Genomic Analysis Research Articles

Transcriptomics reveals dynamic changes in the “gene profiles” of rat supraspinatus tendon at three different time points after diabetes induction

ObjectiveThere is increasing evidence that type 2 diabetes mellitus (T2DM) is an independent risk factor for the occur of tendinopathy. Therefore, this study is the first to explore the dynamic changes of the “gene profile” of supraspinatus tendon in rats at different time points after T2DM induction through transcriptomics, providing potential molecular markers for exploring the pathogenesis of diabetic tendinopathy.MethodsA total of 40 Sprague-Dawley rats were randomly divided into normal (NG, n = 10) and T2DM groups (T2DM, n = 30) and subdivided into three groups according to the duration of diabetes: T2DM-4w, T2DM-8w, and T2DM-12w groups; the duration was calculated from the time point of T2DM rat model establishment. The three comparison groups were set up in this study, T2DM-4w group vs. NG, T2DM-8w group vs. NG, and T2DM-12w group vs. NG. Differentially expressed genes (DEGs) in 3 comparison groups were screened. The intersection of the three comparison groups’ DEGs was defined as key genes that changed consistently in the supraspinatus tendon after diabetes induction. Cluster analysis, gene ontology (GO) functional annotation analysis and Kyoto encyclopedia of genes and genomes (KEGG) functional annotation and enrichment analysis were performed for DEGs.ResultsT2DM-4w group vs. NG, T2DM-8w group vs. NG, and T2DM-12w group vs. NG detected 519 (251 up-regulated and 268 down-regulated), 459 (342 up-regulated and 117 down-regulated) and 328 (255 up-regulated and 73 down-regulated) DEGs, respectively. 103 key genes of sustained changes in the supraspinatus tendon following induction of diabetes, which are the first identified biomarkers of the supraspinatus tendon as it progresses through the course of diabetes.The GO analysis results showed that the most significant enrichment in biological processes was calcium ion transmembrane import into cytosol (3 DEGs). The most significant enrichment in cellular component was extracellular matrix (9 DEGs). The most significant enrichment in molecular function was glutamate-gated calcium ion channel activity (3 DEGs). The results of KEGG pathway enrichment analysis showed that there were 17 major pathways (p < 0.05) that diabetes affected supratinusculus tendinopathy, including cAMP signaling pathway and Calcium signaling pathway.ConclusionsTranscriptomics reveals dynamic changes in the“gene profiles”of rat supraspinatus tendon at three different time points after diabetes induction. The 103 DEGs identified in this study may provide potential molecular markers for exploring the pathogenesis of diabetic tendinopathy, and the 17 major pathways enriched in KEGG may provide new ideas for exploring the pathogenesis of diabetic tendinopathy.

Transcriptome profile of sex-related gene expression and effects of cyp19a1 siRNA-silencing on sex differentiation and determination in yellow perch

In this study, we first sequenced the whole genome of the yellow perch (YP, Perca favencens) and screened transcripts of brain, liver, and gonadal tissues from fish of different sexes. By comparing known female sex-related genes in fish such as cyp19a1 and dmrt1 with YP genome, we obtained full-length sequences through genomic homology analysis. Then an experiment was conducted to evaluate the effects of cyp19a1-siRNA interference on gonadal development and sex differentiation and determination in YP. Transcriptome analysis revealed that the gonadal tissue of female YP exhibited predominantly and differentially up-regulated expression of genes than males, and cyp19a1 was predominantly expressed in the gonads while cyp19a2 showed predominant expression in the brain. KEGG pathway enrichment analysis illustrated the metabolic pathways of the top 25 enriched genes and confirmed CYP19 (P450) pathway in YP. For the cyp19a1-siRNA interference experiment, four intermittent in vivo injections of siRNA were administered to interfere with cyp19a1 gene expression in an all-female group of YP. The results collected at 180 dph revealed that the siRNA interference resulted in an incomplete masculinization and hermaphroditism of all-female monosex YP, displaying the presence of male spermatocytes along with female oocytes. In contrast, tissues from the blank control and negative control groups only contained oocytes. The appearance of the gonads in the experimental group appeared more irregular, lacking the smooth surface and typical histoarchitecture observed in the blank control and negative control groups. In addition, the cyp19a1-siRNA treatment group had significantly (P < 0.05) lower body weight than the control groups, indicating that the interference with cyp19a1 might have affected the expression of estrogen, leading to a trend similar to the growth of males; similarly, the gonad weight and GSIs of the cyp19a1 group were significantly (P < 0.05) lower than those in the control groups, suggesting that the cyp19a1-siRNA interference might have led to a trend of male type of gonadal development. Meanwhile, the treatment group had significantly (P < 0.05) higher HSI and VSI levels compared to control groups, indicating that stimulation of external siRNA might have resulted in more vigorous liver metabolism processes. Furthermore, the expression level of the dmrt1 in the treatment group was significantly (P < 0.05) upregulated compared to the control groups, suggesting the correlation roles between these two genes in the regulation of sex development. The findings provide valuable insights for future research into the underlying mechanisms involved and hold significant promise for sex control in aquaculture.

Truncating the spliceosomal ‘rope protein’ Prp45 results in Htz1 dependent phenotypes

ABSTRACT Spliceosome assembly contributes an important but incompletely understood aspect of splicing regulation. Prp45 is a yeast splicing factor which runs as an extended fold through the spliceosome, and which may be important for bringing its components together. We performed a whole genome analysis of the genetic interaction network of the truncated allele of PRP45 (prp45(1–169)) using synthetic genetic array technology and found chromatin remodellers and modifiers as an enriched category. In agreement with related studies, H2A.Z-encoding HTZ1, and the components of SWR1, INO80, and SAGA complexes represented prominent interactors, with htz1 conferring the strongest growth defect. Because the truncation of Prp45 disproportionately affected low copy number transcripts of intron-containing genes, we prepared strains carrying intronless versions of SRB2, VPS75, or HRB1, the most affected cases with transcription-related function. Intron removal from SRB2, but not from the other genes, partly repaired some but not all the growth phenotypes identified in the genetic screen. The interaction of prp45(1–169) and htz1Δ was detectable even in cells with SRB2 intron deleted (srb2Δi). The less truncated variant, prp45(1–330), had a synthetic growth defect with htz1Δ at 16°C, which also persisted in the srb2Δi background. Moreover, htz1Δ enhanced prp45(1–330) dependent pre-mRNA hyper-accumulation of both high and low efficiency splicers, genes ECM33 and COF1, respectively. We conclude that while the expression defects of low expression intron-containing genes contribute to the genetic interactome of prp45(1–169), the genetic interactions between prp45 and htz1 alleles demonstrate the sensitivity of spliceosome assembly, delayed in prp45(1–169), to the chromatin environment.

A chromosome-scale genome provides new insights into the typical carotenoid biosynthesis in the important red yeast Rhodotorula glutinis QYH-2023 with anti-inflammatory effects

Rhodotorula spp. has been studied as one powerful source for a novel cell factory with fast growth and its high added-value biomolecules. However, its inadequate genome and genomic annotation have hindered its widespread use in cosmetics and food industries. Rhodotorula glutinis QYH-2023, was isolated from rice rhizosphere soil, and the highest quality of the genome of the strain was obtained at chromosome level (18 chromosomes) than ever before in red yeast in this study. Comparative genomics analysis revealed that there are more key gene copies of carotenoids biosynthesis in R. glutinis QYH-2023 than other species of Rhodotorula spp. Integrated transcriptome and metabolome analysis revealed that lipids and carotenoids biosynthesis was significantly enriched during fermentation. Subsequent investigation revealed that the over-expression of the strain three genes related to carotenoids biosynthesis in Komagataella phaffii significantly promoted the carotenoid production. Furthermore, in vitro tests initially confirmed that the longer the fermentation period, the synthesized metabolites controlled by R. glutinis QYH-2023 genome had the stronger anti-inflammatory properties. All of the findings revealed a high-quality reference genome which highlight the potential of R. glutinis strains to be employed as chassis cells for biosynthesizing carotenoids and other active chemicals.

Genomic analysis of Clostridium perfringens type D isolates from goat farms

C. perfringens type D strains are the leading cause of enterotoxaemia in ruminants such as goats, sheep, and cattle. However, there has been no prior research on the genomic characteristics of C. perfringens type D strains from various regions in China. Here, we investigated the antibiotic resistance, genomic characteristics, and phylogenetic relationship of C. perfringens type D isolates recovered from goat farms in Shaanxi, Gansu, and Ningxia provinces. The antibiotic resistance test indicated that the isolates displayed high minimum inhibitory concentration (MIC) values to sulfafurazole, whereas the other antibiotics tested, such as penicillin, enrofloxacin, and florfenicol, worked well on them. Additionally, only tetracycline resistance genes [tetA(P) and tetB(P)] were identified from the isolates. A collective of 13 toxin genes, including etx and cpe were detected among the isolates. Sequence comparison revealed that the etx and cpe genes shared high sequence identities, and they could coexist on a pCW3-like plasmid, representing a potential risk to both animal breeding and public health. Phylogenetic analysis using core genome multi-locus sequence typing (cgMLST) and core genome single nucleotide polymorphisms (SNPs) revealed the close genetic relationship and potential regional/transregional transmission of the C. perfringens type D isolates in Shaanxi and Gansu provinces. Furthermore, pan-genomic analysis suggested the functional differences at the protein-coding gene level, although isolates from the same source shared a close genetic relationship. In conclusion, this study indicated the antibiotic resistance, virulence markers, potential transregional transmission, and genomic diversity of C. perfringens type D strains from various regions in China, which could provide references for the prevention of C. perfringens foodborne diseases and further research.

Enhancing biomethanation performance through co-digestion of diverse organic wastes: a comprehensive study on substrate optimization, inoculum selection, and microbial community analysis.

A blend of organic municipal solid waste, slaughterhouse waste, fecal sludge, and landfill leachate was selected in different mixing ratios to formulate the best substrate mixture for biomethanation. Individual substrates were characterized, and the mixing ratio was optimized with the help of a response surface methodology tool to a value of 1:1:1:1 (with a C/N ratio of 28±0.769 and total volatile fatty acid (VFA) concentration of 2500±10.53 mg/L) to improve the overall biomethanation. The optimized blend (C/N ratio: 28.6, VFA: 2538 mg/L) was characterized for physicochemical, biological, and microbial properties and subjected to anaerobic digestion in lab-scale reactors of 1000 mL capacity with and without the addition of inoculum. The biogas yield of individual substrates and blends was ascertained separately. The observed cumulative biogas yield over 21 days from the non-inoculated substrates varied between 142±1.95 mL (24.6±0.3 ml/gVS) and 1974.5±21.72 mL (270.4±3.1 ml/gVS). In comparison, the addition of external inoculation at a 5% rate (w/w) of the substrate uplifted the minimum and maximum cumulative gas yield values to 203±9.9 mL (35.0±1.6 mL/gVS) and 3394±13.4 mL (315.3±1.2 mL/gVS), respectively. The inoculum procured from the Defence Research and Development Organisation (DRDO) was screened in advance, considering factors such as maximizing VFA production and consumption rate, biogas yield, and digestate quality. A similar outcome regarding biogas yield and digestate quality was observed for the equivalent blend. The cumulative gas yield increased from 2673±14.5 mL (373.7±2.2 mL/gVS) to 4284±111.02 mL (391.47±20.02 mL/gVS) over 21 days post-application of a similar dosage of DRDO inoculum. The 16S rRNA genomic analysis revealed that the predominant bacterial population belonged to the phylum Firmicutes, with the majority falling within the orders Clostridiales and Lactobacillales. Ultimately, the study advocates the potential of the blend mentioned above for biomethanation and concomitant enrichment of both biogas yield and digestate quality.

Comparative analysis of asian carps parvalbumin reveals the divergence pattern of major fish allergen.

Asian carps, a popular freshwater fish globally, are valued for their flavor and serve as a crucial protein source, especially for infants. However, grass carp parvalbumin is highly allergenic, surpassing the allergenicity of fish like salmon and cod. The allergenic potential of parvalbumin in other Asian carps remains unknown, underscoring the need for allergen identification to improve the precision of fish allergy diagnosis and treatment. To identify all parvalbumin homologs in Asian carps and investigate the role of gene divergence in allergenic homolog formation. Three annotated genomes of Asian carp, including grass carp, black carp and bighead carp, were constructed using a hybrid assembly approach. Through sequence homology at the genomic level, all the homologs of major fish allergens were identified. Bioinformatics tools were then employed to reveal the gene structures, expression levels, and protein conformations of parvalbumin. Grass carp genome analysis showed nine parvalbumin homologs, with Cid_PV2 most similar to Cten i 1. Bighead and black carp genomes had ten homologs, including potentially allergenic Mpi_PV7 and Hno_PV7. Tissue-specific expression patterns revealed alternative usage of parvalbumin homologs. Gene duplication events expanded parvalbumin copies in bony fish, with two gene clusters identified in Asian carp genomes. All the homologs of Asian carps' parvalbumin were accurately identified and gene divergence contributed to the formation of allergenic homologs. Together with a comprehensive gene sequence profile of carps' parvalbumin, those could be applied to achieve a more precise clinical diagnostic test.

Identification and genomic insights into a strain of Bacillus velezensis with phytopathogen-inhibiting and plant growth-promoting properties

The use of biological agents offers a sustainable alternative to chemical control in managing plant diseases. In this study, Bacillus velezensis IFST-221 was isolated from the rhizosphere of a healthy maize plant amidst a population showing severe disease symptoms. The investigation demonstrated a broad-spectrum antagonistic activity of IFST-221 against eight species of pathogenic ascomycetes and oomycetes, suggesting its potential utility in combating plant diseases like maize ear rot and cotton Verticillium wilt. Additionally, our study unveiled that IFST-221 has demonstrated significant plant growth-promoting properties, particularly in maize, cotton, tomato, and broccoli seedlings. This growth promotion was linked to its ability to produce indole-3-acetic acid, nitrogen fixation, phosphate and potassium solubilization, and biofilm formation in laboratory conditions. A complete genome sequencing of IFST-221 yielded a genome size of 3.858 M bp and a GC content of 46.71%. The genome analysis identified 3659 protein-coding genes, among which were nine secondary metabolite clusters with known antimicrobial properties. Additionally, three unknown compounds with potentially novel properties were also predicted from the genomic data. Genome mining also identified several key genes associated with plant growth regulation, colonization, and biofilm formation. These findings provide a compelling case for the application of B. velezensis IFST-221 in agricultural practices. The isolate’s combined capabilities of plant growth promotion and antagonistic activity against common plant pathogens suggest its promise as an integrated biological agent in disease management and plant productivity enhancement.

Comparative genomics of plant growth promoting phosphobacteria isolated from acidic soils.

Despite being one of the most abundant elements in soil, phosphorus (P) often becomes a limiting macronutrient for plants due to its low bioavailability, primarily locked away in insoluble organic and inorganic forms. Phosphate solubilizing and mineralizing bacteria, also called phosphobacteria, isolated from P-deficient soils have emerged as a promising biofertilizer alternative, capable of converting these recalcitrant P forms into plant-available phosphates. Three such phosphobacteria strains-Serratia sp. RJAL6, Klebsiella sp. RCJ4, and Enterobacter sp. 198-previously demonstrated their particular strength as plant growth promoters for wheat, ryegrass, or avocado under abiotic stresses and P deficiency. Comparative genomic analysis of their draft genomes revealed several genes encoding key functionalities, including alkaline phosphatases, isonitrile secondary metabolites, enterobactin biosynthesis and genes associated to the production of indole-3-acetic acid (IAA) and gluconic acid. Moreover, overall genome relatedness indexes (OGRIs) revealed substantial divergence between Serratia sp. RJAL6 and its closest phylogenetic neighbours, Serratia nematodiphila and Serratia bockelmanii. This compelling evidence suggests that RJAL6 merits classification as a novel species. This in silico genomic analysis provides vital insights into the plant growth-promoting capabilities and provenance of these promising PSRB strains. Notably, it paves the way for further characterization and potential application of the newly identified Serratia species as a powerful bioinoculant in future agricultural settings.

Global survey of hgcA-carrying genomes in marine and freshwater sediments: Insights into mercury methylation processes

Mercury (Hg) methylation is a microbially mediated process that produces methylmercury (MeHg), a bioaccumulative neurotoxin. A highly conserved gene pair, hgcAB, is required for Hg methylation, which provides a basis for identifying Hg methylators and evaluating their genomic composition. In this study, we conducted a large-scale omics analysis in which 281 metagenomic freshwater and marine sediment samples from 46 geographic locations across the globe were queried. Specific objectives were to examine the prevalence of Hg methylators, to identify horizontal gene transfer (HGT) events involving hgcAB within Hg methylator communities, and to identify associations between hgcAB and microbial biochemical functions/genes. Hg methylators from the phyla Desulfobacterota and Bacteroidota were dominant in both freshwater and marine sediments while Firmicutes and methanogens belonging to Euryarchaeota were identified only in freshwater sediments. Novel Hg methylators were found in the Phycisphaerae and Planctomycetia classes within the phylum Planctomycetota, including potential hgcA-carrying anammox metagenome-assembled genomes (MAGs) from Candidatus Brocadiia. HGT of hgcA and hgcB were identified in both freshwater and marine methylator communities. Spearman's correlation analysis of methylator genomes suggested that in addition to sulfide, thiosulfate, sulfite, and ammonia may be important parameters for Hg methylation processes in sediments. Overall, our results indicated that the biochemical drivers of Hg methylation vary between marine and freshwater sites, lending insight into the influence of environmental perturbances, such as a changing climate, on Hg methylation processes.

Variant Analysis of XDR Salmonella Typhi Strains Using Global Alignment Tool Kit in South Asian Region

Objective: To concisely compare genomic profiles of XDR Salmonella Typhi isolates from Lahore withantimicrobial-resistant Salmonella Typhi isolates from other developing nations.Study Design: Comparative analysis of whole genome sequences.Place and Duration of Study: The study was conducted from December 2020 to September 2021 at KauserAbdulla Malik (KAM) School Life Sciences, Forman Christian College University, Lahore, Pakistan.Methods: The Galaxy pipeline was run to obtain detailed information at the nucleotide level regardingmutations that lead to the emergence of XDR strains. Whole genome sequences were analyzed to compare thegenomes of selected three developing nations.Results: The Pakistani isolates had a significantly higher mutation rate, higher proportion of modifiers, andsilent mutations as compared to isolates of Bangladesh and India.Conclusion: Cases of Salmonella Typhi XDR are rapidly rising in Asian countries such as Pakistan, Bangladesh,and India emphasizing the need to analyze and compare its genome with relevant strains. Our study highlightsthe unique profile of the Lahore (Pakistan) isolate with the highest mutation rate suggesting the potentialregional differences in selective pressure. Further spotlights the necessity to elucidate the functionalconsequences of the identified mutations in S. Typhi isolates. How to cite this: Niaz M, Mumtaz Z, Ahmed M, Ahmad S, Ali A, Yousaf MZ. Variant Analysis of XDR Salmonella Typhi Strains Using Global Alignment Tool Kit in South Asian Region. Life and Science. 2024; 5(2): 244-250. doi: http://doi.org/10.37185/LnS.1.1.432

Genome-Wide Identification and Evolutionary Analysis of Receptor-like Kinase Family Genes Provides Insights into Anthracnose Resistance of Dioscorea alata.

Dioscorea alata, commonly known as "greater yam", is a vital crop in tropical and subtropical regions of the world, yet it faces significant threats from anthracnose disease, mainly caused by Colletotrichum gloeosporioides. However, exploring disease resistance genes in this species has been challenging due to the difficulty of genetic mapping resulting from the loss of the flowering trait in many varieties. The receptor-like kinase (RLK) gene family represents essential immune receptors in plants. In this study, genomic analysis revealed 467 RLK genes in D. alata. The identified RLKs were distributed unevenly across chromosomes, likely due to tandem duplication events. However, a considerable number of ancient whole-genome or segmental duplications dating back over 100 million years contributed to the diversity of RLK genes. Phylogenetic analysis unveiled at least 356 ancient RLK lineages in the common ancestor of Dioscoreaceae, which differentially inherited and expanded to form the current RLK profiles of D. alata and its relatives. The analysis of cis-regulatory elements indicated the involvement of RLK genes in diverse stress responses. Transcriptome analysis identified RLKs that were up-regulated in response to C. gloeosporioides infection, suggesting their potential role in resisting anthracnose disease. These findings provide novel insights into the evolution of RLK genes in D. alata and their potential contribution to disease resistance.

Combinatorial metabolic engineering of Streptomyces sp. CB03234-S for the enhanced production of anthraquinone-fused enediyne tiancimycins

BackgroundAnthraquinone-fused enediynes (AFEs) are excellent payloads for antibody-drug conjugates (ADCs). The yields of AFEs in the original bacterial hosts are extremely low. Multiple traditional methods had been adopted to enhance the production of the AFEs. Despite these efforts, the production titers of these compounds are still low, presenting a practical challenge for their development. Tiancimycins (TNMs) are a class of AFEs produced by Streptomyces sp. CB03234. One of their salient features is that they exhibit rapid and complete cell killing ability against various cancer cell lines.ResultsIn this study, a combinatorial metabolic engineering strategy guided by the CB03234-S genome and transcriptome was employed to improve the titers of TNMs. First, re-sequencing of CB03234-S (Ribosome engineered mutant strains) genome revealed the deletion of a 583-kb DNA fragment, accounting for about 7.5% of its genome. Second, by individual or combined inactivation of seven potential precursor competitive biosynthetic gene clusters (BGCs) in CB03234-S, a double-BGC inactivation mutant, S1009, was identified with an improved TNMs titer of 28.2 ± 0.8 mg/L. Third, overexpression of five essential biosynthetic genes, including two post-modification genes, and three self-resistance auxiliary genes, was also conducted, through which we discovered that mutants carrying the core genes, tnmE or tnmE10, exhibited enhanced TNMs production. The average TNMs yield reached 43.5 ± 2.4 mg/L in a 30-L fermenter, representing an approximately 360% increase over CB03234-S and the highest titer among all AFEs to date. Moreover, the resulting mutant produced TNM-W, a unique TNM derivative with a double bond instead of a common ethylene oxide moiety. Preliminary studies suggested that TNM-W was probably converted from TNM-A by both TnmE and TnmE10.ConclusionsBased on the genome and transcriptome analyses, we adopted a combined metabolic engineering strategy for precursor enrichment and biosynthetic pathway reorganization to construct a high-yield strain of TNMs based on CB03234-S. Our study establishes a solid basis for the clinical development of AFE-based ADCs.

The phased Solanum okadae genome and Petota pangenome analysis of 23 other potato wild relatives and hybrids

Potato is an important crop in the genus Solanum section Petota. Potatoes are susceptible to multiple abiotic and biotic stresses and have undergone constant improvement through breeding programs worldwide. Introgression of wild relatives from section Petota with potato is used as a strategy to enhance the diversity of potato germplasm. The current dataset contributes a phased genome assembly for diploid S. okadae, and short read sequences and de novo assemblies for the genomes of 16 additional wild diploid species in section Petota that were noted for stress resistance and were of interest to potato breeders. Genome sequence data for three additional genomes representing polyploid hybrids with cultivated potato, and an additional genome from non-tuberizing S. etuberosum, which is outside of section Petota, were also included. High quality short reads assemblies were achieved with genome sizes ranging from 575 to 795 Mbp and annotations were performed utilizing transcriptome sequence data. Genomes were compared for presence/absence of genes and phylogenetic analyses were carried out using plastome and nuclear sequences.

Biodegradation of aniline blue dye by salt-tolerant Bacillus thuringiensis DHC4 isolated from soil-feeding termite guts

The existence of aniline blue in aquatic environments poses a large threat to aquatic organisms. Microbial degradation of aniline blue has been frequently used in remediation because of its effectiveness, low cost, and environmental friendliness. However, the mechanisms by which the dye can be biodegraded are not well understood. In this study, Bacillus thuringiensis DHC4 was isolated from the gut of a soil-feeding termite, and its ability to degrade aniline blue was investigated. The results showed that DHC4 was highly resistant to both aniline blue and salt, with the decolorization rate reaching 78% and 67% after 96 h of incubation at initial dye concentrations of 1 g/L and 7% salinity, respectively. Aniline blue degradation products were identified based on UV–vis, FTIR, and GC–MS analyses, and genome analysis confirmed that DHC4 contained many potential dye-degrading genes. Transcriptome analysis revealed significant up-regulation of methyltransferase, nitroreductase, arylamine N-acetyltransferase and oxidoreductase genes on exposure to aniline blue, indicating their active role in its biodegradation. A new degradation pathway of aniline blue by DHC4 is proposed, based on analysis of metabolites and differentially expressed genes. This mechanism involves cleavage of aniline blue by oxidoreductases into mono- and diphenyl ring molecules, followed by further methylation and alkylation through methyltransferases. The phytotoxicity results showed that aniline blue was degraded by DHC4 into less toxic metabolites. These results provide new insights into the mechanism of biodegradation of aniline blue by microorganisms.

Metagenomic nanopore sequencing for exploring the nature of antimicrobial metabolites of Bacillus haynesii

Multidrug-resistant (MDR) pathogens are a rising global health worry that imposes an urgent need for the discovery of novel antibiotics particularly those of natural origin. In this context, we aimed to use the metagenomic nanopore sequence analysis of soil microbiota coupled with the conventional phenotypic screening and genomic analysis for identifying the antimicrobial metabolites produced by promising soil isolate(s). In this study, whole metagenome analysis of the soil sample(s) was performed using MinION™ (Oxford Nanopore Technologies). Aligning and analysis of sequences for probable secondary metabolite gene clusters were extracted and analyzed using the antiSMASH version 2 and DeepBGC. Results of the metagenomic analysis showed the most abundant taxa were Bifidobacterium, Burkholderia, and Nocardiaceae (99.21%, followed by Sphingomonadaceae (82.03%) and B. haynesii (34%). Phenotypic screening of the respective soil samples has resulted in a promising Bacillus isolate that exhibited broad-spectrum antibacterial activities against various MDR pathogens. It was identified using microscopical, cultural, and molecular methods as Bacillus (B.) haynesii isolate MZ922052. The secondary metabolite gene analysis revealed the conservation of seven biosynthetic gene clusters of antibacterial metabolites namely, siderophore lichenicidin VK21-A1/A2 (95% identity), lichenysin (100%), fengycin (53%), terpenes (100%), bacteriocin (100%), Lasso peptide (95%) and bacillibactin (53%). In conclusion, metagenomic nanopore sequence analysis of soil samples coupled with conventional screening helped identify B. haynesii isolate MZ922052 harboring seven biosynthetic gene clusters of promising antimicrobial metabolites. This is the first report for identifying the bacteriocin, lichenysin, and fengycin biosynthetic gene clusters in B. haynesii MZ922052.

Nontraditional Roles of Magnesium Ions in Modulating Sav2152: Insight from a Haloacid Dehalogenase-like Superfamily Phosphatase from Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) infection has rapidly spread through various routes. A genomic analysis of clinical MRSA samples revealed an unknown protein, Sav2152, predicted to be a haloacid dehalogenase (HAD)-like hydrolase, making it a potential candidate for a novel drug target. In this study, we determined the crystal structure of Sav2152, which consists of a C2-type cap domain and a core domain. The core domain contains four motifs involved in phosphatase activity that depend on the presence of Mg2+ ions. Specifically, residues D10, D12, and D233, which closely correspond to key residues in structurally homolog proteins, are responsible for binding to the metal ion and are known to play critical roles in phosphatase activity. Our findings indicate that the Mg2+ ion known to stabilize local regions surrounding it, however, paradoxically, destabilizes the local region. Through mutant screening, we identified D10 and D12 as crucial residues for metal binding and maintaining structural stability via various uncharacterized intra-protein interactions, respectively. Substituting D10 with Ala effectively prevents the interaction with Mg2+ ions. The mutation of D12 disrupts important structural associations mediated by D12, leading to a decrease in the stability of Sav2152 and an enhancement in binding affinity to Mg2+ ions. Additionally, our study revealed that D237 can replace D12 and retain phosphatase activity. In summary, our work uncovers the novel role of metal ions in HAD-like phosphatase activity.

UDP-glycosyltransferase gene family expansion and functional perspectives in five tenebrionid beetles

UDP-glycosyltransferases (UGTs) are widely distributed biotransformation enzymes crucial for various physiological processes. Through genomic analysis, 216 UGT genes were identified in five tenebrionoid beetles: Tebebrio molitor (46), Tribolium castaneum (43), Asbolus verrucosus (30), Hycleus cichorii (60), and Hycleus phaleratus (37), classified into 14 families, with 13 being coleopteran-specific. Lineage-specific expansion of the UGT321, UGT323, UGT324, and UGT347 families was observed, with evidence of gene duplications within these families. Notably, phytophagous blister beetles showed higher UGT321 gene counts compared to omnivorous darkling beetles, while UGT324 family expansion was noted in the latter. Investigation of temporal and tissue-specific UGT gene expression in T. molitor revealed over 60% of TmUGTs were highly expressed in the salivary gland, fat body, midgut, and Malpighian tubule, indicating roles in metabolism and detoxification. Particularly, UGT321 family members exhibited high expression across various tissues, suggesting diverse functions, including potential involvement in olfaction and detoxification. These findings emphasize the multifunctionality of UGTs and provide a basis for further research, utilizing T. molitor as a model organism.

Immune checkpoint inhibitors for POLE or POLD1 proofreading-deficient metastatic colorectal cancer.

POLE and POLD1 proofreading deficiency (POLE/D1pd) define a rare subtype of ultramutated metastatic colorectal cancer (mCRC; over 100 mut/Mb). Disease-specific data about the activity and efficacy of immune checkpoint inhibitors (ICIs) in POLE/D1pd mCRC are lacking and it is unknown whether outcomes may be different from mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) mCRCs treated with ICIs. In this global study, we collected 27 patients with mCRC harboring POLE/D1 mutations leading to proofreading deficiency and treated with anti-programmed cell death-ligand 1 alone+/- anti-cytotoxic T-lymphocyte antigen-4 agents. We collected clinicopathological and genomic characteristics, response, and survival outcomes after ICIs of POLE/D1pd mCRC and compared them with a cohort of 610 dMMR/MSI-H mCRC patients treated with ICIs. Further genomic analyses were carried out in an independent cohort of 7241 CRCs to define POLE and POLD1pd molecular profiles and mutational signatures. POLE/D1pd was associated with younger age, male sex, fewer RAS/BRAF driver mutations, and predominance of right-sided colon cancers. Patients with POLE/D1pd mCRC showed a significantly higher overall response rate (ORR) compared to dMMR/MSI-H mCRC (89% versus 54%; P= 0.01). After a median follow-up of 24.9 months (interquartile range: 11.3-43.0 months), patients with POLE/D1pd showed a significantly superior progression-free survival (PFS) compared to dMMR/MSI-H mCRC [hazard ratio (HR)= 0.24, 95% confidence interval (CI) 0.08-0.74, P= 0.01] and superior overall survival (OS) (HR= 0.38, 95% CI 0.12-1.18, P= 0.09). In multivariable analyses including the type of DNA repair defect, POLE/D1pd was associated with significantly improved PFS (HR= 0.17, 95% CI 0.04-0.69, P=0.013) and OS (HR= 0.24, 95% CI 0.06-0.98, P= 0.047). Molecular profiling showed that POLE/D1pd tumors have higher tumor mutational burden (TMB). Responses were observed in both subtypes and were associated with the intensity of POLE/D1pd signature. Patients with POLE/D1pd mCRC showed more favorable outcomes compared to dMMR/MSI-H mCRC to treatment with ICIs in terms of tumor response and survival.

Improvement of skin wound healing by giant salamander skin mucus gel wrapped with bone marrow mesenchymal stem cells via affecting integrin family molecules.

Traditional bandages, gauze, and cotton balls are increasingly insufficient for addressing complex war injuries characterized by severe bleeding and diverse wound conditions. The giant salamander, a species of high medical value, secretes a unique mucus when stimulated, which has potential applications in wound care. Giant salamander skin mucus gel dressing wrapped with bone marrow mesenchymal stem cells (BMSCs-GSSM-gel) was prepared and validated. Skin wound injury of rabbit and mouse models were established. Hematoxylin and Eosin, Masson's trichrome, and Sirius red staining were performed. The platelet aggregation rate and coagulation items were measured. Transcriptome sequencing was performed to find potential differential expression genes. Preparation and characterization of BMSCs-GSSM-gel were performed, and BMSCs-GSSM-gel particles with a diameter of about 200 nm were obtained. BMSCs-GSSM-gel accelerated wound healing in both rabbit and mouse models. BMSCs-GSSM-gel significantly promoted hemostasis via increasing platelet aggregation rate and fibrinogen, but decreasing activated partial thromboplastin time, thrombin time, and prothrombin time. BMSCs-GSSM-gel treatment significantly impacted several genes associated with cell adhesion, inflammatory response, collagen-containing extracellular matrix, and the positive regulation of cell migration based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Integrin Subunit Beta 4 (ITGB4), Integrin Subunit Alpha 3 (ITGA3), and Laminin Subunit Beta 3 (LAMB3) might be involved in the wound healing process by BMSCs-GSSM-gel. We proved the BMSCs-GSSM-gel greatly improved the skin wound healing, and it might play a crucial role in the application fields of skin damage repair.