Results Of Quantitative Reverse Transcription PCR Research Articles

Rhodopseudomonas palustris Atp2 Protein Exerts Antifungal Effects by Targeting the Ribosomal Protein MoRpl12 in Magnaporthe oryzae.

Rice blast is one of the most hazardous diseases affecting rice production. Previously, we discovered that the Atp2 protein of Rhodopseudomonas palustris could significantly inhibit the appressorium formation and pathogenicity of Magnaporthe oryzae. However, the molecular mechanism of this fungus has remained unknown. This study revealed that Atp2 can enter the cell and interact with the ribosomal protein MoRpl12 of M. oryzae, directly affecting the expression of the MoRpl12 protein. Silencing the MoRPL12 gene can affect cell wall integrity, growth, conidiogenesis, and fungal pathogenicity. The quantitative reverse transcription PCR results showed significant changes in the expression of conidiation-related genes in the MoRPL12 gene-silenced mutants or in the Atp2 protein-treated plants. We further found that Atp2 treatment can influence the expression of ribosomal-related genes, such as RPL, in M. oryzae. Our study revealed a novel antifungal mechanism by which the Atp2 protein binds to the ribosomal protein MoRpl12 and inhibits the pathogenicity of rice blast fungus, providing a new potential target for rice blast prevention and control.

Development of two step reverse transcription droplet digital PCR (RT-ddPCR) for simultaneous identification of saliva and semen

Determination of the type of body fluids is essential for crime scene reconstruction and for improving the reliability of expert testimonies. Messenger RNA (mRNA) analysis by reverse transcription quantitative PCR (RT-qPCR) has been used in forensic genetics, particularly for body fluid identification. It is a relative quantification method that compares the Ct values of target and reference gene. Thus, the method is unsuitable for determining exact copy numbers of the target gene. To address this limitation, this study performed body fluid-specific mRNA analysis using two-step reverse transcription droplet digital PCR (RT-ddPCR), which is capable of absolute quantification. We found that RT-ddPCR was accurate and sensitive enough to detect as little as 1.5 copies/μl of complementary DNA (cDNA), making it suitable for application using casework samples. It was also highly specific for body fluids, as non-specific amplification did not occur. In addition, saliva-semen mixtures with ratios ranging from 1:50 to 50:1 were successfully identified. When comparing the results of RT-qPCR and RT-ddPCR, some samples were difficult to interpret because of the high Ct values of RT-qPCR. However, when the same samples were analyzed using RT-ddPCR, saliva and semen were distinctly identified. Thus, RT-ddPCR is useful for mixed samples (e.g., in sexual assault cases) with low amounts of DNA, which often leads to ambiguous results when using RT-qPCR. Other body fluids (e.g., vaginal fluid and menstrual blood) can also be identified by including additional markers. This study demonstrates the potential of RT-ddPCR for applications in forensic science.

Apple Pomace Extract Induces Cell Proliferation and Increases Type I Collagen and Hyaluronan Production in Human Skin Fibroblasts In Vitro.

Apple pomace is the residue left after apples are squeezed. The majority of pomace produced worldwide is produced by the apple manufacturing industry, however, most of the pomace produced by the industry is discarded. Apple pomace contains functional ingredients, such as polyphenols and triterpenoids, and exerts several beneficial effects on human health; however, studies on its cosmetic effects on the skin are lacking. Therefore, herein, we investigated the effects of apple pomace extract (APE) on human skin fibroblasts (HSFs) in vitro. When HSFs were cultured with the extract for 72h, the number of HSFs increased at concentrations of 10 and 20µg/mL. Transcriptome analysis and reverse transcription-quantitative PCR results revealed that the extract upregulated the expression of hyaluronan synthase (HAS) 1, HAS2, and HAS3 and downregulated the expression of HYAL1, a gene encoding the hyaluronan-degrading enzyme, in HSFs. Additionally, enzyme-linked immunosorbent assay revealed increased amounts of factors related to skin extracellular matrix, such as type I collagen and hyaluronic acid, secreted in the culture supernatant. The western blotting results suggested that the extract induced extracellular signal-regulated kinase and protein kinase B phosphorylation in HSFs. Additionally, several GO_Terms related to mitosis were detected in the Gene Ontology analysis. This is the first study to show that APE induces the proliferation of HSFs and production of factors related to skin anti-aging.

Time-course transcriptome data of silk glands in day 0–7 last-instar larvae of Bombyx mori (w1 pnd strain)

Time-course transcriptome expression data were constructed for four parts of the silk gland (anterior, middle, and posterior parts of the middle silk gland, along with the posterior silk gland) in the domestic silkworm, Bombyx mori, from days 0 to 7 of the last-instar larvae. For sample preparation, silk glands were extracted from one female and one male larva every 24 hours accurately after the fourth ecdysis. The reliability of these transcriptome data was confirmed by comparing the transcripts per million (TPM) values of the silk gene and quantitative reverse transcription PCR results. Hierarchical cluster analysis results supported the reliability of transcriptome data. These data are likely to contribute to the progress in molecular biology and genetic research using B. mori, such as elucidating the mechanism underlying the massive production of silk proteins, conducting entomological research using a meta-analysis as a model for lepidopteran insect species, and exploring medical research using B. mori as a model for disease species by utilising transcriptome data.

Multiple exposures to low-dose ionizing radiation induced the initiation and progression of pro-atherosclerotic phenotypes in mice and vascular endothelial cell damage.

This study aimed to investigate the effects of low-dose radiation on the abdominal aorta of mice and vascular endothelial cells. Wild-type and tumor-bearing mice were exposed to 15 sessions of low-dose irradiation, resulting in cumulative radiation doses of 187.5, 375, and 750 mGy. The effect on the cardiovascular system was assessed. Immunohistochemistry analyzed protein expressions of PAPP-A, CD62, P65, and COX-2 in the abdominal aorta. Microarray technology, Gene Ontology analysis, and pathway enrichment analysis evaluated gene expression changes in endothelial cells exposed to 375 mGy X-ray. Cell viability was assessed using the Cell Counting Kit 8 assay. Immunofluorescence staining measured γ-H2AX levels, and real-time polymerase chain reaction quantified mRNA levels of interleukin-6 (IL-6), ICAM-1, and Cx43. Hematoxylin and eosin staining revealed thickening of the inner membranes and irregular arrangement of smooth muscle cells in the media membrane at 375 and 750 mGy. Inflammation was observed in the inner membranes at 750 mGy, with a clear inflammatory response in the hearts of tumor-bearing mice. Immunohistochemistry indicated increased levels of PAPP-A, P65, and COX-2 post-irradiation. Microarray analysis showed 425 up-regulated and 235 down-regulated genes, associated with processes like endothelial cell-cell adhesion, IL-6, and NF-κB signaling. Cell Counting Kit 8 assay results indicated inhibited viability at 750 mGy in EA.hy926 cells. Immunofluorescence staining demonstrated a dose-dependent increase in γ-H2AX foci. Reverse transcription quantitative PCR results showed increased expression of IL6, ICAM-1, and Cx43 in EA.hy926 cells post 750 mGy X-ray exposure. Repeated low-dose ionizing radiation exposures triggered the development of pro-atherosclerotic phenotypes in mice and damage to vascular endothelial cells.

Reduced expression of secretogranin VGF in laryngeal squamous cell carcinoma.

Laryngeal cancer accounts for one-third of all head and neck tumors, with squamous cell carcinoma (SCC) being the most predominant type, followed by neuroendocrine tumors. Chromogranins, are commonly used as biomarkers for neuroendocrine tumors, including laryngeal cancer. It has been reported that secretogranin VGF, a member of the chromogranin family, can be also used as a significant biomarker for neuroendocrine tumors. However, the expression and role of VGF in laryngeal carcinomas have not been previously investigated. Therefore, the present study aimed to determine the expression levels of VGF in laryngeal SCC (LSCC). The present study collected tumor tissues, as well as serum samples, from a cohort of 15 patients with LSCC. The results of reverse transcription-quantitative PCR, western blot analysis and immunofluorescence assays showed that the selective VGF precursor was downregulated in patients with LSCC. Notably, in tumor tissue, the immunoreactivity for VGF was found in vimentin-positive cells, probably corresponding to T lymphocytes. The current preliminary study suggested that the reduced expression levels of VGF observed in tumor tissue and at the systemic level could sustain LSCC phenotype. Overall, VGF could be a potential biomarker for detecting neoplastic lesions with a higher risk of tumor invasiveness, even in non-neuroendocrine tumors.

Evaluation of Reference Genes for Normalizing RT-qPCR and Analysis of the Expression Patterns of WRKY1 Transcription Factor and Rhynchophylline Biosynthesis-Related Genes in Uncaria rhynchophylla.

Uncaria rhynchophylla (Miq.) Miq. ex Havil, a traditional medicinal herb, is enriched with several pharmacologically active terpenoid indole alkaloids (TIAs). At present, no method has been reported that can comprehensively select and evaluate the appropriate reference genes for gene expression analysis, especially the transcription factors and key enzyme genes involved in the biosynthesis pathway of TIAs in U. rhynchophylla. Reverse transcription quantitative PCR (RT-qPCR) is currently the most common method for detecting gene expression levels due to its high sensitivity, specificity, reproducibility, and ease of use. However, this methodology is dependent on selecting an optimal reference gene to accurately normalize the RT-qPCR results. Ten candidate reference genes, which are homologues of genes used in other plant species and are common reference genes, were used to evaluate the expression stability under three stress-related experimental treatments (methyl jasmonate, ethylene, and low temperature) using multiple stability analysis methodologies. The results showed that, among the candidate reference genes, S-adenosylmethionine decarboxylase (SAM) exhibited a higher expression stability under the experimental conditions tested. Using SAM as a reference gene, the expression profiles of 14 genes for key TIA enzymes and a WRKY1 transcription factor were examined under three experimental stress treatments that affect the accumulation of TIAs in U. rhynchophylla. The expression pattern of WRKY1 was similar to that of tryptophan decarboxylase (TDC) under ETH treatment. This research is the first to report the stability of reference genes in U. rhynchophylla and provides an important foundation for future gene expression analyses in U. rhynchophylla. The RT-qPCR results indicate that the expression of WRKY1 is similar to that of TDC under ETH treatment. It may coordinate the expression of TDC, providing a possible method to enhance alkaloid production in the future through synthetic biology.

DegQ associated with the degS/degU two-component system regulates biofilm formation, antimicrobial metabolite production, and biocontrol activity in Bacillus velezensis DMW1.

The gram-positive bacterium Bacillus velezensis strain DMW1 produces a high level of antimicrobial metabolites that can suppress the growth of phytopathogens. We investigated the mechanism used by degQ and the degS/degU two-component system to regulate the biocontrol characteristics of DMW1. When degQ and degU were deleted, the biofilm formation, cell motility, colonization activities, and antifungal abilities of ΔdegQ and ΔdegU were significantly reduced compared to wild-type DMW1. The expression levels of biofilm-related genes (epsA, epsB, epsC, and tasA) and swarming-related genes (swrA and swrB) were all down-regulated. We also evaluated the impact on secondary metabolites of these two genes. The degQ and degU genes reduced surfactin and macrolactin production and up-regulated the production of fengycin, iturin, bacillaene, and difficidin metabolites. The reverse transcription-quantitative PCR results were consistent with these observations. Electrophoretic mobility shift assay and microscale thermophoresis revealed that DegU can bind to the promoter regions of these six antimicrobial metabolite genes and regulate their synthesis. In conclusion, we provided systematic evidence to demonstrate that the degQ and degU genes are important regulators of multicellular behaviour and antimicrobial metabolic processes in B. velezensis DMW1 and suggested novel amenable strains to be used for the industrial production of antimicrobial metabolites.

The diagnostic or prognostic values of FADD in cancers based on pan‑cancer analysis.

Previous studies have determined that aberrant expression of the fas-associated death domain (FADD) contributes to the development of cancer. However, no pan-cancer analysis has been reported to explore the relationship between FADD and various cancers. Multiple databases were screened to identify cancer datasets for the present study and to validate the expression of FADD in various tumors. The association of FADD alteration with cancer prognosis, clinical features and tumor immunity was also evaluated. Reverse transcription-quantitative PCR (RT-qPCR) was utilized to confirm the expression of FADD in breast, colon, liver and gastric cancer cells. Analysis of Gene Expression Omnibus database and The Cancer Genome Atlas database indicated that FADD was highly expressed in breast invasive carcinoma (BRCA), cervical squamous cell carcinoma and endocervical adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma (COAD), esophageal carcinoma (ESCA), kidney renal clear cell carcinoma, kidney renal papillary cell carcinoma, liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD) and prostate adenocarcinoma, whereas RT-qPCR results revealed that FADD was highly expressed in breast cancer and colon cancer. Further analyses demonstrated that FADD expression was significantly altered in ESCA, head and neck squamous cell carcinoma (HNSC), lung squamous cell carcinoma and BRCA. FADD expression was observed to be a risk factor of the overall survival in patients with HNSC, LIHC and LUAD as demonstrated by Kaplan-Meier and Cox regression analyses. The results of the present study demonstrated that FADD is highly expressed in numerous malignancies and can be utilized as a biomarker for the diagnosis of BRCA, COAD, LIHC and stomach adenocarcinoma. Moreover, FADD expression is a predictive risk factor for the development of HNSC, LIHC and LUAD and can potentially be used as a prognostic marker for these cancers.

Abnormally high expression of D1-like dopamine receptors on lupus CD4+ T cells promotes Tfh cell differentiation

ObjectiveSLE is a chronic autoimmune disease that places a great burden on human society. T follicular helper (Tfh) cells play a critical role in the pathological process of SLE. Therefore,...

CRP‑1 promotes the malignant behavior of hepatocellular carcinoma cells via activating epithelial‑mesenchymal transition and Wnt/β‑catenin signaling.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. It has been reported that cysteine rich protein 1 (CRP-1) is dysregulated in several types of human cancer; however, its role in HCC is poorly understood. Therefore, the current study aimed to investigate the role of CRP-1 in HCC. Western blotting and reverse transcription-quantitative PCR results showed that CRP-1 was upregulated in HCC cell lines. Furthermore, for in vitro experiments, CRP-1 was knocked down and overexpressed in the HCC cell lines Hep 3B2.1-7 and BEL-7405, respectively. c-Myc and proliferating cell nuclear antigen upregulation, and cleaved caspase 3 and poly(ADP-ribose) polymerase downregulation suggested that CRP-1 silencing could inhibit the proliferation and colony-forming ability of HCC cells, and induce apoptosis. In addition, CRP-1 overexpression promoted the malignant behavior of HCC cells and induced epithelial-mesenchymal transition (EMT), as verified by E-cadherin downregulation, and N-cadherin and vimentin upregulation. Additionally, CRP-1 overexpression promoted the nuclear translocation of β-catenin, and activated the expression of cyclin D1 and matrix metalloproteinase-7. Furthermore, inhibition of Wnt/β-catenin signaling, following cell treatment with XAV-939, an inhibitor of the Wnt/β-catenin signaling pathway, abrogated the effects of CRP-1 on enhancing the proliferation and migration of HCC cells. These findings indicated that the regulatory effect of CRP-1 on HCC cells could be mediated by the Wnt/β-catenin signaling pathway. Overall, CRP-1 could promote the proliferation and migration of HCC cell lines, partially via promoting EMT and activating the Wnt/β-catenin signaling pathway.

Screening and identification of cyprinid herpesvirus 2 (CyHV-2) ORF55-interacting proteins by phage display

BackgroundCyprinid herpesvirus 2 (CyHV-2) is a pathogenic fish virus belonging to family Alloherpesviridae. The CyHV-2 gene encoding thymidine kinase (TK) is an important virulence-associated factor. Therefore, we aimed to investigate the biological function of open reading frame 55 (ORF55) in viral replication.MethodsPurified CyHV-2 ORF55 protein was obtained by prokaryotic expression, and the interacting peptide was screened out using phage display. Host interacting proteins were then predicted and validated.ResultsORF55 was efficiently expressed in the prokaryotic expression system. Protein and peptide interaction prediction and dot-blot overlay assay confirmed that peptides identified by phage display could interact with the ORF55 protein. Comparing the peptides to the National Center for Biotechnology Information database revealed four potential interacting proteins. Reverse transcription quantitative PCR results demonstrated high expression of an actin-binding Rho-activating protein in the latter stages of virus-infected cells, and molecular docking, cell transfection and coimmunoprecipitation experiments confirmed that it interacted with the ORF55 protein.ConclusionDuring viral infection, the ORF55 protein exerts its biological function through interactions with host proteins. The specific mechanisms remain to be further explored.

Chemical Composition and Hypnotic Effect of Picea mariana Essential Oils

With rapid economic development, social competition has become increasingly intense. Stress experienced by individuals has increased dramatically, resulting in a steady rise in insomnia. The present study investigated the effect of different concentrations of Picea mariana essential oils (PMEOs) on sleep improvement in mice exhibiting p-chlorophenylalanine (PCPA)-induced insomnia. First, the main constituents of PMEOs were analyzed by gas chromatography-mass spectrometry (GC-MS). The mice were weighed daily, and the sleep latency and duration were measured in each mouse group in the inverted reflex experiment. The number of neurons in the mouse brain was measured using Nissl staining. The expression level of 5-hydroxytryptamine-1A (5-HT1A) was determined by immunohistochemistry, whereas the expression level of gamma-aminobutyric acid A receptor subunit alpha-1 (GABAARɑ1) was detected by immunohistochemistry, western blotting, and reverse transcription-quantitative PCR (RT-qPCR). GC-MS identified the top five components comprising PMEOs as bornyl acetate, camphene, α-pinene, β-pinene, and 3-carene. Inhalation of PMEOs resulted in a progressive increase in the rate of body weight change, a decrease in sleep latency, and prolonged sleep duration in experimental mice. In Nissl staining, the number of neurons was significantly reduced in the PCPA group when compared with the elevated number in the PMEOs group. Immunohistochemistry revealed increased expression levels of GABAARα1 and 5-HT1A in the PMEOs group. Western blotting and RT-qPCR results showed increased levels of GABAARα1 protein and gene expression, respectively. PMEOs could increase GABAARα1 and 5-HT1A protein levels in mice, resulting in hypnotic effects.

Virtual Screening and In Vitro Experimental Verification of LuxS Inhibitors for Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli O157:H7 is an important foodborne pathogen that forms biofilms. In this study, three quorum-sensing (QS) inhibitors (M414-3326, 3254-3286, and L413-0180) were obtained through virtual screening, and their in vitro antibiofilm activities were validated. Briefly, the three-dimensional structure model of LuxS was constructed and characterized using the SWISS-MODEL. High-affinity inhibitors were screened from the ChemDiv database (1,535,478 compounds) using LuxS as a ligand. Five compounds (L449-1159, L368-0079, M414-3326, 3254-3286, and L413-0180) with a good inhibitory effect (50% inhibitory concentration <10 μM) on type II QS signal molecule autoinducer-2 (AI-2) were obtained using a AI-2 bioluminescence assay. The absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties predicated that the five compounds had high intestinal absorption levels (high) and plasma protein binding (absorbent strong) and did not inhibit the metabolism of CYP2D6 metabolic enzymes. In addition, molecular dynamics simulation showed that compounds L449-1159 and L368-0079 could not stably bind with LuxS. Thus, these compounds were excluded. Furthermore, surface plasmon resonance results showed that the three compounds could specifically bind to LuxS. IN addition, the three compounds could effectively inhibit the biofilm formation without affecting the growth and metabolism of the bacteria. Finally, the reverse transcription-quantitative PCR results showed that the three compounds downregulated the expression of the LuxS gene. Overall, these results revealed that the three compounds obtained through virtual screening could inhibit biofilm formation of E. coli O157:H7 and are potential LuxS inhibitors that can be used to treat E. coli O157:H7 infections. IMPORTANCE E. coli O157:H7 is a foodborne pathogen of public health importance. Quorum sensing (QS) is a form of bacterial communication that can regulate various group behaviors, including biofilm formation. Here, we identified three QS AI-2 inhibitors (M414-3326, 3254-3286, and L413-0180) that can stably and specifically bind to LuxS protein. The three QS AI-2 inhibitors inhibited biofilm formation without affecting the growth and metabolic activity of E. coli O157:H7. The three QS AI-2 inhibitors are promising agents for treating E. coli O157:H7 infections. Further studies to identify the mechanism of the three QS AI-2 inhibitors are needed to develop new drugs to overcome antibiotic resistance.

Evaluation of P-glycoprotein-targeting circulating microRNAs as peripheral biomarkers for medically intractable epilepsy

BackgroundEarly diagnosis of medically intractable epilepsy is challenging in clinical work. P-glycoprotein (P-gp) is one of the most important multidrug efflux transporters, which has been demonstrated to contribute to the drug resistance of intractable epilepsy. The present study was aimed to explore the diagnostic value of microRNAs (miRNAs) targeting P-gp for medically intractable epilepsy.MethodsThirty-six patients with intractable epilepsy and 36 epilepsy patients responsive to anti-epilepsy drugs, who visited Jinshan Hospital of Fudan University from September 2014 to September 2016, were enrolled in this study. Clinical information of the patients was obtained by retrospectively reviewing medical records. MiRNAs with differential serum expression between the two groups of patients were detected by microarray assay. Meanwhile, miRNAs that were confirmed to regulate P-gp in vitro by western blot were selected for further validation. In the validation phase, reverse transcription quantitative PCR (RT-qPCR) was conducted to confirm the differential expression of the candidate miRNAs in the epilepsy cohorts. Receiver operating characteristic (ROC) curve analysis was carried out to evaluate the diagnostic value of the miRNAs for intractable epilepsy.ResultsThree miRNAs including miR-6514-3p, miR-6076-5p, and miR-6855-3p were identified to be candidate miRNAs by microarray assay. The results of western blotting validated that miR-146a-5p and miR-138-5p could regulate P-gp expression in vitro, so they were included in the candidate miRNAs for further validation. In the validation phase, the results of RT-qPCR indicated that compared with drug-responsive patients, the patients with intractable epilepsy showed decreased level of miR-138-5p and increased level of miR-146a-5p. The results of ROC curve analysis indicated that miR-138-5p (AUC = 0.877) and miR-146a-5p (AUC = 0.866) had high diagnostic value for intractable epilepsy. In addition, the miR-panel composed of miR-138-5p and miR-146a-5p showed higher diagnostic value (AUC = 0.926) than the miRNAs selected by microarray assay.ConclusionsOur results indicated that the dysregulated miR-138-5p and miR-146a-5p which target P-gp expression have high potential as peripheral biomarkers for medically intractable epilepsy.

Evaluation of microRNA let-7b-3p expression levels in methamphetamine abuse

In this study, we aimed to identify a microRNA expression signature that could be used to distinguish methamphetamine from control samples. We also utilized the existing bioinformatics tools in order to predict the candidate microRNAs that could play potential key roles in regulating drug addiction-related genes. Methamphetamine samples from 21 ventral tegmental area and 21 nucleus accumbens samples and their control regions were obtained from the Council of Forensic Medicine (Istanbul). Quantitative analysis of let-7b-3p was studied using quantitative reverse transcription PCR. Statistical analysis was carried out using Student's t-test. The receiver operating characteristic curves were plotted with Statistical Package for the Social Sciences (SPSS 20.0). Our quantitative reverse transcription PCR results revealed that let-7b-3p was significantly overexpressed in brain tissues of the methamphetamine-user group. Let-7b-3p had significant power to discriminate methamphetamine from control samples in the ventral tegmental area (AUC; 0.922) and nucleus accumbens (AUC; 0.899) regions. We have shown for the first time in the literature the differential expression of let-7b-3p in samples from methamphetamine-addicted individuals. We suggest that let-7b-3p could be a powerful marker for the diagnosis of methamphetamine addiction. Our results showed that differentially expressed let-7b-3p in methamphetamine users could be used as a diagnostic and therapeutic marker.

Genomic footprints of Kam Sweet Rice domestication indicate possible migration routes of the Dong people in China and provide resources for future rice breeding

The Dong people are one of China’s 55 recognized ethnic minorities, but there has been a long-standing debate about their origins. In this study, we performed whole-genome resequencing of Kam Sweet Rice (KSR), a valuable, rare, and ancient rice landrace unique to the Dong people. Through comparative genomic analyses of KSR and other rice landraces from south of the Yangtze River Basin in China, we provide evidence that the ancestors of the Dong people likely originated from the southeast coast of China at least 1000 years ago. Alien introgression and admixture in KSR demonstrated multiple migration events in the history of the Dong people. Genomic footprints of domestication demonstrated characteristics of KSR that arose from artificial selection and geographical adaptation by the Dong people. The key genes GS3, Hd1, and DPS1 (related to agronomic traits) and LTG1 and MYBS3 (related to cold tolerance) were identified as domestication targets, reflecting crop improvement and changes in the geographical environment of the Dong people during migration. A genome-wide association study revealed a candidate yield-associated gene, Os01g0923300, a specific haplotype in KSR that is important for regulating grain number per panicle. RNA-sequencing and quantitative reverse transcription–PCR results showed that this gene was more highly expressed in KSR than in ancestral populations, indicating that it may have great value in increasing yield potential in other rice accessions. In summary, our work develops a novel approach for studying human civilization and migration patterns and provides valuable genomic datasets and resources for future breeding of high-yield and climate-resilient rice varieties.

In vitro and in vivo antimicrobial activity of antimicrobial peptide Jelleine-I against foodborne pathogen Listeria monocytogenes

As a human foodborne pathogen, Listeria monocytogenes can cause severe human listeriosis and develop resistance to antibiotics. Antimicrobial peptides (AMPs) are produced from all kingdoms of life and regarded as promising alternatives to conventional antibiotics. Jelleine-I is an AMP identified from honeybees royal jelly. In this study, we explored the activity and action mechanism of Jelleine-I against L. monocytogenes. We found its minimum inhibitory concentration to be 12.5 μg/mL. Membrane permeability analysis revealed that Jelleine-I increased L. monocytogenes cell membrane permeability, causing calcium leakage. Scanning, transmission electron microscopy and fluorescence microscopy revealed that Jelleine-I destroyed membrane integrity, disrupted intracellular structures and interacted with the bacterial DNA. DNA binding analysis demonstrated that Jelleine-I bound to bacterial genomic DNA. Results of reverse transcription-quantitative PCR revealed that Jelleine-I affected bacterial DNA replication gene expression levels. Moreover, Jelleine-I induced cellular reactive oxygen species (ROS) production from fluorescence intensity analysis, and inhibited bacterial biofilm formation. Results of immunomodulation in Galleria mellonella revealed that Jelleine-I increased host hemocyte counts, upregulated host AMP gene (Gloverin and Cecropin D) expression, and inhibited proinfammatory cytokine (tumor necrosis factor α and interleukin 6) production induced by bacterial infection. It efficiently killed bacteria and increased the survival rate of infected insects to 70 %. Furthermore, Jelleine-I increased the G1 to S phase transition in mammalian cells from cells cycle analysis, and cytotoxicity assay results indicated that it promoted cell proliferation without hemolysis or cytotoxicity. Collectively, Jelleine-I possesses antimicrobial, immunomodulatory and cell proliferative activities, and is a promising candidate for preventing L. monocytogenes emergence and dissemination.

MiR-20b-5p is a novel biomarker for detecting prostate cancer.

Prostate cancer (PCa) is the second most frequently diagnosed solid tumor and the fifth leading cause of cancer mortality among men worldwide. The prostate specific antigen (PSA) test for PCa remains controversial. Therefore, the development of more effective non-invasive biomarkers for PCa is necessary. The present study evaluated the diagnostic value of microRNA (miR)-20b-5p in PCa. Tissue miR-20b-5p expression levels and their correlation with clinical parameters were assessed using The Cancer Genome Atlas (TCGA) datasets, and the diagnostic value of the miR-20b-5p expression levels in PCa tissues was assessed using receiver operating characteristic curve analysis. Reverse transcription-quantitative PCR (RT-qPCR) was used to assess the relative expression levels of miR-20b-5p in PCa tissues compared with benign prostate hyperplasia (BPH) tissues. In addition, miR-20b-5p expression levels in PCa cell lines and non-tumorigenic prostate epithelial cells were compared. In this study, exosomes were extracted from the prostatic fluid as a source of liquid biopsy for the detection of PCa. The prostatic fluid exosomal miR-20b-5p expression levels between patients with PCa and the biopsy-negative patients were compared, and the diagnostic efficiency of prostatic fluid exosomal miR-20b-5p expression levels in PCa was compared with PSA and with the European Randomized Study of Screening for Prostate Cancer (ERSPC) risk calculator. The mechanism by which miR-20b-5p may function in PCa was assessed using bioinformatic analysis and validation experiments. miR-20b-5p was expressed at a markedly higher level in PCa tissues compared with normal prostate tissues with high diagnostic efficiency (area under the curve: 0.826). The expression levels of miR-20b-5p were also significantly higher in PCa tissues compared with BPH tissues; similarly, miR-20b-5p was more highly expressed in PCa cells compared with non-tumorigenic prostate epithelial cells. Prostatic fluid exosomal miR-20b-5p expression levels in patients with PCa were significantly higher compared with confirmed to be biopsy-negative, and the diagnostic performance of miR-20b-5p was superior to PSA and ERSPC risk calculator. The results of RT-qPCR and western blotting following transfection of DU145 cells with miR-20b-5p mimics and inhibitor showed that miR-20b-5p reduced the expression of retinoblastoma-associated protein 1 (RB1). Therefore, RB1 may be a significant target gene for miR-20b-5p. In conclusion, the present study demonstrated that miR-20b-5p was upregulated in PCa at the tissue and cellular levels, as well as in prostatic fluid exosomes. Therefore, miR-20b-5p may be a promising early diagnostic biomarker for PCa and an important tool to guide the decision-making of prostate biopsy.

Positive regulation of the DLT operon by TCSR7 enhances acid tolerance of Lactococcus lactis F44.

Lactococcus lactis, a lactic acid bacterium, has been widely used in the fermented dairy products. The acid tolerance of L. lactis is of great importance to food fermentation and probiotic applications. As the first barrier of bacteria, the cell wall has a protective effect on strains under many stress conditions, whereas the regulatory mechanism has rarely been reported. Here, based on the transcription analysis of 9 cell wall or membrane-related genes of L. lactis F44 under acid stress, the transcription levels of DACB, DLTD, YLBA, HRTA, WP_080613266.1 (1610), and ERFK genes were significantly increased. We constructed 9 overexpressing strains with the cell wall or membrane-related genes, respectively. It was demonstrated that the survival rates under acid stress of DACB, DLTD, and ERFK were significantly higher than that of wild-type F44. To investigate the regulatory mechanism, a DNA pull-down assay was used to identify the transcriptional regulators of these 3 genes. It was discovered that the 2-component system (TCS) transcriptional regulator TCSR7 bound to the upstream region of DLTD involved in the teichoic acid (TA) alanylation. The combination was confirmed through an electrophoretic mobility shift assay in vitro. Reverse-transcription quantitative PCR results indicated that TCSR7 upregulated the expression of DLTD gene. In addition, the transcription level of TCSR7 increased approximately 1.8-fold (log2 fold change) under acidic conditions. In summary, this study found that TCSR7 was induced by acid stress to upregulate the transcription level of the DLT operon genes, which might increase the positive charge on the cell membrane surface to increase the acid tolerance of the strain. This study lays the foundation for the regulatory mechanism of TA alanylation under acid stress.