Real-time Quantitative Reverse Transcription-polymerase Research Articles

Rotavirus vaccine effectiveness and coverage among children younger than 5years old in Shanghai, China: A test-negative case control study.

The number of post-marketing studies assessing the vaccine effectiveness (VE) of the Lanzhou lamb rotavirus vaccine (LLR, licensed in 2000 exclusively in China) and the oral human attenuated pentavalent rotavirus vaccine (RotaTeq, licensed in China in 2018) in China is limited. A test-negative case-control study based on prospective surveillance was conducted among diarrhea patients aged 5years and younger at five hospitals in Shanghai, China. Cases and controls were defined based on the results of real-time fluorescent quantitative reverse transcription polymerase chain reaction (rRT-PCR) of fecal samples for rotavirus. Both matched and unmatched case-control study designs were employed using logistic regression models, with adjustments for age at onset age and the rotavirus epidemic season. In the LLR-specific analysis (247 cases, 2191 controls), the VE of partial LLR vaccination (2 doses) was 49.09% (95% CI: 1.69%∼73.64%) in multivariate analyses. In the RotaTeq-specific analysis (42 cases and 523 controls), the VE of complete RotaTeq vaccination was 87.13% (95% CI: 45.87%∼96.94%), 89.46% (95% CI: 55.03%∼97.53%), and 85.69% (95% CI: 33.43%∼96.93%) respectively in univariate, multivariate, and matched analyses, respectively. The vaccination coverage for any dose among 2893 patients with rotavirus-negative diarrhea born between 2011 and 2022 was 49.78%. Following the licensure of RotaTeq in 2018, this coverage increased from 45.02% to 61.77%. RotaTeq demonstrates a robust protective effectiveness, while LLR provides a certain level of protection against mild to moderate rotavirus diarrhea in children in Shanghai. For privately purchased (non-NIP) vaccines, we estimate that the coverage for rotavirus vaccines among children in Shanghai is high. Complete rotavirus vaccination is recommended for age-eligible children. Further post-marketing research on rotavirus vaccines is necessary to inform decision-making regarding the introduction of rotavirus vaccination in China.

Screening semen samples for Zika virus infection: role for serologic and RT-PCR testing

IntroductionZika virus (ZIKV) is primarily transmitted through mosquito bites though may act as a sexually transmitted infection. Men can shed the virus in their semen for extended periods, with reports indicating shedding for up to six months after the initial infection. This poses a concern for couples planning pregnancy, whether through natural or assisted methods due to the risk of congenital Zika disease. Human reproductive clinics typically perform serologic tests to screen for Zika infection.MethodsIn this study, we evaluated semen samples stored in a human reproduction clinic in Salvador, Brazil, during the Zika virus outbreak, using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) technique.Results36 sperm samples from 23 donors were analyzed, including 5 with positive IgG serology for ZIKV and all negative for IgM. Despite these serologic findings, all analyzed samples were negative by RT-qPCR ZIKV.DiscussionDespite the limited number of samples, this study raises further concern that the use of serology may be an unreliable surrogate method to predict the presence of ZIKV in semen.

LncRNAs in serum-derived extracellular vesicles are potential biomarker and correlated with immune infiltration in gastric cancer

BackgroundLong non-coding RNAs (lncRNAs) in extracellular vesicles (EVs) have been confirmed as effective non-invasive biomarkers for multiple diseases. However, their expression and clinical value in gastric cancer (GC) remain poorly understood.Materials and methodsSerum EV RNA was extracted from four patients with GC and four healthy controls, followed by high-throughput RNA sequencing. LncRNAs were further validated in training and validation sets using quantitative real-time reverse transcription polymerase chain reaction.ResultsA total of 37,684 lncRNAs were obtained, and 10 lncRNAs were selected based on the criteria (P < 0.05 and |log2FoldChange| ≥1). Serum EV lncRNA RMRP, RPPH1, and linc-ROR were significantly higher in patients with GC than in those with chronic gastritis, atypical hyperplasia, or healthy control (all P < 0.05). Three lncRNAs were also significantly correlated with tumor diameter, lymphatic metastasis, distal metastasis, and TNM stage (all P < 0.05). The area under the curve (AUC) values for lncRNA RMRP, RPPH1, and linc-ROR were 0.727, 0.774, and 0.811, respectively. Corresponding sensitivity and specificity were 63.4% and 85.4%, 50.7% and 89.6%, and 78.5% and 66.7%. The combination of these three lncRNAs with carcinoembryonic antigen (CEA) yielded an AUC of 0.909, with a sensitivity and specificity of 83.3% each. Furthermore, high EV linc-ROR and RMRP expression levels were associated with worse disease-free survival and overall survival (OS). Univariate and multivariate Cox regression analyses confirmed that linc-ROR was the only independent prognostic factor for GC. Finally, the lncRNA-miRNA-mRNA network showed that three lncRNAs were predicted to interact with 15 miRNAs and 69 mRNAs. In addition, lncRNA RMRP and linc-ROR were correlated with immune cell infiltration, including neutrophils, central memory CD4 T cells, macrophage, and natural kill T cells.ConclusionEV lncRNAs are prospective biomarker and correlated with immune cell infiltration in GC. It provides a foundation for the development of serum EV-targeted novel biomarkers and immunotherapy targets of GC.

Dynamic changes in RNA integrity, gene expression, and tissue pathomorphology of experimental mice in the postmortem period

Aim. To examine the pattern of morphological changes, RNA quality number, and gene expression in mouse tissues sampled at autopsy under controlled experimental conditions.Materials and methods. Balb/c mice were euthanized and subsequently subjected to necropsy at 0, 3, 12, 24, 48, and 72 hours of the postmortem period. During the first three hours following euthanasia, the mice were maintained at room temperature, after which they were transferred to a refrigerator (4 º С). Total RNA was extracted from tissue samples taken from the kidney, liver, and brain; the integrity of the RNA samples was assessed by capillary electrophoresis, and the RNA quality number (RQN) was calculated. The expression levels of Actb, Epas1, and Rps18 housekeeping genes were evaluated by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) with original primers and probes using the TaqMan assay. The histologic examination was performed according to standard techniques.Results. Degradation of RNA extracted from mouse kidney tissues appeared to be greater than that of RNA taken from the liver. In the meantime, a negative linear correlation was observed between RQN and the duration of the postmortem interval for liver and kidney samples. In contrast, no significant changes in the RQN score were observed for brain RNA samples at any of the time points. The expression of the Epas1 and Rps18 genes was significantly decreased in mouse kidney and liver tissues. However, the level of Epas1 and Rps18 gene expression in the brain remained stable at all time points and did not exhibit a significant decrease at 72 hours after euthanasia. No obvious morphological changes were detected by the histologic examination, which does not exclude the presence of ultrastructural pathological changes.Conclusion. RQN in autopsy tissues serves as a crucial predictor of sample quality for molecular biology studies, including gene expression analysis.

Promotion of microfracture-mediated cartilage repair by the intra-articular injection of Mg2.

Magnesium ions (Mg2+) play an important role in promoting cartilage repair in cartilage lesions. However, no research has focused on the role of Mg2+ combined with microfracture (MFX) in hyaline-like cartilage repair mediated by cartilage injury. This study aimed to investigate the beneficial effects of the combination of MFX and Mg2+ in cartilage repair. A total of 60 rabbits were classified into five groups (n = 12 each): sham, MFX, and three different doses of Mg2+ treatment groups (0.05, 0.5, and 5 mol/L). Bone cartilage defects were created in the trochlear groove cartilage of rabbits. MFX surgery was performed after osteochondral defects. Mg2+ was injected into knee joints immediately and two and four weeks after surgery. At six and 12 weeks after surgery, the rabbits were killed. Cartilage damage was detected by gross observation, micro-CT, and histological analysis. The expression levels of related genes were detected by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). The histological results showed that the 0.5 mol/L Mg2+ group had deeper positive staining in haematoxylin-eosin (H&E), safranin O, Alcian blue, and type II collagen staining. The new cartilage coverage in the injury area was more complete, and the regeneration of hyaline cartilage was higher. The RT-qPCR results showed that sirtuin 1/bone morphogenetic protein-2/sex-determining region Y box 9 (SIRT1/BMP-2/SOX-9) and hypoxia-inducible factor 1-alpha (HIF-1α) messenger RNA levels were up-regulated after Mg2+ injection. MFX combined with Mg2+ treatment has a positive effect on cartilage repair. The Mg2+ injection dose of 0.5 mol/L is most effective in enhancing microfracture-mediated cartilage repair.

LncRNA MANCR is downregulated in non-small cell lung cancer and predicts poor survival

BackgroundIt is known that genomic instability contributes to cancer development. Mitotically associated long non-coding RNA (MANCR) has been reported to promote genomic stability, suggesting its involvement in cancers. Therefore, this study was conducted to investigate the role of MANCR in non-small cell lung cancer (NSCLC).MethodsAfter NSCLC (n = 60) and control (healthy subjects, n = 60) plasma samples, as well as NSCLC and paired non-tumor tissues from patients were collected, the levels of MANCR expression in plasma and tissues was detected using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Then the correlations of MANCR expression with clinical stages were confirmed. The diagnostic values of MANCR in both plasma and tissue samples for stage I/II NSCLC were analyzed using Receiver Operating Characteristic (ROC) curves. All NSCLC patients were monitored for 5 years to investigate the role of MANCR in the prediction of patients’ survival.ResultsMANCR expression was downregulated in both NSCLC plasma and tissue of NSCLC patients compared to controls (P < 0.05). Decreased MANCR expression levels from stage I to IV were observed. However, MANCR expression in non-tumor tissue was not significantly different between different stages (P > 0.05). Additionally, stage I/II NSCLC patients were separated from controls using MANCR in plasma and tumor tissues as biomarkers. Lower MANCR levels in plasma and tumor were closely correlated with patients’ higher mortality rate.ConclusionMANCR is down-expressed in NSCLC patients and may serve as a diagnostic and prognostic biomarker for NSCLC.

Identification and validation of reference genes for quantitative gene expression analysis under 409 and 415 nm antimicrobial blue light treatment.

Reverse transcription quantitative real-time polymerase chain reaction Q7 (RT‒qPCR) is a commonly used tool for gene expression quantification. Because the qPCR method depends on several variables that can influence the analysis process, stably expressed genes should be selected for relative gene expression studies. To date, there is insufficient information on the selection of appropriate reference genes for antimicrobial photodynamic inactivation (aPDI) and antimicrobial blue light (aBL) treatment. Therefore, the purpose of the present study was to determine the most stable reference gene under treatment with aBL under sublethal conditions and to evaluate differences in the expression of the selected gene after aBL treatment in comparison to the nontreated control. Selection of stable reference genes was performed using 4 programs: BestKeeper, geNorm, NormFinder and RefFinder under 409 and 415nm aBL treatment. The results revealed that the gene encoding the integration host factor β subunit (ihfB) in Escherichia coli was the most stably expressed gene after both 409 and 415nm aBL treatment. Three programs, RefFinder, geNorm, and NormFinder, indicated that this gene had the most stable expression in comparison to the other reference gene candidates. The next best candidates were cysG, uidA, and gyrA. NormFinder revealed ihfB as the single gene and cysG - gyrA as the combination of reference genes with the best stability. Universal reference genes are characterized by stable expression that remains consistent across various stress conditions. Consequently, it is essential to evaluate reference genes for each specific stress factor under investigation. In the case of aBL at different wavelengths, we identified genes that maintain stable expression following irradiation.

Gene Expression Signatures of Smoking and Acute Myocardial Infarction: A Blood Transcriptome Analysis.

Background: Tobacco smoke is known to contain numerous harmful chemicals, and epidemiological evidence has firmly established smoking as a potent risk factor for hypertension and myocardial infarction (MI). However, the precise mechanisms by which smoking contributes to cardiovascular disease are not fully understood. The aim of this study is to identify common molecular signatures in blood that link smoking to acute MI (AMI). Methods: We extracted transcriptome data from seven blood microarray datasets in the Gene Expression Omnibus (GEO) database, encompassing a total of 403 patients. Employing both individual dataset analysis and a combined meta-analysis approach, we conducted a thorough examination of blood transcriptome profiles associated with AMI and smoking, uncovering numerous differentially expressed genes (DEGs). Results: Functional enrichment analysis indicated that DEGs associated with AMI and smoking were significantly enriched in overlapping biological processes, such as immune response and inflammation. Moreover, three genes-PTGDR, PYHIN1, and PRSS23-were consistently altered in both conditions and were validated as dysregulated in AMI using an independent GEO dataset. Furthermore, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) validation further confirmed the differential expression of PYHIN1 and PRSS23 in AMI patients. Conclusions: Our findings suggest that gene expression changes induced by smoking in blood may contribute to the heightened risk of AMI. These identified genes are likely to play critical roles in the pathogenesis of AMI. Given the accessibility of peripheral blood samples, the expression levels of these genes could potentially serve as biomarkers for assessing cardiovascular health, particularly in individuals with a history of long-term exposure to cigarette smoke.

TFDP1 overexpression promotes apoptosis of nucleus pulposus cells in intervertebral disc degeneration through regulating ADAM15/MMP9 axis.

Intervertebral disc degeneration (IVDD) is a common contributor for low back pain, which is featured by loss of extracellular matrix and nucleus pulposus cells (NPCs). Hence, our current study is undertaken to explore the potential mechanism of NPC apoptosis during IVDD. Transcription factor Dp-1 (TFDP1) expression in degenerative and non-degenerative intervertebral disc tissues was analyzed by bioinformatics. After transfection as needed, viability and apoptosis of NPCs were evaluated by cell counting kit-8 assay and flow cytometry, respectively. Western blot or quantitative real-time reverse transcription polymerase chain reaction was applied to assess expressions of TFDP1, matrix metallopeptidase 9 (MMP9), a disintegrin and metalloproteinase 15 (ADAM15), and apoptosis-associated proteins. TFDP1 expression was upregulated in degenerative intervertebral disc tissues. TFDP1 overexpression repressed viability, promoted apoptosis, increased expressions of Bax, Cleaved caspase 3, MMP9 and ADAM15, and decreased Bcl-2 expression in NPCs, while TFDP1 silencing did conversely. ADAM15 silencing promoted viability, inhibited apoptosis, increased Bcl-2 expression, and decreased Bax, Cleaved caspase 3, and MMP9 expressions in NPCs, which were reversed by TFDP1 overexpression. TFDP1 overexpression promotes apoptosis of NPCs in IVDD through regulating ADAM15/MMP9 axis, highlighting its role as a molecular target for the treatment of low back pain.

Histone Deacetylase 2 Stabilizes SPARC-related Modular Calcium Binding 2 to Promote Metastasis and Stemness in Gallbladder Cancer.

We aimed to investigate the relationship between histone deacetylase 2 (HDAC2) and SPARC-related modular calcium binding 2 (SMOC2) and the role of SMOC2 in gallbladder cancer (GBC). The expression of HDAC2 and SMOC2 in GBC and normal cells was detected by quantitative real-time reverse transcription polymerase chain reaction (qRTPCR), which was also used to detect the mRNA stability of SMOC2. The combination between HDAC2 and SMOC2 was detected by Chromatin immunoprecipitation (ChIP) assay. After silencing and/or overexpressing HDAC2 and SMOC2, cell viability, migration, invasion, and stemness were respectively tested by the Cell Counting Kit-8 (CCK-8), cell scratch, transwell, and sphere-formation assay. In GBC cells, HDAC2 and SMOC2 were highly expressed. HDAC2 combined with SMOC2 promoted mRNA stability of SMOC2. HDAC2 or SMOC2 overexpression promoted GBC cell metastasis and stemness. SMOC2 overexpression rescued the negative effects of silencing HDAC2 in GBC. HDAC2 stabilizes SMOC2 to promote metastasis and stemness in gallbladder cancer.

Optimization of the in vitro culture system for chicken small intestinal organoids

In order to establish a stable in vitro culture platform for chicken small intestine three-dimensional (3D) organoids, in this study, crypt cells were collected from the small intestine of 18-day-old embryos of AA broilers. On the basis of the L-WRN conditioned medium, we optimized the culture conditions of chicken small intestinal organoids by adjusting the proportions of nicotinamide, N-acetylcysteine, LY2157299, CHIR99021, Jagged-1, FGF, and other cytokines to select the medium suitable for the long-term stable growth of the organoids. The optimization results showed that the addition of 1.5 µmol/L CHIR99021 significantly improved the organoid formation efficiency and organoid diameter. When 0.5 µmol/L Jagged-1 was added, a small amount of bud-like tissue appeared in organoids. After the addition of 50 ng/mL FGF-2, the rate of organoid germination was significantly increased. The 1.5 µmol/L CHIR99021, 0.5 µmol/L Jagged-1, and 50 ng/mL FGF-2 added in the medium can cooperate with each other to improve the formation and speed up the proliferation and differentiation of organoids, while improving the stemness maintenance of cells. The morphology, cell types, and culture characteristics of chicken small intestinal organoids were studied by HE staining, transmission electron microscopy, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), indirect immunofluorescence, and immunohistochemistry. The results showed that the 3D organoids of the chicken small intestine cultured in vitro were morphologically consistent with the chicken intestinal tissue and contained differentiated epithelial cells. In summary, we successfully established an in vitro culture system for chicken small intestinal organoids, providing a new method for the subsequent research on chicken intestinal physiology, pathology, and host-pathogen interaction mechanism and the development of relevant drugs.

RBIS regulates ribosome biogenesis to affect progression in lung adenocarcinoma.

Increased ribosome biogenesis is required for tumor growth. In this study, we investigated the function and underlying molecular mechanism of ribosome biogenesis factor (RBIS) in the progression of non-small cell lung cancer (NSCLC). In our study, we conducted a comprehensive analysis to identify key genes implicated in ribosome biogenesis by leveraging a Gene Set Enrichment Analysis (GSEA) dataset. Subsequently, we performed a comparative analysis of gene expression profiles by utilizing data from the Gene Expression Omnibus (GEO) datasets to ascertain differentially expressed genes (DEGs) between cancerous and adjacent non-cancerous tissues. Through the intersection of gene sets derived from GSEA and GEO, we identified a cohort of ribosome-associated genes that might exert a substantial influence on the progression of lung adenocarcinoma. Following an extensive literature review, we have identified the RBIS gene as an interesting candidate for further investigation. To elucidate the in vitro functional role of RBIS, several assays was employed, including the Transwell migration and invasion assay, wound healing assay, Cell Counting Kit-8 (CCK-8) proliferation assay, and colony formation assay. Subcutaneous and tail vein injection-based lung metastasis xenograft tumor models were used in evaluating the tumorigenic potential, growth, and metastatic spread of lung cancer cells. Flow cytometry analysis was employed to investigate cell cycle distribution and apoptotic rates. Additionally, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was utilized to quantify the mRNA expression levels ofgenes. To comprehensively assess the translational efficiency of nascent proteins, we employed polysome profiling analysis to provide insights into the cellular translational landscape. Furthermore, we quantified global protein synthesis using a fluorescence-based assay to measure protein synthesis rates. The immunofluorescence technology was utilized to study the subcellular reorganization of the nucleolus. We conducted co-immunoprecipitation (Co-IP) assays followed by Western blot analysis to identify potential proteins interacted with RBIS. The half maximal inhibitory concentration (IC50) was used for evaluating the chemosensitivity of lung cancer cells to gemcitabine. Additionally, the colony formation assay was employed to assess the survival and proliferative capacity post-treatment of gemcitabine. The database analysis showed that RBIS was upregulated in lung adenocarcinoma, and its high expression was associated with poor prognosis; Knockdown of RBIS significantly inhibited NSCLC cell migration, invasion and proliferation in vitro and xenograft tumor growth and metastasis in vivo. Additionally, knockdown of RBIS led to G0/G1 phase arrest and significantly increased apoptosis in lung adenocarcinoma cells. Mechanistically, downregulation of RBIS significantly decreased the expression of 47S ribosomal RNA (rRNA), a component associated with ribosome assembly. Polysome profiling analysis indicated that RBIS knockdown affected protein translation efficiency, and global protein synthesis assay further verified that RBIS knockdown inhibited synthesis of newborn proteins. Additionally, the ribosomal biogenesis-targeting drugs CX-5461 and the loss of RBIS exhibited synergistic effects in inhibiting cell cycle progression and inducing apoptosis. Furthermore, the ribosomal maturation factor GNL2 was identified as the key downstream regulator of RBIS in ribosome biogenesis. Notably, knockdown of RBIS substantially increased the sensitivity of lung adenocarcinoma cells to the chemotherapeutic drug gemcitabine, highlighting its l role in chemotherapy. Collectively, these studies suggested the close involvement of RBIS in the progression of lung adenocarcinoma, providing new insights for targeted therapeutic interventions involving ribosomes.

Evaluation of Reference Gene Stability for Investigations of Intracellular Signalling in Human Cancer and Non-Malignant Mesenchymal Stromal Cells.

Real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a powerful tool for analysing target gene expression in biological samples. To achieve reliable results by RT-qPCR, the most stable reference genes must be selected for proper data normalisation, particularly when comparing cells of different types. We aimed to choose the least variable candidate reference genes among eight housekeeping genes tested within a set of human cancer cell lines (HeLa, MCF-7, SK-UT-1B, A549, A431, SK-BR-3), as well as four lines of normal, non-malignant mesenchymal stromal cells (MSCs) of different origins. The reference gene stability was evaluated using four algorithms (BestKeeper, NormFinder, geNorm and the comparative ΔCt method) and ranked with the RefFinder web-based tool. We found increased variability in the housekeeping genes' expression in the cancer cell lines compared to that in normal MSCs. POP4 and GAPDH were identified as the most suitable reference genes in cancer cells, while 18S and B2M were the most suitable in MSCs. POP4 and EIF2B1 were shown to be the least variable genes when analysing normal and cancer cell lines together. Epidermal growth factor receptor (EGFR) mRNA relative expression was normalised by the three most stable or three least stable reference genes to demonstrate the reliability of reference genes validation. We analysed and selected stable reference genes for RT-qPCR analysis in the wide panel of cancer cell lines and MSCs. The study provides a reliable tool for future research concerning the expression of genes involved in various intracellular signalling pathways and emphasises the need for careful selection of suitable references before analysing target gene expression.

Clinical characteristics and genetic analysis of four pediatric patients with Kleefstra syndrome

BackgroundKleefstra syndrome spectrum (KLEFS) is an autosomal dominant disorder that can lead to intellectual disability and autism spectrum disorders. KLEFS encompasses Kleefstra syndrome-1 (KLEFS1) and Kleefstra syndrome-2 (KLEFS2), with KLEFS1 accounting for more than 75%. However, limited information is available regarding KLEFS2. KLEFS1 is caused by a subtelomeric chromosomal abnormality resulting in either deletion at the end of the long arm of chromosome 9, which contains the EHMT1 gene, or by variants in the EHMT1 gene and the KMT2C gene that cause KLEFS2.MethodsThis study was a retrospective analysis of clinical data from four patients with KLEFS. Exome sequencing (ES) and Sanger sequencing techniques were used to identify and validate the candidate variants, facilitating the analysis of genotype‒phenotype correlations of the EHMT1 and KMT2C genes. Protein structure modeling was performed to evaluate the effects of the variants on the protein’s three-dimensional structure. In addition, real-time quantitative reverse transcription‒polymerase chain reaction (RT‒qPCR) and western blotting were used to examine the protein and mRNA levels of the KMT2C gene.ResultsTwo patients with KLEFS1 were identified: one with a novel variant (c.2382 + 1G > T) and the other with a previously reported variant (c.2426 C > T, p.Pro809Leu) in the EHMT1 gene. A De novo deletion at the end of the long arm of chromosome 9 was also reported. Furthermore, a patient with KLEFS2 was identified with a novel variant in the KMT2C gene (c.568 C > T, p.Arg190Ter). The RT‒qPCR and western blot results revealed that the expression of the KMT2C gene was downregulated in the KLEFS2 sample.ConclusionThis study contributes to the understanding of both KLEFS1 and KLEFS2 by identifying novel variants in EHMT1 and KMT2C genes, thereby expanding the variant spectrum. Additionally, we provide the first evidence of how a KMT2C variant leads to decreased gene and protein expression, enhancing our understanding of the molecular mechanisms underlying KLEFS2. Based on these findings, children exhibiting developmental delay, hypotonia, distinctive facial features, and other neurodevelopmental abnormalities should be considered for ES to ensure early intervention and treatment.

Cannabichromene as a Novel Inhibitor of Th2 Cytokine and JAK/STAT Pathway Activation in Atopic Dermatitis Models.

Cannabichromene (CBC) is one of the main cannabinoids found in the cannabis plant, and although less well known than tetrahydrocannabinol (THC) and cannabidiol (CBD), it is gaining attention for its potential therapeutic benefits. To date, CBC's known mechanisms of action include anti-inflammatory, analgesic, antidepressant, antimicrobial, neuroprotective, and anti-acne effects through TRP channel activation and the inhibition of inflammatory pathways, suggesting that it may have therapeutic potential in the treatment of inflammatory skin diseases, such as atopic dermatitis (AD), but its exact mechanism of action remains unclear. Therefore, in this study, we investigated the effects of CBC on Th2 cytokines along with the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways involved in AD pathogenesis. We used a 2,4-Dinitrochlorobenzene (DNCB)-induced BALB/c mouse model to topically administer CBC (0.1 mg/kg or 1 mg/kg). The results showed that skin lesion severity, ear thickness, epithelial thickness of dorsal and ear skin, and mast cell infiltration were significantly reduced in the 0.1 mg/kg CBC-treated group compared with the DNCB-treated group (p < 0.001). In addition, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed a significant decrease in the mRNA expression of Th2 cytokines (TSLP, IL-4, IL-13) and inflammatory mediators (IFN-γ, IL-1β, IL-6, IL-17, IL-18, and IL-33) (p < 0.05). Western blot analysis also revealed a significant decrease in JAK1, JAK2, STAT1, STAT2, STAT3, and STAT6 protein expression (p < 0.05). These results suggest that CBC is a promising candidate for the treatment of AD and demonstrates the potential to alleviate AD symptoms by suppressing the Th2 immune response.

CircFNDC3B inhibits vascular smooth muscle cells proliferation in abdominal aortic aneurysms by targeting the miR-1270/PDCD10 axis

Objectives. This study investigated the role and underlying regulatory mechanisms of circular RNA fibronectin type III domain containing 3B (circFNDC3B) in abdominal aortic aneurysm (AAA). Methods. The expression of circFNDC3B in AAA and normal tissues was assessed by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). To evaluate the biological functions of circFNDC3B, assays were employed including 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), flow cytometry, and Caspase-3 activity assays. Additionally, RNA immunoprecipitation (RIP), dual-luciferase reporter assay, Western blotting, and rescue experiments were utilized to elucidate the molecular mechanism of circFNDC3B. Results. Our findings revealed a significant upregulation of circFNDC3B expression in AAA clinical specimens compared to normal tissues. Functionally, overexpression of circFNDC3B inhibited vascular smooth muscle cells (VSMCs) proliferation and induced apoptosis, contributing to AAA formation in the Ang II-induced AAA model. Mechanistically, circFNDC3B acted as a molecular sponge for miR-1270, leading to the upregulation of programmed cell death 10 (PDCD10). Decreased expression of PDCD10 abrogated the -promoting effects of circFNDC3B overexpression on AAA development. Conclusions. This study demonstrates that circFNDC3B promotes the progression of AAA by targeting the miR-1270/PDCD10 pathway. Our findings suggest that circFNDC3B as well as miR-1270/PDCD10 pathway may serve as a potential therapeutic target for AAA treatment.

SUPPRESSOR OF FRIGIDA 4 cooperates with the histone methylation reader EBS to positively regulate root development.

Maintenance and homeostasis of the quiescent center (QC) in Arabidopsis (Arabidopsis thaliana) root apical meristems are critical for stem cell organization and root development. Despite great progress in relevant research, the molecular mechanisms that determine the root stem cell fate and QC still need further exploration. In Arabidopsis, SUPPRESSOR OF FRIGIDA 4 (SUF4) encodes a C2H2-type zinc finger protein that represses flowering by transcriptional activation of FLOWERING LOCUS C (FLC) through the FRIGIDA (FRI) pathway, and EARLY BOLTING IN SHORT DAYS (EBS) is a bivalent histone reader that prevents premature flowering. Here, we found that SUF4 directly interacts with EBS in vivo and in vitro. Loss of function of SUF4 and/or EBS resulted in disorganization of the QC, aberrant cell division, and stunted root growth. RNA-seq and reverse transcription quantitative real-time polymerase chain reaction analysis revealed that SUF4 and EBS coregulate many root development-related genes. A series of biochemical analyses demonstrated that SUF4 directly binds to the promoter of SCARECROW (SCR), which encodes a key regulator of root development. Chromatin immunoprecipitation assay indicated that both SUF4 and EBS are recruited to the SCR locus in an interdependent manner to promote H3K4me3 levels and suppress H3K27me3 levels, thereby activating the expression of SCR. These findings improve our understanding of the function of SUF4 and EBS and provide insights into the molecular mechanism that couples a transcription factor and a histone methylation reader to modulate QC specification and root development in Arabidopsis.

Circulating miRNAs and Machine Learning for Lateralizing Primary Aldosteronism.

Distinguishing between unilateral and bilateral primary aldosteronism, a major cause of secondary hypertension, is crucial due to different treatment approaches. While adrenal venous sampling is the gold standard, its invasiveness, limited availability, and often difficult interpretation pose challenges. This study explores the utility of circulating microRNAs (miRNAs) and machine learning in distinguishing between unilateral and bilateral forms of primary aldosteronism. MiRNA profiling was conducted on plasma samples from 18 patients with primary aldosteronism taken during adrenal venous sampling on an Illumina MiSeq platform. Bioinformatics and machine learning identified 9 miRNAs for validation by reverse transcription real-time quantitative polymerase chain reaction. Validation was performed on a cohort consisting of 108 patients with known subdifferentiation. A 30-patient subset of the validation cohort involved both adrenal venous sampling and peripheral, the rest only peripheral samples. A neural network model was used for feature selection and comparison between adrenal venous sampling and peripheral samples, while a deep-learning model was used for classification. Our model identified 10 miRNA combinations achieving >85% accuracy in distinguishing unilateral primary aldosteronism and bilateral adrenal hyperplasia on a 30-sample subset, while also confirming the suitability of peripheral samples for analysis. The best model, involving 6 miRNAs, achieved an area under curve of 87.1%. Deep learning resulted in 100% accuracy on the subset and 90.9% sensitivity and 81.8% specificity on all 108 samples, with an area under curve of 86.7%. Machine learning analysis of circulating miRNAs offers a minimally invasive alternative for primary aldosteronism lateralization. Early identification of bilateral adrenal hyperplasia could expedite treatment initiation without the need for further localization, benefiting both patients and health care providers.

FOXF2 may inhibit esophageal squamous cell carcinoma growth and metastasis by regulating the EZR-ERBB2 axis.

FOXF2, a member of the transcription factor FOX family proteins, plays a key role in tumorigenesis and tumor aggressiveness. However, the potential molecular mechanism of FOXF2 in esophageal squamous cell carcinoma (ESCC) remains largely unknown. Exploring its role and mechanism in ESCC progression may help identify new diagnostic markers and therapeutic targets. The aim of this study is to investigate the potential functions of the FOXF2 gene within the context of ESCC and to elucidate the underlying molecular pathways involved. Using the GoMiner database, GeneCard database, Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database, and the COMPARTMENTS subcellular localization database, we identified the most likely downstream molecule of the FOXF2 gene, EZR; the subcellular locations of FOXF2 and EZR; the possible biological pathways [Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)]; and the protein interactions networks of the EZR gene enriched from the OMICS datasets via Metascape. We also used The Cancer Genome Atlas database to analyze the correlation between EZR and ERBB signaling pathway. In addition, we verified the RNA and protein expression of the target genes using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, we employed Western blot analysis and plasmid transfection and lentiviral infection techniques to gene edit FOXF2 and EZR in different EC cells to obtain stable overexpression or knockdown of the cell lines. This was followed by ex vivo and in vivo experiments including migration assay, cell scratch assay, clone formation assay, and a xenotransplantation mouse model to validate the functional phenotype of the gene-edited cells. We found that knockdown of FOXF2 expression significantly enhanced the growth, invasion, and metastasis of ESCC cells both in vitro and in vivo. Moreover, we demonstrated that FOXF2 was predominantly expressed in the nucleus and directly interacted with EZR, thereby inhibiting EZR transcriptional expression, resulting in suppressed ERBB2 signal function, ultimately halting ESCC growth and metastasis. Taken together, these results reveal the tumor-suppressive functions of FOXF2 in inhibiting EZR-mediated ERBB2 activation, suggesting that FOXF2 could serve as a potential novel predicting prognostic biomarker for ESCC.

A New Perspective for Improving COPD: Ginsenoside Rg3 Links SIRT1 to Inhibit Mitochondrial Autophagy.

Chronic obstructive pulmonary disease (COPD) is a prevalent yet manageable respiratory condition. However, treatments presently used normally have side effects and cannot cure COPD, making it urgent to explore effective medications. The ginsenoside Rg3 (Rg3) has been shown to have anti-inflammatory and anti-tumor properties and can improve COPD. The primary objectives of this investigation were to explore the impact of Rg3 on COPD and delve into the associated mechanisms. In vitro models exposed human bronchial epithelial cells (BEAS-2B) to cigarette smoke extract (CSE), and in vivo models induced COPD in mice through chronic inhalation of cigarette smoke (CS). Sirtuin 1 (SIRT1) expression was regulated via cell transfection or mice infection with recombinant lentiviruses. SIRT1 mRNA levels were quantified using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), and SIRT protein levels were assessed by western blot or enzyme-linked immunosorbent assays (ELISA). Mitophagy was evaluated by light chain 3 (LC3) II/I and phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) levels, and apoptosis was determined using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Lung function was measured with the Buxco system, and inflammation was assessed via interleukin 6 (IL-6) and keratinocyte-derived cytokine (KC) levels in bronchial alveolar lavage fluid. Lung morphological impairments were determined through Hematoxylin and Eosin (H&E) staining and mean linear intercept (MLI) measurement. In BEAS-2B cells, CSE treatment caused a decrease in SIRT1 expression (p < 0.01) and an increase in LC3 II/I (p < 0.01) and PINK1 (p < 0.01), which were all reversed by Rg3 (p < 0.01), with 20 μM Rg3 performing the best and being used subsequently. CSE increased apoptosis of BEAS-2B cells (p < 0.01), which was reversed by Rg3 (p < 0.01). Upregulated SIRT1 further decreased levels of LC3 II/I (p < 0.001), PINK1 (p < 0.001), and cell apoptosis (p < 0.001) for CSE- and Rg3-treated cells, whereas downregulated SIRT1 reversely increased levels of LC3 II/I (p < 0.001), PINK1 (p < 0.001), and cell apoptosis (p < 0.001). The establishment of COPD caused a decrease in SIRT1 mRNA (p < 0.001), SIRT1 protein (p < 0.001), and lung functions (p < 0.001) whereas IL-6 (p < 0.001), KC (p < 0.001), lung impairment, and MLI (p < 0.001) were increased; all of these effects were reversed by Rg3 (p < 0.001). Moreover, the Rg3-induced reversion was furthered by SIRT1 upregulation (p < 0.001) and was disrupted by SIRT1 downregulation (p < 0.001). Rg3, through activation of SIRT1, suppresses mitophagy and apoptosis, ameliorates COPD, and improves lung functions.