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We have evaluated the effects of different fish feeds on the body composition, growth, and enzyme activities of Labeo rohita (Rohu). In total, 240 fishes between the average weights of 24.77±2.15g were studied. The treatments were applied in a completely randomized design, with 4 treatments of 60 fishes each. Treatments consisted of four different fish feeds [Oryza (T1), AMG (T2), Aqua (T3), and Supreme (T4)]. Body composition, growth performance, and enzyme activities were evaluated. There was a significant variation in performance of fishes fed with different type of feed; as fishes having Oryza feed showed the highest weight gain, specific growth rate (SGR), and best feed conversion ratio (FCR) as compared to other groups that were considered to be significant (P ≤ 0.05). High net weight gain was obtained in T4 when compared with T2 and T3. FCR value of T4 was less than T1 but higher than T2, T3 and T2, which showed the lowest values. The specific growth rate was recorded as average in T4, but T2 led a high SGR than T3. Similarly, crude protein level and digestive enzymes activity was recorded significantly highest in fed with Oryza (T1) as compared to AMG (T2), Aqua (T3), and Supreme (T4). Water quality parameters were recorded significant in all treatments except pH and DO of treatment (T1), significantly different from other treatments. It was concluded that Rohu (Labeo rohita) could show a promising growth rate and protease enzyme activity when fed with the Oryza feed of 25% protein. Resumo Avaliamos os efeitos de diferentes alimentos para peixes em relação à composição corporal, crescimento e atividades enzimáticas de Labeo rohita (Rohu). No total, foram estudados 240 peixes com pesos médios de 24,77 ± 2,15 g. Os tratamentos foram aplicados em delineamento inteiramente casualizado, com quatro tratamentos de 60 peixes cada. Os tratamentos consistiram em quatro alimentos diferentes para peixes: Oryza (T1), AMG (T2), Aqua (T3) e Supreme (T4). Foram avaliados a composição corporal, o desempenho de crescimento e as atividades enzimáticas. Houve uma variação significativa no desempenho dos peixes alimentados com diferentes tipos de ração. Peixes com alimentação Oryza apresentaram maior ganho de peso, taxa de crescimento específico (SGR) e melhor taxa de conversão alimentar (FCR) em comparação com outros grupos que foram considerados significativos (P ≤ 0,05). Elevado ganho de peso líquido foi obtido em T4 quando comparado com T2 e T3. O valor da FCR de T4 foi menor que T1, mas maior que T2 e T3, que apresentaram os menores valores. A taxa de crescimento específico foi registrada como média em T4, mas T2 teve uma SGR alta do que T3. Da mesma forma, o nível de proteína bruta e a atividade das enzimas digestivas foram registrados significativamente mais altos nos peixes alimentados com Oryza (T1) em comparação com AMG (T2), Aqua (T3) e Supreme (T4). Os parâmetros de qualidade da água foram registrados como significativos em todos os tratamentos, exceto pH e OD do tratamento (T1), significativamente diferente dos demais tratamentos. Concluiu-se que Rohu (Labeo rohita) pode apresentar uma taxa de crescimento promissora e atividade da enzima protease quando alimentado com Oryza de 25% de proteína.

Aim: To find out the epidemiology of spine trauma involving various spinal level by categorization based on CT scan and association of various factors on spine trauma in tertiary care centre in Rajasthan Material and Method: This is a observational study based on patients admitted in a tertiary care centre from 1st June 2019 to 31st May 2020 Results: During this study 204 patients are admitted and treated, out of which 167(81.86%) were male and 37(18.14%) were female. The most common mode of injury was Road traffic accident and most common injury was at thoracic spine level followed by lumber spine level. And most common affected age group was between 30 to 45 yrs. Most common associated injury was head injury followed by chest injury. Conclusion: In spine trauma cases there is not much variation than developed country as there is Road traffic accident also the most common mode of injury but differs from North east India where fall from height is most common mode of injury. Road traffic accident are more common in younger age group and fall from height is common in elderly group.

Understanding published unstructured textual data using traditional text mining approaches and tools is becoming a challenging issue due to the rapid increase in electronic open-source publications. The application of data mining techniques in the medical sciences is an emerging trend; however, traditional text-mining approaches are insufficient to cope with the current upsurge in the volume of published data. Therefore, artificial intelligence-based text mining tools are being developed and used to process large volumes of data and to explore the hidden features and correlations in the data. This review provides a clear-cut and insightful understanding of how artificial intelligence-based data-mining technology is being used to analyze medical data. We also describe a standard process of data mining based on CRISP-DM (Cross-Industry Standard Process for Data Mining) and the most common tools/libraries available for each step of medical data mining.

Proteomics instrumentation and the corresponding bioinformatics tools have evolved at a rapid pace in the last 20 years, whereas the exploitation of deep learning techniques in proteomics is on the horizon. The ability to revisit proteomics raw data, in particular, could be a valuable resource for machine learning applications seeking new insight into protein expression and functions of previously acquired data from different instruments under various lab conditions. We map publicly available proteomics repositories (such as ProteomeXchange) and relevant publications to extract MS/MS data to form one large database that contains the patient history and mass spectrometric data acquired for the patient sample. The extracted mapped dataset should enable the research to overcome the issues attached to the dispersions of proteomics data on the internet, which makes it difficult to apply emerging new bioinformatics tools and deep learning algorithms. The workflow proposed in this study enables a linked large dataset of heart-related proteomics data, which could be easily and efficiently applied to machine learning and deep learning algorithms for futuristic predictions of heart diseases and modeling. Data scraping and crawling offer a powerful tool to harvest and prepare the training and test datasets; however, the authors advocate caution because of ethical and legal issues, as well as the need to ensure the quality and accuracy of the data that are being collected.

Intramolecular coevolution of amino acid sites has repeatedly been studied to improve predictions on protein structure and function. Thereby, the focus was on bacterial proteins with available crystallographic data. However, intramolecular coevolution has not yet been compared between protein sets along a gradient of functional proximity to fertilization. This is especially true for the potential effect of external selective forces on intraprotein coevolution. In this study, we investigated both aspects in equally sized sets of mammalian proteins representing spermatozoa, testis, entire body, and liver. For coevolutionary analyses, we derived the proportion of covarying sites per protein from amino acid alignments of 10 mammalian orthologues each. In confirmation of the validity of our coevolution proxy, we found positive associations with the nonsynonymous or amino acid substitution rate in all protein sets. However, our coevolution proxy negatively correlated with the number of protein interactants (node degree) in male reproductive protein sets alone. In addition, a negative association of our coevolution proxy with protein hydrophobicity was significant in sperm proteins only. Accordingly, the restrictive effect of protein interactants was most pronounced in male reproductive proteins, and the tendency of sperm proteins to form internal structures decreased the more coevolutionary sites they had. Both aspects illustrate that the share of outward and thus functional coevolution increases with greater proximity to fertilization. We found this conclusion confirmed by additional comparisons within sperm proteins. Thus, sperm proteins with high hydrophobicity had the lowest proportions of covarying sites and, according to gene annotations, localized more frequently to internal cellular structures. They should therefore be less exposed to postcopulatory forms of sexual selection. Their counterparts with low hydrophobicity had larger proportions of covarying sites and more often resided at the cell membrane or were secreted. At the cellular level, they are thus closer to externally induced forces of postcopulatory selection which are known for their potential to increase substitution rates. In addition, we show that the intermediary status of the testicular protein set in correlation analyses is probably due to a special combination of reproductive and somatic involvements.

Various information technology (IT) infrastructures for biobanking, networks of biobanks and biomaterial management are described in the literature. As pre-analytical variables play a major role in the downstream interpretation of clinical as well as research results, their documentation is essential. A description for mainly automated documentation of the complete life-cycle of each biospecimen is lacking so far. Here, the example taken is from the University Medical Center Göttingen (UMG), where the workflow of liquid biomaterials is standardized between the central laboratory and the central biobank. The workflow of liquid biomaterials from sample withdrawal to long-term storage in a biobank was analyzed. Essential data such as time and temperature for processing and freezing can be automatically collected. The proposed solution involves only one major interface between the main IT systems of the laboratory and the biobank. It is key to talk to all the involved stakeholders to ensure a functional and accepted solution. Although IT components differ widely between clinics, the proposed way of documenting the complete life-cycle of each biospecimen can be transferred to other university medical centers. The complete documentation of the life-cycle of each biospecimen ensures a good interpretability of downstream routine as well as research results.

Atherosclerosis is an important risk factor in the development of cardiovascular diseases. In addition to increased plasma lipid concentrations, irregular/oscillatory shear stress and inflammatory processes trigger atherosclerosis. Inhibitors of the transcription modulatory bromo- and extra-terminal domain (BET) protein family (BETi) could offer a possible therapeutic approach due to their epigenetic mechanism and anti-inflammatory properties. In this study, the influence of laminar shear stress, inflammation and BETi treatment on human endothelial cells was investigated using global protein expression profiling by ion mobility separation-enhanced data independent acquisition mass spectrometry (IMS-DIA-MS). For this purpose, primary human umbilical cord derived vascular endothelial cells were treated with TNFα to mimic inflammation and exposed to laminar shear stress in the presence or absence of the BRD4 inhibitor JQ1. IMS-DIA-MS detected over 4037 proteins expressed in endothelial cells. Inflammation, shear stress and BETi led to pronounced changes in protein expression patterns with JQ1 having the greatest effect. To our knowledge, this is the first proteomics study on primary endothelial cells, which provides an extensive database for the effects of shear stress, inflammation and BETi on the endothelial proteome.

The paper aims to investigate the impact of COVID-19 pandemic on the perception of travelers and identify new policies and intervention areas for improving urban bus service. This was carried out based on the perception data collected from travelers in the years 2018 (before pandemic) and 2020(in post lockdown scenario). Data was collected from the state of Kerala, India, and Importance-Satisfaction Analysis (ISA) with fuzzy c-means clustering was carried out to identify improvement areas for both scenarios. Results show a substantial increase in the number of priority areas as compared to the pre-pandemic scenario. Bus service attributes such as cleanliness, crowding level, and pedestrian environment emerged as new intervention areas of bus service during the pandemic. This is a clear reflection of the increased awareness of travelers about the prevailing pandemic scenario and the need for improvement in attributes related to social distancing and hygiene. The higher priority to information related attributes of bus service clearly shows the need for improvement in such attributes for the safe planning of trips during the pandemic. The work also estimated desired service levels of quantitative attributes to enhance the satisfaction levels of users. An evaluation of five bus routes in Kochi based on the desired levels shows the underperformance of attributes such as headway, punctuality, and crowding level. While the findings are city-specific, the methodology and experience may be adopted to enhance the service quality of buses and other public transport.

The study was designed to investigate the effect of mannan-oligosaccharide (MOS) supplementation on intestinal histomorphology, immunity against Newcastle disease virus (NDV) and productive parameters of broilers. A total of 1800, day old broiler chicks of Cobb-500 strain were selected and randomly assorted into 6 treatment groups: T1 (basal diet without antibiotics as negative control); T2 (basal diet plus antibiotics as positive control group); T3 (basal diet plus 200g/ton MOS); T4 (basal diet plus 400g/ton MOS); T5 (basal diet plus 600g/ton MOS) and T6 (basal diet plus 800g/ton MOS). Each treatment was having 6 replicates and the feed intake, body weight gain and feed conversion ratio (FCR) were recorded on weekly basis. Results showed that, MOS supplemented birds have significantly higher feed intake, weight gain and FCR (P < 0.05). Similarly, supplementation of MOS showed positive effect on villus height and crypt depth both in jejunum and ilium. Goblet cell density was unaffected by MOS addition (P < 0.05). Furthermore, birds fed with diets containing MOS, exhibited better productive performance in comparison to positive and negative control groups. In conclusion, MOS can replace antibiotic growth promoters (AGPs) as non-microbial performance-enhancing feed advocates. Resumo O estudo foi desenhado para investigar o efeito da suplementação de mananoligossacarídeo (MOS) na histomorfologia intestinal, imunidade contra o vírus da doença de Newcastle (NDV) e parâmetros produtivos de frangos de corte. Um total de 1.800 pintos de corte de um dia da linhagem Cobb-500 foram selecionados e distribuídos aleatoriamente em 6 grupos de tratamento: T1 (dieta basal sem antibióticos como controle negativo); T2 (dieta basal mais antibióticos como grupo controle positivo); T3 (dieta basal mais 200g/ton MOS); T4 (dieta basal mais 400g/ton MOS); T5 (dieta basal mais 600g/ton MOS) e T6 (dieta basal mais 800g/ton MOS). Cada tratamento tinha 6 repetições e o consumo de ração, ganho de peso corporal e conversão alimentar foram registrados semanalmente. Os resultados mostraram que as aves suplementadas com MOS apresentam consumo de ração, ganho de peso e CA significativamente maiores (P < 0,05). Da mesma forma, a suplementação de MOS mostrou efeito positivo na altura das vilosidades e na profundidade das criptas tanto no jejuno quanto no íleo. A densidade de células caliciformes não foi afetada pela adição de MOS (P < 0,05). Além disso, as aves alimentadas com dietas contendo MOS apresentaram melhor desempenho produtivo em comparação aos grupos controle positivo e negativo. Em conclusão, o MOS pode substituir os promotores de crescimento de antibióticos (AGPs) como defensores de alimentos não microbianos que melhoram o desempenho.

Rhizobacteria, a plant growth promoting rhizobacteria (PGPR) as beneficial microorganism which helps in defense from abiotic and abiotic stresses, colonizes in rhizosphere and played a major role in promoting plant growth and also provides enhance soil fertility. In the highly contaminated soil, the content of metal exceeds the limits of plant tolerance. It is also possible that treatment of plant with PGPR, here increasing the biomass of plant, stabilizing and the remediation of metal polluted soil. The use of rhizobacteria plays and important role in increasing the tolerance of plant towards toxic effects of heavy metals like arsenic, sulphur, mercury, chromium, cadmium, nickel, lead and copper etc. Heavy metal accumulation results in deterioration of soil fertility while PGPR helps to restore soil fertility. The process of phytoremediation has been proved to be the best way to remediate heavy metals from soil. The use of rhizobacteria with plants provides highly efficiency phytoremediation. However, there is still need to understanding the concept of microbial ecological study in rhizosphere and mechanism of detoxification of heavy metals form rhizosphere.

Alzheimer’s disease (AD) is a complex neurodegenerative disorder with impaired protein activities. Proteins in the form of complexes have a ubiquitous role in diverse range of cellular functions. The key challenge is to identify novel disease associated protein complexes and their potential role in the progression of AD pathology. Protein complexes were obtained from AD brain prefrontal cortex and age matched controls by Blue Native-Polyacrylamide Gel Electrophoresis. A proteomic analysis was performed using second dimension SDS-PAGE followed by nano LC–MS/MS. Differentially expressed proteins were mapped to existing biological networks by Ingenuity Pathway Analysis (IPA). A total of 13 protein complexes with their interacting proteins were resolved on SDS-PAGE. We identified 34 protein spots and found significant abundance difference between the two experimental samples. IPA analysis revealed degeneration of neurons and cell death as a major consequence of protein dysregulation. Furthermore, focused network analysis suggested an integrated regulation of the identified proteins through APP and MAPT dependent mechanisms. The interacting differentially expressed proteins in AD were found to be part of concomitant signaling cascades terminating in neuronal cell death. The identified protein networks and pathways warrant further research to study their actual contribution to AD pathology.

Multi-omic approaches are promising a broader view on cellular processes and a deeper understanding of biological systems. with strongly improved high-throughput methods the amounts of data generated have become huge, and their handling challenging. Area Covered: New bioinformatic tools and pipelines for the integration of data from different omics disciplines continue to emerge, and will support scientists to reliably interpret data in the context of biological processes. comprehensive data integration strategies will fundamentally improve systems biology and systems medicine. to present recent developments of integrative omics, the göttingen proteomics forum (gpf) organized its 6th symposium on the 23rd of november 2017, as part of a series of regular gpf symposia. more than 140 scientists attended the event that highlighted the challenges and opportunities but also the caveats of integrating data from different omics disciplines. Expert commentary: The continuous exponential growth in omics data require similar development in software solutions for handling this challenge. Integrative omics tools offer the chance to handle this challenge but profound investigations and coordinated efforts are required to boost this field.

Chlamydia trachomatis (CT) and the Chlamydophila species (CS) Chlamydophila pneumoniae (CPn), and Chlamydophila psittaci (CPs) are suggested to induce autoantibodies causative of several human autoimmune disorders like rheumatoid arthritis and systemic lupus erythematosus (SLE). The aim of the present study was therefore to identify cellular protein interaction partners with antisera to CT (α-CT) or CS (α-CS) and to identify functional consequences of such interaction in vitro. As detected with a commercial first trimester human prenatal brain multiprotein array (hEXselect, Engine, Germany), the most frequent interaction partner with both α-CT and α-CS was the ribosomal small subunit protein RPS27a. This could be confirmed by Western blot analysis with a recombinant RPS27a sample. In addition, immunocytochemistry with both antisera in the human choroid plexus papilloma cell line HIBCPP revealed a granular cytoplasmic staining, and Western blot analysis with whole-cell protein samples of HIBCPP cells revealed both antisera to label protein bands of different molecular weights and intensity. By 2D Western blot analysis and mass spectrometry, one of the protein spots interacting with α-CT could be identified as the RPS27a. Finally, two different methods for the detection of protein synthesis activity, the SUnSET technique and an HPG fluorescence assay revealed both antisera to cause reduced translational activity in HIBCPP cells. Together with previous findings of RPS27a as an autoimmune target in a mouse model of systemic lupus erythematosus (SLE), these results suggest that infections with CT and/or CS could induce SLE-associated immune modifications. However, direct evidence for a pathogenic role of these interactions for SLE demands further investigations.

Background:COVID-19 pandemic had an unprecedented impact on the delivery of patient care. Rheumatology services had to rapidly adapt to virtual consultations at the onset of the pandemic. However, providing a high quality and effective service in a virtual setting can be challenging and therefore its prudent to do a formal review and gain patient feedback to ensure that these clinics are fit for purpose.Objectives:To evaluate the impact of first wave of COVID-19 pandemic on patients with autoimmune rheumatic conditions, assess delivery of rheumatology outpatient care and record patient feedback.Methods:This study included patients on the Rheumatology clinic lists between 3rd and 31st August 2020. An electronic survey questionnaire was developed and the survey link was sent to patients via a text message using secure IT platform. Data was collected on patient demographics, diagnosis, comorbidities, treatments, clinical/ laboratory confirmed COVID-19 diagnosis, treatment interruption, impact on work, personal protective measures taken and views on virtual consultations.Results:307 patients responded with 287 complete responses. 73.1%(223) were female and 32.4% (99) were >65 years of age. Rheumatoid arthritis was the most common diagnosis 41.6%(127). Hypertension was the commonest comorbidity 21.4%(64) followed by Chronic lung disease 17.3%(52). 28.8%(85) were on Hydroxychloroquine, 26.7%(79) Methotrexate, 14.2%(42) Sulfasalazine and 13.2%(39) on Prednisolone. 22.3%(66) were on Biologics: Anti TNF 12.8%(38), Tocilizumab 3.7%(11) and Rituximab 3%(9). 52.6%(161) shielded, 16.9%(55) self-isolated and 30.3%(93) only maintained social distance. 197 patients self reported as being vulnerable but based on their treatment,only 167 patiemts met the clinically extremely vulnerable (CEV) criteria and all of those received government shielding letter.3.6%(11) had lab confirmed COVID-19, 3.2%(10) had clinically suspected COVID-19 infection. 14.3% (43) had their treatment interrupted. 4.6%(14) were unable to work from home or maintain social distancing at work. 59.8%(182) had face-to-face consultation changed to virtual. 63.2%(189) were satisfied, 28%(84) neutral and 8.7%(26) reported dissatisfaction with their consultation. 50.5%(153) were happy to continue with virtual consultation but with an option of face to face only if necessary.For consultations post COVID-19, 59.4%(182) preferred a mixture of face to face and virtual appointments.Conclusion:Majority of our patients seem happy with virtual consultations as long as they are assured of a face-to-face consultation if needed. A minority(8.7%) however, were dissatisfied. Some of the suggestions were, use of video consultations and improvement in communication before the virtual appointments. Our survey also shows that our patients have adapted well to virtual consultations and many are keen to have virtual consultation in the longer term. In our survey, only 6.8%(21) patients reported definite or clinically suspected COVID-19. Possible explanations for this include strict compliance with government advice on social distancing/shielding and limited testing at the onset of the pandemic. More patients assumed themselves to be clinically CEV than those who were actually CEV based on their treatment which is not surprising because of high level of anxiety among patients due to rapidly spreading pandemic and multiple sources of information.This feedback provides useful data which will help us to plan the delivery of rheumatology services post COVID-19 pandemic. While face-to-face patient contact is needed for comprehensive disease assessment, teaching and training, a model for the future is likely to include a combination of face-to-face and virtual consultations. This could allow a greater capacity to see new patients and reduce waiting lists. Patients with uncomplicated and stable disease could be followed up in virtual clinics. There is also a need to formally incorportate the virtual consultations into the curriculum for Rheumatology trainees.Disclosure of Interests:None declared.

Human spine curvature data are required to detect spinal deformity/diagnose irregular spine curvature. The shape of the spine assisting for the flexibility and weight distribution of the body. Around the world, many countries have started collecting spine curvature data for more than five decades; however, India lacks in this field. Few countries have also developed various instruments to gather spine curvature data; on the other hand, they still seem inaccessible to Indian markets. A device which can collect the data is very much required as it has application in vast fields like product development on ergonomics principles. This study developed an instrument (simple and low cost) to measure coordinates of spine curvature. A wooden specimen (resembles human spine shape) was designed with various angles to validate the measurement accuracy of the developed instrument. The selected curves of the template were measured using three machines, including CMM machine, FARO arm machine, and developed device. T-Test confirms that there was no significant difference in the measured curvature data among instruments. The error percentage was 2.57 % (maximum).

There was an error published in J. Cell Sci. (2013) 126, [3649–3663][1] ([doi:10.1242/jcs.125088][2]). The affiliations for Marwa Eltoweissy were incorrect. The correct affiliations are as given below. Hassan Dihazi1, Gry Helene Dihazi1, Asima Bibi1, Marwa Eltoweissy1,2, Claudia A. Mueller3

Background and Purpose— Recently, we reported high seroprevalence (age-dependent up to >19%) of N -methyl- d -aspartate-receptor subunit NR1 (NMDAR1) autoantibodies in both healthy and neuropsychiatrically ill subjects (N=4236). Neuropsychiatric syndrome relevance was restricted to individuals with compromised blood–brain barrier, for example, apolipoprotein E4 (APOE4) carrier status, both clinically and experimentally. We now hypothesized that these autoantibodies may upon stroke be protective in individuals with hitherto intact blood–brain barrier, but harmful for subjects with chronically compromised blood–brain barrier. Methods— Of 464 patients admitted with acute ischemic stroke in the middle cerebral artery territory, blood for NMDAR1 autoantibody measurements and APOE4 carrier status as indicator of a preexisting leaky blood–brain barrier was collected within 3 to 5 hours after stroke. Evolution of lesion size (delta day 7–1) in diffusion-weighted magnetic resonance imaging was primary outcome parameter. In subgroups, NMDAR1 autoantibody measurements were repeated on days 2 and 7. Results— Of all 464 patients, 21.6% were NMDAR1 autoantibody–positive (immunoglobulin M, A, or G) and 21% were APOE4 carriers. Patients with magnetic resonance imaging data available on days 1 and 7 (N=384) were divided into 4 groups according to NMDAR1 autoantibody and APOE4 status. Groups were comparable in all stroke-relevant presenting characteristics. The autoantibody+/APOE4− group had a smaller mean delta lesion size compared with the autoantibody−/APOE4- group, suggesting a protective effect of circulating NMDAR1 autoantibodies. In contrast, the autoantibody+/APOE4+ group had the largest mean delta lesion area. NMDAR1 autoantibody serum titers dropped on day 2 and remounted by day 7. Conclusions— Dependent on blood–brain barrier integrity before an acute ischemic brain injury, preexisting NMDAR1 autoantibodies seem to be beneficial or detrimental.

Mycophenolic acid (MPA) is prescribed to prevent allograft rejection in organ transplanted patients. However, its use is sporadically linked to leak flux diarrhea and other gastrointestinal (GI) disturbances in around 75% of patients through yet unknown mechanisms. Recently, we identified Midkine as a modulator of tight junctions (TJs) permeability in MPA treated Caco-2 monolayer. In the present study, we investigated the possible involvement of Midkine dependent PI3K pathway in alteration of TJs under MPA treatment. Caco-2 cells were grown as monolayer to develop TJs and were treated for 72 h with DMSO (control) or MPA in presence and absence of Midkine inhibitor (iMDK) or PI3K inhibitors (LY/AMG). Caco-2 monolayer integrity was assessed by transepithelial electrical resistance (TEER) and FITC-dextran assays. Our functional assays showed that PI3K inhibitors (LY/AMG) can significantly inhibit the compromised TJs integrity of MPA-treated Caco-2 cells monolayer. Chromatin immunoprecipitation analyses showed a significant epigenetic activation of Midkine, PI3K, Cdx-2, and Cldn-2 genes and epigenetic repression of Cldn-1 gene after MPA treatment. The MPA-induced epigenetic alterations were further confirmed by mRNA and protein expression analysis. Collectively, our data shows that PI3K pathway as the downstream target of Midkine which in turn modulates p38MAPK and pAKT signaling to alter TJs permeability in Caco-2 cell monolayers treated with MPA. These results highlight the possible use of either Midkine or PI3K inhibitors as therapeutic agents to prevent MPA induced GI disturbances.

The vascular endothelium lining the luminal surface of all blood vessels is constantly exposed to shear stress exerted by the flowing blood. Blood flow with high laminar shear stress confers protection by activation of antiatherogenic, antithrombotic and anti-inflammatory proteins, whereas low or oscillatory shear stress may promote endothelial dysfunction, thereby contributing to cardiovascular disease. Despite the usefulness of proteomic techniques in medical research, however, there are relatively few reports on proteome analysis of cultured vascular endothelial cells employing conditions that mimic in vivo shear stress attributes. This review focuses on the proteome studies that have utilized cultured endothelial cells to identify molecular mediators of shear stress and the roles they play in the regulation of endothelial function, and their ensuing effect on vascular function in general. It provides an overview on current strategies in shear stress-related proteomics and the key proteins mediating its effects which have been characterized so far.

Children of mothers with prenatal gonococcal infections are of increased risk to develop schizophrenic psychosis in later life. The present study hypothesizes an autoimmune mechanism for this, investigating interactions of a commercial rabbit antiserum directed to Neisseria gonorrhoeae (α-NG) with human NTera2/D1 cells, an established in vitro model for human neuronal differentiation. Immunocytochemistry demonstrated α-NG to label antigens on an intracellular organelle, which by Western blot analysis showed a molecular weight shortly below 72 kDa. An antiserum directed to Neisseria meningitidis (α-NM) reacts with an antigen shortly below 95 kDa, confirming antibody specificity of these interactions. Two-dimensional gel electrophoresis and partial Western transfer, allowed to localize an α-NG reactive protein spot which was identified by LC-Q-TOF MS/MS analysis as mitochondrial heat shock protein Hsp60. This was confirmed by Western blot analysis of α-NG immunoreactivity with a commercial Hsp60 protein sample, with which α-NM failed to interact. Finally, analysis of neurite outgrowth in retinoic acid-stimulated differentiating NTera2-D1 cells, demonstrates that α-NG but not α-NM treatment reduces neurite length. These results demonstrate that α-NG can interact with Hsp60 in vitro, whereas pathogenetic relevance of this interaction for psychotic symptomatology remains to be clarified.

The vast majority of patients with end-stage renal disease are treated with intermittent hemodialysis as a form of renal replacement therapy. To investigate the impact of hemodialysis membrane material on vital protein removal, dialysates from 26 well-characterized hemodialysis patients were collected 5 min after beginning, during 5h of treatment, as well as 5 min before ending of the dialysis sessions. Dialysis sessions were performed using either modified cellulose (n=12) (low-flux and high flux) or synthetic Polyflux (n=14) (low-flux and high-flux) dialyzer. Protein removal during hemodialysis was quantified and the dialysate proteome patterns were analyzed by 2-DE, MS and Western blot. There was a clear correlation between the type of membrane material and the amount of protein removed. Synthetic Polyflux membranes exhibit strong interaction with plasma proteins resulting in a significantly higher protein loss compared to modified cellulosic membrane. Moreover, the proteomics analysis showed that the removed proteins represented different molecular weight range and different functional groups: transport proteins, protease inhibitors, proteins with role in immune response and regulations, constructive proteins and as a part of HLA immune complex. The effect of this protein removal on hemodialysis treatment outcome should be investigated in further studies.

Prenatal maternal infections with Neisseria gonorrhoeae (NG) correlate with an increased lifetime probability for the offspring to develop psychosis. We could previously demonstrate that in human choroid plexus papilloma cells, anti-NG antibodies (α-NG) bind to mitochondrial proteins HSP60 and ATPB, and interfere with cellular energy metabolism. To assess the in vivo relevance for this, especially during prenatal neural development, we investigated here interactions of NG-specific antisera (α-NG1, α-NG2) with brain, choroid plexus and other non-neural tissues in pre- and perinatal samples of the nonhuman primate (NHP) Callithrix jacchus (CJ), a NHP model for preclinical research. In histological sections at embryonic day E75, immunohistochemistry revealed α-NG1 and -2-staining in choroid plexus, ganglionic hill, optic cup, heart, and liver. Within the cells, organelle-like structures were labeled, which could be identified by immunohistochemical double-labeling as mitochondria. Both one- and two-dimensional Western blot analysis revealed tissue specific patterns of α-NG1 immunoreactive bands and spots, respectively, which were subsequently characterized by mass spectrometry. Thereby we could confirm the interactions of α-NG1 with human HSP60 and ATPB also in CJ choroid plexus and liver. Even more important, in the CJ brain, several new targets, including NCAM1, CRMP2, and SYT1, were identified, which by unrelated studies have been previously suggested to correlate with an increased schizophrenia risk. These findings support the idea that the marmoset monkey is a useful NHP model to investigate the role of maternal bacterial infections during prenatal brain development, and thereby might improve the understanding of this important aspect of schizophrenia pathology.

Anti-apoptotic properties of physiological and elevated levels of the cellular prion protein (PrPc) under stress conditions are well documented. Yet, detrimental effects of elevated PrPc levels under stress conditions, such as exposure to staurosporine (STS) have also been described. In the present study, we focused on discerning early apoptotic STS-induced proteome and phospho-proteome changes in SH-SY5Y human neuroblastoma cells stably transfected either with an empty or PRNP-containing vector, expressing physiological or supraphysiological levels of PrPc, respectively. PrPc-overexpression per se appears to stress the cells under STS-free conditions as indicated by diminished cell viability of PrPc-overexpressing versus control cells. However, PrPc-overexpression becomes advantageous following exposure to STS. Thus, only a short exposure (2 h) to 1 μM STS results in lower survival rates and significantly higher caspase-3 activity in control versus PrPc-overexpressing cells. Hence, by exposing both experimental groups to the same apoptotic conditions we were able to induce apoptosis in control, but not in PrPc-overexpressing cells (as assessed by caspase-3 activity), which allowed for filtering out proteins possibly contributing to protection against STS-induced apoptosis in PrPc-overexpressing cells. Among other proteins regulated by different PrPc levels following exposure to STS, those involved in maintenance of cytoskeleton integrity caught our attention. In particular, the finding that elevated PrPc levels significantly reduce profilin-1 (PFN-1) expression. PFN-1 is known to facilitate STS-induced apoptosis. Silencing of PFN-1 expression by siRNA significantly increased viability of PrPc-overexpressing versus control cells, under STS treatment. In addition, PrPc-overexpressing cells depleted of PFN-1 exhibited increased viability versus PrPc-overexpressing cells with preserved PFN-1 expression, both subjected to STS. Concomitant increase in caspase-3 activity was observed in control versus PrPc-overexpressing cells after treatment with siRNA- PFN-1 and STS. We suggest that reduction of PFN-1 expression by elevated levels of PrPc may contribute to protective effects PrPc-overexpressing SH-SY5Y cells confer against STS-induced apoptosis.

In the pathogenesis of renal fibrosis, oxidative stress (OS) enhances the production of reactive oxygen species (ROS) leading to sustained cell growth, inflammation, excessive tissue remodelling and accumulation, which results in the development and acceleration of renal damage. In our previous work (Eltoweissy et al., 2011) we established protein DJ-1 (PARK7) as an important ROS scavenger and key player in renal cell response to OS. In the present study we investigated the impact of profibrogenic agonists on DJ-1 and shed light on the role of this protein in renal fibrosis. Treatment of renal fibroblasts and epithelial cells with the profibrogenic agonist ANG II or PDGF resulted in a significant up-regulation of DJ-1 expression parallel to an increase in the expression of fibrosis markers. Monitoring of DJ-1 expression in kidney extract and tissue sections from a renal fibrosis mouse model (Col4a3-deficient) revealed a disease grade dependent regulation of the protein. Overexpression of DJ-1 prompted cell resistance to OS in both fibroblasts and epithelial cells. Furthermore overexpression of DJ-1, involved in ROS scavenging, in which glutamic acid 18 (E18) is mutated to either to aspartic acid (D) or glutamine (Q) resulted in a significant increase in cell death under OS in the case of E18D mutation, whereas E18Q mutation did not impact significantly the cell response to OS, revealing the importance of the acidic group for the ROS scavenging activity of the DJ-1 protein more than the nature of the amino acid itself. Affinity precipitation of interaction partners of DJ-1 and its mutants revealed an important role of annexin A1 and A5 in the mechanism of action of DJ-1 in anti-oxidative stress response.

Acute kidney injury (AKI) is a common condition with significant associated morbidity and mortality. The insensitivity and non-specificity of traditional markers of renal dysfunction prevent timely diagnosis, estimation of the severity of renal injury, and the administration of possible therapeutic agents. Here, we determine the prognostic ability of urinary liver-type fatty acid-binding protein (L-FABP), and further characterize its sensitivity and specificity as a biomarker of AKI. Initial western blot studies found increased urinary L-FABP in patients with confirmed AKI. A more extensive cross-sectional study found significant increases in urinary L-FABP, normalized to urinary creatinine, in 92 patients with established AKI compared with 62 patients without clinical evidence of AKI. In hospitalized patients, the diagnostic performance of urinary L-FABP for AKI, assessed by the area under the receiver operating characteristic curve, was 0.93. This compares favorably with other established biomarkers of AKI such as kidney injury molecule-1, neutrophil gelatinase-associated lipocalin, N-acetyl-β-glucosaminidase, and interleukin-18. Our study shows that age-adjusted urinary L-FABP levels were significantly higher in patients with poor outcome, defined as the requirement for renal replacement therapy or the composite end point of death or renal replacement therapy.

Thiopurine S-methyltransferase (TPMT) is an excellent example of an enzyme whose pharmacogenetic polymorphisms affect efficacy and toxicity of a drug. The association between TPMT activity and thiopurine-related myelosuppression is well recognized. To study the significance of TPMT deficiency in thiopurine metabolism and immunosuppressive activity in vitro, we established RNA interference-based TPMT knockdown (kd) in a Jurkat cell line.In Jurkat TPMT kd cells, TPMT expression was reduced to 73% at the RNA level and 83% at the protein level. TPMT kd cells were more sensitive to 6-mercaptopurine (6-MP) (10 μmol/L) and 6-thioguanine (6-TG) (8 μmol/L) than wild-type (wt) cells, (32% versus 20%) and (18% versus 9%), respectively. Both Jurkat wt and kd cells were more sensitive to 6-TG-induced apoptosis than to 6-MP. 6-TG activity was also more affected by TPMT levels than was 6-MP as reflected by IC60, concentrations that is, 6-MP [4.6 μmol/L (wt) and 4.7 μmol/L (kd)], 6-TG [2.7 μmol/L (wt) and 0.8 μmol/L (kd)]. IC60 concentrations induced significant apoptosis in both Jurkat wt and kd cells (257%, versus 314%) with 6-MP and (323% versus 306%) with 6-TG, respectively. At IC60 (6-MP) 6-thioguanine nucleotides (6-TGN) accumulation in cells was 518 versus 447 pmol/million cells in wt and kd cells, respectively. On the other hand 6-TGN accumulation at IC60 (6-TG) was 477 versus 570 pmol/million cells in wt and kd cells, respectively. 6-Methylated mercaptopurine (6-MeMP) concentrations were more affected than 6-TGN by TPMT kd (194 versus 10 pmol/million cells) in wt and kd cells, respectively.We conclude that TPMT kd cells are an appropriate in vitro model to investigate the significance of TPMT deficiency with thiopurine therapy and could be helpful in understanding possible clinical consequences of TPMT polymorphism.

J. Neurochem. (2012) 121, 954–963. Abstract Alzheimer’s disease (AD) is the most common form of dementia and cognitive impairment usually characterized by widespread neurodegeneration throughout the association cortex, limbic system and hippocampus. Aberrant protein phosphorylation is a defining pathological hallmark of AD and implicated in the dysregulation of major cellular processes through highly dynamic and complex signaling pathways. Here in, we demonstrate 81 proteins, of 600 spots selected, unambiguously identified as phosphorylated, providing a partial phosphoproteome profile of AD substantia nigra and cortex and respective control brain regions. More importantly, abnormal phosphorylation signal intensity of nine physiologically important proteins observed can profoundly affect cell metabolism, signal transduction, cytoskeleton integration, and synaptic function and accounts for biological and morphological alterations. Our studies employed two-dimensional gel electrophoresis for protein separation, Pro-Q® Diamond phosphoprotein staining and electrospray ionization quadrupole time of flight tandem MS for protein identification. NetPhosk 1.0 is used for the confirmation of protein modification status as well known/putative phosphoproteins. A further insight into the links among the identified phosphoproteins and functional roles STRING 8.3, KEGG and REACTOME pathway databases were applied. The present quantitative phosphoproteomic analysis can be supportive in establishing a broad database of potential protein targets of abnormal phosphorylation in AD brain.

Renal cell carcinoma (RCC) is the most common renal neoplasm in adults. Considering that chemoresistance is a typical feature of RCC, every effort should be made in order to identify mechanisms of drug resistance. We used two-dimensional gel electrophoresis and mass spectrometry to study changes in protein expression levels that occur in primary resistant LN78 RCC cells when treated with therapeutic concentrations of cisplatin. Expression differences of selected proteins were confirmed by immunoblot. Up-regulation of heat-shock proteins can block apoptosis indirectly by altered protein folding and by direct interaction with apoptosis regulatory proteins. Cyclophilin A and stratifin can modify cell cycle control and enable tumor cells to escape and further proliferate despite DNA damage caused by cisplatin. Increased activity of glycolytic enzymes reflect metabolic adaptations to increased energy requirements as well as converting to alternative energy sources because of cisplatin-induced disturbed mitochondrial oxidation. Changes in cytoskeletal proteins may change the handling of cisplatin by altering transport and increasing cellular efflux of the drug. Repression of vimentin and disturbance of antioxidative mechanisms may represent vulnerable points in tumor cellular defense against cisplatin. The involvement of these proteins in cisplatin resistance and their potential as therapeutic targets requires further evaluation.

The present study was undertaken to identify proteins interacting with PrP(C) that could provide new insights into its physiological functions and pathological role. Human PrP(C) was expressed in prion protein-deficient murine hippocampus (HpL3-4) neuronal cells. The PrP(C) along with its interacting proteins were affinity purified using STrEP-Tactin-chromatography, in-gel digested, and identified by Q-TOF MS/MS analysis. Forty-three proteins appeared to interact with PrP(C) in this neuronal cell line. Of these, 15 were already known for their interaction with PrP(C) or PrP(Sc), while 28 new proteins were identified. Interaction of a novel interacting partner of GTPase family-Rab7a, having a suggested role in vesicle trafficking, was further investigated using confocal laser scanning microscopy and reverse coimmunoprecipitation. Both reverse coimmunoprecipitation and immunofluorescence results confirmed potential interaction of Rab7a with the PrP(C). siRNA against the Rab7a gene decreased expression of Rab7a protein, in PrP(C) expressing HpL3-4 and SH-SY5Y cells. This depleted Rab7a expression led to the enhanced accumulation of PrP(C) in Rab9 positive endosomal compartments and consequently an increased colocalization between PrP(C)/Rab9. However, the Rab9 accumulated PrP(C) remained sensitive to proteinase-K digestion. The work described demonstrated for the first time that Rab7a interacts with PrP(C) and highlighted the involvement of endosomal compartments in the trafficking and regulation of PrP(C).

Multipotent adult germline stem cells (maGSCs) are pluripotent cells that can be differentiated into somatic cells of the three primary germ layers. To highlight the protein profile changes associated with stem cell differentiation, retinoic acid (RA) treated mouse stem cells (maGSCs and ESCs) were compared to nontreated stem cells. 2-DE and DIGE reference maps were created, and differentially expressed proteins were further processed for identification. In both stem cell types, the RA induced differentiation resulted in an alteration of 36 proteins of which 18 were down-regulated and might be potential pluripotency associated proteins, whereas the other 18 proteins were up-regulated. These might be correlated to stem cell differentiation. Surprisingly, eukaryotic initiation factor 5A (Eif5a), a protein which is essential for cell proliferation and differentiation, was significantly down-regulated under RA treatment. A time-dependent investigation of Eif5a showed that the RA treatment of stem cells resulted in a significant up-regulation of the Eif5a in the first 48 h followed by a progressive down-regulation thereafter. This effect could be blocked by the hypusination inhibitor ciclopirox olamine (CPX). The alteration of Eif5a hypusination, as confirmed by mass spectrometry, exerts an antiproliferative effect on ESCs and maGSCs in vitro, but does not affect the cell pluripotency. Our data highlights the important role of Eif5a and its hypusination for stem cell differentiation and proliferation.

Aim: The aim of this study was to investigate the effect of mycophenolate mofetil (MMF) using differential kidney proteome profiling of COL4A3-deficient mice as a model of progressive renal disease. Methods: Histological evaluation of kidney sections was performed. Total protein lysate from kidneys of placebo- and MMF-treated COL4A3-deficient mice was studied for significant differences in protein abundance using 2-dimensional electrophoresis and mass spectrometry. Results: While tubulointerstitial fibrosis in COL4A3-deficient mice was inhibited by MMF, 19 proteins in the kidneys were regulated: 12 with lower (ATPO, TAGL2, CAH1, TPD52, VA0D1, SERPH, GNAL, PSB6, EF1D, OTUB1, NDUS8, and NAPSA) and 7 with higher (ACADM, ACY3, CK054, ACTB/G, ACTB, UBP5, and ACY1) spot intensity. Nine differentially expressed proteins showed interaction potential (ATPO, TPD52, PSB6, EF1D, OTUB1, NAPSA, ACTB, ACTG, and UBP5). Conclusions: The identified proteins take part in different signaling pathways. With the highest probability, the VEGF signaling pathway (TAGL2, EF1D, and ACTB) and hypoxia (CAH1, PSB6, and ACTG) were influenced by MMF in fibrotic conditions. Moreover, MMF may modulate antifibrotic and apoptotic pathways as well as epithelial-mesenchymal transition (EMT). Different signaling pathways may be influenced by MMF therapy. These results suggest an inhibitory effect of MMF on renal EMT in COL4A3-deficient mice. Further studies are necessary to validate these findings.

Cerebrospinal fluid (CSF) contains a dynamic and complex mixture of proteins, which can reflect a physiological and pathological state of the central nervous system. In our present study, we show CSF protein patterns from patients with the two most frequent subtypes of sporadic Creutzfeldt-Jakob disease (sCJD) defined by the codon 129 genotype (MM, MV, and VV) and the protease-resistant form of prion protein (type 1 and type 2). The densitometric analysis of 2D gels showed up-regulation of 27 and down-regulation of 3 proteins in the MM-sCJD as well as the up-regulation of 24 proteins in the VV-sCJD as compared to nondemented control. Almost 40% of sCJD specific regulated proteins in CSF are involved in glucose metabolism, regardless of the codon 129 polymorphism. The increase in CSF levels of lactate dehydrogenase (LDH), glucose-6-phosphate isomerase (G6PI), and fructose-bisphosphate aldolase A (ALDOA) were validated on a larger group of sCJD patients including three possible codon 129 polymorphism carriers and three control groups consisting of nondemented, neurological cases as well as patients suffering from Alzheimer's disease or vascular dementia. Subsequently, the abundance of these glycolytic enzymes in the brain as well as their cellular localization were determined. This study demonstrates for the first time the implication of G6PI in prion-induced pathology as well as its cellular translocalization in sCJD. The identification of sCJD-regulated proteins in CSF of living symptomatic patients in our study can broaden our knowledge about pathological processes occurring in sCJD, as they are still not fully understood.

Aspergillus fumigatus is the common cause of allergic broncho-pulmonary aspergillosis (ABPA) and most of the allergens have been described from its secreted fraction. In the present investigation, germinating conidial cytosolic proteins of A. fumigatus were extracted from a 16 h culture. The proteome from this fraction was developed, and immuno-blots were generated using pooled ABPA patients' sera. Well separated Immunoglobulin-E (IgE) and Immunoglobulin-G (IgG) reactive spots were picked from corresponding 2DE gels and subjected to mass spectrometric analysis. As a result, 66 immuno-reactive proteins were identified from two geographically different strains (190/96 and DAYA) of A. fumigatus. Only 3 out of 66 proteins reacted with IgG, and the remaining 63 proteins were found to be IgE reactive. These 63 IgE-reactive cytosolic proteins from germinating conidia included 2 already known (Asp f12 and Asp f22) and 4 predicted allergens (Hsp88, Hsp70, malate dehydrogenase, and alcohol dehydrogenase) based on their homology with other known fungal allergens. In view of this, the panel of presently identified IgE-reactive novel proteins holds the potential of providing a basis for the wider diagnostic application in assay for allergic aspergillosis. We could demonstrate that recombinantly expressed proteins from this panel showed consistent reactivity with IgE of individual sera of ABPA patients. The recombinantly expressed proteins may also be useful in desensitization therapy of allergic disorders including ABPA.

The secreted proteomes of a three week old culture of an Indian (190/96) and a German (DAYA) Aspergillus fumigatus isolate were investigated for reactivity with IgG and/or IgE antibodies derived from pooled allergic broncho-pulmonary aspergillosis (ABPA) patients' sera. Two dimensional Western blotting followed by mass spectrometric analysis of the reactive protein spots revealed 35 proteins from the two A. fumigatus strains. There were seven known A. fumigatus allergens among them (Asp f1-4, Asp f9, Asp f10, and Asp f13/15), whereas three proteins displaying significant sequence similarity to known fungal allergens have been assigned as predicted allergens (Dipeptidyl-peptidase-V precursor, Nuclear transport factor 2, and Malate dehydrogenase, NAD-dependent). Eight IgG and IgE reactive proteins were common in both strains; however, 12 proteins specifically reacted in 190/96 and 15 in DAYA. Further testing with sera of 5 individual ABPA patients demonstrated that 12 out of 20 immunoreactive proteins of 190/96 strain of A. fumigatus had consistent reactivity with IgE. Seven of these proteins reacted with IgG also. The 25 of 35 identified proteins are novel with respect to immuno-reactivity with ABPA patients' sera and could form a panel of molecules to improve the currently existing less-sensitive diagnostic methods. Through expressing recombinantly, these proteins may also serve as a tool in desensibilization strategies.

Objectives: The present study was designed to investigate early proteome and phosphoproteome changes during inhibition of lymphocyte proliferation induced by sirolimus (SRL). Materials and methods: Proliferation assays were conducted using human CCRF-CEM T lymphoblasts under different SRL concentrations. Total protein lysates after SRL treatment were used to identify significantly regulated proteins and phosphorylated proteins by 2-DE and Q-TOF Ultima Global mass spectrometer. Results and conclusions: Incubation with 2.5 μmol/l SRL resulted in a ∼ 70% inhibition of cell proliferation. Cells incubated with 2.5 μmol/l for 30 min showed a differential phosphorylation pattern with one higher (TCPQ) and six lower phosphorylation signals (TBA1B, VIME, HNRPD, ENPL, SEPT9, PLSL). On investigating the differential protein expression, five proteins were found to be up-regulated (ECHB, PSB3, MTDC, LDHB and NDKA) and four were down-regulated (EHD1, AATC, LMNB1 and MDHC). Nine of these differentially regulated proteins/phosphoproteins (TCPQ, TBA1B, VIME, HNRPD, ENPL, ECHB, PSB3, LDHB and LMNB1) showed significant interaction potential, through binding protein YWHAZ using MINT software. Conclusions: We report for the first time the simultaneous early influence of SRL on phosphorylation status and on protein expression in the total proteome of CCRF-CEM T lymphoblasts and predict that 56% of the proteins interact with each other, highlighting significance of these results.

Malfunction of the ubiquitin-proteasome system has been implicated as a causal factor in the pathogenesis of aggregation-related disorders, e.g. Parkinson's disease. We show here that Transforming growth factor-beta 1 (TGF-beta), a multifunctional cytokine and trophic factor for dopaminergic (DAergic) neurons modulates proteasome function in primary midbrain neurons. TGF-beta differentially inhibited proteasomal subactivities with a most pronounced time-dependent inhibition of the peptidyl-glutamyl peptide hydrolyzing-like and chymotrypsin-like subactivity. Regulation of proteasomal activity could be specifically quantified in the DAergic subpopulation. Protein blot analysis revealed an accumulation of ubiquitinated proteins after TGF-beta treatment. The identity of these enriched proteins was further analyzed by 2D-gel electrophoresis and mass spectrometry. We found epidermal fatty acid binding protein (EFABP) to be strongly increased and ubiquitinated after TGF-beta treatment and confirmed this finding by co-immunoprecipitation. While application of TGF-beta increased neurite regeneration in a scratch lesion model, downregulation of EFABP by siRNA significantly decreased this effect. We thus postulate that a differential regulation of proteasomal function, as demonstrated for TGF-beta, can result in an enrichment of proteins, such as EFABP, that mediate physiological functions, such as neurite regeneration.

Invasive aspergillosis caused by the mould Aspergillus fumigatus is a life-threatening lung or systemic infection in the immunocompromised host. In this study, a protective immune response against the disease was achieved in two infected rabbits, and the cellular fungal antigenic proteome that mediated such a response was investigated against the background of vaccine development efforts. Altogether, 59 different Aspergillus proteins were found becoming reactive in the course of the developing immunity, many of which are described in this context for the first time. These included proteins related to oxidative stress management, glycolysis and other metabolic pathways. As oxidative stress is suspected to be one of the major defense mechanisms, the results may indicate at least in part a continuous response of the pathogen to evade the host's immune system. In addition, proteins with suspected cell surface association or crucial function for fungal cell development were identified. As these antigens are newly recognized during the process of the developing immunoprotection, they may not only represent valuable infection markers but also importantly broaden the list of possible vaccine candidates.

Methotrexate was first introduced as a cytotoxic agent that inhibits nucleotide biosynthesis in various cancer disorders; its molecular mechanism remains elusive. To understand the molecular mechanism by which methotrexate induces apoptosis, we analyzed the resulting intracellular protein changes in methotrexate-treated acute promyelocytic leukaemia (HL-60) cells by cysteine-labeled differential in-gel electrophoresis (CL-DIGE) combined with mass spectrometry. Initial CL-DIGE analysis revealed that 24 proteins were differentially expressed (p0.05) in the HL-60 cell proteome after treatment with 2.5microM methotrexate for 72h. We found that three structural alpha4, alpha5, alpha7 proteasome subunits, a non-catalytic beta3 and two 26S regulatory proteasome subunits were down-regulated in methotrexate-treated HL-60 cells. Western blot analyses further showed that the inhibition of proteasome subunits is accompanied by suppression of NF-kappaB subunits and promotes the accumulation of ubiquitinated proteins. Furthermore, methotrexate activated unfolded protein response by inducing the expression of endoplasmic reticulum-resident proteins such as calreticulin, protein disulphide isomerase A3 and A4, and 78kDa glucose regulated protein in a time-dependent manner. Altogether, our findings demonstrated that targeting NF-kappaB, structural and regulatory proteasome subunits with methotrexate may provide new insight into understanding methotrexate-induced apoptotic activities in HL-60 cells.

Background: Identification of markers for prediction of the clinical course of diabetic nephropathy remains a major challenge in disease management. We established a proteomics approach for identification of diabetic nephropathy-related biomarkers in urine. Methods: We used SELDI-TOF mass spectrometry and SAX2 protein arrays to compare protein profiles from urine of 4 defined patient groups. Samples from patients with type 2 diabetes (DM; n = 45) without nephropathy and without microalbuminuria (DM-WNP), patients with DM with macro- or microalbuminuria (DM-NP; n = 38), patients with proteinuria due to nondiabetic renal disease (n = 34), and healthy controls (n = 45) were analyzed. Anionic exchange, reversed-phase fractionation, gel electrophoresis, and mass spectrometry were used to isolate and identify proteins with high discriminatory power. Results: A protein with m/z 6188 (P Conclusion: The release of high amounts of UbA52 in urine of DM-NP patients could serve as a diagnostic marker, whereas the lack of the short form of ubiquitin raises interesting questions about the pathophysiology.

Sex hormones influence the development of female (F) and male (M) specific traits and primarily affect the structure and function of gender-specific organs. Recent studies also indicated their important roles in regulating structure and/or function of nearly every tissue and organ in the mammalian body, including the kidneys, causing gender differences in a variety of characteristics. Clinical observations in humans and studies in experimental animals in vivo and in models in vitro have shown that renal structure and functions under various physiological, pharmacological, and toxicological conditions are different in M and F, and that these differences may be related to the sex-hormone-regulated expression and action of transporters in the apical and basolateral membrane of nephron epithelial cells. In this review we have collected published data on gender differences in renal functions, transporters and other related parameters, and present our own microarray data on messenger RNA expression for various transporters in the kidney cortex of M and F rats. With these data we would like to emphasize the importance of sex hormones in regulation of a variety of renal transport functions and to initiate further studies of gender-related differences in kidney structure and functions, which would enable us to better understand occurrence and development of various renal diseases, pharmacotherapy, and drug-induced nephrotoxicity in humans and animals.

Covalent binding of the acyl glucuronide (AcMPAG) metabolite of the immunosuppressant mycophenolic acid (MPA) to proteins is considered a possible initiating event for organ toxicity. Since the kidney is involved in the formation and excretion of AcMPAG, it can be hypothesized that this tissue may be exposed to relatively high concentrations of this metabolite and would, therefore, be a particularly suitable organ to investigate AcMPAG protein targets. In the present study we identified potential AcMPAG target proteins in kidney tissues from Wistar rats treated with mycophenolate mofetil (40 mg/kg/day over 21 days). Proteins were separated by 2-DE and covalent protein adducts were detected by Western blotting with an antibody specific for MPA/AcMPAG. The corresponding coomassie blue stained proteins from parallel gels were subjected to in-gel tryptic digestion and peptides were characterized on a Q-TOF Ultima Global. The protein targets were further verified by immunoprecipitation with anti-MPA/AcMPAG antibody to purify the modified proteins followed by 1-DE and MS analysis. Database searches revealed several AcMPAG target proteins that could be related to ultrastructural abnormalities, metabolic effects, and altered oxidative stress/detoxification responses. Predominately cytosolic proteins such as selenium binding protein, protein disulfide isomerase, aldehyde dehydrogenase, triosephosphate isomerase, and kidney aminoacylase were involved in adduct formation. Two cytoskeletal proteins tropomyosin 1 and 4 as well as the antioxidant proteins peroxiredoxin 3 and 6 were also targets of AcMPAG. Functional consequences from these protein modifications remain to be demonstrated.

For most cancers, the patient's prognosis improves dramatically if the disease is detected at an early stage. Although advancements in imaging technology have improved early detection, many cancers remain undetected until it is too late for curative intervention. We have established, for the first time, expression difference mapping analysis of whole cell proteins from renal cell carcinoma (RCC) cell lines using ProteinChip technology. A total of 20 different RCC cell lines were cultured in vitro directly on ProteinChip arrays for 24 h. Direct MS analysis of proteins from the attached cells showed identical protein profiles by all analysed RCC lines. Comparative on-chip analysis of isolated malignant cells from native tumour specimens revealed protein patterns highly similar to those from the continuous RCC lines. However, cultured primary cortex cells showed specific protein differences. Differential protein profiling of isolated cytosolic and enriched membrane fractions from the RCC lines revealed that the protein pattern of the membrane proteins included or were identical to those of the entire cells. Proteomics analysis of the chip-binding membrane fractions allowed the identification of three forms of galectin-1 as potential RCC marker. ProteinChip analysis with a bound-specific antibody certified that galectin-1 could be an RCC marker. Immunostaining methods confirmed the overexpression of galectin-1 in renal carcinoma in comparison to healthy tissue.

Although a physiological function of the cellular prion protein (PrP(c)) is still not fully clarified, a PrP(c)-mediated neuroprotection against hypoxic/ischemic insult is intriguing. After ischemic stroke prion protein knockout mice (Prnp(0/0)) display significantly greater lesions as compared to wild-type (WT) mice. Earlier reports suggested an interaction between the glycolytic enzyme lactate dehydrogenase (LDH) and PrP(c). Since hypoxic environment enhances LDH expression levels and compels neurons to rely on lactate as an additional oxidative substrate for energy metabolism, we examined possible differences in LDH protein expression in WT and Prnp(0/0) knockout models under normoxic/hypoxic conditions in vitro and in vivo, as well as in a HEK293 cell line. While no differences are observed under normoxic conditions, LDH expression is markedly increased after 60-min and 90-min of hypoxia in WT vs. Prnp(0/0) primary cortical neurons with concurrent less hypoxia-induced damage in the former group. Likewise, cerebral ischemia significantly increases LDH levels in WT vs. Prnp(0/0) mice with accompanying smaller lesions in the WT group. HEK293 cells overexpressing PrP(c) show significantly higher LDH expression/activity following 90-min of hypoxia as compared to control cells. Moreover, a cytoplasmic co-localization of LDH and PrP(c) was recorded under both normoxic and hypoxic conditions. Interestingly, an expression of monocarboxylate transporter 1, responsible for cellular lactate uptake, increases with PrP(c)-overexpression under normoxic conditions. Our data suggest LDH as a direct PrP(c) interactor with possible physiological relevance under low oxygen conditions.

Several important physiological functions are regulated by cortisol. Previously, we demonstrated the involvement of human organic anion transporter 3 (hOAT3) in cortisol release. In the present study, we investigated the influence of dehydroepiandrosterone sulfate (DHEA-S) and estrone sulfate on cortisol release in a human adrenocortical cell line (NCI-H295R) compared with forskolin stimulation. Additionally, we examined the impact of forskolin and DHEA-S on the expression of key enzymes in steroid biosynthesis and expression of hOAT3 and -4 in NCI-H295R cells. The cortisol release was increased 10-fold after 24-h incubation with DHEA-S, but incubation with estrone sulfate did not show any significant change in cortisol release. When cells were incubated with DHEA-S in the presence of forskolin, an additive influence of DHEA-S stimulation of cortisol was recorded over forskolin alone. The 24-h stimulation of NCI-H295R cells with forskolin increased the expression of steroidogenic acute regulatory protein (StAR), CYP17, CYP21A2, and CYP11A1, whereas only StAR mRNA expression was increased significantly by incubation with DHEA-S. Immunofluorescence analyses revealed strongly elevated expression of hOAT3 by forskolin as well as by DHEA-S stimulation. We conclude that the increased cortisol release of adrenocortical cells by DHEA-S and forskolin stimulation is probably due to high expression of the key enzymes of steroid biosynthesis and hOAT3.

The secreted proteolytic activity of Aspergillus fumigatus is of potential importance as a virulence factor and in the industrial hydrolysis of protein sources. The A. fumigatus genome contains sequences that could encode a five-member gene family that produces proteases in the sedolisin family (MEROPS S53). Four putative secreted sedolisins with a predicted 17- to 20-amino-acid signal sequence were identified and termed SedA to SedD. SedA produced heterologously in Pichia pastoris was an acidic endoprotease. Heterologously produced SedB, SedC, and SedD were tripeptidyl-peptidases (TPP) with a common specificity for tripeptide- p -nitroanilide substrates at acidic pHs. Purified SedB hydrolyzed the peptide Ala-Pro-Gly-Asp-Arg-Ile-Tyr-Val-His-Pro-Phe to Arg-Pro-Gly, Asp-Arg-Ile, and Tyr-Val-His-Pro-Phe, thereby confirming TPP activity of the enzyme. SedB, SedC, and SedD were detected by Western blotting in culture supernatants of A. fumigatus grown in a medium containing hemoglobin as the sole nitrogen source. A degradation product of SedA also was observed. A search for genes encoding sedolisin homologues in other fungal genomes indicates that sedolisin gene families are widespread among filamentous ascomycetes.

Aspergillus fumigatus is a mold causing most of the invasive fungal lung infections in the immunocompromised host. In addition, the species is the causative agent of certain allergic diseases. Both in invasive and in allergic diseases, the conidial surface mediates the first contact with the human immune system. Thus, conidial surface proteins may be reasonable vaccine candidates as well as important allergens. To broaden the list of those antigens, intact viable Aspergillus conidia were extracted with mild alkaline buffer at pH 8.5 in the presence of a 1,3-beta-glucanase. The proteome of this fraction was separated by two- dimensional gel electrophoresis (2-DE) and analyzed by liquid chromatography coupled with tandem mass spectrometry. Altogether 26 different A. fumigatus proteins were identified, twelve of which contain a signal for secretion. Among these were the known major conidial surface protein rodlet A, one acid protease PEP2, one lipase, a putative disulfide isomerase and a putative fructose-1,6-biphosphatase. The known allergen Aspf 3 was identified among the proteins without a signal for secretion. On the basis of the recently annotated A. fumigatus genome (Nature 2005, 438, 1151-1156), proteome analysis is now a powerful tool to confirm expression of hypothetical proteins and, thereby to identify additional vaccine candidates and possible new allergens of this important fungal pathogen.

Epithelial cells of the thick ascending limb of Henle's loop (TALH cells) play a major role in the urinary concentrating mechanism. They are normally exposed to variable and often very high osmotic stress, which is particularly due to high sodium and chloride reabsorption and very low water permeability of the luminal membrane. It is already established that elevation of the activity of aldose reductase and hence an increase in intracellular sorbitol are indispensable for the osmotic adaptation and stability of the TALH cells. To identify new molecular factors potentially associated with the osmotic stress-resistant phenotype in kidney cells, TALH cells exhibiting low or high levels of resistance to osmotic stress were characterized using proteomic tools. Two-dimensional gel analysis showed a total number of 40 proteins that were differentially expressed in TALH cells under osmotic stress. Twenty-five proteins were overexpressed, whereas 15 proteins showed a down-regulation. Besides the sorbitol pathway enzyme aldose reductase, whose expression was 15 times increased, many other metabolic enzymes like glutathione S-transferase, malate dehydrogenase, lactate dehydrogenase, alpha enolase, glyceraldehyde-3-phosphate dehydrogenase, and triose-phosphate isomerase were up-regulated. Among the cytoskeleton proteins and cytoskeleton-associated proteins vimentin, cytokeratin, tropomyosin 4, and annexins I, II, and V were up-regulated, whereas tubulin and tropomyosins 1, 2, and 3 were down-regulated. The heat shock proteins alpha-crystallin chain B, HSP70, and HSP90 were found to be overexpressed. In contrast to the results in oxidative stress the endoplasmic reticulum stress proteins like glucose-regulated proteins (GRP78, GRP94, and GRP96), calreticulin, and protein-disulfide isomerase were down-regulated under hypertonic stress.

Since the organic anion transporter-1 (OAT1) has been implicated in cortisol release from bovine and rat adrenal zona fasciculata cells, we addressed the question of whether OATs are present in human adrenal cortical cells. In the human adrenal cell line NCI-H295R, 24-h cortisol secretion increased up to 30-fold on exposure to forskolin. Incubation of forskolin-treated cells for 24 h with the OAT substrates probenecid, p-aminohippurate (PAH), glutarate or cimetidine inhibited cortisol release partly. RT-PCR did not reveal expression of human OAT1 and OAT2, but OAT3 and OAT4 mRNAs were detected in both NCI-H295R cells and human adrenal tissue. When human OAT3 (hOAT3) and hOAT4 were expressed in Xenopus laevis oocytes, only hOAT3 showed [3H]cortisol uptake in excess of that of water-injected control oocytes. Cortisol uptake via OAT3 was saturable with an apparent Kt of 2.4 microM. In NCI-H295R cells, [3H]estrone sulphate uptake was saturable, cis-inhibited by OAT substrates and trans-stimulated by preloading with glutarate or cortisol. Likewise, [3H]PAH uptake was cis-inhibited by estrone sulphate and trans-stimulated by preloading the cells with PAH, glutarate or cortisol, indicating functional expression of OATs in the plasma membrane of NCI-H295R cells.

Human gastrointestinal tract is covered by a monolayer of specialized epithelial cells that constitute a protective barrier surface to external toxic and infectious agents along with metabolic and digestive functions. Intercellular junctions, among epithelial cells, such as desmosomes, adherens, gap, and tight junctions (TJs), not only provide mechanical integrity but also limit movement of molecules across the monolayer. TJ is a complex structure composed of approximately 35 different proteins that interact with each other at the apical side of two adjacent epithelial cells. Claudin family proteins are important members of TJ with so far 24 known isoforms in different species. Claudins are structural proteins of TJ that help to control the paracellular movement by forming fence and barrier across the epithelial monolayer. Altered function of claudins is implicated in different form of cancers, inflammatory bowel diseases (IBDs), and leaky diarrhea. Based on their significant role in the molecular architecture of TJ, diversity, and disease association, further understanding about claudin family proteins and their genetic/epigenetic regulators is indispensable.