Metabolomic analyses reveal new stage-specific features of COVID-19.

The current pandemic of coronavirus disease 2019 (COVID-19) has affected >160 million individuals to date, and has caused millions of deaths worldwide, at least in part due to the unclarified pathophysiology of this disease. Identifying the underlying molecular mechanisms of COVID-19 is critical to overcome this pandemic. Metabolites mirror the disease progression of an individual and can provide extensive insights into their pathophysiological significance at each stage of disease. We provide a comprehensive view of metabolic characterisation of sera from COVID-19 patients at all stages using untargeted and targeted metabolomic analysis. As compared with the healthy controls, we observed different alteration patterns of circulating metabolites from the mild, severe and recovery stages, in both the discovery cohort and the validation cohort, which suggests that metabolic reprogramming of glucose metabolism and the urea cycle are potential pathological mechanisms for COVID-19 progression. Our findings suggest that targeting glucose metabolism and the urea cycle may be a viable approach to fight COVID-19 at various stages along the disease course.

MicroRNA-197-3p mediates damage to human coronary artery endothelial cells via targeting TIMP3 in Kawasaki disease.

Kawasaki disease (KD) causes cardiovascular system injury in children. However, the pathogenic mechanisms of KD have not been well defined. Recently, strong correlation between aberrant microRNAs and KD nosogenesis has been revealed. A role of microRNA-197-3p (miR-197-3p) in the pathogenesis of KD is identified in the present study. Cell proliferation assay showed human coronary artery endothelial cells (HCAECs) were suppressed by serum from KD patients, which was correlated with high levels of miR-197-3p in both KD serum and HCAECs cultured with KD serum. The inhibition of HCAECs by miR-197-3p was confirmed by cells expressing miR-197-3p mimic and miR-197-3p inhibitor. Comparative proteomics analysis and Ingenuity Pathway Analysis (IPA) revealed TIMP3 as a potential target of miR-197-3p, which was demonstrated by western blot and dual-luciferase reporter assays. Subsequently, by detecting the endothelium damage markers THBS1, VWF, and HSPG2, the role of miR-197-3p/TIMP3 in KD-induced damage to HCAECs was confirmed, which was further validated by a KD mouse model in vivo. The expressions of miR-197-3p and its target, TIMP3, are dramatically variational in KD serum and HCAECs cultured with KD serum. Increased miR-197-3p induces HCAECs abnormal by restraining TIMP3 expression directly. Hence, dysregulation of miR-197-3p/TIMP3 expression in HCAECs may be an important mechanism in cardiovascular endothelium injury in KD patients, which offers a feasible therapeutic target for KD treatment.

Analysis of single-cell microbial mass spectra profiles from single-particle aerosol mass spectrometry.

RATIONALE: Single-particle aerosol mass spectrometry is a practical method for studying microbial aerosols. However, the related mass spectral characteristics of single-cell microorganisms have not yet been studied systematically; hence, further investigations are necessary. METHODS: Different microbial cells were grown and directly aerosolized in the laboratory. These aerosols were then drawn into a single-particle mass spectrometer platform, and single-cell mass spectra profiles were obtained in real-time. The biological characteristics, ion variation trends, and microbial types were analyzed with either laser pulse energy or laser fluence. RESULTS: The single-particle mass spectra contained prominent peaks that could be attributed to the presence of biological matter, such as organic phosphate and nitrogen, amino acids, and spore-associated calcium complexes. Limited types of average mass spectral patterns were present, and a significant correlation was found between the ion intensity trend (presence and absence of peaks) and laser ionization energy (expressed by the total positive ion intensity). Although a single spectral data point does not contain all the peaks of the average spectrum, it covers most of the characteristic peaks and could be identified using a machine learning algorithm. After the analysis of single-particle mass spectra, we found that using multi-group features (e.g., peak intensity ratio of m/z +47 and +41, peak intensity ratio of 59 N(CH3 )3 + and 74 N(CH3 )4 + , and 12 peak variables) led to an identification accuracy of approximately 92.4% with the random forest algorithm. CONCLUSIONS: The results indicate that single-cell mass spectral profiles can be used to distinguish microbial aerosols and further illustrate their origin in a laboratory setting.

Clinical characteristics and viral shedding kinetics of 38 asymptomatic patients with coronavirus disease 2019: A retrospective observational study.

ABSTRACT: This study aims to investigate the clinical characteristics and viral shedding kinetics of asymptomatic patients with coronavirus disease 2019 (COVID-19).The data of 38 asymptomatic patients positive for SARS-CoV-2 nucleic acid were collected from February to March 2020 in Tuanfeng County, Huanggang, Hubei, China. The epidemiology, laboratory examination, chest imaging, viral nucleic acid test results, clinical characteristics, and viral shedding time were summarized in this retrospective study.The study included 20 family members of patients with COVID-19, 10 medical personnel participating in COVID-19 treatment or working in a fever clinic, 6 personnel from quarantine places, 1 individual with a close contact history with confirmed patients, and 1 local epidemic prevention personnel. All were positive for SARS-CoV-2 nucleic acid. The white blood cell (WBC) count, the absolute value of lymphocytes, C-reactive protein (CRP), and D-dimer were normal. Pneumonia manifestations were not found in the chest computed tomography (CT) scan of 36 patients; the remaining 2 cases included a 1-year-old child and a pregnant woman, and they did not undergo chest CT. The viral shedding time was 6 days.All asymptomatic patients with COVID-19 had a history of close contact or exposure. Laboratory tests were normal. Chest imaging did not show any pneumonia manifestation. The viral shedding time was <10 days, which is shorter than that of patients with COVID-19. A timely discovery of such asymptomatic infections is crucial for blocking the spread of the virus and strengthening the prevention and control measures.

SPINK1 promotes cell growth and metastasis of lung adenocarcinoma and acts as a novel prognostic biomarker.

Serine protease inhibitor Kazal type 1 (SPINK1) plays a role in protecting the pancreas against premature activation of trypsinogen and is involved in cancer progression. SPINK1 promoted LAC cells growth, migration, and invasion. Mechanistically, we found that SPINK1 promoted LAC cells migration and invasion via up-regulating matrix metalloproteinase 12 (MMP12). We observed that SPINK1 expression was only up-regulated in lung adenocarcinoma (LAC) tissues, and was an independent prognostic factor for poor survival. Our results indicate that SPINK1 might be a potential biomarker for LAC that promotes progression by MMP12. [BMB Reports 2018; 51(12): 648-653].

MicroRNA­106a regulates the proliferation and invasion of human osteosarcoma cells by targeting VNN2.

MicroRNAs (miRs) serve an essential role in tumorigenesis and are able to act as tumor suppressor genes or oncogenes. miR­106a has been identified generally as an oncogene in multiple types of human cancer; however, its association with osteosarcoma has not previously been understood. Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) was used to detect miR­106a expression in 18 osteosarcoma tissues compared with paired non­cancerous adjacent tissues as well as osteosarcoma cell lines (U2OS, Saos­2 and MG63) compared with a normal osteoblast cell line (hFOB1.19). The biological function of U2OS cells was assessed by using a Transwell cell invasion assay, MTS proliferation assay and flow cytometric analysis following the transfection with lentivirus­mediated small interfering RNA (miR­106a­inhibitor). Western blotting and Luciferase reporters were used to investigate whether VNN2 was a target of miR­106a in osteosarcoma cells. Based on the RT­qPCR data, miR­106a was significantly upregulated in osteosarcoma tissues and osteosarcoma cell lines compared with their control counterparts (P<0.01). The knockdown of miR­106a resulted in cell proliferation and invasion inhibition. Furthermore, apoptosis enhancement and G2/M cell cycle arrest were detected by flow cytometry. The western blot analysis indicated that U2OS cells infected with miR­106a­inhibitor lentivirus had a higher VNN2 protein expression level compared with cells infected with miR­106a­negative control lentivirus. Luciferase reporters containing the 3'­untranslated region sequence of VNN2 messenger RNA demonstrated VNN2 may be a target of miR­106a. In addition, a negative correlation was confirmed between the expression of VNN2 and miR­106a in the tumor samples. The results of the present study indicate that the knockdown of miR­106a overexpressed VNN2 to inhibiting the proliferation, migration and invasion as well as inducing the apoptosis of human osteosarcoma cells.

Molecular basis of coronary artery dilation and aneurysms in patients with Kawasaki disease based on differential protein expression.

Kawasaki disease (KD) is an acquired cardiac disease with a high incidence that affects children. KD has various complications, including coronary artery dilation (CAD) and coronary artery aneurysms (CAA). The identification of differentially expressed proteins and the underlying mechanisms may be the key to understanding differences between these KD complications. In the present study, isobaric tags for relative and absolute quantitation were used to identify variations in serum proteins between KD patients with CAD and CAA. In total, 87 (37 upregulated and 50 downregulated) and 65 (33 upregulated and 32 downregulated) significantly differentially­expressed proteins were identified in comparisons between control samples (healthy individuals) and those obtained from patients with KD and with CAD or CAA. Investigation into the underlying biological process revealed that variations between the two complications were associated with the wound healing response, as well as lipoprotein­ and cholesterol­associated processes. Important proteins involved in the formation of the wound healing signaling network were identified via enriched biological processes and pathway analysis using ClueGo and ReactomeFIViz software. In the present study, 5 significantly differentially­expressed proteins, including mannose binding lectin 2 (MBL2), complement factor H (CFH), kininogen 1 (KNG1), serpin family C member 1 (SERPINC1) and fibronectin 1 (FN1), were selected and confirmed by western blotting. Analysis indicated that these proteins were associated to immunity, inflammation and metabolism, serving a key role within each module, which has never been reported previously. The present study proposed that MBL2, CFH, KNG1, SERPINC1 and FN1 may be a potentially excellent indicator group for distinguishing the two major KD complications, CAD and CAA.

Sets of serum exosomal microRNAs as candidate diagnostic biomarkers for Kawasaki disease.

Although Kawasaki disease is the main cause of acquired heart disease in children, no diagnostic biomarkers are available. We aimed to identify candidate biomarkers for diagnosing Kawasaki disease using serum exosomal microRNAs (miRNAs). Using frozen serum samples from a biobank, high-throughput microarray technologies, two-stage real-time quantitative PCR, and a self-referencing strategy for data normalization, we narrowed down the list of biomarker candidates to a set of 4 miRNAs. We further validated the diagnostic capabilities of the identified miRNAs (namely, CT(miR-1246)-CT(miR-4436b-5p) and CT(miR-197-3p)-CT(miR-671-5p)) in 79 samples from two hospitals. We found that this 4-miRNA set could distinguish KD patients from other febrile patients as well as from healthy individuals in a single pass, with a minimal rate of false positives and negatives. We thus propose, for the first time, that serum exosomal miRNAs represent candidate diagnostic biomarkers for Kawasaki disease. Additionally, we describe an effective strategy of screening for biomarkers of complex diseases even when little mechanistic knowledge is available.

MicroRNA expression profile in exosome discriminates extremely severe infections from mild infections for hand, foot and mouth disease.

BACKGROUND: Changes of miRNAs in exosome have been reported in different disease diagnosis and provided as potential biomarkers. In this study, we compared microRNA profile in exosomes in 5 MHFMD and 5 ESHFMD as well as in 5 healthy children. METHODS: Different expression of miRNAs in exosomes across all the three groups were screened using miRNA microarray method. Further validated test was conducted through quantitative real-time PCR assays with 54 exosome samples (18 ESHFMD, 18 MHFMD, and 18 healthy control). The judgment accuracy was then estimated by the receiver operating characteristic (ROC) curve analysis; and the specificity and sensitivity were evaluated by the multiple logistic regression analysis. RESULTS: There were 11 different miRNAs in exosomes of MHFMD and ESHFMD compared to healthy children, of which 4 were up-regulated and 7 were down-regulated. Further validation indicated that the 4 significant differentially expressed candidate miRNAs (miR-671-5p, miR-16-5p, miR-150-3p, and miR-4281) in exosome showed the same changes as in the microarray analysis, and the expression level of three miRNAs (miR-671-5p, miR-16-5p, and miR-150-3p) were significantly different between MHFMD or ESHFMD and the healthy controls. The accuracy of the test results were high with the under curve (AUC) value range from 0.79 to 1.00. They also provided a specificity of 72%-100% and a sensitivity of 78%-100%, which possessed ability to discriminate ESHFMD from MHFMD with the AUC value of 0.76-0.82. CONCLUSIONS: This study indicated that the exosomal miRNA from patients with different condition of HFMD express unique miRNA profiles. Exosomal miRNA expression profiles may provide supplemental biomarkers for diagnosing and subtyping HFMD infections.

Monitoring of the serum proteome in Kawasaki disease patients before and after immunoglobulin therapy.

Kawasaki disease (KD) is a systemic vasculitis that mainly affects children younger than 5 years. The causal pathogen is unknown, therefore specific diagnostic biomarkers and therapy are unavailable. High-dose intravenous immunoglobulin (IVIG) is considered as the most effective therapy to reduce the prevalence of coronary artery lesion (CAL) in KD; however, it has side effects. This study aimed to (1) determine whether IVIG therapy is effective at the molecular level; (2) provide the first serum proteomic profile of KD under IVIG therapy; and (3) screen for monitoring biomarker candidates. We extracted serum proteins from samples of healthy individuals and from KD patients before and after IVIG therapy, and employed two-dimensional electrophoresis and MALDI-TOF/TOF mass spectrometry to identify differentially expressed proteins. The identifications were validated by Western blotting. We identified 29 differentially expressed proteins in KD patients and found that IVIG therapy restored most of these proteins to near-normal levels. Tracing the protein levels of single patients before and after IVIG therapy showed that the proteins, especially Transthyretin (TTR), are potential markers for therapeutic monitoring. Functional analyses of these proteins by PANTHER and String suggested that the key influence of KD lay in the immune system, which was targeted by IVIG.

Proteomic analysis of extremely severe hand, foot and mouth disease infected by enterovirus 71.

BACKGROUND: To clarify the molecular mechanisms that participate in the severe hand, foot and mouth disease (HFMD) infected by Enterovirus 71 and to detect any related protein biomarkers, we performed proteomic analysis of protein extracts from 5 extremely severe HFMD children and 5 healthy children. METHODS: The protein profiles of them were compared using two-dimensional electrophoresis. Differentially expressed proteins were identified using mass spectrometry. Functional classifications of these proteins were based on the PANTHER. The interaction network of the differentially expressed protein was generated with Pathway Studio. RESULTS: A total of 38 differentially expressed proteins were identified. Functional classifications of these proteins indicated a series of altered cellular processes as a consequence of the severe HFMD. These results provided not only new insights into the pathogenesis of severe HFMD, but also implications of potential therapeutic designs. CONCLUSIONS: Our results suggested the possible pathways that could be the potential targets for novel therapy: viral protection, complement system and peroxide elimination.

Identification of ubiquitinated proteins from human multiple myeloma U266 cells by proteomics.

OBJECTIVE: To identify ubiquitinated proteins from complex human multiple myeloma (MM) U266 cells, a malignant disorder of differentiated human B cells. METHODS: Employing a globally proteomic strategy combining of immunoprecipitation, LC-MS/MS and SCX-LC-MS analysis to identified ubiquitination sites, which were identified by detecting signature peptides containing a GG-tag (114.1 Da) and an LRGG-tag (383.2 Da). RESULTS: In total, 52 ubiquitinated proteins containing 73 ubiquitination sites of which 14 and 59 sites contained LRGG-tag and GG-tag were identified, respectively. CONCLUSION: Classification analysis by of the proteins identified in the study based on the PANTHER showed that they were associated with multiple functional groups. This suggested the involvement of many endogenous proteins in the ubiquitination in MM.

[Inhibiting GDF-8 expression by retrovirus-based RNAi stably].

We cloned human U6 promoter from pAVU6 + 27 vector into pXSN to transcripe small RNA. Meanwhile, a shRNA targeting GDF-8 was cloned down-stream of the hU6 promoter to construct recombinant vector. Then the packing cell GP-293 was co-transfected the recombinant with pVSV-G to gernarate virus particle. Resistant C2C12 cell pools were screened using G418. Levels of mRNA and protein of GDF-8 were tested by Real-Time PCR and western blotting. Cell proliferation and cell cycle were analyzed using MTT and FACS. The expression of GDF-8 was dramatically decreased by the retrovirus-based system in C2C12 cells. Cells proliferated effectively after integrating the recombinant. The cells in G0/G1 phase decreased by 13.7%, while cells in S phase increased by 14.9%. In conclusion, the retrovirus-based RNAi could be used to stably silence GDF-8. It can be a powerful tool in curing muscle atrophy.