Genome-wide identification, phylogeny and expression analysis of Hsf gene family in Verbena bonariensis under low-temperature stress.

BACKGROUND: The heat shock transcription factor (Hsf) is a crucial regulator of plant stress resistance, playing a key role in plant stress response, growth, and development regulation. RESULTS: In this study, we utilized bioinformatics tools to screen 25 VbHsf members, which were named VbHsf1-VbHsf25. We used bioinformatics methods to analyze the sequence structure, physicochemical properties, conserved motifs, phylogenetic evolution, chromosome localization, promoter cis-acting elements, collinearity, and gene expression of Hsf heat shock transcription factor family members under low-temperature stress. The results revealed that the majority of the Hsf genes contained motif1, motif2, and motif3, signifying that these three motifs were highly conserved in the Hsf protein sequence of Verbena bonariensis. Although there were some variations in motif deletion among the members, the domain remained highly conserved. The theoretical isoelectric point ranged from 4.17 to 9.71, with 21 members being unstable proteins and the remainder being stable proteins. Subcellular localization predictions indicated that all members were located in the nucleus. Phylogenetic analysis of the Hsf gene family in V. bonariensis and Arabidopsis thaliana revealed that the Hsf gene family of V. bonariensis could be categorized into three groups, with group A comprising 17 members and group C having at least two members. Among the 25 Hsf members, there were 1-3 exons located on seven chromosome fragments, which were unevenly distributed. Collinearity analysis demonstrated the presence of seven pairs of homologous genes in the VbHsf gene family. The Ka/Ks ratios were less than one, indicating that the VbHsf gene underwent purification selection pressure. Additionally, nine genes in V. bonariensis were found to have collinearity with A. thaliana. Promoter analysis revealed that the promoters of all VbHsf genes contained various types of cis-acting elements related to hormones and stress. Based on RNA-seq data, qRT-PCR analysis of six highly expressed genes was performed, and it was found that VbHsf5, VbHsf14, VbHsf17, VbHsf18, VbHsf20 and VbHsf21 genes were highly expressed at 12 h of low-temperature treatment, and the expression decreased after 24 h, among which VbHsf14 was up-regulated at 12 h of low-temperature by 70-fold. CONCLUSIONS: Our study may help reveal the important roles of Hsf in plant development and show insight for the further molecular breeding of V. bonariensis.

Genome-wide identification and characterization of NAC transcription factor family members in Trifolium pratense and expression analysis under lead stress.

BACKGROUND: The NAC TF family is widely involved in plant responses to various types of stress. Red clover (Trifolium pratense) is a high-quality legume, and the study of NAC genes in red clover has not been comprehensive. The aim of this study was to analyze the NAC gene family of red clover at the whole-genome level and explore its potential role in the Pb stress response. RESULTS: In this study, 72 TpNAC genes were identified from red clover; collinearity analysis showed that there were 5 pairs of large fragment replicators of TpNAC genes, and red clover was found to be closely related to Medicago truncatula. Interestingly, the TpNAC genes have more homologs in Arabidopsis thaliana than in soybean (Glycine max). There are many elements in the TpNAC genes promoters that respond to stress. Gene expression analysis showed that all the TpNAC genes responded to Pb stress. qRT-PCR showed that the expression levels of TpNAC29 and TpNAC42 were significantly decreased after Pb stress. Protein interaction network analysis showed that 21 TpNACs and 23 other genes participated in the interaction. In addition, the TpNAC proteins had three possible 3D structures, and the secondary structure of these proteins were mainly of other types. These results indicated that most TpNAC members were involved in the regulation of Pb stress in red clover. CONCLUSION: These results suggest that most TpNAC members are involved in the regulation of Pb stress in red clover. TpNAC members play an important role in the response of red clover to Pb stress.

Zebrafish cardiac repolarization does not functionally depend on the expression of the hERG1b-like transcript.

Zebrafish provide a translational model of human cardiac function. Their similar cardiac electrophysiology enables screening of human cardiac repolarization disorders, drug arrhythmogenicity, and novel antiarrhythmic therapeutics. However, while zebrafish cardiac repolarization is driven by delayed rectifier potassium channel current (IKr), the relative role of alternate channel transcripts is uncertain. While human ether-a-go-go-related-gene-1a (hERG1a) is the dominant transcript in humans, expression of the functionally distinct alternate transcript, hERG1b, modifies the electrophysiological and pharmacologic IKr phenotype. Studies of zebrafish IKr are frequently translated without consideration for the presence and impact of hERG1b in humans. Here, we performed phylogenetic analyses of all available KCNH genes from Actinopterygii (ray-finned fishes). Our findings confirmed zebrafish cardiac zkcnh6a as the paralog of human hERG1a (hKCNH2a), but also revealed evidence of a hERG1b (hKCNH2b)-like N-terminally truncated gene, zkcnh6b, in zebrafish. zkcnh6b is a teleost-specific variant that resulted from the 3R genome duplication. qRT-PCR showed dominant expression of zkcnh6a in zebrafish atrial and ventricular tissue, with low levels of zkcnh6b. Functional evaluation of zkcnh6b in a heterologous system showed no discernable function under the conditions tested, and no influence on zkcnh6a function during the zebrafish ventricular action potential. Our findings provide the first descriptions of the zkcnh6b gene, and show that, unlike in humans, zebrafish cardiac repolarization does not rely upon co-assembly of zERG1a/zERG1b. Given that hERG1b modifies IKr function and drug binding in humans, our findings highlight the need for consideration when translating hERG variant effects and toxicological screens in zebrafish, which lack a functional hERG1b-equivalent gene.

Seasonal Stability Assessment of Reference Genes for Quantitative Real-Time Polymerase Chain Reaction Normalization in Bombus terrestris.

Bumblebees (B. terrestris) play a crucial role as highly efficient biological agents in commercial pollination. Understanding the molecular mechanisms governing their adaptation to diverse seasonal environments may pave the way for effective management strategies in the future. With the burgeoning advancement in post-genetic studies focusing on B. terrestris, there is a critical need to normalize quantitative real-time PCR (qRT-PCR) data using suitable reference genes. To address this necessity, we employed RefFinder, a software-based tool, to assess the suitability of several candidate endogenous control genes, including actin (ACT), arginine kinase (AK), elongation factor 1 alpha (EF1), glyceraldehyde-3-phosphate (GAPDH), phospholipase (PLA2), and ribosomal proteins (S18, S28). These genes were evaluated for their efficacy as biological endogenous controls by examining their expression patterns across various environmental conditions corresponding to different seasons (Spring, Summer, Autumn, Winter) and tissues (ovary, fat body, thorax, head) in bumblebees. Moreover, the study investigated the significance of selecting appropriate reference genes for three key genes involved in the juvenile hormone (JH) signaling pathways: Krüppel homolog 1 (Kr-h1), methyl farnesoate epoxidase (MFE), and Vitellogenin (Vg). Our research identifies specific genes suitable for normalization in B. terrestris, thereby offering valuable insights into gene expression and functional metabolic genetics under varying seasonal conditions. This catalog of reference genes will serve as a valuable resource for future research endeavors.

Identification and characterization of the Dmrt1B gene in the oriental river prawn, Macrobrachium nipponense.

Dmrt (doublesex and mab-3 related transcription factor) is a protein family of transcription factors implicated in sexual regulation. Dmrt proteins are widely conserved and known for their involvement in sex determination and differentiation across species, from invertebrates to humans. In this study, we identified a novel gene with a DM (doublesex/Mab-3)-domain gene in the river prawn, Macrobrachium nipponense, which we named MniDmrt1B due to its similarities and close phylogenetic relationship with Dmrt1B in Macrobrachium rosenbergii. Through amino acid alignments and structural predictions, we observed conservation and identified putative active sites within the DM domain. qRT-PCR analysis revealed that MniDmrt1B exhibited high expression levels in the testis, with consistently higher expression in males compared to females during development. Additionally, similar to other sex-regulated genes, the MniDmrt1B gene exhibited high expression levels during the sex differentiation-sensitive periods in M. nipponense. These results strongly indicated that MniDmrt1B probably plays an important role in testis development and sex differentiation in M. nipponense.

Genome-wide identification and expression-pattern analysis of sulfate transporter (SULTR) gene family in cotton under multiple abiotic stresses and fiber development.

Sulfate transporter (SULTR) proteins are in charge of the transport and absorption on sulfate substances, and have been reported to play vital roles in the biological processes of plant growth and stress response. However, there were few reports of genome-wide identification and expression-pattern analysis of SULTRs in Hibiscus mutabilis. Gossypium genus is a ideal model for studying the allopolyploidy, therefore two diploid species (G. raimondii and G. arboreum) and two tetraploid species (G. hirsutum and G. barbadense) were chosen in this study to perform bioinformatic analyses, identifying 18, 18, 35, and 35 SULTR members, respectively. All the 106 cotton SULTR genes were utilized to construct the phylogenetic tree together with 11 Arabidopsis thaliana, 13 Oryza sativa, and 8 Zea mays ones, which was divided into Group1-Group4. The clustering analyses of gene structures and 10 conserved motifs among the cotton SULTR genes showed the consistent evolutionary relationship with the phylogenetic tree, and the results of gene-duplication identification among the four representative Gossypium species indicated that genome-wide or segment duplication might make main contributions to the expansion of SULTR gene family in cotton. Having conducted the cis-regulatory element analysis in promoter region, we noticed that the existing salicylic acid (SA), jasmonic acid (JA), and abscisic acid (ABA) elements could have influences with expression levels of cotton SULTR genes. The expression patterns of GhSULTR genes were also investigated on the 7 different tissues or organs and the developing ovules and fibers, most of which were highly expressed in root, stem, sepal, receptacel, ovule at 10 DPA, and fiber at 20 and 25 DPA. In addition, more active regulatory were observed in GhSULTR genes responding to multiple abiotic stresses, and 12 highly expressed genes showed the similar expression patterns in the quantitative Real-time PCR experiments under cold, heat, salt, and drought treatments. These findings broaden our insight into the evolutionary relationships and expression patterns of the SULTR gene family in cotton, and provide the valuable information for further screening the vital candidate genes on trait improvement.

Integrated metabolome and transcriptome analyses reveal the role of BoGSTF12 in anthocyanin accumulation in Chinese kale (Brassica oleracea var. alboglabra).

BACKGROUND: The vivid red, purple, and blue hues that are observed in a variety of plant fruits, flowers, and leaves are produced by anthocyanins, which are naturally occurring pigments produced by a series of biochemical processes occurring inside the plant cells. The purple-stalked Chinese kale, a popular vegetable that contains anthocyanins, has many health benefits but needs to be investigated further to identify the genes involved in the anthocyanin biosynthesis and translocation in this vegetable. RESULTS: In this study, the purple- and green-stalked Chinese kale were examined using integrative transcriptome and metabolome analyses. The content of anthocyanins such as cyanidin-3-O-(6″-O-feruloyl) sophoroside-5-O-glucoside, cyanidin-3,5-O-diglucoside (cyanin), and cyanidin-3-O-(6″-O-p-hydroxybenzoyl) sophoroside-5-O-glucoside were considerably higher in purple-stalked Chinese kale than in its green-stalked relative. RNA-seq analysis indicated that 23 important anthocyanin biosynthesis genes, including 3 PAL, 2 C4H, 3 4CL, 3 CHS, 1 CHI, 1 F3H, 2 FLS, 2 F3'H, 1 DFR, 3 ANS, and 2 UFGT, along with the transcription factor BoMYB114, were significantly differentially expressed between the purple- and green-stalked varieties. Results of analyzing the expression levels of 11 genes involved in anthocyanin production using qRT-PCR further supported our findings. Association analysis between genes and metabolites revealed a strong correlation between BoGSTF12 and anthocyanin. We overexpressed BoGSTF12 in Arabidopsis thaliana tt19, an anthocyanin transport mutant, and this rescued the anthocyanin-loss phenotype in the stem and rosette leaves, indicating BoGSTF12 encodes an anthocyanin transporter that affects the accumulation of anthocyanins. CONCLUSION: This work represents a key step forward in our understanding of the molecular processes underlying anthocyanin production in Chinese kale. Our comprehensive metabolomic and transcriptome analyses provide important insights into the regulatory system that controls anthocyanin production and transport, while providing a foundation for further research to elucidate the physiological importance of the metabolites found in this nutritionally significant vegetable.

Genome-wide analyses of member identification, expression pattern, and protein-protein interaction of EPF/EPFL gene family in Gossypium.

BACKGROUND: Epidermal patterning factor / -like (EPF/EPFL) gene family encodes a class of cysteine-rich secretory peptides, which are widelyfound in terrestrial plants.Multiple studies has indicated that EPF/EPFLs might play significant roles in coordinating plant development and growth, especially as the morphogenesis processes of stoma, awn, stamen, and fruit skin. However, few research on EPF/EPFL gene family was reported in Gossypium. RESULTS: We separately identified 20 G. raimondii, 24 G. arboreum, 44 G. hirsutum, and 44 G. barbadense EPF/EPFL genes in the 4 representative cotton species, which were divided into four clades together with 11 Arabidopsis thaliana, 13 Oryza sativa, and 17 Selaginella moellendorffii ones based on their evolutionary relationships. The similar gene structure and common motifs indicated the high conservation among the EPF/EPFL members, while the uneven distribution in chromosomes implied the variability during the long-term evolutionary process. Hundreds of collinearity relationships were identified from the pairwise comparisons of intraspecifc and interspecific genomes, which illustrated gene duplication might contribute to the expansion of cotton EPF/EPFL gene family. A total of 15 kinds of cis-regulatory elements were predicted in the promoter regions, and divided into three major categories relevant to the biological processes of development and growth, plant hormone response, and abiotic stress response. Having performing the expression pattern analyses with the basic of the published RNA-seq data, we found most of GhEPF/EPFL and GbEPF/EPFL genes presented the relatively low expression levels among the 9 tissues or organs, while showed more dramatically different responses to high/low temperature and salt or drought stresses. Combined with transcriptome data of developing ovules and fibers and quantitative Real-time PCR results (qRT-PCR) of 15 highly expressed GhEPF/EPFL genes, it could be deduced that the cotton EPF/EPFL genes were closely related with fiber development. Additionally, the networks of protein-protein interacting among EPF/EPFLs concentrated on the cores of GhEPF1 and GhEPF7, and thosefunctional enrichment analyses indicated that most of EPF/EPFLs participate in the GO (Gene Ontology) terms of stomatal development and plant epidermis development, and the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways of DNA or base excision repair. CONCLUSION: Totally, 132 EPF/EPFL genes were identified for the first time in cotton, whose bioinformatic analyses of cis-regulatory elements and expression patterns combined with qRT-PCR experiments to prove the potential functions in the biological processes of plant growth and responding to abiotic stresses, specifically in the fiber development. These results not only provide comprehensive and valuable information for cotton EPF/EPFL gene family, but also lay solid foundation for screening candidate EPF/EPFL genes in further cotton breeding.

An improved method to study Phytophthora cinnamomi Rands zoospores interactions with host.

Phytophthora cinnamomi Rands is a highly prevalent phytopathogen worldwide, ranking among the top ten in terms of distribution. It inflicts crown rot, canker, and root rot on numerous plant species, significantly impacting the biodiversity of both flora and fauna within affected environments. With a host range spanning over 5,000 species, including important plants like Quercus suber, Quercus ilex, Castanea sativa, and commercially significant crops such as avocado (Persea americana), maize (Zea mays), and tomato (Solanum lycopersicum), Phytophthora cinnamomi poses a substantial threat to agriculture and ecosystems. The efficient dissemination of the oomycete relies on its short-lived asexually motile zoospores, which depend on water currents to infect host roots. However, managing these zoospores in the laboratory has long been challenging due to the complexity of the life cycle. Current protocols involve intricate procedures, including alternating cycles of growth, drought, and flooding. Unfortunately, these artificial conditions often result in a rapid decline in virulence, necessitating additional steps to maintain infectivity during cultivation. In our research, we sought to address this challenge by investigating zoospore survival under various conditions. Our goal was to develop a stable stock of zoospores that is both easily deployable and highly infective. Through direct freezing in liquid nitrogen, we have successfully preserved their virulence. This breakthrough eliminates the need for repeated culture transfers, simplifying the process of plant inoculation. Moreover, it enables more comprehensive studies of Phytophthora cinnamomi and its interactions with host plants.

Integrated transcriptomic and metabolomic analyses reveals anthocyanin biosynthesis in leaf coloration of quinoa (Chenopodium quinoa Willd.).

BACKGROUND: Quinoa leaves demonstrate a diverse array of colors, offering a potential enhancement to landscape aesthetics and the development of leisure-oriented sightseeing agriculture in semi-arid regions. This study utilized integrated transcriptomic and metabolomic analyses to investigate the mechanisms underlying anthocyanin synthesis in both emerald green and pink quinoa leaves. RESULTS: Integrated transcriptomic and metabolomic analyses indicated that both flavonoid biosynthesis pathway (ko00941) and anthocyanin biosynthesis pathway (ko00942) were significantly associated with anthocyanin biosynthesis. Differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) were analyzed between the two germplasms during different developmental periods. Ten DEGs were verified using qRT-PCR, and the results were consistent with those of the transcriptomic sequencing. The elevated expression of phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), 4-coumarate CoA ligase (4CL) and Hydroxycinnamoyltransferase (HCT), as well as the reduced expression of flavanone 3-hydroxylase (F3H) and Flavonol synthase (FLS), likely cause pink leaf formation. In addition, bHLH14, WRKY46, and TGA indirectly affected the activities of CHS and 4CL, collectively regulating the levels of cyanidin 3-O-(3'', 6''-O-dimalonyl) glucoside and naringenin. The diminished expression of PAL, 4CL, and HCT decreased the formation of cyanidin-3-O-(6"-O-malonyl-2"-O-glucuronyl) glucoside, leading to the emergence of emerald green leaves. Moreover, the lowered expression of TGA and WRKY46 indirectly regulated 4CL activity, serving as another important factor in maintaining the emerald green hue in leaves N1, N2, and N3. CONCLUSION: These findings establish a foundation for elucidating the molecular regulatory mechanisms governing anthocyanin biosynthesis in quinoa leaves, and also provide some theoretical basis for the development of leisure and sightseeing agriculture.

A novel Microbacterium strain SRS2 promotes the growth of Arabidopsis and MicroTom (S. lycopersicum) under normal and salt stress conditions.

MAIN CONCLUSION: Microbacterium strain SRS2 promotes growth and induces salt stress resistance in Arabidopsis and MicroTom in various growth substrates via the induction of the ABA pathway. Soil salinity reduces plant growth and development and thereby decreases the value and productivity of soils. Plant growth-promoting rhizobacteria (PGPR) have been shown to support plant growth such as in salt stress conditions. Here, Microbacterium strain SRS2, isolated from the root endosphere of tomato, was tested for its capability to help plants cope with salt stress. In a salt tolerance assay, SRS2 grew well up to medium levels of NaCl, but the growth was inhibited at high salt concentrations. SRS2 inoculation led to increased biomass of Arabidopsis and MicroTom tomato in various growth substrates, in the presence and in the absence of high NaCl concentrations. Whole-genome analysis revealed that the strain contains several genes involved in osmoregulation and reactive oxygen species (ROS) scavenging, which could potentially explain the observed growth promotion. Additionally, we also investigated via qRT-PCR, promoter::GUS and mutant analyses whether the abscisic acid (ABA)-dependent or -independent pathways for tolerance against salt stress were involved in the model plant, Arabidopsis. Especially in salt stress conditions, the plant growth-promotion effect of SRS2 was lost in aba1, abi4-102, abi3, and abi5-1 mutant lines. Furthermore, ABA genes related to salt stress in SRS2-inoculated plants were transiently upregulated compared to mock under salt stress conditions. Additionally, SRS2-inoculated ABI4::GUS and ABI5::GUS plants were slightly more activated compared to the uninoculated control under salt stress conditions. Together, these assays show that SRS2 promotes growth in normal and in salt stress conditions, the latter possibly via the induction of ABA-dependent and -independent pathways.

Phenotypic, molecular, and in silico characterization of coumarin as carbapenemase inhibitor to fight carbapenem-resistant Klebsiella pneumoniae.

BACKGROUND: Carbapenems represent the first line treatment of serious infections caused by drug-resistant Klebsiella pneumoniae. Carbapenem-resistant K. pneumoniae (CRKP) is one of the urgent threats to human health worldwide. The current study aims to evaluate the carbapenemase inhibitory potential of coumarin and to test its ability to restore meropenem activity against CRKP. Disk diffusion method was used to test the antimicrobial susceptibility of K. pneumoniae clinical isolates to various antibiotics. Carbapenemase genes (NDM-1, VIM-2, and OXA-9) were detected using PCR. The effect of sub-MIC of coumarin on CRKP isolates was performed using combined disk assay, enzyme inhibition assay, and checkerboard assay. In addition, qRT-PCR was used to estimate the coumarin effect on expression of carbapenemase genes. Molecular docking was used to confirm the interaction between coumarin and binding sites within three carbapenemases. RESULTS: K. pneumoniae clinical isolates were found to be multi-drug resistant and showed high resistance to meropenem. All bacterial isolates harbor at least one carbapenemase-encoding gene. Coumarin significantly inhibited carbapenemases in the crude periplasmic extract of CRKP. The checkerboard assay indicated that coumarin-meropenem combination was synergistic exhibiting a fractional inhibitory concentration index ≤ 0.5. In addition, qRT-PCR results revealed that coumarin significantly decreased carbapenemase-genes expression. Molecular docking revealed that the binding energies of coumarin to NDM1, VIM-2, OXA-48 and OXA-9 showed a free binding energy of -7.8757, -7.1532, -6.2064 and - 7.4331 Kcal/mol, respectively. CONCLUSION: Coumarin rendered CRKP sensitive to meropenem as evidenced by its inhibitory action on hydrolytic activity and expression of carbapenemases. The current findings suggest that coumarin could be a possible solution to overcome carbapenems resistance in CRKP.

Unveiling promising breast cancer biomarkers: an integrative approach combining bioinformatics analysis and experimental verification.

BACKGROUND: Breast cancer remains a significant health challenge worldwide, necessitating the identification of reliable biomarkers for early detection, accurate prognosis, and targeted therapy. MATERIALS AND METHODS: Breast cancer RNA expression data from the TCGA database were analyzed to identify differentially expressed genes (DEGs). The top 500 up-regulated DEGs were selected for further investigation using random forest analysis to identify important genes. These genes were evaluated based on their potential as diagnostic biomarkers, their overexpression in breast cancer tissues, and their low median expression in normal female tissues. Various validation methods, including online tools and quantitative Real-Time PCR (qRT-PCR), were used to confirm the potential of the identified genes as breast cancer biomarkers. RESULTS: The study identified four overexpressed genes (CACNG4, PKMYT1, EPYC, and CHRNA6) among 100 genes with higher importance scores. qRT-PCR analysis confirmed the significant upregulation of these genes in breast cancer patients compared to normal samples. CONCLUSIONS: These findings suggest that CACNG4, PKMYT1, EPYC, and CHRNA6 may serve as valuable biomarkers for breast cancer diagnosis, and PKMYT1 may also have prognostic significance. Furthermore, CACNG4, CHRNA6, and PKMYT1 show promise as potential therapeutic targets. These findings have the potential to advance diagnostic methods and therapeutic approaches for breast cancer.

Determining the potential targets of silybin by molecular docking and its antibacterial functions on efflux pumps and porins in uropathogenic E. coli.

BACKGROUND: One of the causes of antibiotic resistance is the reduced accumulation of antibiotics in bacterial cells through pumping out the drugs. Silybin, a key component of the Silybum marianum plant, exhibits various beneficial properties, including anti-bacterial, anti-inflammatory, antioxidant, and hepatoprotective effects. METHODS AND RESULTS: Clinical isolates of E. coli were procured from 17 Shahrivar Children's Hospital in Rasht, Guilan, located in northern Iran. Their susceptibility to six antibiotics was assessed using disc diffusion and broth dilution (MIC) methods. The antibacterial effects of silybin-loaded polymersome nanoparticles (SPNs) were investigated with broth dilution (MIC) and biofilm assays. Molecular docking was utilized to evaluate silybin's (the antibacterial component) binding affinity to efflux pumps, porins, and their regulatory elements. Additionally, qRT-PCR analysis explored the expression patterns of acrA, acrB, tolC, ompC, and ompF genes in both SPNs (sub-MIC) and ciprofloxacin (sub-MIC)-treated and untreated E. coli isolates. The combined use of SPNs and ciprofloxacin exhibited a notable reduction in bacterial growth and biofilm formation, in ciprofloxacin-resistant isolates. The study identified eight overlapping binding sites of the AcrABZ-TolC efflux pump in association with silybin, demonstrating a binding affinity ranging from -7.688 to -10.33 Kcal/mol. Furthermore, the qRT-PCR analysis showed that silybin upregulated AcrAB-TolC efflux pump genes and downregulated ompC and ompF porin genes in combination with ciprofloxacin in transcriptional level in uropathogenic E. coli. CONCLUSIONS: Silybin, a safe herbal compound, exhibits potential in inhibiting antibiotic resistance within bacterial isolates, potentially through the regulation of gene expression and plausible binding to target proteins.

Assessment of bacteriocin production by clinical Pseudomonas aeruginosa isolates and their potential as therapeutic agents.

INTRODUCTION: Bacterial infections and the rising antimicrobial resistance pose a significant threat to public health. Pseudomonas aeruginosa produces bacteriocins like pyocins, especially S-type pyocins, which are promising for biological applications. This research focuses on clinical P. aeruginosa isolates to assess their bacteriocin production, inhibitory spectrum, chemical structure, antibacterial agents, and preservative potential. METHODS: The identification of P. aeruginosa was conducted through both phenotypic and molecular approaches. The inhibitory spectrum and antibacterial potential of the isolates were assessed. The kinetics of antibacterial peptide production were investigated, and the activity of bacteriocin was quantified in arbitrary units (AU ml-1). Physico-chemical characterization of the antibacterial peptides was performed. Molecular weight estimation was carried out using SDS-PAGE. qRT-PCR analysis was employed to validate the expression of the selected candidate gene. RESULT: The antibacterial activity of P. aeruginosa was attributed to the secretion of bacteriocin compounds, which belong to the S-type pyocin family. The use of mitomycin C led to a significant 65.74% increase in pyocin production by these isolates. These S-type pyocins exhibited the ability to inhibit the growth of both Gram-negative (P. mirabilis and P. vulgaris) and Gram-positive (S. aureus, S. epidermidis, E. hirae, S. pyogenes, and S. mutans) bacteria. The molecular weight of S-type pyocin was 66 kDa, and its gene expression was confirmed through qRT-PCR. CONCLUSION: These findings suggest that S-type pyocin hold significant potential as therapeutic agents against pathogenic strains. The Physico-chemical resistance of S-type pyocin underscores its potential for broad applications in the pharmaceutical, hygiene, and food industries.

Development and evaluation of suitable reference genes for qRT-PCR normalization of hybrids derived from Lycium barbarum and Lycium ruthenicum.

BACKGROUND: A correct and stably expressing reference gene is prerequisite for successful quantitative real-time PCR (qRT-PCR). Investigating gene expression profiling during flower development could enhance our understanding of the molecular mechanisms of flower formation and fertility in Lycium. METHODS AND RESULTS: In this study, 11 candidate reference genes in Lycium flower development were selected from transcriptome sequence data and evaluated with five traditional housekeeping genes from previous studies based on qRT-PCR amplification. Comparing the expression stability result of 16 candidate genes using GeNorm, NormFinder, BestKeeper, and Delta Ct algorithms, Lba04g01649 and Lba12g02820 were validated as the optimal reference genes for the flower development of Lycium. CONCLUSIONS: The reference genes identified in this study would improve the accuracy of qRT-PCR quantification of target gene expression in Lycium flower development and facilitate future functional genomics studies on flower development. This research could lay the foundation for the study of the reproduction and development of the Lycium flower.

Comparative transcriptome profiling of contrasting finger millet (Eleusine coracana (L.) Gaertn) genotypes under heat stress.

BACKGROUND: Eleusine coracana (L.) Gaertn is a crucial C4 species renowned for its stress robustness and nutritional significance. Because of its adaptability traits, finger millet (ragi) is a storehouse of critical genomic resources for crop improvement. However, more knowledge about this crop's molecular responses to heat stress needs to be gained. METHODS AND RESULTS: In the present study, a comparative RNA sequencing analysis was done in the leaf tissue of the finger millet, between the heat-sensitive (KJNS-46) and heat-tolerant (PES-110) cultivars of Ragi, in response to high temperatures. On average, each sample generated about 24 million reads. Interestingly, a comparison of transcriptomic profiling identified 684 transcripts which were significantly differentially expressed genes (DEGs) examined between the heat-stressed samples of both genotypes. The heat-induced change in the transcriptome was confirmed by qRT-PCR using a set of randomly selected genes. Pathway analysis and functional annotation analysis revealed the activation of various genes involved in response to stress specifically heat, oxidation-reduction process, water deprivation, and changes in heat shock protein (HSP) and transcription factors, calcium signaling, and kinase signaling. The basal regulatory genes, such as bZIP, were involved in response to heat stress, indicating that heat stress activates genes involved in housekeeping or related to basal regulatory processes. A substantial percentage of the DEGs belonged to proteins of unknown functions (PUFs), i.e., not yet characterized. CONCLUSION: These findings highlight the importance of candidate genes, such as HSPs and pathways that can confer tolerance towards heat stress in ragi. These results will provide valuable information to improve the heat tolerance in heat-susceptible agronomically important varieties of ragi and other crops.

Precision mapping and expression analysis of recessive bacterial blight resistance gene xa-45(t) from Oryza glaberrima.

BACKGROUND: Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most devastating diseases of rice leading to huge yield losses in Southeast Asia. The recessive resistance gene xa-45(t) from Oryza glaberrima IRGC102600B, mapped on rice chromosome 8, spans 80 Kb with 9 candidate genes on Nipponbare reference genome IRGSP-1.0. The xa-45(t) gene provides durable resistance against all the ten Xanthomonas pathotypes of Northern India, thus aiding in the expansion of recessive bacterial blight resistance gene pool. Punjab Rice PR127, carrying xa-45(t), was released for wider use in breeding programs. This study aims to precisely locate the target gene among the 9 candidates conferring resistance to bacterial blight disease. METHODS AND RESULTS: Sanger sequencing of all nine candidate genes revealed seven SNPs and an Indel between the susceptible parent Pusa 44 and the resistant introgression line IL274. The genotyping with polymorphic markers identified three recombinant breakpoints for LOC_Os08g42370, and LOC_Os08g42400, 15 recombinants for LOC_Os08g423420 and 26 for LOC_Os08g42440 out of 190 individuals. Relative expression analysis across six time intervals (0, 8, 24, 48, 72, and 96 h) after bacterial blight infection showed over expression of LOC_Os08g42410-specific transcripts in IL274 compared to Pusa 44, with a significant 4.46-fold increase observed at 72 h post-inoculation. CONCLUSIONS: The Indel marker at the locus LOC_Os08g42410 was found co-segregating with the phenotype, suggesting its candidacy towards xa-45(t). The transcript abundance assay provides strong evidence for the involvement of LOC_Os08g42410 in the resistance conferred by the bacterial blight gene xa-45(t).

Effect of human umbilical cord blood-mesenchymal stem cells on cisplatin-induced nephrotoxicity in rats.

BACKGROUND: Cisplatin-containing regimen is an effective treatment for several malignancies. However, cisplatin is an important cause of nephrotoxicity. So, many trials were performed to transplant stem cells systemically or locally to control cisplatin-induced nephrotoxicity. Stem cell therapeutic effect may be dependent on the regulation of inflammation and oxidant stress. AIM: To investigate the effect of human umbilical cord blood-mesenchymal stem cells (hUCB-MSCs) on the histological structure, the oxidant stress, and the inflammatory gene expression in an experimental model of cisplatin-induced nephrotoxicity in rats. METHOD: The rats were divided into 6 equal groups (each of 10 rats): Group I included normal rats that received no treatment. Group II included healthy rats that received IV hUCB-MSCs. Group III included untreated cisplatin-induced nephrotoxic rats. Group IV included cisplatin-induced nephrotoxic rats that received magnesium (Mg) injections after injury. Group V was injected with hUCB-MSCs after injury. Group VI received both Mg and hUCB-MSCs after injury. In tissue homogenates, reduced glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) activities were measured. Quantitative real-time-polymerase chain reaction (qRT-PCR) was performed to assess iNOS, TLR4, and NF-kB gene expression. Hematoxylin and eosin (H&E) staining was performed to study the histological structure of the kidney. Immunohistochemical staining of iNOS and NF-κB was performed, as well. RESULTS: Disturbed kidney functions, oxidative status, and histological structure were seen in the rats that received cisplatin. Treated groups showed improvements in kidney functions, oxidative status, and histological structure, particularly in the combined treatment group. CONCLUSION: In the cisplatin-induced nephrotoxicity model, hUCB-MSCs could improve the functional and morphological kidney structure by modulation of oxidative and inflammatory status.

Genomic insight of phosphate solubilization and plant growth promotion of two taxonomically distinct winter crops by Enterobacter sp. DRP3.

AIMS: Study of rhizospheric microbiome-mediated plant growth promotional attributes currently highlighted as a key tool for the development of suitable bio-inoculants for sustainable agriculture purposes. In this context, we have conducted a detailed study regarding the characterization of phosphate solubilizing potential by plant growth-promoting bacteria that have been isolated from the rhizosphere of a pteridophyte Dicranopteris sp., growing on the lateritic belt of West Bengal. METHODS AND RESULTS: We have isolated three potent bacterial strains, namely DRP1, DRP2, and DRP3 from the rhizoids-region of Dicranopteris sp. Among the isolated strains, DRP3 is found to have the highest phosphate solubilizing potentiality and is able to produce 655.89 and 627.58 µg ml-1 soluble phosphate by solubilizing tricalcium phosphate (TCP) and Jordan rock phosphate, respectively. This strain is also able to solubilize Purulia rock phosphate moderately (133.51 µg ml-1). Whole-genome sequencing and further analysis of the studied strain revealed the presence of pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase gdh gene along with several others that were well known for their role in phosphate solubilization. Further downstream, quantitative reverse transcriptase PCR-based expression study revealed 1.59-fold upregulation of PQQ-dependent gdh gene during the solubilization of TCP. Root colonization potential of the studied strain on two taxonomically distinct winter crops viz. Cicer arietinum and Triticum aestivum has been checked by using scanning electron microscopy. Other biochemical analyses for plant growth promotion traits including indole acetic acid production (132.02 µg ml-1), potassium solubilization (3 mg l-1), biofilm formation, and exopolymeric substances productions (1.88-2.03 µg ml-1) also has been performed. CONCLUSION: This study highlighted the active involvement of PQQ-dependent gdh gene during phosphate solubilization from any Enterobacter group. Moreover, our study explored different roadmaps for sustainable farming methods and the preservation of food security without endangering soil health in the future.