ability to nongenetically reactivate . In nongenetic reactivation, secondly, poxviruses (like many other viruses) tend to aggregate in solutions . The aggregated viruses that cannot be neutralized by antibodies are called the persistent fraction or nonneutralizable fraction . Studies have shown that vaccinia virus treated with nitrogen mustard bis (-chloroethyl) methylamine (nitrogen mustard), a chemical known for virus inactivation, can survive treatment with this agent when the particles are aggregated . In fact, jolik showed that rabbitpox virus treated with nitrogen mustard is still able to act as an uncoating agent for other viruses that have been inactivated, but still genetically intact, which is the phenomenon described above for nongenetic reactivation . Based on the data and published research, it could be concluded that fowlpox possibly undergoes greater aggregation than vaccinia virus in both 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol, resulting in nongenetic reactivation and therefore, a second - order reaction . Thus, the second - order reaction might be due to the fact that fowlpox virus particles in the center of a viral aggregate might be protected from the disinfectant and have the ability to reactive the genetically intact, but inactivated fowlpox virus particles . Kinetic analysis further elucidated the inactivation similarities and differences between vaccinia and fowlpox viruses . The rate constant, k, is calculated from the starting concentration of virus and the concentration of virus at time, t. the k values differed by no more than 0.7 1/s for the vaccinia and fowlpox viruses treated with 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether, 0.025% sodium hypochlorite, 0.05% sodium hypochlorite, and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride . However, the vaccinia and fowlpox viruses treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol show large differences in k values . These observed differences could be due to the fact that when treated with 40% ethanol, vaccinia undergoes a first - order reaction and fowlpox undergoes a second - order reaction . When treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether, the data suggests that fowlpox virus is more stable in that disinfectant . Although the k value is an important variable for comparison, for the purpose of this work, the virus half - life (t1/2) value is a better indicator of simulant suitability . The purpose of this work is to determine if fowlpox is a suitable decontamination simulant for variola minor, thus the amount of time the virus will survive is more important than the rate of the reaction . Based on the data presented in table 1, we can conclude that fowlpox is a suitable decontamination simulant for variola major when using the following disinfectants at concentrations used for inactivation kinetics: 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether, 0.025% sodium hypochlorite, 0.05% sodium hypochlorite and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride . The similar virus half - life and reaction orders indicate that fowlpox and vaccinia virus undergo similar reactions at similar times . However, fowlpox is not a suitable decontamination simulant for variola major when using 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol . The differences in reaction order for the two viruses for 40% ethanol are important factors . Additionally, vaccinia is two times more stable in that disinfectant than fowlpox . Fowlpox virus is 20 times more stable than vaccinia virus in 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether . The studies presented in this paper suggest that fowlpox is a suitable decontamination simulant for variola major when using the following full strength disinfectants: 70% ethanol, 50% isopropyl alcohol, 0.5% sodium hypochlorite, 30% formaldehyde, 10% benzalkonium chloride, a mixture of 6.67% cetyltrimethylammonium chloride and 3.33% benzalkonium chloride and a mixture of 1.75% iodine and 10% polyethyleneglycol nonylphenyl ether . Fowlpox virus is also a suitable inactivation kinetics simulant when treated with the following chemicals: 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether, 0.025% sodium hypochlorite, 0.05% sodium hypochlorite and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride . Fowlpox virus is not a suitable inactivation kinetics simulant for variola major when treated with the following chemicals: 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol . Knowing the genetic and decontamination similarities of variola major and vaccinia from previous studies, fowlpox virus has been shown to be a suitable variola major simulant through identical decontamination results using vaccinia virus as the common link between the studies . Like the decontamination studies, vaccinia virus served as the common link between variola virus and fowlpox virus for the thermal stability studies . Hahon and kozikowski determined the h and s of variola major virus in various buffers at temperatures from 40c to 55c . Previous studies with vaccinia virus demonstrated that vaccinia virus has a range of h values from 20,000 to 90,000 calories per mole . The calculated h values for the vaccinia virus and fowlpox virus described in this paper fall into the range described kaplan, with the exception of vaccinia virus heated in brain heart infusion broth . The difference in h values in this paper with vaccinia virus in brain heart infusion broth might be due to the decreased virus aggregation, which might have affected the inactivation and plaque counts . Hahon and kozikowski did not mention decreased aggregation in the variola major virus heated in brain heart infusion broth; however, their variola major sample was stored in brain heart infusion broth, while the viruses used in this work were stored in dmem containing 10% fbs . The h values calculated for vaccinia virus in 0.85% saline, ph 4.5, pbs, ph 7.4, and 10% skim milk were in the range described for both vaccinia and variola major virus . The h values calculated for fowlpox virus in 0.85% saline, ph 4.5, pbs, ph 7.4, 10% skim milk, and brain heart infusion broth were in the range described for both vaccinia (kaplan) and variola major virus . It is common to find values of s of the order of 10 to 100 calories per mol per degree for the denaturation of proteins . The s values described in this work for both vaccinia and fowlpox viruses fall well within the 10100 calories per mol per degree celsius range . These studies suggest that fowlpox can serve as a thermal stability simulant for variola major in 0.85% saline, ph 4.5, pbs, ph 7.4, 10% skim milk, and heart infusion broth at temperatures ranging from 40c to 55c . At temperatures above, 55c, fowlpox does not appear to be as stable as vaccinia virus . As the storage conditions studies demonstrate, even when stored at 80c, fowlpox quickly denatures when heated at 56c when compared to vaccinia virus.
According to world health organization, sadness, loss of confidence, low self - esteem, and less energy are more common among females . In india, about one - fourth (27.7%) of the female population falls in the 15 - 29 years age - group . This age is a transition phase of life associated with spurt of physical, mental, emotional, and social development . Some degrees of premenstrual problems are experienced especially in the initial years of reproductive life by majority of young women . Epidemiologic surveys have estimated that as many as 80% of women of reproductive age experience some symptoms attributed to the premenstrual phase of the menstrual cycle . Premenstrual dysphoric disorder (pmdd) is the severe form of premenstrual syndrome (pms). Somatic symptoms include edema, weight gain, mastalgia, headache, syncope, and paresthesia . They appear about 1 week before the onset of menses and disappear soon after onset of menses . The morbidity associated with pms is because of severity of symptoms, chronicity, the resulting emotional distress or impairment in work, relationships, and activities . The level of impairment of pms is significantly higher than community norms on assessment by standard measures and similar to that of major depression . Women with pms report significant impairment in personal relationships, compromised work levels and increased absence from work, school, or college . We considered that pms is relatively under - investigated area of psychiatry in india; hence, this study was planned . The objectives of the study are to find the prevalence of pms and pmdd among college students of bhavnagar (gujarat), study its associated demographic and menstrual factors, rank common premenstrual symptoms, and compare premenstrual symptom screening tool (psst) with structured clinical interview for dsm - iv - tr defined pmdd (scid - pmdd) for sensitivity and specificity . A cross - sectional study was done among undergraduate students of five colleges of bhavnagar city (gujarat) including government nursing college, excel school of nursing, shree sahajan and institute of nursing, mahila arts and commerce college, and government medical college bhavnagar from january to august, 2012 . The sample size was calculated using the openepi version 2 (atlanta, ga, usa) open source epidemiological calculator with hypothesized 15% 4% prevalence of pms and pmdd at 99% confidence level . The design effect was kept one . The minimal sample size which came on estimation at 95% college students of 1730 years, having regular menstrual cycles (2135 days) and willing to give written informed consent, were invited to participate in the study . Those who could not participate in the research due to medical and gynecological illnesses such as anemia, diabetes, hypothyroidism, asthma, migraine, epilepsy, pelvic inflammatory disease, endometriosis, and amenorrhea were excluded . It is the screening tool developed by steiner et al ., which reflects and translates categorical dsm - iv - tr criteria into a rating scale with degrees of severity . It includes 14 items assessing premenstrual symptoms of mood, anxiety, sleep, appetite, and physical symptoms . Participants rated their experience of each symptom and functional impairment item on four - point likert scale as not at all, pmdd, moderate to severe pms, and no / mild pms subjects were identified using psst scoring criteria . The translation was validated by back translation and comparison with original form by group of experts who were well - versed with both languages . Scid - pmdd is a clinician - administered structured interview developed and validated by accortt et al . It includes 11 psychological and physical symptoms of criterion a phrased in a detailed layperson format similar to items on the scid to facilitate the assessment of the specific dsm - iv - tr criteria for pmdd . Section ii included menstrual history, premenstrual symptoms, hormonal pills use, and family history of pms symptoms in first - degree relatives . Written informed consent was obtained from each participant after explaining the objectives and procedure of the study . The questions and meaning of related terms total 170 participants including all moderate to severe pms and pmdd group and 80 participants (20% of no / mild pms group) were called for the structured clinical interview for pmdd (scid - pmdd) according to the dsm - iv - tr criteria . Thirty - four participants did not come for the interview (80% response rate for structured interview). Flowchart of research methodology the data was analyzed with openepi version 2 open source calculator . Data was expressed as mean standard deviation for continuous variables and percentage for categorical variables . Prevalence of pms and pmdd according to dsm - iv - tr research criteria was calculated . Prevalence of premenstrual tension syndrome according to the international classification of diseases, 10 edition (icd-10) criteria was also calculated . Chi - square test was used for qualitative variables to find the significance of difference between proportion among respective groups: no / mild pms, moderate to severe pms, and pmdd . Analysis of variance test with post hoc analysis (kruskal wallis test) was used to find out the significance of difference between the means of respective groups with graph pad instat version 3.06 (graphpad software inc . ). Psst was compared with scid - pmdd between moderate to severe pms and pmdd versus no / mild pms group in terms of sensitivity and specificity . It is the screening tool developed by steiner et al ., which reflects and translates categorical dsm - iv - tr criteria into a rating scale with degrees of severity . It includes 14 items assessing premenstrual symptoms of mood, anxiety, sleep, appetite, and physical symptoms . Participants rated their experience of each symptom and functional impairment item on four - point likert scale as not at all, pmdd, moderate to severe pms, and no / mild pms subjects were identified using psst scoring criteria . The translation was validated by back translation and comparison with original form by group of experts who were well - versed with both languages . Scid - pmdd is a clinician - administered structured interview developed and validated by accortt et al . It includes 11 psychological and physical symptoms of criterion a phrased in a detailed layperson format similar to items on the scid to facilitate the assessment of the specific dsm - iv - tr criteria for pmdd . Section ii included menstrual history, premenstrual symptoms, hormonal pills use, and family history of pms symptoms in first - degree relatives . Written informed consent was obtained from each participant after explaining the objectives and procedure of the study . The questions and meaning of related terms total 170 participants including all moderate to severe pms and pmdd group and 80 participants (20% of no / mild pms group) were called for the structured clinical interview for pmdd (scid - pmdd) according to the dsm - iv - tr criteria . Thirty - four participants did not come for the interview (80% response rate for structured interview). Flowchart of research methodology the data was analyzed with openepi version 2 open source calculator . Data was expressed as mean standard deviation for continuous variables and percentage for categorical variables . Prevalence of pms and pmdd according to dsm - iv - tr research criteria was calculated . Prevalence of premenstrual tension syndrome according to the international classification of diseases, 10 edition (icd-10) criteria was also calculated . Chi - square test was used for qualitative variables to find the significance of difference between proportion among respective groups: no / mild pms, analysis of variance test with post hoc analysis (kruskal wallis test) was used to find out the significance of difference between the means of respective groups with graph pad instat version 3.06 (graphpad software inc . ). Psst was compared with scid - pmdd between moderate to severe pms and pmdd versus no / mild pms group in terms of sensitivity and specificity . Pmdd is associated with relatively young age group as compared to no / mild pms . There was no statistically significant difference among three groups with respect to residence, religion, and marital status . Commerce students had higher rates of pms and pmdd compared to nursing and mbbs students [table 1]. Characteristics of participants on screening by psst, 18 participants (3.7%) met the dsm - iv - tr criteria for the diagnosis of pmdd . Seventy - two participants (14.7%) marginally missed the dsm - iv tr criteria for the diagnosis of pmdd . Hence, they were identified as moderate to severe pms . Remaining 399 participants (81.6%) experienced no / mild pms . According to icd-10, 91.4% participants (n = 447) had, at least, one premenstrual symptom of any given severity (mild to severe) in at least more than or equal to half of the menstrual cycles during last 12 months duration . They were identified as having premenstrual tension syndrome according to icd-10 . Almost all participants (98.6%) of moderate to severe pms group and majority of pmdd group (94.4%) reported fatigue / lack of energy as moderate to severe . All participants of pmdd group and majority of moderate to severe pms group (94.4%), and near two - third of all (60.1%) participants reported decrease interest in work . Anger / irritability remained on third rank, its overall prevalence was 59.9% which was almost similar to the prevalence of decrease interest in work group, and large number (94.4%) of pmdd group reported anger / irritability [table 2]. Frequency of premenstrual symptoms among three groups total 298 participants reported, at least, one area of impaired functioning . It was seen among 76.8% of the total respondents, 90.3% of moderate to severe pms, and 16 of 18 among pmdd group [table 3]. Frequency of functional impairment among groups tables 4 and 5 show the menstrual characteristics and demographic correlates among groups, respectively . Menstrual correlates among three groups demographic correlates among three groups the mean menstrual cycle length was 28.9 2.3 days with mean 4.6 1.6 days of bleeding . There was no statistically significant difference among groups with respect to age of menarche, length of the menstrual cycle, and days of the menstrual bleeding . Group experienced more number of days and years with premenstrual symptoms as compared to no / mild pms group . There was no significant difference among groups with respect to regularity of cycles at the time of menarche (p = 0.84). There was statistically significant difference among groups with respect to menstrual cramps, positive family history in first - degree relative, and symptoms onset since menarche . More number of participants in moderate to severe pms and pmdd groups reported that they had symptoms since menarche as compared to no / mild pms group (p <0.00001). There was no statistically significant difference among groups with respect to regular exercise (p = 0.1143) or regular games (p = 0.7316). Eighteen of them were from moderate to severe pms group and two participants from no / mild pms group . Sensitivity of psst is 90.9%, specificity 57.01%, predictive value of positive psst 28.98%, and predictive value of the negative psst 97.01% [table 6]. Premenstrual symptom screening tool results compared with structured clinical interview for dsm - iv - tr - premenstrual dysphoric disorder (n=136) the sample of this study is comparable with previous similar studies because mean age of all participants is 18.9 1.6 years and the majority of them are urban resident and unmarried . The participants from previous studies were also from similar age group college students, had urban residence, and were unmarried . According to dsm - iv - tr research criteria, the prevalence of pms is 18.4% (14.7% for moderate to severe pms and 3.7% for pmdd) among college students in our study . It is in agreement with the study done by rapkin and mikacich and other studies from asian countries among this population . The prevalence of severe pms and pmdd in this study is not in agreement with the study by steiner et al . Who reported 21.3% and 8.3%, respectively, and also differs from study by chayachinda et al . Who reported 25.1% of pmdd among thai nurses the lower prevalence in our study can be explained by influence of cultural beliefs, socialization factors, actual experiences, and concomitant life stresses about menstruation in indian context which affects both experiencing and reporting of the premenstrual symptoms . The prospective study design, relatively smaller sample size (n = 62), and higher age group of that study can explain the difference of prevalence from our study . According to icd-10 diagnostic criteria of premenstrual tension syndrome, the prevalence of pmts in our study is 91.4% . Reported 86%, and tabassum et al . Reported 53% among young girls according to icd-10 . Bakhshani et al . Reported that 98.2% had, at least, one mild to severe premenstrual symptom in their study . A study done by cleckner - smith et al . In an adolescent sample showed that all participants (n = 78) reported, at least, one premenstrual symptom of minimal severity . The prevalence rates are higher according to icd-10 criteria as the criteria are relatively less stringent and do not require the prospective diary for confirmation of the diagnosis . The study indicates that overall most commonly reported symptom is fatigue / lack of energy (68.3%). It was the third most common symptom reported by tabassum et al ., nisar et al ., and pearlstein et al . Other highest reported symptoms were decreased interest in work (60.1%) and anger / irritability (59.9%) on the second and third rank, respectively . Anger / irritability has been reported as most common symptom by previous several studies . Decreased energy and being irritable were most common reported premenstrual symptoms in a community - based nonpatient sample of 321 black and 462 white women studied by stout et al . A cross - sectional study done among indian college students by singh et al . Reported that the most common symptom reported by subjects not having any impairment was irritability and those having impairment, the most common symptom was tiredness and lack of energy . There is repositioning of symptoms in diagnostic criteria of pmdd within the revised manual of dsm-5 . Markedly depressed mood was at the top in the dsm - iv tr research criteria of pmdd . Hence, finding of this study is consistent with this context of change in dsm-5 . This study reports that the most frequent functional impairment item reported was school / work efficiency and productivity (46.8%). Steiner et al . Found that nearly three - quarters of the pmdd and almost half of the severe pms cases reported symptoms interfered with their relationships with friends, classmates, and/or coworkers, and/or school / work efficiency / productivity . Although both groups had body mass index (bmi) within normal range, the study suggests association of relatively higher bmi with moderate to severe pms and pmdd group than no / mild pms group (p = 0.0004). This is in agreement with review article from india by bansal et al . And literature . No statistically significant difference in length of the menstrual cycle (p = 0.3) as well as days of the menstrual bleeding (p = 0.45) was found by us among all the three groups . The study findings admit that pmdd group and moderate to severe pms group experienced statistically significant more number of days as well as more number of years with premenstrual symptoms than no / mild pms group . This can be explained with finding of association of pms with onset of symptoms since menarche as well as chronic, relapsing, and remitting nature of illness . Steiner et al . Found that moderate to severe pms group and pmdd group experienced more number of days of pms per cycle as compared to no / mild pms group . Moderate to severe pms and pmdd group, the frequency of students reporting the psychological symptoms of tiredness, irritability, depression / tension, and decrease interest in work tended to increase with severity of abdominal pain and may be a psychological influence of pain on these affective symptoms . The study reports significant association of positive family history of pms in mother / sibling with both found that more than half of the study participants (57%) had family history of pms . The study findings indicate that more participants in moderate to severe pms and pmdd group reported the onset of symptoms when they first began to have periods . According to the literature and study by steiner et al ., 69.3% and 54.5% adolescents reporting severe pms and pmdd often said that their symptoms began with menarche which is replicated in this study . This study found no statistically significant association between groups with regards to physical activities such as regular exercise and games . Small trials have suggested aerobic exercise to be beneficial for pms sufferers, and one trial found high - intensity aerobic exercise to be superior to low - intensity one for pms treatment . Fewer premenstrual complaints have been found in women participating in sports than in nonathletic women . Exercise training has been associated with decreased breast, fluid, and stress complaints, but not necessarily with improvements in anxiety or depression . The association between exercise and premenstrual symptoms needs to be further investigated by exploring the type and intensity of physical exercise . The structured clinical interview is a clinician - administered interview which is more reliable than psst . The interview excluded the possibility of major depressive disorder, panic disorder, dysthymic disorder, or personality disorder among provisionally the structured clinical interview also identified two participants with provisional diagnosis of pmdd who were found to have no / mild pms on psst . Psst is highly sensitive tool (sensitivity 90.9%). We can reasonably conclude that psst is a very useful screening instrument in this population . Predictive value of the negative psst is also high (97.01%), so the likelihood of in fact not having pmdd when found negative on psst is very high . Hence, it can be easily excluded those who are not having pmdd . The predictive value of positive hence, it should be followed by more specific tests such as daily record of severity of problems or structured clinical interview for confirmation of diagnosis and planning further interventions . Findings of this study need to be confirmed by large - scale community cross - sectional survey including young girls for obtaining more precise prevalence rates among this age group . Comparison of psst with gold standard prospective daily diaries of symptoms for diagnostic agreement can be done . Psst is a useful and easy to administer screening test for probable diagnosis of pms and pmdd . The reporting of premenstrual symptoms was based on retrospective recall of the participants adding a recall bias in data collection . As there was no prospective diary charting of pms symptoms, the prevalence rates are of provisional diagnosis according to dsm - iv tr research criteria . Differentiation between mild pms and no pms was not possible because both were in the same group . Pms occurs at similar rates in college students of bhavnagar as in other asian countries . Pms is associated with menstrual cramps, positive family history, symptom onset since menarche, and relatively higher bmi . The most common symptoms are fatigue / lack of energy, decrease interest in work followed by anger / irritability . Psst is a useful, sensitive screening tool among this population for provisional diagnosis of pms and pmdd.
The surgical management of cholesteatoma has still remained a controversial subject even over 100 years after the first successful mastoidectomy . Throughout the past decade, the controversy of canal - wall - up mastoidectomy (cwum) versus canal - wall - down mastoidectomy (cwdm) in cholesteatoma was addressed . There seem to be inherent limitations and advantages in both cwum and cwdm including the ease of disease removal, incidence of recurrence, and the extent of postoperative care . If possible, intact - canal - wall mastoidectomy (icwm) is viewed as superior to cwdm due to its better aesthetic consequences and aural hygiene . The higher rate of residual disease is the drawback of icwm rather than cwdm (19 - 47.7% vs 5 - 12%), which seemed to be because of the poor visualization of particular areas in middle ear during surgery . The number of criteria, published in the literature, using for choosing the technique in cholesteatoma surgery is few . It includes patient factors such as age, anatomic factors such as the degree of cellularity of the mastoid, pathologic factors that depend on the degree of extension of cholesteatoma, functional factors that involve hearing affection, and social factors regarding the accessibility of the patient for the follow - up . According to many otologists, canal - wall - down procedures provide better visualization in the most part of middle ear than intact - canal - wall procedures . The study of hulka and mcelveen (1998) on 12 cadaveric temporal bones confirmed this idea . They suggested that cwdm showed significantly superior visualization of the lateral epitympanum, posterior crus of the stapes, and sinus tympani rather than icwm . During the past 40 years, a number of techniques were suggested for getting better intraoperative visualization of middle ear cavity structures in sergeons by sergeries while keeping the posterior canal wall safe . Most of these techniques were intended to make view of the posterior tympanic cavity better particularly, the sinus tympani . The techniques included reversing the surgeon orientation, using mirrors to see around bony ridges, and temporarily removing the posterior canal wall . Although these methods enhance the visualization approximately, they all have some limitations and none of them has entirely been accepted . In 1967, mer et al . First depicted the use of fiberoptic endoscopes for observing the middle ear cavity . However, those early otoendoscopes were short of maneuverability since they were connected to surgical microscope stands . Since then, technological advances have made preparations for handheld scopes that coming higher resolution and smaller diameters . At present, rigid otoendoscopes are used for primary cholesteatoma surgery and for second - stage organized investigative surgeries . It has been proved that the 2.7-mm 30 and 70 otoendoscopes are most practical during the primary surgery . Several reports have been published on endoscopy - aided otologic surgery and its associated lower rate of residual cholesteatoma . For example, yung reported a 9.4% rate of residual cholesteatoma later than icwm aided by 0 and 30 endoscopy . Similarly on a second look, badr - el - dine (2002) discovered that using otoendoscopy for the primary icwm surgery, only 8.6% of his operations displayed residual disease . (1993) found that the rate of residual cholesteatoma following icwm decreased from 47.7% to 5.5% when they start using adjunctive otoendoscopy . The rates of residual cholesteatoma using endoscopy - aided icwm are comparable to those of cwdm using microscopy . While it is widely accepted and logical that this trend is due to increased visualization of the endoscopes, none of the known studies have quantitatively compared visualization through both the approaches in a randomized, blinded manner . In a study by rehl et al . (2012), 30 and 70 otoendoscopy on 10 cadaveric temporal bone showed better visualization on sinus tympani and eustachian tube while there is no significant difference among the lateral epitympanum, posterior stapes crus, and round window niche . In this study, we tried to compare the quality of visualization in different middle ear structures with otoendoscopy compared with cwdm . Patients with the diagnosis of cholesteatoma signed a written consent form after being explained about the study procedure . They underwent tympanomastoidectomy (by retroauricular approach routinely done at our institute) and then those selected for cwdm were included in the study (25 patients11 females and 14 males). After removing the cholesteatoma from the involved areas (using otomicroscope in routine manner in all middle ear structures to the extent that was possible regarding visualization before lowering the posterior canal wall), otoendoscopic examination was done with a 4 mm, 0 endoscope by a neurootologist . In all cases, the scutum was removed for better vision to the extent that stapedius muscle could be seen . Also the incus was removed after recognizing erosion (resulting in better vision and freedom to act). All five middle ear structures suspected of occult cholesteatoma were evaluated in terms of having or lacking the pathology (cholesteatoma). These five anatomic sites were lateral epitympanum, sinus tympani, posterior crus of the stapes, round window niche, and eustachian tube orifice . Then, cwdm was performed and all of the mentioned sites were reevaluated for the diagnosis of occult cholesteatoma . The accurate diagnosis of pathology using endoscopy in these sites was compared with that of cwdm . Statistical analyses were performed with statistical package for the social sciences (spss) version 16 (spss - inc ., results were reported as sensitivity, specificity, positive predictive value (ppv), and negative predictive value (npv). Twenty - five patients (11 female and 14 male) were studied . On average, they had suffered from chronic otitis media for 16 years (3 - 40 9.3). The mean age was 46 years (range from 13 years to 70 years). Of these patients, 56% presented with granulation tissue, 24% presented with polyp, 80% presented with an ossicular chain destruction, and 28% with tympanosclerotic plaque . Considering cwdm as a gold standard for detecting occult cholesteatoma during surgery, sensitivity, specificity, ppv, and npv of otoendoscope is presented for each site in table 1 . Sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv), and symmetric measures of otoendoscopy in different sites of middle ear, comparing cwd in lateral epitympanum, the otoendoscope showed 93.8% sensitivity and 77.8% specificity with a symmetric measure of 73% (p <0.001), demonstrating that there is no significant difference between two techniques visualization . All of the negative sites in sinus tympani were also detected as negative by cwdm (100% specificity). In this area, in one case, we diagnosed cholesteatoma in sinus tympani with otoendoscope, but after the removal of canal wall, the observed pathology was tympanosclerotic plaque . The sensitivity, specificity, and symmetric measures were 77.8%, 85.7%, and 63%, respectively . The specificity for round window niche (as well as sinus tympani and eustachian tube orifice) was 100% . Its sensitivity was 75% and its symmetric measure was 81% (p <0.001). In all the patients, otoendoscope detected the involved eustachian tube correctly (sensitivity and specificity 100%) with a symmetric measure of 100% . This study showed that otoendoscopy, compared with cwdm, would provide a good visualization of middle ear structures that are suspected for occult cholesteatoma . The anatomic sites evaluated in this study were lateral epitympanum, sinus tympani, posterior crus of the stapes, round window niche, and eustachian tube orifice . These anatomic sites were known as areas with difficult visualization and cholesteatoma removal in icwm . Thus, these areas are high risk for residual or recurrent diseases . In this study, instead of we hypothesized that in relatively shallow structures of middle ear, maneuvering 0 endoscope would replace lateral vision endoscopes . We can detect a good degree of visualization in lateral epitympanum and round window niche as cwdm . (2003) reported that most sites of cholesteatoma remnants were seen in sinus tympani . In this study, we found the symmetric measure of this site desirable (92% with p - value less than 0.001 and specificity of 100%). Previous studies reported that otoendoscopy provided better visualization of sinus tympani than cwdm, since cwdm did not address the obstruction caused by the overlying facial canal . The poor symmetric measure for posterior crus of the stapes may be due to conservative manipulation of this anatomic site fearing facial nerve or hearing damage . However, some researchers state that the stapedial tendon will block visualization of the posterior crus . For this reason, posterior crus of the stapes would be best visualized by placing the endoscope inferiorly in the middle ear and then viewing superiorly, seeing the posterior crus of the stapes under the stapedial tendon . The sensitivity, specificity, and symmetric measure of eustachian tube orifice are all 100% . However, generally, in our population the number of involved eustachian tubes was four, and we diagnosed all of them correctly using an otoendoscope . The little amount of involvement can be due to the fact that this anatomic site is more involved in congenital cholesteatomas than acquired cholesteatomas . However, the access to eustachian tube can theoretically be affected by the distance between umbo and promontorium . One of the disadvantages of otoendoscope is fogging the lens of the endoscope, but this can easily be handled by using an antifog solution . Moreover, smearing of blood on the tip of an otoendoscope can significantly obstruct one's view or hinder interpretation of middle ear anatomy . (2012) on 10 cadaver temporal bones which has used 30 and 70 otoendoscopes . It showed that otoendoscopy was significantly better than cwdm for sinus tympani, and there was no significant difference for lateral epitympanum, posterior crus of stapes, and round window niche . In a prospective study comparing cwdm, cwum with or without otoendoscopy, meselaty et al . (2003) reported that after a period of 12 - 48 months of follow - up, all cholesteatoma residuals were from groups of patients with no otoendoscopy . (2010) reported a recurrence rate of 67% for cholesteatoma in patients treated with 4.3-mm 0, 30, 70 otoendoscopy following 3 months to 2 years . This is comparable with that of cwdm . Despite of these results, saravanappa et al . (2003) in a survey in uk, showed that only 2 - 10% of surgeons use otoendoscope routinely and 7 - 38% use it occasionally . It should be emphasized that this method has to be used much more in the future by more surgeons, reducing cwdm in selected patients . The results of the present study confirmed that otoendoscope could be applied in the surgical management of middle ear cholesteatoma . In fact, the otoendoscopy offers a less invasive procedure with a comparable detecting power of cholesteatoma in anatomic structure that could be hidden in otomicroscopy . Hra contributed in the conception of the work, conducting the study, interpretation of data and revising the draft and approval of the final version of the manuscript . Fa contributed in the conception of the work, responsible for the first draft of the manuscript, acquisition, analysis, interpretation of the data and approval of the final version of the manuscript . Mr cotributed in the conception of the work revising the draft and agreed for all aspects of the work . Nb cotributed in the conception of the work revising the draft and agreed for all aspects of the work.
Insulin continues to be the main treatment for type 1 diabetes . As effective as insulin is in addition, its dosage is highly variable through the course of a day, depending on diet as well as activity level . Ideally, for insulin to be most effective, it would be administered in amounts that would vary minute by minute, as blood glucose itself varies minute by minute . This is impractical currently, as even a regimen of 45 variable injections per day is a challenge for the typical patient . Because of these issues, there has been significant interest in the development of an artificial pancreas, a system that can administer variable amounts of insulin in response to blood glucose information . Multiple solutions to this problem have been explored, including (1) exogenous implanted beta cells, (2) sugar responsive insulin releasing polymers, and (3) a continuous glucose monitor (cgm) combined with an insulin pump . The latter approach is the most mature and is the subject of human clinical trials . One of the advantages of the cgm / pump approach is that the function of blood glucose monitoring is separated from the function of insulin delivery, so that each piece of the final system can be optimized independently . One of the main challenges associated with insulin pumps is that they require a physical connection between the outside of the patient (i.e., insulin reservoir) and the inside of the patient . This leads to multiple problems: the point of insertion gets rapidly biofouled, leading to variation in delivery or complete blockage, requiring cannula replacement every 2 days . In addition, the pump and cannula in the course of a normal active life can get snagged or removed . What we have sought is a method of insulin delivery that retains the advantages of a pump (continuous, variable insulin release), while eliminating the physical connection between insulin reservoir and the patient . Ideally it would allow the insulin to be initially administered in a fashion identical to normal insulin via injection, but allow for continuously variable release in response to blood glucose information . We introduced such an approach recently, the so - called photoactivated depot or pad approach . This prior work described a material in which insulin was linked to an insoluble polymer via a photocleavable linker . We demonstrated in vitro that insulin was released from this material in a controlled and predictable fashion using pulses of light from an led . The long - term aim for such a material is that it be injected in a fashion identical to insulin (i.e., cutaneously) and then be irradiated through the skin by a small light source that is guided by blood glucose information (figure 1). Thus, insulin release could be varied continuously without the need for a physical connection that is inherently vulnerable and invasive . The success of this material in vitro suggested multiple critical questions that we sought to address in this work: (1) can sufficient light cross the upper layers of skin to allow photolysis of the pad in vivo? (2) will the released insulin retain its biological activity, despite the synthetic processing required to originally create the pad? (3) can sufficient moles of insulin be released to stimulate blood glucose reduction? (4) can insulin release be varied by varying the amount of light applied? Pad material consisting of insulin linked to polymer via photocleavable linker injected under the skin . This is followed by transcutaneous irradiation, photolysis of the insulin pad bond, and uptake into circulation . In this work after intradermal injection of the material to form the insoluble depot site in the skin of diabetic rats, light from a compact led source is capable of stimulating release of insulin into the bloodstream . We demonstrate that additional pulses of led illumination result in additional pulses of insulin released into the bloodstream . Finally, we show that insulin release is followed by a reduction in blood glucose . All the observed effects are specific to the irradiation of the depot and are not seen in control animals, similarly treated . The results confirm the potential of remotely controlled drug release, which will be particularly useful in the case of diseases like diabetes that require highly varying amounts and highly varying timing of administration . In addition to the following, a complete description of methods and materials, including characterization of photoactivated depot materials and intermediates, is contained in detail in the supporting information . The light source was constructed from a nichia ncsu033b led, with a 365 nm peak irradiation . This was driven by a 6.5 v power source using a current limiting power resistor . The light source holds the led 0.32 cm from the skin surface, and the measured absolute irradiance at the skin was 0.71 w / cm . The absolute irradiance of the light source was determined using a calibrated usb2000 spectrophotometer (ocean optics) and a cc-3uv - s cosine corrector via an optic fiber . The spectrasuite software was used to analyze absolute irradiance in the range of 350400 nm that brackets the led output . Spague dawley male rats (250300 g) were obtained from harlan laboratories (indianapolis, in). Chemical diabetes was induced by treatment with 65 mg / kg streptozotocin (sigma - aldrich, st . Diabetes was defined as blood glucose concentrations> 250 mg / dl on 3 consecutive days using a one touch ii glucometer and blood obtained from the tail vein . This study was carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health, eighth edition . The protocol was approved by the university of missouri kansas city institutional animal care use committee protocol #1401 . The upper backs of rats were shaved prior to injection of the pad materials . Injections of 80 l of pad material were made using a 1/2 cc syringe and a 27 gauge needle . Due to limitations of the volume of material that can be injected into the dermal layer of skin at one time, two injections (40 l each) of pad materials were made side by side . The compact led light source was anchored to the skin over the injection sites by two small dots of superglue . Blood samples were collected in microvette 100 l li - hep tubes (sarstedt) from the tail - vein using a glass capillary . After collecting all time points from an experiment the supernatant was removed and stored at 20 c until the elisa analysis was performed . Glucose measurements were made using one touch ii lifescan glucometer and strips (johnson & johnson, milpitas, ca) using plasma from blood samples obtained from the tail vein . The insulin pad material was synthesized in an analogous fashion to our previously described material . Specifically, human insulin was joined to a photocleavable linker via an ester linkage formed between carboxyl groups on insulin and a diazo group on the linker . This linker was terminated in an azide . In parallel, we synthesized a base resin that would ultimately attach to the linked insulin . Our previous insulin pad material was built using chemmatrix resin as the base upon which insulin was attached via a photocleavable linker . While this resin was sufficient for in vitro demonstration of the approach, it had a particle size (150500 m) that was too large to be conveniently injected during in vivo experiments . We therefore created a new pad material using small diameter tentagel rink amide beads (rapp polymere). These were 10 m in diameter, which allowed them to be injected using a standard 27 g needle . The amine group of the resin was coupled with dibenzocyclooctyne (dbco) acid . Click reaction with insulin monoazide, which was synthesized as previously described (figure 2). The final synthesized material was characterized in two ways, by cleavage of the entire species (dbco - linker - photocleavable group - insulin) from the resin using tfa . This was confirmed to have the expected molecular weight (6597.0 observed, 6593.4 calculated). Synthesis of pad material using a 10 m rink - amide tentagel resin coupled with strained cyclo - octyne, then reacted with i m a (insulin monoazide) containing one photocleavable group . The material was also photolyzed in vitro using light from a 365 nm led (figure 3). In this experiment, test and control samples were treated identically, except that light was blocked from irradiating the control sample by aluminum foil . For the 5 min prior to sampling, we gently vortexed the resin, followed by centrifugation and sample withdraw at the time point . We observed no detectable insulin release for the light - blocked control sample (figure 3 left) and significant insulin release in the light - irradiated test sample . A majority of the released insulin was detected in the first time point after irradiation, but we continued to see additional release approaching a plateau in later time points . Because we see no insulin release in the control samples, we associate this additional insulin detected to a slower kinetic process such as diffusion from the resin following photolysis . At 65 min, we again irradiated the samples for 2 min and saw a similar pattern: no detectable insulin in the light - blocked control sample and a spike in insulin release, followed by a slower and plateauing evolution of insulin . The material released from the resin in response to irradiation showed an hplc retention time identical to insulin (figure 3, upper right). In addition, esi mass spectrometry confirmed this, showing a native molecular weight (5808.0 calculated, 5808.0 observed) (figure 3, lower right). In vitro insulin pad photolysis . Pad material was exposed to two 2 periods of 365 nm led light (blue bars). Material released showed a retention time in hplc consistent with insulin (upper right), and this was confirmed to be insulin via esi - ms (lower right). This pad material has two key components and attributes: (1) an insoluble, but injectable polymer that keeps the material at the site of injection; (2) a linkage to insulin that is cleaved with light and releases native insulin . Complete synthetic schemes and characterization are contained within the supporting information . Because the insulin was exposed to multiple synthetic steps, some involving organic solvents, we wanted to ensure that it retains biological activity in vivo after this processing . We previously have demonstrated that insulin photoreleased from resin retains the molecular weight and hplc retention time of native insulin . In addition, it is recognized by anti - insulin antibodies in an elisa sandwich assay . Both of these data suggest that our processed insulin has retained its native conformation . To confirm that this in vitro activity persists in vivo, we tested our processed insulin in a diabetic rat model . For all studies, we have used a streptozotocin induced rat model of diabetes . Diabetes was induced with a one - time injection of streptozotocin resulting in rats with average blood glucose levels of 450 mg / dl by day 2 postinjection . For all experiments, we lightly anesthetized the rats to reduce distress during the procedures . In - vitro, we photolyzed the insulin pad material, and collected the released insulin in the supernatant above the resin . The dermal layer has multiple advantages, including that it is shallow and therefore more accessible to light as well as leading to faster uptake of insulin.figure 4 shows the change in blood insulin and glucose levels in response to these injections . This is accompanied by a rapid decrease in blood glucose levels, demonstrating that, despite synthetic processing, in vitro photoreleased insulin retains biological activity . Confirmation of in vivo activity of in vitro photolyzed insulin from the pad . Insulin isolated from photolyzed pad material was injected into the dermal layer of diabetic rats (n = 3). Blood glucose (purple squares) was reduced, confirming in vivo activity of in vitro photolyzed pad insulin . The blood glucose reduction that we observed by injecting photoreleased insulin is very similar to what is expected from unprocessed human insulin . Studies with unprocessed human insulin injected intradermally follow similar kinetics with respect to insulin concentration and% blood glucose reduction (data not shown). These matched what is reported elsewhere, specifically that a 23 iu / kg dose of human insulin causes an approximately 70% reduction in glucose levels in sprague dawley rats when injected subcutaneously . We have utilized a 2.36 iu / kg (14.2 nmol / kg) dose here and observe a similar result when injected intradermally . We then examined the ability of the pad material to release insulin into the systemic circulation after injection and transcutaneous irradiation of the injection site . Diabetic rats were injected with 80 l of pad material, containing 140 nmoles of covalently bound insulin, into the dermal layer . Both experimental and control animals were fitted with a compact led light source that uses a nichia 365 nm led, with an estimated output of 0.71 w / cm at the skin (figure 5). Control animals were also irradiated but had the light blocked by a layer of aluminum foil, thus allowing for the control of any heat effects (we observe a 9 c increase in temperature during the irradiation period). The only difference between experimental and control animals was the presence of light on the skin of the experimental animals . We did not observe any surface changes such as scabbing in the skin several days postexperiment . We monitored both blood insulin levels and blood glucose levels (via blood obtained from the tail vein). Blood glucose was determined in real time using test strips, and blood insulin levels were determined using a human insulin elisa assay (alpco). The elisa assay shows minimal cross reaction with rat insulin, allowing us to track specifically the human insulin released from our material . Figure 6 shows the results of these experiments performed in triplicate experimental and control animals . Light source shown from top and bottom (left and middle panels) and in place on rat back (right panel). Blood insulin levels, as determined by elisa assay (top panel), and blood glucose levels (bottom panel) before and after a 2 min period of led activation (indicated by blue bar). Prior to irradiation, almost undetectable amounts of insulin are observed in the blood of both experimental and control animals . This persists in the control animal over a 2 h period, demonstrating no leaching of insulin from the pad material due to biochemical degradation . The experimental animals, however, showed a sharp increase in plasma insulin levels immediately after irradiation, with a peak level at 25 min, followed by a slow decrease . This decrease is likely due to normal degradative and absorptive processes . To our knowledge, this is the first demonstration of insulin release in a live animal stimulated by light . Following an increase in plasma insulin we observed a modest decrease in blood glucose, with only the 45 time point showing significance (p <0.05). We then investigated whether we could further decrease blood glucose by stimulating the release of two pulses of insulin from the depot with a second pulse of led light from the light source . As before, we injected 80 l of the insulin pad material containing 140 nmol of insulin covalently linked to the polymer, and then irradiated the skin over the depot site . Control animals again were treated identically except the light was blocked from the skin by aluminum foil . Blood insulin levels, as determined by elisa assay (top panel), and blood glucose levels (bottom panel) before and after 2 min periods of led activation at time 0 and 65 min (indicated by blue bar). * indicates p <0.05 for differences between control and experimental points . We measured both the blood insulin and glucose levels, starting at 20 min prior to the first irradiation . We observed little to no leaching of insulin from the depot site into the blood . At time 0, we irradiated for 2 min . As before we saw a sharp increase in blood insulin level only in the experimental animals, peaking at 15 min, and a modest drop in blood glucose . At 65 min, we irradiated the injection site for an additional 2 min period and saw an additional release of insulin into the blood, peaking at 85 min . With this additional irradiation step, we now observed a robust drop in blood glucose in the experimental vs control animals . This demonstrates that we can meter insulin release with light and that by doing so we can meter the control of blood glucose . In this work we have described for the first time an injectable form of insulin that is controlled with light in a living animal . The insulin pad material described is a first generation material, and each element in it (polymer, photocleavable linker, insulin) is amenable to optimization to improve performance . For example, there is the potential for immunogenicity, and this can be modulated by the nature of the polymer carrier . We currently use a peg based polymer, which is likely not to be cleared easily from the dermal site, as it is biocompatible, not biodegradable . Second generation materials under development are based on biodegradable polymers or methods that require no polymer . These will eliminate the potential for toxicity associated with the matrix portion of the materials . In addition, the di - methoxy nitrophenyl ethyl (dmnpe) photocleavable group currently used requires 365 nm light to cleave, which has the potential for phototoxicity . Previous studies that utilize 350365 nm light for light triggered release in vivo, or light activated transdermal polymerization purposes have not revealed cell toxicity after a 23 min exposure . Given this, we are developing materials that release insulin using visible light, to eliminate any residual phototoxicity and to increase dermal light penetration . This has the potential to significantly reduce the amount of light needed to photolyze . This resulted in 140 nmol of insulin per depot injection or about 0.8 mg of insulin . Given that the loading efficiency of this material is in the low single digits, it suggests that second generation, higher loading materials (> 50%) will have sufficient material to last for several days in a human . Increasing insulin loading and release efficiency can reduce the volume of injection needed, increase the lifetime of the depot, and reduce the amount of light needed to stimulate insulin release . An optimized insulin pad has the potential to allow continuously variable release of insulin in response to blood glucose information using a familiar injectable material . As such, it can form the foundation of an artificial pancreas system in which insulin delivery is not controlled by an unwieldy pump and cannula, but rather through pulses of light delivered through a light source . When combined with blood sugar information provided by a continuous glucose monitor (cgm), such a system has the potential to provide a convenient and usable route to control of blood glucose and the health benefits that result from this control.
Neurodegeneration in multiple sclerosis (ms) is a multifactorial process manifesting from the very onset of the disease [1, 2]. While, in the early stages of ms, neurodegeneration is mainly driven by inflammation, later in the course of the disease several interacting factors are involved . Among well - known and less well - documented players, mitochondrial dysfunction seems to have a crucial role [4, 5]. Mitochondrial changes in ms include altered distribution and structure, together with biochemical and molecular abnormalities [4, 69]. Oxidative stress can arise in a biological environment whenever there is an imbalance between reactive oxygen species (ros) production and the cell's buffering capacity; this imbalance results in oxidation of proteins, lipids, and dna [9, 15]. Ros are natural bioproducts of oxidative phosphorylation [9, 16] but can also be generated by activated inflammatory cells, including macrophages and microglia [1719]. Just as activated macrophages and microglia are an important source of ros, oxidative stress can, in turn, activate key factors (such as nuclear factor k beta) that upregulate proinflammatory gene expression . Accordingly, studies of oxidative stress in ms have dramatically increased in recent years [10, 22, 23]. Even though evidence of oxidative stress damage in ms appears unequivocal, the assessment of oxidative stress biomarkers has yielded inconsistent results . The concentration of glutathione, a major antioxidant agent, is increased according to some authors but decreased according to others . Similarly, uric acid, a powerful nonenzymatic antioxidant, was reported normal by kastenbauer et al ., these discrepancies may be due to a number of factors: first, the use of different samples (csf, plasma, and peripheral blood cells) and the application of different laboratory techniques; second, the choice of selected groups of oxidant or antioxidant markers is likely to offer a limited and biased view rather than a general overview of the oxidative stress phenomenon . Because of the high redundancy of the antioxidant system and the dual role played by some antioxidant scavengers, the decrease of one marker could be secondary and possibly a compensatory phenomenon, rather than primary . Finally, some inconsistencies could be due to the different clinical phenotypes of the patients investigated . Different clinical phenotypes of ms are characterized by distinct histopathological features and, we hypothized, also by distinctive oxidative stress patterns . To investigate this hypothesis while avoiding, as much as possible, the aforementioned confounding factors, we have performed a pilot trial study comparing the levels of several oxidant and antioxidant biomarkers in a large sample of patients with different clinical variants of ms and in healthy control subjects . Furthermore, we assessed the same markers in patients exposed or nonexposed to immunosuppressive treatment . Consistent with a previous study of oxidative stress molecules in other neurodegenerative diseases, we chose a panel composed of the following markers: coenzyme - q10 (coq10); total (gstot), oxidized (gssg), and reduced (gsh) forms of glutathione; malondialdehyde (mda); reactive - oxygen - species (ros); anti - oxidized - low - density lipoproteins antibodies (anti - oxldl); and antioxidant power (pao). Eighty - seven patients affected by well - established relapsing - remitting (rr, n = 32), benign (bb, n = 13), primary (pp, n = 20), or secondary progressive (sp, n = 22) ms were recruited for the present study (ms center fondazione don carlo gnocchi, milan, italy, and cam polidiagnostic center, monza, italy) between july 2011 and february 2013 . Patients with relapsing - remitting course and an expanded disability status scale (edss) score 3.0 after 15 years of disease were classified as benign ms patients . All ms patients had to be free of relapse or disease progression in the past 30 days . We excluded patients who were treated with mitoxantrone, cyclophosphamide, or steroids, or supplemented with nutraceutical drugs or vitamins during the 3 months before blood drawing . We also excluded patients with clinically or radiologically isolated syndromes . At the time of the study, 20 patients were being treated with beta - interferons, 7 with glatiramer acetate, and 7 were under other treatments (4 on natalizumab, 2 on azathioprine, and 1 on low dose naltrexone). Subjects with serious or unstable medical conditions, including cardiovascular, pulmonary, hepatic, gastrointestinal, renal, and metabolic diseases, malignancies, or diabetes, were excluded from the study . After obtaining informed consent, blood samples were collected in the morning after breakfast and immediately delivered to the central laboratory . There were no significant age differences among ms patients subgroups (table 2) or between hc subjects and ms patients (table 1). The gender distribution was analyzed using a chi - square test and there was no significant difference between ms patients and hc (p = 0.5). Table 2 shows a different gender and (as expected) drug distribution across ms subgroups . We also studied seventy - seven healthy age- and sex - matched controls (hc). Whole blood was collected in vacutainer tubes containing ethylenediaminetetraacetic acid (edta) (becton dickinson & co., rutherford, nj, usa). Blood sample was centrifuged at 2500 rpm for 5 minutes to obtain serum for the detection of coq10, mda, and anti - oxldl . An aliquot of whole blood was used for detection of gstot, gssg, and gsh . Glutathione (gstot), in its reduced (gsh) and oxidized (gssg) form, was measured by hplc with fluorescence detection . One aliquot is derivatised immediately for the determination of gsh; the second aliquot is reduced chemically before derivatisation, which leads to the detection of both oxidized and reduced glutathione . Roms (primarily hydroperoxides and rooh), in the presence of iron (which is released from plasma proteins by an acidic buffer kit), generate alkoxyl (r - o) and peroxyl (r - oo) radicals through the fenton's reaction . Such radicals, in turn, oxidized an alkyl - substituted aromatic amine which acquires a t photometrically detectable pink color . Serum antioxidant levels were measured using the total antioxidant power kit (oxford biomedical research, oxford, mi, usa). The evaluation of serum antioxidant levels is based on the reduction of cu++ into cu+ . The reduced form of copper gives rise to a stable complex with a chromogenic reagent and shows maximum absorbance at 450 nm . The values are expressed as m copper reducing equivalents (cre). To assess anti - oxidized low density lipoproteins antibodies, we used imtec - oxldl - antibodies ig (gm) (imtec - human wiesbaden, germany). The test is based on simultaneous incubation of serum samples with oxldl immobilized into microtiter wells and native ldl immobilized on pins of the microplate cover . The binding of antibodies anti - oxidized ldl (anti - oxldl) from patient serum is detected by an anti - human hrp conjugate and the subsequent reaction of a chromogenic substrate . The normal distribution of all measured data was ascertained using the kolmogorov - smirnov test . Each oxidative stress biomarker was normally distributed (all p> 0.05). To assess group differences in oxidative stress biomarkers between ms and hc, group differences between different ms courses and hc concerning oxidative stress biomarkers were assessed with a one - way anova, followed by tukey hsd or dunnett t post hoc analyses . The relationships between 2 continuous variables were examined by pearson's correlation (r). All statistical analyses were performed with the spss statistical software package version 15.0 (spss, chicago, il, usa). The levels of oxidative stress biomarkers in the studied population are shown in table 3 . Coq10, a potent antioxidant involved in energy metabolism, was lower in ms patients than in hc subjects (p = 0.001) (figure 1(a)). Post hoc analysis showed a significant difference between bb and rr (p <0.05) (figure 1(b)). Anti - oxldl, natural antibodies reacting with bioproducts of lipid peroxidation, were higher in ms patients than in hc (p = 0.038) (figure 1(d)). Post hoc analysis revealed a significant difference between hc and bb patients (p = 0.013) (figure 1(e)). No statistically significant differences between patients and controls were found for gstot, gssg, gsh, mda, and ros, and there was only a trend toward decrease of serum - antioxidant power (pao) in ms patients as compared to hc (p = 0.055). In ms patients, we found significant correlations between levels of pao and coq10 (r = 0.36, p = 0.01), pao and gstot (r = 0.43, p <0.01), and pao and gsh (r = 0.44, p <0.01). Moreover, there was a negative correlation between mda and anti - oxldl levels (r = 0.29, p = 0.04), suggesting these autoantibodies as contributory factor in scavenging oxidized - lipids . It has been reported that oxidative stress naturally increases with aging, and it is known that antioxidant factors are modulated by gender [3234]. Coq10 was lower in ms patients younger than 49 years than in age - matched hc (p = 0.005) (table 4 and figure 1(c)). Conversely, anti - oxldl level was higher in ms patients younger than 49 years than in age - matched hc (p = 0.024) (table 4 and figure 1(f)). No significant differences were found for pao levels and other oxidative biomarkers (data not shown). Interestingly, we found lower antioxidant molecules (coq10, p = 0.007; pao, p <0.001) and higher bioproducts of oxidative stress (ros, p <0.001) in healthy females than in male counterparts (figure 2(a)). Similar gender - related differences were found in ms patients for coq10, pao (ms females lower than ms males, p = 0.034 and p <0.001, resp . ), and ros (ms females higher than ms males p = 0.005) (figure 2(b)). Comparing hc and patients, coq10 was lower in ms females than in hc females (p = 0.013) (figure 2(c)). Guided by the hypothesis that higher oxidative stress damage contributes to higher disease severity, we investigated a possible correlation between ms severity and antioxidant levels . We used edds scale as rough index of ms severity and we measured a correlation with any of the markers investigated . No statistical difference was found both using parametric (pearson) and nonparametric tests (spearman) (see supplementary tables 7 and 8 in supplementary material available online at http://dx.doi.org/10.1155/2014/961863). In addition, we measured disease progression rate (defined as the ratio between edss score and years of disease duration) and grouped patients according to the median of this value (0.36). We investigated a possible correlation between progression rate and coq10 or anti - oxldl (previously reported as different between studied groups). Coq10 levels showed a trend toward increase in patients with progression rate <0.36 (coq10 = 530.67 281.54) than in patients with progression rate 0.36 (coq10 = 452.51 218.54) but it was not significant (p = 0.164). Concerning anti - oxldl, no statistically significant difference (p = 0.539) between patients with progression rate <0.36 (anti - oxldl 36.15 22.43) and patients with progression rate 0.36 (anti - oxldl 33.45 16.76) was found (data reported in figure 3). Coq10 levels showed just a trend toward increase in patients with progression rate <0.36 (coq10 = 530.67 281.54) than in patients with progression rate 0.36 (coq10 = 452.51 218.54, p: n.s . ). However, we found no statistically significant difference in anti - oxldl between patients with progression rate <0.36 (anti - oxldl 36.15 22.43) and patients with progression rate 0.36 (anti - oxldl 33.45 16.76). Because some interferons can modulate cellular antioxidant responses, we assessed a possible influence of disease - modifying drugs (i.e., ifnb and glatimer acetate) on oxidative stress parameters, but we found no differences in oxidative stress biomarker levels between patients treated with immunomodulant (n = 27) or immunosuppressive drugs (natalizumab and azathioprine, n = 7) or under no treatment (table 4). Inflammation and neurodegeneration are intertwined processes present since the early stages of ms . Among the several factors that have been involved in these mechanisms, oxidative stress damage has been the focus of numerous studies [10, 22, 23, 28, 3639]. However, the timing, the degree, and the mechanisms by which oxidative stress contributes to ms tissue damage are still unclear . Histopathological data of haider and colleagues show that oxidative damage is massively present inside active lesions in areas of initial demyelination, at a stage regarded as a prephagocytic . The same group described a deregulation of mitochondrial genes involved in redox homeostasis, which was more evident in initial lesions than in established demyelinated lesions, suggesting that oxidative stress damage associated with early mitochondria dysfunction occurs during the first stages of the disease . At the beginning of ms however, this homeostasis is lost as oxidation processes increase, typically during systemic inflammation or if the antioxidant buffer system is depleted (i.e., due to energetic failure) and tissue damage ensues . The severity of the imbalance between oxidative agents and antioxidant defenses may thus contribute to determine disease severity . In fact, our results show that a higher antioxidant factor (coq10) is associated with a less disabling course of ms (i.e., a benign phenotype). This finding cannot be simply ascribed to a different demographic profile of benign ms (table 2). The treatment regimen, which is clearly milder in benign ms patients, does not have any influence on coq10 or anti - oxldl levels, because there was no difference between treated and untreated ms patients (table 4). This finding is bolstered by the fact that the higher female / male ratio in benign ms (compared to other subgroups) should have, if anything, lowered coq10 because females have lower coq10 levels both in health controls and in ms . Coq10 is a constituent of the proton\electron transport chain, crucially involved in energy production . Moreover, it acts as a primary scavenger of free radicals, protecting membrane phospholipids, proteins, and mtdna from oxidative damage and favoring the regeneration of other antioxidants, such as tocopherol and ascorbate . It was also documented that coq10 is a calcium stabilizer, capable of alleviating calcium overload, and it has anti - inflammatory properties because it inhibits metalloproteinases and il-6 production . Recently, coq10 supplementation in 45 rr ms resulted in an increase of superoxide dismutase activity and a decrease in mda levels compared with controls over 12 weeks of a randomized, double - blinded, placebo - controlled trial . The higher levels of coq10 detected by our study in benign ms are consistent with a greater antioxidant buffering ability in these patients . Interestingly, coq10 difference between hc and ms patients has been seen in subjects younger than 49 years whereas it disappears in older patients . Of note, the first group shows also a shorter disease duration (p = 0.005) compared to the latter . This data supports the hypothesis that oxidative stress is crucial since the early stages of ms as the previously collected histopathological findings have shown [9, 11]. The different ability of patients to cope with oxidative stress may be fundamental in determining their long - term disease course . The finding that coq10 levels are lower in females than in males, irrespective of disease status, may suggest that lower antioxidant protection can be a contributing factor in the well - known female prevalence of ms . The low levels of coq10 we measured are only apparently inconsistent with the results of de bustos et al ., who found no difference between ms and hc subjects . The ms population studied by these authors corresponds exactly to the one that we excluded from our study (patients during ms exacerbation). The normal coq10 levels they found in patients compared to hc could be explained by an attempt by the organism to increase antioxidant mechanisms during an inflammatory phase of disease . Antibodies directed against oxldl react with bioproducts of lipid peroxidation, such as oxidized lipoproteins . These antibodies are thought to have a protective effect in atherosclerosis, where oxidation of lipoproteins is a critical event in the progression of atherogenesis [4749]. Oxldl colocalizes with proinflammatory cells in atherosclerotic lesions and possesses a wide spectrum of highly immunogenic oxidation - specific epitopes (both lipid and protein components of ldl). Recent studies show that oxidized phospholipids favor monocyte binding to endothelial cells, thus promoting diapedesis from the blood stream . In addition, oxldl is known to affect the integrity of the blood - brain barrier [5254] and may contribute to the formation of perivascular infiltrates . Thus, increased levels of anti - oxldl may also impair cells extravasation and inhibit the mechanisms leading to the infiltration of inflammatory cells into the brain . Considering the high polyreactivity of anti - oxldl antibodies, it is reasonable to think that they may also bind lipid epitopes on myelin debris . Myelin debris scavenging activity could reduce an important chemoattractant signal for inflammatory cells and facilitate remyelination processes at the same time . Unfortunately, the lack of detailed data concerning these antibodies (i.e., class, immune - phenotyping of secreting b cells) makes these hypotheses only speculative at the moment . Here, we can only report the association between better prognosis and higher levels of anti - oxldl antibodies . We cannot clarify whether these autoantibodies are an epiphenomenon of demyelination (but in this case, they should be increased in more severe cases) or are part of a healing attempt facilitating remyelination . The higher anti - oxldl antibodies levels that we found in benign ms are in line with the higher coq10 levels that we documented in this subgroup, suggesting that, in general, a greater antioxidant ability is associated with a milder ms course . Curiously, neither ms severity (measured by edds) nor disease progression (measured by progression rate) impacts these oxidative markers . This finding could favor the hypothesis that a depletion in antioxidant is not merely a consequence of mobility constraint (we should have found lower coq10 and another antioxidant associated with higher edss). In fact, this finding sounds to suggest antioxidant depletion as an intrinsic, and probably causative, factor associated with severe ms courses rather than its consequence . This finding confirms that these variables are independent, suggesting that the mechanisms counteracting oxidative damage are diverse . Conversely, lower values of anti - oxldl were fairly well associated with higher mda, a marker of lipid peroxidation, suggesting the involvement of these autoantibodies in the clearance of oxidized lipids . Another interesting finding to be highlighted is a trend toward a relative pao deficiency in ms patients as compared to healthy controls (p = 0.055). Decreased plasma antioxidant capacity has been reported by several authors [10, 26, 39, 56], confirming the hypothesis that the antioxidant system is defective in ms . Ms is a cns disorder and it is still a matter of debate how the immunological, biochemical, and oxidative abnormalities occurring in the periphery reflect the central phenomenon . To address this issue, we tried to investigate the same panel of oxidative biomarkers in a subgroup of patients undergoing a diagnostic spinal tap . Unfortunately, given the small amount of csf that we considered ethical to utilize for this research, we could not obtain consistent results (data available for further analyses). Arguably, coq10 levels were below the detectability threshold even with the sophisticated method employed . Notwithstanding these technical restrictions, we believe that our findings should trigger prospective studies aimed at addressing the potential role of an oxidative panel as a biomarker of disease course . Oxidative stress markers can be different across ms courses . Indeed, benign ms patients show higher antioxidant factors, including coq10 and anti - oxldl autoantibodies, which may confer protection against oxidative stress - driven mechanisms of neurodegeneration . Several studies suggest that antioxidant enzymes activity is associated with the presence of neuroinflammation and oxidative damage . Enhanced antioxidant mechanisms may represent a natural compensatory mechanism protecting against direct oxidative damage and avoiding an indirect inflammatory enhancing system . Higher coq10 and anti - oxldl antibodies found in benign course ms can disclose an alternative mechanism explaining the better prognosis of this phenotype . These preliminary data should prompt additional and possibly longitudinal studies to investigate the potential role of these molecules as biomarkers and predictors of disease course.
Sexual dysfunction is a common condition in women with pelvic floor disorders, especially with stress urinary incontinence [13]. Urinary incontinence has a negative impact on quality of life, social, physiological, physical, and sexual well - being in women [3, 57]. In 1995, ulmsten et al . Initially described the development of a minimally invasive tension - free vaginal tape (tvt). The transobturator tape (tot) procedure was developed by delorme to reduce the complications associated with the tvt, including bladder perforation, urinary retention, and bowel injury . Nowadays, suburethral slings have become a standard surgical procedure for treatment of stress urinary incontinence when conservative therapy has failed . The effect of tvt on sexual functioning has been studied, but the results are still inconsistent [1015]. Treatment of stress urinary incontinence by a suburethral sling could have a positive influence on sexual functioning by decreasing urinary leakage during intercourse and improving body image as a result of declining urinary incontinence [10, 13, 16, 17]. Suburethral sling procedures have a potential risk of neurovasculair injuries . On the other hand, sexual function may worsen by developing dyspareunia after the suburethral sling procedure . De novo dyspareunia could be explained by the position of the tape, especially in the paraurethral folds, vaginal narrowing, or erosions [1921]. The aim of this study was to evaluate the effect of tot for stress urinary incontinence on sexual functioning and sexual satisfaction in women . A prospective observational study was performed in the departments of gynecology and urology of the rijnstate hospital, arnhem, the netherlands . All patients who underwent a tension - free tot outside - in procedure (uretex to, bard, new jersey, usa) for stress urinary incontinence from april 2005 till june 2006 were invited to participate in the study . Patients were selected on basis of symptoms of stress urinary incontinence and the diagnosis was confirmed by urodynamic investigation . Exclusion criteria were inability to communicate in dutch, a maximum bladder capacity of less than 200 ml, severe complaints of urge urinary incontinence and/or detrusor overactivity on urodynamic investigation, and urinary tract infection not responding on therapy . All women underwent gynecological examination, urine dipstick, urodynamics including filling cystometry, pressure flow studies, and urethral profilometry prior to surgery . All patients enrolled were invited to fill out the incontinence quality of life questionnaire (i - qol), the urinary distress inventory (udi) and the nine questions on sexual functioning (nsf-9), prior to surgery, 6 weeks postoperatively and 12 months after surgery . Data on udi scores after 12 months are missing as a result of problems with filling out the questionnaires . The i - qol is a 22-item quality of life instrument specific to persons with stress and mixed types of urinary incontinence . There are three subscales; avoidance and limiting behaviors, psychosocial impacts, and social embarrassment . Furthermore, data on urinary incontinence are obtained using the udi [24, 25]. The udi consists of 19 items and every item consists of two parts: whether or not a symptom is present and the amount of bother related to that symptom . Five subscales are identified: discomfort / pain, urinary incontinence, overactive bladder, genital prolapse, and obstructive micturition . Total scores are converted to a score range of 0300 with higher scores representing greater symptom distress . The nsf-9, a dutch - language self - reported standardized questionnaire, contains questions on sexual desire, frequency of sexual activity, lubrication, orgasm, pain during or after sexual activity, and sexual satisfaction this questionnaire is originally developed to measure the influence of medication on sexual functioning, however is also been used in treatment modalities like percutaneous tibial nerve stimulation . Non - parametric tests (wilcoxon signed - rank test) were applied as the data were not normally distributed . A prospective observational study was performed in the departments of gynecology and urology of the rijnstate hospital, arnhem, the netherlands . All patients who underwent a tension - free tot outside - in procedure (uretex to, bard, new jersey, usa) for stress urinary incontinence from april 2005 till june 2006 were invited to participate in the study . Patients were selected on basis of symptoms of stress urinary incontinence and the diagnosis was confirmed by urodynamic investigation . Exclusion criteria were inability to communicate in dutch, a maximum bladder capacity of less than 200 ml, severe complaints of urge urinary incontinence and/or detrusor overactivity on urodynamic investigation, and urinary tract infection not responding on therapy . All women underwent gynecological examination, urine dipstick, urodynamics including filling cystometry, pressure flow studies, and urethral profilometry prior to surgery . All patients enrolled were invited to fill out the incontinence quality of life questionnaire (i - qol), the urinary distress inventory (udi) and the nine questions on sexual functioning (nsf-9), prior to surgery, 6 weeks postoperatively and 12 months after surgery . Data on udi scores after 12 months are missing as a result of problems with filling out the questionnaires . The i - qol is a 22-item quality of life instrument specific to persons with stress and mixed types of urinary incontinence . There are three subscales; avoidance and limiting behaviors, psychosocial impacts, and social embarrassment . Furthermore, data on urinary incontinence are obtained using the udi [24, 25]. The udi consists of 19 items and every item consists of two parts: whether or not a symptom is present and the amount of bother related to that symptom . Five subscales are identified: discomfort / pain, urinary incontinence, overactive bladder, genital prolapse, and obstructive micturition . Total scores are converted to a score range of 0300 with higher scores representing greater symptom distress . The nsf-9, a dutch - language self - reported standardized questionnaire, contains questions on sexual desire, frequency of sexual activity, lubrication, orgasm, pain during or after sexual activity, and sexual satisfaction . This questionnaire is originally developed to measure the influence of medication on sexual functioning, however is also been used in treatment modalities like percutaneous tibial nerve stimulation . Non - parametric tests (wilcoxon signed - rank test) were applied as the data were not normally distributed . Of all 59 women who underwent a tot procedure in the study period, 54 (92%) agreed to participate in the study . Nulliparity applied to two women (3.8%), while median parity was two (range 15). All women had stress urinary incontinence, in 83% daily stress urinary incontinence was reported . Mean operative time was 24 min (standard deviation, sd: 6). In seven cases, the tot procedure was combined with another operation: in four women, a vaginal wall correction; in two women, a laparoscopy; and in one, a curettage and balloon ablation . The mean blood loss was 50 ml (sd 93; range 0500).the subjective cure rate of stress urinary incontinence was 85% and the other women (15%) reported an improvement of the stress urinary incontinence at 6 weeks . Six weeks postoperatively, this score significantly decreased to 70 (sd 70; p <0.001). The i - qol questionnaire parameters were significantly improved 6 weeks postoperatively (65.8% (sd 18.1) versus 91.8% (sd 14.7); p <0.001). No further changes occurred in the i - qol score 1 year after surgery (91.0% (sd 18.5)). Five patients (9.3%) reported postoperative complications for which reoperation was indicated . Voiding difficulties requiring tape section occurred in two patients (3.7%). Two patients (3.7%) had a partial removal of the tape within 1 year of the initial operation because of vaginal erosions; one of them had a spontaneous drainage of a vaginal abscess before reoperation . One patient was reoperated at 21 days after primary surgery for persistent vaginal blood loss from wound dehiscention . Of all 54 women, 40 (78%) were sexually active in the month prior to surgery . Sixteen women (31%) had feelings of sexual desire once a week or more . One fourth of the women had experienced lubrication problems, while 40% of the women never or just occasionally achieved an orgasm . Pain during and after sexual activity no significant differences were reported on sexual desire, frequency of sexual activity, and problems with lubrication and orgasm 6 weeks postoperatively and 12 months after surgery . However, urinary leakage during sexual activity was significantly improved after the tot procedure . Six weeks postoperatively, 88.5% had no urinary loss during sexual activity and 12 months after surgery 93.5% of the women mentioned no urinary loss . Compared to prior to surgery, satisfaction with current sexual life was increased at 6 weeks (52%; p = 0.048) and 12 months (58%; p = 0.029). Pain during and after sexual activity was significantly diminished 12 months after surgery (p = 0.019). Our findings suggest that the tot procedure seems to have a positive influence on female sexual functioning by reducing the urinary leakage during sexual activity and declining the experience of pain during or after sexual activity . The overall sexual satisfaction with current sexual life seems to improve after the tot procedure . Few studies have evaluated sexual function following sling procedures and the results are not consistent . For the tot procedure, the tvt procedure and its effect on sexual functioning has been the subject of several studies . Some studies showed an overall positive effect on sexual function after surgery [10, 12], while other studies suggest that sexual function is not changed [11, 15] or worsened by the tvt procedure . The tot procedure could have a positive outcome on female sexual function by reducing the urinary leakage during sexual activity . Our data showed a significant decline in urinary leakage during sexual activity after surgery . In a study by serati et al ., between 10% and 27% of women with urinary incontinence reported urinary leakage during intercourse . They suggested an association between urinary leakage at penetration and stress urinary incontinence as well as urinary leakage during orgasm and detrusor overactivity . Unfortunately, the nsf does not differentiate between the moment of urinary leakage during intercourse . . Showed that women with coital incontinence had a higher improvement in sexual intercourse after surgery compared to women without coital incontinence . Also reported that women with coital incontinence were more likely to report improvement of their sexual function after the tvt procedure . Both studies only reported improvement in sexual intercourse or sexual function; however, they do not mention sexual satisfaction . It is good to know that sexual function enhances, but it seems even better that sexual satisfaction improves after sling procedures . Another explanation for the positive effect of sling procedures on sexual function is the enhancement of the body image of women just by reducing stress urinary incontinence . All women in our study had improvement of their stress urinary incontinence and the total subjective cure rate was 85% . Achtari suggested that reducing urinary incontinence improves overall sexual function by increasing body image and self esteem . It must be assumed that sling procedures can damage vascular and neural structures causing worsening of the sexual function . Caruso et al . Studied the effect of tvt and tot on clitoral blood flow using color doppler ultrasonography . They found that clitoral blood flow negatively changed after tvt, whereas tot had no influence at all . The anatomical course of tot showed that the tape is not in contact with major neurovascular structures . Delmas suggested that tot constitutes an anatomically safer approach than minimally invasive retropubic tape techniques . Nevertheless, an altered vaginal anatomy as a result of the tot procedure may result in sexual dysfunction . Elzevier et al . Reported that tot provided more sexual dysfunction than tension - free vaginal tape obturator because the tot procedure causes more pain during intercourse as a result of vaginal narrowing . One of their explanations for vaginal narrowing is that more vaginal tissue in the outside - in procedure is included . Ten percent of the partners of women receiving a tot experienced vaginal narrowing . However, weber et al . Showed that patients symptoms did not correlate with objective measures of vaginal dimensions of introital caliber and vaginal length after prolapse and incontinence surgery . Further research could be done on subjective and objective measures of vaginal dimensions after tot procedures . Postoperatively, anterior vaginal wall banding in the paraurethral folds immediately adjacent to the midurethral placement of the sling was felt . Erosions are a concern for inorganic synthetic sling materials and happen in 5% of cases using polypropylene slings . Our results showed one woman with an increase in pain during and after sexual activity, but there was no vaginal erosion . Firstly, it should be taken into account that inconsistent definitions of sexual function make it difficult to compare prevalence with other studies . Secondly, there was no dutch - validated questionnaire available to evaluate the sexual functioning in women with pelvic floor disorders, like stress urinary incontinence . Rogers et al . Developed a condition - specific validated instrument to measure sexual function in women with urinary incontinence or pelvic organ prolapsed, called pelvic organ prolapse this instrument contains 31 items divided in three domains, labeled behavioral / emotive, physical, and partner - related and it can be useful in further research on sexual function and urinary incontinence . The questionnaire that was used in our study, nsf-9, was validated and used in previous studies on the effect of percutaneous tibial nerve stimulation on sexual functioning in patients with lower urinary tract dysfunction . Additional, larger prospective studies using validated condition - specified instruments are needed to support our preliminary findings and to compare sexual function after tvt versus tot procedures for stress urinary incontinence . In conclusion, we showed that the tot procedure improves female sexual functioning and overall sexual satisfaction mainly by reducing the urinary leakage during sexual activity and declining the experience of pain during or after sexual activity.
A 60-year - old woman visited daegu catholic university medical center due to periumbillical pain lasting several days . The patient had no medical history except a benign ovarian tumor which she had under gone surgery 13 years earlier . According to physical examination findings, the sound of a regular heart beat and clear breathing were observed . The blood pressure was 108/69 mmhg, pulse 81 beats per minute, respiratory rate 20 breaths per minute, and body temperature 36.5. on laboratory test, only the cholesterol was high at 205 mg / dl . Old pulmonary tuberculosis was observed through a simple chest x - ray . On a computed tomography (ct) scan of the abdomen, a lesion suspected being a mass on the right atrium was found (fig . 1). On trans - thoracic and trans - esophageal echocardiograms, a 2.653.06 cm mobile cystic mass was visible on the right atrium and some small calcified masses were also seen inside (fig . Normal cardiac function was observed, and no abnormal observations were found in cardiac motility or the heart valves . Positron emission tomography - ct was performed due to her gynecologic history, but there was no evidence of malignancy and the ca-125 was within normal range . After median sternotomy, an arterial cannula was inserted into the ascending aorta and, a venous cannula into the superior and inferior vena cava . We also started extracorporeal circulation, and decreased the central temperature to 28. cardioplegia was induced by infusing retrograde cardioplegic solution into the coronary sinus, an incision was made in the right atrium, and a 3 cm cystic mass was identified on the lower margin of the limbus fossa ovalis, which had been connected by a stalk . The cystic mass was completely removed, and three 0.5 cm calcified masses and old blood without clotting were found in the cyst (fig . Chiari's network is a congenital disease characterized by a remnant of the right valve of the sinus venosus . It was first described in 1897 by hans chiari, who described 11 cases in which the valve of the inferior vena cava, the eustachian valve, represented by networks with attachments to the superior vena cava and to the tubercle of lower . In normal embryology, the right valve of the sinus venosus is regressed, while the thebesian and eustachian valves are remain at 15 weeks of gestational age . Chiari's networks are present in 1.5% to 4% of the population, and they rarely have clinical significance [2 - 4]. Some reports have described the network as highly mobile, with an oscillating or whiplike motion pattern during each cardiac cycle . The close relationship between chiari's networks and patent foramen ovales (pfos) has been well documented . According to schneider et al ., pfo was detected in 83% of patients with chiari's networks compared with 28% of controls . However, in patients with chiari's network, a pfo was significantly more common in arterial embolic events . They also found that right - to - left shunting of a pfo was significantly more common in patients with chiari's network than in control patients . For this reason, pfos may become a source of cerebral and peripheral arterial emboli, although a right atrial thrombus is usually asymptomatic in chiari's network . . Identified atrial aneurysms in 7 out of 29 patients with chiari's networks, who were associated with a pfo . In the same context, among the patients with atrial aneurysms, 21% there are reports of chiari's networks acting as a physical barrier of right - sided catheters and pacemakers . There are also rare reports of chiari's network associated with valvular endocarditis . The diagnosis of chiari's network is usually made incidentally . The echocardiographic finding of chiari's network is a mobile curvilinear structure on the right atrium . Ct and magnetic resonance imaging also differentiate chiari's network from other cardiac masses . In our case, the patient was not found to have pfo, atrial thrombus, or atrial aneurysm . We presumed that the right atrial mass was a teratoma as her history of ovarian tumor and echocardiographic findings were cystic and calcified lesions . On pathologic study the mass was confirmed as chiari's network . As cystic form of chiari's network have not been reported before, it is the first report of cystic form of chiari's network.
Phosphorylation of the smooth muscle 20 kda myosin light chain (mlc) regulates smooth muscle contraction or vascular tone (1, 2), and mlc phosphorylation is determined by the balance of the activities of mlc kinase and mlc phosphatase (3). There are a number of important signaling pathways in smooth muscle that regulate vascular tone, and the vast majority influence the activity of mlc phosphatase (4, 5). The signaling pathways converging on mlc phosphatase either increase phosphatase activity to decrease mlc phosphorylation and force, which is referred to as ca desensitization, or decrease phosphatase activity to increase mlc phosphorylation and force, which is referred to as ca sensitization (4). Mlc phosphatase is a holoenzyme consisting of enzymatic, 20 kda and myosin targeting (mypt1) subunits (5). Alternative mrna splicing of a central and a 3' exon produce a mypt1 central insert (ci) and a cooh - terminal leucine zipper (lz), respectively (5); there are four mypt1 isoforms (ci+lz+, ci+lz-, ci - lz+ and ci - lz-), and mypt1 isoform expression is developmentally regulated, tissue specific (6,7,8), and is modulated in disease (9,10,11,12,13). The mypt1 lz domain is important for mediating ca desensitization during no mediated vasodilatation: during no / cgmp signaling, lz+, but not lz-, mypt1 isoforms are phosphorylated by pkg (14, 15). Therefore, lz+/lz- mypt1 isoform expression, in part, underlies the heterogenous response of the vasculature to no and no based vasodilators (14,15,16,17). For g - protein coupled agonists, the stimulation of gq and g11 activates phospholipase c, and the subsequent increase in intracellular ca activates mlc kinase (4). However, a number of agonists also activate rhoa / rho kinase through g12 and g13 (4). Rho kinase phosphorylates mypt1 at both thr696 and thr853 (18), which decreases the activity of the mlc phosphatase (18,19,20,21,22) to produce ca sensitization (21, 23, 24). However, similar to ca desensitization, there is variability in the response of the vasculature to agonists; in smooth muscles, there is heterogeneity in the magnitude and sensitivity of ca sensitization (25, 26), and the mechanism to explain this variability is unknown . Therefore, this study was designed to determine if mypt1 isoforms are differentially phosphorylated by rho kinase . To investigate this question, we determined the time course of mypt1 phosphorylation (thr696 and thr853) following activation of rho kinase with gtps in hek293 t cells lines that express each of the mypt1 isoforms . For the present study, we used our human embryonic cell (hek293 t) lines that express the four avian mypt1 isoforms (ci+lz+, ci+lz-, ci - lz+, ci - lz-). These hek293 t cell lines have the sv40 large t - antigen, which allows an episomal plasmid to be replicated, and the cloning and transfection techniques to generate these hek293 t cell lines have been previously described (15). Untransfected hek293 t cells expresses the four isoforms of human mypt1 (15), and in the present experiments, the density of the ratio of ci+/ci- bands was 1.22x higher for the band containing the exogenous avian mypt1 isoform, which is consistent with an overexpression of the exogenous avian mypt1 isoform and our previous data (15). The sequences of the avian and mammalian mypt1 are highly homologous and there are multiple regions of sequence identity (27). Rho kinase mediated mypt1 phosphorylation occurs at thr696 and thr853 of the mammalian sequence, which corresponds to thr695 and thr850 of the avian sequence . For consistency, we will refer to the numbering of the mammalian sequence throughout the manuscript . As previously described (15), hek293 t cells lines were grown in p100 plates using 1x dulbecco's modified eagle's media (dmem) supplemented with 10% fetal bovine serum (fbs), 1% penicillin / streptomycin and 0.004% zeocin at 37 c with 5% co2, and cells were split at 80100% confluence . To determine the time course of mypt1 phosphorylation, cells were grown to 7090% confluence, and then the hek293 t cells were starved for 24 h in low serum media (1% fbs, 1% penicillin / streptomycin and 0.004% zeocin) at 37 c . As described in previous publications (28), cells were placed in skinning solution (0.1% triton x-100) for 10 min and then the solution was changed to a low ca solution and the cells were treated at 37 c with 0.1 mm guanosine 5'-o-(3-thiotriphosphate) tetralithium (gtps). At 0 min, 1 min, 5 min and 30 min of gtps treatment, cells scraped off the plate, sonicated, spun and placed in fresh tubes, and stored at 80 c prior to immunoblotting . As we have described (15), mypt1 phosphorylation was determined using phospho - specific antibodies and normalized to mypt1 expression . Gels were run in pairs, one for mypt1 and the other for phospho - mypt1 . Mypt1 was detected using a polyclonal anti - mypt1 antibody (epitomics), while mypt1 phosphorylation at thr696 or thr853 was detected using phospho - specific antibodies to either phospho - thr696 or phospho - thr853 (cell signaling). To determine relative thr696 and thr853 mypt1 phosphorylation, images were scanned (epson perfection v750 pro) and band density was determined using imagequant tl software (ge healthcare). Relative phosphorylation was computed as the density of the phosphorylated signal (thr696 or thr853) divided by the density of the mypt1 protein band(s). The data for relative phosphorylation for both thr696 and thr853 were normalized . To normalize the data for each analysis, the density of the ratio of phospho - mypt1/mypt1 for the 30 min time point was averaged and all time points were subsequently normalized to this mean . The student's t - test was used to determine the significance of the difference between the relative phosphorylation between the 0 and 30 min time points, and p<0.05 was considered significant . As previously described (15), hek293 t cells lines were grown in p100 plates using 1x dulbecco's modified eagle's media (dmem) supplemented with 10% fetal bovine serum (fbs), 1% penicillin / streptomycin and 0.004% zeocin at 37 c with 5% co2, and cells were split at 80100% confluence . To determine the time course of mypt1 phosphorylation, cells were grown to 7090% confluence, and then the hek293 t cells were starved for 24 h in low serum media (1% fbs, 1% penicillin / streptomycin and 0.004% zeocin) at 37 c . As described in previous publications (28), cells were placed in skinning solution (0.1% triton x-100) for 10 min and then the solution was changed to a low ca solution and the cells were treated at 37 c with 0.1 mm guanosine 5'-o-(3-thiotriphosphate) tetralithium (gtps). At 0 min, 1 min, 5 min and 30 min of gtps treatment, cells scraped off the plate, sonicated, spun and placed in fresh tubes, and stored at 80 c prior to immunoblotting . As we have described (15), mypt1 phosphorylation was determined using phospho - specific antibodies and normalized to mypt1 expression . Gels were run in pairs, one for mypt1 and the other for phospho - mypt1 . Mypt1 was detected using a polyclonal anti - mypt1 antibody (epitomics), while mypt1 phosphorylation at thr696 or thr853 was detected using phospho - specific antibodies to either phospho - thr696 or phospho - thr853 (cell signaling). To determine relative thr696 and thr853 mypt1 phosphorylation, images were scanned (epson perfection v750 pro) and band density was determined using imagequant tl software (ge healthcare). Relative phosphorylation was computed as the density of the phosphorylated signal (thr696 or thr853) divided by the density of the mypt1 protein band(s). The data for relative phosphorylation for both thr696 and thr853 were normalized . To normalize the data for each analysis, the density of the ratio of phospho - mypt1/mypt1 for the 30 min time point was averaged and all time points were subsequently normalized to this mean . The student's t - test was used to determine the significance of the difference between the relative phosphorylation between the 0 and 30 min time points, and p<0.05 was considered significant . Mypt1 phosphorylation at both thr853 and thr696 was detected prior to treatment with gtps . However, the signal representing phospho - thr853 was faint . For the hek293 t cells over expressing ci+ mypt1 isoforms (ci+lz+mypt1 & ci+lz - mypt1), gtps did not increase relative mypt1 phosphorylation at thr853 (phospho - thr853/mypt1; table 1table 1 . Relative thr853 mypt1 phosphorylationmypt1 isoform0 min30 minci+lz - mypt10.7 0.31.0 0.4ci+lz+mypt10.7 0.11.0 0.3ci - lz - mypt10.9 0.11.0 0.4ci - lz+mypt10.7 0.11.0 0.3following treatment with gtps, relative thr853 phosphorylation, computed as the density of both bands on the western blot in fig . 1 (total phospho - thr853/total mypt1) did not change (, p<0.05, n=46). (a) western blots demonstrating time course of thr853 phosphorylation for ci+lz - mypt1 and ci+lz+mypt1 . (b) western blots demonstrating time course of thr853 phosphorylation for the ci - lz - mypt1 and ci - lz+mypt1 . Relative total mypt1 thr853 phosphorylation (total phospho - thr853/total mypt1) did not significantly change after gtps for any mypt1 isoform (table 1).). Similarly, for the hek293 t cells expressing ci- mypt1 isoforms, there was no significant increase in relative thr853 mypt1 phosphorylation with gtps treatment (table 1, fig . Following treatment with gtps, relative thr853 phosphorylation, computed as the density of both bands on the western blot in fig . 1 (total phospho - thr853/total mypt1) did not change (, p<0.05, n=46). (a) western blots demonstrating time course of thr853 phosphorylation for ci+lz - mypt1 and ci+lz+mypt1 . (b) western blots demonstrating time course of thr853 phosphorylation for the ci - lz - mypt1 and ci - lz+mypt1 . Relative total mypt1 thr853 phosphorylation (total phospho - thr853/total mypt1) did not significantly change after gtps for any mypt1 isoform (table 1). In contrast to the results for thr853 mypt1 phosphorylation, the phospho - thr696 bands were readily apparent . (a) western blots demonstrating time course of thr696 phosphorylation for ci+lz - mypt1 and ci+lz+mypt1 . (b) western blots demonstrating time course of thr696 phosphorylation for the ci - lz - mypt1 and ci - lz+mypt1 . Relative total mypt1 thr696 phosphorylation (total phospho - thr696/total mypt1) increased after gtps for all mypt1 isoforms, except ci - lz+ mypt1 (table 2)., both bands). In the hek293 t cells expressing ci+ mypt1 isoforms (ci+lz+mypt1 & ci+lz - mypt1), there was a significant increase in total relative thr696 mypt1 phosphorylation following treatment with gtps (table 2table 2 . Relative thr696 mypt1 phosphorylationmypt1 isoform0 min30 minci+lz - mypt10.2 0.11.0 0.3ci+lz+mypt10.6 0.21.0 0.3ci - lz - mypt10.5 0.11.0 0.2ci - lz+mypt10.8 0.21.0 0.2following treatment with gtps, relative thr696 phosphorylation, computed as the density of both bands on the western blot in fig . 2 (total phospho - thr696/total mypt1) increased for every mypt1 isoform, except ci - lz+mypt1 (, p<0.05, n=46). Note, the data in this table represent the results for both bands in fig . 2, 3 is only for the single mypt1 band expressing the exogenous mypt1 isoform (ci+ or ci-), which is indicated by the arrowhead . ). However in the hek293 t cells expressing ci- mypt1 isoforms, the results were variable; in hek293 t cells expressing ci - lz - mypt1, gtps stimulation increased total relative mypt1 phosphorylation at thr696, while in the hek293 t cells expressing ci - lz+mypt1, total relative thr696 mypt1 phosphorylation did not change (table 2). (a) western blots demonstrating time course of thr696 phosphorylation for ci+lz - mypt1 and ci+lz+mypt1 . (b) western blots demonstrating time course of thr696 phosphorylation for the ci - lz - mypt1 and ci - lz+mypt1 . Relative total mypt1 thr696 phosphorylation (total phospho - thr696/total mypt1) increased after gtps for all mypt1 isoforms, except ci - lz+ mypt1 (table 2). Following treatment with gtps, relative thr696 phosphorylation, computed as the density of both bands on the western blot in fig . 2 (total phospho - thr696/total mypt1) increased for every mypt1 isoform, except ci - lz+mypt1 (*, p<0.05, n=46). Note, the data in this table represent the results for both bands in fig . 2, while the time course of thr696 mypt1 phosphorylation in fig . 3 is only for the single mypt1 band expressing the exogenous mypt1 isoform (ci+ or ci-), which is indicated by the arrowhead . To further define isoform specific mypt1 phosphorylation in response to gtps, we examined the time course of thr696 phosphorylation for only the single mypt1 band containing the exogenous mypt1 isoform (indicated by the arrowhead in fig . 2). For both lz- mypt1 isoforms (ci+lz- & ci - lz-), gtps produced a significant increase in relative thr696 mypt1 phosphorylation (fig . 3fig . The time course of relative thr696 mypt1 phosphorylation was computed for only the single band containing the exogenous mypt1 isoform (ci+ or ci-), which is indicated by the arrowhead in fig 2 . (a) time course of relative thr696 phosphorylation for ci+lz - mypt1 (, n=4) and ci+lz+mypt1 (, n=4). The increase in thr696 phosphorylation was significant for ci+lz- mypt1 (p<0.05), but not the ci+lz+mypt1 isoform . (b) time course of relative thr696 phosphorylation of ci - lz - mypt1 (, n=6) and ci - lz+mypt1 (, n=4). There is a significant increase in thr696 phosphorylation for ci - lz- mypt1 (p<0.05) and a significant decrease (p<0.05) in thr696 phosphorylation for ci - lz+ mypt1 . The solid lines represent a single exponential fit of the time course of phosphorylation . ). For the ci+lz+ mypt1, 3a, p>0.05), while for the ci - lz+ mypt1 isoform, relative thr696 phosphorylation decreased (p<0.05) following gtps (fig . The time course of relative thr696 mypt1 phosphorylation was computed for only the single band containing the exogenous mypt1 isoform (ci+ or ci-), which is indicated by the arrowhead in fig 2 . (a) time course of relative thr696 phosphorylation for ci+lz - mypt1 (, n=4) and ci+lz+mypt1 (, n=4). The increase in thr696 phosphorylation was significant for ci+lz- mypt1 (p<0.05), but not the ci+lz+mypt1 isoform . (b) time course of relative thr696 phosphorylation of ci - lz - mypt1 (, n=6) and ci - lz+mypt1 (, n=4). There is a significant increase in thr696 phosphorylation for ci - lz- mypt1 (p<0.05) and a significant decrease (p<0.05) in thr696 phosphorylation for ci - lz+ mypt1 . The solid lines represent a single exponential fit of the time course of phosphorylation . In smooth muscle, in addition to mlc kinase (1) and rhoa / rho kinase (21, 24), there are multiple other signaling pathways that modulate force including pkc (29,30,31), zip kinase (32), integrin - linked kinase (33) and rac1 (34), and further, the physiologically important signaling pathways that mediate ca sensitization are both agonist as well as tissue specific (4, 35). The rho kinase inhibitor fasudil reduces blood pressure in animal models of hypertension (36,37,38) and in humans, is effective in treating both cerebral vasospasm (39, 40) and pulmonary hypertension (41, 42), which suggests that rho kinase mediated signaling is important in both health and disease . Rho kinase has been demonstrated to phosphorylate both mypt1 as well as cpi-17 (43, 44), and similar to the other signaling pathways which influence mlc phosphatase activity, the importance of each of these proteins for ca sensitization is tissue specific (44). In the present study, we examined whether gtps treatment results in isoform specific mypt1 phosphorylation . Others have demonstrated rho kinase is expressed in hek293 t cells (45, 46) and additionally, in hek293 t cells, gtps stimulates rho (47, 48). Further, rho has been demonstrated to activate rho kinase, pkn and pip5 kinase (49). Therefore, the gtps stimulated increase in mypt1 phosphorylation could be mediated by rho kinase, pkn, pip5-kinase or another unknown kinase . However, only rho kinase and ilk have been demonstrated to phosphorylate mypt1 at thr696 and thr853 (50), and ilk is not activated by rho kinase, pkn or pip5 kinase (51). These data strongly suggest that following gtps treatment of hek293 t cells, mypt1 phosphorylation at thr696 and/or thr853 is mediated by a gtps induced activation of rho kinase . We have previously demonstrated that an overexpressed exogenous mypt1 isoform replaces the endogenous isoform in the mlc phosphatase holoenzyme (17) and alters the activity of mlc phosphatase (15, 17). Similar to previous reports (15, 17), in the present experiments, the ratio of ci+/ci- changed consistent with overexpression of the exogenous mypt1 . These data suggest that mypt1 phosphorylation determined in these hek293 t cell lines reflects the phosphorylation of the exogenous mypt1 isoform (15). However compared to our prior results, the expression of the exogenous avian mypt1 isoform is lower in the present study, which could result in an underestimation of the magnitude of the changes in mypt1 phosphorylation . In the present study, mypt1 phosphorylation at thr696 increased after treatment with gtps for the ci+lz - mypt1 and ci - lz - mypt1 isoforms (fig . Others have demonstrated that activation of pkg increases mypt1 phosphorylation at both ser695 and ser852, which then inhibits rho kinase mediated mypt1 phosphorylation at thr696 and thr853 (52, 53). The lz domain is hypothesized to mediate the interaction of mypt1 and pkg (16) and is required for pkg mediated mypt1 phosphorylation (14, 15, 17). These data could suggest that lz- mypt1 isoforms, but not lz+ mypt1 isoforms, are phosphorylated by rho kinase, which is consistent with our results . For the ci+lz+ mypt1 isoform, gtps treatment increased thr696 phosphorylation, but the increase did not reach statistical significance (fig . 3), which could suggest the ci domain modulates rho kinase phosphorylation . For the ci - lz+mypt1 isoform, it is unclear why relative thr696 phosphorylation decreased with gtps stimulation (fig . One possibility is that rho kinase cannot access thr696 due to differences in the three - dimensional structure of ci - lz+mypt1 and the other mypt1 isoforms, and thus for ci - lz+ mypt1, thr696 is only subject to autodephosphorylation (54). Alternatively, this may be due to an interaction of ci - lz+mypt1 and another protein, such as m - rip (55) or prostate apoptosis response 4 (56), which could alter the kinetics of rho kinase mediated mypt1 phosphorylation . In contrast to the signal for thr696 phosphorylation, the signal for phospho - thr853 mypt1 was low, and we could not detect a significant increase in relative thr853 mypt1 phosphorylation after gtps treatment (table 1, fig . These results contrast to the significant increase in mypt1 thr853 phosphorylation observed in smooth muscle (44, 57). The mechanism to explain the lack of thr853 phosphorylation in the hek293 t cells expressing avian mypt1 isoforms is beyond the scope of the present study . In our hek293 t cells, gtps could have activated a g - protein pathway that inactivated rho or even activated a kinase other than rho kinase . Since gtps treatment increased phosphorylation at thr696, a well documented mypt1 residue for rho kinase (4), this possibility is unlikely . In isolated 500 aa mypt1 protein fragments, we have demonstrated that rho kinase does not phosphorylate thr853 (14). However, in untransfected control hek293 t cells, we have previously demonstrated that the phosphorylation of the endogenous human ci+ mypt1 at both thr696 and thr853 (15), which differs from the present results . These data could also suggest that for avian mypt1, phospho - thr853 is poorly recognized by the anti - phosphothr853 antibody . However, there is a high degree of identity between the aa sequences of avian and mammalian mypt1 (27); 85% identity for the 60 aa flanking the thr850/thr853 phosphorylation site and we also detected a phospho - thr853 signal, albeit faint (fig . Similar to our results, there is also considerable variability in thr696 vs. thr853 mypt1 phosphorylation in mammalian smooth muscle; mypt1 phosphorylation is both tissue and agonist dependent . Studies have demonstrated that 1) a rho kinase mediated mypt1 phosphorylation at thr853, but not thr696, is responsible for the sustained phase of the force response in rat uterine smooth muscle (58), 2) a rho kinase mediated increase in both thr696 and thr853 mypt1 phosphorylation occurs during serotonin induced vasoconstriction of cerebral arteries (59), 3) a rho kinase mediated increase in mypt1 phosphorylation at thr853, but not thr696, contributes to the myogenic response of cerebral vessels (60), 4) a rho kinase mediated increase in thr696 mypt1 phosphorylation occurs during ca sensitization of rat ileal smooth muscle (61) and 5) a rho kinase mediated increase in thr696 mypt1 phosphorylation is responsible for pgf2 induced ca sensitization in rabbit aorta (62). Additionally, during activation of mouse bladder smooth muscle, mypt1 phosphorylation at thr853 is mediated predominantly by rho kinase, while phosphorylation at thr696 did not change (63). Using mypt1 ala mutants, these investigators demonstrated that the increase in thr853 phosphorylation did not contribute to the force response, while the force maintenance was reduced in the ala696thr mypt1 mutant, suggesting that mypt1 phosphorylation at thr696 is important for the regulation of force maintenance (63). Our current results demonstrate that gtps treatment of hek293 t cells increased thr696 phosphorylation of ci+lz- and ci - lz-, but not ci+lz+ and ci - lz+, mypt1 isoforms . These data suggest that following agonist activation, rho kinase will differentially phosphorylate mypt1 isoforms . We have previously demonstrated that mypt1 isoform expression determines the sensitivity of no mediated vasodilatation (14, 15, 17); our previous results show that smooth muscle tissues expressing lz- mypt1 isoforms (ci+lz-, ci - lz-) are not phosphorylated by pkg (14, 15). Taken together, these results suggest that the lz mypt1 domain is an important determinant for the regulation of mlc phosphatase; lz+ mypt1 isoforms are phosphorylated by pkg (14, 15), but poor substrates for rho kinase (fig . 3), while lz- mypt1 isoforms are phosphorylated by rho kinase (fig . The ci domain appears to modulate rho kinase phosphorylation of lz+ mypt1 isoforms; following gtps treatment mypt1 phosphorylation decreased for ci - lz+mypt1, but did not change for ci+lz+mypt1 (fig . 3), which provides another mechanism to tune the vasculature's response to vasoactive agents . These data demonstrate that alternative splicing to produce ci+/ci- and lz+/lz- mypt1 isoforms could contribute to the molecular mechanism producing the variable sensitivity of smooth muscle to signaling pathways for both ca sensitization (rhoa / rho kinase) and desensitization (no / cgmp / pkg). Changes in relative ci+/ci- mypt1 isoform expression have only been examined in an animal model of portal hypertension (10), but relative lz+/lz- mypt1 isoform expression is well documented to decrease in a number of diseases including heart failure (9, 11, 64, 65) and pulmonary hypertension (13, 66). Therefore, modulation of mypt1 isoform expression may represent a mechanism to tune the vasculature's response to both no / pkg and g - protein coupled agonists / rho kinase signaling, which will influence vascular tone and/or resistance in both health and disease.
Exercise - induced muscle damage occurs after high - intensity resistance and endurance exercise (santos et al ., 2004; veggi et al ., exercise - induced muscle damage is classified into primary and the secondary damage (howatson and van someren, 2008). Primary muscle damage is related to morphological changes, including sarcomere (z - disc, i, and a band), sarcolemma, sarcoplasmic reticulum, and cytoskeletal elements (clarkson and hubal, 2002). Secondary muscle damage occurs due to impaired calcium homeostasis and the inflammatory response (beaton et al ., 2002; impaired calcium homeostasis due to sarcoplasmic reticulum dysfunction is activated by calpain-3, which is a calcium - activated neutral protease (beaton et al ., 2002), that increases muscle damage and protein degradation (murphy, 2010). Neutrophils and macrophages invade the damaged site, facilitate phagocytosis, and secrete substances that induce oxidative stress (tidball, 2005). This phenomenon may lead to a decrease in maximal strength and increase delayed - onset muscle soreness and muscle proteins, such as creatine kinase (ck) and lactate dehydrogenase (ldh) in the blood (clarkson and hubal, 2002). Use of dietary supplements is a recommended scheme to attenuate exercise - induced muscle damage (sousa et al ., 2014). Creatine has been used as a dietary supplement for a long time by many athletes and others (bird, 2003). Creatine (n - aminoiminomethyl - n - methylglycine) is a naturally generated endogenous guanidine compound synthesized in the kidneys, pancreas, and liver from methionine, glycine, and arginine (bemben and lamont, 2005) and released into the blood (dantona et al ., 2014). Most creatine is localized in skeletal muscle and stored as creatine phosphate (pcr). Ck and pcr play a pivotal role in short - term (only a few seconds) exercise (bird, 2003). High creatine levels are found naturally in meat and fish (dantona et al ., 2014) and intramuscular pcr levels can be increased by approximately 20% by using a creatine supplement (harris et al ., 1992). The ergogenic effect of creatine is well - known to improve exercise performance such as explosive muscle power (claudino et al ., 2014; zuniga et al ., 2012) and increased lean body mass after resistance exercise (candow et al ., 2014; chilibeck et al ., several studies have reported that creatine promotes recovery by attenuating muscle damage after eccentric exercise (cooke et al ., 2009; rosene et al ., 2009 (2009) indicated that creatine supplementation may help rescue maximal strength and inhibit ck release due to high intensity exercise . In contrast, other studies have reported that creatine does not reduce muscle damage after eccentric exercise (mckinnon et al ., 2012; rawson et al ., 2001). Despite that creatine potentially reduces muscle damage, it has not generally been used in the sports rehabilitation field . The aim of this review was to introduce the effects of taking creatine on exercise - induced muscle damage . (2000) verified the effect of creatine on exercise - induced muscle damage . In this study mice performed 150 eccentric muscle contractions in response to electric stimulation after ingesting 0.5 or 1% creatine for 14 days . As shown in tables 1 and 2, several studies have reported that creatine attenuates exercise - induced muscle damage (bassit et al ., 2010; cooke et al ., 2009; rosene et al ., 2009; veggi et al ., g / kg / day, four servings / day, 5 days; maintenance period: 0.1 g / kg / day, one serving / day, 14 days) beginning 5 days before exercise until 14 days after exercise improved maximal isometric strength and decreased ck compared with those who consumed a carbohydrate placebo only . (2010) also reported that ingesting 20 g / day creatine over 5 days decreases ck and ldh after a triathlon competition . 2009) reported the acute (20 g / day, 7 days) and chronic (6 g / day, after 7 days followed for 23 days) effects of creatine on exercise - induced muscle damage . This study demonstrated that chronic ingestion of creatine effectively increased maximal isometric strength after resistance exercise . (2013) suggested that taking 20 g / day creatine for 6 days between the first and the second exercise phase contributed to decreased muscle soreness, inhibited the elevation in ck, and enhanced of range of motion . 2013), suggested that taking creatine may increase the repeated bout effect after initial exercise - induced muscle damage . The repeated bout effect is protective against subsequent muscle damage through neural, mechanical, and cellular adaptations after exercise (mchugh, 2003). However, several studies suggested that creatine had no benefit on exercise - induced muscle damage (mckinnon et al ., 2012; rawson et al . Rawson et al . (2001) demonstrated that creatine (20 g / day) taken for 5 days before and after exercise does not change the levels of muscle damage markers after exercise between subjects taking creatine and a placebo . Rawson et al . (2007) reported that creatine (loading period: 0.3 g / kg / day, three servings / day, 5 days; maintenance period: 0.03 g / kg / day, one serving / day, 5 days) does not change muscle damage marker levels after exercise . (2012) reported that taking creatine (loading dose: 40 g, two servings / day, 5 days; maintenance period: 10 g, two servings / day, 5 days) had no effect on exercise - induced muscle damage . These conflicting results may be partly explained by differences in exercise protocols used in the studies . A number of potential mechanisms explain the effect of creatine on exercise - induced muscle damage, including the inflammatory response, oxidative stress, calcium homeostasis, and satellite cells activities in damaged muscle (fig . The first potential mechanism of creatine is that it reduces the inflammatory response after exercise - induced muscle damage . Santos et al . (2004) demonstrated that 20 g / day creatine for 5 days before 34 male marathon runners raced significant reduced ldh, prostaglandin e2 (pge2), and tumor necrosis factor - a (tnf-) after the 30 km race . (2008) reported that 11 male triathletes who ingested 20 g / day creatine for 5 days prior to a half - ironman competition had significant decreases in tnf-, interferon- (inf-), interleukin-1 (il-1), and pge2 after the competition compared to those in the placebo group . (2013) also reported that ingesting 0.3 g / kg creatine for 7 days abolishes the increase in tnf- after a repeated running - based anaerobic test . Pge2 and tnf- facilitate the inflammatory response and pain sensation after exercise - induced muscle damage (tidball, 2005). Interestingly, all three studies showed a decrease in the inflammatory response (bassit et al . Santos et al . (2004) particularly showed a decrease in ldh and inflammation . The inflammatory response is associated with markers of sarcolemma damage (kanda et al ., (2013) reported a positive correlation between neutrophil migratory activity and myoglobin after exercise . These results indicate that the reduction of inflammatory response factors by creatine may decrease disruption of sarcolemma due to exercise - induced muscle damage . (2010) reported that taking creatine (5 g / kg / day, 5 days) significant decreases outflux of intracellular enzymes after continuous muscle contraction . 2013) found that ingesting creatine (300 mg / kg / day, 15 days) does not significantly reduce inflammatory response markers, such as tnf-, il-1, and nuclear factor kappa - light - chain - enhancer of activated b cells (nf-b) in mice . The second potential creatine mechanism is diminished oxidative stress (lawler et al ., 2002; rahimi, 2011). Lawler et al . (2002) reported the first evidence for the antioxidant capacity of creatine . Deminice and jordao (2012) found that ingesting 2% creatine during the 28 days before acute exercise decreases thiobarbituric acid - reactive substances (tbars) and lipid hydroperoxides but increases the glutathione (gsh) and glutathione disulfide (gssg) ratio and total antioxidant capacity . However, these studies were limited to cultured cells models, and animals . According to a human study by rahimi (2011), taking 20 g / day creatine for 7 days decreases malonyldialdehyde (mda) and 8-hydroxy-2-deoxyguanosine (8-ohdg) levels after resistance exercise compared to those taking a placebo . In contrast, several studies have reported that creatine does not decrease oxidative stress after exercise - induced muscle damage (deminice et al ., 2013; silva et al ., 2013). Impaired sarcoplasmic reticulum due to muscle damage may increase calcium concentrations in the cytosol, causing secondary muscle damage (beaton et al . Creatine assists in maintaining the sarcoplasmic reticulum calcium pump function by phosphorylating adp to atp, which decreases cytosolic calcium levels (cooke et al . 1996) suggested that increasing muscle pcr accelerates atp homeostasis, leading to reduced secondary damage due to an increase in calcium concentration . Finally, creatine has been associated with satellite cells or so - called muscle stem cells (olsen et al ., 2006; safdar et al ., 2008). Satellite cells play a critical regenerating role after muscle damage (paulsen et al ., 2012). Olsen et al . (2006) demonstrated that ingesting creatine (loading period: 24 g / day, 6 g / serving, four servings / day, 7 days; maintenance period: 6 g / day, one serving / day, 15 weeks) and performing resistance exercise increases the number of satellite cells and myonuclei concentration in human muscle . (2008) reported that taking creatine (loading period: 20 g / day, 10 g / serving, two servings / day, 3 days; maintenance period: 5 g / day, one serving / day, 7 days) promotes proliferation and differentiation of satellite cells and activate cytoskeletal remodeling genes . Creatine may be a useful dietary supplement for preventing muscle damage and facilitating recovery from high - intensity exercise, which is applicable to the sports rehabilitation field . However, several mechanisms of how creatine prevents exercise - induced muscle damage need to be examined in future well - designed studies.
The chemokine receptor cxcr3 is a class a seven - transmembrane - domain or g protein - coupled receptor (gpcr) that is involved primarily in chemotaxis of certain immune cells, inhibition of angiogenesis, and th1 cell polarization [13]. Cxcr3 is expressed by various effector t lymphocytes, including cd4 t helper 1 (th1) cells, cd8 cytotoxic t lymphocytes (ctl), and cd4 and cd8 memory t cells, as well as monocytes, m1 macrophages, natural killer (nk) cells, subsets of b - cells, mast cells, endothelial cells, and vascular smooth muscle cells [14]. Cxcr3 couples to gi protein [5, 6] and although not extensively studied, it has been shown to activate a number of signaling pathways that are generally associated with gpcrs such as increases in intracellular calcium and activation of map kinases and pi3k / akt signaling [4, 7, 8]. The principal agonists of cxcr3 are cxcl9 (mig), cxcl10 (ip-10), and cxcl11 (i - tac). The human equivalent of the murine form of cxcr3 is cxcr3a and unless noted otherwise cxcr3 is used in this review to include both murine and human isoforms . Cxcr3b, which couples to gs, is the receptor isoform expressed in microvascular endothelial cells and is linked to inhibition of angiogenesis and induction of apoptosis [2, 3]. Besides cxcl9, cxcl10, and cxcl11, cxcr3b and cxcr3 are activated by cxcl4 and cxcl4l1, chemokines that are released by platelets and have been implicated in atherogenesis and acute coronary syndrome [3, 9, 10]. Cxcr3-alt is a truncated form of cxcr3 that is selectively activated by cxcl11 [24]. Cxcr3 is associated with the pathophysiology of th1-type diseases, including infections of various etiologies and autoimmune disorders [1, 3]. Although cxcr3 is activated by cxcl9, cxcl10, and cxcl11, the outcome is different with growing evidence that cxcl9 and cxcl10 are essentially proinflammatory, while cxcl11 has anti - inflammatory actions [11, 12]. Over the last decade, numerous studies have documented elevated circulating levels of cxcl10 in wide - ranging infectious and autoimmune diseases, autoimmune encephalomyelitis, crohn's disease, tuberculosis, thyroid autoimmune diseases, and type 1 diabetes, as well as several cancers [1318]. Recent evidence from us and others has revealed the importance of the cxcl10/cxcr3 axis in cardiovascular diseases . As discussed elsewhere, cxcl9 and cxcl10 in addition, the homing signature for memory t cells to the heart from mediastinal lymph nodes is c - metccr4cxcr3 . While c - met triggering supports cardiotropic t cell recirculation, cxcr3 and ccr4 engagement via tissue - released cxcl10 and ccl4, respectively, sustains recruitment in heart inflammation, we present an overview of the role of cxcl9 and cxcl10 in infectious and noninfectious diseases of the heart and its implications for immunotherapy . Recently, zohar et al . Showed that cxcl9 and cxcl10 drive effector th1/th17 cell polarization via stat1, stat4, and stat5 activation, thereby promoting inflammation . In contrast, cxcl11, which exhibits relatively higher binding affinity for cxcr3, drives development of foxp3 (forkhead box p3)-negative il-10 t regulatory 1 (tr1) cells and il-4 th2 cells via stat3 and stat6 activation and was demonstrated to dampen inflammation . The opposite actions of the cxcr3 agonists are likely the consequence of the biased signaling that is a fixture of gpcrs, which can activate both g protein - dependent and protein - independent signaling cascades, the latter occurring via -arrestin 2 recruitment [1, 11, 12]. Biased allosteric agonists of cxcr3 that selectively activate -arrestin or g protein - dependent signaling are in development and may have utility in immunotherapy . Coronary artery disease (cad), which progresses to coronary heart disease, is a leading cause of death in the usa and globally [23, 24]. Cad is caused by atherosclerosis within the arteries of the heart, a chronic inflammatory condition associated with waxy plaque buildup . Cxcr3 expressing monocytes / macrophages, th1 cells, nk cells, and ctl cells play a critical role in atheromatous plaque progression and eventual disruption . Ruptured or ulcerated plaques cause formation of a thrombus that precipitates an acute coronary syndrome, such as unstable angina or a heart attack . Endothelial dysfunction, increased vascular permeability, increased expression of adhesion molecules on endothelial cells for leukocytes, and increased plasma levels of low density lipoprotein (ldl) are initiating factors in atherosclerosis . Ldl, which accumulates in the intima, undergoes oxidation by macrophages and endothelial cells, as well as by vsmc that migrate into the intima from the media and proliferate . In response to the oxidized ldl and plasma ldl, endothelial cells secrete proinflammatory cytokines and chemokines (mcp-1/ccl2, fractalkine, and cxcr3 ligands) that attract monocytes, which differentiate into dendritic cells or macrophages that accumulate oxidized ldl to become foam cells . T cells are recruited into the intima, and dendritic and nk cells help induce the cd4 th1 phenotype, which is the most abundant t cell population in human atherosclerotic plaques . Th1 cells, as do nk cells, produce ifn-, which contributes to th1 polarization, activates proinflammatory m1 macrophages, and induces apoptosis . The atheromatous plaque that builds up in the artery wall is made up of an accumulation of lipids, fibrous connective tissue, macrophages, and cellular debris that arises from the cytolytic actions of oxidized ldl, nk cells, ifn-, and ctl cells on macrophages, foam cells, vsmc, and endothelial cells . A fibrous coat of extracellular matrix proteins produced by vsmc stabilizes the plaque, but proinflammatory m1 macrophages secrete metalloproteinases in response to ifn- that degrade the fibrous cap and enhance its vulnerability to rupture . Cxcl10 is reported to be expressed by endothelial cells, smooth muscle cells, and macrophages during the formation of atherosclerotic lesions in both preclinical and clinical studies [28, 29]. Suppression of cxcl10 bioactivity in apo - e deficient mice resulted in a more stable plaque phenotype with less macrophage activation, along with more smooth muscle cells and collagen abundance . The mechanistic role of cxcl10 in the pathogenesis of atherosclerotic plaque growth and destabilization is not yet resolved . Of note, unstable plaques have increased levels of th1, nk, and ctl cells and decreased levels of anti - inflammatory regulatory t (treg) cells . Recent studies show that the relative levels of treg cells are reduced and their functionality is impaired in patients with cad [32, 33]. Knockout of cxcl10 in the apolipoprotein e - deficient mouse model of atherosclerosis was associated with increased treg cell numbers and activity, along with a reduction in lesion formation . Circulating levels of cxcl10 notably, cxcl10 was also reported to be produced by the endothelium of mouse coronary blood vessels infused with angiotensin ii, human coronary artery endothelial cells treated with tnf-, and rat cardiac microvascular endothelial cells subjected to hypoxia / ischemia . Patients with acute myocardial infarction (ami) showed significantly higher serum levels of cxcl10 than control subjects and patients with stable angina pectoris . Although serum cxcl10 levels were negatively correlated with infarct size, these results in terms of pathogenic implications and determining cause versus effect relationships have limitations . First, during ami there is a massive systemic inflammatory insult in which cxcl10 levels are expected to be high . It would be interesting to test blood concentration of cxcl10 within the first 3 hours after angina onset during ami when systemic activation is not yet started secondly, the pathogenic mechanisms of plaque rupture may involve factors acting locally without necessarily showing a high systemic blood concentration . It would be interesting to analyze cxcl10 in samples of blood obtained by thrombus - aspiration during coronary artery percutaneous intervention (pci) in patients with unstable coronary artery disease . In patients with first - time st - segment elevation ami, high circulating levels of ccl4, cxcl16, cxcl8, and cxcl10 within the first week after pci were found to be positively correlated with the degree of myocardial damage . Kawasaki disease is an autoimmune disease that manifests as a systemic vasculitis with a predilection for coronary arteries . The disease occurs in children under 5 years of age and a preexisting viral infection may have a role in its development . During the acute phase of kawasaki disease the immune system is highly activated and includes both th1 and th2 subsets . Recently, ko et al . Reported that cxcl10 is a good biomarker / predictor of kawasaki disease and, furthermore, that cxcr3 is activated in the t cells of patients with acute kawasaki disease . In addition, several studies report that numbers and functionality of treg cells are reduced in kawasaki patients [4548]. Myocarditis or inflammation of the myocardium is a heterogeneous group of disorders initiated by various pathogens, including worms, bacteria, protozoa, rickettsia, and most commonly viruses . Autoimmunity after viral myocarditis is thought to cause dilated cardiomyopathy, which is characterized by ventricular dilation and contractile dysfunction [50, 51]. Cxcl10 is elevated in the heart following viral and nonviral infection and has the characteristics of a biomarker in rodent models of myocarditis [52, 53]. Reported findings showing that cxcl10 contributes to the pathogenesis of viral myocarditis . In their study, myocarditis was induced with coxsackievirus b3 (cvb3), the primary cause of viral myocarditis, in mice that overexpressed a cxcl10 mutant protein without functional activity in order to antagonize endogenous cxcl10 . These mice exhibited ameliorated disease progression, including reduced cardiac thickening (due to inflammatory edema), inflammation, and cell death, as well as improved survival when compared to wild - type mice . The authors concluded that cxcl10 plays a crucial role in recruitment of th1 cells to the heart, leading to the increase in detrimental proinflammatory th1 cytokines . These findings have implications for interferon treatment, which is beneficial for some forms of viral myocarditis . Following cvb3 infection, the rise in ifn- stimulates cxcl10 expression in cardiac myocytes and other cardiac cells . Yuan et al . Reported that cxcl10 inhibits cvb3 replication at early stage of infection, consequently protecting cardiac myocytes from damage and improving heart function . This antiviral activity of cxcl10 entails the regulation of natural killer (nk) cell infiltration into the myocardium and associated ifn- expression . However, the transient antiviral effect of cxcl10 was shown to be insufficient for viral clearance and in preventing death during acute inflammation stages in their mouse model . Other chemokines or cytokines were proposed to play an important role in clearance of viruses . No simple explanation seems to explain the disparate findings of yuan et al . And yue et al . On whether myocardial cxcl10 is harmful or beneficial in the context of acute cvb3 myocarditis, although timing and dosage levels of cxcl10 and effective viral clearance versus th1 recruitment are likely contributing factors . Evidence indicates the major contribution of autoimmunity to the etiology of myocarditis and thus adoptive transfer of tregs and/or stimulating their differentiation are promising therapeutic approaches [49, 50]. Both th1 and th17 cells drive myocarditis, with th17 cells playing an important part in the development of dilated cardiomyopathy . Chagas disease is a tropical disease that results from infection with the protozoan parasite trypanosoma cruzi and affects ~10 million individuals worldwide but is most prevalent in latin america [58, 59]. In some, 2030%, of infected individuals, chronic infection leads to a potentially fatal cardiomyopathy known as chagas heart disease, generally 1020 years after the initial infection [60, 61]. Chagas heart disease is characterized by marked inflammation and fibrosis of the heart, along with cardiac edema, myofibrillar destruction, chamber dilation, and loss of contractile function . The etiology of chagas heart disease is not fully understood and likely multifactorial, with a contribution of an autoimmune response due in part to molecular mimicry between antigenic determinants of t. cruzi and human (cardiac) antigens [62, 63]. With heart failure in chagas disease, myocardial levels of cd8 and cd4 t cells are increased, with a predominance of cd8 t cells [61, 64, 65]. In addition, the myocardium exhibits a strong th1 cytokine profile with increased expression of ifn- and il-18 genes that correlate with ventricular dilation [64, 65]. Circulating levels of ifn- are elevated during chronic chagas disease and were reported to be inversely correlated to left ventricular ejection fraction (lvef). In contrast, chagas - related heart failure is associated with reduced myocardial levels of treg cells [61, 64], and circulating treg activity was reported to be reduced in moderate or severe cardiomyopathy with activity directly correlated to lvef . Were detected in patients with chronic chagas disease, and lv mrna expression levels of cxcl10 were found to be elevated in patients with chagas cardiomyopathy . Recently, evidence was provided that polymorphisms in the cxcl9 and cxcl10 genes controlled the expression of chemokines in the myocardium and the degree of myocarditis in chagas cardiomyopathy . Behet's disease is an autoimmune or autoinflammatory disorder common in the middle east, asia, and japan . The basis for the pathogenesis of behet's disease is not known, although a number of factors have been proposed to have a role, including viral, bacterial, environmental, genetic, and immune factors . Behet's is caused by small - vessel systemic vasculitis that very often affects the heart in diverse ways, including endomyocardial fibrosis, intracardiac thrombus, endocarditis, pericarditis, myocarditis, coronary arteritis, myocardial infarction, and valvular disease . The cardiomyopathy may be ischemic, nonischemic, or inflammatory in nature and may manifest as asymptomatic systolic or diastolic dysfunction or overt systolic or diastolic heart failure . Recently, monocytes of behet's patients were found to have dysfunctional posttranscriptional regulation of cxcl10 mrna that resulted in overexpression of cxcl10 protein with ifn- stimulation . In general, the role of cxcl10 in immune - mediated and autoimmune myocarditis is little studied; however, based on studies of infective myocarditis, a critical role for cxcl10 is likely . After cad, hypertension is the most common risk factor for heart failure and accounts for ~25% of heart failure cases . In the elderly, as many as 68% of heart failure cases are linked to hypertension and community - based studies indicate that hypertension contributes to heart failure in 60% of patients . Hypertension causes a number of adverse remodeling events at the cellular and tissue level of the heart, including cardiac myocyte hypertrophy and gene reprograming, activation of cardiac fibroblasts, interstitial and perivascular fibrosis, and capillary refraction [7375]. These alterations ultimately cause marked changes in the overall geometry of the heart that may progress to heart failure and the inability of the heart to adequately meet the oxygen and energy demands of the body . Heart failure (hf) may manifest clinically with either preserved or reduced lvef, which are designated hfpef (so - called diastolic heart failure) and hfref (systolic heart failure), respectively . Hypertension results in concentric lv hypertrophy, which may progress to ventricular dilation and eventual hfref because of poorly understood means that may include ischemic injury [76, 77]. Related to this, volume overload due to fluid retention and impaired kidney function may cause a dilated pattern of eccentric lv hypertrophy with hypertension that leads to hfref . Generally, cardiac remodeling with hypertension reflects a combination of both concentric and eccentric patterns of remodeling . Concentric hypertrophy is also a characteristic of hfpef, which typically has hypertension as the major comorbidity . In addition, microvascular dysfunction concomitant to hypertension is thought to be a contributing factor for hfpef . The relative importance of cxcl10 in concentric versus eccentric lv hypertrophy, as well as their progression to heart failure, is not known . Numerous preclinical and clinical studies have implicated marked activation of neurohormonal drive to the heart in the pathoetiology of lv hypertrophy and its progression to heart failure . Neurohormonal drive, namely, activation of the sympathetic and renin - angiotensin - aldosterone systems, is generally thought to directly cause adverse remodeling of the heart . At the same time, there is evidence for indirect actions of neurohormonal stimulation on cardiac remodeling via activation of innate immunity and inflammation, especially the induction of heart - derived proinflammatory cytokines . In chronic heart failure, an increased th1/th2 ratio is seen, but whether increased th1 cell levels contribute to heart failure progression or simply are a consequence of heart failure is unresolved . Exciting new findings have now implicated adaptive immunity and t cells more directly as causal agents in hypertensive lv hypertrophy and resultant heart failure by poorly understood means . These preclinical studies employed the mouse model of transverse aortic constriction- (tac-) induced heart failure to mimic the impact of high blood pressure on the heart . Laroumanie et al . Reported increased recruitment of activated cd4 and cd8 t cells and elevated levels of several chemokines for t cells and monocytes, including cxcl10, in ventricular tissues from mice with tac - induced heart failure . Tac - induced ventricular dilation and fibrosis was prevented and contractile dysfunction was attenuated in mice deficient in mature b and t lymphocytes due to knockout of rag2, although cardiac hypertrophy was still observed . T cell replenishment in rag2 knockout mice restored the tac - induced heart failure phenotype . In addition, elimination of cd4 t cells (mhcii knockout) but not cd8 t cells (cd8 knockout) prevented tac - induced cardiac fibrosis and failure, suggesting a critical involvement of t helper cells . This conclusion was further supported by the observation that mice with transgenic t cell receptor specific for ovalbumin did not develop heart failure and fibrosis with tac . Altogether these findings suggest that activation of cd4 t cells in hypertension causes interstitial and perivascular fibrosis that leads to functional and morphological changes in the heart conducive to the development of heart failure . However, it should be noted that an earlier study reported that coronary vessels of rag1 knockout mice exhibited more intimal hyperplasia and perivascular fibrosis compared to wild - type mice following tac . More recently, nevers et al . Also investigated the role of t cells in cardiac remodeling in response to tac - induced pressure overload . They observed that the development of systolic dysfunction was associated with the kinetics of t cell infiltration into the left ventricle and evidence was provided that most of the infiltrating t cells were ifn- secreting th1 cells . Lv systolic and diastolic function were preserved with tac in t cell deficient mice (t cell receptor (tcr) knockout), and lv hypertrophy, fibrosis, and inflammation were markedly attenuated . In addition, t cell depletion with an anti - cd3 antibody prevented heart failure in wild - type mice . Unresolved at present is the identity of the antigen(s) responsible for t cell activation in lv hypertrophy and heart failure, and the potential contribution played by the loss of regulatory mechanisms that normally protect the heart from t cells . In contrast to the involvement of adaptive immunity in tac, ma et al . Provided evidence that cd8 t cells play a critical role in perivascular and interstitial fibrosis in the angiotensin ii infusion model of hypertensive cardiac remodeling through the recruitment and activation of macrophages . They found that cd8 t cells are recruited to the heart and activated by ifn- secreting myocardial cells; recruited macrophages in turn are activated by cd8 t cells in contact - dependent, but tcr - independent means . A possible contribution of cd4 t cells to the actions of cd8 t cells will need to be explored . Circulating levels of cxcl10 are elevated in patients with untreated essential hypertension . In a small cohort, we observed that the cxcr3 chemokines, including cxcl10, were present in elevated concentrations in the plasma of patients with symptomatic diastolic lv dysfunction indicative of hfpef or early stage hfref . The magnitude of their increase was independent of the extent of hypertension and the cxcr3 agonists enhanced diagnostic accuracy over and beyond nt - pro bnp . More recently, we reported that circulating cxcl10, mip-1, and cd40 ligand were the best indicators for differentiating healthy and heart failure subjects . We found that serum cxcl10 levels were increased in patients with symptomatic heart failure as indexed by nyha classification ii through iv and were positively correlated with serum levels of th1 proinflammatory cytokines . The findings of these two studies are consistent with the idea that inflammation is involved in the pathogenesis of heart failure with cxcl10 playing a central role . Numerous preclinical studies and recent genome - wide association studies (gwas) support a role for both cytotoxic (cd8) t cells and th (cd4) lymphocytes in human hypertension [92, 93]. However, accumulating evidence from experimental studies indicates that increasing treg cell levels in hypertension is an effective strategy to preserve cardiac function, attenuate cardiac hypertrophy and fibrosis, and prevent heart failure progression, independent of any blood pressure lowering effects [9496]. Reduced circulating levels of treg cells in heart failure patients have been reported in several studies [9799]. The role of cxcl10 in other forms of nonischemic heart failure with reduced ejection fraction, such as restrictive cardiomyopathy, ion channelopathies, and diabetic cardiomyopathy, awaits investigation . Recently, di luigi et al . Reported that the phosphodiesterase type 5 inhibitor sildenafil decreased elevated circulating cxcl10 levels in subjects with diabetic cardiomyopathy, suggesting that sildenafil could be used pharmacologically to mitigate cxcl10-associated inflammation in diabetic cardiomyopathy . Recent findings support a role for cxcl10 in right ventricular (rv) remodeling as well . . Found that several chemokines, most notably cxcl10, are upregulated in the pressure - overloaded right ventricle and play a role in myocardial extracellular matrix remodeling in an animal model of pulmonary stenosis . Cxcl10 is implicated also in rv dysfunction and inflammation following experimental pulmonary embolism in rats . The chemokine receptor cxcr3 and its agonist cxcl10 are potential drug targets to treat various cardiovascular diseases . Potential immunotherapies for cardiac inflammation are as follows: treg stimulation il-2/anti - il-2 complex treatment to enhance treg number and activity targeted cytokine - infused nanoparticles to stabilize and expand tregs in vivointravenous immunoglobulin (ivig) therapy to boost treg activity vitamin d to modulate formation and activity of tregs atorvastatin to enhance treg number and activity fty720 to increase treg levels and activityadoptive treg cell transfer il-2/anti - il-2 complex treatment to enhance treg number and activity targeted cytokine - infused nanoparticles to stabilize and expand tregs in vivo intravenous immunoglobulin (ivig) therapy to boost treg activity vitamin d to modulate formation and activity of tregs atorvastatin to enhance treg number and activity fty720 to increase treg levels and activity adoptive treg cell transfer immunosuppression immunoadsorption to remove circulating antibodies and boost treg activityphosphodiesterase type 5 inhibitor to decrease cxcl10 formation cxcl11 to stimulate biased gpcr anti - inflammatory signaling ppar- agonists to block cxcl9, cxcl10, and cxcl11 formation immunoadsorption to remove circulating antibodies and boost treg activity phosphodiesterase type 5 inhibitor to decrease cxcl10 formation cxcl11 to stimulate biased gpcr anti - inflammatory signaling ppar- agonists to block cxcl9, cxcl10, and cxcl11 formation levels of cxcl10 are generally elevated with chronic cardiac inflammation, which is associated with enhanced th1 polarization and infiltration into the myocardium . Cxcr3 plays a key role in recruiting various leukocytes to the heart, including monocytes, effector lymphocytes, and ctl cells . Peroxisome proliferator - activated receptor- (ppar-) agonists may be a potential pharmacological treatment to block cxcl9, cxcl10, and cxcl11 formation in patients, as ppar- agonists show a strong inhibitory effect on their expression and production in vitro . Pioglitazone, which lacks the adverse cardiovascular effects of older thiazolidinediones and may be cardiovascular protective, looks promising in this regard, although pioglitazone is contraindicated in heart failure patients likely due to fluid retention [104107]. Another potential therapeutic approach is the phosphodiesterase type 5 inhibitor sildenafil, which was recently reported to decrease cxcl10 gene expression and protein secretion in human cardiac myocytes and decrease circulating cxcl10 in subjects with diabetic cardiomyopathy . There is substantial evidence that sildenafil has protective effects against adverse remodeling of the heart . Although cxcl9, cxcl10, and cxcl11 all bind to cxcr3, there is evidence that these agonists activate opposing responses due to biased signaling that is a fixture of g protein - coupled receptors [1, 11, 12]. Whereas cxcl9/cxcl10/cxcr3 interactions drive effector th1 polarization, cxcl11/cxcr3 binding seems to induce an immunotolerant state characterized by t lymphocyte polarization into regulatory tr1 lymphocytes that produce anti - inflammatory il-10 [11, 12]. Biased agonists have been developed that exert cxcl11-like actions at cxcr3 but have not as yet been assessed in experimental models of cardiovascular diseases . Cxcl11 has a short half - life in vivo . To address this shortcoming, zohar et al . Generated a stabilized form of cxcl11 by creating a fusion protein in which cxcl11 was linked to igg1 . When administered during ongoing autoimmune encephalomyelitis, the fusion protein suppressed the disease by increasing the number of il-10-secreting tr1-like cells (direct effect) and reducing th1 polarization . Targeting cxcr3 might be more effective in treating chronic heart inflammation in combination with approaches to enhance treg numbers or activity, which are generally reduced in cardiovascular diseases (table 1). Regulatory t cells are immunosuppressive and anti - inflammatory, and a growing number of experimental studies have shown their beneficial effects on the heart in various experimental models of coronary artery disease [109, 110], kawasaki disease, myocarditis and dilated cardiomyopathies [112117], chagas heart disease [118, 119], hypertensive lv hypertrophy [95, 96], and nonischemic heart failure [94, 109, 120]. Although not discussed here, boosting treg numbers or activity in the heart is reported to be beneficial as well in experimental models of infarction - driven remodeling [121124]. Increasing levels of il-10-secreting treg / tr1 cells may be particularly advantageous, as il-10 has anti - inflammatory and protective actions on the vasculature and heart [125132]. Intravenous immunoglobulin (ivig) therapy has been shown to be effective in treating acute kawasaki disease in 8090% of kawasaki patients with rapid resolution of clinical symptoms and reduced risk of coronary disease . Although the exact basis for the effectiveness of ivig therapy is not clear, ivig therapy is thought to modulate the inflammatory process and recent evidence indicates that ivig therapy acts in part by stimulating an immature myeloid population of dendritic cells that secretes il-10 and favors expansion of fc - specific natural treg cells . Ivig may have promise for treating viral myocarditis and might be effective in treating chagas, as well as chronic heart failure, including both ischemic and idiopathic dilated cardiomyopathies . Adoptive transfer of tregs and/or stimulating their differentiation are promising therapeutic approaches to target cardiac inflammation [49, 50], although the concerns that must be overcome to make adaptive transfer routine therapy in humans are considerable . However, the safety and efficacy of treg immunotherapy in humans is supported by preliminary clinical trials for treating graft versus host disease [137, 138]. Immunoadsorption, which is a promising approach for treating myocarditis and dilated cardiomyopathy, may have beneficial effects by not merely removing circulating antibodies, but increasing treg activity [49, 139141]. The prodrug fty720 (fingolimod) is phosphorylated in vivo and has been shown to trap nave and memory t cells in the thymus and secondary lymphoid organs by downregulating sphingosine 1-phosphate receptor 1 (s1p1). Activation of s1p1 is linked to inhibition of treg cell differentiation; however, fty720 and phosphorylated fty720 increase treg levels and activity in vivo and in vitro [143146]. The inhibitory effects of atorvastatin on inflammation in acute coronary syndrome (acs) may be due to its beneficial effects on natural tregs . In patients with acs, il-2 plays a critical role in treg cell activity, growth, and survival, but there may be insufficient il-2 to sustain their in vivo potentiation . Emerging evidence indicates that enhancing treg cell numbers and activity with low - dose il-2 treatment is effective in treating autoimmune and inflammatory diseases [12, 148151], although there may be intrinsic risk as il-2 is also a key growth factor for effector cd4 t cells and nk cells . An il-2/anti - il-2 immune complex is reported to preferentially expand treg cells with little or no effect on other cells . Delivery of the complex to mice before tac was recently reported to attenuate lv hypertrophy, inflammation, and contractile dysfunction, while increasing lv levels of treg cells . Recently reported success in using nanoparticles loaded with the treg inducers il-2 and tgf- and targeted to cd4 t cells with conjugated antibodies . These nanoparticles were demonstrated to induce cd4 tregs in vitro, even in the presence of proinflammatory cytokines, and expand their number in mice in vivo . Vitamin d status has been linked to chronic heart failure and vitamin d supplementation improves lv structure and function in heart failure patients . Recent evidence indicates a modulatory role of the vitamin d system in the formation and activity of regulatory t cells . Vitamin d deficiency was associated with reduced numbers and impaired function of nave cd45ra regulatory t cell in chronic heart failure patients . In addition, the vitamin d receptor agonist bxl-01 - 0029 was shown to inhibit ifn- and tnf--induced cxcl10 secretion by fetal human cardiac myocytes and reduce cxcl10 protein secretion and gene expression by cd4 t cells . Whether a vitamin d receptor agonist or supplementation increases treg levels and reduces cxcl10 levels in heart failure patients will need to be assessed . The last decade has witnessed an impressive advance in our understanding of the role of the immune system in the pathophysiology of cardiovascular diseases . Beyond an expected role in allograft disease and the development and progression of atherosclerosis, an abundant body of evidence supports a significant contribution of the immune system to many other cardiovascular settings, ranging from the development and maintenance of hypertension to cardiac and vessel remodeling (whether constrictive or expansive) in response to hemodynamic stress, in both health and disease . The challenge now is to translate this knowledge for the benefit of patients suffering from cardiovascular diseases . Targeting the cxcl10/cxcr3 axis and cardiac inflammation may open new pharmacological venues for treating heart failure or coronary artery disease to supplement current drugs that target the sympathetic or renin - angiotensin systems, or platelets . This will require selectively targeting the most critical immune pathways in order to optimize interference with pathological processes, while preserving protective and homeostatic immune functions . Antigen - specific modulation of the immune system, for example, through systemic delivery of nanoparticles coated with disease - relevant peptides bound to major histocompatibility complex class ii (pmhcii) to expand endogenous antigen - specific tregs, is an optimal strategy in settings where an antigen - specific adaptive immune response has been involved in disease development or progression . However, several cardiovascular diseases will probably resist the identification of a specific pathogenic antigen and will require a broader, although targeted, regulation of the immune response, for example, through administration of low - dose il-2 to promote endogenous tregs . With regard to cxcr3 pathway, besides the therapeutic possibilities listed above, we believe that the identification and development of selective evasins or evasin - like peptides that differentially bind and neutralize cxcl9 or cxcl10 versus cxcl11 may provide an interesting therapeutic strategy to limit pathogenic while preserving regulatory cxcr3 functions.
The columnar organization of neocortex at the minicolumnar (2050 m) and macrocolumnar (300600 m) scales has long been known (see mountcastle, 1997; horton and adams, 2005 for reviews). Minicolumn - scale organization has been demonstrated on several anatomical bases (lorente de no, 1938; defelipe et al ., 1990; peters and sethares, 1996). There has been substantial debate as to whether this highly regular minicolumn - scale structure has some accompanying generic dynamics or functionality . I.e., one that would apply equally well to all cortical areas and species has been determined . One basis upon which a functionality for the minicolumn has been suggested is possession of highly similar receptive field characteristics, or tuning, by the cells comprising the minicolumn, e.g., v1 orientation columns (hubel and wiesel, 1962, 1968) and minicolumn - sized regions innervating cutaneous zones (favorov and diamond, 1990). The reasoning here appears to be that because a group of cells all have very similar tuning to a particular feature,, e.g., an edge at a particular orientation, they form a unit whose function is to recognize . However, in searching for a possible generic minicolumn function, we need not limit ourselves to considering only recognition functions . Furthermore, possession of highly similar tuning cannot be a basis for a generic minicolumn functionality since in many cortical areas, the cells encountered along vertical penetrations can have quite variable tuning (cf . On closer analysis, this is true in orientation column data as well (hetherington and swindale, 1999; ringach et al ., 2002). If there is a generic minicolumn functionality, then it must be compatible with varying degrees of tuning correlation amongst the minicolumn's cells . I propose that the minicolumn does have a generic functionality (given shortly), but one that only becomes clear when seen in the context of the function of the higher - level, subsuming unit, namely, the macrocolumn, which has been demonstrated anatomically (goldman and nauta, 1977; lbke et al ., 2003; egger et al ., 2008) and functionally (mountcastle, 1957; woolsey and van der loos, 1970; hubel and wiesel, 1974; albright et al ., 1984). I propose that the function of a macrocolumn (e.g., hypercolumn, segregate, barrel) is to store sparse distributed representations of its overall input patterns, and to act as a recognizer of those patterns . By overall input pattern, i mean the macrocolumn's overall input at a given moment, including not only its bottom - up (bu) inputs from thalamus or lower cortical areas, but also its top - down (td) inputs from higher cortical areas and its horizontal (h) inputs, which i propose carry temporal (sequential) context information (recurrently) from the representations previously active in the same and nearby macrocolumns . Thus, an overall input pattern can equally well be termed, a context - dependent input . Thus, it is in fact the macrocolumn whose generic functionality is appropriately characterized as recognition; i.e., recognition of a class determined by the history of context - dependent inputs to which it has been exposed . A distributed representation of an item of information is one in which multiple units collectively represent that item, and crucially, such that each of those units generally participates in the representations of other items as well . Distributed representations are also referred to as cell assemblies, population codes, or ensembles . In this paper, representation and a sparse distributed code (sdc) is one in which only a small fraction of the pool of available representing units is part of any particular code (palm, 1982; lynch et al ., 1986; kanerva, 1988). If the macrocolumn stores sdcs, then there must be some mechanism that enforces sparseness and this, i propose, is the generic function of the minicolumn . Specifically, i propose that small, physically localized pools of l2/3 pyramidals, e.g., 20 such cells, act as winner - take - all (wta) competitive modules (cms). This implies that macrocolumnar codes should consist of about 6080 active l2/3 cells, one per minicolumn: for simplicity, assume 70 minicolumns per macrocolumn hereafter . Defined in this way, the minicolumn has a more flexible relation to the ontogenetic column, the apical dendrite bundle, the dbc horsetail, etc . For example, a given minicolumn might include l2/3 pyramidals from more than one apical dendrite bundle, consistent with the findings of yoshimura and callaway (2005) of fine - scale networks of preferentially interconnected l2/3 pyramidals . There is increasing evidence for the use of sdc in the cortex and other brain structures; e.g., auditory cortex (hromdka et al ., 2008), visual areas (young and yamane, 1992; vinje and gallant, 2000; waydo et al ., 2006; quian quiroga et al ., 2008), zebra finch neopallium (hahnloser et al ., 2002), olfactory structures (jortner et al ., 2007; linster and cleland, 2009; poo and isaacson, 2009), and hippocampus (leutgeb et al ., 2007). Particularly supportive of the proposed hypothesis is the reid lab's calcium imaging data of rat v1 during stimulation by drifting square - wave gratings (ohki et al ., their movie (http://reid.med.harvard.edu/movies.html) shows sparse collections of l2/3 cells extending over an approximately macrocolumn - sized region synchronously turning on and off in response to particular grating orientations . (two frames from the movie) show distinct sets of cells, i.e., codes, responding to bars moving left and right, respectively, and emphasize that individual units may participate in multiple codes (red - circled cells). In terms of the proposed model, the active neurons would be the winners in their respective minicolumns, as suggested in figures 1c, d . Figure s1 in supplementary material provides another view of how the proposed model maps onto cortex . Calcium (tangential) images of l2/3 of rat visual cortex showing sparse sets of cells activating in unison (see movie link in text) in response to leftward (a) and rightward (b) drifting gratings . From ohki (c, d) sketch of proposed sparse distributed coding concept, which could plausibly give rise to data like (a) and (b). : to make the sketches look more like the calcium images, black is used for inactive units and white for active: this is reversed in subsequent figures . If the macrocolumn does indeed function as a sdc field in the way suggested here, then we must answer two key questions regarding its dynamics . How is any particular set of winners, one in each of the 70 minicolumns, initially chosen in response to an input pattern and bound into a holistic code? That is, how are macrocolumnar codes learned? How is a previously learned code reactivated in response to future presentations of the input pattern that it was initially chosen to represent? That is, how are stored macrocolumnar codes retrieved (reactivated)? In the next section, i describe an algorithm, referred to as the code selection algorithm (csa), which answers both questions . A key property of the csa is that it causes similar inputs to be assigned to similar, i.e., more highly intersecting, codes . This property, which will be referred to as sisc (similar inputs map to similar codes), is very important in terms of computational efficiency (see discussion) and is possessed by most distributed coding schemes . However, the csa achieves it in a novel, probabilistic fashion, which can be summarized as follows: determine the familiarity of a macrocolumn's input . To a first approximation, an input's familiarity is its maximum similarity to any input previously stored in the macrocolumn . Set the amount of randomness (noise) in the process of selecting winners in the wta modules in inverse proportion to the input's familiarity . The algorithm's rationale is described in detail in the next section, but broadly, the idea is as follows . When an input, 1, is familiar, we want to reactivate the code, 1, to which 1 was previously assigned . The cells comprising 1 will have had their synapses (from the cells comprising 1) increased during learning . Thus, if 1 is presented again, the cells of 1 will have the highest synaptic input summations in their respective wta modules . In this case, winners should be chosen on the deterministic basis of these summations: no noise should be present in the winner selection process . On the other hand, increasingly novel inputs should be assigned to increasingly distinct codes, i.e., codes having progressively smaller intersection with existing codes . This can be achieved by increasing the noisiness of the winner selection process in each wta module, which can be achieved by suppressing the influence of the deterministic synaptic inputs (which reflect prior learning) relative to baseline random (spontaneous) activity . By adjusting parameters that control the global (i.e., across the whole macrocolumn) noise level, we can modulate the expected intersection between the set of cells which have the maximal input summations in their respective wta modules and the set of winners that are actually chosen, thus implementing sisc . Many experimental and theoretical studies implicate neuromodulators, notably norepinephrine (ne) and acetylcholine (ach), in functionality similar to the above, which can be described generally as modulating signal - to - noise ratio (snr). Doya (2002) proposed that ne levels control the amount of noise in a process of choosing output actions . However, doya's model assumes a localist representation of the choices, which precludes possession of the sisc property (see discussion). In addition, increased ach has been shown to selectively increase the strength of afferent relative to intrinsic inputs in piriform cortex (hasselmo and bower, 1992) and other brain structures (see hasselmo, 2006 for review). These ach findings have been summarized as showing that increased ach adjusts network dynamics to favor encoding new memories compared to retrieving old memories, which fits well with the proposed csa functionality . Following the model description, i offer a speculative mapping of my proposed model onto neural circuitry and discuss evidence for novelty - contingent noise modulation by both ne and ach . However, the specifics of this mechanism are a subject of ongoing research and likely will involve interactions between neuromodulatory systems (cf . Any discussion of columnar function of course centrally concerns cortical circuitry, and more specifically, the putative canonical cortical microcircuit (rockland and pandya, 1979; douglas et al ., 1989; douglas and martin, 1991). We have made huge progress in understanding many of the components of cortical microcircuitry a tiny sample of which includes (defelipe et al ., 1990;, 2004; feldmeyer et al ., 2006; fukuda et al ., 2006;, 2008; hirata and castro - alamancos, 2008; berger et al ., 2009; murayama et al ., 2009; symes and wennekers, 2009; briggs, 2010; and see thomson et al ., 2002; bannister, 2005; silberberg et al ., 2005 for reviews) nevertheless, we remain far from any sort of comprehensive and consensual understanding of how cortical columnar circuitry manipulates, i.e., stores and retrieves, specific items of information . In the main, only very broad conclusions regarding information processing are asserted in the experimental literature, e.g., horizontal connections between fast - spiking l4 interneurons and pyramidals are involved in formation of l4 assemblies and sharpening of tuning (sun et al ., 2006); that both the populations of l4 pyramidals and of l5a pyramidals have transcolumnar connectivity patterns allowing them to act as integrators of information coming in from multiple vibrissae in parallel, or in close sequence (schubert et al ., 2007); that the receptive fields of barrel - related l2/3 pyramids are dynamic and thus may reflect learning to recognize spatiotemporal patterns of vibrissae deflections (brecht et al ., 2003); that wta competition occurs in the supra- and infragranular layers (douglas and martin, 2004); and that local (100 m) l2/3-to - l2/3 connections might serve to synchronize firing of l2/3 cell assemblies (lbke and feldmeyer, 2007); etc . I believe the hypothetical model described herein to be a significant contribution because it goes beyond broad conclusions and offers a mechanistic explanation of how specific informational items are learned and retrieved and in so doing, proposes a generic function for the minicolumn, i.e., that it functions as a wta module in support of manipulating sdcs at the next higher, i.e., macrocolumnar, scale . In particular, it was stated in the introduction that sparse macrocolumnar codes are chosen in response to a macrocolumn's overall input, which includes its bu, h, and td inputs . However, illustrations of the model in operation in that general case become rather complex, particularly since the h (and td) weights carry temporal information, which necessitates showing the model at multiple successive time steps while processing spatiotemporal patterns . More importantly, the core elements of the hypothesis which are: (a) that the macrocolumn stores sdcs consisting of one winning l2/3 cell per minicolumn; and (b) that the sisc property is achieved by modulating the amount of randomness (noise) present in the winner selection process in inverse proportion to input familiarity can be clearly and more simply described for the bu - only case (i.e., where inputs are purely spatial patterns). Therefore, the model description in this paper will be limited to the bu - only case . However, the generalized model (with bu, h, and td inputs) and the accompanying generalized version of the csa are given in figures s2 and table s1 in supplementary material . Functional architecture . The input field (f1) consists of binary feature detectors: a particular input consisting of five active features is shown . It consists of winner - take - all competitive modules (cms) proposed as analogous to minicolumns . Bottom - up connectivity is all - to - all: gray lines signify initially 0 weights . The familiarity (g) of the input is proposed to be computed via a subcortical, neuromodulator - based mechanism, which then modulates the f2 unit activation function parameters (e.g., sigmoid height) contingent on g (purple arrows). The input field, f1, is comprised of 12 binary units and can be considered analogous to a specific thalamic nucleus, though topographical organization is not assumed . The coding field, f2, consists of q = 4 wta cms, each containing k = 3 binary units . Complete (all - to - all) bu connectivity from f1 to f2 is assumed for simplicity . These bu weights (synapses) are binary, initially 0, and are permanently set to a weight of 1 the first time the pre- and postsynaptic units are co - active (i.e., hebbian learning). The model's core algorithm, the csa, determines which cells are chosen to represent an input, during both learning and retrieval . A single iteration of the algorithm involves two rounds of competition in the cms of f2 . The first round is a hard wta competition (represented by boxes labeled max in figure 2). The purpose of the first round is to compute a global familiarity measure, g, of the input pattern . G then drives a global modulation of the f2 unit activation function (figure 2: purple arrows) in preparation for the second competitive round, which is a soft wta competition, the intent of which is that: as g goes to 1 (indicating a completely familiar input), the probability that the unit with the highest input summation in a cm wins approaches 1, and as g goes to 0 (indicating a completely novel input), all units in a cm become equally likely to win (regardless of their input summations). (1)u(i)=j nw(j, i) where n is the current input (f1) pattern . Because unit activations are binary, we can simply sum the weights, w(j, i), which are also binary . Normalize u(i) to [0 .. 1], yielding v(i). V(i) is a local measure of support, or likelihood, that f2 unit i should be activated . It reflects how well unit i's receptive field (rf), specified by its afferent weight vector, matches the current input vector . (round 1 competition) the maximum v(i), v^x, is found in each of the q cms . (3)v^x = maxi cx{v(i)} where x indexes the cms and i indexes the units in a cm, cx . Average the q v^xvalues, yielding g, a global measure of the familiarity of the current input . (4)gx=1qv^x / q the expansivity,, of the probabilistic activation function (which is implemented via steps 68) is set as an increasing nonlinear function of g (eq . 5, expressed as a table). The idea is to increase the range of relative win likelihoods, (i) (defined in step 6) over any given cm's units as g goes to 1 . This in turn, serves to nonlinearly exaggerate the difference in the final win probabilities (eq . The specific parameters of any instance of the g - to- mapping will determine the specifics of the relation between input similarity and code similarity, i.e., the expected code intersection as a function of input similarity . 5 were chosen to yield the -distributions in the examples of figures 3 and 4 . Circled numbers refer to algorithm steps in text . Here, i show the case of the first input, 1, presented to the model . The algorithm detects that 1 is completely unfamiliar, i.e., g is computed to be 0, and sets the f2 activation function to a constant function (red line), which makes the choice of winner in each cm completely random, and thus, the overall f2 code, 1, also completely random . Green f1 (f2) units denote units not in common with 1 (1). Circled numbers refer to algorithm steps in text . Here, i show the case of the first input, 1, presented to the model . The algorithm detects that 1 is completely unfamiliar, i.e., g is computed to be 0, and sets the f2 activation function to a constant function (red line), which makes the choice of winner in each cm completely random, and thus, the overall f2 code, 1, also completely random . The csa and the sisc property . Green f1 (f2) units denote units not in common with 1 (1). The v values of all units in all cms are then passed through the sigmoidal activation function (eq . 6) whose shape / scale reflects g. again, particular parameter values affect the relation of input similarity to code similarity (and therefore, storage capacity): values of = 28 and = 5 produce the v - to- mappings in figure 4 . As noted above, within each cm, the output variable, (i), can be viewed as a relative likelihood that unit i should be chosen winner . (6)(i)=1+e (v(i) +)+1 when g = 1 (perfectly familiar), is maximized (in eq . 7) probabilities of winning for units with the maximum v value in their respective cms . In contrast, when g = 0 (completely novel), = 0, which collapses the sigmoid to the constant function, = 1, thus making all units in a cm equally likely to win . This causes the expected intersection of the code being chosen in the current instance with any previously assigned code to be at chance level . In general, this modulation of the sigmoid activation function tends toward code completion in proportion to the familiarity of the input and code separation in proportion to its novelty . Transform relative likelihood distribution () in each cm to true probability distribution (). (7)(i)=(i)k cm(k) (round 2 competition) choose an f2 code by drawing a winner from the -distribution (soft max) in each cm . Thus, choosing an f2 code is actually performed as q separate draws . When g = 0, these draws are statistically independent, as in figures 3 and 4d . As we consider increasingly familiar inputs, i.e., for g approaching 1 (and, assuming the model is still operating in a regime where crosstalk is sufficiently low), the draws become increasingly correlated (dependent), as can be seen in going from figure 4c to 4b to 4a . Figure 3 graphically illustrates the operation of the csa in the case of the model being presented with its first input, 1 . The gray arrows indicate that the bu signals propagating from the active f1 units will be traversing connections with zero synaptic strength . This leads to unnormalized (u) and normalized (v) input summations of 0 for all 12 f2 units (steps 1,2). In step 3, the max v, v^, in each cm is found (ties broken at random). In step 4, g is computed as the average of the v^ values: in this case all the v^ are 0, so g = 0 . In step 5, the value, g = 0, maps to = 0, which causes the activation function of the f2 units to collapse to the constant function, = 1 . In step 6, each f2 unit applies this activation function to its v value, yielding the uniform relative likelihood distribution in each cm . In step 7, the relative likelihood function in each cm is normalized to a true probability () distribution, which in this case, is again uniform . Finally, in step 8, a winner is drawn in each cm, resulting in a random f2 code, e.g., 1 . Figure 4 demonstrates that the csa realizes the sisc property by considering four possibilities (a d) for the second input presented to the model of figure 3 . These four inputs, 25, range from being identical to 1 (completely familiar) to having zero overlap with 1 (completely unfamiliar). To save space, the panels of figure 4 use an abbreviated version of the format of figure 3 . Most noticeably, the intermediate variable, (relative likelihood), is not shown . Black bu connections are ones that were increased to one when 1 was learned (figure 3). Working from figure 4a to 4d, the inputs have progressively lower similarity (intersection) with 1: f1 units not in common with 1 are shown in green . As g drops, the sigmoid expansivity drops (note the changing scale). Thus, the -distributions become progressively flatter, which in turn results in f2 codes, 25, having progressively smaller intersection with 1 . Figure 4a shows the case of presenting a completely familiar input again, and is thus a recognition test trial, demonstrating retrieval . This leads, via csa steps 3 and 4, to g = 1, which yields, via steps 5 and 6, the expansive nonlinear v - to- mapping shown (red sigmoid). This nonlinearity is applied to every f2 unit, yielding the highly peaked -distributions shown . Finally, one unit is drawn in each cm . The probability of drawing the correct unit in any single cm is approximately 98% . Of course, what's crucial in this case, i.e., when the input is completely familiar (g = 1), is that the entire correct f2 code is reactivated . In this case, that probability is (0.98) 92% . Thus, the familiarity, g, which depends on the entire f2 layer and is thus global information, influences the local activation functions so as to produce the desired overall result, in this case, reactivation of the code (memory trace), 1, of the familiar input pattern, 1 . The explanations of the remaining panels follow that of figures 3 and 4a . In going from figure 4b to 4d, one can readily see decreasing intersection with 1, decreasing u and v values, decreasing g, decreasing sigmoid expansivity, progressively flatter -distributions, and ultimately, decreasing intersection with 1 . Given a desired probability, r, of correctly reactivating an entire code (i.e., of choosing the correct unit in each cm), when g = 1, given values for q and k, we could compute the needed value of . However, the macrocolumn model is a content - addressable associative memory and in actual usage, multiple sparse codes will be stored in superposition . Any thorough analysis of the model's expected retrieval error must include the effect of overlap between the stored codes (i.e., cross - talk): this influences the shapes of the -distributions and thus, the expected retrieval accuracy for any given number of stored codes . Such an analysis will be conducted empirically and reported in a separate paper . Before leaving figure 4 first, while the -distributions become flatter as g decreases, the units comprising the code of the most similar previously learned input (here, 1) remain most likely to win in their respective cms . If we simply deterministically chose the unit with maximum v(i) in each cm, we would have chosen the same f2 code, 1, in response to all four inputs, 25 . Thus, the computation of a quantity, g, which depends on all the cms is essential to achieving the sisc property . It constitutes a channel through which information transfers between all f2 units throughout the whole macrocolumn . As noted earlier, the full model also assumes direct h connections between f2 units, analogous to the horizontal matrix of l2/3 (see figure s2 in supplementary material). These also mediate communication, but of the prior code active in the macrocolumn, not of the simultaneous state of all f2 units throughout the macrocolumn . Second, learning is single - trial and involves only one iteration of the csa . This is largely facilitated by the fact that when a given input - code association, jj, is learned, each of j's f2 units simultaneously has its afferent weight from all of j's f1 units increased . The effect of these simultaneous correlated potentiations allows a rapid, even single - trial, formation of an association, even if the individual synaptic potentiations are small, consistent with the recent characterization of thalamocortical learning described in bruno and sakmann (2006). Third, figure 4a shows that recognizing an exact instance of a previous input also requires only one iteration of the csa . Although this example does not directly show it, this holds for recognition of non - exact matches as well . Evidence for this will be presented in a separate work . That both learning and recognition require only a single csa iteration is especially significant since, as can readily be seen, none of the csa steps involves iterations over stored codes: thus, the time it takes for the csa to either store a new input or retrieve the closest matching stored input remains constant as the number of stored codes increases . This does not imply that an infinite number of codes can be stored: of course, the model has finite storage capacity . This capacity will be characterized in future research, but should be similar to other sparse associative memories (willshaw et al ., 1969; palm, 1982; moll and miikkulainen, 1995; knoblauch et al ., 2010). There remain huge gaps in our knowledge of the intrinsic physiological, synaptic, morphological, and connectional properties of all classes of cortical cell and of functional relationships between cortical and sub - cortical structures . Nevertheless, figure 5 shows a possible neural realization of the model's wta cm, i.e., minicolumn, and dynamics (csa). I have attempted to respect known constraints but the realization is admittedly speculative and ongoing modifications will undoubtedly be required . Figures 5a e show the critical events transpiring in a single minicolumn at five points in time during the csa's computational cycle . Note that due to space limitations figure 5 cannot depict the true extents of the various axonal and dendritic systems of the cells involved . (a e) depict critical events transpiring in a single minicolumn at five points in time during the csa's computational cycle, which i propose corresponds to one gamma cycle: approximate timings relative to start of csa cycle are shown across top . The units labeled c (b) represent the local chandelier (basket cell) populations, respectively; see text for detailed explanation . Figure 5a shows the initial csa steps 1 and 2 when the l2/3 pyramidals (here only two cells, but in reality, 20) integrate their inputs . While the csa explanation in the prior section included only the bu inputs, all three input classes are included here: bu inputs (labeled 2) are mediated via l4 (rockland and pandya, 1979) td inputs (1) are mediated via l2/3 apical tufts (rockland and drash, 1996) h inputs (3) are mediated via the horizontal matrix of l2/3 (gilbert and wiesel, 1989; feldmeyer et al ., 2006) all three input vectors arrive in parallel and collectively give rise to postsynaptic potentials (psps) in the l2/3 pyramidals . The three (normalized) inputs are combined multiplicatively; see the generalized version of the csa (table s1 in supplementary material). C) are firing at this time, preventing the l2/3 pyramids from firing . In figure 5b, the chandeliers shut off (grayed out) and the l2/3 pyramid with the highest psp (cell 2) is assumed to be the first to spike (csa step 3). This winning cell, and more specifically, its psp value (v in eq . 2), represents this minicolumn's local judgment of how similar the macrocolumn's closest - matching stored input is to the current overall (i.e., bu, h, and td) input . The output of cell 2 is then communicated to some locus where it is averaged with the round 1 winners from the other 70 minicolumns of the macrocolumn, yielding g (csa step 4). As noted in the introduction, the functionality related to g seems most consistent with the phenomenology of neuromodulatory systems, especially ach and ne . Note that the communication of cell 2 s psp value is hypothesized to be mediated via l5, one of whose pyramidal cells is seen integrating here (light green); this is based loosely on evidence that l5 cells, specifically l5b pyramidals, almost exclusively target pontine areas (with collaterals to thalamus) (deschnes et al ., 1994; krieger et al ., 2007). L2/3 pyramidals are the primary source of bu signals to higher cortical areas (rockland and pandya, 1979; and see thomson et al ., 2002; brecht et al ., 2003; schubert et al ., 2003; bannister, 2005; helmstaedter et al ., 2007; lbke and feldmeyer, 2007; petersen, 2007; egger et al ., 2008;, 2009 for wider background on cortical columnar circuitry relevant to the current proposal). In addition, as stated earlier, the horizontal l2/3-to - l2/3 connections are proposed to communicate this macrocolumn's final hypothesis regarding its total input pattern in the current csa cycle recurrently back to the same (and surrounding) macrocolumns on the next csa cycle . Hence, it is crucial that since that final hypothesis is not present until the second round of competition completes (figure 5e), the output pathways carrying those signals must be closed (red xs on paths 4 and 5). Though not depicted here, one possible mechanism for selectively preventing horizontal signaling in l2/3 is activation of the double bouquet cells (defelipe et al ., 1990, 2006; peters and sethares, 1997). Axons, being interdigitated, nearly one - to - one with minicolumns would allow them to make contact with a substantial portion of the horizontally (and obliquely) oriented dendritic and axonal processes, in l2/3, and thus prevent passage of horizontal signals . In figure 5c, the l5 pyramidal mediating the winning l2/3 cell's psp value has reached threshold and sends that output to the sub - cortical averaging locus (path 6). In addition, the winning cell itself has activated the local basket cell network (electrically coupled, cf . B, which rapidly deactivates and re - polarizes (resets) the l2/3 pyramidals (grayed out). This reset need not be back to a completely even baseline: some remnant of the relative psp distribution prior to basket cell activation might remain, biasing the second round of competition . In figure 5d, the result of the subcortical computation of g is returned to the macrocolumn (path 7) in the form of neuromodulator release (purple cloud surrounding the l2/3 pyramidals). Cloud actually extends across a broad, i.e., macrocolumnar (or wider), expanse of l2/3, not just within a single minicolumn as this figure may suggest . The chandeliers are again firing to prevent any l2/3 from firing as the second round of integration occurs . The basket cells are inactive, allowing this integration to take place . In figure 5e, in general, the pyramidal cell winning on this second round of competition may differ from the first round winner . In particular, the probability that the second round winner is the same as the first round winner increases with g. the set of l2/3 winners, one per minicolumn, across the whole macrocolumn represents that macrocolumn's final decision (hypothesis) as to identity of its current overall (td, h, and bu) input . Thus, the output of the winning l2/3 cell in each minicolumn is now communicated to: lower cortical regions via l5 and its backprojections to the lower regions l1 (labeled 8) (rockland and drash, 1996). L2/3 pyramids in the same and neighboring macrocolumns via the local horizontal l2/3 matrix (5) (gilbert and wiesel, 1989; feldmeyer et al ., 2006), thus delivering temporal context information (recurrently) to the local region to be integrated on the next csa cycle . The l4 layer of higher cortical regions (4) (rockland and pandya, 1979). Note that the output of the winning l2/3 cell should be prevented from recurring to the local basket network at this time so as to allow the integration period to occur at the beginning of the next computational cycle; hence, the red x on the link to basket cell . I reiterate that the above possible cortical realization of the proposed sdc model is highly speculative . It clearly lacks numerous details, especially regarding processing in the intermediate processing stages, e.g., l4, and output processing involving l5 (and l6). Nevertheless, it is a starting point and can be falsified, especially as experimental methods mature . We still have decidedly little in the way of hard constraints on the time courses of activation of the many cell types involved in cortical (and hippocampal) circuits, though progress is being made (klausberger et al ., 2003, 2004; silberberg et al ., 2005; silberberg and markram, 2007; klausberger and somogyi, 2008; otsuka and kawaguchi, 2009; woodruff et al ., 2009). Moreover, the proposed theory's key assumption that the l2/3 pyramidals are the core repository of information in cortex and that the codes laid down in l2/3 are the context - dependent memories of our experiences, is subject to challenge . Specifically, the anatomy of the l5 thick tufted cells suggests that they too have access to bu (via l4), td (via their apical tufts in l1), and h (via an extensive intra - l5 horizontal network, schubert et al ., 2007) inputs, and therefore that l5 might store the most detailed and context - dependent codes in cortex, a view supported by findings such as (de kock et al ., 2007). In the end, for the purpose of this hypothesis and theory paper, i believe the architecture and algorithm (csa) to be more important than the specifics of any particular neural realization . In this section, i consider evidence relating to six model predictions: a}signals generated in the macrocolumn [i.e., the v^x (eq . Strictly interpreted, figure 2 suggests that the model can only be true of cortical areas that have direct projections to the activation function modulator (afm), hypothesized to be instantiated in midbrain neuromodulator source areas, e.g., basal forebrain (bf, source of ach) and locus coeruleus (lc, source of ne). Direct cortical afferents to bf arise mainly from prepyriform, anterior insula, orbitofrontal, entorhinal and medial temporal areas (mesulam and mufson, 1984). Direct cortical afferents to lc arise from dorsal prefrontal cortex (arnsten and goldman - rakic, 1984), medial prefrontal cortex (jodo et al ., 1998). While direct projections are limited, a much larger fraction of cortex may be able to influence the hypothesized afm via multi - synaptic pathways involving thalamus and other subcortical structures, especially via pathways interconnecting bf, lc, and other neuromodulator source areas . For example, bf cholinergic neurons are excited by lc (jones et al ., 2004), which allows dorsal and medial prefrontal areas indirect influence on bf . Similarly, lc receives input from the raphe nuclei (reviewed in samuels and szabadi, 2008) which would allow further extension of the sphere of cortical influence upon the afm . However, it is clearly possible that my macrocolumnar model applies only to the smaller set of cortical areas suggested above . After all, there would be some advantage to deferring the decision as to the overall familiarity / novelty of the current input (moment) to the very highest cortical levels, which are in position to make the most informed decisions . In this case, once g is computed, it is then broadcast pan - cortically, i.e., to all levels of the hierarchy, allowing the local choice of code to proceed accordingly, i.e., with a level of randomness appropriate to g. figure s2 in supplementary material illustrates this view . 5), which correlates with the detection of familiarity, and/or inversely with the detection of novelty . Such correlations have been shown for both ach (miranda et al ., 2000, 2003) and ne (sara et al ., 1994; vankov et al ., lc projects to all of cortex (foote and morrison, 1987; berridge and waterhouse, 2003; samuels and szabadi, 2008). Bf projects to almost all cortical areas (reviewed in briand et al ., 2007). The amount of randomness to be added to the winner selection process is a global parameter, which applies non - specifically to all the minicolumns . This is consistent with volume transmission believed to be used by neuromodulatory systems (descarries et al ., 1997; see sarter et al . D}the signal determines (eqs 68) the amount of noise (randomness) in the selection (activation) of cortical (i.e., macrocolumnar) codes . Controlling the noisiness of a process of choosing a winner from a competing group of neurons can be achieved by some combination of two actions: (i) increasing the spike probability of cells with high input summations relative to those with low summations and (ii) lowering the spike probability of low - input cells relative to high - input cells . Both of these actions can be achieved by uniformly (i.e., to all competing cells in a wta module) modulating intrinsic cell properties such as excitability . Numerous studies have demonstrated both excitatory and suppressive effects on target cell responses (both principal neurons and interneurons) for both ach (kawasaki and avoli, 1996; shalinsky et al ., 2002; cobb and davies, 2005; tateno et al ., 2005; isakova and mednikova, 2007; lawrence, 2008; eggermann and feldmeyer, 2009) and ne (foote et al ., 1975; kawaguchi and shindou, 1998; harley and helen, 2007; moxon et al ., 2007). It is not my intention here to argue for a precise realization of this mechanism . As suggested in many reviews (berridge and waterhouse, 2003; lucas - meunier et al ., 2003; sara, 2009), the landscape of this research is very complex and we are far from a comprehensive understanding of the how the various neuromodulatory systems affect high - level cognitive processing either alone or in concert (briand et al ., 2007). Nevertheless, the large and varied body of evidence at least admits the possibility that one or more of these neuromodulators could implement the familiarity - contingent afm mechanism (csa steps 58; see doya, 2002, p. 502 e}decreased, i.e., increased noise, leads to greater code separation (decreased intersection). Recently, goard and dan (2009) showed that increased bf stimulation decreased the correlation amongst a population of rat v1 cells . This decreased correlation essentially shows increased separation between population codes, which in the model proposed here, would manifest as decreased intersection between sparse codes . F}disabling of the brain's ability to produce high noise, i.e., causing to be permanently high, should reduce the ability to learn new inputs, while sparing or having much less effect on recognition of known items . Looking at figure 4, if the afm was stuck in the highly expansive sigmoid condition (low noise), all four inputs, 25, would have high probability of mapping to the same code, 1 . However, in general, inputs that were mapped to unique codes prior to such a disabling event will reliably activate those codes on future presentations . In accord with this, browning et al . (2010) found that severely diminishing cholinergic inputs to inferotemporal cortex severely reduced macaques performance on a visual episodic memory task, while having little effect on a dnms task . (2005) found a similar effect: cholinergic deafferentation of entorhinal cortex reduced performance on dnms tasks involving novel odors but not familiar odors . The model's core algorithm, the csa, determines which cells are chosen to represent an input, during both learning and retrieval . A single iteration of the algorithm involves two rounds of competition in the cms of f2 . The first round is a hard wta competition (represented by boxes labeled max in figure 2). The purpose of the first round is to compute a global familiarity measure, g, of the input pattern . G then drives a global modulation of the f2 unit activation function (figure 2: purple arrows) in preparation for the second competitive round, which is a soft wta competition, the intent of which is that: as g goes to 1 (indicating a completely familiar input), the probability that the unit with the highest input summation in a cm wins approaches 1, and as g goes to 0 (indicating a completely novel input), all units in a cm become equally likely to win (regardless of their input summations). (1)u(i)=j nw(j, i) where n is the current input (f1) pattern . Because unit activations are binary, we can simply sum the weights, w(j, i), which are also binary . Normalize u(i) to [0 .. 1], yielding v(i). V(i) is a local measure of support, or likelihood, that f2 unit i should be activated . It reflects how well unit i's receptive field (rf), specified by its afferent weight vector, matches the current input vector . (round 1 competition) the maximum v(i), v^x, is found in each of the q cms . (3)v^x = maxi cx{v(i)} where x indexes the cms and i indexes the units in a cm, cx . Average the q v^xvalues, yielding g, a global measure of the familiarity of the current input . (4)gx=1qv^x / q the expansivity,, of the probabilistic activation function (which is implemented via steps 68) is set as an increasing nonlinear function of g (eq . The idea is to increase the range of relative win likelihoods, (i) (defined in step 6) over any given cm's units as g goes to 1 . This in turn, serves to nonlinearly exaggerate the difference in the final win probabilities (eq . The specific parameters of any instance of the g - to- mapping will determine the specifics of the relation between input similarity and code similarity, i.e., the expected code intersection as a function of input similarity . 5 were chosen to yield the -distributions in the examples of figures 3 and 4 . Circled numbers refer to algorithm steps in text . Here, i show the case of the first input, 1, presented to the model . The algorithm detects that 1 is completely unfamiliar, i.e., g is computed to be 0, and sets the f2 activation function to a constant function (red line), which makes the choice of winner in each cm completely random, and thus, the overall f2 code, 1, also completely random . Green f1 (f2) units denote units not in common with 1 (1)., i show the case of the first input, 1, presented to the model . The algorithm detects that 1 is completely unfamiliar, i.e., g is computed to be 0, and sets the f2 activation function to a constant function (red line), which makes the choice of winner in each cm completely random, and thus, the overall f2 code, 1, also completely random . The csa and the sisc property . Green f1 (f2) units denote units not in common with 1 (1). The v values of all units in all cms are then passed through the sigmoidal activation function (eq . 6) whose shape / scale reflects g. again, particular parameter values affect the relation of input similarity to code similarity (and therefore, storage capacity): values of = 28 and = 5 produce the v - to- mappings in figure 4 . As noted above, within each cm, the output variable, (i), can be viewed as a relative likelihood that unit i should be chosen winner . (6)(i)=1+e (v(i) +)+1 when g = 1 (perfectly familiar), is maximized (in eq . 7) probabilities of winning for units with the maximum v value in their respective cms . In contrast, when g = 0 (completely novel), = 0, which collapses the sigmoid to the constant function, = 1, thus making all units in a cm equally likely to win . This causes the expected intersection of the code being chosen in the current instance with any previously assigned code to be at chance level . In general, this modulation of the sigmoid activation function tends toward code completion in proportion to the familiarity of the input and code separation in proportion to its novelty . Transform relative likelihood distribution () in each cm to true probability distribution (). (7)(i)=(i)k cm(k) (round 2 competition) choose an f2 code by drawing a winner from the -distribution (soft max) in each cm . Thus, choosing an f2 code is actually performed as q separate draws . When g = 0, these draws are statistically independent, as in figures 3 and 4d . As we consider increasingly familiar inputs, i.e., for g approaching 1 (and, assuming the model is still operating in a regime where crosstalk is sufficiently low), the draws become increasingly correlated (dependent), as can be seen in going from figure 4c to 4b to 4a . Figure 3 graphically illustrates the operation of the csa in the case of the model being presented with its first input, 1 . The gray arrows indicate that the bu signals propagating from the active f1 units will be traversing connections with zero synaptic strength . This leads to unnormalized (u) and normalized (v) input summations of 0 for all 12 f2 units (steps 1,2). In step 3, the max v, v^, in each cm is found (ties broken at random). In step 4, g is computed as the average of the v^ values: in this case all the v^ are 0, so g = 0 . In step 5, the value, g = 0, maps to = 0, which causes the activation function of the f2 units to collapse to the constant function, = 1 . In step 6, each f2 unit applies this activation function to its v value, yielding the uniform relative likelihood distribution in each cm . In step 7, the relative likelihood function in each cm is normalized to a true probability () distribution, which in this case, is again uniform . Finally, in step 8, a winner is drawn in each cm, resulting in a random f2 code, e.g., 1 . Figure 4 demonstrates that the csa realizes the sisc property by considering four possibilities (a d) for the second input presented to the model of figure 3 . These four inputs, 25, range from being identical to 1 (completely familiar) to having zero overlap with 1 (completely unfamiliar). To save space, the panels of figure 4 use an abbreviated version of the format of figure 3 . Most noticeably, the intermediate variable, (relative likelihood), is not shown . Black bu connections are ones that were increased to one when 1 was learned (figure 3). Working from figure 4a to 4d, the inputs have progressively lower similarity (intersection) with 1: f1 units not in common with 1 are shown in green . As g drops, the sigmoid expansivity drops (note the changing scale). Thus, the -distributions become progressively flatter, which in turn results in f2 codes, 25, having progressively smaller intersection with 1 . F2 units not in common with 1 also shown in green . Figure 4a shows the case of presenting a completely familiar input again, and is thus a recognition test trial, demonstrating retrieval . This leads, via csa steps 3 and 4, to g = 1, which yields, via steps 5 and 6, the expansive nonlinear v - to- mapping shown (red sigmoid). This nonlinearity is applied to every f2 unit, yielding the highly peaked -distributions shown . The probability of drawing the correct unit in any single cm is approximately 98% . Of course, what's crucial in this case, i.e., when the input is completely familiar (g = 1), is that the entire correct f2 code is reactivated . In this case, thus, the familiarity, g, which depends on the entire f2 layer and is thus global information, influences the local activation functions so as to produce the desired overall result, in this case, reactivation of the code (memory trace), 1, of the familiar input pattern, 1 . The explanations of the remaining panels follow that of figures 3 and 4a . In going from figure 4b to 4d, one can readily see decreasing intersection with 1, decreasing u and v values, decreasing g, decreasing sigmoid expansivity, progressively flatter -distributions, and ultimately, decreasing intersection with 1 . Given a desired probability, r, of correctly reactivating an entire code (i.e., of choosing the correct unit in each cm), when g = 1, given values for q and k, we could compute the needed value of . However, the macrocolumn model is a content - addressable associative memory and in actual usage, multiple sparse codes will be stored in superposition . Any thorough analysis of the model's expected retrieval error must include the effect of overlap between the stored codes (i.e., cross - talk): this influences the shapes of the -distributions and thus, the expected retrieval accuracy for any given number of stored codes . Such an analysis will be conducted empirically and reported in a separate paper . Before leaving figure 4 first, while the -distributions become flatter as g decreases, the units comprising the code of the most similar previously learned input (here, 1) remain most likely to win in their respective cms . If we simply deterministically chose the unit with maximum v(i) in each cm, we would have chosen the same f2 code, 1, in response to all four inputs, 25 . Thus, the computation of a quantity, g, which depends on all the cms is essential to achieving the sisc property . It constitutes a channel through which information transfers between all f2 units throughout the whole macrocolumn . As noted earlier, the full model also assumes direct h connections between f2 units, analogous to the horizontal matrix of l2/3 (see figure s2 in supplementary material). These also mediate communication, but of the prior code active in the macrocolumn, not of the simultaneous state of all f2 units throughout the macrocolumn . Second, learning is single - trial and involves only one iteration of the csa . This is largely facilitated by the fact that when a given input - code association, jj, is learned, each of j's f2 units simultaneously has its afferent weight from all of j's f1 units increased . The effect of these simultaneous correlated potentiations allows a rapid, even single - trial, formation of an association, even if the individual synaptic potentiations are small, consistent with the recent characterization of thalamocortical learning described in bruno and sakmann (2006). Third, figure 4a shows that recognizing an exact instance of a previous input also requires only one iteration of the csa . Although this example does not directly show it, this holds for recognition of non - exact matches as well . Evidence for this will be presented in a separate work . That both learning and recognition require only a single csa iteration is especially significant since, as can readily be seen, none of the csa steps involves iterations over stored codes: thus, the time it takes for the csa to either store a new input or retrieve the closest matching stored input remains constant as the number of stored codes increases . This does not imply that an infinite number of codes can be stored: of course, the model has finite storage capacity . This capacity will be characterized in future research, but should be similar to other sparse associative memories (willshaw et al ., 1969; palm, 1982; moll and miikkulainen, 1995; knoblauch et al ., 2010). There remain huge gaps in our knowledge of the intrinsic physiological, synaptic, morphological, and connectional properties of all classes of cortical cell and of functional relationships between cortical and sub - cortical structures . Nevertheless, figure 5 shows a possible neural realization of the model's wta cm, i.e., minicolumn, and dynamics (csa). I have attempted to respect known constraints but the realization is admittedly speculative and ongoing modifications will undoubtedly be required . Figures 5a e show the critical events transpiring in a single minicolumn at five points in time during the csa's computational cycle . Note that due to space limitations figure 5 cannot depict the true extents of the various axonal and dendritic systems of the cells involved . (a e) depict critical events transpiring in a single minicolumn at five points in time during the csa's computational cycle, which i propose corresponds to one gamma cycle: approximate timings relative to start of csa cycle are shown across top . The units labeled c (b) represent the local chandelier (basket cell) populations, respectively; see text for detailed explanation . Figure 5a shows the initial csa steps 1 and 2 when the l2/3 pyramidals (here only two cells, but in reality, 20) integrate their inputs . While the csa explanation in the prior section included only the bu inputs, all three input classes are included here: bu inputs (labeled 2) are mediated via l4 (rockland and pandya, 1979) td inputs (1) are mediated via l2/3 apical tufts (rockland and drash, 1996) h inputs (3) are mediated via the horizontal matrix of l2/3 (gilbert and wiesel, 1989; feldmeyer et al ., 2006) all three input vectors arrive in parallel and collectively give rise to postsynaptic potentials (psps) in the l2/3 pyramidals . The three (normalized) inputs are combined multiplicatively; see the generalized version of the csa (table s1 in supplementary material). C) are firing at this time, preventing the l2/3 pyramids from firing . In figure 5b, the chandeliers shut off (grayed out) and the l2/3 pyramid with the highest psp (cell 2) is assumed to be the first to spike (csa step 3). This winning cell, and more specifically, its psp value (v in eq . 2), represents this minicolumn's local judgment of how similar the macrocolumn's closest - matching stored input is to the current overall (i.e., bu, h, and td) input . The output of cell 2 is then communicated to some locus where it is averaged with the round 1 winners from the other 70 minicolumns of the macrocolumn, yielding g (csa step 4). As noted in the introduction, the functionality related to g seems most consistent with the phenomenology of neuromodulatory systems, especially ach and ne . Note that the communication of cell 2 s psp value is hypothesized to be mediated via l5, one of whose pyramidal cells is seen integrating here (light green); this is based loosely on evidence that l5 cells, specifically l5b pyramidals, almost exclusively target pontine areas (with collaterals to thalamus) (deschnes et al . L2/3 pyramidals are the primary source of bu signals to higher cortical areas (rockland and pandya, 1979; and see thomson et al ., 2002; brecht et al ., 2003; schubert et al ., 2003; bannister, 2005; helmstaedter et al ., 2007; lbke and feldmeyer, 2007; petersen, 2007; egger et al ., 2008;, 2009 for wider background on cortical columnar circuitry relevant to the current proposal). In addition, as stated earlier, the horizontal l2/3-to - l2/3 connections are proposed to communicate this macrocolumn's final hypothesis regarding its total input pattern in the current csa cycle recurrently back to the same (and surrounding) macrocolumns on the next csa cycle . Hence, it is crucial that since that final hypothesis is not present until the second round of competition completes (figure 5e), the output pathways carrying those signals must be closed (red xs on paths 4 and 5). Though not depicted here, one possible mechanism for selectively preventing horizontal signaling in l2/3 is activation of the double bouquet cells (defelipe et al ., 1990, 2006; peters and sethares, 1997). Axons, being interdigitated, nearly one - to - one with minicolumns would allow them to make contact with a substantial portion of the horizontally (and obliquely) oriented dendritic and axonal processes, in l2/3, and thus prevent passage of horizontal signals . In figure 5c, the l5 pyramidal mediating the winning l2/3 cell's psp value has reached threshold and sends that output to the sub - cortical averaging locus (path 6). In addition, the winning cell itself has activated the local basket cell network (electrically coupled, cf . B, which rapidly deactivates and re - polarizes (resets) the l2/3 pyramidals (grayed out). This reset need not be back to a completely even baseline: some remnant of the relative psp distribution prior to basket cell activation might remain, biasing the second round of competition . In figure 5d, the result of the subcortical computation of g is returned to the macrocolumn (path 7) in the form of neuromodulator release (purple cloud surrounding the l2/3 pyramidals). Cloud actually extends across a broad, i.e., macrocolumnar (or wider), expanse of l2/3, not just within a single minicolumn as this figure may suggest . The chandeliers are again firing to prevent any l2/3 from firing as the second round of integration occurs . The basket cells are inactive, allowing this integration to take place . In figure 5e, the chandeliers again deactivate ., the pyramidal cell winning on this second round of competition may differ from the first round winner . In particular, the probability that the second round winner is the same as the first round winner increases with g. the set of l2/3 winners, one per minicolumn, across the whole macrocolumn represents that macrocolumn's final decision (hypothesis) as to identity of its current overall (td, h, and bu) input . Thus, the output of the winning l2/3 cell in each minicolumn is now communicated to: lower cortical regions via l5 and its backprojections to the lower regions l1 (labeled 8) (rockland and drash, 1996). L2/3 pyramids in the same and neighboring macrocolumns via the local horizontal l2/3 matrix (5) (gilbert and wiesel, 1989; feldmeyer et al ., 2006), thus delivering temporal context information (recurrently) to the local region to be integrated on the next csa cycle . The l4 layer of higher cortical regions (4) (rockland and pandya, 1979). Note that the output of the winning l2/3 cell should be prevented from recurring to the local basket network at this time so as to allow the integration period to occur at the beginning of the next computational cycle; hence, the red x on the link to basket cell . I reiterate that the above possible cortical realization of the proposed sdc model is highly speculative . It clearly lacks numerous details, especially regarding processing in the intermediate processing stages, e.g., l4, and output processing involving l5 (and l6). Nevertheless, it is a starting point and can be falsified, especially as experimental methods mature . For example, the many timing relationships in the circuit can be tested . We still have decidedly little in the way of hard constraints on the time courses of activation of the many cell types involved in cortical (and hippocampal) circuits, though progress is being made (klausberger et al . Silberberg and markram, 2007; klausberger and somogyi, 2008; otsuka and kawaguchi, 2009; woodruff et al ., 2009). Moreover, the proposed theory's key assumption that the l2/3 pyramidals are the core repository of information in cortex and that the codes laid down in l2/3 are the context - dependent memories of our experiences, is subject to challenge . Specifically, the anatomy of the l5 thick tufted cells suggests that they too have access to bu (via l4), td (via their apical tufts in l1), and h (via an extensive intra - l5 horizontal network, schubert et al ., 2007) inputs, and therefore that l5 might store the most detailed and context - dependent codes in cortex, a view supported by findings such as (de kock et al ., 2007). In the end, for the purpose of this hypothesis and theory paper, i believe the architecture and algorithm (csa) to be more important than the specifics of any particular neural realization . In this section, i consider evidence relating to six model predictions: a}signals generated in the macrocolumn [i.e., the v^x (eq . Strictly interpreted, figure 2 suggests that the model can only be true of cortical areas that have direct projections to the activation function modulator (afm), hypothesized to be instantiated in midbrain neuromodulator source areas, e.g., basal forebrain (bf, source of ach) and locus coeruleus (lc, source of ne). Direct cortical afferents to bf arise mainly from prepyriform, anterior insula, orbitofrontal, entorhinal and medial temporal areas (mesulam and mufson, 1984). Direct cortical afferents to lc arise from dorsal prefrontal cortex (arnsten and goldman - rakic, 1984), medial prefrontal cortex (jodo et al ., 1998). While direct projections are limited, a much larger fraction of cortex may be able to influence the hypothesized afm via multi - synaptic pathways involving thalamus and other subcortical structures, especially via pathways interconnecting bf, lc, and other neuromodulator source areas ., 2004), which allows dorsal and medial prefrontal areas indirect influence on bf . Similarly, lc receives input from the raphe nuclei (reviewed in samuels and szabadi, 2008) which would allow further extension of the sphere of cortical influence upon the afm . However, it is clearly possible that my macrocolumnar model applies only to the smaller set of cortical areas suggested above . After all, there would be some advantage to deferring the decision as to the overall familiarity / novelty of the current input (moment) to the very highest cortical levels, which are in position to make the most informed decisions . In this case, once g is computed, it is then broadcast pan - cortically, i.e., to all levels of the hierarchy, allowing the local choice of code to proceed accordingly, i.e., with a level of randomness appropriate to g. figure s2 in supplementary material illustrates this view . 5), which correlates with the detection of familiarity, and/or inversely with the detection of novelty . Such correlations have been shown for both ach (miranda et al ., 2000, 2003) and ne (sara et al ., 1994; vankov et al ., lc projects to all of cortex (foote and morrison, 1987; berridge and waterhouse, 2003; samuels and szabadi, 2008). Bf projects to almost all cortical areas (reviewed in briand et al ., 2007). The amount of randomness to be added to the winner selection process is a global parameter, which applies non - specifically to all the minicolumns . This is consistent with volume transmission believed to be used by neuromodulatory systems (descarries et al ., 1997; see sarter et al ., 2009 for discussion of the complexities of the evidence regarding volume transmission). D}the signal determines (eqs 68) the amount of noise (randomness) in the selection (activation) of cortical (i.e., macrocolumnar) codes . Controlling the noisiness of a process of choosing a winner from a competing group of neurons can be achieved by some combination of two actions: (i) increasing the spike probability of cells with high input summations relative to those with low summations and (ii) lowering the spike probability of low - input cells relative to high - input cells . Both of these actions can be achieved by uniformly (i.e., to all competing cells in a wta module) modulating intrinsic cell properties such as excitability . Numerous studies have demonstrated both excitatory and suppressive effects on target cell responses (both principal neurons and interneurons) for both ach (kawasaki and avoli, 1996; shalinsky et al ., 2002; cobb and davies, 2005; tateno et al ., 2005; isakova and mednikova, 2007; lawrence, 2008; eggermann and feldmeyer, 2009) and ne (foote et al ., 1975; kawaguchi and shindou, 1998; harley and helen, 2007; moxon et al . It is not my intention here to argue for a precise realization of this mechanism . As suggested in many reviews (berridge and waterhouse, 2003; lucas - meunier et al ., 2003; sara, 2009), the landscape of this research is very complex and we are far from a comprehensive understanding of the how the various neuromodulatory systems affect high - level cognitive processing either alone or in concert (briand et al ., 2007). Nevertheless, the large and varied body of evidence at least admits the possibility that one or more of these neuromodulators could implement the familiarity - contingent afm mechanism (csa steps 58; see doya, 2002, p. 502 e}decreased, i.e., increased noise, leads to greater code separation (decreased intersection). Recently, goard and dan (2009) showed that increased bf stimulation decreased the correlation amongst a population of rat v1 cells . This decreased correlation essentially shows increased separation between population codes, which in the model proposed here, would manifest as decreased intersection between sparse codes . F}disabling of the brain's ability to produce high noise, i.e., causing to be permanently high, should reduce the ability to learn new inputs, while sparing or having much less effect on recognition of known items . Looking at figure 4, if the afm was stuck in the highly expansive sigmoid condition (low noise), all four inputs, 25, would have high probability of mapping to the same code, 1 . This would prevent the model from being able to distinguish them in future presentations . However, in general, inputs that were mapped to unique codes prior to such a disabling event will reliably activate those codes on future presentations . In accord with this, browning et al . (2010) found that severely diminishing cholinergic inputs to inferotemporal cortex severely reduced macaques performance on a visual episodic memory task, while having little effect on a dnms task . (2005) found a similar effect: cholinergic deafferentation of entorhinal cortex reduced performance on dnms tasks involving novel odors but not familiar odors . In this section, i consider evidence relating to six model predictions: a}signals generated in the macrocolumn [i.e., the v^x (eq . Strictly interpreted, figure 2 suggests that the model can only be true of cortical areas that have direct projections to the activation function modulator (afm), hypothesized to be instantiated in midbrain neuromodulator source areas, e.g., basal forebrain (bf, source of ach) and locus coeruleus (lc, source of ne). Relatively few cortical areas project directly to bf or lc . Direct cortical afferents to bf arise mainly from prepyriform, anterior insula, orbitofrontal, entorhinal and medial temporal areas (mesulam and mufson, 1984). Direct cortical afferents to lc arise from dorsal prefrontal cortex (arnsten and goldman - rakic, 1984), medial prefrontal cortex (jodo et al ., 1998). While direct projections are limited, a much larger fraction of cortex may be able to influence the hypothesized afm via multi - synaptic pathways involving thalamus and other subcortical structures, especially via pathways interconnecting bf, lc, and other neuromodulator source areas . For example, bf cholinergic neurons are excited by lc (jones et al ., 2004), which allows dorsal and medial prefrontal areas indirect influence on bf . Similarly, lc receives input from the raphe nuclei (reviewed in samuels and szabadi, 2008) which would allow further extension of the sphere of cortical influence upon the afm . However, it is clearly possible that my macrocolumnar model applies only to the smaller set of cortical areas suggested above . After all, there would be some advantage to deferring the decision as to the overall familiarity / novelty of the current input (moment) to the very highest cortical levels, which are in position to make the most informed decisions . In this case, once g is computed, it is then broadcast pan - cortically, i.e., to all levels of the hierarchy, allowing the local choice of code to proceed accordingly, i.e., with a level of randomness appropriate to g. figure s2 in supplementary material illustrates this view . 5), which correlates with the detection of familiarity, and/or inversely with the detection of novelty . Such correlations have been shown for both ach (miranda et al ., 2000, 2003) and ne (sara et al ., 1994; vankov et al ., lc projects to all of cortex (foote and morrison, 1987; berridge and waterhouse, 2003; samuels and szabadi, 2008). Bf projects to almost all cortical areas (reviewed in briand et al ., 2007). The amount of randomness to be added to the winner selection process is a global parameter, which applies non - specifically to all the minicolumns . This is consistent with volume transmission believed to be used by neuromodulatory systems (descarries et al ., 1997; see sarter et al ., 2009 for discussion of the complexities of the evidence regarding volume transmission). D}the signal determines (eqs 68) the amount of noise (randomness) in the selection (activation) of cortical (i.e., macrocolumnar) codes . Controlling the noisiness of a process of choosing a winner from a competing group of neurons can be achieved by some combination of two actions: (i) increasing the spike probability of cells with high input summations relative to those with low summations and (ii) lowering the spike probability of low - input cells relative to high - input cells . Both of these actions can be achieved by uniformly (i.e., to all competing cells in a wta module) modulating intrinsic cell properties such as excitability . Numerous studies have demonstrated both excitatory and suppressive effects on target cell responses (both principal neurons and interneurons) for both ach (kawasaki and avoli, 1996; shalinsky et al ., 2002; cobb and davies, 2005; tateno et al ., 2005; isakova and mednikova, 2007; lawrence, 2008; eggermann and feldmeyer, 2009) and ne (foote et al ., 1975; kawaguchi and shindou, 1998; harley and helen, 2007; moxon et al ., 2007). It is not my intention here to argue for a precise realization of this mechanism . As suggested in many reviews (berridge and waterhouse, 2003; lucas - meunier et al ., 2003; sara, 2009), the landscape of this research is very complex and we are far from a comprehensive understanding of the how the various neuromodulatory systems affect high - level cognitive processing either alone or in concert (briand et al ., 2007). Nevertheless, the large and varied body of evidence at least admits the possibility that one or more of these neuromodulators could implement the familiarity - contingent afm mechanism (csa steps 58; see doya, 2002, p. 502 e}decreased, i.e., increased noise, leads to greater code separation (decreased intersection). Recently, goard and dan (2009) showed that increased bf stimulation decreased the correlation amongst a population of rat v1 cells . This decreased correlation essentially shows increased separation between population codes, which in the model proposed here, would manifest as decreased intersection between sparse codes . F}disabling of the brain's ability to produce high noise, i.e., causing to be permanently high, should reduce the ability to learn new inputs, while sparing or having much less effect on recognition of known items . Looking at figure 4, if the afm was stuck in the highly expansive sigmoid condition (low noise), all four inputs, 25, would have high probability of mapping to the same code, 1 . This would prevent the model from being able to distinguish them in future presentations . However, in general, inputs that were mapped to unique codes prior to such a disabling event will reliably activate those codes on future presentations . (2010) found that severely diminishing cholinergic inputs to inferotemporal cortex severely reduced macaques performance on a visual episodic memory task, while having little effect on a dnms task . (2005) found a similar effect: cholinergic deafferentation of entorhinal cortex reduced performance on dnms tasks involving novel odors but not familiar odors . I have described a theoretical model of cortical function that explains the functional relationship between the minicolumn and macrocolumn . Specifically: a}the macrocolumn (in any of its forms) is proposed to store information in the form of sdcs, and b}the minicolumn (specifically, its l2/3 pool of pyramidals) is proposed to operate as a wta cm, the purpose of which is to enforce the sparseness of the macrocolumnar code . Two key motivations for this model are the computational advantages of sdc and the increasingly strong evidence for sdc in the brain, cited in the section introduction . One important advantage of sdc over a localist code is that the number of unique items that can be stored can be far larger than the number of representing units . For example, the 12 f2 units of the model in figure 2 allow 3 = 81 unique codes, though in realistic systems, e.g., with less than complete connectivity leading to and from a coding field like f2, the number of those codes that can safely (i.e., while maintaining retrieval error rates below some acceptable criterion) be assigned will be substantially lower than 81 . Nevertheless, if the number of input items that will need to be distinguished is not known a priori, sdc is more flexible . A second computational advantage of sdc is that, if used in conjunction with an appropriate storage / retrieval algorithm it possesses the sisc property . I demonstrated, with the small but statistically reasonable example of figures 3 and 4, that the csa yields the sisc property . The sisc property strongly differentiates sdc from localist models: it is not even defined for a localist model since every code is formally disjoint with every other code . Hence, there is no structural way to represent degrees of similarity in a localist code . If there is no way to represent a measure, e.g., similarity, structurally, then whenever that measure is required e.g., when the closest matching stored item in a database (i.e., macrocolumn) to an input must be returned it must be computed, which takes time and energy . In contrast, when items codes are stored in physically overlapped fashion such that the degree of code overlap represents item similarity, as is the case for the proposed model, the most closely matching stored item will be returned immediately, i.e., without requiring any serial search through the stored items . Figure s4 in supplementary material shows test retrievals of the four unique codes stored in the model of figures 3 and 4, demonstrating possession of this immediate access property for this small example . I consider the representation and the csa to be the most important contributions of this paper because of the computational advantages just described . However, i believe the suggestion that the minicolumn's generic function is to act as a wta cm is also important . Saying only that a group of l2/3 units forms a wta cm places no a priori constraints on what their tuning functions or receptive fields should look like . This is what gives that functionality a chance of being truly generic, i.e., of applying across all areas and species, regardless of the observed tuning profiles of closely neighboring units . Indeed, a recent calcium imaging study of mouse auditory cortex by rothschild et al . (2010) shows highly heterogeneous small - scale (even immediately adjacent cells) tuning even though the large - scale tuning is tonotopic . Experimental methods are only now just reaching the point where this hypothesis might be directly testable, e.g., modifying calcium imaging methods to have millisecond temporal granularity; see ohki and reid (2007). In a sense, the main point of this paper is that a generic minicolumnar function becomes apparent as soon as we postulate that what the cortex, i.e., a macrocolumn, generally does is store and retrieve (access) sdcs of specific context - dependent inputs . As noted in the section introduction, the experimental literature contains little in the way of proposals linking the formation and retrieval of specific sdcs (i.e., of specific input items, especially of temporal context - dependent items) to the cortical microcircuitry . My proposed model goes beyond broad conclusions and offers a mechanistic explanation of how specific informational items are learned and retrieved and in so doing, proposes a generic function for the minicolumn, i.e., that it functions as a wta module in support of manipulating sdcs at the next higher, i.e., macrocolumnar, scale . There have been several recent models linking formation / retrieval of specific items to cortical circuitry and which describe specific roles for the minicolumn (kupper et al ., 2007; george and hawkins, 2009; litvak and ullman, 2009; schrader et al ., however, all of these models use localist representations and therefore would not possess the advantages of sdc described above . The cortext model (kupper et al ., 2007; schrader et al ., 2009) assumes that each minicolumn in a hypercolumn represents one particular input feature . On each computational cycle, a wta process runs within each hypercolumn, such that exactly one minicolumn wins, which would be strongly at odds with the calcium image data (ohki et al ., 2005). A second problem is that the assumption that whole minicolumns compete with each other implies that any given hypercolumn (at any level of the cortical hierarchy) can represent only 70 unique features (equivalence classes), which seems severely restrictive, especially for hypercolumns at higher cortical levels, e.g., it . The litvak and ullman (2009) model also postulates that the l2/3 pool of neurons in a minicolumn implements a max function . However, their model proposes that each single minicolumn (specifically, its l2/3 pool) is partitioned into several disjoint groups (cliques) of cells, each representing a different item . Since any particular cell can participate in only one clique, this constitutes a localist code . George and hawkins (2009) also assume that minicolumns represent informational items in a localist fashion . Note however that both george and hawkins (2009) and litvak and ullman (2009) explicitly mention moving to a more general combinatorial code, a.k.a . A core principle of the proposed model is this notion of controlling the amount of noise in the process of choosing (activating) a macrocolumnar code as an inverse function of input similarity . Doya (2002) uses the same principle, referred to as boltzmann selection, to modulate the amount of noise in the process of choosing amongst output action actions . Doya specifically hypothesizes that ne controls the noise whereas i can assert only that it is some neuromodulator - based mechanism . In doya's model, as ne levels increase, the action with the greatest expected reward is chosen with probability approaching 1 . This is suggested as corresponding to the exploitation end of the exploitation exploration continuum . As ne levels drop to 0, all actions become equally probable, i.e., exploration, which is appropriate if no single action has a particular high expected reward, which generally correlates with the condition of novelty, i.e., of being in a novel environment wherein it is harder to anticipate the outcome of known actions . The analogy to high expected reward in my model is a highly familiar input (g 1) in which case we want the stored code for that familiar input to be reactivated with probability approaching 1; the condition where no action has a high expected reward is analogous to low familiarity, i.e., where no stored input is very similar to the current input, in which case we want to lay down a new memory trace for the novel input . Despite the similarities, doya's model also assumes a localist representation of the choices and, like the other models just mentioned, cannot realize the advantages of sdc . I have identified several avenues of active and future research at various points in the text and as noted in the previous section, the prospective neural realization is highly speculative and very incomplete . Several additional questions / issues for future research are: is the current proposal that the l2/3 cells engage in two rounds of competition in each computational (putatively, gamma) cycle plausible? For simplicity, i have described the model in the simplest case of having only one internal coding field (f2) and processing only purely spatial input patterns . However the core model was originally developed for the spatiotemporal pattern (sequence) case (rinkus, 1996) and was generalized some time ago to have an arbitrarily deep hierarchy of coding fields (rinkus and lisman, 2005). See figure s2 in supplementary material . How do these generalized versions of the model map to neural structures? Is there evidence that chandeliers become active twice as frequently as baskets, as the proposed realization predicts? Is there evidence for the converse? Although not elaborated herein, the proposed mini-/macrocolumn model is easily generalized to allow substantial overlap between minicolumns (see figure s5 in supplementary material) and multiple winners in a minicolumn on each computational cycle . We know of the fast, phasic, time scales of operation for ne (rajkowski et al ., 2004) and da (schultz, 1998) and of slightly slower but still phasic mode for ach (gulledge and kawaguchi, 2007), but these have been proposed as signaling other measures besides pure novelty (redgrave et al ., 1999; bouret and sara, 2005; dayan and yu, 2006). How might all these signals conspire to implement a pure novelty signal? The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Ulcerative colitis (cu) and crohn's disease (cd) are known as chronic inflammatory bowel diseases (ibd). There is still some controversy about ibd etiology . Among factors associated with inflammatory bowel disease it has been shown that exacerbation of ibd increases angiogenesis especially in ulcerative colitis [1, 2]. This finding produced a concept of antiangiogenic therapy in ibd, as presented by danese et al . . It was reported that anti - angiogenesis in the development of ibd can be measured by detection of thrombospondin [4, 5]. Since anti - angiogenesis is almost always associated with angiogenesis, numerous angiogenic factors have been indicated as possible counterparts to thrombospondin . Among them, vascular endothelial growth factor (vegf) is thought to play a crucial role by stimulating migration and proliferation of endothelial cells (ecs) and the expression of angiogenesis - related genes . There are 7 types of vegf family and vegf - a with two receptors vegfr-1 and vegfr-2 that are most important in angiogenesis [6, 7]. Thrombospondin (tsp) is a 450 kd adhesive glycoprotein that was initially discovered in platelet -granule . Thrombospondin is one of the extracellular matrix adhesive molecules, including also laminin, fibronectin, fibrinogen, and von willebrand factor . Thrombospondins are secreted glycoproteins that modulate cell - matrix interactions, influence platelet aggregation, and support neutrophil chemotaxis and adhesion . There are several articles about the role of tsp-1 in ibd, most of these based upon animal models [4, 5]. The purpose of our study was to assess angiogenesis in correlation with expression of vegfr-1 and thrombospondin-1 to see if they mediate in maintaining balance between angiogenesis and anti - angiogenesis in the course of ibd . We have decided to use computer - assisted morphometrics to facilitate the analysis of vascular density and immunohistochemically highlighted expression of angiogenic factors . We have analyzed full - thickness intestinal wall histological sections routinely prepared out of formalin fixed, paraffin embedded tissue samples . All our ibd - affected intestines were resected because of an active phase of inflammation . Cu bowels were presented with grade 5 of the disease, according to grading scale for histological assessment of inflammation in ulcerative colitis by geboes et al . . Only one tissue fragment containing full thickness of intestinal wall was collected from each patient . In ibd groups the control group was formed with the use of fragments that were collected from macroscopically unchanged bowel wall . All tissue samples were fixed in formalin, embedded in paraffin, and sectioned into 4 micrometer sections, according to standard histopathology protocol . One section in each case was stained routinely with hematoxylin & eosin to evaluate the extent of the disease or to confirm the lack of pathology (in the control group) (see figures 1, 2, and 3). Other sections were immunostained with antibodies against cd-31 (to highlight vessel walls), thrombospondin-1 and vegfr-1 (to show the expression of both). Vascular density was evaluated based upon two parameters immunostaining against cd31 per square mm and percentage of section area covered by vessels . Vegfr-1 expression was evaluated by counting the vessels that showed immunohistochemical reaction in endothelial cells . Statistical analysis of the results (mann - whitney u test, kruskall - wallis test, correlation analysis) was performed with the use of r programming language . Figures 57 show vascular density values in tissue layers of bowel wall in our study . We found statistically significant differences in both mean vessel count and mean vessel area fraction in crohn's disease when compared to normal bowel and ulcerative colitis in comparison to normal bowel (see table 3, figures 6 and 8). Differences in vascular area percentage between ulcerative colitis and control were insignificant for submucosa and muscularis propria . It is interesting, that our results showed significantly higher vascular density in subserosa of control group patients when compared to ulcerative colitis as shown in figures 6 and 8 . We have also shown significant differences in both mean vessel count and mean vessel area fraction throughout the all layers between crohn's disease and ulcerative colitis (p <0.002). Our study showed no statistically significant correlation between ibd type and vegfr-1 (flt1) expression and between vascular density and vegfr-1 expression (see table 4 and figure 7). Tsp-1 was sporadically found (4 cases, expression within intestinal epithelium, see figure 9) only in samples from patients with ibd (3 in cu and 1 in cd), and it seems to be rather artifactual and insufficient to make any statistical correlation between tsp-1 expression and vascular density or vegfr-1 expression . Answering the title question, we could not find a strict correlation between thrombospondin and vascular endothelial growth factor receptor expression . The results of our study support macro- and microscopic pattern in cases with active phase of inflammatory bowel disease we have also revealed significantly increased number of blood vessels in almost all of the four layers of bowel wall while comparing crohn's disease and ulcerative colitis with normal bowel wall . Staining with cd31 was shown to be of great importance not only allowing the assessment of the number of vessels but also showing blood vessels essentially dilated . However, in our opinion, flt1 (vegfr1) is not a marker of angiogenesis this study not revealed statistically significant reaction in normal and ibd bowels . This is contrary to konno et al . Who stated that expression of flt-1 (among others) we believe that a better marker of angiogenesis could be the expression of another vegf receptor - vegfr2 (kdr / flk-1), which was also studied by the same authors . Importance of vegfr2 in angiogenesis was also described by ferrara et al . And usui et al . . We share the opinion of scaldaferri et al . Who stated that, after the induction of colitis, the expression of both vegf - a and vegfr-2 was markedly enhanced, whereas no increase in the expression of vegfr-1 was observed . Only four positive thrombospondin expression findings in our research could be explained by several mechanisms . All our cases with ibd were collected from patients with acute and severe phases of the diseases . This could explain inflammation - induced changes in tsp-1 expression . Also, increased stimulation of angiogenic pathways could completely suppress anti - angiogenesis (inhibition of thrombospondin expression). It is also possible that the pathways of anti - angiogenesis in ibd rely on mediators other than tsp-1 . We also cannot exclude that our antibody reacted with different epitopes (nonspecific for that of tsp-1) leading to artifactual staining . Punekar et al . [4, 5] have studied tsp-1-deficient mice with experimental colitis and suggested that tsp-1 might decrease angiogenesis . Studied only mucosal samples and found that the expression of tsp-1 was higher in ibd groups relative to healthy control group . We could not find such a correlation because tsp-1 tissue reaction was sporadic and weak . We would like to emphasize that we studied the full thickness of bowel wall in contrast to most reports dealing with samples from the intestinal mucosa only . According to our findings, the number of blood vessels increased, and they became dilated in all layers of bowel wall (especially in the course of crohn's disease) showing that angiogenesis is one of the main pathological processes occurring not only within mucosa but also within other layers of bowel wall.
Femtosecond laser - assisted cataract surgery (flacs) is superior to conventional phacoemulsification in terms of precise wound construction, capsulotomy, intraocular lens (iol) centration, and decreased effective phacoemulsification time.14 flacs is comparable to traditional cataract surgery in terms of intraoperative complications, although further research is needed to evaluate its long - term safety aspects.57 it may be especially advantageous in white cataracts, where achieving an adequately sized continuous curvilinear capsulorhexis is the major surgical challenge . The increased incidence of capsulorhexis - related complications in white cataracts is often associated with posterior capsular rent, vitreous loss, and nucleus drop.8 various maneuvers have been used to facilitate capsulorhexis, such as use of high - molecular - weight viscoelastic,9 trypan blue dye to stain the anterior capsule,10 two - stage capsulorhexis,11 frequent aspiration of liquefied cortical matter,12 use of vannas scissors,12 and endoilluminator.9 however, limited literature exists on the application of femtosecond lasers in white cataracts.13 comparative evaluation of flacs and conventional phacoemulsification has been done for varying grades of nuclear sclerosis; however, there are no studies that have compared the outcomes of flacs with conventional phacoemulsification in cases with white cataract.5,7 we herein report our results of comparison of femtosecond laser capsulotomies with conventional capsulorhexis in cases of white cataract . In this prospective comparative study, 80 eyes of 80 patients with white cataract were enrolled at dr rp centre for ophthalmic sciences, all india institute of medical sciences, new delhi, india . Ethical clearance was obtained from the all india institute of medical sciences institutional review board . The white cataracts were classified into type i, ii, and iii according to the classification provided by brazitikos et al.8 type i intumescent, white cataracts are characterized by the presence of liquefied cortex, type ii white cataracts have a voluminous nucleus with little amount of solid white cortex, and type iii white cataracts have anterior capsule fibrosis . Type iii white cataracts with calcific and fibrotic capsules were excluded from the study.8 eyes with preexisting glaucoma, retinal pathology, pseudoexfoliation syndrome, or pupillary dilatation <6 mm were also excluded . The patients were divided into two groups, each consisting of 40 eyes (40 patients). Group i (n=40) underwent flacs and group ii (n=40) underwent conventional phacoemulsification performed by an experienced phacoemulsification surgeon (jst). The patients with white cataract scheduled to undergo flacs or conventional phacoemulsification (based on patient s choice and affordability of the procedure) were compared . The preoperative detailed examination included uncorrected distance visual acuity (udva), anterior segment evaluation, slit - lamp biomicroscopy, applanation tonometry, biometry (tonoref iii, nidek co., ltd . And ocuscan rxp, alcon laboratories, inc . ), and ultrasonography b - scan (master - vu ophthalmic ultrasound, sonomed escalon). The secondary outcome measures were intraoperative phacoemulsification parameters, intraoperative complications, and postoperative visual acuity . In group i, the preoperative planning of the corneal incisions, capsulotomy, and nucleotomy was done on lensx version 2.23 femtosecond laser system (alcon lensx, inc ., a 4.9 mm capsulotomy was planned, with a 2.2 mm temporal clear corneal incision and two 1.1 mm side ports at 90 and 240. the chop pattern of nucleotomy was selected, with three chops of length 6 mm each . The anterior segment structures were imaged with intraoperative optical coherence tomography and the planned treatment was verified . The femtosecond laser parameters were the same as those used in our routine cases (nonwhite cataract cases) of flacs . The energy was kept at 6 j for corneal incisions, 8 j for capsulotomy, and 12 j for nucleotomy . The spot and layer separation was 5 m for the side port incisions, 6 m for 2.2 mm corneal incision, and 14 m for the nucleotomy . For capsulotomy, the spot separation was 5 m, the layer separation was 4 m, and the anterior and posterior delta value was 350 m each . After femtosecond laser application, the corneal incisions were opened with the help of flap lifter . Sodium hyaluronate 1% was injected to maintain the anterior chamber and trypan blue 0.06% (visionblue, dorc international, the netherlands) was injected to stain the anterior capsule . The capsulotomies were lifted and removed with the microcapsulorhexis forceps, and microadhesions, if any, were released (figure 1, video s1). Gentle hydrodissection was performed, followed by phacoemulsification, irrigation aspiration (ia), and implantation of single - piece hydrophobic acrylic iol . A hybrid technique of ia using a combination of coaxial and bimanual ia hand pieces was frequently employed to aspirate the subincisional cortical matter in cases with difficulty during removal of cortical matter.14 the corneal wounds were hydrated at the end of the procedure . In group ii, a 2.2 keratome was used to construct a temporal corneal incision and two side ports were constructed using a 20 g microvitreoretinal blade . Capsulorhexis was performed with a 26-gauge needle cystotome; microcapsulorhexis forceps was used if required . In cases with intumescence, an initial smaller capsulorhexis was followed by the aspiration of liquefied cortex, thus decreasing the raised intralenticular pressure . The aim was to provide ~0.5 mm iol coverage all around as the optic of the iol was 6 mm . After iol implantation, the capsulorhexis was further enlarged if required to achieve an adequate size . Intraoperatively, the total surgical time, cumulative dissipated energy, total ultrasound time, and total aspiration time were noted . Intraoperative complications such as anterior capsular tears / extension, posterior capsular tears, vitreous loss, need for anterior vitrectomy, and inability to implant iol were noted . Postoperatively, clinical photographs were captured with the help of slit - lamp - mounted camera on postoperative day 30 and after 6 months, and the capsulorhexis size, shape, continuity, and iol optic coverage were evaluated . Image - processing software (imagej 1.46r, national institutes of health, md, usa) was used to measure the diameter of the capsulorhexis and calculate the circularity index . The diameter of the implanted iol was used as a scale to calibrate the image . The circularity index was given by the formula 4 (area / perimeter2) and a circularity index of 1 implied a perfect circle.3 the iol centration and coverage was assessed on the basis of coverage of iol optic in all four quadrants by the capsulotomy / capsulorhexis margin . Continuity of the capsulorhexis refers to the absence of radial extension / tear of the capsulorhexis edge . Udva and corrected distance visual acuity (cdva) were assessed on postoperative day 30 and at 6 months . Statistical analysis was done using statistical package for the social sciences (spss 11.0; spss inc ., chicago, il, usa). Normally distributed continuous variables were expressed as mean standard deviation and were compared using independent samples t - test . Nominal data were compared using chi - square test or fisher s exact test as appropriate . A p - value less than 0.05 was considered significant . In group i, the preoperative planning of the corneal incisions, capsulotomy, and nucleotomy was done on lensx version 2.23 femtosecond laser system (alcon lensx, inc ., a 4.9 mm capsulotomy was planned, with a 2.2 mm temporal clear corneal incision and two 1.1 mm side ports at 90 and 240. the chop pattern of nucleotomy was selected, with three chops of length 6 mm each . The anterior segment structures were imaged with intraoperative optical coherence tomography and the planned treatment was verified . The femtosecond laser parameters were the same as those used in our routine cases (nonwhite cataract cases) of flacs . The energy was kept at 6 j for corneal incisions, 8 j for capsulotomy, and 12 j for nucleotomy . The spot and layer separation was 5 m for the side port incisions, 6 m for 2.2 mm corneal incision, and 14 m for the nucleotomy . For capsulotomy, the spot separation was 5 m, the layer separation was 4 m, and the anterior and posterior delta value was 350 m each . After femtosecond laser application, the corneal incisions were opened with the help of flap lifter . Sodium hyaluronate 1% was injected to maintain the anterior chamber and trypan blue 0.06% (visionblue, dorc international, the netherlands) was injected to stain the anterior capsule . The capsulotomies were lifted and removed with the microcapsulorhexis forceps, and microadhesions, if any, were released (figure 1, video s1). Gentle hydrodissection was performed, followed by phacoemulsification, irrigation aspiration (ia), and implantation of single - piece hydrophobic acrylic iol . A hybrid technique of ia using a combination of coaxial and bimanual ia hand pieces was frequently employed to aspirate the subincisional cortical matter in cases with difficulty during removal of cortical matter.14 the corneal wounds were hydrated at the end of the procedure . In group ii, a 2.2 keratome was used to construct a temporal corneal incision and two side ports were constructed using a 20 g microvitreoretinal blade . Capsulorhexis was performed with a 26-gauge needle cystotome; microcapsulorhexis forceps was used if required . In cases with intumescence, an initial smaller capsulorhexis was followed by the aspiration of liquefied cortex, thus decreasing the raised intralenticular pressure . The aim was to provide ~0.5 mm iol coverage all around as the optic of the iol was 6 mm . After iol implantation, the capsulorhexis was further enlarged if required to achieve an adequate size . Intraoperatively, the total surgical time, cumulative dissipated energy, total ultrasound time, and total aspiration time were noted . Intraoperative complications such as anterior capsular tears / extension, posterior capsular tears, vitreous loss, need for anterior vitrectomy, and inability to implant iol were noted . Postoperatively, clinical photographs were captured with the help of slit - lamp - mounted camera on postoperative day 30 and after 6 months, and the capsulorhexis size, shape, continuity, and iol optic coverage were evaluated . Image - processing software (imagej 1.46r, national institutes of health, md, usa) was used to measure the diameter of the capsulorhexis and calculate the circularity index . The diameter of the implanted iol was used as a scale to calibrate the image . The circularity index was given by the formula 4 (area / perimeter2) and a circularity index of 1 implied a perfect circle.3 the iol centration and coverage was assessed on the basis of coverage of iol optic in all four quadrants by the capsulotomy / capsulorhexis margin . Continuity of the capsulorhexis refers to the absence of radial extension / tear of the capsulorhexis edge . Udva and corrected distance visual acuity (cdva) were assessed on postoperative day 30 and at 6 months . Statistical analysis was done using statistical package for the social sciences (spss 11.0; spss inc ., chicago, il, usa). Normally distributed continuous variables were expressed as mean standard deviation and were compared using independent samples t - test . Nominal data were compared using chi - square test or fisher s exact test as appropriate . A p - value less than 0.05 was considered significant . The mean age of the patients was 62.99.1 years in group i and 64.88.3 years in group ii (p=0.34). Group i consisted of 20 males and 20 females; group ii had 17 males and 23 females . A continuous capsulotomy / capsulorhexis was achieved in 97.5% (39/40) of eyes, each in groups i and ii . Type i (free floating), type ii (microadhesions; ie, an area of complete cut mixed with minute areas of adhesions), type iii (incomplete treatment pattern no visible femtosecond laser cut in the anterior capsule), and type iv (irregular complete pattern). In group i, free - floating capsulotomies (type i) were achieved in 52.5% (21/40) of eyes . However, 47.5% (19/40) of eyes needed manual completion of the capsulotomy, of these 37.5% (15/40) had microadhesions (type ii capsulotomy) and 10% (4/40) had incomplete area of capsulotomy in 12 clock hours (type iii capsulotomy). In group the mean diameter of the capsulotomy / capsulorhexis was 4.90.1 mm in group i and 5.30.4 mm in group ii (p<0.001, difference 0.39; 95% confidence interval [ci]: 0.27, 0.52). The mean circularity index was 0.9960.003 in group i and 0.9100.047 in group ii (p - value <0.001, difference: 0.09 [95% ci: 0.07, 0.10]). Inadequate coverage of iol by the capsulotomy / capsulorhexis edge was noted in 7.5% (3/40) cases in group i and 12.5% (5/40) cases in group ii (p=0.712). At 6 months, the mean diameter of the capsulotomy / capsulorhexis was 4.90.1 mm in group i and 5.30.4 mm in group ii (p<0.001, difference: 0.37 [95% ci: 0.25, 0.50]). The mean circularity index was 0.9910.023 in group i and 0.9100.047 in group ii (p - value <0.001, difference: 0.08 [95% ci: 0.06, 0.10]). The cumulative dissipated energy, total ultrasound time, total aspiration time, and total surgical time were comparable between the two groups (table 2). There was no case of posterior capsular rent, vitreous loss, nucleus drop, or inability to implant iol in group i or ii . The mean logarithm of the minimum angle of resolution (logmar) udva was 0.1280.122 in group i and 0.1150.127 in group ii (p=0.655) at 1 month postoperatively . The mean logmar cdva was 0.0100.044 in group i and 0.0150.053 in group ii (p=0.649) at 1 month postoperatively . At 6 months, mean logmar udva was 0.130.12 in group i and 0.100.12 in group ii (p=0.315). At 6 months, mean logmar cdva was 0.0050.031 in group i and 0.0100.044 in group ii (p=0.562). The white cataracts in each group were divided into two subgroups based on the release of white milky fluid on initiation of the capsulotomy / capsulorhexis . The assessment of milky fluid egress was qualitative, and no attempt was made to quantify the amount of fluid by the surgeon . In group i, 17 cases had release of white milky fluid (subgroup a) and 23 cases did not (subgroup b). In group ii, 19 cases were in the fluid group (subgroup a) and 21 cases were in the no - fluid group (subgroup b). The capsulotomy / capsulorhexis characteristics in the fluid cases were compared with the no - fluid cases in each group . Continuous capsulotomies were achieved in 94.1% (16/17) cases with fluid and 100% (23/23) cases with no - fluid (p=0.42). In the cases with fluid, free - floating (type i capsulotomy) capsulotomies were achieved in 23.5% (4/17) cases . Manual completion of the capsulotomy was needed in 76.5% (13/17) cases, of these 52.9% (9/17) had microadhesions (type ii capsulotomy) and 23.5% (4/17) had incomplete area of capsulotomy in 12 clock hours (type iii capsulotomy). In cases with no - fluid, approximately 26.1% (6/23) of eyes needed manual completion of the capsulotomy and release of the microadhesions (type ii capsulotomy). The incidence of residual adhesions and incomplete capsulotomies was significantly more in cases with release of white milky fluid (p=0.003). Anterior capsular extension with radial tear was observed in one eye in fluid release group only (p=0.42). The mean diameter of the capsulotomy was 4.90.1 mm in fluid and no - fluid group (p=0.9). The mean circularity index was 0.9960.003 in the fluid group and 0.9970.003 in the no - fluid group (p=0.1). Inadequate coverage of iol by the capsulotomy edge was noted in 17.6% (3/17) cases in fluid group only (p=0.09). A continuous capsulorhexis was achieved in 100% (19/19) cases with fluid and 95.2% (20/21) cases with no - fluid (p=1.0). A multistep capsulorhexis was performed in 84.2% (16/19) cases with fluid and 57.1% (12/21) cases with no - fluid . There was one case of anterior capsular extension with radial tear in no - fluid group only (p=1.00). The mean diameter of the capsulorhexis was 5.30.3 mm in the fluid group and 5.40.4 mm in the no - fluid group (p=0.7, difference: 0.05 [95% ci: 0.3, 0.2]). The mean circularity index was 0.880.04 in the fluid group and 0.940.04 in the no - fluid group (p - value <0.001, difference: 0.05 [95% ci: 0.02, 0.08]). Inadequate coverage of iol by the capsulorhexis edge was noted in 15.8% (3/19) cases with fluid and 9.5% (2/21) cases with no - fluid (p=0.65). Continuous capsulotomies were achieved in 94.1% (16/17) cases with fluid and 100% (23/23) cases with no - fluid (p=0.42). In the cases with fluid, free - floating (type i capsulotomy) capsulotomies were achieved in 23.5% (4/17) cases . Manual completion of the capsulotomy was needed in 76.5% (13/17) cases, of these 52.9% (9/17) had microadhesions (type ii capsulotomy) and 23.5% (4/17) had incomplete area of capsulotomy in 12 clock hours (type iii capsulotomy). In cases with no - fluid, approximately 26.1% (6/23) of eyes needed manual completion of the capsulotomy and release of the microadhesions (type ii capsulotomy). The incidence of residual adhesions and incomplete capsulotomies was significantly more in cases with release of white milky fluid (p=0.003). Anterior capsular extension with radial tear was observed in one eye in fluid release group only (p=0.42). The mean diameter of the capsulotomy was 4.90.1 mm in fluid and no - fluid group (p=0.9). The mean circularity index was 0.9960.003 in the fluid group and 0.9970.003 in the no - fluid group (p=0.1). Inadequate coverage of iol by the capsulotomy edge was noted in 17.6% (3/17) cases in fluid group only (p=0.09). A continuous capsulorhexis was achieved in 100% (19/19) cases with fluid and 95.2% (20/21) cases with no - fluid (p=1.0). A multistep capsulorhexis was performed in 84.2% (16/19) cases with fluid and 57.1% (12/21) cases with no - fluid . There was one case of anterior capsular extension with radial tear in no - fluid group only (p=1.00). The mean diameter of the capsulorhexis was 5.30.3 mm in the fluid group and 5.40.4 mm in the no - fluid group (p=0.7, difference: 0.05 [95% ci: 0.3, 0.2]). The mean circularity index was 0.880.04 in the fluid group and 0.940.04 in the no - fluid group (p - value <0.001, difference: 0.05 [95% ci: 0.02, 0.08]). Inadequate coverage of iol by the capsulorhexis edge was noted in 15.8% (3/19) cases with fluid and 9.5% (2/21) cases with no - fluid (p=0.65). The main challenge in phacoemulsification in cases of white cataracts is achieving a continuous curvilinear capsulorhexis . The absence of a red reflex, raised intralenticular pressure, and a fragile anterior capsule complicate the management of white cataracts, and an incomplete capsulorhexis has been reported in 3.85%28.3% cases of white cataracts.912,16 an ideal capsulorhexis is a well - centered circular opening with smooth margins and a slightly smaller diameter than the iol providing 360 iol coverage . Inadequate coverage of iol may lead to posterior capsular opacification and change in the effective lens position,17 whereas an excessively small capsulorhexis may cause anterior capsular contraction.18 in our study, reproducible circular capsulotomies were achieved with the help of femtosecond laser, providing adequate iol coverage in 92.5% (37/40) of cases . The femtosecond laser - assisted capsulotomies may be of four types based on the pattern of completion.15 in our study, type i free - floating capsulotomies were achieved in 52.5% (21/40) cases . Type ii capsulotomy with microadhesions was observed in 37.5% (15/40) cases and 10% (4/40) had type iii capsulotomy with incomplete treatment pattern in 12 clock hours . A significantly higher incidence of residual adhesions (type ii and type iii capsulotomies) was observed in the subgroup with release of white milky fluid after femtosecond laser delivery (p<0.003). The reason for microadhesions / incomplete capsulotomies was the explosive egress of white milky fluid on initiation of the capsulotomy, which obscures the visual field and hampers the subsequent delivery of the laser at the same plane . The release of the intralenticular pressure after initiation of the capsulotomy causes a slight change in the position of the anterior capsular plane and hence may result in the occurrence of incomplete capsulotomy . During the lifting of the capsulotomy flap with the microcapsulorhexis forceps, the areas obscured by the white milky fluid during femtosecond laser application should be carefully assessed, as they are likely to have microadhesions . It is imperative to use trypan blue dye to stain the anterior capsule in all cases to delineate the area of microadhesions or incomplete treatment pattern . In each of these cases, the free edge of the capsulotomy was grasped with the help of microcapsulorhexis forceps and a circumferential motion tracing the capsulotomy margins was performed, which was akin to the maneuver that one does while performing a manual capsulorhexis (figure 1, video s1). This crucial step ensures the effective release of the underlying residual adhesions in femtosecond laser - assisted capsulotomies . Despite the presence of microadhesions and incomplete treatment patterns, the continuity and circularity of the capsulotomies was achieved in 97.5% (39/40) of the eyes . The incidence of incomplete capsulotomies and microadhesions is more in white hypermature cataracts as compared to immature senile cataracts . In a case series of 1,300 cases of senile cataract undergoing flacs, 96% capsulotomies were free floating or had small areas of nonperforations.19 in our study, one case of anterior capsular extension with radial tear was noted during flacs . The radial extension occurred while polishing the anterior capsule after iol implantation (figure 3). This case was characterized by raised intralenticular pressure as evidenced by a gush of white milky fluid after the initial capsulotomy opening and had a type ii capsulotomy with microadhesions . In cases with microadhesions, the capsular edge is fragile and careful anterior capsular polishing is needed to prevent extension and radial tears . In our experience, a raised intralenticular pressure with release of white milky fluid is the most significant factor affecting the femtosecond laser application . The milky fluid results in microadhesions and incomplete sectors of capsulotomy, which may increase the chances of tear extension during subsequent steps of phacoemulsification . This is in contrast to the manual capsulorhexis where the release of fluid is associated with a subsequent decrease in the intralenticular pressure and more safety in completion of capsulorhexis . A two - stage femtosecond laser capsulotomy has also been described in six cases of intumescent white cataract; however, remnant tissue bridges were present in one - third of the mini - capsulotomies.20 in cases undergoing conventional phacoemulsification, a multistep capsulorhexis was performed in 70% cases to achieve an adequately sized capsulorhexis . The circularity of the capsulorhexis was compromised, more so in cases with release of milky fluid . Microadhesions if present can be easily released during capsulotomy removal with forceps, without jeopardizing the size, circularity, and continuity of the capsulotomy . A previous study has demonstrated the safety and efficacy of femtosecond laser - assisted capsulotomy in 25 cases of white cataracts, with radial anterior tears in two eyes, an adherent tongue - like capsule adhesion in nine eyes, and an incomplete capsulotomy in three eyes.13 the total aspiration time and total surgical duration was longer in flacs, but the difference between the two groups was not statistically significant . A hybrid technique of ia using a combination of coaxial and bimanual ia hand pieces was frequently employed to aspirate the subincisional cortical matter in cases undergoing flacs, which may explain the longer aspiration times.14 to conclude, femtosecond laser creates single - step, circular, adequately sized capsulotomies and eliminates the difficulty associated with capsulorhexis in white cataracts . The release of white milky fluid during femtosecond laser delivery is the most important factor affecting the creation of a free - floating capsulotomy, and microadhesions and incomplete capsulotomies may be present in up to 47.5% cases of flacs . There is no significant difference in terms of the intraoperative complications and the final visual outcome between flacs and conventional phacoemulsification . To the best of our knowledge, this is the first prospective study comparing conventional phacoemulsification with flacs in white cataracts in terms of intraoperative parameters and postoperative outcomes . Further randomized control trials with a larger sample size should be undertaken to further evaluate the long - term superiority of femtosecond laser over conventional phacoemulsification.
The development of behavioral addictions (bas, also referred to as impulse control disorders) in association with dopamine agonists (das, commonly used to treat parkinson s disease) has been reported (dodd et al ., 2005; driver - dunckley, samanta, & stacy, 2003; szarfman, doraiswamy, tonning, & levine, 2006; weintraub et al ., 2010). (2014) present evidence that these reported associations are evident in the us food & drug administration s (fda) adverse event reporting system (faers), a database containing information on adverse drug event (ade) and medication error reports submitted to the fda 2014) conducted a retrospective disproportionality analysis based on the 2.7 million serious domestic and foreign ades reported to the faers from 2003 to 2012 . A threshold consisting of a proportional reporting ratio (prr) 2 was used to detect signals (drug - associated adverse events) involving any of six das and any of 10 terms (or ades) in the medical dictionary for regulatory activities characterized as bas . They identified 1,580 reports containing at least one ba, 710 of which also contained a da, and reported a prr for reports containing both a ba and a da (bas w / das) of 277.6 (p <0.001). The authors concluded that their findings confirm prior reports of a ba / da association . However, given three factors, it is possible that their findings could have been affected by publicity . First, vulnerability to publicity - stimulated reporting has been described as a limitation of spontaneous reporting systems like the faers (bate & evans, 2009; moore et al ., 2003). Second, between 2003 and 2012, the development of bas in association with das received considerable media coverage on multiple occasions . Third, a 2014 research report, which, in its analysis of faers reporting cast a wider net than moore et al . (2014) with respect to both number of bas analyzed (16 versus 10) and type of report analyzed (both serious and non - serious reports were included), found no or only very weak associations prior to publicity between das and bas that could be grouped under the general headings binge eating, compulsive shopping, and hypersexuality (pre - publicity data on reports of gambling to the faers are not freely available) (gendreau & potenza, 2014). (2014) briefly allude to the issue of publicity in their discussion and suggest that it cannot account for their findings . Specifically, they characterize growth in ba w / da reporting (dopamine receptor agonists in figure 1) between 2003 and 2012 as steady and conclude that it is therefore unlikely that a burst of publicity or specific events explain their findings . We contend that growth in ba w / da reporting between 2003 and 2012 was not steady, but rather it fluctuated, with upticks in reporting in 2004 (approximately 133%) and 2011 (approximately 49%), and a sustained period of growth between 2005 and 2009 (approximately 588%). Given the observations listed in the previous paragraph, we hypothesized that these fluctuations may coincide with publicity . Trend over time for reports of pathological gambling, hypersexuality, and related impulse control disorders . Total domestic and foreign reports of serious injury received by the us food and drug administration containing any of 10 terms in the medical dictionary for regulatory activities that characterize the impulse control disorder under study . (reproduced with permission from the american medical association: jama internal medicine (moore et al . Note: in the current study, impulse control disorders are referred to as behavioral addictions, or bas to investigate the possibility that these fluctuations may coincide with media coverage, we conducted internet searches of 16 major us and international english - language media outlets between 2003 and 2012 for the keywords parkinson s and gambling . News stories were excluded if they only briefly mentioned a ba / da association (26 stories); keywords were found among comments on a story (four stories); they were in question and answer or dear doctor format (two stories); or the webpage or article was no longer accessible (one story). Both authors have conformed to the highest standards of ethical conduct in the submission of accurate data, acknowledging the work of others and divulging potential conflicts of interest . As the study involved the use of publicly accessible, de - identified data, signed informed consent was not necessary for this study . Given the use of publicly accessible, de - identified data, the study is exempted from irb review under code of federal regulations 45 cfr 46.101(b). Both authors have conformed to the highest standards of ethical conduct in the submission of accurate data, acknowledging the work of others and divulging potential conflicts of interest . As the study involved the use of publicly accessible, de - identified data, signed informed consent was not necessary for this study . Given the use of publicly accessible, de - identified data, the study is exempted from irb review under code of federal regulations 45 cfr 46.101(b). Of the 35 included news stories, 15 were precipitated by scientific publications (43%); 10 by lawsuits (29%); 7 by conference presentations (20%); 1 by a change in pharmaceutical company advertising (3%); 1 by a patient s story (3%); and 1 for which the catalyst was unclear (3%). Results are summarized in figure 2 . The uptick in ba w / da reporting in 2004 seen in figure 1 follows a news story in the new york times in august 2003 (oneil, 2003), precipitated by the publication in the same month of research conducted by driver - dunckley et al . The uptick in 2011 follows four news stories in january and february of 2011 triggered by lawsuits, as well as a cbs news story in june 2010 (edwards, 2010) precipitated by a scientific publication (weintraub et al ., 2010). The approximately 588% growth in ba w / da reporting observed between 2005 and 2009 coincides with the publication of 26 news stories (74% of the 35 news stories included) between july 2005 and april 2009, most of which were prompted by scientific publications (bostwick, hecksel, stevens, bower, & ahlskog, 2009; dodd et al ., 2005; szarfman et al ., 2006; voon et al ., 2007), conference presentations, and lawsuits . Ba = behavioral addiction, da = dopamine agonist . Note: news stories data were compiled from internet searches of 16 major us and international english - language media outlets for the terms parkinson s and gambling (abc news, cbs news, cnbc, cnn, the globe & mail, the guardian, huffington post, msnbc, nbc news, the new york times, npr, scientific american, time, usa today, the wall street journal, and the washington post) upticks in ba w / da reporting to the faers appear to either follow or coincide with publication of news stories . Moreover, fluctuations in ba w / da reporting are not reflected in the trajectories plotted for all faers reports or ba w / non - da reports (figure 3), which suggests the presence of some influence on the ba w / da reporting rate specifically . That fluctuations in news coverage may coincide with fluctuations in ba w / da reporting suggests the possibility that publicity may have influenced reporting . Trend over time for ba w / non - da and all faers reports, 20032012 . Faers = us food & drug administration s (fda) adverse event reporting system, ba = behavioral addiction, da = dopamine agonist . Note: ba w / non - da reports data were estimated based on data presented in moore et al . All faers reports data were obtained from the fda s website at http://www.fda.gov/drugs/guidancecomplianceregulatoryinformation/surveillance/adversedrugeffects/ucm083765.htm given the possibility that publicity could have influenced their findings, it would have been helpful if moore et al . (2014) had provided prrs for clinically related groupings of the 10 bas analyzed . This would make it easier to assess the degree to which the overall prr of 277.6 may be driven by pathological gambling / gambling (two separate ades in the faers), which, in 2003, was the first ba to be reported in association with das in the mainstream media . Pathological gambling / gambling was also the ba most frequently reported to the faers between 2003 and 2012, accounting for 42.7% of the 1,902 reports of bas identified by (moore et al ., 2014). It would also have been interesting to see a comparison between the 2003 prrs (pre - publicity for all but pathological gambling) and the 2012 prrs (post - publicity for all bas) for clinically related groupings of the 10 bas analyzed . It should be noted that significant complexities exist in identifying the specific factors that might cause ades and their reporting . While publicity may influence reporting, it is also possible that faers reporting may have led to increased publicity, perhaps indirectly through analysis and reporting of findings . Possible examples may include through the fda s quarterly reporting of potential drug safety issues identified via analysis of faers data; updates to product labeling or product advertising resulting from analysis of faers reporting; or scientific publication of analysis of faers reporting . Of the 35 news stories included in the analysis, one cites as its catalyst an update to possible adverse events listed in a product s television advertisements, a change the product manufacturer says was prompted by a request from the fda, and two stories cite as an impetus scientific publication of analysis of faers reporting . We also note the gradual nature of the sizable uptick in ba w / da reporting between 2005 and 2009, which occurs in the midst of several years of increased media coverage of a ba / da association . This finding would be consistent with a possible lag, with information taking time to circulate and lead to reporting, although other possibilities also exist . It is possible that the ba w / da data presented in figure 1 are influenced by multiple factors, some of which are indicated above . While we do not cover an exhaustive list, the notion that reporting of bas in association with das might be influenced by publicity has important clinical and public health implications, and is thus not a notion that should be prematurely dismissed . In summary, a ba / da association has received considerable publicity since it was first reported in 2003 . Data suggest that publicity may stimulate ade reporting, and in this case it may coincide with fluctuations in ba w / da reporting . It is important to evaluate the potential effect of publicity on ade reporting to guard against mistaking an increased risk of an ade being reported for an increased risk of an ade occurring (moore et al ., 2003). Kg: contributed study concept and design, collected, analyzed and interpreted data, and performed initial drafting of the manuscript . Mp: obtained funding and aided in analysis and interpretation of data, manuscript review and editing . Both authors have participated sufficiently in the research endeavor in order to qualify for authorship, and take public responsibility for its content . The corresponding author has had access to all data from the study and both authors have had complete freedom to direct its analysis, interpretation and reporting without influence from funding agencies . Potenza has consulted for and advised lundbeck, ironwood, insys, shire and rivermend health for issues relating to impulse control disorders, gambling disorder, eating disorders, substance addictions and gender - related differences; has received research support from the national institutes of health, veteran s administration, mohegan sun casino, the national center for responsible gaming, and pfizer pharmaceuticals; has participated in surveys, mailings or telephone consultations related to drug addiction, impulse control disorders or other health topics; has consulted for gambling and legal entities on issues related to addictions or impulse control disorders; has provided clinical care in the connecticut department of mental health and addiction services problem gambling services program; has performed grant reviews for the national institutes of health and other agencies; has guest - edited journal sections; has given academic lectures in grand rounds, cme events and other clinical or scientific venues; and has generated books or book chapters for publishers of mental health texts.
Breast cancer is a leading cause of mortality and morbidity all over the world . In 2008, close to 1.4 million cases were diagnosed with breast cancer worldwide . The incidence varies among different populations with high rates seen in developed countries compared to developing countries [2, 3]. In general, breast cancer rates are highest in white european and lowest in east asian populations [1, 4, 5]. The estimated incidence rate for women living in the south - east asia region of world health organization' is 26.1 per 100000 population and this figure is 89.7 for women living in western europe . The established risk factors of breast cancer are, mainly, early age at menarche, late age at menopause, nulliparity, number of live birth, and age at first live birth . Contrary to the large variations seen in incidence between population of europe and asia, the prevalence of the established risk factors is not very much different between the two populations [5, 6]. A higher risk of breast cancer among american and european women has been blamed for so - called western lifestyle characterized [7, 8] by the combination of early menarche, decreased parity, delayed childbearing, and a sedentary lifestyle . Studies of migrants have confirmed the relative importance of environment and lifestyle in the etiology of breast cancer [911]. The so - called western lifestyle is now very common in asian countries such as japan, korea, taiwan, and hong kong and is spreading fast in the economic booming region of east asia . In addition to the major differences in magnitude of rates between asians and europeans, there is a distinct difference in the shape of age specific rates between the two populations . In asian population, the age specific incidence curve peaks at 4550 and then plateaus and even slightly decreases so that the rates after 60 years are less than or close to the rates at age group 4555 . In european population, the age specific curve increases steadily with no change of pace around age 50 and the increase continues up to age 80 with a peak around 65 years . A lower risk and the distinct pattern of age specific incidence rates among the asian population (even in countries with great extent of similar lifestyle with western population such as japan, taiwan, and hong kong) have been a challenging issue among epidemiologists and cancer scientists to the extent that some have labeled breast cancer in the population of asia as a different disease . A recent symposium in montreal, canada, specifically addressing the same topic, highlighted the younger age at onset as one of the chief characteristics of breast cancer natural history in asian population . What contributes to the peculiar phenomenon of younger age at onset has been the subject of inertest to epidemiologists, and it has been hypothesized that a cohort effect among asian population causes this phenomenon . The aim of this study was to use the incidence rates reported in the cancer incidence in five continents for the two populations of asia and europe in order to address the nature of the age specific rate differences between the two populations using age - period - cohort analysis . Registered cases of female breast cancer and corresponding person years were ascertained from the ci5plus, cancer incidence in five continents annual dataset (an online data repository of international agency on research on cancer, iarc) for 29 registries in europe and 9 registries in asia for a duration from 1953 to 2002 . Cases from europe included cases registered for the period from 1953 to 2002 and cases from asia included cases registered for the period from 1963 to 2002 . Cases and their corresponding person years were pooled for each population to make two distinct populations, referred to hereafter as asian and european . Age specific rates were addressed both cross - sectionally (period wise) and longitudinally (generational birth cohort wise). For period wise age specifics, the rates were constructed and described based on five years period (1955, 1960, 1965, 2000). For the cohort wise, age specific rates were estimated from age - period - cohort analysis constructed over each five years cohort . In addition, the trends in rates expressed as annual percentage change (apc) and their 95% confidence intervals were estimated using the age - period - cohort model . For analytical part, the age - period - cohort model was used . For this, the periods and cohorts were constructed in intervals of 5 years . The five - year age groups were truncated to age more than 25 years with the last interval (85 and over) included all cases more than 85 years (13 five - year age groups were constructed). The constrained generalized linear model (cglm), the most utilized approach in the epidemiology literature dealing with age - period - cohort analysis, was used . For this purpose, a log - linear model with the general form that includes a (age), p (period), and c (cohort) was applied as follows: (1)log{(a, p)}=f(a)+g(p)+h(c), where a, p, and c represent the mean age, period, and cohort and f, g, and h are parametric functions fitted to the data . In this model, in addition to estimating the main effect of age, other components contributing to magnitude of rates specially the secular changes of rate across study periods and birth cohorts are estimated . The secular change or net drift corresponds, interchangeably, to hazard due to period or cohort, and it has been used to estimate the annual percent changes of rates over a period of time [14, 15]. As the purpose of our study was to tackle the difference between the two populations' age specific rates, it was assumed that mainly the cohort effect explains the changing of rates across aging intervals during the study period in both populations . With this assumption, the model estimates the age function presented as the log of the age specific rates for the reference cohort (longitudinal age specific or age specifics across cohorts) and the cohort effect as log of rate ratio relative to a reference cohort while period effect constrained to be zero on average with zero slopes . The estimated logs of age specific rates were transformed to rate scale (number per 100000 population) for better realization . In the model, the cohort born during 1970 was considered as the reference cohort and the period of 1970 was considered as the reference period . The longitudinal age specific raters were estimated and reported for cohort born on 1885, 1910, 1930, 1950, and 1970 . For details of the modeling please refer to age - period - cohort models for the lexis diagram by carstensen . Data were analyzed using the r 2.14.1 statistical software utilizing epi 1.1.9 package (r development core team, 2009). A total of 236,851 cases of breast cancer registered in the 29 european registries and a total of 188,630 cases registered in the 9 asian registries were included in the analysis (table 4 presents details of the included registries for the two populations). There was a constant increasing of rates for both populations during the last 50 years with an estimated annual percent of change 1.03 (with 95% ci of 1.029, 1.031) for asians and 1.016 (95% ci of 1.015, 1.017) for europeans . The incidence rates across all age groups in europeans were higher than asians, especially in older age groups . During the study period, the magnitude of rates increased for both populations for each succeeding five - year period for all age groups . The shape of the age specific rates (period wise) showed basic differences between the two populations . For asian population, the age specific rates for all periods peaked around 50 years and then decreased and plateaued afterward (figure 1). For the european population, the age specific rates increased up to the last age group for periods ending 1985 and for the periods after 1985, the age specific rates peaked between 55 to 75 years and then slightly decreased (figure 1). The fitting of the age - period - cohort model to data indicated that the model that included all the main effects (age, period, and cohort) has the greatest reduction of deviance, indicating the best model to explain the observed rates in both populations; table 1 presents the goodness of fit of the models along with their parameters . There were cohort effects present in incidence rates of both populations during the study period; however, the cohort effects in asians were much stronger than european . In the asian population, the rate ratios presenting the cohort effects ranged from a low of 0.06 (95% ci 0.05, 0.08) for those born in 1870 to 0.94 (95% ci, 0.93, 0.96) for those born in 1965 . In the european population, the rate ratios presenting the cohort effect ranged from 0.33 (95% ci, 0.32, 0.35) for cohort born in 1865 to 1.03 (95% ci, 1.02, 1.04) for the cohort born in 1965 (table 2 and figure 2). There were residual period effects in the asian population around 1975 (rate ratio of 0.89 and 95% ci 0.86, 0.92) and 1985 (rate ratio of 1.12 95% ci of 1.09, 1.13), figure 2 . For both populations, the estimated age specific rates expressed as longitudinal age specific indicated the same pattern for both populations; the age specific rates increased sharply before the age of 50, and the increase slowed down pace with the last age groups (over 75 years), still the groups with highest incidence rates . The pattern of longitudinal age specific rate is presented in figure 2 along with the other effects, cohorts, and periods . The estimated longitudinal age specific rates (in an increment of 20 years) and their corresponding confidence intervals are presented numerically in table 3 and graphically in figure 3 . As table 3 and figure 3 indicate, the estimated age specific rates have steadily increased in all age groups for both populations but the increase is more in asians compared to europeans . There is a large difference in the magnitude of rates between the two populations in early cohort (1890) when they are compared with the most recent cohort (1970), table 3 and figure 3 . The difference in age specific rates between early and late cohorts is indicative of the cohort effects that cause the distinct pattern of age specific rates observed between the two populations . In addition, comparing the magnitude of the cohort effects between the two populations (figure 2), they indicate that, though, the cohort effects are decreasing along succeeding cohorts for both populations, but the decrease in cohort effects in asians is far larger than those of europeans . This difference in decreasing rates of cohort effects between the two populations indicates that both populations may experience similar rates in the future if there are no other major changes to the underlying cause of the disease in future years . Our study proved a steady increase of breast cancer rate with similar pace during the last 50 years for both populations . We demonstrated that there is no difference between the patterns of age specific rates between the two populations when rates are measured as longitudinal age specific rates . It was demonstrated that a strong cohort effect contributes to the differences in pattern of age specific rates between the two populations . The difference in breast cancer rates with low rates for asians versus high rates for europeans has been documented since registries in asia started reporting population rates [17, 18]. While several studies have demonstrated marked differences in magnitude and the pattern of age specific rates among different countries of europe and asia, no study systematically and collectively has addressed the age specific rate differences in the two populations as our study did . An overall increasing trend of morbidity from breast cancer has been reported for all populations of the world and the increase has been attributed to ageing and increasing median age of women [15, 17, 1922]. This similarity in slope of increase indicates that despite the fact that the two populations are basically different in terms of culture, ethnicity, lifestyle, and social attributes, the breast cancer epidemic enforces its own pace of epidemic projection . Any increase in incidence of breast cancer rates is due to either changing of risk factors or implementation of mass screening (especially mammographic screening). The difference in the pattern of the age specific rates between the two populations is well recognized and several studies have addressed this discrepancy; a study comparing the shape of the age specific rates between taiwanese and caucasian american reported that the age specific rates of breast cancer differed between the two populations and the study concluded that the difference is due to a cohort effect presented in taiwanese . Another study comparing breast cancer rates among populations of singapore and sweden attributed the difference in rates to a large cohort effect and concluded that this effect will decrease in future generations causing similar incidence rates between the two population in coming decades . In addition to comparative studies, it has been demonstrated that the pattern of age specific rates for the populations of japan, korea, china, singapore, thailand, and philippine has changed in the recent years attributing this change to changing of life style toward more westernization and implementation of mammographic screening . The pattern and magnitude of age specific rates of breast cancer have been affected by screening mammography specially in the european population, and this effect has been mainly presented as increase of incidence in the age group of 50 to 70 [2628]. This is compatible with our finding as it was demonstrated in figure 1 that age specific rates for european population increased in that age group 50 to 70 and the change happened after 1985 when the wide spread use of mammographic screening started [27, 29]. Mammographic screening started in asia in late 1990 [30, 31] and its effect on the shape of age specific incidence cannot be assessed in our study . Compatible with previous studies comparing the age specific rates of breast cancer between population of europe and asia, our study proved that a large cohort effect in asian population rates plays a major role in the differences in age specific rates between the two populations . The age specific rates can be defined both cross - sectionally (period wise) and longitudinally (cohort wise). If there are no cohort or period effects, the two definitions will show similar magnitude and pattern of age specific rates . The distinct pattern of age specific rates (cross - sectional rates) between the two populations is in fact due to the cohort effect that was demonstrated in our study . In the other world, what contributes to the observed pattern of period wise age specific rates in asian population is the additive nature of the cross - sectional definition of age specific rates in the presence of decreasing cohort effects . Since the rates for two sequential age groups come from two different cohorts, when the older cohort has lower risk compared to younger cohort, the cross sectional patterns of age specifics will decrease . The strong cohort effect that exists in the asian breast cancer is responsible for a distinct pattern of cross - sectional age specifics seen in the asian population . In applying the cglm model, the choice of constraint (period or cohort) is based on an external knowledge of the underlying cause of change of rates in a population . Our choice of cohort instead of period as constraint was based on previous studies that attributed the changes of the rates to a cohort effect [15, 19, 32]. The period effect has been mainly attributed when change of health policy (e.g., introduction of more sensitive detection techniques or availability of certain diagnostic procedures) causes the changing of rates . In the light of the nature of breast cancer risk factors that are mainly of hormonal and sociobehavioral nature this study enjoyed data of adequate quality as the data were utilized from the registries that met acceptable degree of validity and reliability to be published in the international agency in research on cancer (iarc) official report . In addition, the quality of the data for both populations is comparable owing to the efforts and quality assurances and control protocols that iarc requires for different registries contributing data to the cancer incidence in five continents reports; one may ask . We would have been able to draw the same conclusion on analyzing just the data of asian population without the need of european population . The main reason for using european for comparison was that the fact that the specificity attributed to asian breast cancer age specific rates has been defined as it has contrasted to the well - sestablished breast cancer epidemiology in europe and western countries . The methodology we used is a very established and routine way of analyzing rates at population level when the data of calendar time exist . The age - period - cohort analysis has been a major tool in the hands of demographers and, in recent decades well utilized by epidemiologists . The methodology, while very common in use, suffers major problem especially when it is utilized to attribute the underlying cause of changes of a rate to period versus cohort (the nonidentifiability problem). In our study, this problem was not a concern as our assumption was that the nature of risk factors in breast cancer would translate into cohort effect other than period effect . It was concluded that no differences in the pattern of age specific rates exist between the two populations when the age specifics are measured cohort wise, and the difference seen in the period wise age specific rate is due to a strong cohort effect present in the asian population rates.
An investigation was initiated to characterize the virus and identify the patient s infection source . Phylogenetic inference was based on 9 genes located within the central, conserved region of the genome (supplementary methods). Methods used to identify the patient s infection source included interviews with the patient, contact tracing and analysis of serum samples for the presence of anti - orthopoxvirus immunoglobulin (ig) g and igm, and a visit to the patient s residence to conduct environmental sampling and peridomestic small - mammal trapping . Given the uncertainty about an incubation period for this novel poxvirus, a conservative threshold of 4 weeks was assumed based on the incubation period of human monkeypox . The patient was asked to identify persons with whom she had regular or close contact during the 4 weeks before and after symptom onset . The patient and each contact were interviewed to ascertain whether they had ever received a smallpox vaccination, had traveled recently, or had experienced any unusual health events during the presumptive incubation period . Serum samples were obtained from these persons and tested with enzyme - linked immunosorbent assays to determine the presence of anti - orthopoxvirus igm and igg antibodies . On 8 september, environmental samples were collected in and around the patient s home during a site visit . Household surfaces that the patient indicated had been contacted by wild small mammals that periodically entered the home and possible fomites associated with international travel by the patient s partner were swabbed by using hydraflock dacron swab samples (puritan medical). Approximately 6 weeks later, small mammals were trapped around the perimeter of the patient s home and at a site approximately 1 km away where she and her partner were building a new home (supplementary methods). Environmental and nonblood small - mammal samples (oral swab, liver tissue, and feces) were tested using real - time pcr assays that target specific orthopoxvirus generic sequences [35]; small - mammal blood samples were tested using enzyme - linked immunosorbent assays for anti - orthopoxvirus igg . Phylogenetic analyses of the concatenated sequence alignment (28037 base pairs in length) indicate that the virus isolate represents a distinct genetic lineage of orthopoxvirus that is highly divergent from congeners included in our analysis . A well - supported topology was recovered in which the grouping of taterapox virus and variola virus as sister taxa (bayesian posterior probability, 0.5983) was the only node with a bayesian posterior probability <0.999 (figure 2, supplementary figure 1, and supplementary_table_1). Ak2015_poxvirus was grouped within the genus orthopoxvirus and recovered as sister to a monophyletic clade containing all old world orthopoxviruses with high support; it was estimated to be 6.1%7.3% divergent from different species of old world orthopoxviruses, and 12.3%12.6% divergent from isolates within the north american clade . The genetic distances estimated between examined isolates of recognized old world orthopoxvirus species varied from 0.6% (taterapox virus to camelpox virus) to 3.2% (ectromelia virus to variola virus). Results of bayesian phylogenetic inference, indicating the position of ak2015_poxvirus within the genus orthopoxvirus . Analysis was based on 9 genes located within the central, conserved region of the genome (vaccinia virus copenhagen strain homologues a7l, a10l, a24r, d1r, d5r, e6r, e9l, h4l, and j6r). Electron microscopic observation of cells infected with ak2015_poxvirus demonstrated the presence of different morphological forms, including crescents (figure 1c) and immature virus particles (figure 1d). In addition, infected cells had inclusion bodies formed by accumulation of a - type inclusion proteins . The inclusion bodies were embedded with mature virus particles and surrounded by ribosomes (figure 1e). During interviews, the patient reiterated a lack of sick contacts during the 4 weeks before symptom onset . During the 4 weeks after symptom onset, she reported contact with 4 persons . Of 3 household contacts (adult male partner and 2 teenaged children), all had regular, direct physical contact with her and with common household items, but none reported any unusual health events . One social contact (adult female friend) reported a rash on her chest within 1 week of being in physical contact with the patient in the days after the patient s symptom onset . A history of smallpox vaccination was reported by both adult contacts (vaccination before 2003) but neither adolescent contact . Serological tests performed on specimens collected from these persons did not identify evidence of recent exposure among the contacts; anti - orthopoxvirus igm was only detected in serum from the patient (table 1). Consistent with their self - reported smallpox vaccination histories, anti - orthopoxvirus igg was detected in the serum from both adult contacts but neither adolescent contact . Anti - orthopoxvirus igg and igm results for serum samples from the patient and 4 contacts abbreviation: ig, immunoglobulin . Serum optical density (od) cutoff values (od value 3 standard deviations of negative control) at 1:50 and 1:100 dilutions were considered positive for igm and igg, respectively . The patient did not recall being vaccinated and did not have a vaccination scar . The sample from the patient s older child was positive for anti - orthopoxvirus igm, but because this value was low and the corresponding anti - orthopoxvirus igg result was negative, the result was interpreted as equivocal . The patient reiterated that no out - of - state travel occurred during the 4 weeks before symptom onset . She reported working intermittently in the petroleum industry in the north slope region of alaska, during february her cohabitating partner was also employed in the petroleum industry and had worked intermittently (5 weeks on and 5 weeks off) on oil - drilling platforms in azerbaijan, from october 2013 to march 2015 . He had no other out - of - state travel during this period and while overseas he remained almost exclusively on oil platforms with limited in - country travel . He returned to alaska from his last trip to azerbaijan in march 2015 (approximately 4 months before the patient s symptom onset) and did not report any unusual health events during or within 4 weeks after the trip . When he returned from a trip to azerbaijan in october 2014 (approximately 9 months before the patient s symptom onset), he brought back souvenirs for the patient (a wooden jewelry box containing a cloth jewelry pouch) and a collared work jacket that he wore regularly during the trip and that the patient wore periodically after his return to alaska . Swab samples of the surfaces of these items were tested to determine whether any might have served as fomites . No evidence of orthopoxvirus dna was detected . The home that the patient shared with her partner and 2 children was located in a forested, low - density area, within 50 miles of fairbanks . She reported that wild small mammals (eg, shrews, voles, squirrels) were abundant in this boreal forest environment, were regularly observed around the home s perimeter, and entered the home on occasion . She also reported that her children periodically handled the carcasses of squirrels that they shot near the home using a pellet rifle . When asked about construction of her new home, the patient reported that she and her partner had used scrap wood from an abandoned shed located near her home that had been occupied by wild small mammals . All 23 environmental samples collected during the site visit, including swab samples of scrap wood from the abandoned shed at the new home construction site, tested negative for orthopoxviruses by pcr (supplementary_table_2). At the time of the site visit to the patient s residence (approximately 45 days after symptom onset), her symptoms included an active lesion that had decreased in size since initial presentation but remained raised, tender, and warm (approximated in figure 1b). Thirty - one small - mammal samples, collected from 12 individual animals belonging to 2 species (sorex cinereus, n = 3; myodes rutilus, n = 9), tested negative for orthopoxviruses by pcr (supplementary_table_3). No evidence of transmission from the index patient, or fatalities associated with infection, was reported . Epidemiologic information gathered during the investigation provides inconclusive evidence for 2 general hypotheses concerning the patient s route of exposure . The first involves importation of an old world orthopoxvirus into alaska, either as an active infection in a person with whom the patient came into contact or by way of one or more fomites . The limited number of contacts identified by the patient, and the apparent absence of additional cases, indicates that the probability of unidentified secondary spread is remote . Work - related travel to azerbaijan by the patient s partner represents a possible fomite - associated importation scenario, although sampling and testing of travel - related fomites returned negative results . Although the recent discovery of a novel, zoonotic orthopoxvirus in the nearby republic of georgia indicates that unidentified orthopoxviruses might be circulating in this region, the delay between the arrival of these fomites in alaska and the timing of onset of the patient s symptoms indicates that infection by this route is unlikely . The duration of viability of ak2015_poxvirus on fomites is uncertain; however, a laboratory study of vaccinia virus demonstrated retention of viability on environmental surfaces for up to 56 days . The presence of a - type inclusion bodies occluded with mature virus in the ak2015_poxvirus isolate observed by electron microscopy might be indicative of enhanced environmental resilience and prolonged viability . Alternatively, ak2015_poxvirus might be endemic to alaska, perhaps circulating within one or more wildlife reservoir population, and infection might have occurred through an animal exposure . Different species of wild small mammals occur in the boreal forest environment surrounding the patient s residence, and infection by indirect animal contact (eg, contact with household surfaces, squirrels shot by the patient s children, or handling of potentially contaminated wood from the shed occupied by wild small mammals during construction of the new home) might be implicated . Rodents and other small mammals are known or suspected reservoirs for multiple orthopoxviruses, and evidence of infection has been produced by serosurveys in eurasia [1114], africa [5, 15], south america [16, 17], and the continental united states . Among the limited number of wildlife serosurveys of terrestrial mammals conducted in alaska, the majority have focused on large mammals [1921], and those that sampled small mammals did not test for evidence of a poxvirus infection or exposure [2224]. Small - mammal serosurveys in regions of northern europe that are ecologically similar to alaska have identified seropositive animals [2527]. Although results of our small - mammal trapping and testing were negative, the sample was limited and taxonomically restricted . Phylogenetic analyses indicate that ak2015_poxvirus is more closely related to the old world orthopoxviruses than to north american congeners . However, that the virus is known only from north america creates a discordance that precludes its assignment to one or the other of these geographically defined groups with confidence . The global distribution of other genera and unassigned isolates within chordopoxvirinae, and incomplete sampling of potential reservoir taxa as part of our investigation, create additional uncertainty regarding this assignment . Nevertheless, given the inconsistent timelines of contact travel and patient symptom onset, negative results of travel - associated fomite testing, and the potential for regular and close contact with wild small mammals in and around the patient s home, the most parsimonious explanation of infection is exposure to ak2015_poxvirus near fairbanks . Evidence of virus circulation in alaska or elsewhere in north america (ie, infections in persons or reservoir species) would indicate either a new world origin of orthopoxviruses or an old world origin with multiple introductions to the new world . Both scenarios run counter to the present characterization of north american and old world orthopoxviruses as representing reciprocally monophyletic lineages, and challenge the currently accepted hypothesis of an old world origin of the genus orthopoxvirus with a solitary introduction of the new world orthopoxviruses to north america . This discovery of a novel orthopoxvirus is the latest in a growing number of reports of human poxvirus infection published in recent years . These include the emergence of novel poxviruses [7, 2931] and the increased incidence of human monkeypox, an orthopoxvirus illness historically associated with relatively low incidence . Because smallpox vaccination has been demonstrated to provide cross - protection against other orthopoxviruses [33, 34], these observations have been attributed to the cessation of routine smallpox vaccination after eradication of variola virus in 1980 and the subsequent waning of population - level vaccine - derived immunity [7, 32, 35]. Continued emergence and reemergence of orthopoxviruses is expected . To effectively treat persons infected by orthopoxviruses, clinicians should remain vigilant for signs of poxvirus infections and immediately alert public health officials when infection is suspected so that prompt diagnostic testing and appropriate control measures can be implemented . Within alaska, populations that might represent foci for surveillance include persons, such as the patient s partner, who travel to geographic regions associated with the emergence or reemergence of orthopoxviruses, and persons with regular direct or indirect contact with wildlife (eg, residents of rural settings, scientists, environmental consultants, hunters, and adventure guides). The latter population in alaska might be relatively large, given the high proportion of state residents and visitors who live, work, or recreate in wilderness areas . Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.
Female sexual function is complex and affected by physical, psychological and social factors.1 the prevalence of female sexual dysfunction is high, ranging from 43% to 88%,2,3 and it may significantly affect self - esteem and quality of life . When chronic, it may lead to anxiety and depression, harm relationships, and cause problems in other aspects of life.1,4 moreover, the clinical effects of sexual dysfunction can be augmented by the intensity of the full range of climacteric symptomatology.5 diseases and factors such as aging, arterial hypertension, smoking and pelvic surgery have been associated with female sexual dysfunction.6 personality,7 lifestyle and culture - dependent variables should also be taken into consideration.8 although the epidemiology of male and female sexual function has been investigated in depth, the majority of studies continue to be confined to europe, the united states (us) and australasia.9 few studies have been carried out on the sexuality of climacteric women in latin american populations,1012 and data on this subject in women with high school or university educations is particularly sparse . The aim of this study was to collect information on the prevalence of sexual dysfunction and its associated factors in brazilian women of 40 to 65 years of age with eleven or more years of formal education . The target population was the female population of belo horizonte in the state of minas gerais, brazil, aged 4065 years, with at least 11 years of formal education, which consisted of 44,313 women in the year 2000 . The necessary minimum sample size for a similar study was calculated to be 377 women, based on the assumption that 43% of the female population experiences sexual dysfunction, with an expected difference of 5% between the sample and the general population and a type i error () of 0.05.13 in the present study, which was limited to women who answered all five questions used to calculate the sexual dysfunction score, the sample size was recalculated to evaluate any possible loss of precision . A sample of 315 women is expected to result in an absolute difference of 5.5% . This cross - sectional, population - based study was carried out using a self - response questionnaire that was anonymously completed by participants at home between may and september 2005 in the city of belo horizonte, minas gerais, brazil . For the purposes of this study, the municipality of belo horizonte was stratified into nine regions . From these regions, the weighted area consisted of a geographical area that was considered as the primary sampling unit (psu). Each wa consisted of various census sectors.14 in each selected wa, five census sectors were randomly chosen (secondary sampling unit). The variables of the sampling plan, strata and psu were included in the data analysis . The city of belo horizonte consists of 62 weighted areas (wa), containing a total of 2,563 census sectors.15 all sectors were included in the randomization process . Research assistants initiated the selection of women at each of the five randomly selected corners in each sector, guided by maps of the location . They went to each household and verified whether there were any brazilian - born women of 4065 years of age living in the home and whether they had at least 11 years of formal education . If there were eligible women living at that address, they were invited to participate in the study . If they agreed to participate, a questionnaire was left to be answered and a date was scheduled for the completed questionnaire to be collected . If the eligible women were not at home, they were contacted by telephone and, if they agreed to participate, the questionnaire was delivered to their home . Completed questionnaires were collected by a messenger, placed in an unlabeled envelope, and put into a sealed post - box to be delivered to the principal investigator . When questionnaires were delivered to the principal investigator, the weighted area and the education level (11 years of schooling) were noted to homogenize the response . A total of 420 questionnaires were distributed . The reasons given by the women for not participating were: lack of time, that they did not feel comfortable answering the questions, or that their husbands did not want them to participate in the study . Thus, 378 questionnaires were filled out and delivered to principal investigator; of these, 315 (83.3%) contained answers to all questions used to calculate the sexual dysfunction score . The delivered questionnaires that returned incomplete thus, 315 middle - aged women took part in the present study . The questionnaire used in the study consisted of two parts . In the first part, the participants answered questions regarding their sociodemographic characteristics (age, marital status, ethnic group, income, schooling and paid employment), clinical characteristics (previous surgical history, body mass index, depression, arterial hypertension, diabetes, urinary incontinence, history of cancer, hot flashes, nervousness, insomnia, hormone therapy and self - perception of state of health), reproductive characteristics (menopausal status, number of pregnancies) and behavioral characteristics (physical activity, smoking and presence of sexual partner). The study protocol was conducted in accordance with the helsinki declaration and approved by the internal review board of the institution . The instrument used to evaluate sexual dysfunction was based on the short personal experiences questionnaire (speq).16 the original version of this instrument was made available by investigators at the university of melbourne, australia . The questionnaire was independently translated from its original english into brazilian portuguese by two translators fluent in both languages . Next, the two versions were compared and a final version was obtained with the approval of both translators . This version was then tested in a group of 50 brazilian - born women of 4065 years of age with 11 years or more of schooling . To ensure cultural equivalence, all questions that generated doubt were once again adapted and tested until the questionnaire was completely comprehensible to all women in the pilot group . A final version of the questionnaire in brazilian portuguese was thus obtained . The variable sexual dysfunction was calculated from the mean of the sum of the scores of 1) sexual responsiveness, which evaluated pleasure in sexual activities (graded from 1 to 6, where 1 reflected an absence of pleasure and 6 maximum pleasure), excitation (16) and orgasm (16); 2) frequency of sexual activities (1=never, 2=less than once a week; 3=once or twice a week, 4=several times a week, and 5=once a day or more); and 3) libido (1=it never took place, 2=it took place less than once a week, 3=it took place once or twice a week, 4=it took place several times a week, 5= it took place once a day or more). A score 7 was considered indicative of sexual dysfunction and a score> 7 was considered indicative of no sexual dysfunction.16 age was dichotomized into <50 years or 50 years of age . Menopausal status was defined as premenopausal when the women had regular menstrual cycles or a menstrual cycle similar to the pattern that had been predominant throughout their reproductive life . Women were considered to be in perimenopause if they had menstrual cycles during the previous 12 months, but with some alterations in their menstrual pattern . Women were considered postmenopausal if their last menstrual period was at least 12 months prior to the interview.17 in women who been to hysterectomy, menopausal status was classified as follows: women aged 4044 years who had regular menstruation prior to hysterectomy were considered to be premenopausal; women aged 4548 years who had irregular menstruation prior to hysterectomy were considered to be in perimenopause; and women of more than 48 years of age or as well as those who had undergone hysterectomy with bilateral oophorectomy were considered to be postmenopausal, based on a previous report that the mean age at menopause in latin america is 48.6 years.18 marital status was dichotomized into married / living with a partner, or other; ethnic group into white or non - white; schooling into 11 years or more than 11 years of formal education; family income into us $1300 or> us $1300 per month; physical activity into none/ <3 times a week or 3 times a week; and number of pregnancies into> 2 or 2 . Body mass index (bmi) was dichotomized into <25 or 25 kg / m . Paid employment was dichotomized into none/ 20 hours per week or> 20 hours per week . Smoking was dichotomized into never smoked or current / past smoker . The presence or absence (yes or no) of the following variables was also investigated: depression, arterial hypertension, diabetes, urinary incontinence, history of cancer, hot flashes, nervousness, insomnia and the presence of a sexual partner in the previous month . Self - perception of the state of health was classified as terrible / poor / average or good / excellent . A bivariate analysis was performed to evaluate sexual dysfunction as a function of the independent variables . The chi - square test was applied followed by yates correction or fisher s exact test.19 finally, poisson multiple regression analysis was applied to the model to calculate the prevalence ratio (pr) and the respective 95% confidence intervals (95%ci).20 the backward criterion strategy was used to select the variables.21 for this analysis, the strata and the cluster / smallest geographical unit of the sampling plan were used . Stata software version 7.0 (stata corporation, college station, texas, usa) was used for the analysis . The criterion used for the inclusion of independent variables in multiple regression analysis consisted of a p - value of <0.25 in bivariate analysis or in a simple logistic regression . The target population was the female population of belo horizonte in the state of minas gerais, brazil, aged 4065 years, with at least 11 years of formal education, which consisted of 44,313 women in the year 2000 . The necessary minimum sample size for a similar study was calculated to be 377 women, based on the assumption that 43% of the female population experiences sexual dysfunction, with an expected difference of 5% between the sample and the general population and a type i error () of 0.05.13 in the present study, which was limited to women who answered all five questions used to calculate the sexual dysfunction score, the sample size was recalculated to evaluate any possible loss of precision . A sample of 315 women is expected to result in an absolute difference of 5.5% . This cross - sectional, population - based study was carried out using a self - response questionnaire that was anonymously completed by participants at home between may and september 2005 in the city of belo horizonte, minas gerais, brazil . For the purposes of this study, the municipality of belo horizonte was stratified into nine regions . From these regions, the weighted area consisted of a geographical area that was considered as the primary sampling unit (psu). Each wa consisted of various census sectors.14 in each selected wa, five census sectors were randomly chosen (secondary sampling unit). The variables of the sampling plan, strata and psu were included in the data analysis . The city of belo horizonte consists of 62 weighted areas (wa), containing a total of 2,563 census sectors.15 all sectors were included in the randomization process . Research assistants initiated the selection of women at each of the five randomly selected corners in each sector, guided by maps of the location . They went to each household and verified whether there were any brazilian - born women of 4065 years of age living in the home and whether they had at least 11 years of formal education . If there were eligible women living at that address, they were invited to participate in the study . If they agreed to participate, a questionnaire was left to be answered and a date was scheduled for the completed questionnaire to be collected . If the eligible women were not at home, they were contacted by telephone and, if they agreed to participate, the questionnaire was delivered to their home . Completed questionnaires were collected by a messenger, placed in an unlabeled envelope, and put into a sealed post - box to be delivered to the principal investigator . When questionnaires were delivered to the principal investigator, the weighted area and the education level (11 years of schooling) the reasons given by the women for not participating were: lack of time, that they did not feel comfortable answering the questions, or that their husbands did not want them to participate in the study . Thus, 378 questionnaires were filled out and delivered to principal investigator; of these, 315 (83.3%) contained answers to all questions used to calculate the sexual dysfunction score . The questionnaire used in the study consisted of two parts . In the first part, the participants answered questions regarding their sociodemographic characteristics (age, marital status, ethnic group, income, schooling and paid employment), clinical characteristics (previous surgical history, body mass index, depression, arterial hypertension, diabetes, urinary incontinence, history of cancer, hot flashes, nervousness, insomnia, hormone therapy and self - perception of state of health), reproductive characteristics (menopausal status, number of pregnancies) and behavioral characteristics (physical activity, smoking and presence of sexual partner). The study protocol was conducted in accordance with the helsinki declaration and approved by the internal review board of the institution . The instrument used to evaluate sexual dysfunction was based on the short personal experiences questionnaire (speq).16 the original version of this instrument was made available by investigators at the university of melbourne, australia . The questionnaire was independently translated from its original english into brazilian portuguese by two translators fluent in both languages . Next, the two versions were compared and a final version was obtained with the approval of both translators . This version was then tested in a group of 50 brazilian - born women of 4065 years of age with 11 years or more of schooling . To ensure cultural equivalence, all questions that generated doubt were once again adapted and tested until the questionnaire was completely comprehensible to all women in the pilot group . The variable sexual dysfunction was calculated from the mean of the sum of the scores of 1) sexual responsiveness, which evaluated pleasure in sexual activities (graded from 1 to 6, where 1 reflected an absence of pleasure and 6 maximum pleasure), excitation (16) and orgasm (16); 2) frequency of sexual activities (1=never, 2=less than once a week; 3=once or twice a week, 4=several times a week, and 5=once a day or more); and 3) libido (1=it never took place, 2=it took place less than once a week, 3=it took place once or twice a week, 4=it took place several times a week, 5= it took place once a day or more). A score 7 was considered indicative of sexual dysfunction and a score> 7 was considered indicative of no sexual dysfunction.16 menopausal status was defined as premenopausal when the women had regular menstrual cycles or a menstrual cycle similar to the pattern that had been predominant throughout their reproductive life . Women were considered to be in perimenopause if they had menstrual cycles during the previous 12 months, but with some alterations in their menstrual pattern . Women were considered postmenopausal if their last menstrual period was at least 12 months prior to the interview.17 in women who been to hysterectomy, menopausal status was classified as follows: women aged 4044 years who had regular menstruation prior to hysterectomy were considered to be premenopausal; women aged 4548 years who had irregular menstruation prior to hysterectomy were considered to be in perimenopause; and women of more than 48 years of age or as well as those who had undergone hysterectomy with bilateral oophorectomy were considered to be postmenopausal, based on a previous report that the mean age at menopause in latin america is 48.6 years.18 marital status was dichotomized into married / living with a partner, or other; ethnic group into white or non - white; schooling into 11 years or more than 11 years of formal education; family income into us $1300 or> us $1300 per month; physical activity into none/ <3 times a week or 3 times a week; and number of pregnancies into> 2 or 2 . Body mass index (bmi) was dichotomized into <25 or 25 kg / m . Paid employment was dichotomized into none/ 20 hours per week or> 20 hours per week . Smoking was dichotomized into never smoked or current / past smoker . The presence or absence (yes or no) of the following variables was also investigated: depression, arterial hypertension, diabetes, urinary incontinence, history of cancer, hot flashes, nervousness, insomnia and the presence of a sexual partner in the previous month . Self - perception of the state of health was classified as terrible / poor / average or good / excellent . A bivariate analysis was performed to evaluate sexual dysfunction as a function of the independent variables . The chi - square test was applied followed by yates correction or fisher s exact test.19 finally, poisson multiple regression analysis was applied to the model to calculate the prevalence ratio (pr) and the respective 95% confidence intervals (95%ci).20 the backward criterion strategy was used to select the variables.21 for this analysis, the strata and the cluster / smallest geographical unit of the sampling plan were used . Stata software version 7.0 (stata corporation, college station, texas, usa) was used for the analysis . The criterion used for the inclusion of independent variables in multiple regression analysis consisted of a p - value of <0.25 in bivariate analysis or in a simple logistic regression . In this sample, 46.7% of participants were 50 years of age or more; 65.9% were married or living with a partner; 70.8% reported having a sexual partner; 50.8% stated that they had more than 11 years of schooling; 59.5% had a family income us$1300; 51% had more than two children; and 33.5% reported practicing physical activity regularly, three or more times a week (data not presented as a table). The prevalence of sexual dysfunction among premenopausal women was 24.2%, perimenopausal women 37.3% and postmenopausal women 45.3% . The 315 women who answered the five questions comprising the sexual dysfunction variable were compared to the 63 who failed to answer these questions . Women who were unmarried or not living with a partner (p<0.001) and those who reported hypertension (p=0.002) or urinary incontinence (p=0.036) were more likely to answer all questions contained in the section on sexual dysfunction . In the bivariate analysis, women with greater probability of having sexual dysfunction (score 7) had the following characteristics: they were in peri- or postmenopause (p=0.007), had a sedentary lifestyle (p=0.019), reported depression (p=0.003), arterial hypertension (p<0.002), diabetes (p=0.046), hot flashes (p=0.009), nervousness (p=0.048) or insomnia (p=0.003), and had a poor perception of their own health (p<0.002), (table 1). Multiple regression analysis showed that more advanced age (pr = 1.04; 95%ci: 1.011.07) and the presence of hot flashes (pr = 1.37; 95%ci: 1.041.80) were significantly associated with sexual dysfunction . Conversely, women who felt well (pr = 0.68; 95%ci: 0.520.88) and women with sexual partners (pr = 0.47; 95%ci: 0.340.65) were less likely to have a score of 7 for sexual dysfunction (table 2). The objective of this study was to evaluate the prevalence of sexual dysfunction and to identify its associated factors in middle - aged, brazilian - born women . The profile of the population of the present study should be emphasized in view of the scarcity of population - based studies on female sexuality, particularly among middle - aged women . In the present study, overall sexual dysfunction was evaluated in women with or without a sexual partner . In a study carried out in a us population,22 kinsey (1948) showed that the search for sexual pleasure involves the mind and entire body of individuals, not only their genitals . Given women s greater longevity, it is very probable that many will grow old alone; however this does not imply the end of their sexuality or loss of their need for intimacy, touch and affection.23 the use of an internationally recognized questionnaire, the speq,16 which was adapted for use in brazil, is another factor that deserves particular mention . As direct interviews on sexual activity during menopause may lead to constraints in responses,24 it was important for the questionnaire to be self - response and anonymous . Various measures were taken to ensure that the women selected would feel at ease to respond honestly to the questions . For instance, research assistants were selected who had a similar profile to that of the women in the study sample in order to encourage empathy, and reassurance was given to participants that all answers would remain confidential . In the present study, the prevalence of sexual dysfunction was 35.9%, increasing from the pre- to the postmenopause . These data are in agreement with findings from other studies carried out in different populations . In a prospective observational study, dennerstein et al 3, 25 also found that from the beginning to the latter phase of the menopausal transition, the proportion of women with sexual dysfunction increased from 42% to 88% . In a study carried out in chile, castelo - branco et abdo et al.27 found at least one type of sexual dysfunction in 57.4% of women 41 years of age or older . Nevertheless, in our sample, multiple analysis showed no association between menopausal status and sexual dysfunction . One possible explanation for this finding may be that in women with a higher educational level, the percentage affected by sexual dysfunction is lower.27 sexual dysfunction was not significantly associated with age, which was dichotomized into <50 years and> 50 years for the bivariate analysis; however, in the multiple regression analysis, when the variable was considered in a continuous manner, the correlation between sexual dysfunction and increasing age was significant . In a previous study13 carried out in this same sample population, albeit with a more general evaluation of sexuality, a decline in sexuality as a function of increased chronological age was also found . A longitudinal cohort study carried out in australia reported a significant negative effect of aging on the frequency of sexual activity, as well as on aspects of responsiveness (sexual excitation, pleasure and orgasm).3,25 the women s international study of health and sexuality, a multinational, cross - sectional study carried out in europe and in the united states, reported a decline in all aspects of sexual function with aging.28 in a longitudinal study, ford et al 29 also reported that an increase in age was associated with poorer sexuality . Castelo - branco et al26 evaluated 534 healthy, middle - aged chilean women and found that the prevalence of sexual dysfunction increased from 22% in the 4044-year old age - group to 66% in the 6064 year olds . Hot flushes, depression, nervousness and insomnia were associated with scores 7 for sexual dysfunction in the present analysis . These results are in agreement with those of previous studies in which depression was associated with difficulties in vaginal lubrication in women in some regions of the world, whereas stress was associated with inability to achieve orgasm.30 other studies have also reported that psychological symptoms, stress and emotional problems may be related to a decline in sexual activity.31 it was also found that psychological symptoms were common in climacteric women and were associated with hot flashes . This observation connects psychological symptoms with the menopausal transition and suggests that their cause may be biological.32 hot flashes are known to constitute a triggering factor for insomnia, which, in turn, leads to a reduction in energy and to depression, which has a negative effect on sexual function.33 the women in the present sample who reported leading a sedentary lifestyle or who had a poor perception of their own health were more likely to be affected by sexual dysfunction . Greendale et al34 reported that an increase in physical activity and satisfaction with life were related to improved sexual function . Mental health,30,35 emotional well - being and positive self - image were associated with improved sexual function.30,35,36 on the other hand, additional studies have found correlations between chronic diseases and an increase in sexual dysfunction28,37 due to circulatory problems,38 neurological function, hormone balance or systemic health.39 it has been found that both an increase in systolic arterial pressure and the administration of beta blockers to treat hypertension may be detrimental to sexual function.40 the present analysis shows that arterial hypertension was associated with the occurrence of sexual dysfunction, although the use of medication for hypertension was not evaluated in this study . This finding is in agreement with other studies that reported that women with diabetes had a higher prevalence of sexual dysfunction compared to non - diabetic women.41,42 in the present study, women who had a sexual partner were less likely to have sexual dysfunction . This is consistent with other studies on middle - aged women conducted in different geographical locations, emphasizing the relevance of the sexual partner as a protective factor against sexual dysfunction.13,36,43 nevertheless, previous studies have also described the importance of feelings for the partner with respect to sexuality.44 these data suggest the need to carry out further studies to investigate, in addition to the simple presence of a partner, the quality of relationship of these couples . The questionnaires that were returned incomplete were not taken into consideration in the evaluation of sexual dysfunction . The group composed of unmarried women or those not living with a partner and who reported hypertension or urinary incontinence, was associated with answering the entire block of questions concerning sexual dysfunction . Women with these characteristics who may have sexual dysfunction may be more motivated to answer all the questions . The data, which include information on hypertension, psychological symptoms and the use of hormone therapy, were obtained by participant self - report . Although recall bias cannot be ruled out, previous studies using sef - reports suggest a high validity of health information, indicating that women with higher education levels provide more reliable data.45 sexual dysfunction was found in more than one - third of women that were 4065 years of age with 11 years or more of formal education . Informing women, giving them the tools necessary to change their own lifestyle, and treating climacteric symptoms and existing comorbidities may lead to improved sexuality and, consequently, to a better quality of life . Future studies are required to evaluate the effects of hormones and other drugs on the sexuality of climacteric women.
Assessing susceptibility to diseases based on an individual's genotype has long been a central theme of genetics studies . Among inherited gene variations in humans, nonsynonymous single nucleotide polymorphisms (nssnps) that lead to an amino acid change in the protein product are most relevant to human inherited diseases (1). Nssnps can be classified into two categories according to their phenotypic effects: those that cause deleterious effects on protein functions and are hence disease - associated and those that are functionally neutral . Given the huge number of nssnps already discovered (2,3), a major challenge is to predict which of them are potentially disease associated . Computational tools have been developed to predict the nssnp's phenotypic effect, e.g. The sift server (4) and the polyphen server (5). Recently, studies have shown that combining information obtained from multiple sequence alignment and three - dimensional protein structure can increase the prediction accuracy (6). Nssnpanalyzer server integrates multiple sequences alignment and protein structure analysis to identify disease - associated nssnps . Nssnpanalyzer takes a protein sequence and the accompanying nssnp as inputs and reports whether the nssnp is likely to be disease - associated or functionally neutral . Nssnpanalyzer also provides additional useful information about the nssnp to facilitate the biological interpretation of results, e.g. Structural environment class and multiple sequence alignment . Briefly, on receiving the input sequence, nssnpanalyzer searches the astral database (7) for homologous protein structures . Nssnpanalyzer calculates three types of information from user's input: (i) the structural environment of the snp, including the solvent accessibility, environmental polarity and secondary structure (8); (ii) the normalized probability of the substitution in the multiple sequence alignment (9); and (iii) the similarity and dissimilarity between the original amino acid and mutated amino acid . Nssnpanalyzer then uses a machine learning method called random forest (10) to classify the nssnps . The random forest classifier was trained to optimally combine the heterogeneous sources of predictors using a curated training dataset prepared from the swissprot database (11). Several recent studies have demonstrated the better performance of random forest over other machine learning approaches (1214). For the nssnp phenotypic effect prediction, we also found that random forest gave the best results on this training dataset . In a cross - validation test, the false positive rate is 38% and the false negative rate is 21% (15). The nssnpanalyzer web server is implemented on a linux redhat 8.0 platform with the common gateway interface scripts written in php . Two inputs are mandatory: protein sequence in fasta format and the nssnp identities to be analyzed . An nssnp is denoted as x#y, where x is the original amino acid in one letter, #is the position of the substitution (starting from 1), and y is the mutated amino acid in one letter . In addition to the two mandatory inputs, users may also upload an accompanying protein structure file in pdb format if they want their own structure to be used . Finally, because the calculation usually takes a while users can use the sample data to learn the input format and perform a demo run . The results of nssnpanalyzer are displayed on a web page and stored on the server for a week . A link to the results page can also be sent to the user via email . The output includes several calculated features of the nssnp: (i) predicted phenotypic class (disease - associated versus neutral); (ii) a hyperlink to the homologous structure with a scop identifier (7); (iii) the normalized probability of the substitution calculated by the sift program (4); (iv) area buried score, a measure of the solvent accessibility; (v) fraction polar score, a measure of environmental polarity related to hydrogen bond formation; (vi) secondary structure (helix, sheet and coil); and (vii) the structural environment class, a discrete environment class definition by combining features (iv)(vi) (8). The area buried score and fraction polar score are calculated by the environment program (8), and the secondary structure is calculated by the stride program (16). The user can click the view alignment button to see the local sequence alignment spanning the substitution sites and get a direct sight on the mutability of the substitution . The original amino acid is highlighted in blue, and the mutated amino acid is highlighted in red . Briefly, on receiving the input sequence, nssnpanalyzer searches the astral database (7) for homologous protein structures . Nssnpanalyzer calculates three types of information from user's input: (i) the structural environment of the snp, including the solvent accessibility, environmental polarity and secondary structure (8); (ii) the normalized probability of the substitution in the multiple sequence alignment (9); and (iii) the similarity and dissimilarity between the original amino acid and mutated amino acid . Nssnpanalyzer then uses a machine learning method called random forest (10) to classify the nssnps . The random forest classifier was trained to optimally combine the heterogeneous sources of predictors using a curated training dataset prepared from the swissprot database (11). Several recent studies have demonstrated the better performance of random forest over other machine learning approaches (1214). For the nssnp phenotypic effect prediction, we also found that random forest gave the best results on this training dataset . In a cross - validation test, the false positive rate is 38% and the false negative rate is 21% (15). The nssnpanalyzer web server is implemented on a linux redhat 8.0 platform with the common gateway interface scripts written in php . Two inputs are mandatory: protein sequence in fasta format and the nssnp identities to be analyzed . An nssnp is denoted as x#y, where x is the original amino acid in one letter, #is the position of the substitution (starting from 1), and y is the mutated amino acid in one letter . In addition to the two mandatory inputs, users may also upload an accompanying protein structure file in pdb format if they want their own structure to be used . Finally, because the calculation usually takes a while users can use the sample data to learn the input format and perform a demo run . The results of nssnpanalyzer are displayed on a web page and stored on the server for a week . A link to the results page the output includes several calculated features of the nssnp: (i) predicted phenotypic class (disease - associated versus neutral); (ii) a hyperlink to the homologous structure with a scop identifier (7); (iii) the normalized probability of the substitution calculated by the sift program (4); (iv) area buried score, a measure of the solvent accessibility; (v) fraction polar score, a measure of environmental polarity related to hydrogen bond formation; (vi) secondary structure (helix, sheet and coil); and (vii) the structural environment class, a discrete environment class definition by combining features (iv)(vi) (8). The area buried score and fraction polar score are calculated by the environment program (8), and the secondary structure is calculated by the stride program (16). The user can click the view alignment button to see the local sequence alignment spanning the substitution sites and get a direct sight on the mutability of the substitution . The original amino acid is highlighted in blue, and the mutated amino acid is highlighted in red . Considering the remarkable cpu cost of calculation, we are planning to provide precalculated results for all human nssnps in the dbsnp (17) with homologous structures available . We will also test the applicability of extracting structural predictors from predicted structures to eliminate the requirement of having experimentally determined structures available . (a) the main output page of nssnpanalyzer . The user can click the icon to see the interpretation of each field . (b) an example of local sequence alignment spanning the nssnp (d7n). The original amino acid (d) is highlighted in blue, and the mutated amino acid (n) is highlighted in red.
Renal artery stenosis is a cause of secondary hypertension which can be cured by surgical or radiological intervention such as percutaneous transluminal renal artery stent placement1). Hyperperfusion syndrome is a rare complication following revascularization of the internal carotid artery2). In case of renal artery stenosis subcapsular hematoma of the kidney, an accumulation of blood between the renal parenchymal surface and the surrounding fibrous capsule, occurs most often secondary to post - traumatic bleeding . Although the incidence of subcapsular hematoma following percutaneous transluminal stent placement has been considered rare, we experienced a case whose radiological signs and clinical symptoms were consistent with subcapsular hematoma of the kidney following the procedure . This report is presented to propose the possibility of such a complication associated with the procedure, especially under the state of chronic hypoxia . A 59-year - old man was admitted for a follow - up evaluation and treatment of his renal artery stenosis, which was diagnosed one year prior to his admission . At that time, narrowing of the proximal portion of the right renal artery was discovered by abdominal magnetic resonance imaging . His hypertension was classified as the seventh report of the joint national committee on the prevention, detection, evaluation, and treatment of high blood pressure (jnc 7) stage 2 . He had been medicated with the following antihypertensive drugs: nifedipine, 90 mg; carvediol, 25 mg; eprosartan, 600 mg; doxazocin, 8 mg; hydralazine, 25 mg; metolazone, 5 mg; and aspirin, 100 mg . On admission an abdominal bruit was heard at his epigastric area and the rest of the findings were unremarkable . The vital signs on admission were as follows: blood pressure, 150/90 mmhg with medication; temperature, 36.5; pulse, 55 bpm; and respiratory rate, 23/min . The laboratory findings on admission are unremarkable except for a 2 positive proteinuria on his urinalysis . Arteriography of abdominal aorta showed more than 90% narrowing of his right renal artery at the ostium (fig . Pressure gradient of the aorta and both renal arteries were as follows: aorta, 179/80 mmhg; left renal artery, 153/76 mmhg; and right renal artery, 35/23 mmhg . An 8 fr guiding catheter was placed at the distal portion of the right renal artery, and a 5 mm14 mm corinthian stent (cordis endovascular / johnson & johnson, miami, fl, usa) was placed at the stenotic area of the right renal artery (fig . Pressure gradient of the aorta and right renal artery were reexamined; they were 205/95 mmhg and 192/90 mmhg, respectively . Approximately 2 hours after the procedure, the patient complained of severe pain in his right flank with diaphoresis . An emergency contrast enhanced computed tomography (ct) revealed a large right perirenal subcapsular hematoma and compression of that kidney (fig . Three hours after the stenting procedure, his hemoglobin level dropped to 7.5 g / dl . Three units of blood were transfused and an angiotensin receptor blocker (eprosartan 600 mg) for hypertension was prescribed . Ten days later, a follow - up contrast enhanced ct examination revealed that the compression of renal parenchyma was much improved . His blood pressure fell to 120/75 mmhg without medication . Following the treatment, the patient's general condition became stable and he was discharged without antihypertensive medication . Aging and coexisting atherosclerosis are also known to have significant effects on the prevalence of renovascular hypertension in this population3). Percutaneous transluminal renal artery stent placement, when performed by experienced hands, has been regarded as a safe and effective procedure4). The technical success rate ranges from 96% to 100%, with complications occurring in 13% of the cases5). Mortality related to this procedure is extremely rare, ranging 0 - 0.5% immediately following the procedure and 1% in the 30-day follow up period1, 3, 5). Axelrod et al.6) have reported a case of guide wire perforation that led to a fatal perirenal hemorrhage from the transcortical collaterals after renal artery stent placement . Since there was no evidence of injury provoked by the guide wire in this case, the cause for the subcapsular hematoma was postulated to be secondary to the reperfusion injury, such as seen in increased intrarenal pressure . The pressure gradient of right renal artery before and after stenting were 35/23 mmhg and 192/90 mmhg, respectively . The intrarenal pressure was markedly increased after renal artery stent placement . In case of cerebral hyperperfusion syndrome, the risk factors are considered as long standing high grade carotid artery stenosis, loss of cerebral reserve capacity, and marked increased post - intervention flow velocities in the ipsilateral cerebral artery2). In our case, the patient had long - standing severe renal artery stenosis and high pre- and post - procedure pressure gradient ., this case suggests the possibility of reperfusion injury to the kidney after percutaneous transluminal renal artery stent placement of severe long - standing renal artery stenosis.
The patient was a 46-year - old saudi woman who had chronic hepatitis c, end - stage renal disease of undetermined etiology, and a renal transplant in 1993 . Seizures were usually preceded by epigastric pain and a sensation of nausea for few seconds, followed by left arm posturing and loss of consciousness . She lived in a rural area, was involved in animal husbandry, and consumed unpasteurized milk products . Her medications included mycophenolate mofetil (500 mg 2/d since 1993), prednisone (5 mg 1/d since 1993), levetiracetam (500 mg 2/d for 5 mo), and phenytoin (200 mg every night for 1 mo). Neurologic examination showed left homonymous hemianopia, increased deep tendon reflexes in the left hemibody, and the babinski sign on the left hallux . Initial laboratory test results, including those for complete blood count, erythrocyte sedimentation rate, c - reactive protein, and liver and renal profiles, were within references ranges . Results of serologic analysis for hiv and hepatitis b virus were negative . A standard agglutination tube (sat) test result for brucella spp . Was positive (titer 1:320), and a 2-mercaptoethanol test result for brucella spp . Cerebrospinal fluid (csf) had a leukocyte count of 21 (90% lymphocytes). Levels of protein, glucose, and lactate dehydrogenase in csf were within references ranges . Gram staining of a csf sample and cultures for bacteria, virus, fungi, and acid - fast bacilli (afb) showed negative results . Serologic analysis of csf showed brucella igg (titer <1:20) and antibodies against brucella (titer 1:320). Test results were negative for antibodies against aspergillus, aspergillus galactomannan, blastomyces, borrelia, coccidia, cryptococcus, histoplasma, and toxoplasma . An electroencephalogram showed sharp waves over the right temporal region and continuous slow activity over the right temporooccipital region . Magnetic resonance imaging (mri) of the brain showed diffuse t2/fluid - attenuated inversion recovery hyperintense white matter lesions involving the right frontal, parietal and temporal lobes (figure 1). No appreciable mass effect or enhancement after administration of gadolinium was observed . Positive emission tomography of the brain showed hypometabolic cerebral activity involving a large area of right cerebral hemisphere . Magnetic resonance spectroscopy shows a low peak of n - acetyl aspartate (2.2 ppm). Magnetic resonance imaging of the brain of a 46-year - old immunocompromised woman with central nervous system brucellosis granuloma and white matter disease, saudi arabia . A) axial t2 images showing hyperintensity in the right frontoparietal lobe and right temporal lobe . B) axial fluid - attenuated inversion recovery (flair) and c) coronal flair images showing that hypersensitivity extends to u - fibers without involvement of the cortex . D) gadolinium - enhanced image showing that no appreciable mass effect and no central or peripheral enhancement after administration of gadolinium were observed . Each image within each panel shows involvement in different levels of frontal, parietal, and temporal lobes . A brain biopsy specimen of cerebral cortex and superficial white matter showed a moderate lymphoplasmacytic and focally histiocytic infiltrate that involved deep cortex, white matter, and leptomeninges . The inflammatory reaction, including granulomas, was mainly perivascular with some angiocentric patterns and focal parenchymal involvement . There were no morphologic signs of a specific etiology: no viral inclusions, and staining results microorganisms (afb, fungi, epstein - barr virus, and jc polyomavirus) were negative (figure 2). Histologic analysis of a brain biopsy specimen from a 46-year - old immunocompromised woman with central nervous system brucellosis granuloma and white matter disease, saudi arabia . A) low magnification view of cerebral cortex showing infiltration by perivascular lymphocytes and histiocytes . B) high magnification view showing an angiocentric epithelioid granuloma cuffed by mature lymphocytes (original magnification 200). Was sensitive to gentamicin, streptomycin, tetracycline, trimethoprim / sulfamethoxazole, and rifampin . The patient received intravenous ceftriaxone (2 g every 12 h), oral doxycycline (100 mg every 12 h), oral rifampin (600 mg 1/d), and trimethoprim / sulfamethoxazole (1 tablet [160 mg/800 mg] every 12 h) for 2 wk . After discharge, she was receiving oral doxycycline (100 mg every 12 h), rifampin (600 mg, 1/d), trimethoprim / sulfamethoxazole (1 tablet every 12 h), and ciprofloxacin (500 mg every 12 h) for 6 mo: she was also receiving levitiracetam (750 mg 2/d), carbamazepine (200 mg 2/d), mycophenolate mofetil (500 mg 2/d), and prednisone (5 mg 1/d). Three months later, repeat mri of the brain showed decreased t2 hyperintensity associated with volume loss and ex vacuo dilatation of the subjacent right lateral ventricle . Neurobrucellosis can affect the central or peripheral nervous systems and lead to diverse clinical syndromes (4). Diagnosis of neurobrucellosis depends on clinical manifestations, csf findings suggestive of pinocytosis, high protein levels, low or standard glucose levels, and a positive antibody titer for brucella spp . Although the patient had mild pleocytosis with a predominance of lymphocytes and high antibody titers against brucella spp . In csf, however, low levels of antibodies might not be detected by sat . In suspicious cases in which the sat result is negative, sat and a coombs test, elisa, and pcr are helpful in making a diagnosis . 77the clinicalradiologic correlation for neurobrucellosis ranges from uneventful results for imaging studies, despite positive clinical findings, to imaging abnormalities (3). Neurobrucellosis with a focal brain mass has been rarely observed in imaging studies (7,8). Radiologic results in this case suggested an infectious disease, autoimmune disease, or malignancy in an immunocompromised patient . Because we deemed it necessary to exclude other conditions, such as progressive multifocal leukoencephalopathy or lymphoma the diagnosis was established by detecting antibodies against a brucella sp . In serum and csf and confirmed by isolation of a brucella sp . From brain tissue . We found that the patient had epilepsy and extensive white matter changes secondary to brucellosis . Mri of the brain showed abnormal results (prominent white matter disease and focal encephalomalacia). Inflammation can cause permanent cellular biochemical dysfunction, which can lead to electrically irritable tissue and parenchymal damage despite successful treatment . Longitudinal studies of white matter hyperintensities caused by vascular, noninfectious, infectious, and inflammatory conditions showed white matter hyperintensities over time despite effective treatment . Reported that white matter changes in neurobrucellosis were sequelae of demyelination, as confirmed by the pathologic analysis (9). We believe that unresolved white matter hyperintensities in this patient were a sequela of the inflammatory process . A case report documented similar clinical features in a patient with seizures caused by chronic neurobrucellosis for 2.5 years (10). Solitary cysticercus granuloma and calcified lesion are 2 common neuroimaging abnormalities in patients with epilepsy . Treatment for underlying cysticercosis does not cure epilepsy (12). Seizures associated with central nervous system tuberculomas the underlying pathogenesis for relapsing epilepsy in neurocysticercosis is probably related to abnormal neurons and their arrangement within calcified nodules (13). The epilepsy prognosis for neurobrucellosis is probably similar to that for central nervous system neurocysticercosis (13). Our findings indicate the need for suspecting neurobrucellosis as a cause of epilepsy and white matter disease in immunocompromised patients in disease - endemic areas.
A functional fifth metacarpus and functional ulnar fingers are important for the locking grip and supporting grip . The reverse digital artery island flap is a safe and reliable procedure with a high survival rate . The hypothenar area is used as a vascularized flap donor site very infrequently in the clinical practice . The aim of this study was to present our experience using the reverse hypothenar island flap for finger reconstruction and to evaluate clinical benefits of this safe and easy surgical technique . Hypothenar skin is vascularized by different types of perforators that come out from the ulnar palmar digital artery of the little finger that emerges from the superficial palmar arterial arch . In the most distal region of this area, fasciocutaneous perforators are the dominant vascular supply, but in the proximal segment, musculocutaneous perforators are more dominant . A constant sizeable perforator was identified within 0.7 cm from the proximal margin of the a1 pulley . Neural supply of this area is from the dorsal or palmar cutaneous branches of the ulnar nerve . The average distances from the superficial palmar arch and deep palmar arch to the carpometacarpal joint of the ring finger were 32.2 6.33 mm and 18.3 4.64 mm, respectively . Ulnar digital artery of the little finger is a direct branch of the superficial arch . A finger has three transverse palmar arches and the proximal transverse palmar arch is located at the level of the neck of the proximal phalanx . Flap dissection is performed in the subfascial plane of medial side of the abductor digiti minimi muscle . Multiple perforating branches running transversely and arising from the ulnar palmar digital artery are identified . After ligating the ulnar palmar digital artery and concomitant veins at the proximal side of the flap, they are retrogradely dissected at the distal point of rotation which supplies reverse flow of the flap . The distal point of rotation is the neck of proximal phalanx, which is related to the proximal transverse palmar arch between the ulnar and radial palmar arteries . A perivascular cuff of tissue is preserved that aids in venous drainage [figure 1]. The average distance from the superficial pal mar arch to the carpometacarpal joint of the ring finger was 32.2 mm 6.33 mm . A constant sizeable perforator was identified within 0.7 cm from the proximal margin of the a1 pulley . In the most distal region of this area, we consider that is unsafe to pass the pedicle under a tunnel, so we recommend a brunner incision from the donor site to the defect [figure 2]. The donor site is closed primarily . Hypothenar skin is vascularized by different types of perforators that come out from the ulnar palmar digital artery of the little finger that emerges from the superficial palmar arterial arch . In the most distal region of this area, fasciocutaneous perforators are the dominant vascular supply, but in the proximal segment, musculocutaneous perforators are more dominant . A constant sizeable perforator was identified within 0.7 cm from the proximal margin of the a1 pulley . Neural supply of this area is from the dorsal or palmar cutaneous branches of the ulnar nerve . The average distances from the superficial palmar arch and deep palmar arch to the carpometacarpal joint of the ring finger were 32.2 6.33 mm and 18.3 4.64 mm, respectively . Ulnar digital artery of the little finger is a direct branch of the superficial arch . A finger has three transverse palmar arches and the proximal transverse palmar arch is located at the level of the neck of the proximal phalanx . Flap dissection is performed in the subfascial plane of medial side of the abductor digiti minimi muscle . Multiple perforating branches running transversely and arising from the ulnar palmar digital artery are identified . After ligating the ulnar palmar digital artery and concomitant veins at the proximal side of the flap, they are retrogradely dissected at the distal point of rotation which supplies reverse flow of the flap . The distal point of rotation is the neck of proximal phalanx, which is related to the proximal transverse palmar arch between the ulnar and radial palmar arteries . A perivascular cuff of tissue is preserved that aids in venous drainage [figure 1]. The average distance from the superficial pal mar arch to the carpometacarpal joint of the ring finger was 32.2 mm 6.33 mm . A constant sizeable perforator was identified within 0.7 cm from the proximal margin of the a1 pulley . In the most distal region of this area, we consider that is unsafe to pass the pedicle under a tunnel, so we recommend a brunner incision from the donor site to the defect [figure 2]. The donor site is closed primarily . Since 2013, nine patients with soft - tissue defects and amputation injuries of the little fingerwere treated using a vascularized flap from the hypothenar area [table 1]. Because the flap gains its vascular supply from the opposite digital artery by retrograde perfusion, preoperative assessment of the integrity of the digital arteries there were one female and eight male patients and their ages at the time of surgery averaged 28 years . The size of the soft - tissue defects ranged from 12 mm 12 mm to 20 mm 13 mm . The soft - tissue defects were at the level of distal inter - phalangeal joint crease or distal to it . The size of the flaps ranged from 15 mm 10 mm to 20 mm 15 mm ., we asked all the patients two questions (question 1 and 2) after 6 months postoperatively [table 2]. We assessed moving two - point discrimination (2 pd) and cold intolerance of all the patients . The follow - up periods ranged from 10 months to 25 months, with an average of 14 months . All patients expressed satisfaction with the flap procedure and the aesthetic results [figure 3]. (a) preoperative view, (b) immediate postoperative view and (c) postoperative view, after 3 months five of the patients answered to question 2 saying that the result was excellent . Two patients answered to question 2 saying that the result was good and the other two patients answered to question 2 saying that the result was fair . Four patients who did not answer to question 2 said that the result was excellent; however, they showed the longitudinal scar at the donor site and in one of them secondary multiple z - plasties was required at the follow - up after 6 months because of limited active extension of proximal inter - phalangeal (pip) joint of little finger which has 20 loss of active extension . Eight of nine patients had normal active extension of pip joint at the sixth month, postoperatively . We used the modified american society for surgery of the hand guidelines to stratify the 2 pd measurements (excellent, 6 mm; good, 6 - 10 mm; fair, 11 - 15 mm; poor, 15 mm). We assessed the cold intolerance of all the patients using the self - administered cold intolerance severity score questionnaire that was rated into mild, moderate, severe and extreme (0 - 25, 26 - 50, 51 - 75 and 76 - 100) [table 3]. Digital soft - tissue reconstruction has been developed using the knowledge of the small digital arteries and the different angiosomes located on specific zones of the hand . The reverse hypothenar flap anatomy has been described earlier, including the number of perforants found and the maximal size of the skin island permitted . The allen test is necessary because the ulnar palmar digital vessel is divided at the proximal end and vascularity of the finger entirely depends on the integrity of radial digital vessel . Pan - deng hao et al . Recommended that using cutaneous perforator flaps is a better option than sacrificing a digital artery . But ulnar palmar perforator flap cannot reach the distal level of the little finger . The procedure is easy when flap dissection is performed in the subfascial plane over the abductor digiti minimi muscle . Recommended that subfascial dissection of the flap should be performed from the dorsal side of the hand . On the contrary, in our study wolff et al . Stated that venous congestion is a disadvantage of hypothenar island flap . Adipofascial pedicle of the flap seemed bulky at first gaze but patients are satisfied with the result after a few months . All the patients are satisfied because they do not have a short finger . Like postburn sequel of the fifth finger, nonfunctioning fifth finger this flap provides all reconstructive goals including stable coverage, with colour, texture, sensibility and volume similar to the normal local tissue . Although normal sensation is not completely restored, we believe that this flap provides enough sensation for the little finger without neurotization . However, in this study, the double sensibility phenomenon which is a constant problem of littler's flap was not seen . The only absolute contraindication for using this flap is severe injury to the little finger with vascular compromise of the digits . Other relative contraindications are elderly patients or those suffering from vascular diseases or long - standing diabetes . The success of flap survival of our study can be related to the age group of the patients . Potential disadvantages of the hypothenar flap are contracture and limited extension of little finger because of continuous incisions of palmar side of the hand . We recommend brunner incisions or littler diamond incisions which we used in seven of them . S incision only in two of the nine patients at the proximal metacarpophalangeal joint crease . One of them was operated for multiple z - plasties because of limited active extension of the proximal interphalangeal (pip) joint . Loss of active extension of the pip joint was measured 20 at the sixth month of the hypothenar island flap operation although scar massage and range of motion exercises were advised . After multiple z - plasties, the pip joint gained 15 active extension . Both patient compliance with rehabilitation and choice of appropriate incision technique can overcome the potential disadvantage of this flap which may require secondary procedure of multiple z - plasties.
Diabetes mellitus comprises a group of common metabolic disorders that share the phenotype of hyperglycemia . Several distinct types of diabetes mellitus exist and are caused by a complex interaction of genetic, environmental factors and life style choices . Type-2 diabetes is due to predominantly insulin resistance with relative insulin deficiency niddm, which is much more common than iddm was discovered by chance . It is typically gradual in onset and occurs mainly in the middle aged and elderly . The metabolic dysregulation associated with dm causes secondary patho - physiological changes in multiple organ systems . In general the risks of chronic complication increases as a function of the duration of hyperglycemia, they usually become apparent in the second decade of hyperglycemia . Since type-2 dm often has a long asymptomatic period of hyperglycemia, so far most of the clinical and diagnos - tic studies on diabetic neuropathy have concerned only peripheral and autonomic nerve but recently with the refinement of evoked potential techniques detailed explora - tion of sensory pathway in central nervous system has been possible . Also pathological studies by reske - nielson and ludback (1965) and makishima and tanaka (1971) have shown involvement of brain parenchyma in patients of long standing dm . The brainstem auditory electric responses represent a useful, non invasive and simple procedure to detect both acoustic nerve and cns damage . Abr is a far field recording of the synchronized response of a large number of neurons in the lower portion of auditory pathway . It was first described by shemer and femmesser in 1967.a full description of aep is given by hyde (1987). At present this evoked potential has become a routine part of the standard audio logical test battery . It is recorded by placing active electrodes positioned at vertex and reference electrode at the mastoid or earlobe . Wave i and ii represent activity in cochlear nerve, wave iii in cochlear nucleus, wave iv in superior olivary complex, wave v which is biggest and most consistent represent activity in nuclei of lateral lemniscus while wave vi and vii in the inferior colhculus . In this way the amplitude of peaks are variable with in subjects while the latencies of peaks are stable and are well documented . In detecting evidence of central neuropathy in dm, coshum durmus et al (2004), toth et al(2001), virtamemi j et al(1993) found evidence of central auditory pathway involvements . A very distinct advantage of using abr as a diagnostic modality that it is resistant to the effect of sleep, sedation and anesthesia . It is suitable for children and for adult who are not able to co - operate . The present study was carried at finding out central auditory pathway involvement in diabetes mellitus by bera . The study was carried out in the department of ent, jnmc from 2008 -2010 . The study included two groups, (i) diabetic group (n=25) (ii) control group (n=25). Exclusion criteria: patient who gave history of ear disease, exposure to prolonged loud noise, intake of ototoxic drug, stroke, head injury or family history of deafness were not included . Patient taking any medication which might be expected to interfere with the functioning of central nervous system . (methyldopa, reserpine, phenytoin, antipsychotic, antidepressants) were excluded from the study . In the control group 17 (68%) were males where as 8 (32%) were females . Mean age of control group was 45.7 years . In the study group 13 (52%) camparison of absolute latencies and interpeak latencies in controls and in patients with diabetes mellitus at 70, 80 and 90 db significant difference was found in latencies of wave iii and interpeak iii - v while highly significant difference was found in latencies of wave v and interpeak i - iii, i - v between control and study groupat70db . Highly significant difference was found in latencies of wave iii, v and interpeak i - iii and i - v while significant difference was found in interwave iii - v between control and study group at 80 db . Significant difference was found in latencies of wave v and interpeak iii - v while highly significant difference was found in wave iii and interpeak i - iii, i - v between control and studygroupat90db . The duration of dm was 5 - 10 years in 13 (52%) patients and among these patients bera was delayed in 7 (53.84%) subjects . 12 (48%) patients had history of diabetes for> 10 years and among these patients, bera was delayed in (91.66%) subjects . In the study group 13/25 cases had neuropathy, 1/25 cases had nephropathy and 2/25 cases had retinopathy in the study group among 25 patients, 13 (52%) patients had peripheral neuropathy and the bera was delayed in 12(92.3%) patients . While the incidence of delayed bera was 50% in patients without peripheral neuropathy . Both the study group and control group were tested 2 - 3 times, so as to confirm the reproducibility of the results . The recordings were tabulated and statistical analysis of the data was done by applying unpaired student's t - test to the data . A p - value of less than 0.05 and less than <0.001 was considered as significant and highly significant respectively . The maximum number of cases i.e., 11 (44%) were from 41 - 50years age group . The mean age of study group was 46.8years and mean age of controls was 45.5years . The latency of wave i was found to be equal in the diabetics and controls . This suggests that the eight nerve transmission till the level of cochlear nucleus was not altered in the diabetics . The latency of wave iii was delayed by 0.39 msec, 0.42 msec and 0.42 msec at 70, 80 and 90 db respec - tively in diabetic group as compared to control group . The latency of wave v was delayed by 0.48 msec, 0.47 msec and 0.50 msec at 70, 80 and 90 db respectively in diabetic group as compared to control group . The interpeak latency i - iii, iii - v and i - v was delayed in the diabetic group . This suggests delayed transmission of the auditory stimulus in the auditory pathway of diabetics at the level of brainstem and midbrain . The delay in the latency of wave iii and v in the diabetics indicate neuropathy at brainstem and midbrain level in the auditory pathway . Fidele et al in 1984 found the latencies (ms) of abr waves were significantly impaired in diabetic subjects as compared with normal . Peripheral transmission time (wave i) and central transmission time (wave i - v) kunen et al in 1989 evaluated hearing threshold of 30 diabetic patients and 30 healthy controls by using puretone audiometry . All age group with diabetes showed a significant high frequency hearing loss as compared to the control population . Sharma et al in (2000) found the incidence of delayed wave latencies in diabetics was 64%, 72% and 84% at 2 khz, 4 khz and 6 khz respectively, suggesting that if brainstem evoked response audiometry is conducted at higher fre - quency like 6 khz in diabetic patients, and the involvement of central neural axis can be detected earlier . Diabetes for more than 10 years duration was present in 12 (48%) patients, among these bera was delayed in 11 (91.66%) cases . So longer duration of diabetes is a definite risk factor for development of central neuropathy . Diabetics with peripheral neuropathy (n= 13) had more incidence of delayed bera (n=12) tracings . So we concluded that diabetic subjects could suffer not only from peripheral and autonomic neuropathy but also from central neuropathy . Bayazit y et al in 2000 also concluded the likelihood of encountering a diabetic complication increases as the abr results become abnormal . Bera is a simple, non - invasive procedure to detect early impairment of acoustic nerve, and cns pathways, even m the absence of specific symptoms . This study suggests that if bera is carried out in diabetic patients; involvement of central neuronal axis can be detected earlier . So we strongly recommend that bera should be done in all patients with diabetic mellitus.
The aim of this study is to describe the use of electroconvulsive therapy (ect) in the treatment of methamphetamine - induced withdrawal delirium and craving in a single case . A 44-year - old male presented to the hospital in fars province, iran, with methamphetamine - induced withdrawal delirium who responded to ect . The electroconvulsive therapy can be a suitable option for the treatment of methamphetamine withdrawal delirium and craving . Also, it can be usefully employed in these very serious conditions which may represent a risk to life . We describe a patient with methamphetamine - induced delirium and craving which responded to electroconvulsive therapy (ect). Methamphetamine abuse was a minor problem in iran (1, 2) until the last couple of years . Recently, there has been a marked increase in the use of methamphetamine, especially among the young, with an increase in methamphetamine related psychiatric presentations to hospitals . Formerly, methamphetamine was illegally smuggled in from the abroad, but it is now synthesized in iran in underground laboratories . The methamphetamine synthesized in iran is of higher potency and is commonly associated with psychosis . A 44-year - old male employee presented to the hospital in fars province, iran, with decreased level of consciousness . The patient had no personal or family history of medical problems, and no history of head trauma . The patient began occasional smoking of opium 14 years prior to admission, which increased to daily smoking for the decade leading up to admission . He had a distant history of occasional use of alcohol; however, he stopped using it eight years prior to admission . Moreover, the patient had smoked heroine for six years prior to admission; however, there was no history of cannabis or cocaine use in the past . Eight months prior to admission, the patient commenced smoking methamphetamine, twice weekly, which then increased to daily use in the two months leading up to admission . From the time he commenced daily smoking of methamphetamine, the patient developed persecutory delusions, believing he was being followed by an agent from . He also experienced persecutory delusions regarding his neighbors and had a visual hallucination of a man on his roof . In short, when he was brought to hospital by his wife, he had been smoking heroine for six years and methamphetamine daily for two months . Drug screening was positive for methamphetamine and morphine (consistent with opium and heroin use) and negative for cannabis, ecstasy, methadone, buprenorphine, benzodiazepine and alcohol . The patient had taken methamphetamine on the morning of admission . By that evening his level of consciousness was reduced and he was drowsy . He was given buprenorphine 2 mg sublingually twice daily, to reduce opioid withdrawal, and closely monitored . On the second day of admission his drowsiness had increased and it was difficult to arouse him to answer questions . His condition was further deteriorated and could not speak or take food . On the fourth day of admission, the patient was considered to be in mortal danger and he was given emergency ect using a thymatron tm (system iv, company ind, usa). The electrodes were applied bilaterally, he was administered two seizures during the same anesthetic multiple monitored ect . Fourteen hours later he was alert and orientated, although still with some psychotic thinking and hallucinations . Five hours later the patient was alert and orientated in time, place and person, and free of psychotic features . Single ect was administered on 9th, 11th and 13th days and he was discharged on the 14th day . He received further single ect on the 16th and 18th days (as an out - patient) and remained well . Within a week or so, however, he again began to smoke amphetamine and was soon psychotic as before, with hallucinations and delusions . Multiple monitored ect was again administered, and some hours later, he showed partial improvement . He was administered double ect on two further occasions over the next 5 days and was discharged free of psychiatric symptoms . This case illustrates that ect may be beneficial in treating methamphetamine - induced psychosis and methamphetamine withdrawal delirium . Its use in these conditions, a systematic prospective trial of ect in delirium is yet to be published, and this report is an important addition to the literature . It is of interest that mmect continues to be used with good effect in leading centers in iran.
High - frequency oscillatory ventilation (hfov), as used by bojan and colleagues, is, at least in theory, an ideal tool for lung - protective ventilation as it allows effective pulmonary gas exchange with the delivery of a very small tidal volume (vt) below dead space and diminished risk of atelectrauma . Numerous animal and clinical studies have shown clearly that mechanical ventilation (mv) itself can initiate or exacerbate lung injury, termed ventilator - induced lung injury . The application of a vt and the repetitive opening and closure of alveoli have been identified as important pathophysiological mechanisms . Until now, only two randomized controlled trials investigating the effect of hfov on patient outcome have been performed and the larger of these included only 58 children . The main finding of this study was that hfov did not significantly improve survival (66% with hfov versus 59% with conventional mv) or total ventilator days (20 22 days with hfov versus 22 17 days with conventional mv). As a consequence, hfov is not universally employed in pediatric critical care . Also, it seems very unrealistic that any new pediatric hfov trial will be initiated within the next few years . This means that well - designed observational studies are needed to shed light on some of the many other aspects of pediatric hfov which remain to be explored, including the identification of patients who are most likely to benefit from hfov, the timing of hfov (early versus rescue), optimal oscillator settings, and monitoring during hfov . In the previous issue of critical care, bojan and a total of 120 patients were treated with hfov on the day of surgery, thus excluding rescue hfov use . Patients were transitioned to hfov when hypoxemia and acidosis occurred despite increasing alveolar ventilation on conventional mv (if vt exceeded 10 ml / kg) or when there was evidence of pulmonary hypertension and right ventricular (rv) failure . The main finding was that the duration of mv was significantly shorter in patients in whom hfov was initiated on the day of surgery . However, there are some concerns related to the methodology of the study and the practical use of hfov in the authors' institution . Some of the limitations inherent to their work have been properly addressed . But their study was designed as a retrospective one introducing confounding by indication . Hence, the authors have calculated a propensity score to minimize this, although the variables chosen for calculating it raise some concern . Included were all demographic and post - operative variables that yielded a p value of less than 0.10 in the univariate analysis between patients with hfov and those without hfov . However, it seems more rational to define, beforehand, which variables influence the decision of the attending physician to transition to hfov and therefore which variables should be used for calculating the propensity score . As a consequence of this approach, hemodynamic and if the authors had taken these into account when calculating their propensity score, the results of the study might have been even more firm . Also, it is not customary in their institution to routinely use an open lung strategy . Arterial partial pressure of oxygen (pao2) increases linearly with lung volume during hfov . Animal work has shown improved lung compliance and less hyaline membrane formation when such a strategy was applied . The effect of increased lung volume on pao2 is even more profound when oscillating on the deflation limb of the pressure - volume curve [9 - 11]. Furthermore, there is more simultaneous alveolar recruitment and overstretching on the inspiratory than on the expiratory limb of the pressure - volume curve . The hysteresis of the lungs provides an additional benefit: when oscillating on the expiratory limb, less pressure is required to maintain lung volume in comparison with when oscillating on the inspiratory limb . Nevertheless, what is especially interesting about the work of bojan and colleagues is that they used hfov when there was evidence of pulmonary hypertension or rv failure in their patients . This is an interesting approach as it is often assumed that high intra - thoracic pressures increase rv afterload and thus may enhance rv dysfunction . However, in a small study of 5 children after fontan cardiac surgery, the use of hfov did not result in an increase in pulmonary vascular resistance (pvr). So how can the positive findings of the study by bojan and colleagues be explained? This typically u - shaped curve shows that the pvr is high at both residual volume and total lung capacity . The curve is at its optimum (that is, lowest pvr) at' normal' functional residual capacity . It is not uncommon for ventilated patients after cardiac surgery to suffer from pulmonary dysfunction caused by atelectasis or even acute lung injury . One of the main approaches in such disease conditions is to maintain sufficient end - expiratory lung volume by the application of positive end - expiratory pressure (peep). However, the required level of peep is difficult to establish and may coincide with the delivery of high inspiratory pressures (> 30 cm h2o). Hfov is, in fact, nothing more than a continuous positive airway pressure system with superimposed small oscillations delivering the small vt . As a consequence, it is important to stress that the findings of bojan and colleagues warrant prospective confirmation in a well - designed randomized controlled trial . In the mean time, the authors are to be congratulated for their work as their study has provided arguments not to rule out the early use of hfov in pediatric cardiac surgery patients . Hfov: high - frequency oscillatory ventilation; mv: mechanical ventilation; pao2: arterial partial pressure of oxygen; peep: positive end - expiratory pressure; pvr: pulmonary vascular resistance; rv: right ventricular; vt: tidal volume.
Intravascular lymphoma (ivl) is a rare disease form of malignant lymphoma, and it is characterised by the selective growth of lymphoma cells within the lumina of vessels . Identification of this disease at an early stage is difficult because of its non - specific clinical symptoms and neuroradiological findings . Survival time is less than 1 year in most patients (mean, 5 months). We report the case of a patient with ivl who first presented with status epilepticus (se). His condition followed a fulminant course, culminating in death 21 days after the onset of symptoms . The radiological and pathological characteristics of the patient's brain lesions and the mechanism of seizures are also discussed here . A 76-year - old man with pulmonary emphysema was admitted to our institution following acute onset of convulsions . Several hours before seizure onset, he was asymptomatic and was able to drive his motorbike independently . On physical examination, he was comatose, his pupils were isocoric, and persistent clonic seizures of the head were observed . His peripheral white blood cell count and haemoglobin level were 13,400/l and 14.1 g / dl, respectively . Blood chemistry results were as follows: aspartate aminotransferase, 41 iu / l; alanine aminotransferase, 14 iu / l and serum c - reactive protein, 0.5 mg / dl . Serum albumin, blood sugar and sodium levels were 3.4 g / dl, 94 mg / dl, and 133 mmol / l, respectively . Cerebrospinal fluid analysis showed 3 white blood cells / mm and a protein level of 57 mg / dl; no bacteria or malignant cells were found . The patient was diagnosed with se and was administered immediate treatment with intravenous phenytoin followed by repeated intravenous administrations of diazepam . The seizures were not controlled by phenytoin; however, they ceased after continuous administration of intravenous midazolam . Brain diffusion - weighted imaging (dwi) performed on admission showed a hyperintense lesion in the right fronto - temporal cortex (fig . This lesion was almost completely restricted to the cortex and did not comprise a single vascular territory . Additional magnetic resonance imaging (mri) sequences such as t2-weighted imaging were not performed because his condition was critical . In addition to continuous midazolam infusion, valproic acid and carbamazepine were administered through nasogastric tubes from the time of admission . The patient was intubated the day after admission, and he subsequently developed pneumonia that was treated with antibiotics . Electroencephalography performed 3 days after admission under continuous midazolam administration showed diffuse dysrhythmic theta - delta activity without epileptic discharge . The patient's respiratory condition deteriorated, and he was put on a respirator 9 days after admission . Hypoalbuminaemia ensued, and he was administered total parenteral nutrition . Despite treatment with anti - epileptics, antibiotics and simultaneous general care, the patient's condition deteriorated further . Rapid progression of respiratory failure followed, and he died 21 days after the onset of symptoms . Autopsy findings suggested that the patient died from respiratory failure and heart failure resulting from acute purulent pericarditis . Microscopic examination revealed large malignant lymphoma cells in the small blood vessels of the brain, heart, liver, prostate, adrenal gland and bladder . These abnormal lymphoid infiltrates were entirely intravascular and positive for b - cell antigen cd20 but negative for cd3, cd5, cd10 and bcl-2 (fig . Similar lymphoid infiltrations were observed predominantly in the cortex in the bilateral frontal, parietal, temporal and occipital lobes, and most significantly, in the right frontal lobe of the brain . The cerebral tissue at the frontal area showed some petechial haemorrhages around the capillaries, and this tissue was characterized by the presence of neuronal changes and mild spongiosis, absence of neuronal ferrugination, coagulative necrosis, chronic inflammation, macrophages, hemosiderin pigmentation, neo - vascularization and cavitation . We report the clinical course of a patient who developed acute se as the first manifestation of ivl in the absence of other symptoms . Although ivl generally presents as a subacute progressive disease, this case was characterized by the presence of intractable se from its initial presentation . It followed a fulminant and rapidly progressive course that resulted in death of the patient 21 days after onset . Ivl is characterized by a variety of symptoms resulting from occlusion of small vessels by tumour cells in different organ systems, especially the nervous systems . A previous study suggested that all patients with ivl have one or more of four presentations: progressive, multifocal cerebrovascular events; spinal cord and nerve root vascular syndrome; subacute encephalopathy and peripheral or cranial neuropathies . Common to all these presentations are multiple areas of capillary, arteriole and venule occlusion . Regarding the occurrence of seizures associated with ivl, epileptic seizures have been reported in 8.825% patients with ivl [4, 5, 6]. Epileptic seizures in patients with ivl reportedly occur at a more advanced stage in most cases [6, 7]; however, patients suffering from epileptic seizures at ivl onset have also been reported [4, 6, 8, 9]. Case reports and literature reviews have shown that epileptic seizures as the first manifestation of ivl are found in 34% patients with ivl [4, 6]. This case highlights the importance of making an accurate diagnosis of ivl in patients who initially present with se . Post - mortem examination of the patient revealed a phase of acute neural injury, which may have occurred 12 days after an acute infarction . Most previous reports of patients with ivl accompanied by epileptic seizures have documented the presence of radiologically detectable infarct lesions at the onset of epilepsy or ischemic necrosis at autopsy; however, some ivl cases with epilepsy have shown no infarcted regions at autopsy . Although this case showed se as the first manifestation of ivl, autopsy at 21 days after se onset revealed acute - phase lesions that had probably occurred 12 days after acute infarction; however, there were no chronic or subacute lesions, suggesting that infarctions did not occur at se onset . Moreover, dwi suggested no infarcted lesion at se onset because the lesion seen on dwi did not comprise a single vascular territory . Both dwi and autopsy findings were suggestive of a pathogenesis different from that of post - stroke seizures . Although the mechanism of seizures associated with stroke is not completely understood, metabolic changes, acute glutamate release, changes in the penumbra zone and anoxic depolarization may provoke early seizures . In this case, hypoperfusion due to small vessel stenosis by ivl cells, small petechial haemorrhages from capillaries, meningeal involvement of ivl cells or metabolic effects are possible explanations for the mechanism underlying se due to ivl . Dwi has proved to be a useful tool for the diagnosis of acute ischemia caused by ivl . A previous study has revealed that dwi changes in ivl possibly reflect both ivl - associated ischemia and the movement of tumour cells through the vessel wall to the cerebral white matter . A dynamic pattern of mri lesions with resolution of some dwi lesions may indicate the diagnosis of ivl through neuroimaging . In this case, the patient's critical condition did not allow serial dwi studies; therefore, ivl was not considered . In conclusion, se can present as the initial manifestation of ivl and should be included in the differential diagnosis for etiologically undiagnosed se in elderly patients . Ivl recognition is of clinical importance because the treatment and prognosis of acute se resulting from ivl are different from those of se with different origins . Early detection, aggressive attempts at early diagnosis and appropriate treatment may prove beneficial to these patients.
One of the grand objectives in microbiome research is understanding how these communities of microorganisms interact with their environment . In the case of the human gut microbiome changes in the host s health status or the ecology of the gut can differentially influence the taxa that comprise the microbiome and restructure the community composition . Correspondingly, changes in microbiome structure can affect the ecology of the gut or health of the host . Understanding these interactions facilitates the development of clinical diagnostics in the case that community composition or the abundances of specific taxa are predictive of disease . It can also yield novel therapeutics, such probiotics, in the case that changes in specific taxa are shown to modulate health . The standard approach for characterizing these interactions is to use tests of association . In this approach, the abundance of each taxonomic group is quantified in a variety of microbiome samples and then statistical tests are performed to determine whether changes in the abundance of each group are related to changes in the microbiome s environment (e.g., test of correlation). This approach simplifies the discovery of potential interactions by analyzing each group independently of the others under the assumption that meaningful interactions between taxa and their environment will be robust despite variation in the surrounding structure of the microbiome . This approach has been widely applied to microbiome studies, especially those with simple study designs (e.g., case versus control), and has yielded tremendous insight into how specific groups of taxa associate with environmental parameters, including human disease and nutritional state . However, in complex systems, context may matter . Because the aforementioned approach isolates the analysis of each taxon from the rest, it fails to resolve complex associations that arise through the interactions among taxa that comprise the community . As a toy example, consider a taxon may that positively associate with disease when lactobacillus is absent from the community but does not associate with disease when lactobacillus is present . This change in association could possibly be due to the contextual nature of cellular phenotype; for example, lactobacillus may excrete a compound that changes the metabolic state of the taxon to behave in a commensal rather than pathogenic manner . The frequency of these types of interactions is not well understood, and they may explain some of the inconsistent observations made by different studies of the microbiome in similar disease models (e.g., see reference 1). If our goal is to produce clinically meaningful tools, we should seek to appropriately model these context - dependent interactions . The discovery of robust patterns of association between microbiota and their environment is important to our ability to combat human disease, such as clostridium difficile infection . C. difficile is an environmentally acquired enteropathogen that causes debilitating and life - threatening diarrhea . The gut microbiome appears to play an important role in protecting the host from infection . C. difficile rates of infection are elevated in individuals who have recently been treated with antibiotics, which has contributed to its success as a nosocomial infectious agent . Additionally, manipulation of the microbiome via transplantation from healthy donors is a highly effective form of therapy, even in cases where all other treatments have failed (2, 3). While we have learned quite a bit about the microbiome s relationship with c. difficile infection, there appear to be complex relationships that influence health outcomes . For example, it is not understood why antibiotic administration results in disease in some patients but not others . Nor is it understood why different types of antibiotics appear to be associated with different disease risks . It has also been difficult to determine which microbiota are associated with protecting against c. difficile infection . For example, antibiotic intervention is associated with a loss in c. difficile colonization resistance and a decrease in the abundance of lachnospiraceae and barnesiella (4), while microbiome transplantations are instead associated with a bloom in bacteroidetes (5). These observations intimate that multiple groups of taxa may contribute to colonization resistance and that the overall context of the community may be an important factor in determining resistance . In a recent report, schubert et al . (6) discuss a series of investigations aimed at disentangling the complex web of interactions that comprise the microbiome in order to model the context - dependent associations between the microbiome and clostridium difficile infection . The authors adopted a mouse model study design that allowed them to tune a variety of experimental parameters, including antibiotic treatment, the specific class and concentration of antibiotic, exposure to c. difficile, and the time between administration of the antibiotic and infection . By modulating these various parameters, the authors were able to explore how different types of community perturbations or different microbiome starting states affected the association between the abundance of specific microbiota and resistance to c. difficile intestinal colonization . To quantify these associations, the authors applied a machine learning technique, known as a random forest regression, to mathematically model how antibiotic exposure, structural variation in the microbiome, and time to infection interact to influence c. difficile colonization resistance . Briefly, the authors constructed a series of regression trees that predicted c. difficile colonization levels based on an assemblage of bacterial populations and experimental parameters (e.g., antibiotic class). In effect, this model uses a complex series of if, then statements to estimate c. difficile colonization levels given knowledge of the initial community composition and experimental parameters . The model constructed by the authors was able to explain ~77% of the variation observed in c. difficile colonization levels across samples . However, when the authors instead predicted whether an individual would be colonized by c. difficile or not, they observed an error rate of only 10.7% . The analysis advanced by schubert et al . For example, they found that exposure to different classes of antibiotics resulted in different community compositions and that, within these resulting communities, c. difficile colonization resistance was associated with different taxa . Furthermore, they identified cases where the same taxon produces opposite correlations with c. difficile colonization resistance depending upon the antibiotic exposure . For example, an operational taxonomic unit (otu) from bacteroides was positively associated with c. difficile colonization in streptomycin - treated mice but was negatively associated in mice treated with cefoperazone . Furthermore, according to their model, considering the interactions between taxa is important for predicting colonization resistance . The authors found that decreases in otus associated with porphyromonadaceae, lachnospiraceae, lactobacillus, alistipes, and turicibacter are associated with increased susceptibility to infection when coupled with increases in escherichia or streptococcus otus . When analyzed in isolation, many of these taxa are not predictive of disease (e.g., an otu within akkermansia) or are highly predictive under some conditions (e.g., an escherichia otu in ampicillin - treated mice) but not others (e.g., the same escherichia otu in streptomycin - treated mice). In short, schubert et al.s (6) regression models provide a more robust prediction of c. difficile colonization resistance than traditional analyses would provide . Additionally, their findings indicate that the phenotype of colonization resistance is complex and depends upon a variety of parameters . For example, extensions of their models may ultimately be used to screen clinical patients microbiomes prior to administration of antibiotics and predict their likelihood of developing an infection . Similarly, they may help doctors select patient - appropriate antibiotics or those that minimize the risk of infection . Furthermore, these models may help identify ideal microbiome donor matches to improve the efficacy of microbiome transplantations for treating c. difficile infection . Second, these models provide insight into those taxa that are consistently associated with c. difficile resistance, which can improve the development of effective probiotics based on a complex of organisms . Finally, these models provide insight into how the microbiome operates and can be used to establish hypotheses about the ecological interactions between specific groups of taxa or how perturbations to a community affect the functional relationship between taxa . Of course, the application of these models would benefit from additional investigation, such as the consideration of additional variables that may be relevant to colonization, including the initial immunological state of the host . Furthermore, it may be valuable to compare alternative regression or modeling approaches, with the aim of improving the accuracy of the predictions . Finally, the extension of these models to clinical settings requires validation in human populations, which are subject to sources of variation beyond those in an experimental mouse model, including genetic and nutritional variance . Other studies have applied similar analytical methods to investigate the complex of dependencies that influence the microbiome (7). For example, belzer et al . Identified groups of enteric microbiota that exhibit consistent temporal patterns of variation in response to citrobacter rodentium - induced colitis (10). Applied linear models to understand how dietary variation perturbs a defined gut microbiome (8). These mathematical model - based frameworks provide insight into the mechanisms through which microbiomes operate and will likely be essential in developing a comprehensive view of how hosts and their microbiomes interact . Future investigations should similarly consider establishing study designs that enable robust, predictive modeling of the microbiome.
Acute myeloid leukemia (aml) accounts for 80% of acute leukemia in adults with a median age at diagnosis of 65 years . The standard upfront induction regimen for fit adult individuals remains 7 + 3, consisting of 7 days of continuous infusion cytarabine and 3 days of an anthracycline, either daunorubicin or idarubicin . Variations of induction and re - induction regimens in the refractory / relapsed disease setting often include either idarubicin or mitoxantrone in conjunction with high dose cytarabine and sometimes purine analogues . Medical comorbidities, specifically cardiovascular morbidity, have been shown to negatively impact the clinical outcomes of aml induction therapy in older adults . Cardiotoxicity constitutes the most frequently feared adverse event associated with the use of anthracyclines (> 10%), and compromised cardiac function may compromise the use of potentially curative upfront and subsequent aml regimens in a patient's course . Prevention or mitigation of anthracycline - induced cardiotoxicity, particularly in older adults who constitute the majority of new aml diagnoses, is therefore an area of tremendous clinical relevance . Cardiotoxicity of anthracyclines is dose - dependent and can occur at any time in the treatment course with acute, subacute, and late - onset presentations . Clinical symptoms include arrhythmias, myopericarditis, cardiomyopathy, and congestive heart failure with reduced left ventricular ejection fraction (lvef),, . For example, the incidence of cardiotoxic events with use of doxorubicin was less than 5% at a cumulative dose of 400 mg / m, but increased in a dose - dependent manner to 16% at a cumulative dose of 500 mg / m, 26% at 550 mg / m, and 48% at 700 mg / m, . For this reason, current recommendations limit the cumulative lifetime dose of doxorubicin to no more than 450 mg / m . Although the precise mechanisms of anthracycline - induced cardiac toxicities are not well elucidated, the most commonly proposed mechanism is drug - induced generation of iron - anthracycline complex mediated reactive oxygen species (ros) which cause mitochondrial dysfunction leading to adenosine triphosphate depletion, lipid peroxidation, dna damage, and subsequent myocardial injury, ., ny, ny) is a cyclic derivative of a strong metal - chelating agent which interferes with site - specific iron - based oxidative damage to cardiac mitochondria to exert its cardioprotective activity . By chelating free iron, dexrazoxane prevents the formation of iron - anthracycline complexes that lead to the formation of superoxide free radicals during redox reactions, thereby limiting cardiac injury . Food and drug administration (fda) in 1995 for use as a cardioprotective agent in the treatment regimen of patients with metastatic or advanced breast cancer who have reached a cumulative anthracycline dose of 300 mg / m and who are continuing to receive doxorubicin . Additionally, dexrazoxane was approved in 2007 by the fda to decrease damage that may occur in the setting of inadvertent extravasation of anthracyclines into skin and subcutaneous tissue . Although not approved in the pediatric setting, dexrazoxane has also been extensively utilized in children with leukemia and lymphoma in order to mitigate the long - term cardiovascular effects of intensive chemotherapy regimens incorporating anthracyclines . Despite case reports of aml and myelodysplastic syndrome (mds) developing in some children receiving dexrazoxane in conjunction with chemotherapy, the children's oncology group recently reported that dexrazoxane did not appear to compromise long term survival of over 1000 pediatric patients treated with this agent on multiple clinical trials, . Evidence supporting the benefit of dexrazoxane as cardioprotective therapy in adults with acute leukemia are scant . However, there is a significant need for cardioprotective therapy, particularly in older individuals with preexisting cardiovascular disorders and/or prior anthracycline exposure, who are otherwise considered good candidates for potentially remission - inducing intensive chemotherapy . Here, we present the cases of six older adults with newly diagnosed or relapsed aml at high risk for cardiovascular morbidity who received dexrazoxane in conjunction with anthracycline containing induction / re - induction chemotherapy regimens . Pharmacy database search at roswell park cancer institute was conducted to identify all adult patients (age 18 years) who had received any number of dexrazoxane doses during a 16 year time frame (january 2000 to october 2016). Two patients were then excluded (one patient had ongoing treatment while another patient had inadequate follow up due to death relatively soon after dexrazoxane from aml and its complications but unrelated to dexrazoxane). Deidentified data were collected on the remaining six patients by systematic electronic medical chart review . Patient characteristics like age, gender, race, and pertinent medical history such as the specifics of aml diagnosis and therapy course, cardiac and other medical histories, dose and reason for selection of dexrazoxane administration, echocardiogram and multigated acquisition scan findings, and post dexrazoxane outcomes were reviewed and recorded in microsoft word document and excel sheet on a secure server . This retrospective study was approved by the institutional review board of the roswell park cancer institute, buffalo, ny . Table 1summary of adult patients with aml treated with dexrazoxane.table 1:caseage, sexdiagnosis, chemotherapy regimenprior cardiac historyanthracycline dose (mg / m)lvefclinical outcomes following dexprior to dexwith /after dexprior to dexafter dex159, frelapsed aml, hidac + mitonone30016050%20% -> 55%transient decreased lvef, died of relapsed disease264, frelapsed aml, clag - mnone34013255%55%died from treatment related complications367, mrelapsed and refractory aml, adecad s / p stent placementnone33840%50% -> 30%*decreased lvef after additional anthracycline without dex, died from refractory aml455, mde novo aml, adecad s / p cabg, htnnone31040%50%relapsed aml, alive after allosct557, fde novo aml, adeviral cardiomyopathynone27040%30%-> 66%rbbb and mobitz type ii block 3 yrs later, relapsed aml, alive after allosct671, frelapsed aml, hidac + mitonone37015850%40%decreased lvef and global hypokinesis, died of sepsis and refractory amlall anthracycline doses have been converted to daunorubicin equivalent . Abbreviations: aml: acute myeloid leukemia; ade: cytarabine, daunorubicin, etoposide; allosct: allogeneic stem cell transplantation; cad: coronary artery disease; cabg: coronary artery bypass graft; dex: dexrazoxane; hidac: high - dose cytarabine; htn: hypertension; lvef: left ventricular ejection fraction; lvh: left ventricular hypertrophy; mito: mitoxantrone; rbbb: right bundle branch block summary of adult patients with aml treated with dexrazoxane . Abbreviations: aml: acute myeloid leukemia; ade: cytarabine, daunorubicin, etoposide; allosct: allogeneic stem cell transplantation; cad: coronary artery disease; cabg: coronary artery bypass graft; dex: dexrazoxane; hidac: high - dose cytarabine; htn: hypertension; lvef: left ventricular ejection fraction; lvh: left ventricular hypertrophy; mito: mitoxantrone; rbbb: right bundle branch block while four patients (cases 1, 3, 5, 6) experienced a drop in lvef post - dexrazoxane, two of those patients (cases 1, 5) eventually recovered lvef . The other two patients had a drop in lvef that corresponded with development of sepsis (cases 3, 6). Additionally, the drop in lvef for one patient (case 3) occurred later in the therapy course when dexrazoxane was not used . Besides lvef reduction, conduction abnormality (right bundle branch block and mobitz type ii block) was noted in one patient (case 5). The two patients alive (cases 4, 5) had de novo aml with good risk cytogenetics and both underwent allogeneic stem cell transplant for relapsed / refractory nature of their aml . Table 2temporal changes of left ventricular function (lvef) in patients with improvement in lvef post dexrazoxane.table 2:case 1case 4case 5baseline lvef of 70%baseline lvef=40%baseline lvef=40%post - transplant lvef of 50%, day 1561dexrazoxane on days 13dexrazoxane on days 13dexrazoxane on days 15611563lvef of 55% on day 33lvef of 30% on days 7, 14lvef of 20% on day 1692lvef of 50% on day 728lvef of 41% on day 538lvef of 55% on day 2119lvef of 50% on day 839lvef of 30% on day 656post - transplant lvef of 50% on days 866, 873, 944lvef of 44% on day 694post - transplant lvef of 55% on days 781, 832lvef of 66% on day 1084day 1 is the day of first anthracycline administration in the patient's overall treatment course of acute myeloid leukemia . Abbreviations: lvef: left ventricular ejection fraction temporal changes of left ventricular function (lvef) in patients with improvement in lvef post dexrazoxane . Day 1 is the day of first anthracycline administration in the patient's overall treatment course of acute myeloid leukemia . Abbreviations: lvef: left ventricular ejection fraction see supplementary data for details of each patient's clinical course . Here we describe six older adult patients (ages 5571, median age 61.5 years) who received dexrazoxane to mitigate the cardiotoxicity of daunorubicin or mitoxantrone - based induction / re - induction chemotherapy for aml . All of our patients would have been either considered at very high risk for cardiotoxicities due to preceding cardiovascular morbidities and/or were ineligible for anthracyclines due to concern for exceeding cumulative dose limits . Baseline lvef in five out of six patients was borderline and ranged from 40% to 50% . Of note, half of these patients had aml characterized by favorable karyotype at diagnosis whose disease would be expected to potentially benefit from standard intensive 7 + 3 chemotherapy . Five of the six patients had stable / recovered lvef back to baseline pre - chemotherapy levels following concomitant dexrazoxane and chemotherapy . This includes two patients who experienced a transient drop in lvef in the setting of sepsis . Although four patients have since died, cardiotoxicity was not the leading factor in their demise . Importantly, two patients who received induction chemotherapy with dexrazoxane had preserved lvef afterwards and were able to successfully undergo allogeneic hematopoietic stem cell transplant at the time of relapse . Characterizing the precise clinical effects of dexrazoxane in patients with aml can be difficult because of concurrent use of cardiotoxic agents other than anthracyclines, the general aggressiveness of aml, and the presence of cardiac and non - cardiac comorbidities in older adults . For example, cases 3 and 6, encountered a drop in lvef in the context of ongoing sepsis, which has been shown to result in global left - ventricular hypokinesis (defined as lvef <45%) within 72 h of presentation in the majority of patients . However it is notable that one patient (case 3) who had a stable / improved lvef of 50% following dexrazoxane and anthracycline chemotherapy subsequently developed progressive heart failure with a drop in the lvef of 30% following administration of additional anthracycline therapy without cardioprotection . Although the cardioprotective benefits of dexrazoxane have been clearly demonstrated in both pediatric (mostly hematologic malignancies) and adult (mostly breast cancer) patients, this agent is not currently approved for aml patients receiving anthracycline therapy . One concern was the possibility of dexrazoxane decreasing chemotherapeutic efficacy based on preclinical data showing that dexrazoxane can antagonize the cytotoxicity induced by topoisomerase ii directed drugs, daunorubicin and etoposide, in aml cells . Although dexrazoxane exhibits only weak cytotoxicity against leukemia cells, synergistic anti - tumor effects were reported following combination therapy with dexrazoxane plus anthracycline or dexrazoxane plus daunorubicin and cytarabine in multiple human aml cell lines (hl60, hl60/dox, oci / aml3, aml-193, crf - sb, and molt-4), . Similarly, another study showed that dexrazoxane sensitized k562 and hl60 cells to daunorubicin treatment . Dexrazoxane may also prevent the acquisition of the multi - drug export receptor, mdr1, in k562 aml cell lines following doxorubicin, thereby delaying the emergence of multidrug resistance . Another issue was the potentially increased incidence of secondary malignant neoplasms (smns), specifically aml and mds, in patients receiving dexrazoxane . Although one study reported a statistically significantly increased risk of smns following dexrazoxane therapy, this study has since come under scrutiny because of the controversial statistical methodology leading to the conclusions,, . Moreover a recent meta - analysis of 1561 patients (mostly adults with breast cancer) enrolled in eight trials showed that dexrazoxane significantly prevented the development of heart failure (rr=0.18; 95%-ci: 0.100.32) without any increased risk of smns or negative effects on treatment response, progression - free or overall survival . Several other major studies have similarly not shown any association between dexrazoxane and smns or poor treatment outcomes, . In sum, the available preclinical and clinical data not only support the safety of dexrazoxane as cardioprotective therapy for aml patients but also offer the tantalizing possibility that this agent may enhance anti - leukemic effects . Overall, the potential benefits of dexrazoxane therapy appear to far outweigh the risks, particularly in patients with life threatening malignancies such as aml . Aside from this report, only two other studies of dexrazoxane use in adult aml patients have been published . In a series of seven patients with aml, lemez and maresova reported that the administration of dexrazoxane 30 min before daunorubicin (dose 8 - 13x higher than daunorubicin) or mitoxantrone (dose 40 - 60x higher than mitoxantrone) allowed them to give cumulative anthracycline doses in the range of 5501300 mg / m of daunorubicin without signs of cardiac toxicity . Complete remission was achieved in all patients with relapsed aml treated with high dose cytarabine, anthracycline, and dexrazoxane . However, almost all of these patients were significantly younger (ages 2158, median 35 years) than our cases (ages 5571, median 61.5 years), and all had much higher baseline lvef (6270%) than our patients (lvef 4050%). In another study, woodlock and colleagues safely administered dexrazoxane to two aml patients: prior to mitoxantrone in a 70 year old patient with ischemic heart disease (lvef 30%), and prior to idarubicin in a 43 year old patient with concurrent myocardial infarction (lvef 70%). Strategies to mitigate the cardiac toxicities of anthracycline therapy are urgently needed to allow for the safe administration of effective, intensive chemotherapy regimens in adult aml patients . A recent cooperative group trial demonstrated that higher daunorubicin doses (90 mg / m vs. 45 mg / m) during induction therapy improved clinical outcomes for aml patients across cytogenetic and molecular subgroups . In another large multicenter study, higher - dose daunorubicin (90 mg / m vs. 45 mg / m) improved complete remission rate (52% vs. 35%, p<0.001) in adults over the age of 60 years and overall survival (38% vs. 23%, p<0.001) for those aged 6065 years . Older individuals who constitute the majority of new diagnoses are at particular risk for cardiotoxic complications . Based on our clinical experience and the current data, we believe that the use of dexrazoxane as standard cardioprotection during intensive chemotherapy should be further investigated in large randomized controlled clinical trials . Until such high - quality evidence emerges, we would suggest strongly considering dexrazoxane in adults with known significant cardiac comorbidities and/or who are fit for intensive anthracycline - containing therapy but are approaching or have crossed their lifetime cumulative dose of anthracycline . Dexrazoxane should be administered intravenously 15 min before every dose of mitoxantrone / idarubicin (50:1 dexrazoxane: anthracycline) or daunorubicin (10:1 dexrazoxane: daunorubicin). It is important to note that dexrazoxane is renally eliminated and therefore would require a 50% dose reduction in the setting of renal insufficiency . These include concomitant beta - blockers, angiotensin - converting enzyme inhibitors, angiotensin ii receptor blockers, and statins, all of which have previously been shown in various studies to improve a variety of cardiac outcomes in patients receiving anthracyclines . Of note, in patients with metastatic breast cancer, it remains to be seen whether cpx-351, a new liposomal formulation of cytarabine: daunorubicin which has shown improvement in response and overall survival in older patients with secondary aml, represents a safer, less cardiotoxic, induction choice than standard 7 + 3 . Cardiotoxicity is one of its most serious adverse effects though, occasionally precluding its usage even in potentially curative cases . There is preclinical evidence in aml cell lines demonstrating that it may even have anti - leukemic effects . However, there is limited data of its usage in adult aml patients . Of our six cases of older adults (ages 5571, median 61.5 years) at high risk of anthracycline - induced toxicity who received dexrazoxane as cardioprotective therapy during their therapy for aml, five had preserved / recovered lvef at or above baseline following treatment while two patients proceeded onto subsequent allogeneic stem cell transplantation . Dexrazoxane should be considered especially in adults with known significant cardiac comorbidities and who are fit for intensive anthracycline - containing therapy but are approaching or have crossed their lifetime cumulative dose of anthracycline . Additional clinical investigation of dexrazoxane in adult leukemia therapy is warranted to better define its role particularly in older individuals at highest risk for cardiovascular mortality.
Propofol infusion syndrome (pris) was initially described in 1992 as a syndrome with fatal outcome in critically ill children . Further investigation showed that critically ill adults suffer from identical symptoms after administration of propofol . The syndrome was defined by bray in 1998, describing a sudden onset of bradycardia, resistant to treatment and progression to asystole plus one of the following: hyperlipidemia, fatty infiltration of the liver, severe metabolic acidosis or muscle involvement with rhabdomyolysis and myoglobinuria . The findings of a multicenter study with 1017 medical and neurosurgical adult patients was published in 2009 . Incidence slightly exceeded 1%, but the short exposure of a maximum 24 h in this study has to be taken into account . A previous retrospective study of 55 patients receiving propofol for sedation during catheter radiofrequency ablation suggests a higher incidence: 24% (13 of 55) of the patients showed a metabolic acidosis (base excess (be) 2) which was interpreted as the initial indicator of the syndrome . An alert and well - oriented 7-year old boy presented after being involved in a road traffic accident . The computed tomography (ct) scan of the brain showed a depression of the os parietale with small subcortical contusions, perifocal edema, little intracranial air, but no midline shift . Intracranial pressure (icp) monitoring was installed with a combined liquor drain . At the same time elevated icp (25 mm hg) called for a postoperative ct scan; results were unchanged . After surgery, the intubated patient was sedated with propofol (12 mg / kg / h) and transferred to the intensive care unit . Ph and serum lactate levels were monitored several times daily, and creatine kinase levels once daily . During sedation with propofol, inotropic support with low - dose norepinephrine was necessary to reach the goal of 60 mm hg cranial perfusion pressure . Propofol infusion was stopped after 39 h. eight hours later, the patient had a glasgow coma score of 10, and his trachea was extubated . At that time, generalized muscle weakness and slow awakening were observed . Two hours after successful weaning and extubation, the patient was in respiratory distress with aggravating metabolic acidosis (ph 7.19, be 13.2, lactate sedation with propofol was reinstalled for another 11 h (7.3 mg / kg / h) after which the regimen was changed to midazolam and nalbuphine . On the following day, blood tests showed decreasing metabolic acidosis (ph 7.31, be 6.6, lactate 1.8 mmol / l) but elevated creatine kinase (1023 u / l, reference range 38 - 157 u / l) and mildly elevated liver enzymes (asat 179 u / l, reference range 11 - 34 u / l, alat 174 u u / l, reference range 10 - 37 twenty hours after cessation of propofol, generalized seizure was observed under sedation with midazolam and nalbuphine, which was successfully treated with intravenous thiopentone . No change was observed in another ct brain scan performed immediately afterwards . During the following 6 h, cardiac instability with supraventricular tachycardia up to 180 bpm and peracute renal failure developed . After immediate mechanical and medical resuscitation, the patient presented with unreactive and bilateral dilated pupils and bradyarrhythmia that were resistant to therapy . A further ct brain scan (10 h after the previous scan) showed a generalized edema of the brain with obliteration of the basal cisterns and transtentorial herniation . Nevertheless, renal replacement therapy was installed and cardiac performance improved, and metabolic acidosis could be normalized . Due to persistent rhabdomyolisis (creatine kinase> 100,000 u / l), an open muscle biopsy of the m. quadriceps followed by in vitro muscle contracture testing (ivct) and histological analysis were performed . These investigations were carried out because the clinical signs involved skeletal muscle dysfunctions and because the patient was intubated using succinylcholine, a known trigger agent for malignant hyperthermia (mh). While histology revealed completely normal values, the muscle could not be stimulated electrically for ivct but presented with spontaneous muscle contracture (figure 1). A blood sample was taken to isolate genomic dna and investigate for mutations in the gene encoding for the skeletal muscle type 1 ryanodine receptor (ryr1) using molecular genetic investigations, but no ryr1 variant was identified . Figure 1original recordings from freshly biopsied skeletal muscle were obtained from the vastus medialis of the quadriceps muscle . Muscle strips approximately 2.5 cm long and approximately 3 mm wide were mounted in a tissue bath and slowly stretched to a pre - load force of approximately 25 mn . The tissue bath, containing krebs - ringer solution, was bubbled with carbogen at 37c . Muscle strips were stimulated electrically in order to produce repeated muscle contractions (twitches). (a) a muscle strip of the patient showing a continuously increasing muscle contracture with absolutely no response to electrical stimulation, resulting from the loss of function of excitation - contraction coupling . (b) a muscle strip from a healthy subject . Note the stabilization of the baseline as well as the muscle contractions upon electrical stimulation . Original recordings from freshly biopsied skeletal muscle were obtained from the vastus medialis of the quadriceps muscle . Muscle strips approximately 2.5 cm long and approximately 3 mm wide were mounted in a tissue bath and slowly stretched to a pre - load force of approximately 25 mn . The tissue bath, containing krebs - ringer solution, was bubbled with carbogen at 37c . Muscle strips were stimulated electrically in order to produce repeated muscle contractions (twitches). (a) a muscle strip of the patient showing a continuously increasing muscle contracture with absolutely no response to electrical stimulation, resulting from the loss of function of excitation - contraction coupling . (b) a muscle strip from a healthy subject . Note the stabilization of the baseline as well as the muscle contractions upon electrical stimulation . Despite high doses of inotropic support, the patient developed progressive cardiac and pulmonary failure . U / l), rhabdomyolysis (creatine kinase> 100,000 u / l), and coagulopathy (thrombin time> 120 s, prothrombin time 48%). The boy died of brain death and multiple organ failure four days and 6 h after discontinuation of the propofol infusion . Although millions of patients worldwide are sedated with propofol, there have been only a few reports of pris . This implies a particular susceptibility of some patients and is, therefore, highly suggestive of a pharmacogenetic disease . Hypothesize that the key pathogenetic mechanism is an imbalance between energy demand and utilization through the impairment of free fatty acid utilization and mitochondrial activity, which is generated in the presence of propofol . Propofol seems to produce a cytochrome oxidase deficiency in muscle tissue, as observed in a child after prolonged high - dose infusion . The link between free fatty acid metabolism and myocytolysis was found after plasma analysis of a 2-year old boy which showed raised concentrations of malonyl carnitine and c5 acylcarnitine; these returned to normal after recovery . State that these findings indicate that altered long - chain free fatty acid entry into the mitochondrion and uncoupling of beta - spiral oxidation and respiratory chain at complex ii are the critical events in pris . Bray and vasile et al . Defined the triggering factors to be catecholamines, steroids, cytokine production, and metabolic stress; all these are present or administered for conditions including polytrauma, multiple organ failure, and severe brain or burn injury . Showed a greater risk with high - dose propofol (5 mg / kg / h) or prolonged sedation (> 48 h). The postulated risk factors in our patient are young age, brain injury, norepinephrine application, high - dose propofol and a total of 49 h of therapy . The hypermetabolic state, rhabdomyolysis, and muscle weakness of our patient suggested some parallels to episodes of mh . Therefore, a muscle sample was taken after obtaining written informed consent from his parents . This muscle sample did not twitch in vitro following supramaximal electrical stimulation, but showed spontaneous contracture (figure 1). The enzyme - histochemical examination showed neither a structural congenital myopathy nor an abnormal accumulation of mitochondria . As the involved organs included the brain, heart, and skeletal muscle, and the main symptoms of our patient were metabolic acidosis, seizure, cardiac conduction abnormalities and rhabdomyolysis, we postulate that the key mechanism for the observed dysfunctions in all three organs involves calcium regulation and, therefore, ryanodine receptors . As a different isoform of the ryanodine receptor (ryr1, ryr2, and ryr3) is involved for each of the organs mentioned above (brain, heart, and skeletal muscle), a common modulator of each of these subtypes of receptors could be the missing link . This view is supported by the fact that ryr1 is present in all 3 organs and that all 3 isoforms of ryanodine receptors are very similar . Whether the common final pathway of an altered intracellular calcium regulation via a dysfunction of ryanodine receptors is the result of a decreased energy supply due to impaired oxidation in the mitochondria or by alternative direct or indirect effect(s) of propofol on the function of ryanodine receptors are factors in patients presenting with pris remains to be clarified . The basis for this disorder is the triggering of a sustained rise in intracellular ca in skeletal muscle due to hyperactivation of mutated ryr1 channels . The variability of the clinical presentation and the time differences between the first laboratory findings in published cases with pris, as well as the fact that only a small number of individuals in all age groups developed pris after anesthesia or sedation using propofol, support the view of pris being a pharmacogenetic disorder . The involvement of an altered calcium regulation in pris may be an interesting and attractive approach for identifying potential genetic alterations in patients presenting with pris . Pris could well be an entity of disorders with an identical common pathway caused by a variety of causative genetic mutations.
A headline such as depression is associated with increased inflammation reflects a failure to consider that studies generally indicate that the mean value for a given inflammatory biomarker tends to be higher in depressed groups than in groups of non - depressed individuals . This does not mean that all subjects with depression have higher inflammatory levels than all non - depressed comparison subjects, or that the inflammatory levels in depressed patients are abnormal . Indeed, the mean differences in inflammation between depressed and nondepressed individuals are modest, and mean values for inflammatory markers in groups of depressed individuals are typically in the normal range when such norms are established, as is the case with c - reactive protein . By way of illustrating this point, levels of c - reactive protein in people with autoimmune and inflammatory conditions or acute infection range from 10 to over 100 mg / l,24 versus depression or no diagnosis, where c - reactive protein ranges from 0 to 10 (with a crp <3 mg / l generally considered the upper limit of normal).5 figure 1 (see below) illustrates the other truth about inflammation and depression that is not always adequately emphasized . Despite differences in mean levels of c - reactive protein or il-6 in this case, there is a huge overlap between people with depression and those without, and in any given study, the highest inflammatory value might be found in a control subject, while the lowest might be found in the depressed group.6 the first point that the inflammatory activation observed in people with depression is modest might tempt us to dismiss the potential relevance of inflammation to the pathophysiology of depression . But this would be both a serious mistake and a profound misunderstanding of the huge effect that small physiological differences can have over time if they are consistently skewed in one direction . As it turns out, depression is far from being alone as a condition characterized by reliable but other modern illnesses with evidence of moderately increased inflammatory signaling include cardiovascular disease, stroke, cancer, diabetes, and dementia . Conversely, even minor increases in inflammation such as the ones observed in depression are enough to strongly predict the development over time of many of these modern disease states, including depression . 5,7 the second point that there is a high level of overlap of inflammatory biomarker levels between depressed and nondepressed groups raises a more complex issue . When we say that groups of depressed people tend to have elevated levels of inflammatory biomarkers, what we really mean is that within any depressed group, there are individuals with levels that are significantly higher than those seen in the vast majority of healthy, nondepressed people, whereas there are many other depressed people with perfectly normal values . It is a dirty little secret of sorts that the one - third or so of depressed individuals with elevated inflammation have been pulling all their noninflamed, depressed colleagues along with them in publication after publication, giving the world a slightly misguided sense that depression as a whole is driven by increased inflammation . The critical question is whether inflammation is relevant to depression as a whole or only to individuals with chronically elevated inflammatory biomarkers . And if depression is relevant only to those with increased inflammation, how much of an increase needs to exist before it reliably contributes to depressive pathogenesis? And might it be the case that people with depression and low levels of inflammation are just more sensitive to the depressogenic effects of inflammatory activity, so that even low levels disrupt brain functioning in ways that promote the disorder? A recent study from our group provides some surprising, tentative answers to these questions . Determined to see if peripheral inflammatory processes really contribute to depressive pathogenesis, we decided to put the theory to the test by examining whether blocking the inflammatory cascade would eradicate depression in patients who were otherwise medically stable . To test this as rigorously as possible, we elected to use a medication called infliximab, which is not believed to cross the blood - brain barrier a tight layer of cells and tissue that separates the brain from the rest of the body and has no biological effects other than to potently block the activity of tnf, the cytokine that along with il-1beta is most responsible for initiating the inflammatory response.8 we measured pretreatment levels of peripheral inflammation in 60 patients with treatment - resistant depression, which has been shown to have a special relationship with increased inflammation, in part related to the ability of cytokines to sabotage and circumvent the mechanism of action of antidepressants . Patients were then randomized to receive three infusions of either infliximab or saline in a blinded manner over a six - week period . We followed depressive symptoms during this period and for six weeks following the final infusion . For the group as a whole, infliximab was no better than placebo in fact, it was nearly identical . This strongly suggests that inflammatory processes are not directly causal for the entire spectrum of pathology that we currently characterize as depression . Interestingly, however, we found a significant relationship between pretreatment levels of inflammation and clinical response . Indeed, for people with baseline levels of c - reactive protein above 5 mg / l, infliximab outperformed placebo to the same degree that standard antidepressants do . What we were also not expecting was that for depressed individuals with low levels of peripheral inflammation, placebo far outperformed infliximab, suggesting that blocking inflammation was not just neutral, but was actually doing something profoundly negative in these individuals . At the least it was blocking people s ability to respond to placebo . Animal work by psychobiologist raz yirmiya and others had already suggested that at lower concentrations, inflammatory molecules such as tnf play important roles in processes associated with positive brain functioning, including neurogenesis, synaptic scaling, and long - term potentiation.9 more recently, the antidepressant - like effect of deep brain stimulation (dbs) in rodents has been shown to be associated not with electrode placement or even with the passage of electric current, but rather with the local brain inflammation induced by needle placement.10 consistent with this, the use of anti - inflammatory agents in humans seems to block the efficacy of dbs in patients with treatment - resistant depression . This negative association between anti - inflammatory agents and response was also noted with standard antidepressants in the star*d trial.11 taken together, these results suggest that the relationship between inflammation and depression may be u shaped, with both very low levels and high levels of activity posing risks, albeit for different reasons . Nevertheless, the modern world is so replete with depression risk factors that also promote inflammation including obesity, sedentary lifestyle, sleep loss, psychosocial stress, processed foods, air pollution, and perhaps hygienic practices (separating us from exposure to a range of coevolved microorganisms and parasites with powerful immunomodulatory and anti - inflammatory effects12)that the association between inflammation and depression is hardly surprising . This is especially true given that relatively mild elevations in peripheral inflammatory activity when chronic seem sufficient to set in motion physiological mechanisms that are depressogenic, particularly in individuals made vulnerable by early environment and/or genetic inheritance.13 it is to these mechanisms that we now turn . When we started exploring how inflammation produces depression, we were convinced that depressogenic pathways unique to the immune system would be identified; leading to the conclusion that depression that occurs in response to stress or comes out of the blue is biologically different from depression induced by immune activation . One of the most surprising, and consistent, findings over the last decade has been that inflammatory processes induce depression because they are capable of tapping in to every known risk pathway . In this regard, inflammation functions like psychological stress, which has similar wide - ranging, and generally depressogenic, biological effects . In fact, inflammation causes most of the same changes in the brain and body that have been repeatedly observed in animals and humans exposed to stress, especially when the stress is chronic and of a psychosocial nature.1 one of the best models for understanding the impact of chronic inflammation on humans has been treatment with the cytokine interferon (ifn)-alpha for either cancer or hepatitis c virus infection . Ifn - alpha causes full - blown depression in a respectable minority of patients, produces depressive symptoms in a far higher percentage, and produces an improvement in mood in no one but the unfortunate few who develop mania in response to the treatment.14 our understanding of the central - nervous - system (cns) effects of peripheral inflammation also have been augmented by studies of humans who became acutely inflamed as a result of receiving a dose of lipopolysaccharide or a typhoid vaccine . Figure 2 (see below) provides a schematic of the neurobiological pathways known to be influenced by both acute and chronic inflammatory stimuli in humans, ultimately leading to alterations in neurocircuitry and behavior . Neuroimaging studies reliably indicate that peripheral inflammation targets brain regions repeatedly implicated in the pathophysiology of depression, especially the anterior cingulate cortex (acc) and basal ganglia.15 depending on the nature of the stimulus, the impact of cytokines on the acc is seen most strongly in either the subgenual or the dorsal area.16,17 cytokine - induced increases in neural activity in these regions have been associated with the development of mood and anxiety symptoms . On the other hand, peripheral immune activation has been shown repeatedly to impair basal ganglia functioning in ways that are consistent with the known inhibitory effects of cytokines on dopamine signaling in the cns.18,19 reductions in basal ganglia activity have been noted in more posterior regions, where they associate with fatigue, and in more ventral regions (such as the nucleus accumbens), where they have been associated with the development of anhedonia, a psychological condition characterized by an inability to experience enjoyment in normally pleasurable acts.18 regarding the mechanisms of the effects of cytokines on these and other brain regions, cytokines have been shown repeatedly to alter neurotransmitter signaling in the cns in ways that are relevant to the pathophysiology of depression and its treatment . For example, through activation of the intracellular signaling pathway mitogen - activated protein kinase, cytokines can increase the number and function of the reuptake pumps for serotonin, norepinephrine, and dopamine, which in turn can reduce the availability of these neurotransmitters within the synaptic cleft . This is relevant to depression and its treatment given that most currently available antidepressants act by blocking these reuptake pumps to increase neurotransmitter availability in the synapse . Cytokines have other effects known to impact neurotransmitter availability . Indeed, by activating enzyme indoleamine 2, 3-dioxygenase, cytokines can shunt tryptophan away from the production of serotonin and into the production of kynurenine . Kynurenine is transported to the brain and can be converted by activated microglia (innate cells in the brain) to the neurotoxic metabolite quinolinic acid . The clinical relevance of this process has been shown by the association between cerebrospinal fluid levels of kynurenine and quinolinic acid, and the development of depression during treatment with ifn - alpha.1 moreover, increased quinolinic acid has been found in activated microglia in the acc of suicide victims who were depressed . Quinolinic acid can impact glutamate signaling in ways relevant to depression, including the stimulation of extrasynaptic n - methyl - d - aspartate (nmda) receptors, which lead to the downregulation of the production of brain - derived neurotrophic factor (bdnf), a potent inducer of neurogenesis . Consistent with this and other activities of inflammatory cytokines, animal and human studies have demonstrated that increased inflammatory cytokines can reduce central levels of bdnf and neurogenesis, leading to depressive - like behavior.20 depression is not just a brain disease . Indeed, many of the depression - related physiological abnormalities identified at the dawn of biological psychiatry involved the body s stress system and especially the hypothalamic - pituitary - adrenal (hpa) axis . As a group, depressed individuals have been repeatedly reported to demonstrate increased circulating cortisol and concomitant glucocorticoid resistance (e.g. Decreased sensitivity to the inhibitory effects of glucocorticoids on hpa axis regulation and inflammation).21 depressed patients also show a flatter diurnal pattern of cortisol secretion than do healthy control subjects . Strikingly, inflammatory cytokines have been shown to be capable of producing all these abnormalities, including glucocorticoid resistance, and in the context of treatment with ifn - alpha, flattening of the cortisol slope strongly predicts the development of depression.22 further suggesting a link between inflammation and depression is the fact that psychosocial stress which is a primary risk factor for depression development this activation is measured either as increases in plasma concentrations of inflammatory cytokines such as il-1beta and il-6 or as increased activation of the intracellular inflammatory transcription element nuclear factor - kappa beta (nfkb).1 although not established in humans, psychological stressors in laboratory animals have been shown repeatedly to increase levels of proinflammatory cytokines in the cns, especially in areas, such as the hippocampus, that are integrally involved in the mammalian stress response and repeatedly implicated in depression . Importantly, in these animal models, the behavioral and biological effects of stress (such as reductions in bdnf) can be made more tolerable by blocking the stress - induced increases in cns inflammatory signaling.20 in animal models, stress has been shown to activate cns microglial cells, which take on an inflammatory phenotype when activated.23 of relevance to depression, several postmortem studies point to evidence of microglial activation in individuals who died by suicide.24 also linking inflammation to depression is the fact that individuals at high risk for depression such as those exposed to early - life adversity respond to laboratory psychosocial stressors with more robust inflammatory responses than do others.25 inflammatory responses to these types of stressors have, in turn, been shown to predict the future development of depression . The science is clear: while depression is not an inflammatory condition, inflammation can cause depression, and inflammatory cytokines are clearly a factor . It suggests that inflammatory processes will be highly relevant to some individuals with depression, irrelevant to others, and perhaps even of benefit to a minority of depressed individuals . Recent data suggest that something as simple as the widely available blood test for c - reactive protein may identify individuals with greater or lesser likelihood of responding to anti - inflammatory therapeutic strategies and, by extension, individuals for whom inflammation is more or less of a causative factor . Such identification of patients with depression and increased inflammation represents a critical first step to the personalization of care, allowing specific treatments to be directed to specific pathologies that can then be monitored as a function of response . Such a development represents a game changer in psychiatry and truly emphasizes that when it comes to treatment for depression, one size does not fit all.
Sodium picosulfate / magnesium citrate (sp / mc) powder has been widely used for colon cleansing because it has an acceptable taste and a small volume of liquid is required after dissolving it . The effectiveness, safety, and ease of administration of this orange - flavored powder are well established . Adverse effects are rare in clinical practice, although it may induce mucosal inflammation . Here, we report the case of a patient who directly ingested sp / mc powder without first dissolving it in water, resulting in severe mucosal injury that extended from the oral cavity to the upper esophagus and caused airway compromise . A 59-year - old man was brought to the emergency department (ed) in an ambulance with a chief complaint of sore throat . He was scheduled to undergo colonoscopy that day, and 2 h prior, he had unintentionally ingested a pack of undissolved sp / mc powder (picolight powder; pharmbio korea co., seoul, korea) that had been prescribed for bowel evacuation . Upon arrival in the ed, he was hemodynamically stable and did not have respiratory distress, but he complained of progressive hoarseness . The emergency physician noted gross mucosal injury of the oropharynx, and examination using a fiberoptic laryngoscope revealed diffuse mucosal swelling and erosion extending from the oral cavity to the epiglottis, which obstructed the view of the vocal cords (fig . Endotracheal intubation was performed because his respiratory status was predicted to deteriorate, although he was well oxygenated and ventilated at that time . After admission to the intensive care unit, the upper gastrointestinal tract was examined using a flexible endoscope, and mucosal hyperemia, swelling, hemorrhage, and some necrotic changes extending from the oral cavity to the pharynx and epiglottis were observed . Supportive therapy was maintained, including intravenous steroid administration to reduce airway swelling, and he was examined regularly to evaluate the airway status and assess the feasibility of extubation . Early tracheostomy was considered, but it was not performed in the first week after admission because he refused the procedure; therefore, we decided to wait for spontaneous recovery . However, on day 12 of hospitalization, fiberoptic examination and computed tomography of the neck revealed persistent stenosis of the supraglottic and subglottic areas (fig . Therefore, tracheostomy was performed on day 14 of admission, and remnant mucosal damage was still present (fig . The tracheostomy tube was successfully removed after confirming recovery, and the stoma was closed on day 21 of hospitalization . He visited the outpatient clinic for follow - up 1 month after discharge, and no functional or structural sequelae were noted . Sp / mc powder, composed of sodium picosulfate, magnesium oxide, and citric acid, is widely prescribed for bowel preparation . When the powder is dissolved in water, magnesium oxide and citric acid react to form magnesium citrate . In solution, sodium picosulfate acts as a laxative resulting in augmented peristalsis, and magnesium citrate acts as an osmotic laxative and is not absorbed in the gastrointestinal tract . Side effects of sp / mc are uncommon; however, it has been reported to induce nausea, vomiting, abdominal pain, dehydration, and electrolyte disturbances . The most serious known adverse effect is severe electrolyte imbalance that includes hyponatremia, and rapid worsening of hyponatremia can lead to death . To our knowledge, this is the first report of life - threatening upper airway mucosal damage induced by direct ingestion of sp / mc powder . Reported esophageal and gastric mucosal ulcers that resulted from the inadvertent ingestion of sp / mc powder . However, it may be related to direct thermal injury from heat generated when the powder mixes with a small volume of water or secretions . Citric acid may also cause caustic injury to the mucosa . Considering that sp / mc powder is commonly used for bowel cleansing, ed physicians and those who prescribe sp / mc powder should be aware that serious mucosal injury can occur if the powder is directly ingested without dissolving it in water in particular, health care providers should emphasize that sp / mc powder should be dissolved in an adequate volume of water.
New targeted inhibitors of bcell receptor, such as ibrutinib, yield remarkable responses in patients with cll . An 83yearold man with cll refractory to rituximab and bendamustine presented a massive enlargement of mesenteric lymph nodes without histologic transformation into richter syndrome . A transient lymphocytosis up to 85 10/l after 1 month of therapy was considered to be treatment related 1 . On the other hand, sequential ct scanning demonstrated a progressive shrinkage of the abdominal lymph nodes with the best overall response obtained at 12 months (fig . The concomitant minimal residual disease (mrd) negativity in the blood or bone marrow denoted complete response (international workshop on cll criteria) 2 . Progressive changes in abdominal lymph nodes by ct scan in a patient with cll treated with ibrutinib . Images refer to baseline (panel a) and to evaluation after 3 (panel b), 6 (panel c), 12 (panel d), and 24 (panel e) months of therapy.
Urinary tract infection (uti) is the commonest bacterial infectious disease in community practice with a high rate of morbidity and financial cost . It has been estimated that 150 million people were infected with uti per annum worldwide which costing global economy more than 6 billion us dollars . The term cystitis has been used to define the lower uti infection and is characterized by symptoms such as dysuria, frequency, urgency, and suprapubic tenderness . The presence of the lower uti symptoms does not exclude the upper uti which is often present in most uti cases . The treatment of uti can be classified into uncomplicated and complicated on the basis of their choice of treatment . Uti is more common in females than in males as female urethra structurally found less effective for preventing the bacterial entry . It may be due to the proximity of the genital tract and urethra and adherence of urothelial mucosa to the mucopolysaccharide lining . The other main factors which make females more prone to uti are pregnancy and sexual activity . In pregnancy, the physiological increase in plasma volume and decrease in urine concentration develop glycosuria in up to 70% women which ultimately leads to bacterial growth in urine . Also in the nonpregnant state the uterus is situated over the bladder whereas in the pregnant state the enlarged uterus affects the urinary tract . Sexual activity in females also increases the risk of urethra contamination as the bacteria could be pushed into the urethra during sexual intercourse as well as bacteria being massaged up the urethra into the bladder during child birth [11, 12]. Using a diaphragm also causes uti as it pushes against the urethra and makes the urethra unable to empty the bladder completely and the small concentration of urine left in the bladder leads to the growth of bacteria which ultimately causes uti . The spectrum of bacteria causing complicated uti is much broader than of those causing uncomplicated uti . However, the most commonly encountered microorganisms are gram negative bacteria including escherichia coli, citrobacter spp ., enterobacter aerogenes, pseudomonas aeruginosa, and proteus vulgaris whereas klebsiella spp . The infectious disease society of america (idsa) identified some microorganisms for new effective therapies . Increasing drug resistance in uti needs regular monitoring of the antibiotic susceptibility of uropathogens in a particular area . Various factors such as the type of uti (complicated or uncomplicated), gender, age, and previous history of antibiotic therapy of each uti patient should also be considered to find out the correct global data on susceptibility . The distribution of antimicrobial susceptibility data of uti - causing microorganisms changes from time to time and from place to place . The susceptibility data provided by regional microbiology laboratories helps to choose the empirical choice of antimicrobials to treat uti; however, these conditions are limited to complicated uti as the samples of uncomplicated uti are rarely sent to laboratories [16, 17]. Generally, the antimicrobial treatment is initiated before the laboratories results which may lead to the frequent misuse of antibiotics . The resistance pattern of community acquired uropathogens has not been extensively studied in india [1921]. To the best of our knowledge, no data regarding the bacterial resistance in utis from meerut district (uttar pradesh), india, has been documented . Since most utis are treated empirically, the criteria for the selection of antimicrobial agents should be determined on the basis of the most likely pathogen and its expected resistance pattern in a geographic area . Therefore there is a need for periodic monitoring of etiologic agents of uti and their resistance pattern in the community . This study was undertaken in view of paucity of reports of utis in patients of meerut district (uttar pradesh), india . The aim of the study is to determine the prevalence of uti in male and female patients as well as the effect of gender and age on its prevalence . The uti - causing microorganisms, their distribution among different ages and genders, and their antimicrobial susceptibility will also be determined . The study was carried out in the microbiology laboratory of the department of botany, meerut college, meerut (uttar pradesh), india . The urine samples were collected from the opds (outpatients departments) section of three major hospitals (meerut kidney hospital, pyarelal hospital, and jaswant rai hospital) of meerut city . These sample collection sites were chosen as they mostly covered the urban area of the city . The duration of the study was one and a half year from july 2011 to january 2013 . The urine samples of 288 patients, comprised of 148 males and 140 females, who attended the outpatient departments (opds) of three hospitals and had clinical evidence of urinary tract infection, determined by treating physicians, were included in this study . Patients with history of hospital admission a week before their presentation in opds were excluded from the study to rule out hospital - acquired infections . Clean catch midstream urine was collected from each patient into a 20 ml calibrated sterile screw - capped universal container which was distributed to the patients . The specimens were labeled, transported to the laboratory, and analyzed within 6 hours . In each container boric acid (0.2 mg) was added to prevent the growth of bacteria in urine samples . All patients were well instructed on how to collect sample aseptically prior to sample collection to avoid contaminations from urethra . The study was conducted after due ethical approval which was subjected to the hospital administrations . A calibrated loop method was used for the isolation of bacterial pathogens from urinary samples . A sterile 4.0 mm platinum wired calibrated loop was used which delivered 0.001 ml of urine . A loopful urine sample was plated on cystine - lactose - electrolyte deficient (cled) agar, macconkey agar, and blood agar medium (hi media laboratories, mumbai, india). The inoculated plates were incubated at 37c for 24 h and for 48 h in negative cases . The number of isolated bacterial colonies was multiplied by 1000 for the estimation of bacterial load / ml of the urine sample . A specimen was considered positive for uti if an organism was cultured at a concentration of 10 cfu / ml or when an organism was cultured at a concentration of 10 cfu / ml and> 5 pus cells per high - power field were observed on microscopic examination of the urine . Identification of bacterial isolates was done on the basis of their cultural and biochemical characteristics . Gram negative bacteria were identified by the standard biochemical tests [14, 23] and gram positive microorganisms were identified with the corresponding laboratory tests: catalase, coagulase, and mannitol test for staphylococcus aureus . Identified and pure isolates were maintained in nutrient agar slants and incubated at 37c for 24 hrs . Isolates were tested for antimicrobial susceptibility testing by the standard kirby bauer's disc diffusion method . Standard inoculums adjusted to 0.5 mcfarland was swabbed on mueller hinton agar and was allowed to soak for 2 to 5 minutes . Mueller hinton agar plates were then incubated at 37c for 24 h. after 24 h the inhibition zones were measured and interpreted by the recommendations of clinical and laboratory standards . The following standard antibiotic discs were used for the isolates, ciprofloxacin (cip), moxifloxacin (mox), ofloxacin (ofl), sparfloxacin (spr), levofloxacin (lev), nalidixic acid (nal), gatifloxacin (gtx), tobramycin (tob), amikacin (amk), gentamycin (get), ceftazidime (ctz), cefotaxime (ctx), ceftriaxone (cfx), imipenem (imp), meropenem (mrp), nitrofurantoin (ntf), netillin (ntl) and co - trimoxazole (cot). Standard strains of e. coli (atcc 25922), s. aureus (atcc 25923), and p. aeruginosa (atcc 27853) were used routinely in this study as control . The mean of triplicates was considered and standard error of mean was calculated by microsoft excel ver . The following formula was used for the calculation of mar index of antibiotics: mar index for an antibiotic = [number of antibiotics resistant to the isolates/(number of antibiotics number of isolates)]. The data were analyzed using chi - square () test, confidence interval (ci), odds ratio (or) analysis, and student's t - test for paired samples . Relative risk and odds ratio were performed to compare the risk factors in the different groups of interest (male and female patients), and the chi square test was conducted to find out the significant difference between the isolated uropathogens, infected male and female patients related to different age groups, and statistical comparisons for the mar indices group; however, test for trend was conducted for antimicrobial resistance and sensitivity variables among all isolated uropathogens . A p value of <0.05 was considered as statistically significant for all tests and at 95% level of confidence interval . All statistical tests were performed by statistical package for social sciences (spss) software, inc . 233 south wacker drive, 11th floor chicago, il 60606 - 6412, usa, for windows, version 20 . The test for trend and graphs were prepared by graphpad prism software (version 5.03), inc . The overall prevalence of uti in both male and female patients was found to be 53.82% . Total 155 urine samples showed the significant bacterial growth which were comprised of 52 (35.14%) samples from males and 103 (73.57%) from females . These results indicated that the prevalence of uti was higher in female patients than in males . The p value and the odds ratio showed a the significant variation between male and female patients (table 1). The highest susceptible age group of patients to uti was 48 years (63.51%) followed by 2636 years (58.11%), 1525 years (54.55%), and 3747 years (39.19%). Comparatively, however, more cases of uti were observed in females than in males in all age groups . The highest prevalence of uti in females was found in the age group of 2636 years (90.69%); however in males the highest susceptible age group to uti was 48 years (71.15%). The chi square test showed statistically significant variations (p <0.05) at 95% level of confidence interval for the infected and not infected male and female patients variables among all age groups . For the infected and not infected male patients variable the chi - square test values were = 13.081; degree of freedom = 1; p = 0.000 and the values for infected and not infected female patients were = 31.114; degree of freedom = 1; p = 0.000 (table 2). The highest female to male ratio for the occurrence of uti was found in the age group of 1525 years (17: 1) followed by 2636 years (9.75: 1), 3747 years (2.22: 1), and 48 years (0.27: 1). The test for trend results showed significant variations (p <0.05) between the female to male ratio variables in all age groups at 95% confidence interval level (= 5.228; degree of freedom = 1; p = 0.0222) (figure 1). A total of 155 bacterial uropathogens comprised of 140 (90.32%) gram negative and 15 (9.68%) gram positive were isolated from positive urine samples . Escherichia coli was found the dominant bacteria among all isolated uropathogens with the prevalence rate of 42.58% . The second most prevalent isolate was klebsiella pneumoniae (18.71%) followed by pseudomonas aeruginosa (12.90%), staphylococcus aureus (9.68%), proteus spp . There was no statistically significant variation (p> 0.05) was found among the isolates (table 3). Out of 140 gram negative bacteria 50 (35.71%) were isolated from males and 90 (64.29%) were from female patients . Only 2 (13.33%) gram positive bacteria were isolated from male and 13 (86.67%) were isolated from female patients . The highest number of gram positive and negative uropathogens (39) was found in the female patients of the age group 2636 years followed by 37 uropathogens which were isolated from the male patients with the age group of 48 years (table 4). The highest to lowest prevalence rate for the occurrence of different isolated uropathogens within the age groups were as follows: e. coli48 years (36.36%); 1525 years (24.24%); 2636 years (21.21%); 3747 years (18.18%): k. pneumoniae1525 years (37.93%); 2636 years (27.59%); 48 years (24.14%); 3747 years (10.34%): p. aeruginosa2636 years (35.00%); 48 years (30.00%); 3747 years (25.00%); 1525 years (10.00%): proteus spp.3747 years (35.71%); 48 years and 2636 years (28.57%); 1525 years (7.14%): enterobacter spp.2636 years (45.45%); 48 years and 2636 years (27.27%); 3747 years (0.00%): s. aureus2636 years (33.33%); 3747 years (26.67%); 48 years and 1525 years (20.00%) (figure 2). Overall nal was found the most resistant drug as 122 (78.71%) uropathogens were found resistant against nal . The second most resistant drug was ctz (71.61%) followed by ctx (67.74%); however, the most sensitive drug against all uropathogens was mrp (92.26%) followed by imp (84.52%), lev, and ntl each showing 74.84% sensitivity (figure 3). The test for trend results showed a statistically significant variation (p <0.05) between the resistant and sensitive variables (= 9.152; degree of freedom = 1; p = 0.0025). Tob was found the highest resistant drug against 96.97% e. coli followed by nal (90.91%) and ctx (87.88%); however, both carbapenems imp and mrp showed the highest sensitivity against 98.45% and 95.45% e. coli . 79.31% of k. pneumoniae were resistant against ctz and lev was found the most susceptible drug with the rate of 89.66% . In case of p. aeruginosa the highest resistant and susceptible antibiotics were spr (100%), and mrp (100%) respectively . Were resistant against cfx and 100% sensitive against both carbapenems (imp and mrp). . Showed 81.82% resistance against ntf; however, all (100%) were sensitive to ofl, spr, lev, imp, and mrp . All s. aureus (100%) showed resistance against nal and ctx; however, imp was found 100% sensitive followed by spr, cfx, and ntl (each showed 93.33% sensitivity against s. aureus isolates) (table 5). The results of the paired t - test showed that there was no statistical significance between e. coli resistant versus sensitive variables (p = 0.876), k. pneumoniae resistant versus sensitive variables (p = 0.232), p. aeruginosa resistant versus sensitive variables (p = 0.950), proteus spp . Resistant versus sensitive variables (p = 0.162) and s. aureus resistant versus sensitive variables (p = 0.072), however, enterobacter spp . Showed the significant variations between resistant versus sensitive variables (p = 0.000). The highest mar index was found for nal (0.044) followed by ctz (0.039) and ctx (0.038) indicating that these antibiotics were highly resistant among all tested uropathogens; however, the lowest mar index was found for both carbapenems mrp and imp which were 0.004 and 0.008, respectively, indicating the highest sensitivity against uropathogens . The test results showed no statistically significant variation among the mar indices of all tested antibiotics (= 1.556; degree of freedom = 15; p = 1.000). This study provides valuable data to compare and monitor the status of antimicrobial resistance among uropathogens to improve efficient empirical treatment . Increasing antimicrobial resistance has been documented globally [2733]. The prevalence of uti was found to be 53.82% in this study and this rate of prevalence is higher than in the other studies which accounts for 25.6%, 22%, 38.6%, 35.5%, 4.2%, 17.19%, 10.86%, 34.5%, and 36.68% in india; however, the prevalence rate of uti in our study correlates with other studies done in south trinidad, and in the mexican population which showed such more highly significant uropathogens 49% and 97.3%, respectively . Our study showed a high prevalence of uti in females (73.57%) than in males (35.14%) which correlates with other findings which revealed that the frequency of uti is greater in females as compared to males [6, 30, 4044]. The reason behind this high prevalence of uti in females is due to close proximity of the urethral meatus to the anus, shorter urethra, sexual intercourse, incontinence, and bad toilet [4547]. The occurrence of uti recorded among the elderly (48 years, 63.51%) compared to young age patients (2637 years, 58.11%; 1525 years, 54.55%) and middle - age patients (3747 years, 39.19%) in this study differs from the other studies done in kuwait and nigeria in which the highest incidence of uti was recorded among the age group 20 to 50 years (63.4 and 74.7%, resp .) And lowest among the age group> 50 years (13.3 and 10.3%, resp . ). However, our results agree with the study done in japan with a 20-year period in which a trend of increasing complicated uti was reported in elderly patients . In our study it was found that the elderly males (48 years) had a higher incidence of uti (71.15%) when compared with the elderly females (45.45%). This finding is similar to a study conducted at a tertiary care hospital in jaipur, rajasthan, india . The main cause behind this increasing incidence of uti with advancing age in males is due to prostate enlargement and neurogenic bladder . This factor is also reported by other authors whose studies showed that the prostate disease in males is responsible for the increase in incidence of uti and decrease in female: male ratio in patients above 50 years . Females of the age group 2636 years were found more susceptible (90.69%) to uti followed by 1525 years (82.93%), 3747 years (58.82%), and 48 years (45.45%). These findings correlate with other reports which showed that females are more prone to utis than males during adolescence and adulthood [12, 18, 20, 44, 5358]. The factors of this increasing incidence of uti in young age females are associated with high sexual activity, recent use of a diaphragm with spermicide, and a history of recurrent utis . The highest incidence of utis among female to male ratio was found in the age group of 1525 years (17: 1) followed by 2636 years (9.75: 1), 3747 years (2.22: 1), and 48 years (0.27: 1). These findings differ from other reports [57, 60] which stated a lower female to male ratio in neonates and young children . The prevalence rate of uti in boys depends on many factors including congenital malformations and uncircumcised genitalia which are often contaminated . In this study, the gram negative bacilli constituted 90.32% of the total bacterial isolates while gram positive cocci constituted 9.68% . Escherichia coli (42.58%) was found the most prevalent gram negative bacteria in the positive urine samples of uti . This result is consistent with reports from other studies [38, 48, 49, 53, 6163] but differs from the reports in which p. aeruginosa and klebsiella spp . Other isolated bacteria from uti cases in this study were k. pneumoniae (18.71%), p. aeruginosa (12.90%), s. aureus (9.68%), proteus spp . These findings were not correlate with other reports in which p. aeruginosa was reported as the second most common bacterial isolate in uti studies in india and lafia, nigeria; however, these results correlates with others in which klebsiella spp . Was reported as the second most frequently isolated organism in uti [32, 54, 63, 66, 67]. The studies on uti in other places of the world also showed that e. coli and klebsiella spp . Higher incidence of gram negative bacteria, related to enterobacteriaceae, in causing uti has many factors which are responsible for their attachment to the uroepithelium . In addition, they are able to colonize in the urogenital mucosa with adhesins, pili, fimbriae, and p-1 blood group phenotype receptor . In females of all age categories, e. coli is the most frequently isolated uropathogen which correlates with other studies [7173] but not with others which found that e. coli causes most male utis, followed by other enterobacteriaceae and enterococci [74, 75] whereas proteus mirabilis was more frequently isolated in the younger female patients of uti and k. pneumoniae in the elderly patients . Both carbepenems (mrp and imp) used in this study were found to be the most sensitive drugs against all isolated uropathogens . The sensitivity rate of carbepenems among uropathogens was as follows: e. coli (mrp; 95.45% and imp; 98.89%), p. aeruginosa (mrp; 100% and imp; 95.00%), proteus spp . (mrp; 100% and imp; 100%), and s. aureus (mrp; 80% and imp; 100%), followed by lev and ntl each of which showed 74.84% sensitivity, however, k. pneumonia did not show a high susceptibility to imp (24.14%) but it was susceptible to mrp (86.21%). Another study conducted in india showed that meropenem was highly sensitive against gram negative bacilli whereas cephalosporin showed highest resistance against gram negative rods . In other study, meropenem and imipenem were found to be 98% and 100% sensitive, respectively, against highly resistant gram negative bacilli . A study done in king fahd hospital, saudi arabia showed that meropenem was 95.8% sensitive followed by amikacin (93.7%) and imipenem (91.71%) against extended spectrum lactamase producing e. coli . Tested fluoroquinolones in this study showed the highest resistance among uropathogens as in e. coli; nal (90.91%): k. pneumoniae; cip (79.31%), p. aeruginosa; spr (100%), and s. aureus; nal (100%); however, iii generation cephalosporin showed the highest resistance in k. pneumoniae; ctz (79.31%) the proteus spp . ; . This high rate of resistance against fluoroquninolones was also suggested by other studies done in spain, europe, and iran [33, 81] and also by other studies done in india [21, 44, 82]. Another study done in spain also showed the reduced susceptibility of e. coli isolates from patients with uti to fluoroquinolones (16%). In several studies it has been shown that the highly prescribing habits of the physicians are the driving factor for the antibiotic resistance for this group of antibiotic [8385]. Mcewen et al . Found that 37% of physicians actually prescribe trimethoprim - sulphamethoxazole closely followed by fluoroquinolones (32%) and the average duration of antibiotic therapy is 8.6 days in the united states which is the best example of this problem; empiric use of fluoroquinolones should be restricted and founding the strategies against increasing resistance of pathogens to these antibiotics should be done . Our finding about the fluoroquinolones did not correlate with others which showed that they were highly effective (sensitive) [11, 55, 64, 87, 88]. For these organisms, drugs with inhibitors like augmentin may be tried but such drugs should be reserved for the last line of treatment . The alarming finding in this study is the resistance to third - generation cephalosporin; the highest resistance was seen against ctz (71.61%) followed by ctx (67.74%) among all uropathogens . The other possible explanation behind this situation is that the iii generation cephalosporin has been in use for a long period and must have been abused and over time organisms have developed resistant mechanisms due to changing their mode of action . The inappropriate usage of wide spectrum antibiotics, insufficient hygiene, immunosuppression, and a prolonged stay in the hospital are some other major etiological factors that elevate the chances of mdr infections . Against the background of paucity of reports of uti in meerut city (uttar pradesh), india, this is the first study conducted to determine the prevalence of uti, the effect of gender and age on its prevalence, and their susceptibility profile in the community of meerut city . This study provides valuable laboratory data to monitor the status of antimicrobial resistance among uropathogens and to improve treatment recommendations in a specific geographical region . The study also allows comparison of the situation in meerut with other regions within and outside the state as well as in the country.
No clear etiology has been associated with most ependymomas to date . Unlike many other cancers for which existing familial cancer syndromes provided important clues for our initial understanding of tumorigenic mechanisms, there are few known familial ependymoma syndromes . We do know, however, that there is increased incidence of spinal intramedullary ependymomas in patients with neurofibromatosis type 2 (nf2),.the nf2 gene is located on chromosome 22q, which is frequently lost in patients with spinal ependymomas. However, many of these tumors, especially those that occur intracranially, do not harbor nf2 mutations . Thus, despite that the nf2 gene may be important in the formation of some spinal ependymomas, it is probably not the critical tumor suppressor gene on chromosome 22q that is involved in sporadic intracranial ependymoma tumorigenesis. Ependymoma has also been reported in patients with li - fraumeni syndrome, i.e. Germline mutation of the tp53 tumor suppressor gene, but such occurrences as well as somatic mutations of tp53 in sporadic ependymomas are rare, thus diminishing the role of p53 in ependymoma tumorigenesis . There has been one report of a patient with turcot syndrome, i.e. Germline mutation of the adenomatous polyposis coli (apc) gene, whose loss of function activates the wnt pathway and predisposes the patient to colorectal cancer, who developed multiple ependymomas located intracranially and spinally, . Both intracranial and spinal ependymomas have also been observed in patients with the multiple endocrine neoplasia type i (men1) syndrome. However, the role of the apc gene / wnt signaling activation and that of men1 in sporadic ependymoma tumorigenesis remain unknown . Furthermore, there are a few families with increased ependymoma incidence but without any currently known familial cancer syndromes. Two such families have loss of 22q but lack nf2 mutation, further suggesting the presence of another crucial tumor suppressor gene at that chromosomal region, . Dna sequences similar to sv40 virus and the virus - encoded large t - antigen have also been found in some ependymomas. Furthermore, ependymoma can be induced in rodents through intracerebral inoculation of the sv40 virus, . Nevertheless, several studies have disqualified the sv40 tumor virus as a causative agent of ependymoma. The strongest opposing argument is based on epidemiologic studies that showed no increase in the incidence of ependymoma and other cancers in the years following the massive introduction of sv40-contaminated polio vaccines into the human population, . To date, knowledge of whether sv40 virus contributes to ependymoma tumorigenesis remains unknown; and if it does, the oncogenic pathways on which it acts remain to be elucidated . Over the years, cytogenetic studies using karyotyping and comparative genomic hybridization (cgh) have reported numerous broad chromosomal abnormalities in ependymoma . Results varied considerably among early studies largely due to their small sample sizes and the variations among sample sets in terms of patient age and anatomical tumor location . In fact, increasing evidence supports that ependymomas are heterogeneous and can be classified as distinct disease subtypes based on patient age, anatomical tumor location, and genetic alterations,, . Frequently observed genomic anomalies in pediatric ependymomas include loss of chromosomes 1p, 2, 3, 6/6q, 9p, 13q, 17, and 22 as well as gains of 1q, 5, 7, 8, 9, 11, 18, and 20, with the gain of 1q occurring in over 20% of cases being the most common . In adult ependymomas, chromosomes 6, 10, 13q, 14q, 16, and 22/22q are frequently lost whereas chromosomes 2, 5, 7, 9, 12, 18, and x are gained, with gains of 7 and 9 and loss of 22q being the most frequently observed, though with each occurring in only 30% of cases or less . Location - specific genomic anomalies observed in intracranial versus spinal ependymomas roughly correspond to those seen in children versus adults, as most pediatric ependymomas occur intracranially whereas adult cases predominantly occur within the spinal cord . Aside from genomic gains and losses, cytogenetic studies have also identified translocations within the ependymoma genome, often involving chromosomes 1, 11, and 22. adult ependymomas have been observed to display more frequent and broader chromosomal aberrations than pediatric tumors . Based on a meta - analysis of all cgh studies performed on more than 300 primary ependymomas, kilday et al . Calculated that there are on average 7.5 and 3.8 genomic anomalies per adult and pediatric tumor, respectively . This finding is reinforced given that over 40% of pediatric ependymomas exhibit balanced genetic profiles, whereas a balanced genomic profile is observed in less than 10% of adult cases,, . Interestingly, the large number of genomic aberrations often seen in adult spinal ependymomas is associated with tumors of lower histological grades and favorable patient outcome,, . Furthermore, according to the cgh analysis done by dyer et al . On pediatric intracranial ependymomas, tumors can be subdivided into three distinct subgroups based on the number of chromosomal anomalies detected per tumor . Tumors with a balanced genetic profile make up the balanced group, which is significantly associated with an infant age at diagnosis . The second structural group shows few and mainly partial genomic imbalances . Lastly, the third numerical group exhibits 13 or more primarily whole chromosome imbalances similar to those often seen in adult ependymomas . These subdivisions are significantly associated with prognosis, with the numerical group demonstrating the best patient outcome and the structural group doing the worst . Consistent with this observation, almost all recurrent ependymomas exhibit genetic profiles characteristic of the structural group,, . Despite the identification of the aforementioned common genomic gains and losses in ependymomas and their cytogenetic profile - based stratification, few insights into the oncogenes, tumor suppressors, and molecular pathways responsible for the development of ependymoma could be obtained from these findings . Furthermore, specific genetic events could not be identified as prognostic markers for this disease at only chromosome - level resolution . These chromosome - level aberrations are broad and typically span numerous genes, making it difficult to discriminate driver genetic events from passenger events . Recently, array cgh (acgh) has been adopted by the research community to fine - map copy number variations in cancer at much higher resolutions . The list of genes within the common regions of amplification or deletion identified using acgh can be further narrowed through correlation with their expression levels . This permits the discovery of candidate driver genes for ependymoma development, with putative oncogenes and tumor suppressor genes exhibiting copy number - driven expression . Indeed, the advances in microarray and next generation sequencing technologies have permitted the examination of ependymoma genetics in terms of copy number variations and gene expression levels in much greater detail . Irrespective of anatomical tumor location or patient age, monosomy 22 and allelic losses on chromosome 22q have been found in numerous studies to be the most common genetic abnormalities in sporadic ependymoma, with frequencies ranging from 26% to 71%, . Initial quests for tumor suppressor genes present on 22q focused on nf2 located at 22q12; however, nf2 mutation is not associated with the majority of ependymoma cases.another potential tumor suppressor gene is hsnf5/ini1 at 22q11.23 . Kraus et al . Found no mutations or homozygous deletions of this gene in a series of 53 ependymomas, and this gene has not been shown to be silenced by dna promoter methylation . Mapping of deletions and translocation breakpoints on 22q using high - resolution techniques revealed 22pter22q11.2, 22q11, 22q11.2112.2, and 22q13.113.3 to be the hotspots where the elusive tumor suppressor gene is likely to be found,. Within the frequently deleted region 22q12.3q13.33, karakoula et al . Found rac2 and c22orf2 to be deleted in 38% and 32% of the 47 pediatric intracranial ependymomas analyzed, respectively . In over 60% of these ependymomas, c22orf2 was found to be transcriptionally inactive, indicating its potential importance in the development of pediatric intracranial ependymomas . Loss of rac2, on the other hand, was shown to be a prognostic factor significantly associated with shorter overall survival in patients younger than 2 years . Using gene expression microarray technology, suarez - merino et al . Found the transcripts of four genes mapping to 22q12.322q13.33, namely c22orf2, as identified by karakoula et al . Mentioned above, fbx7, cbx7, and sbf1, to be under - expressed in pediatric ependymomas as compared to normal brain controls . Allelic loss of one of these genes, cbx7 located at 22q13.1, could be detected in 55% of ependymoma cases . Interestingly, cbx7 controls cellular lifespan through regulating both the p16/rb and the arf / p53 pathways . The role of these pathways in ependymoma is unclear, though their deregulation is central to many types of cancer, including gliomas . Furthermore, deletion and hypermethylation of cdkn2a / p16 at 9q21.3 and rb at 13q14.2 have been reported in ependymomas. In the same study by suarez - merino et al . Schip-1 is known to interact with the nf2 gene product merlin, and their interaction is regulated by conformational changes in merlin induced by post - translational modifications, alternative splicing, or mutations . Furthermore, by integrating the genomic and expression profiles of 24 primary intracranial ependymomas, modena et al . Identified down - regulation of the sult4a1 gene located at 22q13.3 . In pediatric intracranial ependymomas, importantly, this genetic aberration is preferentially associated with tumors in the posterior fossa location in children and with anaplastic histological features . It is also a significant predictor of tumor aggressiveness and poor patient outcome,,,,,. Interestingly, 1q gain is occasionally the only observable alteration, with few other chromosome imbalances detected in ependymomas,,,; yet, in some cases, it marks tumor recurrence, . This suggests the presence of genes located on 1q that may be involved in the initiation, progression, and/or therapeutic resistance of ependymoma . Thus, efforts have been made to determine the critical region on chromosome 1q for the identification of these crucial genes . Reported a minimal overlapping region with high - copy amplification at 1q2431 in pediatric ependymomas . Subsequently, an acgh study done on 49 sporadic intracranial ependymomas by mendrzyk et al . Identified two commonly gained regions on 1q, one at 1q21.323.1 and another at 1q31.131.3 . They also found that gains of 1q21.132.1 were correlated with tumor recurrence and identified the gain of 1q25 as an independent prognostic marker for significantly lower recurrence - free or overall survival rate . Additionally, they identified dusp12, found to be over - expressed in all their tested samples, as a candidate gene located at 1q23.3 . The mrna level of dusp12 correlates with that of cyclin d1 throughout the cell cycle, suggesting its role in regulating cell division and potentially in neoplastic transformation, . Dusp12 was also found to be important for cell survival in response to heat - shock - induced cell death, which further supports its proposed oncogenic function . Gene expression analyses correlated with copy number variations have since uncovered additional candidate oncogenes located within 1q2132, including laminin, prelp, hspa6, gac1, chi3l1, tpr, jtb, shc1, and s100a10 and other s100 family members,,,, . Among these, gac1 amplification - driven over - expression has also been implicated in the pathogenesis of other malignant gliomas, suggesting its likely importance in ependymoma development . In addition to chromosome 22q loss and 1q gain, other commonly identified chromosomal aberrations include deletion of chromosomes 6q and 9 and gain of chromosome 7, notably the region from 7q11.2322.1, which is associated almost exclusively with spinal ependymomas,, . Candidate oncogenes proposed by analyzing recurrent gains on chromosome 7 include egfr (epidermal growth factor receptor) at 7p11.2, twist1 and hdac9 at 7p21.1, and arhgef5 at 7q34, egfr in particular exhibits frequent gains and high - level amplifications in intracranial ependymomas, and its over - expression predicts poor patient outcome . Loss of chromosome 6q is found mostly in infratentorial tumors, whereas deletions on chromosome 9 occur more frequently in supratentorial tumors,,, . With microsatellite analysis, loh hotspots on chromosome 6 were determined to be 6q1516, 6q2122.1, and 6q24.325.3, which were further limited to 6q24.3 and 6q25.225.3, . Locus 6q25.3, containing the snx9 and synj2 genes, was found to be the most frequently deleted . However, loss of 6q25.3 was a favorable prognostic marker for overall survival of patients with anaplastic intracranial ependymomas, as the deletion of the snx9 and synj2 genes, which are known to regulate cell migration and invasion, could inhibit tumor progression . Furthermore, the polyamine biosynthesis gene amd1 and the cyclin - dependent kinase cdk11, both located at 6q21, as well as the tumor suppressor gene sash1 at 6q24.3 were found to be under - expressed by suarez - merino et al . Which is also frequently deleted in patients with ependymomas, homozygous deletion spanning the cdkn2a locus at 9q21.3 has been detected and is a characteristic of anaplastic supratentorial tumors, the molecular staging system developed by korshunov et al . Highlighted that cdkn2a deletion together with young age at diagnosis and gain of 1q comprise the most reliable independent indicators of unfavorable patient outcome . In contrast, gains of chromosomes 9, 15q, and 18 and loss of chromosome 6 are features indicating excellent chance of survival . Furthermore, detection of the expression of p14 protein by immunohistochemistry in 103 intracranial ependymomas revealed that decreased p14 expression is associated with tumor aggressiveness in terms of higher tumor grade, elevated growth fraction, and p53 protein accumulation . Closely examined the aberrations on chromosome 9 in both adult and pediatric ependymomas and identified 9p21.122.3 and 9q31.333.2 to be the potential tumor suppressor genes located within 9q31.333.2 include dbc1, which is frequently deleted in bladder cancer and also exhibits markedly reduced mrna expression in gliomas,; dec1, whose down - regulation driven by copy number loss is frequently seen in esophageal cancer and contributes to tumor cell motility,; lpar1, which is known to mediate cell proliferation, differentiation, and migration among other functions; and txn, which inhibits apoptosis and enhances drug resistance in cancer cells, . Other frequently occurring regions of genomic imbalances have been revealed by profiling the ependymoma genome at high resolution,,,,, and are summarized in table 1 . Among these imbalances, the combined presence of 6p22-pter and 13q14.3-qter losses predicted significantly reduced survival in intracranial pediatric ependymomas . Puget et al . Found that gains of 1q and 9qter and loss of 6q occurred more often in recurrent tumors . Interestingly, the specific 9qter region linked to tumor recurrence is associated with posterior fossa ependymomas, whereas chromosome 9 deletion is usually associated with supratentorial ependymomas . Candidate oncogenes and tumor suppressor genes proposed based on these copy number variation hotspots,,, are listed in table 2 . Among the putative oncogenes in ependymoma are notch1, notch4, and jag1, which are two of the membrane receptors and one of the ligands, respectively, of the notch signaling pathway, suggesting the involvement of notch signaling in ependymoma tumorigenesis . Furthermore, recurrent gains at 5p15.33, which includes the human telomerase reverse transcriptase (htert) gene, were validated by immunohistochemistry . Elevated htert expression has been shown to be associated with ependymoma progression and recurrence and is currently the most important predictor of survival for pediatric intracranial ependymomas independent of other clinicopathologic prognostic features,. Furthermore, htert expression relates with telomerase activity . Recently, wong et al . Proposed telomerase inhibition as a novel therapy for ependymoma after demonstrating its effects on reducing ependymoma cell viability by increasing dna damage, decreasing proliferation, and increasing apoptosis . In addition to fine - mapping genomic aberrations to identify candidate genes involved in ependymoma development, profiling studies have also been used to divide ependymomas into distinct subgroups that correlate with tumor location . Categorized these tumors into three molecularly distinct subgroups that correlate with the anatomical location of the tumor, namely the supratentorial region, the posterior fossa, or the spine . Although ependymomas from these different anatomical regions are histologically indistinguishable, they are in fact molecularly distinct diseases that should be separately examined to determine the genetic events involved in tumorigenesis and progression, as well as prognostic factors and patient outcome . According to the results of their acgh experiment, taylor et al . Found that cdkn2a deletion occurred in> 90% of supratentorial ependymomas but was rare in tumors from other regions of the central nervous system (cns). Furthermore, posterior fossa ependymomas could be further classified into three subgroups: tumors harboring multiple concurrent dna amplifications, tumors with chromosome 1q gain, and tumors exhibiting a balanced genomic profile . Although acgh analyses have considerably advanced our understanding of the genetic events in ependymoma tumorigenesis, almost half of ependymomas present a balanced acgh profile, making it imperative to interrogate alternative mechanisms of gene regulation . Epigenetics in the form of promoter dna (cpg) hypermethylation is an important route by which transcriptional inactivation can be achieved and, as in other cancers, likely plays a significant role in silencing tumor suppressor genes involved in ependymoma development . Unfortunately, epigenetic studies on ependymoma have been limited to candidate gene approaches, with the genes in question selected based on their roles as tumor suppressor genes and methylation status in other malignancies . Therefore investigated the methylation status of the hypermethylated in cancer 1 (hic-1) putative tumor suppressor gene, which exhibits hypermethylation and loss of expression in various tumors such as medulloblastoma and gliomas . They detected hic-1 hypermethylation and down - regulation in 83% and 81% of ependymomas, respectively, and found that hic-1 hypermethylation was significantly correlated with nonspinal localization and pediatric age . The ras association domain family 1 isoform a (rassf1a) gene has also been found, independent of clinical and histological subtype, to be frequently silenced by methylation in ependymoma, with an incidence of 86% . Rassf1a is a recently well - recognized tumor suppressor gene whose inactivation through promoter methylation is implicated in the development of many human cancers . Rna interference experiments have shown that down - regulation of rassf1a, an effector of ras, results in loss of cell cycle control, enhanced genetic instability and cell motility, and resistance to k - ras and tumor necrosis factor (tnf)-induced apoptosis . Identified the trail apoptosis pathway - related genes casp8, tfrsf10c, tfrsf10d, and tnfrsf10c to be methylated in ependymoma, with incidences of 30%, 9.5%, 36.4%, and 9.5%, respectively . Other commonly methylated genes in ependymoma include dapk, thbs1, timp3, tp73, mgmt, gstp1, cdkn2a, fhit, rarb, blu, and mcj, with incidence ranging from 10% to 57%,,,, . Gene expression profiling employs microarray technology to capture gene expression levels of thousands of genes simultaneously . Integration of gene expression with copy number data allows one to determine the genes demonstrating copy number - driven expression as putative oncogenes and tumor suppressor genes . Moreover, by applying ontological analysis on the gene expression profiles, it is possible to uncover those aberrant cellular processes and pathways that contribute to ependymoma . Using microarray - based gene expression profiling to compare ependymoma with normal brain controls, suarez - merino et al . Identified 112 abnormally expressed genes in ependymoma . Genes with increased expression included the oncogene wnt5a, tp53 homologue tp63, and several cell cycle, proliferation, adhesion, and extracellular matrix genes such as the transcription factor zic1, the angiogenesis factor vegf, and fibronectin 1 (fn1). Other putative oncogenes identified in this study that have been implicated in other cancers are col4a1, ibp2, hox7, wee1, and gac1 . Genes that were found to be down - regulated included the a / f2-interacting gene schip-1, the apc - associated gene eb1, and genes that are involved in vesicle trafficking and recycling such as npc1, rab40b, tj2, and sh3gl3 . Consistent with ependymoma subgroups based on acgh profiles, ependymoma gene expression profiles are significantly associated with tumor location, patient age at disease onset, grade, and retrospective risk for relapse,,, . Found that supratentorial ependymomas expressed markedly elevated levels of members of the ephb - ephrin (ephb2/3/4 and ephrin a3/4) and notch (jagged 1/2) signaling pathways, as well as genes involved in cell cycle regulation (cyclin b2/d1/g2, cdk2/4, and cdkn1c/2c). On the other hand, the highly expressed genes that distinguished posterior fossa ependymomas were inhibitors of differentiation (id1/2/4) and members of the aquaporin family (aqp1/3/4). Spinal ependymomas are characterized by the up - regulation of multiple homeobox (hox) family members (hoxa7/9, hoxb6/7, and hoxc6/10) and insulin - like growth factor 1 (igf1). Subsequently, gene expression profiling studies performed by modena et al . And palm et al . Confirmed that intracranial ependymomas are indeed characterized by high expression levels of genes involved in notch signaling and that spinal ependymomas are defined by over - expression of numerous hox genes . Additionally, up - regulation of the sonic hedgehog (shh) and bone morphogenetic protein (bmp) pathway members were also evident in intracranial ependymomas, . Deregulated notch signaling, which is crucial for neural development, is believed to play a significant role in ependymoma tumorigenesis, especially at the supratentorial location, since oncogenesis is thought to mirror normal development gone awry . In addition to over - expression of the notch ligands jagged 1/2 shown by taylor et al ., there is consistent up - regulation of the notch receptors (notch1/2), ligands (jagged 1/2 and dll1/3), and target genes (hes1/5, hey2, c - myc, and erbb2), whereas fbxw7, the major repressor of the notch pathway, is consistently down - regulated,,, . In an early study, missense mutations of notch1, either in the heterodimerization domain c or the transactivation domain, were detected in 8.3% of pediatric intracranial ependymomas from the posterior fossa, thus making notch1 the first oncogene found to be mutated in ependymomas . These mutations cause the notch1 receptor to be constitutively active in a ligand - independent manner . Moreover, inhibition of notch signaling with -secretase inhibitor gsi - ix impaired ependymoma primary cell culture growth . Gilbertson et al . Further demonstrated that high - level expression of erbb receptors (erbb2/4), which are direct targets of notch signaling, could be found in over 75% of pediatric ependymomas and were significantly correlated to tumor proliferative activity as measured by the ki-67 labeling index . Functional studies proved that activating erbb receptor signaling in short - term ependymoma cell cultures resulted in akt phosphorylation and cell proliferation, which could be effectively blocked in a dose - dependent manner with an inhibitor of erbb2 tyrosine kinase activity . Coincidentally, we have recently learned that the sv40 virus, which was thought to be a causative agent for ependymoma, can in fact induce oncogenic transformation of human mesothelial cells through direct induction of notch1 over - expression . Currently, neither cell lines nor animal models are available to elucidate the sequential events in ependymoma development . Thus, researchers have compared the gene expression profiles of low grade versus high grade ependymomas and primary tumors versus recurrent tumors to better understand the molecular genetics of ependymoma progression . Palm et al . Revealed that who grade 3 anaplastic ependymomas differed from grade 2 tumors by the over - expression of genes implicated in wnt/-catenin signaling activation, cell cycle regulation / cell proliferation (cyclin - dependent kinases cdk2/4, cell division cycle proteins cdc25a/25b/25c/2, and minimal chromosome maintenance proteins mcm2/3/5/6/7), apoptosis (tumor necrosis factor super family members tnfrsf11a/21 and caspases casp1/4), angiogenesis (vegf, vegfr2, vegfb, tnip2, and doc2), and remodeling of adherens junctions through e - cadherin destruction (met, mn23h1, caveolin, rab5/7 gtpases), as well as up - regulation of the transcription factors e2f1 and dp1 (tfdp1). Wnt signaling activation in grade 3 ependymomas is indicated by the over - expression of wnt ligand (wnt11), frizzled receptors (fzd2/5/8), and dishevelled genes (dvl2/3). Furthermore, increased expression was detected for -catenin (ctnnb1) and its associated transcription factor tcf3 and the wnt target genes birc5, ccnd1, fosl1, c - myc, and tp53 . Similarly, to comprehend the molecular mechanisms underlying ependymoma recurrence, peyre et al . Performed a dual - color gene expression microarray analysis on 17 tumors at diagnosis co - hybridized with 27 corresponding tumors at first or subsequent relapses they identified 87 genes collectively as the expression signature of ependymoma recurrence . Like the gene expression characteristics of high grade ependymomas noted by palm et al ., the signature of ependymoma recurrence was also marked by wnt pathway activation with over - expression of sfrp1, sfrp2, fzd2, fzd8, and wnt10b . Other frequently over - expressed genes in recurrent ependymomas included cd133, members of the notch signaling pathway, and genes involved in the kinetochore (kif14, kif11, kif1c, kif2c, prc1, bub1b, zwint, aspm, kntc2, and cenpf). The genes that were significantly down - regulated were metallothionein genes (mt1l, mt1 g, mt1e, mt1x, mt1b, mt2a, mts), with reduced expression in up to 80% of recurrences, and genes involved in the immune system (cxcl5, cx3cl1, traf3ip2, itgbl1, serping1, ift20, entpd3, hp, and hpr). The importance of immune function in hindering ependymoma progression and recurrence was also recognized through the study by donson et al .. their ontological analysis on gene expression profiles from pediatric ependymomas correlated with clinical outcome revealed that the up - regulation of immune function - related genes was associated with non - recurrent ependymomas and a longer time to progression in recurrent ependymomas . In addition, increased infiltration of cd4 t cells were observed by immunohistochemistry in non - recurrent ependymoma samples . Furthermore, like the primary ependymomas which can be subgrouped based on location, peyre et al . Found that supratentorial versus infratentorial ependymomas showed distinct changes in expression profile at recurrence . Recurrent supratentorial ependymomas were characterized by the up - regulation of genes related to cytoskeleton organization (gelsolin, sema5a, contactin-1, sarcoglycan, villin - like, scinderin) and extracellular matrix - cell interactions (gliomedin, extl1, galectin-9, desmuslin, tetranectin, versican, col21a1, col16a1, cxcl12), which are functionally involved in the mesenchymal transition . Infratentorial ependymoma recurrences, on the other hand, were associated with over - expression of ribosomal protein genes, which are markers of oncogenic transformation in many human tumor types, . One of the key questions to answer in the field of cancer research is to determine the normal cell type that gives rise to a particular malignancy . This is a crucial step towards functionally identifying the successive oncogenic events leading to tumor onset and progression, which would be indispensable for developing targeted therapies and finding keys to prevention . Growing evidence suggests that tumor subgroups may arise due to deregulation of cell signaling pathways involved in normal development of different precursor cell populations . Thus, the unique gene expression signatures of ependymoma subgroups might provide insight into their cells of origin . Indeed, taylor et al . Showed that the signature genes which characterize supratentorial, posterior fossa, and spinal ependymomas are expressed in the matching regions in the developing cns of embryonic mice . Moreover, many of these signature genes are members of signaling pathways that modulate neural precursor cell proliferation and differentiation in the corresponding regions of the cns, . This confirmed the hypothesis that subgroups of ependymoma either maintain or recapitulate the developmental expression profiles of anatomically restricted progenitor cells, which were then identified to be radial glial cells (rgcs).taylor et al . Further demonstrated that rgcs are likely the cells of origin for ependymoma by isolating a rare population of self - renewing and multipotent cancer stem cells from fresh samples of ependymoma . These cancer stem cells exhibited bipolar morphology resembling rgcs, expressed the rgc immunophenotype cd133/ nestin/ rc2/brain lipid - binding protein (blbp), and were both required and sufficient to recapitulate the original tumor when transplanted into immunocompromised mice . Rgcs are a pivotal cell type in the developing cns of all vertebrates and are a specific group of neural stem cells . They serve as ubiquitous precursors that generate neurons and glia, as guide cells for subsequent neuronal migration, and as key elements in patterning and region - specific differentiation of the cns . Studies have also shown that ependymal cells, from which ependymoma arises, are derived from rgcs during embryogenesis . Genetic mutations in rgcs may therefore lead to their transformation into cancer stem cells of pediatric ependymomas, . Since supratentorial ependymomas are characterized by elevated expression of members of the notch and ephb - ephrin signaling pathways, it is likely that over - activation of these pathways may induce neoplastic transformation of rgcs in the cerebral subventricular zone . Likewise, up - regulation of the hox family of transcription factors may be involved in spinal ependymoma development by transforming rgcs in the spinal region . Furthermore, there is evidence that rg - like cells are present not only during development but also persist in the adult cns, specifically in the subventricular zone and the spinal cord . Thus, these rg - like cells may serve as the cells of origin for adult ependymomas. Recently, johnson et al . Catalogued dna copy number alterations among 204 tumor samples, which is the largest cohort of ependymomas ever examined at the highest resolution . They further generated mrna and microrna expression profiles for 83 and 64 of these tumors, respectively . These profiles segregated ependymomas by cns location and unmasked additional subgroups among supratentorial, posterior fossa, and spinal ependymomas . To test that distinct subgroups of ependymoma might arise due to oncogenic transformation of regionally and developmentally restricted populations of rgcs by characteristic genetic mutations, the gene expression profile of a subset of human supratentorial ependymomas was matched with that of embryonic cerebral rgcs taken from ink4a / arf (cdkn2a)-null mice, as the cdkn2a locus is frequently deleted from human supratentorial ependymomas . These embryonic cerebral ink4a / arf (cdkn2a)-null rgcs were first transduced with ephb2, which has been shown to be focally amplified in a subgroup - specific manner and to exhibit copy number - driven over - expression in supratentorial ependymomas, and were subsequently implanted into the cerebrum of immunocompromised mice . This established the first highly penetrant (over 70% incidence) murine allograft model of supratentorial ependymoma that accurately recapitulates the histological features and gene expression profile of the human tumor . Comparative gene expression analysis of matched mouse and human tumors revealed deregulation of genes in neural differentiation and maintenance, particularly ion transport and synaptogenesis, thus highlighting the importance of these events in the formation of this ependymoma subgroup . Thus, this study provided functional confirmation that ependymoma variants indeed arise from their matched populations of rgcs transformed with the subgroup - specific mutations . Over the past decade, research has significantly advanced our knowledge on the molecular genetics of ependymoma . Early cytogenetic studies identified broad chromosomal gains and losses, with loss of 22q being the most common . Nf2 is recognized as a putative tumor suppressor gene in spinal ependymomas based on mutational analysis and increased incidence of ependymoma in patients with nf2 familial syndrome . It is, however, rarely mutated in pediatric intracranial ependymomas, for which much effort is still being made in identifying the elusive tumor suppressor gene(s) on chromosome 22q . Other common genomic imbalances include gain of 1q and losses of 6q and 9 in intracranial ependymomas, and gain of 7 in spinal ependymomas, among many others . With the advent of acgh technology permitting the identification of genomic imbalances at much greater resolution, it became possible to uncover putative oncogenes and tumor suppressor genes, such as htert and cdkn2a, respectively, by testing candidates found in focal regions of amplifications and deletions for copy number - driven expression . Importantly, over the years, ependymoma tumor heterogeneity has become progressively more appreciated at the genetic level and can be subgrouped based on chromosomal abnormalities, acgh, and, recently, gene expression profiles . It is now generally recognized that ependymomas from different regions of the cns, i.e. The supratentorium, posterior fossa, and the spinal cord, are genetically distinct diseases marked by unique gene expression signatures, indicating the deregulation of different developmental pathways involved in tumorigenesis . Supratentorial ependymomas are characterized by notch and ephb - ephrin signaling, whereas spinal ependymomas show specific over - expression of hox family transcription factors . Furthermore, comparing the expression profiles of ependymomas at first diagnosis versus at relapse and at low grade versus high grade revealed that ependymoma recurrence and progression likely result from the up - regulation of wnt signaling and down - regulation of immune function - related genes . Recently, rcgs at various locations throughout the cns have been identified to be the cells of origin for the corresponding ependymoma subgroups, as illustrated in figure 1 . The notion that subgroups of ependymoma arise from regionally and developmentally distinct rgcs that have undergone transformation by subgroup - specific genetic mutations was further confirmed functionally in the case of supratentorial ependymomas . Despite these achievements in ependymoma research, greater progress is still urgently needed if we are to realize the ultimate goal of improving clinical outcome for patients . With newly developed microarray platforms able to detect copy number changes and gene expressions at even higher resolution, next - generation sequencing technologies and high - throughput techniques for unbiased epigenetic profiling posterior fossa ependymomas in particular deserve our attention, as they frequently occur in children of very young age, and complete surgical resection is often difficult to achieve owing to the involvement of multiple cranial nerves and branches of the vertebrobasilar arterial system at this location . In addition, up to half of posterior fossa ependymomas present a balanced genomic profile, making the identification of genetic events contributing to their tumorigenesis especially challenging . Consequently, it is important to examine the genetics of posterior fossa ependymomas at a greater resolution to identify very focal amplifications and deletions, as well as to concentrate on decoding its epigenome . Candidate oncogenes and tumor suppressor genes discovered to date should be promptly assessed for their diagnostic and therapeutic potential, with the aim to effectively translate our knowledge from laboratory to clinic . At present, however, ependymoma research is severely hampered by the lack of in vitro and in vivo systems to functionally examine the genetic events identified through acgh and gene expression studies that potentially contribute to ependymoma development . Indeed, the identification of rgcs as the cells of origin for ependymoma was a significant breakthrough towards mapping out the pathogenic mechanisms of ependymoma . Similar to what has been done for one subset of supratentorial ependymomas, the next step will be to identify the distinct populations of rgcs for all ependymoma variants and functionally determine the subgroup - specific driver mutations necessary for transforming corresponding rgcs to ependymoma . This approach will allow us to functionally identify the key genetic events involved in the initiation and progression of all ependymoma subgroups, as well as to model these subgroups in vitro and in vivo . Unlike end - stage tumor samples which provide little information on the chronology and relative importance of the uncovered genetic events in the process of ependymoma pathogenesis, these functional models will be instrumental in deciphering the pathogenic mechanisms of the ependymoma subgroups, as well as in uncovering and verifying potential targets for therapy.
Fe(ii)-oxidizing bacteria (feob) gain energy from the chemical oxidation of fe(ii) coupled to reduction of oxygen or nitrate or using light energy coupled to reduction of co2, e.g., anoxygenic photosynthesis . At the near neutral ph of many surface waters, the oxidation of fe(ii) for that reason, cyanobacteria, which generate oxygen as a result of oxygenic photosynthesis, can act as indirect fe(ii)-oxidizing bacteria where anoxic and fe(ii)-containing waters encounter to sunlit surface environments . The contribution of cyanobacteria to fe(ii) oxidation has been quantitatively addressed in fe(ii)-rich hot spring environments and in benthic photosynthetic communities living at the sediment water interface . Although the modern oceans are predominantly oxygenated to great depths, promoting the speciation of iron as ferric [fe(iii)] rather than ferrous [fe(ii)], fe(ii) may be increasingly mobilized out of sediments and stabilized in the marine water column due to expanding low - oxygen conditions in so - called oxygen minimum zones (omz). Where omz intersect with the photic zone, fe(ii) oxidation by planktonic oxygen - producing cyanobacteria could contribute to the marine iron cycle . Furthermore, anoxic and fe(ii)-rich bottom waters are a pervasive feature of oceans in the precambrian era [before about 500 million years (my) ago] at a time when oxygen was building up in the surface oceans as a result of cyanobacteria and other oxygenic phototrophs . Therefore, redox interfaces between anoxic and fe(ii)-containing waters and photosynthetically produced oxygen were likely common throughout much of earth s history . Iron redox processes fractionate the naturally occurring isotopes of iron dependent on their mass (e.g., fe, fe, fe, and fe), such that the quantitative contribution of biotic and abiotic iron cycling at the earth s surface may be recorded in sediments composed of iron - rich minerals . Due to the large fractionations between fe(ii) and fe(iii) species, fe(ii) oxidation generally produces a solid iron phase that is enriched in heavy isotopes of iron relative to aqueous fe(ii), regardless of the mechanism of oxidation . This makes it difficult to parse the contribution of enzymatic fe(ii)-oxidizing bacteria from abiotic fe(ii) oxidation, not to mention indirect fe(ii) oxidation by oxygen - producing cyanobacteria by using iron isotopes . However, subtle differences in the mechanism of oxidation and precipitation, and in the characteristics of the iron minerals or phases (e.g., mineralogy, particle size, or presence of impurities) formed, can influence the overall fractionation between aqueous fe(ii) and iron minerals . Furthermore, the role of cyanobacteria in direct or indirect redox cycling of iron at the cell surface is increasingly recognized and may be associated with distinct isotope fractionation . Therefore, detailed mechanistic studies of iron isotope fractionation during different pathways of fe(ii) oxidation are warranted and may help to define isotopic, mineralogical, or microscopic signatures associated with certain biological processes . Furthermore, the isotopic composition of iron minerals is known to be modified by electron and atom exchange between aqueous fe(ii) and fe(iii) (oxyhdr)oxide minerals . These processes have been most effectively characterized under reducing conditions, when a supply of aqueous fe(ii) is produced by, for instance, microbial fe(iii) reduction . However, at fe(ii)o2 interfaces with a flux of aqueous fe(ii), electron and atom - exchange could also occur on newly formed fe(iii) (oxyhydr)oxide minerals . Although the effect of some organics as well as si and low ph on blocking electron and atom exchange have been investigated, the effect of cell surfaces and microbially produced organics on this reaction and via blocking sites on fe(iii) minerals, particularly in an oxidizing system, are not known . In this contribution, we tracked the iron isotope composition of different pools of iron during fe(ii) oxidation by the marine planktonic cyanobacterium synechococcus pcc 7002 . Several prior studies have characterized the interaction of this oxygen - producing strain with fe(ii), which gives us a body of work to aid in interpreting the nature of different iron phases in the system, and their mechanism of transformation . Additional microscopy and mineral characterization in this study are used to build the picture of how iron is processed during indirect fe(ii) oxidation resulting from oxygenic photosynthesis . The results have implications for understanding the reactivity of iron minerals as well as identifying isotopic signatures associated with biological activity . Synechococcus pcc 7002 was routinely cultivated on ph 6.8 marine phototroph (mp) medium containing 6 mg l ferric ammonium citrate as the fe(iii) source at 24 c under an irradiance of 12.8 mol photons m s from a standard 40w tungsten light bulb as measured by a li-250a light probe (li - cor, inc . ). For fe(ii) fe(ii) amendments were added from a sterile, anoxic fecl2 stock solution, and the medium was filtered twice through a 0.22 m filter in an anoxic glovebox (100% n2), separated by 48 h incubations at 4 c to ensure that all fe(ii) precipitated as carbonate and phosphate minerals with growth media components were removed . The final fe(ii) concentration in the medium after filtration was 2 mm as measured by the spectrophotometric ferrozine assay . A log - phase culture of synechococcus pcc 7002 grown with ferric ammonium citrate was degassed for 5 min with sterile n2/co2 (90%:10%) and inoculated into the 2 mm fe(ii)-containing medium to a final concentration of 5 10 cells ml . Glass media bottles were acid washed in 1 m hcl for 24 h and then soaked in fresh ultrapure water (resistivity of 18.2 m cm) for two successive 24 h treatments before use . All anoxic bottles were closed with butyl rubber stoppers that had been washed in 1 n hcl for 24 h and then thrice boiled in ultrapure water . This concentration of 2 mm fe(ii) was chosen for experiments because a freshly inoculated culture of synechococcus pcc 7002 took about 10 days to oxidize this, during which time we could sample sufficiently often to have resolution on the evolution of the isotopic composition of different iron pools . Despite the fact that this strain grows more slowly at 2 mm fe(ii) than at lower fe(ii) concentrations, due to fe(ii) toxicity, sufficient growth did occur to fully oxidize all fe(ii). Although this concentration is at the upper end of fe(ii) concentrations in modern sunlit environments, it is within the range documented for environments where cyanobacteria have been documented as having a role in fe(ii) oxidation . During fe(ii) oxidation, which lasted about 10 days, volumes of 2 ml were repeatedly removed with a syringe from the bottles of two contemporaneous replicate experiments (bottles 1 and 2) in an anoxic glovebox . Before extracting, the bottles were shaken to yield a homogeneous slurry of iron precipitates . The aliquots were subsequently centrifuged for 10 min at 16 000 g, and the supernatants were filtered through a nylon 0.22 m centrifuge tube filter (costar spin - x, corning, international) to yield particle - free aqueous fe(ii), henceforth fe(ii)aq . The solids were washed with anoxic ultrapure water to remove any loosely bound iron . A second wash utilized anoxic 0.5 m sodium acetate (adjusted to ph 4.85 using acetic acid) to recover sorbed iron (fenaac) from the solids (24 h incubation in the dark). The concentrations of fe(ii) and total iron in the four different iron fractions were measured with the ferrozine assay . The fe(ii) in the fe(ii)aq, water wash, and fenaac was stabilized in a final concentration of anoxic 1 m hcl prior to measurements . Fe(iii) was determined as the difference between fe(ii) measurement and total iron measurements (after reduction of iron by hydroxylamine hydrochloride). Purification of the fe(ii)aq, fenaac, and feppt fractions was performed in positively pressured clean laboratories of the isotope geochemistry group at the university of tuebingen under conditions and with reagents that have previously been described . The concentrations of iron in the water washes of feppt were below the detection limit of the ferrozine assay (<0.01 mm, 0.56 g ml), and so, these samples were not purified . Sample aliquots of the separated iron fractions containing 5 g of iron were purified for iron isotope measurements using anion exchange chromatography according to prior methodology . An adequate amount of fe fe double spike was added to the samples prior to fe purification to ensure accurate correction of the instrumental mass bias and possible fe isotope fractionation during anion chromatography caused by the organic matrix of the samples . Iron isotope analyses were performed on the thermofisher scientific neptuneplus multicollector inductively coupled plasma mass spectrometer (mc - icp - ms) of the isotope geochemistry group of the university of tuebingen . Polyatomic interferences, such as arn on fe or aro on fe were resolved using the high mass - resolution mode (16 m slit). The four iron isotope beams were simultaneously detected with 90 integration cycles at 8 s each during the runs . Background corrections for sample signals were based on on - peak - zero measurements on the pure analyte solution (0.3 m hno3) run before and after each sample . Iron isotope data are reported relative to the isotopically certified international reference material irmm-014 (institute for reference materials and measurements in gent, belgium) using the -notation: the results are reported in units of per mil (). The reproducibility of the double - spike measuring method as determined by repeated fe measurements of the irmm-014 reference material in between sample runs was 0.00 0.032 (2sd; n = 18). Interspersed measurements of our in - house iron standard, hanfe, yielded fe = 0.282 0.039 (2sd; n = 12), which is in excellent agreement with previously published values of 0.28 0.05 (2sd; n = 19) and 0.279 0.030 (2sd; (f) of fe(ii) remaining were utilized to determine the isotopic enrichment factor (fe) using the following equations:12 fe(ii)aq-0 indicates the fe(ii)aq at the beginning of the experiment . The isotopic fractionation fe () is related to fe by the equation:3 the fitting parameters were determined by minimizing the sum of values . Data were also fit by linear regression, with slope and intercept determined by minimizing the sum of values . High energy synchrotron x - ray scattering experiments were performed on the solid, dry products of fe(ii) oxidation by synechococcus pcc 7002 at the advanced photon source at argonne national laboratory, beamline 11-id - b . 5 mm fe(ii), freeze - dried, and washed three times with millipore water to remove excess salt . Cells of synechococcus pcc 7002 were grown under similar conditions as described above until the initial ca . Mineral aggregates were imaged by confocal laser scanning microscopy (clsm; leica spe, mannheim, germany). A 635 nm laser was used for excitation autofluorescence of synechococcus pcc 7002, with a maximum emission peak at 660 nm (detected range of emission, 640700 nm). The fe(iii) minerals were visualized using the reflection signal of the 488 nm laser . Alexa dye conjugates were screened in order to optimize visualization of exopolysaccharides (eps) without overlap with the autofluorescence emission maximum of the pigments of synechococcus pcc 7002 (660 nm). Alexa 488 (maximum emission peak at 520 nm) was chosen because sba bound to the eps in higher amounts, resulting in brighter fluorescence than the other lectins screened [wheat germ agglutinin alexa fluor 555 conjugate (wga-555) and lectin pna from arachis hypogaea (peanut), alex fluor 568 conjugate (pna-568)]. Brighter fluorescence at lower laser power was observed with sba-488, which binds terminal - and -n - acetylgalactosamine and galactopyranosyl residues, compared to wga-555 and pna-568, which are specific to sialic acid and n - acetylglucosaminyl residues and terminal -galactose, respectively . A turn - on type selective probe for fluorescent labeling of dissolved, sorbed, or ligand - bound fe(iii) was previously used to visualize the relationship of fe(iii) synechococcus pcc 7002 cells and minerals from this same incubation . Because of spectral overlap, the lectin and fe(iii)-binding probe could not be combined in a single experiment here, and therefore, we compare new results to prior data . The auto - quant deconvolution algorithm implemented in the leica las af software was applied to blind deconvolute the 3d image stacks . The spatial relationships of species detected using fluorescence dyes and cell autofluorescence in clsm image stacks were analyzed using scatterj, a plugin for correlation analysis of species - specific maps for use in imagej and fiji . The fe(ii)aq values from two replicated experiments evolved from an initial value near 0 to lighter values during oxidation (table 1, figure 1). The fe(ii)aq fraction, measured after the sample was centrifuged and filtered, consisted of only fe(ii). All iron concentration and speciation data (measured by ferrozine) the first feppt samples analyzed, at about 40% fe(ii) oxidized, had feppt of about 2, trending toward 0 at 100% fe(ii) oxidized . The speciation of feppt, which was measured after washing with water and sodium acetate, consisted of predominantly fe(iii), with generally <10% fe(ii). Iron in the water wash of the precipitates was below the detection limit of the ferrozine assay (<0.01 mm, 0.56 g ml). The sodium acetate wash removed sorbed iron, which contained both fe(ii) and fe(iii). The fenaac fraction represented 1020% of total iron in the system after feppt began to form . The fenaac had an intermediate isotopic composition between fe(ii)aq and feppt but was variable and generally lighter than 0. (a) bottle 1 and (b) bottle 2 are biological replicates of the fe(ii) oxidation experiment with synechococcus pcc 7002 . Green circles are fe(ii)aq data; orange squares are feppt data; blue diamonds are fenaac data . The solid green lines are the rayleigh fits of the fe(ii)aq data, with an fe for fe(ii)aq of 1.79 (a) to 2.15 (b). The solid orange lines are the rayleigh fits of the feppt data, with fe for feppt of 2.44 (a) and 2.98 (b). Samples from b2 and e2 had anomalous values for fenaac and feaq, respectively . These samples were most likely lost after drying due to electrostatic charging of the teflon beakers . The fast reaction between fe(ii) and oxygen favors the heavy isotopes of iron in the resulting fe(iii) minerals that precipitate . Abiotic and biotic fe(ii) oxidation both follow this trend, resulting in fe of 24 between aqueous fe(ii) and fe(iii) minerals, with minerals enriched in heavy isotopes . Our fe for fe(ii)aq (1.792.15 for bottles 1 and 2, respectively; table 1), determined from a rayleigh fit of the fe(ii)aq data, is on the low end of this range, similar to what was previously documented for fe(ii) oxidation by anoxygenic phototrophs . The rayleigh fit of the feppt data from both replicates resulted in fe of 2.44 and 2.98, larger than that attained for the fe(ii)aq data (table 1) and within the literature range . Prior explanation for the offset in fe between these two fractions is that, following precipitation, the feppt underwent partial equilibration with another phase of iron in the system, possibly a ligand - bound or sorbed fe(iii) phase or fe(ii)aq . Below, we use our mineralogical and microscopic characterizations of the experiment to explore possible exchange processes in this system . The third, quantitatively significant fraction of iron in the system in addition to fe(ii)aq and feppt was fenaac (up to 18% of total fe). The fenaac data had an intermediate isotopic composition between fe(ii)aq and feppt, from 0.10 to 0.73 throughout the experiment, and contained both fe(ii) and fe(iii) (supplementary table 1). The presence of fe(iii) in fenaac has previously been observed in fe(ii) oxidation experiments with anoxygenic phototrophs but not with nitrate - dependent fe(ii)-oxidizing bacteria . The fenaac must have been sorbed onto one of the surfaces, either feppt or cells, on the basis of its extraction with sodium acetate . In order to infer whether equilibration processes were occurring between fenaac and feppt, it is necessary to know (1) where fenaac was in our experiments and (2) what type of iron species [i.e., fe(ii) or fe(iii)] that it was . Our use of a lectin - binding dye in confocal microscopy documents that eps was also forming during fe(ii) oxidation with synechococcus pcc 7002 (figure 3). We can use this data set to first determine whether eps was important in binding / sorbing iron extracted as fenaac and then to determine whether iron was associated with the surface of cells and/or feppt . An overlay of figure 3a c, which show the location of cells, eps, and feppt, indicates that eps is colocalized with feppt (figure 3d). The correlation analysis in figure 3e implies there is no spatial overlap of eps with cells . In previous work with synechococcus pcc 7002 under identical growth conditions as in figure 3, a fluorescent sensor for soluble or ligand - bound fe(iii) was used in clsm, and fluorescence was localized directly at the synechococccus pcc 7002 cell surfaces . While spectral interferences prevented us from simultaneously labeling eps and fe(iii) in our current clsm experiments, we can infer from comparing our data set with the previously published one that there was fe(iii) sorbed to the surface of cells but not eps or feppt . In support of this, eps is expected to stay with the aqueous phase during filtration through a 0.2 m filter or be washed off of feppt in the water wash . We did not detect any fe(iii) in the fe(ii)aq fraction or measure any detectable iron in the water wash . From these results, we exclude eps as having a major role in binding soluble fe(iii) in the current system . Previous experiments with synechococcus pcc 7002 cells demonstrated that sorption to cells is a major fate for aqueous iron, although the oxidation state of sorbed iron was not determined in those experiments, so we cannot rule out that some fe(ii) was also sorbed to cells . However, sorption onto cells has previously been documented as a fate for aqueous iron with diverse cyanobacteria, with fe(iii) more commonly detected at the cell surface than fe(ii), via attachment of fe o fe polymers to phosphoryl groups, strengthening the inferences made from clsm that synechococcus pcc 7002 cells sorbed fe(iii). (a) x - ray diffraction (xrd) pattern obtained from x - ray total scattering data of the feppt phase after complete fe(ii) oxidation, freeze - drying, and water washing . The indexed reflections for lepidocrocite (lp) and goethite (gt) are shown . (b) a 3-component linear combination fit of 58% ferrihydrite, 22% goethite, and 20% lepidocrocite (supplementary table 4). (a) autofluorescent cells, (b) stained with the lectin - binding dye sba-488, (c) the reflection signal from fe(iii) minerals, and (d) an overlay of (a c). Correlation plot of the fluorescence intensity in individual pixels from (e) autofluorescence (a) vs sba-488 (b) and (f) sba-488 (b) vs fe(iii) minerals (c). This analysis demonstrates that eps, which is bound by sba-488, is coating fe(iii) minerals but is not spatially associated with cells . The other surface in our experiments that could have sorbed iron extracted as fenaac was feppt . The three techniques we used to address mineralogy indicate that our feppt was a mixture of 58% ferrihydrite, 22% goethite, and 20% lepidocrocite (figure 2), and ferrihydrite was likely the predominant mineral present during the experiments (see supporting information). Minerals such as ferrihydrite and goethite, similar to what was present in our experiments, can sorb fe(ii). Both fe(ii) and fe(iii) were detected in the fenaac (supplementary table 1), raising the possibility that fe(iii) was extracted from the mineral . However, we verified that no fe(iii) was extracted from synthetic ferrihydrite with our 0.5 m sodium acetate solution prior to beginning experiments (data not shown), consistent with previous reports that used a 1 m sodium acetate solution . A further inference in support of sorbed fe(ii) being extracted from feppt by sodium acetate is that feppt still contained some fe(ii) after extraction, as measured by ferrozine (supplementary table 1). We take this as evidence that sorbed iron associated with the mineral was predominantly fe(ii), although we cannot exclude that some fe(iii) may also be sorbed to the mineral surface . We observed evidence for three reactions in our experiments that are essential for understanding the observed fractionations of iron isotopes, and these are summarized in figure 4 . They are (1) fe(ii) oxidation to fe(iii), which forms feppt, (2) sorption of fe(iii) to cells, and (3) sorption of fe(ii) to feppt . These observations fit a two step - model of fe(ii) oxidation, where fe(ii) is oxidized and undergoes rapid isotopic equilibration with a pool of fe(iii), which then precipitates as fe(iii) minerals . We suggest, however, that in our experiments, feppt undergoes subsequent partial equilibration with fe(ii)aq . Controls on the overall iron isotope fractionation in the system are (1) fe(ii) oxidation and precipitation of fe(iii) as feppt; (2) sorption of fe(iii) to cells; (3) partial equilibrium atom and electron exchange after sorption of fe(ii)aq to feppt . (1) generates feppt (solid orange line) that is 23 heavier than fe(ii)aq (solid green line). (2) produces sorbed fe(iii) on cells with an estimated equilibrium fefenaac - fe(ii)aq of 1.84. (3) produces fe(ii) sorbed on goethite with an estimated fefenaac - fe(ii)aq of 0.8. the resulting fenaac predicted from (2) and (3) are denoted by the light blue diamonds . The fitting of our fe(ii)aq and feppt with rayleigh equations representing isolation of the feppt pool from fe(ii)aq after precipitation and a linear equation representing complete isotopic equilibrium are helpful in interpreting the fractionation mechanisms taking place . The larger values for linear fits of all data as compared to rayleigh fits indicate that complete isotopic equilibrium between fe(ii)aq and feppt is not occurring during fe(ii) oxidation and precipitation (table 1). The smaller fe values for rayleigh fits of the fe(ii)aq (1.79 and 2.15 as compared to 2.44 and 2.98 for feppt) are on the order of fractionation observed in other biological fe(ii) oxidation experiments in batch at circumneutral ph: 1.5, 1.52, and 12. such small net fractionations have been noted when the presence of significant quantities of sorbed or ligand - bound fe(iii) has been detected or observed . Up to a few percent of total iron was found as fe(iii) in the fenaac fraction (supplementary table 1). On the basis of detection of iron sorbed to cells with a dye that is specific for an aqueous or ligand - bound fe(iii), we suggest that this fe(iii) could equilibrate with fe(ii)aq . In experiments with synechococcus sp . Cells at ph 6, added fe(ii) [which was adsorbed as fe(iii)] was 1.84 heavier than aqueous fe(ii), and equilibrium with fe(ii)aq was inferred from the data . This fractionation is very similar to our fe values derived from rayleigh fits of fe(ii)aq (1.79 and 2.15). Because of the similar type of organism that we used, it is likely that equilibrium fractionation between fe(ii)aq and fe(iii) sorbed to cells is a relevant process in our experiments . While the feppt data are not well fit by a linear model representing complete equilibrium exchange with fe(ii)aq, the range of the fe determined from rayleigh fits of the two feppt data sets (2.44 and 2.98) is of the same magnitude expected for equilibrium between feppt and fe(ii)aq . Wu et al . Inferred a feferrihydrite - fe(ii)aq (where feferrihydrite - fe(ii)aq = feferrihydrite fefe(ii)aq) of 3.2, while beard et al . And frierdich et al . Reported a fegoethite - fe(ii)aq of 1.1. considering the 58% ferrihydrite, 22% goethite, and 20% lepidocrocite in our precipitates as determined from x - ray scattering (figure 2), the equilibrium fefeppt - fe(ii)aq for our minerals could range from 2.3 to 2.7, depending on whether the assumed fractionation for lepidocrocite is the same as for goethite or ferrihydrite, respectively . The larger fe we calculate for our second data set (2.98) may reflect that ferrihydrite, with a larger fe value, was likely the mineral present during active fe(ii) oxidation (see supporting information). In our batch experiments, fe(ii)aq may continue to react with feppt, given their proximity and the time frame of experiments (10 days). Sorption of fe(ii) on feppt provides a likely mechanism for partial isotope equilibrium and is supported by our detection of fe(ii) in feppt (supplementary table 1). Sorption of fe(ii) is an important pathway in recrystallization of fe(iii) (oxyhydr)oxide minerals, particularly ferrihydrite . During this process, equilibrium atom and electron exchange occur between sorbed fe(ii) and fe(iii) minerals, with complete equilibrium attained within 2 weeks for goethite, for instance (a similar time frame as our 10 day experiment). In this model, fe(ii) sorbs to fe(iii) minerals and donates an electron into the bulk mineral structure, adding to the fe(iii) mineral, and causing the desorption of a newly produced fe(ii) from the mineral . This is also consistent with the shifts in mineralogy we see during the course of oxidation (see supporting information). We do not see evidence for complete equilibrium between fe(ii)aq and feppt, given the poor linear fit of feppt (table 1). Atom and electron exchange between feppt and sorbed fe(ii) is expected to be diminished in the presence of organic compounds . Our clsm data indicate that eps is colocalized to minerals (figure 2). It is possible that atom and electron exchange can still occur when fe(iii) minerals are coprecipitated with organics, as retardation of this process seems to result from blockage of surface sites when organics coat already formed fe(iii) minerals, or if long chain carbon molecules are present . Therefore, we suggest that only partial atom and electron exchange occurred in our system as a result of the eps coating the feppt . During atom exchange, the fractionation when fe(ii)aq sorbs onto goethite varies among experiments . One study reports sorbed fe(ii) is 0.73 heavier than fe(ii)aq, and another reported sorbed fe(ii) was 1.24 heavier . Crosby et al . Directly measured sorbed fe(ii) extracted with sodium acetate during microbial fe(iii) reduction experiments, which was just 0.3 heavier than fe(ii)aq for experiments using hematite and up to 0.8 heavier for experiments using goethite as the sorbing surface . Our fefenaac - fe(ii)aq ranged from 0.45 to 2.66, which is much larger and more variable than the experiments of crosby et al ., which may be in part because our fenaac includes both fe(ii) and fe(iii). Another factor is that, in our experiments, the less crystalline mineral ferrihydrite was likely the sorbing surface present during experiments (see supplementary figures 1 and 2 and supporting information). Several studies have noted a trend of larger fractionations during sorption to less crystalline minerals or higher surface area minerals . The feppt in our experiments had a surface area of 122.1 m g. we calculated fenaac considering that the fe(iii) fraction of fenaac should be 1.84 heavier than fe(ii)aq due to adsorption of fe(iii) at cell surfaces, and the sorbed fe(ii) fraction of fenaac should be at least 0.8 heavier than fe(ii)aq . The calculated fenaac is a good model of our actual fenaac values (figure 4). This calculation supports the model presented here, in which fe(ii) is oxidized to fe(iii), which is sorbed onto cells and equilibrates with fe(ii)aq, and full equilibration of fe(ii)aq with feppt via atom and electron exchange is hindered by the presence of eps on feppt . Our experiments provide evidence that iron isotope fractionation during microbially influenced fe(ii) oxidation by cyanobacteria is not a simple reaction, controlled only by the abiotic oxidation of fe(ii) with oxygen and rapid precipitation of fe(iii) at circumneutral ph . Multiple secondary processes generate a significant fraction of sorbed iron that is isotopically distinct from either residual fe(ii)aq or feppt, and subsequent equilibration between the iron pools can further modify the isotopic composition of these phases . Our data indicate that sorption of fe(iii) at cell surfaces likely further fractionates the fe(ii)aq pool . In addition, abiotic sorption of fe(ii) to fe(iii) mineral surfaces can also fractionate fe(ii)aq, through equilibrium atom and electron exchange subsequent to fe(ii) sorption, despite the presence of eps . Follow - up experiments could investigate atom and electron exchange in this system . Specifically, isotopically enriched fe(ii)aq solutions mixed with preformed cells and minerals would be useful for monitoring atom exchange between the fe(ii)aq and feppt . The processes and phases described here can overprint the anticipated fractionations and compositions of fe(iii) minerals and organic - associated iron present in the environment and can challenge interpretation of the genesis of fe(iii) minerals in the geological record, where the residual fe(ii)aq pool is no longer present . Although iron atom and electron exchange has received the most attention as a process relevant to fe(iii)-reducing systems where fe(ii) is in contact with fe(iii) minerals, our data suggest this process could also be relevant in environments where fe(ii) is abundant during fe(ii) oxidation and fe(iii) mineral formation . This includes oxidizing environments with high enough fluxes of fe(ii) for fe(ii) to persist even in the face of rapid oxidation, such as fe(ii)-rich springs or seeps and marine upwelling zones that tap ferruginous bottom waters, past or present . Furthermore, our work documents atom and electron exchange in the presence of iron minerals whose formation pathways are biologically induced and when organic phases coat iron minerals . Finally, iron redox cycling and sorption of iron at the surface of cyanobacteria may be an important component of modern and ancient aquatic iron cycling, and our work highlights the effect of such processes on iron isotope systematics.
Parkinson's disease (pd) is a neurodegenerative disease of dopamine (da) neurons in substantia nigra characterized predominantly by resting tremors, bradykinesia, muscular rigidity, and postural instability, along with several nonmotor symptoms . The disease is associated with a loss of antioxidants or increase in prooxidant levels and mitochondrial dysfunction . The neurotoxin 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (mptp) is known to cause a similar loss of dopaminergic neurons in the human midbrain with corresponding parkinsonian symptoms . Several animal species have also shown sensitivity to mptp, including primates, mice, goldfish, and, most recently, zebrafish . Mptp is metabolized to 1-methyl-4-phenyl pyridinium (mpp) in glial cells in the brain . After release from the glia, mpp is transported into dopaminergic neurons via the dopamine transporter (dat). Mpp accumulates in the mitochondria and induce neuronal cell death via several pathways, including the inhibition of complex i activity of the respiratory chain . The excessive production of reactive oxygen species, such as superoxide anion, hydroxyl radical and hydrogen peroxide, may either directly damage the cellular macromolecule to cause cell necrosis or affect normal cellular signaling pathways and gene regulation to induce apoptosis . Lately, several studies demonstrated the free radical scavenging activity, reducing the concentration of reactive oxygen, and nitrogen species of artificial antioxidants include inorganic nanoparticles possessing intrinsic antioxidant properties and nanoparticles functionalized with natural antioxidants or antioxidant enzymes . Recent studies have proposed that platinum in the form of nanoparticles has an activity that is similar to that of oxidizing nicotinamide adenine dinucleotide (nadh) and reducing ubiquinone (coq10). Platinum nanoparticles also provide dual functions as mitochondrial complex i to lower reactive oxygen species (ros) generation and as superoxide dismutases (sod)/catalase mimetics to scavenge ros including superoxide anion (o) as well as hydrogen peroxide (h2o2) and free radicals [8, 9]. This suggests that platinum nanoparticles can mimic a part of the enzymatic functions of the complex i and indicates their possible use in medical treatments for parkinson's disease . With such important applications, it is imperative to develop platinum nanoparticles through environmentally sound and nonpolluting technologies . While a number of chemical methods are currently available and are extensively used, the collaborations are often energy intensive and employ toxic chemicals, thereby precluding biomedical application . With the flourishing demand on green nanotechnological processes, biosynthetic methods employing either biological microorganisms or plant extracts have emerged as simple and viable alternative to chemical synthetic procedures . Using plants for nanoparticle synthesis can be advantageous over other biological processes, because they eliminate the elaborate process of maintaining cell cultures and can also be suitably scaled up for large - scale nanoparticle synthesis . In this work presented here, protocol for the production of well - defined platinum nanoparticles by utilising aqueous extract of bacopa monnieri (bm) leaves . We have chosen bacopa monnieri (bm) leaves because it is often used to treat people with parkinson's disease . Studies suggest that they improve circulation to the brain, as well as improving mood, cognitive function, and general neurological function . The presence of bacoside a and b in bm leaves attributes to their neurobeneficial function . In addition to the neurobeneficial effects they exert antiamnesic, antioxidant, antistress, anxiolytic, memory enhancing, and antiulcerogenic activities . Hosamani and muralidhara have reported the neuroprotective efficacy of bacopa monnieri against rotenone - induced oxidative stress and neurotoxicity in drosophila melanogaster . Recent studies have demonstrated that pretreatment with the bm extract protected the human neuroblastoma cell line sk - n - sh against h2o2 and acrolein by modulating the activity of several redox regulated proteins, that is, nf - kappab, sirt1, erk1/2, and p66shc, so as to favor cell survival in response to oxidative stress . Based on these stipulations, we used bm extract in the present study to prove its synergistic reduction potential in reducing platinum salts and for the creation of robust coating on platinum nanoparticles to produce stable phyto platinum nanoparticles for potential applications in medicine and technology . Since recent research in antioxidant nanomaterial has opened a new era in pharmaceutical industries, we, hence forth finally, propose to determine the potentiality of platinum nanoparticles coated with phytochemicals of bacopa monnieri leaf (bme - ptnps) extract endowed with antioxidants to mitigate the oxidative damage induced by mptp in zebrafish (danio rerio). Thereby, in the present, we aimed to explore the neuroprotective role of bme - ptnps in mptp - induced zebrafish pd model . The components (natural constructs) used in the synthesis of platinum nanoparticles (ptnps) were procured from standard vendors . Mptp and chloroplatinic acid (h2ptcl6) was purchased from bio corporals, vadapalani, chennai . Nicotinamide adenine dinucleotide (nadh), 5, 5-dithio - bis (2 nitro benzoic acid) (dtnb), nitro blue tetrazolium, perchloric acid, potassium dihydrogen phosphate, acetonitrile, citric acid, 5 - 5-dithiobis - p - nitrobenzoic acid, potassium dihydrogen phosphate (kh2po4), ethylene diamine tetra acetic acid (edta), and octanesulfonic acid were obtained from southern scientific corporation, chennai . The bacopa monnieri leaves extract (bme) was prepared by weighing 50 g of bacopa monnieri leaves in 250 ml beaker along with 100 ml of distilled water and maintained at 80c for 2 hrs before decanting it . The solution was filtered by 0.45 m milipore membrane filter and followed by 0.2 m millipore membrane filter . For the synthesis of platinum nanoparticles, 40 ml of 1 mm was reacted with 10 ml of the bacopa monnieri leaves extract in erlenmeyer flask at room temperature . The formation of platinum nanoparticles was confirmed by the change in the color of the mixture from pale yellow color to dark brown . The final concentration of metal nanoparticles in the solution was around 6 10 m. the nps synthesized were found to be stable for more than a month when stored in closed containers, and no visible change was observed for several days . The stability and identity of the bme - ptnps were measured by recording uv absorbance . The absorbance peak at ~330380 nm confirmed the retention of nanoparticulates in all the above mixtures . The size and shape of the nanoparticles were determined by using tem on a jeol temscan2000ex model operating at accelerating voltage at 80 kev . The sample for tem was prepared by putting one drop of the suspension onto standard carbon - coated copper grids and then drying under an ir lamp for 30 min . Ftir spectra of freeze - dried bme - ptnps were investigated by analyzing the sample under brukere tensor 27 ftir spectrometer in attenuated total reflection mode using the spectral range of 2000400 cm with the resolution of 4 cm . The energy dispersive x - ray analysis of isolated nanoparticles was carried out by means of jeol edx model - jsm-5610 lv . To investigate the chemical change of bme - ptnps by nadh oxidation, uv - vis surface plasmon resonance absorption spectra of bme - ptnps bme - ptnps at 50 g / ml were incubated with 100 m nadh in water at room temperature for 12 h. subsequently, the incubated mixture was centrifuged to remove nadh and nad which impede the spectrum measurement . This washing process was repeated 10 times . Wild - type adult (<8 months old) zebrafish were obtained from specialized supplier . Animals were kept in aged tap water at 28c under a 14: 10 h light: dark cycle . A) experimental parkinsonism - induced group (a single dose of mptp (225 mg / kg bwt) injected intraperitoneally (i.p)) this dosage was based on a previous study that has demonstrated impaired locomotor activity in adult zebrafish bme - ptnps -control group (various concentrations such 0.3 mol, 0.4 mol, and 0.5 mol / kg body weight suspended in physiological saline (ps) were administered once in alternative days for 5 days). (b) bme - ptnps pretreated group (bme - ptnps + mptp), various concentrations of bme - ptnps were administered once in alternative days for 5 days while mptp was given on the 4th day 24 hours after the injection of bme - ptnps . (c) control group (ps - injected but otherwise identically treated fish served as control group): the injections were conducted using hamilton syringes with a mean injection volume of 5 l / g body weight . The fish were sacrificed after mptp injections to observe the effects of bme - ptnps, 24 hours for lipid peroxidation, the activities of antioxidant systems and complex i and 5 days for the locomotor activity, content of dopamine and its metabolites in zebrafish brain . The whole brain tissue homogenates were prepared in 0.1 m phosphate buffer and centrifuged at 3000 g for 30 min . The brain supernatants were then subjected to the measurement of malondialdehyde (mda), glutathione (gsh), superoxide dismutase (sod), glutathione peroxidase (gsh - px), catalase (cat), and total antioxidant capability by spectrophotometric methods . The thiobarbituric acid reactive substance (tbars) was measured to analyse the mda levels . Gsh content was measured according to the method of based on the reacting with 5, 5-dithio - bis (2 nitro benzoic acid) (dtnb or ellman's reagent) to give a yellow colour compound that absorbs at 412 nm . The activity of sod was assayed by monitoring the inhibition of the reduction of nitro blue tetrazolium by the sample at 560 nm . Catalase was analysed as the rate of decrease in absorbance of h2o2 at 240 nm / min / mg protein . Complex i activity in crude mitochondrial preparation from zebrafish whole brain was monitored by the decrease in absorbance at 340 nm due to the oxidation of nadh . The contents of dopamine and its metabolites were determined according to the method of luo et al ., 2009 . Briefly, whole brain tissue was homogenised in 0.5 ml of cold perchloric acid (0.4 m). Subsequently, the sample was centrifuged at 20,000 g for 10 min at 4c, and the supernatant was transferred to a clean tube and measured for volume . One - half volume of a solution containing 0.02 m potassium citrate, 0.3 m potassium dihydrogen phosphate, and 0.002 m na2edta was added and mixed thoroughly to deposit perchloric acid . After incubation in an ice bath for 60 min, the mix was centrifuged at 15,000 g, for 20 min at 4c . Supernatants were analyzed for catecholamines, especially dopamine and its metabolite 3.4-dihydroxyphenylacetic acid (dopac), by hplc (125 mm 3 mm i.d . Column, packed with nucleosil 100 c 18; 3 m particle size) and electrochemical detection (intro, antec leyden, the netherlands; cell potential = 800 mv). The mobile phase consisted of 5% acetonitrile, 10 g / l citric acid, 4 g / l kh2po4, 0,1 g / l edta, and 0,175 the locomotor activity of zebrafish was measured as per the protocol followed by xia et al ., 2010 with slight modifications . A small experimental tank (30 cm 10 cm 15 cm) contained 3 l water was used to assess the locomotor activity of zebrafish . A transparent plastic film was placed in front of the tank and divided the tank into four segments . Fish were placed individually in the tank and their behavior was video recorded for 5 min after a 10 min habituation period . Spontaneous swimming activity was measured by recording the distance traveled, mean speed, and number of times the subject moved from one section into another during a 5 min observation . Our new process for the production of bacopa monnieri phytochemicals coated platinum nanoparticles (bme - ptnps) uses a direct interaction of chloroplatinic acid with bacopa monnieri leaf extract in aqueous media without the intervention of any external man - made chemicals and, hence, 100% biogenic . The colloidal solution of bme - ptnps showed a very intense brown color which indicates the reduction of platinum ions (figure 1(a) (inset)). The formation of bme - ptnps was further confirmed by tracing the reaction with uv - visible spectroscopy . The absorption spectrum of the brown platinum collides prepared by biogenic process showed a surface plasmon absorption band with a maximum of ~340 nm (figure 1(a)). Tem (figure 1(b)) analysis exposes mostly spherical shaped platinum nanoparticles of approximate size of 520 nm . Under careful observation, it was evident that the edges of the particles were lighter than the centers, suggesting that some bioorganic compounds such as proteins in bacopa monnieri leaf extract capped the platinum nps contributing to excellent robustness against agglomeration . The result obtained in the synthesis and characterization of nanoparticles is strongly supported by previously published report on the synthesis of platinum nanoparticles using phytochemicals . The compositional analysis through energy dispersive x - ray (edx) spectrometers illustrated the purity of the platinum, with the spectra showing a strong pt signal (figure 1(c)). Prominent bands were observed in the ftir spectra (figure 1(d)) at 616, 887, 1015, 1049, 1270, 1389, and 1705 cm, these peaks are assigned to alcohols c n stretching vibration of aromatic amines, germinal methyls, c = c groups or aromatic rings, and carbonyl groups, respectively . Significant peaks were not observed in the amide i (1640 cm) or amide ii (1540 cm) regions that are characteristic of proteins / enzymes accountable for the reduction of metal ions to nanoparticles by biological processes . These results indicate that phytochemicals of bm leaf extract like flavanoids that have functional groups of amines, alcohols, ketones, aldehydes, and carboxylic acid are robustly coated over the platinum nanoparticles synthesized . Because of the phytochemical coating and the redox chemistry of bme - ptnps, it is possible that they are biologically active as antioxidants . To determine if bme - ptnps can oxidize nadh, 100 m nadh was incubated with 50 g / ml bmept nps for 3 h and 6 h, respectively . The absorbance decreased and increased with time at 340 and 260 nm, respectively (figure 2). This is because the bands at 340 and 260 nm are from the n- transition of dihydronicotinamide part and - transition of the adenine ring, respectively . This result demonstrates that bme - ptnps have an activity similar to mitochondrial nadh: ubiquinone oxidoreductase, which is concurrence with the earlier published results of pectin protected platinum nanoparticles . This suggests that bme - ptnps are a potential medicinal substance for oxidative stress mediated disease with suppressed mitochondrial complex i, namely, parkinson's disease (pd). Oxidative stress was generated in zebrafish by exposure to mptp, which is an intracellular free radical - generating compound resulting in corresponding parkinsonian symptoms . The intraperitoneal administration of a single dose of mptp (225 mg / kg bwt) resulted in a profound increase in the levels of mda, diminished activities of antioxidant defense mechanism in charge for scavenging free radicals and maintaining redox homeostasis such as sod, cat, gpx, gsh, and complex i were observed in experimental parkinsonism - induced group (mptp) (figures 3(a) and 3(b)). The bme - ptnps concentrations tested were 0.3, 0.4, and 0.5 mol, respectively . The mda levels were significantly decreased by 0.4 mol of bme - ptnps (figure 3(a)). This makes clear the inhibitory effect of bme - ptnps over ros generation during mptp - induced oxidative stress . Glutathione (gsh) is a tripeptide with a free reductive thiol functional group, responsible for the detoxification of peroxides such as hydrogen peroxide or lipid peroxides and acting as an important antioxidant in cells . During the detoxification process, gsh (reduced form) becomes oxidized glutathione (gssg) which is then recycled to gsh by the enzyme glutathione reductase present in cells . The increased mda levels in mptp could be due to their increased production and/or decreased destruction by antioxidants such as gsh, sod, catalase, and glutathione peroxidase . The activities of antioxidant defense enzymes in charge for scavenging free radicals and maintaining redox homeostasis such as gsh, sod, catalase, and glutathione peroxidase are diminished during oxidative stress induced by mptp . In the present study, a statistically significant increase in the levels of gsh, sod, catalase, and glutathione peroxidase in the mptp treated zebrafish with 0.4 mol of bme - ptnps is being proved . This study demonstrates that bme - ptnps act as reductive catalyst, by the ability to scavenge ros, superoxide anion radicals (o2), and hydrogen peroxide (h2o2). This data is in good accordance with the previous studies where platinum nanoparticles are known to act as a sod / catalase mimetics to extend the lifespan of c. elegans, extended the lifespan of the roundworm caenorhabditis elegans[3941], inhibited pulmonary inflammation in mice exposed to cigarette smoke, inhibited cell growth of human tongue carcinoma cells, and furthermore ameliorated neurological function and brain damage after ischemic stroke . The mitochondrial respiratory chain, especially at complexes i, is thought of as a primary site of ros generation . In some oxidative stress diseases such as parkinson's disease, excessive ros generation is responsible for pathogenesis due to the suppression of complex i. in the current study a significant inhibition of complex i activity was observed in the experimental parkinsonism - induced group (figure 3(b)), which was attenuated by the pretreatment of various concentrations of bme - ptnps (figure 3(b)). However, 0.4 mol of bme - ptnps demonstrated a noteworthy effect on restoring the complex i activity as well as the levels of gsh, sod, catalase, and glutathione peroxidase in the mptp treated zebrafish . This result demonstrates that bme - ptnps serve dual functions as mitochondrial complex i to lower ros generation and as sod / catalase mimetics to scavenge generated excessive ros . Postmortem studies provided evidence for the decrease in the content of dopamine (da) and its metabolites dihydroxyphenylacetic acid (dopac), and homovanillic acid (hva) in the brains of parkinson's disease . Our results showed that the da, dopac, and hva contents in mptp zebrafish were markedly lower than those of control fish, and bme - ptnps increased da, dopac, and hva levels (figure 4). In parkinson's disease, mptp administration results in a significant reduction in the total movement distance, mean velocity, and mean distance per movement in zebrafish compared to the control animals . However, these reductions were partially rescued in bme - ptnps (0.4 mol / kg body weight) treatment animals, while bme - ptnps alone did not affect the locomotor activity (data not shown). Our current results suggest that bme - ptnps may be potentially effective in protecting against ros - mediated disease, by scavenging ros under pathophysiological conditions . In conclusion, platinum nanoparticles (bme - ptnps) have been synthesized from bacopa monnieri leaf extract (bme). The analysis of the chemical composition by edax strongly suggests the formation of elemental platinum nanoparticles instead of their oxides . From the tem analysis, ftir measurements provided strong evidence for proteins to form a coat covering the platinum nanoparticles to stabilize and prevent the agglomeration of the particles . This simple procedure for the biosynthesis of platinum nanoparticles has several advantages such as cost effectiveness, compatibility, and eco friendliness for biomedical and pharmaceutical applications . In addition, the ecofriendly method will be a competitive alternative to the existing methods for producing nanoscale inorganic materials . The antioxidant and neurorescue activities of nanosized bacopa monnieri phytochemicals coated platinum nanoparticles (bme - ptnps) were ascertained by alleviating the ros generation and scavenging free radicals, thus increasing the levels of dopamine, its metabolites, gsh, and activities of gpx, catalase, sod, and complex i and reducing levels of mda along with enhanced locomotor activity . These results provide ample evidence pertaining to the neuroprotective ability of bme - ptnps in mptp - induced neurotoxicity in zebrafish model of parkinson's disease via its dual functions as mitochondrial complex i and antioxidant activity (sod and catalase mimic activities). Further study is necessary to manifest the exact mechanism of action of bme - ptnps on neuroprotection in mptp - induced experimental parkinsonism in zebrafish model . Such a biocompatible and ecofriendly nanoparticle holds promise as a potential therapeutic option for parkinson's disease.

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