Integrating Magnetic-Bead-Based Sample Extraction and Molecular Barcoding for the One-Step Pooled RT-qPCR Assay of Viral Pathogens without Retesting.

Pooling multiple samples prior to real-time reverse-transcription polymerase chain reaction (RT-PCR) analysis has been proposed as a strategy to minimize expenses and boost test throughput during the COVID-19 pandemic. Nevertheless, the traditional pooling approach cannot be effectively deployed in high-prevalence settings due to the need for secondary tests in the case of a positive pool. In this study, we present a pooling test platform with high adaptability and simplicity that allows sample-specific detection of multiple-tagged samples in a single run without the need for retesting. This was accomplished by labeling distinct samples with predefined ID-Primers and identifying tagged pooled samples using one-step RT-PCR followed by melting curve analysis with rationally designed universal fluorescence- and quencher-tagged oligo probes. Using magnetic beads (MBs), nucleic acid targets from different individuals can be tagged and extracted concurrently and then pooled before RT, eliminating the need for extra RNA extraction and separate RT and enzyme digestion steps in the recently developed barcoding strategies. Pools of six samples (positive and negative) were successfully identified by melting temperature values under two fluorescent channels, with a detection sensitivity of 5 copies/µL. We validated the reproducibility of this assay by running it on 40 clinical samples with a hypothetical infection rate of 15%. In addition, to aid the scenario of large-scale pooling tests, we constructed a melting curve autoreadout system (MCARS) for statistical analysis of melting curve plots to eliminate error-prone manual result readout. Our results suggest that this strategy could be a simple and adaptable tool for alleviating existing bottlenecks in diagnostic pooling testing.

Efficient large-scale screening of viral pathogens by fragment length identification of pooled nucleic acid samples (FLIPNAS).

The necessity for the large-scale screening of viral pathogens has been amply demonstrated during the COVID-19 pandemic. During this time, SARS-CoV-2 nucleic acid pooled testing, such as Dorfman-based group testing, was widely adopted in response to the sudden increased demand for detection. However, the current approach still necessitates the individual retesting of positive pools. Here, we established an efficient method termed the fragment-length identification of pooled nucleic acid samples (FLIPNAS), where all subsamples (n = 8) can be uniquely labelled and tested in a single-time detection among pools of samples. We used a novel and simple design of unique primers (UPs) to generate amplicons of unique lengths after reverse transcription and polymerase chain reaction to reach this aim. As a result, the unique lengths of the amplicons can be recognized and traced back to the corresponding UPs and specific samples. Our results demonstrated that FLIPNAS could recognize one to eight positive subsamples in a single test without retesting positive pools. The system also showed sufficient sensitivity for the mass monitoring of SARS-CoV-2 and no cross-reactivity against three common respiratory diseases. Moreover, the FLIPNAS results of 40 samples with a positive ratio of 7.8% were in 100% agreement with their individual detection results using the gold standard. Collectively, this study shows that the efficiency of nucleic acid pooling detection can be further improved by FLIPNAS, which can speed up testing and mitigate the urgent demand for resources.

Systematic analysis of the prognostic and immunological role of DHX33 in pan-cancer.

DEAH-box helicase 33 (DHX33) is a potent oncogenic agent on patients with hepatocellular carcinoma and colon cancer. Nevertheless, the prognostic significance of DHX33 in human pan-cancers remains unclear. Various bioinformatics tools have been used to clarify the oncogenic effects of DHX33 in pan-cancers. The Cancer Genome Atlas (TCGA) and Human Protein Atlas (HPA) were used to evaluate DHX33 at the mRNA and protein levels, respectively. The pan-cancer prognostic prediction value of DHX33 was evaluated using the Kaplan-Meier plotter. The association between DHX33 levels and clinicopathological features was then determined using the UALCAN database. In addition, gene set enrichment analysis and nomogram prediction models were constructed. The association between DHX33 levels and immune cell infiltration was then assessed using TISIDB. We determined that DHX33 is aberrantly overexpressed in pan-cancers and related to lung adenocarcinoma (LUAD) clinical stage (p < .001). Moreover, DHX33 overexpression was indicative of poor overall survival, disease-free survival, and progression-free interval in patients with LUAD (p < .05). Furthermore, DHX33 levels were associated with age, N stage, T stage, and pathologic stage in patients with LUAD (p < .001). Additionally, multivariate Cox analysis revealed that DHX33 was an independent risk factor for OS in patients with LUAD. A nomogram model between DHX33 levels and characteristic clinical parameters was developed. Additionally, DHX33 levels correlated with immunomodulators and chemokines. Finally, gene set enrichment analysis revealed that the amyloid fiber formation pathway and the WICH complex positively regulates RNA expression pathway, which was the most enriched in LUAD. Our data revealed oncogenic effects of DHX33 in various cancers. We suggest that DHX33 may serve as a biomarker for poor prognosis in LUAD.

Label-free and self-assembled fluorescent DNA nanopompom for determination of miRNA-21.

A label-free fluorescence method based on self-assembled DNA nanopompom has been developed for miRNA-21 detection. In the presence of miRNA-21, three DNA hairpin probes with split G-quadruplex assemble the DNA nanopompom. Based on the isothermal toehold-mediated DNA strand displacement reaction, the target miRNA can be catalytically recycled and trigger three DNA hairpin probes to self-assemble the DNA nanopompom and release the G-quadruplex. The formation of the G-quadruplex increases the fluorescence emission intensity of thioflavin. For thioflavin-based miRNA-21 detection, the excitation and emission wavelengths are set to 425 nm and 490 nm, respectively. The limit of detection for miRNA-21 is 0.8 pM according to F/F0 = 0.0031 × CmiRNA-21 + 1.0382 (R2 = 0.9978). This sensing system provides a low-cost, effective, and convenient method for miRNA detection, which holds great potential in biochemical diagnosis and clinical practice. Graphical abstract Label-free and self-assembled fluorescent DNA nanopompom for miRNA detection.

Implementation of emergency general practitioner management of patients during the complete lockdown consequent to the COVID-19 Omicron outbreak in Shanghai.

The outbreak of the Omicron strain of COVID-19 led to the most restrictive lockdowns ever implemented in Shanghai.

Disposable and low-cost pen-like sensor incorporating nucleic-acid amplification based lateral-flow assay for at-home tests of communicable pathogens.

Rapid at-home test is a good alternative to the gold standard quantitative polymerase chain reaction (qPCR) for early identification and management of infected individuals in pandemic. However, the currently available at-home rapid antigen kits and nucleic acid tests (NATs) are prone to false results. Although some CRISPR-mediated NATs enhanced accuracy, long turnaround time (ca. 1 h) and aerosol contamination due to additional open-lid reaction hinder its applicability for self-tests. Moreover, the accuracy of at-home NATs is also impacted by interference of sample matrix due to lack of sample purification. Here we report a Fast, Low-cost, Aerosol contamination-free and Sensitive molecular assay for at-Home tests of communicable pathogens (FLASH) incorporating oLAMP, a recently reported isothermal and target-specific NATs by our group, and a visible lateral-flow readout. The integrated platform enabled sample-to-result SARS-CoV-2 RNA detection in 20-30 min achieving a sensitivity of 0.5 copies/µL in a blinded experiment with a high accuracy comparable with the qPCR. Its prototype consists of two disposable pen-like instruments for single-step sample preparation and contamination-free NATs, respectively. The simplified workflow of the FLASH enabled detection to be readily conducted by untrained users for at-home tests. All in all, the FLASH prototype demonstrates itself to be a promising home-use assay platform for effective mitigation of the pandemic.

Association between NF-κB Signal Pathway-Related Gene Polymorphisms and Response to Alendronate Treatment in Postmenopausal Chinese Women with Low Bone Mineral Density.

Background: Osteoporosis is a systemic bone disease characterized by reduction of bone content. Bisphosphonates are first-line treatments for osteoporosis, but they have variable effectiveness. Genetic factors may explain these differences. The NF-κB signaling pathway plays a key role in the regulation of bone metabolism. We aimed to determine whether genetic variations in the NF-κB signaling pathway affect the effectiveness of alendronate in postmenopausal Chinese women with low bone mass. Methods: We recruited 455 postmenopausal Han Chinese women with primary osteoporosis or osteopenia aged 48-90 yrs who had experienced no spontaneous menses for at least 1 yr. All participants had dual X-ray absorptiometry (DEXA) bone mineral density (BMD) measurement at baseline and 1 yr after treatment. Treatment involved 1 yr administration of 70 mg oral alendronate weekly and 600 mg calcium and 125 IU of vitamin D daily. Thirteen tagSNPs in NF-κB1 (rs28362491, rs3774937, rs230521, rs230510, and rs4648068), RELA (rs7119750, rs11820062), and NLRC5 (rs289747, rs1566439, rs1684575, rs289726, rs289723, and rs41383) were chosen from the NCBI Locus Link and HapMap and genotyped individually. Genetic variation in these genes and the corresponding therapeutic response to alendronate treatment were analyzed. Results: Among the 13 tagSNPs, rs289747 was significantly correlated with the BMD change rate at the femoral neck (P=0.048). This significance no longer existed after Bonferroni correction. We then performed principal component analysis (PCA) and found NLRC5 (rs289747 and rs1566439) were strongly correlated with alendronate efficacy in femoral phenotypes and were major components of BMD change values, particularly total hip and intertrochanteric phenotypes. Furthermore, the PLINK linear regression GLM model revealed that haplotype TT of RELA (rs7119750 and rs11820062) and ICCTA of NF-κB1 (rs28362491, rs3774937, rs230521, rs230510, and rs4648068) were associated with BMD of the total hip among each haplotype after 1 yr of treatment. Conclusion: The NF-κB1, RELA, and NLRC5 genetic variations affect the therapeutic response of alendronate treatment for postmenopausal osteoporosis.

Circular RNA circ_0001287 inhibits the proliferation, metastasis, and radiosensitivity of non-small cell lung cancer cells by sponging microRNA miR-21 and up-regulating phosphatase and tensin homolog expression.

As a type of non-coding RNA, circular RNA (circRNA) figures prominently in human cancer progression. Nonetheless, the expression, function, and regulatory mechanism of circ_0001287 in non-small cell lung cancer (NSCLC) remain obscure. In this work, quantitative real-time polymerase chain reaction (qRT-PCR) was implemented to quantify circ_0001287 and miR-21 expressions in NSCLC tissues and cells. The relationship between circ_0001287 expression and the clinicopathological parameters of NSCLC patients was examined. Cell counting kit-8 (CCK-8), 5-bromo-2©-deoxyuridine (BrdU), and Transwell experiments were conducted to detect the multiplication, migration, and invasion of NSCLC cells after circ_0001287 was overexpressed or knocked down. The survival of NSCLC cells was studied using colony formation experiment under different doses of radiation. RNA immunoprecipitation (RIP) experiment and luciferase reporter gene experiment verified the binding relationship between circ_0001287 and miR-21. Western blot was employed to examine the regulatory effects of circ_0001287 and miR-21 on phosphatase and tensin homolog (PTEN) expression. We reported that circ_0001287 expression was down-modulated in NSCLC tissues and cell lines. Besides, circ_0001287 low expression was associated with low differentiation and positive lymph node invasion of NSCLC. Circ_0001287 overexpression suppressed the multiplication, migration, invasion, and radioresistance of NSCLC cells, whereas circ_0001287 knockdown promoted the above phenotypes. Circ_0001287 could adsorb miR-21 and repress its expression, and indirectly up-modulate PTEN expression in NSCLC cells. Taken together, circ_0001287/miR-21/PTEN axis is probably involved in regulating NSCLC cell multiplication, metastasis, and radioresistance.

The Impact of COVID-19 on Primary Care General Practice Consultations in a Teaching Hospital in Shanghai, China.

Background: The COVID-19 (2019 novel coronavirus disease) pandemic is deeply concerning because of its massive mortality and morbidity, creating adverse perceptions among patients likely to impact on their overall medical care. Thus, we evaluated the impact of the COVID-19 pandemic on the pattern of primary care consultations within a Shanghai health district. Methods: A retrospective observational cohort study was performed, with data analyzed concerning the pattern of patient visits to general practitioners within the Tongren Hospital network (the sole provider of general practice to the population of 700,000). Data from all general practice consultations for adults were collected for the first 6 months of 2020, which included a 60-day lockdown period (January 24-March 24, 2020) and compared to corresponding data from the first 6 months of 2019. We evaluated changes to the numbers and patterns of primary care consultations, including subgroup analysis based on age, sex, and primary diagnosis. Results: A substantial reduction in patient visits, associated with increased median age, was observed during the first wave of the pandemic in the first 6 months of 2020, compared to the same interval during 2019. Additionally, reduced reappointments and waiting times, but increased costs per visit were observed. When analyzed by primary disease diagnosis, patient visits were reduced for all the major systems. The most striking visit reductions were in cardiovascular, respiratory, endocrine, and gastrointestinal diseases. However, psychological disorders were increased following lockdown, but there was also a dramatic fall in consultations for depression. Reduced monthly patient numbers correlated with both rate of reappointment and average waiting time during the first 6 months of both 2019 and 2020, but an inverse correlation was observed between cost per visit and monthly patient numbers. Specifically during the lockdown period, there was ~50% reduced patient visits. Conclusions: The lockdown has had a serious impact on patients' physical and psychological health. Our analysis provides objective health-related data that may inform the current controversy concerning the balance between the detrimental effects of the use of lockdown vs. the use of a more targeted approach to eliminate viral transmission. These data may improve decision-making in medical practice, policy, and education.

Peptidomics analysis revealed that a novel peptide VMP­19 protects against Ang II­induced injury in human umbilical vein endothelial cells.

Vascular endothelial dysfunction is a vital pathological change in hypertension, which is mainly caused by apoptosis and oxidative stress injury of vascular endothelial cells. Peptidomics is a method for the direct analysis of small bioactive peptides in various biological samples using liquid chromatography­mass spectrometry (MS)/MS. Given the advantages of the low molecular weight, optimum targeting and easy access to cells, peptides have attracted extensive attention in the field of drug research. However, to the best of our knowledge, little is currently known regarding the role of peptides in vascular endothelial injury. In order to investigate the peptides involved in vascular endothelial protection, MS was used to analyze the peptide profiles in the supernatant of human umbilical vein endothelial cells (HUVECs) stimulated by Ang II. The results revealed that 211 peptides were identified, of which six were upregulated and 13 were downregulated when compared with the control group. Subsequently, the present study analyzed the physical and chemical properties and biological functions of identified peptides by bioinformatics, and successfully screened a peptide (LLQDSVDFSLADAINTEFK) named VMP­19 that could alleviate the apoptosis and oxidative stress injury of HUVECs induced by Ang II. In conclusion, to the best of our knowledge, the present study was the first to use peptidomics to analyze the peptide profiles of supernatant secreted by HUVECs, and revealed that the novel peptide VMP­19 could protect HUVECs from apoptosis and oxidative stress injury. The results of the present study could provide novel insights into treatment strategies for hypertension.

The Immune Subtypes and Landscape of Gastric Cancer and to Predict Based on the Whole-Slide Images Using Deep Learning.

Background: Gastric cancer (GC) is a highly heterogeneous tumor with different responses to immunotherapy. Identifying immune subtypes and landscape of GC could improve immunotherapeutic strategies. Methods: Based on the abundance of tumor-infiltrating immune cells in GC patients from The Cancer Genome Atlas, we used unsupervised consensus clustering algorithm to identify robust clusters of patients, and assessed their reproducibility in an independent cohort from Gene Expression Omnibus. We further confirmed the feasibility of our immune subtypes in five independent pan-cancer cohorts. Finally, functional enrichment analyses were provided, and a deep learning model studying the pathological images was constructed to identify the immune subtypes. Results: We identified and validated three reproducible immune subtypes presented with diverse components of tumor-infiltrating immune cells, molecular features, and clinical characteristics. An immune-inflamed subtype 3, with better prognosis and the highest immune score, had the highest abundance of CD8+ T cells, CD4+ T-activated cells, follicular helper T cells, M1 macrophages, and NK cells among three subtypes. By contrast, an immune-excluded subtype 1, with the worst prognosis and the highest stromal score, demonstrated the highest infiltration of CD4+ T resting cells, regulatory T cells, B cells, and dendritic cells, while an immune-desert subtype 2, with an intermediate prognosis and the lowest immune score, demonstrated the highest infiltration of M2 macrophages and mast cells, and the lowest infiltration of M1 macrophages. Besides, higher proportion of EVB and MSI of TCGA molecular subtyping, over expression of CTLA4, PD1, PDL1, and TP53, and low expression of JAK1 were observed in immune subtype 3, which consisted with the results from Gene Set Enrichment Analysis. These subtypes may suggest different immunotherapy strategies. Finally, deep learning can predict the immune subtypes well. Conclusion: This study offers a conceptual frame to better understand the tumor immune microenvironment of GC. Future work is required to estimate its reference value for the design of immune-related studies and immunotherapy selection.

Identification of a Set of Genes Improving Survival Prediction in Kidney Renal Clear Cell Carcinoma through Integrative Reanalysis of Transcriptomic Data.

BACKGROUND: With an enormous amount of research concerning kidney cancer being conducted, various treatments have been applied to its cure. However, high recurrence and metastasis rates continue to pose a threat to the survival of patients with kidney renal clear cell carcinoma (KIRC). METHODS: Data from The Cancer Genome Atlas were downloaded, and a series of analyses were performed, including differential analysis, Cox analysis, weighted gene coexpression network analysis, least absolute shrinkage and selection operator analysis, multivariate Cox analysis, survival analysis, and receiver operating characteristic curve and functional enrichment analysis. RESULTS: A total of 5,777 differentially expressed genes were identified from the differential analysis. The Cox analysis showed 1,853 significant genes (P < 0.01). Weighted gene coexpression network analysis revealed that 226 genes in the module were related to clinical parameters, including Tumor-Node-Metastasis (TNM) staging. Least absolute shrinkage and selection operator and multivariate Cox analyses suggested that four genes (CDKL2, LRFN1, STAT2, and SOWAHB) had a potential function in predicting the survival time of patients with KIRC. Survival analysis uncovered that a high risk of these four genes was associated with an unfavorable prognosis. Receiver operating characteristic curve analysis further confirmed the accuracy of the risk score model. The analysis of clinicopathological parameters of the four identified genes revealed that they were associated with the progression of KIRC. CONCLUSION: The gene expression model consisting of CDKL2, LRFN1, STAT2, and SOWAHB is a promising tool for predicting the prognosis of patients with KIRC. The results of this study may provide insights into the diagnosis and treatment of KIRC.

LncRNA Blnc1 expression and its effect on renal fibrosis in diabetic nephropathy.

BACKGROUND AND OBJECTIVES: Diabetic nephropathy (DN) is one of the commonest microvascular complications of diabetes and has been the major cause of end-stage renal disease in many countries. It is of great clinical significance to further explore more efficacious therapeutic strategies for DN. This study aims to explore the effect of Blnc1 on renal fibrosis in diabetic nephropathy. METHODS: In this study, mRNA level of Blnc1 was examined by RT-PCR. HE staining and Masson staining were adopted to detect kidney damage and renal fibrosis. The renal fibrosis was evaluated by the levels of PTEN, fibronectin, collagen I and collagen IV with immunofluorescence assay and western blot analysis. Oxidative Stress and inflammatory response were detected by ELISA assay. At the same time, western blot was performed to detect the proteins related to NRF2/HO-1 and NF-κB pathways. RESULTS: Blnc1 has higher expression in serum of DN patients, STZ-induced DN model and HG-induced HK2 cells. Blnc1 interference significantly attenuated renal fibrosis, inflammation and oxidative stress via NRF2/HO-1 and NF-κB pathways. CONCLUSION: Our present study suggested that Blnc1 can affect inflammation, oxidative stress and renal fibrosis by Nrf2/HO-1 and NF-κB pathways in DN.