Quantitative Reverse Transcription-polymerase Chain Reaction Research Articles

Snake Venom-Inspired Novel Peptides Protect Caenorhabditis elegans against Paraquat-Induced Parkinson's Pathology.

The in vivo protective mechanisms of two low-molecular-mass (∼1.4 kDa) novel custom peptides (CPs) against paraquat-induced neurodegenerative dysfunction in the Caenorhabditis elegans model were deciphered. CPs prevented the paraquat from binding to the nerve ring adjacent to the pharynx in C. elegans (wild-type) by stable and high-affinity binding to the tyrosine-protein kinase receptor CAM-1, resulting in significant inhibition of paraquat-induced toxicity by reducing the production of reactive oxygen species, mitochondrial membrane depolarization, and chemosensory dysfunction. The CPs inhibited paraquat-induced dopaminergic neuron degeneration and alpha-synuclein protein expression, the hallmarks of Parkinson's disease, in transgenic BZ555 and NL5901 strains of C. elegans. Transcriptomic, functional proteomics, and quantitative reverse transcription-polymerase chain reaction analyses show that CPs prevented the increased expression of the genes involved in the skn-1 downstream pathway, thereby restoring paraquat-mediated oxidative stress, apoptosis, and neuronal damage in C. elegans. The ability of CPs to repair paraquat-induced damage was demonstrated by a network of gene expression profiles, illustrating the molecular relationships between the regulatory proteins.

IL-22/IL-22R1 pathway enhances cholangiocarcinoma progression via ERK1/2 activation.

IL-22 plays a pivotal role in the processes of inflammation and tissue healing., but its role in cholangiocarcinoma (CCA) remains unclear. our study explored the IL-22/IL-22R1 pathway and its impact on CCA progression through the ERK1/2 signaling cascade. To determine the mechanism of the IL-22/IL-22R1 pathway in CCA and provide new directions for its clinical treatment. IL-22R1 expression was assessed in human and rat CCA tissues utilizing immunohistochemical techniques, Western blot analysis, and quantitative reverse transcription PCR. The impact of IL-22 on CCA cells was assessed in vitro via tests for proliferation, migration, invasion, and apoptosis assays. The rat models of thioacetamide-induced CCA and subcutaneous xenografts in nude mice were used to assess the in vivo effects. ERK1/2 inhibitors were applied to elucidate the mechanistic role of the pathway. IL-22R1 was overexpressed in CCA cell lines and tissues. IL-22 treatment increased the phosphorylation of ERK1/2, promoting tumor cell proliferation, migration, invasion, and resistance to apoptosis. ERK1/2 inhibition considerably reversed these effects both in vitro and in vivo. The IL-22/IL-22R1 axis promotes CCA progression by activating ERK1/2 signaling. Targeting this pathway with ERK1/2 inhibitors offers potential therapeutic strategies for CCA.

Integrative bioinformatics analysis of high-throughput sequencing and in vitro functional analysis leads to uncovering key hub genes in esophageal squamous cell carcinoma

BackgroundEsophageal squamous cell carcinoma (ESCA) is a type of cancer that starts in the cells lining the esophagus, the tube connecting the throat to the stomach. It is known for its aggressive nature and poor prognosis. Understanding the key factors that drive this cancer is crucial for developing better diagnostic tools and treatments.MethodsGene expression profiles of ESCA were analyzed using Gene Expression Omnibus (GEO) datasets (GSE23400, GSE29001, GSE92396, and GSE1420) from the GEO database. Differentially expressed genes (DEGs) were identified using the limma package, and a protein-protein interaction (PPI) network was constructed using the STRING database. Hub genes were identified based on the degree method. Further validation was performed through reverse transcription quantitative PCR (RT-qPCR), mutational and copy number variation (CNV) analysis via the cBioPortal database, promoter methylation analysis using the OncoDB and GSCA databases, survival analysis, immune infiltration analysis through the GSCA database, and functional assays, including knockdown of key genes.ResultsWe identified four key hub genes, COL3A1, COL4A1, COL5A2, and CXCL8 that play significant roles in ESCA. These genes were highly expressed in ESCA tissues and cell lines, with expression levels significantly (p-value < 0.001) elevated compared to normal controls. Receiver operating characteristic (ROC) curve analysis revealed exceptional diagnostic performance for all four genes, with area under the curve (AUC) values of 1.0, indicating perfect sensitivity and specificity in distinguishing ESCA from normal controls. Mutational analysis revealed that COL3A1 was altered in 67% of ESCA samples, primarily through missense mutations, while COL5A2 exhibited alterations in 50% of the samples, including splice site and missense mutations. Additionally, gene amplification patterns were observed in all four hub genes, further validating their oncogenic potential in ESCA progression. A significant (p-value < 0.05) promoter hypomethylation was detected in these genes, suggesting a potential regulatory role in their expression. Functional assays demonstrated that knocking down COL3A1 and COL4A1 led to decreased cell proliferation, colony formation, and migration, indicating their critical roles in tumor progression. Additionally, these genes were involved in pathways related to the extracellular matrix and immune system modulation.ConclusionCOL3A1, COL4A1, COL5A2, and CXCL8 are crucial in ESCA development and progression, particularly in remodeling the extracellular matrix, modulating the immune system, and promoting metastasis. These findings suggest that these genes could serve as potential biomarkers for diagnosing ESCA and targets for future therapies. Future research should focus on in vivo validation of these findings and clinical testing to assess the therapeutic potential of targeting these genes in ESCA treatment.

Loss of miR-424 and miR-503 promotes decidualization of human endometrial stromal cells by increasing SCARA5 expression.

This study aims to investigate the function of miR-424 and miR-503, identified as putative regulatory miRNAs of FOXO1, a key factor for decidualization. The expression of both miR-424 and miR-503 in human endometrial stromal cells (HESCs) were measured before and after decidualization. Then, HESCs were transfected with both miR-424 and miR-503 before decidualization. Quantitative reverse transcription PCR, actin staining analysis, migration assay, fluorescence immunostaining, and luciferase assay were performed. MiR-424 and miR-503 expression was decreased after decidualization. Overexpression of both miR-424 and miR-503 inhibited major decidual maker genes, including FOXO1, PRL, IGFBP1, WNT4, and SCARA5, and altered F-actin's subcellular distribution from the periphery to all over the cytoplasm, concomitantly increasing cell mobility. Moreover, immunohistochemical analysis revealed overexpression of both miRNAs resulted in FOXO1 protein accumulation in the cytoplasm. Knocking down FOXO1 decreased SCARA5 expression, revealing SCARA5 is a downstream target of FOXO1. In addition, a luciferase reporter assay confirmed that the 3'-untranslated region of FOXO1 mRNA is targeted by miR-424. These results suggest that both miRNAs may play an important role in endometrial decidualization by regulating transcriptional activity of FOXO1, which alters decidualization-related gene expression such as SCARA5.Abstract: Journal standard instruction requires an unstructured abstract; hence structured abstract changed to unstructured.Thank you for the correction. I approve this change.

Role of metallothionein gene in Cd and Pb detoxification in Chironomus kiiensis.

Heavy metal pollution in the aquatic environment is a growing concern. Consequently, it is crucial to promptly and accurately assess the level of contamination. Metallothionein (MT), an omnipresent protein, plays a pivotal role in regulating metal balance and detoxifying harmful substances. This study focused on CkMT, the metallothionein gene from the aquatic insect Chironomus kiiensis. The full-length cDNA of the CkMT gene was identified and cloned from C. kiiensis, revealing a 138-base-pair sequence that encodes 45 amino acids, with the CkMT protein being rich in cysteine. The CkMT was expressed throughout all developmental stages and in all tissues, as determined by quantitative reverse transcription PCR (qRT-PCR), with the highest expression observed in the larval stage and the midgut. Exposure to different concentrations of heavy metals (Cd, Pb, or a mixture of both) significantly induced the expression of CkMT. The functions of CkMT in C. kiiensis were investigated using RNA interference (RNAi). This knockdown resulted in increased larval mortality and decreased pupation and eclosion rates. Additionally, the overexpression of recombinant CkMT in Escherichia coli enhanced tolerance to Cd and Pb. Together, this study reveals that CkMT plays an important physiological role in the growth and development of C. kiiensis and suggests that CkMT is involved in the detoxification and tolerance of Cd and Pb in C. kiiensis. Furthermore, these findings highlight CkMT's potential as a valuable biomarker for monitoring Cd and Pb contamination in aquatic ecosystems.

Usefulness of gibberellin-regulated protein specific IgE measurement in patients with systemic symptoms of apple allergy with exercise.

Patients with peach allergy who experience severe symptoms, including anaphylaxis, reportedly have a higher positivity for peach gibberellin-regulated protein (GRP)-specific immunoglobulin (Ig) E than those with only oral symptoms. However, a study in Italy investigating apple allergy (another Rosaceae fruit) found no clear association between apple GRP-specific IgE levels and clinical disease types. This study aimed to evaluate the clinical utility of GRP-specific IgE measurement in Japanese patients with apple allergy. We collected sera from apple-allergic patients in Japan and measured their IgE levels specific to apple GRP. Apple-allergic patients (14 with oral reactions and 14 with systemic reactions) and seven non-allergic controls were examined. The specific IgE levels against apple, Mal d 1, Mal d 4, Japanese cedar, Japanese alder, Japanese white birch, Bet v 1, and Bet v 2 were also determined using 3gAllergy™. Positive results for apple-GRP-specific IgE by enzyme-linked immunosorbent assay were obtained in one patient with oral reactions and in seven cases of systemic reactions. Exercise as a cofactor was involved in cases with high apple GRP-specific IgE. GRP expression was considerably lower in apples than in peaches, as detected by reverse transcription-quantitative polymerase chain reaction testing. Thus, GRP-specific IgE may be an important marker for diagnosing systemic reactions triggered by exercise in fruits with low GRP expression, such as apples.

Two-photon polymerization of miniaturized 3D scaffolds optimized for studies on glioblastoma multiforme in spaceflight-like microgravity conditions

The obtainment of innovative models recalling complex tumour architectures and activitiesin vitrois a challenging drive in the understanding of pathology molecular bases, yet it is a crucial path to the identification of targets for advanced oncotherapy. Cell environment recapitulation by 3D scaffolding and gravitational unloading of cell cultures represent powerful means in tumour biomimicry processes, but their simultaneous adoption has consistently been explored only in the latest decade. Here, an unprecedented bioengineering approach capitalizing on spaceflight biology practice is proposed for modelling of glioblastoma multiforme, a highly aggressive neoplasm that affects the central nervous system and has poorly effective pharmacological and radiological countermeasures. Tumour modelling was pursued by the original implementation of two-photon polymerization in fast prototyping of 3D scaffolds on flexible substrates for U87-MG glioma cell culture, and by the exposure of cell-laden scaffolds to simulated microgravity (s-μg). Realistic spaceflight conditions were applied to collect preliminary information suitable for testing of U87-MG cell-laden scaffold in low Earth orbit. Responses of glioma cells anchored to 3D scaffolds were investigated by microscopy, quantitative reverse transcription-polymerase chain reaction and proteomic analyses, revealing synergic regulatory effects of cell scaffolding and s-μg on markers of tumour cell growth, metabolism and invasiveness.

Echinocystic acid ameliorates ischemic acute kidney injury in neonatal rats by attenuating ferroptosis via the Nrf2/GPX4 pathway.

Acute kidney injury (AKI) is the most common complication in neonates with hypoxic-ischemic encephalopathy (HIE), significantly contributing to both morbidity and mortality, and targeting key pathological processes, such as inflammation, ferroptosis and apoptosis, could be an effective approach to improving survival outcomes in these patients. In this context, echinocystic acid (EA), a pentacyclic triterpene, has shown promising anti-inflammatory, antioxidant, and anti-apoptotic effects in various disease models, suggesting its potential as a therapeutic agent for AKI in HIE. To evaluate the therapeutic potential and underlying mechanisms of EA in ameliorating ischemia/reperfusion (IR)-induced AKI in neonatal rats. Seven-day-old neonatal rat pups were subjected to an IR injury model to induce AKI and treated with EA via intraperitoneal injection. The effects of EA on renal injury were assessed using western blotting, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), immunofluorescence, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), and hematoxylin and eosin (H&E) staining. Treatment with EA significantly reduced IR-induced renal pathology and injury scores, as well as serum levels of blood urea nitrogen (BUN) and creatinine (Cr). In addition, EA diminished the release of pro-inflammatory cytokines and reduced the levels of F4/80, a macrophage marker, in the IR-treated pups. EA also attenuated ferroptosis, as evidenced by decreased levels of iron (Fe2⁺), reactive oxygen species (ROS), myeloperoxidase (MPO), and malondialdehyde (MDA), while simultaneously increasing the activity of antioxidant enzymes such as catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), and glutathione (GSH). Furthermore, EA reduced apoptosis, as demonstrated by lower levels of cleaved caspase 3 and cleaved poly(ADP-ribose) polymerase (PARP). Mechanistically, EA activated the Nrf2/GPX4 pathway, and inhibition of Nrf2 with ML385 reversed EA's beneficial effects on ferroptosis, inflammation, and renal injury. EA may relieve ischemic AKI in neonatal rats by modulating inflammation, ferroptosis and apoptosis, through the activation of the Nrf2/GPX4 pathway, indicating that it could be a promising therapeutic agent for AKI in neonates.

Long Non-Coding RNA Osr2 Promotes Fusarium solani Keratitis Inflammation via the miR-30a-3p/ Xcr1 Axis.

Fungal keratitis (FK) is a challenging and sight-threatening corneal disease caused by fungal infections. Although long noncoding RNAs (lncRNAs) have been explored in various infectious diseases, their specific roles in FK remain largely unexplored. A mouse model of FK was created by infecting corneal stromal cells with Fusarium solani. High-throughput lncRNA expression profiling was conducted on FK-affected corneal tissues to identify differentially expressed lncRNAs. Reverse transcription quantitative PCR (RT-qPCR) was used to validate the results. A competing endogenous RNA (ceRNA) network was constructed. Additionally, a specific antisense oligonucleotide (ASO) targeting lncRNA ENSMUST00000226838/Osr2 (Osr2) was developed for therapeutic evaluation. Inflammatory markers IL-1β, IL-6, and TNF-α were measured, and corneal inflammation was assessed through histological analysis and slit-lamp examination. Fluorescent in situ hybridization (FISH) was used to confirm Osr2 localization, whereas a dual-luciferase reporter assay verified interactions between Osr2 and miR-30a-3p. We identified 1143 differentially expressed lncRNAs in FK, with 701 upregulated and 442 downregulated. The ceRNA network analysis indicated that lncRNA Osr2 regulates Xcr1 expression through miR-30a-3p. Treatment with ASO-Osr2 significantly reduced corneal inflammation, and FISH confirmed lncRNA Osr2 distribution in both the nucleus and cytoplasm. Dual-luciferase assays demonstrated the interaction between Osr2 and miR-30a-3p, highlighting their potential roles in the progression of FK. This study outlined the lncRNA expression profile in FK and established a ceRNA regulatory network, identifying lncRNA Osr2 as a crucial modulator of FK pathogenesis through its interaction with miR-30a-3p. These findings highlighted lncRNA Osr2 as a promising therapeutic target for the treatment of FK.

The Anti-PEDV Effects and Mechanisms of Forsythia Essential Oil Based on Network Pharmacology and Experimental Validation.

Porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae, is responsible for acute diarrhea, vomiting, and dehydration, which can lead to high mortality in neonatal piglets. Previous research has indicated the antiviral potential of forsythia essential oil (FEO); however, its active components and mechanisms of action remain inadequately defined. This study aims to investigate the antiviral effects of FEO and elucidate its potential mechanisms for treating PEDV. The primary components of FEO were identified using gas chromatography-mass spectrometry (GC/MS) in conjunction with the National Institute of Standards and Technology Standard Spectrum (NIST) Database. Network pharmacology and weighting coefficients were employed to determine the key signaling pathways associated with PEDV-related diseases. Molecular docking simulations were conducted to explore the interactions between the active ingredients and their corresponding targets. The safety profile of FEO was assessed through cell viability assays utilizing the CCK8 method. Subsequently, immunofluorescence assays (IFA) and reverse transcription-quantitative polymerase chain reaction (RT-Q-PCR) were performed to provide evidence of the anti-PEDV effects. Additionally, the viral replication cycle was analyzed to identify the stages at which FEO exerts its antiviral effects. Finally, key targets were validated through RT-Q-PCR to further investigate the anti-PEDV mechanisms of FEO. The IL-17 signaling pathway was identified as a critical pathway for the treatment of PEDV with FEO based on network pharmacology and weighting coefficient analyses. Furthermore, results from RT-Q-PCR and IFA demonstrated that FEO influenced the replication of PEDV during the attachment and internalization phases. Specifically, during the viral attachment phase, FEO significantly upregulated the expression of HSP90AA1 while downregulating MAPK14 expression, leading to a reduction in associated inflammatory factors. At the high dose of FEO, the expression of HSP90AA1 was higher than that of the model group by about 5-fold, and the expression of MAPK14 was lower than that of the model group by about 2-fold. Cell viability assay showed no significant cytotoxicity of FEO at 0.63 μL/mL, thus confirming its safety. The findings of this study suggest that FEO possesses potential antiviral properties against PEDV. Its novel mechanisms of action warrant further investigation, which may contribute to the development of effective therapeutic strategies for managing PEDV infections.

Introduction of AGPAT3 gene as a regulator of cisplatin resistance in A2780 ovarian endometrioid carcinoma cell line.

Ovarian cancer therapy remains a challenge for human health, partly due to chemotherapy resistance. Understanding the molecular mechanisms underlying this resistance is crucial. Therefore, to identify genes involved in cisplatin resistance in ovarian cancer, RNA-seq analysis of A2780cp (cisplatin-resistant) and A2780 (cisplatin-sensitive) cell lines was performed, revealing 1-acylglycerol-3-phosphate O-acyltransferase 3 (AGPAT3) as a differentially expressed candidate gene. First, MTT analysis confirmed the drug resistance of A2780cp and the sensitivity of A2780 cell lines. Subsequent reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting analyses revealed elevated AGPAT3 and mTOR expression in A2780cp cells compared with A2780 cells. Additionally, western blotting showed increased p-mTOR (phospho-mTOR)/mTOR and p-S6K (phospho-S6K)/S6K ratios in A2780cp cells. The overexpression of AGPAT3 in A2780 cells led to increased p-mTOR/mTOR and p-S6K/S6K ratios and increased IC50 values, as shown by RT-qPCR, western blotting, and MTT analysis. Conversely, shRNA-mediated downregulation of AGPAT3 resulted in reduced p-mTOR/mTOR and p-S6K/S6K ratios. At the cellular level, AGPAT3 overexpression in A2780 cells increased survival rates, decreased apoptosis, and caused G2/M cell cycle arrest under cisplatin treatment, as detected by apoptosis assay, and cell cycle flow cytometry analysis. Overall, we conclude that AGPAT3 is involved in cisplatin resistance in A2780cp cells and propose that targeting this gene or its enzymatic product could help overcome drug resistance.

Identification and role of CmLhcb2.1 in regulating low-light stress resistance in Chinese chestnut (Castanea mollissima)

Chinese chestnut (Castanea mollissima) is a significant woody food plant that has garnered increasing attention due to its potential role in addressing food security challenges. However, low yield remains a critical issue facing the Chinese chestnut industry. One contributing factor to this low yield is insufficient light, particularly since Chinese chestnuts predominantly grow in mountainous regions. Therefore, the present study aims to investigate the intrinsic mechanisms underlying chestnut resistance to light stress, identify and validate genes associated with low light stress tolerance, and provide a foundation for targeted breeding of chestnut varieties that can withstand light stress. Studies have demonstrated that the light-harvesting chlorophyll a/b (Lhca/b) proteins play key roles in regulating the adaptation of plants to low-light stress. However, there have been no reports on the role of the Lhca/b gene family in the chestnut under light stress. We initially identified 17 CmLhca/b gene members across the chestnut genome and constructed a phylogenetic tree that divided them into five subgroups: the Lhca, the Lhcb, the CP24, the CP26, and the CP29 groups. CmLhcb2.1 and CmLhcb2.2 were grouped on the same branch with GhLhcb2.3 of upland cotton that involved in chlorophyll synthesis.The chestnut leaves exhibited phenotypic and transcriptomic differences under low and normal light conditions. By the 10th day of shading treatment, the leaves showed signs of damage, with the extent of damage intensifying as shading intensity increased. Additionally, the leaf color darkened due to the gradual increase in chlorophyll b content, which was correlated with increased shading intensity. The gene CmLhcb2.1 was upregulated across all shading intensities. Specifically, quantitative reverse transcription PCR (qRT-PCR) confirmed the upregulation of CmLhcb2.1 in chestnut under low-light stress. Overexpression studies in tobacco indicated that CmLhcb2.1 enhances chestnut resistance to low-light stress by promoting chlorophyll b synthesis. Finally, yeast one-hybrid and dual-luciferase reporter assays confirmed that the transcription factor CmGLK positively regulated CmLhcb2.1. These findings lay a theoretical foundation for exploring how CmLhcb2.1 regulates chestnut resistance to low-light stress.

Plasma Circulating lncRNAs: MALAT1 and NEAT1 as Biomarkers of Radiation-Induced Adverse Effects in Laryngeal Cancer Patients

Background: The majority of head and neck cancers (HNCs) occur in the larynx. In clinical practice, adverse effects are frequently observed in laryngeal cancer (LC) patients undergoing radiotherapy (RT). Therefore, investigating markers that can predict these unfavorable events is of interest. Long non-coding RNAs (lncRNAs) have emerged as potential biomarkers for the early identification of patients susceptible to post-RT toxicity. MALAT1 and NEAT1 regulate various cellular processes, the inflammatory response, and resistance to anti-cancer treatments; however, their impact on the portability of post-RT adverse effects remains unknown. The aim of this study was to evaluate the clinical value of two plasma-circulating lncRNAs, MALAT1 and NEAT1, as predictive biomarkers for post-RT adverse effects in LC patients. Methods: The expression levels of the studied lncRNAs were determined using real-time quantitative reverse transcription PCR (qRT-PCR) in plasma samples obtained from 70 LC patients before the initiation of RT. These levels were then correlated with patient outcomes. Results: A low expression of MALAT1 was associated with a significantly higher probability of anemia, liver failure, and severe malnutrition (OR = 5.36; p = 0.040, OR = 6.07; p = 0.037, OR = 9.75; p &lt; 0.001, respectively) after the completion of RT. Similarly, patients with low NEAT1 expression had a significantly higher risk of anemia, liver failure, and mild or severe malnutrition (OR = 5.26; p = 0.020, OR = 5.70; p = 0.016, OR = 13.09; p = 0.002, respectively). Simultaneous lower expression levels of both lncRNAs were significantly associated with shorter median overall survival (OS) in RT-treated LC patients (HR = 5.44; p = 0.001). Conclusions: The analysis of MALAT1 and NEAT1 expression indicates clinical utility in predicting toxic events induced by RT-based therapy.

Identification of the SARS-CoV-2 Genome in Various Systemic Samples from the Laboratory of Confirmed COVID-19 Patients in Iran

Background: COVID-19 is a highly contagious respiratory illness caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While nasopharyngeal swab testing remains the primary method for identifying SARS-CoV-2 carriers, the virus’s genetic material can be detected in various bodily samples, suggesting potential transmission through non-respiratory routes. Objectives: This study aimed to investigate the presence and quantity of SARS-CoV-2 RNA in stool, endotracheal tube (ETT) samples, cerebrospinal fluid (CSF), bronchoalveolar lavage (BAL), and sputum. The research sought to correlate these findings with clinical characteristics and the severity of illness. Methods: We retrospectively collected 1,567 samples, including 550 BAL samples, 464 ETT samples, 45 fecal samples, 21 CSF samples, and 487 sputum samples from patients admitted to Namazi Hospital between April 1, 2020, and the end of September 2022. Clinical characteristics, patient demographics, radiographic, and laboratory results were among the medical data gathered and assessed. We used reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect and measure SARS-CoV-2 RNA shedding in different samples of individuals infected with COVID-19. Results: Among the 550 BAL samples collected from patients, 20.7% tested positive for SARS-CoV-2. Additionally, the virus’s genome was detected in 17.8% of 45 CSF samples, 11% of 464 ETT samples, 4.76% of 21 fecal samples, and 2.9% of 487 sputum samples. Notably, patients with SARS-CoV-2-positive BAL samples exhibited a significantly higher frequency of cough and dyspnea than those who tested negative. Conclusions: These findings revealed that SARS-CoV-2 has been detected in diverse specimens collected from the upper and lower respiratory systems (sputum, ETT, and BAL samples), the central nervous system (CSF), and the digestive tract (fecal samples) during the infection’s progression. This widespread presence significantly impacts our understanding of the disease’s pathogenesis and enhances diagnostic capabilities, proving a valuable asset in managing the infection.

KMT2D Induces M1 Macrophage Polarization to Repress Non-small Cell Lung Cancer Progression via Transcription Activation of ITGAL

Background: Recent evidence has demonstrated the crucial role of macrophage polarization in promoting non-small cell lung cancer (NSCLC) progression within the tumor microenvironment. Objectives: This study investigated the possible regulatory mechanism of macrophage polarization during NSCLC development. Methods: The proportion of M1/M2 macrophages was examined by flow cytometry. The expression of macrophage markers and target molecules was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR), western blotting, and immunohistochemical staining. Non-small cell lung cancer cells were treated with conditioned medium (CM) from THP-1 macrophages. Cell counting kit-8 (CCK-8), scratch, and transwell assays were used to assess NSCLC cell growth and metastasis. Gene promoter activity was evaluated by dual-luciferase reporter assay. A xenograft model was adopted to determine NSCLC growth in vivo. Results: Histone-lysine N-methyltransferase 2D (KMT2D) and integrin subunit alpha L (ITGAL) were lowly expressed in NSCLC tissues and cells. The KMT2D overexpression facilitated the polarization of macrophages from M2 to M1 type, which repressed the growth, migration, and invasion of NSCLC cells. Mechanistically, KMT2D promoted the transcription and expression of ITGAL. Inhibition of ITGAL abrogated KMT2D overexpression-mediated M1 macrophage polarization and its anti-cancer effects on NSCLC. Conclusions: The KMT2D transcriptionally activated ITGAL to trigger M1 macrophage polarization, thereby delaying NSCLC progression. Our findings suggest KMT2D as a potential therapeutic target for NSCLC.

Prevention of ribozyme catalysis through cDNA synthesis enables accurate RT-qPCR measurements of context-dependent ribozyme activity.

Self-cleaving ribozymes are important tools in synthetic biology, biomanufacturing, and nucleic acid therapeutics. These broad applications deploy ribozymes in many genetic and environmental contexts, which can influence activity. Thus, accurate measurements of ribozyme activity across diverse contexts are crucial for validating new ribozyme sequences and ribozyme-based biotechnologies. Ribozyme activity measurements that rely on RNA extraction, such as RNA sequencing or reverse transcription-quantitative polymerase chain reaction (RT-qPCR), are generalizable to most applications and have high sensitivity. However, the activity measurement is indirect, taking place after RNA is isolated from the environment of interest and copied to DNA. So these measurements may not accurately reflect the activity in the original context. Here we develop and validate an RT-qPCR method for measuring context-dependent ribozyme activity using a set of self-cleaving RNAs for which context-dependent ribozyme cleavage is known in vitro. We find that RNA extraction and reverse transcription conditions can induce substantial ribozyme cleavage resulting in incorrect activity measurements with RT-qPCR. To restore the accuracy of the RT-qPCR measurements, we introduce an oligonucleotide into the sample preparation workflow that inhibits ribozyme activity. We then apply our method to measure ribozyme cleavage of RNAs produced in Escherichia coli (E. coli). These results have broad implications for many ribozyme measurements and technologies.

Clinical characteristics, molecular epidemiology and mechanisms of colistin heteroresistance in Enterobacter cloacae complex

IntroductionColistin has emerged as the last resort for treating multidrug-resistant Enterobacter cloacae complex (ECC) infections. The primary purposes of this study were to demonstrate the presence of colistin heteroresistance in ECC and to further investigate their clinical characteristics, molecular epidemiology and mechanisms.MethodsPopulation analysis profiles (PAP) were performed to confirm the heteroresistance phenotype. Average nucleotide identity (ANI) was determined to classify ECC species. Phylogenetic analysis based on core genome single nucleotide polymorphisms (cg-SNPs), multilocus sequence typing (MLST) and core genome MLST (cg-MLST). Risk factors and clinical outcomes of infections were analyzed through a retrospective case-control study. Potential mechanisms of colistin heteroresistance were evaluated using polymerase chain reaction (PCR), efflux pump inhibition assays and reverse transcription quantitative PCR (RT-qPCR).ResultsA high proportion (24.4%) of the non-resistant strains were colistin-heteroresistant isolates. Among the several ECC species, Enterobacter kobei had the largest percentage (29.4%) of colistin-heteroresistant isolates, followed by Enterobacter hormaechei (20.5%) and Enterobacter bugandensis (20.0%). Notably, only one strain (0.8%; 1/132) of Enterobacter hormaechei was fully resistant to colistin. Different ECC species showed varying heteroresistance levels: Enterobacter roggenkampii, Enterobacter kobei, Enterobacter asburiae and Enterobacter bugandensis displayed high heteroresistance levels (MIC ≥ 128 mg/L). 75% of all ST116 and ST56 strains were heteroresistant to colistin. The infection of ST116 and ST56 strains as well as exposure to cephalosporin antibiotics were independent risk factors for colistin-heteroresistant ECC infections. Mechanistic analysis revealed that heteroresistance strongly correlated with the overexpression of arnA, regulated by the PhoPQ two-component system (TCS). Notably, mgrB had minimal impact. AcrAB-TolC efflux pump genes showed unsynchronized expression; High acrB expression was strongly associated with colistin heteroresistance, while acrA and tolC were not.DiscussionColistin heteroresistance showed species-dependent variations in levels and prevalence rates. The colistin-heteroresistant mechanisms were complex, involving coordinated regulation of multiple genes. These results highlighted the need for tailored antimicrobial stewardship. In addition, the development of direct, reliable and rapid clinical methods for detecting heteroresistance is essential for improving infection management and prevention.

HOXC-AS1: A Key Biomarker for Prognosis and Immunotherapy in Lung Adenocarcinoma.

The function of HOXC antisense RNA 1 (HOXC-AS1) in lung adenocarcinoma (LUAD) remains largely unexplored. The objective of this research was to examine the relationship between HOXC-AS1 levels and LUAD through both bioinformatics analysis and experimental validation. We employed statistical methods and bioinformatics to evaluate the correlation between HOXC-AS1 expression and various clinical features, survival predictors, regulatory mechanisms, and immune cell infiltration in LUAD. The levels of HOXC-AS1 in LUAD cell lines were ascertained through quantitative reverse transcription PCR. HOXC-AS1 displayed significantly increased expression in individuals with LUAD. There was a significant correlation between high HOXC-AS1 levels and diminished overall survival in LUAD patients, characterized by a hazard ratio of 0.66, a 95% confidence interval of 0.49 to 0.88, and a statistically significant P-value (0.005). An elevated expression of HOXCAS1 was found to be a standalone predictor of poor overall survival in LUAD patients, with a Pvalue of 0.002. HOXC-AS1 was found to be implicated in various pathways, such as neuroactive ligand-receptor interaction and asthma, among others. The study revealed a substantial link between high HOXC-AS1 expression and unfavorable outcomes in LUAD, including poor survival and altered immune cell infiltration. LUAD cell lines exhibited a marked increase in HOXC-AS1 expression compared to the Beas-2B normal lung cell line. The research indicated a strong association between higher levels of HOXC-AS1 and negative outcomes in LUAD, such as reduced survival rates and the presence of immune cell infiltration. HOXC-AS1 could potentially be utilized as a biomarker to anticipate patient prognosis and their likelihood of responding to immunotherapies in LUAD.

PD-1IR2 promotes tumor evasion via deregulating CD8+ T cell function

BackgroundThe programmed cell death 1 (PD-1) is an immune checkpoint that mediates immune evasion of tumors. Alternative splicing (AS) such as intron retention (IR) plays a crucial role in the...

The Histone Demethylase Inhibitor GSK-J4 Attenuates Periodontal Bone Loss and Inflammation in a Rat Model of Periodontitis.

To investigate the treatment effect of the histone demethylase inhibitor GSK-J4, a small molecule that inhibits the demethylase activity of Jumonji domain-containing protein 3 (JMJD3), in the treatment of periodontitis. Gingival tissues from patients with moderate to severe chronic periodontitis and healthy controls were collected to evaluate JMJD3 expression via real-time quantitative reverse transcription PCR (RT-qPCR) and immunohistochemistry (IHC). Next, Sprague-Dawley (SD) rats were used to investigate the effect of GSK-J4 in vivo. The experimental periodontitis model was induced by upper first molar ligation and gingival sulcus injection of Porphyromonas gingivalis. The rats were divided into a healthy group, a periodontitis group, periodontitis plus GSK-J4 treatment groups (P + GSK-J415 mg/kg or 25mg/kg), and a periodontitis plus dimethyl sulfoxide (DMSO) group (P + DMSO). After 4weeks, maxillary molar segments were assessed via micro-computed tomography (CT) and hematoxylin and eosin (HE) staining. Serum tumor necrosis factor-α (TNF-α) levels were measured by enzyme-linked immunosorbent assay (ELISA). Higher expression of the Jmjd3 gene and JMJD3 protein was detected in human inflamed gingiva than in healthy gingiva (P < 0.05). GSK-J4 administration reversed alveolar bone absorption [i.e., reduced alveolar bone crest (ABC)-cementoenamel junction (CEJ) distance], reduced inflammatory cell accumulation at the crest of the alveolar bone, and alleviated serum TNF-α levels in rats with periodontitis. Moreover, the number of H3K27me3-positive nuclei was greater in model rats treated with GSK J4 than in model rats. The histone demethylase inhibitor GSK-J4 attenuated periodontal bone loss and inflammation in a rat periodontitis model by targeting JMJD3.