RT-PCR Research Articles

Influenza Vaccine Effectiveness Against Illness and Asymptomatic Infection in 2022-2023: A Prospective Cohort Study.

Previous estimates of vaccine effectiveness (VE) against asymptomatic influenza virus infection based on seroconversion have varied widely and may be biased. We estimated 2022-2023 influenza VE against illness and asymptomatic infection in a prospective cohort. In the HEROES-RECOVER cohort, adults at increased occupational risk of influenza exposure across 7 US sites provided weekly symptom reports and nasal swabs for reverse transcription-polymerase chain reaction (RT-PCR) influenza testing. Laboratory-confirmed influenza virus infections were classified as symptomatic (≥1 symptom) or asymptomatic during the week of testing. Participants reported demographic information and vaccination through surveys; most sites verified vaccination through medical record and immunization registry review. Person-time was calculated as days from the site-specific influenza season start (September-October 2022) through date of infection, study withdrawal, or season end (May 2023). We compared influenza incidence among vaccinated versus unvaccinated participants overall, by symptom status, and by influenza A subtype, using Cox proportional hazards regression adjusted for site and occupation. We estimated VE as (1 - adjusted hazard ratio) × 100%. In total, 269 of 3785 (7.1%) participants had laboratory-confirmed influenza, including 263 (98%) influenza A virus infections and 201 (75%) symptomatic illnesses. Incidence of laboratory-confirmed influenza illness among vaccinated versus unvaccinated participants was 23.7 and 33.2 episodes per 100 000 person-days, respectively (VE: 38%; 95% CI: 15%-55%). Incidence of asymptomatic influenza virus infection was 8.0 versus 11.6 per 100 000 (VE: 13%; 95% CI: -47%, 49%). Vaccination reduced incidence of symptomatic but not asymptomatic influenza virus infection, suggesting that influenza vaccination attenuates progression from infection to illness.

Loss of miR-634 contributes to the formation FOXA1-positive triple negative breast cancer subtype

BackgroundTriple-negative breast cancer (TNBC), the most aggressive subtype of breast cancer, lacks targeted therapies, posing a substantial challenge for treatment. Therefore, investigating its pathogenesis is a crucial research focus. FOXA1 and miR-634 are involved in tumorigenesis. However, the molecular mechanisms underlying the aberrant upregulation of FOXA1 expression in TNBC remain unclear. Therefore, we explore the role of miR-634 in the FOXA1-positive TNBC subtype.MethodsQuantitative reverse transcription polymerase chain reaction was used to detect miR-634 expression in breast cancer tissues and cell lines. Aberrantly activated signaling pathways and related genes in TNBC were analyzed using The Cancer Genome Atlas. The potential target of miR-634 was predicted by TargetScan, the TNBC cell proliferation rate was detected using an MTT assay, the in vitro metastatic capacity was determined by transwell assay and the cell cycle distribution was tested using flow cytometry. Western blotting was performed to measure the expression of proteins involved in the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway.ResultsThe expression of miR-634 was significantly down-regulated in both TNBC tissues and cells, compared with adjacent non-cancerous tissues and MCF10A, respectively. Ectopic expression of miR-634 inhibits breast cancer cell proliferation and in vitro metastasis. The TCGA-based expression profile analysis of TNBC revealed that aberrantly activated PI3K/AKT signaling may contribute to its malignant phenotype. FOXA1, the top hit of aberrantly upregulated genes in TNBC, was a direct target of miR-643. Moreover, forced expression of miR-643 drastically suppressed FOXA1 expression by the inactivation PI3K/AKT signaling.ConclusionMiR-634 suppresses FOXA1 to inhibit the proliferation and metastasis of TNBC cells by inactivating the PI3K/AKT signaling pathway.

Single-Cell Analysis Reveals the Cellular and Molecular Changes of Liver Injury and Fibrosis in Mice During the Progression of Schistosoma japonicum Infection

Schistosomiasis is a parasitic disease that poses a serious threat to human health. However, the pathogenic mechanism during the progression of Schistosoma japonicum infection remains unclear. In order to elucidate this mechanism, we used single-cell RNA sequencing (scRNA-seq) to investigate the transcriptome characteristics of the cellular (single-cell) landscape in the livers of mice infected with Schistosoma japonicum, which were divided into three groups: uninfected mice (0 week (w)), infected mice at 6 w post-infection (the acute phase), and infected mice at 10 w post-infection (the chronic phase). A total of 31,847 liver cells were included and clustered into 21 groups. The cells and T-cells had high heterogeneity in the liver during the progression of schistosome infection. The number and intensity of the intercellular interactions significantly increased at 6 w after infection but decreased at 10 w. The inflammatory signaling pathways chemoattractant cytokine ligand (CCL)5-chemokine C-C-motif receptor (CCR)5 between macrophages and T-cells were predominant at 6 w post-infection; the CCL6-CCR2 signaling pathway between macrophages was predominant at 10 w. The CD80 signaling pathway related to T-cell activation was increased at 6 w after infection, and increased expression of its receptor CD28 on the surfaces of CD4+ and CD8+ T-cells was confirmed by flow cytometry, suggesting an increase in their activation. In addition, scRNA-seq and quantitative reverse transcription polymerase chain reaction (qRT-PCR) confirmed that the intercellular communication between secretory phosphoprotein 1 (SPP1)-cluster of differentiation (CD44), insulin-like growth factor (IGF)-1-IGF1r and visfatin-insulin receptor (Insr) associated with bone metabolism and insulin metabolism was increased and enhanced in the liver at 6 w post-infection. Overall, we provide the comprehensive single-cell transcriptome landscape of the liver in mice during the progression of schistosome infection and delineate the key cellular and molecular events involved in schistosome infection-induced liver injury and fibrosis. The elevated CCL5-CCR5 and CCL6-CCR2 signaling pathways in the liver may be a drug target for liver injury and fibrosis caused by schistosome infection, respectively.

Apolipoprotein-B mRNA-editing complex 3B could be a new potential therapeutic target in endometriosis

This study investigated the correlation of Apolipoprotein-B mRNA-editing complex 3B (APOBEC3B) expression with hypoxia inducible factor 1α (HIF-1α), Kirsten rat sarcoma virus (KRAS) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) in endometriosis patients, and the inhibitory effects of APOBEC3B knockdown in a human endometriotic cell line. Here, APOBEC3B, HIF-1α, KRAS, and PIK3CA were examined in patients with and without endometriosis using reverse transcription polymerase chain reaction (RT-PCR). The apoptosis, cell proliferation, invasion, migration, and biological function of APOBEC3B knockdown were explored in 12Z immortalized human endometriotic cell line. We observed APOBEC3B, HIF-1α, KRAS and PIK3CA expressions were significantly higher in endometriosis patients (p < 0.001, p < 0.001, p = 0.029, p = 0.001). Knockdown of APOBEC3B increased apoptosis, which was 28.03% and 22.27% higher than in mock and control siRNA samples, respectively. APOBEC3B knockdown also decreased PIK3CA expression and increased Caspase 8 expression, suggesting a potential role in the regulation of apoptosis. Furthermore, knockdown of APOBEC3B significantly inhibited cell proliferation, invasion, and migration compared to mock and control siRNA. (Cell proliferation: mock: p < 0.001 and control siRNA: p = 0.049. Cell invasion: mock: p < 0.001 and control siRNA: p = 0.029. Cell migration: mock: p = 0.004, and control siRNA: p = 0.014). In conclusion, this study suggests that APOBEC3B may be a new potential therapeutic target for endometriosis.

Evaluation of Fecal Sample Pooling for Real-Time RT-PCR Testing SARS-CoV-2 in Animals

During the COVID-19 pandemic, veterinary diagnostic laboratories tested both human and animal samples and needed to ensure that they could accurately perform large numbers of diagnostic tests in a timely manner. Sample pooling, a methodology used effectively for over 80 years as a surveillance tool for screening large numbers of potentially infected individuals, was employed. Given its sensitivity, real-time polymerase chain reaction (PCR) is more suitable for employing this strategy, as compared to other less sensitive testing methods. In this study, we evaluated the capability of detecting SARS-CoV-2 in both 5-sample and 10-sample pools of feces using real-time reverse transcriptase polymerase chain reaction (rRT-PCR) as well as determined the level of sensitivity. A blinded method test (BMT) by an independent laboratory was conducted to assess the five-sample fecal pool. To complement detection capability, the stability of the genome within a PBS fecal suspension was measured under various time and temperature conditions across a 28-day period. Our results showed that the limit of detection for 5-sample and 10-sample fecal pools is 12.8 and 6.4 genome copies in a 25 µL PCR, respectively. The 5-sample and 10-sample pooling resulted in a cycle threshold (Ct) value loss of 2.35 and 3.45, as compared to Ct values of known positive individual samples, but consistent detection was still achieved in pools containing positive samples with an original Ct below 36 and 34, respectively. The simulation of clinical five-sample pooling showed that all positive samples could be detected regardless of the number (1–3) of positive samples in each pool. The BMT results demonstrated excellent sensitivity (100 copies/reaction) in five-sample pools for the detection of SARS-CoV-2 RNA even though a fecal matrix effect was observed. Finally, our results show that the SARS-CoV-2 genome remains stable over a wide range of time and temperature variations. Overall, our findings provide solid data to scale up SARS-CoV-2 testing capacity in veterinary diagnostic laboratories.

First report of tomato ringspot virus infection of Krymsk 86 (Prunus cerasifera × P. persica), a hybrid rootstock, in California.

California produces 99% of prunes (Prunus domestica) in the U.S.A., valued at $148 million and accounting for 40% of world prune production. In the last decade, the rootstock Krymsk 86 (K86; P. cerasifera x P. persica) is increasingly used in prune production because of its high vigor, excellent anchorage and graft compatibility with a wide variety of Prunus crops. An estimated >50% of new prune plantings in the Sacramento Valley are on K86. In late spring of 2023, 'Improved French' prune trees in two Northern California counties, grafted on K86 and Lovell (P. persica) rootstocks, were declining with a pale colored canopy. One orchard was ~ 9-years-old and in the other, trees varied from ~ 20 years to newly planted. The percentage of affected trees in one orchard was 3.6% (n=1,824) and 4.6% (n=1,295) in trees on K86 and Lovell, respectively. Symptomatic trees were scattered throughout this orchard, with a higher density of affected trees in the northeast quadrant. Examination of the trunk revealed a necrotic brown line at the graft union, typical of prune brown line (PBL) disease (Mircetich and Hoy, 1981), caused by infection of rootstock by tomato ringspot virus (ToRSV), a member species of Nepovirus lycopersici (family Secoviridae. The virus is vectored by dagger nematodes (Teliz et al. 1967) and its presence was confirmed in one of the orchards. To confirm infection by ToRSV in declining trees, total RNA from leaf samples, feeder roots, and cambial tissue from bark samples from scion and rootstock of one tree each on K86 and Lovell, was obtained using RNeasy Plant Mini kit (www.qiagen.com) and tested by one step reverse transcription-polymerase chain reaction (RT-PCR) assay using primers previously described (Tang et al., 2014). The reaction conditions included RT at 54oC using random hexamers and Superscript III (Thermo Fisher Scientific, USA), followed by a denaturation step at 95°C for 5 min, and 35 amplification cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 45 sec; and a final extension of 72°C for 5 min. The expected size amplicons (176 bp) were visualized by agarose gel electrophoresis only from RNA obtained from the cambial tissue below the graft union confirming the ToRSV infection in K86 rootstock. Extracts from bark scrapings of the rootstocks of three symptomatic trees also tested positive for ToRSV with immunostrips (www.agdia.com). In subsequent RT-PCR tests, RNA extracts from cambial tissue from 9 of 10 symptomatic trees on K86 tested positive for ToRSV. The amplicons were purified using a gel extraction kit (Qiagen Inc., Valencia, CA), and subjected to Sanger sequencing (Azenta Life Sciences (South Plainfield, NJ, USA). The sequence of the ToRSV isolates 316 (GenBank accession PQ282959) and 318 (GenBank accession PQ2829560 exhibited 99.4% (175/176 base pairs) and 98.9 % (175/177) sequence identity, respectively, with ToRSV isolate Rasp1-2014 segment RNA2 (GenBank accession KM083895). Our results indicate that ToRSV can infect K86 and cause PBL. ToRSV and its nematode vectors have a wide host range that includes several crop plants and weeds, but this virus is not currently part of registration and phytosanitory certification of Prunus species in the state of California. This is the first report of ToRSV infection of the K86 rootstock. This information is important for the selection of a rootstock in prune orchards where the virus is endemic.

LncRNA MSTRG.13,871/miR155-5p/Grip1 network involved in the post-cardiac arrest brain injury

Post-cardiac arrest brain (PCABI) is a severe medical condition characterized by a significant risk of neurological impairment and death. Nevertheless, the specific process and essential molecules responsible for its development are not fully understood. Profiling based on competing endogenous RNAs (ceRNA) has been implicated in the onset and progression of neurological disorders, yet its role in PCABI remains unclear. In this study, we performed RNA transcriptome sequencing analysis to identify differentially expressed genes in the rat model for cardiac arrest and cardiopulmonary resuscitation (CA/CPR). A hub ceRNA regulatory network was constructed using miRWalk 2.0 and Cytohubba plug-in in Cytoscape. Subsequently, real-time quantitative reverse transcription-polymerase chain reaction and dual-luciferase activity assays validated MSTRG.13,871, miR-155-5p, and Grip1 as differentially expressed in CA/CPR group, with MSTRG.13,871 capable of targeting both miR-155-5p and Grip1. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed the ceRNA network enrichment in immunoregulation mechanisms such as mitochondrion, apoptotic process, and negative regulation cell death. Our research highlights the mechanism of PCABI by revealing a critical regulatory axis involving MSTRG.13,871-miR-155-5p-Grip1 in the hippocampus CA1 region after CA/CPR in rats, proposing a feasible controlled mechanism, which may serve as a theoretical basis for designing innovative therapies.

Poly (ADP-Ribose) Polymerase 1 Induces Cyclic GMP-AMP Synthase-stimulator of Interferon Genes Pathway Dysregulation to Promote Immune Escape of Colorectal Cancer Cells

Colorectal cancer (CRC) ranks third globally in cancer incidence and mortality, posing a significant human concern. Recent advancements in immunotherapy are noteworthy. This study explores immune modulation for CRC treatment. Initially targeting poly (ADP-ribose) polymerase 1 (PARP-1), a gene overexpressed in CRC tissues per The Cancer Genome Atlas, we examined its correlation with immune cell infiltration using the Tumor Immune Estimation Resource tool. Quantitative reverse transcription polymerase chain reaction assessed PARP-1 mRNA and inflammation-related gene expression in tumor tissues and cells. Assessing CD8+ T-cell proliferation and cytotoxicity towards HCT116 cells involved carboxyfluorescein diacetate succinimidyl ester and lactate dehydrogenase kits. Chemotaxis was gauged using a Transwell system in a CD8+ T-cell coculture setup, with immunofluorescence revealing cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) levels in HCT116 cells. Enzyme-linked immunosorbent assay kits measured CD8+ T-cell cytokine secretion. The findings suggested that PARP-1 was overexpressed in CRC tissues and cells and this overexpression was positively correlated with Treg cell infiltration. Overexpression of PARP-1 could significantly reduce the proportion of cGAS and STING-positive cells in HCT116 cells, dampen the proliferation, tumor-killing capacity, and chemotaxis of CD8+ T cells, and inhibit the secretion of related cytokines. The introduction of STING agonists could reverse the effects caused by overexpressed PARP-1. In vivo experiments affirmed the independent anti-tumor effects of PARP-1 inhibitors and STING agonists, synergistically inhibiting tumor growth. Silencing PARP-1 in HCT116 cells potentially boosts CD8+ T-cell activity against these cells through the cGAS-STING pathway.

Epidemiology and population-based incidence of influenza in two communities, Bandung district, West Java, Indonesia, 2008–2011

Influenza surveillance is important for monitoring influenza virus circulation and disease burden to inform influenza prevention and control measures. The aim of this study was to describe the epidemiology and to estimate the incidence of influenza in two communities in West Java, Indonesia, before and after the 2009 H1N1 pandemic. A population-based surveillance study in the community health care setting was conducted to estimate the annual incidence of influenza. A real-time reverse transcription polymerase chain reaction (RT-PCR) assay was used for influenza case ascertainment. A population census was implemented to calculate the population at risk and estimate community health care utilization rate. The mean annual incidence of influenza A and B, adjusted for healthcare utilization, was 1.6 (95%CI: 1.3–2.0) and 0.7 (95%CI: 0.5–1.0) per 1000 persons, respectively, with the most affected group being young and school-age children. The annual cumulative incidence of influenza A for children under five in 2009, 2010, and 2011 was 7.0 (95%CI: 4.4–11.2), 10.6 (95%CI: 7.3–15.4), and 6.3 (95%CI: 3.8–10.2). For influenza B was 4.3 (95%CI: 2.4–7.8), 2.0 (95%CI: 0.8–4.7), and 0.4 (95%CI: 0.1–2.8), respectively. This study highlights that the incidence of influenza among young and school-age children is consistently higher compared to adults and the elderly throughout these periods. These populations are potential targets for influenza vaccination in Indonesia.

Global regulator AdpA directly binds to tunicamycin gene cluster and negatively regulates tunicamycin biosynthesis in Streptomyces clavuligerus.

Since a transcriptional regulator has yet to be identified within the tunicamycin biosynthetic gene cluster in Streptomyces clavuligerus, we conducted a comprehensive investigation by focusing on the possible function of the pleiotropic regulator AdpA on tunicamycin. The genes encoding early steps of tunicamycin biosynthesis were significantly upregulated in S. clavuligerus ΔadpA. At the same time, they were downregulated in adpA overexpressed strain as shown by RNA-sequencing (RNA-seq) and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) analysis. The tunicamycin gene cluster's co-transcription pattern was understood by reverse transcriptase polymerase chain reaction (RT-PCR). Our Electrophoretic Mobility Shift Assay (EMSA) data clearly showed AdpA's binding to the upstream sequence of the tunA gene, asserting its regulatory control. In addition to its direct negative regulation of tunicamycin biosynthesis, AdpA operates at a global level by orchestrating various regulatory genes in S. clavuligerus, such as wblA, whiB, bldM, arpA, brp, and adsA involved in morphological differentiation and secondary metabolite biosynthesis as depicted in RNA-seq data. This study represents a significant milestone by unveiling the AdpA regulator's pathway-specific and global regulatory effect in S. clavuligerus.

Evaluation of the Effect of Vaccination Status on Lung Involvement and Hospitalization in COVID-19 Patients

Background: The spectrum of COVID-19 in adults ranges from asymptomatic infection to mild respiratory tract symptoms to severe pneumonia with acute respiratory distress syndrome (ARDS) and multiorgan dysfunction. In our study, it was aimed to investigate the effects of COVID- 19 vaccine on lung involvement and hospitalization. Materials and Methods: Cases over the age of 18, who had a history of contact with cases infected with COVID-19 or had clinical findings suggestive of COVID-19, who applied to the emergency department and who were diagnosed with COVID-19 by reverse-transcriptase-polymerase chain reaction (RT-PCR) were included in the study. Age, gender, comorbid diseases, hospitalization status, thorax tomography findings, and vaccination status in the last 3 months of the patients included in the study were obtained by examining the hospital information system and patient files. Results: Our study included 496 patients. One hundred eighty-three of the cases were vaccinated and 42.6% (n = 78) of the vaccinated patients were male. Of the 313 unvaccinated patients, 36% (n = 113) were male. Comorbid disease was observed in 55.2% (n = 101) of the vaccinated patients and 38.7% (n = 121) of the unvaccinated patients. Of the 313 unvaccinated patients, 72.2% (n = 226) were discharged from the emergency department, 19.8% (n = 62) were admitted to the service, and 8% (n = 25) were admitted to the intensive care unit. Of the 183 vaccinated patients, 78.1% (n = 143) were discharged from the emergency department, 16.4% (n = 30) were admitted to the service, and 5.5% (n = 10) were admitted to the intensive care unit. In the vaccinated patient group, both the service admission rate and the intensive care hospitalization rate were found to be significantly lower than the non-vaccinated patient group (p &lt; 0.05). Thoracic tomography findings of 32.9% (n = 103) of 313 unvaccinated patients were reported as CORADS 1-2-3, and thoracic tomography of the remaining 210 patients was reported as CORADS 4-5. The number of patients whose thorax tomography report was CORADS 4-5 was found to be significantly higher in the unvaccinated group than in the vaccinated group (p &lt; 0.05). Conclusion: In our study, it was found that vaccination significantly reduced hospitalization in COVID-19 patients. Although the mean age was higher in the vaccinated patient group, both the service admission rate and the intensive care admission rate were found to be lower.

Assessment of equine intestinal epithelial junctional complexes and barrier permeability using a monolayer culture system

Gastrointestinal disease is a leading cause of death in mature horses. A lack of in vitro modeling has impeded the development of novel therapeutics. The objectives of this study were to develop and further characterize a small intestinal monolayer cell culture derived from equine jejunum including establishing normal measurements of intestinal permeability and restitution. Three-dimensional enteroids, derived from postmortem sampling of equine jejunum, were utilized to develop confluent epithelial monolayers. The presence of differentiated intestinal epithelial cell types and tight junctions were confirmed using histology, reverse transcription PCR (RT-PCR), RNAscope, protein immunofluorescence and transmission electron microscopy. Transepithelial resistance (TER) and macromolecule flux were assessed as measurements of paracellular and transcellular permeability. Scratch assays were utilized to model and assess intestinal restitution. Monolayer cell cultures reached 100% confluency by ~5–7 days. Equine jejunum monolayers were confirmed as epithelial in origin, with identification of differentiated intestinal epithelial cell types and evidence of tight junction proteins. Function of the intestinal barrier was supported by acquisition of physiologically normal TER values (179.9 ± 33.7 ohms*cm2) and limited macromolecule flux (22 ± 8.8% at 60 min). Additionally, following a scratch wound, epithelial cell monolayers migrated to close gap defects within 24 h. In conclusion, this study describes the development of a novel intestinal epithelial monolayer cell culture for equine jejunum, and provides evidence of intestinal epithelial cell differentiation, formation of physiologically relevant barrier function and use as a model of intestinal restitution to test potential therapeutics for equine colic.

Optical Genome Mapping for Detection of BCR::ABL1—Another Tool in Our Toolbox

Background: BCR::ABL1 fusion is mostly derived from a reciprocal translocation t(9;22)(q34.1;q11.2) and is rarely caused by insertion. Various methods have been used for the detection of t(9;22)/BCR::ABL1, such as G-banded chromosomal analysis, fluorescence in situ hybridization (FISH), quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) and optical genome mapping (OGM). Understanding the strengths and limitations of each method is essential for the selection of appropriate method(s) of disease diagnosis and/or during the follow-up. Methods: We compared the results of OGM, chromosomal analysis, FISH, and/or RT-PCR in 12 cases with BCR::ABL1. Results: BCR:ABL1 was detected by FISH and RT-PCR in all 12 cases. One case with ins(22;9)/BCR::ABL1 was cryptic by chromosomal analysis and nearly missed by OGM. Atypical FISH signal patterns were observed in five cases, suggesting additional chromosomal aberrations involving chromosomes 9 and/or 22. RT-PCR identified the transcript isoforms p210 and p190 in seven and five cases, respectively. Chromosomal analysis revealed additional chromosomal aberrations in seven cases. OGM identified extra cytogenomic abnormalities in 10 cases, including chromoanagenesis and IKZF1 deletion, which were only detected by OGM. Conclusions: FISH offers rapid and definitive detection of BCR::ABL1 fusion, while OGM provides a comprehensive cytogenomic analysis. In scenarios where OGM is feasible, chromosomal analysis and RT-PCR may not offer additional diagnostic value.

MYL9 binding with MYO19 suppresses epithelial-mesenchymal transition in non-small cell lung cancer.

The elusive function of myosin light chain 9 (MYL9) in cancer is an area ripe for further investigation. Bioinformatics was utilized to compare the expression levels of MYL9 in non-small cell lung cancer (NSCLC) and normal tissues. Gene set enrichment analysis (GSEA) was employed to investigate the pathways associated with MYL9. BioGRID database was utilized to screen for potential targets of MYL9. The expression of MYL9 and myosin 19 (MYO19) mRNA was quantified using quantitative reverse transcriptase PCR. Cell migration was assessed using a scratch wound healing assay. Protein levels of MYL9, MYO19, and epithelial-mesenchymal transition (EMT) biomarkers were examined using western blot (WB). EpCAM expression in different cell groups was profiled using flow cytometry analysis. Co-immunoprecipitation assays were performed to determine the binding affinity between MYL9 and MYO19. Additionally, direct protein interaction between MYL9 and MYO19 was explored using a GST-pull-down assay. In NSCLC patients, MYL9 was significantly downregulated in vivo and in cell cultures, showing high enrichment in the EMT pathway. Scratch assays indicated its inhibitory effect on cell migration. Western blotting revealed that MYL9 suppresses EMT marker protein expression in NSCLC cells. Flow cytometry showed that MYL9 reduced EpCAM levels on the cell surface. MYO19 was identified as a potential target of MYL9 through CoIP and GST-pull-down assays. Rescue experiments demonstrated that MYO19 enhances cell migration, EMT marker expression, and EpCAM levels, but these effects were countered by MYL9 overexpression. MYL9 impedes the migration and EMT in NSCLC cells by binding to MYO19.

The Immunomodulatory Effects of Curcumin on Forkhead Box O1 and MicroRNA-873 in Patients with Osteoarthritis

Osteoarthritis (OA) is among the most prevalent articular disorders, whose incidence is directly related to aging. Due to the antiinflammatory potential of curcumin as the active component of turmeric, the present study evaluated the effects of curcumin on the expression of genes related to T helper 17 (Th17), including forkhead box p3 (FOXP3), forkhead box o1 (FOXO1), transforming growth factor-β (TGFB1) and microRNA-873, human (HSA-MIR-873), in OA patients. Female patients with knee OA (n=30) were randomly categorized into 2 groups, including the intervention group who received curcumin (n=15) and the placebo (n=15) in a double-blind clinical trial for 3 months. The expression of FOXO1, FOXP3, TGFB1, and HSA-MIR-873 genes was evaluated by SYBR Green real-time reverse transcription polymerase chain reaction. In the curcumin group, FOXO1 gene expression was significantly increased, while the increase in FOXP3 gene expression was not significant. Moreover, the expression level of the HSA-MIR-873 gene showed a significant increase in the curcumin group. The modulatory effects of curcumin on Th17 function might be associated with the expression of FOXO1 and HSA-MIR-873 genes.

Fish collagen sponge with human umbilical cord mesenchymal stem cells for diabetic wound repair in rats.

Stem cell therapy offers a novel approach to treating difbetic foot ulcers. Fish skin decellularized matrix, a type I collagen, provides a promising carrier for stem cells, creating a supportive microenvironment that enhances cell survival and therapeutic potential. This study aims to investigate the effects and mechanisms of human umbilical cord mesenchymal stem cells (HUCMSCs) loaded onto a fish collagen sponge for wound healing in diabetic rats. The study evaluates stem cell-loading efficiency with fish collagen sponge in vitro, assesses material distribution on diabetic rat wounds, and establishes a wound model. Rats are divided into the Self-healing group, Fish collagen sponge group, and Sponge loaded with HUCMSCs group. Therapeutic effects are evaluated through various analyses, including histopathology and reverse transcription polymerase chain reaction for collagen-related gene expression levels. Compared to the self-healing group, both the fish collagen group and the composite group show faster wound repair and improved healing outcomes. The composite group exhibits superior wound healing quality, with fish collagen contributing to enhanced tissue regeneration through collagen regulation at the wound site. Loading HUCMSCs onto a fish collagen sponge shows promise for treating diabetic wounds by addressing nutrient deficiency and cell supply issues, offering potential benefits for patients undergoing treatment.

Ribosomal S6 kinase (RSK) plays a critical role in DNA damage response via the phosphorylation of histone lysine demethylase KDM4B

BackgroundEpigenetic dysregulation affecting oncogenic transcription and DNA damage response is a hallmark of cancer. The histone demethylase KDM4B, a factor regulating these processes, plays important roles in estrogen receptor-mediated transcription and DNA repair in breast cancer. However, how oncogenic phospho-signal transduction affects epigenetic regulation is not fully understood. Here we found that KDM4B phosphorylation by ribosomal S6 kinase (RSK), a downstream effector of the Ras/MAPK pathway, is critical for the function of KDM4B in response to DNA damage.MethodsKDM4B-knockout breast cancer cell lines were generated via CRISPR/Cas9-mediated gene editing. Re-expression of wild-type or phospho-site mutated KDM4B in knockout cells was performed by lentivirus-mediated gene transfer. Gene knockdown was achieved by RNA interference. DNA double-strand breaks (DSBs) were induced by ionizing radiation or laser-microirradiation. Protein accumulation at DSB sites was analyzed by immunofluorescence. KDM4B phosphorylation by RSK was assessed by in vitro and in vivo kinase assays. Gene and protein expression levels were analyzed by RT‒PCR and western blotting. The sensitivity of cells to ionizing radiation was examined by a clonogenic survival assay.ResultsRSK phosphorylated KDM4B at Ser666, and inhibition of the phosphorylation by RSK depletion or RSK inhibitors abrogated KDM4B accumulation at the sites of DNA double-strand breaks (DSBs). DSB repair was significantly delayed in KDM4B-knockout cells or cells treated with RSK inhibitors. The replacement of endogenous KDM4B with the phosphomimetic mutant S666D restored KDM4B accumulation and DSB repair that had been inhibited by RSK inhibitors, suggesting a critical role for RSK at the specific serine residue of KDM4B in the effect of RSK inhibitors on DSB repair. As a consequence of these aberrant responses, inhibition of KDM4B phosphorylation increased the sensitivity of the cells to ionizing radiation.ConclusionsOverall, the present study uncovered a novel function of RSK on the DNA damage response, which provides an additional role of its inhibitor in cancer therapy.

The impact of antibiotic induction on virulence and antibiotic resistance in Klebsiella pneumoniae: a comparative study of CSKP and CRKP strains

BackgroundKlebsiella pneumoniae is an opportunistic pathogen causing nosocomial infections, classified into carbapenem-sensitive and carbapenem-resistant strains. Understanding the virulence factors and antibiotic resistance of these strains is essential for effective clinical management.ObjectiveThis study compared the virulence genes and antibiotic resistance profiles of 50 CSKP and 50 CRKP strains, examining their expression under antibiotic pressure and the mechanisms contributing to their pathogenicity.MethodsVirulence genes (rmpA, rmpA2, iucA, iutA, Peg-344, ybts, iroB) were detected in both strains using polymerase chain reaction (PCR). Antibiotic susceptibility testing established minimum inhibitory concentrations (MICs) for key antibiotics. Gene expression analysis was performed with quantitative reverse transcription PCR (qRT-PCR) after 10 days of antibiotic exposure.ResultsCSKP strains exhibited significantly higher positivity rates for virulence genes compared to CRKP strains. CRKP strains predominantly expressed resistance genes KPC, SHV, and CTX-M3, whereas no resistance genes were found in CSKP. Antibiotic susceptibility tests showed increased MICs, particularly for ciprofloxacin and imipenem, following antibiotic induction. CSKP demonstrated elevated expression of rmpA and rmpA2, while CRKP showed increased expression of SHV, and KPC after antibiotic exposure.ConclusionThis study highlights the intricate relationship between virulence and resistance in Klebsiella pneumoniae. CSKP strains show strong virulence factor expression, while CRKP strains adapt to antibiotic pressure through altered gene expression patterns. These findings underscore the urgent need for continuous surveillance and innovative therapeutic strategies to combat multidrug-resistant Klebsiella pneumoniae infections.

Serum Exosomal MiR-874 as a Potential Biomarker for Nonsmall Cell Lung Cancer Diagnosis and Prognosis

AbstractLung cancer, primarily nonsmall cell lung cancer (NSCLC), is a leading cause of cancer-related fatalities globally. Due to late detection, the 5-year survival rate for NSCLC remains low. Therefore, the current research aimed to assess the diagnostic and prognostic value of serum exosomal miR-874 levels in NSCLC patients. This study involved 161 NSCLC patients and 80 control subjects. Blood samples were collected from all participants, and serum exosomal MiR-874 levels were quantified using quantitative reverse transcription-polymerase chain reaction. The study revealed a significant decrease in MiR-874 levels among NSCLC patients compared to controls. The receiver operating characteristic analysis demonstrated the diagnostic value of serum exosomal MiR-874 in effectively distinguishing NSCLC patients from controls.Furthermore, associations were observed between serum exosomal MiR-874 expression and adverse clinical factors such as young age, male sex, smoking, high tumor grade, squamous cell carcinoma histopathology, advanced tumor stage, and lymphatic involvement. Patients with high levels of MiR-874 had significantly longer overall and disease-free survival compared to those with lower levels. The study demonstrates that levels of serum exosomal miR-874 are considerably lower in NSCLC patients, indicating its potential as a diagnostic biomarker. The study's findings suggest that the expression of MiR-874 may predict the prognosis of NSCLC patients based on clinical features.

Genome-Wide Identification and Expression Analysis of the Melon Aldehyde Dehydrogenase (ALDH) Gene Family in Response to Abiotic and Biotic Stresses

Through the integration of genomic information, transcriptome sequencing data, and bioinformatics methods, we conducted a comprehensive identification of the ALDH gene family in melon. We explored the impact of this gene family on melon growth, development, and their expression patterns in various tissues and under different stress conditions. Our study discovered a total of 17 ALDH genes spread across chromosomes 1, 2, 3, 4, 5, 7, 8, 11, and 12 in the melon genome. Through a phylogenetic analysis, these genes were classified into 10 distinct subfamilies. Notably, genes within the same subfamily exhibited consistent gene structures and conserved motifs. Our study discovered a pair of fragmental duplications within the melon ALDH gene. Furthermore, there was a noticeable collinearity relationship between the melon’s ALDH gene and that of Arabidopsis (12 times), and rice (3 times). Transcriptome data reanalysis revealed that some ALDH genes consistently expressed highly across all tissues and developmental stages, while others were tissue- or stage-specific. We analyzed the ALDH gene’s expression patterns under six stress types, namely salt, cold, waterlogged, powdery mildew, Fusarium wilt, and gummy stem blight. The results showed differential expression of CmALDH2C4 and CmALDH11A3 under all stress conditions, signifying their crucial roles in melon growth and stress response. RT-qPCR (quantitative reverse transcription PCR) analysis further corroborated these findings. This study paves the way for future genetic improvements in melon molecular breeding.