Polymerase Chain Reaction Kit Research Articles

Detection of Bordetella spp. in children with pertussis-like illness from 2018 to 2024 in China

ObjectiveTo evaluate the role of of Bordetella pertussis (B. pertussis), B. parapertussis, B. holmesii, and B. bronchiseptica on pertussis resurgence in China, particularly the sharp rise since the latest winter. MethodsNasopharyngeal swabs collected from children with pertussis-like illness from January 2018 to March 2024 were cultured to detect B. pertussis, B. parapertussis, B. holmesii, and B. bronchiseptica, and tested for all of these except for B. bronchiseptica using a pooled real-time polymerase chain reaction (PCR) kit targeting insertion sequences ptxS1, IS481, IS1001, and hIS1001. ResultsOut of the collected 7732 nasopharyngeal swabs, 1531 cases tested positive for B. pertussis (19.8%, 1531/7732), and 10 cases were positive for B. parapertussis (0.1%, 10/7732). B. holmesii and B.bronchiseptica was not detected. The number of specimens and the detection rate of B. pertussis were 1709 and 26.9% (459/1709) in 2018, 1936 and 20.7% (400/1936) in 2019, which sharply declined to 308 and 11.4% (35/308) in 2020, 306 and 4.2% (13/306) in 2021, and then notably increased to 754 and 17.6% (133/754) in 2022, 1842 and 16.0% (295/1842) in 2023, 196 and 22.4% (196/877) in the first quarter of 2024. The proportion of children aged 3 to less than 6 years (preschool age) and 6 to 16 years (school age) in pertussis cases increased significantly during the study period, especially the proportion of school-aged children increased from 2.0% (9/459) in 2018 to 40.8% (80/196) in 2024. ConclusionsB. pertussis was the predominant pathogen among children with pertussis-like illness in China, with sporadic detection of B. parapertussis and no detection of B. holmesii or B.bronchiseptica. The preschool and school-age children are increasingly prevalent in B. pertussis infection cases, which may be associated with the latest rapid escalation of pertussis outbreak.

Exploring Melezitose as a Potential Therapeutic Agent in Lung Cancer: Inhibitory Effects on Cell Proliferation and EMT-Mediated Signaling in A549 Cells

Background Lung cancer is one of the most common cancers worldwide and a leading cause of cancer-related deaths. The study delves into melezitose, a naturally occurring compound known for its biocompatibility. Purpose This study aims to uncover its therapeutic potential and molecular mechanisms within lung cancer, particularly in A549 cells. Melezitose’s impact on inhibiting cell proliferation and influencing epithelial-mesenchymal transitions (EMT) was the primary focus. Material and Methods In a time-dependent manner, A549 cells, representative of lung adenocarcinoma (LUAD), underwent melezitose treatment. Analysis of cytotoxicity by MTT assay, cell migration assay, and its responsible genes were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) kit method. Results The MTT assay indicated a significant reduction in A549 cell growth after 48 hours of melezitose treatment. Additionally, melezitose induced G1 phase cell arrest and stimulated apoptosis in A549 cells. Subsequent determination of IC50 values represents the concentration at which melezitose inhibits 50% of cell growth. The study also investigated EMT-related gene expression like claudin 1 (CLD1), E-cadherin (ECADH), SNAIL1, SLUG, and vimentin (VIM) through RT-PCR. The findings revealed strong binding associations between melezitose and these EMT targets, suggesting a potential regulatory role of melezitose in impeding EMT processes. Conclusion Overall, this study illuminates the significant role of melezitose in lung cancer. Its observed inhibition of lung cancer cell proliferation and its influential impact on EMT-related gene expression highlights. Melezitose has potential as a therapeutic agent, particularly in the context of NSCC. The multifaceted effects of melezitose on A549 cells open promising avenues for advancing our understanding of this disease and developing innovative therapeutic strategies.

Preliminary study on the diversity and quantity analysis of oral bacteria according to the sampling methods

Objectives: Oral bacterial samples included subgingival, supragingival, and saliva plaques. As the diversity and number of microorganisms deffer depending on the area of the oral cavity and the method used, an appropriate and reliable collection method is important. The present study investigated oral bacterial sampling methods. Methods: Supragingival dental plaque was collected from the buccal and lingual tooth surfaces of study participants using sterilized cotton swabs. Plaques were collected from the subgingival area using a sterilized curette. Bacterial genomic DNA was extracted using MagNA Pure 96 DNA and Viral NA low-volume kits. Real-time polymerase chain reaction (PCR) was performed using the PowerCheck™ Periodontitis Pathogens Multiplex Real-time PCR kit. Results: Aggregatibacter actinomycetemcomitans, Prevotella intermedia, and Fusobacterium nucleatum of the orange complex were not observed in the subgingival biofilms of all study participants. For Porphyromonas. gingivalis, a significant correlation was observed between supragingival, subgingival, and total tooth surface biofilms. Compared to the supragingival and subgingival biofilmss, total tooth surface biofilm exhibited the highest bacterial count when the inswabbing method was used. Conclusions: Based on these findings, the supragingival swab method is recommended for oral bacterial research.

Prevalence and predictors of post-acute COVID syndrome among infected healthcare workers at University Malaya Medical Centre.

Post Acute COVID Syndrome (PACS), a complex and poorly understood condition characterised by persistent symptoms following the acute phase of COVID-19 infection, has emerged as a significant global health concern. Healthcare workers who had been at the forefront of the pandemic response are at heightened risk of contracting the virus and subsequently developing PACS. Therefore, we aim to determine the prevalence and risk factors for PACS among healthcare workers infected with COVID-19. A cross-sectional study was conducted between October 2022 and August 2023 using an online REDCap electronic data capture tool questionnaire. PACS was defined as new or persistent symptoms lasting more than 28 days after a positive SARS-CoV-2 polymerase chain reaction or rapid test kit antigen test. Multivariable logistic regression was performed to determine predictors associated with PACS. Among 609 infected healthcare workers, they were predominantly female (71.8%), Malays (84.6%), and aged 18-39 years (70.1%). 50.7% of infected healthcare workers experienced PACS. The most common PACS symptoms experienced were fatigue (27.9%), cough (25.1%), decreased physical strength (20.5%), and musculoskeletal pain (19.2%). Those who are more likely to develop PACS were females, underlying asthma, and COVID-19 severity category 3. On the other hand, those who received booster vaccinations were less likely to develop PACS. PACS is prevalent among healthcare workers with COVID-19 at the University Malaya Medical Centre. These findings emphasise the critical need for those with higher risk to receive regular health monitoring and checkups to detect any early signs of PACS. It underscores the need for continuous support and healthcare interventions to mitigate the impacts of PACS and ensure the physical and mental well-being of healthcare workers.

Evaluation of analytical performance of the STANDARDTM M10 MPX/OPX assay for the simultaneous DNA detection and clade attribution of Monkeypox virus

ABSTRACT Monkeypox virus (MPXV) infection confirmation needs reliable polymerase chain reaction (PCR) assays; in addition, viral clade attribution is a key factor in containment measures, considering a more severe syndrome in clade I and the possibility of simultaneous circulation. This study evaluates the performance of all-in-one STANDARD M10 MPX/OPX (SD BIOSENSOR, South Korea – M10). Frozen samples from 205 subjects were selected and stratified according to routine test results (RealStar® Orthopoxvirus PCR Kit 1.0, Altona DIAGNOTICS, Germany – RS; RS-1): in detail, 100 negative skin lesions (SL) and 200 positive samples at the variable stage of infection were analysed. Positive samples were retested with RS (RS-2). Positive and Negative Percent Agreements (PPA, NPA) were calculated. The median (IQR) C t values of RS and M10 (OPXV target) assays were highly similar. The PPA of M10 compared to RS-1 was 89.5% considering system interpretation, and 96.0% when the operator classified results as positive if any target was detected; NPA was 100%. Comparing the RS-2 run and M10, an overall concordance of 95.3% between assays was found; however, considering operator interpretation, M10 returned more positive results than RS-2. The occurrence of False-Negative results was likely associated with the influence of thawing on low viral concentration; no False-Positive tests were observed. All samples collected at the time of Mpox diagnosis were positive and M10 correctly attributed the clade (West-Africa/II). The M10 MPX/OPX assay demonstrated high reliability in confirming MPXV infection and clade attribution.

PCR in Forensic Science: A Critical Review.

The polymerase chain reaction (PCR) has played a fundamental role in our understanding of the world, and has applications across a broad range of disciplines. The introduction of PCR into forensic science marked the beginning of a new era of DNA profiling. This era has pushed PCR to its limits and allowed genetic data to be generated from trace DNA. Trace samples contain very small amounts of degraded DNA associated with inhibitory compounds and ions. Despite significant development in the PCR process since it was first introduced, the challenges of profiling inhibited and degraded samples remain. This review examines the evolution of the PCR from its inception in the 1980s, through to its current application in forensic science. The driving factors behind PCR evolution for DNA profiling are discussed along with a critical comparison of cycling conditions used in commercial PCR kits. Newer PCR methods that are currently used in forensic practice and beyond are examined, and possible future directions of PCR for DNA profiling are evaluated.

Use of the Investigator ESSplex SE QS Kit (QIAGEN) at Half PCR Reaction Volumes for the Analysis of Forensic Samples

The Investigator ESSplex SE QS Kit (Qiagen) is a next-generation polymerase chain reaction (PCR) kit that, in 60 min, amplifies 17 Short Tandem Repeat (STR) markers, including the five European Standard Set (ESS) loci (D10S1248, D12S391, D1S1656, D22S1045, D2S441), the SE33 marker, and the locus Amelogenin for sex determination. Two quality sensors (QS1 and QS2) are also co-amplified to check PCR performance. Since forensic laboratories carry out hundreds of DNA typings annually, we verified the kit’s performance using half reaction volumes with the aim of improving the number of samples that may be amplified with a single kit and consequently reducing laboratory costs. In the present study, intended as a technical note rather than internal validation, some control samples (oral swabs) with known DNA profiles and 40 real casework samples were analyzed. We observed that reducing the total reaction volume, while keeping all component ratios unaltered, yields DNA profiles comparable to those obtained using standard reaction volumes and with allele peaks higher than those with regular volumes. Using half volumes for PCR amplification enables the analysis of a larger number of samples compared to the standard protocol, thereby reducing laboratory costs without compromising the quality of the analysis.

Pre-admission virus detection during the COVID-19 pandemic in children with and without symptoms of infection.

Pre-admission viral screening is used only in exceptional situations such as pandemics. We therefore evaluated pre-admission screening for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respiratory syncytial virus (RSV) and influenza during the COVID-19 pandemic, comparing epidemiology and clinical features of admitted children. Children were screened at a paediatric emergency department from 1 March 2020 to 30 June 2022 by nasopharyngeal sampling and polymerase chain reaction kit. We retrospectively retrieved positive results from the laboratory and scrutinised charts of admitted children. Out of 15 927 screened children, 522, 127 and 572 were positive and admitted with RSV, influenza A or SARS-CoV-2, respectively. Of these, 29 (5.6%), 26 (24.1%) and 245 (44.8%) were incidental findings, lacking symptoms of infection. RSV and influenza A were initially absent but re-emerged in the autumn of 2021. The rate of COVID-19 rose when the Omicron variant emerged in December 2021. The median age of children with RSV was 0.3 years, of those with influenza A 6.7 years and of those with COVID-19 1.6 years. Major complications were rare. Frequent incidental detections of SARS-CoV-2 likely reflected widespread presence of a mild infection. Clinically, COVID-19 was like other viral respiratory infections in children.

Investigation of gut microbiota diversity according to infectious agent in pediatric infectious acute gastroenteritis in a Korean university hospital

BackgroundAcute gastroenteritis (AGE) is a common cause of pediatric morbidity and mortality worldwide. AGE can cause an imbalance in the intestinal microbiota. This study aimed to investigate the diversity of the gut microbiome in Korean children hospitalized for infectious AGE at a university hospital. MethodsA total of 23 stool samples from patients aged 5 months to 11 years with AGE were analyzed. Thirteen convalescent stool samples were collected 1 month after discharge. Multiplex polymerase chain reaction (PCR) for the five viruses and 16 bacteria-specific AGE pathogens (PowerChek Multiplex Real time PCR Kit, Seoul, Korea), and 16s rRNA sequencing (Illumina MiSeq Sequencing system, Illumina, USA) were performed. ResultsAccording to the results of multiplex PCR for causative pathogens, the microbiome taxonomic profile (MTP) of the gut microbiome in three groups of AGE, norovirus AGE (n = 11), Campylobacter AGE (n = 7) and Salmonella AGE (n = 5) was compared. The phylum Actinobacteria was significantly more abundant in the norovirus AGE (P = 0.011), whereas the phylum Proteobacteria was significantly more abundant in Salmonella AGE (P = 0.012). Alpha diversity, which indicates species richness and diversity, showed no statistical differences. However, beta diversity, representing the similarity in MTP between norovirus, Campylobacter, and Salmonella AGE, was significantly different (P = 0.007). In convalescence, compared with their corresponding AGE samples, the phylum Firmicutes; and the lower taxa Christensenellaceae (P = 0.0152) and Lachnospiraceae (P = 0.0327) were significantly increased. ConclusionsIn pediatric AGE, the type of infectious agent can affect the diversity and dominance of gut microbiota in pediatric patients. Furthermore, healthy gut bacteria increased during the period of 1 month after infection, allowing a return to a healthy state without causing long-term dysbiosis.

Clinical use of molecular methods for Trypanosoma cruzi infection in endemic and non-endemic countries: Benefits, limitations and challenges

Trypanosoma cruzi infection is diagnosed by parasitological, molecular, and serological tests. Molecular methods based on DNA amplification provide a more sensitive alternative to classical parasitological techniques for detecting evidence of T. cruzi parasitemia, and are the preferred tests for congenital and oral transmission cases and parasite reactivation in chronically infected immunosuppressed individuals. In newborns at risk of vertical transmission, simplified diagnostic algorithms that provide timely results can reduce the high follow-up losses observed with current algorithms. Molecular methods have also proved useful for monitoring T. cruzi infection in solid organ transplantation recipients, regardless of host immune status, allowing parasite detection even before symptom manifestation. Furthermore, in the absence of other biomarkers and a practical test of cure, and given the limitations of serological methods, recent clinical guidelines have included polymerase chain reaction (PCR) to detect therapeutic failure after antiparasitic treatment in chronically infected adults. Increasing evidence supports the use of molecular tests in a clinical context, given the improved sensitivity and specificity of current assays – characteristics which largely depend on epidemiological factors and genetic and antigenic variability among T. cruzi strains. Further development and registration of commercial PCR kits will improve the use of molecular tests. We discuss the attributes of PCR and other molecular tests for clinical management in people with T. cruzi infection.

Congenital cytomegalovirus infection cases and follow-up findings in Antalya, Turkey

Objective: To investigate the presence of cCMV infection and the CMV-DNA virus in the newborns who applied for newborn hearing
 screening test (NHST) and CMV-DNA viruria with physical, mental-motor development and hearing status of cCMV cases in the
 second year of age.
 Patients and Methods: CMV-DNA was investigated in 1150 newborns’ oral swabs (0-21 days) by polymerase chain reaction kit and
 urine of patients with positive CMV-DNA in saliva. Transient Evoked Otoacoustic Emission test was performed for NHST.
 Results: CMV-DNA was posititve in saliva of 38 (3.3%) newborns and urine of 10 out of 37 newborns. The prevalence of cCMV
 was 0.87% (95% CI=0.697-1.042). All newborns passed the NHST. In newborns with cCMV:jaundice in 60% (6/10), low birthweight
 in 40% (4/10), small for gestational age in 50% (5/10) of them. Jaundice was the most significant variable (P

Development of a Reagent Kit for the Quantitative Determination of Hepatitis B Virus (HBV) DNA in Clinical Material by PCR with Hybridization-Fluorescence Detection

Relevance.Hepatitis B virus (HBV) is one of the most common viral infections affecting people worldwide and can lead to chronic hepatitis, cirrhosis and hepatocellular carcinoma. Currently 3% of the world's population are infected with hepatitis B virus and are at risk of developing life-threatening liver disease. Immunological and molecular biological methods of detection of HBV are currently used in laboratory diagnostics. The polymerase chain reaction (PCR) is currently the most sensitive method for the detection and quantification of HBV. HBV DNA quantification is widely used to monitor the antiviral treatment of HBV infection.Aim.To develop a real-time PCR kit for the quantification of HBV DNA.Materials and methods.A total of 200 plasma and serum samples positive and negative for HBV were used in the development. The performance of the developed kit was compared with the use of other commercially registered HBV diagnostic kits in Russia. Additionally, the nucleotide sequences of all existing virus genotypes analysed for the selection of primers using GeneBank system.Results and discussion.Comparison analysis of the results of quantitative determination by real-time PCR in 200 clinical serum and blood plasma samples showed that the diagnostic sensitivity of the developed kit was 100% and specificity 100%. The primers developed specific to the POL gene region. The kit is capable of detecting all types of virus genotypes.Conclusions.The developed reagent kit allows detection of hepatitis B virus and determination of its quantity within 70 minutes. In addition to a large number of genotypes and subgenotypes, the virus is characterized by mutational changes in the genome, which complicates its diagnosis and, as a consequence, the ongoing therapy with drugs. Conservative regions for primer and probe selection taken into account in the development, and the sequencing results obtained are applicable to all HBV genotypes. The reagent kit is designed to monitor HBV infected patients and will allow the analysis of different HBV viral loads.

Lessons from Real Life Experience: Importance of In-House Sequencing and Smart Ratio-Based Real-Time PCR Outperform Multiplex Ligation-Dependent Probe Amplification in Prenatal Diagnosis for Spinal Muscular Atrophy: Bench to Bedside Diagnosis.

Spinal muscular atrophy (SMA) is a rare, recessively inherited neurodegenerative disorder caused by the presence of pathogenic variants in the SMN gene. As it is the leading inherited cause of infant mortality, identification of SMN gene pathogenic variant carriers is important for diagnostic purposes with effective genetic counseling. Multiple ligation probe analysis (MLPA), a probe-based method, is considered as the gold standard for SMA carrier analysis. However, MLPA might give false-negative results in cases with variations in the probe-binding regions. Here, we present a case born to consanguineous SMA carrier parents. Prenatal diagnosis with MLPA failed to detect the compound heterozygous mutant state of the proband and she was born unfortunately with SMA phenotype. Further analysis with a real-time polymerase chain reaction kit was able to detect the compound heterozygous state of the patient and was confirmed with targeted next-generation sequencing technology.

Testicular fat deposition attenuates reproductive performance via decreased follicle-stimulating hormone level and sperm meiosis and testosterone synthesis in mouse.

Testicular fat deposition has been reported to affect animal reproduction. However, the underlying mechanism remains poorly understood. The present study explored whether sperm meiosis and testosterone synthesis contribute to mouse testicular fat depositioninduced reproductive performance. High fat diet (HFD)-induced obesity CD1 mice (DIO) were used as a testicular fat deposition model. The serum hormone test was performed by agent kit. The quality of sperm was assessed using a Sperm Class Analyzer. Testicular tissue morphology was analyzed by histochemical methods. The expression of spermatocyte marker molecules was monitored by an immuno-fluorescence microscope during meiosis. Analysis of the synthesis of testosterone was performed by real-time polymerase chain reaction and reagent kit. It was found that there was a significant increase in body weight among DIO mice, however, the food intake showed no difference compared to control mice fed a normal diet (CTR). The number of offspring in DIO mice decreased, but there was no significant difference from the CTR group. The levels of follicle-stimulating hormone were lower in DIO mice and their luteinizing hormone levels were similar. The results showed a remarkable decrease in sperm density and motility among DIO mice. We also found that fat accumulation affected the meiosis process, mainly reflected in the cross-exchange of homologous chromosomes. In addition, overweight increased fat deposition in the testis and reduced the expression of testosterone synthesis-related enzymes, thereby affecting the synthesis and secretion of testosterone by testicular Leydig cells. Fat accumulation in the testes causes testicular cell dysfunction, which affects testosterone hormone synthesis and ultimately affects sperm formation.

A multiplex real-time polymerase chain reaction (qPCR) kit targeting VP664 and VP28 genes of white spot syndrome virus (WSSV)

The implementation of a precise and sensitive detection method is crucial to prevent the spread of the white spot syndrome virus (WSSV) within and between regions. The present study developed and validated the analytical and diagnostic performances of a multiplex real-time polymerase chain reaction kit (AccuPower® WSSV detection qPCR kit: AWqPCR kit) that simultaneously detects two distinct genomic sequences of WSSV (VP664 and VP28). The analytical sensitivity of the kit, defined as 95% limit of detection (LoD95%), was 1.15 copies of WSSV plasmid. No cross-reactivity was found against five shrimp pathogens (yellow head virus genotype 1, Taura syndrome virus, infectious myonecrosis virus, infectious hypodermal and hematopoietic necrosis virus, and Vibrio parahaemolyticus). Moreover, no significant PCR inhibition was observed while using nine potential interfering materials (antibiotics and immunostimulants). Diagnostic performance was evaluated using 200 known WSSV-infected whiteleg shrimp (Litopenaeus vannamei). Compared to the IQ2000™ WSSV Detection and Prevention System (IQ2000™ WSSV DPS), the diagnostic sensitivity (Dsn) and specificity (Dsp) of the AWqPCR kit were found to be 100% (95% confidence interval (CI): 96.38–100%) and 100% (95% CI: 96.38–100%), respectively. In addition, using 50 shrimp samples of unknown WSSV infection status, the Dsn and Dsp of the AWqPCR kit were compared to the two available gold standard WSSV detection methods, including the qPCR assay using WOAH primers and probe (WOAH qPCR) and IQ2000™ WSSV DPS. The evaluated Dsn and Dsp of the kit were 100% (95% CI: 87.66–100%) and 72.73% (95% CI: 49.78–89.27%) compared to the WOAH qPCR, whereas the Dsn and Dsp were 100% (95% CI: 25–100%) and 32.65% (95% CI: 19.95–47.54%) when compared to the IQ2000™ WSSV DPS. All six samples that were detected as WSSV-positive by the AWqPCR kit and confirmed by sequencing were found to be WSSV-negative by WOAH qPCR. Furthermore, the blind sample analysis of 50 shrimp samples carried out by three laboratories showed no significant difference in the test results. Overall, AWqPCR kit was found to be a simple and reliable method for WSSV detection with high sensitivity and specificity. Therefore, it could be applied in quarantine, surveillance, and monitoring programmes as well as for the confirmation of WSSV in apparently healthy shrimps.

Visualization test strip reading of urine miRNA via cascade selective recognition reactions for non-invasive diagnosis of bladder cancer

This study proposed a homogeneous enzyme-free and nucleic acid-based catalytic hairpin assembly and cascade recognition reactions strategy that used calcein as the signal reporter for dual visualization (color and length) and fluorescence signal detection of bladder cancer marker miRNA-126 in clinical urine. This strategy was based on the ability of calcein and CuS nanoparticles (NPs) to recognize C-Ag+-C structure/Ag+/Cu2+/CuS NPs and C-Ag+-C structure/Ag+, respectively. Due to the integration of cascade amplification and material cation exchange reaction (CER), the sensitivity of homogeneous enzyme-free miRNA analysis at room temperature was significantly improved. This study achieved the limits of detections (LODs) as low as 0.25 aM of miRNA-126 and single-cell level of T24 cells with fluorescence mode. And the reading mode of color and length changes could detect miRNA and cells with concentrations as low as 5 aM and 1 cell/mL without instruments, respectively. And less reduction of fluorescence signal was observed when target miRNA was replaced with these base-mismatched RNAs. Then miRNA-126 in 66 clinical urine samples was quantified. The experimental results were consistent with the polymerase chain reaction (PCR) kit, computed tomography (CT), and pathological findings. The receiver operating characteristic (ROC) curve exhibited an area under the curve (AUC) value of 0.954, and the assay showed 100% specificity and 90% sensitivity.

CircRNA SCAR Improves High-Glucose-Induced Mitochondrial Dysfunction and Permeability Damage in Retinal Microvascular Endothelial Cells.

This study was designed to assess the role and mechanism of circRNA SCAR in human retinal microvascular endothelial cells (hRMVECs) treated with high glucose. Quantitative real-time polymerase chain reaction (qRT-PCR) and cell counting kit 8 (CCK-8) were used to detect the effects of different concentrations of glucose on circRNA SCAR expression and cell proliferation in hRMVECs. Cell viability, levels of oxygen species (ROS), malondialdehyde (MDA) and adenosine triphosphate (ATP), as well as activities of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) in the transfected hRMVECs in each group were detected using CCK-8 and their corresponding detection kits. Changes in mtDNA copy number in high-glucose-induced hRMVECs were observed by qRT-PCR. Additionally, western blot was applied to detect effect of overexpressing circRNA SCAR on the expression levels of mitochondrial function-related proteins (Drp1 and Fis1) and cell permeability-related proteins (claudin-5, occludin and ZO-1) in hRMVECs under high-glucose concentration. According to experimental results, high glucose significantly downregulated circRNA SCAR expression and inhibited cell proliferation in hRMVECs. Instead, overexpression of this circRNA SCAR promoted cell proliferation, reduced levels of ROS, MDA and ATP, and increased SOD and CAT activities in hRMVECs under high-glucose concentration. Also, circRNA SCAR overexpression reversed the high-glucose-induced decrease in mtDNA copy number as well as, high-glucose-induced upregulation of Drp1 and Fis1 protein expression and downregulation of claudin-5, occludin and ZO-1 protein expression in hRMVECs. In summary, circRNA SCAR promotes the proliferation of hRMVECs under high-glucose concentration, alleviates oxidative stress induced by high glucose, and improves mitochondrial function and permeability damage.

ERBB2 mRNA expression to distinguish HER2-low/neg breast cancer prognosis.

569 Background: Antibody-drug conjugates such as Trastuzumab deruxtecan significantly improved progression-free and overall survival for the HER2 low expression (HER2-low) breast cancer (BC) patients, pushing the research field of HER2-low to a new climax. In fact, the traditional immunohistochemistry (IHC) to identify the HER2-low or HER2-negative expression (HER2-neg) has been challenged, and it cannot predict the prognosis of HER2-low/neg patients well. Methods: Three pathologists evaluated the HER2 expression status of the subjects via IHC. More than two pathologists judged HER2 0-2 and FISH negative samples were included in this trial. ERBB2 mRNA level of formalin-fixed, paraffin-embedded BC post-surgery specimens were detected by MammaTyper quantitative real-time polymerase chain reaction (qRT-PCR) kit. The consistency of HER2 immunohistochemistry (IHC) and MammaTyper results were analyzed by interclass correlation coefficients (ICC). Differences in disease-free survival (DFS) were assessed by Kaplan Meier analysis. Results: 793 cases were included. The ICC value between IHC and MammaTyper, assessed for HER2-neg vs HER2-low is 0.28. With a median follow up of 2.5 years, there were 36 events in the whole subjects. In the whole subjects, MammaTyper successfully showed that the prognosis of HER2-low was significantly better than HER2-neg (P < 0.001, HR = 3.497, 95%CI,1.807-6.77), but IHC could not distinguished them (P = 0.1071, HR = 1.776, 95%CI, 0.9194-3.43). Although the follow-up time is short, HER2-low subjects also showed better prognosis than HER2-neg in TNBC subjects distinguished by MammaTyper (P = 0.0271, HR = 2.912, 95%CI,1.129-7.511), not by IHC (P = 0.1032, HR = 2.213, 95%CI, 0.8512-5.756). However, in 291 cases of IHC and MammaTyper detection discordancy group, IHC results showed that the prognosis of HER2-low was worse, which was contrary to the MammaTyper results and the results of the above overall test results, suggesting that IHC could not verily reflect the amount of HER2 expression in some samples, and the prognosis of HER2-low population might be more related to the mRNA expression of HER2. Conclusions: Compared with IHC/FISH, MammaTyper qRT-PCR assay may actually reflect the prognosis of HER2-neg/low, which is expected to become a diagnostic beacon for HER2-low target drugs.

Nanoarchitectonics-Assisted Simultaneous Fluorescence Detection of Urinary Dual miRNAs for Noninvasive Diagnosis of Prostate Cancer.

Herein, we report a fluorescence strategy for the homogeneous and simultaneous analysis of urine miRNA-375 and miRNA-148a. The target miRNAs in urine bonded the devised dumbbell-shaped "C-Ag+-C" and "T-Hg2+-T" hairpin structures that could trigger cascade enzyme-free amplification. Then, the fluorescent CdTe quantum dots (QDs) and carbon dots (CDs) could selectively recognize Ag+ and Hg2+, to quantify the dual miRNAs concurrently. Under optimized conditions, the linear range was from 0.1 to 1000 fM and the limits of detection (LOD) for dual miRNAs reached 30 and 25 aM, respectively. The practicality was further evaluated with 45 clinical urine samples including prostate cancer (PC) and other patients, and the results were consistent with the clinical polymerase chain reaction (PCR) kit and ultrasonic and pathological findings. The receiver operating characteristic (ROC) curve analysis showed that the estimates of the area under the curve (AUC) were 0.739 for the serum prostate-specific antigen (PSA) and 0.941 for miRNA-375 and 0.946 for miRNA-148a. The sensitivity and specificity reached 75 and 100% for miRNA-375 and 71 and 94% for miRNA-148a, respectively, which was better than serum PSA. This strategy constructed a reliable system for dual miRNA detection in urine samples and proposed new insights into the rapid and noninvasive diagnosis of PC.

First report of Choanephora rot on Lettuce (Lactuca sativa L.) caused by Choanephora cucurbitarum in Korea.

Lettuce (Lactuca sativa L.) is a vegetable belonging to the family Asteraceae, and one of the major leafy vegetables in Korea. Lettuce has long been consumed as a fresh vegetable. In 2020, 96,774 tons were produced in about 3,800 ha according to Statistics Korea, and various types of lettuce have been bred for year-round production in line with consumer needs (Jang 2007). In August 2020, symptoms of a disease on young leaves of green lettuce (cv. Cheongpungyeoreumchima) were observed in the commercial greenhouse, Jeongeup (35°37'17.5"N, 126°53'40.4"E), Korea. About 60% of plants in 500 m2 of the cultivation area were infected. The tips of infected leaves appeared water-soaked, with brown to black lesions and were covered with dark masses of sporangiophores. Two fungal isolates NC20860 and NC20861 were isolated from monosporous sporangiola in infected leaves. Two isolates produced fast-growing colonies on potato dextrose agar medium (PDA) with abundant white mycelium at the top and bright yellow pigmentation at the underside in only 2 days. Morphological characteristics of the two fungal isolates were investigated from 30 days after inoculation on PDA. Sporangiophores bearing sporangia were erect, non-septate, unbranched, hyaline, and straight. Sporangia were hyaline to brown, globose to subglobose, 28.4 to 33.6 µm diameter. Sporangiospores from sporangia were brown to dark brown, ellipsoid to ovoid, distinctly longitudinally striate, with hyaline appendages at each pole, 6.3 to 7.5 µm wide and 13.0 to 16.0 µm long. Sporangiophores bearing sporangiola were erect, unbranched, hyaline and sporangiola were fusiform, ellipsoid to broadly ellipsoid, 7.6 to 9.8 µm wide, 13.3 to 17.8 µm long. Based on the morphological and cultural characteristics, this fungus was identified as Choanephora cucurbitarum (Berk.&Ravenel) Thaxt (Kirk 1984). To identify the species of the two isolates, DNA was extracted by using the Maxwell® RSC PureFood GMO and Authentication Kit (Promega). The internal transcribed spacer (ITS) region of ribosomal DNA was amplified using nTaq-Tenuto polymerase chain reaction (PCR) kit and therein specified protocol (Enzynomics). The primers ITS1F and ITS4 were used for PCR and sequencing (Gardes 1991). The ITS sequences of the isolates NC20860 and NC20861 from Korea (NCBI GenBank accession no.MZ960299, MZ960300) and the sequences of C. cucurbitarum strains CBS 150.51 (MH856791.1), and KUS-F29113 (KU316934.1) were compared. The sequences of the isolates were identical to those of the reference strains of C. cucurbitarum CBS 150.51 (reference/isolates base pairs, 566/566) and KUS-F29113 (572/572) at the 100% of sequence similarity. Pathogenicity test was conducted using seedlings of 8-week-old green lettuce (cv. Cheongsimchima) by spraying spore suspension (2⨉104 spores/ml). The plants were covered with plastic bags and incubated at 25 ℃ and 70 to 90% RH with a 12-h photoperiod for 20 days. The symptoms were observed only on the inoculated plants within 20 days after inoculation. The inner leaves of the inoculated lettuces had water-soaked lesions. No symptoms were observed in controls. C. cucurbitarum was reisolated from inoculated lettuce, fulfilling Koch's postulates. This fungus has been reported as the causal pathogen of fruit and blossom rot in cucurbits and other plants (Akwaji 2014). However, to our knowledge, occurrence of this pathogen on lettuce has not been reported as yet. This disease is crucial for producers because infected leaves directly results in yield loss of lettuce. This is the first report of Choanephora rot on lettuce caused by C. cucurbitarum in Korea and worldwide.