Retrospective Analysis to Optimize the Detection of MET Exon 14 Skipping Mutations in Non-Small Cell Lung Cancer.

Our study optimized METex14 skipping mutation detection by analyzing 223 Oncomine™ Focus Assay-positive cases using Pan Lung Cancer PCR Panel and reverse transcription (RT)-PCR. Among the 11 METex14 skipping mutation-positive cases (average read counts: 1390), 2 with Oncomine™ Focus Assay read counts of 2540 and 10,177 were positive on all platforms. Those with Oncomine™ Focus Assay read counts ranging from 179 to 612 tested negative elsewhere. Specimens with low ratios (average ratio: 0.12% for nine cases) may yield false-positive results. Our results suggested that monitoring read counts and ratios and validating the results with RT-PCR are crucial to prevent false positives.

Effect of phenylalanine arginyl ß-naphthylamide on the imipenem resistance, elastase production, and the expression of quorum sensing and virulence factor genes in Pseudomonas aeruginosa clinical isolates.

Pseudomonas aeruginosa is one of the most important nosocomial pathogens that possess the ability to produce multiple antibiotic resistance and virulence factors. Elastase B (LasB) is the major factor implicated in tissue invasion and damage during P. aeruginosa infections, whose synthesis is regulated by the quorum sensing (QS) system. Anti-virulence approach is now considered as potential therapeutic alternative and/or adjuvant to current antibiotics' failure. The aim of this study is primarily to find out the impact of the efflux pump inhibitor (EPI) phenylalanine arginyl ß-naphthylamide (PAßN) on the production of elastase B and the gene expression of lasI quorum sensing and lasB virulence factor in clinical isolates of P. aeruginosa. Five P. aeruginosa isolates recovered from patients with respiratory tract infections were examined in this study. Antimicrobial susceptibility of isolates was performed by the disk agar diffusion method. Effect of the PAßN on imipenem susceptibility, bacterial viability, and elastase production was evaluated. The expression of lasB and lasI genes was measured by quantitative real-time PCR in the presence of PAßN. All isolates were identified as multidrug-resistant (MDR) and showed resistance to carbapenem (MIC = 64-256 µg/mL). Susceptibility of isolates to imipenem was highly increased in the presence of efflux inhibitor. PAßN significantly reduced elastase activity in three isolates tested without affecting bacterial growth. In addition, the relative expression of both lasB and lasI genes was diminished in all isolates in the presence of inhibitor. Efflux inhibition by using the EPI PAßN could be a potential target for controlling the P. aeruginosa virulence and pathogenesis. Furthermore, impairment of drug efflux by PAßN indicates its capability to be used as antimicrobial adjuvant that can decrease the resistance and lower the effective doses of current drugs.

Molecular phylogeny and secondary structure analysis of hop stunt viroid (HSVd) associated with Mulberry (Morus alba) in India.

Hop stunt viroid (HSVd), a small, single stranded, circular, non-coding infectious RNA known to cause infection in various economically important crop plants. In the present investigation, a study was conducted in the southern part of Karnataka districts of India to detect the possible association of HSVd infection in mulberry plants. A total of 41 mulberry plants showing typical viroid-like symptoms along with asymptomatic samples were collected and screened using conventional Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) using a specific set of HSVd-Fw/ HSVd-Re primers. Out of 41 samples, the study confirmed the presence of HSVd in six samples of mulberry collected from Ramanagara (1 sample), Chikkaballapur (3 samples) and Doddaballapura (2 samples) regions with an expected HSVd amplicon size of ∼ 290-300 nucleotides. The mechanical transmission of HSVd was also confirmed on cucumber (cv. Suyo) seedlings through bioassay, which was reconfirmed by RT-PCR. The amplicons were cloned, sequenced, and the representative nucleotide sequences were deposited in the NCBI GenBank. Subsequently, molecular phylogenetic analysis showed that HSVd mulberry isolates from this study were most closely related to grapevine isolates, indicating a common origin. On the other hand, it was shown to belong to a different group from mulberry isolates so far reported from Iran, Italy, Lebanon, and China. The secondary structure analysis of HSVd mulberry Indian isolates exhibited substitutions in the terminal left, pathogenicity, and variable regions compared to those of the Indian grapevine isolates. As far as this study is concerned, HSVd was detected exclusively in some mulberry plants with viral-like symptoms, but the pathogenesis and symptom expression needs to be further investigated to establish the relationship between HSVd and the disease symptoms in the mulberry plants.

Determination of the cycle threshold value of the Xpert Xpress SARS-CoV-2/Flu/RSV test that corresponds to the presence of infectious SARS-CoV-2 in anterior nasal swabs.

Despite having high analytical sensitivities and specificities, qualitative SARS-CoV-2 nucleic acid amplification tests (NAATs) cannot distinguish infectious from non-infectious virus in clinical samples. In this study, we determined the highest cycle threshold (Ct) value of the SARS-CoV-2 targets in the Xpert Xpress SARS-CoV-2/Flu/RSV (Xpert 4plex) test that corresponded to the presence of detectable infectious SARS-CoV-2 in anterior nasal swab samples. A total of 111 individuals with nasopharyngeal swab specimens that were initially tested by the Xpert Xpress SARS-CoV-2 test were enrolled. A healthcare worker subsequently collected anterior nasal swabs from all SARS-CoV-2-positive individuals, and those specimens were tested by the Xpert 4plex test, viral culture, and laboratory-developed assays for SARS-CoV-2 replication intermediates. SARS-CoV-2 Ct values from the Xpert 4plex test were correlated with data from culture and replication intermediate testing to determine the Xpert 4plex assay Ct value that corresponded to the presence of infectious virus. Ninety-eight of the 111 (88.3%) individuals initially tested positive by the Xpert Xpress SARS-CoV-2 test. An anterior nasal swab specimen collected from positive individuals a median of 2 days later (range, 0-9 days) tested positive for SARS-CoV-2 by the Xpert 4plex test in 39.8% (39/98) of cases. Of these samples, 13 (33.3%) were considered to contain infectious virus based on the presence of cultivable virus and replication intermediates, and the highest Ct value observed for the Xpert 4plex test in these instances was 26.3. Specimens that yielded Ct values of ≤26.3 when tested by the Xpert 4plex test had a likelihood of containing infectious SARS-CoV-2; however, no infectious virus was detected in specimens with higher Ct values.IMPORTANCEUnderstanding the correlation between real-time PCR test results and the presence of infectious SARS-CoV-2 may be useful for informing patient management and workforce return-to-work or -duty. Further studies in different patient populations are needed to correlate Ct values or other biomarkers of viral replication along with the presence of infectious virus in clinical samples.

Expression profile analysis of the transient receptor potential (TRPM) channel, a possible target of praziquantel in Schistosoma japonicum.

The WHO considers schistosomiasis, which is controlled by the mass administration of the drug praziquantel (PZQ), to be a neglected tropical disease. Despite its clinical use for over four decades, PZQ remains the only choice of chemotherapy against this disease. Regarding the previous studies that demonstrated that PZQ activates the transient receptor potential (TRP) channel in Schistosoma mansoni (Sm.TRPMPZQ), the expression profile of the ortholog of this channel gene (Smp_246790.5) in S. japonicum (EWB00_008853) (Sj.TRPMPZQ) was analyzed. The relative expression of this gene in various stages of the parasite lifecycle was analyzed by quantitative real-time reverse transcription-PCR (qRT-PCR), and the expression of Sj.TRPMPZQ was observed by immunohistochemical staining using anti-serum against the recombinant Sj.TRPMPZQ protein. qRT-PCR revealed the significantly lower mRNA expression in the snail stage in comparison to other stages (p < 0.01). The relative quantity of the Sj.TRPMPZQ expression for paired females, unpaired males, and eggs was 60%, 56%, and 68%, respectively, in comparison to paired males that showed the highest expression (p < 0.05). Interestingly, immunostaining demonstrated that Sj.TRPMPZQ is expressed in the parenchyma which contains muscle cells, neuronal cells and tegument cells in adult worms. This may support the two major effects of PZQ-worm paralysis and tegument disruption-induced by channel activation. Moreover, the channel was expressed in both the eggshell and the miracidia inside, but could not be observed in sporocyst. These results suggest that the expression of Sj.TRPMPQZ corresponds to the known sensitivity of S. japonicum to PZQ.

Validating post-enrichment steps in environmental RNA analysis for improving its availability from water samples.

Environmental RNA (eRNA) analysis is expected to inclusively provide the physiological information of a population and community without individual sampling, having the potential for the improved monitoring of biodiversity and ecosystem function. Protocol development for maximizing eRNA availability is crucial to interpret its detection and quantification results with high accuracy and reliability, but the methodological validation and improvement of eRNA collection and processing methods are scarce. In this study, the technical steps after eRNA extraction, including genomic DNA (gDNA) removal and reverse transcription, were focused on and their performances were compared by zebrafish (Danio rerio) aquarium experiments. Additionally, this study also focused on the eRNA quantification variabilities between replicates and compared them between the PCR and sample levels. Results showed that (i) there was a trade-off between gDNA removal approaches and eRNA yields and an excess gDNA removal could lead to the false-negative eRNA detection, (ii) the use of the gene-specific primers for reverse transcription could increase the eRNA yields for multiple mitochondrial and nuclear genes compared with the random hexamer primers, and (iii) the coefficient of variation (CV) values of eRNA quantifications between PCR replicates were substantially lower for those between samples. Including the study, further knowledge for the sensitive and precise detection of macro-organismal eRNA should be needed for increasing the reliability and robustness of eRNA-based biomonitoring.

A sensitive mass spectrometry-based method to identify common respiratory pathogens in children.

Public health threats posed by emerging respiratory infections are a significant concern, particularly in children and infants. Traditional culture-based detection methods are time-consuming and typically require 1-3 days. Herein, we developed and evaluated a 23-plex common respiratory pathogen mass spectrometry assay that enables the simultaneous detection of 18 common respiratory pathogens in children. This assay combines matrix-assisted laser desorption/ionization time of flight mass spectrometry with multiplex reverse transcription-PCR and targets 11 bacterial and 7 viral pathogens (including 10 subtypes), and two internal controls. The detection limit of the common respiratory pathogen mass spectrometry assay was as low as 1 copy/µL, with no cross-reactivity with other organisms. We assessed the clinical performance of the common respiratory pathogen mass spectrometry assay using respiratory samples from 450 children. The total 450 clinical specimens underwent analysis via matrix-assisted laser desorption/ionization time of flight mass spectrometry, and the outcomes were juxtaposed with those derived from real-time reverse-transcriptase PCR conducted concurrently. The concordance between these methods was 96.0%, and the multiple infection identification rate was 7.1%. This innovative approach enables the simultaneous analysis of numerous outcomes from a solitary examination across 192 specimens within a timeframe of approximately 7 hours, with a dramatically reduced sample use and cost. In summary, the common respiratory pathogen mass spectrometry assay is a sensitive, accurate, and cost-effective method for detecting common respiratory pathogens in children and has the potential to revolutionize the diagnosis of respiratory tract infections. IMPORTANCE This study aimed to present and evaluate a novel co-detection method that enables the simultaneous identification of 11 bacterial and 7 viral pathogens in about 7 hours using matrix-assisted laser desorption/ionization time of flight mass spectrometry. Our approach utilizes a combination of multiplex reverse transcription-PCR and matrix-assisted laser desorption/ionization time of flight mass spectrometry, which overcomes the limitations of conventional assays, which include a long assessment time, technical difficulty, and high costs. As a screening method for common respiratory pathogens in children, common respiratory pathogen mass spectrometry assay has the potential to revolutionize the diagnosis of respiratory tract infections by providing an accurate etiological diagnosis. The common respiratory pathogen mass spectrometry assay is expected to be a critical tool for the diagnosis of respiratory infections in children, offering a more efficient, cost-effective, and accurate approach for the detection of common respiratory pathogens.

A Young Adult With Multisystem Inflammatory Syndrome Following Weeks of Initial COVID-19 Respiratory Infection, With No Prior COVID-19 Vaccination: A Case Report.

Multisystem inflammatory syndrome (MIS) following COVID-19, a condition primarily diagnosed in children, has also been observed less frequently in adults. It usually presents with a multitude of symptoms, mimicking a shock-like state characterized by multiple organ failure. Diagnosis often involves ruling out other conditions and timely management to mitigate morbidity and mortality. In this case, a 39-year-old unvaccinated Caucasian male patient reported symptoms of fever, chills, night sweats, diarrhea, headache, nasal congestion, and facial pain. Despite treatment with antipyretics, the fever persisted. The patient had tested positive for COVID-19 via polymerase chain reaction (PCR) six weeks prior. Clinical findings included low oxygen saturation, sinus tachycardia, abnormal liver function, elevated inflammatory markers, a negative respiratory viral panel, a negative immunologic workup, and a positive Clostridium difficile (C. difficile) PCR. Following complaints of chest pain which quickly escalated to cardiac arrest, he was diagnosed with myopericarditis. These manifestations met the multisystem inflammatory syndrome in adults (MIS-A) diagnostic criteria as stipulated by the Centers for Disease Control and Prevention. The diagnosis of MIS-A was reached through exclusion. Notably, the patient responded well to symptomatic management. Given the infrequent occurrence of MIS-A cases, even in 2023, it remains a challenging diagnosis. Despite existing guidelines for management, the recovery of this patient solely through symptomatic treatment prior to the consideration of conventional treatment is striking. The patient had concurrent infections, including a C. difficile infection, but these did not account for the overall clinical presentation, particularly the myopericarditis and positive laboratory findings.

A Comprehensive Phylogenetic Analysis of SARS-CoV-2: Utilizing a Novel and Convenient In-House RT-PCR Method for Characterization without Virus Culture and BSL-3 Facilities.

We developed a convenient method for amplifying the complete SARS-CoV-2 sequence using in-house RT-PCR without virus culture. Forty-one stored throat swabs and blood specimens were collected from eight SARS-CoV-2 infections at multiple time points. Total RNA was extracted using the QIAamp viral RNA mini kit and pooled for higher RNA levels. Only those positive specimens by commercial real-time RT-PCR (RT-qPCR) were selected and amplified by in-house RT-PCR for complete sequences, followed by sequencing. Phylogenetic trees and exploratory analyses were performed using MEGA 11 and Simplot 3.5.1 software. Swab samples had significantly higher total RNA concentrations than plasma (p < 0.01). Positive results were found mainly in swabs, but one was found in plasma. Successful gene amplification depended on Ct values (Ct < 38). A non-synonymous substitution was found in ORF1ab/Nsp3 (at NC045512.2 position 6312, C to A) and most spike protein mutations occurred in the S1 subunit (residues 14-685). The proposed method is time-saving and reliable for rapid genomic analysis. Increasing sample volume and pooling them for RNA extraction increases RNA concentration without culture. Combining nucleotide sequences from specific variable regions of the genome is more efficient than conventional methods.

Identification and validation of new MADS-box homologous genes in 3010 rice pan-genome.

KEY MESSAGE: Identification and validation of ten new MADS-box homologous genes in 3010 rice pan-genome for rice breeding. The functional genome is significant for rice breeding. MADS-box genes encode transcription factors that are indispensable for rice growth and development. The reported 15,362 novel genes in the rice pan-genome (RPAN) of Asian cultivated rice accessions provided a useful gene reservoir for the identification of more MADS-box candidates to overcome the limitation for the usage of only 75 MADS-box genes identified in Nipponbare for rice breeding. Here, we report the identification and validation of ten MADS-box homologous genes in RPAN. Origin and identity analysis indicated that they are originated from different wild rice accessions and structure of motif analysis revealed high variations in their amino acid sequences. Phylogenetic results with 277 MADS-box genes in 41 species showed that all these ten MADS-box homologous genes belong to type I (SRF-like, M-type). Gene expression analysis confirmed the existence of these ten MADS-box genes in IRIS_313-10,394, all of them were expressed in flower tissues, and six of them were highly expressed during seed development. Altogether, we identified and validated experimentally, for the first time, ten novel MADS-box genes in RPAN, which provides new genetic sources for rice improvement.

Evaluation of the AdvanSure One-Stop COVID-19 Plus Kit for SARS-CoV-2 Detection Using a Streamlined RNA Extraction Method.

Real-time reverse transcription (rRT)-PCR, which is the reference standard for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, generally involves a time-consuming and costly RNA extraction step prior to amplification. We evaluated the performance of the AdvanSure One-Stop COVID-19 Plus Kit (LG Chem, Seoul, Korea), a novel rRT-PCR assay that can detect SARS-CoV-2 within 90 minutes using a streamlined RNA extraction method. In total, 509 nasopharyngeal swab (NPS) specimens (SARS-CoV-2 positive: N=205; SARS-CoV-2 negative: N=304) previously tested using the PowerChek SARS-CoV-2 Real-time PCR Kit (Kogene Biotech, Seoul, Korea) were tested using the AdvanSure assay. The limit of detection (LOD) of the AdvanSure assay was determined using serially diluted inactivated SARS-CoV-2. The positive and negative percent agreements between the AdvanSure and PowerChek assays were 99.5% (204/205) and 99.3% (302/304), respectively. The LODs of the AdvanSure assay for SARS-CoV-2 nucleocapsid and spike/RNA-dependent RNA polymerase genes were 672 and 846 copies/mL, respectively. The results show that the performance of the AdvanSure assay is comparable to that of the PowerChek assay used for routine SARS-CoV-2 testing, suggesting that the AdvanSure assay is a useful diagnostic tool for rapid and accurate detection of SARS-CoV-2 infection.

Small molecule drugs promote repopulation of transplanted hepatocytes by stimulating cell dedifferentiation.

Background & Aims: Hepatocyte transplantation has emerged as a possible treatment option for end-stage liver disease. However, an important obstacle to therapeutic success is the low level of engraftment and proliferation of transplanted hepatocytes, which do not survive long enough to exert therapeutic effects. Thus, we aimed to explore the mechanisms of hepatocyte proliferation in vivo and find a way to promote the growth of transplanted hepatocytes. Methods: Hepatocyte transplantation was performed in Fah -/- mice to explore the mechanisms of hepatocyte proliferation in vivo. Guided by in vivo regeneration mechanisms, we identified compounds that promote hepatocyte proliferation in vitro. The in vivo effects of these compounds on transplanted hepatocytes were then evaluated. Results: The transplanted mature hepatocytes were found to dedifferentiate into hepatic progenitor cells (HPCs), which proliferate and then convert back to a mature state at the completion of liver repopulation. The combination of two small molecules Y-27632 (Y, ROCK inhibitor) and CHIR99021 (C, Wnt agonist) could convert mouse primary hepatocytes into HPCs, which could be passaged for more than 30 passages in vitro. Moreover, YC could stimulate the proliferation of transplanted hepatocytes in Fah -/- livers by promoting their conversion into HPCs. Netarsudil (N) and LY2090314 (L), two clinically used drugs which target the same pathways as YC, could also promote hepatocyte proliferation in vitro and in vivo, by facilitating HPC conversion. Conclusions: Our work suggests drugs promoting hepatocyte dedifferentiation may facilitate the growth of transplanted hepatocytes in vivo and may facilitate the application of hepatocyte therapy. Impact and implications: Hepatocyte transplantation may be a treatment option for patients with end-stage liver disease. However, one important obstacle to hepatocyte therapy is the low level of engraftment and proliferation of the transplanted hepatocytes. Herein, we show that small molecule compounds which promote hepatocyte proliferation in vitro by facilitating dedifferentiation, could promote the growth of transplanted hepatocytes in vivo and may facilitate the application of hepatocyte therapy.

Quantifying red complex bacteria, oral hygiene condition, and inflammation status in elderly: A pilot study.

Introduction: Periodontitis is an inflammation of the periodontal apparatus leads to destruction of connective tissue attachment and tooth loss. Red complex bacteria may contribute to disease initiation. Bacterial infection in periodontitis leads to a low-grade chronic infection and inflammation in distant organs. Notably, aging can affect the immune response. Objectives: The aim of this study was to analyze the effect of aging on oral hygiene and inflammation condition. Moreover, to evaluate the correlation between the oral hygiene condition and red complex bacterial load in subgingival plaque. Materials and methods: In this cross-sectional study, we examined 20 adult and 20 elderly subjects with periodontitis. Clinical parameters included Oral Hygiene Index Simplified (OHI-S) and Papillary Bleeding Index (PBI) were recorded. Subgingival plaque was collected from the tooth with a probing depth of 5-7 mm and analyzed with a reverse transcription polymerase chain reaction (RT-PCR) for red complex bacteria quantification. Statistical analysis was performed, respectively. Results: Both groups had poor oral hygiene conditions, reflected by high OHI-S and PBI. The quantity of red complex bacteria (P. gingivalis, T. denticola, T. forsythia) in the elderly group was significantly higher in comparison to the adult group. There was significant strong linear relationship between OHI-S and red complex bacteria (r < 1, p < 0.05). Only P. gingivalis bacteria with PBI values had a strong linear relationship and statistically significant. (r < 1, p < 0.05). P. gingivalis load was significantly higher than T. denticola and T. forsythia load, and it correlated with poor oral hygiene in the adult and elderly groups and with PBI in the elderly group. Conclusions: Aging affects to the red complex bacterial load and oral hygiene condition, but not the inflammation. These findings contribute to the development of novel treatment strategies focusing on bacterial aspect for periodontitis in the elderly.

Measles Outbreak Response Activity in Japan, and a Discussion for a Possible Strategy of Outbreak Response Using Cycle Threshold Values of Real-Time Reverse Transcription PCR for Measles Virus in Measles Elimination Settings.

Measles is a highly contagious, but vaccine-preventable disease caused by the measles virus (MeV). Although the administration of two doses of measles vaccines is the most effective strategy to prevent and eliminate measles, MeV continues to spread worldwide, even in 2022. In measles-eliminated countries, preparedness and response to measles outbreaks originating from imported cases are required to maintain elimination status. Under these circumstances, real-time reverse transcription (RT) PCR for MeV could provide a diagnostic method capable of strengthening the subnational capacity for outbreak responses. Real-time RT-PCR can detect MeV RNA from patients with measles at the initial symptomatic stage, which can enable rapid public health responses aimed at detecting their contacts and common sources of infection. Furthermore, low cycle threshold (Ct) values (i.e., high viral load) of throat swabs indicate high infectiousness in patients with measles. The high basic reproduction number of measles suggests that patients with high infectiousness can easily become super-spreaders. This opinion proposes a possible strategy of rapid and intensive responses to counter measles outbreaks caused by super-spreader candidates showing low Ct values in throat swabs. Our strategy would make it possible to effectively prevent further measles transmission, thereby leading to the early termination of measles outbreaks.

Sensitive immunoassay of Legionella using multivalent conjugates of engineered VHHs.

VHH antibodies or nanobodies, which are antigen-binding domains of heavy chain antibodies from camelid species, have several advantageous characteristics, including compact molecular size, high productibility in bacteria and easy engineering for functional improvement. Focusing on these advantages of VHHs, we attempted to establish an immunoassay system for detection of Legionella, the causative pathogen of Legionnaires' disease. A VHH phage display library was constructed using cDNA from B cells of alpacas immunized with Legionella pneumophila serogroup1 (LpSG1). Through biopanning, two specific VHH clones were isolated and used to construct a Legionella detection system based on the latex agglutination assay. After engineering the VHHs and improving the assay system, the sensitive detection system was successfully established for the LpSG1 antigen. The immunoassay developed in this study should be useful in easy and sensitive detection of Legionella, the causative agent of Legionnaires' disease, which is a potentially fatal pneumonia.

A genomic DNA-based NGS method for the simultaneous detection of multiple fusion genes in pediatric leukemia.

Fusion genes are products of chromosomal translocations that generate either a dysregulated partner gene or a chimeric fusion protein with new properties, and contribute significantly to leukemia development and clinical risk stratification. However, simultaneous detection of several hundreds of fusion genes has always been a challenge in a clinical laboratory setting. In the present study, a total of 182 pediatric patients with leukemia were screened for fusion genes by employing a novel genomic DNA-, instead of RNA-, based next-generation sequencing (NGS) method. This involved the comparison of the multiply targeted capture sequencing method with a detection panel of 270 fusion genes (MTCS-270) with an RNA-based multiplex reverse transcription-PCR technique with a detection panel of 57 fusion genes (MRTP-57). MRTP-57 has been well established in the clinical lab at Beijing Hightrust Diagnostics, Co. (Beijing, China) for an up-front leukemia diagnosis and served as the control technique in the present study. In the series, MTCS-270 and MRTP-57 yielded a positive fusion gene detection rate of 50.0% (91/182) and 41.8% (76/182), respectively, indicating an advantage of MTCS-270 over MRTP-57 in overall detection sensitivity. Specifically, all the fusion genes detected by MRTP-57 were also identified by MTCS-270, clearly signifying the respectable detection accuracy of MTCS-270. Notably, across the patients screened, MTCS-270 identified more samples with fusion genes than MRTP-57, illustrating a broader fusion gene detection coverage by MTCS-270. The present study provides solid evidence that this DNA-based NGS approach can be used as a potential detection tool together with other well-established molecular cytogenetic methods for leukemia management, and to the best of our knowledge, represents the largest leukemia fusion gene identification analysis by genomic NGS.

Clinical profile and molecular typing of viral etiological agents associated with Hand, Foot and Mouth Disease (HFMD): A study from Udhampur, Northern India.

PURPOSE: Hand, Foot and Mouth disease (HFMD) is a contagious pediatric viral disease caused due to enteroviruses (EV) of the family Picornaviridae. Cases of HFMD were reported from a tertiary care health centre, Udhampur, (Jammu and Kashmir), Northern India. The present study highlights the clinical and molecular virological aspects of HFMD cases. MATERIAL AND METHODS: Cases reported during August 2016-September 2017, and clinically diagnosed as HFMD of all age groups were included. Clinical, Biochemical and molecular virology aspects were compared. Clinical samples (n â€‹= â€‹50) such as vesicle swab, buccal and throat swabs were collected for enterovirus detection. EV-RNA was detected by 5'NCR based RT-PCR and genotyping by VP1 gene amplification and cycle sequencing. RESULTS: Of the cases of HFMD enrolled (n â€‹= â€‹50), highest (84%) were of children aged <5 years, presented either or both anathemas and exanthemas with prodromal symptoms (fever, irritability). Clinical presentations involved mainly oral ulcers on lips and tongue (48%). Oral erosions were either single or multiple in numbers. Exanthemas were seen on hand and palm, widely spread up to buttocks, legs, arms and trunk. Of these, six patients were found anemic. Complete blood count (CBC) indicated lymphocytosis and C-reactive protein (n â€‹= â€‹10) in children aged <5 years. EV-RNA was detected in 78% (39/50) of the clinical samples. VP1 gene based typing indicated the presence of CV-A16, CVA6 and EV-A71 types. CONCLUSIONS: The study highlights association of EVs in HFMD cases in the reported region. CV-A16, CV-A6 and EV-A71 types were reported for the first time from Udhampur (J&K), Northern India. No differences were observed in the clinical profile of EV strains detected. Circulation of the strains warrant and alarm outbreaks. More focused studies on HFMD and monitoring of viral strains is mandatory.

Bellidifolin from Gentianella acuta (Michx.) Hulten protects H9c2 cells from hydrogen peroxide-induced injury via the PI3K-Akt signal pathway.

Cardiovascular disease is the most common disease in the world and the first among the causes of human death. Its morbidity and mortality increase annually, but no effective treatment is available. Therefore, new drugs should be developed to treat cardiovascular disease. Gentianella acuta (Michx.) Hulten (G. acuta) is an important Mongolian medicine in China and elicits protective effects on cardiovascular health. In this study, liquid chromatography-mass spectrometry (LC-MS) combined with network pharmacology was used to screen the main active ingredients and confirm that bellidifolin was one of the main components for the treatment of ischemic heart disease. Then, rat myocardial (H9c2) cells injury model induced by hydrogen peroxide (H2O2) in vitro was established to verify the effect of bellidifolin on oxidative stress stimulation, including determination of antioxidant enzyme activity and apoptosis. Transcriptome sequencing, qRT-PCR, and western blot were performed to further verify the antioxidant stress mechanism of bellidifolin. Results showed that bellidifolin pretreatment decreased the rate of apoptosis and the levels of lactate dehydrogenase (LDH), creatine kinase (CK), and alanine aminotransferase (ALT). Conversely, it increased the contents of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in a dose-dependent manner, indicating that bellidifolin caused a protective effect on cardiomyocyte injury. Bellidifolin minimized the H2O2-induced cell injury by activating the PI3K-Akt signal pathway and downregulating glycogen synthase kinase-3ß (GSK-3ß) and p-Akt1/Akt1. Therefore, this work revealed that G. acuta has a good development prospect as an edible medicinal plant in cardiovascular disease. Its bellidifolin component is a potential therapeutic agent for cardiovascular disease induced by oxidative stress damage.

IGF2BP3 is an essential N6-methyladenosine biotarget for suppressing ferroptosis in lung adenocarcinoma cells.

A lack of promising targets leads to poor prognosis in patients with lung adenocarcinoma (LUAD). Therefore, it is urgent to identify novel therapeutic targets. The importance of the N6-methyladenosine (m6A) RNA modification has been demonstrated in various types of tumors; however, knowledge of m6A-related proteins in LUAD is still limited. Here, we found that insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), an m6A reader protein, is highly expressed in LUAD and associated with poor prognosis. IGF2BP3 desensitizes ferroptosis (a new form of regulated cell death) in a manner dependent on its m6A reading domain and binding capacity to m6A-methylated mRNAs encoding anti-ferroptotic factors, including but not limited to glutathione peroxidase 4 (GPX4), solute carrier family 3 member 2 (SLC3A2), acyl-CoA synthetase long chain family member 3 (ACSL3), and ferritin heavy chain 1 (FTH1). After IGF2BP3 overexpression, expression levels and mRNA stabilities of these anti-ferroptotic factors were successfully sustained. Notably, significant correlations between SLC3A2, ACSL3, and IGF2BP3 were revealed in clinical LUAD specimens, further establishing the essential role of IGF2BP3 in desensitizing ferroptosis. Inducing ferroptosis has been gradually accepted as an alternative strategy to treat tumors. Thus, IGF2BP3 could be a potential target for the future development of new biomaterial-associated therapeutic anti-tumor drugs.

Direct SARS-CoV-2 Detection System Utilizing Simple-to-Use Capillary Gel Electrophoresis Sample-to-Result.

We present a Direct SARS-CoV-2 Detection System that achieves sample-to-results in less than two hours in three simple steps. The Detection System includes Direct one-step Reverse Transcription PCR (RT-PCR) reagents (Qexp-MDx kit), a portable thermal cycler (Qamp-mini) with a pre-programmed chip and a simple-to-use Capillary Gel Electrophoresis system &amp;#40;Qsep Series Bio-Fragment Analyzer&amp;#41; with high fluorescence detection sensitivity to solve the problems associated with traditional real-time PCR (qPCR) systems which produces inaccurate test results with high false negative and false positive rates. The proposed simple-to-use detection platform can provide high detection sensitivity (identify less than 20 copies), fast results (less than 120 minutes) and cost-effective results which should be suitable for decentralized testing application of COVID-19.