Quantitative Real-time PCR Analysis Research Articles

Integration of Genome-Wide Identification and Transcriptome Analysis of Class III Peroxidases in Paeonia ostii: Insight into Their Roles in Adventitious Roots, Heat Tolerance, and Petal Senescence

As a plant-specific gene family, class III peroxidases (PODs) play an important role in plant growth, development, and stress responses. However, the POD gene family has not been systematically studied in Paeonia ostii. In this study, a total of 57 PoPOD genes were identified in the P. ostii genome. Subsequently, phylogenetic analysis and chromosome mapping revealed that PoPODs were classified into six subgroups and were unevenly distributed across five chromosomes. The gene structure and conserved motifs indicated the potential for functional divergence among the different subgroups. Meanwhile, four PoPODs were identified as tandem duplicated genes, with no evidence of segmental duplication. Using RNA-seq data from eight different tissues, multiple PoPODs exhibited enhanced expression in apical and adventitious roots (ARs). Next, RNA-seq data from AR development combined with trend analysis showed that PoPOD30/34/43/46/47/57 are implicated in the formation of ARs in tree peony. Through WGCNA based on RNA-seq, two key genes, PoPOD5/15, might be involved in heat tolerance via ABA and MeJA signaling. In addition, real-time quantitative PCR (qRT-PCR) analysis indicated that PoPOD23 may play an important role in flower senescence. These findings deepened our understanding of POD-mediated AR development, heat tolerance, and petal senescence in tree peony.

XRCC1 is linked to poor prognosis in adenocarcinoma of the esophagogastric junction after radiotherapy: transcriptome and alternative splicing events analysis.

This study aimed to (i) investigate the relationship between X-ray repair cross-complementing protein 1 gene (XRCC1) and prognosis in patients with adenocarcinoma of the esophagogastric junction (AEG), and (ii) analyze the roles of XRCC1 in human gastric adenocarcinoma (AGS) cells following X-ray radiation. A total of 46 AEG patients were enrolled and examined for XRCC1 protein by immunohistochemistry. XRCC1 was knocked down in AGS cells by transfection, and AGS cells were subsequently exposed to 6Gy of X-ray radiation. XRCC1 mRNA and protein expression was examined via quantitative real-time PCR (qRT-PCR) and Western blot analysis. The apoptosis of AGS cells was examined by flow cytometer. RNA-sequencing technology was used to identified differentially expressed genes and alternative splicing events following XRCC1 knockdown and radiation exposure. XRCC1 positivity was strongly associated with distant metastasis, pathological tumor-node-metastasis (pTNM) classification, and radiotherapy resistance in AEG patients. A significant difference in progression-free survival was observed between AEG patients with low and high XRCC1 protein expression. The knockdown of XRCC1 notably exacerbated the effects of X-ray radiation on apoptosis in AGS cells. Additionally, X-ray radiation modified the expression of genes related to apoptosis and immune response in XRCC1-knockdown AGS cells. Furthermore, the generation of splice variants was influenced by XRCC1 knockdown in AGS cells. XRCC1 may serve as a key oncogene that elucidates the role of alternative splicing events in the progression of AEG following X-ray treatment.

Evaluation and Validation of Reliable Reference Genes for Quantitative Real-Time PCR Analysis of the Gene Expression in Macadamia integrifolia

Macadamia is an economically significant crop, with its kernel oil being abundant in monounsaturated fatty acids (MUFA). Analyzing the expression of genes related to MUFA biosynthesis is essential for understanding the complex regulatory networks in Macadamia. However, there are few reports on the identification of suitable reference genes for use as internal controls in this species. Consequently, selecting a reliable reference gene for gene expression studies under various conditions is critical. In this study, we evaluated the expression stability of 11 traditional housekeeping genes: α-tubulin (TUBa), β-tubulin (TUBb), malate dehydrogenase (MDH), 18S ribosomal RNA (18S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), α-elongation factor 1 (EF1a), β-elongation factor 1 (EF1b), ubiquitin (UBQ), ubiquitin-conjugating enzyme (UBC), cyclophilin (CYP), and actin (ACT) under abiotic stresses, hormonal treatments and in variety of plant tissues using the online tool RefFinder, which integrates four commonly used software programs: ΔCt, geNorm (version 3.4), NormFinder (version 0953), and BestKeeper (version 1.0). A comprehensive expression stability ranking was established by integrating results from these four methods based on the geometric mean. The findings indicated that ACT was the most stable gene across all samples, including those subjected to cold stress, NaCl stress, PEG stress, ABA treatment, MeJA treatment, and both stem and leaf tissues. EF1b was identified as the most stable gene in GA treatment and heat stress samples, while UBC and CYP were ranked highest in ethrel treatment and root tissue samples, respectively. Finally, the reliability of these findings was further validated using the target gene SAD through qRT-PCR. In summary, this study evaluated and validated appropriate reference genes for qRT-PCR, which will facilitate future investigations into the molecular mechanisms in Macadamia.

Genome-Wide Identification of the bHLH Gene Family in Callerya speciosa Reveals Its Potential Role in the Regulation of Isoflavonoid Biosynthesis

Callerya speciosa (Champ. ex Benth.) Schot is a significant leguminous plant valued for its edible tuberous roots, which are a plentiful source of isoflavonoids. Basic helix–loop–helix (bHLH) transcription factors (TFs) have been reported to regulate secondary metabolism in plants, especially flavonoid biosynthesis. However, the bHLH genes in C. speciosa have not yet been reported, and their regulatory role in isoflavonoid biosynthesis remains unexplored. Here, 146 CsbHLH genes were identified in the C. speciosa genome, classifying them into 23 subfamilies based on the gene structures and phylogenetic relationships. All the CsbHLH proteins contained both motifs 1 and 2, whereas motif 8 was only distributed in subgroup III (d + e). Collinearity analysis demonstrated that fragmental replications are the primary driver of CsbHLH evolution, with the majority of duplicated CsbHLH gene pairs experiencing selective pressure. Nine candidate CsbHLH genes were found to play a potential role in regulating isoflavonoid biosynthesis through a combination of gene-to-metabolite correlation analysis and weighted gene co-expression network analysis (WGCNA). Additionally, the cis-regulatory elements and response to MeJA of these nine genes were characterized and confirmed through quantitative real-time PCR (qRT-PCR) analysis. Among them, three CsbHLHs (CsbHLH9, CsbHLH89, and CsbHLH95) were selected for further investigation. Yeast two-hybrid (Y2H), dual-luciferase (LUC) assays, bimolecular fluorescence complementation (BiFC) assays, and transient transformation demonstrated that CsbHLH9 acted as a transcriptional activator through its interaction with CsMYB36 and binding to the promoters of isoflavonoid biosynthesis genes in a MeJA-induced manner, such as CsIFR2, CsI3′H2, and CsCHS4, to promote isoflavonoid (calycosin, calycosin-7-o-glucoside, and formononetin) accumulation. Our results establish a basis for the functional analysis of bHLH genes and investigations into the molecular mechanisms underlying isoflavonoid biosynthesis in C. speciosa.

Demonstration of T-Cell Monotypia Using Anti-TCRbeta1/2 (TRBC1/2) Immunostaining as a Rapid and Cost-Effective Alternative to PCR-Based Clonality Studies for the Diagnosis of T-Cell Lymphoma

Background/Objectives: T-cell lymphomas are often histologically indistinguishable from benign T-cell infiltrates, and diagnosis typically relies on slow, complex, and expensive multiplexed PCR reactions, requiring significant training and experience to interpret them. We aimed to raise highly specific antibodies against the two alternatively used and very similar T-cell receptor beta constant regions, TCRbeta1 and TCRbeta2, encoded by the TRBC1 and TRBC2 gene segments, respectively. We sought to demonstrate the feasibility of detecting TCRbeta1 and TCRbeta2 immunohistochemically in routine clinical (formalin-fixed, paraffin-embedded (FFPE)) tissue sections as a novel diagnostic strategy for T-cell lymphomas. Methods: Recombinant rabbit antibodies were validated using Western blotting and FFPE immunostaining of T-cell leukemia lines. The immunostaining of FFPE tissue containing benign and lymphomatous T-cell populations was undertaken, with corroboration by BaseScopeTM high-sensitivity in situ hybridization and quantitative real-time PCR (Q-PCR). An additional Q-PCR literature review and analysis of publicly available RNAseq data was used to determine the TCRbeta2/TCRbeta1 ratio cut-off to separate benign and malignant T-cell populations. Results: Our TCRbeta1/TCRbeta2 antibody pair gave highly specific FFPE tissue staining. All benign samples analyzed (immunohistochemically, by BaseScopeTM, by Q-PCR, and by RNAseq data analysis) had TCRbeta1/TCRbeta2 or TRBC1/TRBC2 ranges well within the previously published flow cytometric benign range (TCRbeta2/TCRbeta1 = 0.18:1–5.7:1), while samples of T-cell lymphoma did not. One out of thirteen (7.7%) lymphoma samples showed some detectable TCRbeta1/TCRbeta2 protein co-expression, and 4 out of 13 (30.8%) T-cell lymphomas showed a TRBC1/TRBC2 transcript co-expression using BaseScopeTM. Conclusions: Analyzing T-cell monotypia immunohistochemically, analogous to B-cell monotypia (kappa: lambda ratio for B-cell and plasma cell neoplasms), could make the diagnosis of T-cell lymphomas cheaper, quicker, and more accurate. Larger studies are needed to validate our antibodies for clinical use.

PlFabG improves high-temperature resistance in herbaceous peony by increasing saturated fatty acids

The herbaceous peony (Paeonia lactiflora Pall.) is renowned for its striking flowers, which are highly valued in the cut flower industry. However, in the middle and lower reaches of the Yangtze River, the elevated summer temperatures negatively affect the plant's flowering capacity in the subsequent year. 3-oxoacyl-ACP reductase (FabG) is a component of the type II fatty acid synthesis (FAS II) system, where it plays a role in facilitating the production of saturated fatty acids. However, its role in conferring resistance to high-temperature stress remains unclear. In order to investigate the function of PlFabG in dealing with high-temperature stress, we isolated PlFabG from P. lactiflora. The gene contains an open reading frame (ORF) of 780 bp, which encodes 259 amino acids. Quantitative real-time PCR (qRT-PCR) analysis showed that the expression levels of PlFabG increased with prolonged exposure to high temperature. Additionally, plants overexpressing PlFabG maintained a relatively healthy phenotype under high-temperature stress, whereas plants with silencing PlFabG exhibited more severe leaf scorching and wilting under the same conditions. Various physiological indices indicated that PlFabG reduced reactive oxygen species (ROS) accumulation and enhanced the saturation of photosystem II. Most importantly, PlFabG helped P. lactiflora withstand high-temperature stress by increasing the proportion of saturated fatty acids, thereby maintaining cell membrane integrity. These findings elucidate the beneficial role of PlFabG in enhancing plant tolerance to high temperature and provide a strong theoretical support for the development of high-temperature tolerance in transgenic P. lactiflora.

Selective Dry Cow Therapy: Clinical Field Trial on Prevention and Cure of Intramammary Infections

Intramammary antibiotic dry cow therapy (DCT) at the end of lactation is one of the key measures in the management and control of bovine mastitis. Currently, livestock production is under pressure to reduce antibiotic consumption, emphasizing the need to avoid medicating all cows at dry-off, and instead to treat only infected cows. The study objective was to evaluate IMI cure risk, new IMI risk, and post-calving IMI risk between DCT-treated and untreated quarters over the dry period. The sample totaled 269 cows from 12 Finnish automatic milking system herds entering the dry period between 2019 and 2021. Cows eligible for the study had a final DHI test-day SCC ≥ 100,000 cells/mL and had no clinical signs of mastitis. Based on odd or even identification numbers, cows with an SCC between 100,000 and 250,000 cells/mL were sequentially assigned to either receive DCT at dry-off (DCT100-250) or remain untreated (NoDCT100-250). All cows with an SCC > 250,000 cells/mL received DCT (DCT > 250). No quarters received teat sealants. Farmers collected aseptic quarter-milk samples for microbiological quantitative real-time PCR (qPCR) analysis 0 to 4 d before dry-off and 0 to 4 d after calving. The outcome of the statistical model was the odds of a quarter having an IMI at freshening 0 to 4 d after calving. The statistical tool was a generalized linear mixed-effects model with logit link function and 2-level random intercepts, cows nested within herds. Quarter-level IMI prevalence at dry-off was NoDCT100-250 17.6% (45/256), DCT100-250 22.2% (83/374), and DCT > 250 32.0% (132/413). Quarter-level IMI prevalence at freshening was NoDCT100-250, 36.2% (84/232), DCT100-250, 13.6% (50/369), and DCT > 250, 11.6% (46/397). The untreated quarters in the NoDCT100-250 group had a 1.1 times higher risk of failing to cure over the dry period than did the treated quarters in the DCT100-250 group (unadjusted RR 1.10, 95% CI 0.94-1.28) and a 1.2 times higher risk than the treated quarters in the DCT > 250 group (unadjusted RR 1.15, 95% CI 1.00-1.33). The untreated quarters in the NoDCT100-250 group had a 1.5 times higher risk of having a new IMI over the dry period than did the treated quarters in the DCT100-250 group (unadjusted RR 1.48, 95% CI 1.35-1.63) and a 1.4 times higher risk than the treated quarters in the DCT > 250 group (unadjusted RR 1.35, 95% CI 1.23-1.49). The principal cause of new IMI was NAS, followed by Streptococcus dysgalactiae. Quarters in the NoDCT100-250 group had 6.6 times higher odds for having an IMI at freshening than did quarters in the DCT100-250 group (95% CI 3.52-12.21). According to our findings within the framework of this experiment, selectively DCT-treated cows with an SCC between 100,000 and 250,000 cells/mL at dry-off demonstrated that, although the group-specific cure risks over the dry period were high, untreated quarters had a higher risk of acquiring a new IMI over the dry period than did treated quarters, highlighting the effectiveness of DCT in reducing IMI risks. The prophylactic use of antibiotics, however, is not recommended, and in some countries is even prohibited.

Differential gliding motility responses of Chryseobacterium sp. strain PMSZPI isolated from uranium ore deposit on hard and soft substrates

The Bacteroidota bacterium, Chryseobacterium sp. strain PMSZPI isolated from sub-surface soil of uranium ore deposit was shown to move on solid surfaces via gliding motility resulting in the formation of thin spreading colonies. In this study, we attempted to understand the influence of the surfaces, soft or hard/rigid, on the motility behaviour of PMSZPI cells. The computational tool T9GPred in combination with LC-MS/MS analysis established the presence of orthologs of vital gliding motility proteins in PMSZPI. We analyzed the single cell or population motility phenotypes of PMSZPI under spreading and non-spreading conditions. A low percentage of agar or soft agar (0.35%) with low nutrient levels induced more active gliding motility in individual cells leading to increased colony spreading. Microscopic analyses indicated the self-assembly of the gliding cells into irregular edged or spherical microcolonies based on the agar concentration. Cells moved at a speed of 0.2 to 0.6 µm s-1 on low-percentage gliding permissive agar (0.35%) surface in contrast to significant inhibition of motility on rigid or hard agar (1.5%) surface. RNA sequencing and real-time quantitative PCR (qPCR) analysis revealed increased expression of gliding motility genes under low agar conditions consistent with increased spreading behaviour. These findings provide the first glimpse into the gliding motility behaviour of a Bacteroidota bacterium from metal enriched environment that apparently could have implications on bacterial adaptation to changing surface environments.

Testosterone replacement has a beneficial effect on the hemostatic system by altered gene expression of coagulation factors

This study aimed to investigate the effects of testosterone replacement therapy on hemostasis and some procoagulant gene expression in mice. 42 mice were randomly divided into two groups of non-orchiectomized (non-ORX) and orchiectomized (ORX) with three subgroups (n = 7) each, were subcutaneously administered with sesame oil (control), 2 and 20 mg/kg/week testosterone enanthate. Orchiectomized mice were allowed to recover for one week before treatment. On the 7th week of treatment, blood samples were collected for coagulation parameters analysis and measurement of plasma testosterone levels. Moreover, quantitative real-time PCR analysis was performed on liver samples to assess the expression of factor IX, factor X, and prothrombin genes. The results showed that supraphysiological doses (20 mg/kg) of testosterone significantly increased plasma testosterone levels in all groups, while physiological doses (2 mg/kg) only increased testosterone levels in non-ORX animals. Although testosterone administration had no effect on prothrombin time (PT) and activated partial thromboplastin time (aPTT), supraphysiological doses reduced bleeding time and clotting time. Furthermore, platelet count increased in a dose-dependent manner with testosterone enanthate treatment. The expression of coagulation factors was also decreased with supraphysiological doses of testosterone. In conclusion, testosterone had significant effects on primary hemostasis and common coagulation pathway, including increased platelet number and aggregation, decreased clotting time, and altered gene expression of coagulation factors.

Extracellular vesicle-incorporated long noncoding RNA PUNISHER is increased in coronary artery disease and regulates endothelial cell function

Abstract Aims Long noncoding RNAs (lncRNAs) have emerged as biomarkers and regulators of cardiovascular disease. Circulating lncRNAs can be incorporated into extracellular vesicles (EVs). Our aim was to study the expression pattern of circulating EV-incorporated lncRNAs in patients with and without coronary artery disease (CAD). The regulation of the lncRNA PUNISHER was further assessed as an intercellular messenger in endothelial cell (EC) biology. Methods and results Circulating EVs were isolated from the plasma of patients using ultracentrifugation. Electron microscopy, western blot, and NTA were used to determine the size and origin of the EVs. PCR-based human lncRNA array analysis revealed that certain EV-lncRNAs are significantly different in patients with CAD compared to patients without CAD. To validate the lncRNA array results, 60 patients with (n=30) or without (n=30) CAD were prospectively studied. Four atherosclerosis-related lncRNAs (PUNISHER, GAS5, MALAT1, and H19) were quantified in the circulating EVs by using real-time quantitative PCR (RT-qPCR). Among these, PUNISHER (p=0.002) and GAS5 (0.02) were significantly increased in patients with CAD, compared to non-CAD patients. Analysis of RNA degradation revealed that circulating PUNISHER is mainly incorporated within EVs. In vitro, atherosclerotic stimuli consisting of oxLDL or TNF-a treatment caused an upregulation of PUNISHER levels in human coronary artery endothelial cells (HCAEC) and in the corresponding EVs. Labeling of EVs followed by RT-qPCR demonstrated that functional PUNISHER was transported into the recipient ECs, which accelerated cell migration, proliferation, and tube formation. Mechanistically, the RNA-binding protein hnRNPK was identified by an RNA immunoprecipitation (RIP) assay as an interaction partner of PUNISHER, regulating its loading into small EVs. Knockdown of PUNISHER abrogated the EV-mediated effects on EC migration, proliferation, and tube formation. PCR-based gene profiling showed that the expression of VEGF RNA was significantly increased in ECs by treatment with EVs. Protein stability and RNA-immunoprecipitation followed by RT-qPCR analysis indicated that the PUNISHER-hnRNPK axis regulates the stability and binding of VEGFA mRNA to hnRNPK. Knockdown of PUNISHER in EVs abrogated the EV-mediated promotion of VEGFA gene- and protein expression. Conclusion The circulating lncRNA PUNISHER is increased in CAD patients. Intercellular transfer of EV-incorporated PUNISHER promotes a pro-angiogenic phenotype via a VEGFA-dependent mechanism.

Immunostimulation Signaling via Toll-like Receptor 2 Activation: A Molecular Mechanism of Lactococcus lactis OTG1204 In Vitro and In Vivo.

The immune system's defense against pathogens involves innate and adaptive responses, crucial in maintaining overall health. Immunosuppressed states render individuals more susceptible to potential diseases, indicating the need for effective strategies to bolster immune functions. Although the immunostimulatory effects of various probiotics have been studied, the specific effects and molecular mechanisms of Lactococcus lactis OTG1204 (OTG1204) remain unknown. In this study, the aim was to investigate the molecular mechanisms of OTG1204 in RAW 264.7 macrophages, the key effector cells of the innate immune system involved in host defense and inflammatory responses. Additionally, in this study, the effects of OTG1204 on cyclophosphamide (CTX)-induced immunosuppression states were investigated, thereby demonstrating its potential as an immune stimulant. To assess the macrophage activation ability and underlying mechanisms of OTG1204, RAW 264.7 cells were utilized with transfection, enzyme-linked immunosorbent assay, and quantitative real-time PCR analyses. Furthermore, to evaluate the immunostimulatory effects under immunosuppressed conditions, CTX-induced immunosuppression mice model was employed, and analyses were performed using hematoxylin and eosin staining, flow cytometry, and microbiota examination. OTG1204 activated RAW 264.7 macrophages, leading to increased production of nitric oxide, prostaglandin E2, and cytokines. This immune activation was mediated through the upregulation of toll-like receptor 2, which subsequently activated the nuclear factor-κB (NF-kB) and mitogen-activated protein kinase (MAPK)/activator protein 1 (AP-1) pathways, thereby stimulating the immune response. In CTX-treated mice, OTG1204 recovered body weight, spleen, and mesenteric lymph node indices, and natural killer cell activity. It re-established populations of innate and adaptive immune cells and activated T cells to secrete cytokines. We also examined the gut barrier integrity and microbiota composition to assess OTG1204's impact on intestinal health, as these factors play a significant role in immune enhancement. OTG1204 enhanced gut barrier integrity by upregulating mucin 2 and tight junction proteins and modulated the gut microbiota by restoring the Firmicutes/Bacteroidetes balance and reducing the abundance of Actinobacteria and Tenericutes. These results suggest that OTG1204 may serve as an effective probiotic for immune enhancement and gut health management by targeting the NF-κB and MAPK/AP-1 pathways, with minimal side effects.

Overexpression of miR-451a Aggravates Renal Ischemia–Reperfusion Injury by Targeting KLF1-ACSL4 to Promote Ferroptosis

Ischemia–reperfusion injury (IRI) is a predominant factor leading to delayed graft function (DGF) following kidney transplantation. MicroRNAs (miRNAs) play a pivotal role in the pathogenesis of renal IRI, with ferroptosis being a critical driving force throughout the process. In this study, we utilized bioinformatics methods to construct a network diagram of differentially expressed miRNAs, transcription factors (TFs), and ferroptosis-related genes. An I/R-induced renal injury model in mice and an in vitro H/R-induced HK-2 cell injury model were established. Quantitative real-time PCR (qRT-PCR) and Western blot analysis were used to measure the mRNA and miRNA levels in cells and tissues. The MDA concentration, iron levels, and GSH concentration were measured to evaluate the ferroptosis levels. CCK-8 assays were performed to assess cell viability. Luciferase reporter assays were conducted to validate the downstream targets of miRNA, and chromatin immunoprecipitation assays were performed to verify the interaction between TFs and mRNAs. Both the in vivo and in vitro results demonstrate that miR-451a was significantly enriched in the IRI renal tissues and cells, exacerbating ferroptosis. MiR-451a was found to reduce the expression of Kruppel-like factor 1 (KLF1) by directly binding to the 3′UTR of KLF1 mRNA. Additionally, KLF1 was identified as a negative transcription factor for acyl-CoA synthetase long-chain family member 4 (ACSL4). We demonstrated that IRI induced the upregulation of miR-451a, which reduced KLF1 expression, thereby promoting ferroptosis by upregulating ACSL4 expression, ultimately aggravating IRI-induced renal damage.

Differentiation of human umbilical cord mesenchymal stem cells into functional intestinal epithelial cells via conditioned medium co-culture

BackgroundThe induction of stem cell differentiation to generate intestinal epithelial cells (IECs) with absorptive functions offers significant therapeutic potential for treating conditions such as Crohn’s disease, ulcerative colitis, radiation enteritis, and other refractory intestinal epithelial injuries. Human umbilical cord mesenchymal stem cells (hUC-MSCs) are capable of differentiating into functional IEC-like cells. ObjectiveThis study aimed to induce the differentiation of hUC-MSCs into IECs using a conditioned medium co-culture method. MethodA culture medium derived from human IECs was used as the inductive medium to facilitate the differentiation of hUC-MSCs into IECs. The cellular morphology was assessed using inverted microscopy, and the expression of IEC markers, including Villin, CK20, CK8, and CK18 proteins, was analyzed via immunofluorescence staining. Furthermore, the expression levels of IEC markers, such as KRT18, were quantified using real-time quantitative PCR analysis. The functionality of the differentiated IECs in terms of sucrase secretion was assessed through sucrase activity assays. ResultsBy the 14th day of induction, hUC-MSCs exhibited a morphology similar to IECs and exhibited the expression of IEC markers, including the KRT18 gene and Villin, CK20, CK8, and CK18 proteins. Sucrase activity assays further confirmed that the differentiated cells demonstrated significant sucrase activity. ConclusionThe conditioned medium co-culture method effectively induced the differentiation of hUC-MSCs into functional IECs.

Impact of bariatric surgery on gut microbiota composition in obese patients compared to healthy controls

Bariatric surgery is vital for sustainable weight loss and metabolic improvement in obese individuals, but its effects on gut microbiota and their role in these benefits require further investigation. Investigate the temporal changes in gut microbiota in obese patients undergoing bariatric surgery (gastric sleeve gastrectomy or Roux-en-Y Gastric Bypass (RYGB)) compared to healthy controls, aiming to understand their role in weight loss and metabolic health improvement. A case-control study included 30 obese patients aged 65–95 undergoing bariatric surgery, and 18 matched healthy controls. Selection criteria were based on age, race, BMI, history of antibiotics, probiotics, and prebiotics usage. Stool samples were collected at baseline, three months, and six months post-surgery for DNA extraction and quantitative real-time PCR analysis to assess gut microbiota changes. Physical activity and dietary intake were evaluated using standardized questionnaires. Statistical analyses were performed using R. Post-surgery, patients showed significant reductions in weight and BMI, with changes in dietary habits and physical activity. Quantitative real-time PCR analysis revealed substantial alterations in bacterial groups such as Bacteroides and Fusobacterium. However, some groups showed no significant changes, indicating a complex interaction between gut microbiota and bariatric surgery. Notable correlations were found between body weight, BMI, and specific bacterial groups like the C. cluster IV and Lactobacillus, particularly in RYGB patients. Bariatric surgery significantly alters gut microbiota, aiding weight loss and metabolic regulation in obese patients. Understanding these changes is crucial for developing effective obesity management strategies, requiring further research to optimize outcomes.

Exogenous Melatonin Reinforces Photosynthesis, Antioxidant Defense and Gene Expression to Ameliorate Na2CO3 Stress in Maize.

Salt stress can seriously affect the growth and development of maize (Zea mays L.), resulting in a great yield loss. Melatonin (MT), an indole hormone, is a potential enhancer of plant tolerance against salt stress. However, the complex mechanisms of MT application in enhancing maize salt tolerance are still unclear. Herein, three-leaf seedlings of salt-susceptible P138 and its salt-resistant ethyl methane sulfonate (EMS)-104 mutant were cultured with or without 150 μM MT application under 0 and 100 mM Na2CO3 treatments for seven days, to systematically explore the response mechanisms of exogenous MT in improving the salt tolerance of maize. The results showed that salt stress triggered an escalation in reactive oxygen species production, enhanced multiple antioxidant enzymes' activities, impaired cellular membrane permeability, inhibited photosynthetic pigment accumulation, and ultimately undermined the vigor and photosynthetic prowess of the seedlings. While suitable MT application counteracted the detrimental impacts of Na2CO3 on seedlings' growth and photosynthetic capacity, the seedling length and net photosynthetic rate of P138 and EMS-104 were increased by 5.5% and 18.7%, and 12.7% and 54.5%, respectively. Quantitative real-time PCR (qRT-PCR) analysis further showed that MT application activated the expression levels of antioxidant enzyme-related genes (Zm00001d025106, Zm00001d031908, Zm00001d027511, and Zm00001d040364) and pigment biosynthesis-related genes (Zm00001d011819 and Zm00001d017766) in both maize seedlings under Na2CO3 stress; they then formed a complex interaction network of gene expression, multiple physiological metabolisms, and phenotype changes to influence the salt tolerance of maize seedlings under MT or Na2CO3 stress. To sum up, these observations underscore that 150 μM MT can alleviate salt injury of maize seedlings, which may provide new insights for further investigating MT regulation mechanisms to enhance maize seedlings' salt resistance.

Singapore grouper iridovirus VP12 evades the host antiviral immune response by targeting the cGAS-STING signalling pathway.

The emergence of Singapore grouper iridovirus (SGIV) has caused huge losses to grouper farming. SGIV is a DNA virus and belongs to the genus Ranavirus. Groupers infected with SGIV showed haemorrhaging and swelling of the spleen, with a mortality rate of more than 90% within a week. Therefore, it is of great significance to study the escape mechanism of SGIV from host innate immunity for the prevention and treatment of viral diseases in grouper. In this study, the viral proteins that interact with EccGAS were identified by mass spectrometry, and the SGIV VP12 protein that inhibits cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-mediated antiviral innate immunity was screened by the dual-luciferase reporter gene assay. VP12 belongs to the late gene of the virus. The immunofluorescence analysis demonstrated that VP12 was aggregated and distributed in the cytoplasm during the early stage of virus infection and translocated into the nucleus at the late stage of virus infection. VP12 inhibited the activation of IFN3, ISRE and NF-κB promoter activities mediated by cGAS-STING, EcTBK1 and EcIRF3. Quantitative real-time PCR analysis showed that VP12 inhibited the expression of interferon-related genes, including those mediated by cGAS-STING. VP12 enhanced the inhibition of IFN3, ISRE and NF-κB promoter activity by EccGAS, EccGAS-mab-21 and EccGAS-delete-mab21. The interaction between VP12 and EccGAS was found to be domain independent. The immunoprecipitation results demonstrated that VP12 interacted and co-localized with EccGAS, EcTBK1 and EcIRF3. VP12 degraded the protein levels of EcTBK1 and EcIRF3 and degraded EcIRF3 through the protease pathway. These results suggest that SGIV VP12 protein escapes the cGAS-STING signalling pathway and degrades EcIRF3 protein expression through the protease pathway.

Identification and expression profiling of neuropeptides and neuropeptide receptor genes in a natural enemy, Coccinella septempunctata.

Neuropeptides and their receptors constitute diverse and abundant signal molecules in insects, primarily synthesized and released primarily from neurosecretory cells within the central nervous system Neuropeptides act as neurohormones and euromodulators, regulating insect behavior, lifecycle, and physiology by binding to receptors on cell surface. As a typical natural predator of agricultural pests, the lady beetle, Coccinella septempunctata, has been commercially mass-cultured and widely employed in pest management. Insect diapause is a physiological and ecological adaptative strategy acquired in adverse environments. In biological control programs, knowledge about diapause regulation in natural enemy insects provides important insight for improving long-term storage, transportation, and field adoption of these biological control agents. However, little is known about the function of neuropeptides and their receptors in controlling reproductive diapause of C. septempunctata. It is unclear which neuropeptides affect diapause of C. septempunctata. In this study, RNA-seq technology and bioinformatics were utilized to investigate genes encoding neuropeptides and their receptors in female adults of C. septempunctata. Quantitative real-time PCR (qRT-PCR) analysis was employed to examine gene expression across different development/diapause stages. A total of 17 neuropeptide precursor genes and 9 neuropeptide receptor genes were identified, implicated in regulating various behaviors such as feeding, reproduction, and diapause. Prediction of partial mature neuropeptides from precursor sequences was also performed using available information about these peptides from other species, conserved domains and motifs. During diapause induction, the mRNA abundance of AKH was notably higher on the 10th day compared to non-diapause females, but decreased by the 20th day. In contrast, GPHA showed lower expression levels on the 5th day of diapause induction compared to non-diapause females, but increased significantly by the 15th and 20th days. NPF was higher expressed in head and midgut while DH showed higher expression in the fat body and midgut. Additionally, NPF expression remained consistently lower throughout all stages of diapause induction compared to non-diapause conditions in females. This study represents the first sequencing, identification, and expression analysis of neuropeptides and neuropeptide receptor genes in C. septempunctata. Our results could provide a foundational framework for further investigations into the presence, functions, and potential targets of neuropeptides and their receptors, particularly in devising novel strategies for diapause regulation in C. septempunctata.

Lysine (K)-specific methyltransferase 2A (KMT2A) rearrangements among Iraqi de novo acute myeloid leukemia

Abstract: BACKGROUND: The classification of acute myeloid leukemia (AML) has evolved extensively over the last 20 years significantly, impacting the diagnosis and prognosis of the patients. The lysine (K)-specific methyltransferase 2A (KMT2A) gene, which has more than 80 gene fusions, is present in approximately 10% of all leukemias. Most KMT2A rearrangements are associated with adverse prognosis and need heavy chemotherapy protocol upfront. OBJECTIVE: This study aims to study the prevalence of KMT2A gene fusion among Iraqi patients with AML and its association with clinical and hematological parameters and patients’ outcomes. PATIENTS, MATERIALS, AND METHODS: A prospective cohort study conducted between December 2020 and May 2022 enrolled 115 Iraqi adults newly diagnosed with AML at the Hematology Unit of Baghdad Teaching Hospital. The patients were also monitored at this facility during the study period. Genetic rearrangements were detected using the Leukemia Q-Fusion Screening Kit through Real-Time Quantitative Reverse Transcription PCR (RT-qPCR) analysis. RESULTS: KMT2A rearrangements were identified in 23 (20%) patients. The most common was t(10;11) which presented in 15 (13%) patients, followed by t(9;11) in 5 (4.3%) patients and t(11;17) in 3 (2.6%) patients. Patients with KMT2A rearrangements were significantly older and more likely to have splenomegaly. At 1-month posttreatment, they had significantly lower red blood cell counts and hemoglobin levels and higher blast percentages. Only 4.3% achieved complete remission (CR) compared to 76.1% without KMT2A rearrangements, with a significantly higher mortality rate (30.4% vs. 5.4%, P = 0.0001). Regarding the treatment response, no significant differences were observed among the different fusion types of KMT2A rearrangements. CONCLUSION: KMT2A rearrangements are more prevalent among Iraqi AML patients compared to the global trend and are associated with older age, higher rates of splenomegaly, poorer hematological recovery, and worse outcomes, regardless of the KMT2A rearrangement fusion type.

Characterization and Expression of the Cytochrome P450 Genes in Daphnia magna Exposed to Cerium Oxide Nanoparticles.

As cerium oxide nanoparticles (nCeO2) continue to infiltrate aquatic environments, the resulting health risks to exposed aquatic organisms are becoming evident. Cytochrome P450 (CYP) enzymes are integral to the detoxification processes in these species. Herein, we conducted a genomic analysis of CYPs in Daphnia magna, encompassing phylogenetic relationships, gene structure, and chromosomal localization. We identified twenty-six CYPs in D. magna, categorizing them into four clans and seven families, distributed across six chromosomes and one unanchored scaffold. The encoded CYP proteins varied in length from 99 to 585 amino acids, with molecular weights ranging from 11.6 kDa to 66.4 kDa. A quantitative real-time PCR analysis demonstrated a significant upregulation of CYP4C1.4, CYP4C1.5, CYP4C1.6, CYP4c3.3, and CYP4c3.6 in D. magna exposed to 150 mg/L nCeO2 for 24 h. The transcript levels of CYP4C1.3, CYP18a1, CYP4C1.1, and CYP4c3.9 were notably downregulated in D. magna exposed to 10 mg/L nCeO2 for 48 h. A further transcriptomic analysis identified differential expression patterns of eight CYP genes, including CYP4C1.3, in response to nCeO2 exposure. The differential regulation observed across most of the 26 CYPs highlights their potential role in xenobiotic detoxification in D. magna, thereby enhancing our understanding of CYP-mediated toxicological responses to metal nanoparticles in aquatic invertebrates.

Comprehensive analysis of ankyrin repeat gene family revealed SbANK56 confers drought tolerance in sorghum

Ankyrin repeat (ANK) proteins are crucial for cell growth, development, and response to hormones and environmental stress. However, there has been little research done to clarify the roles of ANK proteins in sorghum. In this study, 142 ANK genes of sorghum were identified and classified into 12 subfamilies according to the conserved domains. The cis-elements analysis revealed a substantial presence of stress-responsive elements within the promoter region of SbANK genes. After treated with drought, salt, and abscisic acid, SbANK56 showed the highest expression levels among family members by using quantitative real-time PCR (qRT-PCR) analysis. The survival rate was significantly improved by the overexpression of SbANK56 compared to wild type (WT) under drought conditions. SbANK56 overexpressing plants displayed lower malondialdehyde and higher proline contents compared to WT plants under drought conditions. Additionally, the expression levels of drought-associated genes were significantly increased in SbANK56 transgenic plants. Importantly, the analysis of natural variation in SbANK56 revealed a significant positive correlation between SbANK56Hap4 and both its differential expression and drought stress tolerance. Taken together, our results provide some evidence for improving drought tolerance in sorghum through breeding initiatives while also advancing our knowledge of the evolutionary trends and functional mechanisms underlying ANK genes.