Real-time Fluorescence Quantitative PCR Analysis Research Articles

Functional Verification of Transcription Factor comp54181_c0 in Monascus purpureus.

Monacolin K is a valuable secondary metabolite produced after a period of fermentation by Monascus purpureus; however, our current understanding of the regulatory mechanisms of its synthesis remains incomplete. This study conducted functional analysis on the key transcription factor, comp54181_c0, that is involved in the synthesis of monacolin K in Monascus. Mutant strains with either knockout or overexpression of comp54181_c0 were constructed using CRISPR/Cas9. A comparison between the knockout and overexpression strains revealed changes in fungal morphology and growth, with a significant increase in the production of Monascus pigments and monacolin K when comp54181_c0 was absent. Real-time fluorescence quantitative PCR analysis revealed that comp54181_c0 significantly influenced the transcription of key genes related to monacolin K biosynthesis in Monascus. In conclusion, our study elucidates the crucial role of comp54181_c0 in Monascus, enriches our understanding of fungal secondary metabolite development and regulation, and provides a foundation for the development and regulation of Monascus and monacolin K production.

RNAi-mediated silencing of transferrin promotes entomopathogens lethality in Odontotermes formosanus (Shiraki)

Transferrin (Tsf) is a highly conserved multifunctional protein involved in insect physiology, defense and development that has been developed as a novel RNA interference (RNAi)-based target for pest control. The function study of the Tsf gene in Odontotermes formosanu (Shiraki) was evaluated for synergistic control of this agroforestry pest with Serratia marcescens (SM1), Bacillus thuringiensis (Bt) or Beauveria bassiana (Bb). The Tsf gene of O. formosanus was identified and characterized. Real-time fluorescent quantitative PCR (qPCR) analysis demonstrated that OfTsf was most highly expressed in the male dealate of O. formosanus, and OfTsf was highly expressed in the hemolymph. OfTsf expression was considerably elevated after SM1, Bt or Bb infection. Furthermore, dsOfTsf treatment was effective in increasing the virulence of entomopathogens to O. formosanus. In addition, OfTsf expression was markedly upregulated in O. formosanus fed with oxidative stress inducers; reactive oxygen species (ROS) levels were significantly increased after dsOfTsf treatment. Therefore, OfTsf gene played an important role in defending against entomopathogen infection and antioxidant stress. Most importantly, our work suggested OfTsf as a potential RNAi target for the control of O. formosanus.

Ge-Zhi-Jie-Jiu decoction alleviates alcoholic liver disease through multiple signaling pathways

Ethnopharmacological relevanceAlcoholic liver disease (ALD) is a growing public health concern caused by excessive alcohol consumption, but effective treatments are limited. Ge-Zhi-Jie-Jiu decoction (JJY) is a modified traditional Chinese herbal remedy that aims to alleviate ALD. This formula contains various components such as Ge Hua, Ge Gen, Zhi Ju Zi, and other medicinal-food herbs. However, the specific pharmacotherapeutic compounds of JJY and its pharmacological mechanisms remain unclear. Aim of the studyThis study aimed to elucidate the molecular mechanism and pharmacodynamic basis of JJY in treating ALD. Materials and methodsUPLC-Q-Orbitrap HRMS, HPLC fingerprinting, and LC-MS techniques were used for the composition identification and quality control of JJY. The pharmacological components and molecular mechanisms of JJY in anti-ALD were then predicted using network pharmacology and molecular docking approaches. Ultimately, an acute alcoholic liver injury mouse model was developed, and the potential mechanisms were verified by hematoxylin-eosin (H&E), Oil Red O, and TUNEL staining, real-time fluorescence quantitative PCR (RT-qPCR), Western blot (WB) and molecular docking analysis. ResultsThe results showed that the main components of JJY are organic acids, flavonoids, and isoflavonoids, in which puerarin, daidzein, glycitein, ononin, quercetin, and tectorigenin can be used as the indicator components of JJY. In addition, JJY might ameliorate ALD through several pathways, including potentially promoting alcohol metabolism via alcohol-metabolizing enzymes, and possibly inhibiting oxidative stress, inflammation and apoptosis via the Nrf2/Keap1/HO-1 and MAPK signaling pathways. Furthermore, JJY may also alleviated hepatic lipid accumulation through the PPARα signaling pathway. ConclusionsJJY has significant anti-ALD efficacy with multiple mechanisms. This study offers a solid experimental foundation for JJY's development as a medicine with anti-ALD characteristics and elucidates its probable active components.

Transcriptome-wide identification of the Hsp70 gene family in Pugionium cornutum and functional analysis of PcHsp70-5 under drought stress.

The PcHsp70-5 enhances drought stress tolerance in transgenic Arabidopsis thaliana by upregulating stress tolerance genes and antioxidant enzyme activities. Heat shock proteins (HSPs) constitute a class of evolutionarily conserved proteins synthesized by organisms in response to various adverse environmental stimuli such as elevated temperatures, drought, hormonal fluctuations, high salt concentrations, and mechanical stress. However, research on HSPs has predominantly focused on model plants and crops, whereas their functions in desert plants have not been well investigated. This study analyzed the transcriptome of Pugionium cornutum and identified the complete ORFs of 25 genes of the PcHsp70 family genes. Their expression levels under drought stress were investigated using existing RNA-seq data. PcHsp70-5 genes exhibited high expression levels in both roots and leaves under drought stress. Consequently, the PcHsp70-5 genes were cloned and transformed into Arabidopsis thaliana for further analysis of their roles in drought stress response. Real-time fluorescence quantitative PCR (qRT-PCR) analysis demonstrated that both, drought stress and ABA, induced PcHsp70-5 expression. Under drought conditions, transgenic Arabidopsis plants exhibited markedly enhanced growth compared to wild-type plants, as evidenced by improved survival rates, root length, fresh weight, chlorophyll content, and reduced levels of malondialdehyde (MDA) and hydrogen peroxide (H2O2) in leaves, indicating that PcHsp70-5 overexpression mitigated growth inhibition and oxidative damage induced by drought stress. Subsequent research revealed that PcHsp70-5 overexpression significantly augmented the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and increased the proline content in transgenic Arabidopsis under drought conditions, alongside a significant increase in the expression levels of genes related to stress tolerance. This suggests that PcHsp70-5 enhances drought stress tolerance in transgenic Arabidopsis by upregulating stress tolerance genes and antioxidant enzyme activities.

Prognostic value of KLFs family genes in renal clear cell carcinoma

Numerous studies have shown that the Krüppel-like factors (KLFs) family of transcription factors regulate various eukaryotic physiological processes including the proliferation, differentiation, senescence, death, and carcinogenesis of animal cells. In addition, they are involved in the regulation of key biological processes such as cell cycle, DNA repair, and immune response. Current studies focus on investigating the role of KLFs in normal physiological conditions and the incidence and development of diseases. However002C the significance of KLFs family genes in clear cell renal cell carcinoma (ccRCC) remains partly understood; therefore, an in-depth investigation of their role and clinical value in this cancer is desired. The study aimed to investigate the role of KLF family genes in the incidence, development, and prognosis of ccRCC, and to identify the related potential biomarkers and therapeutic targets. The expression of KLFs in the RNA sequencing data of 613 ccRCCs from the TCGA database was analyzed using R software, and UALCAN and GEPIA assessed the expression of KLF genes in ccRCC. Real-time fluorescence quantitative PCR analysis was performed using 10 pairs of paired ccRCC sample tissues and renal cancer cell lines from the First Affiliated Hospital of Nanchang University. Overall survival (OS), progression-free interval (PFI), and disease-specific survival (DSS) of Kidney Clear Cell Carcinoma (KIRC) samples at differential expressions of KLFs in the TCGA database were analyzed using the R software, followed by generating a nomogram prediction model. GSCALite assessed the interactions of KLF genes with miRNAs and generated network maps. Protein interaction network maps of 50 neighboring genes associated with KLF mutations were analyzed using STRING with GO and KEGG functional enrichment analyses. The cBioPortal determined the probability of KLF gene mutations and their impact on OS and disease-free survival (DFS) in patients with ccRCC. Immune cell infiltration of KLFs was analyzed using TIMER. Finally, GSCALite was used to analyze the drug sensitivity and associated pathways of action of KLFs. Correlation validation using cellular experiments. KLF3/5/9/15 were significantly downregulated in ccRCC tissues, whereas KLF16/17 were upregulated compared with the adjacent tissues. Patients with high mRNA levels of KLF16/17 showed significantly lower OS, PFI, and DSS, whereas KLF3/5/9 showed a reverse trend. In patients with ccRCC, a significant correlation was observed between KLF mutations and OS and DSS. Furthermore, the correlation of KLF3/5/9 with immune cell infiltration was stronger than that of KLF15/16, while KLF17 was significantly associated with the Epithelial-Mesenchymal Transition (EMT) pathway. Overexpression of KLF5 inhibits the proliferative and migratory capacity of renal cancer cells (786-O and OS-RC-2), as well as their sensitivity to relevant small molecule drugs. Our research revealed the expression levels and biological significance of KLF genes in ccRCC, particularly highlighting the potential of KLF5 as a promising biomarker and therapeutic target for effective prognosis and diagnosis of ccRCC.

Repair of Mechanical Cartilage Damage Using Exosomes Derived from Deer Antler Stem Cells.

Articular cartilage has limited self-repair capacity, and current clinical treatment options for cartilage defects are inadequate. However, deer antler cartilage possesses unique regenerative properties, with the ability to rapidly repair itself. This rapid self-repair process is closely linked to the paracrine factors released by deer antler stem cells. These findings present potential for the development of cell-free therapies for cartilage defects in clinical settings. The aim of this study was to investigate a novel method for repairing cartilage. A rat model with articular cartilage defects was established through surgery. Hydrogels loaded with exosomes (Exos) derived from antler stem cells (ASC-Exos) were implanted into the rat cartilage defects. The extent of cartilage damage repair was assessed using histological methods. The effects of ASC-Exos on chondrocytes and rat bone marrow mesenchymal stem cells (BMSCs) were evaluated using cell viability assays, proliferation assays, and scratch assays. Additionally, the maintenance of the chondrocyte phenotype by ASC-Exos was assessed using real-time fluorescence quantitative PCR (qPCR) and western blot analysis. The protein components contained of the Exos were identified using data-independent acquisition (DIA) mass spectrometry. ASC-Exos significantly promoted the repair of cartilage tissue damage. The level of cartilage repair in the experimental group (ASC-Exos) was higher than that in the positive control (human adipose-derived stem cells, hADSC-Exos) and negative control (dulbecco's modified eagle medium) groups (p < 0.05). In vitro experiments demonstrated that ASC-Exos significantly enhanced the proliferation abilities of chondrocytes and the proliferation abilities and the migration abilities of BMSCs (p < 0.05). ASC-Exos up-regulated the expression levels of Aggrecan, Collagen II (COLII), and Sox9 mRNA and proteins in chondrocytes. Analysis of ASC-Exos protein components revealed the presence of active components such as Serotransferrin (TF), S100A4, and Insulin-like growth factor-binding protein 1 (IGF1). ASC-Exos have a significant effect on cartilage damage repair, which may be attributed to their promotion of chondrocyte and BMSCs proliferation and migration, as well as the maintenance of chondrocyte phenotype. This effect may be mediated by the presence of TF, S100A4, and IGF1.

Analysis of the relationship between intestinal segmented filamentous bacteria and rotavirus infection in children

Objective: To investigate the association between intestinal colonization of segmented filamentous bacteria (SFB) and the risk of rotavirus infection, and the possible mechanisms by which SFB resist rotavirus infection. Methods: This case-control study enrolled 50 children aged 0 to 5 years who present to the outpatient Department of Children's Hospital, Zhejiang University School of Medicine with diarrhea and positive stool tests for rotavirus. The children were divided into rotavirus enteritis group and control group consisting of 55 children with non-gastrointestinal and non-infectious surgical diseases.The age and sex composition of the two groups was matched. The DNA of the fecal flora was extracted and SFB was detected by real-time fluorescence quantitative PCR analysis. The children in the rotavirus enteritis group and the control group were subgrouped by age and sex to analyze the differences in SFB positivity rates between different groups, and further compare and analyze the differences in SFB positivity rates between these two groups of children in the ≤2 years old subgroup and the >2-5 years old subgroup. Neutralization test was performed with p3340 protein and rotavirus to determine the relationship between rotavirus infection rate and p3340 concentration in Vero cells. χ2 test or Fisher's exact probability method was used for comparison between the two groups. Results: There were 50 children in the rotavirus enteritis group with an age of (1.7±0.9) years, and 55 children in the control group with an age of (1.8±1.1) years. The positive rate of SFB in children with rotavirus enteritis showed a declining trend across ages groups, with the highest rate of 10/14 in the ≤1 year old group, followed by 67% (14/21) in the >1-2 years old group, 9/15 in the >2-5 years old group, and there was no statistically significant difference (P=0.867). The positive rate of SFB in the control group was 12/15 in the ≤1 year old group, 95% (19/20) in the >1-2 years old group, 50% (10/20) in the >2-5 years old group, with statistical significance (P=0.004). The positive rate of SFB in children with rotavirus enteritis was 74% (20/27) in males and 56% (13/23) in females (χ2=1.71, P=0.192). In the control group, it was 79% (22/28) in males and 70% (19/27) in females (χ2=0.49, P=0.485). The positive rate of SFB was 66% (33/50) in the rotavirus enteritis group and 75% (41/55) in the control group, with no statistically significant (χ2=0.56, P=0.454). In the children ≤2 years old, the SFB positivity rate was 69% (24/35) in the rotavirus enteritis group and 89% (31/35) in the control group, with a statistically significant difference (χ2=4.16, P=0.041). However, in the children >2-5 years old, no statistically significant difference was observed, with the positive rate of SFB being 9/15 in the rotavirus enteritis group and 50% (10/20) in the control group (P=0.734). Pearson correlation analysis revealed a negative correlation between rotavirus infection and SFB positivity (r=-0.87,P<0.001). As the concentration of the p3340 specific protein increased, the luminescence intensity of the luciferase in the Vero cells, which were suitable for cultivating rotavirus, exhibited a decreasing trend (F=4.17, P=0.001). Conclusions: SFB colonization in infants less than 2 years old is associated with a reduced risk of rotavirus infection. Cloning of specific SFB functional protein p3340 neutralizes rotavirus infection of Vero cells, and this mechanism of targeting rotavirus infection differs from the common antiviral mechanism.

Identification of the CsFtsH genes from Camellia sinensis reveals its potential role in leaf color phenotype

The filamentation temperature-sensitive H (FtsH) metalloprotease participates in the chloroplast photosystem II (PSII) repair cycle, playing a crucial role in regulating leaf coloration. However, the evolutionary history and biological function of the FtsH family in albino tea plants are still unknown. In this study, 35 CsFtsH members, including 7 CsFtsH-like (CsFtsHi1-CsFtsHi7) proteins, mapping onto 11 chromosomes in 6 subgroups, were identified in the ‘Shuchazao2′ tea genome, and their exon/intron structure, domain characteristics, collinearity, protein interaction network, and secondary structure were comprehensively analyzed. Furthermore, real-time fluorescence quantitative PCR (RT-qPCR) analysis revealed that the expression levels of CsFtsH1/2/5/8 were significantly positively correlated with the leaf color of tea plants. The subcellular localization revealed that they were located in the chloroplast. The transgenic Arabidopsis has demonstrated that CsFtsH2 and CsFtsH5 could restore the chlorophyll content and chlorophyll fluorescence intensity in var1 and var2 mutants, respectively. Moreover, protein–protein interactions have confirmed that CsFtsH1 with CsFtsH5, and CsFtsH2 with CsFtsH8 could form a hetero-comples and function in chloroplasts. In summary, this study aims to not only increase the understanding of the underlying molecular mechanisms of CsFtsH but also to provide a solid and detailed theoretical foundation for the breeding of albino tea plant varieties.

Gallnut tannic acid alleviates gut damage induced by Salmonella pullorum in broilers by enhancing barrier function and modulating microbiota.

Pullorum disease (PD) is a bacterial infection caused by Salmonella pullorum (S. pullorum) that affects poultry. It is highly infectious and often fatal. Antibiotics are currently the mainstay of prophylactic and therapeutic treatments for PD, but their use can lead to the development of resistance in pathogenic bacteria and disruption of the host's intestinal flora. We added neomycin sulfate and different doses of tannic acid (TA) to the drinking water of chicks at 3 days of age and infected them with PD by intraperitoneal injection of S. pullorum at 9 days of age. We analyzed intestinal histopathological changes and the expression of immune-related genes and proteins by using the plate smear method, histological staining, real-time fluorescence quantitative PCR, ELISA kits, and 16S rRNA Analysis of intestinal flora. The results demonstrate that S. pullorum induces alterations in the immune status and impairs the functionality of the liver and intestinal barrier. We found that tannic acid significantly ameliorated S. pullorum-induced liver and intestinal damage, protected the intestinal physical and chemical barriers, restored the intestinal immune barrier function, and regulated the intestinal flora. Our results showed that TA has good anti-diarrhoeal, growth-promoting, immune-regulating, intestinal barrier-protecting and intestinal flora-balancing effects, and the best effect was achieved at an additive dose of 0.2%.

Study of proanthocyanidin promotes osteogenic differentiation of human periodontal ligament stem cells through the transcription factor EB-induced autophagy-lysosome pathway

Objective: To investigate the mechanism of proanthocyanidin (PA) in regulating the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs), and to explore the effects of PA on the expression and nuclear translocation of transcription factor EB (TFEB) and on the autophagy-lysosome pathway. Methods: PDLSCs were divided into control group and PA group, which were subjected to RNA sequencing analysis (RNA Seq) to detect differentially expressed genes. The osteogenic differentiation ability and autophagy level were observed by real-time fluorescence quantitative PCR (RT-qPCR) analysis, alkaline phosphatase (ALP) staining and transmission electron microscope (TEM), respectively. Scratch assay and Transwell assay were used to detect the migration ability of PDLSCs. Lysotracker and immunofluorescence staining were used to detect the biogenesis of lysosomes. The total protein expression of transcription factor EB (TFEB) as well as that in cytoplasm and nucleus were detected by Western blotting. Confocal laser scanning microscope (CLSM) was used to observe the nuclear translocation of TFEB. The PDLSCs were treated with small interfering RNA (siRNA) technology to knock down the expression levels of TFEB gene with or without PA treatment. Western blotting was used to analyze the expressions of autophagy-related proteins Beclin1 and microtubule-associated protein 1 light chain 3 (LC3B), as well as osteogenic-related proteins runt-related transcription factor 2 (RUNX2), ALP, and osteocalcin in PDLSCs. Results: Compared with the control group, the osteogenic-related and autophagy-related genes showed differential expression in PDLSCs after PA treatment (P<0.05). The mRNA expression levels of osteogenic-related genes RUNX2 (2.32±0.15) and collagen type Ⅰ alpha 1 (COL1α1) (1.80±0.18), as well as the autophagy related genes LC3B (1.87±0.08) and Beclin1 (1.63±0.08) were significantly increased in the PA group, compared with the control group (1.01±0.16, 1.00±0.10, 1.00±0.07, 1.00±0.06, respectively, all P<0.01). Compared with the control group, the PA group had higher ALP activity, and more autophagosomes and autophagolysosomes observed by TEM. PA promoted the migration of PDLSCs (P<0.05) and the increased number of lysosomes and the expression of lysosomal associated membrane protein 1 (LAMP1). In the PA group, the relative expression level of total TFEB protein (1.49±0.07) and the nuclear/cytoplasmic expression of TFEB protein (1.52±0.12) were significantly higher than the control group (1.00±0.11, 1.00±0.13, respectively) (t=6.43, P<0.01; t=5.07, P<0.01). The relative nuclear/cytoplasmic fluorescence intensity of TFEB in the PA group (0.79±0.09) was increased compared with the control group (0.11±0.08) (t=8.32, P<0.01). Knocking down TFEB significantly reduced the expression of TFEB (1.00±0.15 vs 0.64±0.04), LAMP1 (1.00±0.10 vs 0.69±0.09), Beclin1 (1.00±0.05 vs 0.60±0.05), and LC3B Ⅱ/Ⅰ (1.00±0.06 vs 0.73±0.07) in PDLSCs (P<0.05, P<0.05, P<0.01, P<0.01). When TFEB gene was knocked down, the expression levels of Beclin1 (1.05±0.11), LC3B Ⅱ/Ⅰ (1.02±0.09), RUNX2 (1.04±0.10), ALP (1.04±0.16), and osteocalcin (1.03±0.15) proteins were significantly decreased in the PA group compared with the pre-knockdown period (1.28±0.03, 1.44±0.11, 1.38±0.11, 1.62±0.11, 1.65±0.17, respectively) (P<0.05, P<0.01, P<0.05, P<0.01, and P<0.01, respectively). Conclusions: PA promotes the osteogenic differentiation of PDLSCs through inducing the expression and nuclear translocation of TFEB and activating the autophagy-lysosome pathway.

Effect of moxibustion on the SIRT1/FOXO3a signaling pathway in ApoE-/- mice with atherosclerosis.

To observe the effects of moxibustion on blood lipid metabolism, pathological morphology of thoracic aorta, and the expression of silent information regulator 1 (SIRT1) and forkhead box transcription factor O3a (FOXO3a) in ApoE-/- atherosclerosis (AS) mice, so as to explore the potential mechanism of moxibustion in preventing and treating AS. Ten C57BL/6J mice were fed a normal diet as the control group, and 30 ApoE-/- mice were fed a high-fat diet to establish the AS model, which were randomly divided into the model group, simvastatin group, and moxibustion group, with 10 mice in each group. From the first day of modeling, mice in the moxibustion group received mild moxibustion treatment at "Shenque"(CV8), "Yinlingquan"(SP9), bilateral "Neiguan"(PC6) and "Xuehai"(SP10) for 30 min per time；the mice in the simvastatin group were given simvastatin orally (2.5 mg·kg-1·d-1), with both treatments given once daily, 5 times a week, with a total intervention period of 12 weeks. The body weight and general condition of the mice were observed and recorded during the intervention period. After the intervention, the contents of serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured using an automated biochemistry analyzer. Hematoxylin eosin (HE) staining was used to observe the pathological morphology of the thoracic aorta. ELISA was used to measure the contents of serum oxidized low-density lipoprotein (ox-LDL) and superoxide dismutase (SOD) activity. Western blot and real-time fluorescent quantitative PCR analysis were used to detect the expression levels of SIRT1 and FOXO3a protein and mRNA in the thoracic aorta. Compared with the control group, body weight at the 8th and 12th week, serum TC, TG, LDL-C, and ox-LDL contents of the model group mice were significantly increased(P<0.05, P<0.01), while the HDL-C contents, SOD activity, and the expression levels of SIRT1 protein and mRNA in the thoracic aorta were significantly decreased(P<0.05, P<0.01). HE staining showed thickening of the aortic intima, endothelial cell degeneration, swelling, and shedding. Compared with the model group, body weight at the 8th and 12th week, serum TC, TG, LDL-C, and ox-LDL contents of mice in the simvastatin group and moxibustion group were significantly decreased(P<0.01), while the serum SOD activity, expression levels of SIRT1 protein and mRNA in the thoracic aorta were significantly increased(P<0.01). The HDL-C contents were significantly increased in the simvastatin group(P<0.05). The thoracic aortic structure was more intact in both groups, with a more regular lumen and orderly arrangement of the elastic membrane in the media, and a slight amount of endothelial cell degeneration and swelling in the intima. There was no significant difference in the evaluated indexes between the moxibustion group and the simvastatin group and the pathological changes in the thoracic aorta were similar between the two groups. Moxibustion can reduce the body weight of AS model mice, regulate lipid levels, repair vascular intima, and alleviate endothelial damage. Its mechanism of action may be related to the regulation of the SIRT1/FOXO3a signaling pathway to improve oxidative damage.

Inhibitory effect of downregulating G protein-coupled receptor class C group 5 member A expression on lipopolysaccharide-induced inflammatory response in human gingival fibroblasts

Objective: To clarify the effect and the mechanism of G protein-coupled receptor class C group 5 member A (GPRC5A) on lipopolysaccharide (LPS)-induced inflammatory response in human gingival fibroblasts (GFs), thus to provide a foundation for delving into the role of G protein coupled receptor (GPCR) in periodontitis. Methods: Gingival tissue samples were collected from 3 individuals periodontally healthy (health group) and 3 patients with periodontitis (periodontitis group) in Shandong Stomatological Hospital from December 2022 to February 2023. The expressions of GPRC5A of the two groups were detected by immunohistochemistry staining. GFs used in this study were isolated from a portion of gingiva for the extraction of impacted teeth in School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University from December 2022 to February 2023. GFs were isolated with enzymic digestion and transfected with 30, 50 and 80 μmol/L small interfering RNA-GPRC5A (siGPRC5A) or small interfering RNA-negative control (siNC), regarded as the experimental group and the negative control one, respectively. The silencing efficiency of siGPRC5A was evaluated by real-time fluorescence quantitative PCR (RT-qPCR). Experiments were then conducted using these cells which were divided into four groups of negative control (NC), LPS, siGPRC5A+LPS and siGPRC5A. The mRNA and protein levels of GPRC5A in GFs under 1 mg/L LPS-induced GFs inflammatory state were evaluated by RT-qPCR and Western blotting analysis after GPRC5A knockdown. RT-qPCR was used to detect the gene expression levels of the inflammatory cytokines in GFs induced by LPS, namely, interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α, prostaglandin endoperoxide synthase 2 (PTGS2) after GPRC5A knockdown. Western blotting analysis and immunofluorescence staining were used to further investigate the activation of nuclear factor-kappa B (NF-κB) signaling pathway. Results: Immunohistochemistry staining showed that the expression of GPRC5A in gingival tissues of periodontitis group (0.132±0.006) increased compared with that in periodontally healthy group (0.036±0.019) (t=8.24, P=0.001). Meanwhile, RT-qPCR results showed that the gene expression levels of GPRC5A at different time point (2, 6, 12, 24 h) in LPS-induced GFs (0.026±0.002, 0.042±0.005, 0.004±0.000, 0.016±0.000) were upregulated compared with those in the NC group (0.004±0.000, 0.004±0.000, 0.002±0.000, 0.007±0.000) (all P<0.001), respectively, and peaked at 6 h. The 50 μmol/L group displayed the most significant decrease in siGPRC5A expression (31.16±3.29) compared with that of the siNC group (100.00±4.88) (F=297.98, P<0.001). The results of RT-qPCR and Western blotting analysis showed that siGPRC5A (0.27±0.03, 0.71±0.00) suppressed the expressions of GPRC5A at both gene and protein levels, while LPS (1.30±0.10, 1.43±0.03) was able to promote the expressions of GPRC5A compared with those of the NC group (1.00±0.01, 1.00±0.00)(all P<0.001). The siGPRC5A+LPS group (0.39±0.03, 1.06±0.16) also inhibited the increase of GPRC5A at both gene and protein levels induced by LPS (1.30±0.10, 1.43±0.03) (F=208.38, P<0.001; F=42.04, P<0.001). RT-qPCR results showed that the expressions of IL-8, IL-1β, IL-6, TNF-α, and PTGS2 at the gene level in LPS group were highly increased compared with those in the NC group (all P<0.001). siGPRC5A significantly suppressed LPS-induced expressions of these inflammatory cytokines in GFs (all P<0.001). Western blotting analysis showed that the levels of p65 and IκBα protein phosphorylation in the LPS group were highly increased compared with those in the NC group, and siGPRC5A could effectively suppressed LPS-induced protein phosphorylation (all P<0.01). Furthermore, immunofluorescence staining showed that NF-κB p65 in the control group was mainly concentrated in the cytoplasm, and partially translocated to the nucleus under the stimulation of LPS. siGPRC5A was able to inhibit LPS-induced intranuclear translocation of p65 to a certain extent. Conclusions: GPRC5A expression was upregulated in periodontitis, and GPRC5A knockdown inhibited LPS-induced inflammation. Moreover, GPRC5A played a role in inflammation regulation by interacting with NF-κB signaling pathway.

Overexpression of the potato VQ31 enhances salt tolerance in Arabidopsis.

Plant-specific VQ proteins have crucial functions in the regulation of plant growth and development, as well as in plant abiotic stress responses. Their roles have been well established in the model plant Arabidopsis thaliana; however, the functions of the potato VQ proteins have not been adequately investigated. The VQ protein core region contains a short FxxhVQxhTG amino acid motif sequence. In this study, the VQ31 protein from potato was cloned and functionally characterized. The complete open reading frame (ORF) size of StVQ31 is 672 bp, encoding 223 amino acids. Subcellular localization analysis revealed that StVQ31 is located in the nucleus. Transgenic Arabidopsis plants overexpressing StVQ31 exhibited enhanced salt tolerance compared to wild-type (WT) plants, as evidenced by increased root length, germination rate, and chlorophyll content under salinity stress. The increased tolerance of transgenic plants was associated with increased osmotic potential (proline and soluble sugars), decreased MDA accumulation, decreased total protein content, and improved membrane integrity. These results implied that StVQ31 overexpression enhanced the osmotic potential of the plants to maintain normal cell growth. Compared to the WT, the transgenic plants exhibited a notable increase in antioxidant enzyme activities, reducing cell membrane damage. Furthermore, the real-time fluorescence quantitative PCR analysis demonstrated that StVQ31 regulated the expression of genes associated with the response to salt stress, including ERD, LEA4-5, At2g38905, and AtNCED3. These findings suggest that StVQ31 significantly impacts osmotic and antioxidant cellular homeostasis, thereby enhancing salt tolerance.

Adaptive laboratory evolution to obtain furfural tolerant Saccharomyces cerevisiae for bioethanol production and the underlying mechanism.

Furfural, a main inhibitor produced during pretreatment of lignocellulose, has shown inhibitory effects on S. cerevisiae. In the present study, new strains named 12-1 with enhanced resistance to furfural were obtained through adaptive laboratory evolution, which exhibited a shortened lag phase by 36 h, and an increased ethanol conversion rate by 6.67% under 4 g/L furfural. To further explore the mechanism of enhanced furfural tolerance, ADR1_1802 mutant was constructed by CRISPR/Cas9 technology, based on whole genome re-sequencing data. The results indicated that the time when ADR1_1802 begin to grow was shortened by 20 h compared with reference strain (S. cerevisiae CEN.PK113-5D) when furfural was 4 g/L. Additionally, the transcription levels of GRE2 and ADH6 in ADR1_ 1802 mutant were increased by 53.69 and 44.95%, respectively, according to real-time fluorescence quantitative PCR analysis. These findings suggest that the enhanced furfural tolerance of mutant is due to accelerated furfural degradation. Importance: Renewable carbon worldwide is vital to achieve "zero carbon" target. Bioethanol obtained from biomass is one of them. To make bioethanol price competitive to fossil fuel, higher ethanol yield is necessary, therefore, monosaccharide produced during biomass pretreatment should be effectively converted to ethanol by Saccharomyces cerevisiae. However, inhibitors formed by glucose or xylose oxidation could make ethanol yield lower. Thus, inhibitor tolerant Saccharomyces cerevisiae is important to this process. As one of the main component of pretreatment hydrolysate, furfural shows obvious impact on growth and ethanol production of Saccharomyces cerevisiae. To get furfural tolerant Saccharomyces cerevisiae and find the underlying mechanism, adaptive laboratory evolution and CRISPR/Cas9 technology were applied in the present study.

Genome-wide analysis of WRKY gene family and the dynamic responses of key WRKY genes involved in cadmium stress in Brassica juncea.

The WRKY transcription factors comprise one of the most extensive gene families and serve as pivotal regulators of plant responses to heavy metal stress. They contribute significantly to maintaining plant growth and development by enhancing plant tolerance. However, research on the role of WRKY genes in response to cadmium (Cd) stress in mustard is minimal. In this study, we conducted a genome-wide analysis of the mustard WRKY gene family using bioinformatics. The results revealed that 291 WRKY putative genes (BjuWRKYs) were identified in the mustard genome. These genes were categorized into seven subgroups (I, IIa-e and III) through phylogenetic analysis, with differences in motif composition between each subgroup. Homology analysis indicated that 31.62% of the genes originated from tandem duplication events. Promoter analysis revealed an abundance of abiotic stress-related elements and hormone-related elements within the BjuWRKY genes. Transcriptome analysis demonstrated that most BjuWRKY genes exhibited differential expression patterns at different Cd treatment stages in mustard. Furthermore, 10 BjuWRKY genes were confirmed to respond to Cd stress through the construction of a BjuWRKY protein interaction network, prediction of hub genes, and real-time fluorescence quantitative PCR analysis, indicating their potential involvement in Cd stress. Our findings provide a comprehensive insight into the WRKY gene family in mustard and establish a foundation for further studies of the functional roles of BjuWRKY genes in Cd stress response.

Application of CD25 and CTLA4 gene transcription levels in early prediction of acute graft-versus-host disease.

Our study investigated the potential of peripheral blood T cell CD25, CD28, and CTLA-4 gene transcription levels as predictive biomarkers for acute graft-versus-host disease (aGVHD) following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Real-time reverse transcription fluorescent quantitative PCR (RT-qPCR) analysis was conducted on day +7, +14, and +21 post-transplantation in patients undergoing allo-HSCT. Elevated levels of CD25 and CTLA-4 mRNA were found to be associated with the occurrence of aGVHD, as well as severe and gastrointestinal aGVHD. Receiver operating characteristic (ROC) curve analysis was utilized to assess the predictive value of each biomarker. Combined analysis of CD25 and CTLA-4 mRNA levels demonstrated promising predictive potential for aGVHD. Our results confirmed that the transcription levels of CD25 and CTLA-4 genes could be used as early predictive biomarkers for aGVHD post-allo-HSCT.

Spatio-temporal variation of toxin-producing gene abundance in Microcystis aeruginosa from Poyang Lake.

Microcystis aeruginosa (M. aeruginosa) causes massive blooms in eutrophic freshwater and releases microcystin. Poyang Lake is the largest freshwater lake in China and has kept a mid-nutrient level in recent years. However, there is little research on microcystin production in Poyang Lake. In this study, water and sediment samples from ten sampling sites in Poyang Lake were collected from May to December in 2020, and from January to April in 2021 respectively. Microcystis genes (mcyA, mcyB, 16s rDNA) were quantified by real-time fluorescence quantitative PCR analysis, and then the spatial and temporal variation of mcy genes, physicochemical factors, and bacterial population structure in the lake was analyzed. The relationship between the abundance of mcy genes and physicochemical factors in water column was also revealed. Results indicated that the microcystin-producing genes mcyA and mcyB showed significant differences in spatial and temporal levels as well, which is closely related to the physicochemical factors especially the water temperature (p < 0.05) and the nitrogen content (p < 0.05). The abundance of mcy genes in the sediment in December affected the abundance of mcy genes in the water column in the next year, while the toxic Microcystis would accumulate in the sediment. In addition to the toxic Microcystis, we also found a large number of non-toxic Microcystis in the water column and sediment, and the ratio of toxic to non-toxic species can also affect the toxicity production of M. aeruginosa. Overall, the results showed that M. aeruginosa toxin-producing genes in Poyang Lake distributed spatially and temporally which related to the physicochemical factors of Poyang Lake.

Effects of Light and Feeding on Expressions of Clock Genes and Metabolic Genes in the Ridgetail Shrimp, Exopalaemon carinicauda

The changes of the circadian clock genes expression, growth, and muscle composition in Exopalaemon carinicauda under different photoperiods and feeding time were investigated by real-time fluorescence quantitative PCR and muscle biochemical composition analysis, respectively. Results showed that four clock genes of Clock, Cry1, Tim, and Per2 showed significant circadian rhythms in both eyestalk and hepatopancreas. Their expression rhythms were highly correlated with the photoperiod in the eyestalk, but in the hepatopancreas, it was significantly correlated with the feeding time. Moreover, the expressions of several metabolic genes were also variable; adipose triglyceride lipase (ATGL) and amyloglucosidase (AGL) changed significantly with the shift of feeding time, whereas methionine synthase (MS) and succinate dehydrogenase (SDH) changed significantly with the photoperiod. This reflected the close relationship between the metabolic and circadian clock in E. carinicauda; Alterations in photoperiod or feeding time lead to changes in the biological clock systems, which affects the daily expression of metabolic genes at the downstream metabolic pathway and closely correlates their expression rhythms with epi-environmental factors such as feeding. It can be seen from the results that the feeding was also a significant factor that affected the crude protein and crude fat contents in muscle tissue of E. carinicauda; their highest values (18.69% ± 0.13%, 1.81% ± 0.02%) were also occurred in the treatment with feeding at 19:00. Differences in muscle composition through a similar mechanism of action, i.e., the biological clock system are affected by the feeding and then regulated changes in downstream metabolic processes. As a result, the optimized conditions for the cultivation of E. carinicauda were a short light-phase photoperiod (8L : 16D) with feeding after sunset (19:00).

Anti-oxidant effects of herbal residue from Shengxuebao mixture on heat-stressed New Zealand rabbits

Heat stress can lead to hormonal imbalances, weakened immune system, increased metabolic pressure on the liver, and ultimately higher animal mortality rates. This not only seriously impairs the welfare status of animals, but also causes significant economic losses to the livestock industry. Due to its rich residual bioactive components and good safety characteristics, traditional Chinese medicine (TCM) residue is expected to become a high-quality feed additive with anti-oxidative stress alleviating function. This study focuses on the potential of Shengxuebao mixture herbal residue (SXBR) as an anti-heat stress feed additive. Through the UPLC (ultra performance liquid chromatography) technology, the average residue rate of main active ingredients from SXBR were found to be 25.39%. SXBR were then added into the basal diet of heat stressed New Zealand rabbits at the rates of 5% (SXBRl), 10% (SXBRm) and 20% (SXBRh). Heat stress significantly decreased the weight gain, as well as increased neck and ear temperature, drip loss in meat, inflammation and oxidative stress. Also, the hormone levels were disrupted, with a significant increase in serum levels of CA, COR and INS. After the consumption of SXBR in the basal diet for 3 weeks, the weight of New Zealand rabbits increased significantly, and the SXBRh group restored the redness value of the meat to a similar level as the control group. Furthermore, the serum levels T3 thyroid hormone in the SXBRh group and T4 thyroid hormone in the SXBRm group increased significantly, the SXBRh group showed a significant restoration in inflammation markers (IL-1β, IL-6, and TNF-α) and oxidative stress markers (total antioxidant capacity, HSP-70, MDA, and ROS) levels. Moreover, the real-time fluorescence quantitative PCR analysis found that, the expression levels of antioxidant genes such as Nrf2, HO-1, NQO1, and GPX1 were significantly upregulated in the SXBRh group, and the expression level of the Keap1 gene was significantly downregulated. Additionally, the SXBRm group showed significant upregulation in the expression levels of HO-1 and NQO1 genes. Western blot experiments further confirmed the up-regulation of Nrf2, Ho-1 and NQO1 proteins. This study provides a strategy for the utilization of SXBR and is of great significance for the green recycling of the TCM residues, improving the development of animal husbandry and animal welfare.

Genome-wide identification and characterization of the kinesin gene family in upland cotton (Gossypium hirsutum L.)

Kinesin (KIN) genes play vital functions in plant development and the stress response. However, no comprehensive analysis of this gene family has been reported for cotton. In the present study, we performed a genome-wide investigation and comprehensive analysis of KIN genes in Gossypium hirsutum. A total of 172 putative GhKIN genes were identified in the cotton genome, classified into eight subfamilies (KIN1-KIN8) according to a phylogenetic tree. The GhKINs showed an uneven distribution on chromosomes. Gene duplication analysis indicated that segmental duplication was the only driving force for the expansion of the GhKIN gene family. Segmental duplication gene pairs all have undergone purification selection in the process of evolution. Different subfamily genes had differences in gene structure and motif composition, but the same subfamily showed some similarity. The promoter regions of GhKIN genes contained abundant stress- and hormone-related cis-elements. Based on the published RNA-seq data, 56 common potential candidate genes among these 172 GhKINs were used to identify the spatiotemporal expression patterns in different tissues and abiotic stress response patterns under different treatments (drought, salt, heat and cold). Real-time fluorescence quantitative PCR (RTqPCR) analysis further revealed the expression patterns of 15 GhKINs under drought and salt stress. GhKIN14 and 27 were downregulated under both stress conditions, while GhKIN18, 65, 101, 106, and 137 were upregulated at certain time points. Weighted gene coexpression network analysis (WGCNA) showed that five GhKIN genes, GhKIN13, GhKIN14, GhKIN27, GhKIN140 and GhKIN152, may be important genes regulating the salt stress response. Our study comprehensively analyzes the GhKIN genes in Gossypium hirsutum, providing a new perspective on the functional roles of GhKINs in cotton development and stress adaptation.