In-Depth Bioinformatic Study of the CLDN16 Gene and Protein: Prediction of Subcellular Localization to Mitochondria

Background and Objectives: The defects in the CLDN16 gene are a cause of primary hypomagnesemia (FHHNC), which is characterized by massive renal magnesium wasting, resulting in nephrocalcinosis and renal failure. The mutations occur throughout the gene’s coding region and can impact on intracellular trafficking of the protein or its paracellular pore forming function. To gain more understanding about the mechanisms by which CLDN16 mutations can induce FHHNC, we performed an in-depth computational analysis of the CLDN16 gene and protein, focusing specifically on the prediction of the latter’s subcellular localization. Materials and Methods: The complete nucleotide or amino acid sequence of CLDN16 in FASTA format was entered and processed in 14 databases. Results: One CpG island was identified. Twenty five promoters/enhancers were predicted. The CLDN16 interactome was found to consist of 20 genes, mainly involved in kidney diseases. No signal peptide cleavage site was identified. A probability of export to mitochondria equal to 0.9740 and a cleavable mitochondrial localization signal in the N terminal of the CLDN16 protein were predicted. The secondary structure prediction was visualized. Νo phosphorylation sites were identified within the CLDN16 protein region by applying DISPHOS to the functional class of transport. The KnotProt database did not predict any knot or slipknot in the protein structure of CLDN16. Seven putative miRNA binding sites within the 3’-UTR region of CLDN16 were identified. Conclusions: This is the first study to identify mitochondria as a probable cytoplasmic compartment for CLDN16 localization, thus providing new insights into the protein’s intracellular transport. The results relative to the CLDN16 interactome underline its role in renal pathophysiology and highlight the functional dependence of CLDNs-10, 14, 16, 19. The predictions pertaining to the miRNAs, promoters/enhancers and CpG islands of the CLDN16 gene indicate a strict regulation of its expression both transcriptionally and post-transcriptionally

Study of the frequency and incidence of Herpesviruses in healthy blood donors and multiply transfused patients in the wider area of Greece

Introduction: Human Herpesviruses (HHVs) are double-stranded DNA viruses that are characterized by the ability to persist for a life in the host cells mainly through mechanisms capable of avoiding immune responses. Both primary and secondary HHVs infections are associated with high morbidity and mortality in non immunocompetent patients. In addition, HHVs types 4 and 8 are known for their tumorigenicity. Aim: This study aimed: 1. To investigate the prevalence of HHVs types 1-6 and 8 in a population of Greek blood donors 2. To investigate the prevalence of HHVs types 4 and 5 in a population of Greek patients with thalassemia 3. To compare the two populations with respect to the detection rate of HHVs 4-5 DNA. 4. To correlate HHVs molecular detection with the demographic and clinical features of the studied population and, 5. To identify and functionally analyze novel genes that could be targetd by HHV-4. Materials and Methods: Βy the method of random sampling, a population of N1 = 401 healthy blood donors and a population of N2 = 76 patients with thalassemia were studied. Whole blood DNA was extracted. Specific gene amplification reactions were used for each virus by standardized commercial assays. To find new host genes, potential targets of HHV-4, the interaction network of the tumor suppressor BIM which is epigenetically silenced by HHV-4 was built and functionally analysed by using contemporary bioinformatics tools. Results: The detection rates (%) of HHVs 4-6 and 8 in healthy Greek blood donors was 21.2, 0.25, 3.5 and 0.5 respectively. HHVs 1-3 were not detected in any of the 401 samples of population N1. Residency was found to affect the probability of HHV-4 DNA detection both in univariate and multivariate analysis (p=0.037 and p=0.025 respectively). Regarding patients population, HHV-4 DNA was detected in 14 of the 76 samples (18.42%) while HHV-5 DNA was detected in one sample (1.315%). The molecular prevalence of HHV-4 did not differ between patients and controls in a statistically significant manner. Ιt was not considered appropriate to compare the molecular prevalence of HHV-5 due to the very low frequency of detection. Fifty nine unique genes were found to compose the BIM regulatory network. Ontological analysis at the pathway level highlighted cancer, infectious diseases like tuberculosis and toxoplasmosis and neuropathies such as Alzheimer disease, Amyotrophic lateral sclerosis, Huntington disease and Parkinson disease.Εισαγωγή: Oι ανθρώπινοι ερπητοϊοί (HHVs) είναι δίκλωνοι DNA ιοί, που χαρακτηρίζονται από την ικανότητα να λαθροβιούν στα κύτταρα του ξενιστή τους κυρίως μέσω μηχανισμών αποφυγής της ανοσιακής του απόκρισης. Τόσο οι πρωτοπαθείς όσο και οι δευτεροπαθείς HHV λοιμώξεις έχουν συσχετιστεί με μεγάλη νοσηρότητα και θνητότητα στους μη ανοσοϊκανούς ασθενείς. Παράλληλα οι HHVs τύπου 4 και 8 είναι γνωστοί για την ογκογονικότητα τους. Σκοπός: Ο σκοπός διεξαγωγής της παρούσας μελέτης ήταν: 1. Η διερεύνηση του μοριακού επιπολασμού των HHVs 1-6 και 8 σε πληθυσμιακό δείγμα Ελλήνων αιμοδοτών 2. Η διερεύνηση του μοριακού επιπολασμού των HHVs 4-5 σε πληθυσμιακό δείγμα Ελλήνων ασθενών με μεσογειακή αναιμία 3. Η σύγκριση των δύο πληθυσμών ως προς το ποσοστό μοριακής ανίχνευσης των HHVs 4-5 ώστε να διερευνηθεί η πιθανότητα μετάδοσης τους μέσω των μεταγγίσεων 4. Η συσχέτιση της μοριακής ανίχνευσης των HHVs με δημογραφικά και κλινικά χαρακτηριστικά των συμμετεχόντων και, 5. Η αναζήτηση και λειτουργική ανάλυση νέων γονιδίων-πιθανών στόχων του HHV-4. Υλικά και Μέθοδοι: Μελετήθηκε με τη μέθοδο της τυχαίας δειγματοληψίας πληθυσμός Ν1=401 υγιών αιμοδοτών καθώς και πληθυσμός Ν2=76 ασθενών με μεσογειακή αναιμία. Το DNA των HHVs αναζητήθηκε σε δείγματα ολικού αίματος. Για κάθε ιό χρησιμοποιήθηκε ειδική αντίδραση ενίσχυσης της γονιδιακής ακολουθίας με προτυποποιημένες εμπορικές δοκιμασίες. Για την ανεύρεση νέων γονιδίων-στόχων του HHV-4 κατασκευάστηκε το δίκτυο αλληλεπίδρασης του διαμεσολαβητή της απόπτωσης BIM που καταστέλλεται επιγενετικά από τον HHV-4 ενώ πραγματοποιήθηκε και ανάλυση της πιθανής λειτουργίας των εμπλεκόμενων στο δίκτυο γονιδίων με την εφαρμογή σύγχρονων εργαλείων βιοπληροφορικής. Αποτελέσματα: Τα ποσοστα ανίχνευσης (%) των HHVs 4-6 και 8 στον υγιή πληθυσμό ήταν 21.2, 0.25, 3.5 και 0.5 αντίστοιχα. Οι HHVs 1-3 δεν ανιχνεύτηκαν σε κανένα από τα 401 δείγματα του πληθυσμού Ν1. Τόσο σε μονοπαραγοντική όσο και σε πολυπαραγοντική ανάλυση διαπιστώθηκε ότι ο τόπος κατοικίας σχετίζεται με την πιθανότητα ανίχνευσης του HHV-4 DNA (p=0.037 και p=0.025 αντίστοιχα). Στους πάσχοντες, σε 14 από τα 76 εξεταζόμενα δείγματα ανιχνεύτηκε HHV-4 DNA (18.42%) ενώ HHV-5 DNA ανιχνεύτηκε σε ένα δείγμα (1.315%). Δεν προέκυψε στατιστικά σημαντική διαφορά στον μοριακό επιπολασμό του HHV-4 μεταξύ ασθενών και μαρτύρων. Δεν κρίθηκε σκόπιμη η σύγκριση του μοριακού επιπολασμού του HHV-5 λόγω της πολύ μικρής συχνότητας ανίχνευσης. Από την βιοπληροφορική ανάλυση βρέθηκε, ότι συνολικά 59 γονίδια συνθέτουν το ρυθμιστικό δίκτυο του BIM. Τα γονίδια αυτά όπως προέκυψε από την οντολογική ανάλυση συμμετέχουν σε κυτταρικά μονοπάτια, που σχετίζονται με την καρκινογένεση, τις λοιμώξεις από άλλους μολυσματικούς παράγοντες όπως η φυματίωση και η τοξοπλάσμωση καθώς και με νευροεκφυλιστικές ασθένειες όπως η νόσος του Alzheimer, η νόσος του Parkinson, η πλάγια μυατροφική σκλήρυνση και η ασθένεια Huntington

Identification of Genes and miRNAs Associated with TAFI-Related Thrombosis: An in Silico Study

Thrombin-Activatable Fibrinolysis Inhibitor (TAFI) is a carboxypeptidase B-like proenzyme encoded by the CPB2 gene. After thrombin activation, TAFI downregulates fibrinolysis, thus linking the latter with coagulation. TAFI has been shown to play a role in venous and arterial thrombotic diseases, yet, data regarding the molecular mechanisms underlying its function have been conflicting. In this study, we focused on the prediction and functional enrichment analysis (FEA) of the TAFI interaction network and the microRNAs (miRNAs) targeting the members of this network in an attempt to identify novel components and pathways of TAFI-related thrombosis. To this end, we used nine bioinformatics software tools. We found that the TAFI interactome consists of 28 unique genes mainly involved in hemostasis. Twenty-four miRNAs were predicted to target these genes. Co-annotation analysis of the predicted interactors with respect to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and transcription factors (TFs) pointed to the complement and coagulation cascades as well as neutrophil extracellular trap formation. Cancer, stroke, and intracranial aneurysm were among the top 20 significant diseases related to the identified miRNAs. We reason that the predicted biomolecules should be further studied in the context of TAFI-related thrombosis

Prediction and enrichment analyses of the Homo sapiens-Drosophila melanogaster COPD-related orthologs: potential for modeling of human COPD genomic responses with the fruit fly

The significant similarities in airway epithelial cells between mammals and the fruit fly Drosophila melanogaster have rendered the latter an important model organism for studies of chronic inflammatory lung diseases. Focusing on the chronic obstructive pulmonary disease (COPD), we here mapped human gene orthologs associated with this disease in D. melanogaster to identify functionally equivalent genes for immediate, further screening with the fruit fly model. The DIOPT-DIST tool was accessed for the prediction of the COPD-associated orthologs between humans and Drosophila. Enrichment analyses with respect to pathways of the retrieved functional homologs were performed using the ToppFun and FlyMine tools, identifying 73 unique human genes as well as 438 fruit fly genes. The ToppFun analysis verified that the human gene list is associated with COPD phenotypes. Furthermore, the FlyMine investigation highlighted that the Drosophila genes are functionally connected mainly with the “ABC-family proteins mediated transport” and the “β-catenin-independent WNT signaling pathway.” These results suggest an evolutionarily conserved role toward responses to inhaled toxicants and CO2 in both species. We reason that the predicted orthologous genes should be further studied in the Drosophila models of cigarette smoke-induced COPD

Natural autoimmunity in oligoarticular juvenile idiopathic arthritis

Abstract Background Oligoarticular juvenile idiopathic arthritis (oligo-JIA) is considered as an antigen-driven lymphocyte-mediated autoimmune disease. Natural antibodies (NAbs) are pre-immune antibodies produced in the absence of exogenous antigen stimulation, participating in both, innate and adaptive immunity. Considering their major immunoregulatory role in homeostasis and autoimmune pathogenesis, we designed this study to further elucidate their role in oligo-JIA pathogenesis. Methods Seventy children with persistent oligo-JIA and 20 healthy matched controls were enrolled in the study. Serum IgM and IgA antibodies against human G-actin, human IgG F(ab΄)2 fragments and the hapten TriNitroPhenol (TNP) as well as the total concentration of serum IgM and IgA were measured by in-house enzyme-immunoassays. Kolmogorov–Smirnov normality test, Kruskal–Wallis H and Mann–Whitney tests were used to assess data distribution, and significant differences of non-parametric data between groups of the study. Backward regression analysis was used to analyze the effect of multiple factors (age, gender, disease activity, anti-nuclear antibody positivity, presence of uveitis) on continuous dependent variables (activities and activity/ concentration ratios of IgM and IgA NAbs). Results The ratios of IgA anti-TNP, anti-actin and anti-F(ab΄)2 levels to total serum IgA concentration were found to be significantly increased in patients with oligo-JIA compared to healthy subjects. Significantly elevated levels of IgM anti-TNP antibodies were also found in children with inactive oligo-JIA compared to those of children with active disease and of healthy controls. In the presence of anterior uveitis, IgM anti-TNP levels were significantly higher than in patients without uveitis or in healthy controls. Backward regression analysis revealed that the disease activity and the presence of anterior uveitis independently affect IgM anti-TNP levels. Conclusuions Our findings are in accordance with the hypothesis that NAbs contribute to the pathogenesis of autoimmune diseases and provide additional evidence that disturbances in natural autoimmunity may contribute to the as yet unclarified pathogenesis of oligo-JIA

Incorporating Biomarkers in COPD Management: The Research Keeps Going

Globally, chronic obstructive pulmonary disease (COPD) remains a major cause of morbidity and mortality, having a significant socioeconomic effect. Several molecular mechanisms have been related to COPD including chronic inflammation, telomere shortening, and epigenetic modifications. Nowadays, there is an increasing need for novel therapeutic approaches for the management of COPD. These treatment strategies should be based on finding the source of acute exacerbation of COPD episodes and estimating the patient’s own risk. The use of biomarkers and the measurement of their levels in conjunction with COPD exacerbation risk and disease prognosis is considered an encouraging approach. Many types of COPD biomarkers have been identified which include blood protein biomarkers, cellular biomarkers, and protease enzymes. They have been isolated from different sources including peripheral blood, sputum, bronchoalveolar fluid, exhaled air, and genetic material. However, there is still not an exclusive biomarker that is used for the evaluation of COPD but rather a combination of them, and this is attributed to disease complexity. In this review, we summarize the clinical significance of COPD-related biomarkers, their association with disease outcomes, and COPD patients’ management. Finally, we depict the various samples that are used for identifying and measuring these biomarkers

The Drosophila septate junctions beyond barrier function: Review of the literature, prediction of human orthologs of the SJ‐related proteins and identification of protein domain families

The involvement of Septate Junctions (SJs) in critical cellular functions that extend beyond their role as diffusion barriers in the epithelia and the nervous system has made the fruit fly an ideal model for the study of human diseases associated with impaired Tight Junction (TJ) function. In this study, we summarized current knowledge of the Drosophila melanogaster SJ‐related proteins, focusing on their unconventional functions. Additionally, we sought to identify human orthologs of the corresponding genes as well as protein domain families. The systematic literature search was performed in PubMed and Scopus databases using relevant key terms. Orthologs were predicted using the DIOPT tool and aligned protein regions were determined from the Pfam database. 3‐D models of the smooth SJ proteins were built on the Phyre2 and DMPFold protein structure prediction servers. A total of 30 proteins were identified as relatives to the SJ cellular structure. Key roles of these proteins, mainly in the regulation of morphogenetic events and cellular signalling, were highlighted. The investigation of protein domain families revealed that the SJ‐related proteins contain conserved domains that are required not only for cell‐cell interactions and cell polarity but also for cellular signalling and immunity. DIOPT analysis of orthologs identified novel human genes as putative functional homologs of the fruit fly SJ genes. A gap in our knowledge was identified regarding the domains that occur in the proteins encoded by eight SJ‐associated genes. Future investigation of these domains is needed to provide functional information

Predictors of SARS-CoV-2 IgG Spike Antibody Responses on Admission and Clinical Outcomes of COVID-19 Disease in Fully Vaccinated Inpatients: The CoVax Study

Background: SARS-CoV-2 vaccines have shown high efficacy in protecting against COVID-19, although the determinants of vaccine effectiveness and breakthrough rates are yet to be determined. We aimed at investigating several factors affecting the SARS-CoV-2 IgG Spike (S) antibody responses on admission and clinical outcomes of COVID-19 disease in fully vaccinated, hospitalized patients. Methods: 102 subjects were enrolled in the study. Blood serum samples were collected from each patient upon admission for the semiquantitative determination of the SARS-CoV-2 IgG S levels with lateral flow assays. Factors influencing vaccine responses were documented. Results: 27 subjects had a negative antibody test upon hospital admission. Out of the 102 patients admitted to the hospital, 88 were discharged and 14 died. Both the absence of anti-S SARS-CoV-2 antibodies and poor clinical outcomes of COVID-19 disease were associated with older age, lower Ct values, and a shorter period between symptom onset and hospital admission. Ct values and time between symptom onset and hospitalization were independently associated with SARS-CoV-2 IgG S responses upon admission. The PaO2/FiO2 ratio was identified as an independent predictor of in-hospital mortality. Conclusions: Host- and disease-associated factors can predict SARS-CoV-2 IgG S responses and mortality in hospitalized patients with breakthrough SARS-CoV-2 Infection