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Abstract 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.

Abstract 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.

Deep learning (DL) algorithms have shown incredible potential in many applications. The success of these data-hungry methods is largely associated with the availability of large-scale datasets, as millions of observations are often required to achieve acceptable performance levels. Recently, there has been an increased interest in applying deep learning methods to geophysical applications where electromagnetic methods are used to map the subsurface geology by observing variations in the electrical resistivity of the subsurface materials. To date, there are no standardized datasets for electromagnetic methods, which hinders the progress, evaluation, benchmarking, and evolution of deep learning algorithms due to data inconsistency. Therefore, we present a large-scale electrical resistivity model database (RMD) with a wide variety of geologically plausible and geophysically resolvable subsurface structures for the commonly deployed ground-based and airborne electromagnetic systems. Potentially, the presented database can be used to build surrogate models of well-known processes and to aid in labour-intensive tasks. The geophysically constrained property of this database will not only achieve enhanced performance and improved generalization but, more importantly, incorporate consistency and credibility into deep learning models. We show the effectiveness of the presented database by surrogating the forward-modelling process, and we urge the geophysical community interested in deep learning for electromagnetic methods to utilize the presented database. The dataset is publicly available at https://doi.org/10.5281/zenodo.7260886 (Asif et al., 2022a).

Abstract Emergence of plasmid mediated colistin and extended spectrum β-lactamases (ESBL) resistant genes has been impacted the efficacy of colistin and β-lactams drugs like 3rd, 4th generation cephalosporin. Current study was aimed to investigate antimicrobial resistance genes (ARGs) among Escherichia coli isolates from meat producing commercial broilers in Pakistan. Two hundred (n=200) fecal samples were collected during January-2018 to August-2019. For isolation of E. coli, pink colonies on MacConkey agar were transferred to EMB agar. Metallic sheen color colonies were tested biochemically using API-20E kit. The molecular identification of E. coli (n=153) was targeted by amplification of uid gene through polymerase chain reaction (PCR) and different ARGs i.e. gentamicin, streptomycin, tetracycline, colistin, β-lactams drugs, quinolone and ampicillin followed by sequence analysis. Genotypically, followed by phenotypically of resistant ARGs of isolated PCR-confirmed E. coli (153) shoed resistant against gentamicin (aac(3)-IV), streptomycin (aadA1), tetracycline (tetA), colistine (mcr-1), ampicillin (bla-TEM) and bla-CTX-M were 86%, 88%, 86%, 88%, 83% & 77% respectively. 33/38 (86%) of the isolate was positive for quinolone resistance. Colistine (mcr-1), ESBLs (bla-TEM) and (bla-CTX-M) resistance genes were 88%, 83% and 77% respectively. About 33 isolated E. coli harbored the both mcr-1 and ESBLs genes. All of E. coli isolates were found sensitive to ceftriaxone (CTX-30) and imipenem (IMP-10). The Isolated E. coli showed single or multi clade decadency. The E. coli and ARGs sequences showed single or multi clade decadency. This is first comprehensive study from Pakistan that described the molecular evidences of ARGs and their co-existence in single isolates originated from commercial poultry. Commercial chicken (Broilers) can act as melting pot of antibiotic resistance genes for human being. It is alarming situation for surveillance of antibiotic resistance program because of more regulated prescription of antimicrobial agents in Pakistan

NF-κB signaling is a key regulator of inflammation and atherosclerosis. NF-κB cooperates with bromodomain-containing protein 4 (BRD4), a transcriptional and epigenetic regulator, in endothelial inflammation. This study aimed to investigate whether BRD4 inhibition would prevent the proinflammatory response towards TNF-α in endothelial cells. We used TNF-α treatment of human umbilical cord-derived vascular endothelial cells to create an in vitro inflammatory model system. Two small molecule inhibitors of BRD4—namely, RVX208 (Apabetalone), which is in clinical trials for the treatment of atherosclerosis, and JQ1—were used to analyze the effect of BRD4 inhibition on endothelial inflammation and barrier integrity. BRD4 inhibition reduced the expression of proinflammatory markers such as SELE, VCAM-I, and IL6 in endothelial cells and prevented TNF-α-induced endothelial tight junction hyperpermeability. Endothelial inflammation was associated with increased expression of the heparin-binding growth factor midkine. BRD4 inhibition reduced midkine expression and normalized endothelial permeability upon TNF-α treatment. In conclusion, we identified that TNF-α increased midkine expression and compromised tight junction integrity in endothelial cells, which was preventable by pharmacological BRD4 inhibition.

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.

A major drawback in deploying beamforming scheme in orthogonal frequency division multiplexing (OFDM) is to obtain the optimal weights that are associated with information beams. Two beam weighting methods, namely co-phasing and singular vector decomposition (SVD), are considered to maximize the signal beams for such beamforming scheme. Initially the system performance with and without interleaving is investigated using coded fast Fourier transform (FFT)-OFDM and wavelet-based OFDM. The two beamforming schemes are applied to the wavelet-based OFDM as confirmed to perform better than the FFT-OFDM. It is found that the beam-weight by SVD improves the performance of the system by about 2dB at the expense of the co-phasing method. The capacity performances of the weighting methods are also compared and discussed.

The present study was undertaken to identify proteins that interact with the mammalian target of rapamycin complex 1 (mTORC1) to enable it to carry out its crucial cell signaling functions. Endogenous and myc-tag mTORC1 was purified, in-gel tryptic digested and then identified by nano-LC ESI Q-TOF MS/MS analysis. A total of nine novel interacting proteins were identified in both endogenous and myc-tag mTORC1 purifications. These new mTORC1 interacting partners include heterogeneous nuclear ribonucleoproteins A2/B1, enhancer of mRNA decapping protein 4, 60S acidic ribosomal protein, P0, nucleolin, dynamin 2, glyceraldehyde 3 phosphate dehydrogenase, 2-oxoglutarate dehydrogenase, glycosyl transferase 25 family member 1 and prohibitin 2. Furthermore hnRNP A2/B1 and dynamin 2 interaction with mTORC1 was confirmed on immunoblotting. The present study has for the first time identified novel interacting partners of mTORC1 in human T lymphoblasts (CCRF-CEM) and human embryonic kidney (HEK293) cells. These new interacting proteins may offer new targets for therapeutic interventions in human diseases caused by perturbed mTORC1 signaling.

Background: Mycophenolic acid (MPA) is an important immunosuppressive drug (ISD) prescribed to prevent graft rejection in the organ transplanted patients, however, its use is also associated with adverse side effects like sporadic gastrointestinal (GI) disturbances. Recently, we reported the MPA induced tight junctions (TJs) deregulation which involves MLCK/MLC-2 pathway. Here, we investigated the global histone acetylation as well as gene-specific chromatin signature of several genes associated with TJs regulation in Caco-2 cells after MPA treatment.Results: The epigenetic analysis shows that MPA treatment increases the global histone acetylation levels as well as the enrichment for transcriptional active histone modification mark (H3K4me3) at promoter regions of p38MAPK, ATF-2, MLCK, and MLC-2. In contrast, the promoter region of occludin was enriched for transcriptional repressive histone modification mark (H3K27me3) after MPA treatment. In line with the chromatin status, MPA treatment increased the expression of p38MAPK, ATF-2, MLCK, and MLC-2 both at transcriptional and translational level, while occludin expression was negatively influenced. Interestingly, the MPA induced gene expression changes and functional properties of Caco-2 cells could be blocked by the inhibition of p38MAPK using a chemical inhibitor (SB203580).Conclusions: Collectively, our results highlight that MPA disrupts the structure of TJs via p38MAPK-dependent activation of MLCK/MLC-2 pathway that results in decreased integrity of Caco-2 monolayer. These results led us to suggest that p38MAPK-mediated lose integrity of epithelial monolayer could be the possible cause of GI disturbance (barrier dysfunction) in the intestine, leading to leaky style diarrhea observed in the organ-transplanted patients treated with MPA.

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-β), a multifunctional cytokine and trophic factor for dopaminergic (DAergic) neurons modulates proteasome function in primary midbrain neurons. TGF-β 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-β 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-β treatment and confirmed this finding by co-immunoprecipitation. While application of TGF-β 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-β, can result in an enrichment of proteins, such as EFABP, that mediate physiological functions, such as neurite regeneration.

Thiopurines are extensively used immunosuppressants for the treatment of inflammatory bowel disease (IBD). The polymorphism of thiopurine S-methyltransferase (TPMT) influences thiopurine metabolism and therapy outcome. We used a TPMT knockdown (kd) model of human Jurkat T-lymphocytes cells to study the effects of treatment with 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) on proteome and phosphoproteome. We identified thirteen proteins with altered expression and nine proteins with altered phosphorylation signals. Three proteins (THIO, TXD17, and GSTM3) with putative functions in cellular oxidative stress responses were altered by 6-TG treatment and another protein PRDX3 was differentially phosphorylated in TPMT kd cells. Furthermore, reactive oxygen species (ROS) assay results were consistent with a significant induction of oxidative stress by both TPMT knockdown and thiopurine treatments. Immunoblot analyses showed treatment altered expression of key antioxidant enzymes (i.e., SOD2 and catalase) in both wt and kd groups, while SOD1 was downregulated by 6-TG treatment and TPMT knockdown. Collectively, increased oxidative stress might be a mechanism involved in thiopurine induced cytotoxicity and adverse effects (i.e., hepatotoxicity) and an antioxidant cotherapy might help to combat this. Results highlight the significance of oxidative stress in thiopurines’ actions and could have important implications for the treatment of IBD patients.

Rheumatoid arthritis (RA) is a chronic, autoimmune and inflammatory joint disease with a poorly understood etiology. Despite widespread diagnostic use of anti-citrullinated protein antibodies and rheumatoid factor proteins there is a strong demand for novel serological biomarkers to improve the diagnosis this disease. The present study was aimed to identify novel autoantigens involved in rheumatoid arthritis (RA) pathogenesis through immune-proteomic strategy. Synovial fluid samples from clinically diagnosed RA patients were separated on two-dimensional gel electrophoresis (2-DE). Samples from patients with non-RA rheumatisms (osteoarthritis and trauma) were used as controls. Immunoreactive proteins were spotted by Western blotting followed by identification through Q-TOF mass spectrometer analysis. Forty Western blots were generated using plasma from ten individual RA patients and 33 reactive spots were identified, 20 from the high molecular weight (HMW) gel and 13 from the low molecular weight (LMW) gel. Among the 33 common immunogenic spots, 18 distinct autoantigens were identified, out of which 14 are novel proteins in this context. Expression analysis of five important proteins, vimentin, gelsolin, alpha 2 HS glycoprotein (AHSG), glial fibrillary acidic protein (GFAP), and α1B-glycoprotein (A1BG) by Western blot analysis using their specific antibodies revealed their higher expression in RA synovial fluid as compared to non-RA samples. Recombinantly expressed GFAP and A1BG protein were used to develop an in-house ELISA to quantify the amount of autoantibodies in the RA patients. RA patients revealed an increase in the expression of GFAP and A1BG in the plasma as compared to osteoarthritis patients. Therefore, GFAP and A1BG can be proposed as potential new autoantigens of diagnostic importance for RA subjects. Further characterization of these proteins in rheumatoid arthritis will be helpful in understanding the role of these proteins in the disease pathogenesis providing new diagnostic tool with better specificity and accurate detection of the disease.

Abstract Background The effects of fetal calf serum (FCS) heat inactivation and bacterial lipopolysaccharide (LPS) contamination on cell physiology have been studied, but their effect on the proteome of cultured cells has yet to be described. This study was undertaken to investigate the effects of heat inactivation of FCS and LPS contamination on the human T lymphoblast proteome. Human T lymphoblastic leukaemia (CCRF-CEM) cells were grown in FCS, either non-heated, or heat inactivated, having low (< 1 EU/mL) or regular (< 30 EU/mL) LPS concentrations. Protein lysates were resolved by 2-DE followed by phospho-specific and silver nitrate staining. Differentially regulated spots were identified by nano LC ESI Q-TOF MS/MS analysis. Results A total of four proteins (EIF3M, PRS7, PSB4, and SNAPA) were up-regulated when CCRF-CEM cells were grown in media supplemented with heat inactivated FCS (HE) as compared to cells grown in media with non-heated FCS (NHE). Six proteins (TCPD, ACTA, NACA, TCTP, ACTB, and ICLN) displayed a differential phosphorylation pattern between the NHE and HE groups. Compared to the low concentration LPS group, regular levels of LPS resulted in the up-regulation of three proteins (SYBF, QCR1, and SUCB1). Conclusion The present study provides new information regarding the effect of FCS heat inactivation and change in FCS-LPS concentration on cellular protein expression, and post-translational modification in human T lymphoblasts. Both heat inactivation and LPS contamination of FCS were shown to modulate the expression and phosphorylation of proteins involved in basic cellular functions, such as protein synthesis, cytoskeleton stability, oxidative stress regulation and apoptosis. Hence, the study emphasizes the need to consider both heat inactivation and LPS contamination of FCS as factors that can influence the T lymphoblast proteome.

Abstract 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.

Abstract Cancer is a fatal malignancy and its increasing worldwide prevalence demands the discovery of more sensitive and reliable molecular biomarkers. To investigate the GINS1 expression level and its prognostic value in distinct human cancers using a series of multi-layered in silico approach may help to establish it as a potential shared diagnostic and prognostic biomarker of different cancer subtypes. The GINS1 mRNA, protein expression, and promoter methylation were analyzed using UALCAN and Human Protein Atlas (HPA), while mRNA expression was further validated via GENT2. The potential prognostic values of GINS1 were evaluated through KM plotter. Then, cBioPortal was utilized to examine the GINS1-related genetic mutations and copy number variations (CNVs), while pathway enrichment analysis was performed using DAVID. Moreover, a correlational analysis between GINS1 expression and CD8+ T immune cells and a the construction of gene-drug interaction network was performed using TIMER, CDT, and Cytoscape. The GINS1 was found down-regulated in a single subtypes of human cancer while commonly up-regulated in 23 different other subtypes. The up-regulation of GINS1 was significantly correlated with the poor overall survival (OS) of Liver Hepatocellular Carcinoma (LIHC), Lung Adenocarcinoma (LUAD), and Kidney renal clear cell carcinoma (KIRC). The GINS1 was also found up-regulated in LIHC, LUAD, and KIRC patients of different clinicopathological features. Pathways enrichment analysis revealed the involvement of GINS1 in two diverse pathways, while few interesting correlations were also documented between GINS1 expression and its promoter methylation level, CD8+ T immune cells level, and CNVs. Moreover, we also predicted few drugs that could be used in the treatment of LIHC, LUAD, and KIRC by regulating the GINS1 expression. The expression profiling of GINS1 in the current study has suggested it a novel shared diagnostic and prognostic biomarker of LIHC, LUAD, and KIRC.

Chromatin is a dynamic structure comprising of DNA and proteins. Its unique nature not only help to pack the DNA tightly within the cell but also is pivotal in regulating gene expression DNA replication. Furthermore it also protects the DNA from being damaged. Various proteins are involved in making a specific complex within a chromatin and the knowledge about these interacting partners is helpful to enhance our understanding about the pathophysiology of various chromatin associated diseases. Moreover, it could also help us to identify new drug targets and design more effective remedies. Due to the existence of chromatin in different forms under various physiological conditions it is hard to develop a single strategy to study chromatin associated proteins under all conditions. In our current review, we tried to provide an overview and comparative analysis of the strategies currently adopted to capture the DNA bounded protein complexes and their mass spectrometric identification and quantification. Precise information about the protein partners and their function in the DNA-protein complexes is crucial to design new and more effective therapeutic molecules against chromatin associated diseases.

The mammalian target of rapamycin complex 1 (mTORC1) is involved in the cellular transcription and translation processes. The undertaken study characterized the enhancer of mRNA decapping protein 4 (Edc4) as mTORC1 interacting protein. Human T lymphoblast (CCRF-CEM) cells were used for mTORC1 purification. Co-immunoprecipitation coupled with immunoblotting analysis was used to confirm the interaction of Edc4 in mTORC1 specific purifications. Further assays were incorporated to conclude the role of mTORC1 in mRNA decapping via Edc4. Edc4 was identified as a new interacting protein with mTORC1 in both the endogenous and myc-tag raptor component mTORC1 specific purifications. Quantitative co-localization using confocal microscopy demonstrated that raptor component of mTORC1 coexists with Edc4 in processing (P) bodies, a site for mRNA degradation. Incubation of cells with rapamycin, a known inhibitor of mTOR kinase activity, increased the total Edc4 protein expression but at the same time decreased the Edc4 interaction with mTORC1. Moreover, rapamycin treatment resulted in a significant decrease in total serine phosphorylated Edc4 protein signal and the total 5'-capped mRNA. These findings provide the first evidence for the pivotal role of mTORC1 in Edc4 regulation. Further in-depth studies are required to get a complete understanding of molecular crosstalk between mTORC1 signaling and mRNA decapping pathway.

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.

Mass spectrometry-based proteomics methods are finding increasing use in structural biology research. Beyond simple interaction networks, information about stable protein-protein complexes or spatially proximal proteins helps to elucidate the biological functions of proteins in a wider cellular context. To shed light on new developments in this field, the Göttingen Proteomics Forum organized a one-day symposium focused on complexome profiling and proximity labeling, two emerging technologies that are gaining significant attention in biomolecular research. The symposium was held in Göttingen, Germany on 23 May, 2019, as part of a series of regular symposia organized by the Göttingen Proteomics Forum.

Upon publication of the original article [1], Marwa Eltoweissy noticed that her affiliation: “3. Department of Zoology, Faculty of Science, Alexandria University, Alexandria, Egypt” was missing. This affiliation has now been added in this correction article.

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.

Mycophenolic acid (MPA) is prescribed to maintain allografts in organ-transplanted patients. However, gastrointestinal (GI) complications, particularly diarrhea, are frequently observed as a side effect following MPA therapy. We recently reported that MPA altered the tight junction (TJ)-mediated barrier function in a Caco-2 cell monolayer model system. This study investigates whether MPA induces epigenetic changes which lead to GI complications, especially diarrhea. Methods: We employed a Chromatin Immunoprecipitation-O-Proteomics (ChIP-O-Proteomics) approach to identify proteins associated with active (H3K4me3) as well as repressive (H3K27me3) chromatin histone modifications in MPA-treated cells, and further characterized the role of midkine, a H3K4me3-associated protein, in the context of epithelial monolayer permeability. Results: We identified a total of 333 and 306 proteins associated with active and repressive histone modification marks, respectively. Among them, 241 proteins were common both in active and repressive chromatin, 92 proteins were associated exclusively with the active histone modification mark, while 65 proteins remained specific to repressive chromatin. Our results show that 45 proteins which bind to the active and seven proteins which bind to the repressive chromatin region exhibited significantly altered abundance in MPA-treated cells as compared to DMSO control cells. A number of novel proteins whose function is not known in bowel barrier regulation were among the identified proteins, including midkine. Our functional integrity assays on the Caco-2 cell monolayer showed that the inhibition of midkine expression prior to MPA treatment could completely block the MPA-mediated increase in barrier permeability. Conclusions: The ChIP-O-Proteomics approach delivered a number of novel proteins with potential implications in MPA toxicity. Consequently, it can be proposed that midkine inhibition could be a potent therapeutic approach to prevent the MPA-mediated increase in TJ permeability and leak flux diarrhea in organ transplant patients.

The identification of new biomarkers is of high interest for the prediction of the disease course and also for the identification of pathomechanisms in multiple sclerosis (MS). To specify markers of the chronic disease phase, we performed proteome profiling during the later phase of myelin oligodendrocyte glycoprotein induced experimental autoimmune encephalomyelitis (MOG-EAE, day 35 after immunization) as a model disease mimicking many aspects of secondary progressive MS. In comparison to healthy controls, high resolution 2 dimensional gel electrophoresis revealed a number of regulated proteins, among them glial fibrilary acidic protein (GFAP). Phase specific up-regulation of GFAP in chronic EAE was confirmed by western blotting and immunohistochemistry. Protein levels of GFAP were also increased in the cerebrospinal fluid of MS patients with specificity for the secondary progressive disease phase. In a next step, proteome profiling of an EAE model with enhanced degenerative mechanisms revealed regulation of alpha-internexin, syntaxin binding protein 1, annexin V and glutamate decarboxylase in the ciliary neurotrophic factor (CNTF) knockout mouse. The identification of these proteins implicate an increased apoptosis and enhanced axonal disintegration and correlate well the described pattern of tissue injury in CNTF -/- mice which involve oligodendrocyte (OL) apoptosis and axonal injury.In summary, our findings underscore the value of proteome analyses as screening method for stage specific biomarkers and for the identification of new culprits for tissue damage in chronic autoimmune demyelination.