Detection Kit Research Articles

Dihydroartemisinin inhibits liver cancer cell migration and invasion by reducing ATP synthase production through CaMKK2/NCLX.

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) and mitochondrial sodium/calcium exchanger protein (NCLX) are key regulatory factors in calcium homeostasis. Finding natural drugs that target regulators of calcium homeostasis is critical. Dihydroartemisinin (DHA) is considered to have anticancer effects. The present study aimed to investigate the mechanism of DHA in regulating liver cancer migration and invasion. The present study used HepG2 and HuH-7 cells and overexpressed CaMKK2 and knocked down CaMKK2 and NCLX. The antiproliferative activity of DHA on liver cancer cells was assessed through colony formation and EdU assays. Cell apoptosis was detected through YO-PRO-1/PI staining. The levels of reactive oxygen species (ROS) were measured using a ROS detection kit (DCFH-DA fluorescent probe). Cell migratory and invasive abilities were examined using wound healing and Transwell assays. The ATP production of liver cancer cells was detected using ATP fluorescent probes. Cell microfilaments were monitored for changes using Actin-Tracker Green-488. The effects of DHA on the expression of CaMKK2, NCLX, sodium/potassium-transporting ATPase subunit α-1 (ATP1A1) and ATP synthase subunit d, mitochondrial (ATP5H) were determined by western blotting and reverse transcription-quantitative PCR. The results revealed that DHA significantly inhibited proliferation, reduced ROS levels and promoted apoptosis in liver cancer cells. CaMKK2 overexpression significantly enhanced the invasive and migratory ability of liver cancer cells, whereas DHA inhibited the pro-migratory effects of CaMKK2 overexpression. DHA significantly reduced the mitochondrial ATP production and altered the arrangement of microfilaments in liver cancer cells. In addition, DHA significantly decreased the expression of CaMKK2, NCLX, ATP1A1 and ATP5H. Furthermore, by knockdown experiments of NCLX the results demonstrated that CaMKK2 downregulated the expression of ATP1A1 and ATP5H in liver cancer cells through NCLX. In conclusion, DHA may reduce ATP synthase production via the CaMKK2/NCLX signaling pathway to inhibit the invasive phenotype of liver cancer cells. It is essential to further investigate the effectiveness of DHA in the anticancer mechanism of liver cancer cells.

MicroRNA-124a regulates the differentiation of bone marrow mesenchymal stem cells into neurons

Objective This study investigated the effects of microRNA-124a on the differentiation of bone marrow mesenchymal stem cells (BMSCs) and its underlying mechanism. Methods Flow cytometry was used for isolation and identification of BMSCs. Real-time polymerase chain reaction (RT-PCR) was used to detect gene mRNA expression. Apoptosis was detected using Annexin V-FITC/PI Apoptosis Detection Kit. Cell proliferation ability was tested using Cell Counting Kit-8 (CCK-8). The differentiation of BMSCs into neuron inducers β-thiol ethanol or baicalin formed the basis of the study. Results β-thiol ethanol markedly suppressed the microRNA-124a expression of BMSCs, baicalin markedly induced the microRNA-124a expression of BMSCs and β-thiol ethanol or baicalin promoted apoptosis and reduced the growth of BMSCs. Only the microRNA-124a inhibitor did not affect apoptosis or the differentiation of BMSCs, and it increased the effects of β-thiol ethanol or baicalin on the apoptosis of BMSCs. Conclusion β-thiol ethanol and baicalin treatment could affect microRNA-124a expression in BMSCs. We demonstrated that microRNA-124a promoted the differentiation of BMSCs into neurons.

Melatonin attenuates sevoflurane-induced hippocampal damage and cognitive deficits in neonatal mice by suppressing CypD in parvalbumin neurons

BackgroundSevoflurane, a commonly administered inhaled anesthetic, is found to induce synaptic and mitochondrial damage in neonatal mice. Mitochondrial membrane potential (MMP) changes, mediated by Cyclophilin D (CypD), are implicated in mitochondrial function. Melatonin, known for its significant neuroprotective properties, was investigated in this study to elucidate its mechanisms in mitigating the cognitive impairment caused by sevoflurane. MethodsThe mice were categorized into several groups, including the control, vehicle, sevoflurane, vehicle plus sevoflurane, and melatonin plus sevoflurane groups. From postnatal day 6 to day 8, the mice were administered inhaled sevoflurane or intraperitoneal melatonin. MMP and reactive oxygen species (ROS) were measured using appropriate detection kits. The protein expression levels of PSD95, Synapsin Ⅰ, and CypD in the hippocampus were analyzed through western blotting in acute and prolonged terms. Immunofluorescence staining was used to assess the co-localizations of PSD95 or CypD in parvalbumin (PV) neurons. Cognitive ability was evaluated through novel object recognition, social interaction experiment, and the Morris water maze. ResultsThe findings revealed that repeated exposure to sevoflurane in neonatal mice resulted in cognitive and synaptic impairment. Furthermore, melatonin administration suppressed the ROS and CypD protein expression, enhanced the MMP in mitochondria and synaptic protein expression in PV neurons, and ameliorated cognitive deficits. ConclusionMelatonin alleviated sevoflurane-induced cognitive deficits by suppressing CypD and promoting synaptic development in hippocampal PV neurons. These results provide valuable insights into a promising therapeutic approach for preventing neurotoxic injuries caused by general anesthetics.

Melatonin induces RAW264.7 cell apoptosis via the BMAL1/ROS/MAPK-p38 pathway to improve postmenopausal osteoporosis.

Currently, the effect of drug treatment for osteoporosis is relatively poor, and the side effects are numerous and serious. Melatonin is a potential drug to improve bone mass in postmenopausal women. Unfortunately, the mechanism by which melatonin improves bone metabolism remains unclear. The aim of this study was to further investigate the potential mechanism of melatonin in the treatment of osteoporosis. The effects of melatonin on mitochondrial apoptosis protein, bmal1 gene, and related pathway proteins of RAW264.7 (mouse mononuclear macrophage leukaemia cells) were analyzed by western blot. Cell Counting Kit-8 was used to evaluate the effect of melatonin on cell viability. Flow cytometry was used to evaluate the effect of melatonin on the apoptosis of RAW264.7 cells and mitochondrial membrane potential. A reactive oxygen species (ROS) detection kit was used to evaluate the level of ROS in osteoclast precursors. We used bmal1-small interfering RNAs (siRNAs) to downregulate the Bmal1 gene. We established a postmenopausal mouse model and verified the effect of melatonin on the bone mass of postmenopausal osteoporosis in mice via micro-CT. Bmal1 lentiviral activation particles were used to establish an in vitro model of overexpression of the bmal1 gene. Melatonin promoted apoptosis of RAW264.7 cells and increased the expression of BMAL1 to inhibit the activation of ROS and phosphorylation of mitogen-activated protein kinase (MAPK)-p38. Silencing the bmal1 gene weakened the above effects of melatonin. After that, we used dehydrocorydaline (DHC) to enhance the activation of MAPK-p38, and the effects of melatonin on reducing ROS levels and promoting apoptosis of RAW264.7 cells were also blocked. Then, we constructed a mouse model of postmenopausal osteoporosis and administered melatonin. The results showed that melatonin improves bone loss in ovariectomized mice. Finally, we established a model of overexpression of the bmal1 gene, and these results suggest that the bmal1 gene can regulate ROS activity and change the level of the MAPK-p38 signalling pathway. Our study confirmed that melatonin promotes the apoptosis of RAW264.7 cells through BMAL1/ROS/MAPK-p38, and revealed the therapeutic effect and mechanism of melatonin in postmenopausal osteoporosis. This finding enriches BMAL1 as a potential target for the treatment of osteoporosis and the pathogenesis of postmenopausal osteoporosis.

Hepatitis C and B prevalence in Faisalabad and its correlation to gender distribution.

Objective: To estimate the prevalence of hepatitis C and B in Faisalabad district with its association to gender difference. Study Design: Cross Sectional study. Setting: Department of Gastroenterology, General Hospital Ghulam-Muhammadabad, Faisalabad. Period: 01-08-2021 to 31-10-2021. Material & Methods: A sample size of 1827 population fulfilling the inclusion criteria was selected by using the probability sampling method. A blood sample of 5ml taken from each subject, centrifuged for 15 min according to manufacturer’s recommended speed (usually 1000-2000 RCF) and serum was stored at -20°C. The HBsAg kit (HBsAg2 from Abbott pharmaceuticals) for detection of HBsAg in serum and Hepatitis C kit (SD rapid test, SD BIOLINE. Data was analyzed with statistical analysis program (SPSS version 26). Results: Hepatitis C was found in 248 people (13.5%), while Hepatitis B was found in 95 people (5.1%). In a hepatitis gender study, we observed that a higher proportion of females were seropositive for hepatitis C. Conclusion: Hepatitis C is more common than hepatitis B in the Faisalabad district, but it's far less common than it was in earlier surveys, which may be because there are more facilities available.

Ferrostatin-1 attenuates hypoxic-ischemic brain damage in neonatal rats by inhibiting ferroptosis.

Hypoxic-ischemic brain damage (HIBD) is a type of brain damage that is caused by perinatal asphyxia and serious damages the central nervous system. At present, there is no effective drug for the treatment of this disease. Besides, the pathogenesis of HIBD remains elusive. While studies have shown that ferroptosis plays an important role in HIBD, its role and mechanism in HIBD are yet to be fully understood. The HIBD model of neonatal rats was established using the Rice-Vannucci method. A complete medium of PC12 cells was adjusted to a low-sugar medium, and the oxygen-glucose deprivation model was established after continuous hypoxia for 12 h. Laser Doppler blood flow imaging was used to detect the blood flow intensity after modeling. 2,3,5-triphenyl tetrazolium chloride staining was employed to detect ischemic cerebral infarction in rat brain tissue, and hematoxylin and eosin staining and transmission electron microscopy were used to observe brain injury and mitochondrial damage. Immunofluorescence was applied to monitor the expression of GFAP. Real-time quantitative polymerase chain reaction, western blot, and immunofluorescence were utilized to detect the expression of messenger RNA and protein. The level of reactive oxygen species (ROS) in cells was detected using the ROS detection kit. The results showed that ferrostatin-1 (Fer-1) significantly alleviated the brain injury caused by hypoxia and ischemia. Fer-1 significantly increased the expression of SLC3A2, SLC7A11, ACSL3, GSS, and GPX4 (P<0.05) and dramatically decreased the expressions of GFAP, ACSL4, TFRC, FHC, FLC, 4-HNE, HIF-1α, and ROS (P<0.05). Fer-1 inhibits ferroptosis and alleviates HIBD by potentially targeting the GPX4/ACSL3/ACSL4 axis; however, its specific mechanism warrants further exploration.

Calixarene: The Dawn of a New Era in Forensic Chemistry

Abstract: Globally, rapid, ultrasensitive, and ultraselective detection of forensic evidence is critically required for social security and support. In the past four decades, calixarenes have been the focus of intensive research in the invention of several extractants, transporters, stationary phases, electrode ionophores, and optical and electrochemical sensors. This review is ardent about calixarene, a new class of selective and sensitive forensic sensors. It has become clear that calix[n]arenes are particularly attractive building blocks for supramolecular chemistry due to their synthesis on a large scale, distinctive concave molecular architecture, tunable inner cavity size, exceptional derivatization capabilities, and a broad range of applications. This paper opens up new avenues for the creation of on-site detection kits for forensic evidence, which will speed up the detection procedure.

GGT1 Suppresses the Development of Ferroptosis and Autophagy in Mouse Retinal Ganglion Cell Through Targeting GCLC.

Glaucoma is a neurodegenerative disorder characterized with optic nerve injury and the loss of retinal ganglion cells (RGCs). Ferroptosis has been proved to be associated with the degradation of RGCs. The aim of this study is to elucidate the relationship between ferroptosis and glaucoma pathogenesis, and unveil the underlying mechanism. Methyl thiazolyl tetrazolium (MTT) assay was used to evaluate the proliferation of RGCs. The accumulation of cellular iron was measured by Iron assay kit, and the level of reactive oxygen species (ROS) was detected by fluorescence probe. The mitochondrial morphology and autophagosomes were analysed by using transmission electron microscopy (TEM). The contents of glutathione (GSH) and malondialdehyde (MDA) were tested by a GSH assay kit and an MDA detection kit, respectively. The expression of autophagy-related proteins was detected by Western blotting. A serious cell damage, aberrant iron homeostasis, and oxidative stress was shown in RGC-5 after oxygen-glucose deprivation/reoxygenation (OGD/R) treatment and gamma-Glutamyl transpeptidase 1 (GGT1) knockdown, but these effects were significantly alleviated by overexpression of GGT1 or ferroptosis inhibitors. The TEM and immunofluorescent results indicated that mitochondria impairment and autophagosome accumulation in OGD/R-treated cells was improved after GGT1 overexpression, while the phenomenon in GGT1-silenced cells was aggravated. Furthermore, we found that GGT1 can interact with glutamate cysteine ligase catalytic subunit (GCLC) to inhibit autophagy and ferroptosis in RGC-5 cells. GGT1 represses autophagy in RGC-5 cells by targeting GCLC, which further restrains the development of ferroptosis in cells.

A Novel Approach on the Sensitivity of Preserved Plasmodium falciparum Positive RDT kits for the Molecular Detection of Pfmdr 1 and Pfcrt Genes in Pregnant Women

Storing blood samples for molecular screening have become increasingly difficult due to unstable electricity and high cost of generators fuels. Samples often deteriorate giving false negative upon molecular analysis. This research was aimed at determining the sensitivity of preserved Plasmodium falciparum positive RDT kits for the molecular detection of Pfmdr 1 and Pfcrt genes among pregnant women in From February 2020 to May 2020, Sokoto, Nigeria. 200 pregnant patients who visited the hospital for ANC had their blood samples taken, and a questionnaire was given out. To detect Plasmodium falciparum, RDT kits tailored to its monoclonal antibody were utilized. P. falciparum positive Rapid Diagnostic Test Kits were air tightened with nylon and silica-preserved for three months in a closed cabinet. The preserved positive samples were utilized to extract DNA, and the Pfmdr1 and Pfcrt genes' existence was then checked using a PCR procedure. 4 samples tested positive for both genes, suggesting a 21.05 % prevalence, according to the results of the inquiry into the Pfcrt and Pfmdr1 genes. This study has revealed the sensitivity of preserved RDT kits (with silica) for DNA extraction. Researchers and molecular laboratories should be preserving positive RDT kits meant for DNA extraction and other molecular analysis as it is much easier to preserve and there is no cost involved.

Effect of miR-182-5p on apoptosis in myocardial infarction

ObjectiveThis study aimed to delineate the diagnostic significance of miR-182-5p by investigating its influence on myocardial apoptosis and function, employing both in vivo and in vitro myocardial infarction models. MethodsA rat myocardial infarction model was established. Myocardial infarction area was detected using the 2,3,5-chlorotriphenyltetrazolium (TTC) method, myocardial enzyme spectrums were measured using enzyme-linked immunosorbent assay (ELISA), myocardial structure was detected by hematoxylin and eosin (HE) staining, myocardial apoptosis was detected using the TUNEL method, and expression levels of miR-182-5p and apoptosis-related molecules were detected using real-time fluorescence quantitative PCR (qPCR) and Western blot. miR-182-5p mimics and inhibitor were transfected into rat H9C2 cardiomyocytes and mouse HL-1 cardiomyocytes to establish a hypoxia model. Cardiomyocyte viability was detected using the CCK-8 method, expression levels of apoptosis-related indicators were detected using Western blot, and caspase-3/7 activity was detected using a caspase-3/7 activity detection kit. AAV9 adeno-associated virus was used to construct an miR-182-5p overexpression virus, which was injected into mice through the tail vein to create a mouse myocardial infarction model. TTC, ELISA, HE staining, echocardiography, real-time fluorescence qPCR, and Western blot methods were used to detect the effects of AAV9-miR-182-5p on myocardial injury, myocardial function, and myocardial apoptosis levels in myocardial infarction. ResultsThe rat model displayed reduced miR-182-5p expression concurrent with an increase in apoptosis. The in vitro H9C2 and HL-1 hypoxia models revealed that miR-182-5p augmented the hypoxia-induced decrease in myocardial cell viability, suppressed Bcl-2 expression, and increased Bax, Bnip3, and caspase-3/7 activity levels. The injection of AAV9-miR-182-5p significantly exacerbated myocardial tissue damage, impaired myocardial function, and enhanced apoptosis. ConclusionmiR-182-5p escalates myocardial injury during myocardial infarction by fostering apoptosis. Interventions that aim to reduce miR-182-5p levels might be crucial in halting the progression of myocardial infarction.

Cytochrome P450 2B6 6 distribution among patients living with HIV in Burkina Faso

The function of cytochrome P450 (CYP) enzymes, one of the major catalysts in drug metabolism, is significantly influenced by genetic polymorphisms leading to substantial inter-individual variability in drug response and/ or adverse reactions activity. The aim of this study was to determine the frequency of the CYP2B6 516G&gt;T allele in patients living with HIV receiving treatment at the Medical Center of Camp General Aboubacar Sangoule Lamizana for whom the treatment was changed after the initiation of treatment because of the appearance of side effects. 155 HIV-positive patients were enrolled. Genomic Deoxyribonucleic acid (DNA) extraction from blood was performed using the GenJET® Genomic DNA according to the manufacturer’s instructions. Genotyping for the cytochrome P450 variant alleles was performed using predesigned primers. The amplification was carried out by Polymerase Chain Reaction (PCR), while the differentiation between the alleles was carried out after digestion with restriction enzymes by electrophoresis. The results obtained showed. Among these patients, 61 changed treatments during their follow-up. The majority of the 61 patients, 32 patients, received an initial treatment regimen based on the AZT (3'-Azido-3'-Deoxythymidine)/3TC (Lamivudine)/ NVP(Nevirapine) combination, followed by 18 patients for the TDF (tenofovir)/3TC (lamivudine)/ EFV(Efavirenz) regimens. Currently, the majority of patients, 48 patients, have benefited from a therapeutic regimen based on the TDF (tenofovir)/3TC (lamivudine) /DTG(Dolutegravir) combination, followed by 11 patients for the TDF (tenofovir)/3TC (lamivudine)/EFV(Efavirenz) regimens. The distribution of the major allele CYP2B6*T was 68%, and the minor CYP2B6*G allele was 32%. Heterozygote (GT genotype) and Homozygous mutants (TT genotype) and Heterozygote were 31% and 52,5%. Studies could be continued to set up cytochrome mutation detection kits to anticipate the onset of side effects and adapt treatments.

Characteristics of respiratory viruses' circulation through a six-year period (2016-2022) in a pediatric population in Normandy, France, and the impact of COVID-19 pandemic.

The impact of COVID-19 on the circulation of respiratory viruses has highlighted the need for ongoing surveillance and preparedness for emerging infectious diseases. The aim of this study was to provide information on the dynamics of respiratory viruses in pediatric populations to inform public health policies and strategies for the prevention and control of respiratory infections. This study analyzed data on the detection of respiratory viruses in pediatric populations from 2016 to 2022. The weekly number of tests and positive results for respiratory viruses were collected and analyzed from the two major university hospitals, Caen and Rouen. Different assays, including multiplex panels and rapid detection kits, were used for testing. The study analyzed the epidemiological trends of respiratory viruses in a French region during the COVID-19 pandemic. The data set included 37,969 tests, with a significant increase in the number of tests in 2020-2021 and 2021-2022. The rate of positive tests fell below 50% in 2021-2022. Enteroviruses (EV)/rhinoviruses (RV) and respiratory syncytial viruses (RSV) were the most frequently identified viruses, except in 2021-2022, when Flu ranked second. The Flu epidemic appeared slightly earlier in 2021-2022 and lasted for 25 weeks, with a doubling time of 4.03. The RSV epidemic occurred earlier in 2021-2022 and had similar dynamics to pre-pandemic years. EV/RV continued to circulate throughout the lockdowns, while low numbers of other viruses were detected. Our results emphasize the importance of continuing active surveillance of respiratory virus circulation and the need for new recommendations for respiratory virus detection in pediatric patients.IMPORTANCEThe report highlights an epidemiological change in the circulation of respiratory viruses in pediatric populations due to strategies adopted against COVID-19 pandemic. COVID-19 has resulted in a significant increase in requests for multiplex respiratory research to identify the virus responsible for the symptoms. The diagnostic needs have increased, and the number of samples analyzed in 2021-2022 is equal to the samples analyzed over the four epidemic periods preceding the pandemic. The report suggests the importance of active surveillance of respiratory viruses' circulation and new recommendations for respiratory virus detection in pediatric patients.

Evaluation of a novel lyophilized-pellet-based 2019-nCoV nucleic acid detection kit for the diagnosis of COVID-19.

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has swept the world and poses a serious threat to human health. In the post-pandemic-era, we must remain vigilant against the co-infection of SARS-CoV-2 and other respiratory viruses. More accurate and convenient detection methods are required for the diagnosis of SARS-CoV-2 due to its prolonged existence. In this study, the application value of a novel lyophilized-pellet-based 2019-nCoV nucleic acid diagnostic kit (PCoV-Kit) was evaluated by comparing it with a conventional liquid diagnostic kit (LCoV-Kit). We assessed the sensitivity, precision, accuracy, specificity, and amplification efficiency of PCoV-Kit and LCoV-Kit using diluted SARS-CoV-2 RNA reference materials. The results showed that both kits had high sensitivity, precision, accuracy, and specificity. A total of 2,033 oropharyngeal swab specimens collected during mass screening in Fuzhou in December 2022 were applied for the consistency analysis of the two reagents. In the detection of clinical oropharyngeal swab specimens, although the positive rate of PCoV-Kit (19.28%) was slightly lower than that of LCoV-Kit (20.86%), statistical analysis demonstrated a high degree of consistency between the test results obtained using both kit (χ2 = 1.57, P>0.05; Kappa coefficient = 0.90, 95%CI: 0.88-0.93). In conclusion, the use of lyophilized PCoV-Kit provides a non-inferior assay for the diagnosis of COVID-19.

Performance of a microfluidic immunofluorescence assay kit for equine influenza virus antigen detection

Equine influenza virus (EIV) infection is one of the most important respiratory diseases in the equine industry around the world. Rapid diagnosis, facilitated by point-of-care testing, is essential to implement movement restrictions and control disease outbreaks. This study evaluated a microfluidic immunofluorescence assay kit, which detects influenza virus and SARS-CoV-2 antigens in human specimens with a 12 min turnaround time, for its potential use in detecting EIV. The microfluidic immunofluorescence assay kit succeeded in detecting 11 EIV strains. Using the real-time reverse transcription polymerase chain reaction as a reference assay, the microfluidic immunofluorescence assay kit showed a sensitivity of 60.7% when evaluating nasopharyngeal swab samples of three horses experimentally infected with EIV. Comparing with the other two rapid antigen detection kits based on immunochromatography and silver amplification immunochromatography, the microfluidic immunofluorescence assay kit exhibited higher sensitivity than the former assay (53.6%) and the same sensitivity as the latter (60.7%). The microfluidic immunofluorescence assay kit did not detect nine non-EIV viruses including one equine coronavirus strain and seven bacteria, suggesting a high specificity for EIV antigens. Similar to other rapid antigen detection kits, the microfluidic immunofluorescence assay kit could be an effective diagnostic tool to detect EIV in the field.

Establishment and application of national reference panels for SARS-CoV-2 antigen detection kit

To develop a national reference panel for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen detection kit and establish a quality standard. The cultures of SARS-CoV-2 and other pathogens were collected to establish a national reference panel for SARS-CoV-2 antigen detection. The stability and homogeneity of the reference panel were evaluated. Based on World Health Organization (WHO) guidance and nucleic acid quantitative results, a quality standard reference panel was established. Currently, three generations of SARS-CoV-2 antigen national reference materials with batch numbers 370095–202001, 370095–202202, and 370095–202203 have been successfully established. These national reference panels comprised 8 positive samples, 20 negative samples, 1 repetitive sample, and 1 lower detection limit sample. The stability and homogeneity of the reference panel meet the requirements. The quality standards are as follows: the positive and negative coincidence rates are 8/8 and 20/20, respectively. The 10 test results of the medium and low-concentration repetitive reference materials should be positive, and the color rendering should be uniform (or the coefficient of variance should not be higher than 20.0%). The lower detection limit should be at least 5 × 105 U/mL (equivalent to copies/mL), and higher concentrations above the lower detection limit must be positive. A national reference panel for the SARS-CoV-2 antigen detection kit has been established. As the standard of SARS-CoV-2 antigen reagents, the reference panel has played a crucial role in the pre-marketing quality evaluation and post-marketing quality supervision in China.

Evaluation of Y Chromosome Microdeletion and Chromosome Analysis Results in Infertile Male Patients

Objective: Genetic testing for male infertility is rarely performed in our country. Male infertility is caused by chromosome number or structural problems, Y chromosome deletions and gene alterations. Infertility is a problem seen in 15% of couples. Genetic causes are responsible for the etiology of 3-10% of those diagnosed with male infertility due to oligozoospermia and azoospermia. In this retrospective study, we aimed to determine both the chromosomal structure and the microdeletion of the azoospermic factor (AZF) region on the Y chromosome in infertile men admitted to our center before the application of assisted reproductive techniques.&#x0D; Method: We studied 327 patients who applied to our laboratory for routine analysis. Chromosome analysis was performed from peripheral blood by conventional cytogenetic method. DNA was isolated from peripheral blood and Y chromosome microdeletion was analyzed by fragment analysis method with Y chromosome microdeletion detection kit.&#x0D; Results: Out of 327 patients, 32 had cytogenetic and 18 had molecular abnormalities and 4 had both cytogenetic and molecular abnormalities. Numerical and structural anomalies were detected in patients with anomalous karyotype. Among the patients with Y microdeletions, 1 patient had AZFa, 2 patient had AZFb, 6 patients had AZFc, 3 patients had AZFc+d, 2 patients had AZFb+c+d, 1 patient had AZFb+c+sY160, 1 patient had AZFa+b+d+c+sY90, and 2 patient had AZFb+d+c+sY90.&#x0D; Conclusion: Our study shows that chromosomal abnormalities and Y chromosome microdeletions are important causes of male infertility and that chromosome analysis and Y chromosome microdeletion tests should be performed to explain these abnormalities. It also emphasizes the importance of genetic counseling in explaining male infertility.

Establishment of real-time fluorescence and visual LAMP for rapid detection of Escherichia coli O157: H7 and kits construction.

E. coli O157: H7 is a common pathogenic bacterium in food and water that can pose a threat to human health. The aim of this study was to develop LAMP method for the detection of E. coli O157: H7 in food based on the specific gene Ecs_2840, and to construct rapid detection kits based on the established methods. Specifically, we established two methods of Real-time fluorescent LAMP (RT-LAMP) and visual LAMP with calcein as indicator. In pure bacterial culture, the cell sensitivity and genomic sensitivity of the RT-LAMP kit were 8.8×10°CFU·mL-1 and 4.61 fg·µL-1 respectively. The sensitivity of the visual LAMP kit were 2.35×10°CFU·mL-1 and 4.61 fg·µL-1. Both kits had excellent specificity and anti-interference performance. In addition, milk inoculated with 2.26×10°CFU·mL-1 E. coli O157: H7 could be detected within the reaction time after enrichment for 3 hours. The results showed that the LAMP kits were rapid, sensitive and specific for the detection of E. coli O157: H7 in food and had good application prospects in food safety surveillance.

Curcumol reverses temozolomide resistance in glioma cells by regulating the UTX/MGMT axis

To explore the mechanism through which curcumol reverses primary drug resistance in glioma cells. The inhibitory effect of 10, 20, and 40 μg/mL curcumol were observed in human glioma cell lines A172 and U251. UTX-overexpressing glioma cells constructed by lentiviral transfection were treated with curcumol (40 μg/mL), temozolomide (TMZ; 10 μg/mL), or both, and the changes in cell viability, clone formation capacity and apoptosis were assessed using MTT assay, cell clone formation experiment, and flow cytometry; UTX activity in the cells was determined using a UTX detection kit, and the enrichment of UTX and H3K27me3 in the MGMT promoter region was detected with ChiP-qPCR. The protein expressions in glioma cells were detected using Western blotting and immunohistochemistry. In a nude mouse model bearing glioma xenografts, the effects of curcumol (20 mg/kg), TMZ (20 mg/kg) and their combination on tumor growth and expressions of UTX, H3K27me3 and MGMT were evaluated. Curcumol significantly inhibited the proliferation (P<0.05) and promoted apoptosis of cultured glioma cells (P<0.01). Curcumol, but not TMZ, produced significant inhibitory effect on tumor growth in the tumor-bearing mice (P<0.01). Curcumol significantly inhibited UTX activity and increased the expression level of H3K27me3 protein in the glioma cells. UTX overexpression obviously decreased H3K27me3 protein expression and reversed the effects of curcumol on glioma cell proliferation and apoptosis (P<0.01). Curcumol reduced the enrichment of UTX and H3K27me3 in the MGMT promoter region (P<0.05) and decreased MGMT protein expression, which was reversed by UTX overexpression. In both the in vivo and in vitro experiments, curcumol combined with TMZ significantly increased H3K27me3 protein expression in the glioma cells, reduced the expression of its downstream target gene MGMT, and enhanced TMZ sensitivity of the glioma cells. Curcumol can enhance glioma cell sensitivity to TMZ by regulating the UTX/MGMT axis.

GSDME has prognostic and immunotherapeutic significance in residual hepatocellular carcinoma after insufficient radiofrequency ablation

BackgroundHeat stress can induce programmed cell death (PCD). Pyroptosis is a gasdermin-mediated PCD. This study hypothesized that insufficient radiofrequency ablation (IRFA) induced pyroptosis in hepatocellular carcinoma (HCC) and investigated its underlying mechanism and clinical significance. MethodsThermostatic water bath was used to stimulate IRFA in vitro. Cell viability was assessed by MTT assay. IL-1β and HMGB1 were measured by ELISA assay. LDH level was measured by LDH cytotoxicity detection kit. Permeability of cell membrane was assessed by Hoechst33342/PI fluorescence staining. RNA expression was evaluated by qRT-PCR, and protein was assessed by Western Blotting or immunofluorescence or immunohistochemistry. Gene expression with clinicopathological characteristics from HCC patients treated by RFA were analyzed for associations between GSDME expression and prognosis. ResultsOur study revealed that IRFA induced pyroptosis in HCCLM3 and HepG2 cells. GSDME, rather than GSDMD, was cleaved in heat stress-induced pyroptosis in HCCLM3 and HepG2 cells due to caspase-3 activation. However, GSDME overexpression promoted HCC growth in vivo and predicted poor PFS and OS in HCC patients treated by RFA. Heat stress modulated gene expression related to PD-L1 signaling and caspase inhibitors inhibited heat-induced PD-L1 expression in residual HCC after IRFA. Gsdme overexpression caused resistance to PD-L1 inhibitor in residual HCC after IRFA by increasing infiltrating of CD3+PD-1+ or CD3+CTLA-4+ exhausted T cells. ConclusionsThis study indicated that GSDME could serve as a potential prognostic biomarker and help to prescribe personalized sequential immunotherapy for HCC patients receiving RFA.

Dry eye disease among patients infected with the SARS-CoV-2 Omicron variant: a cross-sectional study.

To investigate the incidence of dry eye disease (DED) and relevant risk factors among patients infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant. This cross-sectional, observational analysis included 993 patients with corona virus disease 2019 (COVID-19) treated at the National Exhibition and Convention Center (Shanghai) Fangcang Shelter Hospital, from April 10 to May 26, 2022. Totally 944 uninfected control participants were recruited. All participants completed ocular surface disease index (OSDI) questionnaires, and DED symptoms were determined using OSDI scores. The demographic characteristics, length of hospital stay and in nasopharyngeal swabs were performed using questionnaires. SARS-CoV-2 Omicron variant infection was confirmed by nucleic acid-based detection in nasopharyngeal swabs using a 2019-nCoV nucleic acid detection kit. The risk factors for DED symptoms among patients with COVID-19 and control participants were determined by logistic regression analysis. Patients with COVID-19 showed a higher incidence of DED than controls (64.9% vs 55.1%, P<0.001). SARS-CoV-2 infection [odds ratios (ORs) (95%CI): 1.271 (1.038, 1.556)], use of contact lenses [ORs (95%CI): 9.350 (3.676, 23.783)], history of corneal refractive surgery [ORs (95%CI): 2.047 (1.494, 2.804)], poor sleep quality [ORs (95%CI): 2.657 (2.029, 3.480)], and video display terminal (VDT) use for more than 8h per day [ORs (95%CI): 6.348 (4.720, 8.538)] were found to be risk factors for DED symptoms in patients with COVID-19 as well as controls. For patients with COVID-19, the length of hospital stay [ORs (95%CI): 1.196 (1.134, 1.262)], use of contact lenses [ORs (95%CI): 20.423 (2.680, 155.632)], history of corneal refractive surgery [ORs (95%CI): 2.166 (1.321, 3.553)], poor sleep quality [ORs (95%CI): 3.650 (2.381, 5.597)], and VDT use for more than 8h per day [ORs (95%CI): 7.740 (4.918, 12.180)] were significant risk factors for DED symptoms. Patients with COVID-19 are more prone to develop symptomatic DED. SARS-CoV-2 infection and length of hospital stay are important risk factors for DED symptoms.