Assessment of reference genes for qRT-PCR normalization to elucidate host response to African swine fever infection.

Viral infection disrupts the normal regulation of the host gene's expression. In order to normalise the expression of dysregulated host genes upon virus infection, analysis of stable reference housekeeping genes using quantitative real-time-PCR (qRT-PCR) is necessary. In the present study, healthy and African swine fever virus (ASFV) infected porcine tissues were assessed for the expression stability of five widely used housekeeping genes (HPRT1, B2M, 18 S rRNA, PGK1 and H3F3A) as reference genes using standard algorithm. Total RNA from each tissue sample (lymph node, spleen, kidney, heart and liver) from healthy and ASFV-infected pigs was extracted and subsequently cDNA was synthesized, and subjected to qRT-PCR. Stability analysis of reference genes expression was performed using the Comparative delta CT, geNorm, BestKeeper and NormFinder algorithm available at RefFinder for the different groups. Direct Cycle threshold (CT) values of samples were used as an input for the web-based tool RefFinder. HPRT1 in spleen, 18 S rRNA in liver and kidney and H3F3A in heart and lymph nodes were found to be stable in the individual healthy tissue group (group A). The majority of the ASFV-infected organs (liver, kidney, heart, lymph node) exhibited H3F3A as stable reference gene with the exception of the ASFV-infected spleen, where HPRT1 was found to be the stable gene (group B). HPRT1 was found to be stable in all combinations of all CT values of both healthy and ASFV-infected porcine tissues (group C). Of five different reference genes investigated for their stability in qPCR analysis, the present study revealed that the 18 S rRNA, H3F3A and HPRT1 genes were optimal reference genes in healthy and ASFV-infected different porcine tissue samples. The study revealed the stable reference genes found in healthy as well as ASF-infected pigs and these reference genes identified through this study will form the baseline data which will be very useful in future investigations on gene expression in ASFV-infected pigs.

microRNA-106b-5p and Rab10: Potential Markers of Acute Myeloid Leukemia.

This study focuses on acute myeloid leukemia (AML), a condition with a 5-year survival rate below 30% despite various treatment options. Recent strides in targeted therapies have shown promise, leading to better outcomes with minimal toxicity. These advances underscore the importance of discovering new diagnostic and prognostic targets for AML. In this context, the authors investigated the expression of microRNA-106b-5p (miR-106b-5p), Rab10 mRNA, and Rab10 proteins in peripheral blood and bone marrow (BM) samples from both healthy individuals and AML patients at different stages of the disease (initial diagnosis, recurrence, and complete remission). This examination aimed to identify potential biomarkers for AML diagnosis, treatment, and prognosis. From June 2021 to December 2022, they collected 100 BM and peripheral blood samples. The relative expression of miR-106b-5p and Rab10 mRNA in the BM of AML patients was measured using Real-time polymerase chain reaction (qRT-PCR), while the relative expression of Rab10 protein in serum was determined using the ELISA method. The chromosomal karyotype of initially diagnosed patients was analyzed using the R tape. The qRT-PCR results revealed that the expression of miR-106b-5p and Rab10 mRNA were significantly higher in patients at initial diagnosis and recurrence compared with healthy individuals and those in complete remission (p < 0.001). They observed a significant reduction in the expression of miR-106b-5p, Rab10 mRNA, and Rab10 protein in the BM and peripheral blood of patients during complete remission (p < 0.05), as demonstrated by dynamic monitoring of five patients in the initial group. Furthermore, they found a close association between the expression of miR-106b-5p and the number of white blood cells at the initial diagnosis in AML patients (p < 0.05). Spearman correlation analysis revealed a positive correlation among miR-106b-5p, Rab10 mRNA, and Rab10 proteins (p < 0.05). The diagnostic potential of miR-106b-5p and Rab10 proteins was underscored by Receiver Operating Characteristic (ROC) curve analysis, which demonstrated their high accuracy in AML diagnosis (AUC: 0.944 and 0.853, respectively; p < 0.0001). Additionally, Kaplan-Meier survival analysis suggested that lower expression of these markers was associated with better prognoses (p < 0.05). In summary, their findings propose miR-106b-5p and Rab10 proteins as promising biomarkers for AML, offering insights for diagnosis, treatment, and prognosis.

A Transcriptomic and Metabolomic Study on the Biosynthesis of Iridoids in Phlomoides rotata from the Qinghai-Tibet Plateau.

Phlomoides rotata is a traditional Chinese herbal medicine that grows in the Qinghai-Tibet Plateau region at a 3100-5000 m altitude. Iridoid compounds are the main active compounds of the P. rotata used as medical ingredients and display anti-inflammatory, analgesic, and hepatoprotective properties. To better understand the biological mechanisms of iridoid compounds in this species, we performed a comprehensive analysis of the transcriptome and metabolome of P. rotata leaves from four different regions (3540-4270 m). Global metabolome profiling detected 575 metabolites, and 455 differentially accumulated metabolites (DAMs) were detected in P. rotata leaves from the four regions. Eight major DAMs related to iridoid metabolism in P. rotata leaves were investigated: shanzhiside methyl ester, 8-epideoxyloganic acid, barlerin, shanzhiside, geniposide, agnuside, feretoside, and catalpin. In addition, five soil physical and chemical indicators in P. rotata rhizosphere soils were analyzed. Four significant positive correlations were observed between alkaline nitrogen and geniposide, exchangeable calcium and geniposide, available potassium and shanzhiside, and available phosphorus and shanzhiside methyl ester. The transcriptome data showed 12 P. rotata cDNA libraries with 74.46 Gb of clean data, which formed 29,833 unigenes. Moreover, 78.91% of the unigenes were annotated using the eight public databases. Forty-one candidate genes representing 23 enzymes involved in the biosynthesis of iridoid compounds were identified in P. rotata leaves. Moreover, the DXS1, IDI1, 8-HGO1, and G10H2 genes associated with iridoid biosynthesis were specifically expressed in P. rotata. The integration of transcriptome and metabolome analyses highlights the crucial role of soil physical and chemical indicators and major gene expression related to iridoid metabolism pathways in P. rotata from different areas. Our findings provide a theoretical foundation for exploring the molecular mechanisms underlying iridoid compound accumulation in P. rotata.

Broadly neutralizing antibodies and monoclonal V2 antibodies derived from RV305 inhibit capture and replication of HIV-1.

An important approach to stopping the AIDS epidemic is the development of a vaccine that elicits antibodies that block virus capture, the initial interactions of HIV-1 with the target cells, and replication. We utilized a previously developed qRT-PCR-based assay to examine the effects of broadly neutralizing antibodies (bNAbs), plasma from vaccine trials, and monoclonal antibodies (mAbs) on virus capture and replication. A panel of bNAbs inhibited primary HIV-1 replication in PBMCs but not virus capture. Plasma from RV144 and RV305 trial vaccinees demonstrated inhibition of virus capture with the HIV-1 subtype prevalent in Thailand. Several RV305 derived V2-specific mAbs inhibited virus replication. One of these RV305 derived V2-specific mAbs inhibited both virus capture and replication, demonstrating that it is possible to elicit antibodies by vaccination that inhibit virus capture and replication. Induction of a combination of such antibodies may be the key to protection from HIV-1 acquisition.

Expression of the Folate Receptor Proteins FOLR1 and FOLR2 in Correlation With Clinicopathological Variables of Gastric Cancer.

INTRODUCTION: Gastric cancer (GC) remains a leading cause of cancer-related mortality worldwide, owing to its aggressive nature and poor prognosis. The role of folate receptors, particularly folate receptor 1 (FOLR1) and folate receptor 2 (FOLR2), in cancer has been increasingly recognized due to their overexpression in various malignancies including gastric cancer, and its potential implications in cancer progression, treatment resistance and as therapeutic targets. OBJECTIVE: To evaluate the expression patterns of FOLR1 and FOLR2 in GC patients' tissue and blood specimens and to correlate these patterns with clinicopathological variables. METHODS: A total of 58 gastric cancer patients were enrolled at the Regional Cancer Centre (RCC) from March 2017 to March 2020. Immunohistochemical analysis was performed to examine the expression of FOLR1 and FOLR2 in formalin-fixed paraffin-embedded (FFPE) tissue samples. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to analyze FOLR1 and FOLR2 expression in blood samples. Statistical analyses were conducted using chi-square tests, independent T-tests, and Kaplan-Meier survival analysis. RESULTS: FOLR1 and FOLR2 were overexpressed in 82.76% and 70.69% of gastric cancer tissues, respectively. High expression levels of FOLR1 were significantly associated with the diffuse type of gastric cancer (p<0.005). qRT-PCR showed significant overexpression of FOLR1 in gastric cancer blood samples compared to control samples, with a median fold change of approximately 14.18 times. Conversely, FOLR2 was significantly underexpressed in gastric cancer samples, with a fold change of 0.30. However, no significant correlation was found between FOLR2 expression and the clinicopathological features. The overall survival analysis did not show a significant difference in survival rates based on the expression levels of FOLR1 and FOLR2. CONCLUSIONS: This study highlights the differential expression patterns of FOLR1 and FOLR2 in gastric cancer and underscores the complexity of their roles in cancer biology. While FOLR1 shows potential as a biomarker for gastric cancer due to its overexpression, further studies are needed to fully elucidate the therapeutic and prognostic implications of folate receptors in gastric cancer.

Long non-coding RNAs in ferroptosis and cuproptosis impact on prognosis and treatment in hepatocellular carcinoma.

Ferroptosis and cuproptosis are recently discovered forms of cell death that have gained interest as potential cancer treatments, particularly for hepatocellular carcinoma. Long non-coding RNAs (lncRNAs) influence cancer cell activity by interacting with various nucleic acids and proteins. However, the role of ferroptosis and cuproptosis-related lncRNAs (FCRLs) in cancer remains underexplored. Ferroptosis and cuproptosis scores for each sample were assessed using Gene Set Variation Analysis (GSVA). Weighted correlation network analysis identified the FCRLs most relevant to our study. A risk model based on FCRLs was developed to categorize patients into high-risk and low-risk groups. We then compared overall survival (OS), tumor immune microenvironment, and clinical characteristics between these groups. The IPS score and ImmuCellAI webpage were used to predict the association between FCRL-related signatures and immunotherapy response. Finally, we validated the accuracy of FCRLs in hepatocellular carcinoma cell lines using induction agents (elesclomol and erastin). Patients in different risk subgroups showed significant differences in OS, immune cell infiltration, pathway activity, and clinical characteristics. Cellular assays revealed significant changes in the expression of AC019080.5, AC145207.5, MIR210HG, and LINC01063 in HCC cell lines following the addition of ferroptosis and cuproptosis inducers. We created a signature of four FCRLs that accurately predicted survival in HCC patients, laid the foundation for basic research related to ferroptosis and cuproptosis in hepatocellular carcinoma, and provided therapeutic recommendations for HCC patients.

Consuming royal jelly alters several phenotypes associated with overwintering dormancy in mosquitoes.

Introduction: Females of the Northern house mosquito, Culex pipiens, enter an overwintering dormancy, or diapause, in response to short day lengths and low environmental temperatures that is characterized by small egg follicles and high starvation resistance. During diapause, Culex pipiens Major Royal Jelly Protein 1 ortholog (CpMRJP1) is upregulated in females of Cx. pipiens. This protein is highly abundant in royal jelly, a substance produced by honey bees (Apis mellifera), that is fed to future queens throughout larval development and induces the queen phenotype (e.g., high reproductive activity and longer lifespan). However, the role of CpMRJP1 in Cx. pipiens is unknown. Methods: We first conducted a phylogenetic analysis to determine how the sequence of CpMRJP1 compares with other species. We then investigated how supplementing the diets of both diapausing and nondiapausing females of Cx. pipiens with royal jelly affects egg follicle length, fat content, protein content, starvation resistance, and metabolic profile. Results: We found that feeding royal jelly to females reared in long-day, diapause-averting conditions significantly reduced the egg follicle lengths and switched their metabolic profiles to be similar to diapausing females. In contrast, feeding royal jelly to females reared in short-day, diapause-inducing conditions significantly reduced lifespan and switched their metabolic profile to be similar nondiapausing mosquitoes. Moreover, RNAi directed against CpMRJPI significantly increased egg follicle length of short-day reared females, suggesting that these females averted diapause. Discussion: Taken together, our data show that consuming royal jelly reverses several key seasonal phenotypes of Cx. pipiens and that these responses are likely mediated in part by CpMRJP1.

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.

Proteomic and transcriptomic analyses of Cutibacterium acnes biofilms and planktonic cultures in presence of epinephrine.

Transcriptomic and proteomic analysis were performed on 72 h biofilms of the acneic strain Cutibacterium acnes and planktonic cultures in the presence of epinephrine. Epinephrine predominantly downregulated genes associated with various transporter proteins. No correlation was found between proteomic and transcriptomic profiles. In control samples, the expression of 51 proteins differed between planktonic cultures and biofilms. Addition of 5 nM epinephrine reduced this number, and in the presence of 5 µM epinephrine, the difference in proteomic profiles between planktonic cultures and biofilms disappeared. According to the proteomic profiling, epinephrine itself was more effective in the case of C. acnes biofilms and potentially affected the tricarboxylic acid cycle (as well as alpha-ketoglutarate decarboxylase Kgd), biotin synthesis, cell division, and transport of different compounds in C. acnes cells. These findings are consistent with recent research on Micrococcus luteus, suggesting that the effects of epinephrine on actinobacteria may be universal.

Full-length transcriptome sequencing of Arabidopsis plants provided new insights into the autophagic regulation of photosynthesis.

Autophagy is a highly conserved eukaryotic pathway and plays a crucial role in cell survival under stress conditions. Here, we applied a full-length transcriptome approach to study an Arabidopsis autophagy mutant (atg5-1) subjected to nitrogen-starvation, using Oxford Nanopore Technologies. A total of 39,033 transcripts were identified, including 11,356 new transcripts. In addition, alternative splicing (AS) events and lncRNAs were also detected between Col-0 (WT) and atg5-1. Differentially expressed transcript enrichment showed that autophagy upregulates the expression of many stress-responsive genes and inhibits the transcription of photosynthesis-associated genes. The qRT-PCR results showed that the expression patterns of photosynthesis-related genes in the atg5-1 differed under the conditions of nitrogen starvation and carbon starvation. Under nitrogen starvation treatment, many genes related to photosynthesis also exhibited AS. Chlorophyll fluorescence images revealed that the Fv/Fm and ΦPSII of old atg5-1 leaves were significantly reduced after nitrogen starvation treatment, but the Y(NPQ) indices were significantly increased compared to those of the WT plants. The results of qRT-PCR suggest that autophagy appears to be involved in the degradation of genes related to photodamage repair in PSII. Taken together, the full-length transcriptiome sequencing provide new insights into how new transcripts, lncRNAs and alternative splicing (AS) are involved in plant autophagy through full-length transcriptome sequencing and suggest a new potential link between autophagy and photosynthesis.

Transcriptional and biochemical changes in mouse liver following exposure to a metal/drug cocktail. Attenuating effect of a selenium-enriched diet.

Real-life pollution usually involves simultaneous co-exposure to different chemicals. Metals and drugs are frequently and abundantly released into the environment, where they interact and bioaccumulate. Few studies analyze potential interactions between metals and pharmaceuticals in these mixtures, although their joint effects cannot be inferred from their individual properties. We have previously demonstrated that the mixture (PC) of the metals Cd and Hg, the metalloid As and the pharmaceuticals diclofenac (DCF) and flumequine (FLQ) impairs hepatic proteostasis. To gain a deeper vision of how PC affects mouse liver homeostasis, we evaluated here the effects of PC exposure upon some biochemical and morphometric parameters, and on the transcriptional profiles of selected group of genes. We found that exposure to PC caused oxidative damage that exceeded the antioxidant capacity of cells. The excessive oxidative stress response resulted in an overabundance of reducing equivalents, which hindered the metabolism and transport of metabolites, including cholesterol and bile acids, between organs. These processes have been linked to metabolic and inflammatory disorders, cancer, and neurodegenerative diseases. Therefore, our findings suggest that unintended exposure to mixtures of environmental pollutants may underlie the etiology of many human diseases. Fortunately, we also found that a diet enriched with selenium mitigated the harmful effects of this combination of toxicants.

Molecular Evolution of SNAREs in Vitis vinifera and Expression Analysis under Phytohormones and Abiotic Stress.

SNARE proteins (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) play a key role in mediating a variety of plant biological processes. Currently, the function of the SNARE gene family in phytohormonal and abiotic stress treatments in grapevine is currently unknown, making it worthwhile to characterize and analyze the function and expression of this family in grapevine. In the present study, 52 VvSNARE genes were identified and predominantly distributed on 18 chromosomes. Secondary structures showed that the VvSNARE genes family irregular random coils and α-helices. The promoter regions of the VvSNARE genes were enriched for light-, abiotic-stress-, and hormone-responsive elements. Intraspecific collinearity analysis identified 10 pairs collinear genes within the VvSNARE family and unveiled a greater number of collinear genes between grapevine and apple, as well as Arabidopsis thaliana, but less associations with Oryza sativa. Quantitative real-time PCR (qRT-PCR) analyses showed that the VvSNARE genes have response to treatments with ABA, NaCl, PEG, and 4 °C. Notably, VvSNARE2, VvSNARE14, VvSNARE15, and VvSNARE17 showed up-regulation in response to ABA treatment. VvSNARE2, VvSNARE15, VvSNARE18, VvSNARE19, VvSNARE20, VvSNARE24, VvSNARE25, and VvSNARE29 exhibited significant up-regulation when exposed to NaCl treatment. The PEG treatment led to significant down-regulation of VvSNARE1, VvSNARE8, VvSNARE23, VvSNARE25, VvSNARE26, VvSNARE31, and VvSNARE49 gene expression. The expression levels of VvSNARE37, VvSNARE44, and VvSNARE46 were significantly enhanced after exposure to 4 °C treatment. Furthermore, subcellular localization assays certified that VvSNARE37, VvSNARE44, and VvSNARE46 were specifically localized at the cell membrane. Overall, this study showed the critical role of the VvSNARE genes family in the abiotic stress response of grapevines, thereby providing novel candidate genes such as VvSNARE37, VvSNARE44, and VvSNARE46 for further exploration in grapevine stress tolerance research.

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.

Molecular characterization and expression analysis of thyroid hormone receptors in protogynous rice field eel, Monopterus albus.

Thyroid hormones (THs) play important roles in growth, development, morphogenesis, reproduction, and so on. They are mainly meditated by binding to thyroid hormone receptors (TRs) in vertebrates. As important members of the nuclear receptor superfamily, TRs and their ligands are involved in many biological processes. To investigate the potential roles of TRs in the gonadal differentiation and sex change, we cloned and characterized the TRs genes in protogynous rice field eel (Monopterus albus). In this study, three types of TRs were obtained, which were TRαA, TRαB and TRß, encoding preproproteins of 336-, 409- and 415-amino acids, respectively. Multiple alignments of the three putative TRs protein sequences showed they had a higher similarity. Tissue expression analysis showed that TRαA mainly expressed in the gonad, while TRαB and TRß in the brain. During female-to-male sex reversal, the expression levels of all the three TRs showed a similar trend of increase followed by a decrease in the gonad. Intraperitoneal injection of triiodothyronine (T3) stimulated the expression of TRαA and TRαB, while it had no significant change on the expression of TRß in the ovary. Gonadotropin-releasing hormone analogue (GnRHa) injection also significantly upregulated the expression levels of TRαA and TRαB after 6 h, while it had no significant effect on TRß. These results demonstrated that TRs were involved in the gonadal differentiation and sex reversal, and TRα may play more important roles than TRß in reproduction by the regulation of GnRHa in rice field eel.

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.

Integration of food raw materials, food microbiology, and food additives: systematic research and comprehensive insights into sweet sorghum juice, Clostridium tyrobutyricum TGL-A236 and bio-butyric acid.

Introduction: Sweet sorghum juice is a typical production feedstock for natural, eco-friendly sweeteners and beverages. Clostridium tyrobutyricum is one of the widely used microorganisms in the food industry, and its principal product, bio-butyric acid is an important food additive. There are no published reports of Clostridium tyrobutyricum producing butyric acid using SSJ as the sole substrate without adding exogenous substances, which could reach a food-additive grade. This study focuses on tailoring a cost-effective, safe, and sustainable process and strategy for their production and application. Methods: This study modeled the enzymolysis of non-reducing sugars via the first/second-order kinetics and added food-grade diatomite to the hydrolysate. Qualitative and quantitative analysis were performed using high-performance liquid chromatography, gas chromatography-mass spectrometer, full-scale laser diffraction method, ultra-performance liquid chromatography-tandem mass spectrometry, the cell double-staining assay, transmission electron microscopy, and Oxford nanopore technology sequencing. Quantitative real-time polymerase chain reaction, pathway and process enrichment analysis, and homology modeling were conducted for mutant genes. Results: The treated sweet sorghum juice showed promising results, containing 70.60 g/L glucose and 63.09 g/L fructose, with a sucrose hydrolysis rate of 98.29% and a minimal sucrose loss rate of 0.87%. Furthermore, 99.62% of the colloidal particles and 82.13% of the starch particles were removed, and the concentrations of hazardous substances were effectively reduced. A food microorganism Clostridium tyrobutyricum TGL-A236 with deep utilization value was developed, which showed superior performance by converting 30.65% glucose and 37.22% fructose to 24.1364 g/L bio-butyric acid in a treated sweet sorghum juice (1:1 dilution) fermentation broth. This titer was 2.12 times higher than that of the original strain, with a butyric acid selectivity of 86.36%. Finally, the Genome atlas view, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and evolutionary genealogy of genes: Non-supervised Orthologous (eggNOG) functional annotations, three-dimensional structure and protein cavity prediction of five non-synonymous variant genes were obtained. Conclusion: This study not only includes a systematic process flow and in-depth elucidation of relevant mechanisms but also provides a new strategy for green processing of food raw materials, improving food microbial performance, and ensuring the safe production of food additives.

Systematic selection of suitable reference genes for quantitative real-time PCR normalization studies of gene expression in Lutjanus erythropterus.

Quantitative real-time PCR (qRT-PCR) has been widely employed for the study of gene expression in fish, and accurate normalization is crucial. In this study, we aimed to identify the most stably expressed genes in various tissues, different developmental stages, and within astaxanthin treatment groups in Lutjanus erythropterus. Twelve candidate genes (EEF1A, CYB5R3, DLD, IDH3A, MRPL17, MRPL43, NDUFS7, PABPC1, PAGR1, PFDN2, PSMC3, and RAB10) were examined via qRT-PCR. We employed geNorm and NormFinder to assess their stability. The results revealed that RAB10 and PFDN2 exhibited relatively stable expression patterns across different tissue and astaxanthin treatment groups, while NDUFS7 and MRPL17 proved to be the most reliable reference gene combinations across various developmental stages. The stability of these selected genes was further validated by assessing the expression of two target genes, CRADD and CAPNS1, across developmental stages, reinforcing the reliability of NDUFS7 as it closely aligned with transcriptome-wide expression patterns at these stages. The present results will help researchers to obtain more accurate results in future qRT-PCR analysis in L. erythropterus.

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.

Determination of Differential Alternative Splicing Under Stress Conditions.

Alternative splicing (AS) is an important mechanism contributing to stress-induced regulation of gene expression and proteome diversity. Massive sequencing technologies allow the identification of transcripts generated via stress-responsive AS, potentially important for adaptation to stress conditions. Several bioinformatics tools have been developed to identify differentially expressed alternative splicing events/transcripts from RNA-sequencing results. This chapter describes a detailed protocol for differential alternative splicing analysis using the rMATS tool. In addition, we provide guidelines for validation of the detected splice variants by qRT-PCR based on the obtained output files.