Search Results (87)

Background: Membrane potential represents one of the fundamental physiological characteristics of cells, playing a critical role in cellular function. Studies have shown that membrane hyperpolarization positively regulates the osteogenic differentiation of mesenchymal stem cells. Optogenetic technology based on the Natronomonas pharaonis halorhodopsin (NpHR) light-activated channel can induce membrane hyperpolarization through optical methods. Given the working principle of optogenetic technology, this study aimed to investigate whether optogenetic activation of NpHR channels could induce membrane hyperpolarization in human dental follicle stem cells (hDFCs)—mesenchymal stem cells derived from dental follicle tissue—to regulate their osteogenic differentiation. Methods: hDFCs were isolated and cultured. Engineered hDFCs expressing the NpHR channels were constructed through lentiviral transduction. Patch clamps were performed to observe the effects of optogenetic activation of NpHR channels on membrane potentials of hDFCs. Single-cell Ca²⁺ imaging were performed to observe the effects of membrane hyperpolarization via modulation of extracellular K⁺ concentration ([K⁺]_e) on the intracellular Ca²⁺ levels of hDFCs. Cell viability assay, transwell chamber assay, wound healing assay, osteogenic differentiation induction, alizarin red staining, alkaline phosphatase (ALP) staining, real-time reverse transcriptase polymerase chain reaction (RT-qPCR) and Western blot (WB) were performed to observe the effects of optogenetic activation of NpHR channels on proliferation, migration, and osteogenic differentiation of NpHR-hDFCs. Results: Reversing membrane hyperpolarization via modulation of extracellular K⁺ concentration ([K⁺]_e) was shown to suppress osteogenic differentiation of hDFCs, whereas promoting membrane hyperpolarization via opening ATP-sensitive K⁺ channels was shown to enhance osteogenic differentiation of hDFCs. Hyperpolarizing cells by decreasing [K⁺]_e increased intracellular Ca²⁺ levels of hDFCs. Optogenetic activation of NpHR channels by an optogenetic system induced membrane hyperpolarization and significantly enhanced the proliferation, migration, and osteogenic differentiation abilities of NpHR-hDFCs. Conclusions: Hyperpolarization by optogenetic activation of NpHR channels can promote hDFCs’ proliferation, migration, and osteogenic differentiation abilities. Full article

The aim of this preliminary study was to determine the effect of taurine on the expression of genes involved in glycolysis, oxidative phosphorylation, inflammation, autophagy, and regenerative activity in cultured peripheral blood mononuclear cells (PBMCs) and articular cartilage explants from patients with end-stage rheumatoid arthritis (RA). PBMCs and knee articular cartilage were obtained from 20 patients with RA (3 men and 17 women) aged 62.2 ± 10.9 years, with a mean disease duration of 17.5 years (range: 2–43), prior to arthroplasty. PBMCs and cartilage explants were cultured in the presence of 50 µM taurine. Gene expression was determined using real-time reverse transcriptase polymerase chain reaction (RT-PCR). Protein expression of the examined genes in PBMCs was quantified using ELISA. In the presence of 50 µM taurine PBMCs from patients with RA demonstrated a significant increase in the expression of genes encoding pyruvate kinase (PKM2), succinate dehydrogenase (SDHB), uncoupler of oxidation and phosphorylation (UCP2), ATP synthase (ATP5B), and unc-51-like kinase 1 (ULK1). At the same time a significant decrease in tumor necrosis factor (TNF)α and interleukin (IL)-1β expression was noted. In cartilage explants, taurine upregulated SDHB, UCP2, ULK1, and type 2 collagen gene (COL2A1), and decreased TNFα expression. We concluded that, under in vitro conditions, taurine can influence the expression of genes involved in glycolysis, oxidative phosphorylation, inflammation, autophagy, and regenerative processes in PBMCs and articular chondrocytes from patients with end-stage RA. Full article

Measles, also known as rubeola, is a highly contagious and potentially life-threatening disease caused by the measles virus. It classically presents with fever, cough, coryza, conjunctivitis, and a maculopapular rash. Despite the availability of an effective vaccine for decades, measles outbreaks continue to occur globally, largely driven by declining vaccination coverage and increased international travel. With no specific antiviral therapy available, rapid and accurate diagnosis remains essential for timely clinical management and effective outbreak control. Diagnostic methods have evolved from traditional virus isolation in cell culture to serologic assays and, more recently, to molecular techniques such as real-time reverse transcriptase polymerase chain reaction (rRT-PCR). Each diagnostic method has unique advantages and limitations influenced by specimen type, timing of collection, and laboratory capacity. This minireview summarizes the progress of measles virus diagnostics, outlines current laboratory detection strategies, and discusses emerging technologies and ongoing challenges amid global measles resurgence and increasing public health demands. Full article

Human rhinovirus (HRV) is the most common cause of upper respiratory tract infections and can cause substantial morbidity in children. Because its clinical features are nonspecific, differentiation from influenza virus and respiratory syncytial virus is often difficult, underscoring the diagnostic importance of real-time reverse transcriptase polymerase chain reaction (Real-Time RT-PCR)-based detection. This study aimed to characterize long-term epidemiological patterns of HRV in the Republic of Korea and assess their clinical and public health implications. We retrospectively analyzed 23,284 nasopharyngeal swab specimens collected between 2007 and 2024 from outpatients and inpatients presenting with influenza-like illness at a tertiary care hospital. HRV RNA was detected by Real-Time RT-PCR, and positivity rates were compared by year, month, and age group. Annual detection peaked in 2015 (31.3%) and 2016 (28.6%), then dropped sharply during the COVID-19 pandemic (2020–2021, 4.2–11.0%) and remained low through 2024. Seasonally, rates were highest in July (24.4%) and September (24.1%) and lowest in January (6.9%). Age-specific analysis showed peak positivity in children (26.1%) and infants (20.3%), with lower rates in adults (3.9%) and older adults (3.3%). These findings underscore the diagnostic value of HRV detection and provide evidence for pediatric-focused prevention, outbreak preparedness, and climate-informed surveillance strategies. Full article

We aimed to explore SARS-CoV-2 evolution during in vitro neutralisation using next generation sequencing, and to determine whether sera from individuals immunised with two doses of the Pfizer-BioNTech vaccine (BNT162b2) were as effective at neutralising the variant of concern (VOC) Delta (B.1.617.2) compared to the earlier lineages Beta (B.1.351) and wild-type (A.2.2) virus. Using a live-virus SARS-CoV-2 neutralisation assay in Vero E6 cells, we determined neutralising antibody titres (nAbT) against three SARS-CoV-2 strains (wild type, Beta, and Delta) in 14 participants (vaccine-naïve (n = 2) and post-second dose of BNT162b2 vaccination (n = 12)), median age 45 years [IQR 29–65]; the median time after the second dose was 21 days [IQR 19–28]. The determination of nAbT was based on cytopathic effect (CPE) and in-house quantitative reverse transcriptase real-time quantitative polymerase chain reaction (RT-qPCR) to confirm SARS-CoV-2 replication. A total of 110 representative samples including inoculum, neutralisation breakpoints at 72 h, and negative and positive controls underwent genome sequencing. By integrating live-virus neutralisation assays with deep sequencing, we characterised both functional antibody responses and accompanying viral genetic changes. There was a reduction in nAbT observed against the Delta and Beta VOC compared with wild type, 4.4-fold (p ≤ 0.0006) and 2.3-fold (p = 0.0140), respectively. Neutralising antibodies were not detected in one vaccinated immunosuppressed participant and the vaccine-naïve participants (n = 2). The highest nAbT against the SARS-CoV-2 variants investigated was obtained from a participant who was vaccinated following SARS-CoV-2 infection 12 months prior. Limited consensus level mutations occurred in the various SARS-CoV-2 lineage genomes during in vitro neutralisation; however, consistent minority allele frequency variants (MFV) were detected in the SARS-CoV-2 polypeptide, spike (S), and membrane protein. Findings from countries with high COVID-19 incidence may not be applicable to low-incidence settings such as Australia; as seen in our cohort, nAbT may be significantly higher in vaccine recipients previously infected with SARS-CoV-2. Monitoring viral evolution is critical to evaluate the impact of novel SARS-CoV-2 variants on vaccine effectiveness, as mutational profiles in the sub-consensus genome could indicate increases in transmissibility and virulence or suggest the development of antiviral resistance. Full article

Crimean–Congo hemorrhagic fever (CCHF) is a zoonotic viral disease endemic to parts of Africa, Asia and southeastern Europe. Bulgaria is one of the few European countries with the consistent annual reporting of human CCHF cases. This study provides a descriptive overview of 24 confirmed CCHF cases in Bulgaria between 2015 and 2024. Laboratory confirmation was performed by an enzyme-linked immunosorbent assay (ELISA) and/or real-time reverse transcriptase polymerase chain reaction (RT-qPCR) testing. Common findings included fever, fatigue, gastrointestinal symptoms, thrombocytopenia, leukopenia, liver dysfunction and coagulopathy. Two fatal cases were recorded. Two samples collected in 2016 and 2024 were subjected to whole-genome sequencing. Phylogenetic analysis showed that both strains clustered within the Turkish branch of the Europe 1 genotype and shared high genetic similarity with previous Bulgarian strains, as well as strains from neighboring countries. These findings suggest the long-term persistence of a genetically stable viral lineage in the region. Continuous molecular and clinical surveillance is necessary to monitor the evolution and public health impact of CCHFV in endemic areas. Full article

Reverse transcription polymerase chain reaction (RT-PCR) is an important tool for the detection of target RNA molecules and the assay of RNA pathogens. Coupled RT-PCR is performed with an enzyme mixture containing a reverse transcriptase and a thermostable DNA polymerase. To date, several biotechnological companies offer artificial thermostable DNA polymerases with a built-in reverse transcriptase activity for use in the coupled RT-PCR instead of the enzyme mixtures. Here, we compared the artificial DNA polymerases and conventional enzyme mixtures for the RT-PCR by performing end-point and real-time RT-PCR assays using severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV2) RNA and endogenous mRNA molecules as templates. We found that the artificial enzymes were suitable for different RT-PCR applications, including SARS-CoV2 RNA detection but not for long-fragment RT-PCR amplification. Full article

Highly pathogenic avian influenza (HPAI) surveillance for influenza A virus (IAV) in the United States is conducted using a National Animal Health Laboratory Network (NAHLN) real-time reverse transcriptase–polymerase chain reaction (rRT-PCR). Samples showing the presence of IAV are confirmed and characterized at the national reference laboratory. During the H5N1 HPAI outbreak in 2022, our laboratory reported the detection of IAV in a PA commercial chicken flock using rRT-PCR targeting the matrix gene, which was negative for the H5/H7 subtypes. IAV was not detected by additional sampling of the birds the following day with rRT-PCR. The virus detected was characterized as a human seasonal H3N2 with whole-genome sequencing (WGS). Further investigation revealed that the collector who visited the farm was diagnosed with an IAV infection. This case report emphasizes the importance of farm biosafety and biosecurity, of conducting regular reviews of worker safety protocols, and of advanced molecular techniques like WGS for viral characterization and epidemiology. Full article

Background: Tube thoracostomy, utilized through conventional methodologies in the context of pleural disorders such as pleural effusion and pneumothorax, constitutes one of the primary therapeutic interventions. Nonetheless, it is imperative to recognize that invasive procedures, including tube thoracostomy, are classified as aerosol-generating activities during the management of pleural conditions in patients afflicted with COVID-19, thus raising substantial concerns regarding the potential exposure of healthcare personnel to the virus. The objective of this investigation was to assess the SARS-CoV-2 viral load by detecting viral RNA in pleural drainage specimens from patients who underwent tube thoracostomy due to either pleural effusion or pneumothorax. Methods: In this single-center prospective cross-sectional analysis, a real-time reverse transcriptase (RT) polymerase chain reaction (PCR) assay was employed to conduct swab tests for the qualitative identification of nucleic acid from SARS-CoV-2 in pleural fluids acquired during tube thoracostomy between August 2021 and December 2021. Results: All pleural drainage specimens from 21 patients who tested positive for COVID-19 via nasopharyngeal PCR, of which 14 underwent tube thoracostomy due to pneumothorax, 4 due to both pneumothorax and pleural effusion, and 3 due to pleural effusion, were found to be negative for SARS-CoV-2 RNA. Moreover, individuals exhibiting pleural effusion were admitted to the intensive care unit with a notably higher incidence, yet demonstrated significantly more radiological anomalies in patients diagnosed with pneumothorax. Conclusions: The current findings, inclusive of the results from this study, do not furnish scientific evidence to support the notion that SARS-CoV-2 is transmitted via aerosolization during tube thoracostomy, and it remains uncertain whether the virus can be adequately contained within pleural fluids. Full article

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. Full article

Backgound/Objectives: Cycle threshold (Ct) values of SARS-CoV-2 real-time reverse transcriptase-polymerase chain reaction (RT-PCR) tests are associated with infectivity and viral load, and they could be an aid in forecasting the evolution of SARS-CoV-2 outbreaks. The objective was to know the Ct values related to the incidence and reinfection of SARS-CoV-2 in successive outbreaks, which took place in nursing homes in Castellon (Spain) during 2020–2022, and to test its usefulness as an instrument of epidemic surveillance in nursing homes. Methods: a retrospective cohort design with Poisson regression and multinomial logistic regression were used. Results: We studied four nursing home SARS-CoV-2 outbreaks, and the average infection rate, reinfection rate, and case fatality were 72.7%, 19.9%, and 5.5%, respectively; 98.9% of residents were vaccinated with three doses of a mRNA SARS-CoV-2 vaccine. Ct values for first infections and reinfections were 27.1 ± 6.6 and 31.9 ± 5.4 (p = 0.000). Considering Ct values ≥ 30 versus <30, residents with reinfections had Ct values higher than residents with a first infection, an adjusted relative risk of 1.66 (95% Confidence interval 1.10–2.51). A sensitivity analysis confirmed these results. Conclusions: Reinfection and SARS-CoV-2 vaccination (hybrid immunity) could protect against severe disease better than vaccination alone. High Ct values suggest lower transmission and severity. Its value can be useful for surveillance and forecasting future SARS-CoV-2 epidemics. Full article

Radioresistance poses a significant challenge in the effective treatment of cervical cancer, often leading to poor patient outcomes. MicroRNA-21 (miR-21) and MicroRNA-145 (miR-145) are oncogenic micro-RNAs associated with various cancers, including cervical cancer, but their potential as predictive biomarkers for radioresistance remains underexplored. This study aimed to investigate the association between miR-21 and miR-145 expressions and the response to radiation therapy in cervical cancer patients. An analytical cross-sectional study was conducted on 140 subjects with cervical cancer stages IIIB and IVA who received definitive radiotherapy. miR-21 and miR-145 expressions were measured using real-time reverse transcriptase–polymerase chain reaction (RT-qPCR). A total of 102 subjects (72.9%) were classified as having stage III cervical cancer, and 38 subjects (27.1%) were classified as having stage IV cervical cancer. Disease progression occurred in 60.7% of subjects. The cut-off value for miR-21 expression was 0.00088 nmol/(mg/mL) (AUC 0.676, sensitivity 70.8%, specificity 50.8%), and a higher expression was significantly associated with radioresistance (p = 0.010). miR-145, with a cut-off of 0.0239 nmol/(mg/mL) (AUC 0.612, sensitivity 67.5%, specificity 45.5%), showed no significant association with treatment response (p = 0.132). Combining miR-21 and miR-145 (AUC 0.639, sensitivity 68.6%, specificity 46.9%, p = 0.063) did not significantly improve the predictive accuracy. This study suggests that an elevated miR-21 expression is significantly associated with radioresistance in cervical cancer patients, while miR-145 expression shows no significant correlation with treatment response. Additionally, combining miR-21 and miR-145 does not enhance the predictive power. Full article

The urgent need for accurate COVID-19 diagnostics has led to the development of various SARS-CoV-2 detection technologies. Real-time reverse transcriptase polymerase chain reaction (RT-qPCR) remains a reliable viral gene detection technique, while other molecular methods, including nucleic acid amplification techniques (NAATs) and isothermal amplification techniques, provide diverse and effective approaches. Serological assays, detecting antibodies in response to viral infection, are crucial for disease surveillance. Saliva-based immunoassays show promise for surveillance purposes. The efficiency of SARS-CoV-2 antibody detection varies, with IgM indicating recent exposure and IgG offering prolonged detectability. Various rapid tests, including lateral-flow immunoassays, present opportunities for quick diagnosis, but their clinical significance requires validation through further studies. Challenges include variations in specificity and sensitivity among testing platforms and evolving assay sensitivities over time. SARS-CoV-2 antigens, particularly the N and S proteins, play a crucial role in diagnostic methods. Innovative approaches, such as nanozyme-based assays and specific nucleotide aptamers, offer enhanced sensitivity and flexibility. In conclusion, ongoing advancements in SARS-CoV-2 detection methods contribute to the global effort in combating the COVID-19 pandemic. Full article

Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory infections in young children worldwide. RSV-associated deaths in children are underreported in Bangladesh. We analyzed hospital-based surveillance data on severe acute respiratory infections (SARIs) in under-five children before (August 2009–February 2020) and during the COVID-19 pandemic (March 2020–March 2022). Using the World Health Organization definition, we identified SARI cases in 14 tertiary-level hospitals. Nasopharyngeal and oropharyngeal swabs were collected for real-time reverse-transcriptase–polymerase chain reaction (rRT-PCR) testing of six respiratory viruses, including RSV. SARI deaths during the pandemic (2.6%, 66) were higher than pre-pandemic (1.8%, 159; p < 0.001). Nearly half of pandemic deaths (47%) had underlying respiratory viruses, similar to the pre-pandemic rate (45%). RSV detection in deaths was consistent pre-pandemic (13%, 20/159) and during the pandemic (12%, 8/66). Children aged < 6 months constituted 57% (16) of RSV-related deaths. Evaluating interventions like maternal vaccination and infant monoclonal antibody prophylaxis is crucial to address RSV, a major contributor to under-five SARI deaths. Full article

Background: Coagulation disorders are frequently encountered among patients infected with coronavirus disease 2019 (COVID-19), especially among admitted patients with more severe symptoms. This study aims to determine the mortality rate and incidence and risk factors for venous thromboembolism (VTE) in hospitalized patients with COVID-19. Methods: This retrospective observational cohort study was conducted from March to July 2020 using a hospital database. All adult patients (>18 years old) with laboratory-confirmed COVID-19 were included. Laboratory data and the real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) for SARS-CoV-2 were obtained from medical records. The mortality rate and the incidence of VTE were established as study results. A multivariate logistic regression analysis was performed to identify predictors of thrombotic events. Results: rA total of 1024 confirmed COVID-19 patients were treated, of whom 110 (10.7%) were deceased and 58 patients (5.7%) developed VTE. Death occurred more frequently in patients older than 50 years and those admitted to the intensive care unit (ICU, 95%) and who received mechanical ventilation (62.7%). Multivariate analysis revealed that cancer patients were two times more likely to have VTE (adjusted odds ratio = 2.614; 95% CI = (1.048–6.519); p = 0.039). Other chronic diseases, such as diabetes, hypertension, and chronic kidney disease, were not associated with an increased risk of VTE. Conclusions: One-tenth of hospitalized COVID-19 patients were deceased, and VTE was prevalent among patients with chronic conditions, such as cancer, despite anticoagulation therapy. Healthcare professionals should closely monitor individuals with a high risk of developing VTE to prevent unwanted complications. Full article