Musashi-2 potentiates colorectal cancer immune infiltration by regulating the post-translational modifications of HMGB1 to promote DCs maturation and migration.

Post-translational modifications (PTMs) of the non-histone protein high-mobility group protein B1 (HMGB1) are involved in modulating inflammation and immune responses. Recent studies have implicated that the RNA-binding protein (RBP) Musashi-2 (MSI2) regulates multiple critical biological metabolic and immunoregulatory functions. However, the precise role of MSI2 in regulating PTMs and tumor immunity in colorectal cancer (CRC) remains unclear. Here, we present data indicating that MSI2 potentiates CRC immunopathology in colitis-associated colon cancer (CAC) mouse models, cell lines and clinical specimens, specifically via HMGB1-mediated dendritic cell (DC) maturation and migration, further contributes to the infiltration of CD4+ and CD8+ T cells and inflammatory responses. Under stress conditions, MSI2 can exacerbate the production, nucleocytoplasmic transport and extracellular release of damage-associated molecular patterns (DAMPs)-HMGB1 in CRC cells. Mechanistically, MSI2 mainly enhances the disulfide HMGB1 production and protein translation via direct binding to nucleotides 1403-1409 in the HMGB1 3' UTR, and interacts with the cytoplasmic acetyltransferase P300 to upregulate its expression, further promoting the acetylation of K29 residue in HMGB1, thus leading to K29-HMGB1 nucleocytoplasmic translocation and extracellular release. Furthermore, blocking HMGB1 activity with glycyrrhizic acid (Gly) attenuates MSI2-mediated immunopathology and immune infiltration in CRC in vitro and in vivo. Collectively, this study suggests that MSI2 may improve the prognosis of CRC patients by reprogramming the tumor immune microenvironment (TIME) through HMGB1-mediated PTMs, which might be a novel therapeutic option for CRC immunotherapy.

Musashi-2 Deficiency Triggers Colorectal Cancer Ferroptosis by Downregulating the MAPK Signaling Cascade to Inhibit HSPB1 Phosphorylation.

BACKGROUND: Musashi-2 (MSI2) is a critical RNA-binding protein (RBP) whose ectopic expression drives the pathogenesis of various cancers. Accumulating evidence suggests that inducing ferroptosis of tumor cells can inhibit their malignant biological behavior as a promising therapeutic approach. However, it is unclear whether MSI2 regulates cell death in colorectal cancer (CRC), especially the underlying mechanisms and biological effects in CRC ferroptosis remain elusive. METHODS: Experimental methods including qRT‒PCR, immunofluorescence, flow cytometry, western blot, co-immunoprecipitation, CCK-8, colony formation assay, in vitro cell transwell migration and invasion assays, in vivo xenograft tumor experiments, liver and lung CRC metastasis models, CAC mice models, transmission electron microscopy, immunohistochemistry, histopathology, 4D label-free proteomics sequencing, bioinformatic and database analysis were used in this study. RESULTS: Here, we investigated that MSI2 was upregulated in CRC and positively correlated with ferroptosis inhibitor molecules. MSI2 deficiency suppressed CRC malignancy by inhibiting cell proliferation, viability, migration and invasion in vitro and in vivo; and MSI2 deficiency triggered CRC ferroptosis by changing the intracellular redox state (ROS levels and lipid peroxidation), erastin induced cell mortality and viability, iron homeostasis (intracellular total irons and ferrous irons), reduced glutathione (GSH) levels and mitochondrial injury. Mechanistically, through 4D-lable free proteomics analysis on SW620 stable cell lines, we demonstrated that MSI2 directly interacted with p-ERK and MSI2 knockdown downregulated the p-ERK/p38/MAPK axis signaling pathway, which further repressed MAPKAPK2 and HPSB1 phosphorylation, leading to decreased expression of PCNA and Ki67 and increased expression of ACSL4 in cancer cells. Furthermore, HSPB1 could rescue the phenotypes of MSI2 deficiency on CRC ferroptosis in vitro and in vivo. CONCLUSIONS: This study indicates that MSI2 deficiency suppresses the growth and survival of CRC cells and promotes ferroptosis by inactivating the MAPK signaling pathway to inhibit HSPB1 phosphorylation, which leads to downregulation of PCNA and Ki67 and upregulation of ACSL4 in cancer cells and subsequently induces redox imbalance, iron accumulation and mitochondrial shrinkage, ultimately triggering ferroptosis. Therefore, targeted inhibition of MSI2/MAPK/HSPB1 axis to promote ferroptosis might be a potential treatment strategy for CRC.

dbCNV: deleteriousness-based model to predict pathogenicity of copy number variations.

BACKGROUND: Copy number variation (CNV) is a type of structural variation, which is a gain or loss event with abnormal changes in copy number. Methods to predict the pathogenicity of CNVs are required to realize the relationship between these variants and clinical phenotypes. ClassifyCNV, X-CNV, StrVCTVRE, etc. have been trained to predict the pathogenicity of CNVs, but few studies have been reported based on the deleterious significance of features. RESULTS: From single nucleotide polymorphism (SNP), gene and region dimensions, we collected 79 informative features that quantitatively describe the characteristics of CNV, such as CNV length, the number of protein genes, the number of three prime untranslated region. Then, according to the deleterious significance, we formulated quantitative methods for features, which fall into two categories: the first is variable type, including maximum, minimum and mean; the second is attribute type, which is measured by numerical sum. We used Gradient Boosted Trees (GBT) algorithm to construct dbCNV, which can be used to predict pathogenicity for five-tier classification and binary classification of CNVs. We demonstrated that the distribution of most feature values was consistent with the deleterious significance. The five-tier classification model accuracy for 0.85 and 0.79 in loss and gain CNVs, which proved that it has high discrimination power in predicting the pathogenicity of five-tier classification CNVs. The binary model achieved area under curve (AUC) values of 0.96 and 0.81 in the validation set, respectively, in gain and loss CNVs. CONCLUSION: The performance of the dbCNV suggest that functional deleteriousness-based model of CNV is a promising approach to support the classification prediction and to further understand the pathogenic mechanism.

TIPE2 knockout reduces myocardial cell damage by inhibiting IFN-γ-mediated ferroptosis.

Acute rejection of the transplanted heart is mediated by oxidative programmed cell death through the synergistic effects of the innate and adaptive immune systems. However, the role of ferroptosis, a newly discovered form of oxidative cell death, has not been widely evaluated. Tumor necrosis factor-α-induced protein-8 like 2 (TNFAIP8L2), also known as TIPE2, is required for maintaining immune homeostasis. To characterize the role of TIPE2 in mediating heart allografts, BALB/c hearts were transplanted into C57BL/6 wild-type (WT) and TIPE2-/- recipient mice. In TIPE2-/- recipient mice, allograft injury in BALB/c allograft hearts was significantly reduced through the inhibition of allograft ferroptosis. On day 3 and day 6 post-transplantation, the numbers of CD3+, CD4+, and CD8+ cells among splenocytes and draining lymph node cells were significantly decreased, and the activation of CD4+ and CD8+ cells in grafts was decreased in TIPE2-/- recipient mice compared with WT mice. Moreover, CD4+ and CD8+ T cells in TIPE2-/- recipient mice were characterized by deficient capacities for interferon-γ (IFN-γ) production through the TBK1 signaling axis and increased glutathione peroxidase 4 (GPX4). In cell experiments, treatment with IFN-γ enhanced ferroptosis-specific lipid peroxidation in myocardial cells and correlated inversely with GPX4 expression. Mechanistically, IFN-γ administration decreased the expression of GPX4 by inhibiting MEK/ERK phosphorylation. In summary, our findings demonstrated that TIPE2 deficiency inhibits T-cell production of IFN-γ to reduce ferroptosis in allografts by restraining lipid peroxidation.

Increased expression of the RNA-binding protein Musashi-2 is associated with immune infiltration and predicts better outcomes in ccRCC patients.

RNA-binding proteins (RBPs) mainly contribute to abnormalities in posttranscriptional gene regulation. The RBP Musashi-2, an evolutionarily conserved protein, has been characterized as an oncoprotein in various tumors. However, the prognostic value and potential roles of Musashi-2 in clear cell renal cell carcinoma (ccRCC) have not yet been elucidated. In this study, we found that Musashi-2 was mainly expressed in the normal distal tubular cells and collecting duct cells of the kidneys, while its expression was significantly decreased in ccRCC. And higher expression levels of Musashi-2 indicated better overall survival (OS) in ccRCC. Furthermore, immunohistochemistry demonstrated that PD-L1 expression was negatively correlated with Musashi-2 expression, and Musashi-2 was found to be remarkably correlated with multiple immune cells and immune inhibitors, including CD8+ T cells, CD4+ T cells, regulatory T (Treg) cells, PDCD1, CTLA4, Foxp3, and LAG3. Functional enrichment analysis revealed that Musashi-2 might be involved in ccRCC metabolic reprogramming and immune infiltration and further predicted the therapeutic sensitivity of ccRCC. Taken together, Musashi-2 is a prognostic biomarker for ccRCC patients that may provide novel insights into individualized treatment strategies and guide effective immunotherapy.

The Lysine Acetylation Modification in the Porin Aha1 of Aeromonas hydrophila Regulates the Uptake of Multidrug Antibiotics.

Protein lysine acetylation (Kac) modification plays important roles in diverse physiological functions. However, there is little evidence on the role of Kac modification in bacterial antibiotic resistance. Here, we compared the differential expressions of whole-cell proteins and Kac peptides in oxytetracycline sensitive and oxytetracycline resistance (OXYR) strains of Aeromonas hydrophila using quantitative proteomics technologies. We observed a porin family protein Aha1 downregulated in the OXYR strain, which may have an important role in the OXY resistance. Interestingly, seven of eight Kac peptides of Aha1 decreased abundance in OXYR as well. Microbiologic assays showed that the K57R, K187R, and K197R Aha1 mutants significantly increased antibiotic resistance to OXY and reduced the intracellular OXY accumulation in OXY stress. Moreover, these Aha1 mutants displayed multidrug resistance features to tetracyclines and ß-lactam antibiotics. The 3D model prediction showed that the Kac states of K57, K187, and K197 sites located at the extracellular pore vestibule of Aha1 may be involved in the uptake of specific types of antibiotics. Overall, our results indicate a novel antibiotic resistance mechanism mediated by Kac modification, which may provide a clue for the development of antibiotic therapy strategies.

Liver transplantation for metastatic non-resectable gastrointestinal stromal tumor after molecular targeted therapies: A case report.

INTRODUCTION AND IMPORTANCE: Metastatic GIST (gastrointestinal stromal tumor) is most commonly seen in the liver. Surgical resection and Imatinib administration are the preferred treatment for localized and potentially resectable GIST. However, it is still a matter of debate about the optimal therapeutic management for unresectable, liver-confined, metastatic GIST even after the administration of imatinib. The present case illustrates the possibility of LT surgery maybe for unresectable GIST. CASE PRESENTATION: A 56-year-old man revealed clinical symptoms such as abdominal pain, eating little, fullness of abdomen, and fatigue. A 6.0 cm tumor in the fundus of the stomach was found by gastroscopy, which was confirmed to be GIST by pathological biopsy and molecular testing. Abdominal enhanced CT scanning showed multiple hepatic mass occupying synchronous. Then the patient initiated on targeted drug therapy of Imatinib (400 mg daily). A year later, a follow-up abdominal enhanced CT scanning showed that no tumor was found in the gastric fundus except the thickened gastric wall with poor dilatation, and the liver tumors were significantly smaller than before. When the patient showed symptoms of drug resistance, he was refered to our hospital for LT. The surgery was very successful, and the patient is disease-free and there is no evidence of recurrence until the paper was finished. DISCUSSION AND CONCLUSION: Metastatic GIST to the whole liver is a rare clinical entity requiring unique considerations for treatment. Treatment based on LT might be the last resort therapy for unresectable, liver-confined, metastatic GIST in transplant oncology.

MSI2 deficiency in ILC3s attenuates DSS-induced colitis by affecting the intestinal microbiota.

Background: The etiology and pathogenesis of inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), are generally believed to be related to immune dysfunction and intestinal microbiota disorder. However, the exact mechanism is not yet fully understood. The pathological changes associated with dextran sodium sulfate (DSS)-induced colitis are similar to those in human UC. As a subgroup of the innate immune system, group 3 innate lymphoid cells (ILC3s) are widely distributed in the lamina propria of the intestinal mucosa, and their function can be regulated by a variety of molecules. Musashi2 (MSI2) is a type of evolutionarily conserved RNA-binding protein that maintains the function of various tissue stem cells and is essential for postintestinal epithelial regeneration. The effect of MSI2 deficiency in ILC3s on IBD has not been reported. Thus, mice with conditional MSI2 knockout in ILC3s were used to construct a DSS-induced colitis model and explore its effects on the pathogenesis of IBD and the species, quantity and function of the intestinal microbiota. Methods: Msi2flox/flox mice (Msi2fl/fl ) and Msi2flox/floxRorcCre mice (Msi2ΔRorc ) were induced by DSS to establish the IBD model. The severity of colitis was evaluated by five measurements: body weight percentage, disease activity index, colon shortening degree, histopathological score and routine blood examination. The species, quantity and function of the intestinal microbiota were characterized by high-throughput 16S rRNA gene sequencing of DNA extracted from fecal samples. Results: MSI2 was knocked out in the ILC3s of Msi2ΔRorc mice. The Msi2ΔRorc mice exhibited reductions in body weight loss, the disease activity index, degree of colon shortening, tissue histopathological score and immune cells in the peripheral blood compared to those of Msi2fl/fl mice after DSS administration. The 16S rRNA sequencing results showed that the diversity of the intestinal microbiota in DSS-treated Msi2ΔRorc mice changed, with the abundance of Firmicutes increasing and that of Bacteroidetes decreasing. The linear discriminant analysis effect size (LEfSe) approach revealed that Lactobacillaceae could be the key bacteria in the Msi2ΔRorc mouse during the improvement of colitis. Using PICRUST2 to predict the function of the intestinal microbiota, it was found that the functions of differential bacteria inferred by modeling were mainly enriched in infectious diseases, immune system and metabolic functions. Conclusions: MSI2 deficiency in ILC3s attenuated DSS-induced colonic inflammation in mice and affected intestinal microbiota diversity, composition, and function, with Lactobacillaceae belonging to the phylum Firmicutes possibly representing the key bacteria. This finding could contribute to our understanding of the pathogenesis of IBD and provide new insights for its clinical diagnosis and treatment.

miR-539 activates the SAPK/JNK signaling pathway to promote ferropotosis in colorectal cancer by directly targeting TIPE.

Colorectal cancer (CRC) is a common tumor that harms human health with a high recurrence rate. It has been reported that the expression of microRNA-539 (miR-539) is low in several types of cancer, including CRC. Tumor necrosis factor (TNF)-α-induced protein 8 (TNFAIP8/TIPE) is highly expressed in CRC and promotes the proliferation, migration and angiogenesis of CRC. However, the relationship between miR-539 and TIPE and the mechanisms by which they regulate the proliferation of CRC remain to be explored. We aimed to investigate the functions and mechanisms of miR-539 in CRC proliferation. Functionally, miR-539 can bind to and regulate the expression of TIPE, and miR-539 activates SAPK/JNK to downregulate the expression of glutathione peroxidase 4 (GPX4) and promote ferroptosis. Our data reveal the novel role of miR-539 in regulating ferroptosis in CRC via activation of the SAPK/JNK axis, providing new insight into the mechanism of abnormal proliferation in CRC and a novel potential therapeutic target for advanced CRC.

An aberrant DNA methylation signature for predicting the prognosis of head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a common malignancy worldwide with a poor prognosis. DNA methylation is an epigenetic modification that plays a critical role in the etiology and pathogenesis of HNSCC. The current study aimed to develop a predictive methylation signature based on bioinformatics analysis to improve the prognosis and optimize therapeutic outcome in HNSCC. Clinical information and methylation sequencing data of patients with HNSCC were downloaded from The Cancer Genome Atlas database. The R package was used to identify differentially methylated genes (DMGs) between HNSCC and adjacent normal tissues. We identified 22 DMGs associated with 246 differentially methylated sites. Patients with HNSCC were classified into training and test groups. Cox regression analysis was used to build a risk score formula based on the five methylation sites (cg26428455, cg13754259, cg17421709, cg19229344, and cg11668749) in the training group. The Kaplan-Meier survival curves showed that the overall survival (OS) rates were significantly different between the high- and low-risk groups sorted by the signature in the training group (median: 1.38 vs. 1.57 years, log-rank test, p < 0.001). The predictive power was then validated in the test group (median: 1.34 vs. 1.75 years, log-rank test, p < 0.001). The area under the receiver operating characteristic curve (area under the curve) based on the signature for predicting the 5-year survival rates, was 0.7 in the training and 0.73 in test groups, respectively. The results of multivariate Cox regression analysis showed that the riskscore (RS) signature based on the five methylation sites was an independent prognostic tool for OS prediction in patients. In addition, a predictive nomogram model that incorporated the RS signature and patient clinical information was developed. The innovative methylation signature-based model developed in our study represents a robust prognostic tool for guiding clinical therapy and predicting the OS in patients with HNSCC.

Proteomics Analysis Reveals Bacterial Antibiotics Resistance Mechanism Mediated by ahslyA Against Enoxacin in Aeromonas hydrophila.

Bacterial antibiotic resistance is a serious global problem; the underlying regulatory mechanisms are largely elusive. The earlier reports states that the vital role of transcriptional regulators (TRs) in bacterial antibiotic resistance. Therefore, we have investigated the role of TRs on enoxacin (ENX) resistance in Aeromonas hydrophila in this study. A label-free quantitative proteomics method was utilized to compare the protein profiles of the ahslyA knockout and wild-type A. hydrophila strains under ENX stress. Bioinformatics analysis showed that the deletion of ahslyA triggers the up-regulated expression of some vital antibiotic resistance proteins in A. hydrophila upon ENX stress and thereby reduce the pressure by preventing the activation of SOS repair system. Moreover, ahslyA directly or indirectly induced at least 11 TRs, which indicates a complicated regulatory network under ENX stress. We also deleted six selected genes in A. hydrophila that altered in proteomics data in order to evaluate their roles in ENX stress. Our results showed that genes such as AHA_0655, narQ, AHA_3721, AHA_2114, and AHA_1239 are regulated by ahslyA and may be involved in ENX resistance. Overall, our data demonstrated the important role of ahslyA in ENX resistance and provided novel insights into the effects of transcriptional regulation on antibiotic resistance in bacteria.

A Fully Automated and Integrated Microfluidic System for Efficient CTC Detection and Its Application in Hepatocellular Carcinoma Screening and Prognosis.

Analysis of circulating tumor cells (CTCs) is regarded as a useful diagnostic index to monitor tumor development and guide precision medicine. Although the immunoassay is a common strategy for CTC identification and heterogeneity characterization, it is challenged by poor reaction efficiency and laborious manipulations in microdevices, which hinder the sensitivity, throughput, simplification, and applicability. To meet the need for rapid, sensitive, and simple CTC analysis, we developed an efficient CTC detection system by integrating a 3D printed off-chip multisource reagent platform, a bubble retainer, and a single CTC capture microchip, which can achieve CTC capture and identification within 90 min. Compared with traditional CTC identification methods, this system decreases immunostaining time and antibody consumption by 90% and performs the on-chip immunoassay in a fully automated manner. Using this system, CTCs from the peripheral blood of 19 patients with various cancers were captured, detected, and compared with clinical data. The system shows great potential for early screening, real-time monitoring, and precision medicine for hepatocellular carcinoma (HCC). With the advantages of automation, stability, economy, and user-friendly operation, the proposed system is promising for clinical scenarios.

Proteomics analysis reveals the importance of transcriptional regulator slyA in regulation of several physiological functions in Aeromonas hydrophila.

SlyA is a well-known transcription factor that plays important roles in the regulation of diverse physiological functions including virulence and stress response in various bacterial species. The biological effects of slyA have species-specific characteristics. In this study, a phenotype assay showed that slyA gene deletion in Aeromonas hydrophila (ahslyA) decreased biofilm formation capability but did not affect bacterial hemolytic activity or acid stress response. The differentially expressed proteins between ΔahslyA and wild-type strains were compared by label-free quantitative proteomics to further understand the effects of AhSlyA on biological functions. Bioinformatics assays showed that ΔahslyA may be involved in the regulation of several intracellular metabolic pathways such as galactose metabolism, arginine biosynthesis, and sulfur metabolism. A further phenotypic assay confirmed that AhSlyA plays an important role in the regulation of sulfur and phosphate metabolism. Moreover, ahslyA also directly or indirectly regulated at least eight outer membrane proteins involved in the maintenance of cell permeability. Overall, the results provide insights into the functions of ahslyA and demonstrate its importance in A. hydrophila. BIOLOGICAL SIGNIFICANCE: In this study, we compared the DEPs between the transcriptional regulator slyA-deleted and the wild-type A. hydrophila strains using a label-free quantitative proteomics method. The bioinformatics analysis showed that slyA may be involved in the regulation of several metabolic pathways. Subsequent phenotype and growth assays confirmed that ΔahslyA affected sulfur and phosphate metabolism, and OM permeability. Finally, a ChIP-PCR assay further confirmed that AhSlyA directly binds to the promoters of several candidate genes, including sulfur metabolism-related genes. These results indicated that slyA plays an important regulatory role in pleiotropic physiological functions of A. hydrophila, and these functions may be different from those identified in previous reports from other bacterial species.

The loss of SHMT2 mediates 5-fluorouracil chemoresistance in colorectal cancer by upregulating autophagy.

5-Fluorouracil (5-FU)-based chemotherapy is the first-line treatment for colorectal cancer (CRC) but is hampered by chemoresistance. Despite its impact on patient survival, the mechanism underlying chemoresistance against 5-FU remains poorly understood. Here, we identified serine hydroxymethyltransferase-2 (SHMT2) as a critical regulator of 5-FU chemoresistance in CRC. SHMT2 inhibits autophagy by binding cytosolic p53 instead of metabolism. SHMT2 prevents cytosolic p53 degradation by inhibiting the binding of p53 and HDM2. Under 5-FU treatment, SHMT2 depletion promotes autophagy and inhibits apoptosis. Autophagy inhibitors decrease low SHMT2-induced 5-FU resistance in vitro and in vivo. Finally, the lethality of 5-FU treatment to CRC cells was enhanced by treatment with the autophagy inhibitor chloroquine in patient-derived and CRC cell xenograft models. Taken together, our findings indicate that autophagy induced by low SHMT2 levels mediates 5-FU resistance in CRC. These results reveal the SHMT2-p53 interaction as a novel therapeutic target and provide a potential opportunity to reduce chemoresistance.

Impaired AGO2/miR-185-3p/NRP1 axis promotes colorectal cancer metastasis.

Increasing evidence suggests that global downregulation of miRNA expression is a hallmark of human cancer, potentially due to defects in the miRNA processing machinery. In this study, we found that the protein expression of Argonaute 2 (AGO2), a key regulator of miRNA processing, was downregulated in colorectal cancer (CRC) tissues, which was also consistent with the findings of the Clinical Proteomic Tumor Analysis Consortium (CPTAC). Furthermore, the correlation between the levels of AGO2 and epithelial-mesenchymal transition (EMT) markers (E-cadherin and vimentin) indicated that reduced levels of AGO2 promoted EMT in CRC. Low expression of AGO2 was an indicator of a poor prognosis among CRC patients. Knockdown of AGO2 in CRC cells promoted migration, invasion and metastasis formation in vitro and in vivo but had no influence on proliferation. To provide detailed insight into the regulatory roles of AGO2, we performed integrated transcriptomic, quantitative proteomic and microRNA sequencing (miRNA-seq) analyses of AGO2 knockdown cells and the corresponding wild-type cells and identified neuropilin 1 (NRP1) as a new substrate of AGO2 via miR-185-3p. Our data provided evidence that knockdown of AGO2 resulted in a reduction of miR-185-3p expression, leading to the upregulation of the expression of NRP1, which is a direct target of miR-185-3p, and elevated CRC cell metastatic capacity. Inhibition of NRP1 or treatment with a miR-185-3p mimic successfully rescued the phenotypes of impaired AGO2, which suggested that therapeutically targeting the AGO2/miR-185-3p/NRP1 axis may be a potential treatment approach for CRC.

Analytical performance evaluation of five RT-PCR kits for severe acute respiratory syndrome coronavirus 2.

BACKGROUND: We aimed to evaluate the analytical performance of five commercial RT-PCR kits (Genekey, Daan, BioGerm, Liferiver, and Yaneng) commonly used in China, since such comparison data are lacking. METHODS: A total of 20 COVID-19 confirmed patients and 30 negative nasopharyngeal swab specimens were analyzed by five kits. The detection ability of five RT-PCR kits was evaluated with 5 concentration gradients diluted by a single positive sample. The limit of detection was evaluated by N gene fragment solid standard. Two positive clinical specimens were used to evaluate the repeatability and imprecision. Finally, we used six human coronaviruses plasmid and four respiratory pathogens plasmid to check for cross-reactivity. RESULTS: The positive detection rate was 100% for Genekey, Daan, and BioGerm,and 90% for Liferiver and Yaneng in 20 clinical SARS-CoV-2 infection. The coincidence rate of five kits in 10 negative samples was 100%. The detection rate of target genes for Daan, BioGerm, Liferiver, and Yaneng was 100% from Level 1 to Level 3. In Level 4, only Daan detection rate was 100%. In Level 5, five kits presented poor positive rate. The limit of detection declared by each manufacturer was verified. The repeatability for target genes was less than 5% and so did the total imprecision. There is no cross-reactivity of five kits with six human coronaviruses and four respiratory pathogens for ORF1ab and N gene. CONCLUSIONS: Five RT-PCR kits assessed in this study showed acceptable analytical performance characteristics and are useful tools for the routine diagnosis of SARS-CoV-2.

Consistency evaluation of Liferiver, Yaneng, Darui, and the Cobas 4800 test for high-risk human papillomavirus screening.

BACKGROUND: In recent years, several high-risk human papillomavirus (HR-HPV) tests have been developed. The assay capabilities need to be systematically reviewed. Here, we compared the clinical sample performance of three novel HR-HPV assays (Liferiver, Yaneng, and Darui) based on different platforms with the widely adopted cobas4800 test. METHODS: A total of 346 cervical swabs from women who were screened for cervical cancer were analyzed for the presence of 14 HR-HPV genotypes. The distinction between the four assays was investigated by the genotyping and direct sequencing. RESULTS: The positive rates of the four assays ranged from 61.56% to 64.16%. The overall concordance was 88.15%. The Yaneng assays displayed the best sensitivity (100%) and specificity (98.43%). The sensitivity (98.17%) and specificity (98.43%) of the Darui assay were superior to those of the cobas4800 test (97.72% and 93.70%, respectively). The Liferiver assay displayed comparable sensitivity with the cobas4800 test (95.89% and 97.72%, respectively). The specificity of the cobas4800 was lower than that of the Liferiver assay (93.70% vs. 97.64%). CONCLUSIONS: The three novel HR-HPV assays displayed good agreement with the cobas4800 test. The analytical performance of all four fulfilled the requirements of sensitivity and specificity for HR-HPV detection.

Evaluation on the separated effect of 13 hemoglobin variants by a new automatic Hba1c analyzer.

OBJECTIVE: To evaluate the effect of a new type of automatic glycated hemoglobin analyzer on the separation of abnormal hemoglobin. METHODS: Samples diagnosed as hemoglobin variants by capillary electrophoresis and gene testing were selected, and HbA1c analyzer was used for separation and detection. RESULTS: A total of 13 hemoglobin variants in 40 samples could be separated from the normal peaks. CONCLUSIONS: The variant mode of hemoglobin HbA1c can identify a variety of hemoglobin variants, and the type of variants can be preliminarily determined according to the retention time and characteristic peak shape of the variants.

Development of a novel prognostic signature for predicting the overall survival of bladder cancer patients.

BACKGROUND: Bladder cancer is one of the most common malignancies. So far, no effective biomarker for bladder cancer prognosis has been identified. Aberrant DNA methylation is frequently observed in the bladder cancer and holds considerable promise as a biomarker for predicting the overall survival (OS) of patients. MATERIALS AND METHODS: We downloaded the DNA methylation and transcriptome data for bladder cancer from The Cancer Genome Atlas (TCGA), a public database, screened hypo-methylated and up-regulated genes, similarly, hyper-methylation with low expression genes, then retrieved the relevant methylation sites. Cox regression analysis was used to identify a nine-methylation site signature of a training group. Predictive ability was validated in a test group by receiver operating characteristic (ROC) analysis. RESULTS: We identified nine bladder cancer-specific methylation sites as potential prognostic biomarkers and established a risk score system based on the methylation site signature to evaluate the OS. The performance of the signature was accurate, with area under curve was 0.73 in the training group and 0.71 in the test group. Taking clinical features into consideration, we constructed a nomogram consisting of the nine-methylation site signature and patients' clinical variables, and found that the signature was an independent risk factor. CONCLUSIONS: Overall, the significant nine methylation sites could be novel prediction biomarkers, which could aid in treatment and also predict the overall survival likelihoods of bladder cancer patients.