The aim of this study is to evaluate opportunistic pathogenic bacteria of the genus Pseudomonas in anthropogenically impacted bathing waters, primarily focusing on bathing ponds. The findings include the detection of these bacteria, their susceptibility to selected antibiotics, and the determination of the Exotoxin A (exoA) gene using PCR method. P. aeruginosa was present in most samples, albeit in low concentrations (1-14 CFU/100 mL). The presence of P. otitidis, which is associated with ear infection, in this type of bathing water, was not rare (up to 90 CFU/100 mL). This species would not be detected by the standard methods, including tests on acetamid medium, used for P. aeruginosa in water. The isolated strains of P. otitidis lack the exoA gene and exhibited higher resistance to meropenem compared to P. aeruginosa.
- MeSH
- ADP-ribosatransferasy genetika MeSH
- antibakteriální látky * farmakologie MeSH
- bakteriální léková rezistence MeSH
- bakteriální proteiny genetika MeSH
- bakteriální toxiny genetika MeSH
- exotoxin A z Pseudomonas aeruginosa MeSH
- exotoxiny genetika MeSH
- faktory virulence genetika MeSH
- mikrobiální testy citlivosti * MeSH
- mikrobiologie vody * MeSH
- polymerázová řetězová reakce MeSH
- Pseudomonas * genetika izolace a purifikace klasifikace účinky léků MeSH
- rybníky * mikrobiologie MeSH
- Publikační typ
- časopisecké články MeSH
To explore the effects and underlying mechanisms of Mdivi-1 on three common clinical models of acute kidney injury (AKI). Three common AKI cell models were constructed, classified into the control group (human renal tubular epithelial cells [HK-2] cells), the Iohexol group (HK-2 cells treated with Iohexol), the Genta group (HK-2 cells treated with Gentamicin), and the Cis group (HK-2 cells treated with Cisplatin). To explore the optimal protective concentration of Mdivi-1 for each AKI cell model, the experimental design consisted of the following seven groups: the control group (HK-2 cells cultured in medium), three injury groups (HK-2 cells subjected to Iohexol, Gentamicin, or Cisplatin), and the corresponding protection groups (with a certain concentration of Mdivi-1 added to each injury group). Cellular survival and apoptosis, reactive oxygen species (ROS) levels, and the expression of recombinant Sirtuin 3 (SIRT3) in each group were measured. Mitochondrial fission and fusion dynamics in cells were observed under an electron microscope. To explore relevant pathways, the changes in relevant pathway proteins were analyzed through Western blotting. The half maximal inhibitory concentration (IC50) values were 150.06 mgI/ml at 6 h in the Iohexol group, 37.88 mg/ml at 24 h in the Gentamicin group, and 13.48 microM at 24 h in the Cisplatin group. Compared with the control group, the three injury groups showed increased cell apoptosis rates and higher expressions of apoptotic proteins in HK-2 cells, with an accompanying decrease in cell migration. After the addition of corresponding concentrations of Mdivi-1, the optimal concentrations were 3 μM in the Iohexo-3 group, 1 microM in the Genta-1 group, and 5 μM in the Cis-5 group, HK-2 cells showed the highest survival rate, reduced apoptosis, decreased mitochondrial ROS and SIRT3 expression, and reduced mitochondrial fission and autophagy when compared with each injury group. Further verification with Western blot analysis after the addition of Mdivi-1 revealed a reduction in the expressions of mitochondrial fission proteins DRP1, Nrf2, SIRT3, Caspase-3, Jun N-terminal Kinase (JNK)/P-JNK, NF-kappaB, Bcl2, and autophagic protein P62, as well as reduced ROS levels. Mdivi-1 had protective effects on the three common AKI cell models by potentially reducing mitochondrial fission in cells and inhibiting the production of ROS through the mediation of the NF- B/JNK/SIRT3 signaling pathway, thereby exerting protective effects. Key words AKI, Cisplatin, Gentamicin, Iohexol, Mdivi-1.
- MeSH
- akutní poškození ledvin * metabolismus patologie farmakoterapie MeSH
- apoptóza účinky léků MeSH
- buněčné linie MeSH
- lidé MeSH
- MAP kinasový signální systém účinky léků fyziologie MeSH
- mitochondriální dynamika * účinky léků fyziologie MeSH
- NF-kappa B * metabolismus MeSH
- reaktivní formy kyslíku metabolismus MeSH
- signální transdukce * účinky léků MeSH
- sirtuin 3 * metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Several authors have attributed the explosive outbreak of gastroenteritis that occurred in Czechoslovakia in 1965 to a toxigenic strain of Vibrio cholerae serogroup O37 based on unverified metadata associated with three particular strains from the American Type Culture Collection. Here, by sequencing the original strain preserved at the Czech National Collection of Type Cultures since 1966, we show that the strain responsible for this outbreak was actually a V. cholerae O5 that lacks the genes encoding the cholera toxin, the toxin-coregulated pilus protein and Vibrio pathogenicity islands present in V. cholerae O37 strains.
- MeSH
- cholera * epidemiologie mikrobiologie dějiny MeSH
- cholerový toxin genetika MeSH
- epidemický výskyt choroby * MeSH
- gastroenteritida * mikrobiologie epidemiologie dějiny MeSH
- genomové ostrovy MeSH
- lidé MeSH
- séroskupina MeSH
- Vibrio cholerae * genetika klasifikace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- historické články MeSH
- úvodníky MeSH
- Geografické názvy
- Československo MeSH
In response to DNA damage, the histone PARylation factor 1 (HPF1) regulates PARP1/2 activity, facilitating serine ADP-ribosylation of chromatin-associated factors. While PARP1/2 are known for their role in DNA single-strand break repair (SSBR), the significance of HPF1 in this process remains unclear. Here, we investigated the impact of HPF1 deficiency on cellular survival and SSBR following exposure to various genotoxins. We found that HPF1 loss did not generally increase cellular sensitivity to agents that typically induce DNA single-strand breaks (SSBs) repaired by PARP1. SSBR kinetics in HPF1-deficient cells were largely unaffected, though its absence partially influenced the accumulation of SSB intermediates after exposure to specific genotoxins in certain cell lines, likely due to altered ADP-ribosylation of chromatin. Despite reduced serine mono-ADP-ribosylation, HPF1-deficient cells maintained robust poly-ADP-ribosylation at SSB sites, possibly reflecting PARP1 auto-poly-ADP-ribosylation at non-serine residues. Notably, poly-ADP-ribose chains were sufficient to recruit the DNA repair factor XRCC1, which may explain the relatively normal SSBR capacity in HPF1-deficient cells. These findings suggest that HPF1 and histone serine ADP-ribosylation are largely dispensable for PARP1-dependent SSBR in response to genotoxic stress, highlighting the complexity of mechanisms that maintain genomic stability and chromatin remodeling.
- MeSH
- buněčné linie MeSH
- chromatin metabolismus MeSH
- DNA vazebné proteiny metabolismus genetika MeSH
- histony metabolismus MeSH
- jaderné proteiny metabolismus genetika MeSH
- jednořetězcové zlomy DNA * MeSH
- lidé MeSH
- oprava DNA * MeSH
- poly-ADP-ribosylace MeSH
- poly(ADP-ribosa)polymerasa 1 * metabolismus genetika MeSH
- poly(ADP-ribosa)polymerasy metabolismus genetika MeSH
- protein XRCC1 metabolismus genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Obesity is a major health burden. Preadipocytes proliferate and differentiate in mature adipocytes in the adipogenic process, which could be a potential therapeutic approach for obesity. Deficiency of SIRT6, a stress-responsive protein deacetylase and mono-ADP ribosyltransferase enzyme, blocks adipogenesis. Mutants of SIRT6 (N308K/A313S) were recently linked to the in the long lifespan Ashkenazi Jews. In this study, we aimed to clarify how these new centenarian-associated SIRT6 genetic variants affect adipogenesis at the transcriptional and epigenetic level. METHODS: We analyzed the role of SIRT6 wild-type (WT) or SIRT6 centenarian-associated mutant (N308K/A313S) overexpression in adipogenesis, by creating stably transduced preadipocyte cell lines using lentivirus on the 3T3-L1 model. Histone post-translational modifications (PTM: acetylation, methylation) and transcriptomic changes were analyzed by mass spectrometry (LC-MS/MS) and RNA-Seq, respectively, in 3T3-L1 adipocytes. In addition, the adipogenic process and related signaling pathways were investigated by bioinformatics and biochemical approaches. RESULTS: Overexpression of centenarian-associated SIRT6 mutant increased adipogenic differentiation to a similar extent compared to the WT form. However, it triggered distinct histone PTM profiles in mature adipocytes, with significantly higher acetylation levels, and activated divergent transcriptional programs, including those dependent on signaling related to the sympathetic innervation and to PI3K pathway. 3T3-L1 mature adipocytes overexpressing SIRT6 N308K/A313S displayed increased insulin sensitivity in a neuropeptide Y (NPY)-dependent manner. CONCLUSIONS: SIRT6 N308K/A313S overexpression in mature adipocytes ameliorated glucose sensitivity and impacted sympathetic innervation signaling. These findings highlight the importance of targeting SIRT6 enzymatic activities to regulate the co-morbidities associated with obesity.
- MeSH
- adipogeneze * genetika MeSH
- buňky 3T3-L1 * MeSH
- epigeneze genetická * genetika MeSH
- histony metabolismus genetika MeSH
- lidé MeSH
- mutace MeSH
- myši MeSH
- obezita genetika metabolismus MeSH
- posttranslační úpravy proteinů genetika MeSH
- sirtuiny * genetika metabolismus MeSH
- tukové buňky * metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Coagulation factor replacement therapy for the X-linked bleeding disorder Haemophilia, characterized by a deficiency of coagulation protein factor VIII (FVIII), is severely complicated by antibody (inhibitors) formation. The development of FVIII inhibitors drastically alters the quality of life of the patients and is associated with a tremendous increase in morbidity as well as treatment costs. The ultimate goal of inhibitor control is antibody elimination. Immune tolerance induction (ITI) is the only clinically established approach for developing antigen-specific tolerance to FVIII. This work aims to establish a novel cost-effective strategy to produce FVIII molecules in fusion with cholera toxin B (CTB) subunit at the N terminus using the Bacillus subtilis expression system for oral tolerance, as the current clinical immune tolerance protocols are expensive. Regions of B-Domain Deleted (BDD)-FVIII that have potential epitopes were identified by employing Bepipred linear epitope prediction; 2 or more epitopes in each domain were combined and cDNA encoding these regions were fused with CTB and cloned in the Bacillus subtilis expression vector pHT43 and expression analysis was carried out. The expressed CTB-fused FVIII epitope domains showed strong binding affinity towards the CTB-receptor GM1 ganglioside. To conclude, Bacillus subtilis expressing FVIII molecules might be a promising candidate for exploring for the induction of oral immune tolerance.
- MeSH
- aplikace orální MeSH
- Bacillus subtilis * genetika imunologie metabolismus MeSH
- cholerový toxin * genetika imunologie MeSH
- epitopy * imunologie genetika MeSH
- exprese genu MeSH
- faktor VIII * imunologie genetika MeSH
- imunologická tolerance * MeSH
- lidé MeSH
- rekombinantní fúzní proteiny * imunologie genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Inhibition of hypoxanthine-guanine-xanthine phosphoribosyltransferase activity decreases the pool of 6-oxo and 6-amino purine nucleoside monophosphates required for DNA and RNA synthesis, resulting in a reduction in cell growth. Therefore, inhibitors of this enzyme have potential to control infections, caused by Plasmodium falciparum and Plasmodium vivax, Trypanosoma brucei, Mycobacterium tuberculosis, and Helicobacter pylori. Five compounds synthesized here that contain a purine base covalently linked by a prolinol group to one or two phosphonate groups have Ki values ranging from 3 nM to >10 μM, depending on the structure of the inhibitor and the biological origin of the enzyme. X-ray crystal structures show that, on binding, these prolinol-containing inhibitors stimulated the movement of active site loops in the enzyme. Against TBr in cell culture, a prodrug exhibited an EC50 of 10 μM. Thus, these compounds are excellent candidates for further development as drug leads against infectious diseases as well as being potential anticancer agents.
- MeSH
- inhibitory enzymů * farmakologie chemie chemická syntéza MeSH
- katalytická doména MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- molekulární modely MeSH
- molekulární struktura MeSH
- pentosyltransferasy * antagonisté a inhibitory metabolismus MeSH
- racionální návrh léčiv * MeSH
- Trypanosoma brucei brucei účinky léků enzymologie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Non-alcoholic fatty liver disease (NAFLD), encompassing fatty liver and its progression into nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma (HCC), is one of the rapidly rising health concerns worldwide. SIRT6 is an essential nuclear sirtuin that regulates numerous pathological processes including insulin resistance and inflammation, and recently it has been implicated in the amelioration of NAFLD progression. SIRT6 overexpression protects from formation of fibrotic lesions. However, the underlying molecular mechanisms are not fully delineated. Moreover, new allelic variants of SIRT6 (N308K/A313S) were recently associated with the longevity in Ashkenazi Jews by improving genome maintenance and DNA repair, suppressing transposons and killing cancer cells. Whether these new SIRT6 variants play different or enhanced roles in liver diseases is currently unknown. In this study, we aimed to clarify how these new centenarian-associated SIRT6 genetic variants affect liver metabolism and associated diseases. We present evidence that overexpression of centenarian-associated SIRT6 variants dramatically altered the metabolomic and secretomic profiles of unchallenged immortalized human hepatocytes (IHH). Most amino acids were increased in the SIRT6 N308K/A313S overexpressing IHH when compared to IHH transfected with the SIRT6 wild-type sequence. Several unsaturated fatty acids and glycerophospholipids were increased, and ceramide tended to be decreased upon SIRT6 N308K/A313S overexpression. Furthermore, we found that overexpression of SIRT6 N308K/A313S in a 3D hepatic spheroid model formed by the co-culture of human immortalized hepatocytes (IHH) and hepatic stellate cells (LX2) inhibited collagen deposition and fibrotic gene expression in absence of metabolic or dietary challenges. Hence, our findings suggest that novel longevity associated SIRT6 N308K/A313S variants could favor the prevention of NASH by altering hepatocyte proteome and lipidome.
- MeSH
- hepatocelulární karcinom * metabolismus patologie MeSH
- hepatocyty metabolismus patologie MeSH
- kolagen metabolismus MeSH
- lidé MeSH
- nádory jater * metabolismus patologie MeSH
- nealkoholová steatóza jater * genetika metabolismus patologie MeSH
- senioři nad 80 let MeSH
- sirtuiny * genetika metabolismus MeSH
- století lidé MeSH
- Check Tag
- lidé MeSH
- senioři nad 80 let MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND AND PURPOSE: N-methyl-d-aspartate receptor (NMDAR) and leucine-rich glioma-inactivated protein 1 (LGI1) encephalitis are important types of autoimmune encephalitis (AE) with significant morbidity. In this study, we used a proteomic approach in search of novel clinically relevant biomarkers in these types of encephalitides. METHODS: Swedish and Czech tertiary neuroimmunology centers collaborated in this retrospective exploratory study. Fifty-eight cerebrospinal fluid (CSF) samples of 28 patients with AE (14 definite NMDAR, 14 with definite LGI1 encephalitis) and 30 controls were included. CSF samples were analyzed using proximity extension assay technology (Olink Target 96 Inflammation panel). For each CSF sample, 92 proteins were measured. Clinical variables were retrospectively collected, and correlations with protein levels were statistically analyzed. RESULTS: Patients and controls differed significantly in the following 18 biomarkers: TNFRSF9, TNFRSF12, TNFRSF14, TNFβ, TNFα, IL7, IL10, IL12B, IFNγ, CD5, CD6, CASP8, MMP1, CXCL8, CXCL10, CXCL11, IL20RA, and sirtuin 2 (SIRT2). In LGI1 encephalitis, no clinically useful association was found between biomarkers and clinical variables. In the NMDAR encephalitis group, SIRT2, TNFβ, and CD5 were significantly associated with ovarian teratoma. For SIRT2, this was true even for the first patients' CSF sample (SIRT2 without vs. with tumor, mean ± SD = 2.2 ± 0.29 vs. 2.88 ± 0.48; p = 0.007, 95% confidence interval = -1.15 to -0.22; r statistic in point-biserial correlation (rpb) = 0.66, p = 0.011). SIRT2 was positively correlated with age (rpb = 0.39, p = 0.018) and total hospital days (r = 0.55, p = <0.001). CONCLUSIONS: SIRT2 should be investigated as a biomarker of paraneoplastic etiology in NMDAR encephalitis.
- MeSH
- autoprotilátky MeSH
- biologické markery mozkomíšní mok MeSH
- encefalitida s protilátkami proti NMDA receptorům * mozkomíšní mok MeSH
- lidé MeSH
- novorozenec MeSH
- proteomika MeSH
- retrospektivní studie MeSH
- sirtuin 2 MeSH
- Check Tag
- lidé MeSH
- novorozenec MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
