For the whole 20th century, bovine tuberculosis (BTB) challenged the international community efforts to control this zoonotic disease. Asia and Africa accommodate the largest BTB-infected zebu cattle in the world. Similar to other few Asian nations, Iran has been actively running its BTB-control plan for the last four decades. BTB however, is still a number-one health concern for Iranian veterinary practitioners and also farmers across the country. Why is that? Here we have addressed this question in the light of most recent epidemiological data as well as microbiology and molecular biology observations. Iran (formerly known as Persia) has some of the oldest historical records on cattle farming in the world. The Zagros mountain chain in the west of Iran, a part of the Fertile Crescent with its ideal climate for animal farming, and the historic city of Soukhteh in Eastern Iran, have been recognised as birthplaces for cattle domestication  1)..1).Since 2010, Iran comprises 31 provincial regions which have a diverse range of climatic conditions from the tropical areas on the coast of the Persian Gulf to the deserts and arid lands on the Eastern half of the country . There aThe national cattle herd size in Iran in 2010 was about 8.5 million head  in the suburbs of Tehran . Since the 1960s, when Holstein-Friesian (HF) bovids were imported into Iran for the first time, HF cattle gradually became the predominant and favourite breed for Iranian farmers and were imported from several countries including USA and Canada.Records of BTB in Iran date back to the first half of the last century. In 1931, Carpantier, a French veterinarian employed by the Iranian army was the first to report BTB in zebu cattle slaughtered at Tehran old abattoir. Later in 1936 and 1937, these observations were added to by local veterinary officers from other Iranian cities. Some of the formalin-preserved BTB specimens collected at the time are still present in the veterinary laboratories of the Iranian Army in Tehran.An increasing number of the animals on Heidar Abad State farm were reported tuberculous in the early years after their importation and by 1947, 70 out of the 75 animals now on this farm were confirmed with BTB .Furthermore, in 1945 BTB was confirmed in the cattle herd of the Abadan National Oil Company, the second modern government cattle farm in Iran. The cattle of this herd were also European breeds that had been imported from abroad , 4.M. bovis. Similarly, an epidemic of BTB was reported in the imported cattle at the second government farm of the Abadan National Oil Company in 1945, which had probably imported cattle from the UK   .From 1941 onwards, along with increases in the importation of cattle and the commissioning of new cattle farms, particularly in the suburbs of Tehran, more and more breakdowns of BTB were reported in Tehran. This was believed to be partly due to the ignorance of farmers about the basic hygiene standards required for intensive cattle farming. On the other hand, at Razi Institute in the Eastern suburbs of Tehran, tuberculous lesions were regularly reported in cattle from local breeds which had been brought by the Institute from different Iranian provinces in order to produce Rinderpest vaccine against cattle plague which posed a big threat to bovids in Iran in 1940s , 4.In 1952 the first serious attempt to comprehend the epidemiology of BTB in Iran was made. This research was conducted under Jurgensen, a veterinarian adviser from FAO, in association with IVO and the Razi Institute. In 1967, legislation for the test-and-slaughter program that was compulsory for modern (European style) and semi-modern cattle farms, as well as traditional farms located within their perimeters was brought in. From 1967 until 1977 the test-and-slaughter program was noticeably expanded and apparently lowered the prevalence of BTB in Iran . In 1969M. bovis in this country  and the second isolate represented a new spoligotype (SB2182) .While there are a number workers reporting post-vaccinal complications after BCG-vaccination of children and adults in Iran –7, only  country . In a stBTB has been found repeatedly in cattle of local breeds in Iran. The infection in local breeds does appear to be less frequent, and with a sporadic nature compared to that in European bovids.It is very likely that BTB had a broader spatial distribution than that reported in IVO records of the 1940s and 1950s. The results of geographically-limited tuberculinations in the mid 1950s identified BTB in several Northern and Western provinces of Iran. However, later tuberculinations by IVO after 1980s detected the disease in the vast majority of Iranian provinces, indicating that BTB was epidemiologically endemic throughout much of Iran . WhetherPoor health measures in the husbandry of imported European cattle in 1940s and 1950s may have led to extensive epidemics of BTB particularly in the Tehran suburbs where most of the newly established farms were located. Over these years the highest ever prevalences of BTB were in farms with European cattle (28%).The 4-year lapse in the test-and-slaughter program from 1978 to 1981 due to the nation-wide strikes and establishment of the Islamic Republic of Iran and declaration of war on Iran by Iraq resulted in a rise in the prevalence of BTB from 0.43% in 1977 to 2.74% in 1984. Within a few years of the program starting to operate again, this figure re-commenced its downward trend and is still decreasing . This ‘iThe declining annual reports of BTB breakdowns in Iran has apparently been the result of the test-and-slaughter program but the program has not yet eradicated the disease from any Iranian province. Almost all IVO regional offices in Iran continue to report the BTB every year. The status of BTB in Southern provinces is noteworthy. Boushehr and Hormozgan, which participate in a test-and-slaughter program similar to other Iranian provinces, still report few outbreaks of BTB in some years thus, they are not considered BTB-free. These provinces have low cattle densities and they are not important farming zones compared to other parts of Iran. Further, these two provinces are coastal regions located on the Persian Gulf and thus geographically isolated. Moreover, the neighbouring provinces of Fars, Khouzestan, Kerman and Sistan also commonly have low prevalence of the disease .Almost a quarter of Iran, predominantly the North Western provinces, continues to be a major stronghold of BTB. The test-and-slaughter program has improved the status of BTB in this part of Iran but it still reports the highest rates of the disease.The epidemiology of BTB over the last 80 years in the Iranian cattle herd can be summarised as follows:M. avium D4 and the bovine tuberculin is made from M. bovis AN5.The national test-and-slaughter program in Iran is based on the single comparative intradermal tuberculin test (SICTT) using locally-prepared tuberculins. The bovine tuberculin and avian tuberculins are both provided by the State-run Razi Vaccine and Serum Research Institute (RVSRI). The avian tuberculin is prepared from the standard strain of Despite the successes of the test-and-slaughter program in Iran, it suffers from a number of weaknesses:It is estimated that almost 10% of the national cattle herd in Iran is regularly tuberculin tested by the program.Complete culling of all the identified reactors within the legal deadline of 20 days is not normally achieved leading to a remaining portion of potentially infectious cattle on their original farms, probably in contact with other cattle. The IVO records explain that in the majority of cases farmers are not satisfied with the compensation they would receive for their reactor animals. Consequently, some animals may be slaughtered and sold illegally before the deadline or farmers may have tried to hide or sell them.Farmers in rural areas of Iran tend to farm local, zebu breeds of cattle, they have small numbers of animals in their herds and grazing on pasture is typical. The density of local breeds in rural areas is lower than on the intensive farms of urbanised areas. On the other hand poorer health measures are likely to be in place and access to veterinary services is more difficult. Abattoir observations and the test-and-slaughter program results gathered over several decades show a higher rate of BTB in European breeds of cattle compared to the zebu local breeds . This haIn 1993 a study by Khaladj from IVO on BTB in the traditional cattle farms in Iran showed a 0.14% prevalence of the disease in cattle on these farms , the IVO initiated its first nation-wide system for collection of epidemiological data on animal infectious diseases .With more than 40 years of operating the test-and-slaughter program, there are only a handful of researchers conducting microbiological studies on BTB in Iran. The main reason is a lack of appropriate laboratory infrastructure. This dearth is not specific to Iran but is common over the whole of the Middle East. Occasional microbiology services for the IVO are provided by the Pasteur and Razi Institutes in Tehran and Karaj respectively, but these are not sufficient. The Razi Institute is mainly involved in the production of PPD tuberculins required for the test-and-slaughter program, and although it does provide a veterinary mycobacteriology diagnostic service there is a desperate need for IVO to establish a modern veterinary laboratory which would operate as an accredited reference facility.M. tuberculosis complex pathogens (most probably M. bovis). They may perhaps alternatively be infected with environmental mycobacteria that induce reaction against tuberculin , M. flavescence (9 isolates), M. szulgi (2 isolates), M. terrae (1 isolates), fast growing mycobacteria (5 isolates) and two unidentified isolates. Interestingly, all but four of these beasts had visible lesions in the carcass, which resulted in partial or total condemnation of their carcasses at abattoir. Khavari concluded that environmental mycobacteria in Iran are probably able to produce extensive tuberculous-like lesions in cattle.Cattle without a conclusive test result may or may not be infected with berculin –15. The  lesions . In the Fasciola hepatica) and Schistosomiasis (caused by Schistosoma bovis) can raise false positive reactions in the tuberculin test  and VNTR type (6554334) of one of the three most common clones of M. bovis in Iran. Whether transmission was from cattle to these mice or vice versa is unknown. Buffalos with a population of 650,000 heads (2010) are also farmed in some parts of Iran. West and East Azerbaijans, Khouzestan and Ardebil provinces accommodate 90% of the national buffalo herd. These provinces are also important cattle farming regions. In West and East Azerbaijan many farmers exercise mixed-farming of cattle and buffalo on their farms and pastures. Buffalos are not normally tuberculinated in Iran. Tadayon and co-workers reported collection of a single M. tuberculosis complex bacterium from bacterial cultures of a buffalo carcass slaughtered at Urmia, the central city of West Azerbaijan province. Their work included lymph node specimens from 140 slaughtered buffalos representing majority of province herds . However, until now no microbiologically-confirmed case of buffalo infection with M. bovis has ever been observed in Iran. Sheep and goat (with 54 and 25.7 million heads in 2010 respectively) are also frequently farmed in Iran. The latest records of IVO show that tuberculosis does occasionally affect goats. In 2003 M. tuberculosis complex spp. had been isolated from four goats in Kurdistan in North West of Iran. There are no confirmed reports of BTB in sheep in Iran. In 2003, abattoir specimens from a sheep carcass with typical tuberculosis lesions were processed for bacterial isolation. The culture on specific media for M. tuberculosis complex mycobacteria and incubation of the organism at 37°C successfully yielded bacterial growth. Nevertheless, the very weak bacterial growth hindered further molecular investigations in order to identify this isolate. In 2009, bacterial culture of lymph nodes from a follow deer in Hoveizeh, Khouzestan resulted in collection of a M. tuberculosis isolate that were later understood to hold a specific spoligotype (SIT587). In all, the role of reservoir animal(s) in epidemiology of BTB in Iran is as yet unknown and requires further studies.In some countries such as the UK and Ireland there is significant involvement of animal reservoirs in the epidemiology of f Tehran . A similce herds . This isThere are currently some limitations on cattle movements between different cities in Iran such as permanent IVO check points on major roads, but these are not adequate. Indeed the present farming system of Iran, particularly in rural regions, is hardly amenable to such restrictions unless new legislation is brought in to restrict cattle movements in order to augment the test-and-slaughter program.M. bovis reported that 6 of 7 M. bovis isolates carried a single copy of IS6110 on the discriminative 1.9 kb restriction fragment and one isolate had three copies of IS6110 cattle in the last fifty years. Tadayon et al. (2008) speculates that the absence of adequate initial BTB control measures could have led to the rapid clonal expansion of an SB0120 strain.Feizabadi was first to employ molecular techniques to understand the epidemiology of BTB in Iran. Initial molecular genotyping of f IS6110, 21. Couf IS6110. This woPvu II and Alu I-digestion and RFLP-PGRS typing. All of these methods detected a visibly homogenous population in which variants were generally only detected in a few isolates. Only Alu I-based RFLP was able to clearly differentiate between BCG and field strains of M. bovis. As in previous reports, these findings seem to reflect the recent dispersal of a few strain(s) in Iran following the substantial expansion of HF cattle over the last few decades.These findings have been confirmed by a subsequent study  in whichM. bovis in Iran, raises the interesting question as to the origins of M. bovis strains currently found in Iran. These strains might have been indigenous, exotic or a mixture.At present, BTB is endemic across much of Iran although the current prevalence of infection is low . The recM. bovis isolates from cattle in these countries. The closest spoligotype between Turkey (SB1198) and Iran (SB0288) has deletion of spacers 3, 8, 9, 10, 11, 12, 16, and 39–43, although spacer 23 is exclusively deleted in the Turkish spoligotypes. It is interesting that SB0288 has been reported from West Azerbaijan which is actually the bordering province between Iran and Turkey. However, there is no VNTR data for the Turkish isolates for further analysis.A link between Iranian strains and those of its neighboring countries  is likely. Unfortunately, there is a lack of typing studies on M. bovis as a consequence of European colonization  has been found as a common strain type in most European countries and countries that imported European cattle  Fig. 6)Fig. 6). There are reasons to believe that zebu cattle and West African Dama beasts are more resistant than Jersey/Guernsey cattle to BTB . An abatM. bovis population and the generation over this period of 2 or 3 sequential spoligotype changes is clearly compatible with these timescales. The absence of geographical regionalization of strains in Iran may also reflect the shorter timescale of events than in the UK with insufficient time for the significant diversification of new strains. The similarities of the Iranian isolates to each other at very high level suggest a single, common ancestral strain of the SB0120 spoligotype. Whether such an ancestral strain originated in Iran or had been imported into Iran could only be established through further genotypic comparison of Iranian SB0120 isolates with others from neighboring countries and from around the world.Of the fourteen spoligotypes reported to date in Iran, seven  have only been reported from Iran and these account for over 60% of all the typed isolates until now . Some ofM. bovis strains and would also reduce strain regionalization, as has been seen in Cameroon  and VNTR type (6554334) of this isolate showed it to be one of the three common clones of M. bovis in Iran. Overall, little is known in Iran about other reservoirs of M. bovis and the findings of recent work confirm there is a need for further epidemiological studies on M. bovis in wild, and domestic, animals of Iran and their potential role as hosts for this pathogen.Molecular genetics studies on isolates . TherefoM. bovis isolates from across the world, Smith and co-workers recently identified a globally important clonal complex of M. bovis characterized by deletion of a genomic locus named RDEu1. While this group of M. bovis isolates are frequently found in UK, Ireland and their former commercial partners including South Africa, USA and Australia and even Kazakhstan but they have not been reported from Iran until now (M. bovis seem that are frequently observed in Africa are not presents in Iran (M. bovis clone (European 2) is also not present in Iran, further work is required as lack of spacer 21 which is a characteristic specification of this close has also been detected in Iranian M. bovis isolates (SB0265)   Fig. 6)M. bovis The very recent highly escalated political tensions seen in the international community over Iran's nuclear interests has resulted in imposing the most serious trade sanctions ever seen on Iran. Given the fact that the envisaged reductions in the country's oil revenues might continue for long in the coming years, cuts in funding of health schemes including test-and-slaughter program is likely. Working to develop a bovine Tb vaccine therefore, seems to be a very useful economical way to support the current bovine Tb control scheme in Iran.


Glioblastoma can be classified into four distinct molecular subtypes , based on gene expression profiling. This study aimed to investigate the prevalence, clinicopathologic features and overall survival (OS) of the four molecular subtypes among all malignant gliomas.A total of 941 gene expression arrays with clinical data were obtained from the Rembrandt, GSE16011 and CGGA datasets. Molecular subtypes were predicted with a prediction analysis of microarray.Among 941 malignant gliomas, 32.73% were Proneural, 15.09% Neural, 19.77% Classical and 32.41% Mesenchymal. The Proneural and Neural subtypes occurred largely in low-grade gliomas, while the Classical and Mesenchymal subtypes were more frequent in high-grade gliomas. A survival analysis showed that the Proneural subtype displayed a good prognosis, Neural had an intermediate correlation with overall survival, Mesenchymal had a worse prognosis than Neural, and Classical had the worst clinical outcome. Furthermore, oligodendrocytomas were preferentially assigned to the Proneural subtype, while the Mesenchymal subtype included a higher percentage of astrocytomas, compared with oligodendrocytomas. Additionally, nearly all classical gliomas harbored EGFR amplifications. Classical anaplastic gliomas have similar clinical outcomes as their glioblastoma counterparts and should be treated more aggressively.Molecular subtypes exist stably in all histological malignant gliomas subtypes. This could be an important improvement to histological diagnoses for both prognosis evaluations and clinical outcome predictions. Glioma is the most common brain tumor type and an important cause of cancer mortality in adults and children In the present study, we reviewed 941 glioma samples with gene profiles from three glioma genome databases . Molecular subtypes were assigned by Prediction Analysis for Microarrays (PAM), using the TCGA 840-gene classifier http://www.cgga.org.cn/portal.php), GSE16011 (http://www.ncbi.nlm.nih.gov/geo/) and the Rembrandt databases (https://caintegrator.nci.nih.gov/rembrandt/) were gathered from published studies Microarray data from CGGA  was performed to predict the molecular subtypes of glioma samples from gene profiles, using the Verhaak et al. 840-gene classifier A total of 941 samples with gene profiles from incident cases of glioma were gathered from 3 databases . The molecular subtypes were predicted with PAM . As showThe prevalence and clinical features of the molecular subtypes were further evaluated in each histological glioma subtype. Only samples with precise histological grade and follow-ups were included in the following analysis. As shown in Additionally, oligodendrocytomas had a higher prevalence of Proneural and a lower prevalence of Mesenchymal , compared with astrocytomas .Furthermore, the survival analysis showed that molecular subtypes did not correlate strongly with overall survival. The molecular subtypes significantly stratified the anaplastic gliomas into different prognostic subgroups. In glioblastomas, Proneural had a good prognosis, Classical had the worst clinical outcome and Neural and Mesenchymal had intermediate correlations with overall survival . BesidesClassical glioblastomas are reportedly characterized by EGFR amplifications. In the present study, we first report that EGFR amplification is also enriched in Classical anaplastic glioma samples. As shown in As shown in Gliomas are the most common primary malignant brain tumors in adults, with much heterogeneity in both histopathology and clinical courses Reportedly, the intrinsic gene expression profiles of gliomas are better predictors of survival than histology In glioblastoma, the Proneural class was highly enriched for the oligodendrocytic signature, but not the astrocytic signature, whereas the Mesenchymal class strongly associated with the cultured astroglial signature In summary, the molecular glioblastoma subtypes suggested by the TCGA network are relevant for low-grade and anaplastic gliomas and are associated with different prognoses. The above-described molecular subtyping system could be an important improvement to routine histological diagnoses and might guide therapeutic glioma management.Figure S1The molecular subtypes of glioma samples from GSE16011 and Rembrandt datasets were predicted using Prediction Analysis for Microarrays (PAM).(TIF)Click here for additional data file.


Antibody-mediated rejection (AMR) is a major cause of late kidney transplant failure. It is important to have an understanding of human-leukocyte antigen (HLA) typing including well-designed studies to determine anti-MHC-class-I-related chain A (MICA) and antibody rejection pathogenesis. This can allow for more specific diagnosis and treatment which may improve long-term graft function. HLA-specific antibody detection prior to transplantation allows one to help determine the risk for AMR while detection of DSA along with a biopsy confirms it. It is now appreciated that biopsy for AMR does not have to include diffuse C4d, but does require a closer look at peritubular capillary microvasculature. Although plasmapheresis (PP) is effective in removing alloantibodies (DSAs) from the circulation, rebound synthesis of alloantibodies can occur. Splenectomy is used in desensitization protocols for ABO incompatible transplants as well as being found to treat AMR refractory to conventional treatment. Also used are agents targeted for plasma cells, B cells, and the complement cascade which are bortezomib rituximab and eculizumab, respectively. There are 20 class I genes and in transplantation HLA-A, B, and C are the classic genes referenced to when typing the recipient . Class Iγ-producing cells were produced stimulating CD4+ and NK cells [α, IL-8, and chemokine ligand 2) allowing complement activation. In antibody rejection, C4d is a useful marker of complement activation and is seen commonly in peritubular capillaries with proper immunohistochemical staining technique. Chemoattractants C3a and C5a are a part of the compliment cascade which activate C5b allowing for assembly of membrane attack complex (MAC) which is composed of C5b-C9. The MAC complex once activated will cause local necrosis allowing detachment of endothelial cells from the basement membrane, which is also a characteristic finding upon biopsy indicative of AMR. If not treated properly in a timely manner, sequelae can include thrombotic microangiopathy (TMA) causing hemorrhage with arterial wall necrosis, and ultimately graft loss. Also potentially playing a role in AMR pathogenesis are polymorphic MHC-class-I-related chain A (MICA) antigens [Not only can antibodies form against HLA molecules, but to endothelial-cell antigens and across ABO blood group . SensitiNK cells . With deantigens . MICA anantigens . MICA anAMR occurs in up to 33% of cases undergoing ABO incompatible kidney transplantation. Among cases in which AMR is triggered in the early post-op period, prevalence of transplant glomerulopathy is 22% at one year . This isCrossmatching not only improves long-term graft survival by identifying the presence of antidonor antibody in the recipient, but is used as a predictor of AMR . Flow crThe 1997 Banff classification was used to classify rejection prior to the meeting in 2001, which further defined pathological classification of AMR . Gorer dAcute tubular injury; neutrophils and/or mononuclear cells in peritubular capillaries and/or glomeruli, and/or capillary thrombosis; or arteritis/fibrinoid necrosis in the intima along with intramural/transmural arterial inflammation.C4d evidence for antibody action and/or immunoglobulin in peritubular capillaries, immunoglobulin and complement in arterial fibrinoid necrosis.Anti-HLA antibody (DSA) circulation in serum or other antidonor endothelial antigens.After the Banff meeting in 2009, positive C4d deposition without evidence of rejection was added to criteria suggesting antibody-mediated changes. Several studies had reported this finding including Mark Haas (Los Angeles) who had seen C4d deposition in biopsies without evidence of rejection . In protIn normal kidneys, specifically in the glomerular mesangium as well as arterioles at the vascular pole, turnover of immune complexes occurs (C4d) . Transpln = 173) with DSA and ENDATs had diffuse C4d staining in peritubular capillaries. Investigators also have found that C4d negative form of AMR is less severe than C4d positive AMR but is associated with chronic changes within the graft, such as transplant glomerulopathy. A possible complement-independent mechanism of microcirculation injury in AMR is plausible. It has been postulated that DSA binding to endothelial cells triggers natural-killer cells to release interferon-γ resulting in granule-associated toxicity.There has also been evidence for C4d negative AMR in a couple studies, which in the past few years has brought to the forefront the limitations of C4d as a marker for AMR . In one DSA has been found to play a role in diagnosis of AMR and can be an independent predictor of allograft loss . It was Plasmapheresis (PP) removes alloantibodies from the circulation and is the fastest and most effective method for elimination of DSA . UsuallyStaphylococcus aureus. The protein A coating the column beads has a strong affinity for the Fc portion of immunoglobulin and immune complexes [Immunoadsorption (IA) is a substance derived from the Cowan strain of omplexes . For theIVIG is prepared by human plasma from approximately 50,000–100,000 of healthy donors, composed of 90% intact IgG, a few dimers, Fabs (fragment antigen-binding) and traces of IgM and IgA . there aJin et al. had used Thymoglobulin (ATG) 0.75 mg/kg/day for 5–10 days in combination with plasma exchange in 7 patients with AMR and showed 85% reversal in AMR . ATG is Haemophilus influenza vaccine, pneumococcal, and meningococcal vaccines.The spleen is the largest lymphoid organ in the body and has been found to play a role in alloantibody generation. Splenectomy is used in desensitization protocols for ABO incompatible transplants as well as positive crossmatches, however it has been found to surprisingly treat AMR refractory to treatment . There aMost episodes of AMR are associated by evidence of early complement activation by the presence of C4d+ staining of the peritubular capillaries . The exaThe transplant community is looking towards the use of this medication because of its highly selective mechanism of action . There aThe transplant community has recognized the potential use of an agent that can directly target plasma cells. Traditional treatments have been successful in removing antibodies, inhibiting antibody activity or lowering production but no treatments have been effective in targeting mature antibody production in plasma cells. AMR involves the production of high levels of DSA by plasma cells either newly made from memory or naïve B cells or from those that existed prior to transplant. One mechanism to control AMR is to target DSA production by plasma cells. Bortezomib is a proteasome inhibitor which is FDA approved for the treatment of multiple myeloma. This medication has been shown to cause apoptosis of normal plasma cells which in turn decreases alloantibody production in sensitized patients .Several case reports and series have been published regarding the use of bortezomib. Currently available studies of bortezomib for treatment of AMR are summarized in Rituximab is a chimeric anti-CD 20 (anti-B cell) monoclonal antibody that is currently FDA approved for the treatment of lymphoma. The CD 20 antigen is expressed early in B-cell cycle but is absent on mature plasma cells. The variable region of rituximab binds to CD 20 through three different mechanisms including antibody-dependent cell cytotoxicity, complement-dependent cell killing, and induction of apoptotic cell death. These mechanisms mark cells for destruction and leads to sustained depletion of circulating B cells , 33. Genn = 26) received plasmapheresis and rituximab and Group B (n = 28) received plasmapheresis alone. Patients with low serum IgG levels also received immunoglobulin. The two year graft survival was significantly better in the rituximab group [The first report of using rituximab in AMR evaluated 27 patients with refractory rejection and received a single dose of rituximab. Three grafts were lost and the remaining grafts had good function at the time of discharge . Kaposztab group . Since tab group –40. Withab group .


Kaposi's sarcoma (KS) is a low-grade vascular neoplasm associated with human herpes virus-8 (HHV-8) infection, and, in the epidemic form, with the human immunodeficiency virus (HIV). Although HHV-8 is present in all body fluids and is sexually transmitted, there are surprisingly few reports of anogenital KS. Clinically, especially in HIV/KS endemic areas, dark stained skin patches or nodules are prone to misdiagnosis, especially in dark-skinned individuals. Therefore, a biopsy is recommended. The histologic appearance spans a broad spectrum of KS and non-KS lesions; therefore, the final diagnosis should be confirmed by HHV-8 immunohistochemistry. We report a series of 36 anogenital biopsies from a group of 16 documented HIV-positive patients; in 20 the HIV serostatus was unknown. There were ten KS (five in HIV-positive patients), and 26 non-KS (11 in HIV-positive subjects) lesions. In the era of HIV/AIDS, anogenital lesions may be the first manifestation of KS in immunocompromised individuals and should be biopsied. The histological diagnosis should be confirmed by HHV-8 immunohistochemistry. The human herpes virus-8 (HHV-8), also called Kaposi's sarcoma herpes virus (KSHV), is the causative agent of all types of Kaposi's sarcoma (KS), namely the classic, endemic, iatrogenic immunosuppression, and the epidemic/human immunodeficiency virus (HIV) related forms. The modes of acquisition of the KSHV and its transmission vary with age, gender, geography, and sexual practices . AlthougIn Uganda, HHV-8 DNA was detected in 28 percent of oral swabs and 27 percent of blood samples of healthy asymptomatic subjects –6. It haThe sexual transmission of KSHV is more controversial. In the industrialized world, there is evidence of transmission between men having sex with men (MSM) via anal but not oroanal sex , 12, 13.The purpose of this study was to describe the clinical and histopathological spectrum of anogenital lesions either under- or overdiagnosed as KS in a HIV/AIDS endemic province of South Africa.The study was carried out at the Histopathology Department of the National Health Laboratory Service of the Limpopo Province. The population is mainly rural and is in excess of 5 M. The department receives and reports all the histology specimens from the public hospitals in the province.The cases were retrieved from a prospective survey of 429 biopsy and LAN-1 immunohistochemically stained cases from March 2010 through July 2012 . PatientRoutine streptavidin-biotin-peroxidase immunostaining with diaminobenzidine was performed on formalin-fixed paraffin-embedded tissue using a murine monoclonal antibody directed against the C-terminus of the latent nuclear antigen-1 (LNA-1) molecule of HHV-8 .Patients' anonymity was preserved. Ethical approval was obtained from the institutional research ethics committee.African endemic KS has been identified for many decades in the past. Currently, the increasing prevalence of HIV infection complicates all efforts to clearly distinguish the endemic variant from the HIV-related one . The cliEarly reports on African KS concentrated on the gender differences and histological features , 32. OneIt is now well established that both the clinical and histological diagnosis of KS is fraught with over- and underdiagnosis because of the wide range of mimickers , 40. TheLiterature case reports all illustrate that vulvar KS appeared clinically as a mass, a papilloma, or an abscess; none was initially suspected to be a KS –26. In tIn conclusion, anogenital KS is rare and easily misdiagnosed even in HIV/KS endemic regions. Therefore, a high clinical suspicion threshold should prompt a biopsy, and the diagnosis of KS should be confirmed by LNA-1 immunohistochemistry to avoid misdiagnosis and wrong management.


During 2002–2009, classical non-Nor98 transmissible spongiform encephalopathy had been confirmed among ≈1.7 million small ruminants in France. Five sheep and 2 goats that showed a PrPres pattern consistent with BSE, or with the CH1641 experimental scrapie source, were identified. Later, bioassays confirmed infection by the BSE agent in 1 of the 2 goats. Western blot testing of the 6 other isolates showed an additional C-terminally cleaved PrPres product, with an unglycosylated band at ≈14 kDa, similar to that found in the CH1641 experimental scrapie isolate and different from the BSE isolate.The agent that causes bovine spongiform encephalopathy (BSE) may be infecting small ruminants, which could have serious implications for human health. To distinguish BSE from scrapie and to examine the molecular characteristics of the protease-resistant prion protein (PrP These were the SSBP/1 scrapie isolate, experimentally maintained by serial passages in sheep  or, on the contrary, was similar as in most scrapie cases. PrPres extracts were obtained from animal brains by using Bio-Rad protocol , which is used to confirm suspected TSE-positive samples and includes PK digestion and rapid protein precipitation steps. Denatured samples were then loaded on two 15% bis-polyacrylamide gels with the same gel plan. After electrophoresis (200 volts for 80 min), the proteins were transferred onto a nitrocellulose membrane . Blocking was performed for 1 h in 5% (wt/vol) milk powder in phosphate-buffered saline (PBS) containing 0.1 % (vol/vol) Tween-20 (PBS-T) for the membrane to be treated with monoclonal antibody Bar233 (1/5000) (144-FGNDYEDRYYRE-155 ovine PrP sequence)  or in 3% (wt/vol) bovine serum albumin solution  in PBS-T for the membrane to be treated with monoclonal antibody P4 (0.2 mg/mL) (93-WGQGGSH-99 ovine PrPsequence) (R-Biopharm).All confirmed classical TSE (non–Nor98-like) cases were analyzed to determine whether the PrPres bands with a biotinylated marker (B2787) (Sigma). All samples were compared, during molecular characterization of field isolates, by expressing the molecular mass of the unglycosylated band in terms of differential molecular mass (dmm). The dmm corresponds to the difference measured between the test sample and the control cattle BSE sample always loaded beside it.Both antibodies were incubated on the membranes for 30 min at room temperature. The membranes were then washed for 20 min in PBS-T and incubated with a solution of streptavidine-peroxidase–conjugated antimouse immunoglobulin  in PBS-T for 20 min at room temperature. The membranes were then washed for 30 min in PBS-T and for 5 min in PBS before detection by use of enhanced chemiluminescent substrate . The signals were identified on autoradiographic films (Amersham) after a 3-min exposure. Quantitative studies were performed by using Quantity One software (Bio-Rad), and the apparent molecular masses were determined by comparing the positions of the PrPres products (PrPres #2) (res was detected with SAF84 (0.6 mg/mL) (167-RPVDQY-172 ovine PrP sequence)  mouse monoclonal antibody. The respective proportions of ≈14- and ≈19-kDa bands, observed after PNGase deglycosylation, were quantified by using Quantity One software (Bio-Rad).The possible presence of additional C-terminal PrPSince active surveillance of TSEs in small ruminants began in France in 2002, a total of 1,231 small ruminant  samples have been confirmed as TSE-positive by Western blot using Sha 31 antibody; >1.7 million animals have been tested by rapid tests  . Nearly res patterns similar to those found in experimental ovine BSE and is essentially based on comparison of the PrPres molecular mass with that of cattle BSE, and comparative labeling with 2 antibodies, P4 and Bar233, against either the N terminal end or core part of the PrPres protein, respectively.The other classical TSE cases, i.e., non–Nor98 cases (648 sheep and 53 goats), which typically showed a 3-band pattern between 19–30 kDa, have been further characterized by applying a discriminatory Western blot method, described as the Agence française de sécurité sanitaire des aliments (French Food Safety Agency) discriminatory method in the Technical Handbook for National Reference Laboratories (res bands (using Bar233 antibody) and the P4/Bar233 differential labeling, as well as the proportions of glycoforms, are shown in res in BSE in sheep (–0.6 kDa) and 1 goat (–0.5 kDa), and in CH1641 scrapie (–0.8 kDa), compared with the cattle BSE , monoglycosylated (L), and unglycosylated (U) PrPttle BSE , panel Attle BSE . Like BSres in experimental BSE in sheep (75 ± 9%/18 ± 4%) and goats (71 ± 7%/20 ± 4%), even compared with cattle BSE (64 ± 10%/27 ± 4%) (res (46 ± 6%/32 ± 3%), whereas CH1641 was close to cattle BSE (62 ± 9%/29 ± 5%).Comparison of the proportions of diglycosylated and monoglycosylated bands showed the highest levels of diglycosylated PrP27 ± 4%) , panel DExperimental BSE in a goat showed the same molecular features as those observed in experimental BSE in sheep. Both molecular masses, P4 immunolabeling and glycoforms proportions, were involved . Most of the isolates tested (657) were PrPWe chose to express the molecular masses measures as dmm, which corresponds to the difference of molecular masses measured between the tested sample and the control cattle BSE always loaded beside it. Most (650) samples showed a positive dmm  and strores molecular mass than the bovine BSE control, i.e., a negative dmm (black bar in A few samples (5 sheep and 2 goats) showed a lower PrPres molecular masses and P4/Bar233 differential labeling and estimation of the proportions of PrPres glycoforms. Western blot analyses were also performed with the C-terminal SAF84 antibody to identify possible additional C-terminal PrPres cleavage products, as described for the CH1641 experimental isolate , with regard to PrPres molecular masses  and Italy  were comparable to those previously described for most scrapie cases, in studies in Europe or France , as previously described for the CH1641 experimental scrapie isolate (The other unusual isolates showed molecular characteristics that were partly similar, not only to BSE in small ruminants with a low molecular mass of PrP isolate , panel Bres features are assessed by analyzing a single homogenate prepared from a brain fragment from the animal. Stack et al. described a case in sheep that appeared as CH1641-like after repeated Western blot analysis of a brain stem sample, whereas previous analysis of the caudal medulla at the time of submission had shown the usual scrapie profile (d could be clearly identified by using P4 antibody in some of the brain stem nuclei and in lymphoid tissues (res product in ovine transgenic mice infected with usual scrapie isolates, we hypothesized that a CH1641-like component might be present as a minor component in these scrapie cases that showed usual molecular features (These 6 CH1641-like isolates were identified among 1,153 sheep and 78 goats with confirmed TSE, and the goat case represents, to our knowledge, a spreading of the known species range for natural CH1641 infection. At least in sheep, for which 4.34 cases per million sheep tested were identified in this study, the frequency of CH1641-like scrapie was notably higher compared with other rare TSEs in ruminants such as atypical BSEs, which showed a frequency of 0.76 per million cattle tested during 2001–2007 (


Several genetic parts for cell lysis have been developed and their quantitative characterization is an essential step to enable the engineering of synthetic lytic systems with predictable behavior.Bacterial cell lysis is a widely studied mechanism that can be achieved through the intracellular expression of phage native lytic proteins. This mechanism can be exploited for programmed cell death and for gentle cell disruption to release recombinant proteins when E. coli by assembling the device under the control of a well characterized N-3-oxohexanoyl-L-homoserine lactone (HSL) -inducible promoter and the transfer function, lysis dynamics, protein release capability and genotypic and phenotypic stability of the device have been evaluated. Finally, its modularity was tested by assembling the device to a different inducible promoter, which can be triggered by heat induction.Here, a BioBrick™ lysis device present in the Registry of Standard Biological Parts has been quantitatively characterized. Its activity has been measured in The studied device is suitable for recombinant protein release as 96% of the total amount of the intracellular proteins was successfully released into the medium. Furthermore, it has been shown that the device can be assembled to different input devices to trigger cell lysis in response to a user-defined signal. For this reason, this lysis device can be a useful tool for the rational design and construction of complex synthetic biological systems composed by biological parts with known and well characterized function. Conversely, the onset of mutants makes this device unsuitable for the programmed cell death of a bacterial population. Naturally occurring lytic and temperate bacteriophages have the ability to provoke the host cell lysis through the expression of specific proteins during the lytic cycle. In many phages, like the T4 phage and the lambda phage, these proteins have been identified and widely studied -4. In paThe described lytic mechanism can be exploited for the release of useful recombinant proteins which cannot be secreted by the engineered host strain .Escherichia coli is a widely used organism for recombinant protein production, but its secretion capabilities are limited and recombinant protein targeting to the growth medium has shown to work only with a small set of proteins [proteins . For thiproteins . Other rproteins . The engAnother important application of an inducible lytic system is the programmed cell death of a bacterial population, which might be useful in those processes where the microorganism must be eliminated at a specific time, after having completed its work.E. coli strains to achieve the gentle disruption of cells upon IPTG induction using the lac promoter or the DE3 inducible system [SRRz for high throughput enzyme release [In literature, inducible lysis systems have been proposed. T4 phage holin and T7 phage lysozyme genes have been used to construct lytic e system ,11. The e system . Heat- a release .1 BBa_K112808 consists of a promoterless operon composed by the T4 phage genes t and e, encoding a holin and a lysozyme respectively [rI gene, encoding a t-specific antiholin, is present under the control of a weak constitutive promoter , the HSL-inducible lysis device (pLC-T4LysHSL) and the heat-inducible lysis device (pLC-T4LysHeat) respectively, all of them in a low copy number vector.In this work, BioBrick™ parts have been used to quantitatively characterize the BBa_K112808 lysis device by assembling it to BBa_F2620, a well characterized N-3-oxohexanoyl-L-homoserine lactone (HSL)-inducible device based on the E. coli strains and plasmids used in this study are listed in Table All the pSB1A2 and BBa_J61002 are high copy number vectors with a pUC19-derived pMB1 replication origin (~100-300 molecules per cell) and Ampicillin resistance marker, while pSB4C5 is a low copy number vector with a pSC101 replication origin (~5 molecules per cell) and Chloramphenicol resistance marker . The fulE. coli (Invitrogen) were routinely used both for cloning and for quantitative experiments. Chemically competent DB3.1 E. coli (Invitrogen) were used to propagate pLC-ccdB plasmid, containing a ccdB expression cassette, which is toxic for TOP10 but not for DB3.1. DH5alpha (Invitrogen) and MG1655  were only used for quantitative experiments. TOP10, DH5alpha and DB3.1 were heat shock-transformed according to the manufacturer's protocol. MG1655 were made chemically competent with the protocol described in [Chemically competent TOP10 ribed in  and theyribed in  with Ampribed in  and isolTOP10-rfp-lys strain (see Protein release assays section), which bears two plasmids, pHC-RFP and pLC-T4LysHSL, was built up with the following procedure: competent TOP10 were transformed with pLC-T4LysHSL, then transformed cells were grown in selective LB and made chemically competent again with the protocol described in  and heatLB and M9 supplemented medium   were useDNA sequencing was performed through BMR Genomics  DNA analysis service.- and pHC-HSL cut with EcoRI-PstI to yield pLC-T4Lys- and pLC-HSL respectively. pHC-T4LysHSL was constructed by assembling the insert of pHC-HSL cut with EcoRI-SpeI to pHC-T4Lys- cut with EcoRI-XbaI. pLC-T4LysHSL was constructed by assembling the insert of pHC-T4Lys- cut with XbaI-PstI to pLC-HSL cut with SpeI-PstI. Finally, pLC-T4LysHeat was constructed by assembling the insert of pHC-Heat cut with EcoRI-SpeI to pLC-T4Lys- cut with EcoRI-XbaI.pSB4C5 low copy vector bulk, obtained from pLC-ccdB after EcoRI-PstI cut, was ligated to the inserts of pHC-T4Lys- glycerol stocks were inoculated in 5 ml of selective LB medium and grown at 37°C, 220 rpm overnight. The cultures were diluted 1:100 in 5 ml of selective LB medium and grown for additional 4-5 hours under the same conditions as before. After that time, a 200 μl aliquot of each culture was transferred in a flat-bottomed 96-well microplate (Greiner) and the OD600 was measured with an Infinite F200 microplate reader (Tecan). Based on this measurement, the cultures were diluted to the same OD600 (0.05-0.13) and then six 200 μl aliquots of each culture were transferred in a flat-bottomed 96-well microplate (Greiner). Unless otherwise noted, three wells of each culture were induced with 2 μl of properly diluted HSL (Sigma Aldrich #K3007) and 2 μl of deionized water were added to the other three wells (uninduced wells). If the HSL-inducible lysis device had to be assayed with different HSL concentrations in the same experiment, three 200 μl aliquots of the cultures for each investigated concentration were transferred in the microplate and induced. The microplate was incubated at 37°C in the Infinite F200 microplate reader and assayed every 5 min following this protocol immediately before the measurement: 15 s of linear shaking (amplitude = 3 mm), wait for 5 s, OD600 measurement.Unless otherwise noted, 5 μl of bacteria bearing pLC-T4LysHSL and pLC-T4LysFor lysis assays on pLC-T4LysHeat, the cultures were grown at 30°C instead of 37°C and an automatic temperature shift from 30°C to 42°C was used to induce lysis in the microplate reader instead of HSL.Raw OD600 values measured in the Infinite F200 microplate reader were normalized by subtracting for each time point the mean raw absorbance of the media to compute the actual bacterial optical density.ln(OD600(t)) over time. Then, the exponential phase was identified by visual inspection as the linear region of ln(OD600(t)), the late stationary phase as the constant region and the early stationary phase as the region between the other two.The growth phases of bacterial cultures with the different plasmids used in this study, grown in LB medium in a microplate, have been characterized in each experiment by computing the natural logarithm of the OD600 values ln(OD600(t)) linear region to estimate the curve slope m, which represents the growth rate of the culture. Then the culture doubling time was computed as ln(2)/m.In all the lysis assays, uninduced bacteria doubling time in exponential growth phase was evaluated by performing linear regression over the OD600/refOD600)100* 100*(1-OD600(t)/refODsignal to rise from 10% to 90% of the lysis entity.The lysis entity after induction was computed as - glycerol stocks were inoculated in 5 ml of selective LB medium and grown at 37°C, 220 rpm overnight. TOP10-rfp-lys and TOP10 with pHC-RFP cultures were diluted 1:100 into six 15 ml tubes containing 5 ml of pre-warmed selective LB medium. TOP10 with pLC-T4Lys- culture was diluted 1:100 in one 15 ml tube with 5 ml of pre-warmed selective LB. The 13 resulting cultures were grown to an OD600 of about 0.55 . Three of the six replicates of TOP10-rfp-lys and TOP10 with pHC-RFP were induced with HSL at a final concentration of 100 nM and all the 13 tubes were incubated under the same conditions as before for 125 min. Every 25 min, the OD600 was measured with the NanoDrop ND-1000 and a 300 μl aliquot was taken from each culture. The 300 μl samples were centrifuged at 11000 rpm in a table top centrifuge and the fluorescence of 200 μl of the supernatant was measured in a microplate with the Infinite F200 reader using: 535 nm excitation filter, 620 nm emission filter, excitation bandwidth = 25 nm, emission bandwidth = 20 nm, gain = 55, number of flashes = 25, integration time = 20 μs, top reading. The raw fluorescence measurements of TOP10-rfp-lys and TOP10 bearing pHC-RFP (induced and uninduced) were normalized by subtracting the background fluorescence value of pLC-T4Lys- cells at the same time points.5 μl of TOP10-rfp-lys, TOP10 bearing pHC-RFP and TOP10 bearing pLC-T4Lys- were grown and induced as described above, in 3 ml cultures. At t = 125 min, the cultures were centrifuged  and supernatants and pellets were processed to measure the amount of intracellular and extracellular RFP. Pellets were resuspended in 200 μl of sterile LB broth and 200 μl of 2X lysis buffer  were added to the resuspended pellets [- lysed pellet and supernatant respectively. Finally, the mean RFP values of lysed pellets and supernatants of TOP10-rfp-lys and TOP10 bearing pHC-RFP (induced and uninduced) were corrected by the total culture volume, so the percentage of extracellular RFP molecules was computed for each culture , measuring the OD600 of serial dilutions of a TOP10 culture grown in LB or M9 supplemented medium.21000 = 9.97) [Bacteria bearing pLC-T4LysHSL were propagated for 100 generations as described in  without  = 9.97) . Every dTo analyze mutants, a lysis assay was performed on TOP10 bearing pLC-T4LysHSL induced with HSL 10 nM and when the lysed and re-grown cells reached an OD600 = 0.22 they were diluted 1:1000 in fresh selective LB medium in a new 96-well microplate, incubated in the Infinite F200 reader and induced again with HSL 10 nM when they reached an OD600 of 0.35  to check if they could lyse again. The re-grown cells were also streaked on selective LB agar, then 2 single colonies were inoculated in 5 ml of selective LB and let grow overnight . Plasmid DNA was extracted from the 5 ml overnight cultures and analyzed by restriction enzyme digestion/electrophoresis and DNA sequencing using primers VF2: 5'-TGCCACCTGACGTCTAAGAA-3', VR: 5'-ATTACCGCCTTTGAGTGAGC-3' and C0062VF: 5'-GAATGTTTAGCGTGGGCATG-3'. This procedure was carried out starting from 5 μl of pLC-T4LysHSL glycerol stock and also from single colonies isolated from this stock.- were used as a negative control in all the experiments. Lysis entity, i.e. the maximum percent reduction of OD600 caused by lysis induction, was computed in all the assays as an indirect measurement of the amount of lysed cells.Lysis was assayed in a 96-well microplate by measuring the optical density at 600 nm (OD600) dynamics of TOP10 bearing HSL-inducible lysis device pLC-T4LysHSL, induced with HSL and uninduced. Induced and uninduced TOP10 bearing the promoterless lysis device pLC-T4LysA typical lysis profile is reported in Figure - was 43 ± 1.3 min, thus demonstrating that the HSL-inducible lysis device gives a reasonably low metabolic burden and allows the cells to grow at a rate comparable to their negative control , the lysis device in high copy plasmid has not been considered for further studies.Lysis was also assayed for TOP10 bearing pLC-T4LysHeat low copy vector containing a heat-inducible lysis device. Induction was triggered in a 96-well microplate by shifting the incubation temperature from 30°C to 42°C. The results are shown in Figure E. coli strains  grown in LB, giving consistent results when compared to the TOP10 strain in LB medium results described in this section  and in two other - were chosen as controls in this assay.A co-transformed TOP10 strain  bearing both a high copy plasmid with a Red Fluorescent Protein (RFP) constitutive expression cassette pHC-RFP and the HSL-inducible lysis device pLC-T4LysHSL was induced with HSL to study the RFP release in the medium. Uninduced TOP10-rfp-lys, TOP10 bearing pHC-RFP (induced and uninduced) and TOP10 bearing pLC-T4LysFigure gly culture), while the other three cultures were obtained starting from single colonies streaked from the glycerol stock . In all the mutant clones the pSB4C5 vector band (~3.2 kbp) was present, but the insert was highly different from the unmutated culture (~2.8 kbp), suggesting that mutations occurred just in the HSL-inducible lysis device. The sequencing of the clones disclosed all the mutations, as shown in Figure gly and colony 2 of mutsc3) showed the insertion sequence IS10R after the nucleotide 576 of luxR (BBa_C0062) gene  showed the insertion of IS10R in the same place as before, but in the opposite direction  showed the deletion of the DNA comprising the lux promoter and the holin-lysozyme operon  showed the insertion of IS10R after the nucleotide 318 of the holin (BBa_K112805) gene  had two different mutated plasmids in the same colony: one of them showed the insertion of IS10R before the nucleotide 1 of the luxR RBS (BBa_B0034), while the other one showed the insertion of IS5 before the start codon of luxR . When the re-grown cultures of TOP10 bearing pLC-T4LysHSL were diluted 1:1000 in fresh selective LB medium and let grow to an OD600 = 0.35 , they did not lyse upon induction with HSL 10 nM, suggesting that the cells have completely lost the inducible lysis phenotype (data not shown). The restriction analysis of the mutant plasmids after DNA digestion with EcoRI-PstI is reported in Figure e Figure ; two clon Figure ; two clon Figure ; one cloe Figure ; one cloR Figure . All theIn order to study the reliability of the lysis device when cell disruption is not triggered, bacteria bearing pLC-T4LysHSL were propagated for 100 generations without HSL and their inducible lysis phenotype was tested over time in terms of lysis entity. Supplementary Figure E. coli using a well characterized HSL-inducible promoter and the transfer function, lysis dynamics, protein release capability, modularity and genotypic and phenotypic stability of the device have been evaluated.In this work, the quantitative characterization of a BioBrick™ lysis device of the Registry of Standard Biological Parts is reported. Its activity has been measured in E. coli membrane disruption mediated by T4 phage lysis gene t or e was more efficient when the genes were expressed in the exponential growth phase than in the stationary phase, where less or almost no disruption occurred [lux promoter and the holin and lysozyme genes was deleted from the plasmid most probably by a replication slippage mechanism between two identical transcriptional terminators (BBa_B0015) of 129 bp flanking the fragment. These mutations were consistent with published DNA mutations commonly occurring in E. coli [Low copy number has been found to be the optimal working condition, as lysis could be triggered in all the growth phases of the bacterial culture and the cells grew with a relatively low metabolic burden, according to their doubling time. Lysis entity in late stationary phase was lower than in the other growth phases. These results are consistent with the published findings for which occurred ,11,13. 9 E. coli , in fact E. coli . On the  E. coli . The HSLE. coli strains and different growth media, thus providing parameters that can be used in models to aid future biological systems design and to facilitate the re-usability of this lysis device.All the results have been confirmed in different The lysis device in high copy number plasmid gave worse performance, in fact lysis entity was lower than in low copy plasmid, the metabolic burden was much higher and the device was strongly unstable, as cell lysis induction usually failed to occur.The authors declare that they have no competing interests.LP, SZ, ML and PM designed the experiments. LP, SZ and ML performed all the plasmid constructions and validation experiments. LP, SZ and PM analyzed the data. LP, MGCDA and PM wrote the manuscript. All authors read and approved the final manuscript.1 BioBrick™ is a trademark of The BioBricks Foundation. (http://www.biobricks.org)


Escherichia coli K-12 is able to synthesize three active [NiFe]-hydrogenases (Hyd1-3). Two of these hydrogenases are respiratory enzymes catalysing hydrogen oxidation, whereby Hyd-1 is oxygen-tolerant and Hyd-2 is considered a standard oxygen-sensitive hydrogenase. Hyd-3, together with formate dehydrogenase H (Fdh-H), forms the formate hydrogenlyase (FHL) complex, which is responsible for H2 evolution by intact cells. Hydrogen oxidation activity can be assayed for all three hydrogenases using benzyl viologen  as an artificial electron acceptor; however ascribing activities to specific isoenzymes is not trivial. Previously, an in-gel assay could differentiate Hyd-1 and Hyd-2, while Hyd-3 had long been considered too unstable to be visualized on such native gels. This study identifies conditions allowing differentiation of all three enzymes using simple in-gel zymographic assays.When grown under anaerobic conditions, Eo′ = -80 mV) it was demonstrated that Hyd-1 reduces this redox dye in a hydrogen-dependent manner, while neither Hyd-2 nor Hyd-3 could couple hydrogen oxidation to NBT reduction. Hydrogen-dependent reduction of NBT was also catalysed by an oxygen-sensitive variant of Hyd-1 that had a supernumerary cysteine residue at position 19 of the small subunit substituted for glycine. This finding suggests that tolerance toward oxygen is not the main determinant that governs electron donation to more redox-positive electron acceptors such as NBT.Using a modified in-gel assay hydrogen-dependent BV reduction catalyzed by Hyd-3 has been described for the first time. High hydrogen concentrations facilitated visualization of Hyd-3 activity. The activity was membrane-associated and although not essential for visualization of Hyd-3, the activity was maximal in the presence of a functional Fdh-H enzyme. Furthermore, through the use of nitroblue tetrazolium (NBT; The utilization of particular electron acceptors at different hydrogen concentrations and redox potentials correlates with the known physiological functions of the respective hydrogenase. The ability to rapidly distinguish between oxygen-tolerant and standard [NiFe]-hydrogenases provides a facile new screen for the discovery of novel enzymes. A reliable assay for Hyd-3 will reinvigorate studies on the characterisation of the hydrogen-evolving FHL complex. Escherichia coli synthesizes three membrane-associated [NiFe]-hydrogenases (Hyd), although its genome has the capacity to encode four of these enzymes -hydrogenases (FTD147 and DHP-F2), it was shown that only strains able to synthesize Hyd-1 were capable of reducing nitroblue tetrazolium (NBT) in a hydrogen-dependent manner  was employed and a crude extract derived from this strain displayed a Hyd-1 activity band of similar intensity to that in MC4100 but the extract lacked the slower migrating activity band confirming that this was due to Fdh-N and Fdh-O .As a final control, we replaced the electron donor HO Figure B and thiE. coli cells only Hyd-1 can reduce NBT in a hydrogen-dependent manner. One of the major differences between Hyd-1 and the other enzymes is its oxygen tolerance  cluster-containing subunit, HybA, and the HybB integral membrane protein -cluster-containing small subunit of the hydrogenase must be associated with the large subunit in order for hydrogen-dependent BV reduction to occur . It is pE. coli, only Hyd-1 was capable of the specific, hydrogen-dependent reduction of PMS/NBT. Notably, both respiratory Fdhs also showed a strong NBT-reducing activity, which correlates well with previous findings for these enzymes -cluster [R. eutropha[Eh value of -80 mV). The similar redox potential of NBT in our assay buffer with and without PMS  and TTC (Eh = -80 mV), along with 5% hydrogen in the headspace, of the assay was sufficient to maintain a low redox potential to detect Hyd-2 readily. This also explains why long incubation times are required for visualization of Hyd-1 activity with the BV/TTC assay. Increasing the hydrogen concentration in the assay to 100% drives the redox potential below -320 mV and explains why the Hyd-3 activity was readily detectable at hydrogen concentrations above 25%  Triton X-100 prior to application of the solubilized sample  to the gels. Alternatively, for neutral pH analyses the barbitone gel system was used. This system uses final concentrations of 34 mM Tris-phosphate buffered stacking gel, pH 5.5 and 62.5 mM Tris-HCl resolving gel pH 7.5. The running buffer consists of 82.5 mM Tris and 26.8 mM diethylbarbituric acid, pH 7.0. Hydrogenase activity-staining was done as described in [Non-denaturing PAGE was performed using a discontinuous system with 7.5% (w/v) polyacrylamide separating gels in 250 mM Tris/HCl buffer, pH 8.5 including 0.1% (w/v) Triton X-100 . As runnribed in  with 0.5ribed in . When fo2+ was carried out as described [Western blotting was performed as described in  by transescribed . Determiescribed .Aliquots of 50 mM MOPS buffer pH 7.0 containing the concentrations of the respective redox dyes indicated above were either incubated overnight in an anaerobic chamber with an atmosphere containing 5% hydrogen for 6 h or was bubbled with hydrogen gas (100% atmosphere) for 30 min and the redox potential determined using a EMC 30-K010-D redox micro-electrode  attached to a Lab850 pH/redox meter . The electrode was standardized using a redox buffer provided by the company. Measurements were performed two times.FHL, Formate hydrogenlyase; Hyd, Hydrogenase; Fdh, Formate dehydrogenase; BV, Benzyl viologen; TTC, 2,3,5-triphenyltetrazolium chloride; NBT, Nitroblue tetrazolium; PMS, Phenazine methosulfate.The authors declare that they have no competing interests.CP carried out the experimental studies and drafted the manuscript. MJ conducted the redox potential measurements and the gel staining experiments, RGS and FS conceived and coordinated the study and drafted the manuscript. All authors read and approved the final manuscript.


Silk sericin (SS)/silk fibroin (SF) blend nanofibers have been produced by electrospinning in a binary SS/SF trifluoroacetic acid (TFA) solution system, which was prepared by mixing 20 wt.% SS TFA solution and 10 wt.% SF TFA solution to give different compositions. The diameters of the SS/SF nanofibers ranged from 33 to 837 nm, and they showed a round cross section. The surface of the SS/SF nanofibers was smooth, and the fibers possessed a bead-free structure. The average diameters of the SS/SF  blend nanofibers were much thicker than that of SS and SF nanofibers. The SS/SF  blend nanofibers were easily dissolved in water, while the SS/SF (25/75 and 0/100) blend nanofibers could not be completely dissolved in water. The SS/SF blend nanofibers could not be completely dissolved in methanol. The SS/SF blend nanofibers were characterized by Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry, and differential thermal analysis. FTIR showed that the SS/SF blend nanofibers possessed a random coil conformation and ß-sheet structure. Electrospinning is an interesting and effective process for producing nanoscale fibers with diameters in the nanometer to micrometer range. These nanofibers have high functionality, high specific surface area, and high porosity with very small pore size ,2. ThereBombyx mori silkworm are composed of SS and SF. SS is bio-synthesized exclusively in the middle silk gland and is about 25-30% of silk fiber mass. Sericin coats fibroin threads to glue them together and fills the gaps to enhance the toughness of the cocoon fiber [Silk cocoon filaments from the on fiber . Sericinon fiber ,9. SF haon fiber -12. Theron fiber ,14, but In this study, we present a novel way to prepare SS/SF nanofibers. Our results demonstrate the successful preparation of SS/SF blend nanofibers with smooth surfaces via electrospinning from SS/SF blend solutions, and we analyze their molecular conformation and physical properties.Sericin as the powder form of silk sericin (SS) was purchased from Wako Pure Chemical Industries, Ltd.  (Lot. CDR4258). Silk fibroin (SF) film was prepared by casting 2 wt.% SF solution in flat-bottomed polystyrene dishes at room temperature in a flow cabinet. SF solution was prepared by dissolving SF fibers in 8 M LiBr solution at 60°C for 30 min and then dialyzed against water at 5°C for 4 days using cellulose dialysis tubes.w/w: 75/25, 50/50, and 25/75) blend solutions were prepared. The SS/SF (75/25) blend nanofibers were produced from solutions containing 75 wt.% SS and 25 wt.% SF. The pure and mixed solutions were stirred for 3 h. These sample solutions were stored in the refrigerator (4°C) for 12 h, while the electrospinning solution was prepared. The sample nanofibers were produced by electrospinning the SS/SF solutions, having different sample compositions .SS solution (20 wt.%) and SF solution (10 wt.%) were prepared by stirring the samples in trifluoroacetic acid (TFA) at 25°C for 3 h. Solutions of 0.45, 0.3, and 0.15 g SS were mixed with 0.3, 0.6, and 0.9 g SF solutions, respectively, so that the SS/SF , needle , aluminum collecting screen, and syringe pump. The spinning speed of the syringe pump was adjusted within the range 0.003-0.320 cm/min. High voltage power , 26, Nishi 9-jou, Minami-ku, Kyoto city, 601-8447, Japan) was supplied in the range 0 to 40 kV. The sample solution was placed into the 1-ml syringe, with a 21-gauge needle (inner diameter 0.3 mm). The voltage was applied between the end of the needle and collecting screen. Nanofiber mats were produced on the aluminum collecting screen. The electrospinning was conducted with values of working distance, applied voltage, and flow rate at 15 cm, 25 kV, and 0.06 cm/min, respectively.The morphology and the diameter of nanofibers were determined with a scanning electron microscope (SEM)  spectroscopy was measured with a Shimadzu FT-IR-8400S infrared spectrometer  by the ATR method in the region of 1800-700 cm-1 and spectral range of 4,000-500 cm-1. Infrared spectra were recorded from 16 scans per sample.The spectra of samples were acquired in transmittance mode with a resolution of 4 cmDifferential scanning calorimetry (DSC) curves of samples were measured with a DSC instrument  under normal atmospheric conditions, at a heating rate of 10°C/min. The temperature ranged from room temperature to 300°C, and the sample weight was 3 mg.Differential thermal analysis (DTA) measurements were carried out using a DTA instrument  under a nitrogen atmosphere in the range from room temperature to 300°C and at a heating rate of 10°C/min. The sample weight was 6 mg.Figure We prepared blend solutions by the following procedures. The SS/SF blend solution was firstly stirred at 25°C for 6 h, then stored in the refrigerator (4°C) for 12 h, and stirred again at 25°C for 222 h . Immediately after stirring, sample nanofibers were produced by electrospinning.Figure It is of important to note that the average diameter of nanofibers (100/0) is about 155 nm Figure , whereasWe examined the solubility of the SS/SF blend nanofibers in water and in methanol. The SS nanofibers were completely dissolved in water after a short immersion time (1 h), while the SF nanofibers were not dissolved in water. The SS/SF blend nanofibers, containing higher amounts of SS, were more easily dissolved in water. The SS/SF  blend nanofibers were completely dissolved in water within 1 h. However, when the SF component was greater than the SS component, as in the SS/SF blend nanofibers (25/75), they could not be completely dissolved in water. Over the same time period, the SS/SF blend nanofibers could not be dissolved in methanol.-1, which were attributed to the random coil conformation. SF showed at 1,625 cm-1 (amide I), and the SS/SF nanofibers showed absorption peaks at around 1,512 cm-1 (amide II), attributed to the ß-sheet conformation. Amide III showed in SS and SF at 1,238 and 1,233 cm-1 attributable to ß-sheet structure, while the absorption bands of amide III of the SS/SF  blend nanofibers . However, according to Figure -1 is due to the trace amount of TFA contained in the SS/SF blend nanofibers and that the molecular interaction between the trace amount of TFA and SS is slightly stronger than that between TFA and SF.The IR spectra of the SS/SF blend nanofibers Figure  were sims Figure . Accordi-1. The SS/SF blend nanofibers and SF showed weak absorption bands at 1,408 and 1,334 cm-1. SS displayed a distinct absorption band at 1,390 cm-1, and SF showed a weak peak at 1,386 cm-1. However, this peak had disappeared in the SS/SF (100/0 and 75/25) blend nanofibers' spectra  blend nanofibers, heat-treated at 160°C for 30 min shown in Figure It was of interest to note that this absorption band was detected in the SS/SF blend nanofibers Figure . The SS/a Figure . Around F Figure  showed ws Figure  had the In order to analyze the thermal behavior of the samples, DSC measurements were conducted. Figure The SS/SF  blend nanofibers Figure  showed bThe SS/SF (50/50) blend nanofibers had a broad decomposition peak from 138°C to 213°C Figure , while tAll the samples showed endothermic peaks at around 60°C to 70°C that are due to the evaporation of water or solvent contained in the samples Figure . The SS DTA curves of the SS/SF 50/50) Figure  and SS/S0/50 FiguWe succeeded in producing SS/SF blend nanofibers by electrospinning with a SS/SF TFA blend solution. The "as-spun" nanofibers exhibited smooth surfaces, round cross sections, and bead-free structures. The average diameters of SS/SF  blend nanofibers were thicker than those of SS or SF nanofibers. The mean diameter of these blended nanofibers decreased, and the number of beads slightly increased with increasing dissolving time of the SS/SF blend solution prior to electrospinning. The number of beads also increased with increasing SF content.The SS/SF  blend nanofibers were easily dissolved in water, while the SS/SF (25/75 and 0/100) blend nanofibers were not completely dissolved in water. The SS/SF blend nanofibers were not dissolved in methanol. From the FTIR measurements, the SS/SF blend nanofibers were shown to possess a random coil conformation and ß-sheet structure. According to FTIR, DSC, and DTA measurements, "as-spun" nanofibers, including SS, SS/SF, and SF nanofibers, contained small amounts of TFA used as the solvent to dissolve the samples. For the SS/SF (50/50) blend nanofibers heat-treated at 160°C, the TFA contained in the sample nanofibers was almost completely removed.These SS/SF nanofibers are of considerable interest for different kinds of application because these proteins have specific functions, as mentioned in the introduction part, and unique properties including comparatively high specific surface area, etc.The authors declare that they have no competing interests.XZ carried out experimental works on SEM, DSC, FTIR etc. MT participated in the desigin of the study and drafted the manuscript. HM excised efficient supervision of this work. KA carried out the DSC measurement of the samples. GZ participated in the average diameter distribution analysis. MM carried out the statistical analysis of average diameter distribution. All authors read and approved the final manuscript.


Network medicine has been applied successfully to elicit the structure of large-scale molecular interaction networks. Its main proponents have claimed that this approach to integrative medical investigation should make it possible to identify functional modules of interacting molecular biological units as well as interactions themselves. This paper takes a significant step in this direction. Based on a large-scale analysis of the nervous system molecular medicine literature, this study analyzes and visualizes the complex structure of associations between diseases on the one hand and all types of molecular substances on the other. From this analysis it then identifies functional co-association groups consisting of several types of molecular substances, each consisting of substances that exhibit a pattern of frequent co-association with similar diseases. These groups in turn exhibit interlinking in a complex pattern, suggesting that such complex interactions between functional molecular modules may play a role in disease etiology. We find that the patterns exhibited by the networks of disease – molecular substance associations studied here correspond well to a number of recently published research results, and that the groups of molecular substances identified by statistical analysis of these networks do appear to be interesting groups of molecular substances that are interconnected in identifiable and interpretable ways. Our results not only demonstrate that networks are a convenient framework to analyze and visualize large-scale, complex relationships among molecular networks and diseases, but may also provide a conceptual basis for bridging gaps in experimental and theoretical knowledge. Information science can help to identify interesting approaches and provide new perspectives for scientific research P value, highlighting a complete lack of support for the validity of hypothesis that any of the variants previously reported in scores of publications are associated with acute coronary syndromes The molecular basis of a disease is very complex, especially so for the aptly-named complex diseases. For example, there is no ‘cancer gene’. A typical cancer patient has mutations in a few dozen of about 300 genes, an elusive combinatorial problem whose complexity is increasingly a worry to the medical community On the other hand, current research suggests that it is not enough to know a precise list of “disease genes”, but rather that diseases should be viewed as the breakdown of specific functional modules rather than single or small groups of genes, where discernible modules consist of an interlocking network of genes, transcription factors, RNAs, enzymes, and metabolites In addition, disease-proteins have been found to exhibit more protein-protein interactions than do non-disease proteins de rigueur in identifying breakthrough discoveries in science from the seemingly infinite volumes of data generated using modern technologies Nervous system diseases include many complex diseases and syndromes, which are involved in several systems and cause disorders of activities of human body. Recent research has found that there exist networks between neuro-degenerative diseases In this study, we experiment with a novel non-invasive, information science-based approach to providing an overview of a wide range of disease-associated molecular substances (rather than focusing on associated proteins only) in nervous system diseases. Based on 28,652 records of nervous system disease research published during the period 1965–2012, we identify and visualize major interrelationships in disease-molecular substance association networks derived from these.Instead of focusing on biological network itself only, the objectives of this study are as follows:to explore a new approach for mapping networks of disease-gene associated molecular substances in nervous system diseases based on large-scale text-analysis rather than focus on biological networks only, in order to identify major substances that are most strongly or widely associated to clinical diseases and their interrelationships at the molecular level;to reveal networks of diseases associated via their molecular bases, in order to provide a novel integrative perspective for clinical concepts rather than focusing on a single disease as a meta-analysis would do.to construct a bridge for connecting experimental research and clinical medicine in a new integrated perspective, and to present an overview of molecular substances and nervous system diseases in a new pattern to complement traditional review and evidence-based medicine.The results provide a series of visualizations of networks of molecular substances associated with nervous system disease genetics, derived and integrated from a large collection of published research .The visualization identifies different subnetworks, each consisting of a disease (circular node) and its associated substances (square nodes). Alzheimer disease is the largest disease node in the network, brain neoplasms are second, and Parkinson’s disease is third, indicating that they are most extensively associated to molecular substances in their respective subnetworks, which suggests that they are the three most complex of the system diseases in this network.One can identify the closely associated major molecular substances of a disease by the sizes and distances of nodes in each subnetwork. In the Alzheimer disease subnetwork, for example, the apolipoproteins E node is clearly largest, with the closest association to the Alzheimer disease node. This is consistent with published research findings that apolipoproteins E play a crucial role in this disease In addition, one can identify closely related diseases by the distance between their nodes. Multiple sclerosis, spinal muscular atrophy, and dementia nodes are close to Alzheimer disease, suggesting that the molecular substances involved in these diseases are highly associated biologically.Another high density subnetwork that we can identify in Parkinson disease is a complex disease, which involves complex networks of molecular bases Besides helping to identify disease-associated major molecular substances, in the center of the network  one may We determined 15 special proteins that are highly associated with nervous system diseases, each of them related to at least 20 diseases . ClearlyIn contrast to We notice an interesting phenomenon in Around “apolipoproteins E”, one can find apolipoprotein E4, amyloid beta-protein precursor, amyloid beta-peptides, tau proteins, prions and their associated diseases: most of the molecular substances in this group are highly related to diseases to which apolipoproteins E are associated (described above). We also see that the disease most closely associated to prions is Creutzfeldt-Jakob syndrome, consistent with previous findings Clearly, one can identify the “tumor suppressor proteins” associated cluster as the largest and densest subnetwork in this figure, in close vicinity to associated diseases and other major molecular substances. This shows neoplasm DNA, neoplasm proteins, tumor suppressor protein p53, proto-oncogene proteins, highly interrelated with brain neoplasms, tuberous sclerosis, von Hippel-Lindau disease, ataxia telangiectasia, glioblastoma, glioma, astrocytoma, cerebellar neoplasms, neurofibromatosis1, Down syndrome.As the top-left of In addition, we see some groups such as “transcription factors”, “complementary DNA”, “carrier proteins”, “ubiquitin-protein ligases”, “protein-serine-threonine kinases” displayed in the center of network, each of them connected to many nodes belonging to different clusters, indicating that they act as important “mediators” for different diseases.One may also find kidney neoplasms  with nodes with high interconnectivity in the network, to extract 15 clusters by factor analysis, see We use the term “co-association group” here in a cautious sense, as the functions of a molecular substance are very complex in the biological process, but we do label each factor according to its perceived biological function. We find that each co-association group generates a brief story about actions on biological processes involved in nervous system diseases when examining known roles of its member substances. The following outlines the perceived function of each factor in turn.As shown in This co-association group of molecular substances plays vital roles in gene transcription . It is wThis co-association group plays significant roles in the biological process of neurodegeneration. The substances often serves as biomarkers , i.e., tThis group of molecular substances is associated with immune response . The priThis group of molecular substances is associated with reversible regulations in the biological process of nervous system diseases . ElevatiThis group of molecular substances is associated with regulating the biological process of diseases . The VonThis associated group of molecular substances is important for the maintenance of specialized nerve cells called motor neurons . Cyclic This group of molecular substances is associated with catalysis and regulation for metabolism and energy . MutatioThis group of molecular substances acts as a transcriptional repressors and activators . HoweverThe substances in this group are closely associated with proteins implicated in neurodegeneration Direct inhibition of prion protein function by PrP(Sc) may be necessary for neurodegeneration in prion disease This associated group of molecular substances is related to muscle function . FunctioThis group of molecular substances is associated with bioluminescence and can be used as markers for particular characteristics .This group of molecular substances is associated with modifying the transmission of molecular signals from a cell's exterior to its interior . ProteinThis co-association group of molecular substances make up microtubules or regulate their stability . MicrotuThis group of molecular substances is associated with transport proteins . MembranAs the biological process of nervous system diseases is very complex and the function of many molecular substances is still uncertain, newly discovered disease gene associated molecular substances need to be paid more attention to even if they do not meet the threshold of interconnectedness for the integrated networks shown as We recall that one of networks properties is that functionally related components are likely to be found in their network-based vicinity The purpose of present study is to explore a new way to study widely agreed-upon interrelationships between diseases at the molecular level to help bridge the gaps between experimental research and clinical concepts. This consensus is discovered through a census of nearly 30,000 research journal publications as to which links they describe most frequently. Infrequently discussed connections, many of which presumably correspond to “discoveries” that did not pan out, are effectively ignored in the figures that we present as results.This approach, while common in network medicine, is in stark contrast to traditional meta-analysis, which focuses on the optimal evidence for clinical protocols for a single diseases by evaluating a small number of studies, from which irreproducible ones are weeded out one by one. This manual process of separating useful from useless information is usually documented as PRISMA  and is required when publishing systematic reviews and meta-analyses The issue of bias has not really been considered in network medicine as well as in literature data mining for biology yet Nevertheless, we do acknowledge that there is some risk of biases within this study which could influence the validation of results.All study types of 28562 records are included in the data analysis, which would lead to selection bias;All data of 28562 records are collected from the PubMed database, which would lead to information bias.In this study, we apply a network analysis based approach originally developed in information science to a comprehensive set of publications touching the genetics of neural system diseases to identify major molecular substances in an integrated network of associations derived from the literature.Our results demonstrate that it is problematic to consider diseases as being consistently independent of one another, and instead reflect “resonating perturbations” in networks of associated molecular substances and their interrelationships in the disease-molecular substance complex –2. SpeciRemarkably, our network-based analysis reveals functional co-association groups of major molecular substances, such that each co-association group generates a story of its member substances involved in biological processes which are critical in nervous system diseases.First, we find large-scale literature-based evidence that networks of substances operating in biomedical systems are not random, but exhibit interesting structural features, as exhibited in Second, we show that the 20 molecular substances most highly associated to nervous system diseases are closely linked to two large clusters centered on “brain neoplasms” and “Alzheimer disease”, exhibiting high density interrelationships within each group. 15 proteins are particularly highly associated with nervous system diseases, each of them connected to at least 20 diseases . This paThird, we reveal that there exist functional co-association groups in major molecular substances, each group generating an interesting narrative about biological processes involved in nervous system diseases. In summary, our network-based results indicate fundamental frameworks of major molecular substances that are highly associated with nervous system diseases and their interrelationships, derived from integration of a huge literature survey, standing out from a “daunting” number of molecular entities The key findings of our study are that not only revealing functional co-association groups exist in the network of major molecular substances involved in nervous system diseases, but also that the interrelationships within each group and among different groups can be identified. As From these results we can see that the identification of network properties present in biomedical systems can provides a significant basis for studying network medicine in the future, which may help to find valid systemic interpretations of the biological mechanism of human diseases.As the functions of most newly discovered molecular substances are uncertain, and biological processes of many nervous system diseases are very complex and still unclear In conclusion, our study visually reveals a fundamental large-scale consensus framework of major molecular substances associated with nervous system diseases and their interrelationships among one another, suggesting that network-based analysis is a convenient approach to analyzing and visualizing large-scale, complex relationships among molecular substances and diseases. Compared to the model of traditional review and evidence-based medicine, our findings not only have profound implications for improved understanding of disease-molecular substance-association networks, but may also provide a conceptual basis for bridging gaps in experimental and theoretical knowledge, in a new integrated perspective.Here we merely explore a new approach for mapping networks of disease-gene associated molecular substances in nervous system diseases based on large-scale text-analysis. This is necessarily a preliminary study, as network medicine is currently still an emerging field, if a promising one for informing both basic research and clinical medicine in the future. Although there are no similar criteria like PRISMA As a first step in the direction of network medicine, the results of our study is preliminary. It now merely sketches a broad framework of molecular associations in a network of nervous system diseases rather than providing precise scientific conclusion on these interrelationships.The statistical analysis in this study is not multiple-dimensional, which may limit the ability of our study to produce deeper findings.We use PubMed database as the only data source to research, and all study types of 28562 records are included in our research, which could lead to information bias and selection bias in this study. To balance this, only items and connections which frequently occur in these records are extracted for analysis, thus presumably focusing on those for which a broad consensus exists in the scientific literature.The study is conducted under the following hypotheses:Each record in PubMed corresponds to a paper which was assessed by reviewers before publication. As such, it represents at least a unit of knowledge about scientific phenomena resulting from research;In scientific fields, the more important the relation between two topics, the more frequently studies deal with them, and the more journal articles are written that include both topics. If there are plenty of articles that discuss similar conclusions on a topic, then these conclusions are acceptably reliable.In nervous system diseases, the more complex biological processes a molecular substance is involved in, the more frequently other substances will be connected with it, and therefore the more articles will discuss such a connection. Therefore, we can construct meaningful networks from frequently documented connections and visualize their interrelationships in the area of nervous system diseases, based on a high threshold of the number of articles on the same kind of findings.We used PubMed to collect data for the present study. PubMed is a bibliographical database known for its excellent coverage and indexing of journal publications in the biomedical research fields These XML records (our dataset) were then processed by a computer program we developed in order to produce the data we needed. Specifically, we ranked MeSH terms (Descriptors) and Chemicals (Substances) by the number of times they appeared in our dataset, and took the top 1000 from each ranking. We then manually examined the most commonly used descriptors and substances in order to extract the data that can help address our research questions.Substances are particular molecular entities that are registered in the CAS  Database, including drugs, proteins, and enzymes. Descriptors are terms that describe various facets of biomedical research, including diseases, topics, chromosomes, and substances that have been introduced into MeSH. In order to focus on the interrelationships between major diseases and molecular substances, we first removed chemical drugs for nervous system diseases We then took the top 100 substances and top 100 diseases, and calculated two co-occurrence matrices for them: a disease – substance matrix, and a substance – substance matrix. A number in the former for disease x and substance y, for example, is the number of articles in which both x and y appear, i.e., articles that are indexed in PubMed using both x and y, indicating the degree of association between x and y as collectively perceived by the indexers based on the content of the articles indexed. We also calculated a year – substance matrix, recording how many articles contain each of the top 100 substances in each year  a 20×100 disease – substance network that focuses on the top 20 major diseases and represents how these diseases are related to each other and how they relate to the top 100 molecular substances; (b) a 20×100 substance – disease network that focuses on the top 20 molecular substances and represents how these substances are related to each other and how they relate to the top 100 major diseases; (c) a 93×93 substance – substance network that represents the interrelationships among the top 93 molecular substances resulting from removing from the top 100 the “generic substances”  that are associated with almost all diseases; and (d) a 12×43 substance – substance matrix that represents the interrelationships between 12 recently discovered substances and the major substances that are related to them. The 12 “new” substances were chosen from the substances that emerged during the years 2001 and 2012, based on whether they have been studied sufficiently. Their 43 related substances are those among the top 100 substances that co-occurred with them in our dataset.These four networks were then visualized using techniques introduced in previous studies The factor analysis of network (c), i.e., the 93×93 major substance co-occurrence matrix, was performed using SPSS’ Factor Analysis routine in order to reveal the underlying structure of the interrelationships among these substances. Factors were extracted by Principal Component Analysis (PCA), and we took a 15-factor model after exploring different factor models balancing their explanatory powers with their statistical features and model fits including the Scree plot, total variance explained, communalities – how well a variable  is explained by the factor model, and correlation residuals –, the differences between observed correlations and correlations implied by the factor model We applied an oblique rotation (SPSS Direct OBLIMIN) to the factor model, which resulted in a pattern and a structure matrix. As explained in previous studies Pajek is one of the most popular network tools in social network analysis and with excellent functions and widely applications for information science The size of a circular node corresponds to the sum of the weights of links that are sufficiently strong . The width of a line that connects a square node with a circular node is proportional to the weight of this link, as is its grey-scale value, with wider and darker lines signifying higher link weights. The color of a square node indicates the number of circular nodes that this square node links to sufficiently: yellow for squares that only link sufficiently to a single circular node, green for those that link to two circular nodes, red for three, and blue for four.The authors would like to thank Zhu Lijun at the Institute of Neuroscience of the Zhejiang University China for her help with the interpretational labeling of the factor groupings of molecular substances, and Guo Gencheng at the University of Alberta for writing the computer program we used in data processing. This study was carried out during Hu’s visit at the University of Alberta in collaboration with Zhao and Strotmann.Figure S1Flow chart depicting data processing of co-occurrence matrices construction.(TIF)Click here for additional data file.Table S1Items included in our study vs. PRISMA 2009 Checklist.(DOC)Click here for additional data file.