Identifying quantitative sncRNAs signature using global sequencing as a potential biomarker for tuberculosis diagnosis and their role in regulating host response.

OBJECTIVES: The study aimed to identify a quantitative signature of circulating small non-coding RNAs (sncRNAs) as a biomarker for pulmonary tuberculosis disease (active-TB/ATB) and explore their regulatory roles in host-pathogen interactions and disease progression. METHODS: We conducted a cross-sectional study recruiting subjects diagnosed with active-TB (drug-sensitive and drug-resistant) and healthy controls. Sera samples were collected and utilized for preparing small RNA libraries. Quantitative patterns of circulating sncRNAs (miRNAs, piRNAs and tRFs) were identified via high-throughput sequencing and DeSeq2 analysis and validated in independent active-TB cohorts. Functional knockdown for two selected miRNAs were also performed. RESULTS: A diagnostic signature of four sncRNAs for both drug-sensitive and drug-resistant active-TB cases was validated, exhibiting an AUC of 0.96 (95% CI: 0.937-0.996, p < 0.001) with 86.7% sensitivity (95% CI: 0.775-0.932) and 91.7% specificity (95% CI: 0.730-0.990) in ROC analysis. Functional knockdown demonstrated regulatory roles of hsa-miR-223-5p and hsa-miR-10b-5p in Mycobacterium tuberculosis (Mtb) growth and pro-inflammatory cytokine expression (IL-6 and IL-8). CONCLUSION: The study identified a diagnostic tool utilizing a signature of four sncRNAs with high specificity and sensitivity, enhancing our understanding of sncRNAs as ATB diagnostic biomarker. Additionally, hsa-miR-223-5p and hsa-miR-10b-5p demonstrated potential roles in Mtb pathogenesis and host-response to infection.

Latent Tuberculosis Infection Diagnosis among Household Contacts in a High Tuberculosis-Burden Area: a Comparison between Transcript Signature and Interferon Gamma Release Assay.

Diagnosis of latent tuberculosis infection (LTBI) using biomarkers in order to identify the risk of progressing to active TB and therefore predicting a preventive therapy has been the main bottleneck in eradication of tuberculosis. We compared two assays for the diagnosis of LTBI: transcript signatures and interferon gamma release assay (IGRA), among household contacts (HHCs) in a high tuberculosis-burden population. HHCs of active TB cases were recruited for our study; these were confirmed to be clinically negative for active TB disease. Eighty HHCs were screened by IGRA using QuantiFERON-TB Gold Plus (QFT-Plus) to identify LTBI and uninfected cohorts; further, quantitative levels of transcript for selected six genes (TNFRSF10C, ASUN, NEMF, FCGR1B, GBP1, and GBP5) were determined. Machine learning (ML) was used to construct models of different gene combinations, with a view to identify hidden but significant underlying patterns of their transcript levels. Forty-three HHCs were found to be IGRA positive (LTBI) and thirty-seven were IGRA negative (uninfected). FCGR1B, GBP1, and GBP5 transcripts differentiated LTBI from uninfected among HHCs using Livak method. ML and ROC (Receiver Operator Characteristic) analysis validated this transcript signature to have a specificity of 72.7%. In this study, we compared a quantitative transcript signature with IGRA to assess the diagnostic ability of the two, for detection of LTBI cases among HHCs of a high-TB burden population; we concluded that a three gene (FCGR1B, GBP1, and GBP5) transcript signature can be used as a biomarker for rapid screening. IMPORTANCE The study compares potential of transcript signature and IGRA to diagnose LTBI. It is first of its kind study to screen household contacts (HHCs) in high TB burden area of India. A transcript signature (FCGR1B, GBP1, & GBP5) is identified as potential biomarker for LTBI. These results can lead to development of point-of-care (POC) like device for LTBI screening in a high TB burdened area.

Expression, Purification and Characterization of the Hepatitis E Virus Like-Particles in the Pichia pastoris.

Hepatitis E virus (HEV) is associated with acute hepatitis disease, which may lead to chronic disease in immunocompromised individuals. The disease is particularly severe among pregnant women (20-30% mortality). The only licensed vaccine against HEV, which is available in China, is the Escherichia coli purified recombinant virus-like particles (VLPs) encompassing the 368-660 amino acids (aa) of the viral ORF2 protein. The viral capsid is formed by the ORF2 protein, which harbors three glycosylation sites. Baculo virus expression system has been employed to generate a glycosylated VLP, which encompasses 112-608aa of the ORF2 protein. Here, we sought to produce a recombinant VLP containing 112-608aa of the ORF2 protein in Pichia pastoris (P. pastoris) expression system. The cDNA sequence encoding 112-608aa of the ORF2 protein was fused with the α-mating factor secretion signal coding sequence (for release of the fusion protein to the culture medium) and cloned into the yeast vector pPICZα. Optimum expression of recombinant protein was obtained at 72 h induction in 1.5% methanol using inoculum density (A600) of 80 and at pH-3.0 of the culture medium. Identity of the purified protein was confirmed by mass spectrometry analysis. Further studies revealed the glycosylation pattern and VLP nature of the purified protein. Immunization of BALB/c mice with these VLPs induced potent immune response as evidenced by the high ORF2 specific IgG titer and augmented splenocyte proliferation in a dose dependent manner. 112-608aa ORF2 VLPs produced in P. pastoris appears to be a suitable candidate for development of diagnostic and prophylactic reagents against the hepatitis E.

Identification of critical residues in Hepatitis E virus macro domain involved in its interaction with viral methyltransferase and ORF3 proteins.

Hepatitis E virus (HEV) is a major cause of hepatitis in normal and organ transplant individuals. HEV open reading frame-1 encodes a polypeptide comprising of the viral nonstructural proteins as well as domains of unknown function such as the macro domain (X-domain), V, DUF3729 and Y. The macro domain proteins are ubiquitously present from prokaryotes to human and in many positive-strand RNA viruses, playing important roles in multiple cellular processes. Towards understanding the function of the HEV macro domain, we characterized its interaction partners among other HEV encoded proteins. Here, we report that the HEV X-domain directly interacts with the viral methyltransferase and the ORF3 proteins. ORF3 association with the X-domain was mediated through two independent motifs, located within its N-terminal 35aa (amino acids) and C-terminal 63-123aa. Methyltransferase interaction domain was mapped to N-terminal 30-90aa. The X-domain interacted with both ORF3 and methyltransferase through its C-terminal region, involving 66(th),67(th) isoleucine and 101(st),102(nd) leucine, conserved across HEV genotypes. Furthermore, ORF3 and methyltransferase competed with each other for associating with the X-domain. These findings provide molecular understanding of the interaction between the HEV macro domain, methyltransferase and ORF3, suggesting an important role of the macro domain in the life cycle of HEV.

Clinical Implications of a Multivariate Stratification Model for the Estimation of Prognosis in Ventricular Septal Defect.

OBJECTIVES: To derive and validate a multivariate stratification model for prediction of survival free from intervention (SFFI) in ventricular septal defect (VSD). A secondary aim is for this model to serve as proof of concept for derivation of a more general congenital heart disease prognostic model, of which the VSD model will be the first component. STUDY DESIGN: For 12 years, 2334 subjects with congenital heart disease were prospectively and consecutively enrolled. Of these, 675 had VSD and form the derivation cohort. One hundred seven other subjects with VSD followed in another practice formed the validation cohort. The derivation cohort was serially stratified based on clinical and demographic features correlating with SFFI. RESULTS: Six strata were defined, the most favorable predicting nearly 100% SFFI at 10 years, and the least favorable, a high likelihood of event within weeks. Strata with best SFFI had many subjects with nearly normal physiology, muscular VSD location, or prior intervention. In the validation cohort, the relation between predicted and actual SFFI at 6 months, 1 year, 2 years, and 5 years follow-up had areas under the receiver operating characteristic curves 0.800 or greater. CONCLUSIONS: A prediction model for SFFI in VSD has been derived and validated. It has potential for clinical application to the benefit of patients and families, medical trainees, and practicing physicians.

Main pulmonary artery dilation in association with congenital bicuspid aortic valve in the absence of pulmonary valve abnormality.

BACKGROUND: Main pulmonary artery (MPA) dilation is reported to occur in association with pulmonary valve (PV) abnormalities and in genetic diseases of connective tissue. AIM: To determine whether MPA dilation occurs with bicuspid aortic valve (BAV) in the absence of PV abnormality. METHODS: MPA and aortic dimensions were investigated in 194 subjects with BAV and 178 controls matched for age, sex and body surface area (BSA) using transthoracic two-dimensional echocardiography. Using control measurements, linear regression of each parameter versus age, gender, and BSA(2/3) provided expected values. For each subject with BAV, the percentage divergence from expected value ((observed--expected value)/expected value) for each dimension was analysed. RESULTS: MPA and aortic measurements showed normal distribution in all subjects. PV annulus and MPA were larger in BAV; 17.7% positive divergence of MPA dimension from expected was found in diastole, and 16.7% in systole. Aortic dimensions at all levels measured were larger in BAV; divergence was greater distally than proximally (10% divergence at annulus, 31% at ascending aorta). In BAV, divergence of ascending aortic dimensions from expected values increased with age (r(2)=0.142, p=0.02), but MPA divergence from expected values did not increase with age (r(2)=0.001, p=0.296). CONCLUSIONS: MPA dilation occurs in association with BAV in the absence of PV abnormality, suggesting primary vessel wall pathology predisposing to arterial dilation. A systemic abnormality of connective tissue common to both arteries may be responsible, or the dilation may result from a common developmental exposure as both originate from the embryologic cono-truncus.

A novel complication of bariatric surgery.

The incidence of obesity in the US is increasing. This has been associated with an increasing incidence and prevalence of diabetes, hypertension, and obstructive sleep apnea. Diet alone has generally been ineffective in attaining significant weight loss. Bariatric surgery is associated with significant weight loss and improvement in diabetes, hypertension, and obstructive sleep apnea. In this article we describe an unusual complication of bariatric surgery.