Development of a rapid and ultra-sensitive RNA:DNA hybrid immunocapture based biosensor for visual detection of SARS-CoV-2 RNA.

The Development of reliable and field-compatible detection methods is essential to monitoring and controlling the spread of any global pandemic. We herein report a novel anti-RNA:DNA hybrid (anti-RDH) antibody-based biosensor for visual, colorimetric lateral flow assay, using gold nanoparticles, coupled with transcription-mediated-isothermal-RNA-amplification (TMIRA) for specific and sensitive detection of viral RNA. We have demonstrated its utility for SARS-CoV-2 RNA detection. This technique, which we have named RDH-LFA (anti-RNA:DNA hybrid antibody-based lateral flow assay), exploits anti-RDH antibody for immunocapture of viral RNA hybridized with specific DNA probes in lateral flow assay. This method uses biotinylated-oligonucleotides (DNAB) specific to SARS-CoV-2 RNA (vRNA) to generate a vRNA-DNAB hybrid. The biotin-tagged vRNA-DNAB hybrid molecules bind to streptavidin conjugated with gold nanoparticles. This hybrid complex is trapped by the anti-RDH antibody immobilized on the nitrocellulose membrane resulting in pink color signal leading to visual naked-eye detection in 1 minute. Combining RDH-LFA with isothermal RNA amplification (TMIRA) significantly improves the sensitivity (LOD:10 copies/µl) with a total turnaround time of an hour. More importantly, RDH-LFA coupled with the TMIRA method showed 96.6% sensitivity and 100% specificity for clinical samples when compared to a commercial gold standard reverse-transcription quantitative polymerase-chain-reaction assay. Thus, the present study reports a rapid, sensitive, specific, and simple method for visual detection of viral RNA, which can be used at the point-of-care without requiring sophisticated instrumentation.

Novel mutation in Cul7 gene in a family diagnosed with 3M syndrome.

This study evaluates a family with two siblings having severe growth retardation and facial dysmorphism, born to consanguineous normal healthy parents. Affymetrix CytoScan 750K microarray showed a 34-Mb pericentric homozygous region on chromosome 6 for both siblings. CUL7 was one of the 141 genes present in this region. Sanger sequencing of CUL7 gene detected a 2-bp novel deletion in the 15th exon (c.2943_2944delCT of the cDNA). This deletion leads to a frameshift and a premature termination signal much upstream of the wild-type termination signal, leading to a nonsense mediated decay of the mRNA. CUL7 protein plays an important role in formation of 3M complex, ubiquitination, microtubule dynamics and cell cycle regulation. Mutations in CUL7 gene is known to cause a rare 3M syndrome. Information about the novel mutation has been accepted in the ClinVar database with rs1064792895.

Hydroxychavicol, a key ingredient of Piper betle induces bacterial cell death by DNA damage and inhibition of cell division.

Antibiotic resistance is a global problem and there is an urgent need to augment the arsenal against pathogenic bacteria. The emergence of different drug resistant bacteria is threatening human lives to be pushed towards the pre-antibiotic era. Botanical sources remain a vital source of diverse organic molecules that possess antibacterial property as well as augment existing antibacterial molecules. Piper betle, a climber, is widely used in south and south-east Asia whose leaves and nuts are consumed regularly. Hydroxychavicol (HC) isolated from Piper betle has been reported to possess antibacterial activity. It is currently not clear how the antibacterial activity of HC is manifested. In this investigation we show HC generates superoxide in E. coli cells. Antioxidants protected E. coli against HC induced cell death while gshA mutant was more sensitive to HC than wild type. DNA damage repair deficient mutants are hypersensitive to HC and HC induces the expression of DNA damage repair genes that repair oxidative DNA damage. HC treated E. coli cells are inhibited from growth and undergo DNA condensation. In vitro HC binds to DNA and cleaves it in presence of copper. Our data strongly indicates HC mediates bacterial cell death by ROS generation and DNA damage. Damage to iron sulfur proteins in the cells contribute to amplification of oxidative stress initiated by HC. Further HC is active against a number of Gram negative bacteria isolated from patients with a wide range of clinical symptoms and varied antibiotic resistance profiles.

Rabies - epidemiology, pathogenesis, public health concerns and advances in diagnosis and control: a comprehensive review.

Rabies is a zoonotic, fatal and progressive neurological infection caused by rabies virus of the genus Lyssavirus and family Rhabdoviridae. It affects all warm-blooded animals and the disease is prevalent throughout the world and endemic in many countries except in Islands like Australia and Antarctica. Over 60,000 peoples die every year due to rabies, while approximately 15 million people receive rabies post-exposure prophylaxis (PEP) annually. Bite of rabid animals and saliva of infected host are mainly responsible for transmission and wildlife like raccoons, skunks, bats and foxes are main reservoirs for rabies. The incubation period is highly variable from 2 weeks to 6 years (avg. 2-3 months). Though severe neurologic signs and fatal outcome, neuropathological lesions are relatively mild. Rabies virus exploits various mechanisms to evade the host immune responses. Being a major zoonosis, precise and rapid diagnosis is important for early treatment and effective prevention and control measures. Traditional rapid Seller's staining and histopathological methods are still in use for diagnosis of rabies. Direct immunofluoroscent test (dFAT) is gold standard test and most commonly recommended for diagnosis of rabies in fresh brain tissues of dogs by both OIE and WHO. Mouse inoculation test (MIT) and polymerase chain reaction (PCR) are superior and used for routine diagnosis. Vaccination with live attenuated or inactivated viruses, DNA and recombinant vaccines can be done in endemic areas. This review describes in detail about epidemiology, transmission, pathogenesis, advances in diagnosis, vaccination and therapeutic approaches along with appropriate prevention and control strategies.

Sero-epidemiology and molecular detection of Bluetongue virus in Indian ruminants.

Bluetongue (BT) is a non­contagious arthropod­borne viral disease of domestic and wild ruminants. It is endemic to India and clinical outbreaks of disease have been reported mainly in sheep, although BT is often asymptomatic in other ruminant species. In the present serological survey, a total of 576 serum samples, comprising of 416 cattle and 160 sheep, covering different agro­climatic zones of Rajasthan, Uttar Pradesh, and Karnataka states, were screened for the presence of Bluetongue virus (BTV) specific antibodies using competitive enzyme­linked immunosorbent assay (c­ELISA). Overall 73.08% (304/416) of the cattle and 53.30% (87/160) of the sheep serum samples were positive for BTV antibodies. The prevalence of BTV antibodies in cattle in different agro­climatic zones ranged between 60­80% in Rajasthan and 66­70% in Uttar Pradesh. During the study, a nested polymerase chain reaction (PCR) based on the BTV NS1 gene (genome segment 5) was optimized for detection of BTV's nucleic acid from a cell adapted strain of BTV­23, and field derived clinical blood samples. In the present study, 19/70 of cattle and 9/30 of sheep blood samples tested positive for BTV RNA by the nested PCR, which amplified specific products of 274 bp and 101 bp sizes, respectively. From this study, it can be concluded that cattle showed higher percentage of sero­positivity in comparison to sheep. The improved sero­surveillance system for BTV in endemic areas will be of great help to understand the epidemiology of BTV and to formulate effective control and preventive strategies.

Phylogenetic analysis of Indian rabies virus isolates targeting the complete glycoprotein gene.

Rabies a fatal viral zoonosis is endemic in India. There is no report on phylogenetic study of Indian rabies virus isolates based on the complete G gene. In the present study, a total of 25 rabies positive brain samples collected during 2001-2014 from North India (UP, MP, Delhi, Rajasthan), South India (Kerala and Karnataka) and Gujarat states belonging to six different host species were subjected to G gene amplification by RT-PCR as three overlapping fragments of 881 bp, 991 bp and 618 bp. Phylogenetic analysis revealed that all Indian rabies virus isolates are genetically closely related with Arctic-like 1a lineage viruses. However, two distinct clusters were identified namely, India South and India North. All the Indian rabies isolates had 95.5-100% homology related to geography, but not to host species. Deduced amino acids on comparison revealed two amino acid changes, aa 356 in ECTO; N→K and aa 458; M→I, which were found to distinguish between the India South and India North isolates.

Genetic characterisation and phylogenetic analysis of PCV2 isolates from India: indications for emergence of natural inter-genotypic recombinants.

Porcine circovirus type 2 (PCV2), the necessary agent in pathogenesis of porcine circovirus diseases (PCVDs), has a worldwide distribution and is considered as one of the most important emerging viral pathogens of economic importance. PCV2 has been divided into four major genotypes namely PCV2a with five clusters or subtypes (2A-2E), PCV2b with three clusters (1A-1C), PCV2c and PCV2d, based on capsid (cap) gene analysis. PCV2 genome is rapidly evolving through events of recombination and mutation. Though, PCV2a was the predominant genotype initially, PCV2b shared majority of PCV2 sequences submitted to GenBank since 2003. In India, data regarding molecular characterisation of PCV2 is scant or absent. In the present study, we thoroughly analysed genetic heterogeneity of PCV2 strains circulating in Indian pig population. The results revealed that pigs in this region harboured PCV2 viruses of different genotypes including PCV2a-2D, PCV2b-1C and PCV2d. More interestingly, two isolates (PCV2Izn-89-13 and PCV2Izn-218-13) were classified as recombinant strains. Further detailed analysis suggested that these strains evolved from inter-genotypic recombination between PCV2a-2C and PCV2b-1C genotypes within cap gene. This study reports for the first time, the emergence of recombinant PCV2 strains in the Indian pig population.

Nonhemophilic hemosiderotic synovitis of the knee: a case report and review of literature.

Synovium is specialized mesenchymal tissue lining the inner surface of the joint capsule and is the site for a series of pathologic processes that are characteristic, and in some cases specific, to this distinctive tissue. Hemosiderotic synovitis is a rare and inadequately defined synovial proliferative disorder, which develops with recurrent hemorrhages in the joint. The most affected joint from bleeding is the knee whatever the etiology is. Repeated hemarthrosis may produce significant structural alteration of joints leading to chronic osteoarthritis. The most common cause is hereditary clotting factor deficiency diseases like hemophilia. We report a rare case of nonhemophilic hemosiderotic synovitis of the knee joint, in which the patient lacks history of any bleeding diathesis. Its definitive diagnosis was possible only by histopathological examination. The prompt recognition of this distinct subtype of hemosiderotic synovitis and awareness of underlying causes should lead to earlier diagnosis, appropriate therapy, less joint destruction, and better outcomes.

Edible vaccines against veterinary parasitic diseases--current status and future prospects.

Protection of domestic animals against parasitic infections remains a major challenge in most of the developing countries, especially in the surge of drug resistant strains. In this circumstance vaccination seems to be the sole practical strategy to combat parasites. Most of the presently available live or killed parasitic vaccines possess many disadvantages. Thus, expression of parasitic antigens has seen a continued interest over the past few decades. However, only a limited success was achieved using bacterial, yeast, insect and mammalian expression systems. This is witnessed by an increasing number of reports on transgenic plant expression of previously reported and new antigens. Oral delivery of plant-made vaccines is particularly attractive due to their exceptional advantages. Moreover, the regulatory burden for veterinary vaccines is less compared to human vaccines. This led to an incredible investment in the field of transgenic plant vaccines for veterinary purpose. Plant based vaccine trials have been conducted to combat various significant parasitic diseases such as fasciolosis, schistosomosis, poultry coccidiosis, porcine cycticercosis and ascariosis. Besides, passive immunization by oral delivery of antibodies expressed in transgenic plants against poultry coccidiosis is an innovative strategy. These trials may pave way to the development of promising edible veterinary vaccines in the near future. As the existing data regarding edible parasitic vaccines are scattered, an attempt has been made to assemble the available literature.

Epithelial-myoepithelial carcinoma in the hard palate: a case report.

BACKGROUND: Epithelial-myoepithelial carcinoma (EMC) is an uncommon salivary gland tumor. CASE: EMC arising from the minor salivary gland of the hard palate is very rare. A 70-year-old man presented with a nodular swelling in the hard palate. Fine needle aspiration cytology revealed biphasic epithelial (small cell) and myoepithelial (large/clear cell) clusters in a pseudopapillary and trabecular pattern. The cytology was reported as salivary gland neoplasm. The mass was excised and the histomorphology was suggestive of a low grade EMC. Immunohistochemistry demonstrated the biphasic nature of the tumor and confirmed the diagnosis of EMC. Follow-up for 2 years post surgery to date did not show any recurrence or metastases. CONCLUSION: Though exact cytologic typing of EMC was not possible; this case highlights the importance of awareness of this tumor in the differential diagnosis of biphasic tumors of the salivary gland.

Hyperbilirubinemia in normal healthy donors.

The present study was carried out in B.A.R.C. Hospital Blood Bank over a span of five years, and includes 2734 donors. All the bags were screened for HIV, HBsAg, HCV and VDRL and the plasma in the pilot tubes of the blood bags was observed to detect any abnormality in color. In 27 cases plasma was found to be icteric and liver function tests were carried out on these samples. Two donors showed higher SGPT level, and were excluded. No significant increases in liver enzymes were recorded in the others. Causes of icteric plasma in these apparently healthy donors are discussed. Differential diagnosis includes Gilbert's disease, hemolytic anemia, drug-induced anemia and other hepatic causes of hyperbilirubinemia, of which Gilbert's disease is most probable cause with a prevalence of 0.91% in our population. As there are no studies to document the safety of the recipients receiving such abnormal colored plasma as well as to document the hazards in its transfusion, the question arises whether to transfuse such units or not. This study highlights this dilemma. A reassessment of existing policies and regulations is merited.

Modulation of neuronal [Ca2+]i by caffeine is altered with aging.

Voltage-dependent calcium channels play an important role in controlling many neuronal processes such as neuronal excitability and synaptic transmission. Any slight alteration in intracellular calcium concentration ([Ca2+]i) can have a considerable impact on various neuronal functions. The effects of caffeine on [Ca2+]i were studied in CA1 hippocampal neurons of young (2 months) and old (24 months) C57BL mice. Fura 2-AM fluorescence photometry was used to measure [Ca2+]i in the presence and absence of caffeine (100 microM) in response to KCl (26 mM) application. Caffeine enhanced the peak [Ca2+]i as compared to control solution in young mice (control: 325+/-8 nM, caffeine: 402+/-10 nM), but had no effect on the peak [Ca2+]i in old mice (control: 222+/-6 nM, caffeine: 223+/-7 nM). These results indicate that caffeine can impact neuronal functions through the modification of [Ca2+]i. The lack of caffeine-induced modulation of [Ca2+]i in old mice suggests that this role of caffeine has been compromised with aging.

Increased calcium influx through acetylcholine receptors in dunce neurons.

Calcium homeostasis was studied in dunce, a Drosophila mutant that is defective in learning and memory. Fura 2-AM fluorescence photometry was used to measure the intracellular calcium concentration in wild type and dunce cleavage-arrested neurons under resting conditions and in response to neurotransmitters. After acetylcholine application, the peak [Ca2+]i was greater in dunce (693 +/- 125 nM) than in wild type neurons (464 +/- 154 nM), but half decay time was shorter in dunce (66 +/- 15 s) than in wild type neurons (104 +/- 40 s). In contrast, the application of glutamate, NMDA, dopamine, and serotonin had no effect on [Ca2+]i. These results indicate that calcium influx through acetylcholine receptors is increased in dunce, compared to wild type neurons. The results also suggest that calcium extrusion to the outside and/or calcium buffering are enhanced in dunce, compared to wild type neurons. This disturbance in the homeostasis of cytosolic calcium concentration in dunce may be implicated in defective associative learning in Drosophila, and may play a role in acute and chronic neurodegenerative disorders in the mammalian brain.

Drug effects on calcium homeostasis in mouse CA1 hippocampal neurons.

Voltage-dependent Ca2+ channels (VDCC) are important in control of neuronal excitability, synaptic transmission, and many other cellular process. Even the slightest alteration in Ca2+ currents can have a considerable impact on the neuronal function. However, it is still unknown whether Ca2+ currents are affected by neurotoxic drugs such as lead, cobalt, zinc, cadmium, thallium, lanthanum, and aluminum. We have characterized the effects of neurotoxic drugs on Ca2+ homeostasis in CA1 hippocampal C57BL mice. Fura 2-AM fluorescence photometry was used to measure intracellular Ca2+ concentration ([Ca2+]i) in the presence and absence of neurotoxic drugs (10 microM) in response to KCl application. The peak [Ca2+]i due to KCl application was reduced in the presence of lead (60%), cobalt (35%), zinc (62%), cadmium (71%), thallium (27%), and lanthanum (66%). By contrast, in the presence of aluminum the peak [Ca2+]i was either increased (46%) or it was not affected. These results indicate that neurotoxic drugs could block the entry of calcium into CA1 neurons via VDCC.