Real-time PCR Research Articles

Role of m6A methyltransferase METTL3 in keratoconus pathogenesis.

Keratoconus (KC) is the most common ectatic corneal disease with unknown pathogenesis. This study aimed to investigate the role of methyltransferase-like enzyme 3 (METTL3) in KC pathogenesis. In the present study, we examined the levels of METTL3 and other N6-methyladenosine (m6A) modification-related proteins in KC samples and human stromal keratocyte (HTK) cells stimulated by mechanical stretch (MS) using Western blotting and immunohistochemistry. The level of m6A RNA methylation was quantified using the m6A RNA methylation assay kit. Genetic (Mettl3 knockdown mice) and pharmacological (STM2457) approaches were employed to investigate the effect of METTL3 on the expression of metalloproteinases (MMPs) in MS-treated corneal stromal cells (CSCs) via Western blotting and real-time polymerase chain reaction. Moreover, YAP signaling activity was assessed to explore the relationship between METTL3 and MMPs in MS-treated CSCs. Increased expression of METTL3 and decreased expression of METTL14, WTAP, and YTHDF2 were detected in KC samples and MS-stimulated HTK cells. Correspondingly, the m6A levels in KC specimens and MS-stimulated CSCs were significantly higher than those in controls but were significantly reduced when METTL3 activity was genetically and pharmacologically blocked. Inhibition of METTL3 significantly reduced the expression of MMP1 and MMP3 in mechanically stretched CSCs and reduced YAP activity. Furthermore, pharmacologically inhibiting YAP signaling in MS-stimulated HTK cells significantly reduced MMP1 and MMP3 expression. Our findings highlight the pathogenic role of METTL3 in KC. Further investigation is required to investigate the underlying mechanism.

Therapeutic Effects of Crocin Nanoparticles Alone or in Combination with Doxorubicin against Hepatocellular Carcinoma In vitro.

Crocin (CRO), the primary antioxidant in saffron, is known for its anticancer properties. However, its effectiveness in topical therapy is limited due to low bioavailability, poor absorption, and low physicochemical stability. This study aimed to prepare crocin nanoparticles (CRO-NPs) to enhance their pharmaceutical efficacy and evaluate the synergistic effects of Cro-NPs with doxorubicin (DOX) chemotherapy on two cell lines: human hepatocellular carcinoma cells (HepG2) and non-cancerous cells (WI38). CRO-NPs were prepared using the emulsion diffusion technique and characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), Zeta potential, and Fourier transform infrared spectroscopy (FT-IR). Cell proliferation inhibition was assessed using the MTT assay for DOX, CRO, CRO-NPs, and DOX+CRO-NPs. Apoptosis and cell cycle were evaluated by flow cytometry, and changes in the expression of apoptotic gene (P53) and autophagic genes (ATG5 & LC3) were analyzed using real-time polymerase chain reaction. TEM and SEM revealed that CRO-NPs exhibited a relatively spherical shape with an average size of 9.3 nm, and zeta potential analysis indicated better stability of CRO-NPs compared to native CRO. Significantly higher antitumor effects of CRO-NPs were observed against HepG2 cells (IC50 = 1.1 mg/ml and 0.57 mg/ml) compared to native CRO (IC50 = 6.1 mg/ml and 3.2 mg/ml) after 24 and 48 hours, respectively. Annexin-V assay on HepG2 cells indicated increased apoptotic rates across all treatments, with the highest percentage observed in CRO-NPs, accompanied by cell cycle arrest at the G2/M phase. Furthermore, gene expression analysis showed upregulation of P53, ATG5, and LC3 genes in DOX/CRO-NPs co-treatment compared to individual treatments. In contrast, WI38 cells exhibited greater sensitivity to DOX toxicity but showed no adverse response to CRONPs. Although more in vivo studies in animal models are required to corroborate these results, our findings suggest that CRO-NPs can be a potential new anticancer agent for hepatocellular carcinoma. Moreover, they have a synergistic effect with DOX against HepG2 cells and mitigate the toxicity of DOX on normal WI38 cells.

Molecular and serological investigation of Brucella species in kennel and farm dogs in Iran.

Brucellosis is still a significant emerging threat to public health, as it can infect humans, wild, domestic animals, and livestock. Hence, the current study aims to determine the frequency of canine brucellosis (CB), its relationship with clinical findings and reproductive disorders in kennel and farm dogs, and its importance on public health. From January 2022 to December 2023, a total of 150 blood samples were taken from 100 adult dogs in breeding kennels and 50 shepherd dogs in breeding farms in Kerman, Iran. Rose Bengal test (RBT) and Wright tests were used for the primary screening of Brucella abortus (B. abortus) and Brucella melitensis (B. melitensis) species, and conventional IS711-based PCR and real-time polymerase chain reaction (RT-PCR) were performed to determine the Brucella genus and species in all samples. In kennel dogs, 34 % (34/100) had a history of breeding disorders, and 30 % (30/100) were RBT-positive. Moreover, B. canis and B. abortus species were detected in kennel dogs. Among farm dogs, 16 % (8/50) had a history of breeding disorders, and 28 % (14/50) were RBT positive. Additionally, B. canis, B. abortus, and B. melitensis species were detected in farm dogs. Feeding with unpasteurized milk was significantly related to a positive RBT in kennel dogs (p = 0.009), and there was a significant correlation between breeding disorders and seropositivity in kennel (p = 0.045) and farm dogs (p = 0.03). The current study represents a significant advancement in understanding CB in Iran by the first molecular detection of B. canis, revealing B. melitensis and B. abortus as important pathogens in kennel and farm dogs and highlighting the public health significance of disease in Iran.

Topical neurokinin-1 receptor antagonism ameliorates ocular pain and prevents corneal nerve degeneration in an animal model of dry eye disease.

Ocular pain is a common complaint to eye care providers, associated with a variety of ocular conditions, among which dry eye disease (DED) is affecting millions of people worldwide. Despite being highly prevalent, ocular pain is not managed adequately in the clinic. The aim of this study was to investigate the analgesic potential of neurokinin-1 receptor (NK1R) antagonism in DED. Dry eye disease was induced in mice, and an NK1R antagonist L-733,060 was topically administered twice daily throughout the study for 14 days. Hyperalgesia and allodynia were assessed using the eye-wiping test and palpebral ratio measurements. Corneas were collected for measuring substance P (SP) levels by enzyme-linked immunosorbent assay (ELISA) and imaging nerves by immunostaining. Trigeminal ganglions (TG) were collected to determine SP levels by ELISA and transient receptor potential cation channel subfamily V member 1 (TRPV1), transient receptor potential cation channelsubfamily M (melastatin) member 8, c-Fos, and activating transcription factor 3 (ATF3) mRNA levels by real-time polymerase chain reaction. Treating DED mice with L-733,060 resulted in a significant reduction in eye wipe behavior, a significant increase in palpebral ratio, and significant decreases in SP levels in both the cornea and TG compared with the vehicle-treated group. In addition, NK1R antagonist treatment significantly suppressed the upregulation of TRPV1, ATF3, and c-Fos and prevented corneal nerve loss. Neurokinin-1 receptor antagonism effectively reduced ocular nociception, decreased neuronal activation, and preserved corneal nerves in mice with DED. These findings suggest that blockade of SP signaling pathway is a promising therapeutic strategy for managing DED pain.

MicroRNA biomarkers and host response pathways in severe pulmonary hemorrhagic syndrome due to leptospirosis: A multi-omics study.

Severe pulmonary hemorrhagic syndrome (SPHS) remains a fatal complication of leptospirosis with poorly understood mechanisms and an urgent need for effective biomarkers. A nested case-control analysis was conducted using blood specimens from two previous Thai leptospirosis cohorts. Candidate microRNAs were initially discovered through a global profiling of 798 serum microRNAs in five SPHS and seven non-SPHS patients, and then validated using real-time polymerase chain reactions in 168 patients. Pathways enriched from microRNA targets were compared to those from an integrated transcriptomic-proteomic analysis. Proteins pertaining to the key resulting pathway were measured to validate significance and reveal correlation with microRNA biomarkers. Serum microRNA profiling revealed a total of 81 significantly expressed microRNAs, of which seven were selected for further validation in the whole cohort of 168 leptospirosis patients, including 28 in SPHS and 140 nonSPHS groups. Among the selected microRNAs, miR-5010-3p and miR-147b-3p had significantly higher expression in SPHS group compared to nonSPHS group, with consistently higher expression after adjusting for age, sex, days of illness, comorbidity, smoking status or recruitment site. The two had area under the curve (AUC) values of 0.76 (95% CI: 0.67-0.85) and 0.70 (95% CI: 0.56-0.81) for discriminating SPHS, respectively. These microRNAs also exhibited consistent AUC values in patients tested before chest radiograph shadows manifested. Combination of miR-5010-3p with miR-548ai and miR-224-5p, as selected by Bayesian Model Averaging algorithm, substantially boosts the AUC value to 0.86 (95% CI: 0.77-0.94). The miRNA biomarkers also enhanced the predictive values of a previously validated clinical model, increasing AUC value from 0.87 to 0.92 with a significant reclassification net index. Multi-omics pathway analysis incorporating microRNA targets and transcriptomic-proteomic data suggested TNF signaling as among the key pathways. In validation, seven out of ten pathway proteins were significantly different between groups, with principal components correlated with severity and miR-5010-3p. MiR-5010-3p and miR-147b-3p are novel biomarkers with good predictability and potential relevance with TNF signaling pathway, an important host response mechanism in leptospirosis SPHS.

GATAD1 is involved in sphingosylphosphorylcholine-attenuated myocardial ischemia-reperfusion injury by modulating myocardial fatty acid oxidation and glucose oxidation

Modulating the equilibrium between glucose metabolism and lipid metabolism represents highly promising novel strategies for therapy of myocardial ischemia/reperfusion (I/R) injury. Sphingosylphosphorylcholine (SPC), an intermediate metabolite of sphingolipids, has shown cardioprotective roles during myocardial infarction by regulating the activities of various transcript factors. Gene microarray revealed that SPC significantly upregulated the expression of GATA zinc finger domain protein 1 (GATAD1), which is a vital transcript factor affecting heart development and various heart diseases. However, it remains unclear whether SPC is involved in the regulation of cardiac fatty acid and glucose metabolism via GATAD1. In this study, we found that myocardium-specific Gatad1 knockout (Gatad1 CKO) significantly increased the myocardial infarct size, impaired cardiac function in I/R mice, and disrupted the protective effect of SPC on the hearts of I/R mice. Immunofluorescence experiment and western blot evaluation of the nuclear-cytoplasmic fractionation sample showed that GATAD1 acted as a transcription factor and was regulated by SPC. Double fluorescence reporting experiment and quantitative polymerase chain reaction (qPCR) revealed that GATAD1 could inhibit the expression of genes involved in fatty acid oxidation (FAO), i.e., acetyl-coenzyme A acyltransferase 2 (Acaa2) and medium-chain acyl-CoA dehydrogenase (Acadm), and promoted the expression of genes involved in glucose oxidation, i.e., pyruvate dehydrogenase E1 α subunit (Pdha1). Small interfering RNA (SiRNA) or overexpression strategies confirmed the pro-apoptotic roles of Acaa2 and Acadm and anti-apoptotic role of Pdha1 in cardiac myocytes challenged with I/R treatment. In summary, our findings suggest that SPC can be used as a candidate to prevent I/R injury by reshaping fatty acid and glucose metabolism. Transcription factor GATAD1 plays a crucial role in regulating fatty acid oxidation and glucose oxidation homeostasis and is involved in SPC-mediated cardioprotection during I/R of the heart. Our study identifies GATAD1 as a new therapeutic target for clinical treatment of myocardial I/R injury.

Occurrence and risk factors of equine piroplasmosis in Portugal: A five-year retrospective study.

Equine piroplasmosis (EP) is a tick-borne disease of equids caused by Theileria equi, Theileria haneyi, and Babesia caballi. EP is endemic in most tropical and subtropical regions worldwide, and there is a likelihood that it is also endemic in Portugal. This retrospective study aimed to determine the seroprevalence, prevalence, and potential risk factors of EP in our country over the past five years. A total of 3063 diagnostic test records were analysed. Results from the competitive enzyme-linked immunosorbent assay (cELISA) revealed a seroprevalence of 32.7 % and 15.7 % for T. equi and B. caballi, respectively, with a coinfection rate of 7.4 %. For the indirect fluorescent antibody test (IFAT), 38.8 % of the samples were positive for T. equi, 45.7 % for B. caballi, and 23.1 % for both parasites. Prevalence determined using quantitative polymerase chain reaction (qPCR) showed 40.5 % T. equi-positive cases, 8.3 % B. caballi-positive cases, and 3.2 % mixed infections in the studied population. Considering risk factors, age and season appear to be associated with higher seropositivity, and location was also found to play a significant role. This study represents the first retrospective analysis carried out in Portugal, confirming the endemicity of EP in the country. Further studies are needed to corroborate our findings, to determine actual prevalence and seroprevalence in the Portuguese general equine population, and to identify risk factors better, helping breeders and owners to minimise the health and economic impact of EP.

Imported seafood is a reservoir of Enterobacteriaceae carrying CTX-M-encoding genes of high clinical relevance.

We determined the frequency, genotypes, phenotypes, and mobility of extended-spectrum β-lactamase (ESBL)-encoding genes in Enterobacteriaceae isolated from retail seafood products. Overall, 288 samples of fresh shrimps, catfish and seabass imported from Asia were collected from three supermarket chains in the UK (96 each). After enrichment in MacConkey broth supplemented with cefotaxime, total DNA was screened for the presence of CTX-M, SHV and TEM by real-time PCR. Positive samples were cultured on ESBL selective media and presumptive ESBL-producing isolates were confirmed by PCR and identified to the species level by MALDI-TOF-MS. CTX-M-positive isolates were further characterized by whole genome sequencing (WGS), antimicrobial susceptibility testing, and conjugation experiments. Approximately one in thirteen (7.6%) seafood products were contaminated with ESBL-producing Enterobacteriaceae. WGS analysis revealed the presence of CTX-M-15 (n=7), CTX-M-27 (n=7), and CTX-M-55 (n=7), CTX-M-14 (n=4) among Enterobacteriaceae isolated from shrimp (n=21) and catfish (n=4), and FONA-6 in two Serratia fonticola isolates from seabass. The higher rate of contamination in shrimp could be due to post-harvest contamination due to human handling or washing practices during processing. Half (n=13) of the CTX-M-producing isolates transferred blaCTX-M to laboratory E. coli via IncA/C (n=6), IncX2 (n=4), IncFIIK (n=1) or non-typeable plasmids (n=2). All plasmids contained additional resistance genes conferring resistance to antimicrobials used in aquaculture, indicating possible co-selection through the use these antimicrobials. The frequent occurrence of CTX-M-encoding genes of high clinical relevance in imported seafood, particularly shrimp, often on transferrable plasmids, underscores the need for ESBL surveillance on traded seafood, alongside quantitative risk assessment studies aimed at evaluating the potential health risks for consumers who are exposed to these bacteria via consumption of raw seafood.

Gentiopicroside ameliorates synovial inflammation and fibrosis in KOA rats by modulating the HMGB1-mediated PI3K/AKT signaling axis.

Knee osteoarthritis (KOA) is a degenerative joint disease characterized by synovial inflammation and fibrosis. Gentiopicroside (GPS), one of the main active ingredients of Gentiana macrophylla, is widely used in anti-inflammatory and anti-fibrotic therapies. However, the exact mechanism by which GPS treats synovial inflammation and fibrosis in KOA remains unclear. Fibroblast-like synoviocytes (FLSs) were stimulated with lipopolysaccharide (LPS) to induce inflammation and fibrosis, and CCK-8 was performed to determine the viability of GPS-treated FLSs, using immunofluorescence to examine the expression of P-PI3K and P-AKT, confocal microscopy was used to identify intracellular HMGB1 translocation. The KOA rat model was established by anterior cruciate ligament transection (ACLT) and subsequently subjected to GPS intervention. Inflammatory cytokines (TNF-α, IL-1β, and IL-6), fibrosis-related indicators (TGF-β, collagen I, TIMP1, and α-SMA), and HMGB1/PI3K/AKT signaling axis-related proteins and gene expression of fibroblast-like synoviocytes and synovial tissues were detected by Western blotting and real-time PCR. The histopathology of the synovium of the rats was assessed using Hematoxylin-eosin (HE), Sirius Red, and Masson staining. Immunohistochemistry was performed to detect the expression of HMGB1, P-PI3K, and P-AKT. The present study revealed that GPS intervention significantly ameliorated inflammation and fibrosis in LPS-stimulated FLSs and KOA rat synovium. Immunofluorescence demonstrated that GPS inhibited the release of HMGB1 from the nucleus. Furthermore, GPS intervention down-regulates the levels of proteins and gene associated with the HMGB1/PI3K/AKT signaling pathway. GPS ameliorated synovial inflammation and fibrosis in KOA rats, which may involve HMGB1-mediated activation of the PI3K/AKT signaling axis.

SHMT2 regulates CD8+ T cell senescence via the reactive oxygen species axis in HIV-1 infected patients on antiretroviral therapy.

Although antiretroviral therapy (ART) effectively inhibits viral replication, it does not fully mitigate the immunosenescence instigated by HIV infection. Cellular metabolism regulates cellular differentiation, survival, and senescence. Serine hydroxymethyltransferase 2 (SHMT2) is the first key enzyme for the entry of serine into the mitochondria from the de novo synthesis pathway that orchestrates its conversion glutathione (GSH), a key molecule in neutralising ROS and ensuring the stability of the immune system. It remains incompletely understood whether SHMT2 is involved in the senescence of CD8+ T cells, crucial for immune vigilance against HIV. HIV-infected individuals receiving antiretroviral therapy were enrolled in our study. SHMT2-siRNA was electroporated into T cells to disrupt the gene expression of SHMT2, followed by the quantification of mRNA levels of crucial serine metabolism enzymes using real-time PCR. Immunophenotyping, proliferation, cellular and mitochondrial function, and senescence-associated signalling pathways were examined using flow cytometry in CD8+ T cell subsets. Our findings revealed that CD8+ T cells in HIV-infected subjects are inclined towards senescence, and we identified that SHMT2, a key enzyme in serine metabolism, plays a role in CD8+ T cell senescence. SHMT2 can regulate glutathione (GSH) synthesis and protect mitochondrial function, thus effectively controlling intracellular reactive oxygen species (ROS) levels. Moreover, SHMT2 significantly contributes to averting immunosenescence and sustaining CD8+ T cell competence by modulating downstream DNA damage and phosphorylation cascades in pathways intricately linked to cellular senescence. Additionally, our study identified glycine can ameliorate CD8+ T cell senescence in HIV-infected individuals. Decreased SHMT2 levels in HIV-infected CD8+ T cells affect ROS levels by altering mitochondrial function and GSH content. Increased ROS levels activate senescence-related signalling pathways in the nucleus. However, glycine supplementation counteracts these effects and moderates senescence. This study was supported by grants from the National Key R&D Program of China (2021YFC2301900-2021YFC2301901), National Natural Science Foundation of China (82372240), and Department of Science and Technology of Liaoning Province Project for the High-Quality Scientific and Technological Development of China Medical University (2022JH2/20200074).

Enhanced antimicrobial, anti-biofilm, and efflux pump inhibitory effects of ursolic acid-conjugated magnetic nanoparticles against clinical isolates of multidrug-resistant Pseudomonas aeruginosa.

In the present study, we investigate the effect of Fe3O4 nanoparticles conjugated with ursolic acid (Fe3O4NPs@UA) on inhibiting the growth, biofilm-forming ability and efflux pump activity in clinical isolates of Pseudomonas aeruginosa with multiple drug resistance. Iron oxide NPs conjugated with ursolic acid (Fe3O4NPs@UA) were synthesized. Physicochemical features of the NPs were studied by FT-IR, XRD, EDAX, and TEM. The antibacterial and antibiofilm effects of Fe3O4NPs@UA against P. aeruginosa isolates were determined by broth microdilution and microtiter plate methods, respectively. The efflux pump inhibitory effect of Fe3O4NPs@UA was determined using Cartwheel method and through determining the expression level of efflux pump genes, including mexA and oprD in selected P. aeruginosa isolates treated with sub-MIC concentration of Fe3O4NPs@UA by real-time PCR. In investigating the antimicrobial effect of Fe3O4NPs@UA, the MIC of these nanoparticles varied between 0.19 and 0.78mg/mL and in the study of the anti-biofilm effect of Fe3O4NPs@UA, it caused a 68-75% decrease in biofilm formation compared to the control. Moreover, in the Cartwheel test, the anti-efflux effect of these nanoparticles was confirmed at 1/4-MIC concentrations, and the expression of mexA and oprD genes was reduced in bacteria treated with Fe3O4NPs@UA compared to the control. According to the results, the use of Fe3O4NPs@UA can provide a basis for the development of new treatments against drug-resistant bacteria in P. aeruginosa. This substance can improve the concentration of antibiotics in bacterial cells and increase their effectiveness by inhibiting the efflux in P. aeruginosa isolates.

Effects of tumor treating fields (TTFields) with FOLFIRINOX on BRCA WT pancreatic cancer cells.

753 Background: Pancreatic cancer remains one of the most aggressive malignancies, frequently diagnosed at the locally advanced or metastatic stage. In the advanced setting, first-line chemotherapy options include gemcitabine with nab-paclitaxel or FOLFIRINOX, a combination regimen of fluorouracil, oxaliplatin, irinotecan and leucovorin. The FOLFIRINOX regimen is however associated with greater toxicity and is hence used only in patients with good performance status. BRCA-mutated tumors seem to be more sensitive to FOLFIRINOX due to the DNA-damaging, platinum-based component of this treatment regimen. Tumor Treating Fields (TTFields) are electric fields that disrupt cellular processes crucial for cancer cell viability that have shown efficacy in preclinical pancreatic cancer models; and, in various cancer types, have been shown to reduce expression of proteins from the BRCA-dependent DNA repair pathway. The current study examined the potential use of TTFields for sensitization of BRCA wild-type pancreatic cancer cells to treatment with FOLFIRINOX. Methods: Human pancreatic BRCA wild-type cancer cells BxPC3 and AsPC1 were treated with TTFields (150 kHz; 0.7 and 1 V/cm RMS, respectively), using the inovitro device. FOLFIRINOX was administered to the cells at increasing concentrations, with or without co-treatment with TTFields. After 72h of treatment, cell count, colony formation, and apoptosis were measured. For mechanistical insight, gene and protein expression were evaluated following 24, 48, or 72h of TTFields treatment. Results: TTFields application to the pancreatic cells together with FOLFIRINOX elevated the cytotoxic, clonogenic and apoptotic effects induced by FOLFIRINOX or TTFields alone. RNA sequencing data revealed that TTFields downregulated DNA damage repair, DNA replication, and cell cycle related processes. Specifically, real-time PCR and Western blot analysis demonstrated reduced expression of several central players in the BRCA DNA damage repair pathway. Conclusions: TTFields application together with FOLFIRINOX in pancreatic cancer cells lacking background BRCA mutations exhibits potential improvement relative to FOLFIRINOX alone, that may be rationalized based on downregulation of key DNA repair pathways. Future studies should explore the possibility of reducing FOLFIRINOX treatment dose, potentially alleviating FOLFIRINOX-related toxicity.

Contamination of retail market meat with extended-spectrum beta-lactamase genes in Vietnam.

The contamination of retail meat with antibiotic-resistant bacteria poses a substantial public health risk because of the potential spread of these bacteria within communities. The contamination of retail meat with extended-spectrum beta-lactamase (ESBL)-producing bacteria was investigated in four cities in Vietnam using real-time PCR, employing ESBL marker genes. This method provides a more comprehensive assessment of ESBL-producing bacterial contamination in meat samples than culture-based methods because it directly detects resistance genes from the extracted sample DNA. Retail meats in Vietnam were substantially contaminated with ESBL genes [54% (n=46) and 48% (n=49) of chicken and pork samples, respectively]. No significant differences in ESBL gene detection rates were observed between chicken and pork. The most frequently detected ESBL gene was blaTEM, followed by blaSHV, whereas blaCTX-M was found in only 4-8% of the samples. Ho Chi Minh City showed significantly higher contamination rates for both chicken and pork than those in other cities. ESBL-producing Escherichia coli strains were isolated from contaminated meat samples and genomically analyzed. All isolated strains carried blaCTX-M, with some harboring blaTEM, whereas blaSHV was not detected. Although IncFIB plasmids were prevalent among the ESBL-producing E. coli strains, the variability in resistance gene profiles suggested that the endemic spread of specific resistance gene-carrying plasmids was unlikely. Overall, these findings highlight the effectiveness of the ESBL gene detection method and the high levels of ESBL-producing E. coli in retail meat.

The Ataxia-telangiectasia mutated (ATM) is the most important gene for repairing the DNA in Myelodysplastic Neoplasm.

Myelodysplastic Neoplasm (MDS) is a cancer associated with aging, often leading to acute myeloid leukemia (AML). One of its hallmarks is hypermethylation, particularly in genes responsible for DNA repair. This study aimed to evaluate the methylation and mutation status of DNA repair genes (single-strand - XPA, XPC, XPG, CSA, CSB and double-strand - ATM, BRCA1, BRCA2, LIG4, RAD51) in MDS across three patient cohorts (Cohort A-56, Cohort B-100, Cohort C-76), using methods like pyrosequencing, real-time PCR, immunohistochemistry, and mutation screening. Results showed that XPA had higher methylation in low-risk MDS compared to high-risk MDS. For double-strand repair genes, ATM displayed higher methylation in patients who transformed to AML (p = 0.016). ATM gene expression was downregulated in MDS compared to controls (p = 0.042). When patients were classified according to the WHO 2022 guidelines, ATM expression progressively decreased from low-risk subtypes (e.g., Hypoplastic MDS) to high-risk MDS and AML. Patients who transformed to AML had a higher 5mC/5hmC ratio compared to those who didn't (p = 0.045). Additionally, poor cytogenetic risk patients had higher tissue methylation scores than those with good risk (p = 0.035). Analysis using the cBioPortal platform identified ATM as the most frequently mutated DNA repair gene, with various mutations, such as frameshift and missense, most of which were classified as oncogenic. The findings suggest that ATM is frequently silenced or downregulated in MDS due to methylation or mutations, contributing to the progression to AML. This highlights ATM's potential role in the disease's advancement and as a target for future therapeutic strategies.

Role of Ferroptosis in COVID-19 Pathogenesis: Insights from Iron Metabolism and Lipid Peroxidation Markers

Objective: Ferroptosis is a form of programmed cell death characterized by the irondependent accumulation of lipid peroxides, leading to membrane damage and cell rupture. Several lines of evidence suggest that ferroptosis may contribute to the pathogenesis and severity of COVID- 19. This study aimed to measure iron metabolism and lipid peroxidation markers in COVID-19 patients to establish a direct link between ferroptosis and COVID-19. Methods: In the present cross-sectional study, the serum levels of hemoglobin (Hb), ferritin, and iron, total iron binding capacity (TIBC), malondialdehyde (MDA), and glutathione (GSH) were assessed in 100 COVID-19 patients diagnosed using Real-time PCR and compared to the levels in 100 healthy individuals. Results: Our findings revealed a significant increase in serum levels of ferritin and MDA in COVID- 19 patients compared to control subjects. Conversely, TIBC, Hb, Iron, and GSH levels were lower in COVID-19 patients than in control. Conclusion: Our study provides further evidence supporting the significance of ferroptosis in the pathogenesis of COVID-19. Further research aimed at elucidating the exact role of ferroptosis in COVID-19 is warranted, as it may lead to improved strategies for mitigating multi-organ dysfunction associated with this disease.

Comparative Outcomes of Intravenous, Intranasal, and Intracerebroventricular Transplantation of Human Neural Stem Cells in Mice Model of Ischemic Stroke.

Transplantation of neural stem cells improves ischemic stroke outcomes in rodent models and is currently in the clinical test stage. However, the optimal delivery route to achieve improved efficacy remains undetermined. This study aims to evaluate three more clinically feasible delivery routes: intravenous (IV), intranasal (IN), and intracerebroventricular (ICV). We compared the therapeutic efficacies of the three routes of transplanting human neural stem cells (hNSCs) into mice with permanent middle cerebral artery obstruction (pMCAO). Behavioral tests and cresyl violet staining were used to evaluate the therapeutic efficacies of functional recovery and lesion volumes. The expression of proinflammatory cytokines and neurotrophic factors was measured by real-time PCR. The distribution and differentiation of hNSCs were determined by immunofluorescence staining. The effect on endogenous neurogenesis and astrocyte function were determined by immunofluorescence staining and western blot. hNSC transplantation using the three routes improved behavioral outcomes and reduced lesion volumes; IV transplantation of hNSCs results in earlier efficacy and improves the inflammatory microenvironment. The long-term distribution and differentiation of transplanted hNSCs in the peri-infarct areas can only be evaluated using ICV delivery. IV and ICV transplantation of hNSCs promote neurogenesis and modulate the dual function of astrocytes in the peri-infarct areas. IV and IN delivery is suitable for repeated administration of hNSCs to achieve improved prognosis. Comparatively, ICV transplantation provides long-term efficacy at lower doses and fewer administration times.

Functional characterization, transcriptome and metabolome analyses reveal that pacR possesses multifaceted physiological roles in Xanthomonas campestris pathovar campestris

Xanthomonas campestris pathovar campestris (Xcc) is the pathogen responsible for causing black rot in cruciferous plants. In this study, we show that mutation of AAW18_RS04175 (pacR, encodes a hypothetical protein containing a domain of unknown function, DUF1631) of Xcc strain Xc17 had decreased bacterial attachment, exopolysaccharide production, hypersensitive response and virulence. Furthermore, the pacR mutant exhibited reduced cell membrane integrity and outer membrane vesicle production. Transcriptomic analysis indicated that 225 genes were differentially expressed following pacR mutation. These genes can be classified into various functional categories, such as the type three secretion system and membrane component. Among them, genes associated with attachment, exopolysaccharide synthesis, the type three secretion system, and nucleotide metabolism were further verified by quantitative RT-PCR. Metabolomic analysis showed that 81 and 132 metabolites in positive and negative modes, respectively, were altered after pacR mutation. Among the identified metabolites, some are known to belong to different pathways, such as biosynthesis of secondary metabolites, microbial metabolism in diverse environments, and nucleotide and purine metabolism, while others have not been previously documented in microbial systems. Additionally, the transcription initiation point of pacR was mapped, and promoter analysis indicated that pacR expression is influenced by different conditions. Taken together, our findings advance the understanding of PacR function and expression in Xcc and offer new insights into the role of the DUF1631-containing hypothetical protein in bacterial physiology.

Therapeutic effect of low-dose BMSCs-Loaded 3D microscaffold on early osteonecrosis of the femoral head.

The early treatment of Osteonecrosis of Femoral Head (ONFH) remains a clinical challenge. Conventional Bone Marrow Mesenchymal Stem Cell (BMSC) injection methods often result in unsatisfactory outcomes due to mechanical cell damage, low cell survival and retention rates, inadequate cell matrix accumulation, and poor intercellular interaction. In this study, we employed a novel cell carrier material termed "3D Microscaffold" to deliver BMSCs, addressing these issues and enhancing the therapeutic effects of cell therapy for ONFH. We injected 3D microscaffold loaded with low-dose BMSCs or free high-dose BMSCs into the femoral heads of ONFH rats and assessed therapeutic effects using imaging, serology, histology, and immunohistochemistry. To understand the mechanism of efficacy, we established a co-culture model of human osteoblasts and BMSCs, followed by cell proliferation and activity detection, flow cytometry analysis, Quantitative RT-PCR, and Western blotting. Additionally, RNA sequencing was performed on femoral head tissues. Results showed that the 3D microscaffold with low-dose BMSCs had a therapeutic effect comparable to high-dose free BMSCs. Osteoblasts in the 3D microscaffold group exhibited superior phenotypes compared to the non-3D microscaffold group. Furthermore, we have, for the first time, preliminarily validated that the low-dose BMSCs-loaded 3D microscaffolds may promote the repair of femoral head necrosis through the synergistic action of the MAPK and Hippo signaling pathways.

LncRNA-THBS4 affects granulosa cell proliferation and apoptosis in diminished ovarian reserve by regulating PI3K/AKT/mTOR signaling pathway.

Recent studies have found Several lncRNAs were proved differential expression in diminished ovarian reserve (DOR) patients, however, the mechanism of DOR caused by lncRNAs is still largely unclear. High throughput sequencing was performed in ovarian GCs extracted from women with normal ovarian function and women with DOR. Bioinformation analysis was used to analyze the sequencing data and identify the differential expression of lncRNAs. Quantitative RT-PCR (qRT-PCR) was used to verify the sequencing results. Situ fluorescence hybridization (FISH) followed by confocal microscopy and qRT-PCR were used to explore the location and expression of LncRNA-THBS4 in GCs. The significantly enriched signaling pathways of LncRNA-THBS4 were identified by KEGG. The study used RNA interference technology to decipher LncRNA-THBS4 function by silencing LncRNA-THBS4 in GCs. Western blot and qRT-PCR were used to explore the mRNA and protein expressions of key factors of PI3Ks pathway. The pro-apoptotic protein and anti-apoptotic protein were detected by western blot. The proliferation and apoptosis of GCs were detected by MTT assay and Flow cytometry. 197 lncRNAs with significant differences in expression levels were detected between control and DOR group by high throughput sequencing. The study found the expression of LncRNA-THBS4 in GCs was positively correlated with Anti-Mullerian hormone (AMH) (p = 0.0020, r = 0.4742)、antral follicle count (AFC) (p = 0.0007, r = 0.5130)、good embryo rate (p = 0.0006, r = 0.5210), negatively correlated with basal FSH level (p = 0.0007, r = －0.5152). LncRNA-THBS4 was mainly localized in the cytoplasm of GCs. LncRNA-THBS4 silencing could inhibit the PI3Ks pathway; decrease the levels of anti-apoptotic protein, inhibit the proliferation of GCs; increase the levels of apoptosis protein, enhance the apoptosis of GCs. The expression level of lncRNA-THBS4 is correlated with ovarian function indicators and pregnancy outcomes in women. LncRNA-THBS4 may participate in the pathogenesis of DOR by affecting the proliferation and apoptosis of GCs via regulating PI3K/AKT/mTOR signaling pathway.

Simultaneous Activation of Beta-Oxidation and De Novo Lipogenesis in MASLD-HCC: A New Paradigm.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common cause of hepatocellular carcinoma (HCC). In this study, we combine metabolomic and gene expression analysis to compare HCC tissues with non-tumoural tissues (NTT). A non-targeted metabolomic strategy LC-MS was applied to 52 pairs of human MASLD-HCC and NTT separated into 2 groups according to fibrosis severity F0F1-F2 versus F3F4. The expression of genes related to de Novo lipogenesis (DNL) and fatty acid oxidation (FAO) has been analysed by quantitative RT-PCR and/or interrogation of RNA-seq datasets in 259 pairs of tissues (MASLD-HCC vs. VIRUS-HCC). Metabolomic analysis revealed that acylcarnitines were the main discriminating metabolites according to fibrosis severity when we compared MASLD-HCC-F0F1-F2 versus NTT and MASLD-HCC-F3F4 versus NTT. Based on these metabolomic data, the analysis of a panel of 15 selected genes related to DNL and FAO indicated that there is no difference between the 2 groups of MASLD-HCC. In contrast the same comparative gene analysis according to the aetiology of HCC: MASLD-HCC versus VIRUS-HCC showed that both aetiologies shared the same upregulation of genes involved in DNL. However, five genes involved in FAO (HADHA, CRAT, CPT1, CPT2 and PPARA) are upregulated exclusively in MASLD-HCC. This result indicates that FAO and DNL pathways are simultaneously activated in MASLD-HCC in contrast to VIRUS-HCC. These results suggest that, the involvement of adaptive metabolic pathways is different depending on the aetiology of HCC. Moreover, the dogma that simultaneous activation of FAO and DNL is incompatible in cancer would not apply to MASLD-HCC.