BACKGROUND: The complexity of the galaninergic system is still not fully understood, especially under specific pre-existing comorbidities related to metabolic dysfunction. A plant-derived triterpenoid celastrol was demonstrated to exert a complex effect on the galaninergic system and to have hepatoprotective and anti-obesity properties. However, the exact molecular mechanisms responsible for these effects remain unclear. Specifically, there are no data on the impact of celastrol on the heart and liver galaninergic system. Therefore, this study aimed to investigate the effects of celastrol on the galaninergic system expression in the heart and liver of mice suffering from diet-induced obesity and metabolic dysfunction-associated steatotic liver disease and steatohepatitis (MASLD/MASH). METHODS: The male mice C57BL/6J were fed a Western-type high-fat diet for 16 and 20 weeks to induce obesity and MASLD/MASH. Celastrol was administered along with a specific diet for the last 4 weeks to evaluate its impact on the progression of these conditions. Moreover, the inhibitor of sterol regulatory element-binding protein 1/2 (SREBP1/2), fatostatin, was also tested to compare its influence on the galaninergic system with celastrol. RESULTS: The study demonstrates that celastrol treatment was safe and led to a reduction in food and energy intake, body fat and liver weights, and MASLD-to-MASH progression and improved glucose tolerance, serum biochemistry markers, and hepatic lipid peroxidation in mice. Quantitative gene expression originally showed significant regulation of galanin and all three of its receptors (GalR1/2/3) in the heart ventricles and only GalR2 in the liver of obese mice. Celastrol influenced the gene expression of galanin receptors: it downregulated Galr1 in the heart and upregulated Galr2 in the liver and Galr3 in the heart ventricles, potentially affecting energy metabolism, oxidative stress, and inflammation. Fatostatin suppressed gene expression of all the detected members of the galaninergic system in the heart ventricles, depicting the role of SREBP in this process. CONCLUSION: These findings suggest that celastrol may beneficially modulate the galaninergic system under obesity and MASLD-to-MASH progression, indicating its potential as a therapeutic agent for disorders associated with metabolic dysfunction.

• Klíčová slova
• MASH, MASLD, celastrol, fatostatin, galanin receptor, heart, mouse, obesity,
• Publikační typ
• časopisecké články MeSH

Long non-coding RNAs (lncRNAs) serve an important role in cancer progression and may be used as efficient molecular biomarkers. The present study aimed to identify lncRNAs associated with the response to the receptor tyrosine kinase inhibitor sunitinib and transcriptome profile and clinical features of metastatic renal cell carcinoma (mRCC). The gene expression of 84 cancer-associated lncRNAs in tumor and non-malignant tissue samples of 38 patients with mRCC was evaluated using quantitative PCR. In addition, the coding transcriptome was estimated using RNA sequencing in a subgroup of 20 patients and mRNA-lncRNA intersections were identified. In total, 37 and 13 lncRNAs were down- and upregulated, respectively, in tumor compared with non-malignant adjacent tissue samples. A total of 10 and 4 lncRNAs were up- and downregulated, respectively, in good responders to sunitinib compared with poor responders. High expression of HNF1A-AS1 and IPW lncRNAs was associated with prolonged progression-free survival of patients and a high expression of the TUSC7 lncRNA was associated with poor response and worse survival. Significant associations of dysregulated MEG3 and SNHG16 lncRNAs with expression of protein-coding genes representing various pathways, were identified. Furthermore, a significantly higher expression of CLIP4 gene was observed in good responders. The present study revealed promising candidates for predictive and prognostic biomarkers with further therapeutic potential.

• Klíčová slova
• inhibitor, long non-coding RNA, metastatic, prognosis, renal cancer, response, sunitinib, therapy, transcriptome, tyrosine kinase,
• Publikační typ
• časopisecké články MeSH

Leishmania parasites are transmitted to vertebrate hosts by female phlebotomine sand flies as they bloodfeed by lacerating the upper capillaries of the dermis with their barbed mouthparts. In the sand fly midgut secreted proteophosphoglycans from Leishmania form a biological plug known as the promastigote secretory gel (PSG), which blocks the gut and facilitates the regurgitation of infective parasites. The interaction between the wound created by the sand fly bite and PSG is not known. Here we nanoinjected a sand fly egested dose of PSG into BALB/c mouse skin that lead to the differential expression of 7,907 transcripts. These transcripts were transiently up-regulated during the first 6 hours post-wound and enriched for pathways involved in inflammation, cell proliferation, fibrosis, epithelial cell differentiation and wound remodelling. We found that PSG significantly accelerated wound healing in vitro and in mice; which was associated with an early up-regulation of transcripts involved in inflammation (IL-1β, IL-6, IL-10, TNFα) and inflammatory cell recruitment (CCL2, CCL3, CCL4, CXCL2), followed 6 days later by enhanced expression of transcripts associated with epithelial cell proliferation, fibroplasia and fibrosis (FGFR2, EGF, EGFR, IGF1). Dermal expression of IGF1 was enhanced following an infected sand fly bite and was acutely responsive to the deposition of PSG but not the inoculation of parasites or sand fly saliva. Antibody blockade of IGF1 ablated the gel's ability to promote wound closure in mouse ears and significantly reduced the virulence of Leishmania mexicana infection delivered by an individual sand fly bite. Dermal macrophages recruited to air-pouches on the backs of mice revealed that IGF1 was pivotal to the PSG's ability to promote macrophage alternative activation and Leishmania infection. Our data demonstrate that through the regurgitation of PSG Leishmania exploit the wound healing response of the host to the vector bite by promoting the action of IGF1 to drive the alternative activation of macrophages.

• MeSH
• hojení ran účinky léků   MeSH
• insulinu podobný růstový faktor I fyziologie   MeSH
• interakce hostitele a parazita fyziologie   MeSH
• kultivované buňky MeSH
• kůže účinky léků   parazitologie   patologie   MeSH
• Leishmania mexicana metabolismus   MeSH
• leishmanióza kožní parazitologie   patologie   MeSH
• makrofágy účinky léků   metabolismus   parazitologie   patologie   MeSH
• membránové proteiny metabolismus   farmakologie   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• progrese nemoci MeSH
• proteoglykany metabolismus   farmakologie   MeSH
• protozoální proteiny metabolismus   farmakologie   MeSH
• Psychodidae metabolismus   MeSH
• signální transdukce účinky léků   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• insulinu podobný růstový faktor I MeSH
• membránové proteiny MeSH
• Ppg1 protein, Leishmania MeSH Prohlížeč
• proteoglykany MeSH
• protozoální proteiny MeSH

Successful molecular analyses of human solid tissues require intact biological material with well-preserved nucleic acids, proteins, and other cell structures. Pre-analytical handling, comprising of the collection of material at the operating theatre, is among the first critical steps that influence sample quality. The aim of this study was to compare the experimental outcomes obtained from samples collected and stored by the conventional means of snap freezing and by PAXgene Tissue System (Qiagen). These approaches were evaluated by measuring rRNA and mRNA integrity of the samples (RNA Quality Indicator and Differential Amplification Method) and by gene expression profiling. The collection procedures of the biological material were implemented in two hospitals during colon cancer surgery in order to identify the impact of the collection method on the experimental outcome. Our study shows that the pre-analytical sample handling has a significant effect on the quality of RNA and on the variability of qPCR data. PAXgene collection mode proved to be more easily implemented in the operating room and moreover the quality of RNA obtained from human colon tissues by this method is superior to the one obtained by snap freezing.

• MeSH
• DNA nádorová genetika   MeSH
• DNA-topoisomerasy I genetika   MeSH
• dusík MeSH
• fixace tkání metody   MeSH
• karcinom chemie   chirurgie   MeSH
• kolon chemie   MeSH
• kryoprezervace přístrojové vybavení   metody   MeSH
• kvantitativní polymerázová řetězová reakce přístrojové vybavení   metody   MeSH
• lidé MeSH
• nádorové proteiny biosyntéza   genetika   MeSH
• nádory tračníku chemie   chirurgie   MeSH
• ochrana biologická přístrojové vybavení   metody   MeSH
• odběr biologického vzorku přístrojové vybavení   metody   MeSH
• regulace genové exprese u nádorů MeSH
• reprodukovatelnost výsledků MeSH
• ribozomální DNA genetika   MeSH
• řízení kvality MeSH
• RNA nádorová analýza   genetika   izolace a purifikace   MeSH
• RNA ribozomální 18S genetika   MeSH
• roztoky pro uchovávání orgánů MeSH
• rychlé screeningové testy přístrojové vybavení   metody   MeSH
• stanovení celkové genové exprese metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• DNA nádorová MeSH
• DNA-topoisomerasy I MeSH
• dusík MeSH
• nádorové proteiny MeSH
• ribozomální DNA MeSH
• RNA nádorová MeSH
• RNA ribozomální 18S MeSH
• roztoky pro uchovávání orgánů MeSH
• TOP1 protein, human MeSH Prohlížeč

Integrity of the mRNA in clinical samples has major impact on the quality of measured expression levels. This is independent of the measurement technique being next generation sequencing (NGS), Quantitative real-time PCR (qPCR) or microarray profiling. If mRNA is highly degraded or damaged, measured data will be very unreliable and the whole study is likely a waste of time and money. It is therefore common strategy to test the quality of RNA in samples before conducting large and costly studies. Most methods today to assess the quality of RNA are ignorant to the nature of the RNA and, therefore, reflect the integrity of ribosomal RNA, which is the dominant species, rather than of mRNAs, microRNAs and long non-coding RNAs, which usually are the species of interest. Here, we present a novel molecular approach to assess the quality of the targeted RNA species by measuring the differential amplification (ΔAmp) of an Endogenous RNase Resistant (ERR) marker relative to a reference gene, optionally combined with the measurement of two amplicons of different lengths. The combination reveals any mRNA degradation caused by ribonucleases as well as physical, chemical or UV damage. ΔAmp has superior sensitivity to common microfluidic electrophoretic methods, senses the integrity of the actual targeted RNA species, and allows for a smoother and more cost efficient workflow.

• Klíčová slova
• DAmp, Differential amplicons, Endogenous RNase resistant marker, RNA integrity, RNA quality, ΔAmp,
• Publikační typ
• časopisecké články MeSH

The precision and reliability of quantitative nucleic acid analysis depends on the quality of the sample analyzed and the integrity of the nucleic acids. The integrity of RNA is currently primarily assessed by the analysis of ribosomal RNA, which is the by far dominant species. The extrapolation of these results to mRNAs and microRNAs, which are structurally quite different, is questionable. Here we show that ribosomal and some nucleolar and mitochondrial RNAs, are highly resistant to naturally occurring post-mortem degradation, while mRNAs, although showing substantial internal variability, are generally much more prone to nucleolytic degradation. In contrast, all types of RNA show the same sensitivity to heat. Using qPCR assays targeting different regions of mRNA molecules, we find no support for 5' or 3' preferentiality upon post-mortem degradation.

• Klíčová slova
• Degradation, RNA integrity, RNA quality, RQI, RT-qPCR,
• Publikační typ
• časopisecké články MeSH

We report an improved method for total nucleic acids extraction from rumen content samples. The method employs bead beating, and phenol-chloroform extraction followed by saline-alcohol precipitation. Total nucleic acids and RNA yield and purity were assessed by spectrophotometric measurements; RNA integrity was estimated using Agilent RNA 6000 Nano Kit on an Agilent 2100 Bioanalyzer. The method provided total nucleic acids and RNA extracts of good quantity and quality. The extraction is not time consuming and it is valuable for ecological studies of rumen microbial community structure and gene expression.

• MeSH
• bachor mikrobiologie   MeSH
• DNA izolace a purifikace   MeSH
• genomika metody   MeSH
• mikrobiologické techniky metody   MeSH
• molekulární biologie metody   MeSH
• RNA izolace a purifikace   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• RNA MeSH