Real-time PCR Results Research Articles

Selection of vaccine candidates against Pseudomonas koreensis using reverse vaccinology and a preliminary efficacy trial in Empurau (Tor tambroides)

This study marks the first utilization of reverse vaccinology to develop recombinant subunit vaccines against Pseudomonas koreensis infection in Empurau (Tor tambroides). The proteome (5538 proteins) was screened against various filters to prioritize proteins based on features that are associated with virulence, subcellular localization, transmembrane helical structure, antigenicity, essentiality, non-homology with the host proteome, molecular weight, and stability, which led to the identification of eight potential vaccine candidates. These potential vaccine candidates were cloned and expressed, with six achieving successful expression and purification. The antigens were formulated into two distinct vaccine mixtures, Vac A and Vac B, and their protective efficacy was assessed through in vivo challenge experiments. Vac A and Vac B demonstrated high protective efficacies of 100 % and 81.2 %, respectively. Histological analyses revealed reduced tissue damage in vaccinated fish after experimental infection, with Vac A showing no adverse effects, whereas Vac B exhibited mild degenerative changes. Quantitative real-time PCR results showed a significant upregulation of TNF-α and downregulation of IL-1β in the kidneys, spleen, gills, and intestine in both Vac A- and Vac B-immunized fish after challenged with P. koreensis. Additionally, IL-8 exhibits tissue-specific differential expression, with significant upregulation in the kidney, gills, and intestine, and downregulation in the spleen, particularly notable in Vac A-immunized fish. The research underscores the effectiveness of the reverse vaccinology approach in fish and demonstrates the promising potential of Vac A and Vac B as recombinant subunit vaccines.

Green Synthesis of Silver Nanoparticles using Cirsium congestum Extract Modified by Chitosan/Alginate: Bactericidal Activity against Pathogenic Bacteria and Cytotoxicity Analysis in Normal Cell Line.

The study aimed to determine in vitro pharmacological effects of modified Ag nanoparticles (AgNPs). AgNPs are considered antimicrobial agents. However, the cytotoxicity of chemically synthesized AgNPs (cAgNPs) has raised challenges that limit their use. The purpose of the study was to examine the antimicrobial and cytotoxicity effects of AgNPs synthesized using Cirsium congestum extract modified by chitosan/alginate AgNPS (Ch/ALG-gAgNPs). Nanoparticles were characterized using TEM, DLS, XRD, and FTIR. Resistant strains of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were used for the antimicrobial analysis of Ch/ALG-gAgNPs using disc diffusion and microdilution methods. The effects of NPs on cell viability and apoptosis in L929 normal cells were determined using MTT assay and annexin/PI staining, respectively. Physicochemical characterizations confirmed Ch/ALG-gAgNPs to be spherical and uniformly dispersed, and their size ranged from 50 to 500 nm. Ch/ALG-gAgNPs inhibited the growth of microbial strains in a dose-dependent manner. The antibacterial effect of Ch/ALG-gAgNPs was significantly higher than cAgNPs. The Ch/ALG-gAgNPs showed little cytotoxicity against normal cells at concentrations less than 50 μg/ml. Cytotoxicity effects of Ch/ALG-gAgNP were less than cAgNPs. Flow cytometry and real-time PCR results showed a decrease in apoptosis percentage and BAX marker in the presence of Ch/ALG-gAgNPs relative to when the cell was treated with cAgNPs. Current findings introduce novel gAgNPs modified with chitosan/alginate for use in medicine.

Investigation of the effect of low-power, low-frequency ultrasound application on SARS-COV-2

Abstract Objectives Numerous studies have been conducted on the prevention, diagnosis, and treatment of the SARS-CoV-2 pandemic, which remains a global health concern. Low-frequency and low-dose ultrasound can help eradicate the virus from the air and the environment. Our research aims to determine how altering ultrasonic waves frequencies and low power affects the virulence and replication rate of a virus. Methods The virus was identified using atomic force microscopy before the initiation of laboratory tests. The experimental environment was exposed to 8 W of ultrasound at frequencies of 40 KHz, 25, 50, and 110 MHz. The cycle threshold (Ct) of the samples before and after ultrasonography was evaluated using real-time PCR (RT-PCR). Before and during ultrasonography, the VERO E6 Cell line was employed to determine whether the virus was still alive. Results Following the RT-PCR results, the application of 40 KHz ultrasonic waves frequency enhanced the Ct values of the virus while concurrently inhibiting its growth rate in the cell culture. Conclusions Our findings suggest that employing ultrasound to eliminate SARS-CoV-2 and possibly other closed and single-stranded RNA viruses from the environment is feasible.

#1870 Uninephrectomized diabetic rats delay onset of hyperglycemia via down-regulation of SGLT2 mRNA

Abstract Background and Aims The kidney is essential for glucose metabolism. Reabsorption of almost all the glucose filtered by the glomerulus is performed by the sodium-glucose cotransporters (the SGLTs) in the proximal tubules, with SGLT2 responsible for about 90%. In addition, the kidney, along with the liver, plays an important role in gluconeogenesis, especially in the long fasting state. SGLT2 inhibitors originally developed for the treatment of type 2 diabetes mellitus (DM) are clinically very effective drugs halting chronic kidney disease progression. The underlying mechanisms are, however, not fully understood. Furthermore, SGLT2 expression in diabetic patients has been reported to increase and decrease but remains unclear. Therefore, this study aimed to evaluate how uninephrectomy (UNx) and SGLT2 inhibitor treatment affects the dynamics of blood glucose and SGLTs in SDT fatty rats with obese type 2 DM (leptin receptor mutation), which is hyperglycemic from an early stage. Method 24 male SDT fatty rats were divided into four groups: sham + placebo group (n=6); sham + dapagliflozin (dapa, 1.5 mg/kg/day) group (n=6); UNx + placebo group (n=6); UNx + dapa group (n=6). UNx or sham operations were undergone at week 6. Measurements included body weight, food intake, water consumption, urine output, blood pressure, serum creatinine, and blood glucose in the experiment. At week 12, the rats were sacrificed, and the kidneys were sampled to measure SGLT1 and SGLT2 mRNA in the renal cortex by real-time PCR quantification. Results Blood glucose levels continued to rise from week 7 in the sham + placebo group, but the rate of increase was still slower in the UNx + placebo group, and the increase in blood glucose levels was mostly suppressed with dapa administration (Fig. 1). Only the Sham + placebo group showed increased urinary volume and weight loss after week 10 due to hyperglycemia. There were no significant differences in serum creatinine or blood pressure during the study. Real-time PCR results showed that SGLT2 mRNA in the renal cortex was significantly decreased by 26% in the UNx group (P=0.0386), but SGLT1 mRNA was not significantly different. Conclusion Uninephrectomized diabetic rats delayed onset of hyperglycemia and reduced SGLT2 mRNA; SGLT2 inhibitor treatment further delayed the onset of hyperglycemia but did not alter SGLT2 mRNA expression. In animal models of diabetes, renal glucose reabsorption appeared to be a major contributor to the development of hyperglycemia.

Genome-wide characterization of LEA gene family reveals a positive role of BnaA.LEA6.a in freezing tolerance in rapeseed (Brassica napus L.)

BackgroundFreezing stress is one of the major abiotic stresses that causes extensive damage to plants. LEA (Late embryogenesis abundant) proteins play a crucial role in plant growth, development, and abiotic stress. However, there is limited research on the function of LEA genes in low-temperature stress in Brassica napus (rapeseed).ResultsTotal 306 potential LEA genes were identified in B. rapa (79), B. oleracea (79) and B. napus (148) and divided into eight subgroups. LEA genes of the same subgroup had similar gene structures and predicted subcellular locations. Cis-regulatory elements analysis showed that the promoters of BnaLEA genes rich in cis-regulatory elements related to various abiotic stresses. Additionally, RNA-seq and real-time PCR results indicated that the majority of BnaLEA family members were highly expressed in senescent tissues of rapeseed, especially during late stages of seed maturation, and most BnaLEA genes can be induced by salt and osmotic stress. Interestingly, the BnaA.LEA6.a and BnaC.LEA6.a genes were highly expressed across different vegetative and reproductive organs during different development stages, and showed strong responses to salt, osmotic, and cold stress, particularly freezing stress. Further analysis showed that overexpression of BnaA.LEA6.a increased the freezing tolerance in rapeseed, as evidenced by lower relative electrical leakage and higher survival rates compared to the wild-type (WT) under freezing treatment.ConclusionThis study is of great significance for understanding the functions of BnaLEA genes in freezing tolerance in rapeseed and offers an ideal candidate gene (BnaA.LEA6.a) for molecular breeding of freezing-tolerant rapeseed cultivars.

Gluconeogenesis during development of the grass puffer (Takifugu niphobles)

During the development of teleost fish, the sole nutrient source is the egg yolk. The yolk consists mostly of proteins and lipids, with only trace amounts of carbohydrates such as glycogen and glucose. However, past evidence in some fishes showed transient increase in glucose during development, which may have supported the development of the embryos. Recently, we found in zebrafish that the yolk syncytial layer (YSL), an extraembryonic tissue surrounding the yolk, undergoes gluconeogenesis. However, in other teleost species, the knowledge on such gluconeogenic functions during early development is lacking. In this study, we used a marine fish, the grass puffer (Takifugu niphobles) and assessed possible gluconeogenic functions of their YSL, to understand the difference or shared features of gluconeogenesis between these species. A liquid chromatography (LC) / mass spectrometry (MS) analysis revealed that glucose and glycogen content significantly increased in the grass puffer during development. Subsequent real-time PCR results showed that most of the genes involved in gluconeogenesis increased in segmentation stages and/or during hatching. Among these genes, many were expressed in the YSL and liver, as shown by in situ hybridization analysis. In addition, glycogen immunostaining revealed that this carbohydrate source was accumulated in many tissues at segmentation stage but exclusively in the liver in hatched individuals. Taken together, these results suggest that developing grass puffer undergoes gluconeogenesis and glycogen synthesis during development, and that gluconeogenic activity is shared in YSL of zebrafish and grass puffer.

CRISPR/Cas9-mediated knockout of a male accessory glands-specific gene takeout1 decreases the fecundity of Zeugodacus cucurbitae female.

The melon fly, Zeugodacus cucurbitae (Coquillett), is an invasive Tephritidae pest with robust fertility. The male accessory glands (MAGs) form a vital organ that ensures insect reproductive efficiency. Most of the secreted proteins by MAGs exhibit a male bias expression. Takeout, one of these proteins, is abundantly present in the MAGs of many insects. In this study, we identified 32 takeout genes in Z. cucurbitae. The phylogenetic analysis and multiple sequence alignment results showed that Zctakeout1 is the most related homolog to the MAGs-specific takeout in Tephritidae. The real-time quantitative PCR results showed that Zctakeout1 was exclusively expressed in the male adult stage, and its expression level gradually increased with the increase in age and then remained stable at the sexually matured stage. The distribution among tissues demonstrated the specific expression of Zctakeout1 in the MAGs, and fluorescence immunohistochemical results confirmed the presence of Zctakeout1 in close proximity to binuclear cells of the mesoderm epidermal MAGs. In continuation, CRISPR/Cas9-mediated genome editing was employed, resulting in successfully generating a homozygous strain with an +8 bp insertion. The mating experiments with the Zctakeout1-/- males resulted in significant reductions in both the mating rate and egg production of females. These findings prove that the MAGs-specific Zctakeout1 is essential in regulating fecundity in female Z. cucurbitae fruit flies. Our data suggests its utilization in future essential insect-specific gene-directed sterility insect technique (SIT) by the genetic manipulation to keep these important Tephritidae populations under control. © 2024 Society of Chemical Industry.

Comparing the Developmental Toxicity Delay and Neurotoxicity of Benzothiazole and Its Derivatives (BTHs) in Juvenile Zebrafish.

In this study, a semi-static water exposure method was employed to investigate the early developmental and neurotoxic effects of four benzothiazole substances (BTHs), namely benzothiazole (BTH), 2-mercaptobenzothiazole (MBT), 2-hydroxybenzothiazole (BTON), and 2-aminobenzothiazole (2-ABTH), on zebrafish at an equimolar concentration of 10 μM. The findings revealed that all four BTHs exerted certain impacts on early development in zebrafish. MBT stimulated spontaneous movement in juvenile zebrafish, whereas BTON inhibited such movements. Moreover, all four BTHs hindered the hatching process of zebrafish larvae, with MBT exhibiting the strongest inhibition at 24 h post-fertilization (hpf). Notably, MBT acted as a melanin inhibitor by suppressing melanin production in juvenile zebrafish eyes and weakening phototaxis. Additionally, both BTH and BTON exhibited significantly lower speeds than the control group and other test groups under conditions without bright field stimulation; however, their speeds increased to average levels after percussion stimulation, indicating no significant alteration in motor ability among experimental zebrafish groups. Short-term exposure to these four types of BTHs induced oxidative damage in zebrafish larvae; specifically, BTH-, MBT-, and BTON-exposed groups displayed abnormal expression patterns of genes related to oxidative damage. Exposure to both BTH and MBT led to reduced fluorescence intensity in transgenic zebrafish labeled with central nervous system markers, suggesting inhibition of central nervous system development. Furthermore, real-time quantitative PCR results demonstrated abnormal gene expression associated with neural development. However, no significant changes were observed in 2-ABTH gene expression at this concentration. Overall findings indicate that short-term exposure to BTHs stimulates neurodevelopmental gene expression accompanied by oxidative damage.

Impact of Circulating Lymphoma Cells on HLA Typing Outcomes in Patients with Diffuse Large B-Cell Lymphoma: A Case Report

Recipient's high resolution HLA typing is required in allogeneic hematopoietic cell transplantation from unrelated donors, as well as for haploidentical family donors. For these purposes, Next-Generation Sequencing (NGS) methods are the gold standard. We present a case of a patient with an incorrect HLA typing result caused by the population of circulating lymphoma cells. The first HLA examination was performed from peripheral blood (PB) using NGS in the active phase of diffuse large B-cell lymphoma with bone marrow involvement. Because of rare and inconclusive results, confirmed twice for the A* locus (A*02:32N), real-time polymerase chain reaction (RT-PCR)was performed. With RT-PCR method, we obtained more expected results according to the population allele frequency: in HLA-A locus (A*02:01) but also in DQB1 (DQB1*03:01, not as in NGS - DQB1*03:10). For the final verification, we used swab material and we obtained unambiguous NGS result with expected, frequent HLA-A*02:01 and DQB1*03:01 alleles corresponding to the RT-PCR result from PB. To conclude, we suspect that the discrepancies between NGS and RT-PCR results were caused by the presence of a significant amount of circulating lymphoma cells in the peripheral blood sample. Lymphomagenic mutations may involve the histocompatibility antigen coding region and affect HLA expressed on malignant cells. This finding may be relevant for the selection of test material in primary and confirmatory HLA testing in patients with active hematological malignancies because of the strong impact of incorrect HLA typing on the procedure of a donor selection.

Ten different viral agents infecting and co-infecting children with acute gastroenteritis in Southern Italy: Role of known pathogens and emerging viruses during and after COVID-19 pandemic.

Acute gastroenteritis (AGE) represents a world public health relevant problem especially in children. Enteric viruses are the pathogens mainly involved in the episodes of AGE, causing about 70.00% of the cases. Apart from well-known rotavirus (RVA), adenovirus (AdV) and norovirus (NoV), there are various emerging viral pathogens potentially associated with AGE episodes. In this study, the presence of ten different enteric viruses was investigated in 152 fecal samples collected from children hospitalized for gastroenteritis. Real time PCR results showed that 49.3% of them were positive for viral detection with the following prevalence: norovirus GII 19.7%, AdV 15.8%, RVA 10.5%, human parechovirus (HPeV) 5.3%, enterovirus (EV) 3.3%, sapovirus (SaV) 2.6%. Salivirus (SalV), norovirus GI and astrovirus (AstV) 1.3% each, aichivirus (AiV) found in only one patient. In 38.2% of feces only one virus was detected, while co-infections were identified in 11.8% of the cases. Among young patients, 105 were ≤5 years old and 56.0% tested positive for viral detection, while 47 were >5 years old with 40.0% of them infected. Results obtained confirm a complex plethora of viruses potentially implicated in gastroenteritis in children, with some of them previously known for other etiologies but detectable in fecal samples. Subsequent studies should investigate the role of these viruses in causing gastroenteritis and explore the possibility that other symptoms may be ascribed to multiple infections.

Systematic Analysis of Zinc Finger-Homeodomain Transcription Factors (ZF-HDs) in Barley (Hordeum vulgare L.).

Zinc finger-homeodomain transcription factors (ZF-HDs) are pivotal in regulating plant growth, development, and diverse stress responses. In this study, we found 8 ZF-HD genes in barley genome. Theses eight HvZF-HD genes were located on five chromosomes, and classified into ZHD and MIF subfamily. The collinearity, gene structure, conserved motif, and cis-elements of HvZF-HD genes were also analyzed. Real-time PCR results suggested that the expression of HvZF-HD4, HvZF-HD6, HvZF-HD7 and HvZF-HD8 were up-regulated after hormones (ABA, GA3 and MeJA) or PEG treatments, especially HvZF-HD6 was significantly induced. These results provide useful information of ZF-HD genes to future study aimed at barley breeding.

Release of exosomes from injectable silk fibroin and alginate composite hydrogel for treatment of myocardial infarction.

Myocardial infarction (MI) is considered as a significant cause of death globally. Exosomes (EXOs) are essential for intercellular communication and pathophysiology of several cardiovascular diseases. Nevertheless, the short half-life and rapid clearance of EXOs leads to a lack of therapeutic doses delivered to the lesioned area. Therefore, an injectable silk fibroin and alginate (SF/Alg) composite hydrogel was developed to bind folate receptor-targeted EXOs (FA-EXOs) derived from H9C2 cells for the therapy of myocardial injury following myocardial infarction-ischemia/reperfusion (MI-I/R). The resulting composite exhibits a variety of properties, including adjustable gelation kinetics, shear-thinning injectability, soft and dynamic stability that adapts to the heartbeat, and outstanding cytocompatibility. After injected into the damaged rat heart, administration of SF/Alg + FA-EXOs significantly enhanced cardiac function as demonstrated by improved ejection fraction, fractional shortening and decreased fibrosis area. The results of real-time PCR and immunofluorescence staining show a remarkable up-regulation in the expression of proteins (CD31) and genes (VWF and Serca2a) related to the heart. Conversely, expression of fibrosis-related genes (TGF-β1) decreased significantly. Therefore, the obtained SF/Alg + FA-EXOs system remarkably enhanced the intercellular interactions, promoted cell proliferation and angiogenesis, and achieved an outstanding therapeutic effect on MI.

Modulation of IKK/NF-κB Signaling: A Therapeutic Mechanism of Shilajit in Breast Cancer Cells

Background: Shilajit is a natural pale-brown to blackish-brown phytocomplex that exudes from mountain rocks in various parts of the world. In recent years, numerous pharmacological effects of shilajit have been explored through extensive experiments. Shilajit is known as an antioxidant and anti-inflammatory agent. Activation of the nuclear factor kappa B (NF-κB) signaling pathway could be an important player in inflammation-driven tumor progression. Therefore, the current study evaluated the anti-inflammatory effect of shilajit on a breast cancer cell line (MCF7). Methods: In this experimental study, the MCF7 cell line was treated with shilajit, and an MTT assay was applied to analyze the half-maximal inhibitory concentration (IC50) after 72 hours of treatment. Following this, the apoptotic rate was assessed using flow cytometry, and p50, RelB, and IKKα/β gene expression were evaluated using real-time PCR assay. Results: Shilajit had potent cytotoxic activity in a dose- and time-dependent manner with an IC50 of 280 μg/mL. Based on the Annexin-PI analysis, the IC50 concentration of this compound induced significant apoptosis in the cells, possibly through suppression of NF-κB (p50, RelB, and IKKα/β)-regulated genes. The real-time PCR results indicated that treating MCF7 cells at the IC50 dose of shilajit for 72 hours could reduce the mRNA expression levels of p50, RelB, and IKKα/β in the cells. Conclusion: The findings of the present experiment showed that shilajit may have promising anti-breast-tumor and anti-inflammatory activity through inhibition of the NF-κB signaling pathway. Therefore, shilajit can help treat breast cancer in combination with other standard treatments.

Design and in vitro evaluation of curcumin-loaded PLGA nanoparticle-embedded sodium alginate/gelatin 3D printed scaffolds for Alzheimer's disease

BackgroundTargeted nanoparticles (NPs) are aimed at improving clinical outcomes by enhancing the diagnostic and therapeutic efficacy of drugs in the treatment of Alzheimer's disease (AD). MethodsCurcumin (CUR)-loaded poly-lactic-co-glycolic acid (PLGA) NPs (CNPs) were produced to demonstrate a prolonged release and successfully embedded into 3D printed sodium alginate (SA)/gelatin (GEL) scaffolds that can dissolve rapidly sublingually. Characterization and in vitro activity of the NPs and scaffolds were evaluated. ResultsBased on the in vitro drug release studies, 99.6 % of the encapsulated CUR was released in a controlled manner within 18 days for the CNPs. In vitro cell culture studies showed that all samples exhibited cell viability above 84.2 % and no significant cytotoxic effect on SH-SY5Y cells. The samples were analyzed through 2 different pathways by PCR analysis. Real-time PCR results indicated that CNP and CNP-embedded SA/GEL scaffolds (CNPSGS) may show neuroprotective effects by modulating the Wnt/β-catenin pathway. The gene expression level of β-catenin slightly increased compared to the gene expression levels of other proteins and enzymes with these treatments. However, the PI3K/Akt/GSK-3β signaling pathway was regulated at the same time because of the crosstalk between these 2 pathways. ConclusionCNPSGS might be an effective therapeutic alternative for AD treatment.

Genome-wide identification of toll-like receptors in Octopus sinensis and expression analysis in response to different PAMPs stimulation

Toll-like receptors (TLRs) are one of the extensively studied pattern recognition receptors (PRRs) and play crucial roles in the immune responses of vertebrates and invertebrates. In this study, 14 TLR genes were identified from the genome-wide data of Octopus sinensis. Protein structural domain analysis showed that most TLR proteins had three main structural domains: extracellular leucine-rich repeats (LRR), transmembrane structural domains, and intracellular Toll/IL-1 receptor domain (TIR). The results of subcellular localization prediction showed that the TLRs of O. sinensis were mainly located on the plasma membrane. The results of quantitative real-time PCR (qPCR) showed that the detected TLR genes were differentially expressed in the hemolymph, white bodies, hepatopancreas, gills, gill heart, intestine, kidney, and salivary gland of O. sinensis. Furthermore, the present study investigated the expression changes of O. sinensis TLR genes in hemolymph, white bodies, gills, and hepatopancreas in different phases (6 h, 12 h, 24 h, 48 h) after stimulation with PGN, poly(I: C) and Vibrio parahaemolyticus. The expression of most of the TLR genes was upregulated at different time points after infection with pathogens or stimulation with PAMPs, a few genes were unchanged or even down-regulated, and many of the TLR genes were much higher after V. parahaemolyticus infection than after PGN and poly(I:C) stimulation. The results of this study contribute to a better understanding of the molecular immune mechanisms of O. sinensis TLRs genes in resistance to pathogen stimulation.

Histone H2B lysine 122 and lysine 130, as the putative targets of Penicillium oxalicum LaeA, play important roles in asexual development, expression of secondary metabolite gene clusters, and extracellular glycoside hydrolase synthesis.

Core histones in the nucleosome are subject to a wide variety of posttranslational modifications (PTMs), such as methylation, phosphorylation, ubiquitylation, and acetylation, all of which are crucial in shaping the structure of the chromatin and the expression of the target genes. A putative histone methyltransferase LaeA/Lae1, which is conserved in numerous filamentous fungi, functions as a global regulator of fungal growth, virulence, secondary metabolite formation, and the production of extracellular glycoside hydrolases (GHs). LaeA's direct histone targets, however, were not yet recognized. Previous research has shown that LaeA interacts with core histone H2B. Using S-adenosyl-L-methionine (SAM) as a methyl group donor and recombinant human histone H2B as the substrate, it was found that Penicillium oxalicum LaeA can transfer the methyl groups to the C-terminal lysine (K) 108 and K116 residues in vitro. The H2BK108 and H2BK116 sites on recombinant histone correspond to P. oxalicum H2BK122 and H2BK130, respectively. H2BK122A and H2BK130A, two mutants with histone H2B K122 or K130 mutation to alanine (A), were constructed in P. oxalicum. The mutants H2BK122A and H2BK130A demonstrated altered asexual development and decreased extracellular GH production, consistent with the findings of the laeA gene deletion strain (ΔlaeA). The transcriptome data showed that when compared to wild-type (WT) of P. oxalicum, 38 of the 47 differentially expressed (fold change ≥ 2, FDR ≤ 0.05) genes that encode extracellular GHs showed the same expression pattern in the three mutants ΔlaeA, H2BK122A, and H2BK130A. The four secondary metabolic gene clusters that considerably decreased expression in ΔlaeAalso significantly decreased in H2BK122A or H2BK130A. The chromatin of promotor regions of the key cellulolytic genes cel7A/cbh1 and cel7B/eg1 compacted in the ΔlaeA, H2BK122A, and H2BK130A mutants, according to the results of chromatin accessibility real-time PCR (CHART-PCR). The chromatin accessibility index dropped. The histone binding pocket of the LaeA-methyltransf_23 domain is compatible with particular histone H2B peptides, providing appropriate electrostatic and steric compatibility to stabilize these peptides, according to molecular docking. The findings of the study demonstrate that H2BK122 and H2BK130, which are histone targets of P. oxalicum LaeA in vitro, are crucial for fungal conidiation, the expression of gene clusters encoding secondary metabolites, and the production of extracellular GHs.

Circadian rhythm response and its effect on photosynthetic characteristics of the Lhcb family genes in tea plant

BackgroundThe circadian clock, also known as the circadian rhythm, is responsible for predicting daily and seasonal changes in the environment, and adjusting various physiological and developmental processes to the appropriate times during plant growth and development. The circadian clock controls the expression of the Lhcb gene, which encodes the chlorophyll a/b binding protein. However, the roles of the Lhcb gene in tea plant remain unclear.ResultsIn this study, a total of 16 CsLhcb genes were identified based on the tea plant genome, which were distributed on 8 chromosomes of the tea plant. The promoter regions of CsLhcb genes have a variety of cis-acting elements including hormonal, abiotic stress responses and light response elements. The CsLhcb family genes are involved in the light response process in tea plant. The photosynthetic parameter of tea leaves showed rhythmic changes during the two photoperiod periods (48 h). Stomata are basically open during the day and closed at night. Real-time quantitative PCR results showed that most of the CsLhcb family genes were highly expressed during the day, but were less expressed at night.ConclusionsResults indicated that CsLhcb genes were involved in the circadian clock process of tea plant, it also provided potential references for further understanding of the function of CsLhcb gene family in tea plant.

Microglia differential genes and their functions in paraquat-induced Parkinson's disease-like in mice's brains based on single-cell RNA sequencing

Objective: To analyze the differential genes and related signaling pathways of microglia subpopulations in Parkinson's disease (PD) -like mouse brains induced by paraquat (PQ) based on single-cell RNA sequencing, and provide clues to elucidate the mechanism of PQ-induced PD-like changes in the brain of animals. Methods: In September 2021, six male 6-week-old C57BL/6 mice were randomly divided into control group and experimental group (three mice in each group) . The mice were injected with saline, 10.0 mg/kg PQ intraperitoneally, once every three days, and 10 consecutive injections were used for modeling. After infection, the brains of mice were taken and 10×Genomics single-cell RNA sequencing was performed. Microglia subpopulations were screened based on gene expression characteristics, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. The differential genes of microglia subpopulations between the experimental group and control group were further screened, and functional enrichment analysis was performed using bioinformatics tools. Mouse microglia (BV2 cells) were treated with 0, 60, 90 μmol/L PQ solution, respectively. And real-time fluorescence quantitative PCR experiments were conducted to validate the expressions of differential genes hexokinase 2 (Hk2) , ATPase H+ Transporting V0 Subunit B (Atp6v0b) and Neuregulin 1 (Nrg1) . Results: Cluster 7 and Cluster 20 were identified as microglia subpopulations based on the signature genes inositol polyphosphate-5-phosphatase d, Inpp5d (Inpp5d) and transforming growth factor beta receptor 1 (Tgfbr1) , and they reflected the microglia-activated M2 phenotype. The bioinformatics analysis showed that the characteristic genes of identified microglia subpopulations were enriched in endocytosis. In terms of molecular function, it mainly enriched in transmembrane receptor protein kinase activity and cytokine binding. The up-regulated genes of Cluster 7 were mainly enriched in lysosomal pathway, endocytosis pathway, and down-regulated genes were mainly enriched in neurodegenerative disease and other signaling pathways. The up-regulated genes of Cluster 20 were mainly enriched in signaling pathways related to PD, and down-regulated genes were mainly enriched in cyclic adenosine 3', 5'-monophosphate (cAMP) signaling pathways, neurological development, synaptic function and other signaling pathways. The results of real-time fluorescence quantitative PCR showed that the expressions of Hk2 mRNA and Atp6v0b mRNA increased and the expression of Nrg1 mRNA decreased in the 90 μmol/L PQ-treated BV2 cells compared with the 0 μmol/L, and the differences were statistically significant (P<0.05) . Conclusion: Microglia are activated in the PQ-induced PD-like mouse model and polarized toward the M2 phenotype. And their functions are associated with lysosomal (endocytosis) , synaptic functions and the regulation of PD-related pathways.

Evolutionary Characterization of tubulin Gene Family in the Desert Biomass Willow (Salix psammophila) and Expression of the β-tubulin Gene SpsTUB10 during Different Stresses

Microtubules, polymerized from α-tubulin (TUA) and β-tubulin (TUB) monomers, play a pivotal role in shaping plant morphogenesis according to developmental and environmental cues. Salix psammophila C. Wang &amp; C. Y. Yang is an important shrub plant in sand-fixing afforestation in arid regions, with three significantly distinct plant types shaped under various environments, namely, upright, intermediate, and scattered types. However, how tubulin genes respond to the developmental and environmental signs in S. psammophila has been far less studied. Here, based on RNA-seq, Sanger sequencing, and real-time PCR (RT-PCR) data, we analyzed the phylogeny of tubulins and their expression profiles in S. psammophila among the three plant types. Furthermore, we analyzed the genetic structure and expression pattern of SpsTUB10 in S. psammophila under various abiotic stress treatments. In total, we identified 26 SpsTubulin genes in S. psammophila. The homologous alignment and phylogenetic analysis revealed that these SpsTubulin genes can be classified into two groups, corresponding to the TUA and TUB genes. The expression profiles of these SpsTubulin genes in various organs showed that most SpsTubulin genes were mainly expressed in the root. SpsTUB10 is a member of the TUB IIa group, consisting of two intros and three exons. The SpsTUB10 protein contains a typical GTPase domain and a C-terminal domain, with α-helix and random coil dominant in the secondary and tertiary structures. The RT-PCR results of SpsTUB10 showed an extremely significant difference in expression levels among the root and stem-developing organs between the upright and scattered types, and the transcript level of SpsTUB10 had a significantly negative correlation with the crown-height ratio. Under different treatments, we found that cold, osmotic stress, and short daylight could significantly increase SpsTUB10 expression levels compared to those in the controls, thereby supporting the positive role of SpsTUB10 in stress-induced responses. These results will provide evidence for the SpsTubulin genes’ response to the developmental and environmental cues in S. psammophila.

Activated and Naïve Allogenic Human Placental Mesenchymal Stromal Cells Exert an Immunomodulatory Effect on Hidradenitis Suppurativa Patient Peripheral Blood Mononuclear Cells.

This pilot study aimed to evaluate the immunomodulatory effect of placental mesenchymal stem/stromal cells (MSCs) on peripheral blood mononuclear cells (PBMCs) from patients with hidradenitis suppurativa (HS). Blood samples were collected from 3 healthy and 3 patients with HS. Isolated PBMCs were stained with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with phorbol 12-myristate 13-acetate (PMA)/Ionomycin solution. The PBMCs of patients with HS were co-cultured with naïve MSCs (n-MSCs), activated with tumor necrosis factor (TNF)-α (10 ng/mL) and interferon (IFN)-γ (10 ng/mL) MSCs (a-MSCs), or adalimumab (30 μg/mL). The division index (proliferation inhibition) of PBMCs was analyzed by flow cytometry using the Proliferation Modeling tool after 5 days of coculture. The relative inflammatory gene expression dynamics and cytokine secretion were quantified in triplicate using real-time polymerase chain reaction (PCR) and Luminex assays. PBMCs from the HS control group showed statistically significant increases in interleukin (IL)-6 and IFN-γ cytokine concentrations and IL-17A gene expression when compared with healthy subjects. Statistically significant reduction of the division index was found in the a-MSCs group (P = 0.04). Also, the Luminex assay revealed significantly reduced proinflammatory cytokine concentrations of IL-9 (P = 0.022) and IL-17A (P = 0.022) in the a-MSCs group with the same trend of numerical lowering in n-MSCs group when compared to HS control. The results of real-time PCR revealed a numerical increase in the expression of the IL-1β, IL-36α, and TNF-α genes in both the a-MSCs and n-MSCs groups compared with the HS control. In conclusion, our findings suggest that MSCs can effectively curb PBMCs proliferation and suppress the production of inflammatory cytokines. Moreover, the preactivation of MSCs with IFN-γ and TNF-α before use can enhance their therapeutic effectiveness. Nevertheless, a larger sample size is imperative to validate these results.