Real-time PCR Research Articles

AKR1B1 Inhibits Ferroptosis and Promotes Gastric Cancer Progression via Interacting With STAT3 to Activate SLC7A11.

Gastric cancer (GC) is a frequently diagnosed malignant tumor in clinical settings; however, the mechanisms underlying its tumorigenesis remain inadequately understood. In this study, we identified significantly elevated expression levels of AKR1B1 in GC tissues through quantitative polymerase chain reaction (qPCR) and western blotting assays. Furthermore, a negative correlation was established between patient survival probability and AKR1B1 expression levels. Functionally, our experiments, including colony formation, transwell migration, and xenograft assays, demonstrated that the depletion of AKR1B1 inhibited the proliferation and progression of GC cells both in vivo and in vitro. Additionally, the assessment of reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), and mitochondrial morphology confirmed that AKR1B1 depletion induces ferroptosis. Mechanistically, we found that AKR1B1 interacts with STAT3, which subsequently activates SLC7A11. Notably, the ferroptosis induced by AKR1B1 depletion could be reversed by the overexpression of SLC7A11, thereby substantiating these interactions. In conclusion, our findings identify AKR1B1 as a novel oncogene in GC and elucidate the mechanism involving the AKR1B1-STAT3-SLC7A11 pathway and ferroptosis, providing new insights for potential therapeutic strategies in the treatment of GC.

A new signature associated with anoikis predicts the outcome and immune infiltration in nasopharyngeal carcinoma.

Previous studies have confirmed the phenomenon of anoikis resistance in nasopharyngeal carcinoma (NPC). Nevertheless, the prognostic significance of anoikis-related genes (ARGs) in NPC remains incompletely understood. This study aimed to create a predictive risk score using an ARGs signature for NPC patients and to investigate how this score relates to clinicopathologic features and immune infiltration in the tumor microenvironment. By using data from the Gene Expression Omnibus (GEO) database, we employed machine learning methods to discover prognostic ARGs and create a risk score. Key gene expression levels were validated through real-time PCR and immunohistochemical staining. Three differentially expressed ARGs (CDC25C, E2F1 and RBL2) with prognostic value were identified by the intersection of multiple machine learning algorithms. A risk score based on t 3-ARG feature was developed to stratify NPC patients into two distinct risk groups using the optimal model, Random Survival Forest. NPC patients with high-risk scores experienced notably shorter progression-free survival in comparison to those with low-risk scores. Multivariate Cox regression analysis indicated that the risk score served as an independent prognostic factor. The time-dependent ROC and decision curve analyses demonstrated the risk model's strong predictive accuracy and clinical utility. The low-risk score group exhibited features indicative of early clinical stage, immune activation, high immune checkpoint gene's expression, and low Epstein-Barr virus gene's expression. Functional analysis revealed enrichment of immune-related pathways in the low-risk group. Patients with high-risk scores were discovered to be unlikely to benefit from immune checkpoint inhibitor treatment. Moreover, the expression of RBL2, E2F1, and CDC25C were significantly correlated with the expression of caspase family genes. Finally, the lower mRNA and protein expression of RBL2 were validated in NPC cell lines and tissues. Our ARGs-based signature model shows promising results in predicting the prognosis of NPC patients and might be associated with immune cell infiltration.

Downstream Link of Vitamin D Pathway with Inflammation Irrespective of Plasma 25OHD3: Hints from Vitamin D-Binding Protein (DBP) and Receptor (VDR) Gene Polymorphisms

Background: Vitamin D insufficiency/deficiency is a highly prevalent condition worldwide. At the same time, chronic inflammation is a versatile pathophysiological feature and a common correlate of various disorders, including vitamin D deficiency. Methods: We investigated the possible association of inflammation with 25-hydroxyvitamin D3 (25OHD3) levels and its down-stream pathway by exploring vitamin D-binding protein (DBP) and vitamin D receptor (VDR) genes for single-nucleotide polymorphisms (SNPs), in healthy non-elderly Bahraini adults. Plasma levels of 25OHD3 were measured by chemiluminescence, and six SNPs, four in the GC gene (rs2282679AC, rs4588CA, rs7041GT, and rs2298849TC) and two in the VDR gene (rs731236TC and rs12721377AG) were genotyped by real-time PCR. The concentrations of five inflammatory biomarkers, IL6, IL8, procalcitonin (PCT), TREM1, and uPAR, were measured by ELISA. Results: The results showed no association between the 25OHD3 level and any of the inflammatory markers’ levels. However, three tested SNPs were significantly associated with the concentrations of tested biomarkers except for IL6. The TT mutant genotype of rs2298849TC was associated with lower levels of IL8 and higher levels of PCT and TREM1, the AA mutant genotype of rs2282679AC was associated with decreased levels of IL8 (p ≤ 0.001) and increased levels of TREM1 (p = 0.005), and the GG wild genotype of rs12721377AG was associated with increased levels of 25OHD3 (p = 0.026). Conclusions: Although chronic inflammation is not associated with the vitamin D system in the blood, it is downstream, as revealed by DBP and VDR genotyping. Alternatively, DBP and VDR pursue other functions beyond the vitamin D pathway.

Von Willebrand factor polymorphism (rs1063856) as a risk factor for portal vein thrombosis in chronic liver diseases

Abstract Background Due to its well-established involvement in clot formation, von Willebrand factor (vWF) has been held responsible for decades as a risk factor for arterio-venous thrombosis. It is currently unclear how genetic differences in vWF genes relate to the incidence of portal vein thrombosis (PVT). Thus, the aim of this study was to evaluate the significance of vWF:Ag level and vWF gene polymorphism (rs1063856) as risk factors for PVT in patients with chronic liver disease (CLD). Subjects and methods This research of 70 individuals with CLD comprised 30 without PVT and 40 with PVT. Twenty patients with PVT-linked hepatocellular carcinoma (HCC) and 20 patients with non-HCC-PVT comprised the two subgroups from the 40 PVT patients. Additionally, 30 healthy volunteers of the same age and gender as the patients were included in the study as a control group. Tests for liver and renal function, coagulation profile, complete blood count, hepatitis virus markers, and vWF:Ag level were conducted. The identification of the vWF gene polymorphism (rs1063856) was assessed using real-time PCR. Results When comparing CLD patients with PVT to those without PVT and the healthy control group, there was a substantial rise in the vWF: Ag level. Additionally, compared to instances of benign PVT, the vWF:Ag levels were considerably greater in malignant PVT. CLD patients with PVT had higher frequencies of the vWF (rs1063856) AA genotype and A alleles (37.5% and 61.3%, respectively) than did control and non-PVT cases. Patients with the AA genotype showed the highest serum levels of vWF antigen, followed by those with the TA genotype. Conclusion vWF (rs1063856) AA gene polymorphism has a strong correlation with vWF plasma level and may be a risk factor for PVT development in CLD patients. These findings should be included in the risk assessment model to determine and guide management for those patients.

Noncoding RNA profiling in omentum adipose tissue from obese patients and the identification of novel metabolic biomarkers

BackgroundObesity, a prevalent metabolic disorder, is linked to perturbations in the balance of gene expression regulation. Noncoding RNAs (ncRNAs), including long noncoding RNAs (lncRNAs), circular RNAs (circRNAs), and microRNAs (miRNAs), play pivotal roles in regulating gene expression. The aim of this study was to identify additional ncRNA candidates that are implicated in obesity, elucidating their potential as key regulators of the pathogenesis of obesity.MethodsWe identified distinct ncRNA expression profiles in omental adipose tissue in obese and healthy subjects through comprehensive whole-transcriptome sequencing. Subsequent analyses included functional annotation with GO and KEGG pathway mapping, validation via real-time quantitative polymerase chain reaction (qRT‒PCR), the exploration of protein‒protein interactions (PPIs), and the identification of key regulatory genes through network analysis.ResultsThe results indicated that, compared with those in healthy individuals, various lncRNAs, circRNAs, and miRNAs were significantly differentially expressed in obese subjects. Further verifications of top changed gene expressions proved the most genes’ consistence with RNA-sequencing including 11 lncRNAs and 4 circRNAs. Gene network analysis highlighted the most significant features associated with metabolic pathways, specifically ENST00000605862, ENST00000558885, and ENST00000686149. Collectively, our findings suggest potential ncRNA therapeutic targets for obesity, including ENST00000605862, ENST00000558885, and ENST00000686149.

Over-the-counter carrageenan-based sprays may interfere with PCR testing of nasopharyngeal swabs to detect SARS-CoV-2

Carrageenan-containing nasal sprays, available over-the-counter (OTC), are often marketed as having anti-viral effects. Carrageenan belongs to the glycosaminoglycan family alongside heparin, and heparin is known to inhibit real-time quantitative polymerase chain reaction (RT-qPCR) in nasopharyngeal swabs used to detect SARS-CoV-2. As heparin and carrageenan share structural similarities, this work aimed to investigate the interferent effect of carrageenan on RT-qPCR for SARS-CoV-2 detection across 4 different diagnostic platforms. This work demonstrated that in the presence of carrageenan samples return inaccurate and invalid results on the Seegene STARlet, while qualitative accuracy was maintained on the Cepheid GeneXpert, Roche Cobas LIAT, and Hologic Panther Aptima. Evidence of carrageenan interference on SARS-CoV-2 testing was consistent across two OTC brands and research-grade reconstituted iota-carrageenan, with 80% of results returning invalid regardless of the carrageenan formulation added to the samples. Further, a preliminary in vivo interference study demonstrated an increased Ct value within 15 minutes of carrageenan dosage, with Ct values restored 60 minutes post-application. A direct comparison of carrageenan- and heparin-mediated PCR interference demonstrated that carrageenan PCR interference occurs to a lesser degree, but is not reversible by the addition of heparinase I. As carrageenan is available OTC, interference with PCR testing that causes an increase in false negative results could lead to accidental spread of disease and could therefore have significant public health impacts on community testing of respiratory infectious diseases via PCR.

ACSM3 Suppresses Ovarian Cancer Progression by Inactivating the IFN-γ/JAK/STAT3 Signaling Pathway.

This study aimed to investigate the role of Acyl-CoA medium-chain synthetase 3 (ACSM3) in ovarian cancer (OC) progression. ACSM3 expression in OC tissues and cells is detected by real-time quantitative polymerase chain reaction, immunohistochemistry, or western blot. The influences of ACSM3 on OC progression are assessed in vitro and in vivo experiments. Gene set enrichment analysis is performed to find significantly enriched pathways related to ACSM3. In this study, ACSM3 expression in OC tissues and cells is downregulated. ACSM3 inhibited tumor growth in vivo. Knockdown of ACSM3 promoted cell proliferation, migration, and invasion, inhibited cell apoptosis, and activated JAK/STAT3 signaling pathway in SKOV3 cells, while overexpression of ACSM3 in A2780 cells led to the opposite results. Moreover, treatment with interferon-gamma (IFN-γ) in A2780 cells reversed the effects of ACSM3 on cell proliferation, migration, invasion, and apoptosis, but IFN-γ further enhanced the effects of ACSM3 knockdown on SKOV3 cell proliferation, migration, invasion, and apoptosis. In conclusion, ACSM3 inhibited OC cell proliferation, migration, and invasion, and promoted cell apoptosis by suppressing the IFN-γ/JAK/STAT3 signaling pathway, which might provide a promising therapeutic target for OC treatment.

CrRNA Conformation-Engineered CRISPR-Cas12a System for Robust and Ultrasensitive Nucleic Acid Detection.

Despite the widespread application of the CRISPR-Cas12a system in vitro diagnostics due to its high programmability and distinctive trans-cleavage activity, the susceptibility of its crRNA component to degradation and sensitivity to storage and working conditions poses a significant challenge to improving the practical efficacy of these diagnostic systems. Here, we show that engineered crRNA with a covalently closed circular structure (C-crRNA) can replace traditional linear crRNA to form functional complexes with Cas12a protein, significantly enhancing the anti-interference ability of the CRISPR-Cas12a system while maintaining its sensitivity and specificity. Based on this finding, a circular crRNA-mediated CRISPR molecular diagnostic (CRCD) toolkit is developed and successfully integrated with a standard nucleic acid amplification technique to detect synthesized Human Papillomavirus type 16 (HPV-16) plasmids down to 10 aM sensitivity levels. Furthermore, the CRCD system is applied for ultrasensitive detection of 40 HPV-16 and 40 influenza A viruses in clinical samples, with results consistent with those from PANTHER detection and quantitative real-time polymerase chain reaction (qRT-PCR). In conclusion, this strategy introduces a novel paradigm for engineering crRNA to program Cas12a, which has the potential to revolutionize the use of crRNA in CRISPR-based molecular diagnostics.

Association Between Genetically Proxied SLC12A2 Inhibition and Inflammatory Bowel Disease: A Mendelian Randomization Study.

The global rise in hypertension prompts the use of medications to manage blood pressure. However, selecting first-line drugs remains challenging as their efficacy often stems from blood pressure reduction rather than specific pharmacological actions. Evaluating interactions between antihypertensive drugs and common diseases can aid tailored treatment. Here, we assess the potential link between antihypertensives and inflammatory bowel disease (IBD). Summary-level coronary heart disease (CHD) data (184,305 individuals), systolic BP (SBP) data (757,601 individuals), ulcerative ileocolitis data (361,188 individuals), ulcerative colitis data (364,454 individuals), other ulcerative colitis data (361,619 individuals), and ulcerative proctitis data (361,700 individuals) were all from genome-wide association studies (GWASs), FinnGen or eQTL studies publicly accessible. The DrugBank10 and ChEMBL11 databases function to identify genes encoding protein products targeted by active constituents of BP-lowering drugs. Summary-data-based MR (SMR) estimated the associations between expressions of drug target genes and symptoms of IBD. A multivariable MR study was further conducted to examine if the observed association was direct association. Subsequently, we collected blood samples from IBD patients in the Gastroenterology Department of Gastroenterology, the First Affiliated Hospital of Anhui Medical University and blood from healthy individuals at the physical examination center. Real-time quantitative PCR was employed to detect the expression changes of drug target genes in the peripheral blood of patients with IBD. Furthermore, we used Caco2 cells to construct an in vitro model of IBD, examined the expression of the target molecules, and verified the potential of Bumetanide to improve IBD. SMR analysis revealed that enhanced SLC12A2 gene expression in blood (equivalent to a one standard deviation increase) was a risk factor for ulcerative ileocolitis (beta = 0.5861, se = 0.2972, p = 0.0486) and enhanced gene expression of ACE was a protective factor. Additionally, SCNN1D and SLC16A1 played protective roles of IBD, while NR3C1 was identified as a risk factor. However, among these genes, only SLC12A2 was considered to influence the progress of inflammatory bowel disease through systolic blood pressure based on Mendelian randomization analysis results. Other genes may be associated with IBD depending on the expression of their own proteins, independent of changes in blood pressure. In the peripheral blood of IBD patients and in vitro experiments, SCL12A2 has been shown to be highly expressed in IBD. In vitro experiments have confirmed that Bumetanide can inhibit SCL12A2 to improve tight junctions, reduce inflammation levels, and ameliorate IBD symptoms. Therapeutic inhibition of SCL12A2 may benefit patients with IBD. In the future, this study may contribute to the selection of more personalized antihypertensive medications for different subgroups of hypertensive patients.

Assessment of potential candidate genes for partial resistance to Sclerotinia stem rot caused by Sclerotinia sclerotiorum using real-time quantitative PCR.

Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum (Lib) de Bary (S. sclerotiorum), is one of the most important diseases that causes significant soybean [Glycine max (L.) Merr.] seed yield and quality losses in Canada and globally. Initiation of plant defense mechanisms is crucial for establishing partial resistance to the pathogenic fungus. To understand plant response to S. sclerotiorum, we conducted a temporal (1, 3, and 5 days post-inoculation [DPI]) assessment of gene expression changes in the stem of soybean genotypes with contrasting phenotypic response. We focused on four genes that have been previously reported as associated with SSR partial resistance and are known to be involved in defense-related functions such as cell wall modification, signaling, response to wounding, and response to fungus. The results showed a higher and earlier expression of the genes in partially resistant cultivars compared to the susceptible. Expression of some genes increased up to 11- (Glyma.02G059700) to 16-fold (Glyma.09G232100) by 3 DPI in the partially resistant cultivar, OAC Drayton, while the genes were generally downregulated in the susceptible cultivar, OAC Shire, at the same DPI. This study improves our understanding of expression patterns of genes involved in plant defense against fungal pathogens in soybean. More importantly, the knowledge of genes that are essential in defense against S. sclerotiorum can be used to fine-map the quantitative trait loci for SSR resistance and facilitate accelerated breeding of SSR-resistant cultivars through gene-based marker-assisted selection.

Genetic Basis of Seedling Root Traits in Common Wheat (Triticum aestivum L.) Identified by Genome-Wide Linkage Mapping

Common wheat production is significantly influenced by abiotic stresses. Identifying the genetic loci for seedling root traits and developing the available molecular markers are crucial for breeding high yielding and stable varieties. In this study, five wheat seedling root traits, including root length (RL), root surface area (RA), root volume (RV), number of root tips (RT), and root dry weight (RW), were measured in the Wp-072/Wp-119 recombinant inbred line (RIL) population. Genotyping was conducted for the RIL population and their parents using the wheat 90K single-nucleotide polymorphism (SNP) chip. In total, three quantitative trait loci (QTLs) for RL (QRL.gau-1DS, QRL.gau-1DL and QRL.gau-4AL), two QTLs for RA (QRA.gau-1D and QRA.gau-2DL), one locus for RV (QRV.gau-6AS), two loci for RW (QRW.gau-2DL and QRW.gau-2AS), and two loci for RT (QRT.gau-3AS and QRT.gau-6DL) were identified, with each explaining 4.5–8.4% of the phenotypic variances, respectively. Among these, QRT.gau-3AS, QRL.gau-4AL, and QRV.gau-6AS overlapped with the previous reports, whereas the other seven QTLs were novel. The favorable alleles of QRL.gau-1DS, QRL.gau-1DL, QRL.gau-4AL, QRA.gau-1D, QRW.gau-2AS, QRV.gau-6AS, QRT.gau-3AS, and QRT.gau-6DL were contributed by Wp-072, whereas the other two loci originated from Wp-119. Additionally, five kompetitive allele-specific PCR (KASP) markers, KASP-RL-1DL for RL, KASP-RA-1D and KASP-RA-2DL for RA, KASP-RW-2AS and KASP-RW-2DL for RW, were developed and validated successfully in 149 wheat accessions. Furthermore, seven candidate genes mainly for plant hormones were selected and validated by quantitative real-time PCR (qRT-PCR). This study provides new loci, new candidate genes, available KASP markers, and varieties for optimizing wheat root system architecture.

First case of glyphosate resistance in Polypogon fugax: possible involvement of P450-based mechanisms.

Polypogon fugax has evolved resistance to multiple herbicides in China, yet there has been no documented case of glyphosate resistance. A putative glyphosate-resistant P. fugax (HN-R) population was collected from canola fields in Hunan Province, China, surviving glyphosate treatment at the field-recommended rate [540 g acid equivalent (a.e.) ha-1]. The aims of this study were to elucidate the resistance level and mechanism of HN-R population. Dose-response and shikimic acid assays indicated that the HN-R population has developed a low-level resistance (up to 4.6-fold) to glyphosate, compared to two glyphosate-susceptible populations (HN-S and SC-S). No evidence of EPSPS gene mutations and differential EPSPS gene expression were found in association with the resistance. However, pre-treatment with the known cytochrome P450 monooxygenases (P450s) inhibitor, malathion, reversed glyphosate resistance in the HN-R population. Transcriptomic and reverse transcription quantitative polymerase chain reaction (RT-qPCR) analyses showed up-regulation of the PfCYP51 and PfABCG25 genes in the HN-R population. Expression of PfCYP51 in yeast cells significantly enhanced glyphosate tolerance, whereas expression of PfABCG25 did not. Molecular docking experiments suggest that PfCYP51 may catalyze glyphosate metabolism. This is the first report of glyphosate resistance in P. fugax, with evidence suggesting P450 involvement in this resistance. This study enhances our understanding of herbicide-resistant weeds and provides valuable insights into the management of glyphosate-resistant weeds in agriculture. © 2025 Society of Chemical Industry.

Detection of Chlamydia trachomatis ompA DNA in urine by loop-mediated isothermal amplification (LAMP) assay

Abstract Objectives Efficient real-time PCR kits are commercially available for the detection of Chlamydia trachomatis (CT) genetic material, but at a price. As a result, cost-effective, sensitive and specific CT diagnostic tests are essential for resource limited countries. This study aims to describe the optimization of a loop mediated isothermal amplification (LAMP) assay for the detection of CT ompA DNA in urine. Methods Cost-saving modifications included using Bsm polymerase and a nucleic acid gel stain. Crude DNA extraction (method-1) involved centrifuging urine at 14,000 g for 30 min, heating the deposit at 95 °C for 5 min, and centrifuging again at 17,000 g for 1 min. To boost sensitivity, urinary inhibitors were diluted with phosphate-buffered saline washes and a larger urine volume was used (method-2). The LAMP-SYBR GOLD assay was incubated at 56 °C for 60 min, with nucleic acid gel stain color changes observed under UV light. Urine from 326 sexually transmitted diseases clinic attendees was tested with both LAMP-SYBR GOLD and real-time PCR, comparing sensitivity and specificity. Results Analytical sensitivity of the LAMP-SYBR GOLD assay was 0.8 copies per reaction volume. Compared to real-time PCR, LAMP-SYBR GOLD assay had sensitivity, specificity, positive predictive and negative predictive values of 71.4 , 99.7, 96.2, 96.7 % respectively. Five of the seven false negative results obtained with method-1 were re-tested using method-2, providing in all the cases the expected positive results. Conclusions The LAMP-SYBR GOLD assay showed a sensitivity of 71 % and a high specificity for detecting CT in urine with extraction method-1. The use of method-2 could increase this sensitivity, likely due to the removal of urine inhibitors.

Neuroprotective effects of tranexamic acid against hydrogen peroxide-induced cytotoxicity on human neuroblastoma SH-SY5Y cells

ABSTRACT Introduction Tranexamic acid (TA) is an anticoagulant drug that is used worldwide. However, the adverse effects of TA may insult the nervous system. This study aimed to investigate the dual effects of TA on SH-SY5Y cells, including its detrimental and neuroprotective effects. Methods SH-SY5Y cells were treated with various concentrations of TA and exposed to H2O2 for 24 hours. The neuroprotective effects of TA were evaluated in H2O2-challenged cells. To assess the neuroprotective effects of TA, SH-SY5Y cells were pretreated with TA for 12 hours and then exposed to H2O2 for 24 hours. Cell viability was assessed using the MTT assay. Flow cytometry was used to evaluate cellular apoptosis. The expression of Bax, Bcl-2, and Caspase-3 genes was analyzed by real-time PCR. Additionally, Akt phosphorylation was evaluated using western blotting. Results At high concentrations, TA reduced cell viability and induced apoptosis by up-regulating BAX and Caspase-3 gene expression and down-regulating BCL-2 transcript. Furthermore, Akt phosphorylation was reduced following TA treatment. TA exhibited protective effects against H2O2-induced cell stress by down-regulating Bax and Caspase-3 gene expression, up-regulating Bcl-2 expression, and increasing the p-AKT/AKT ratio. Conclusion Our findings demonstrated that TA exerts its neuroprotective effect at lower concentrations, but induces apoptosis in SH-SY5Y cells at high concentrations.

Batryticatus bombyx improved atopic dermatitis in NC/Nga mice and impact on inflammation and recovery of skin barrier via STAT1/P38/NF-κB downregulation and HO-1/Nrf2 activation in HaCaT keratinocytes.

Batryticatus bombyx (BB) (Beauveria bassiana Vuill.) is used as an herbal medicine and food additive. Although BB has neuroprotective and anti-apoptotic effects, its impacts on atopic dermatitis (AD) are unknown. We evaluated the effects of BB in an NC/Nga mouse model of house dust mite (HDM)-induced AD, in TNF-α and IFN-γ (TI)-stimulated HaCaT keratinocytes, and in a 3D human skin model. NC/Nga mice were induced with HDM and treated with BB for 6 weeks. We assessed skin symptoms, serum levels of IgE, histamine, and IL-6, immune cell counts, and performed histological analysis of dorsal skin tissue. Additionally, we induced inflammation in HaCaT cells and a 3D human skin model using TNF-α/IFN-γ. We measured inflammatory cytokine levels, skin hydration factors, and delved into underlying mechanisms via ELISA, real-time PCR, and Western blot. BB improved skin lesions, reduced thickness, and inflammatory cell infiltration in HDM-induced mice. It alleviated scratching, improved moisture retention, and reduced IgE, histamine, and IL-6 levels. In HaCaT cells, BB inhibited thymic stromal lymphopoietin and increased hyaluronan content. It also upregulated aquaporin-3, hyaluronan synthase-1/3, filaggrin, involucrin, and occludin, enhancing skin hydration and tight junction stability. BB decreased STAT1, p38 MAPK, and NF-κB p65 levels in HaCaT cells and exhibited antioxidant effects by increasing HO-1/Nrf2 expression. BB may serve as a therapeutic alternative for AD treatment by inhibiting skin inflammation.

Molecular characterization of a rare TP63 variant associated with split-hand/split-foot malformation 4 and incomplete penetrance: disruption of the p63-Dlx signaling pathway

BackgroundSplit-hand/foot malformation (SHFM) is a congenital disability characterized by the absence or hypoplasia of the central ray of the hands and/or feet. This study reports a causative variant in the TP63 gene in a Chinese family exhibiting limb anomalies associated with SHFM4.MethodsEnrolled in this study was a Chinese family with limb anomalies without any other clinical features. Karyotype analysis and chromosomal microarray analysis (CMA) were conducted to identify chromosomal abnormalities. Whole exome sequencing (WES) was utilized to investigate sequence variants, while RNA sequencing assessed differentially expressed genes, with findings confirmed through quantitative PCR (qPCR).ResultsKaryotype analysis and CMA revealed no chromosomal abnormalities in the family. Subsequently, WES identified a rare heterozygous variant of NM_003722.5: c.956G > A (p.Arg319His) in the TP63 gene in the proband, which was inherited from her father who also presented with limb deformities. However, both of the sister and grandfather of the proband had the same variant but exhibited normal limb morphology. RNA sequencing results demonstrated an increased expression level of TP63 and its downstream genes (PERP, CDH3, and DLX5) compared with the controls, indicating an enrichment of cell adhesion molecules the differentially expressed genes in the patient. However, significant differences were noted only for the CDH3 and DLX5 genes in qPCR analysis (p<0.05).ConclusionThis study identifies, for the first time, the TP63 gene variant c.956G > A (p.Arg319His) as a causative factor for SHFM4 in Chinese individuals with incomplete penetrance. In addition, we hypothesize that the p.Arg319His variant functions as a gain-of-function variant, leading to the upregulation of cell adhesion target genes. Such upregulation then disrupts the p63-Dlx signaling pathway and causes AER stratification failure.

Biophysical and structural insights into AAV genome ejection.

Recombinant adeno-associated virus (rAAV) is comprised of non-enveloped capsids that can package a therapeutic transgene and are currently being developed and utilized as gene therapy vectors. The therapeutic efficiency of rAAV is dependent on successful cytoplasmic trafficking and transgene delivery to the nucleus. It is hypothesized that an increased understanding of the effects of the cellular environment and biophysical properties of the capsid as it traffics to the nucleus could provide insight to improve vector efficiency. The AAV capsid is exposed to increasing [H+] during endo-lysosomal trafficking. Exposure to low pH facilitates the externalization of the viral protein 1 unique region (VP1u). This VP1u contains a phospholipase A2 domain required for endosomal escape and nuclear localization signals that facilitate nuclear targeting and entry. The viral genome is released either after total capsid disassembly or via a concerted DNA ejection mechanism in the nucleus. This study presents the characterization of genome ejection (GE) for two diverse serotypes, AAV2 and AAV5, using temperature. The temperature required to disassemble the virus capsid (TM) is significantly higher than the temperature required to expose the transgene (TE) for both serotypes. This was verified by quantitative PCR (qPCR) and transmission electron microscopy. Additionally, the absence of VP1/VP2 in the capsids and a decrease in pH increase the temperature of GE. Furthermore, cryo-electron microscopy structures of the AAV5 capsid pre- and post-GE reveal dynamics at the twofold, threefold, and fivefold regions of the capsid interior consistent with a concerted egress of the viral genome.IMPORTANCEThe development of recombinant adeno-associated virus (rAAV) capsids has grown rapidly in recent years, with five of the eight established therapeutics gaining approval in the past 2 years alone. Clinical progression with AAV2 and AAV5 represents a growing need to further characterize the molecular biology of these viruses. The goal of AAV-based gene therapy is to treat monogenic disorders with a vector-delivered transgene to provide wild-type protein function. A better understanding of the dynamics and conditions enabling transgene release may improve therapeutic efficiency. In addition to their clinical importance, AAV2 and 5 were chosen in this study for their diverse antigenic and biophysical properties compared to more closely related serotypes. Characterization of a shared genome ejection process may imply a conserved mechanism for all rAAV therapies.

Unraveling the association and regulatory role of miR-146b-5p in coronary artery disease

BackgroundCoronary artery disease (CAD), one of the most prevalent cardiovascular diseases, is a critical health issue that affects millions of individuals worldwide. It has been reported that miR-146b-5p exhibited a strong correlation with inflammatory responses and atherosclerosis. However, its association with the incidence and severity of CAD has not been substantiated in a large cohort. In the study, we focus on the expression of miR-146b-5p in peripheral blood mononuclear cells (PBMCs) of patients with CAD and preliminarily investigate its function and underlying mechanism.Methods and resultsThe study encompassed a total of 452 participants, consisting 295 patients with CAD and 157 individuals without CAD. Quantitative reverse transcription–polymerase chain reaction (qRT–PCR) was performed to assess miR-146b-5p expression in PBMCs. We found that miR-146b-5p was significantly increased in PBMCs of patients with CAD compared with the control group. Binary logistic regression revealed that miR-146b-5p was associated with CAD. Receiver Operation Characteristic (ROC) analysis showed that the sensitivity and specificity of miR-146b-5p in discriminating CAD patients from non-CAD patients were meaningful. Subsequent subgroup analysis showed that miR-146b-5p was related to the severity of CAD. Furthermore, gain- and loss-of-function experiments in THP-1 cells showed that miR-146b-5p inhibited inflammation, cell proliferation, and migration. Mechanically, miR-146b-5p was involved in the classical NF-κB inflammatory pathway by directly targeting IKKβ.ConclusionOur study revealed that miR-146b-5p was higher in the PBMCs of CAD patients than non-CAD individuals, and established a correlation between miR-146b-5p and occurrence and severity of CAD. In addition, the inflammatory role of miR-146b-5p is mediated by targeting IKKβ.

Pattern of Expression of PML-RARα Transcripts in Acute Promyelocytic Leukemia Patients and Its Correlation with Overall Survival: A Retrospective Study from Western India

Abstract Introduction Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia with specific molecular pathogenesis, clinical features, and treatment. It is cytogenetically characterized by translocation t (15;17) (q24;q21). The location of breakpoints within the PML gene determines the formation of distinct promyelocytic leukemia-retinoic acid receptor α (PML-RARα) transcript subtypes which have prognostic significance. Breakpoints in intron 6 result in the long (L or bcr-1) subtype, those in exon 6 produce the variant (V or bcr-2) subtype, and breakpoints in intron 3 lead to the short (S or bcr-3) subtype. Objectives The aim of our study was to determine the frequencies of the different PML-RARα transcripts in patients with APL at baseline and to investigate the impact of bcr-3 transcript as a prognostic factor on survival in newly diagnosed patients with APL. Materials and Methods A retrospective study was conducted that included 54 newly diagnosed patients with APL. Clinicopathological parameters were evaluated in all cases for prognostic significance with overall survival. Real-time quantitative polymerase chain reaction was used for the quantification of PML-RARα transcripts. Results Out of 54 patients, 53 (98.1%) patients expressed bcr-3 transcript either alone or in combination with either bcr-1 or bcr-2 transcripts. Twenty-one patients (38.9%) in our study showed expression of bcr-3 transcript only. Twenty eight (51.9%) patients in our study expressed all the three transcripts. Out of the 54 patients, 20 (37%) patients died of disease, out of which 19 patients died before completion of induction therapy. Complete remission was obtained in 34 patients (63%) after induction therapy. The survival rate for patients expressing the bcr-3 transcript alone was 66.66% as compared with 60.71% for patients expressing bcr-3 transcript in combination with other two transcripts. The survival rate for patients receiving all-trans retinoic acid (ATRA) in combination with arsenic trioxide was far better than patients receiving ATRA alone. Conclusion The total leucocyte count is an independent prognostic factor in patients with APL as it was statistically significant with overall survival in our study. The bcr transcript either alone or in combination with bcr-1 and/or bcr-2 transcripts was the most frequent pattern observed.

Stachydrine targeting tumor-associated macrophages inhibit colorectal cancer liver metastasis by regulating the JAK2/STAT3 pathway

IntroductionColorectal cancer (CRC) represents the third most prevalent form of cancer worldwide, with liver metastasis representing a significant contributor to mortality. The interaction between tumor-associated macrophages (TAMs) and tumor cells plays a pivotal role in the development of colorectal cancer liver metastases (CRLM) and represents a promising avenue for therapeutic intervention. Stachydrine (STA), a compound derived from the Leonurus heterophyllus plant, has been shown to effectively inhibit tumor growth through a range of mechanisms.MethodsThe study employed imaging and histopathology to evaluate the efficacy of STA monotherapy in preventing CRLM. The inhibition of M2 macrophage polarization by STA was confirmed through the use of flow cytometry and immunofluorescence. Subsequently, a series of assays, including quantitative reverse transcription polymerase chain reaction (qRT-PCR), flow cytometry, scratch, invasion, and tube formation assays, were conducted to confirm STA’s capacity to impede tumor cell migration, invasion, and angiogenesis in vitro. Western blotting and flow cytometry were employed to elucidate the mechanisms through which STA exerts its effects on tumor metastasis.ResultsIn our research, STA has been shown to attenuate liver metastasis in CRC mouse models by inhibiting the polarization of macrophages to the M2 phenotype. This anti-metastatic effect is dependent on the presence of macrophages. In vitro, STA has been found to impede tumor cell migration, invasion, and angiogenesis by preventing TAMs from polarizing to the M2 phenotype via the JAK2/STAT3 signaling pathway. Moreover, the combination of STA with anti-PD-1 therapy has been observed to restore immune infiltration within the tumor microenvironment and inhibit tumor progression.ConclusionThe findings of this study demonstrate that STA exerts an inhibitory effect on colorectal cancer liver metastasis by targeting macrophages and impeding their M2 polarization via the JAK2/STAT3 pathway. Furthermore, the combination of STA with anti-PD-1 therapy has been observed to enhance the effectiveness of immune checkpoint blockade and reduce tumor spread, indicating the potential of STA to improve the efficacy of immunotherapy for liver metastases.