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OBJECTIVES: The term "inflammageing" describes the process of inflammation-induced aging that leads living cells to a state of permanent cell cycle arrest due to chronic antigenic irritation. This in vitro study aimed to shed light on the mechanisms of "inflammageing" on human oral cells. METHODS: Primary cultures of human gingival fibroblasts (hGFs) were exposed to variable pro-inflammatory stimuli, including lipopolysaccharide (LPS), Tumor Necrosis Factor-alpha (TNFa), and gingival crevicular fluid (GCF) collected from active periodontal pockets of systemically healthy patients. Inflammageing was studied through two experimental models, employing either late-passage ("aged") cells (p. 10) that were exposed to the pro-inflammatory stimuli or early-passage ("young") cells (p. 1) continuously exposed during a period of several passages (up to p. 10) to the above-mentioned stimuli. Cells were evaluated for the expression of beta-galactosidase activity (histochemical staining), senescence-associated genes (qPCR analysis), and biomarkers related to a Senescence-Associated Secretory Phenotype (SASP), through proteome profile analysis and bioinformatics. RESULTS: A significant increase (p < 0.05) in beta-galactosidase-positive cells was observed after exposure to each pro-inflammatory stimulus. The senescence-associated gene expression included upregulation for CCND1 and downregulation for SUSD6, and STAG1, a profile typical for cellular senescence. Overall, pro-inflammatory priming of late-passage cells caused more pronounced effects in terms of senescence than long-term exposure of early-passage cells to these stimuli. Proteomic analysis showed induction of SASP, evidenced by upregulation of several pro-inflammatory proteins (IL-6, IL-10, IL-16, IP-10, MCP-1, MCP-2, M-CSF, MIP-1a, MIP-1b, TNFb, sTNF-RI, sTNF-RII, TIMP-2) implicated in cellular aging and immune responses. The least potent impact on the induction of SASP was provoked by LPS and the most pronounced by GCF. CONCLUSION: This study demonstrates that long-term exposure of hGFs to various pro-inflammatory signals induced or accelerated cellular senescence with the most pronounced impact noted for the late-passage cells. The outcome of these analyses provides insights into oral chronic inflammation as a potential confounder of age-related diseases.
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Lipopolissacarídeos , Proteômica , Humanos , Lipopolissacarídeos/toxicidade , Envelhecimento , Inflamação , beta-GalactosidaseRESUMO
Even though Listeria monocytogenes is an extensive-studied foodborne pathogen, genome analysis of isolates from snails that may represent a reservoir of L. monocytogenes are still scarce. Here, we use whole-genome sequencing (WGS) to assess the genomic diversity of hypervirulent, virulent and non-virulent phenotypes of 15 L. monocytogenes isolated from snails to unveil their survival, virulence, and host-pathogen mechanisms of interactions in a snail infection model. Most of isolates (66.7%) were characterized as multidrug resistant (MDR) and belonged to clonal complexes (CCs) which are strongly associated with cases of human infection. All isolates contained intact genes associated with invasion and infection while hypervirulent isolates are adapted to host environment, possessing genes which are involved in teichoic acid biosynthesis, peptidoglycan modification and biofilm formation, correlating with their tolerance to haemolymph plasma phenotype and biofilm formation ability. A snail infection model showed that hypervirulent isolates triggered programmed host cell death pathway by increasing up to 30% the circulating apoptotic hemocytes in combination with induced nitrate production and reactive oxygen species (ROS) generation in snails' haemolymph. In contrast, the administration of the non-virulent strain which possesses a truncated mogR gene that regulates flagellar motility gene expression led only to an increase of necrotic non-apoptotic cells. Overall, this study provides significant insights into the genetic diversity of L. monocytogenes from snails, the genomic features of them linked to their hypervirulent/non-virulent phenotype, and the mechanisms of host-pathogen interactions.
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Listeria monocytogenes , Listeriose , Animais , Interações Hospedeiro-Patógeno , Carne , Sequenciamento Completo do GenomaRESUMO
Diagnosis of Mycosis Fungoides (MF) may be challenging, due to its polymorphic nature. The use of miRNAs as biomarkers to assist in diagnosis has been investigated, mainly in skin lesion biopsies. The purpose of this study is to evaluate the plasma levels of miR-146a and miR-155 in MF patients and to investigate their association with SNPs of their genes. Plasma miRNAs were quantified by RT-qPCR. Genomic DNA was used for SNPs' genotyping by Sanger sequencing. Plasma levels of miR-146a and miR-155 were significantly higher in patients vs. controls, in early MF patients vs. controls, and in advanced vs. early MF patients. Both miRNAs' levels were significantly higher in stage IIB vs. early-stage patients. miR-155 plasma levels were significantly higher in patients with skin tumors or erythroderma. CC genotype (rs2910164 C>G) was significantly more frequent in healthy controls and associated with lower MF risk and lower miR-146a levels. The AA genotype (rs767649 T>A) was significantly more frequent in patients and correlated with increased MF risk and increased miR-155 levels. The combination of GG+AA was only detected in patients and was correlated with higher MF susceptibility. Increased mir-146a and mir-155 plasma levels in MF is an important finding to establish putative noninvasive biomarkers. The presence of SNPs is closely associated with miRs' expression, and possibly with disease susceptibility.
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MicroRNAs , Micose Fungoide , Neoplasias Cutâneas , Humanos , Biomarcadores , Estudos de Casos e Controles , Predisposição Genética para Doença , Genótipo , MicroRNAs/genética , Micose Fungoide/genética , Polimorfismo de Nucleotídeo Único , Neoplasias Cutâneas/genéticaRESUMO
Lactococcosis is a disease encountered in a wide variety of fish species causing mortalities and having great economic impact on farmed fish. In this study, we report for the first time the isolation of a strain of the recently described novel species Lactococcus petauri, from rainbow trout suffering from lactococcosis. The aim of this study was to determine the complete genome sequence of L. petauri strain LG_SAV_20 and to characterize its antimicrobial resistance and virulence. The genome of L. petauri LG_SAV_20 consists of 2,078,949 base pair (bp) with a GC content of 38.05%, 1950 predicted coding sequence (CDS), and 60 RNAs (51 tRNAs, 3 ncRNAs, and 6 rRNAs). Phylogenetic analysis revealed that L. petauri LG_SAV_20 shares most of its genome with L. garvieae strains isolated from rainbow trout. Detection of genes associated with antimicrobial resistance indicated that the isolate possesses the multidrug transporter mdt(A) gene, while using comparative analysis we identified several genes that might be related to bacterial pathogenesis. This genomic information provides new insights into the role of this novel species as an etiological agent of lactococcosis.
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Surtos de Doenças , Doenças dos Peixes/epidemiologia , Doenças dos Peixes/microbiologia , Genoma Bacteriano/genética , Lactococcus/isolamento & purificação , Oncorhynchus mykiss/microbiologia , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Composição de Bases , Sequência de Bases , DNA Bacteriano/genética , Farmacorresistência Bacteriana/genética , Lactococcus/classificação , Lactococcus/efeitos dos fármacos , Lactococcus/genética , Testes de Sensibilidade Microbiana , Filogenia , Virulência/genéticaRESUMO
Chronic lymphocytic leukemia (CLL) stereotyped subsets #6 and #8 include cases expressing unmutated B cell receptor immunoglobulin (BcR IG) (U-CLL). Subset #6 (IGHV1-69/IGKV3-20) is less aggressive compared to subset #8 (IGHV4-39/IGKV1(D)-39) which has the highest risk for Richter's transformation among all CLL. The underlying reasons for this divergent clinical behavior are not fully elucidated. To gain insight into this issue, here we focused on epigenomic signatures and their links with gene expression, particularly investigating genome-wide DNA methylation profiles in subsets #6 and #8 as well as other U-CLL cases not expressing stereotyped BcR IG. We found that subset #8 showed a distinctive DNA methylation profile compared to all other U-CLL cases, including subset #6. Integrated analysis of DNA methylation and gene expression revealed significant correlation for several genes, particularly highlighting a relevant role for the TP63 gene which was hypomethylated and overexpressed in subset #8. This observation was validated by quantitative PCR, which also revealed TP63 mRNA overexpression in additional nonsubset U-CLL cases. BcR stimulation had distinct effects on p63 protein expression, particularly leading to induction in subset #8, accompanied by increased CLL cell survival. This pro-survival effect was also supported by siRNA-mediated downregulation of p63 expression resulting in increased apoptosis. In conclusion, we report that DNA methylation profiles may vary even among CLL patients with similar somatic hypermutation status, supporting a compartmentalized approach to dissecting CLL biology. Furthermore, we highlight p63 as a novel prosurvival factor in CLL, thus identifying another piece of the complex puzzle of clinical aggressiveness.
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Metilação de DNA/genética , Regulação Neoplásica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Apoptose/genética , Epigenômica/métodos , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Cultura Primária de Células , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/metabolismo , Análise de Sequência de RNA , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/metabolismo , Regulação para CimaRESUMO
DNA and histone methylation are post-transcriptional modifications that have been recently described in gliomas. Indeed, glioma CpG island hypermethylated phenotype has been identified as prognostic biomarker and as a surrogate marker of IDH1/2 mutations. However, the role of DNA methylation in glioblastoma progression is unknown. We sought to analyze DNA methylation levels in paired (initial and recurrent) primary glioblastoma samples to identify candidate pathways that may prone to glioblastoma progression. We have analyzed 12 samples (5 paired samples, two of them with three surgeries) using methylation arrays. We have analyzed differential methylation at probe and at gene region level. Finally, pathway analysis has been performed using differentially methylated regions. All analysis has been performed with R and Bioconductor packages. Mean methylation level at initial sample compared to recurrence was strongly positively correlated (R(2) = 0.98). There was no differentially methylation at probe level. However, at gene level 3080 regions were differentially methylated. Interestingly, pathways analysis showed that the most differentially methylated genes are involved in cellular response to macrophage colony-stimulating factor stimulus (GO:0036006). Methylation levels were strongly conserved when comparing initial to recurrence in primary glioblastomas. Interestingly, differentially methylated pathway analysis suggests that a modulation of methylation in immune response genes may play a role in glioblastoma progression. Further studies are needed to validate the role of methylation of glioblastoma immune response genes in tumor progression.
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Neoplasias Encefálicas/genética , Neoplasias Encefálicas/imunologia , Glioblastoma/genética , Glioblastoma/imunologia , Isocitrato Desidrogenase/genética , Mutação/genética , Ilhas de CpG/genética , Metilação de DNA/genética , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Progressão da Doença , Feminino , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Estatísticas não Paramétricas , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
The therapeutic potential of cold physical gas plasma operated at atmospheric pressure in oncology has been thoroughly demonstrated in numerous preclinical studies. The cytotoxic effect on malignant cells has been attributed mainly to biologically active plasma-generated compounds, namely, reactive oxygen and nitrogen species. The intracellular accumulation of reactive oxygen and nitrogen species interferes strongly with the antioxidant defense system of malignant cells, activating multiple signaling cascades and inevitably leading to oxidative stress-induced cell death. This study aims to determine whether plasma-induced cancer cell death operates through a universal molecular mechanism that is independent of the cancer cell type. Using whole transcriptome data, we sought to investigate the activation mechanism of plasma-treated samples in patient-derived prostate cell cultures, melanoma, breast, lymphoma, and lung cancer cells. The results from the standardized single-cohort gene expression analysis and parallel multi-cohort meta-analysis strongly indicate that plasma treatment globally induces cancer cell death through immune-mediated mechanisms, such as interleukin signaling, Toll-like receptor cascades, and MyD88 activation leading to pro-inflammatory cytokine release and tumor antigen presentation.
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Carbapenemase-producing Pseudomonas aeruginosa strains present a specific geographical distribution regarding the type of carbapenemase-encoding genes that they harbor. For more than twenty years, VIM-type enzymes were the only major carbapenemases that were detected among P. aeruginosa isolates in Greece until the emergence of NDM-1-encoding P. aeruginosa in early 2023. In the present study, we present the rapid reversal of the carbapenemase-producing P. aeruginosa epidemiology from blaVIM- to blaNDM-harbouring isolates that occurred in our hospital since then. Between January 2023 and February 2024, 139 isolates tested positive for carbapenemase production with the NG-Test CARBA 5 immunochromatographic assay. Eight isolates were processed with the Hybrispot antimicrobial resistance direct flow chip molecular assay, and the first NDM-producing isolate was further analyzed through whole genome sequencing and bioinformatics analysis. Multiple resistance genes were detected by molecular techniques in accordance with the extensively drug-resistant phenotype. The isolate that was subjected to whole-genome sequencing belonged to the P. aeruginosa high-risk clone ST308, and the blaNDM was located in the chromosome in accordance with previously reported data. During the study period, NDM-producing isolates were increasingly detected, and only five months after their emergence, they overcame VIM producers. Our results indicate the potential of this new clone to spread rapidly and predominate within healthcare institutions, further restricting the already limited treatment options.
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In the current study, the genotypic characteristics such as antimicrobial resistance and virulence genes, and plasmid replicons and phenotypic characteristics such as biofilm formation and antimicrobial resistance of 87 extended-spectrum beta-lactamase (ESBL)-producing E. coli (ESBL-Ec) isolated from 7 water bodies in northern Greece were investigated. Our data show a high prevalence (60.0 %) of ESBL-Ec in surface waters that exhibit high genetic diversity, suggesting multiple sources of their transmission into the aquatic environment. When evaluating the antimicrobial resistance of isolates, wide variation in their resistance profiles has been detected, with all isolates being multi-drug resistant (MDR). Regarding biofilm formation capacity and phylogenetic groups, the majority (54.0 %, 47/87) of ESBL-Ec were classified as no biofilm producers mainly assigned to phylogroup A (35.6 %; 31/87), followed by B2 (26.5 %; 23/87). PCR screening showed that a high proportion of the isolates tested positive for the blaCTX-M-1 group genes (69 %, 60/87), followed by blaTEM (55.2 %, 48/87), blaOXA (25.3 %, 22/87) and blaCTX-M-9 (17.2 %, 15/87). A subset of 28 ESBL-Ec strains was further investigated by applying whole genome sequencing (WGS), and among them, certain clinically significant sequence types were identified, such as ST131 and ST10. The corresponding in silico analysis predicted all these isolates as human pathogens, while a significant proportion of WGS-ESBL-Ec were assigned to extraintestinal pathogenic E. coli (ExPEC; 32.1 %), and urinary pathogenic E. coli (UPEC; 28.6 %) pathotypes. Comparative phylogenetic analysis, showed that the genomes of the ST131-O25:H4-H30 isolates are genetically linked to the human clinical strains. Here, we report for the first time the detection of a plasmid-mediated mobile colistin resistance gene in ESBL-Ec in Greece isolated from an environmental source. Overall, this study underlines the role of surface waters as a reservoir for antibiotic resistance genes and for presumptive pathogenic ESBL-Ec.
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Escherichia coli , Rios , beta-Lactamases , Escherichia coli/genética , Grécia , beta-Lactamases/genética , Rios/microbiologia , FilogeniaRESUMO
NDM carbapenemase-encoding genes disseminate commonly among Enterobacterales through transferable plasmids carrying additional resistance determinants. Apart from the intra-species dissemination, the inter-species exchange of plasmids seems to play an additional important role in the spread of blaNDM. We here present the genetics related to the isolation of three species (Klebsiella pneumoniae, Proteus mirabilis, and Morganella morganii) harboring the blaNDM-1 gene from a single patient in Greece. Bacterial identification and antimicrobial susceptibility testing were performed using the Vitek2. Whole genome sequencing and bioinformatic tools were used to identify resistance genes and plasmids. BlaNDM-1 harboring plasmids were found in all three isolates. Moreover, the plasmid constructs of the respective incomplete or circular contigs showed that the blaNDM-1 and its neighboring genes form a cluster that was found in all isolates. Our microbiological findings, together with the patient's history, suggest the in vivo transfer of the blaNDM-1-containing cluster through three different species in a single patient.
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Lactococcus garvieae is the etiological agent of lactococcosis, a clinically and economically significant infectious disease affecting farmed rainbow trout. L. garvieae had been considered the only cause of lactococcosis for a long time; however, L. petauri, another species of the genus Lactococcus, has lately been linked to the same disease. The genomes and biochemical profiles of L. petauri and L. garvieae have a high degree of similarity. Traditional diagnostic tests currently available cannot distinguish between these two species. The aim of this study was to use the transcribed spacer (ITS) region between 16S rRNA and 23S rRNA as a potential useful molecular target to differentiate L. garvieae from L. petauri, saving time and money compared to genomics methods currently used as diagnostic tools for accurate discrimination between these two species. The ITS region of 82 strains was amplified and sequenced. The amplified fragments varied in size from 500 to 550 bp. Based on the sequence, seven SNPs were identified that separate L. garvieae from L. petauri. The 16S-23S rRNA ITS region has enough resolution to distinguish between closely related L. garvieae and L. petauri and it can be used as a diagnostic marker to quickly identify the pathogens in a lactococcosis outbreak.
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DNA methylation is presently accepted as a tentative regulatory parameter in splicing. Recently, we reported significant methylation differences among various exonic splicing-enhancing elements and alternative splicing events, based on CpG methylation data from the Human Epigenome Project for chromosomes 6, 20 and 22. Presently, using a different computational approach and the same database, we report: (a) significant increase of hypermethylation in intronic and exonic sequences close to acceptor sites, relative to overall introns and exons, respectively (1,973 CpGs examined); (b) frequent CpGs, mostly hypomethylated, in donors and infrequent CpGs mostly hypermethylated, in acceptors; and (c) hypermethylation in cassette exons which are occasionally spliced and have weaker average splicing potential, relative to constitutive exons (p < 0.0001). CpGs are hypomethylated in non-coding exons (only 16 % hypermethylation). Single-exon genes, similarly to first exons, frequently contain hypomethylated CpGs, while in internal and last exons CpGs are more frequently hypermethylated. Methylation is also more frequent in strange introns and splice sites processed by the minor spliceosome, e.g., ATAC, (p < 0.0001 in all cases), but not in sites of incomplete processing, e.g., retained introns or bleeding exons, (p = 0.706 and p = 0.313, respectively). Most Alus, which are known to contribute to transcript presentation, are heavily methylated, in contrast with other Alus, e.g., AluJo and mammalian interspersed repetitive elements which have been previously associated with alternative expression. These results elucidate the role of intragenic methylation in association with alternative splicing and facilitate the evaluation of genomic variations/polymorphisms and the development of tools for the prediction of alternative splicing events.
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Processamento Alternativo , Metilação de DNA , Sequências Repetitivas de Ácido Nucleico , Cromossomos Humanos Par 20 , Cromossomos Humanos Par 22 , Cromossomos Humanos Par 6 , Biologia Computacional/métodos , Humanos , Sítios de Splice de RNA , Transcrição GênicaRESUMO
OBJECTIVES: In this communication, we describe the emergence of the mcr-1 colistin resistance gene in a blaCTX-M-32 extended-spectrum-ß-lactamase-producing Escherichia coli isolate recovered from a pediatric patient in Greece. METHODS: Bacterial identification and antimicrobial susceptibility testing were performed with the VITEK2 automated system and broth microdilution. Detection of resistance genes, assignment to sequence type, in silico plasmid detection, and virulence factors were carried out using ResFinder, MLST 2.0, PlasmidFinder 2.1., and VirulenceFinder 2.0, respectively. PlasmidSPAdes v3.11.1 was used to assemble the plasmid contigs. The mcr-1.1-containing plasmid was analyzed for insertion sequence elements using ISfinder. Phylogenetically relevant sequences of the plasmid were identified using the Microbe BLASTN suite. RESULTS: The microorganism was assigned to sequence type 48 and carried four plasmids of different incompatibility groups. The specific mcr-1.1 allele was located in a 32.722 bp plasmid belonging to the IncX4 group with no additional resistance genes. CONCLUSION: To the best of our knowledge, this is the first detection of mcr-1 in a human specimen in our country. A potential spread of mcr-1 in Greece is concerning because of the existing high rates of carbapenem resistance and colistin usage as a last resort regimen.
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Proteínas de Escherichia coli , Escherichia coli , Humanos , Criança , Colistina/farmacologia , Proteínas de Escherichia coli/genética , Farmacorresistência Bacteriana/genética , Tipagem de Sequências Multilocus , Grécia , Testes de Sensibilidade Microbiana , Antibacterianos/farmacologiaRESUMO
The precisionFDA Truth Challenge V2 aimed to assess the state of the art of variant calling in challenging genomic regions. Starting with FASTQs, 20 challenge participants applied their variant-calling pipelines and submitted 64 variant call sets for one or more sequencing technologies (Illumina, PacBio HiFi, and Oxford Nanopore Technologies). Submissions were evaluated following best practices for benchmarking small variants with updated Genome in a Bottle benchmark sets and genome stratifications. Challenge submissions included numerous innovative methods, with graph-based and machine learning methods scoring best for short-read and long-read datasets, respectively. With machine learning approaches, combining multiple sequencing technologies performed particularly well. Recent developments in sequencing and variant calling have enabled benchmarking variants in challenging genomic regions, paving the way for the identification of previously unknown clinically relevant variants.
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The presence of 5-hydroxymethyl cytosine in DNA has been previously associated with ageing. Using in silico analysis of normal liver samples we presently observed that in 5-hydroxymethyl cytosine sequences, DNA methylation is dependent on the co-presence of G-quadruplexes and palindromes. This association exhibits discrete patterns depending on G-quadruplex and palindrome densities. DNase-Seq data show that 5-hydroxymethyl cytosine sequences are common among liver nucleosomes (p < 2.2x10-16) and threefold more frequent than nucleosome sequences. Nucleosomes lacking palindromes and potential G-quadruplexes are rare in vivo (1%) and nucleosome occupancy potential decreases with increasing G-quadruplexes. Palindrome distribution is similar to that previously reported in nucleosomes. In low and mixed complexity sequences 5-hydroxymethyl cytosine is frequently located next to three elements: G-quadruplexes or imperfect G-quadruplexes with CpGs, or unstable hairpin loops (TCCCAY6TGGGA) mostly located in antisense strands or finally A-/T-rich segments near these motifs. The high frequencies and selective distribution of pentamer sequences (including TCCCA, TGGGA) probably indicate the positive contribution of 5-hydroxymethyl cytosine to stabilize the formation of structures unstable in the absence of this cytosine modification. Common motifs identified in all total 5-hydroxymethyl cytosine-containing sequences exhibit high homology to recognition sites of several transcription factor families: homeobox, factors involved in growth, mortality/ageing, cancer, neuronal function, vision, and reproduction. We conclude that cytosine hydroxymethylation could play a role in the recognition of sequences with G-quadruplexes/palindromes by forming epigenetically regulated DNA 'springs' and governing expansions or compressions recognized by different transcription factors or stabilizing nucleosomes. The balance of these epigenetic elements is lost in hepatocellular carcinoma.
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Quadruplex G , Neoplasias , 5-Metilcitosina/análogos & derivados , Envelhecimento , Metilação de DNA , Humanos , Fatores de TranscriçãoRESUMO
Staphylococcus aureus is the most common mastitis-related pathogen in dairy cattle, goats, and sheep worldwide. However, the population structure and genomic characteristics of mastitis-associated S. aureus in small ruminants are limited. Furthermore, the genotypic and phenotypic characteristics involved in the pathogenicity of S. aureus have been thoroughly defined, yet their association with the severity of mastitis is not fully established. Here, we performed genotyping by pulsed-field gel electrophoresis (PFGE) and spa analyses to assess the genetic diversity and relatedness of 162 S. aureus strains recovered from clinical mastitis (CM) and subclinical mastitis (SCM) cases from goats, sheep, and bovines. PFGE analysis revealed 108 distinguishable pulsotypes and 3 main clusters that comprised isolates from the three host species, while according to spa typing, 32 different spa types were identified. Genotypic analysis revealed a spreading of genetically related or indistinguishable S. aureus strains among ovine, caprine, and bovine farms of distant geographical regions. In total, 28 different staphylococcal enterotoxin (SE) gene profiles were observed, revealing a diverse range of SE genes among isolates. By evaluating the antimicrobial resistance, we found low phenotypic antimicrobial resistance among all ruminant isolates. We also performed multiple correspondence analysis, which indicated that the presence of the sec gene, biofilm production, and high autoaggregation ability are associated with CM cases.
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Enterotoxinas/genética , Doenças das Cabras/microbiologia , Mastite Bovina/microbiologia , Mastite/veterinária , Doenças dos Ovinos/microbiologia , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/isolamento & purificação , Animais , Antibacterianos/farmacologia , Bovinos , Farmacorresistência Bacteriana/genética , Feminino , Variação Genética , Genótipo , Doenças das Cabras/diagnóstico , Doenças das Cabras/tratamento farmacológico , Cabras , Grécia , Mastite/diagnóstico , Mastite/tratamento farmacológico , Mastite/microbiologia , Mastite Bovina/diagnóstico , Mastite Bovina/tratamento farmacológico , Ovinos , Doenças dos Ovinos/diagnóstico , Doenças dos Ovinos/tratamento farmacológico , Infecções Estafilocócicas/diagnóstico , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , VirulênciaRESUMO
Splice sites define the boundaries of exonic regions and dictate protein synthesis and function. The splicing mechanism involves complex interactions among positional and compositional features of different lengths. Computational modeling of the underlying constructive information is especially challenging, in order to decipher splicing-inducing elements and alternative splicing factors. SpliceIT (Splice Identification Technique) introduces a hybrid method for splice site prediction that couples probabilistic modeling with discriminative computational or experimental features inferred from published studies in two subsequent classification steps. The first step is undertaken by a Gaussian support vector machine (SVM) trained on the probabilistic profile that is extracted using two alternative position-dependent feature selection methods. In the second step, the extracted predictions are combined with known species-specific regulatory elements, in order to induce a tree-based modeling. The performance evaluation on human and Arabidopsis thaliana splice site datasets shows that SpliceIT is highly accurate compared to current state-of-the-art predictors in terms of the maximum sensitivity, specificity tradeoff without compromising space complexity and in a time-effective way. The source code and supplementary material are available at: http://www.med.auth.gr/research/spliceit/.
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Algoritmos , Biologia Computacional/métodos , Reconhecimento Automatizado de Padrão/métodos , Sítios de Splice de RNA , Análise de Sequência de DNA/métodos , Software , Arabidopsis , Bases de Dados Genéticas , Árvores de Decisões , Genes de Plantas , Humanos , Distribuição Normal , Curva ROC , Elementos Reguladores de TranscriçãoRESUMO
The extended and diverse interference of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in multiple host functions and the diverse associated symptoms implicate its involvement in fundamental cellular regulatory processes. The activity of ten-eleven translocase 2 (TET2) responsible for selective DNA demethylation, has been recently identified as a regulator of endogenous virus inactivation and viral invasion, possibly by proteasomal deregulation of the TET2/TET3 activities. In a recent report, we presented a detailed list of factors that can be affected by TET activity, including recognition of zinc finger protein binding sites and bimodal promoters, by enhancing the flexibility of adjacent sequences. In this review, we summarize the TET-associated processes and factors that could account for SARS-CoV-2 diverse symptoms. Moreover, we provide a correlation for the observed virus-induced symptoms that have been previously associated with TET activities by in vitro and in vitro studies. These include early hypoxia, neuronal regulation, smell and taste development, liver, intestinal, and cardiomyocyte differentiation. Finally, we propose that the high mortality of SARS-CoV-2 among adult patients, the different clinical symptoms of adults compared to children, the higher risk of patients with metabolic deregulation, and the low mortality rates among women can all be accounted for by the complex balance of the three enzymes with TET activity, which is developmentally regulated. This activity is age-dependent, related to telomere homeostasis and integrity, and associated with X chromosome inactivation via (de)regulation of the responsible XIST gene expression.
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Mutations in codon 133 of p53, which cause the loss of the Delta 133 isoform(s) expression, are very frequent in the Li-Fraumeni (LF) and Li-Fraumeni-like (LFL) syndromes. In sporadic cancers, silent p53 mutations are correlated with exonic splicing enhancers (ESEs) and exonic methylated sites. The present study shows that mutations in splice sites are also very frequent in LF/LFL syndromes, while missense mutations are less common compared to other familial or sporadic cancers (P = 0 in both cases). Furthermore, it is shown that the codons at which LF/LFL germline missense mutations occur, correlate with CpG-containing ESEs (r = 0.181, P = 0.014) which are all methylated in p53. While both silent and LF/LFL missense mutations correlate with SC35 motifs, only the latter are associated with SRp55. On the contrary, only silent mutations in sporadic cancers correlate with SF2/ASF motifs in p53. Moreover, 12.1% of LF/LFL missense mutations involve the formation of potential splice sites of considerable splicing scores. Finally, mutations that are not at, or adjacent to CpGs (+/-1 codon, 34% of all LF/LFL mutated sites), introduce considerable changes of the ESE scores (>1.3 score change). The above data verify that LF/LFL missense mutations probably result also in splicing deregulation, in addition to any changes of the protein function and are mostly associated with alterations of the exonic methylation landscape. Some of the ESEs affected in LF/LFL syndromes are also genetically unstable in sporadic cancers but non-CpG cytosine instability, which is predominantly associated with specific ESEs, is only common in sporadic cancers.
Assuntos
Metilação de DNA , Éxons/genética , Síndrome de Li-Fraumeni/genética , Mutação/genética , Splicing de RNA/genética , Sequências Reguladoras de Ácido Nucleico/genética , Proteína Supressora de Tumor p53/genética , Códon/genética , Ilhas de CpG , Análise Mutacional de DNA , Elementos Facilitadores Genéticos/genética , Humanos , SíndromeRESUMO
OBJECTIVES: Klebsiella pneumoniae is a well-known pathogen frequently implicated in serious life-threatening nosocomial infections. Here we present a K. pneumoniae isolate (AHEPA1046) co-harbouring blaNDM-1 and blaOXA-48 isolated from a blood sample of an inpatient in Thessaloniki, Greece. METHODS: Whole-genome sequencing (WGS) was performed using an Illumina MiniSeq Sequencing System. Multilocus sequence typing (MLST) was performed using a BLAST-based approach, and antimicrobial resistance genes and plasmid replicons were identified by ResFinder and PlasmidFinder, respectively. The Rapid Annotation using Subsystem Technology (RAST) v.2.0 server was used for genome annotation. RESULTS: WGS analysis revealed the complete resistome of K. pneumoniae AHEPA1046. The strain harboured blaNDM-1 and blaOXA-48 together with 16 additional antimicrobial resistance genes and was resistant to carbapenems, aminoglycosides, quinolones, macrolides, tetracyclines, trimethoprim, fosfomycin and phenicols. Moreover, it was classified as ST11. CONCLUSION: This is the first report of a K. pneumoniae clinical isolate from Greece co-producing NDM-1 and OXA-48 carbapenemases and is one of a few reported worldwide.