Influence factors of metagenomic next-generation sequencing negative results in diagnosed patients with spinal infection.

The aim of this study was to evaluate the influence factors of metagenomic next-generation sequencing (mNGS) negative results in the diagnosed patients with spinal infection. mNGS test was applied in a cohort of 114 patients with suspected spinal infection, among which 56 patients had a final diagnosis of spinal infection. mNGS achieved a sensitivity of 75.0% (95% CI, 61.6% to 85.6%) and a specificity of 84.5% (95% CI, 72.6% to 92.7%), using histopathology and culture results as reference. Diagnosed patients with a negative culture result had lower white blood cell account, percentage of neutrophilic granulocyte, C-reactive protein (all P<0.05) and relatively higher rate of prior antimicrobial treatment history (P=0.059). However, diagnosed patients with a negative mNGS result did not have such difference with mNGS-positive patients, suggesting that mNGS was not strictly limited by the above indicators, which presented the advantages of this technique from another point of view.

Bioorthogonal Reaction-Mediated Enzymatic Elongation-Driven Dendritic Nanoassembly for Genome-Wide Analysis of 5-Hydroxymethyluracil in Breast Tissues.

5-Hydroxymethyluracil (5hmU) is an oxidation derivative of thymine in the genomes of various organisms and may serve as both an epigenetic mark and a cancer biomarker. However, the current 5hmU assays usually have drawbacks of laborious procedures, low specificity, and unsatisfactory sensitivity. Herein, we demonstrate the click chemistry-mediated hyperbranched amplification-driven dendritic nanoassembly for genome-wide analysis of 5hmU in breast cell lines and human breast tissues. The proposed strategy possesses good selectivity, ultralow background, and high sensitivity with a detection limit of 83.28 aM. This method can accurately detect even a 0.001% 5hmU level in the mixture. Moreover, it can determine 5hmU at single-cell level and distinguish the expressions of 5hmU in tissues of normal persons and breast cancer patients, holding great promise in 5hmU-related biological research and clinical diagnosis.

Small non-coding RNAome changes during human chondrocyte senescence as potential epigenetic targets in age-related osteoarthritis.

Chondrocyte senescence is a decisive component of age-related osteoarthritis, however, the function of small noncoding RNAs (sncRNAs) in chondrocyte senescence remains underexplored. Human hip joint cartilage chondrocytes were cultivated up to passage 4 to induce senescence. RNA samples were extracted and then analyzed using small RNA sequencing and qPCR. ß-galactosidase staining was used to detect the effect of sncRNA on chondrocyte aging. Results of small RNA sequencing showed that 279 miRNAs, 136 snoRNAs, 30 snRNAs, 102 piRNAs, and 5 rasiRNAs were differentially expressed in senescent chondrocytes. The differential expression of 150 sncRNAs was further validated by qPCR. Transfection of sncRNAs and ß-galactosidase staining were also performed to further revealed that hsa-miR-135b-5p, SNORA80B-201, and RNU5E-1-201 have the function to restrain chondrocyte senescence, while has-piR-019102 has the function to promote chondrocyte senescence. Our data suggest that sncRNAs have therapeutic potential as novel epigenetic targets in age-related osteoarthritis.

Procyanidin B2 alleviates oxidative stress-induced nucleus pulposus cells apoptosis through upregulating Nrf2 via PI3K-Akt pathway.

Oxidative stress can lead to nucleus pulposus cell (NPC) apoptosis, which is considered to be one of the main contributors to intervertebral disc degeneration (IVDD). Procyanidin B2 is a natural antioxidant that protects against oxidative stress. However, whether procyanidin B2 protects NPCs from oxidative stress remains unknown. In this study, we demonstrated that procyanidin B2 could reduce tert-butyl hydroperoxide-induced reactive oxygen species in rat NPCs and attenuate rat NPC apoptosis. Further experiments revealed that procyanidin B2 upregulated the expression of both nuclear factor erythroid 2-related factor 2 (Nrf2) and phosphorylation of protein kinase B (Akt). We then used silencing of Nrf2 and LY294002 to silence Nrf2 expression and block the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, respectively, and found that the protective roles of procyanidin B2 in NPCs were inhibited. Therefore, we demonstrated that procyanidin B2 alleviated rat NPC apoptosis induced by oxidative stress by upregulating Nrf2 via activation of the PI3K/Akt signaling pathway. This study provides a potential therapeutic approach for procyanidin B2 in IVDD, which might help in the development of new drugs for IVDD treatment.

Development of a quadruple PCR-based gene microarray for detection of vaccine and wild-type classical swine fever virus, African swine fever virus and atypical porcine pestivirus.

BACKGROUND: Classical swine fever (CSF), African swine fever (ASF), and atypical porcine pestivirus (APPV) are acute, virulent, and contagious viral diseases currently hampering the pig industry in China, which result in mummification or stillbirths in piglets and mortality in pigs. Diagnostic assays for the differentiation of infection and vaccination of CSFV, in addition to the detection of ASFV and APPV, are urgently required for better prevention, control, and elimination of these viral diseases in China. METHODS: A quadruple PCR-based gene microarray assay was developed in this study to simultaneously detect wild-type and vaccine CSFV strains, ASFV and APPV according to their conserved regions. Forty-two laboratory-confirmed samples, including positive samples of 10 other swine viral diseases, were tested using this assay to confirm its high specificity. RESULTS: This assay's limit of detections (LODs) for the wild-type and vaccine CSFV were 6.98 and 6.92 copies/µL. LODs for ASFV and APPV were 2.56 × 10 and 1.80 × 10 copies/µL, respectively. When compared with standard RT-PCR or qPCR for CSFV (GB/T 26875-2018), ASFV (MARR issue No.172), or APPV (CN108611442A) using 219 clinical samples, the coincidence was 100%. The results showed that this assay with high sensitivity could specifically distinguish ASFV, APPV, and CSFV, including CSFV infection and immunization. CONCLUSION: This assay provides a practical, simple, economic, and reliable test for the rapid detection and accurate diagnosis of the three viruses and may have good prospects for application in an epidemiological investigation, prevention, and control and elimination of these three diseases.

Site-specific MCM sumoylation prevents genome rearrangements by controlling origin-bound MCM.

Timely completion of eukaryotic genome duplication requires coordinated DNA replication initiation at multiple origins. Replication begins with the loading of the Mini-Chromosome Maintenance (MCM) complex, proceeds by the activation of the Cdc45-MCM-GINS (CMG) helicase, and ends with CMG removal after chromosomes are fully replicated. Post-translational modifications on the MCM and associated factors ensure an orderly transit of these steps. Although the mechanisms of CMG activation and removal are partially understood, regulated MCM loading is not, leaving an incomplete understanding of how DNA replication begins. Here we describe a site-specific modification of Mcm3 by the Small Ubiquitin-like MOdifier (SUMO). Mutations that prevent this modification reduce the MCM loaded at replication origins and lower CMG levels, resulting in impaired cell growth, delayed chromosomal replication, and the accumulation of gross chromosomal rearrangements (GCRs). These findings demonstrate the existence of a SUMO-dependent regulation of origin-bound MCM and show that this pathway is needed to prevent genome rearrangements.

A prognostic survival model based on metabolism-related gene expression in plasma cell myeloma.

Accurate survival prediction of persons with plasma cell myeloma (PCM) is challenging. We interrogated clinical and laboratory co-variates and RNA matrices of 1040 subjects with PCM from public datasets in the Gene Expression Omnibus database in training (N = 1) and validation (N = 2) datasets. Genes regulating plasma cell metabolism correlated with survival were identified and seven used to build a metabolic risk score using Lasso Cox regression analyses. The score had robust predictive performance with 5-year survival area under the curve (AUCs): 0.71 (95% confidence interval, 0.65, 0.76), 0.88 (0.67, 1.00) and 0.64 (0.57, 0.70). Subjects in the high-risk training cohort (score > median) had worse 5-year survival compared with those in the low-risk cohort (62% [55, 68%] vs. 85% [80, 90%]; p < 0.001). This was also so for the validation cohorts. A nomogram combining metabolic risk score with Revised International Staging System (R-ISS) score increased survival prediction from an AUC = 0.63 [0.58, 0.69] to an AUC = 0.73 [0.66, 0.78]; p = 0.015. Modelling predictions were confirmed in in vitro tests with PCM cell lines. Our metabolic risk score increases survival prediction accuracy in PCM.

An Immune Risk Score Predicts Survival of Patients with Acute Myeloid Leukemia Receiving Chemotherapy.

PURPOSE: Prediction models for acute myeloid leukemia (AML) are useful, but have considerable inaccuracy and imprecision. No current model includes covariates related to immune cells in the AML microenvironment. Here, an immune risk score was explored to predict the survival of patients with AML. EXPERIMENTAL DESIGN: We evaluated the predictive accuracy of several in silico algorithms for immune composition in AML based on a reference of multi-parameter flow cytometry. CIBERSORTx was chosen to enumerate immune cells from public datasets and develop an immune risk score for survival in a training cohort using least absolute shrinkage and selection operator Cox regression model. RESULTS: Six flow cytometry-validated immune cell features were informative. The model had high predictive accuracy in the training and four external validation cohorts. Subjects in the training cohort with low scores had prolonged survival compared with subjects with high scores, with 5-year survival rates of 46% versus 19% (P < 0.001). Parallel survival rates in validation cohorts-1, -2, -3, and -4 were 46% versus 6% (P < 0.001), 44% versus 18% (P = 0.041), 44% versus 24% (P = 0.004), and 62% versus 32% (P < 0.001). Gene set enrichment analysis indicated significant enrichment of immune relation pathways in the low-score cohort. In multivariable analyses, high-risk score independently predicted shorter survival with HRs of 1.45 (P = 0.005), 2.12 (P = 0.004), 2.02 (P = 0.034), 1.66 (P = 0.019), and 1.59 (P = 0.001) in the training and validation cohorts, respectively. CONCLUSIONS: Our immune risk score complements current AML prediction models.

Identification of cancer stem cell characteristics in liver hepatocellular carcinoma by WGCNA analysis of transcriptome stemness index.

Cancer stem cells (CSCs) are characterized by self-renewal and -differential potential as compared to common cancer cells and play an important role in the development and therapeutic resistance of liver hepatocellular carcinoma (LIHC). However, the specific pathogenesis of LIHC stem cells is still unclear, and the genes involved in the stemness of LIHC stem cells are currently unknown. In this study, we investigated novel biomarkers associated with LIHC and explored the expression characteristics of stem cell-related genes in LIHC. We found that mRNA expression-based stemness index (mRNAsi) was significantly overexpressed in liver cancer tissues. Further, mRNAsi expression in LIHC increased with the tumor pathological grade, with grade 4 tumors harboring the greatest stem cell features. Upon establishing mRNAsi scores based on mRNA expression of every gene, we found an association with poor overall survival in LIHC. Moreover, modules of interest were determined based on weighted gene co-expression network analysis (WGCNA) inclusion criteria, and three significant modules (red, green, and brown) and 21 key genes (DCN, ECM1, HAND2, PTGIS, SFRP1, SRPX, COLEC10, GRP182, ADAMTS7, CD200, CDH11, COL8A1, FAP, LZTS1, MAP1B, NAV1, NOTCH3, OLFML2A, PRR16, TMEM119, and VCAN) were identified. Functional analysis of these 21 genes demonstrated their enrichment in pathways involved in angiogenesis, negative regulation of DNA-binding transcription factor activity, apoptosis, and autophagy. Causal relationship with proteins indicated that the Wnt, Notch, and Hypoxia pathways are closely related to LIHC tumorigenesis. To our knowledge, this is the first report of a novel CSC biomarker, mRNAsi, to predict the prognosis of LIHC. Further, we identified 21 key genes through mRNA expression network analysis, which could be potential therapeutic targets to inhibit the stemness of cancer cells in LIHC.

Effects of Vaccination with the C-Strain Vaccine on Immune Cells and Cytokines of Pigs Against Classical Swine Fever Virus.

The attenuated C-strain vaccine against classical swine fever virus (CSFV) is one of the safest and most effective attenuated vaccines. However, little is known of the host immune response after vaccination with the C-strain vaccine. Blood samples from vaccinated pigs were collected to evaluate the number of immune cells, the level of specific CSFV antibody, and related cytokines induced by the vaccination of C-strain vaccine. The C-strain nucleic acid was gradually removed and specific antibody to vaccine kept increasing; the amount of the lymphocyte, Tc cell, and Th cell increased; some inflammatory cytokines such as interleukin (IL)-1 and tumor necrosis factor-α mainly showed downregulated trends, but IL-6 and IL-8 were upregulated greatly; IL-2, IL-4, IL-5, IL-12p40, IL-13, interferon (IFN)-I, and Toll-like receptors (TLRs) kept high expression level after 28 days postvaccination (dpv); IFN-γ was upregulated slightly at 5 and 9 dpv, respectively. These results suggest that the C-strain vaccine induces a Th2 cell response to produce the specific antibody. The vaccine virus replicates at very low level. C-strain vaccine burden has close relationship with the expression of TLRs. The overexpression of TLRs initiates the innate immune system to clear up the vaccine. Meanwhile, ILs expressed by immune system induce the differentiation of B cells and produce specific antibody.

Caught in a TRAP: substrate-binding proteins in secondary transport.

Substrate-binding protein (SBP)-dependent secondary transporters are ubiquitous in prokaryotes yet poorly characterised. Recently, the structures of over 10 prokaryotic SBPs have been solved, which we compare here to consider their impact on our understanding of transporter function and evolution. Seven structures are from tripartite ATP-independent periplasmic (TRAP) transporters of the DctP-type, which have similar overall structures distinct from SBPs used by ATP-binding cassette (ABC) transporters, despite recognising a range of substrates. A defining feature of substrate recognition in the DctP-TRAP SBPs is the formation of a salt bridge between a highly conserved arginine and a carboxylate group in the substrate, suggesting that these transporters might have evolved specifically for uptake of diverse organic acids. Remarkably, two of the DctP-TRAP SBPs are clearly dimers and the potential impact of this on transporter function will be discussed. Other SBPs used in secondary transporters are structurally similar to ABC SBPs, demonstrating that multiple families of SBPs have evolved to function with secondary transporters.