DNA Metabolism Research Articles

Real-time imaging of drug-induced trapping of cellular topoisomerases and poly(ADP-ribose) polymerase 1 at the single-molecule level.

Topoisomerases (TOP1, TOP2α, and β) are nuclear enzymes crucial for virtually all aspects of DNA metabolisms. They also are the targets of important anti-tumor chemotherapeutics that act by trapping the otherwise reversible topoisomerase-DNA covalent complex intermediates (TOPccs) that are formed during their catalytic reactions, resulting in long-lived topoisomerase DNA-protein crosslinks (TOP-DPCs) that interfere with DNA transactions. The Poly(ADP-ribose) polymerase (PARP) family protein PARP1 is activated by DNA damage to recruit DNA repair proteins, and PARP inhibitors are another class of commonly used chemotherapeutics, which bind and trap PARP molecules on DNA. To date, the trapping of TOPccs and PARP by their respective inhibitors can only be measured by immune-biochemical methods in cells. Here, we developed an imaging-based approach enabling real-time monitoring of drug-induced trapping of TOPccs and PARP1 in live cells at the single-molecule level. Capitalizing on this approach, we calculated the fraction of self-fluorescence tag-labeled topoisomerases and PARP single-molecules that are trapped by their respective inhibitors in real time. This novel technique should help elucidate the molecular processes that repair TOPcc and PARP trapping and facilitate the development of novel topoisomerase and PARP inhibitor-based therapies.

Structural characterization of human RPA70N association with DNA damage response proteins.

The heterotrimeric Replication protein A (RPA) is the ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein and participates in nearly all aspects of DNA metabolism, especially DNA damage response. The N-terminal OB domain of the RPA70 subunit (RPA70N) is a major protein-protein interaction element for RPA and binds to more than 20 partner proteins. Previous crystallography studies of RPA70N with p53, DNA2 and PrimPol fragments revealed that RPA70N binds to amphipathic peptides that mimic ssDNA. NMR chemical-shift studies also provided valuable information on the interaction of RPA70N residues with target sequences. However, it is still unclear how RPA70N recognizes and distinguishes such a diverse group of target proteins. Here, we present high-resolution crystal structures of RPA70N in complex with peptides from eight DNA damage response proteins. The structures show that, in addition to the ssDNA mimicry mode of interaction, RPA70N employs multiple ways to bind its partners. Our results advance the mechanistic understanding of RPA70N-mediated recruitment of DNA damage response proteins.

Genomic Instability of G-Quadruplex Sequences in Escherichia coli: Roles of DinG, RecG, and RecQ Helicases.

Guanine-rich DNA can fold into highly stable four-stranded DNA structures called G-quadruplexes (G4). Originally identified in sequences from telomeres and oncogene promoters, they can alter DNA metabolism. Indeed, G4-forming sequences represent obstacles for the DNA polymerase, with important consequences for cell life as they may lead to genomic instability. To understand their role in bacterial genomic instability, different G-quadruplex-forming repeats were cloned into an Escherichia coli genetic system that reports frameshifts and complete or partial deletions of the repeat when the G-tract comprises either the leading or lagging template strand during replication. These repeats formed stable G-quadruplexes in single-stranded DNA but not naturally supercoiled double-stranded DNA. Nevertheless, transcription promoted G-quadruplex formation in the resulting R-loop for (G3T)4 and (G3T)8 repeats. Depending on genetic background and sequence propensity for structure formation, mutation rates varied by five orders of magnitude. Furthermore, while in vitro approaches have shown that bacterial helicases can resolve G4, it is still unclear whether G4 unwinding is important in vivo. Here, we show that a mutation in recG decreased mutation rates, while deficiencies in the structure-specific helicases DinG and RecQ increased mutation rates. These results suggest that G-quadruplex formation promotes genetic instability in bacteria and that helicases play an important role in controlling this process in vivo.

Meiosis in budding yeast.

Meiosis is a specialized cell division program that is essential for sexual reproduction. The two meiotic divisions reduce chromosome number by half, typically generating haploid genomes that are packaged into gametes. To achieve this ploidy reduction, meiosis relies on highly unusual chromosomal processes including the pairing of homologous chromosomes, assembly of the synaptonemal complex, programmed formation of DNA breaks followed by their processing into crossovers, and the segregation of homologous chromosomes during the first meiotic division. These processes are embedded in a carefully orchestrated cell differentiation program with multiple interdependencies between DNA metabolism, chromosome morphogenesis, and waves of gene expression that together ensure the correct number of chromosomes is delivered to the next generation. Studies in the budding yeast Saccharomyces cerevisiae have established essentially all fundamental paradigms of meiosis-specific chromosome metabolism and have uncovered components and molecular mechanisms that underlie these conserved processes. Here, we provide an overview of all stages of meiosis in this key model system and highlight how basic mechanisms of genome stability, chromosome architecture, and cell cycle control have been adapted to achieve the unique outcome of meiosis.

The combined signatures of telomere and immune cell landscape provide a prognostic and therapeutic biomarker in glioma.

Gliomas, the most prevalent primary malignant tumors of the central nervous system in adults, exhibit slow growth in lower-grade gliomas (LGG). However, the majority of LGG cases progress to high-grade gliomas, posing challenges for prognostication. The tumor microenvironment (TME), characterized by telomere-related genes and immune cell infiltration, strongly influences glioma growth and therapeutic response. Therefore, our objective was to develop a Telomere-TME (TM-TME) classifier that integrates telomere-related genes and immune cell landscape to assess prognosis and therapeutic response in glioma. This study encompassed LGG patients from the TCGA and CCGA databases. TM score and TME score were derived from the expression signatures of telomere-related genes and the presence of immune cells in LGG, respectively. The TM-TME classifier was established by combining TM and TME scores to effectively predict prognosis. Subsequently, we conducted Kaplan-Meier survival estimation, univariate Cox regression analysis, and receiver operating characteristic curves to validate the prognostic prediction capacity of the TM-TME classifier across multiple cohorts. Gene Ontology (GO) analysis, biological processes, and proteomaps were performed to annotate the functional aspects of each subgroup and visualize the cellular signaling pathways. The TM_low+TME_high subgroup exhibited superior prognosis and therapeutic response compared to other subgroups (P<0.001). This finding could be attributed to distinct tumor somatic mutations and cancer cellular signaling pathways. GO analysis indicated that the TM_low+TME_high subgroup is associated with the neuronal system and modulation of chemical synaptic transmission. Conversely, the TM_high+TME_low subgroup showed a strong association with cell cycle and DNA metabolic processes. Furthermore, the classifier significantly differentiated overall survival in the TCGA LGG cohort and served as an independent prognostic factor for LGG patients in both the TCGA cohort (P<0.001) and the CGGA cohort (P<0.001). Overall, our findings underscore the significance of the TM-TME classifier in predicting prognosis and immune therapeutic response in glioma, shedding light on the complex immune landscape within each subgroup. Additionally, our results suggest the potential of integrating risk stratification with precision therapy for LGG.

Proteomic profiles and leaf antioxidative metabolism in cacao juvenile plants subjected to Pb and Pb+Fe doses and to soil flooding

Under natural conditions, plants can be affected by several types of abiotic stresses, such as flooding and the accumulation of toxic metals in the soil. Pb is a non-essential metallic element for plants and is highly toxic for living beings. Fe, as an essential micronutrient, is involved in fundamental plant processes, such as photosynthesis. The main objective of this work was to evaluate the proteomic profiles and the activity of key enzymes of leaf antioxidant metabolism in juvenile plants of the CCN 51 cacao genotype, grown in flooding soil and subjected to a dose of Pb and to different equimolar doses of Pb+Fe. Doses of Pb and Pb+Fe did not interfere with the predawn leaf water potential (ΨWL) of plants, but soil flooding was a key factor for the decline of ΨWL. There was a differential accumulation of Pb and Fe in the roots and leaves, both in flooded soil and non-flooded. Most of Fe and Pb uptake by the root system predominantly accumulated in the roots and only a small fraction was translocated to the shoot, regardless of the water content in the soil. Flooding and the variation of Pb+Fe doses in the soil did not change the transport of Pb to the plant shoot since there were no major alterations in the levels of Pb in the leaves. Leaf enzymatic antioxidant metabolism was altered, mainly by flooding, in relation to Pb or Pb+Fe doses applied in soil. The time of exposure of plants to stressor factors was important for the changes in the activity of key enzymes of the leaf antioxidant metabolism. Soil flooding, with doses of 2 mmol Pb kg−1 soil + 0.5 mmol Fe kg−1 soil, at 6 and 24 h after treatment application, had a greater influence on ΨWL, antioxidant enzymatic metabolism and leaf proteome, and affected proteins related to photosynthesis, DNA, antioxidative system and carbohydrate metabolism, whose abundance was overexpressed or repressed compared to the control.

Transcriptome Analysis Reveals Changes in Whole Gene Expression, Biological Process, and Molecular Functions Induced by Nickel in Jack Pine (Pinus banksiana).

Understanding the genetic response of plants to nickel stress is a necessary step to improving the utility of plants in environmental remediation and restoration. The main objective of this study was to generate whole genome expression profiles of P. banksiana exposed to nickel ion toxicity compared to reference genotypes. Pinus banksiana seedlings were screened in a growth chamber setting using a high concentration of 1600 mg of nickel per 1 kg of soil. RNA was extracted and sequenced using the Illumina platform, followed by de novo transcriptome assembly. Overall, 25,552 transcripts were assigned gene ontology. The biological processes in water-treated samples were analyzed, and 55% of transcripts were distributed among five categories: DNA metabolic process (19.3%), response to stress (13.3%), response to chemical stimuli (8.7%), signal transduction (7.7%) and response to biotic stimulus (6.0%). For molecular function, the highest percentages of genes were involved in nucleotide binding (27.6%), nuclease activity (27.3%) and kinase activity (10.3%). Sixty-two percent of genes were associated with cellular compartments. Of these genes, 21.7% were found in the plasma membrane, 16.1% in the cytosol, 12.4% with the chloroplast and 11.9% in the extracellular region. Nickel ions induced changes in gene expression, resulting in the emergence of differentially regulated categories. Overall, there were significant changes in gene expression with a total 4128 genes upregulated and 3754 downregulated genes detected in nickel-treated genotypes compared to water-treated control plants. For biological processes, the highest percentage of upregulated genes in plants exposed to nickel were associated with the response to stress (15%), the response to chemicals (11,1%), carbohydrate metabolic processes (7.4%) and catabolic processes (7.4%). The largest proportions of downregulated genes were associated with the biosynthetic process (21%), carbohydrate metabolic process (14.3%), response to biotic stimulus (10.7%) and response to stress (10.7%). For molecular function, genes encoding for enzyme regulatory and hydrolase activities represented the highest proportion (61%) of upregulated gene. The majority of downregulated genes were involved in the biosynthetic processes. Overall, 58% of upregulated genes were located in the extracellular region and the nucleus, while 42% of downregulated genes were localized to the plasma membrane and 33% to the extracellular region. This study represents the first report of a transcriptome from a conifer species treated with nickel.

Generation of Rat Monoclonal Antibody for Human Nucleolin.

Nucleolin (NCL) is a multifunctional phosphoprotein that is mainly localized in the nucleolus, but it is also found in the nucleoplasm, cytoplasm, and cell membrane. The principal functions of NCL involve DNA and RNA metabolism, gene transcription and translation, ribosome biogenesis, and mRNA stability. It was also reported that the localization of human NCL (hNCL) is related to tumor malignancy. Therefore, analyzing the cellular dynamics of NCL could be useful. In this article, we describe rat monoclonal antibody (mAb) 6F9A6 that was generated against a hNCL peptide. This mAb recognizes endogenous human, monkey, dog, and mouse NCL and was shown to be useful in immunofluorescence staining, immunoprecipitation, and immunoblotting experiments in several cancer cell lines. We anticipate that the mAb 6F9A6 will be useful for functional analyses of hNCL in cancer cells.

Interplay between DNA methylation and metabolism: Implications in pathogenesis of vascular diseases

Interplay between DNA methylation and metabolism: Implications in pathogenesis of vascular diseases

GEMIN4, a potential therapeutic targets for patients with basal-like subtype breast cancer

BackgroundBasal-like breast cancer (BLBC) takes up about 10–20% of all breast cancer(BC), what’s more, BLBC has the lowest survival rate among all BC subtypes because of lacks of efficient treatment methods. We aimed to explore the molecules that can be used as diagnostic maker for BLBC at early stage and provide optimized treatment strategies for BLBC patients in this study.MethodsApply weighted gene co-expression network analysis (WGCNA) to identify gene modules related to BLBC;The functional enrichment of candidate genes related to BLBC in the red module of Go data package and KEGG analysis;Overlapping cross analysis of URGs and WGCNA to identify candidate genes in each BC subtype;Divide BCBL patients into high-risk and low-risk groups, and analyze the two groups of overall survival (OS) and relapse free survival (RFS);Screening of GEMIN4 dependent cell lines; QRT PCR was used to verify the expression of GEMIN4 transfected with siRNA; CCK8 was used to determine the effect of GEMIN4 on cell viability; Positive cell count detected by BrdU staining;GO and KEGG enrichment analysis of GEMIN4.ResultsThe "red module" has the highest correlation with BLBC, with 913 promising candidate genes identified from the red module;913 red module candidate genes related to BLBC participated in multiple GO terms, and KEGG enrichment analysis results mainly enriched in estrogen signaling pathways and pathways in cancer;There are 386 overlapping candidate genes among the 913 "red module" genes identified by 1893 common URG and WGCNA;In BLBC patients, 9 highly expressed genes are associated with OS. Five highly expressed genes are associated with RFS. Kaplan Meier survival analysis suggests that high GEMIN4 expression levels are associated with poor prognosis in BLBC patients;The GEMIN4 gene dependency score in HCC1143 and CAL120 cell lines is negative and low; Si-GEMIN4-1 can significantly reduce the mRNA expression of GEMIN4; Si-GEMIN4 can inhibit cell viability; Si-GEMIN4 can reduce the number of positive cells;GO enrichment analysis showed that GEMIN4 is associated with DNA metabolism processes and adenylate binding; KEGG pathway enrichment analysis shows that GEMIN4 is related to ribosome biogenesis in eukaryotes.ConclusionWe hypothesized that GEMIN4 may be the potential target for the treatment of BLBC.

Inhibition of Topoisomerases by Metal Thiosemicarbazone Complexes.

Topoisomerases, common targets for anti-cancer therapeutics, are crucial enzymes for DNA replication, transcription, and many other aspects of DNA metabolism. The potential anti-cancer effects of thiosemicarbazones (TSC) and metal-TSC complexes have been demonstrated to target several biological processes, including DNA metabolism. Human topoisomerases were discovered among the molecular targets for TSCs, and metal-chelated TSCs specifically displayed significant inhibition of topoisomerase II. The processes by which metal-TSCs or TSCs inhibit topoisomerases are still being studied. In this brief review, we summarize the TSCs and metal-TSCs that inhibit various types of human topoisomerases, and we note some of the key unanswered questions regarding this interesting class of diverse compounds.

Xiaoyaosan ameliorates CUMS-induced depressive-like and anorexia behaviors in mice via necroptosis related cellular senescence in hypothalamus

Ethnopharmacological relevanceDepression and anorexia often co-occur and share symptoms such as low mood, lack of energy, and weight loss. Xiaoyaosan is a classic formula comprising of a combination of eight herbs, possessing definitive therapeutic effects, minimal side effects, and economical benefits. It has been extensively employed in clinical treatment of ailments and symptoms such as depression, anxiety, and appetite problems. Nonetheless, its exact pharmacological mechanism with necroptosis remains incompletely explicit. Aim of the studyThe aim of this study is to explore the potential mechanisms of anti-depressive and appetite-regulating effects of the active ingredients in Xiaoyaosan, and to investigate whether there is a correlation with necroptosis. Materials and methodsThe network pharmacology method was conducted to identify active ingredients, which were used to predict the possible targets of Xiaoyaosan and explore the potential targets in treating depression and anorexia by overlapping with differentially expressed genes (DEGs) screened from GEO datasets (GSE125441, GSE198597, and GSE69151). Afterwards, the protein-protein interaction (PPI) network, enrichment analyses, hub gene identification, co-expression study and molecular docking were used to study the potential mechanism of Xiaoyaosan. Then, a mice model of depression was established by chronic unpredictable mild stress (CUMS) and the incidence of necroptosis in the hypothalamus of CUMS mice was investigated, while verifying the key therapeutic target of Xiaoyaosan. ResultsThrough network pharmacology research, it had been discovered that the 145 active ingredients of the 8 herbs in the Xiaoyaosan could regulate 198 disease targets. Through PPI network analysis and functional enrichment analysis, it had been found that the pharmacological mechanism of Xiaoyaosan mainly involved biological processes such as oxidative stress, kinase activity, and DNA metabolism. It is related to various pathways such as cellular senescence, immune inflammation, and the cell cycle, and 9 hub targets had been identified. Further analysis of the 9 hub targets and the key PPI network clusters clarified the key mechanisms by which Xiaoyaosan exerts anti-depressant and appetite regulating effects, possibly related to necroptosis-mediated cellular senescence. Molecular docking of the key indicators of cellular senescence screened by bioinformatics, SIRT1, ABL1, and MYC, revealed that the key component regulating SIRT1 is 2-[3,4-dihydroxyphenyl]-5,7-dihydroxy-6-[3-methylbut-2-enyl]chromone in licorice root, Glabridin in licorice root regulates ABL1, and β-sitosterol found in Chinese angelica, debark peony root, and fresh ginger regulates MYC. Finally, through in vivo experiments, the expression of necroptosis in the hypothalamus of CUMS mice was verified. The regulatory effects of Xiaoyaosan on key substances RIPK1, RIPK3, MLKL, and p-MLKL were determined, while regulating effects on SIRT1, ABL1, and MYC were also observed. ConclusionThe present study have revealed the common mechanism of Xiaoyaosan in treating depression and anorexia, indicating that the active ingredients of Xiaoyaosan may alleviate the symptoms of depression and anorexia by intervening in the pathways related to necroptosis and cellular senescence. The hub genes and common pathways identified by the study also provide new insights into the therapeutic targets of depression and anorexia, as well as the exploration of pharmacological mechanism of Xiaoyaosan.

A FRET-Based Assay for the Identification of PCNA Inhibitors.

Proliferating cell nuclear antigen (PCNA) is the key regulator of human DNA metabolism. One important interaction partner is p15, involved in DNA replication and repair. Targeting the PCNA-p15 interaction is a promising therapeutic strategy against cancer. Here, a Förster resonance energy transfer (FRET)-based assay for the analysis of the PCNA-p15 interaction was developed. Next to the application as screening tool for the identification and characterization of PCNA-p15 interaction inhibitors, the assay is also suitable for the investigation of mutation-induced changes in their affinity. This is particularly useful for analyzing disease associated PCNA or p15 variants at the molecular level. Recently, the PCNA variant C148S has been associated with Ataxia-telangiectasia-like disorder type 2 (ATLD2). ATLD2 is a neurodegenerative disease based on defects in DNA repair due to an impaired PCNA. Incubation time dependent FRET measurements indicated no effect on PCNAC148S-p15 affinity, but on PCNA stability. The impaired stability and increased aggregation behavior of PCNAC148S was confirmed by intrinsic tryptophan fluorescence, differential scanning fluorimetry (DSF) and asymmetrical flow field-flow fractionation (AF4) measurements. The analysis of the disease associated PCNA variant demonstrated the versatility of the interaction assay as developed.

A real-time fluorescent gp32 probe-based assay for monitoring single-stranded DNA-dependent DNA processing enzymes

Single-stranded DNA (ssDNA) generated during DNA replication, recombination and damage repair reactions is an important intermediate and ssDNA-binding proteins that binds these intermediates coordinate various DNA metabolic processes. Mechanistic details of these ssDNA-dependent processes can be explored by monitoring the generation and consumption of ssDNA in real time. In this work, a fluorescein-labeled gp32-based sensor was employed to continuously monitor various aspects of ssDNA-dependent DNA replication and recombination processes in real time. The gp32 protein probe displayed high sensitivity and specificity to a variety of ssDNA-dependent processes of T4 phage. Several applications of the probe are illustrated here: the solution dynamics of ssDNA-binding protein, protein-protein and protein-DNA interactions involving gp32 protein and its mode of interaction, ssDNA translocation and protein displacement activities of helicases, primer extension activity of DNA polymerase holoenzyme and nucleoprotein filament formation during DNA recombination. The assay has identified new protein-protein interactions of gp32 during T4 replication and recombination. The fluorescent probe described here can thus be used as a universal probe for monitoring in real time various ssDNA-dependent processes, which is based on a well-characterized and easy-to-express bacteriophage T4 gene 32 protein, gp32.

Acquisition, loss, and replication of functional modules promote the genetic diversity of Salmonella bacteriophages

Owing to the threats that Salmonella poses to public health and the abuse of antimicrobials, bacteriophage therapy against Salmonella is experiencing a resurgence. Although several phages have been reported as safe and efficient for controlling Salmonella, the genetic diversity and relatedness among Salmonella phages remain poorly understood. In this study, whole-genome sequences of 91 Salmonella bacteriophages were obtained from the National Center for Biological Information genome database. Phylogenetic analysis, mosaic structure comparisons, gene content analysis, and orthologue group clustering were performed. Phylogenetic analysis revealed four singletons and two major lineages (I–II), including five subdividing clades, of which Salmonella phages belonging to morphologically distinct families were clustered in the same clade. Chimeric structures (n = 31), holin genes (n = 18), lysin genes (n = 66), DNA packaging genes (n = 55), and DNA metabolism genes (n = 24) were present in these phages. Moreover, phages from different subdivided clusters harboured distinct genes associated with host cell lysis, DNA packaging, and DNA metabolism. Notably, phages belonging to morphologically distinct families shared common orthologue groups. Although several functional modules of phages SS1 and SE16 shared > 99% nucleotide sequence identity with phages SI2 and SI23, the major differences between these phages were the absence and replication of functional modules. The data obtained herein revealed the genetic diversity of Salmonella phages at genomic, structural, and gene content levels. The genetic diversity of Salmonella phages is likely owing to the acquisition, loss, and replication of functional modules.

Immunotherapy Efficacy-related Risk Classifier Differentiate Prognostic Characteristics of Gastric Cancer-A Large-scale Retrospective Study.

Prognostic signatures related to the efficacy of immunotherapy have not been determined in gastric cancer (GC). We identified the differentially expressed genes between the CR/PR and SD/PD groups with the R package "limma" (false discovery rate <0.05) in the IMvigor210 data set. The GSE13861 (n=65), GSE15459 (n=192), GSE26899 (n=93), GSE26901 (n=109), GSE28541 (n=40), GSE34942 (n=56), and GSE62254 (n=300) cohorts were merged into a training cohort (n=855). Univariate Cox regression analysis, LASSO penalized Cox regression analysis, and multivariate Cox regression analysis were jointly applied to construct the prognostic model. The Cancer Genome Atlas (TCGA)-STAD (n=371), GSE84437 (n=433), GSE26253 (n=432), and IMvigor210 (n=348) cohorts were utilized for external validation. The GC patients were divided into 16 subgroups according to clinical features for universal applicability validation. Repeated validation confirmed that the overall survival of the high-risk (HR) group was significantly reduced compared with that of the low-risk (LR) group. The HR group showed a higher infiltration abundance of regulatory T cells, macrophages, T follicular helper cells, and natural killer T cells, whereas the infiltration levels of activated CD4 T cells and monocytes were upregulated in the LR group. The calcium, TGF-β, MAPK, Hedgehog, and KRAS signaling pathways were overactivated in the HR group, while the hallmarks related to DNA damage repair and metabolism were enriched in the LR group. In addition, the LR group had high tumor mutation burden, FLG, and OBSCN mutations. A prognostic risk classifier for GC patients was identified and validated by carrying out a multicenter retrospective study.

Deinococcus lineage and Rad52 family-related protein DR0041 is involved in DNA protection and compaction

DR0041 ORF encodes an uncharacterized Deinococcus lineage protein. We earlier reported presence of DR0041 protein in DNA repair complexes of Ssb and RecA in Deinococcus radiodurans. Here, we systematically examined the role of DR0041 in DNA metabolism using various experimental methodologies including electrophoretic mobility assays, nuclease assays, strand exchange assays and transmission electron microscopy. Interaction between DR0041 and the C-terminal acidic tail of Ssb was assessed through co-expression and in vivo cross-linking studies. A knockout mutant was constructed to understand importance of DR0041 ORF for various physiological processes. Results highlight binding of DR0041 protein to single-stranded and double-stranded DNA, interaction with Ssb-coated single-stranded DNA without interference with RecA-mediated strand exchange, protection of DNA from exonucleases, and compaction of high molecular weight DNA molecules into tightly condensed forms. Bridging and compaction of sheared DNA by DR0041 protein might have implications in the preservation of damaged DNA templates to maintain genome integrity upon exposure to gamma irradiation. Our results suggest that DR0041 protein is dispensable for growth under standard growth conditions and following gamma irradiation but contributes to protection of DNA during transformation. We discuss the role of DR0041 protein from the perspective of protection of broken DNA templates and functional redundancy.

Comparative transcriptomic analysis provided insights into the local molecular responses of swimming crabs, Portunus trituberculatus and mud crabs, Scylla paramamosain to the infection of Ameson portunus (Microsporidia)

Microsporidiosis has been increasingly causing severe economic losses in aquaculture. Insights into the molecular mechanism of host responses to aquatic microsporidia infection will be beneficial for the formulation of control strategies of microsporidiosis in aquaculture. Ameson portunus is the recently identified causative agent of “toothpaste-like syndrome” in Portunus trituberculatus and “slurry-like syndrome” in Scylla paramamosain. Here, muscle tissues of these two crabs at the early and late disease stages of A. portunus infection were subjected to the comparative transcriptomic analysis to disclose differential molecular responses of this microsporidian infection and characterize the host's responses dynamics against the infection at these two stages. These differentially expressed genes (DEGs) were mainly involved in DNA replication, metabolism, and immunity. Among them, the DEGs associated with DNA replication were significantly up-regulated, indicating that A. portunus can achieve proliferation by regulating the cellular responses of host. The significant upregulation of DEGs associated with recognizing and removing DNA damage, homologous recombination, and mismatch repair suggested that A. portunus damaged to the genetic material of crab muscle cells, and host repaired DNA damage caused by the infection of A. portunus by enhancing homologous recombination. The DEGs associated with amino acid and carbohydrate metabolism were significantly down-regulated, suggesting that muscle metabolic function was impaired in severely infected crabs, and energy supply, regulation of oxidative stress, and resistance to pathogens of host were significantly affected at the late disease stage. It could possibly explain why diseased P. trituberculatus and S. paramamosain exhibited reduced food intake, sluggish movement, and slow growth. In response to the infection, the two crabs exhibited different immune responses. P. trituberculatus responded to the infection of A. portunus by phagocytosis and apoptosis, and S. paramamosain induced cytokine production by recognizing the pathogen and signaling downstream to eventually clear the invading cells of A. portunus.

FAM72 family proteins as poor prognostic markers in clear cell renal carcinoma

PurposeThis study aimed to investigate the prognostic significance of the Family with Sequence Similarity 72 member (FAM72) gene family in clear cell renal carcinoma (ccRCC) using a bioinformatic approach. Patients and methodsTo investigate the association between FAM72 and ccRCC, we utilized various databases and analysis tools, including TCGA, GEPIA, Metscape, cBioPortal, and MethSurv. We conducted an analysis of FAM72 expression levels in ccRCC tissues compared to normal kidney tissues and performed univariate and multivariate Cox analysis to determine the relationship between FAM72 expression and patient prognosis. Furthermore, we carried out Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) to identify enriched biological processes associated with FAM72 expression. Additionally, we analyzed immune cell infiltration and the level of methylation in ccRCC patients. Our bioinformatic analysis revealed that FAM72 expression levels were significantly higher in ccRCC tissues than in normal kidney tissues. High expression of FAM72 was associated with poor prognosis in ccRCC patients and was found to be an independent prognostic factor for ccRCC. GO and GSEA analyses indicated that FAM72 was enriched in biological processes related to mitosis, cell cycle, and DNA metabolism. Moreover, we found a significant correlation between FAM72 and immune cell infiltration and the level of methylation in ccRCC patients. ConclusionOur findings suggest that FAM72 could serve as an unfavorable prognostic molecular marker for ccRCC. A comprehensive understanding of FAM72 could provide crucial insights into tumor progression and prognosis.

Revealing the metabolic characteristics and nitrite accumulation mechanism of glycerol-driven partial denitrification process by batch tests and untargeted metabolomic analysis

Glycerol has been proven as an effective carbon source for partial denitrification (PD), while the underlying mechanism of the nitrite accumulation remains unknown. In this study, the nitrite accumulation characteristics was firstly investigated by batch tests. Denitrifying bacteria exhibited high nitrate to nitrite ratio (NTR) (84.6%) and nitrite accumulation rate (NAR) (30.23 mg N h−1 gMLVSS−1) in a typical PD process with sufficient glycerol supply. But when glycerol was exhausted, denitrifying bacteria entered endogenous PD process driven by polyhydroxyalkanoates (PHA) and glycogen, and the NTR and NAR significantly decreased to 63.9% and 1.74 mg N h−1 gMLVSS−1, respectively. More analysis found that keeping the endogenous carbon source at a relatively high level before PD reaction could further improved the NTR up to 99.7%. Metabolomic analysis showed that most required amino acids were synthesized in nitrite accumulation phase with active RNA metabolism. While in the subsequent nitrite reduction phase with limited glycerol, microbes tended to synthesize those amino acids with lower energetic cost, and activities related to DNA metabolism was more active in this phase. Moreover, reactive oxygen species generated in the glycerol degradation were deduced to be the key factor that induced the efficient nitrite accumulation. This study provided a comprehensive insight into the mechanism of glycerol-driven PD process.