Real-time qRT-PCR Research Articles

Mouse Nucleolus-Like Body Supports Transcription and Enhances Processing of rRna in Porcine Enucleolated Oocytes

Abstract Fully-grown mammalian oocytes contain morphologically evident but transcriptionally inactive nucleoli called “nucleolus-like bodies” (NLBs). These nuclear structures are essential for early embryonic development. Removing oocyte NLBs (enucleolation) before their activation leads to developmental failure, mainly at the time point of embryonic genome activation. The present study examined the developmental and expression dynamics of embryos derived from intra- (pig) and interspecies (mouse) nucleolus (NLB) transferred porcine oocytes after parthenogenetic activation. Activation of rDNA transcription and pre-rRNA processing in NLB re-injected embryos was observed by real-time qRT-PCR analysis, targeting the expression rates of 45S rRNA and levels of 18S rRNA. Reinjection of NLBs from mouse or porcine GV oocytes into the enucleolated MII stage porcine oocytes supported the embryonic development up to the blastocyst stage after their activation. Intra- and intergeneric NLB transferred embryos demonstrated rDNA transcription initiation at the 8-cell stage, corresponding with the embryonic genome activation in porcine parthenogenetic embryos (control). The measured levels of 18S rRNA in these experimental embryos showed delayed initiation of pre-rRNA processing at the blastocysts stage compared with the control (8-cell stage). However, the porcine embryos with re-injected mouse NLB displayed significantly higher levels of 18S rRNA than the control and experimental intraspecies group. In conclusion, NLBs from different mammal species (mouse) can enhance the quality of enucleolated porcine oocytes and thus support their early embryonic development after activation.

Correlation of polymerase chain reaction results with hematocrit levels and platelet counts in dengue patients in Batam City

Background Dengue hemorrhagic fever (DHF) is a viral disease transmitted by Aedes aegypti mosquitoes, posing global public health challenge. The Riau Islands Province has the highest incidence of DHF in Indonesia. Objective This study aimed to investigate the relationship between hematocrit and platelet levels with the cycle threshold (Ct) values of polymerase chain reaction (PCR) results in DHF cases in Batam City, Riau Islands Province. Methods A descriptive correlation study was conducted using data from 102 patients infected with the dengue virus. Hematocrit and platelet counts were measured using a hematology analyzer, while Ct values for DENV1, DENV2, DENV3, and DENV4 were obtained through real-time qRT-PCR. Pearson's correlation test was employed to analyze the relationship between these variables. Results The study found no significant gender difference in DHF incidence (males: 50%, females: 50%). The highest prevalence was observed in the 6–11 years age group (44.1%), followed by the 12–18 years group (25.5%), the >18 years group (24.5%), and the 1–5 years group (11.8%). DENV3 was identified as the dominant serotype. No statistically significant correlation was found between Ct values and hematocrit (p = 0.607) or platelet counts (p = 0.323). Conclusion DHF cases in this study showed no gender disparity, with the most affected group being children aged 6–11 years, and DENV3 was the prevalent serotype. Ct values did not show a statistically significant correlation with hematocrit levels or platelet counts, suggesting that these hematological parameters may not predict viral load in DHF cases.

Dufulin Impacts Plant Defense Against Tomato Yellow Leaf Curl Virus Infecting Tomato.

Tomato yellow leaf curl virus (TYLCV) poses a significant threat to tomato production, leading to severe yield losses. The current control strategies primarily rely on the use of pesticides, which are often nonselective and costly. Therefore, there is an urgent need to identify more environmentally friendly alternatives. Dufulin, a novel compound that has been effective in controlling viral diseases in tobacco and rice, has not yet been tested against TYLCV. This study assessed the efficacy of dufulin in controlling TYLCV over a three-year span from 2021 to 2023 through field trials, by monitoring disease symptoms and viral titers. Additionally, this study assessed the expression levels of genes associated with systemic acquired resistance (SAR), specifically proteinase inhibitor II (PI II) and non-expressor of pathogenesis-related genes 1 (NPR1), using real-time qRT-PCR. The chlorophyll and nitrogen content in the leaves were also measured. Plants treated with dufulin showed reduced symptomatology and lower viral titers compared to the controls. Analysis of gene expression revealed that NPR1 was upregulated in the dufulin-treated plants, whereas PI II expression was consistently downregulated in the TYLCV-infected plants. Interestingly, PI II expression increased in the healthy plants following a seven-day post-treatment with dufulin. Moreover, the treated plants exhibited a higher chlorophyll content than the controls, though no significant differences in the nitrogen levels were observed between the dufulin-treated and water-treated plants. Overall, the application of dufulin significantly bolstered the plant's defense response, effectively reducing TYLCV symptoms and enhancing resistance.

Integrated metabolomic and transcriptomic analyses of flavonoid accumulation in different cultivars of Platostoma palustre

BackgroundPlatostoma palustre is a kind of plant resource with medicinal and food value, which has been differentiated into many different varieties after a long period of breeding. The cultivars of Taiwan(TW) and Pingyuan(PY) are widely grown in Guangdong, but a clear basis for species differentiation has not yet been established, resulting in the mixing of different species which limits their production and application.ResultsRegarding leaf surface morphology, the TW exhibited greater leaf area, non-glandular hairs, and the number of stomata than the PY. Regarding chemical activities, the TW exhibited higher total flavonoid content and antioxidant activity than the PY. In metabolomics, a total of 85 DAMs were detected, among which four flavonoid DAMs were identified, all of which were up-regulated in TW expression. Transcriptome analysis identified 2503 DEGs, which were classified according to their functional roles. The results demonstrated that the DEGs were primarily involved in amino acid metabolism, carbohydrate metabolism, sorting and degradation. A total 536 transcription factors (TFs) were identified, of which bHLH and MYB were the top two most abundant TFs families. Combined analysis of metabolome and transcriptome indicated that the phenylpropanoid pathway plays a significant role in flavonoid synthesis. Furthermore, real-time fluorescence qrt-PCR validation demonstrated that the expression trend of 10 DEGs was consistent with the transcriptomics data.ConclusionThe phenylpropanoid pathway affects the synthesis of secondary metabolites, resulting in functional differences. In this study, metabolomic and transcriptomic analyses were performed to elucidate the regulatory mechanisms of flavonoid synthesis in P. palustre and to provide a theoretical basis for the identification, differentiation and breeding cultivation of different cultivars of P. palustre.

Bacterial products initiation of alpha-synuclein pathology: an in vitro study

Parkinson’s Disease (PD) is a prevalent and escalating neurodegenerative disorder with significant societal implications. Despite being considered a proteinopathy, in which the aggregation of α-synuclein is the main pathological change, the intricacies of PD initiation remain elusive. Recent evidence suggests a potential link between gut microbiota and PD initiation, emphasizing the need to explore the effects of microbiota-derived molecules on neuronal cells. In this study, we exposed dopaminergic-differentiated SH-SY5Y cells to microbial molecules such as lipopolysaccharide (LPS), rhamnolipid, curli CsgA and phenol soluble modulin α-1 (PSMα1). We assessed cellular viability, cytotoxicity, growth curves and α-synuclein levels by performing MTS, LDH, real-time impedance readings, qRT-PCR and Western Blot assays respectively. Statistical analysis revealed that rhamnolipid exhibited concentration-dependent effects, reducing viability and inducing cytotoxicity at higher concentrations, increasing α-synuclein mRNA and protein levels with negative effects on cell morphology and adhesion. Furthermore, LPS exposure also increased α-synuclein levels. Curli CsgA and PSMα-1 showed minimal or no changes. Our findings suggest that microbiota-derived molecules, particularly rhamnolipid and LPS, impact dopaminergic neurons by increasing α-synuclein levels. This study highlights the potential involvement of gut microbiota in initiating the upregulation of α-synuclein that may further initiate PD, indicating the complex interplay between microbiota and neuronal cells.

Establishment of a New Real-Time Molecular Assay for the Detection of Babanki Virus in Africa.

Babanki virus is a subtype of the Sindbis virus, a widespread arthropod-borne alphavirus circulating in Eurasia, Africa, and Oceania. Characterized by rashes and arthritis, clinical infections due to Sindbis were mainly reported in Africa, Australia, Asia, and Europe. However, its sub-type, Babanki virus, was reported in Northern Europe and Africa, where its epidemiology potential remains poorly understood. The diagnosis of alphaviruses is mainly based on serological testing and conventional PCR methods, which have considerable limits. In this study, we developed a real-time qRT-PCR assay for the detection of Babanki virus. The analytical sensitivity and specificity of the newly established assay were evaluated using in vitro standard RNA and related viruses relevant to the African context, respectively. In addition, its diagnostic sensitivity was assessed using a subset of Babanki virus-positive and -negative mosquito pools collected from the field. The new real-time qRT-PCR assay exhibited a 100% specificity, a 95% detection limit of 1 RNA molecule/reaction, and a diagnostic sensitivity of up to 120 pfu/reaction. This newly established assay could be useful not only for the detection of Babanki virus during epidemics but also in future experimental and surveillance studies focusing on their epidemiology and pathogenicity.

Development of RT-qPCR assay for assessing the expression of ACTB and SDHA housekeeping genes in the cell cultures of mammalian hosts of zoonotic infections

Background. The molecular mechanisms behind the maintenance of zoonotic pathogens in nature can be better understood by examining the gene expression in host cells in response to the infection. Reverse transcription and quantitative polymerase chain reaction (RT-qPCR) is a powerful method to study gene expression, especially with the use of endogenous reference genes (RG) to normalize the data. Therefore, it is critical to develop the reliable qRT-PCR assay and validate that selected RGs are stably expressed in the studied organism or cell culture.The aim. In this work, we aimed to develop the real-time qRT-PCR method for detecting mRNA of two candidate RGs, succinate dehydrogenase subunit A (SDHA) and beta-actin (ACTB), in the cell lines of mammalian hosts of zoonotic infections.Materials and methods. The SPEV (porcine embryonic kidney cell line) and ApnK (Korean field mouse kidney cell line) cells were grown in 24-well culture plates. Total RNA/DNA was isolated from trypsin-detached cell monolayers. Genomic DNA in the samples was removed with RNase-free DNase I, and one-step RT-qPCR was performed using primers for SDHA and ACTB gene fragments and the corresponding TaqMan hydrolysis probes. The experiment was performed in 4 independent replicates.Results. In the Korean field mouse cells, the linearity, efficiency, repeatability, and reproducibility of the RT-qPCR for ACTB gene mRNA corresponded to the modern requirements. However, RT-qPCR for SDHA exhibited good linearity and efficiency of the reaction, but CV values for repeatability and reproducibility slightly exceeded the recommended standards. In porcine cells, both assays had acceptable parameters. Thus, to use the SDHA as RG for ApnK cells, a detailed study of the stability of its expression in this particular model is required.Conclusions. New qRT-PCR assay was developed to assess the expression of housekeeping genes ACTB and SDHA in the cells of the Korean field mouse and domestic pig. Further research is necessary to validate these genes as references for quantitative assessment of gene expression in the cells of mammalian hosts of zoonotic infections.

Oxidative damage and mitochondrial dysfunction in cystathionine beta-synthase deficiency

Cystathionine beta-synthase deficiency (CBSD) is the most prevalent inherited disorder of homocysteine metabolism in the transsulphuration pathway. Research have suggested oxidative stress and inflammation as candidate pathogenic mechanisms in CBSD. This study aims to evaluate mitochondrial dysfunction and oxidative stress biomarkers in cystathionine beta-synthase deficiency (CBSD) patients, which may aid in understanding the pathogenesis of CBSD and improving treatment. The study group comprised 23 patients with a diagnosis of CBSD and healthy controls. We analysed serum levels of NAD+ and NADH by fluorometric assay, FGF-21 and GDF-15 by ELISA, mitochondrial DAMPs by real time qRT-PCR, total homocysteine levels in plasma by enzymatic test and compared the results in CBSD group with healthy controls. In patient group, a positive correlation was found between the total homocysteine level and both GDF-15 and NAD+/NADH levels. Furthermore, there was a negative correlation between total homocysteine levels and both total NAD++NADH and NADH levels. The alterations in NAD+, FGF-21, GDF-15 levels, and NAD+/NADH ratio in patients suggest that oxidative damage coexists with mitochondrial dysfunction in CBSD. Assessment of oxidative damage and addition of anti-oxidant therapy together with mitochondrial support may have additional benefits in reducing long-term morbidity in CBSD patients.

Selection and validation of reference genes for quantitative expression analysis of regeneration-related genes in Cheilomenes sexmaculata by real-time qRT-PCR.

Regeneration is a fascinating phenomenon that has intrigued scientists for a long time. Cheilomenes sexmaculata (Fabricius), a zig-zag ladybird beetle, possesses a high capacity for limb regeneration. The molecular mechanics of the zig-zag ladybird beetle are under-explored. Current research trends are focused on uncovering functional genes associated with limb regeneration. Most of these investigations involve quantitative real-time PCR (qRT-PCR) for their rapid and accurate analysis of gene expression levels. Hence, a stable and suitable reference gene is required to normalize the gene expression data. In this study, five housekeeping genes were selected from the transcriptomics data (in-house unpublished data) of C. sexmaculata (Fabricius). The expression stabilities of the selected genes were evaluated under different time intervals post-amputation using geNorm, normFinder, and refFinder software. Actin was revealed to be the most stable housekeeping gene, along with elongation factor 2 and glyceraldehyde-3-phosphate dehydrogenase. A target gene named engrailed (an important segment-forming gene) was used to validate the selected reference genes. The expression levels were found to be consistent with the transcriptomics results. According to our study, actin, along with elongation factor 2 and glyceraldehyde-3-phosphate dehydrogenase, serve as the most stable reference genes and are suitable for regeneration-related research. This study is a groundbreaking effort to identify the most stable reference gene for limb regeneration in C. sexmaculata (Fabricius), and the findings can be applied to other related insect species.

Development of a Triplex Endpoint PCR Assay for the Detection of SARS-CoV-2: Insights on Cost-Efficiency and Method Design

Introduction: Lower respiratory tract infections, including COVID-19, have a substantial global impact, making the development of diagnostic tests crucial. The study aimed to develop a new, accurate, fast, and cost-effective PCR-based detection method for SARS-CoV-2, applicable in limited settings and capable of detecting all current variants and potential future pathogens. Methods: The study was conducted between 2020 and 2022 at the molecular biology department of Mures County Clinical Hospital (MCCH), Romania. Initially, pharyngeal and nasal secretions were collected and processed using the real-time qRT-PCR method for routine COVID-19 diagnosis. Ninety-two samples were randomly selected to develop the assay, including samples from different infection periods and negative controls. Complementary DNA was prepared from the selected samples, and the presence and integrity of the extracted RNA were evaluated by amplifying the GAPDH housekeeping gene. Primers for three specific viral genes (N, ORF1ab, and S) were designed, and their efficiency was evaluated using endpoint PCR and sequencing. Finally, the method was optimized and implemented as a one-step triplex PCR assay for routine diagnostic use. Results: The molecular laboratory at the MCCH analyzed a total of 41,818 samples between June 2020 and December 2022. Among these samples, 26.15% tested positive for SARS-CoV-2, while 70.9% were negative and 2.95% were inconclusive or invalid. Three peaks of positive tests were observed in November 2020, April 2021, and February 2022. The study selected 92 preserved RNA samples for triplex PCR assay development, validating the primers’ specificity and confirming the quality of the nucleic acids. The comparative analysis showed the efficiency and accuracy of the endpoint reverse transcription triplex PCR method (RT-PCR), indicating its potential as a cost-effective alternative to real-time reverse transcription PCR (qRT-PCR) in low-income countries with limited infrastructure for COVID-19 testing. Conclusion: This method has the potential to facilitate large-scale diagnosis of SARS-CoV-2 infections, allowing for rapid and appropriate therapeutic management and ongoing monitoring of patients. Additionally, the method can be easily adapted for the detection of other pathogens.

Detection and Quantification of Soil-Borne Wheat Mosaic Virus, Soil-Borne Cereal Mosaic Virus and Japanese Soil-Borne Wheat Mosaic Virus by ELISA and One-Step SYBR Green Real-Time Quantitative RT-PCR.

Furoviruses are bipartite viruses causing mosaic symptoms and stunting in cereals. Infection with these viruses can lead to severe crop losses. The virus species Furovirus tritici with soil-borne wheat mosaic virus (SBWMV), Furovirus cerealis with soil-borne cereal mosaic virus (SBCMV) and Furovirus japonicum with Japanese soil-borne wheat mosaic virus (JSBWMV) and French barley mosaic virus (FBMV) as members are biologically and genetically closely related. Here, we develop SYBR green-based real-time quantitative RT-PCR assays to detect and quantify the RNA1 and RNA2 of the three virus species. Using experimental data in combination with Tm-value prediction and analysis of primer and amplicon sequences, we determine the capacity of our method to discriminate between the different viruses and evaluate its genericity to detect different isolates within the same virus species. We demonstrate that our method is suitable for discriminating between the different virus species and allows for the detection of different virus isolates. However, JSBWMV RNA1 primers may amplify SBWMV samples, bearing a risk for false positive detection with this primer. We also test the efficiency of polyclonal antibodies to detect the different viruses by ELISA and suggest that ELISA may be applied as a first screening to identify the virus. The real-time qRT-PCR assays developed provide the possibility to screen for quantitative disease resistance against SBCMV, SBWMV and JSBWMV. Moreover, with our method, we hope to promote research to unravel yet unresolved questions with respect to furovirus-host interaction concerning host range and resistance as well as regarding the role of multipartite genomes.

Ecotoxicological effects of paracetamol on the biochemical and molecular responses of spinach (Spinacia oleracea L.)

The widespread use of pharmaceuticals, including paracetamol, has raised concerns about their impact on the environment and non-target species. The aim of this study was to investigate the biochemical and molecular responses of Spinacia oleracea (spinach) to high paracetamol concentrations in order to understand the plant's stress responses and underlying mechanisms. Under controlled conditions, spinach plants were exposed to different paracetamol concentrations (0, 50, 100, and 200 mg/L). The study evaluated the impact of paracetamol exposure on biochemical parameters such as oxidative stress markers (H2O2, MDA), activities of antioxidant enzymes (APX, CAT, GPOD, SOD), levels of non-enzymatic components (phenolics and flavonoids), and phytohormones (ABA, SA, and IAA). Furthermore, the study assessed molecular impacts by analyzing stress-related genetic variation and alterations in the gene expression of the antioxidant enzymes. Results showed that paracetamol exposure significantly increased oxidative stress in spinach, which was evident through the elevated H2O2 and MDA levels. However, the antioxidant defense mechanisms were activated to counteract this effect, as evidenced by increased activity of antioxidant enzymes and higher phenolics and flavonoid levels. Moreover, induction in the phytohormone levels indicated a stress response in paracetamol-treated plants compared to control plants. RAPD analysis revealed polymorphism indicating the DNA damage, and the Real-time qRT-PCR method showed significant upregulation of stress-responsive genes, highlighting the severe impact of paracetamol at the molecular level. The study concludes that high paracetamol concentrations pose a significant threat to spinach growth by affecting both biochemical and molecular processes. These findings underscore the need for strict environmental management practices to mitigate the possible impact of continuous release, accumulation, and long-term exposure of pharmaceutical contaminants to the environment and implement policies to reduce pharmaceutical pollutants to preserve ecological health and biodiversity.

A-064 Improved Insulin production in Advanced glycation end-products (AGEs)-induced RIN-m5F pancreatic β-cells by using a Flavonoid-Rich Medicinal Plant, Kaempferia parviflora treatment

Abstract Background Advanced glycation end-products (AGEs) are glycated proteins that react with reducing sugar which causes the damages of function and cellular signaling of insulin production in insulinoma cells. AGEs also contribute to hyperglycemia-induced insulin resistance in type 2 diabetes. AGEs gradually form during the chronic high blood glucose condition, which is a major risk factor and harmful to β-cells function and survival, and could alter the insulin signaling and downregulate the insulin-responsive transcription factors including PDX-1, Glut-2, GCK, and Pre-INS. Kaempferia parviflora, also known as black ginger and flavonoid-rich medicinal plant, has beneficial properties for type 2 diabetes patients as a counteracts the detrimental effects of AGEs and improves the insulin production in pancreatic β-cells. Thus, we hypothesize that treated β-cells with K. parviflora, which is the most effective flavonoid-containing plant might protect against the damage of AGEs-induced insulin impairment, and these effects are also responsible for improved insulin synthesis and the essential transcription factors in pancreatic β-cells. Methods Rat pancreatic β-cell line RIN-m5F (ATCC CRL-2058) were maintained and cultured in RPMI 1640 medium containing 10% (v/v) FBS, 1% penicillin-streptomycin at 37◦C in a humidified atmosphere containing 5% CO2. RIN-m5F cells were pre-treated with the K. parviflora at concentrations 0, 25, and 50 µg/ml for 48 hours, and afterward, exposure to 250 µg/ml AGEs treatment. To quantify the bioactive compound of K. parviflora, total phenolic and flavonoid content assays were performed and the transcriptional changes of insulin-related genes (PDX-1, Glut-2, GCK, and Pre-INS) were determined by using real-time qRT-PCR. Results K. parviflora has shown a high phenolic content and flavonoid content was 50.43 ± 1.74, 26.66 ± 1.18 mg GAE/g Dry weight of sample, respectively. For the transcriptional changes of insulin-related genes, the levels of gene expression in the regulators of insulin secreting pathway (PDX-1, Glut-2, GCK, and Pre-INS) were significantly downregulated after exposure to AGEs-treated, while the pre-treatment of K. parviflora was showed statistically difference. PDX-1, Glut-2, GCK, and Pre-INS expressions were significantly upregulated after pre-treated with 50 µg/ml K. parviflora. However, under the AGEs-treated alone condition, the transcriptional levels were significantly decreased as compared to the control (untreated). Taken together, these results suggest K. parviflora could be a potential natural medicine to ameliorate the damage of insulin production in AGEs-induced β-cell damage and highlight promising alternatives for therapeutic avenue against diabetes. Conclusions Treated RIN-m5F pancreatic β-cells with K. parviflora, a flavonoid-rich medicinal plant, could upregulate the insulin-responsive transcription factors and improve the insulin production in damaging AGEs-induced pancreatic β-cells. Our findings provide a novel strategy related metabolic disease and insights into the regulation of insulin synthesis by using K. parviflora treatment as a therapeutic agent against type 2 diabetes.

Genome-Wide Analysis Elucidates the Roles of AhLBD Genes in Different Abiotic Stresses and Growth and Development Stages in the Peanut (Arachis hypogea L.).

The lateral organ boundaries domain (LBD) genes, as the plant-specific transcription factor family, play a crucial role in controlling plant architecture and stress tolerance. However, the functions of AhLBD genes in the peanut plant (Arachis hypogea L.) remain unclear. In this study, 73 AhLBDs were identified in the peanut plant and divided into three groups by phylogenetic tree analysis. Gene structure and conserved protein motif analysis supported the evolutionary conservation of AhLBDs. Tandem and segment duplications contributed to the expansion of AhLBDs. The evolutionary relationship analysis of LBD gene family between A. hypogaea and four other species indicated that the peanut plant had a close relationship with the soybean plant. AhLBDs played a very important role in response to growth and development as well as abiotic stress. Furthermore, gene expression profiling and real-time quantitative qRT-PCR analysis showed that AhLBD16, AhLBD33, AhLBD67, and AhLBD72 were candidate genes for salt stress, while AhLBD24, AhLBD33, AhLBD35, AhLBD52, AhLBD67, and AhLBD71 were candidate genes for drought stress. Our subcellular localization experiment revealed that AhLBD24, AhLBD33, AhLBD67, and AhLBD71 were located in the nucleus. Heterologous overexpression of AhLBD33 and AhLBD67 in Arabidopsis significantly enhanced tolerance to salt stress. Our results provide a theoretical basis and candidate genes for studying the molecular mechanism for abiotic stress in the peanut plant.

RNA-binding protein GIGYF2 orchestrates hepatic insulin resistance through STAU1/PTEN-mediated disruption of the PI3K/AKT signaling cascade

BackgroundObesity is well-established as a significant contributor to the development of insulin resistance (IR) and diabetes, partially due to elevated plasma saturated free fatty acids like palmitic acid (PA). Grb10-interacting GYF Protein 2 (GIGYF2), an RNA-binding protein, is widely expressed in various tissues including the liver, and has been implicated in diabetes-induced cognitive impairment. Whereas, its role in obesity-related IR remains uninvestigated.MethodsIn this study, we employed palmitic acid (PA) exposure to establish an in vitro IR model in the human liver cancer cell line HepG2 with high-dose chronic PA treatment. The cells were stained with fluorescent dye 2-NBDG to evaluate cell glucose uptake. The mRNA expression levels of genes were determined by real-time qRT-PCR (RT-qPCR). Western blotting was employed to examine the protein expression levels. The RNA immunoprecipitation (RIP) was used to investigate the binding between protein and mRNA. Lentivirus-mediated gene knockdown and overexpression were employed for gene manipulation. In mice, an IR model induced by a high-fat diet (HFD) was established to validate the role and action mechanisms of GIGYF2 in the modulation of HFD-induced IR in vivo.ResultsIn hepatocytes, high levels of PA exposure strongly trigger the occurrence of hepatic IR evidenced by reduced glucose uptake and elevated extracellular glucose content, which is remarkably accompanied by up-regulation of GIGYF2. Silencing GIGYF2 ameliorated PA-induced IR and enhanced glucose uptake. Conversely, GIGYF2 overexpression promoted IR, PTEN upregulation, and AKT inactivation. Additionally, PA-induced hepatic IR caused a notable increase in STAU1, which was prevented by depleting GIGYF2. Notably, silencing STAU1 prevented GIGYF2-induced PTEN upregulation, PI3K/AKT pathway inactivation, and IR. STAU1 was found to stabilize PTEN mRNA by binding to its 3’UTR. In liver cells, tocopherol treatment inhibits GIGYF2 expression and mitigates PA-induced IR. In the in vivo mice model, GIGYF2 knockdown and tocopherol administration alleviate high-fat diet (HFD)-induced glucose intolerance and IR, along with the suppression of STAU1/PTEN and restoration of PI3K/AKT signaling.ConclusionsOur study discloses that GIGYF2 mediates obesity-related IR by disrupting the PI3K/AKT signaling axis through the up-regulation of STAU1/PTEN. Targeting GIGYF2 may offer a potential strategy for treating obesity-related metabolic diseases, including type 2 diabetes.Graphical

The Accumulation of Abscisic Acid Increases the Innate Pool of Soluble Phenolics through Polyamine Metabolism in Rice Seedlings under Hexavalent Chromium Stress.

Potential toxic element (PTE) pollution has emerged as a significant environmental and social concern in global agriculture. Chromium (Cr) occurs in different oxidation states naturally, among them Cr(VI), which is highly toxic. This study carried out biochemical and molecular tests to elucidate the accumulation of total soluble phenolics (TSPs) in rice plants exposed to Cr(VI) at 2.0, 8.0, and 16.0 mg Cr/L, emphasizing the interaction between polyamines (PAs) and abscisic acid (ABA). The results revealed significant Cr accumulation in different tissues of rice plants, which hindered their growth. Cr(VI) exposure increased the ABA concentration, with higher levels detected in the shoots than in the roots. The TSP concentration in rice tissues showed a positive relationship with the supplied concentrations of Cr(VI). The measured PAs, including spermine (Spm), putrescine (Put), and spermidine (Spd), exhibited varied responses to Cr(VI) stress, with only Spm concentration increasing with Cr(VI) concentrations. Real-time qRT-PCR showed PAs and ABA synthesis-associated genes such as OsADC1, OsAIH, OsCPA1, and OsCPA4 were significantly up-regulated in shoot of rice plants treated with Cr(VI). These genes are associated with the second pathway of Put synthesis, originating from Arg. Almost all genes activated in the Met pathway were significantly up-regulated as well. Moreover, the genes involved in the interconversion among the three species of PAs exhibited completely different responses to Cr(VI) exposure. Overall, the biochemical analysis and gene expression data indicate that the interaction between ABA and Spm is likely to enhance the TSP levels in rice plants subjected to Cr(VI) toxicity.

Investigating the Antifibrotic Effects of β-Citronellol on a TGF-β1-Stimulated LX-2 Hepatic Stellate Cell Model.

Liver fibrosis, a consequence of chronic liver damage or inflammation, is characterized by the excessive buildup of extracellular matrix components. This progressive condition significantly raises the risk of severe liver diseases like cirrhosis and hepatocellular carcinoma. The lack of approved therapeutics underscores the urgent need for novel anti-fibrotic drugs. Hepatic stellate cells (HSCs), key players in fibrogenesis, are promising targets for drug discovery. This study investigated the anti-fibrotic potential of Citrus hystrix DC. (KL) and its bioactive compound, β-citronellol (β-CIT), in a human HSC cell line (LX-2). Cells exposed to TGF-β1 to induce fibrogenesis were co-treated with crude KL extract and β-CIT. Gene expression was analyzed by real-time qRT-PCR to assess fibrosis-associated genes (ACTA2, COL1A1, TIMP1, SMAD2). The release of matrix metalloproteinase 9 (MMP-9) was measured by ELISA. Proteomic analysis and molecular docking identified potential signaling proteins and modeled protein-ligand interactions. The results showed that both crude KL extract and β-CIT suppressed HSC activation genes and MMP-9 levels. The MAPK signaling pathway emerged as a potential target of β-CIT. This study demonstrates the ability of KL extract and β-CIT to inhibit HSC activation during TGF-β1-induced fibrogenesis, suggesting a promising role of β-CIT in anti-hepatic fibrosis therapies.

MRGPRX2-mediated mast cell activation by substance P from overloaded human tenocytes induces inflammatory and degenerative responses in tendons

Mast cells are immune cells minimally present in normal tendon tissue. The increased abundance of mast cells in tendinopathy biopsies and at the sites of tendon injury suggests an unexplored role of this cell population in overuse tendon injuries. Mast cells are particularly present in tendon biopsies from patients with more chronic symptom duration and a history of intensive mechanical loading. This study, therefore, examined the cross talk between mast cells and human tendon cells in either static or mechanically active conditions in order to explore the potential mechanistic roles of mast cells in overuse tendon injuries. A coculture of isolated human tenocytes and mast cells (HMC-1) combined with Flexcell Tension System for cyclic stretching of tenocytes was used. Additionally, human tenocytes were exposed to agonists and antagonists of substance P (SP) receptors. Mast cell degranulation was assessed by measuring β-hexosaminidase activity. Transwell and cell adhesion assays were used to evaluate mast cell migration and binding to tendon extracellular matrix components (collagen and fibronectin), respectively. Gene expressions were analyzed using real time qRT-PCR. Our results indicate that mechanical stimulation of human tenocytes leads to release of SP which, in turn, activates mast cells through the Mas-related G-protein-coupled receptor X2 (MRGPRX2). The degranulation and migration of mast cells in response to MRGPRX2 activation subsequently cause human tenocytes to increase their expression of inflammatory factors, matrix proteins and matrix metalloproteinase enzymes. These observations may be important in understanding the mechanisms by which tendons become tendinopathic in response to repetitive mechanical stimulation.

Kinetics of dengue viremia and its association with disease severity: an ambispective study

BackgroundDengue virus (DENV) infection is an important public health problem and causes significant morbidity and mortality. DENV typically causes a febrile illness that ranges from mild asymptomatic infection to fatal dengue hemorrhagic fever (DHF) and/or dengue shock syndrome (DSS). Early prediction of severe dengue disease is of utmost importance for providing prompt monitoring and treatment. The search for an ideal biomarker (host or viral factors) for early prediction of severe dengue remains elusive.AimTo standardize a real time qRT-PCR for quantifying dengue viremia in serum samples and evaluate the kinetics of dengue viremia and its significance in disease severity.ResultsIn this ambispective study of 126 laboratory confirmed dengue patients, 72 were primary infections and 54 were secondary infections. The most common serotype was serotype 1 (n = 37) followed by serotype 2 (n = 34). According to WHO 1997 dengue case classification, 111 patients were cases of dengue fever (DF), 13 from DHF and 02 from DSS. Day 3 viremia levels were significantly elevated in severe dengue patients (DHF/DSS) as compared to that of DF (p < 0.05). However, no such association was found between viremia levels and serotype or immune status.ConclusionDengue viremia has a significant association with disease severity and day 3 viremia levels may be used as a predictor for dengue disease severity.

Genome-Wide Identification, Characterization, and Expression Analysis of the CYP450 Family Associated with Triterpenoid Saponin in Soapberry (Sapindus mukorossi Gaertn.)

Soapberry (Sapindus mukorossi Gaertn.) is a tree species of the family Sapindaceae, the pericarp of which is rich in triterpenoid saponins, which are important in chemical production, biomedicine, and other fields. Cytochrome P450 monooxygenase (CYP450) is involved in the modification of the skeletons of triterpenoid saponins and is linked to their diversity. We previously identified 323 CYP450 genes in the transcriptome of soapberry and screened 40 CYP450 genes related to the synthesis of triterpenoid saponins by gene annotation and conserved structural domain analysis. The genetic structure and phylogeny of the CYP450 genes were analyzed separately. Phylogenetic analysis categorized the CYP450 genes of soapberry into five subfamilies, the members of which had similar conserved cumulative sequences and intron structures. A cis-acting element analysis implicated several genes in the responses to environmental changes and hormones. The expression of several genes during eight periods of fruit development was analyzed by real-time quantitative qRT-PCR; most showed high expression during the first four periods of fruit development, and their expression decreased as the fruits matured. A co-expression network analysis of SmCYP450s and related genes in the triterpenoid saponin synthesis pathway was performed. Correlation analysis showed that 40 SmCYP450s may be involved in saponin synthesis in soapberry. The triterpenoid saponin synthesis-related candidate genes identified in this study provide insight into the synthesis and regulation of triterpenoid saponins in soapberry.