Humoral immunosuppression of exposure to polycyclic aromatic hydrocarbons and the roles of oxidative stress and inflammation.

Previous studies have indicated adverse health effects of exposure to polycyclic aromatic hydrocarbons (PAHs), but evidence on the association between PAH exposure and immunity is scarce and its underlying mechanism is largely unknown. This study assessed human exposure to PAHs by determining the concentrations of PAHs in serum and their metabolites in paired urine. The oxidative stress and inflammation levels were evaluated by urinary DNA damage biomarker 8-hydroxydeoxyguanosine, white blood cell counts and C-reaction protein. We investigated the relationship between PAH exposure and seven immunological components, and explored the indirect roles of oxidative stress and inflammation by mediation and moderation analysis. Multivariate regression analysis revealed that 1-hydroxynaphthalene and 2-hydroxyfluorene were negatively associated with immunoglobulin A, and 3-hydroxyphenanthrene was negatively correlated with complement component 3. Restricted cubic spline analysis demonstrated nonlinear relationships between some individual PAHs or their metabolites with immunological components. Bayesian kernel machine regression and quantile g-computation revealed significant associations of higher PAH exposure with decreased immunoglobulin G and kappa light chain levels. Phenanthrene was the compound that contributed the most to reduced immunoglobulin G. Mediation analysis demonstrated significant indirect effects of 8-hydroxydeoxyguanosine and white blood cell counts on the association between higher PAH exposure and decreased immunological components. Moderation analysis revealed that PAH exposure and decreased immunological components are significantly associated with higher levels of C-reaction protein and white blood cell counts. The results demonstrated significant immunosuppression of PAH exposure and highlighted the indirect roles of oxidative stress and inflammation. Interventions to reduce systemic inflammation may mitigate the adverse immune effects of PAH exposure.

Utility of metagenomic Next-Generation Sequencing for simultaneously detecting pathogens and neoplasms.

Objectives: Clinicians often face the challenge of differentially diagnosing febrile patients who are suspected of infectious diseases, since the clinical manifestations of infection and cancer may overlap. A single test that can detect both pathogens and tumor could provide timely and accurate diagnostic clues to aid the treatment and management of these patients. Methods: We enrolled eight patients to evaluate the utility of metagenomic Next-Generation Sequencing for simultaneously detecting pathogens and neoplasms using body fluids and tissue samples. Patients were selected by the following criteria: 1) Tumor was not considered upon hospitalization, but mNGS testing indicated neoplasm; 2) Tumor was not excluded, but microbial infection was primarily suspected according to initial clinical assessment. Results: We detected potential pathogens in five patients, three of whom had progressed into critical infections. Moreover, abnormal chromosomal copy numbers were identified in all patients that indicated presence of neoplasms, which were pathologically confirmed. Conclusions: Although copy number variations do not render a definitive cancer diagnosis, it can prompt clinicians to conduct more focused diagnostic testing for cancer, potentially saving time and cost. As a result, integrating copy number analysis with pathogen detection in mNGS may help establish rapid and accurate diagnosis for febrile patients.

RVFScan predicts virulence factor genes and hypervirulence of the clinical metagenome.

Bacterial infections often involve virulence factors that play a crucial role in the pathogenicity of bacteria. Accurate detection of virulence factor genes (VFGs) is essential for precise treatment and prognostic management of hypervirulent bacterial infections. However, there is a lack of rapid and accurate methods for VFG identification from the metagenomic data of clinical samples. Here, we developed a Reads-based Virulence Factors Scanner (RVFScan), an innovative user-friendly online tool that integrates a comprehensive VFG database with similarity matrix-based criteria for VFG prediction and annotation using metagenomic data without the need for assembly. RVFScan demonstrated superior performance compared to previous assembly-based and read-based VFG predictors, achieving a sensitivity of 97%, specificity of 98% and accuracy of 98%. We also conducted a large-scale analysis of 2425 clinical metagenomic datasets to investigate the utility of RVFScan, the species-specific VFG profiles and associations between VFGs and virulence phenotypes for 24 important pathogens were analyzed. By combining genomic comparisons and network analysis, we identified 53 VFGs with significantly higher abundances in hypervirulent Klebsiella pneumoniae (hvKp) than in classical K. pneumoniae. Furthermore, a cohort of 1256 samples suspected of K. pneumoniae infection demonstrated that RVFScan could identify hvKp with a sensitivity of 90%, specificity of 100% and accuracy of 98.73%, with 90% of hvKp samples consistent with clinical diagnosis (Cohen's kappa, 0.94). RVFScan has the potential to detect VFGs in low-biomass and high-complexity clinical samples using metagenomic reads without assembly. This capability facilitates the rapid identification and targeted treatment of hvKp infections and holds promise for application to other hypervirulent pathogens.

Novel Clinical mNGS-Based Machine Learning Model for Rapid Antimicrobial Susceptibility Testing of Acinetobacter baumannii.

Multidrug-resistant (MDR) bacteria are important public health problems. Antibiotic susceptibility testing (AST) currently uses time-consuming culture-based procedures, which cause treatment delays and increased mortality. We developed a machine learning model using Acinetobacter baumannii as an example to explore a fast AST approach using metagenomic next-generation sequencing (mNGS) data. The key genetic characteristics associated with antimicrobial resistance (AMR) were selected through a least absolute shrinkage and selection operator (LASSO) regression model based on 1,942 A. baumannii genomes. The mNGS-AST prediction model was accordingly established, validated, and optimized using read simulation sequences of clinical isolates. Clinical specimens were collected to evaluate the performance of the model retrospectively and prospectively. We identified 20, 31, 24, and 3 AMR signatures of A. baumannii for imipenem, ceftazidime, cefepime, and ciprofloxacin, respectively. Four mNGS-AST models had a positive predictive value (PPV) greater than 0.97 for 230 retrospective samples, with negative predictive values (NPVs) of 100% (imipenem), 86.67% (ceftazidime), 86.67% (cefepime), and 90.91% (ciprofloxacin). Our method classified antibacterial phenotypes with an accuracy of 97.65% for imipenem, 96.57% for ceftazidime, 97.64% for cefepime, and 98.36% for ciprofloxacin. The average reporting time of mNGS-based AST was 19.1 h, in contrast to the 63.3 h for culture-based AST, thus yielding a significant reduction of 44.3 h. mNGS-AST prediction results coincided 100% with the phenotypic AST results when testing 50 prospective samples. The mNGS-based model could be used as a rapid genotypic AST approach to identify A. baumannii and predict resistance and susceptibility to antibacterials and could be applicable to other pathogens and facilitate rational antimicrobial usage.

New comprehensive reference values for kidney function indexes across adult and geriatric ages in Chinese popuplation.

Background and aims: China has the largest number of chronic kidney disease (CKD) patients. Current CKD definition has been challenged recently. We aim to reassess kidney function in healthy Chinese population, to provide a more appropriate reference range (RIs) for diagnosis, treatment, monitoring (or screening) of kidney disease and related research. Materials and methods: A total of 49627 apparently healthy people aged 18-94 years old were enrolled. Age and sex effects were explored for the kidney function indicators and RIs were calculated non-parametrically. Results: Albumin's limits were lower than the national RIs, with 5.7 g/L lower in upper limit (UL) and 0.4 g/L lower in lower limit (LL) [RIs: 39.6-49.3 vs 40-55]. The LL of estimate glomerular filtration rate (eGFR) was 80.4 mL/min/1.73 m2 or 63.3 mL/min/1.73 m2 at the age of <50 or ≥70 years, respectively. Notably, eGFR showed an approximately 0.7 mL/min/1.73 m2 decrease every year. In addition, eGFR increase 0.35 mL/min/1.73 m2 per standard deviation increase in blood glucose when uric acid (UA) exceed the RIs. Conclusion: UA was an important factor affecting eGFR. For healthy elderly in China, albumin's limits were lower than the national RIs, and LLs of eGFR were nearly 60 mL/min/1.73 m2. Using national RIs for healthy elderly may be overly stringent.

Pneumonia Caused by Coinfection with Cytomegalovirus and Pneumocystis jirovecii in an HIV-Negative Infant Diagnosed by Metagenomic Next-Generation Sequencing.

Background: Pneumonia produced by coinfection with Pneumocystis jirovecii (PJ) and cytomegalovirus (CMV) in infants and young children without timely diagnosis and treatment is often fatal due to the limitations of traditional tests. More accurate and rapid diagnostic methods for multiple infections are urgently needed. Case Presentation: Here, we report a case of a 2-month-old boy with pneumonia caused by Pneumocystis jirovecii (PJ) and cytomegalovirus (CMV) without HIV infection. Chest computed tomography (CT) showed massive exudative consolidation in both lungs. Microscopic examination of stained sputum and smear specimens and bacterial and fungal culture tests were all negative, and CMV nucleic acid and antibody tests were positive. After a period of antiviral and anti-infective therapy, pulmonary inflammation was not relieved. Subsequently, sputum and venous blood samples were analysed by metagenomic next-generation sequencing (mNGS), and the sequences of PJ and CMV were acquired. The patient was finally diagnosed with pneumonia caused by PJ and CMV coinfection. Anti-fungal combined with anti-viral therapy was given immediately. mNGS re-examination of bronchoalveolar lavage fluid (BALF) also revealed the same primary pathogen. Therapy was stopped due to the request of the patient's guardian. Hence, the child was discharged from the hospital and eventually died. Conclusion: This case emphasizes the combined use of mNGS and traditional tests in the clinical diagnosis of mixed lung infections in infants without HIV infection. mNGS is a new adjunctive diagnostic method that can rapidly discriminate multiple causes of pneumonia.

Water-Induced Self-Assembly and In Situ Mineralization within Plant Phenolic Glycol-Gel toward Ultrastrong and Multifunctional Thermal Insulating Aerogels.

Biopolymer/silica nanocomposite aerogels are highly attractive as thermally insulating materials for prevailing energy-saving engineering but are usually plagued by their lack of mechanical strength and environmental stability. Lignin is an appealing plant phenolic biopolymer due to its natural abundance, high stiffness, water repellency, and thermostability. However, integrating lignin and silica into high-performance 3D hybrid aerogels remains a substantial challenge due to the unstable co-sol process. In diatoms, the silicic acid stabilization prior to the condensation reaction is enhanced by the intervention of biomolecules in noncovalent interactions. Inspired by this mechanism, we herein rationally design an ultrastrong silica-mineralized lignin nanocomposite aerogel (LigSi) with an adjustable multilevel micro/nanostructure and arbitrary machinability through an unusual water-induced self-assembly and in situ mineralization based on ethylene glycol-stabilized lignin/siloxane colloid. The optimized LigSi exhibits an ultrahigh stiffness (a specific modulus of ∼376.3 kN m kg-1) and can support over 5000 times its own weight without obvious deformation. Moreover, the aerogel demonstrates a combination of outstanding properties, including superior and humidity-tolerant thermal insulation (maintained at ∼0.04 W m-1 K-1 under a relative humidity of 33-94%), excellent fire resistance withstanding an ∼1200 °C flame without disintegration, low near-infrared absorption (∼9%), and intrinsic self-cleaning/superhydrophobic performance (158° WCA). These advanced properties make it an ideal thermally insulating material for diversified applications in harsh environments. As a proof of concept, a dual-mode LigSi thermal device was designed to demonstrate the application prospect of combining passive heat-trapping and active heating in the building.

DYSF promotes monocyte activation in atherosclerotic cardiovascular disease as a DNA methylation-driven gene.

Dysferlin (DYSF) has drawn much attention due to its involvement in dysferlinopathy and was reported to affect monocyte functions in recent studies. However, the role of DYSF in the pathogenesis of atherosclerotic cardiovascular diseases (ASCVD) and the regulation mechanism of DYSF expression have not been fully studied. In this study, Gene Expression Omnibus (GEO) database and epigenome-wide association study (EWAS) literatures were searched to find the DNA methylation-driven genes (including DYSF) of ASCVD. The hub genes related to DYSF were also identified through weighted correlation network analysis (WGCNA). Regulation of DYSF expression through its promoter methylation status was verified using peripheral blood leucocytes (PBLs) from ASCVD patients and normal controls, and experiments on THP1 cells and Apoe-/- mice. Similarly, the expressions of DYSF related hub genes, mainly contained SELL, STAT3 and TMX1, were also validated. DYSF functions were then evaluated by phagocytosis, transwell and adhesion assays in DYSF knock-down and overexpressed THP1 cells. The results showed that DYSF promoter hypermethylation up-regulated its expression in clinical samples, THP1 cells and Apoe-/- mice, confirming DYSF as a DNA methylation-driven gene. The combination of DYSF expression and methylation status in PBLs had a considerable prediction value for ASCVD. Besides, DYSF could enhance the phagocytosis, migration and adhesion ability of THP1 cells. Among DYSF related hub genes, SELL was proven to be the downstream target of DYSF by wet experiments. In conclusion, DYSF promoter hypermethylation upregulated its expression and promoted monocytes activation, which further participated in the pathogenesis of ASCVD.

Atheroprotective mechanism by which folic acid regulates monocyte subsets and function through DNA methylation.

BACKGROUND: Recent studies have suggested that folic acid can restore abnormal DNA methylation and monocyte subset shifts caused by hyperhomocysteinemia (HHcy) and hyperlipidemia (HL). However, the exact mechanism of action is still not fully understood. In this study, we further investigated the reversal effect and underlying mechanism of folic acid on the shift in monocyte subsets induced by aberrant lipids and Hcy metabolism via DNA methylation in vitro and in vivo. RESULTS: Our results showed that intermediate monocytes were significantly increased but had the lowest global 5-methylcytosine (5-mC) levels in coronary artery disease (CAD) patients, which might lead to a decrease in the global 5-mC levels of peripheral blood leukocytes (PBLs). We also discovered that ARID5B might mediate the increased proportion of intermediate monocytes, as this factor was related to the proportion of monocyte subsets and the expression of CCR2. The expression of ARID5B was inversely associated with the hypermethylated cg25953130 CpG site, which was induced by HL and HHcy. ARID5B could also regulate monocyte CCR2, MCP-1, and TNF-α expression, adhesion and migration, macrophage polarization, and monocyte/macrophage apoptosis, which might explain the regulatory effect of ARID5B on monocyte subset shifting. Folic acid reversed HL- and HHcy-mediated aberrant global and cg25953130 DNA methylation, reduced the proportion of intermediate monocytes, and inhibited the formation of atherosclerotic plaques. CONCLUSION: Folic acid plays a protective role against atherosclerosis through the regulation of DNA methylation, ARID5B expression, and monocyte subsets.

Four Types of ST11 Novel Mutations From Increasing Carbapenem-Resistant Klebsiella pneumoniae in Guangdong, 2016-2020.

Objectives: This study aimed to explore changes in carbapenem-resistant Klebsiella pneumoniae (CR-KP) isolates collected in Guangdong over the period of 2016-2020. Methods: Antibacterial susceptibility was quantified through VITEK 2 compact and K-B method. Carbapenemase phenotypes and genotypes were characterized by modified carbapenem inactivation method (mCIM), EDTA-carbapenem inactivation method (eCIM), and polymerase chain reaction (PCR). Molecular characteristics and evolutionary trends were analyzed by multilocus sequence typing and evolutionary tree. Results: Isolates (2,847) of K. pneumoniae were separated in 2016-2020, and the separate rate of CR-KP increased from 5.65 to 9.90% (p = 0.009). The top 3 wards were intensive care unit (ICU) (21.92%), neonatal wards (13.70%), and respiratory wards (12.33%). In 146 CR-KP strains, serine carbapenemase was the main phenotype, and KPC was the main genotype, and 57 contained two resistant genes, and 1 contained three resistant genes. Two polygenic strains were first found: IMP + GES and KPC + NDM + VIM, but all the phenotypes were metalloenzyme, which indicated that metalloenzyme was usually the first choice for CR-KP resistance. In addition, all the ST54 of metalloenzyme type contained IMP, and all the ST45, ST37, and ST76 contained OXA. ST11 was the most prevalent (42.47%); ST11 and its mutants proved the predominant sequence type making up 51.1% of the carbapenemase-producing isolates. A novel type of ST11 mutation, the rpoB was mutated from sequence 1 to sequence 146, was in an independent separate branch on the evolutionary tree and was resistant to all antibacterial agents. The other three mutants, rpoB 1-15, infB 3-148, and infB 3-80, are also resistant to all antibacteria. Of note, all the four mutants produced serine carbapenemase and contained KPC, and indicated that the prevalent strain in China, ST11, has serious consequences and potential outbreaks. Conclusion: The infection rate of CR-KP has increased, and ICU and neonatal wards have become the key infection areas. Producing serine enzyme, the KPC genotype, and ST11 are the predominant CR-KP. Polygenic strains and ST11 mutation made clinical treatment difficult and may become a potential threat.

A novel One-pot rapid diagnostic technology for COVID-19.

Novel coronavirus disease (COVID-19) caused by SARS-CoV-2 is an ongoing global pandemic associated with high rates of morbidity and mortality. RT-qPCR has become the diagnostic standard for the testing of SARS-CoV-2 in most countries. COVID-19 diagnosis generally relies upon RT-qPCR-mediated identification of SARS-CoV-2 viral RNA, which is costly, labor-extensive, and requires specialized training and equipment. Herein, we established a novel one-tube rapid diagnostic approach based upon formamide and colorimetric RT-LAMP (One-Pot RT-LAMP) that can be used to diagnose COVID-19 without the extraction of specific viral RNA. The technique could visually detect SARS-CoV-2 within 45 min with a limit of detection of 5 copies per reaction in extracted RNA, and about 7.66 virus copies per µL in viral transport medium. The One-Pot RT-LAMP test showed a high specificity without cross-reactivity with 12 viruses including SARS-CoV, MERS-CoV, and human infectious influenza virus (H1N1/H3N2 of influenza A and B virus, ect. We validated this One-Pot RT-LAMP approach by its successful use for the analysis of 45 clinical nasopharyngeal swab samples, yielding results identical to those of traditional RT-qPCR analyses, while achieving good selectivity and sensitivity relative to a commercial RT-qPCR approach. As such, this One-Pot RT-LAMP technology may be a valid means of conducting high-sensitivity, low-cost and rapid SARS-CoV-2 identification without the extraction of viral RNA.

Development and Clinical Application of a Rapid and Sensitive Loop-Mediated Isothermal Amplification Test for SARS-CoV-2 Infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak urgently necessitates sensitive and convenient COVID-19 diagnostics for the containment and timely treatment of patients. We aimed to develop and validate a novel reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay to detect SARS-CoV-2. Patients with suspected COVID-19 and close contacts were recruited from two hospitals between 26 January and 8 April 2020. Respiratory samples were collected and tested using RT-LAMP, and the results were compared with those obtained by reverse transcription-quantitative PCR (RT-qPCR). Samples yielding inconsistent results between these two methods were subjected to next-generation sequencing for confirmation. RT-LAMP was also applied to an asymptomatic COVID-19 carrier and patients with other respiratory viral infections. Samples were collected from a cohort of 129 cases (329 nasopharyngeal swabs) and an independent cohort of 76 patients (152 nasopharyngeal swabs and sputum samples). The RT-LAMP assay was validated to be accurate (overall sensitivity and specificity of 88.89% and 99.00%, respectively) and diagnostically useful (positive and negative likelihood ratios of 88.89 and 0.11, respectively). RT-LAMP showed increased sensitivity (88.89% versus 81.48%) and high consistency (kappa, 0.92) compared to those of RT-qPCR for SARS-CoV-2 screening while requiring only constant-temperature heating and visual inspection. The time required for RT-LAMP was less than 1 h from sample preparation to the result. In addition, RT-LAMP was feasible for use with asymptomatic patients and did not cross-react with other respiratory pathogens. The developed RT-LAMP assay offers rapid, sensitive, and straightforward detection of SARS-CoV-2 infection and may aid the expansion of COVID-19 testing in the public domain and hospitals.IMPORTANCE We developed a visual and rapid reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay targeting the S gene for SARS-CoV-2 infection. The strength of our study was that we validated the RT-LAMP assay using 481 clinical respiratory samples from two prospective cohorts of suspected COVID-19 patients and on the serial samples from an asymptomatic carrier. The developed RT-LAMP approach showed an increased sensitivity (88.89%) and high consistency (kappa, 0.92) compared with those of reverse transcription-quantitative PCR (RT-qPCR) for SARS-CoV-2 screening while requiring only constant-temperature heating and visual inspection, facilitating SARS-CoV-2 screening in well-equipped labs as well as in the field. The time required for RT-LAMP was less than 1 h from sample preparation to the result (more than 2 h for RT-qPCR). This study showed that the RT-LAMP assay was a simple, rapid, and sensitive approach for SARS-CoV-2 infection and can facilitate COVID-19 diagnosis, especially in resource-poor settings.

CircRNA ZNF609 in peripheral blood leukocytes acts as a protective factor and a potential biomarker for coronary artery disease.

BACKGROUND: Circular RNAs (circRNAs) have been reported to aberrantly express in coronary artery disease (CAD). Due to their special structures, circRNAs have the potential to be specific and stable markers. We conducted this study to explore circZNF609's function in atherosclerosis and to evaluate its predictive values for CAD. METHODS: About 330 CAD patients and 209 controls were enrolled and the expression of circZNF609 in peripheral blood leukocytes (PBLs) was detected by quantitative real time polymerase chain reaction (RT-PCR). Spearman correlation, multivariate regression, multivariate logistic regression and receiver operating characteristic curve (ROC) were performed. Moreover, circZNF609 was overexpressed in mice macrophage RAW264.7 to investigate its influence on inflammatory cytokines. Finally, bioinformatics analysis was executed to excavate the potential downstream pathway of circZNF609. RESULTS: The expression level of circZNF609 in PBLs of CAD patients was significantly decreased compared with the controls (the fold changes of 0.4133, P<0.0001). The logistic regression analysis showed that decreased circZNF609 expressions were independently associated with increased risks of CAD. The area under the ROC curve was 0.761 (95% CI: 0.721-0.800, P<0.0001). Furthermore, the circZNF609 expression level was correlated with C-reactive protein (r=-0.138, P=0.026) and lymphocyte counts (r=0.16, P=0.01). After overexpression of circZNF609 in RAW264.7 cells, the expression level of IL-6 (P<0.001) and TNF-α (P<0.01) were significantly decreased and IL-10 was significantly increased (P<0.001). Bioinformatics analysis suggested that the abnormal expression of circZNF609 might probably sponge miRNA to modulate the inflammation cytokines. CONCLUSIONS: CircRNA ZNF609 played an anti-inflammatory role and was an independent protective factor for CAD. It represented a moderate diagnostic value and might provide a new therapeutic target for CAD.

[Identification of a novel FBN1 variant in a pedigree affected with Marfan syndrome].

OBJECTIVE: To explore the genetic basis for a pedigree affected with Marfan syndrome (MFS). METHODS: Clinical data of the patients was collected. With genomic DNA extracted from peripheral blood samples, potential mutation was detected by targeted exome sequencing. Candidate variants were validated by Sanger sequencing and bioinformatic analysis. RESULTS: Targeted exome sequencing and Sanger sequencing revealed a missense c.649T to C(p.Trp217Arg) variant in the exon 7 of FBN1 gene, which was unreported previously. Bioinformatics analysis suggested that the variant can cause amino acid replacement and affect the structure and function of fibrillin-1. CONCLUSION: A novel missense variant of the FBN1 gene was identified, which probably underlies the autosomal dominant MFS in this pedigree.

Using circular RNA SMARCA5 as a potential novel biomarker for hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common malignant tumor worldwide. Circular RNAs (circRNAs), a new class of endogenous non-coding RNAs, are widespread and abundant in mammalian cells. Cumulative evidence showed that circRNAs play significant roles in the process of cancer. However, the expression and function of circRNAs in HCC remain to be investigated. METHODS: The expression of circular RNA SMARCA5 (circSMARCA5) in tissues and plasma samples was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The role of circSMARCA5 in HCC progression was assessed by in vitro experiments. A receiver operating characteristic (ROC) curve was established to evaluate the value of circSMARCA5 as a biomarker in HCC. RESULTS: The expression of circSMARCA5 was significantly downregulated in HCC tissues compared with para-carcinoma tissues. CircSMARCA5 levels were correlated with tumor differentiation (p = 0.023), Tumor-node-metastasis (TNM) stage (p = 0.001), cancer invasion (p = 0.004), as well as cancer diameter (p = 0.018). In vitro cell experiments revealed that overexpression of circSMARCA5 resulted in inhibited proliferation, increased apoptosis and suppressed invasion. Moreover, we found that circSMARCA5 expression was down-regulated in plasma samples of patients with HCC. The ROC curve analyses revealed that plasma circSMARCA5 showed a high accuracy (AUC = 0.938, 0.853, 0.711) for diagnosing HCC from healthy controls, hepatitis and cirrhosis. The area under the ROC curve of plasma circSMARCA5 in combination with AFP in diagnosing HCC from hepatitis and cirrhosis was 0.903 and 0.858. Especially, plasma circSMARCA5 presented a high accuracy (AUC = 0.847, 0.706) for detecting HCC with serum AFP below 200 ng/ml from those hepatitis and cirrhosis with AFP below 200 ng/ml. CONCLUSION: Our study revealed that circSMARCA5 may promote apoptosis, inhibit proliferation, invasion and metastasis of HCC cells. CircSMARCA5 may serve as a potential prediction and monitor biomarker for HCC, especially in HCC patients with AFP blow the cutoff value.

The pretreatment lymphocyte to monocyte ratio predicts clinical outcome for patients with urological cancers: A meta-analysis.

BACKGROUND: The lymphocyte to monocyte ratio (LMR), a novel systematic biomarker of inflammation, has been reported to be associated with the progression and prognosis of many malignant cancers. However, the relationship between LMR and survival outcome of urological cancers (UCs) remains controversial. Herein, we conducted a meta-analysis to identify the prognostic value of pretreatment LMR in patients with UCs. METHODS: A literature search was performed in PubMed, Web of Science, Embase, Cochrane Library, Cochrane Central Register of Controlled Trials, Scopus, and CINAHL databases up to July 2018. The pooled hazard ratios (HRs) and odds ratios (ORs) with corresponding 95% confidence intervals (CIs) were calculated to evaluate the association of LMR with survival outcome and clinicopathological characteristics in UCs. RESULTS: A total of 17 articles containing 5552 patients were included in our study. The synthesized analysis showed that elevated pretreatment LMR level could predict favorable overall survival (OS) of UCs patients (pooled HR = 0.82, 95%CI: 0.77-0.87). Additionally, the decreased LMR level was correlated with tumor stage (OR = 1.72, 95%CI: 1.15-2.55), lymph node metastasis (OR = 1.46, 95%CI:1.06-1.99), grade (OR = 1.79, 95%CI:1.41-2.27), tumor size (OR = 2.21, 95%CI:1.81-2.68) and necrosis (OR = 1.71, 95%CI:1.36-2.16). CONCLUSION: The high pretreatment LMR was associated with favorable prognosis, and could be a potential prognostic biomarker in patients with UCs.

Genomic 5-mC contents in peripheral blood leukocytes were independent protective factors for coronary artery disease with a specific profile in different leukocyte subtypes.

Background: Alterations in DNA methylation are demonstrated in atherosclerosis pathogenesis. However, changing rules of global DNA methylation and hydroxymethylation in peripheral blood leukocytes (PBLs) and different blood cell subtypes of coronary artery disease (CAD) patients are still inconclusive, and much less is known about mechanisms underlying. Results: We recruited 265 CAD patients and 270 healthy controls with genomic DNA from PBLs, of which 50 patients and 50 controls were randomly chosen with DNA from isolated neutrophils, lymphocytes and monocytes, and RNA from PBLs. Genomic 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) contents were quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) assay. Genomic 5-mC contents were negatively associated with the serum total cholesterol (TC) level (P = 0.010), age (P = 0.016), and PBL classifications (P = 0.023), explaining 6.8% individual variation in controls. Furthermore, genomic 5-mC contents were inversely associated with an increased risk of CAD (odds ratio (OR) = 0.325, 95% confidence interval (CI) = 0.237~0.445, P = 2.62 × 10- 12), independent of PBL counts and classifications, age, sex, histories of hyperlipidemia, hypertension, and diabetes. Within-individual analysis showed a general 5-mC decrease in PBL subtypes, but significant difference was found in monocytes only (P = 0.001), accompanied by increased 5-hmC (P = 3.212 × 10- 4). In addition, coincident to the reduced DNMT1 expression in patients' PBLs, the expression level of DNMT1 was significantly lower (P = 0.022) in oxidized low-density lipoprotein (ox-LDL) stimulated THP-1-derived foam cells compared to THP-1 monocytes, with decreased genomic 5-mdC content (P = 0.038). Conclusions: Global hypomethylation of blood cells defined dominantly by the monocyte DNA hypomethylation is independently associated with the risk of CAD in Chinese Han population. The importance of monocytes in atherosclerosis pathophysiology may demonstrate via an epigenetic pathway, but prospective studies are still needed to test the causality.

Development of C60-based labeling reagents for the determination of low-molecular-weight compounds by matrix assisted laser desorption ionization mass (I): Determination of amino acids in microliter biofluids.

Quantification of low molecular weight compounds (<800 Da) using matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI MS) is challenging due to the matrix signal interference at low m/z region and poor reproducibility of MS responses. In this study, a C60 labeling-MALDI MS strategy was proposed for the fast, sensitive and reliable determination of amino acids (AAs) in biofluids. An N-hydroxysuccinimide functionalized C60 was synthesized as the labeling reagent and added as an 880 Da tag to AAs; a carboxyl acid containing C60 was employed as the internal standards to normalize MS variations. This solved the inherent problems of MALDI MS for small molecule analysis. The entire analytical procedure-which consisted of simple protein precipitation and 10 min of derivatization in a microwave prior to the MALDI MS analysis-could be accomplished within 20 min with high throughput and great sample matrix tolerance. AA quantification showed good linearity from 0.7 to 70.0 µM with correlation coefficients (R) larger than 0.9954. The limits of detection were 70.0-300.0 fmol. Good reproducibility and reliability of the method were demonstrated by intra-day and inter-day precision with relative standard deviations less than 13.8%, and the recovery in biofluid ranged from 80.4% to 106.8%. This approach could be used in 1 µL of urine, serum, plasma, whole blood, and cerebrospinal fluid. Most importantly, the C60 labeling strategy is a universal approach for MALDI MS analysis of various LMW compounds because functionalized C60 is now available on demand.

Analysis of liposoluble carboxylic acids metabolome in human serum by stable isotope labeling coupled with liquid chromatography-mass spectrometry.

Fatty acids (FAs) are groups of liposoluble carboxylic acids (LCAs) and play important roles in various physiological processes. Abnormal contents or changes of FAs are associated with a series of diseases. Here we developed a strategy with stable isotope labeling combined with liquid chromatography-tandem mass spectrometry (IL-LC-MS) analysis for comprehensive profiling and relative quantitation of LCAs in human serum. In this strategy, a pair of isotope labeling reagents (2-dimethylaminoethylamine (DMED)) and d4-2-dimethylaminoethylamine (d4-DMED) were employed to selectively label carboxyl groups of LCAs. The DMED and d4-DMED labeled products can lose four characteristic neutral fragments of 45 and 49Da or 63 and 67Da in collision-induced dissociation. Therefore, quadruple neutral loss scan (QNLS) mode was established and used for non-targeted profiling of LCAs. The peak pairs of DMED and d4-DMED labeling with the same retention time, intensity and characteristic mass differences were extracted from the two NLS spectra respectively, and assigned as potential LCA candidates. Using this strategy, 241 LCA candidates were discovered in the human serum; 156 carboxylic acid compounds could be determined by searching HMDB and METLIN databases (FAs are over 90%) and 21 of these LCAs were successfully identified by standards. Subsequently, a modified pseudo-targeted method with multiple reaction monitoring (MRM) detection mode was developed and used for relative quantification of LCAs in human serum from type 2 diabetes mellitus (T2DM) patients and healthy controls. As a result, 81 LCAs were found to have significant difference between T2DM patients and healthy controls. Taken together, the isotope labeling combined with tandem mass spectrometry analysis demonstrated to be a powerful strategy for identification and quantification of LCA compounds in serum samples.

DNA hydroxymethylation age of human blood determined by capillary hydrophilic-interaction liquid chromatography/mass spectrometry.

BACKGROUND: Aging is a complex phenomenon and characterized by a progressive decline in physiology and function of adult tissues. However, it hasn't been well established of the correlation between aging and global DNA methylation and hydroxymethylation that regulate the growth and development of higher organisms. RESULTS: We developed an on-line trapping/capillary hydrophilic-interaction liquid chromatography/electrospray ionization-mass spectrometry method for ultra-sensitive and simultaneous quantification of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in genomic DNA from human blood. Limits of detection for 5-mC and 5-hmC were 0.04 and 0.13 fmol, respectively. The imprecision and recovery of the method were determined with the relative standard deviations (RSDs) and relative errors being <11.2 and 14.0 %, respectively. We analyzed the contents of 5-mC and 5-hmC in genomic DNA of blood from 238 healthy people aged from 1 to 82 years. The results showed that 5-hmC content was significantly decreased and highly correlated with aging process, while 5-mC only showed slight correlation with age. We then established a DNA hydroxymethylation age model according to 5-hmC content with a mean absolute deviation (MAD) of approximate 8.9 years. We also calculated the mean relative error (MRE) using the predicted ages based on the age model and the chronological ages. The results showed that the MRE was 18.3 % for samples with ages from 20 to 82 years (95 % confidence interval, N = 190). CONCLUSIONS: The global DNA hydroxymethylation represents a strong and reproducible mark of chronological age, which could be potentially applied in health assessment and prevention of diseases. The identification of biological or environmental factors that influence DNA hydroxymethylation aging rate may permit quantitative assessments of their impacts on health.