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Here we report first two cases of invasive pulmonary aspergillosis caused by Aspergillus lentulus from India, in non-neutropenic, critically ill patients with chronic obstructive pulmonary disease (COPD).

Cryptococcosis is a life-threatening infection caused by Cryptococcus neoformans and C. gattii species complex. In the present study, to understand the molecular epidemiology of 208 clinical isolates of Cryptococcus from different parts of India, multilocus sequence typing (MLST) using ISHAM MLST consensus scheme for C. neoformans/C. gattii species complex was used. MLST analysis yielded a total of 10 Sequence Types (STs)-7 STs for C. neoformans and 3 for C. gattii species complex. The majority of isolates identified as C. neoformans belonged to molecular type VNI with predominant STs 31 and 93. Only 3 isolates of C. gattii species complex were obtained, belonging to ST58 and ST215 of VGI and ST69 of VGIV. Phylogenetic analysis revealed less diversity among the clinical Indian isolates compared to the global MLST database. No association between prevalent STs and HIV status, geographical origin or minimum inhibitory concentration (MIC) could be established.

Introduction.Invasive mucormycosis (IM) is a life-threatening infection caused by fungi belonging to the order Mucorales. Histopathology, culture and radiology are the mainstay of diagnosis but lack sensitivity, leading to a delay in timely diagnosis and intervention. Recently, PCR-based approaches have been shown to be a promising method in diagnosing IM.Hypothesis/Gap Statement.Molecular-based approaches may be a valuable adjunct to standard conventional methods for diagnosing IM, especially among culture negatives and patients on antifungal therapy.Aim.In the present study we aimed to evaluate the clinical utility of panfungal and Mucorales-specific PCR for diagnosing IM from various clinical specimens.Methodology.This was a prospective study in which 239 clinically suspected cases of IM attending our tertiary care hospital from August 2015 to March 2018 were enrolled. All the cases were defined as ‘proven’, ‘probable’ or ‘possible’ based on EORTC/MSGERC guidelines. In addition to conventional diagnostics (KOH-calcofluor stain and culture), panfungal and Mucorales-specific PCR assays were also performed. The amplified products were sequenced for species identification.In vitroantifungal susceptibility was performed on all the culture-positive isolates.Results.Among 239 clinically suspected cases of IM, only 140 cases were diagnosed by the demonstration of aseptate ribbon-like hyphae on direct microscopy. Culture was positive in 35.7 % (54/140) of direct microscopy-positive samples. Among the proven cases (n=11), the sensitivity for both Mucorales-specific nested PCR and panfungal PCR was 100 %, but specificity was 91.9 and 73.7% respectively. In probable cases (n=129), the sensitivity of both the PCRs was 98.5 % and specificity for panfungal PCR was 73.7 and 91.9 % for Mucorales-specific PCR.Conclusion.Pan fungal PCR in combination with Mucorales-specific PCR, followed by sequencing, may play a significant role in IM diagnosis especially among those negative for both direct microscopy and culture.

Background Chronic pulmonary obstructive disease (COPD) prevalence increases with tobacco smoking, suggesting a relationship between disease severity and an increased rate of systemic symptoms. Aim To evaluate IL-1β, IL-8 and NF-κB expression in COPD patients with smoking and non-smoking habits and determine their levels with disease severity compared to respective healthy controls. Methods One hundred seventy-six subjects (88 COPD cases and 88 healthy controls) details demographic smoking, and disease severity enrolled in the study. Interleukin transcript and protein levels were measured by quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA). Statistical Package for the Social Sciences (SPSS) software version 21.0 was used to analyse the data. Statistical significance was set at p Results IL-1β, IL-8 and NF-κB mRNA levels were significantly higher (p Conclusion IL-1β and IL-8 expression correlated with COPD severity, which worsened with pack-years tobacco smoking.

Lupus nephritis (LN) is one of the common complications of systemic lupus erythematosus (SLE). Timely treatment will decrease progression to chronic kidney disease. Treatment varies with different stages for which biopsy is needed. Controversies still exist regarding its requirement in management. This is a retrospective study from September 2014 to August 2016 in B. P. Koirala Institute of Health Sciences, Dharan, Nepal among all patients with SLE and undergone renal biopsy. Of 92 patients, most were female 85 (92.4%) with a median age of 32 years. In this study, 80.4% had some clinical symptomatology. Of the clinical manifestations, 41.3% had polyarthritis, edema (20.7%), and malar rash (17.4%). Anti-nuclear antibody was positive in 80.4% and ds DNA in 70.7%. Renal biopsy showed more number of patients 27 (35%) had Stage IV LN, followed by Stage I, 19 (24%), and Stage II, 16 (20%) LN. Median urinary protein in Class I was 1.05 g, Class II (0.63 g), Class III (1.5 g), Class IV (2.44 g), Class V (3.99 g), and Class VI (4.7 g). Only Stage IV had Kappa of 0.269 {P = 0.003) showing agreement between proteinuria and histological staging which was statistically significant (P

Objectives Chronic obstructive pulmonary disease (COPD) is one of the leading causes of morbidity and mortality around the world. Preliminary studies have evaluated the association between innate immunity including Toll-like receptors (TLRs) and airway samples of patients with COPD. The role of TLRs in peripheral blood neutrophils is poorly understood. Hence, this study aimed to investigate the role of TLR2 and TLR4 in peripheral blood neutrophils of COPD patients. Methods A total of 101 COPD cases and an equal number of healthy controls participated in this case-control study. Peripheral blood neutrophils were isolated from all participants and cultured for 24 hours through lipopolysaccharide (LPS) stimulation. The gene expressions of TLR2 and TLR4 were assessed by real-time polymerase chain reaction. The protein levels of interleukin (IL)-8 and matrix metalloproteinase (MMP)-9 were measured in neutrophils cell culture supernatants using enzyme-linked immunosorbent assay (ELISA). Results The levels of IL-8 and MMP-9 were significantly higher in patients with COPD compared to healthy controls. Similarly, the gene expression of TLR2 and TLR4 were increased in LPS stimulated peripheral blood neutrophils of patients with COPD. Smoke pack years was positively correlated with IL-8 levels and negatively correlated with forced expiratory volume in the first second % (r = -0.33; p = 0.023) and FEV1/forced vital capacity (FVC) (r = -0.27; p = 0.011). Conclusions The increased expression of TLR2 and TLR4 suggests its role in disease pathogenesis of COPD. Smoke pack years was negatively associated with spirometric parameters in COPD patients. This may help to predict the smokers without COPD who risk developing the condition in the future.

Introduction: Chronic Obstructive Pulmonary Disease (COPD) is characterized by partially reversible airflow obstruction and chronic inflammation in the pulmonary as well as in systemic compartment. Interleukin 8 (IL-8) is one of the most potent inflammatory mediators and it is involved in the migration and activation of neutrophils. Previous studies demonstrated the increased levels of IL-8 in plasma and sputum of COPD, but peripheral blood neutrophils mediated IL-8 level including their relationship to smoking pack years and spirometric parameters is still unknown. Aim: To assess the levels of IL-8 in sputum and peripheral blood neutrophils of COPD patients and to compare them with healthy controls. Materials and Methods: This case control study was done from March 2014 to November 2016 in the department of respiratory medicine KGMU. A total of 81 COPD patients and 81 healthy controls were recruited in this study. Peripheral blood neutrophils were isolated from COPD patients and healthy controls and stimulated with Lipopolysaccharide (LPS). The neutrophils cell culture supernatant was assessed for IL-8 level using ELISA techniques, whilst IL-8 mRNA expression was measured using real-time polymerase chain reaction. Induced sputum was obtained from all participants and processed. The sputum supernatant was also assayed for IL-8 level. Results: COPD patients had significantly higher protein level of IL-8 in peripheral blood neutrophils (p=0.018) and sputum supernatant (p=0.011) compared to healthy controls. Gene expression of IL-8 was also significantly higher in peripheral blood neutrophils of COPD patients (p=0.001). In the patients with COPD, the smoke pack year was significantly correlated with the level of IL-8 in peripheral blood neutrophils (r=0.42; p=0.009) and sputum (r=0.48; p=0.002). The smoke pack years was found negatively correlated with FEV1 % predicted (r=- 0.37; p=0.044) and FEV1 /FVC (r=-0.47; p=0.007) in the COPD patients. Conclusion: The increased level of IL-8 in peripheral blood neutrophils and sputum support the hypothesis that COPD is a multicomponent disease. IL-8 level may be helpful in predicting development of COPD in smokers.

Recently, we have demonstrated that the protease domain of NS3 alone can bind specifically to hepatitis C virus (HCV) internal ribosome entry site (IRES) near the initiator AUG, dislodges human La protein and inhibits translation in favor of viral RNA replication. Here, by using a computational approach, the contact points of the protease on the HCV IRES were putatively mapped. A 30-mer NS3 peptide was designed from the predicted RNA-binding region that retained RNA-binding ability and also inhibited IRES-mediated translation. This peptide was truncated to 15 mer and this also demonstrated ability to inhibit HCV RNA-directed translation as well as replication. More importantly, its activity was tested in an in vivo mouse model by encapsulating the peptide in Sendai virus virosomes followed by intravenous delivery. The study demonstrates for the first time that the HCV NS3-IRES RNA interaction can be selectively inhibited using a small peptide and reports a strategy to deliver the peptide into the liver.

With fast evolving technology, Cognitive Science plays a vital role in our day-to-day life. Cognitive science is summed up as the investigation of psyche taking into account logical techniques. It is about the entirety of all interdisciplinary like logic, brain research, semantics, computerized reasoning, mechanical autonomy, and neuroscience. In this paper, we concentrated on the outward appearances or feelings of person as it has a critical part in interpersonal relations. In this strategy, we use back propagation system for image processing. It is a data preparing framework that has been created as a speculation of the numerical model of human acknowledgment. We have proposed a framework to investigate EEG flags and group them into various feelings (upbeat, miserable, outrage, dread and so forth). We have accumulated a dataset with EEG signals. This was finished by measuring EEG signals from individuals that were sincerely invigorated. This system enabled us to instruct our structure the relationship between the characteristics of the psyche activity and the feeling. We have watched diverse cerebrum values(α, β, θ) utilizing single cathode EEG.

Placental like alkaline phosphate (PLAP), an oncofetal antigen, is highly expressed in germ cell, cervical, ovarian and several other tumour types but minimally in normal tissues. The expression of a PLAP promoter based transcriptional unit following antigen mediated cell specific delivery is a possible approach for tumour targeting. PLAP promoter alone or in combination with NFκB DNA response elements was used for expressing shRNA targeting the long control region (LCR) of human papillomavirus (HPV)-16 oncogenes E6 and E7 via transcriptional gene silencing in PLAP expressing cervical cancer cell lines, SiHa and CaSki. This was packaged in a Sendai virus envelope incorporating a single chain variable fragment antibody (scFv) for antibody mediated targeting. Specificity and efficacy of the shRNA was assessed by studying the heterochromatization, down regulation of the HPV-16 E6/E7 genes and subsequent effects on their targets and cell growth properties. Reduction of HPV-16 E6 and E7 expression by TGS led to the activation of the previously suppressed target genes of p53 (PUMA and NOXA) and Rb (cyclins A2 and E). Cell death was seen only in PLAP expressing HPV-16 infected SiHa and CaSki cells but not in the HPV-18 integrated HeLa and non-PLAP CHO cells. There was reduction in the enhancer associated transcripts of the long control region (LCR) of HPV-16 E6/E7 genes. Also, an increase in the enrichment of dimethylated histone three lysine nine (H3K9Me2) and trimethylated histone three lysine twenty-seven (H3K27Me3) was observed by ChIP assay, which decreased upon trichostatin A treatment, indicating a possible mechanism for the heterochromatization of the target LCR region. A combination of novel PLAP promoter and antibody based specificities has the potential for being developed as a possible therapeutic strategy for PLAP positive neoplasia.

Internal ribosome entry site (IRES)-mediated translation of input viral RNA is the initial required step for the replication of the positive-stranded genome of hepatitis C virus (HCV). We have shown previously the importance of the GCAC sequence near the initiator AUG within the stem and loop IV (SLIV) region in mediating ribosome assembly on HCV RNA. Here, we demonstrate selective inhibition of HCV-IRES-mediated translation using short hairpin (sh)RNA targeting the same site within the HCV IRES. sh-SLIV showed significant inhibition of viral RNA replication in a human hepatocellular carcinoma (Huh7) cell line harbouring a HCV monocistronic replicon. More importantly, co-transfection of infectious HCV–H77s RNA and sh-SLIV in Huh7.5 cells successfully demonstrated a significant decrease in viral RNA in HCV cell culture. Additionally, we report, for the first time, the targeted delivery of sh-SLIV RNA into mice liver using Sendai virosomes and demonstrate selective inhibition of HCV-IRES-mediated translation. Results provide the proof of concept that Sendai virosomes could be used for the efficient delivery of shRNAs into liver tissue to block HCV replication.

Asialoglycoprotein receptor (ASGPR)-mediated endocytosis has been used to target genes to hepatocytes in vivo. However, the level and duration of transgene expression have been low because of lysosomal translocation and degradation of the DNA and lack of its integration into the host genome. In this study we packaged the DNA of interest in proteoliposomes containing the fusogenic galactose-terminated F-glycoprotein of the Sendai virus (FPL) for targeted delivery to hepatocytes. After the FPL binds to ASGPR on the hepatocyte surface, fusogenic activity of the F-protein delivers the DNA into the cytosol, bypassing the endosomal pathway. For transgene integration we designed plasmids containing one transcription unit expressing the Sleeping Beauty transposase (SB) and another expressing human uridinediphosphoglucuronate glucuronosyltransferase-1A1 (pSB-hUGT1A1). The latter was flanked by inverted/direct repeats that are substrates of SB. In cell culture, FPL-mediated delivery of the E. coli -galactosidase gene (LacZ) resulted in transduction of ASGPR-positive cells (rat hepatocytes or Hepa1 cell line), but not of ASGPR-negative 293 cells. Intravenous injection of the FPL-entrapped pSB-hUGT1A1 (4-8g/day, 1-4 doses) into UGT1A1-deficient hyperbilirubinemic Gunn rats (model of CriglerNajjar syndrome type 1) resulted in hUGT1A1 expression in 5%-10% of hepatocytes, but not in other cell types. Serum bilirubin levels declined by 30% 4% in 2 weeks and remained at that level throughout the 7-month study duration. With histidine containing FPL, serum bilirubin was reduced by 40% 5%, and bilirubin glucuronides were excreted into bile. No antibodies were detectable in the recipient rats against the F-protein or human UGT1A1. Conclusion: FPL is an efficient hepatocyte-targeted gene delivery platform in vivo that warrants further exploration toward clinical application. (HEPATOLOGY 2009;50:815-824.)