BACKGROUND: Glioblastoma is the commonest malignant brain tumor and has a very poor prognosis. Reduced expression of the MGMT gene (10q26.3), influenced primarily by the methylation of two differentially methylated regions (DMR1 and DMR2), is associated with a good response to temozolomide treatment. However, suitable methods for detecting the methylation of the MGMT gene promoter and setting appropriate cutoff values are debated. RESULTS: A cohort of 108 patients with histologically and genetically defined glioblastoma was retrospectively examined with methylation-specific Sanger sequencing (sSeq) and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) methods. The DMR2 region was methylated in 29% of samples, whereas DMR1 was methylated in 12% of samples. Methylation detected with the MS-MLPA method using probes MGMT_215, MGMT_190, and MGMT_124 from the ME012-A1 kit (located in DMR1 and DMR2) correlated with the methylation of the corresponding CpG dinucleotides detected with sSeq (p = 0.005 for probe MGMT_215; p < 0.001 for probe MGMT_190; p = 0.016 for probe MGMT_124). The threshold for methylation detection with the MS-MLPA method was calculated with a ROC curve analysis and principal components analysis of the data obtained with the MS-MLPA and sSeq methods, yielding a weighted value of 0.362. Thus, methylation of the MGMT gene promoter was confirmed in 36% of samples. These patients had statistically significantly better overall survival (p = 0.003). CONCLUSIONS: Our results show that the threshold for methylation detection with the MS-MLPA method determined here is useful from a diagnostic perspective because it allows the stratification of patients who will benefit from specific treatment protocols, including temozolomide. Detailed analysis of the MGMT gene promoter enables the more-precise and personalized treatment of patients with glioblastoma.

• MeSH
• CpG Islands genetics   MeSH
• DNA Modification Methylases * genetics   MeSH
• Adult MeSH
• DNA Repair Enzymes * genetics   MeSH
• Glioblastoma * genetics   drug therapy   MeSH
• Middle Aged MeSH
• Humans MeSH
• DNA Methylation * genetics   MeSH
• Tumor Suppressor Proteins * genetics   MeSH
• Brain Neoplasms * genetics   MeSH
• Promoter Regions, Genetic * genetics   MeSH
• Retrospective Studies MeSH
• Sequence Analysis, DNA methods   MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Temozolomide therapeutic use   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Validation Study MeSH

BACKGROUND: Juvenile granulosa cell tumor (JGCT) of the ovary is a rare tumor with distinct clinicopathological and hormonal features primarily affecting young women and children. We conducted a complex clinicopathological, immunohistochemical, and molecular analysis of five cases of JGCT. METHODS: The immunohistochemical examination was performed with 32 markers, including markers that have not been previously investigated. Moreover, DNA next-generation sequencing (NGS) and PTEN methylation analysis was performed. RESULT: We found the expression of calretinin, inhibin A, SF1, FOXL2, CD99, CKAE1/3, ER, PR, AR in all cases. WT1 was expressed in one case. Conversely, the expression of p16, OCT3/4, SALL4, GATA3, Napsin A, SATB2, MUC4, TTF1, and CAIX was completely negative. All tumors showed the wild-type pattern of p53 expression. Regarding predictive markers, all tumors were HER2 negative and did not express PD-L1. Mismatch repair proteins (MMR) showed no loss or restriction of expression, similarly to ARID1A, DPC4, BRG1, and INI1. The molecular analysis revealed AKT1 internal tandem duplication in two tumors. Two other cases exhibited mutations in TERT and EP400 and both developed recurrence. All AKT1-wild type tumors exhibited immunohistochemical loss of PTEN expression. However, no mutations, deletions (as assessed by CNV analysis), or promoter hypermethylation in the PTEN gene were detected. CONCLUSION: The results of our study further support the hypothesis that the pathogenesis of JGCT may be driven by activation of the PIK3/AKT/mTOR pathway. These findings could potentially have future therapeutic implications, as treatment strategies targeting the PTEN/mTOR pathways are currently under investigation.

• MeSH
• Child MeSH
• Phosphatidylinositol 3-Kinases genetics   metabolism   MeSH
• PTEN Phosphohydrolase genetics   metabolism   MeSH
• Immunohistochemistry * MeSH
• Humans MeSH
• DNA Methylation MeSH
• Adolescent MeSH
• Granulosa Cell Tumor * pathology   genetics   metabolism   MeSH
• Biomarkers, Tumor * genetics   analysis   metabolism   MeSH
• Ovarian Neoplasms * pathology   genetics   metabolism   MeSH
• Proto-Oncogene Proteins c-akt * metabolism   genetics   MeSH
• Signal Transduction * MeSH
• TOR Serine-Threonine Kinases * metabolism   MeSH
• Check Tag
• Child MeSH
• Humans MeSH
• Adolescent MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

The mechanism of rotator cuff injury remains to be elucidated. And COX-2 plays a dual role in skeletal muscle injury and regeneration, would be associated with the development of rotator cuff injury. Therefore, we chose human skeletal muscle cells (HSKMC) as an in vitro muscle tissue model and transfected lentivirus with overexpressed COX-2 to simulate the in vitro environment of rotator cuff injury. To investigate the specific molecular biological mechanism of COX-2, transcriptome sequencing (RNA-Seq) was used to analyze the differentially expressed mRNAs in HSKMC overexpressing COX-2. Enrichment analysis was performed to analyze these differentially expressed genes and real-time quantitative PCR (RT-qPCR) was used to examine the mRNA levels of genes induced by overexpression. Subsequently, the role of COX-2 in cell proliferation was confirmed by cell counting kit-8 (CCK-8), and focal adhesion kinase (FAK) and signal transducer and activator of transcription 3 (STAT3) phosphorylation induced by COX-2 was utilized by western blotting (WB). The results showed that total of 30,759 differentially expressed genes were obtained, and the expression of CYP4F3 and GPR87 was significantly increased. COX-2 could bind CYP4F3 and GPR87 and co-localize with them in the cytoplasm. Finally, COX-2 promoted the proliferation of human skeletal muscle cells by activating the FAK and STAT3 pathways.

• MeSH
• Cyclooxygenase 2 * metabolism   genetics   MeSH
• Muscle Fibers, Skeletal metabolism   enzymology   pathology   MeSH
• Muscle, Skeletal metabolism   pathology   MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Rotator Cuff Injuries * metabolism   pathology   enzymology   genetics   MeSH
• Cell Proliferation MeSH
• STAT3 Transcription Factor metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

In this study, we employed short- and long-read sequencing technologies to delineate the transcriptional architecture of the human monkeypox virus and to identify key regulatory elements that govern its gene expression. Specifically, we conducted a transcriptomic analysis to annotate the transcription start sites (TSSs) and transcription end sites (TESs) of the virus by utilizing Cap Analysis of gene expression sequencing on the Illumina platform and direct RNA sequencing on the Oxford Nanopore technology device. Our investigations uncovered significant complexity in the use of alternative TSSs and TESs in viral genes. In this research, we also detected the promoter elements and poly(A) signals associated with the viral genes. Additionally, we identified novel genes in both the left and right variable regions of the viral genome.IMPORTANCEGenerally, gaining insight into how the transcription of a virus is regulated offers insights into the key mechanisms that control its life cycle. The recent outbreak of the human monkeypox virus has underscored the necessity of understanding the basic biology of its causative agent. Our results are pivotal for constructing a comprehensive transcriptomic atlas of the human monkeypox virus, providing valuable resources for future studies.

• MeSH
• Genome, Viral MeSH
• Humans MeSH
• Transcription Initiation Site * MeSH
• Promoter Regions, Genetic MeSH
• RNA, Viral genetics   MeSH
• Sequence Analysis, RNA * methods   MeSH
• Gene Expression Profiling MeSH
• Transcriptome * MeSH
• Monkeypox virus genetics   MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Alternative polyadenylation (APA) modulates mRNA processing in the 3'-untranslated regions (3' UTR), affecting mRNA stability and translation efficiency. Research into genetically regulated APA has the potential to provide insights into cancer risk. In this study, we conducted large APA-wide association studies to investigate associations between APA levels and cancer risk. Genetic models were built to predict APA levels in multiple tissues using genotype and RNA sequencing data from 1,337 samples from the Genotype-Tissue Expression project. Associations of genetically predicted APA levels with cancer risk were assessed by applying the prediction models to data from large genome-wide association studies of six common cancers among European ancestry populations: breast, ovarian, prostate, colorectal, lung, and pancreatic cancers. A total of 58 risk genes (corresponding to 76 APA sites) were associated with at least one type of cancer, including 25 genes previously not linked to cancer susceptibility. Of the identified risk APAs, 97.4% and 26.3% were supported by 3'-UTR APA quantitative trait loci and colocalization analyses, respectively. Luciferase reporter assays for four selected putative regulatory 3'-UTR variants demonstrated that the risk alleles of 3'-UTR variants, rs324015 (STAT6), rs2280503 (DIP2B), rs1128450 (FBXO38), and rs145220637 (LDHA), significantly increased the posttranscriptional activities of their target genes compared with reference alleles. Furthermore, knockdown of the target genes confirmed their ability to promote proliferation and migration. Overall, this study provides insights into the role of APA in the genetic susceptibility to common cancers. Significance: Systematic evaluation of associations of alternative polyadenylation with cancer risk reveals 58 putative susceptibility genes, highlighting the contribution of genetically regulated alternative polyadenylation of 3'UTRs to genetic susceptibility to cancer.

• MeSH
• 3' Untranslated Regions * genetics   MeSH
• Genome-Wide Association Study * MeSH
• Genetic Predisposition to Disease * MeSH
• Polymorphism, Single Nucleotide MeSH
• Humans MeSH
• Quantitative Trait Loci MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Cell Line, Tumor MeSH
• Neoplasms * genetics   MeSH
• Polyadenylation * MeSH
• Gene Expression Regulation, Neoplastic MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

AIM: The transcriptional factor HIF-1α is recognized for its contribution to cardioprotection against acute ischemia/reperfusion injury. Adaptation to chronic hypoxia (CH) is known to stabilize HIF-1α and increase myocardial ischemic tolerance. However, the precise role of HIF-1α in mediating the protective effect remains incompletely understood. METHODS: Male wild-type (WT) mice and mice with partial Hif1a deficiency (hif1a +/-) were exposed to CH for 4 weeks, while their respective controls were kept under normoxic conditions. Subsequently, their isolated perfused hearts were subjected to ischemia/reperfusion to determine infarct size, while RNA-sequencing of isolated cardiomyocytes was performed. Mitochondrial respiration was measured to evaluate mitochondrial function, and western blots were performed to assess mitophagy. RESULTS: We demonstrated enhanced ischemic tolerance in WT mice induced by adaptation to CH compared with their normoxic controls and chronically hypoxic hif1a +/- mice. Through cardiomyocyte bulk mRNA sequencing analysis, we unveiled significant reprogramming of cardiomyocytes induced by CH emphasizing mitochondrial processes. CH reduced mitochondrial content and respiration and altered mitochondrial ultrastructure. Notably, the reduced mitochondrial content correlated with enhanced autophagosome formation exclusively in chronically hypoxic WT mice, supported by an increase in the LC3-II/LC3-I ratio, expression of PINK1, and degradation of SQSTM1/p62. Furthermore, pretreatment with the mitochondrial division inhibitor (mdivi-1) abolished the infarct size-limiting effect of CH in WT mice, highlighting the key role of mitophagy in CH-induced cardioprotection. CONCLUSION: These findings provide new insights into the contribution of HIF-1α to cardiomyocyte survival during acute ischemia/reperfusion injury by activating the selective autophagy pathway.

• MeSH
• Hypoxia-Inducible Factor 1, alpha Subunit * metabolism   genetics   MeSH
• Adaptation, Physiological physiology   MeSH
• Hypoxia * metabolism   MeSH
• Myocardial Infarction * metabolism   pathology   genetics   MeSH
• Myocytes, Cardiac metabolism   pathology   MeSH
• Mitophagy * physiology   MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Rhabdomyosarcoma (RMS) of the urinary bladder in adults and elderly is an exceptionally rare neoplasm that displays poorly differentiated solid (alveolar-like) small cell pattern, frequently indistinguishable from small cell neuroendocrine carcinoma (SCNEC). However, the histogenesis of RMS and SCNEC and their inter-relationship have not been well studied and remained controversial. We herein analyzed 23 SCNEC and 3 small round cell RMS of the bladder for neuroendocrine (synaptophysin + chromogranin A) and myogenic (desmin + myogenin) marker expression and for TERT promoter mutations. In addition, the RMS cohort and one SCNEC that was revised to RMS were tested for gene fusions using targeted RNA sequencing (TruSight Illumina Panel which includes FOXO1 and most of RMS-related other genes). Overall, significant expression of myogenin and desmin was observed in one of 23 original SCNEC justifying a revised diagnosis to RMS. On the other hand, diffuse expression of synaptophysin was noted in 2 of the 4 RMS, but chromogranin A was not expressed in 3 RMS tested. TERT promoter mutations were detected in 15 of 22 (68%) SCNEC and in two of three (67%) assessable RMS cases, respectively. None of the four RMS cases had gene fusions. Our data highlights phenotypic and genetic overlap between SCNEC and RMS of the urinary bladder. High frequency of TERT promoter mutations in SCNEC is in line with their presumable urothelial origin. In addition, the presence of TERT promoter mutation in 2 of 3 RMS and lack of FOXO1 and other gene fusions in all 4 RMSs suggest a mucosal (urothelial) origin, probably representing extensive monomorphic rhabdomyoblastic transdifferentiation in SCNEC.

• MeSH
• Cell Differentiation MeSH
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Carcinoma, Small Cell * genetics   pathology   MeSH
• Mutation MeSH
• Biomarkers, Tumor * genetics   analysis   MeSH
• Urinary Bladder Neoplasms * pathology   genetics   MeSH
• Carcinoma, Neuroendocrine * pathology   genetics   MeSH
• Rhabdomyosarcoma * genetics   pathology   MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Telomerase genetics   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH

There is an increasing demand for bioinoculants based on plant growth-promoting rhizobacteria (PGPR) for use in agricultural ecosystems. However, there are still concerns and limited data on their reproducibility in different soil types and their effects on endemic rhizosphere communities. Therefore, this study explored the effects of inoculating the PGPR, Pseudomonas fluorescens strain UM270, on maize growth (Zea mays L.) and its associated rhizosphere bacteriome by sequencing the 16S ribosomal genes under greenhouse conditions. The results showed that inoculation with PGPR P. fluorescens UM270 improved shoot and root dry weights, chlorophyll concentration, and total biomass in the three soil types evaluated (clay, sandy-loam, and loam) compared to those of the controls. Bacterial community analysis of the three soil types revealed that maize plants inoculated with the UM270 strain showed a significant increase in Proteobacteria and Acidobacteria populations, whereas Actinobacteria and Bacteroidetes decreased. Shannon, Pielou, and Faith alpha-biodiversity indices did not reveal significant differences between treatments. Beta diversity revealed a bacterial community differential structure in each soil type, with some variation among treatments. Finally, some bacterial groups were found to co-occur and co-exclude with respect to UM270 inoculation. Considered together, these results show that PGPR P. fluorescens UM270 increases maize plant growth and has an important effect on the resident rhizobacterial communities of each soil type, making it a potential agricultural biofertilizer.

• MeSH
• Bacteria classification   genetics   isolation & purification   growth & development   MeSH
• Biodiversity MeSH
• Biomass MeSH
• Phylogeny MeSH
• Plant Roots * microbiology   growth & development   MeSH
• Zea mays * microbiology   growth & development   MeSH
• Pseudomonas fluorescens * genetics   growth & development   physiology   MeSH
• Soil * chemistry   MeSH
• Soil Microbiology * MeSH
• Rhizosphere * MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Publication type
• Journal Article MeSH

Východiska: N-myc downstream-regulovaný gen 1 (NDRG1) má významnou funkci při progresi nádorů. U karcinomu prostaty (prostate cancer – PCa) však regulační mechanizmus NDRG1 zůstává nejasný. Materiál a metody: Hladiny exprese miR-96-5p a NDRG1 byly hodnoceny v buněčných liniích PCa a v tkáních prostaty a validovány ve veřejných databázích pomocí polymerázové řetězové reakce v reálném čase, analýzy western blot a imunohistochemie. Funkce miR-96-5p a NDRG1 byla zkoumána pomocí testů hojení ran a transwell testů in vitro a testu myšího xenoimplantátu in vivo. Dráha regulovaná pomocí NDRG1 byla testována technikou sekvenování nové generace. K detekci vztahu mezi miR-96-5p, NDRG1 a NF-kB dráhou byl použit imunofluorescenční test a test s luciferázou. Výsledky: Nadměrná exprese NDRG1 potlačuje migraci, invazi a epiteliálně-mezenchymální přechod (EMT) in vitro a inhibuje metastázy in vivo. Navíc miR-96-5p přispívá k deficitu NDRG1 a podporuje migraci a invazi buněk PCa. Kromě toho ztráta NDRG1 aktivuje dráhu NF-kB, která stimuluje fosforylaci p65 a IKBa a indukuje EMT v PCa. Závěr: MiR-96-5p podporuje migraci a invazi PCa tím, že cílí na NDRG1 a reguluje dráhu NF-kB.

Background: The N-myc downstream-regulated gene 1 (NDRG1) has been discovered as a significant gene in the progression of cancers. However, the regulatory mechanism of NDRG1 remained obscure in prostate cancer (PCa). Methods: The miR-96-5p and NDRG1 expression levels were evaluated in PCa cell lines, and prostate tissues, and validated in public databases by real-time polymerase chain reaction, western blot analysis, and immunohistochemistry. The function of miR-96-5p and NDRG1 were investigated by scratch assay and transwell assays in vitro, and mouse xenograft assay in vivo. The candidate pathway regulated by NDRG1 was conducted by the next-generation gene sequencing technique. Immunofluorescence and luciferase assays were used to detect the relation between miR-96-5p, NDRG1, and NF-kB pathway. Results: Overexpressing NDRG1 suppresses the migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro, and inhibits metastasis in vivo. Moreover, miR-96-5p contributes to NDRG1 deficiency and promotes PCa cell migration and invasion. Furthermore, NDRG1 loss activates the NF-kB pathway, which stimulates p65 and IKBa phosphorylation and induces EMT in PCa. Conclusions: MiR-96-5p promotes the migration and invasion of PCa by targeting NDRG1 and regulating the NF-kB pathway.

• Keywords
• NDRG1, miR-96-5p,
• MeSH
• Epithelial-Mesenchymal Transition MeSH
• Genetic Techniques MeSH
• Immunohistochemistry methods   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Prostatic Neoplasms * genetics   physiopathology   MeSH
• NF-kappa B * MeSH
• Cell Movement MeSH
• Polymerase Chain Reaction methods   MeSH
• Sequence Analysis MeSH
• Signal Transduction MeSH
• Transfection methods   MeSH
• Blotting, Western methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Clinical Study MeSH

In the past few decades, the pressure of higher food production to satisfy the demand of ever rising population has inevitably increased the use synthetic agrochemicals which have deterioration effects. Biostimulants containing beneficial microbes (single inoculants and microbial consortium) were found as an ideal substitute of synthetic chemical fertilizers. In recent years, microbial consortium is known as a better bioinoculant in comparison to single inoculant bioformulation because of multifarious plant growth-promoting advantages. Looking at the advantageous effect of consortium, in present investigation, different bacteria were isolated from rhizospheric soil and plant samples collected from the Himalayan mountains on the green slopes of the Shivaliks, Himachal Pradesh. The isolated bacteria were screened for nitrogen (N) fixation, phosphorus (P) solubilization and potassium (K) solubilization plant growth promoting attributes, and efficient strains were identified through 16S rRNA gene sequencing and BLASTn analysis. The bacteria showing a positive effect in NPK uptake were developed as bacterial consortium for the growth promotion of eggplant crop. A total of 188 rhizospheric and endophytic bacteria were sorted out, among which 13 were exhibiting nitrogenase activity, whereas 43 and 31 were exhibiting P and K solubilization traits, respectively. The selected three efficient and potential bacterial strains were identified using 16S rRNA gene sequencing as Enterobacter ludwigii EU-BEN-22 (N-fixer; 35.68 ± 00.9 nmol C2H4 per mg protein per h), Micrococcus indicus EU-BRP-6 (P-solubilizer; 201 ± 0.004 mg/L), and Pseudomonas gessardii EU-BRK-55 (K-solubilizer; 51.3 ± 1.7 mg/mL), and they were used to develop a bacterial consortium. The bacterial consortium evaluation on eggplant resulted in the improvement of growth (root/shoot length and biomass) and physiological parameters (chlorophyll, carotenoids, total soluble sugar, and phenolic content) of the plants with respect to single culture inoculation, chemical fertilizer, and untreated control. A bacterial consortium having potential to promote plant growth could be used as bioinoculant for horticulture crops growing in hilly regions.

• MeSH
• Bacteria * genetics   classification   metabolism   isolation & purification   growth & development   MeSH
• Potassium metabolism   MeSH
• Nitrogen metabolism   MeSH
• Nitrogen Fixation * MeSH
• Phosphorus * metabolism   MeSH
• Phylogeny MeSH
• Plant Roots microbiology   MeSH
• Microbial Consortia * MeSH
• Soil Microbiology * MeSH
• Rhizosphere MeSH
• RNA, Ribosomal, 16S * genetics   MeSH
• Solanum melongena * microbiology   MeSH
• Plant Development MeSH
• Publication type
• Journal Article MeSH