Quantitative Reverse Transcription-polymerase Chain Reaction Research Articles

Electrical signature of heterogeneous human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) have gained traction in transplantation therapy due to their immunomodulatory, paracrine, immune-evasive, and multipotent differentiation potential. The inherent heterogeneity of hMSCs poses a challenge for therapeutic treatments and necessitates the identification of robust biomarkers to ensure reproducibility in both in vivo and in vitro experiments. In this study, we utilized dielectrophoresis (DEP), a label-free electrokinetic phenomenon, to investigate the heterogeneity of hMSCs derived from bone marrow (BM) and adipose tissue (AD). The electrical properties of BM-hMSCs were compared to homogeneous mouse fibroblasts (NIH-3T3), human fibroblasts (WS1), and human embryonic kidney cells (HEK-293). The DEP profile of BM-hMSCs differed most from HEK-293 cells. We compared the DEP profiles of BM-hMSCs and AD-hMSCs and found that they have similar membrane capacitances, differing cytoplasm conductivity, and transient slopes. Inducing both populations to differentiate into adipocyte and osteoblast cells revealed that they behave differently in response to differentiation-inducing cytokines. Histology and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses of the differentiation-related genes revealed differences in heterogeneity between BM-hMSCs and AD-hMSCs. The differentiation profiles correlate well with the DEP profiles developed and indicate differences in the heterogeneity of BM-hMSCs and AD-hMSCs. Our results demonstrate that using DEP, membrane capacitance, cytoplasm conductivity, and transient slope can uniquely characterize the inherent heterogeneity of hMSCs to guide robust and reproducible stem cell transplantation therapies.

Molecular characterization and induced changes of histone acetyltransferases in the tick Haemaphysalis longicornis in response to cold stress

BackgroundEpigenetic modifications of histones play important roles in the response of eukaryotic organisms to environmental stress. However, many histone acetyltransferases (HATs), which are responsible for histone acetylation, and their roles in mediating the tick response to cold stress have yet to be identified. In the present study, HATs were molecularly characterized and their associations with the cold response of the tick Haemaphysalis longicornis explored.MethodsHATs were characterized by using polymerase chain reaction (PCR) based on published genome sequences, followed by multiple bioinformatic analyses. The differential expression of genes in H. longicornis under different cold treatment conditions was evaluated using reverse transcription quantitative PCR (RT-qPCR). RNA interference was used to explore the association of HATs with the cold response of H. longicornis.ResultsTwo HAT genes were identified in H. longicornis (Hl), a GCN5-related N-acetyltransferase (henceforth HlGNAT) and a type B histone acetyltransferase (henceforth HlHAT-B), which are respectively 960 base pairs (bp) and 1239 bp in length. Bioinformatics analysis revealed that HlGNAT and HlHAT-B are unstable hydrophilic proteins characterized by the presence of the acetyltransferase 16 domain and Hat1_N domain, respectively. RT-qPCR revealed that the expression of HlGNAT and HlHAT-B decreased after 3 days of cold treatment, but gradually increased with a longer period of cold treatment. The mortality rate following knockdown of HlGNAT or HlHAT-B by RNA interference, which was confirmed by RT-qPCR, significantly increased (P < 0.05) when H. longicornis was treated at the lowest lethal temperature (− 14 °C) for 2 h.ConclusionsThe findings demonstrate that HATs may play a crucial role in the cold response of H. longicornis. Thus further research is warranted to explore the mechanisms underlying the epigenetic regulation of the cold response in ticks.Graphical

Regulatory mechanism and expression level of PRPS2 in lung cancer.

Lung cancer, with high morbidity and mortality, is the commonest respiratory system neoplasm, which seriously endangers the life safety of patients. In this study, the effect of PRPS2 on cell progression was preliminarily investigated. Immunohistochemical staining, western blot and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were performed to verify the expression level of PRPS2 in lung cancer. Lung cancer cell lines with stable downregulation of PRPS2 were constructed in A549 cells and NCIH460 cells. The function of PRPS2 silencing on the proliferation ability was verified by the EdU and cell colony formation experiment. Scratch and transwell tests were conducted to verify the role of PRPS2 silencing on the migratory and invasive ability of cells. The impact of PRPS2 silencing on cell apoptosis and cell cycle was verified by flow cytometry test. The effects of PRPS2 silencing on apoptosis-associated proteins were assessed by western blot assay. The function of PRPS2 silencing on tumor growth in vivo was studied through xenograft tumor experiment. In comparison with normal tissues, PRPS2 was upregulated in lung cancer tissues. PRPS2 knockdown notably hindered the migratory ability, invasive ability and proliferation, but accelerated cell apoptosis. In vivo experiments confirmed that PRPS2 silencing blocked the growth of transplanted tumors. In lung cancer, PRPS2 silencing suppressed the malignant progression, indicating that PRPS2 might be a novel biomarker for lung cancer treatment and diagnosis.

Identification and Verification of Endoplasmic Reticulum Stress-Related Genes as Novel Signatures for Osteoarthritis Diagnosis and Therapy: A Bioinformatics Analysis-Oriented Pilot Study.

Endoplasmic reticulum stress (ERS) has been reported to be closely associated with the development of osteoarthritis (OA), but the underlying mechanisms are not fully delineated. The present study was designed to investigate the involvement of ERS-related genes in regulating OA progression. The expression profiles of OA patients and normal people were downloaded from the gene expression omnibus (GEO) database. The differentially expressed genes (DEGs) in datasets GSE55457 and GSE55235 were screened and identified by R software with the construction of the protein-protein interaction (PPI) networks. Through the STRING and Venn diagram analysis, hub ERS-related genes were obtained. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were performed. Biomarkers with high diagnostic values of osteoarthritis (OA) were studied. The hematoxylin and eosin (H&E) staining and micro-CT were applied to evaluate the establishment of the OA model. The expression levels of biomarkers were validated with the use of reverse transcription‑quantitative polymerase chain reaction (RT-qPCR) and western blot. Finally, we evaluated the correlations of hub ERS-related genes with the immune infiltration cells via the CIBERSORT algorithm. A total of 60 downregulated and 52 upregulated DEGs were identified, and the following GO and KEGG pathway analyses verified that those DEGs were mainly enriched in biological process (BP), cellular component (CC), molecular function (MF), and inflammation-associated signal pathways. Interestingly, among all the DEGs, six ER stress-associated genes, including activating transcription factor 3 (ATF3), DEAD-Box Helicase 3 X-Linked (DDX3X), AP-1 transcription factor subunit (JUN), eukaryotic initiation factor 4 (EIF4A1), KDEL endoplasmic reticulum protein retention receptor 3 (KDELR3), and vascular endothelial growth factor A (VEGFA), were found to be closely associated with OA progression, and the following RT-qPCR and Western Blot analysis confirmed that DDX3X, JUN, and VEGFA were upregulated, whereas KDELR3, EIF4A1, and ATF3 were downregulated in OA rats tissues compared to the normal tissues, which were in accordance with our bioinformatics findings. Furthermore, our receiver operating characteristic (ROC) curve analysis verified that the above six ER stress-associated genes could be used as ideal biomarkers for OA diagnosis and those genes also potentially regulated immune responses by influencing the biological functions of mast cells and macrophages. Collectively, the present study firstly identified six ER stress-associated genes (ATF3, DDX3X, JUN, EIF4A1, KDELR3, and VEGFA) that may play critical role in regulating the progression of OA.

Stryphnodendron adstringens have a modulatory effect on inflammatory cytokines markers of in vitro activated macrophages

Aim: The purpose of this study is to conduct a comprehensive investigation into the modulatory effects of Stryphnodendron adstringens (Mart.; S. adstringens), a Brazilian wound-healing plant, on the expression of inflammatory cytokines. This will be achieved using an in vitro protocol with the commercial macrophage cell line RAW 264.7. Methods: The macrophage inflammatory response was induced by the natural antigen phytohemagglutinin (PHA), with and without supplementation of different concentrations of S. adstringens extract. The effects on cell proliferation rate and the concentration and production of transcripts of pro-inflammatory cytokines interleukin 1β (IL-1β), IL-6, tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ), as well as the anti-inflammatory cytokine IL-10, were assessed using spectrophotometric, immunoassay, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) techniques. Results: S. adstringens extract at all concentrations tested here reduced the cellular proliferation rate of activated macrophages. Extracts at concentrations of 0.49 mg/mL and 0.99 mg/mL decreased the protein and gene expression of pro-inflammatory cytokines, exhibiting the opposite effect concerning IL-10. Conclusions: The findings suggest that the wound-healing action of S. adstringens may encompass differential modulation of inflammation associated with tissue injury.

Novel Environmentally Friendly RNAi Biopesticides: Targeting V-ATPase in Holotrichia parallela Larvae Using Layered Double Hydroxide Nanocomplexes.

RNA interference (RNAi)-based biopesticides offer an attractive avenue for pest control. Previous studies revealed high RNAi sensitivity in Holotrichia parallela larvae, showcasing its potential for grub control. In this study, we aimed to develop an environmentally friendly RNAi method for H. parallela larvae. The double-stranded RNA (dsRNA) of the V-ATPase-a gene (HpVAA) was loaded onto layered double hydroxide (LDH). The dsRNA/LDH nanocomplex exhibited increased environmental stability, and we investigated the absorption rate and permeability of dsRNA-nanoparticle complexes and explored the RNAi controlling effect. Silencing the HpVAA gene was found to darken the epidermis of H. parallela larvae, with growth cessation or death or mortality, disrupting the epidermis and midgut structure. Quantitative reverse transcription-polymerase chain reaction and confocal microscopy confirmed the effective absorption of the dsRNA/LDH nanocomplex by peanut plants, with distribution in roots, stems, and leaves. Nanomaterial-mediated RNAi silenced the target genes, leading to the death of pests. Therefore, these findings indicate the successful application of the nanomaterial-mediated RNAi system for underground pests, thus establishing a theoretical foundation for developing a green, safe, and efficient pest control strategy.

Integrated transcriptome and metabolome study reveal the therapeutic effects of nicotinamide riboside and nicotinamide mononucleotide on nonalcoholic fatty liver disease

Nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) have received considerable attention as anti-aging and anti-metabolic disease nutraceuticals. However, few studies have focused on their role in ameliorating hepatic metabolic disturbances. In the present study, the effects of NMN and NR on the liver of mice with nonalcoholic fatty liver disease (NAFLD) were investigated via transcriptome and metabolome analyses. NMN and NR reduced body weight gain, improved glucose homeostasis, regulated plasma lipid levels, and ameliorated liver injury, oxidative stress, and lipid accumulation in mice with HFD-induced NAFLD. Integrated transcriptome and metabolome analyses indicated that NMN and NR altered the biosynthesis of unsaturated fatty acids, arachidonic acid metabolism, and linoleic acid metabolism pathways, increased saturated fatty acid (palmitic acid, stearate, and arachidic acid) content, and increased polyunsaturated fatty acid (linoleic acid and eicosapentaenoic acid) content. Quantitative reverse transcription PCR (qRT-PCR) showed that NMN and NR primarily promoted arachidonic acid and linoleic acid catabolism via cytochrome P450 (CYP450) enzymes. This study established a theoretical foundation for the potential use of NMN and NR in future clinical settings.

The Ang-(1-7)/MasR axis ameliorates neuroinflammation in hypothermic traumatic brain injury in mice by modulating phenotypic transformation of microglia.

The Ang-(1-7)/MasR axis is critically involved in treating several diseases; For example, Ang-(1-7) improves inflammatory response and neurological function after traumatic brain injury and inhibits post-inflammatory hypothermia. However, its function in traumatic brain injury (TBI) combined with seawater immersion hypothermia remains unclear. Here, we used a mice model of hypothermic TBI and a BV2 cell model of hypothermic inflammation to investigate whether the Ang-(1-7)/MasR axis is involved in ameliorating hypothermic TBI. Quantitative reverse transcription PCR, western blotting assay, and immunofluorescence assay were performed to confirm microglia polarization and cytokine regulation. Hematoxylin-eosin staining, Nissl staining, and immunohistochemical assay were conducted to assess the extent of hypothermic TBI-induced damage and the ameliorative effect of Ang-(1-7) in mice. An open field experiment and neurological function scoring with two approaches were used to assess the degree of recovery and prognosis in mice. After hypothermic TBI establishment in BV2 cells, the Ang-(1-7)/MasR axis induced phenotypic transformation of microglia from M1 to M2, inhibited IL-6 and IL-1β release, and upregulated IL-4 and IL-10 levels. After hypothermic TBI development in mice, intraperitoneally administered Ang-(1-7) attenuated histological damage and promoted neurological recovery. These findings suggest that hypothermia exacerbates TBI-induced damage and that the Ang-(1-7)/MasR axis can ameliorate hypothermic TBI and directly affect prognosis.

Development of a triplex quantitative reverse transcription-polymerase chain reaction for the detection of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, and porcine rotavirus A

Porcine viral diarrhea is caused by many pathogens and can result in watery diarrhea, dehydration and death. Various detection methods, such as polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR), have been widely used for molecular diagnosis. We developed a triplex real-time quantitative reverse transcription PCR (qRT-PCR) for the simultaneous detection of three RNA viruses potentially associated with porcine viral diarrhea: porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and porcine rotavirus A (PoRVA). The triplex qRT-PCR had R2 values of 0.999 for the standard curves of PEDV, TGEV and PoRVA. Importantly, the limits of detection for PEDV, TGEV and PoRVA were 10 copies/μL. The specificity test showed that the triplex qRT-PCR detected these three pathogens specifically, without cross-reaction with other pathogens. In addition, the approach had good repeatability and reproducibility, with intra-and inter-assay coefficients of variation &amp;lt;1%. Finally, this approach was evaluated for its practicality in the field using 256 anal swab samples. The positive rates of PEDV, TGEV and PoRVA were 2.73% (7/256), 3.91% (10/256) and 19.14% (49/256), respectively. The co-infection rate of two or more pathogens was 2.73% (7/256). The new triplex qRT-PCR was compared with the triplex RT-PCR recommended by the Chinese national standard (GB/T 36871-2018) and showed 100% agreement for PEDV and TGEV and 95.70% for PoRVA. Therefore, the triplex qRT-PCR provided an accurate and sensitive method for identifying three potential RNA viruses for porcine viral diarrhea that could be applied to diagnosis, surveillance and epidemiological investigation.

Fucoidans from Laminaria hyperborea demonstrate bactericidal activity against diverse bacteria

AbstractFucoidans are a heterogenous class of fucose-rich sulfated carbohydrates which have attracted increasing attention in cancer and inflammation research due to their bioactive properties. There are reports that fucoidans may have direct antibacterial effects and synergy with antibiotics. However, the literature is conflicting, potentially due to the limited reporting of origin, characteristics, and extraction methods of the fucoidans tested. Here we report the results of 18 defined fucoidans screened for direct, indirect, and synergistic antibacterial effects. 15 distinct fucoidan fractions, isolated from Laminaria hyperborea using a solvent-free extraction process, were characterised for molecular weight, pH, viscosity, and sulfur content. These, together with three commercially available crude fractions, were assessed at concentrations from 0.03125-24 mg mL-1 for minimum inhibitory concentration against Staphylococcus aureus, Streptococcus mutans and Streptococcus sanguinis. Furthermore, we tested a selection of fucoidans for antibacterial synergy with vancomycin and indirect antibacterial effects in whole blood survival assays. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) was performed to assess the stress response in fucoidan-treated S. aureus cultures. We have identified one fucoidan fraction with bactericidal activity against diverse bacteria. This effect is dose-, fucoidan fraction- and bacteria-specific, and furthermore, not related to osmotic stress. No synergistic effects were observed with fucoidan in combination vancomycin. Fucoidans have exciting potential as antimicrobial agents. Further analysis is required to establish the precise molecular characteristics responsible for their potent bactericidal activity.

Isocucurbitacin B inhibits glioma growth through PI3K/AKT pathways and increases glioma sensitivity to TMZ by inhibiting hsa-mir-1286a

Aim: Glioma accounts for 81% of all cancers of the nervous system cancers and presents one of the most drug-resistant malignancies, resulting in a relatively high mortality rate. Despite extensive efforts, the complete treatment options for glioma remain elusive. The effect of isocucurbitacin B (isocuB), a natural compound extracted from melon pedicels, on glioma has not been investigated. This study aims to investigate the inhibitory effect of isocuB on glioma and elucidate its underlying mechanisms, with the objective of developing it as a potential therapeutic agent for glioma. Methods: We used network pharmacology and bioinformatics analysis to predict potential targets and associated pathways of isocuB in glioma. Subsequently, the inhibitory effect of isocuB on glioma and its related mechanisms were assessed through Counting Kit-8 (CCK-8), wound healing, transwell, Western blot (WB), reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and other in vitro experiments, alongside tumor formation experiments in nude mice. Results: Based on this investigation, it suggested that isocuB might inhibit the growth of gliomas through the PI3K-AKT and MAPK pathways. Additionally, we proposed that isocuB may enhance glioma drug sensitivity to temozolomide (TMZ) via modulation of hsa-mir-1286a. The CCK-8 assay revealed that isocuB exhibited inhibitory effects on U251 and U87 proliferation and outperformed TMZ. Wound healing and transwell experiments showed that isocuB inhibited the invasion and migration of U251 cells by suppressing the activity of MMP-2/9, N-cadherin, and Vimentin. The tunnel and flow cytometry (FCM) assays revealed that isocuB induced cell apoptosis through inhibition of BCL-2. Subsequently, we conducted RT-qPCR and Western blot (WB) experiments, which revealed that PI3K/AKT and MAPK pathways might be involved in the mechanism of the inhibition isocuB on glioma. Additionally, isocuB promoted the sensitivity of glioma U251 to TMZ by inhibiting hsa-mir-1286a. Furthermore, we constructed TMZ-resistant U251 strains and demonstrated effective inhibition by isocuB against these resistant strains. Finally, we confirmed that isocuB can inhibit tumor growth in vivo through experiments on tumors in nude mice. Conclusion: IsocuB may protect against glioma by acting on the PI3K/AKT and MAPK pathways and promote the sensitivity of glioma U251 to TMZ by inhibiting hsa-mir-1286a.

Exosomes Derived from Mesenchymal Stem Cells Increase the Viability of Damaged Endometrial Cells via the miR-99b-5p/PCSK9 Axis.

The aim of this article was to investigate whether exosomes derived from bone marrow mesenchymal stem cells repair damaged endometrial stromal cells (EnSCs) through the miR-99b-5p/PCSK9 axis. Exosomes derived from bone marrow mesenchymal stem cells (BMSC-exos) were isolated by ultracentrifugation and characterized using transmission electron microscopy and nanoflow cytometry. A mifepristone-induced EnSC injury model was established in vitro, and the uptake of BMSC-exos was assessed. EnSCs were divided into three groups: the normal group (ctrl), EnSC injury group (model), and BMSC-exo treatment group. The effects of BMSC-exos on EnSC proliferation, apoptosis, and vascular endothelial growth factor (VEGF) expression were assessed by coculturing MSC-exos with endometrial cells. Furthermore, high-throughput sequencing was used to identify differentially expressed genes (DEGs). Through bioinformatics analysis, reverse transcription-quantitative polymerase chain reaction, western blotting, the CCK8 assay, immunohistochemistry, and dual-luciferase experiments, the potential mechanism by which BMSC-exos-derived miRNAs repair EnSC injury was studied. BMSC-exos expressed the marker proteins CD9 and CD63. Laser confocal microscopy showed that BMSC-exos could enter damaged EnSCs. In the BMSC-exos-EnSC coculture group compared with the model group, BMSC-exos significantly increased the proliferation of damaged EnSCs and inhibited cell apoptosis in a dose-dependent manner. The expression levels of Caspase-3, Caspase-9, Bax, and VEGF mRNA were significantly downregulated in the BMSC-exos-EnSC coculture group, whereas Bcl-2 expression was upregulated. We identified 28 overlapping DEGs between the model and ctrl groups and between the BMSC-exo and model groups. Transfection with miR-99b-5p mimics significantly decreased PCSK9 gene expression and inhibited the expression of the autophagy-related proteins Beclin-1 and LC3-II/I and apoptosis, thereby promoting EnSC proliferation. Transfection with a miR-99b-5p inhibitor showed the opposite effects. Beclin-1, LC3-II/I, and PCSK9 expression in the thin endometrium was significantly increased. miR-99b-5p promoted cell proliferation by targeting PCSK9. BMSC-exos promoted endometrial proliferation, and miR-99b-5p inhibited cell apoptosis and promoted EnSC proliferation by targeting PCSK9, providing a new target for the treatment of thin endometrium.

Therapeutic Potential of Antibody Targeting Neuronal Pentraxin Receptor in Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) has a poor prognosis despite advances in multidisciplinary treatments and immune checkpoint inhibitors. We previously reported that neural pentraxin receptor (NPTXR), a transmembrane protein mainly expressed in the brain and involved in synaptic transmission, is implicated in gastric cancer malignancy. This study evaluated the expression and function of NPTXR in ESCC, the therapeutic potential of monoclonal antibody (mAb) against NPTXR, and its prognostic value in ESCC patients. The study involved analyzing the NPTXR expression in 21 ESCC cell lines and total 371 primary ESCC tissue samples using quantitative reverse-transcription polymerase chain reaction and immunohistochemistry. The impact of NPTXR on the malignant behavior of ESCC was examined using small interfering RNA-mediated knockdown and a subsequent assessment of cell proliferation, apoptosis, and adhesion. This study further investigated the efficacy of anti-NPTXR mAb in vitro and associations between the expression of NPTXR messenger RNA (mRNA) and protein with clinicopathological factors and the prognosis. NPTXR was overexpressed in several ESCC cell lines and primary ESCC tissues. Knockdown of NPTXR in ESCC cells resulted in reduced proliferation, increased apoptosis, and decreased cell adhesion. The mAb against NPTXR significantly inhibited ESCC cell proliferation in vitro. A high NPTXR expression in patient tissues was correlated with a worse overall survival, suggesting its potential as a prognostic biomarker. NPTXR influences the malignant behavior of ESCC cells. Anti-NPTXR mAb may be a promising therapeutic agent, and its expression in ESCC tissues may serve as a prognostic biomarker.

Wastewater monitoring of SARS-CoV-2 gene for COVID-19 epidemiological surveillance in Tucumán, Argentina.

Wastewater-based epidemiology provides temporal and spatial information about the health status of a population. The objective of this study was to analyze and report the epidemiological dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the province of Tucumán, Argentina during the second and third waves of coronavirus disease 2019 (COVID-19) between April 2021 and March 2022. The study aimed to quantify SARS-CoV-2 RNA in wastewater, correlating it with clinically reported COVID-19 cases. Wastewater samples (n = 72) were collected from 16 sampling points located in three cities of Tucumán (San Miguel de Tucumán, Yerba Buena y Banda del Río Salí). Detection of viral nucleocapsid markers (N1 gene) was carried out using one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Viral loads were determined for each positive sample using a standard curve. A positive correlation (p < 0.05) was observed between viral load (copies/mL) and the clinically confirmed COVID-19 cases reported at specific sampling points in San Miguel de Tucumán (SP4, SP7, and SP8) in both months, May and June. Indeed, the high viral load concurred with the peaks of COVID-19 cases. This method allowed us to follow the behavior of SARS-CoV-2 infection during epidemic outbreaks. Thus, wastewater monitoring is a valuable epidemiological indicator that enables the anticipation of increases in COVID-19 cases and tracking the progress of the pandemic. SARS-CoV-2 genome-based surveillance should be implemented as a routine practice to prepare for any future surge in infections.

Improving grape fruit quality through soil conditioner: Insights from RNA-seq analysis of Cabernet Sauvignon roots.

The application of fertilizers and soil quality are crucial for grape fruit quality. However, the molecular data linking different fertilizer (or soil conditioner [SC]) treatments with grape fruit quality is still lacking. In this study, we investigated three soil treatments, namely inorganic fertilizer (NPK, 343.5 kg/hm2 urea [N ≥ 46%]; 166.5 kg/hm2 P2O5 [P2O5 ≥ 64%]; 318 kg/hm2 K2O [K2O ≥ 50%]), organic fertilizer (Org, 9 t/hm2 [organic matter content ≥ 35%, N + P2O5 + K2O ≥ 13%]), and SC (SC, 3 t/hm2 [humic acid ≥ 38.5%; C, 56.1%; H, 3.7%; N, 1.5%; O, 38%; S, 0.6%]), on 4-year-old Cabernet Sauvignon grapevines. Compared with the NPK- and Org-treated groups, the SC significantly improved the levels of soluble solids, tannins, anthocyanins, and total phenols in the grape berries, which are important biochemical indicators that affect wine quality. Furthermore, we conducted RNA-seq analysis on the grapevine roots from each of the three treatments and used weighted gene co-expression network analysis to identify five hub genes that were associated with the biochemical indicators of the grape berries. Furthermore, we validated the expression levels of three hub genes (ERF, JP, and SF3B) and five selected genes related to anthocyanin biosynthesis (UFGT1, UFGT2, UFGT3, GST, and AT) by using quantitative reverse transcription-polymerase chain reaction. Compared to the NPK and Org treatment groups, the SC treatment resulted in a significant increase in the transcription levels of three hub genes as well as VvUFGT1, VvUFGT3, VvGST, and VvAT. These results suggest that the SC can improve grape fruit quality by altering gene transcription patterns in grapevine roots and further influence the biochemical indices of grape fruits, particularly anthocyanin content. This study reveals that the application of SC can serve as an important measure for enhancing vineyard SC and elevating grape quality.

Multiple pathways of vanadate reduction and denitrification mediated by denitrifying bacterium Acidovorax sp. strain BoFeN1

Contamination of aquifers by a combination of vanadate [V(V)] and nitrate (NO3−) is widespread nowadays. Although bioremediation of V(V)- and nitrate-contaminated environments is possible, only a limited number of functional species have been identified to date. The present study demonstrates the effectiveness of V(V) reduction and denitrification by a denitrifying bacterium Acidovorax sp. strain BoFeN1. The V(V) removal efficiency was 76.5 ± 5.41 % during 120 h incubation, with complete removal of NO3− within 48 h. Inhibitor experiments confirmed the involvement of electron transport substances and denitrifying enzymes in the bioreduction of V(V) and NO3−. Cyt c and riboflavin were important for extracellular V(V) reduction, with quinone and EPS more significant for NO3− removal. Intracellular reductive compounds including glutathione and NADH directly reduce V(V) and NO3−. Reverse transcription quantitative PCR confirmed the important roles of nirK and napA genes in regulating V(V) reduction and denitrification. Bioaugmentation by strain BoFeN1 increased V(V) and NO3− removal efficiency by 55.3 % ± 2.78 % and 42.1 % ± 1.04 % for samples from a contaminated aquifer. This study proposes new microbial resources for the bioremediation of V(V) and NO3−contaminated aquifers, and contributes to our understanding of coupled vanadium, nitrogen, and carbon biogeochemical processes.

Serum LINC00339 is a promising biomarker for prognosis prediction of nasopharyngeal carcinoma.

Long non-coding RNAs (lncRNAs) in serum were useful and promising biomarkers for diagnostic and prognostic application. Herein, we investigated the serum lncRNA LINC00339 expression and its role in nasopharyngeal carcinoma. In this study, we recruited a cohort of 129 nasopharyngeal carcinoma patients, 68 patients with nasopharyngitis, and 80 healthy controls. Serum LINC00339 levels were measured by reverse transcription-quantitative polymerase chain reaction. Receiver operating characteristic (ROC) and Kaplan-Meier curve analyses were conducted to evaluate th e clinical role of LINC00339. The effects of linc00339 on cellular activities were measured using CCK-8 and Transwell assays. We observed that serum LINC00339 expression was upregulated in nasopharyngeal carcinoma patients and closely associated with tumor node metastasis stage, lymph node metastasis, and overall survival rate. Meanwhile, ROC analysis showed serum LINC00339 had diagnostic value to distinguish nasopharyngeal carcinoma patients from healthy individuals and nasopharyngitis patients. Silencing of LINC00339 could repress cellular behaviors by targeting miR-152. This study clarified that LINC00339 was upregulated in nasopharyngeal carcinoma, and that serum LINC00339 may act as a diagnostic or prognostic marker, and a hopeful therapeutic target for patients with nasopharyngeal carcinoma.

Identification and map-based cloning of an EMS-induced mutation in wheat gene TaSP1 related to spike architecture.

A candidate gene TaSP1 related to spike shape was cloned, and the gene-specific marker was developed to efficiently track the superior haplotype in common wheat. Spike shape, an important factor that affects wheat grain yield, is mainly defined by spike length (SPL), spikelet number (SPN), and compactness. Zhoumai32 mutant 1160 (ZM1160), a mutant obtained from ethyl methane sulfonate (EMS) treatment of hexaploid wheat variety Zhoumai32, was used to identify and clone the candidate gene that conditioned the spike shape. Genetic analysis of an F2 population derived from a cross of ZM1160 and Bainong207 suggested that the compact spike shape in ZM1160 was controlled by a single recessive gene, and therefore, the mutated gene was designated as Tasp1. With polymorphic markers identified through bulked segregant analysis (BSA), the gene was mapped to a 2.65-cM interval flanked by markers YZU0852 and MIS46239 on chromosome 7D, corresponding to a 0.42-Mb physical interval of Chinese spring (CS) reference sequences (RefSeq v1.0). To fine map TaSP1, 15 and seven recombinants were, respectively, screened from 1599 and 1903 F3 plants derived from the heterozygous F2 plants. Finally, TaSP1 was delimited to a 21.9Kb (4,870,562 to 4,892,493bp) Xmis48123-Xmis48104 interval. Only one high-confidence gene TraesCS7D02G010200 was annotated in this region, which encodes an unknown protein with a putative vWA domain. Quantitative reverse transcription PCR (qRT-PCR) analysis showed that TraesCS7D02G010200 was mainly expressed in the spike. Haplotype analysis of 655 wheat cultivars using the candidate gene-specific marker Xg010200p2 identified a superior haplotype TaSP1b with longer spike and more spikelet number. TaSP1 is beneficial to the improvement in wheat spike shape.

Mechanically strained osteocyte-derived exosomes contained miR-3110-5p and miR-3058-3p and promoted osteoblastic differentiation

BackgroundOsteocytes are critical mechanosensory cells in bone, and mechanically stimulated osteocytes produce exosomes that can induce osteogenesis. MicroRNAs (miRNAs) are important constituents of exosomes, and some miRNAs in osteocytes regulate osteogenic differentiation; previous studies have indicated that some differentially expressed miRNAs in mechanically strained osteocytes likely influence osteoblastic differentiation. Therefore, screening and selection of miRNAs that regulate osteogenic differentiation in exosomes of mechanically stimulated osteocytes are important.ResultsA mechanical tensile strain of 2500 με at 0.5 Hz 1 h per day for 3 days, elevated prostaglandin E2 (PGE2) and insulin-like growth factor-1 (IGF-1) levels and nitric oxide synthase (NOS) activity of MLO-Y4 osteocytes, and promoted osteogenic differentiation of MC3T3-E1 osteoblasts. Fourteen miRNAs differentially expressed only in MLO-Y4 osteocytes which were stimulated with mechanical tensile strain, were screened, and the miRNAs related to osteogenesis were identified. Four differentially expressed miRNAs (miR-1930-3p, miR-3110-5p, miR-3090-3p, and miR-3058-3p) were found only in mechanically strained osteocytes, and the four miRNAs, eight targeted mRNAs which were differentially expressed only in mechanically strained osteoblasts, were also identified. In addition, the mechanically strained osteocyte-derived exosomes promoted the osteoblastic differentiation of MC3T3-E1 cells in vitro, the exosomes were internalized by osteoblasts, and the up-regulated miR-3110-5p and miR-3058-3p in mechanically strained osteocytes, were both increased in the exosomes, which was verified via reverse transcription quantitative polymerase chain reaction (RT-qPCR).ConclusionsIn osteocytes, a mechanical tensile strain of 2500 με at 0.5 Hz induced the fourteen differentially expressed miRNAs which probably were in exosomes of osteocytes and involved in osteogenesis. The mechanically strained osteocyte-derived exosomes which contained increased miR-3110-5p and miR-3058-3p (two of the 14 miRNAs), promoted osteoblastic differentiation.

Transcriptomic and metabolomic dissection of skeletal muscle of crossbred Chongming white goats with different meat production performance

BackgroundThe transcriptome and metabolome dissection of the skeletal muscle of high- and low- growing individuals from a crossbred population of the indigenous Chongming white goat and the Boer goat were performed to discover the potential functional differentially expressed genes (DEGs) and differential expression metabolites (DEMs).ResultsA total of 2812 DEGs were detected in 6 groups at three time stages (3,6,12 Month) in skeletal muscle using the RNA-seq method. A DEGs set containing seven muscle function related genes (TNNT1, TNNC1, TNNI1, MYBPC2, MYL2, MHY7, and CSRP3) was discovered, and their expression tended to increase as goat muscle development progressed. Seven DEGs (TNNT1, FABP3, TPM3, DES, PPP1R27, RCAN1, LMOD2) in the skeletal muscle of goats in the fast-growing and slow-growing groups was verified their expression difference by reverse transcription-quantitative polymerase chain reaction. Further, through the Liquid chromatography-mass spectrometry (LC-MS) approach, a total of 183 DEMs in various groups of the muscle samples and these DEMs such as Queuine and Keto-PGF1α, which demonstrated different abundance between the goat fast-growing group and slow-growing group. Through weighted correlation network analysis (WGCNA), the study correlated the DEGs with the DEMs and identified 4 DEGs modules associated with 18 metabolites.ConclusionThis study benefits to dissection candidate genes and regulatory networks related to goat meat production performance, and the joint analysis of transcriptomic and metabolomic data provided insights into the study of goat muscle development.