Genetic heterogeneity in epilepsy and comorbidities: insights from Pakistani families.

BACKGROUND: Epilepsy, a challenging neurological condition, is often present with comorbidities that significantly impact diagnosis and management. In the Pakistani population, where financial limitations and geographical challenges hinder access to advanced diagnostic methods, understanding the genetic underpinnings of epilepsy and its associated conditions becomes crucial. METHODS: This study investigated four distinct Pakistani families, each presenting with epilepsy and a spectrum of comorbidities, using a combination of whole exome sequencing (WES) and Sanger sequencing. The epileptic patients were prescribed multiple antiseizure medications (ASMs), yet their seizures persist, indicating the challenging nature of ASM-resistant epilepsy. RESULTS: Identified genetic variants contributed to a diverse range of clinical phenotypes. In the family 1, which presented with epilepsy, developmental delay (DD), sleep disturbance, and aggressive behavior, a homozygous splice site variant, c.1339-6 C > T, in the COL18A1 gene was detected. The family 2 exhibited epilepsy, intellectual disability (ID), DD, and anxiety phenotypes, a homozygous missense variant, c.344T > A (p. Val115Glu), in the UFSP2 gene was identified. In family 3, which displayed epilepsy, ataxia, ID, DD, and speech impediment, a novel homozygous frameshift variant, c.1926_1941del (p. Tyr643MetfsX2), in the ZFYVE26 gene was found. Lastly, family 4 was presented with epilepsy, ID, DD, deafness, drooling, speech impediment, hypotonia, and a weak cry. A homozygous missense variant, c.1208 C > A (p. Ala403Glu), in the ATP13A2 gene was identified. CONCLUSION: This study highlights the genetic heterogeneity in ASM-resistant epilepsy and comorbidities among Pakistani families, emphasizing the importance of genotype-phenotype correlation and the necessity for expanded genetic testing in complex clinical cases.

Molecular characterization of plasma virome of hepatocellular carcinoma (HCC) patients.

Hepatocellular carcinoma (HCC) stands as the most common cancer type, arising from various causes, and responsible for a substantial number of cancer-related fatalities. Recent advancements in viral metagenomics have empowered scientists to delve into the intricate diversity of the virosphere, viral evolution, interactions between viruses and their hosts, and the identification of viral causes behind disease outbreaks, the development of specific symptoms, and their potential role in altering the host's physiology. The present study had the objective of "Molecular Characterization of HBV, HCV, anelloviruses, CMV, SENV-D, SENV-H, HEV, and HPV viruses among individuals suffering from HCC." A total of 381 HCC patients contributed 10 cc of blood each for this study. The research encompassed the assessment of tumor markers, followed by molecular characterization of HBV, HCV, Anelloviruses (TTV, TTMV, and TTMDV), SENV-H and SENV-D viruses, HEV, CMV, and HPV, as well as histopathological examinations. The outcomes of this study revealed that majority of the HCC patients 72.4% (276/381) were male as compared to females. HCV infection, at 76.4% (291 out of 381), exhibited a significant association (p < 0.05) with HCC. Most patients displayed singular lesions in the liver, with Child Pugh Score Type B being the predominant finding in 45.2% of cases. Plasma virome analysis indicated the prevalence of TTMDV (75%), followed by TTMV (70%) and TTV (42.1%) among anelloviruses in HCC patients. Similarly, SENV-H (52%) was followed by SENV-D (20%), with co-infections at 15%. The presence of CMV and HEV among the HCC patients was recorded 5% each however 3.5% of the patients showed the presence of HPV. In conclusion, this study underscores that HCC patients serve as reservoirs for various pathogenic and non-pathogenic viruses, potentially contributing to the development, progression, and severity of the disease.

Structural and functional implications of SLC13A3 and SLC9A6 mutations: an in silico approach to understanding intellectual disability.

BACKGROUND: Intellectual disability (ID) is a condition that varies widely in both its clinical presentation and its genetic underpinnings. It significantly impacts patients' learning capacities and lowers their IQ below 70. The solute carrier (SLC) family is the most abundant class of transmembrane transporters and is responsible for the translocation of various substances across cell membranes, including nutrients, ions, metabolites, and medicines. The SLC13A3 gene encodes a plasma membrane-localized Na+/dicarboxylate cotransporter 3 (NaDC3) primarily expressed in the kidney, astrocytes, and the choroid plexus. In addition to three Na + ions, it brings four to six carbon dicarboxylates into the cytosol. Recently, it was discovered that patients with acute reversible leukoencephalopathy and a-ketoglutarate accumulation (ARLIAK) carry pathogenic mutations in the SLC13A3 gene, and the X-linked neurodevelopmental condition Christianson Syndrome is caused by mutations in the SLC9A6 gene, which encodes the recycling endosomal alkali cation/proton exchanger NHE6, also called sodium-hydrogen exchanger-6. As a result, there are severe impairments in the patient's mental capacity, physical skills, and adaptive behavior. METHODS AND RESULTS: Two Pakistani families (A and B) with autosomal recessive and X-linked intellectual disorders were clinically evaluated, and two novel disease-causing variants in the SLC13A3 gene (NM 022829.5) and the SLC9A6 gene (NM 001042537.2) were identified using whole exome sequencing. Family-A segregated a novel homozygous missense variant (c.1478 C > T; p. Pro493Leu) in the exon-11 of the SLC13A3 gene. At the same time, family-B segregated a novel missense variant (c.1342G > A; p.Gly448Arg) in the exon-10 of the SLC9A6 gene. By integrating computational approaches, our findings provided insights into the molecular mechanisms underlying the development of ID in individuals with SLC13A3 and SLC9A6 mutations. CONCLUSION: We have utilized in-silico tools in the current study to examine the deleterious effects of the identified variants, which carry the potential to understand the genotype-phenotype relationships in neurodevelopmental disorders.

Unravelling the genetic basis of retinal dystrophies in Pakistani consanguineous families.

BACKGROUND: Retinitis Pigmentosa (RP) is a clinically and genetically progressive retinal dystrophy associated with severe visual impairments and sometimes blindness, the most common syndromic form of which is Usher syndrome (USH). This study aimed to further increase understanding of the spectrum of RP in the Khyber Pakhtunkhwa region of Pakistan. METHODOLOGY: Four consanguineous families of Pashtun ethnic group were investigated which were referred by the local collaborating ophthalmologists. In total 42 individuals in four families were recruited and investigated using whole exome and dideoxy sequencing. Among them, 20 were affected individuals including 6 in both family 1 and 2, 5 in family 3 and 3 in family 4. RESULT: Pathogenic gene variants were identified in all four families, including two in cone dystrophy and RP genes in the same family (PDE6C; c.480delG, p.Asn161ThrfsTer33 and TULP1; c.238 C > T, p.Gln80Ter) with double-homozygous individuals presenting with more severe disease. Other pathogenic variants were identified in MERTK (c.2194C > T, p.Arg732Ter), RHO (c.448G > A, p.Glu150Lys) associated with non-syndromic RP, and MYO7A (c.487G > A, p.Gly163Arg) associated with USH. In addition, the reported variants were of clinical significance as the PDE6C variant was detected novel, whereas TULP1, MERTK, and MYO7A variants were detected rare and first time found segregating with retinal dystrophies in Pakistani consanguineous families. CONCLUSIONS: This study increases knowledge of the genetic basis of retinal dystrophies in families from Pakistan providing information important for genetic testing and diagnostic provision particularly from the Khyber Pakhtunkhwa region.

Maternal FMR1 alleles expansion in newborns during transmission: a prospective cohort study.

INTRODUCTION: The CGG repeats in the 5' untranslated region of the fragile X mental retardation 1 gene (FMR1) gene shows increased instability upon maternal transmission. Maternal FMR1 intermediate (45-54 repeats) and premutation (PM: 55-<200 repeats) alleles usually expand to full mutation (>200 repeats) alleles in offspring and consequently, cause fragile X syndrome (FXS) in them. METHODS: In a prospective cohort study, Pakistani pregnant women in prenatal care were first screened for FMR1 expanded alleles. In the follow-up, pregnancy outcomes in women carrying FMR1 expanded alleles were recorded and their newborn offspring were also screened for FXS. RESULTS: In a total of 1950 pregnant women, 89 (4.6%) were detected carriers for FMR1 expanded alleles; however, rates of detection of expanded alleles were found significantly high in women with a history of FXS. In addition, miscarriages and birth of affected newborns with FXS were significantly more common in women carrying large size PM alleles and had a history of FXS (P = 0.0494 and P = 0.0494, respectively). CONCLUSIONS: The current study provides the first evidence of screening Pakistani pregnant women for FMR1 expanded alleles in prenatal care. Moreover, the miscarriage was also detected as a clinical predictor for FXS. IMPACT: Offspring would have a higher risk of developing FXS due to maternal FMR1 alleles expansions during transmission. This is the first prospective cohort study in Pakistan for finding FMR1 allelic status of pregnant women and their newborn offspring in follow-up. The robust offspring risk for FXS estimated in this study may be valuable information for genetic counseling of women carriers for FMR1 expanded alleles. The family history and miscarriage were detected as effective indicators for FXS carrier screening in Pakistani women.

Fragile X premutation carrier screening in Pakistani preconception women in primary care consultation.

PURPOSE: Women of reproductive age who carry fragile X premutation (PM) alleles have 56 to 200 CGG repeats in the 5'-untranslated region of FMR1 gene are at increased risk for producing children with intellectual disabilities (ID) or autism spectrum disorders (ASD) due to expansion of PM alleles to full mutation alleles (> 200 repeats) during maternal transmission. METHODS: In present study fragile X PM carrier screening was performed in total 808 women who were consulting primary health care centers for preconception care in Khyber Pakhtunkhwa region of Pakistan between April, 2018 and December, 2020. Polymerase chain reaction (PCR) was performed for detection of PM carrier women and the CGG repeats number was confirmed by Southern blotting and capillary electrophoresis. RESULTS: The prevalence rate for PM carriers among preconception women was found to be 0.7% that was contributed by 0.5% women in risk group (RG1) with family history of ID and 0.2% in risk group 2 (RG2) with family history of ASD. PM carrier women had at least one affected child or sibling. In addition, the preconception women with FMR1 PM alleles were found to be at increased risk for primary ovary insufficiency (RG1: P = 0.0265, RG2: P = 0.0389), postpartum depression (RG1: P = 0.0240, RG2: P = 0.0501) and neuropsychiatric disorders (RG1: P = 0.0389, RG2: P = 0.0432). CONCLUSIONS: Current study provides first evidence of fragile X PM carrier screening in Pakistani preconception women in primary care consultation. Findings of current study may help to improve preconception care and to reduce burden of fragile X associated disorders in our population.

Salmonella Typhi from Northwest Pakistan: Molecular Strain Typing and Drug Resistance Signature.

Objective: To determine the molecular strain typing and drug resistance pattern of Salmonella enterica serovar Typhi prevalent in Northwest Pakistan. Methodology: A total of 2,138 blood samples of suspected typhoid patients from Northwest Pakistan were collected followed by identification of Salmonella Typhi through biochemical, serological, and species-specific fliC-d gene amplification. These isolates were typed by variable-number tandem repeat (VNTR) profiling and investigated for drug resistance. Results: The overall prevalence of Salmonella Typhi was found to be 8.8% (n = 189). Thirty different VNTR strain types of Salmonella Typhi were detected and the most prevalent strain types were T1 and T4, whereas T27 was less prevalent strain. Among the 189 isolates 175 (92.5%) isolates were multidrug resistant, whereas 12 (5.8%) isolates were extensively drug resistant. Resistance to imipenem in Salmonella Typhi was not observed. Most of the isolates have genes encoding for resistance to fluoroquinolones, including gyrA (n = 164), gyrB (n = 160), parC (n = 164), parE (n = 160), ac(6')-ib-cr (n = 163), qnrS (n = 15), and qnrB (n = 3). Similarly, chloramphinicol (cat; n = 147), azithromycin (msrA; n = 3), and co-trimoxazole (dfrA7; n = 145) resistance genes were detected among Salmonella Typhi isolates. Conclusion: In this study, T1 and T4 type Salmonella Typhi strains were predominantly prevalent in Northwest Pakistan. Antibiotic resistance among Salmonella Typhi isolates were observed. Findings of the study would be helpful to devise an appropriate antibiotic policy to control the emergence of drug-resistant Salmonella Typhi in Pakistan.

Novel mutations in PDE6A and CDHR1 cause retinitis pigmentosa in Pakistani families.

AIM: To investigate the genetic basis of autosomal recessive retinitis pigmentosa (arRP) in two consanguineous/ endogamous Pakistani families. METHODS: Whole exome sequencing (WES) was performed on genomic DNA samples of patients with arRP to identify disease causing mutations. Sanger sequencing was performed to confirm familial segregation of identified mutations, and potential pathogenicity was determined by predictions of the mutations' functions. RESULTS: A novel homozygous frameshift mutation [NM_000440.2:c.1054delG, p. (Gln352Argfs*4); Chr5:g.149286886del (GRCh37)] in the PDE6A gene in an endogamous family and a novel homozygous splice site mutation [NM_033100.3:c.1168-1G>A, Chr10:g.85968484G>A (GRCh37)] in the CDHR1 gene in a consanguineous family were identified. The PDE6A variant p. (Gln352Argfs*4) was predicted to be deleterious or pathogenic, whilst the CDHR1 variant c.1168-1G>A was predicted to result in potential alteration of splicing. CONCLUSION: This study expands the spectrum of genetic variants for arRP in Pakistani families.

Current Analytical Strategies in Studying Chromatin-Associated-Proteome (Chromatome).

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.

USH2A gene variants cause Keratoconus and Usher syndrome phenotypes in Pakistani families.

BACKGROUND: Retinitis pigmentosa (RP) is the most common inherited retinal dystrophy, affecting approximately 1 in 4000 individuals worldwide. The most common form of syndromic RP is Usher syndrome (USH) accounting for approximately 20-30 % of RP cases. Mutations in the USH2A gene cause a significant proportion of recessive non-syndromic RP and USH type II (USH2). This study aimed to determine the causative role of the USH2A gene in autosomal recessive inherited ocular diseases and to establish genotype-phenotype correlation associated with USH2A variants. METHODS: We performed direct Sanger sequencing and co-segregation analysis of the USH2A gene to identify disease causing variants in a non-syndromic RP family, two USH2 families and two Keratoconus (KC) families. RESULTS: Disease causing variants in the USH2A gene were identified in two families displayed KC and USH2 phenotypes. A novel variant c.4029T > G, p.Asn1343Lys in the USH2A gene was detected in a Pakistani family with KC phenotype. In addition, a missense variant (c.7334 C > T, p. Ser2445Phe) in the USH2A gene was found segregating in another Pakistani family with USH2 phenotype. Homozygosity of identified missense USH2A variants was found associated with autosomal recessive inherited KC and USH2 phenotypes in investigated families. These variants were not detected in ethnically matched healthy controls. Moreover, the USH2A variants were predicted to be deleterious or potentially disease causing by PolyPhen-2, PROVEAN and SIFT. CONCLUSIONS: This study provided first evidence for association of a novel USH2A variant with KC phenotype in a Pakistani family as well as established the phenotype-genotype correlation of a USH2A variant (c.7334 C > T, p. Ser2445Phe) with USH2 phenotype in another Pakistani family. The phenotype-genotype correlations established in present study may improve clinical diagnosis of affected individuals for better management and counseling.

A novel nonsense variant in SLC24A4 causing a rare form of amelogenesis imperfecta in a Pakistani family.

BACKGROUND: Amelogenesis imperfecta (AI) is a highly heterogeneous group of hereditary developmental abnormalities which mainly affects the dental enamel during tooth development in terms of its thickness, structure, and composition. It appears both in syndromic as well as non-syndromic forms. In the affected individuals, the enamel is usually thin, soft, rough, brittle, pitted, chipped, and abraded, having reduced functional ability and aesthetics. It leads to severe complications in the patient, like early tooth loss, severe discomfort, pain, dental caries, chewing difficulties, and discoloration of teeth from yellow to yellowish-brown or creamy type. The study aimed to identify the disease-causing variant in a consanguineous family. METHODS: We recruited a consanguineous Pashtun family of Pakistani origin. Exome sequencing analysis was followed by Sanger sequencing to identify the pathogenic variant in this family. RESULTS: Clinical analysis revealed hypomaturation AI having generalized yellow-brown or creamy type of discoloration in affected members. We identified a novel nonsense sequence variant c.1192C > T (p.Gln398*) in exon-12 of SLC24A4 by using exome sequencing. Later, its co-segregation within the family was confirmed by Sanger sequencing. The human gene mutation database (HGMD, 2019) has a record of five pathogenic variants in SLC24A4, causing AI phenotype. CONCLUSION: This nonsense sequence variant c.1192C > T (p.Gln398*) is the sixth disease-causing variant in SLC24A4, which extends its mutation spectrum and confirms the role of this gene in the morphogenesis of human tooth enamel. The identified variant highlights the critical role of SLC24A4 in causing a rare AI type in humans.

Step-by-Step Protocol for Superparamagnetic Nanoparticle-Based Plasma Membrane Isolation from Eukaryotic Cell.

Here, we elaborate our detailed protocol for synthesis, functionalization, and application of superparamagnetic nanoparticle (SPMNP) for plasma membrane and lysosome isolation. We used standard thermal decomposition-based synthesis of iron oxide (Fe3O4) core SPMNP 1.0. Using ligand addition methodology, we surface functionalized SPMNP 1.0 with phospholipids and generated phospholipid-SPMNP 2.0. Further we used NH2-phospholipid-SPMNP 2.0 to isolate plasma membrane. Using our SPMNP subcellular fractionation protocol, we are able to isolate high-pure-high-yield plasma membrane using NH2-phospholipid-SPMNP 2.0. As a future perspective, we propose to use SPMNP on clinical patient samples and perform mass spectrometry-based proteomics, lipidomics, and glycomics for early cancer diagnosis.

Step-by-Step Protocol for Superparamagnetic Nanoparticle-Based Endosome and Lysosome Isolation from Eukaryotic Cell.

Here, we report our step-by-step protocol for superparamagnetic nanoparticle (SPMNP)-based endosome and lysosome isolation from HeLa. Briefly, we synthesized SPMNP 1.0 with iron oxide (Fe3O4) core using thermal decomposition method. Further, we performed ligand-exchange strategy for surface functionalization of SPMNP 1.0 with dimercaptosuccinic acid (DMSA). Thus, we generated DMSA-SPMNP 2.0 and used DMSA-SPMNP 2.0 to isolate endosomes and lysosome from HeLa cells. Using our SPMNP subcellular fractionation protocol, we are able to isolate high-pure-high-yield lysosomes using DMSA-SPMNP 2.0 for lysosome proteomics and lipidomics in order to better understand subcellular compartments.

Synthesis of Hybrid Gold Nanoparticle (AuNP) Functionalized Superparamagnetic Nanoparticles (SPMNPs) for Efficient Coupling of Biomolecules.

Recently, we reported our methodology for isolating plasma membrane and lysosome from eukaryotic cell using superparamagnetic nanoparticles (SPMNPs). Here in this article, we report a step-by-step protocol for synthesis of hybrid gold nanoparticle (AuNP), surface functionalization of AuNPs on superparamagnetic nanoparticles (SPMNPs), and potential use of hybrid AuNP-SPMNP for efficient coupling of biomolecules.

BacMam System for Rapid Recombinant Protein Expression in Mammalian Cells.

Baculovirus expression vector system (BEVS) is an established technology for recombinant protein expression in insect cells. Further, BEVS-mediated gene transduction of mammalian cells (BacMam) is emerging as a technique for high level recombinant protein expression in mammalian cells. Here, we describe generic method in using BEVS as a BacMam for rapid recombinant protein expression in mammalian cells.

Surface functionalization dependent subcellular localization of Superparamagnetic nanoparticle in plasma membrane and endosome.

In this article, we elaborate the application of thermal decomposition based synthesis of Fe3O4 superparamagnetic nanoparticle (SPMNP) in subcellular fractionation context. Here, we performed surface functionalization of SPMNP with phospholipids and dimercaptosuccinic acid. Surprisingly, we observed surface functionalization dependent SPMNP localization in subcellular compartments such as plasma membrane, endosomes and lysosomes. By using SPMNP based subcellular localization with pulse-chase methodology, we could use SPMNP for high pure-high yield organelle (plasma membrane, endosomes and lysosome) fractionation. Further, SPMNP that are distinctly localized in subcellular compartments can be used as technology for subcellular fractionation that can complement existing tools for cell biology research. As a future perspective, isolated magnetic organelles can be extended to protein/protein complex purification for biochemical and structural biology studies.

Drug-induced ciliogenesis in pancreatic cancer cells is facilitated by the secreted ATP-purinergic receptor signaling pathway.

Malignant transformation of cells is often accompanied by the loss of the primary cilium, a protruding microtubule-based sensory organelle, suggesting that it plays an "onco-suppressive" role. Therefore, restoration of the primary cilium is being explored as a new therapeutic approach to attenuate tumor growth. Recently, several commonly used chemotherapeutic drugs have been identified to induce the primary cilium in pancreatic cancer cells. The mechanisms by which these drugs re-express the cilium remain, however, enigmatic. Here, evaluation of a panel of diverse ciliogenic drugs on pancreatic cancer cell models revealed a significant positive relationship between drug-induced extracellular ATP, released through pannexin channels, and the extent of primary cilium induction. Moreover, cilium induction by these drugs was hampered in the presence of the ATP degrading enzyme, apyrase, and in the presence of the pan-purinergic receptor inhibitor, suramin. Our findings reveal that ciliogenic drug-induced re-expression of the primary cilium in pancreatic cancer cells is, at least in certain contexts, dependent on a hitherto unrecognized autocrine/paracrine loop involving the extracellular ATP-purinergic receptor signaling pathway that can be exploited in a therapeutic approach targeting at restoring the primary cilium.

MPA Modulates Tight Junctions' Permeability via Midkine/PI3K Pathway in Caco-2 Cells: A Possible Mechanism of Leak-Flux Diarrhea in Organ Transplanted Patients.

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.

Active and Repressive Chromatin-Associated Proteome after MPA Treatment and the Role of Midkine in Epithelial Monolayer Permeability.

UNLABELLED: 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.

Identification of drugs that restore primary cilium expression in cancer cells.

The development of cancer is often accompanied by a loss of the primary cilium, a microtubule-based cellular protrusion that functions as a cellular antenna and that puts a break on cell proliferation. Hence, restoration of the primary cilium in cancer cells may represent a novel promising approach to attenuate tumor growth. Using a high content analysis-based approach we screened a library of clinically evaluated compounds and marketed drugs for their ability to restore primary cilium expression in pancreatic ductal cancer cells. A diverse set of 118 compounds stimulating cilium expression was identified. These included glucocorticoids, fibrates and other nuclear receptor modulators, neurotransmitter regulators, ion channel modulators, tyrosine kinase inhibitors, DNA gyrase/topoisomerase inhibitors, antibacterial compounds, protein inhibitors, microtubule modulators, and COX inhibitors. Certain compounds also dramatically affected the length of the cilium. For a selection of compounds (Clofibrate, Gefitinib, Sirolimus, Imexon and Dexamethasone) their ability to restore ciliogenesis was confirmed in a panel of human cancer cell line models representing different cancer types (pancreas, lung, kidney, breast). Most compounds attenuated cell proliferation, at least in part through induction of the primary cilium, as demonstrated by cilium removal using chloral hydrate. These findings reveal that several commonly used drugs restore ciliogenesis in cancer cells, and warrant further investigation of their antineoplastic properties.