tRNA Function and Dysregulation in Cancer.

Transfer RNA (tRNA) is a central component of protein synthesis and plays important roles in epigenetic regulation of gene expression in tumors. tRNAs are also involved in many cell processes including cell proliferation, cell signaling pathways and stress response, implicating a role in tumorigenesis and cancer progression. The complex role of tRNA in cell regulation implies that an understanding of tRNA function and dysregulation can be used to develop treatments for many cancers including breast cancer, colon cancer, and glioblastoma. Moreover, tRNA modifications including methylation are necessary for tRNA folding, stability, and function. In response to certain stress conditions, tRNAs can be cleaved in half to form tiRNAs, or even shorter tRNA fragments (tRF). tRNA structure and modifications, tiRNA induction of stress granule formation, and tRF regulation of gene expression through the repression of translation can all impact a cell's fate. This review focuses on how these functions of tRNAs, tiRNA, and tRFs can lead to tumor development and progression. Further studies focusing on the specific pathways of tRNA regulation could help identify tRNA biomarkers and therapeutic targets, which might prevent and treat cancers.

An investigation into the effects of infection and ORF expression patterns of the Indian bidensovirus isolate (BmBDV) infecting the silkworm Bombyx mori.

The Indian isolate of Bombyx mori bidensovirus (BmBDV) is a bipartite virus that comprises of a segmented, non-homologous, two linear single-strands of DNA molecules (VD1 and VD2). It is one of the causative agents of the fatal silkworm disease 'Flacherie' that causes severe crop loss for the sericulture farmers. Genome analyses of the Indian isolate of BmBDV revealed that it consists of 6 putative ORFs similar to the Japanese and Chinese isolates. VD1 consists of 4 ORFs while VD2 has 2 ORFs that code for 4 non- structural (NS) and 2 structural (VP) proteins, in total. In this study, we investigated, in detail, the impact of BmBDV pathogenesis on growth and development of the silkworm Bombyx mori, at different developmental stages. Mortality rate and weight uptake analyses were also performed on newly ecdysed 4th instar larvae. BmBDV infection was not found to be developmental stage specific and it occurred at all stages. Onset of mortality took place 8 days post infection (dpi) and 100% mortality occurred at 11 dpi. The infected larvae showed a significant difference in weight uptake wherein from 7 dpi the larvae stopped gaining weight and from 8th dpi started demonstrating the typical symptoms of flacherie. Further, the expression pattern of the 6 viral ORFs were also investigated in the newly ecdysed 4th instar BmBDV infected silkworms. Among all the six ORFs, VD2 ORF 1 and 2 revealed the highest transcript numbers, which was followed by VD1 ORF 4 that encodes for the viral DNA polymerase enzyme. This was the first ever attempt to understand the pathogenesis and the expression pattern of all the six ORF transcripts of the Indian isolate of BmBDV.

Investigation of an outbreak of sheeppox among native sheep breeds in the Western Himalayas of India.

An outbreak of sheeppox was investigated in a cluster of villages situated in Western Himalayan ranges of a Northern Indian state. Non-migratory sheep (n = 80) of native breeds namely Gaddi and Rampur Bushair were infected and 15 have died. The outbreak started after a few animals contracted the disease during the summer grazing period at the highland pastures from migrating flocks of sheep. This initial outbreak resulted in a further spreading of the disease into the valley. Clinical examination revealed varying degree of cutaneous papular lesions and respiratory distress. Upon necropsy, visceral lesions in the lungs, trachea and kidneys were also found. Clinical and morbid samples were found positive for sheeppox virus using group specific P32 gene and I3L gene based multiplex PCR differentiating sheeppox and goatpox viruses. Histopathological, hematological and blood biochemical analysis also supported the pathology of an acute viral infection. The causative sheeppox virus strain was isolated using lamb testicular cell culture and phylogenetic analysis, based upon P32 and RPO30 genes, showed its clustering with other Indian strains reported from neighboring states. This study demonstrated the spread of sheeppox virus to new niches by migratory sheep flocks leading to establishment of endemic infections in many new pockets of higher Western Himalayas.

Theileria orientalis outbreak in an organized cattle breeding farm.

Theileriosis is an important tick borne disease of cattle caused by a haemoprotozoan of genus Theileria. Clinical bovine theileriosis is mainly caused by T. annulata or T. parva but the clinical disease due to T. orientalis is rare. T. orientalis mainly infect RBCs and causes "Oriental theileriosis" or Theileria-associated bovine anaemia in cattle and other livestock species. Two genotypes of T. orientalis (Chitose and Ikeda) are reported to cause severe disease in some countries. In this report, a spontaneous outbreak of Oriental theileriosis was studied in an organized Holstein-Friesian cattle breeding farm situated in the south-eastern Himalayan ranges of Himachal Pradesh State of India. Animal blood and tick samples were tested using cytological and PCR techniques. The disease episode occurred in a protracted manner spanning over 10 to 12 months and association of T. orientalis was confirmed in 93.3% of the blood and 21.7% of Rhipicephalus microplus (tick) samples. No other tick borne pathogen was detected except Anaplasma marginale in two blood samples. Haematological profiling of infected cattle showed characteristic indicators of anaemia like haemoblobin, RBC count, haematocrit value and mean corpuscular volume at either lower than normal or near the lower normal range. The prevailing persistent anaemic changes led to more severe clinical manifestations like abortion and joint inflammation. The detected T. orientalis strains and ticks species were further confirmed by nucleotide sequence analysis of 18S rRNA and 16S rRNA genes. Phylogenetically, T. orientalis strains showed clustering with other reported strains of T. orientalis from the surrounding regions. This first report of clinical Oriental theileriosis from India emphasises the importance of T. orientalis as an emerging tick borne pathogen and role of widely prevalent ticks species in disease transmission and their impact on livestock production.

Evaluation of real-time PCR as an alternative for potency testing of Brucella abortus vaccines.

The aim of the research was to evaluate real-time PCR (qPCR) as an alternate method for quantitative detection of Brucella abortus strain 544 (S544) in the spleen of mice for potency testing of live B. abortus strain 19 (S19) vaccine. IS711 and eryC gene-based qPCR were optimized for calculating copy number. The copy number was further correlated with live Brucella count in the spleen by standard plate count (SPC) method. The mice were immunized with S19 and challenged with S544 on 30th Day post-immunization. The spleen of mice was collected at 15th, 21st, and 30th days post challenge (DPC) for estimation of S19 and S544 load via SPC as well as qPCR. The noteworthy difference was observed between immunized and unimmunized group by both methods at all time points. The maximum correlation between SPC and qPCR method was observed at 15th DPC in both immunized and unimmunized group. Repeated experiments at 15th DPC gave the parallel significant difference between immunized and unimmunized group by both methods. Thus novel, risk-free qPCR method can be used for the indirect culture-free potency evaluation of S19 vaccine in order to preclude the cultivation of zoonotic Brucella organisms from spleen samples.

A review: Lumpy skin disease and its emergence in India.

Lumpy skin disease (LSD) is a viral disease caused by lumpy skin disease virus (LSDV), a member of Capripoxvirus genus of Poxviridae family. It is a transboundary disease of the economic importance affecting cattle and water buffaloes. The disease is transmitted by arthropod vectors and causes high morbidity and low mortality. LSD has recently been reported first time in India with 7.1% morbidity among cattle. Generally, fever, anorexia, and characteristic nodules on the skin mucous membrane of mouth, nostrils, udder, genital, rectum, drop in milk production, abortion, infertility and sometimes death are the clinical manifestations of the disease. The disease is endemic in African and Middle East countries but has started spreading to Asian and other countries. It has been recently reported from China and Bangladesh sharing borders with India. We have summarized occurrence of LSD outbreaks in last 10 years in Asian countries for the first time. In India, currently epidemiological status of the disease is unknown. Vaccination along with strict quarantine measures and vector control could be effective for preventing the spread of the disease. This review aims to summarise the latest developments in the epidemiology with the focus on transboundary spread, aetiology and transmission, clinical presentations, diagnostics and management of the disease.

Potential adjuvants for the development of a SARS-CoV-2 vaccine based on experimental results from similar coronaviruses.

The extensive efforts around the globe are being made to develop a suitable vaccine against COVID-19 (Coronavirus Disease-19) caused by SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2). An effective vaccine should be able to induce high titers of neutralizing antibodies to prevent the virus from attaching to the host cell receptors. However, to elicit the protective levels of antibodies, a vaccine may require multiple doses or assistance from other immunostimulatory molecules. Further, the vaccine should be able to induce protective levels of antibodies rapidly with the least amount of antigen used. This decreases the cost of a vaccine and makes it affordable. As the pandemic has hit most countries across the globe, there will be an overwhelming demand for the vaccine in a quick time. Incorporating a suitable adjuvant in a SARS-CoV-2 vaccine may address these requirements. This review paper will discuss the experimental results of the adjuvanted vaccine studies with similar coronaviruses (CoVs) which might be useful to select an appropriate adjuvant for a vaccine against rapidly emergingSARS-CoV-2. We also discuss the current progress in the development of adjuvanted vaccines against the disease.

Bacillus cereus: public health burden associated with ready-to-eat foods in Himachal Pradesh, India.

The study determined incidence, enterotoxigenecity and antimicrobial susceptibility profiles of Bacillus cereus isolated from ready-to-eat (RTE) milk products (n = 80), RTE meat products (n = 40), beverages (n = 40) and water samples (n = 60, from food preparing and serving outlets/restaurants) collected from eight different tourist places of Himachal Pradesh. 11.4% (25/220) samples were contaminated with Bacillus and isolates were identified as B. cereus (76.0%, n = 19), B. alvei (12.0%, n = 3), B. polymyxa (8.0%, n = 2) and B. firmus (4.0%, n = 1) by conventional and molecular methods. B. cereus incidence was highest in cheese based foods (25.0%) followed by vegetable soups (16.7%), khoa based foods (14.0%), milk based beverages (10.5%), paneer based foods (8.6%), cream based foods (8.3%) and water (8.3%) samples. Multiplex polymerase chain reaction detected enterotoxigenic genes only in B. cereus isolates. nhe complex (encoding non-haemolytic enterotoxins, ABC) genes were detected only in B. cereus isolates. 57.6% (11/19), 36.8% (7/19) and 5.3% (1/19) harboured all three (nheA, nheB, nheC), two (nheB, nheC) and one (nheC) nhe gene, respectively. Among hbl complex genes (encoding haemolytic enterotoxins CAD), only hblC (36.8%, 7/19) was detected. Incidence B. cereus cytK (encoding cytotoxin enterotoxin) was 52.6% (10/19). Each B. cereus isolate harboured two or more enterotoxigenic genes. Seven isolates had at least one gene from haemolytic and non-haemolytic complexes along with cytK. High levels (> 50%) of antimicrobial resistance were recorded for penicillin, amoxicillin, ampicillin cefixime and ceftazidine in tested B. cereus isolates. Two isolates were identified as multidrug resistant isolates with resistance to ≥ 3 antibiotic classes.

Development and comparison of real-time and conventional PCR tools targeting ß-tubulin gene for detection of Nosema infection in silkworms.

Microsporidiosis (Pebrine) caused by the microsporidian parasite is one of the important devastating disease which affect the silk production leading to an unprofitable harvest. Till date ribosomal RNA (rRNA) gene was used as a target for detection of microsporidian species. In this study, we describe conventional and SYBR green based real-time PCR techniques alternatively targeting ß-tubulin gene for quantitative detection of microsporidia infecting both the mulberry and non-mulberry silkworms. The modified DNA extraction method followed in our study was found to be easy, economical and could be used for both conventional and real time PCR as template. The real time qPCR revealed the expression of ß-tubulin gene in different infected tissues of the silkworm Bombyx mori. The sensitivity of the SYBR green based real time PCR was found to be 100 times more than the conventional PCR and PCR was found more sensitive than the microscopic examination. The developed method did not produce any false positive results with the other silkworm pathogens and healthy silkworm. The data suggest that both the developed PCR methods targeting ß-tubulin gene could be used effectively in quarantine process at seed centres for early detection of microsporidian infection in silkworms.

Molecular characterization of Nosema bombycis methionine aminopeptidase 2 (MetAP2) gene and evaluation of anti-microsporidian activity of Fumagilin-B in silkworm Bombyx mori.

Nosema bombycis is a spore-forming parasite causing microsporidiosis in silkworm Bombyx mori. Methionine aminopeptidase 2 (MetAP2), an essential gene of N. bombycis, is a target for the anti-microsporidian drug Fumagillin, an antibiotic derived from Aspergillus fumigatus. In this study, a 1077 bp full-length cDNA of the MetAP2 gene of N. bombycis was cloned and characterized. Furthermore, the expression study of the MetAP2 gene revealed a ubiquitous expression during all the developmental stages of the silkworm B. mori. The phylogenetic analysis of the MetAP2 gene of N. bombycis revealed the MetAP2 gene sequences to be highly conserved in nature. The present study also includes the validation of the anti-microsporidian drug Fumagillin against the MetAP2 gene of N. bombycis. The findings revealed that Fumagilin-B could also suppress the N. bombycis multiplication in the silkworm B. mori, thereby proving the therapeutic role of Fumagillin against microsporidian infection. This is the first-ever report regarding the characterization of the MetAP2 gene in the Indian isolate of N. bombycis and also towards the usage of Fumagillin in the control of microsporidiosis in B. mori.

Characterization and genome comparison of an Indian isolate of bidensovirus infecting the silkworm Bombyx mori.

The bipartite genome of an Indian isolate of Bombyx mori bidensovirus (BmBDV), one of the causative agents of the fatal silkworm disease 'Flacherie', was cloned and completely sequenced. Nucleotide sequence analysis of this Indian isolate of BmBDV revealed two viral DNA segments, VD1 and VD2 as well as a DNA polymerase motif which supports its taxonomical status as the type species of a new family of Bidnaviridae. The Indian isolate of BmBDV was found to have a total of six putative ORFs four of which were located on the VD1 with the other two being on the VD2 DNA segment. The VD1 DNA segment was found to code for three non-structural proteins including a viral DNA polymerase as well as one structural protein, while the VD2 DNA segment was found to code for one structural and one non-structural protein, similar to that of the Japanese and Zhenjiang isolates of BmBDV. A BmBDV ORF expression study was done through real time qPCR wherein the VD2 ORF 1 and 2 showed the maximum transcript levels. This is the first report of the genome characterization of an Indian isolate of BmBDV, infecting silkworm B. mori.

Genome-wide identification, characterization of sugar transporter genes in the silkworm Bombyx mori and role in Bombyx mori nucleopolyhedrovirus (BmNPV) infection.

Sugar transporters play an essential role in controlling carbohydrate transport and are responsible for mediating the movement of sugars into cells. These genes exist as large multigene families within the insect genome. In insects, sugar transporters not only have a role in sugar transport, but may also act as receptors for virus entry. Genome-wide annotation of silkworm Bombyx mori (B. mori) revealed 100 putative sugar transporter (BmST) genes exists as a large multigene family and were classified into 11 sub families, through phylogenetic analysis. Chromosomes 27, 26 and 20 were found to possess the highest number of BmST paralogous genes, harboring 22, 7 and 6 genes, respectively. These genes occurred in clusters exhibiting the phenomenon of tandem gene duplication. The ovary, silk gland, hemocytes, midgut and malphigian tubules were the different tissues/cells enriched with BmST gene expression. The BmST gene BGIBMGA001498 had maximum EST transcripts of 134 and expressed exclusively in the malphigian tubule. The expression of EST transcripts of the BmST clustered genes on chromosome 27 was distributed in various tissues like testis, ovary, silk gland, malphigian tubule, maxillary galea, prothoracic gland, epidermis, fat body and midgut. Three sugar transporter genes (BmST) were constitutively expressed in the susceptible race and were down regulated upon BmNPV infection at 12h post infection (hpi). The expression pattern of these three genes was validated through real-time PCR in the midgut tissues at different time intervals from 0 to 30hpi. In the susceptible B. mori race, expression of sugar transporter genes was constitutively expressed making the host succumb to viral infection.

Genome wide microarray based expression profiles associated with BmNPV resistance and susceptibility in Indian silkworm races of Bombyx mori.

The molecular mechanism involved in BmNPV resistance was investigated using a genome wide microarray in midgut tissue of Indian silkworm Bombyx mori. In resistant race (Sarupat), 735 genes up-regulated and 589 genes down-regulated at 12 h post BmNPV infection. Similarly, in case of susceptible race (CSR-2), 2183 genes up-regulated and 2115 genes down-regulated. Among these, nine up-regulated and eight down-regulated genes were validated using real-time qPCR analysis. In Sarupat, vacuolar protein sorting associated, Xfin-like protein and carboxypeptidase E-like protein genes significantly up-regulated in infected midgut; prominently down-regulated genes were glutamate receptor ionotropic kainite 2-like, BTB/POZ domain and transferrin. Considerably up-regulated genes in the CSR-2 were peptidoglycan recognition protein S6 precursor and rapamycin while the conspicuous down-regulated genes were facilitated trehalose transporter and zinc transporter ZIP1-like gene. The up-regulation of genes in resistant race after BmNPV infection indicates their possible role in antiviral immune response.