Identification of phytochemical inhibitors against main protease of COVID-19 using molecular modeling approaches.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a novel corona virus that causes corona virus disease 2019 (COVID-19). The COVID-19 rapidly spread across the nations with high mortality rate even as very little is known to contain the virus at present. In the current study, we report novel natural metabolites namely, ursolic acid, carvacrol and oleanolic acid as the potential inhibitors against main protease (Mpro) of COVID-19 by using integrated molecular modeling approaches. From a combination of molecular docking and molecular dynamic (MD) simulations, we found three ligands bound to protease during 50 ns of MD simulations. Furthermore, the molecular mechanic/generalized/Born/Poisson-Boltzmann surface area (MM/G/P/BSA) free energy calculations showed that these chemical molecules have stable and favourable energies causing strong binding with binding site of Mpro protein. All these three molecules, namely, ursolic acid, carvacrol and oleanolic acid, have passed the ADME (Absorption, Distribution, Metabolism, and Excretion) property as well as Lipinski's rule of five. The study provides a basic foundation and suggests that the three phytochemicals, viz. ursolic acid, carvacrol and oleanolic acid could serve as potential inhibitors in regulating the Mpro protein's function and controlling viral replication. Communicated by Ramaswamy H. Sarma.

Genetic Changes of P53 and Kras in Gallbladder Carcinoma in Kumaon Region of Uttarakhand.

BACKGROUND: Gallbladder carcinoma is a highly lethal but relatively rare neoplasm of the digestive tract. p53 mutations are one of the most frequent genetic alterations in human cancers and are thought to play a role in pathogenesis of several malignancies. Kras oncogene is responsible for high frequency recognized as an early event in pancreatic and colonic carcinogenesis. OBJECTIVES: We investigated the genetic change of p53, Kras and histopathological changes in gallbladder cancer tissue samples. METHODS: P53 mutation was seen in the axons 5, 6, 7 and 8 of p53 gene and Kras codon 12 mutations in 25 operative specimens of gallbladder carcinoma. The hispathology observations and polymerase chain reaction-based techniques of these patients were used for point mutations study in p53 and in codon 12 of Kras gene. RESULTS: Mutations of p53 analyzed from exons 5 to 8 using the method of PCR-SSCP were 44%, PCR-RFLP was carried out, and incidence of mutation in codon 12 of Kras was 48% in the adenocarcinoma patients. There was a significant correlation between presence of gall stone, histopathological type, cellular differentiation, grade, lymphovascular invasion, perineural invasion, lymph node invasion, involvement of cystic duct end margin, liver invasion, omental tissue invasion, and T.N.M. staging. CONCLUSIONS: Curative surgical resection remains the only effective approach for treatment of GBC. Studies with large number of cases with latest application, next-generation sequencing, DNA microarray, transcriptome analysis, and real-time PCR would help in exploring more targets and better classification of these cancers at genetic level.

Histopathological Examination of Gallbladder Specimens in Kumaon Region of Uttarakhand.

Gall stones are one of the major causes of morbidity and mortality all over the world and common health problems throughout in developing countries. Cholecystectomy is one of the most common surgical practices and postoperative analysis of cholecystectomy specimen has a great value since histopathological reports may document some entities with significant clinical significances. Gallbladder carcinomas in cholecystectomy specimens are received in our histopathology laboratory to analyse their clinicopathological features. This was a descriptive study carried out at the histopathology section of the Department of Pathology at our hospital over a period of two years ranging from November 2016 to October 2018. Both intraoperative and postoperative histological examinations of the excised gallbladder facilitated the diagnosis of gallbladder cancer. Surgery-related variables and surgical approaches were evaluated according to the extent of tumor invasion. Twenty five cholecystectomy specimens of the acute and symptomatic chronic cholecystitis patients were analyzed. Standardization of the reporting were examined. Age, gender, presence of gall stone, cholesterolosis, adenomatous hyperplasia, gastric or intestinal metaplasia, dysplasia, histopathological type of gallbladder carcinoma, cellular differentiation, grading, lympho vascular invision, perineural invasion, lymph node invasion, involvement of cystic duct end margin, liver invasion, omental tissue invasion and T.N.M. staging were investigated. Reported rates of histopathological findings were comparable between patients aged twenty six years to seventy six years. Epithelial hyperplasia and metaplasia were found to be related to age. The correlation between cholesterolosis and gender or metaplasia was noted. We suggest that in India and other nations, high incidences of gallbladder carcinoma, all cholecystectomy specimens must be submitted to routine macroscopic and histopathology examination in the laboratory, as this is the only capability through which malignancies can be detected at an early, potentially curable stage. This incidental finding has altered the management and outcome of this dreadful disease.

Congenital Absence of Unilateral Internal Carotid Artery with an Intracerebral Aneurysm.

The absence of the internal carotid artery (ICA) is a rare congenital anomaly. Diagnosis of this entity is important because of its association with the cerebral aneurysm and also indicated during planned carotid or transsphenoidal surgery in thromboembolic disease and in the surveillance and detection of associated cerebral aneurysms. We report a case of congenital absence of unilateral ICA with associated cerebral aneurysm of the anterior cerebral artery.

Infections, genetic and environmental factors in pathogenesis of autoimmune thyroid diseases.

In Autoimmune disease a combination of infection, genetic and environmental factors causes an autoimmune response to the thyroid gland (characterized by lymphocytic infiltrations), thyroid stimulating hormone receptor (TSHR) and different thyroid antigens. Graves' and Hashimoto disease are autoimmune disorders with genetic predisposition. CD40 that stimulates the proliferation and differentiation of lymphocytes is an essential immunomodulatory component for follicular cells in the thyroid and the cell that present the antigen. CD40, PTPN22 and thyroid-specific genes are immunomodulating genes for the TSH receptor and thyroglobulin. CD40 used to be associated with Graves's disease as positional candidate on the basis of Graves' disease linkage study connecting with 20q11 genome chromosomal region. The PTPN22 gene gives rise to a substantial risk of specific autoimmune phenotypes and frequent disease mechanisms. Infections have been implicated in the pathogenesis of AITD including Coxsackie virus, Yersinia enterocolitica, Borrelia burgdorferi, Helicobacter pylori and retroviruses (HTLV-1, HFV, HIV and SV40). Infectious hepatitis C agents are the strongest proof supporting an affiliation with AITD. The essential environmental triggers of AITD are iodine, drugs, infection, smoking and perhaps stress. Autoimmune disease provide important facts on genetic mechanisms that influence the prognosis and treatment of the disease and by recent molecular techniques through gene expression study by quantitative Real Time-PCR and microarray, we can identify novel genes which are responsible for Graves' and Hashimoto disease.

Staging, Treatment, and Future Approaches of Gallbladder Carcinoma.

BACKGROUND: Gallbladder cancer is the most common malignant cancer of the bile ducts and third most common gastrointestinal malignant in the world for public health. Its relatively low incidence and confused symptoms result in advanced disease at the time of presentation, contributing to poor prognosis and reduced survival associated with this disease. The main function of the gallbladder is to store excreted bile acids from the liver in preparation for a meal. Its main risk factor is prolonged exposure to biliary calculations, although bacterial infections and other inflammatory conditions are associated. Chronic inflammatory bowel conditions are associated with gallbladder cancer. T stage translates to identifying residual disease at reoperation for incidental gallbladder cancer and residual disease negatively affects survival. CONCLUSION: It is the most common cancer of gallbladder, gallbladder cancer remains a rare disease. Gallbladder cancer is a rare disease that can be accidentally diagnosed after cholecystectomy or accidentally, often with more advanced disease. The prognosis is generally extremely poor and improvements in surgical resection of this approach have to be re-evaluated, while the role of chemotherapy and radiotherapy remains controversial.

Pathway analysis of differentially expressed genes in Mycobacterium bovis challenged bovine macrophages.

The immune signalling genes during the challenge of bovine macrophages with bacterial products derived from tuberculosis causing bacteria in cattle were investigated in the present study. An in-vitro cell culture model of bovine monocyte-derived macrophages were challenged to Mycobacterium bovis. Macrophages from healthy and already infected animals can both be fully activated during M. bovis infection. Analysis of mRNA abundance in peripheral blood mononuclear cells from M. bovis infected and non-infected cattle were performed as a controls. Cells of treatment were challenged after six days for six hours incubation at 37 °C, with 5% CO2, to total RNA was extracted then cDNA labelling, hybridization and scanning for microarray methods have been developed for microarray based immune related gene expression analysis. The differential expressions twenty genes (IL1, CCL3, CXCR4, TNF, TLR2, IL12, CSF3, CCR5, CCR3, MAPT, NFKB1, CCL4, IL6, IL2, IL23A, CCL20, IL8, CXCL8, TRIP10, CXCL2 and IL1B) implicated in M. bovis response were examined Agilent Bovine_GXP_8 × 60 K microarray platform. Cells of treatment were challenged after six days for six hours incubation then pathways analysis of Toll like receptor and Chemokine signalling pathway study of responsible genes in bovine tuberculosis. The PBMC from M. bovis infected cattle exhibit different transcriptional profiles compared with PBMC from healthy control animals in response to M. bovis antigen stimulation, providing evidence of a novel genes expression program due to M. bovis exposure. It will guide future studies, regarding the complex macrophage specific signalling pathways stimulated upon phagocytosis of M. bovis and role of signalling pathways in creating the host immune response to cattle tuberculosis.

Differential gene expression in Mycobacterium bovis challenged monocyte-derived macrophages of cattle.

A functional genomics approach was used to examine the immune response for transcriptional profiling of PBMC M. bovis infected cattle and healthy control cattle to stimulation with bovine tuberculin (purified protein derivative PPD-b). Total cellular RNA was extracted from non-challenged control and M. bovis challenged MDM for all animals at intervals of 6 h post-challenge, in response to in-vitro challenge with M. bovis (multiplicity of infection 2:1) and prepared for global gene expression analysis using the Agilent Bovine (V2) Gene Expression Microarray, 8 × 60 K. The pattern of expression of these genes in PPD bovine stimulated PBMC provides the first description of an M. bovis specific signature of infection that may provide insights into the molecular basis of the host response to infection. Analysis of these mapped reads showed 2450 genes (1291 up regulated and 1158 down regulated) 462 putative natural antisense transcripts (354 up-regulated and 108 down regulated) that were differentially expressed based on sense and antisense strand data, respectively (adjusted P-value ≤ 0.05). The results provided enrichment for genes involved top ten up regulated and down regulated panel of genes, including transcription factors proliferation of T and B lymphocytes. The highest differentially-expressed genes were associated to immune and inflammatory responses, immunity, differentiation, cell growth, apoptosis, cellular trafficking and regulation of lipolysis and thermogenesis. Microarray results were confirmed in infected cattle by RT qPCR to identify potential biomarkers TLR2, CD80, NFKB1, IL8, CXCL6 and ADORA3 of bovine tuberculosis.

Intra-abdominal inflammatory myofibroblastic tumour (IMT) mimicking gonadal mass in a male infant with undescended testes.

Inflammatory myofibroblastic tumour (IMT) is a rare benign neoplastic tumour, originally described as an inflammatory pseudotumor. Only a single case of IMT in the spermatic cord associated with undescended testes has been reported. We present a rare case of abdominal IMT in a male infant with undescended bilateral testes. The abdominopelvic mass was suspected to be a gonadal malignancy due to empty scrotal sac but proved to be otherwise on histopathology. Mass was completely resected and orchiopexy was performed for both undescended normal-appearing testes. The patient developed a recurrent mass 2 months later involving the left spermatic cord and the left testicle. Radical surgery was performed with removal of the left testicle and spermatic cord. The patient was disease-free at 1-year follow-up.

Evolutionary and bioinformatic analysis of the spike glycoprotein gene of H120 vaccine strain protectotype of infectious bronchitis virus from India.

The infectious bronchitis virus is a causative agent of avian infectious bronchitis (AIB), and is is an important disease that produces severe economic losses to the poultry industry worldwide. Recent AIB outbreaks in India have been associated with poor growth in broilers, drop in egg production, and thin egg shells in layers. The complete spike gene of Indian AIB vaccine strain was amplified and sequenced using a conventional reverse transcription polymerase chain reaction and is submitted to the GenBank (accession no KF188436). Phylogenetic analysis revealed that the vaccine strain currently used belongs to H120 genotype, an attenuated strain of Massachusetts (Mass) serotype. Nucleotide and amino acid sequence comparisons have shown that the reported spike gene from Indian isolates have 71.8%-99% and 71.4%-96.9% genetic similarity with the sequenced H120 strain. The study identifies live attenuated IBV vaccine strain, which is routinely used for vaccination, for the first time. Based on nucleotide and amino acid relatedness studies of the vaccine strain with reported IBV sequences from India, it is shown that the current vaccine strain is efficient in controlling the IBV infection. Continuous monitoring of IBV outbreaks by sequencing for genotyping and in vivo cross protection studies for serotyping is not only important for epidemiological investigation but also for evaluation of efficacy of the current vaccine.

Protective effects of recombinant glycoprotein D based prime boost approach against duck enteritis virus in mice model.

Duck virus enteritis, also known as duck plague, is an acute herpes viral infection of ducks caused by duck enteritis virus (DEV). The method of repeated immunization with a live attenuated vaccine has been used for the prevention and control of duck enteritis virus (DEV). However, the incidence of the disease in vaccinated flocks and latency reactivation are the major constraints in the present vaccination programme. The immunogenicity and protective efficacy afforded by intramuscular inoculation of plasmid DNA encoding DEV glycoprotein D (pCDNA-gD) followed by DEV gD expressed in Saccharomyces cerevisia (rgD) was assessed in a murine model. Compared with mice inoculated with DNA (pCDNA-gD) or protein (rgD) only, mice inoculated with the combination of gD DNA and protein had enhanced ELISA antibody titers to DEV and had accelerated clearance of virus following challenge infection. Furthermore, the highest levels of lymphocyte proliferation response, IL-4, IL-12 and IFN-γ production were induced following priming with the DNA vaccine and boosting with the rgD protein. For instance, the specially designed recombinant DEV vector vaccine would be the best choice to use in ducks. It offers an excellent solution to the low vaccination coverage rate in ducks. We expect that the application of this novel vaccine in the near future will greatly decrease the virus load in the environment and reduce outbreaks of DEV in ducks.

Adaptation and growth kinetics study of an Indian isolate of virulent duck enteritis virus in Vero cells.

Duck virus enteritis, also known as duck plague, is a viral infection of ducks caused by duck enteritis virus (DEV). The control of the disease is mainly done by vaccination with chicken embryo adapted live virus that is known to be poorly immunogenic and elicits only partial protection. Further, the embryo propagated vaccine virus pose a threat of harboring other infectious agents. Seeing these limitations, the present study reports for the first time regarding propagation and adaptation of a virulent Indian isolate of duck enteritis virus in Vero cell line. In this study isolation of an outbreak virus from Kerala state was done in chicken embryo fibroblast cell culture (CEF). Then adapted the DEV isolate in the Vero cell line. The characteristic cytopathic effects (CPE) of clumping and fusion of Vero cells were observed starting from the 7th passage onwards. The presence of the virus and its multiplication in Vero cells was confirmed by detection of viral specific DNA and antigen by using polymerase chain reaction (PCR) and indirect immuno fluorescent assay (IIFA), respectively. PCR detection of DEV using self designed primers for US4 (gD) and UL30 (DNA Polymerase) gene has been reported for the in the present study. The kinetics of DEV in Vero cells revealed a maximum infectivity titer of 10(5.6) TCID 50/ml after 48hr of viral infection. Compared to chicken embryo adapted DVE vaccine virus, the Vero cell culture system is free from other infectious agents. So it will be a good candidate for cultivation and propagation of duck enteritis virus vaccine strain. Further research studies are suggested to explore the feasibility of utilizing this Vero cell culture adapted DEV isolate for developing an attenuated vaccine virus against duck virus enteritis.

Detection and identification of globally distributed mycobacterial fish pathogens in some ornamental fish in India.

Mycobacteriosis is a progressive disease of a wide range of wild and captive, marine and freshwater fish species. Conventional detection of fish Mycobacteria is based on histopathology, culture, and biochemical characteristics. The present study analyzed the occurrence of Mycobacteria in clinically ill ornamental fish of different species, from different places of India. In first group, 60 fish were examined for presence of granulomatous inflammation and acid-fast bacteria. Thirty-eight (63.34 %) fish were positive for granulomatous inflammations. Presences of acid-fast bacteria were detected in 27 (45 %) fish having granulomatous inflammation and in two (3.33 %) fish without granulomatous inflammation. In total, AFB were found in 29 (48.34 %) of the 60 fish examined. In second group, 20 fish having granulomatous inflammation, 12 (60 %) samples were positive using Ziehl-Neelsen (Z-N) staining and 11 (55 %) of them were culture positive. Eight (40 %) samples were Z-N negative but two (10 %) of them were culture positive. In total, 13 (65 %) of the 20 examined fish were culture positive. On the basis of biochemical tests and 16S rRNA sequencing, 13 isolates were identified: five as Mycobacterium fortuitum, five as Mycobacterium gordonae, and three as Mycobacterium chelonae. In comparison of two decontamination methods, 2 % HCl treatment was better than 4 % NaOH treatment. Mycobacteria recovery from decontaminated samples was significantly high on Lowenstein-Jensen medium compared to Middlebrook 7H11 agar and Stonebrink (SB) media. The disease is transmissible from fish to fish and also from fish to human, so the significance of Mycobacteria in ornamental fish should not be overlooked.