Metachromatic leukodystrophy (MLD) is a recessive autosomal disease which is characterized by an accumulation of sulfatides in the central and peripheral nervous system. It is due to the enzyme deficiency of the sulfatide sulfatase i.e. arylsulfatase A (ASA). we studied 5/200 cases of MLD and clearly distinguished three clinical forms. One of them presented the juvenile form; two presented the late infantile form; and two other presented the adult form. The Magnetic Resonance Imaging (MRI) of these patients showed a diffuse, bilateral and symmetrical demyelination. The biochemical diagnosis of MLD patients evidencing the low activity of ASA and sulfatide accumulation.Virtual slides: The virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/1791578262610232We studied 5/200 MLD patients addressed to us for behavioral abnormalities and progressive mental deterioration. All of them were diagnosed at first by brain MRI evidencing a bilateral demyelination, then the measurement of ASA activity using P-nitrocathecol sulfate as substrate, finally the sulfatiduria was performed using thin-layer chromatography using alpha-naphtol reagent.In this study, from 200 patients presenting behavioral abnormalities and a progressive mental deterioration, we reported just 2 patients were diagnosed as late-infantile form of MLD. Only1 case presented as the juvenile form; and 2 patients with the adult-type of MLD. The brain magnetic resonance imaging (MRI) of all patients showed characteristic lesions of MLD with extensive demyelination. Biochemical investigations of these patients detected a low level of ASA activity at 0°C and 37°C; the excess of sulfatide in sulfatiduria.MRI is required to orient the diagnosis of MLD patients; the latter must be confirmed by the biochemical investigations which is based on the measurement of ASA activity and the excess of sulfatide showed in the sulfatiduria. Metachromatic leukodystrophy or scholz's disease is an autosomal recessively inherited lysosomal storage disorder caused by the deficiency of arylsulfatase A (ASA). This enzyme catalyses the first degradation step of the glycosphingolipid 3-O-sulfogalactosylceramide (sulfatide). Sulfatide accumulates in white matter of the central nervous system and peripheral nerves as a result of ASA deficiency and leads to progressive demyelination and lethal neurological symptoms. Visceral organs with an excretory function such a kidney and gallbladder also store sulfatide but not so much affected functionally .MLD is divided into three major clinical forms according to the age of onset. The most frequent and fatal form is the late-infantile form which starts before 4 years of age and patients die by the end of the first decade. The juvenile form of MLD includes age onset between 4 and 16 years, while symptoms of adult MLD start after puberty. In a few patients, MLD results from deficiency of the activator protein saposin B (SAP-B) .For many years, since the discovery of the defect in ASA for MLD, and the easy enzymatic detection of ASA deficiency, this disease has been considered as affecting only children at the walking period, this neuropathology is characterized by a regression of previous acquisitions related to dys and demyelination . More prThe aim of this study is to underline the value of MRI for MLD diagnosis orientation and the interest of the biochemical study that involves the measurement of ASA activity and sulfatiduria to confirm MLD diagnosis.The patients in this study were unrelated and originated from different geographic areas of Tunisia. This study was approved by ethics committees of the respective Tunisian hospitals, and the families gave informed consent before withdrawal of blood. The studied patients were addressed to us for behavioral abnormalities and a progressive mental deterioration. The patients were always consanguineous.Family and history description of the 5 studied patients were summarized in table This boy was born as a child of healthy first degree consanguineous parents originated from the Sahel of Tunisia (Teboulba). The patient was delivered vaginally after an uncomplicated full-term pregnancy. He was admitted to the pediatric department of CHU Fattouma Bourguiba of Monastir at 3 years. He had difficulty in walking, nystagmus, spontaneous contraction at extremities. He had mental-motor retardation and was diagnosed as late infantile form of MLD with low ASA activity and the excess of sulfatide showed in his sulfatiduria profile. His brain MRI indicated characteristic lesions of MLD in the white matter; Figure This girl of 2 years was referred to the pediatric department of Nabeul hospital in the northern-west region of Tunisia. She was diagnosed as late infantile MLD; she had the same symptoms as the previous case, difficulty in walking, spontaneous contraction at extremities. She also had mental-motor retardation with low ASA activity and the chromatographic profile of sulfatiduria revealed the same excess of sulfatides showed in the first case. Her brain MRI showed the same lesions demonstrated in the first patient; Figure This little girl whose parents were first cousin was the second child, after a normal vaginal delivery. By the age of 5 years, this girl was evaluated for difficulty in walking at pediatric department of Sfax hospital in Southern Tunisia. After that, she lost her abilities of sitting, eating and swallowing. She had epileptic seizures, optic atrophy and spasticity. She was diagnosed as juvenile form of MLD on the basis of clinical analysis and the diagnosis was demonstrated by decreased ASA activity. Her sulfatiduria demonstrated the same results of the patients with late infantile form. Her brain MRI showed characteristic lesions of MLD; Figure A 43-year-old female, originated from the Sahel of Tunisia (Mahdia), presented a history of primary sterility. She was admitted to the neurological service of CHU Fattouma Bourguiba of Monastir for behavioural modifications, she showed gradual obvious dementing symptoms such as memory disturbance and disorientation. We also detected some neurological signs, i.e. pyramidalor cerebellar syndrome and peripheral neuropathy. The cognitive deficits encompassed several cognitive domains including memeory, visio-spatial, and executive functions indicating widespread organic lesions, and based on the initial brain MRI, evidencing a diffuse bilateral demyelination often symmetrical, sometimes limited to the periventricular areas and respecting U fibres; Figure A 34-year-old female was referred to the neurological department of Fattouma Bourguiba Hospital of Monastir (Sahel). She presented both mental deterioration and behavioural modifications at the onset of the disease. These modifications lead her to psychiatric institutions. During evolution, other modifications appeared such as memory dysfunction, ideation difficulties, spatio-temporal disorientation, and loss of sense of judgement. The brain MRI showed the same lesions of the patient S.F; Figure ASA was determined in leukocytes which were isolated from blood samples of patients and homogenized by three times of freeze-thawing . ProteinThe excess of sulfatides must be evidenced to confirm the diagnosis as there are pseudo-deficiencies of ASA: it was either detected on a peripheral nerve biopsy showing characteristic metachromatic deposits, or more easily looking for sulfatiduria in urine .This method was performed as followed: a 10 ml fresh urine sample was collected; 2 drops of concentrated acetic acid were added, after that the sample was left overnight in the cold; the pellet obtained after centrifugation 10 min at 2500 rpm, was collected and extracted by 5 ml chloroform/methanol 2:1 (v/v). After sonication, 1 ml distilled water was added; after mixing throughly and centrifugation, the upper phase contained urinary salts, and the lower phase, the lipid extract. The latter was evaporated to dryness and redissolved in 100 μl of chloroform:methanol 2:1for chromatography on silica-gel thin-later plates in chloroform:methanol:water 70:30:4 by volume; the revelation of the sulfatide glycolipid was done using alphanaphtol reagent .In This study, we estimated for the first time in Tunisia, the incidence of this neuropathology in our studied population; we founded just 5/200 cases representing the three MLD phenotypes. All suspected patients have a collapsed ASA activity in leukocyte homogenate and their chromatographic profiles were similar and revealed the presence of an abnormal band corresponding to the excess of 3-O-sulfogalactosylceramide and their brain MRI showed characteristics lesions of the three MLD forms in the white matter.The MLD is a lysosomal storage disease, resulting mainly from ASA deficiency which is essential for the degradation of sulfatides. Three clinical forms have been described: the infantile form, the juvenile form and the adult form. The disparity of these phenotypes reflects the difficulty of the clinical, biological and paraclinical diagnostic. The study of heterozygous subjects by determination of enzyme activity is limited because there is an overlap between the values of ARSA activity in normal subjects and heterozygous . In someThe prenatal diagnosis is the best way to put heterozygous couple to the disease, especially in a couple who had an affected child (index case). Currently, the identification of fetal genotype and predicting the type of the disease even in the absence of family history has become easier with the use of molecular technologies . It is iThe conventional therapeutic approaches are essentially symptomatic. They were made in order to restore the arylsulfatase A activity and prevent the progression of the pathological accumulation of sulfatides and consequently reduce morbidity associated with MLD. The aim of enzymatic therapy is to administer MLD patient with active enzyme, this approach will be the future of metachromatic leukodystrophy .The lysosomal storage diseases, especially the sphingolipidoses, presented a great clinical variability which complicated the diagnosis. According this study, the brain magnetic resonance imaging (MRI) was required to orient the diagnosis of MLD patients. The biochemical investigations such as the measurement of ASA activity and qualitative sulfatiduria were necessary to confirm MLD diagnosis. It is important to note that according this work, we estimated for the first time in Tunisia, the incidence of this neuropathology in our studied population; we found just 5/200 cases representing the three phenotypes of MLD.ASA: arylsulfatase A; MLD: metachromatic leukodystrophy; MRI: magnetic resonance imaging.The authors declare that they have no competing interests.IB carried out the enzymatic activities and the extraction of urinary sulfatides and drafted the manuscript. SH carried out the clinical diagnosis of patients with late infantile and juvenile form of MLD. SC performed the clinical diagnosis of patients with adult form of MLD. RB.M participated in the development of the used techniques. HC participated in the preparation of reagents. MN.G participated in collecting clinical data of patients with infantile and juvenile form of MLD. MF.A participated in collecting clinical data of patients with adult form of MLD. SF conceived of the study, and participated in its design and coordination. AM also participated in the study design and coordination. All authors read and approved the final manuscript.


Metastases to the supraclavicular region usually originate from the head and neck or from infraclavicular tumors. Ovarian primaries of supraclavicular metastases are extremely rare. The present study reports the case of a 60-year-old patient with a bilateral supraclavicular mass that was diagnosed as a poorly-differentiated squamous cell carcinoma of unknown primary, following a fine-needle aspiration biopsy (FNAB) and comprehensive clinical investigation. The analysis of a peripheral blood sample using the CellSearch system revealed the presence of circulating tumor cells (CTCs) that were positive for epithelial cell adhesion molecule (EpCAM) and cytokeratin (CK) expression. Since EpCAM is usually expressed in adenocarcinoma, an excisional biopsy of the right supraclavicular lymph node was performed. The patient was diagnosed with occult ovarian low-grade serous carcinoma by immunohistochemistry. To the best of our knowledge, this is the first report to demonstrate that CTCs may be detected in the peripheral blood of a patient with cancer of unknown primary (CUP) by using the CellSearch system. A literature review was performed to analyze the diagnostic procedures of CUP metastatic to the cervical lymph nodes and the clinical features of CTCs. Metastases to the supraclavicular region may originate from the head and neck and from infraclavicular tumors. The principal metastatic sites from the head and neck region are the hypopharynx, oropharynx and nasopharynx. Infraclavicular primary sites mostly originate from the lung, breast, lower digestive tract and genitourinary tract . OvarianIn February 2011, a female 60-year-old Chinese patient was admitted to the Otolaryngology-Head and Neck Surgery Department of the Beijing Tongren Hospital  complaining of a growing mass in the right side of the neck that had been present for six months. No other symptoms were observed. The patient’s surgical, medical and family histories were unremarkable. In the ear-nose-throat examination, a 2×2.5 cm solid, fixed mass was identified in the right supraclavicular fossa and multiple 1×1.5 cm solid masses were observed in the left supraclavicular fossa. A fine-needle aspiration biopsy (FNAB) was performed in the right supraclavicular mass and the pathological diagnosis was of a metastatic poorly-differentiated squamous cell carcinoma. Subsequently, the patient underwent a detailed comprehensive examination of the head, neck and upper aerodigestive tract using nasolaryngoscopy, bronchoscopy and gastroenteroscopy and CT scans of the neck and chest for primary malignancy, which demonstrated only lymphadenopathy in the bilateral supraclavicular region . The pat18F-fluorodeoxyglucose (FDG) positron emission tomography combined with computed tomography (PET-CT) revealed an intense uptake of FDG in the bilateral supraclavicular lymph nodes and a right pelvic mass with a diameter of 3.9 cm  Stage IV] was established by histological examination. Subsequent to surgery, the patient was treated with 135 mg/mCUP is defined as the histological diagnosis of metastasis without the detection of a primary tumor. CUP metastatic to the cervical lymph nodes accounts for ∼3–5% of all head and neck cancers . HoweverThe main objectives of the diagnostic evaluation of a patient with CUP are to determine the histology of the metastatic tumor and identify the primary tumor. Following a routine detailed physical examination of the head and neck and of the upper aerodigestive tract using panendoscopy, the initial tissue diagnostic procedure is a FNAB . A FNAB In the majority of cases, the biological material obtained by FNAB is sufficient for diagnosis. However, in certain cases, insufficient immunophenotypic characterization of the available biological material may lead to misdiagnosis . For exaIn the present study, it was shown that the analysis of peripheral blood using the CellSearch system provided additional information on the immunophenotype of the tumor cells. To the best of our knowledge, the present study is the first to demonstrate that CTCs may be identified in patients with CUP. This technology uses positive selection with magnetically labeled anti-EpCAM and immunocytochemical staining for CK8, 18 and 19 to isolate and enumerate CTCs. In this regard, the expression of EpCAM has been shown to occur in almost all adenocarcinomas, including ovarian adenocarcinomas ,14.et al reported a multicenter, randomized, exploratory study that included 216 patients with relapsed/recurrent advanced ovarian cancer (The CellSearch system has been approved by the US Food and Drug Administration (FDA) and is the only validated and standardized CTC detection system to be introduced into clinics. Increasing evidence suggests that CTC numbers are an independent predictor of progression-free survival (PFS) and overall survival (OS) in breast, colorectal and prostate cancer according to the CellSearch system . Poveda et al published a meta-analysis based on 16 studies that included 302 patients with cervical lymph node metastasis from an unknown primary . ER and/or PR are positive in 50–83% of ovarian serous carcinomas. Carcinomas arising in the breast and other sites in the female genital tract are also ER- and PR-positive, whereas carcinomas from other locations are ER- and PR-negative . WT1 andIn conclusion, to the best of our knowledge, the present study is the first to demonstrate that CTC may be detected in the peripheral blood of patients with CUP when using the CellSearch system. The molecular characterization and specific biological properties of CTC may provide important information for the diagnosis of CUP. Further studies on a larger patient population are required to evaluate the significance of CTCs as diagnostic decision markers in CUP. Although ovarian cancer rarely metastasizes to the cervical lymph node, it should be considered in the differential diagnosis of lymphadenopathy in the supraclavicular area of postmenopausal individuals.


Shot noise is one of the most extensively harnessed phenomena used to model different aspects of neural activity in neuroscience. Shot noise processes  display Campbell Limit corresponding to steady-state regime, or to specific post-synaptic responses that make the process Markovian or with the assumption of homogeneous Poisson event arrivals. In this work we go beyond this context to show that the shot-noise formalism can be used to analyze the synaptic activity of neurons beyond the Campbell limit, for any type of bounded post-synaptic response function and non-homogeneous Poisson event arrivals.In general, applications of shot noise -4 have bThis enhancement of technique allows for two main applications.1) Computation of the full density for current synapses and statistical moments for conductance synapses. This yields a statistical description of the subthreshold membrane potential. This includes considering distributions on the afferent populations over biological parameters such as peak intensity, number of independent channels, or dynamical parameters such as distribution of incoming firing rate or variable incoming firing rate. The picture obtained is of particular interest for various applications such as calculating the transfer function.in vivo.2) Under stationarity assumptions, this technique offers the potential to reverse the moments of membrane potential integration. This offers a candidate method to infer characteristics of the synaptic activity (and therefore of the network activity) from the analysis of the subthreshold variations of the membrane potential. This application is of particular interest for the analysis of intracellular recordings We illustrate these techniques by comparing analytic expressions with numerical estimates, as well as to neurons subject to a large range of synaptic activity (including low release rate far from the diffusion limit).In conclusion, due to their flexibility as a modeling tool, shot noise analysis appears as a very useful tool in computational neuroscience to model post-synaptic and sub-threshold membrane potential responses to incoming network activity, and also as a tool for electrophysiological analysis of network activity from intracellular recordings.


Psoas and thigh, although rare localizations, could represent the site of primary or secondary hydatidosis.Muscular hydatidosis should always be considered in patients with previous history of such a disease, especially in cases of previous surgical dissemination.A muscle- or nerve-sparing attempt should always be performed in cases of wide diffusion as well.A 46-year-old male shepherd presented with a mildly painful mass 4.5 cm in diameter localized at the right groin and thigh, diffuse edema involving the right leg lasting for 2 weeks, and fever. The patient had had eight previous operations for diffuse hydatidosis with reported intraperitoneal seeding, but further information was unavailable.Laboratory tests are shown in Piperacillin/tazobactam was administered from the date of admission to the day of surgery. Preoperative prophylaxis with benzimidazole derivatives was not performed due to the extent of the disease, the history of recurrences, and the need to perform the operation promptly to reduce the symptoms.At surgery, the retroperitoneum was accessed and tissues surrounding the cysts were covered with sponges soaked with hypertonic saline. The cystic content was evacuated and the interior of the cyst was repeatedly washed with protoscolicides. Due to the tight adhesions with the peritoneal sac, only the lateral-lower portion of the cystic wall could be resected. No daughter cyst was found. Subsequently, the right thigh was anteriorly incised and the cysts were evacuated. The washing treatment was repeated but extensive resection was avoided to prevent any risk of unnecessary damage.Histopathological examination did not detect viable protoscolices and routine cultures performed to individuate other pathogens were negative.A CT scan performed before discharge showed the integrity of the psoas muscle  and the In secondary hydatidosis, whose reported incidence is between 1.1% and 25% Hydatid cysts of the psoas and thigh are reported very rarely Different types of serological tests may help in diagnosis US represents the preferred diagnostic method Medical therapy with a benzimidazole derivative is preferable whenever there is multi-organ involvement or when patients are not fit for surgery Abnormalities in liver function, leucopenia and alopecia are side effects of the benzimidazoles The treatment of choice is complete surgical excision of the cyst and irrigation of the surrounding tissues with hypertonic saline. World Health Organization guidelines suggest several specific approaches for classic hydatidosis, but there is no mention regarding unusual localizations An alternative to surgery is the US-guided cyst puncture and injection of protoscolicides , indicated for inoperable patients Since our patient had eight prior operations with recurrence each time, perhaps none of these operations was adequately performed and the post-operative treatment was not properly instituted or monitored. The contemporary excision of all the macroscopic localization, the careful protection of the surgical field, and the postoperative administration of mebendazole distinguish our intervention from the eight that preceded it.This report emphasizes the importance of conservative treatment for a benign disease like diffuse recurrent hydatidosis. A muscle- or nerve-sparing attempt should always be performed and prolonged medical therapy is the mainstay of treatment. The risk/benefit ratio should always be analyzed in order to avoid extended surgical resections that may result in possible disability. Rare manifestations of neglected diseases are extremely important, especially for inexperienced physicians.


The striatum is populated by a single projection neuron group, the medium spiny neurons (MSNs), and several groups of interneurons. Two of the electrophysiologically well-characterized striatal interneuron groups are the tonically active neurons (TANs), which are presumably cholinergic interneurons, and the fast spiking interneurons (FSIs), presumably parvalbumin (PV) expressing GABAergic interneurons. To better understand striatal processing it is thus crucial to define the functional relationship between MSNs and these interneurons in the awake and behaving animal. We used multiple electrodes and standard physiological methods to simultaneously record MSN spiking activity and the activity of TANs or FSIs from monkeys engaged in a classical conditioning paradigm. All three cell populations were highly responsive to the behavioral task. However, they displayed different average response profiles and a different degree of response synchronization . TANs displayed the most transient and synchronized response, MSNs the most diverse and sustained response and FSIs were in between on both parameters. We did not find evidence for direct monosynaptic connectivity between the MSNs and either the TANs or the FSIs. However, while the cross correlation histograms of TAN to MSN pairs were flat, those of FSI to MSN displayed positive asymmetrical broad peaks. The FSI-MSN correlogram profile implies that the spikes of MSNs follow those of FSIs and both are driven by a common, most likely cortical, input. Thus, the two populations of striatal interneurons are probably driven by different afferents and play complementary functional roles in the physiology of the striatal microcircuit. We thus recorded and analyzed the simultaneous spiking activity of MSN–TAN or MSN–FSI pairs and used cross-correlation methods to identify the direct synaptic interactions and/or common input drives of these striatal projection—interneurons pairs.Both striatal interneuron cell types have been shown to be imperative to normal striatal functioning  were used in this study. Experimental protocols were conducted in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals and the Hebrew University guidelines for the use and care of laboratory animals in research. The experimental protocols were approved and supervised by the Institutional Animal Care and Use Committee (IACUC) of the Hebrew University and Hadassah Medical Center. The Hebrew University is an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) internationally accredited institute. The behavioral paradigm, surgery procedures, data-recording, and analysis methods were described in previous manuscripts  and video computerized analysis  that were advanced separately  into the different domains of the anterior striatum Figure . The eleThe discharge patterns of the recorded striatal neurons were characterized by the ISI CV and the auto-correlation histograms Figure . The ISISD of 20 ms). The baseline firing rate was calculated by averaging the firing rate in the last 3 s of the ITI and was subtracted from the smoothed PSTH. To determine a significant response in a single PSTH, we calculated the SD of the PSTH of the last 3 s of the ITI using the same number of trials as in the studied PSTH and identified time segments in which the deviation from the baseline firing rate exceeded three times the ITI-SD. A response was considered significant only if the duration of the deviant segment was >60 ms (three times the SD of the smoothing filter).Neural responses to behavioral events were characterized by a post stimulus time histogram (PSTH) starting at cue presentation and ending 2 s after outcome delivery Figure . PSTHs wThe signal correlation Figure . BrieflySD of 10 ms). For the TAN-TAN pairs the selection of the trigger and the reference cell was done randomly. Only cell pairs with minimal isolation quality  by using PSTH (reflecting the average responses of the recorded cell) and shift predictors . As expected for stationary data, PSTH and shift predictors yielded similar results. Only the PSTH correction method is presented here.To determine a significant peak/trough in a single CCH we calculated the SD of the last 0.5 s in both negative and positive lags of the CCH, and identified segments in which the CCH (±1.5 s around zero) exceeded three times the SD. A CCH was considered to a have a significant peak/trough only if the duration of the deviant segment was >30 ms (three times the SD of the smoothing filter). We used additional methods Abeles,  to deterTo better characterize the CCHs we calculated the area under the curve of the normalized CCH at ±1.5 s time lags. Specifically, we summed the number of spikes in the ±1.5 s CCH time bins and divided this sum by the number of bins to obtain the average number of added spikes of the reference cell (FSI or TAN) around the occurrence of a spike of the trigger (MSN) cell. This parameter ranges from negative values (indicating inhibitory correlation) through zero (indicating no correlation) to positive values (indicating positive correlation).Finally, to determine the skewness of a significant CCH we calculated a symmetry index. This index is found by subtracting the number of significant bins in the negative lag of the CCH from those in the positive lag divided by their sum.We recorded the activity of striatal neurons from two monkeys engaged in a well-practiced classical conditioning task Figure . The tasThe three populations of striatal neurons also displayed distinctive firing patterns Figure . TANs haSimilar phenomena were observed in the average auto-correlograms of the three populations Figure  shows a Cells in all three sub-populations were highly modulated by the task, particularly to cue presentation Figure . More thMSNs Figure  typicallUnlike MSNs, TANs Figures , 4B respp < 0.05, f = 75.13, df = 2; MSN response onset time was different from that of TAN and FSI). In terms of similarity of the neural responses of FSI-FSI pairs  from that of MSN-MSN and TAN-TAN pairs.FSIs Figure , like MSs Figure . A One-WTo sum up, all three populations of striatal projection and interneurons were highly modulated by the task; however, they differed considerably in their response profile and response synchronization levels.Figures N = 379 pairs) but three MSN-TAN pairs were not significantly correlated. We further calculated the average number of added spikes of the reference cell (TAN) to the trigger cell (MSN) around the corrected CCH time window of ±1.5 s (see Methods). A negative value would indicate that whenever the trigger cell spiked, the reference cell was more likely to suppress its discharge, a positive value would indicate the opposite, and zero would imply there was no correlation between the two. As predicted by the average flat CCH, we found the distribution of added spikes for the MSN-TAN pairs to be symmetrical around zero and not significantly different from zero  had a positive peak (N = 24 pairs) and only five had a negative trough. This is evident both in the average CCH . We found the correlation between MSN and FSI pairs was dependent on the cells' location within the striatum .Unlike the flat CCHs of MSN-TAN pairs, the MSN-FSI pairs were highly correlated. The average raw CCH of all MSN-FSI pairs  followed those of the reference cell (FSI). We quantified the asymmetry (in the CCHs with significant positive peaks) using a symmetry index (see Methods). Most (20/24) MSN-FSI pairs with a significant CCH had a negative symmetry index with an average of −0.31 ± 0.1  indicating a CCH peak that was shifted to the left. Like MSN-TAN pairs, the correlation between FSIs and MSNs was not dependent on the task event Figure , right. We simultaneously recorded the spiking activity of striatal projection neurons (MSNs) and interneurons (TANs or FSIs) from monkeys engaged in a classical conditioning task involving rewarding, aversive, and neutral cues.All striatal neurons were highly responsive to the behavioral events, but they displayed differential response properties. Striatal MSNs displayed the most sustained Figure  and diveThe TAN-MSN CCHs were flat, suggesting a modulatory rather than a driving effect of the synchronized TAN activity on MSN neurons. The FSI-MSN pairs displayed a broad and asymmetrical peak in their CCHs. Thus, our correlation analysis of the spiking activity of remote (>0.5 mm) FSI-MSN pairs does not reveal evidence for mono-synaptic inhibition, but it shows that generally FSI discharges precede MSN spikes.Striatal TANs constitute a very small percentage of striatal cells  and height of 1 spike/s above the average discharge rate of the reference TAN  was small since FSI make strong and dense projections on MSN neurons within a 0.3 mm radius of their soma  and the spiking activity of pairs of neurons have been reported previously  peak. This very broad peak might explain the discrepancy between our study and a previous rodent study  is very short and the result of the IPSC is often a delaying of MSN spiking. The end result could appear to be MSN firing following FSI firing with a delay that appears to be synchronous  with the FSI discharge. Our results thus imply that the shared cortical drive to both cell types, the faster responses of FSI and the relative delay of MSN discharge are the main processes in striatal microcircuit physiology. Therefore, consistent with growing evidence  method (Aertsen et al., We presented a differential functional relationship between MSNs and two types of striatal interneurons: TANs, the presumably cholinergic interneurons, and FSIs, the presumably PV expressing GABAergic interneurons. We did not find evidence for direct monosynaptic interactions between the MSNs and either striatal interneuron at the level of cross correlation of their spiking activity. However, the flat CCHs of MSN-TAN pairs contrasted with the asymmetric broadly peaked CCHs of MSN-FSI pairs. This suggests that the two interneuron populations play a different role in modulating MSN activity and striatal information processing (Szydlowski et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The use of unlicensed and "off-label" medicines in children is widespread. Between 50-80% of the medicines currently administered to children have neither been tested nor authorized for their use in the paediatric population which represents approximately 25% of the whole European population. On 26 January 2007, entered into force the European Regulation of Paediatric Medicines. It aims at the quality of research into medicines for children but without subjecting the paediatric population to unnecessary clinical trial. This article addresses ethical and legal issues arising from the regulation and makes recommendations for the framework conditions facilitating the development of clinical research with children. The network's primary goal is to contribute to the European Research Area on priority medicines for children by implementing a European joint research programme including development and innovation themes [The Coordination of Research on Priority Medicines for Children (ERA-NET PRIOMEDCHILD) is a network of research funding organisations from eleven different European Union (EU)-member-states, initially founded on January 1Between 2007 and 2010, key national and international organisations including the European Medicine Agency (EMA) and its Paediatric Committee (PDCO) representative members have been consulted to address research, research funding, public-private research cooperation, ethical and regulatory issues. A set of workshops and meetings involving stakeholders, i.e., academic experts from public hospitals, pharmaceutical industry's, insurance company's, parent associations' and health authorities representatives were held. Based on expert consultations, questionnaires sent to stakeholders and a literature review on that topic, ERA-NET PRIOMEDCHILD identified legal and ethical issues arising from the European Regulation of Paediatric Medicines and made recommendations for facilitating clinical research with children.patent protected and off-patent medicinal products.The three legal pillars of the new Regulation  are i) tpatented products and for the development of new medicinal products, pharmaceutical companies (applicants) have to submit a PIP to the PDCO before Marketing Authorization Application (MAA) is submitted. The PIP binds applicants, but it may be amended. Indeed, for products devoid of interest in children, "waivers" of class or indications or specific products can be requested [For new indications, new routes of administration or new formulations of already equested  as well equested .off-patent products for paediatric use. If paediatric indications and formulations - based on studies conducted in line with a previously agreed PIP - are authorized, it is possible for the applicant to get a PUMA approval with 10-year market exclusivity. Moreover, community funding for studies on off-patent products under the Paediatric Research Programme will be possible. Conversely, submitting a PIP and producing data about the paediatric population for off-patent products is an optional procedure.A new type of marketing authorization - Paediatric Use Marketing Authorisation (PUMA) - has been established by the Regulation to stimulate the development of off-patent products which represent an important part of available medicines for the paediatricians [off-patent products for children. The regulation should also have included obligations for applicants regarding off-label drugs  which are very commonly used in ordinary practice. Actually, clinical research on these products is rather complicated and the profit margin being low, pharmaceutical companies generally do not make off-label drugs available for children research [Not many PUMAs are so far being applied for tricians . To reduresearch .The paediatric committee (PDCO) includes representatives from all EU-member-states, health-care professionals, members of patients' associations, permanent and external experts, methodologists and ethics specialists. Evaluation by the PDCO should take into consideration the significant therapeutic benefits of the proposed studies for children, the need to avoid unnecessary studies and delay of authorization for adult populations. The drug safety and the monitoring of the clinical trials involving children by an independent safety monitoring committee is also an important issue to consider ,7. PDCO The United-States (US) approach for paediatric authorization seems pragmatic and more flexible than in Europe. Food and Drug Administration (FDA) asks to pharmaceutical companies a complete Pediatric Development Plan  providing any sufficient safety data base from adult population. When an off-label drug is used for a long period, US authorities give a paediatric authorization based on i) the number of paediatric patients already treated, ii) available efficacy and safety data collected among a rather large paediatric population, iii) the life duration of the off-label product use, iv) adequate safety data base in adults. Specific and justified studies are demanded only if those points are not met. Interactions between FDA and EMA should benefit from each other's experience the priorities for paediatric drug research being obviously set by the need of the patients, not by market considerations ,8-12.Beneficence) and in the Directive-2001/20/EC [Responsibility for the researcher). Article 4 on "Clinical trials on minors" gives specific rules to reinforce the protection of minors participating to clinical research. Most of these stipulations are related to the informed consent process. Specific protection of minors implies strengthened protection and possibly strengthened legal sanctions. Some countries sanction researchers who fail to obtain the informed consent  allowing him/her to take a free decision. Many parents openly admit that the informed consent process is useful, albeit often confusing. They find discussions more helpful than the consent documents . The decspecific rules aimed at assuring that minors' opinions should increasingly carry more weight in the final decision" [Despite the mandatory direct participation of children in a shared-consent procedure, in most cases consent is obtained after discussions with and agreement of parents only while the children view point is often not taken into account . The neoecision" .A clinical trial on minors may be undertaken only if benefit for this group of patients is obtained . HoweverThe application of the rules of good clinical practice when performing clinical trials of drugs for human includes the requirements for insurance policies safeguarding participants ,13. A moThe placebo use is going to be accepted at regulatory agency level in view of limiting the number of experimental subject. Indeed, regulatory and ethical guidelines highlight that in some instances the judicious use of placebo remains essential to demonstrate the efficiency of new medicines Table  26,36,3,336,37. A clinical trial on minors may be undertaken only if the ethics committee has endorsed the protocol . The ethIn case of multicentre clinical trials carried out in more than one member state simultaneously, an ethics committee's single opinion must be given for each EU-member-state implicated with the multicentre trial . Ethics The ERA-NET PRIOMEDCHILD final recommendations to facilitate the development of clinical research with children are summarized in Table In order to move from "research as a potential damage" to "research as health opportunity for children" European paediatric research should change in a multidisciplinary approach, taking advantage of the 30-year experience US approach. A better communication/sensitization of parents concerning the necessity of clinical research with a fair distribution of responsibilities among the stakeholders will increase parents' willingness to participate in trials. Sufficient time to consider pros and cons for child participation into a clinical trial should be given to the family including the suggestion to ask for external advice. To avoid supplementary delays due to the necessity to submit the study to several ethics committees the nomination of an independent ethics coordinator should be an opportunity. Extra funds, human resources, adequate research structures, education and training are necessary to optimise the process of implementing clinical trials with children.The authors declare that they have no competing interests.ALK and PP managed and coordinated the WP3 and wrote the scientific reports for the deliverables of ERA-NET PRIOMEDCHILD Work Package 3 (WP3). SB analysed and evaluated legal and ethical aspects of the regulation. ODCA analysed and evaluated regulatory aspects of the regulation. EB contributed the discussion of ethical and legal aspects of WP3 results. IL contributed the discussion of regulatory aspects of WP3 results. DA coordinated the overall ERA-NET PRIOMEDCHILD project and actively participated to all WP3 meetings. All authors contributed to the intellectual content and approved the final version.


Focal or diffuse inflammation is often present in the vessels of patients with CAD. Mast cells are frequently present in the plaques as well as in the inflammatory infiltrates in the atherosclerotic vessel wall. In the study we wanted to examine whether there are differences in the morphology, number and distribution of mast cells and in their ability to modify the atherosclerotic process in coronary arteries (CA) in the diabetic Coronary artery endarterectomy specimens were obtained from patients with diabetes or hypertension as the only risk factor for CAD. The specimens were stained with haematoxylin-eosin and Sulphated Alcian Blue for mast cells and with immunofluorescent methods for fibrinogen-fibrin and IgG deposits in the vessel wall. Both morphological and stereological assessments were conducted for mast cells and mononuclear cell infiltrates.The histological analysis of the vessel wall of diabetic patients in comparison with hypertensive patients showed a damaged endothelial cells layer and deposits of fibrin-fibrinogen and IgG in the tunica intima and media. The stereological count revealed a diminished numerical density of mast cells and a significantly higher volume density of the mononuclear cells. Mast cells displayed cytoplasmic vacuolization, extracellular extrusion of granule and pyknotic nuclei.This preliminary study suggests that the impaired mast cells might be the reason for more extensive inflammatory and immunologic atherosclerotic changes in the CA vessel wall of CAD patients with type 2 diabetes.134/88;C3-0564-381-92 Coronary artery disease (CAD) can be macroscopically visible as one or more localized atherosclerotic plaques, or as a diffuse, long, concentric thickness of the vessel wall, which protrudes and obstructs the vessel lumen. These changes alter the structure of the vessel wall, ultimately disrupting normal cardiac function. CAD is defined as a chronic inflammatory disease, and several cytokines and growth factors are involved in the pathogenesis of the disease -6. Surgivia many secretory mediators [The differences in the content and recruitment  of varioediators , in the ediators . It was ediators  mast celWe hypothesized that the morphology, number, distribution and probably the function of mast cells may differ in CAD patients with type 2 diabetes or arterial hypertension, as the only risk factor. Thus, we examined endarterectomy specimens of CA from CAD patients with diabetes or with arterial hypertension using histological and histomorphometrical analyses .Coronary endarectomy sequesters were obtained during Coronary Artery Bypass Graft Surgery (CABG) at the University Clinic of Cardiovascular Surgery, Novi Sad, Serbia, from 20 patients with CAD. The study was approved by the National Medical Ethics Committee. In 20 patients with CAD CABG surgery was performed due to angina pectoris, whereas endarectomy was performed in those coronary arteries with diffuse stenotic lesions. Twenty consecutive patients with diffuse CAD were enrolled in the study with either type 2 diabetes or arterial hypertension. The exclusion criteria were type 1 diabetes and the presence of both disorders . Patients were divided in two groups the group of patients with type 2 diabetes only (N = 10) and the group of patients with arterial hypertension only (N = 10)  of mononuclear cells infiltrates in diabetic patients in comparison with hypertensive patients Figure . Moreovevia their paracrine activity, especially through the release of heparin. It is known that in hyperglycemic conditions heparin may protect cells from injury [vs. hypertensive patients.In this preliminary study we demonstrated a clear difference in atherosclerotic lesions in CAD patients with type 2 diabetes in comparison with the patients from the hypertensive group. The samples from diabetic patients showed a greater degree of inflammation compared to the samples of hypertensive patients. The fibrin-fibrinogen and IgG deposits, accompanying abundant mononuclear cells infiltrates, and damaged endothelial cells of coronary arteries may indicate that atherosclerosis in diabetic patients constitutes humoral  and cellm injury . In diabA major limitation of this preliminary study is the relatively low number of CAD cases with either type 2 diabetes or arterial hypertension. We are planning to enroll new CAD cases with either type 2 diabetes or arterial hypertension to increase the reliability of the study. However, there are only some patients with CAD suitable for the endarterectomy procedure. Further studies enrolling larger numbers of CAD are needed to confirm our findings.This preliminary study suggests that the impaired mast cells might be the reason for more extensive inflammatory and immunologic atherosclerotic changes in the CA vessel wall of CAD patients with type 2 diabetes. Moreover, we speculate that mast cells are differently involved in atherosclerotic lesions in CAD patients with type 2 diabetes in comparison with hypertensive patients.CAD: coronary artery disease; CA: coronary arteries; BMI: Body mass index; HDL: high density lipoprotein; LDL: low density lipoprotein; IgG: immunoglobulin G; Vv: volume density; Nvm: numerical density of mast cells; SD: standard deviation;The authors declare that they have no competing interests.AM designed, coordinated and wrote the manuscript. AP and MZ provided the human coronary arteries and wrote the manuscript. DP led the treatment of patients and wrote the manuscript. AP, RZP and OVP made the histological examination, stereological and statistic analyses and wrote the manuscript. All authors have read and approved the final manuscript.


Proteins secreted to the extracellular environment or to the periphery of the cell envelope, the secretome, play essential roles in foraging, antagonistic and mutualistic interactions. We hypothesize that arms races, genetic conflicts and varying selective pressures should lead to the rapid change of sequences and gene repertoires of the secretome. The analysis of 42 bacterial pan-genomes shows that secreted, and especially extracellular proteins, are predominantly encoded in the accessory genome, i.e. among genes not ubiquitous within the clade. Genes encoding outer membrane proteins might engage more frequently in intra-chromosomal gene conversion because they are more often in multi-genic families. The gene sequences encoding the secretome evolve faster than the rest of the genome and in particular at non-synonymous positions. Cell wall proteins in Firmicutes evolve particularly fast when compared with outer membrane proteins of Proteobacteria. Virulence factors are over-represented in the secretome, notably in outer membrane proteins, but cell localization explains more of the variance in substitution rates and gene repertoires than sequence homology to known virulence factors. Accordingly, the repertoires and sequences of the genes encoding the secretome change fast in the clades of obligatory and facultative pathogens and also in the clades of mutualists and free-living bacteria. Our study shows that cell localization shapes genome evolution. In agreement with our hypothesis, the repertoires and the sequences of genes encoding secreted proteins evolve fast. The particularly rapid change of extracellular proteins suggests that these public goods are key players in bacterial adaptation. Prokaryotes secrete effector molecules to the environment and to exposed regions in the cell envelope to change their niche, scavenge resources and to interact with other organisms. Some of such functions require the secretion of proteins across the cell envelope either to the periphery of the cell, the cell wall in monoderms and the outer membrane in diderms, or to the extracellular environment. Secreted proteins perform a variety of important functions. They provide antibiotic resistance Salmonella enterica Typhymurium the expression of the type 3 secretion system 1 (T3SS-1) was found to double the generation time The very large size of typical bacterial populations compensates the reduced impact of a single bacterial cell on its environment. Thus, most of the environmentally relevant bacterial processes are social Escherichia coli, where the density of genes encoding secreted proteins is strongly related to the genetic mobility of the loci, i.e. the highest density of genes encoding secreted proteins is found in the regions of the genome that are gained and lost at higher rates Horizontal transfer of social traits favors the emergence and the stabilization of cooperative behaviors Pyrococcus concerned membrane or secreted proteins E. coli and B. subtilis, cell envelope proteins were found to evolve faster than the average protein after accounting for essentiality and expression levels Pseudomonas aeruginosa extracellular and outer membrane proteins evolve faster than cytoplasmic proteins Photobacterium profundum SS9 and Shewanella benthica KT99 showed more frequent positive selection in genes encoding functions related to motility and transport Mycobacterium tuberculosisStreptococcus pneumoniaeBacteria are constantly engaging in evolutionary arms races with their parasites, their hosts and their predators The previous examples suggest that protein localization shapes the rate of change of gene repertoires and sequences in bacteria. Yet, the pervasiveness of this effect has not been tested. In this work we do a systematic analysis of genetic diversification of proteins in function of their cell localization in both monoderms and diderms. Our dataset includes a large fraction of the most significant bacterial human pathogens and therefore we analyze the diversification of virulence factors in the light of their cellular localization. Thanks to this, we can explicitly link gene repertoires, sequence plasticity and the evolution of virulence factors in bacterial pathogens. Our work is aimed at testing the hypothesis that both key evolutionary processes among prokaryotes, accumulation of substitutions and horizontal gene transfer, drive rapid evolution of the secretome.Bacillus cereus and B. anthracis, but that are generally considered a single species. In order to accommodate the imprecision associated with measures of divergence based on a single gene (the 16S), the sequence similarity threshold was lowered to 98% if and only if the compared genomes had the same species name Salmonella spp. presented S. bongori with a large terminal branch (>0.1 subst/nt) and the genome was thus excluded even if it respected the 16S rRNA similarity criterion. To control for the effect of strain choice, we re-did all major analyses in this work putting together in clades only the genomes with the same species name. We found qualitatively similar results (data not shown). We thus defined 42 groups (clades): 28 of Proteobacteria and 14 of Firmicutes. These clades contain between 4 and 47 genomes (E. coli) to below 40% (e.g. Listeria)  and Proteobacteria (diderms). They include most of the best-studied prokaryotes in terms of secretion and virulence. We put together the gene repertoires of chromosomes  of closely related taxa within these phyla to compute their pan-genomes. A widely accepted concept of species for prokaryotes is lacking  genomes  and S2,  genomes , of whic2 tests on contingency tables after Bonferroni correction). The number of localized proteins per clade varied proportionally with the clade’s average genome size . The slope of the regression of genome size and the number of identified proteins size was not significantly different from 1 . Hence, the fraction of genes that cannot be classed is higher in the accessory genome but is not affected by genome size.To study the evolutionary patterns of the secretome we identified the cell localization of the proteins encoded by the pan-genomes with PsortB The results of PsortB suggest that most proteins are localized in the cytoplasm or in the inner membrane (resp. 60 and 33%). In Firmicutes, cell wall and extracellular proteins account resp. for 1.8% and 3.3% of all localized proteins, whereas in Proteobacteria the outer membrane and extracellular proteins account for resp. 2.5% and 1.8%. Genome size varies widely between and within the clades of our dataset. Larger genomes have higher rates of horizontal gene transfer 2 test on a contingency table) (E. coli and E. fergusonii. At this genetic distance (average 96.6% protein similarity) the genes are becoming too divergent to engage in homologous recombination To assess the possible role of intra-chromosomal recombination in the evolution of secreted proteins we identified the multi-gene families in the pan-genomes see . We founy table) . The inn2 on contingency tables). Genes encoding proteins localized in the cell wall and outer membrane, and especially extracellular proteins, are highly over-represented in the accessory genome relative to the core genome . Extracellular proteins were the most over-represented class in the accessory genome in 75% of the clades, followed by outer-membrane (Proteobacteria) and cell wall proteins (Firmicutes)  . These rThe accessory part of pan-genomes is predominantly composed of proteins encoding genes present in a very small number of strains (as low frequency genes) or in a very large number of strains (as high frequency genes)  was due to a number of reasons. (i) Cultivation and sequencing procedures often exclude plasmids from complete genomes. For example, all We studied the association between substitution rates and cell localization to test the hypothesis that genes encoding secreted proteins evolve faster. To avoid the effects of hidden paralogy and gene conversion we excluded from the analysis of substitution rates the few (6%) multi-gene families and the 2.6% of genes encoded in plasmids. Nine clades had too little genetic diversity, i.e. more than half of the genes had zero synonymous (dS) and/or non-synonymous (dN) substitution rates even when comparing the most distant taxa. At this level of divergence the analysis of substitution rates on a per-gene basis is not meaningful and these clades were excluded. One clade had either too closely related genomes or too divergent genomes. It was also rejected to avoid the problem of saturation of synonymous substitutions see . Hence, In the clades showing statistically significant association between substitution rates and cell localization the extracellular proteins show higher substitution rates than the cytoplasmic proteins . Similar2 test on a contingency table) and are highly over-represented among outer membrane proteins . Genes encoding virulence factors and genes encoding secreted proteins evolve faster than the other genes . The model for dN/dS explained a smaller fraction of the variance . Genes encoding virulence factors and genes encoding secreted proteins were associated with higher dN/dS . While statistically significant, the effects of removing the variable “homology to virulence factors” from the explanatory variables in the linear models were very small (less than 1% of the R2). Furthermore, they were 3 to 30 times smaller than the effects of removing the variable “protein localization”. Hence, the former variable has a much lower explanatory value than the latter. This suggests that the association between secreted proteins and virulence factors is not the major cause of the high substitution rates of the secretome.Many secreted proteins are known virulence factors, and these are often under positive selection in bacteria 2 of the linear models when removing the variable “homology to virulence factors”, which has a smaller impact on the R2 than the variable “protein localization”. Hence, the gene sequences of virulence factors show higher substitution rates per codon, but the effect of this variable is weaker than the effect of the variable protein localization. Interestingly, while the core genes encoding the secretome exhibit higher than expected dN/dS ratios, this is not the case for the genes encoding virulence factors . This reinforces the previous conclusion that higher rates of substitution per codon and higher dN/dS in the genes encoding the secretome are not trivial consequences of the over-representation of virulence factors in these localizations.Higher substitution rates in secreted proteins relative to virulence factors, could result from the over-representation of the former in the accessory genome  and as traits constantly adapting to environmental changes, they are both likely to evolve fast. Thirdly, we showed that genes encoding secreted proteins are over-represented in the accessory genome, evolve fast and show an excess of non-synonymous substitutions, but we have not studied their selection patterns. Notably we have not tried to disentangle in these trends the effects of weaker purifying selection from the effects of positive or diversifying selection. These analyses are very complex at this scale and are ongoing. Fourthly, we have ignored all proteins for which we could not reliably identify cell localization. This left out of the analysis a large fraction of the accessory genome. Nevertheless, substitution rates are higher in the genes encoding secreted proteins even among the core genome, suggesting this should not affect our conclusions.Within membrane-associated proteins, outer membrane proteins are more frequent in the accessory genome and show higher substitution rates than inner membrane proteins. Within the other localizations, extracellular proteins show the highest frequency in the accessory genome and the highest substitution rates. Thus, association to the membrane, which affects protein evolution Rhizobium and Cupriavidus tawanensis, and the free-living Geobacillus and Shewanella. Hence, rapid evolution of the secretome does not depend strictly on pathogenicity. Mechanisms of pathogenesis have naturally been subject to extensive scrutiny by the scientific community, but in many respects they are like many other processes that allow bacteria to scavenge their environment for nutrients Our work shows that virulence factors do not diversify as fast as other secreted proteins: they have lower substitution rates, lower dN/dS and are less frequent in the accessory genome. Naturally, our conclusions are dependent on the quality of the data from VFDB, which previous works indicate as the most accurate available databank on virulence factors ftp://ftp.ncbi.nih.gov/). We excluded pseudogenes. The 16S rDNA sequences were retrieved from the genomes using their annotations and their precise limits manually corrected when needed. Data on 2295 virulence factors was retrieved from VFDB −5, and more than 50% protein similarity along at least 70% of the size of the smallest protein. We discarded hits when one of the proteins was less than half the size of the other. A more permissive definition of homology  provided qualitatively similar results (data not shown). Protein localization of the representative of each protein family was computed with PsortB 3.1 Complete genome sequences and their annotations were retrieved from GenBank RefSeq .A preliminary set of orthologs was defined by identifying unique pairwise reciprocal best hits, with at least 60% similarity in amino acid sequence and less than 20% of difference in protein length. We refined this list using the distribution of similarity of these putative orthologs and gene order conservation as in . The anaWe computed a multiple alignment for each protein family of the core genome using muscle v3.6 (default parameters) Substitution rates between pairs of orthologs were computed using PAML 4 Figure S1Observed/expected ratio of the proteins per localization category among the three classes of core genome, accessory and present in more than 50% of the genomes (high frequency genes) and accessory present in less than 50% of the genomes (low frequency genes). Proteobacteria data on the left and Firmicutes data on the right.(TIF)Click here for additional data file.Table S1Description of clades from Proteobacteria used in the study. The table displays the number of genomes per clade, the average number of proteins per clade, the pangenome size and its decomposition in core and accessory genes, the number of proteins with predicted cell localization, the number of multigenic families, i.e. families with more than one member in any given genome, and the number of homologs to virulence factors.(DOC)Click here for additional data file.Table S2Description of clades from Proteobacteria used in the study. The table displays the number of genomes per clade, the average number of proteins per clade, the pangenome size and its decomposition in core and accessory genes, the number of proteins with predicted cell localization, the number of multigenic families and the number of homologs to virulence factors.(DOC)Click here for additional data file.Table S3Genomes used in the study, classification in clades, pan-genome and its spectrum of frequencies.(XLS)Click here for additional data file.Table S4Contingency table of cell localization by multi-gene families. First line in cell is the count, second line is the percentage relative to the cell localization column and the third line is the expected value. Abbreviations of cell localization: cytoplasm (Cyt), inner membrane (IM), periplasm , cell wall , outer membrane  and extracellular (Extr).(DOC)Click here for additional data file.Table S5Tests regarding genetic repertoires of the pan-genome. Columns depict: (ii) p-value of the test of independence of protein localization, (iii) localization with the lowest fraction of genes in the core (relative to the non-core genome), (iv & v) ratio of genes in core/accessory for extracellular proteins and for outer-membrane .(DOC)Click here for additional data file.Table S6Summary of tests for the substitution rates of the pan-genome.(DOC)Click here for additional data file.


Male pseudohermaphroditism is a sex differentiation disorder in which the gonads are testes and the genital ducts are incompletely masculinized. An 8 years old dog with normal male karyotype was referred for examination of external genitalia abnormalities. Adjacent to the vulva subcutaneous undescended testes were observed. The histology of the gonads revealed a Leydig and Sertoli cell neoplasia. The contemporaneous presence of testicular tissue, vulva, male karyotype were compatible with a male pseudohermaphrodite (MPH) condition. XY embryos will develop testes whereas XX ones will develop ovaries . The process regulating gonadal development is under genetic control and is referred as sex determination. Once gonads are well developed and fully functional, hormones play a key role in phenotypic sex establishment. In male, embryonic testes produce two key hormones: anti-Muellerian hormone  and testosterone. These hormones, together with testosterone derivative dehydrotestosterone, are responsible for the development of internal and external male phenotype. In the XX embryos the absence of these hormones lead to female phenotypic sex development  cell tumor with solid-diffuse and cystic-vascular patterns (right testicle) and a Sertoli [sustentacular] cell tumor (left testicle) .SRY gene, together with a 78, XY chromosome complement, were present, even if in this gene a new SNP (Single Nucleotide Polymorphism) was identified. The SNP discovered is not reported in any public database or in any publication involving dog SRY gene studies  the change occurs outside the HMG box and it is clear that this domain represents the unique important functional domain of this gene ; [SRY sequence analysis on a large number of normal male dog in order to establish the real nature of this SNP (rare polymorphism or mutation?) but in this case we can consider that it is possible a not fully expression of the mutation (as this condition is yet reported in human cases of SRY missense mutation) so we can observe the SNP in normal dog. Nevertheless our hypothesis is that this SRY SNP could not be responsible for the observed phenotype.Male pseudohermaphroditism is a condition of sex differentiation disorder in which the gonads are testes and the genital ducts or external genitalia are incompletely masculinised . In the s Figure . To estatation); ; b) The tation); ,13; c) Btation); , of thistation); -17. One XX) patients are described in human medicine [In addition to the pseudohermaphroditism in the described case, neoplastic growth was found in both testicles, an interstitial cells tumour and a Sertoli cells tumor. Already at ultrasonograpic examination, the increase of blood flow was suggestive of a neoplastic growth . Only femedicine , probablmedicine -22. To tRetained testes lack spermatogenesis and have an increased risk of tumor development , particuDue to the good differentiation of both testicular tumours, immunohistochemistry was only performed for vimentin, CK and Melan A. Histological appearance of most testicular tumours in dogs is highly specific and immunophenotyping of the neoplasia is not necessary. In cases of mixed tumours or poorly differentiated testicular neoplasias the use of 3-beta-hydroxysteroid dehydrogenase (3beta-HSD) and LH receptor markers may help in the identification of the tumour type . Sertolird phase. This may have occurred due to the lack of the scrotum, the particular conformation of the whole genital system and the hormonal metabolism of the dog.Descent of testes, epididymis and spermatic cord is a complex series of events that requires hormonal, constitutive, and nervous control. This process occurs in three main stages: the relative transabdominal migration phase, the intra-inguinal phase and extra-inguinal migration . In thisExamination of the testes revealed the presence of inactive seminiferous tubules and epididymes. In this case there were atrophic seminiferous tubules and epididymes, which is probably due to the compression of the tumour on the surrounding testicular parenchyma.Both testicles also presented a complete tunica albuginea, epididymes, vessels and cremaster muscle but no deferent ducts, probably because the surgical exeresis did not include it.This results confirm that when a congenital problem is suspected, karyotyping should be performed before any other test and if the reproductive problem of intersex is confirmed, surgical resection of genital tract is the treatment of choice to avoid clinical problems and the development of neoplastic diseases .The authors declare that they have no competing interests.BE performed the clinical and ultrasonographic examination, conceived, designed and wrote the paper. PaP and DL carried out the genetic analysis and helped to draft the manuscript. PeP performed the clinical examination and surgery procedures. WP, JS and PB performed the histological and immunohistochemical examination, intellectually contributed and helped to write the paper. CAM was responsible for the histological procedures and reviewed the paper. All authors read and approved the final manuscript.


The surgical treatment of open pilon fractures has a high complication rate especially in diabetic patients. In this article, we present a case of an infected tibial non-union after an open reduction and internal fixation in a diabetic patient, treated with Ilizarov external fixation combined with Papineau technique. Combined use of external fixation and Papineau technique can provide an alternative option for the treatment of septic pseudoarthrosis of the distal tibia. Operative fixation of distal tibial pilon fractures can become quite challenging as the extensive soft tissue compromise associated with such injuries can lead to a higher complication rate. The choices of surgical treatment include a) open reduction and internal fixation, b) external fixation, c) closed reduction with internal fixation and minimal periosteal stripping with preservation of soft tissues. Surgical complication may include ankle stiffness, infection, delayed union, non-union, and arthritis , 2.Wound infection is one of the most common complications of the surgical treatment of pilon fractures , 4. The There are many techniques for the treatment of an infected non-union of the distal tibia but none has been absolutely convincing in their efficacy. Such treatment options include removal of retained hardware, intramedullary and extramedullary debridement of necrotic tissue, placement of bone grafts and antibiotic spacers, use of Papineau technique, and application of an Ilizarov external fixation in a single or multiple reconstructive stages , 8–13.A 63-year-old male presented to the emergency department of our institution in January of 2011 with an open pilon fracture of his left tibia along with a fracture of the distal fibula . His medOne month after the operation, the patient presented with a skin lesion at the side of the incision  with a pStreptococcus mitis, Escherichia coli, and Stenotrophomonas maltophilia, and biopsies were positive for inflammation. Postoperatively, the patient was allowed immediate full weight-bearing and received oral antibiotics for an additional 3 months.Six months after the first operation, the patient was diagnosed with a septic non-union of the tibia. At that time, an extensive surgical osseous and soft tissue debridement was performed in addition to skeletal stabilization and Ilizarov external fixation. The skin defects were treated with the Papineau technique . IntraopSix months after the Ilizarov external fixation, there was radiographic evidence of fracture healing  and 8. TMany surgical protocols have been described to treat infected non-unions of the distal tibia but few exist for the treatment of septic tibial pseudoarthrosis in the diabetic population.In the presence of deep infections with implanted hardware for the treatment of long bone fractures, the surgeon is faced with a dilemma of initial implant preservation that may support the fracture but can certainly be the cause of continuous infection , 15 versEscherichia coli, Streptococcus mitis, and Stenotrophomonas maltophilia. The presence of Escherichia coli has been reported to be responsible for septic tibial non-unions in previous studies (Streptococcus mitis is a commensal oral streptococcus that presents no great immunological threat but has emerged as a significant pathogen in elderly immunocompromised patients and it is implicated in a wide range of diseases (Stenotrophomonas maltophilia is an environmental global emerging multiple-drug-resistant organism that is most commonly associated with respiratory infections in humans but can also cause osteomyelitis (In this case report, intraoperative cultures revealed  studies . Streptodiseases  while Stmyelitis , 21.The Ilizarov method has been used successfully for the treatment of septic tibial non-unions , 23. KarThe placement of antibiotic-loaded bone cement has also been reported for the treatment of septic pseudoarthrosis. The main advantage of this approach is the continuous local release of antibiosis over a longer period of time , 32. HowInitial debridement with incomplete bone resection followed by repeated local soft tissue management was originally described by Papineau in 1973. The technique involves open cancellous bone grafting on a granulated tissue base with delayed soft-tissue coverage by skin grafting or healing by secondary intention . PapineaIn conclusion, the combined use of Ilizarov external fixation and Papineau technique may be a feasible option for the treatment of infective non-union of the tibia, especially in the diabetic population. Further studies are necessary to evaluate the effectiveness of the method in a larger series of patients.


The safety of thyroid operations mainly depends on complete anatomical knowledge. Anatomical and embryological variations of the inferior laryngeal nerve (ILN), of the thyroid gland itself and unusual relations between ILN and the gland threaten operation security are discussed. The patient with toxic multinodular goiter is treated with total thyroidectomy. During dissection of the right lobe, the right ILN which has nonrecurrent course arising directly from cervical vagus nerve is identified and fully isolated until its laryngeal entry. At the operation, we observe bilateral Zuckerkandl's tubercles (ZTs) as posterior extension of both lateral lobes. The left ILN has usual recurrent course in the trachea-esophageal groove. The right ZT is placed between upper and middle third of the lobe points the nonrecurrent ILN. The coincidence of non-recurrent ILN pointed by a ZT is rare anatomical and embryological feature of this case. Based on anatomical and embryological variations, we suggest identification and full exposure of ILN before attempting excision of adjacent structures, like the ZT which has surgical importance for completeness of thyroidectomy. The thyroid has a lot of embryologic and anatomic variations affecting the safety of total thyroidectomy which is the procedure of choice for treatment of surgical disease of the thyroid. Therefore, a thyroid surgeon must have complete knowledge of all anatomic variations of the gland and of adjacent structures. As the inferior (or recurrent) laryngeal nerve (ILN or RLN) is the most important structure in terms of complication, anatomic variations of the nerve may threat the safety of the thyroid surgery. Nonrecurrent ILN is a rare but important example of anatomic variations –3.  Emil Zuckerkandl 1849–1910), an Austrian anatomist, has described posterior extension of lateral lobes of the thyroid gland 9–1910, a. The tub In this manuscript, we present the coincidence of ZT and non-RLN in a patient with toxic multinodular goiter treated with total thyroidectomy. A 66-years-old female patient with goiter had a history of thyroid gland enlargement for a long time. The patient presented our department with symptoms of hyperthyroidism. Physical examination, blood chemistry for hormones profile, ultrasound imaging, and thyroid scintigraphy established the diagnosis of toxic multinodular goiter. Thyroid hyper-function was normalized with antithyroid medication before operation. The right lobe is mobilized medially after ligation and cutting the middle thyroid vein and branches of superior thyroid artery closely to the gland. Between superior and middle third of the lobe, we observe posterior extension of the tissue; a grade 3 (larger than 10 mm) ZT is present in the right lateral lobe. The inferior thyroid artery is identified, isolated, and a loop of silk suture is placed around the artery for traction after freeing and medially mobilizing the thyroid gland. We use this artery as an anatomical landmark to identify the ILN. Using standard lateral approach, the right ILN is explored at anticipated crossing point of the nerve and the artery. The right RLN is not found at its usual course, and the dissection is advanced upward direction. A nonrecurrent laryngeal nerve is identified and exposed near the Berry ligament. The nerve is fully isolated from its originating point on the vagus nerve until its laryngeal entry. The nonrecurrent nerve is showing a parallel course with the inferior thyroid artery. It is placed at 3 cm cephalic position to the artery. The ZT situated at posterior side between upper and middle third of the right lobe is pointing the non-RLN . This is The safety of the thyroid operation mainly depends on the surgical anatomy of the ILN. Complete knowledge of RLN anatomy including all its variations is mandatory for thyroid surgeons. Nonrecurrent ILN is a rare but important variation affecting safety of thyroid operations. The prevalence of nonrecurrent nerve has been reported less than 1% , 3. Full Emile Zuckerkandl has been reported in 1902 “processus posterior glandulae thyroidea” , 5. Many Beside completeness of thyroidectomy, importance of ZT mainly arises from its relation (neighboring) with ILN. An understanding of the relationship between the ZT and RLN is essential for safety of thyroid operations , 10, 12. The ZT and ILN have close relationship increasing their surgical importance. Careful and meticulous dissection around the tubercle, when present, is mandatory for safety of thyroid operations. Anatomical variations of ILN, especially non-RLN, are real and serious threat for operation safety. We suggest identification and full isolation of ILN before attempting excision of adjacent structures, like the Zuckerkandl's tubercle which has surgical importance for completeness of thyroidectomy.


To prove the hypothesis that procedural knowledge might be tested using Key Feature (KF) questions in written exams, the University of Veterinary Medicine Hannover Foundation (TiHo) pioneered this format in summative assessment of veterinary medicine students. Exams in veterinary medicine are either tested orally, practically, in written form or digitally in written form. The only question formats which were previously used in the written e-exams were Type A Single-choice Questions, Image Analysis and Short Answer Questions. E-exams are held at the TiHo using the electronic exam system Q [kju:] by CODIPLAN GmbH.In order to examine less factual knowledge and more procedural knowledge and thus the decision-making skills of the students, a new question format was integrated into the exam regulations by the TiHo and some examiner used this for the first time in the computer based assessment. Following a successful pilot phase in formative e-exams for students, KF questions were also introduced in summative exams. A number of multiple choice questions were replaced by KF questions in four computer based assessment in veterinary medicine. The subjects were internal medicine, surgery, reproductive medicine and dairy science.The integration and linking of KF questions into the computer based assessment system Q [kju:] went without any complications. The new question format was well received both by the students and the teaching staff who formulated the questions.The hypothesis could be proven that Key Feature questions represent a practicable addition to the existing e-exam question formats for testing procedural knowledge. The number of KF questions will be therefore further increased in examinations in veterinary medicine at the TiHo. The University of Veterinary Medicine Hannover Foundation (TiHo) is one of five veterinary educational institutions in Germany. Over 2,400 students, 260 per semester, are enrolled at the TiHo, including PhD students. The 2006 licensure regulations for veterinarians (TAppV) gave veterinary medical educational institutions in Germany more freedom in designing teaching and exams, including the possibility of using new forms of teaching and learning .Each veterinary education institution in Germany has its own exam regulations in which exam requirements and procedures are laid down. Before 2006 most exams were traditionally performed orally. The latest addition to the TAppV allows the use of oral, written and multiple-choice question (MCQ) exams. Due to the alterations in the regulations, written tests in the form of computer based assessment (e-exams) were introduced in the TiHo exam  worked successfully. Both the students and the faculty gave good feedback on this case-based approach.The TiHo changed their exam system from only oral exams to a mixture of written (most electronic) and practical exams (LIT). With educational research the TiHo tries to find and establish more reliable and valid test systems and to carry out exams in a more competence based manner. The TiHo has currently reached the point where it uses computer based assessment  for assessing students of veterinary medicine in addition to traditional exam methods . The aim is not only to test descriptive but also procedural knowledge in written computer based assessment .et al and acceptance by the faculty and students was positive. Nevertheless, before it can be routinely used in exams there is still some work to be done. With the coming exams and the accompanying increase in the share of KF questions, test data such as the difficulty index, selectivity and Cronbach’s α will be collected, presented and discussed.The authors declare that they have no competing interests.ES and JE developed the idea and the study design and collected, analyzed and interpreted the data. ES was the primary author of the paper. JE and AT acted as supervisors of the work. All authors read and approved the final manuscript.


APC, MUTYH, PTEN, BMPR1A, SMAD4 and STK11 genes preconditioning the occurrence of individual diseases was performed employing PCR-SSCP, PCR-HD, DHPLC as well as RFLP techniques and DNA sequencing. At the present time, the DNA Bank comprises the total of 1097 DNA samples derived from 449 families with intestinal polyposis of which 945 samples come from persons in whose families Familial Adenomatous Polyposis (FAP) occurred. In addition, the collected data also contain material derived from 25 families with Peutz-Jeghers syndrome and from 20 families with juvenile polyposis as well as single cases with the Cowden syndrome, Proteus syndrome and desmoid tumors. The performed molecular investigations allowed identification of mutations from 44 to 50% studied families. With regard to the quantity of the material collected for analyses and the efficacy level of the employed molecular methods, the obtained results are in keeping with the results found in the literature from the field of genetics and medicine and do not differ from world standards. The collection of data and materials for investigations in the case of rare diseases allows qualitative, organisational and economic optimisation of the performed investigations.Intestinal polyposis syndromes comprise of a group of diseases conditioned by the occurrence of hereditary mutations. In the Polish DNA bank of hereditary predisposition to polyposis we collected DNA samples derived from persons from families with a diagnosed adenomatous polyposis including familial adenomatous polyposis coli together with its recessive form, Turcot’s syndrome, inherited mixed polyposis as well as persons with recognised hamartomatous polyposis containing juvenile polyposis, Peutz-Jeghers syndrome, Cowden syndrome and Proteus syndrome. Investigations were conducted on DNA isolated from the peripheral blood cells. The search for mutations in


HTLV-1 causes ATLL in 3-5% of infected individuals after a long latent period of 40-60 years. ATLL is divided into four stages: namely, smoldering, chronic, lymphoma and acute types. The smoldering and chronic types are indolent, but the acute and lymphoma types are aggressive ATLL characterized by resistance to chemotherapy and a poor prognosis. Such a long latent period suggests that a multi-step leukemogenic/lymphomagenic mechanism is involved in the development of ATLL, although the critical events in the progression have not been characterized. To determine whether epigenetic abnormalities are playing important roles in the progression of ATLL, we analyzed the methylation profiles, showing that number of CpG island methylated genes increased with disease progression and aberrant hyper-methylation in specific genes was detected even in HTLV-1 carriers and correlated with progression to ATLL. The CpG island methylator phenotype (CIMP) was observed most frequently in lymphoma type ATLL and was also closely associated with the progression and crisis of ATLL. The high number of methylated genes and increase of CIMP incidence were shown to be unfavorable prognostic factors and correlated with a shorter overall survival with the Kaplan-Meyer analysis. Increase of aberrant DNA methylation density was observed during the progression of an ATLL patient. The present findings strongly suggest that the multi-step accumulation of aberrant CpG methylation in specific target genes and the presence of CIMP are deeply involved in the initiation and progression of ATLL not only epidemiologically but also in the clinical course of a specific ATLL patient.


Lymphogranuloma venereum (LGV) is an important re-emerging sexually transmitted infection which is reported to affect particularly HIV-positive men who have sex with men (MSM). The aim of this study is to quantify the association between LGV and HIV in the context of the current emergence of LGV.A systematic review was performed on the emergence of LGV among MSM since 2000. We report the prevalence of HIV infection from descriptive studies of MSM with LGV, and conduct a meta-analysis to produce a summary estimate of the association between LGV and HIV from case-control studies where cases were MSM with LGV and controls were MSM with rectal chlamydia caused by non-LGV serovars.The prevalence of HIV among LGV cases ranges from 67% to 100% in 13 descriptive studies. There is a significant association between HIV and LGV .HIV-positive MSM are disproportionately affected by LGV highlighting the importance of prevention efforts to be targeted to this group. Further research is needed to determine whether the association is due to biological or behavioural factors. Chlamydia trachomatis (CT) which produces infection in humans and includes three biovars [Lymphogranuloma venereum (LGV) is a sexually transmitted infection (STI) which largely disappeared from the Western world after the introduction of antibiotics. LGV is caused by the species  biovars ; serovar biovars ) is tran biovars . Serovar biovars . LGV is  biovars  and, fol biovars -10. Repo biovars , Canada  biovars  and Aust biovars  followedThe clinical features of LGV have historically been divided into three stages: the primary stage involves the site of inoculation where a small papule, ulcer, herpetiform lesion or nonspecific urethritis appears after incubation period of 3-30 days . During In response to the outbreak surveillance systems were established in several affected countries. These have produced a consistent picture of cases among men who have sex with men (MSM), many of whom are co-infected with HIV  [As LGV seems to be circulating mainly among HIV-positive MSM it is important to consider the effect these STIs have on each other . STIs ma. (2001)  both GUDA systematic review was conducted using PubMed of the National Center for Biotechnology Information (NCBI), MEDLINE of the National Library of Medicine (NLM) and Web of Science (the latter two via ISI Web of Knowledge platform). The search was restricted for the years 2000-2009. The searches were performed on 17 June 2009 using the search words "Lymphogranuloma venereum"  and the acronym LGV with Boolean search looking for any of the words. No restrictions were selected on the type of studies, or publication language. An additional search was done in Eurosurveillance  on 18 JuThe grey literature was searched online using the websites of national public health agencies and professional organisations including the Health Protection Agency of England and Wales, the British Association for Sexual Health and HIV, the Public Health Agency of Canada and the Centres for Disease Prevention and Control of the United States. Reference lists of LGV publications were utilised to identify relevant literature and they were also reviewed for completeness of already found publications. No attempt was made to identify unpublished studies. Ethical approval was not sought, since the study relied on published data only.The study selection was done in two stages: during the first phase all publications involving a component of LGV epidemiology or management of LGV were included apart from publications focusing on the molecular biology of LGV or CT which were excluded. The study selection at this point was done based on abstract or the full publication if abstract did not give sufficient information. At the second phase complete publications were reviewed and their suitability in respect to the research objective was assessed. Studies were included if they met the following criteria: the main outcome was LGV and the study population was composed of MSM with data available for the level of HIV co-infection among study subjects. The selection of papers was restricted to MSM as they have been most affected by LGV in developed countries, and because the clinical manifestation of LGV is different among homosexuals than heterosexuals. Case-series and conference abstracts were excluded at the second stage of the review.From the descriptive studies that met all the inclusion criteria, the following variables were obtained: first author, year of publication, purpose and type of the study, study period, sample size, number of confirmed LGV cases (confirmation of LGV status requires detection of L1-L3 serovars from a chlamydia positive sample), age as reported in the paper, number of HIV positive LGV cases as well as number of cases with negative or unknown HIV status. Additionally from case-control studies information on the effect sizes and their confidence intervals and two by two table of the distribution of HIV serostatus was retrieved. When several publications reported data from the same setting, the latest or most complete report was used. The eligible publications were in English and Swedish as the first author is fluent in both languages.To estimate the prevalence of HIV among LGV cases, raw pooled prevalence estimates were calculated based on the numbers of HIV-positive and negative individuals given in the publications. A summary estimate was constructed and heterogeneity of the estimates explored using meta-analysis (using inverse-variance and derSimonian and Laird method ). To calTo analyse LGV-HIV association in case-control studies, a meta-analysis was performed using Mantel-Haenszel fixed-effect method as presented by Petitti (2000) . For tesThe search produced the following results: PubMed 270, Medline 273 and Web of Science 264. Many citations were common to all three search engines and after removal of duplicates there was a total of 368 original publications for the years 2000 to 2009. Eurosurveillance had 21 publications under the subject heading Lymphogranuloma venereum out of which 3 were identified through Eurosurveillance search only,8,10. SeDuring the first stage of the review 140 publications were excluded  and one study in London, UK. Therefore it seems that no large methodological, clinical or geographic sources of heterogeneity can be detected between the studies.We found consistent evidence of a strong association between HIV and LGV across the literature. In thirteen descriptive studies at least two-thirds of MSM with LGV were co-infected with HIV. In a meta-analysis, MSM with LGV were over eight times more likely to have HIV  than those who had non-LGV chlamydia infection. This association is stronger than for other sexually transmitted infections which have also re-emerged in MSM over the past decade . As the A number of detailed reviews have been published on the emergence of LGV ,51. HoweIn this review we did not apply strict quality criteria. This is due to the relatively small number of published studies and our desire to be inclusive in order to increase sample size. This means that we cannot exclude the possibility that sampling and measurement differences may account for some of the heterogeneity. Outcome-level assessment for LGV was performed by including only confirmed cases (identification of LGV serovars required for a confirmed case) which assures the validity of the data for the main outcome. Due to the small number of studies conducted to date and the recent emergence of LGV, publication bias was not thought to be relevant in this analysis. The search and article retrieval was conducted by the first author alone, but the analysis and interpretation was done by both authors.et al. [The strong association between LGV and HIV in an unadjusted model is interesting, and some association would be expected to remain after adjusting for confounders, especially since the association is strong even though the control group represents another high risk group of MSM with a non-LGV CT infection. It is possible that there may be a biological interaction between HIV-infection and LGV. HIV-infection has been reported to cause abnormalities in the structure and function of the gastrointestinal tract as well as abnormal lymphocyte trafficking  that mayet al.  also hypThe strongest confounder for this hypothesis is high-risk sexual behaviour. There have been behavioural changes in MSM communities as a response to HIV epidemic, and serosorting may make HIV-positivity an intermediating factor: individuals participate in certain type of practices partly because of their HIV-infection . SeroadaThe findings from HIV prevalence estimates and case-control studies reveal that HIV-infected MSM are disproportionately affected by the emergence of LGV. LGV prevention efforts should be targeted towards HIV-infected sexually active MSM. Although the majority of LGV infected individuals are HIV-positive, a proportion of diagnosed LGV cases are HIV-negative and this group would benefit from primary HIV prevention as they belong to sexual networks where HIV-seropositivity is common and subsequently they are at high risk of HIV-infection.The strong association between LGV and HIV co-infection identified from case control studies may indicate a biological synergy between LGV and HIV, but further research is required to explore the relationship between HIV-infection, behaviour and acquisition of LGV.The authors declare that they have no competing interests.HW and MR defined the research questions. MR performed the review and conducted the analysis and HW reviewed the results. Both authors wrote or reviewed the article. Both authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/11/70/prepubTable S1 and Table S2. Table S1 presents the descriptive studies selected for the analysis of prevalence of HIV co-infection in LGV cases, and Table S2 presents the case-control studies with estimates of association between HIV-positivity and LGV [ and LGV -67.Click here for file