Trend of viral load during the first, second, and third wave of COVID-19 in the Indian Himalayan region: an observational study of the Uttarakhand state.

India had faced three waves throughout the Coronavirus disease 2019 (COVID-19) pandemic, which had already impacted economic lives and affected the healthcare setting and infrastructure. The widespread impacts have inspired researchers to look for clinical indicators of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection prognosis. Cyclic threshold values have been used to correlate the viral load in COVID-19 patients and for viral transmission. In light of this correlation, a retrospective study was conducted to assess the trend of viral load in clinical and demographic profiles across the three waves. Data of a total of 11,125 COVID-19-positive patients were obtained, which had a Ct value of <35. We stratified Ct values as follows: under 25 (high viral load), 25-30 (moderate viral load), and over 30 (low viral load). We found a significantly high proportion of patients with high viral load during the second wave. A significantly high viral load across the symptomatic and vaccinated populations was found in all three waves, whereas a significantly high viral load across age groups was found only in the first wave. With the widespread availability of real-time PCR and the limited use of genomic surveillance, the Ct value and viral load could be a suitable tool for population-level monitoring and forecasting.

Interference of Antinuclear Antibody (ANA) in Indirect Immunofluorescence Assay (IIFA)-Based Perinuclear Antineutrophil Cytoplasmic Antibody (pANCA) Interpretation.

Background: Indirect immunofluorescence assay (IIFA) based on antineutrophil cytoplasmic antibody (ANCA) testing is a commonly employed test for diagnosing autoimmune vasculitis. Antinuclear antibody (ANA) can give rise to a false interpretation of perinuclear-ANCA (pANCA) in ethanol-fixed granulocyte substrates. Analytical interference could frequently occur in setups where ethanol-fixed substrates are used alone. Here, we intend to investigate this ANA interference in pANCA interpretation. Methods: In this retrospective study, we studied anti-MPO-negative but ANA-positive and pANCA (IIFA based) samples. We also correlated immunoblot results (where data were available) and checked the association between grades of blot positivity (an indicator of the concentration of ANA) and frequency of pANCA interpretation. Data were analyzed by appropriate statistical techniques (Chi-square and kappa statistics). Results: About 19.2% of ANA blot (ENA-blot) positive samples displayed a pANCA positive pattern in the ethanol-fixed substrate, while this positivity in ENA-blot negatives was 6.5%. In positive ANA-IIFA samples, about 14.7% yielded pANCA patterns (on ethanol fixed substrates). Out of this, nuclear homogenous pattern yielding samples gave the highest frequency pANCA, that is, in 31.5% followed by speckled (11.1%), DFS (10.3%), and centromere (6.7%).The association of the nuclear homogenous pattern was statistically significant. Conclusions: ANA-positive results may interfere with the interpretation of pANCA as observed in ANA-IIFA and ENA-blot positive samples. ANA-IIFA patterns like nuclear homogenous may strongly associate this pANCA interpretation. This can help laboratories perform ANCA testing more effectively, ruling out ANA interference in ANCA screening.

Functional characterization of novel variants in SMPD1 in Indian patients with acid sphingomyelinase deficiency.

Pathogenic variations in SMPD1 lead to acid sphingomyelinase deficiency (ASMD), that is, Niemann-Pick disease (NPD) type A and B (NPA, NPB), which is a recessive lysosomal storage disease. The knowledge of variant spectrum in Indian patients is crucial for early and accurate NPD diagnosis and genetic counseling of families. In this study, we recruited 40 unrelated pediatric patients manifesting symptoms of ASMD and subnormal ASM enzyme activity. Variations in SMPD1 were studied using Sanger sequencing for all exons, followed by interpretation of variants based on American College of Medical Genetics and Genomics & Association for Molecular Pathology (ACMG/AMP) criteria. We identified 18 previously unreported variants and 21 known variants, including missense, nonsense, deletions, duplications, and splice site variations with disease-causing potential. Eight missense variants were functionally characterized using in silico molecular dynamic simulation and in vitro transient transfection in HEK293T cells, followed by ASM enzyme assay, immunoblot, and immunofluorescence studies. All the variants showed reduced ASM activity in transfected cells confirming their disease-causing potential. The study provides data for efficient prenatal diagnosis and genetic counseling of families with NPD type A and B.

Ectopic Expression of Rv0023 Mediates Isoniazid/Ethionamide Tolerance via Altering NADH/NAD+ Levels in Mycobacterium smegmatis.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) accounts for nearly 1.2 million deaths per annum worldwide. Due to the emergence of multidrug-resistant (MDR) Mtb strains, TB, a curable and avertable disease, remains one of the leading causes of morbidity and mortality. Isoniazid (INH) is a first-line anti-TB drug while ethionamide (ETH) is used as a second-line anti-TB drug. INH and ETH resistance develop through a network of genes involved in various biosynthetic pathways. In this study, we identified Rv0023, an Mtb protein belonging to the xenobiotic response element (XRE) family of transcription regulators, which has a role in generating higher tolerance toward INH and ETH in Mycobacterium smegmatis (Msmeg). Overexpression of Rv0023 in Msmeg leads to the development of INH- and ETH-tolerant strains. The strains expressing Rv0023 have a higher ratio of NADH/NAD+, and this physiological event is known to play a crucial role in the development of INH/ETH co-resistance in Msmeg. Gene expression analysis of some target genes revealed reduction in the expression of the ndh gene, but no direct interaction was observed between Rv0023 and the ndh promoter region. Rv0023 is divergently expressed to Rv0022c (whiB5) and we observed a direct interaction between the recombinant Rv0023 protein with the upstream region of Rv0022c, confirmed using reporter constructs of Msmeg. However, we found no indication that this interaction might play a role in the development of INH/ETH drug tolerance.

Mce2R/Rv0586 of Mycobacterium tuberculosis is the functional homologue of FadRE. coli.

Lipid metabolism is critical to Mycobacterium tuberculosis survival and infection. Unlike Escherichia coli, which has a single FadR, the M. tuberculosis genome encodes five proteins of the FadR sub-family. While the role of E. coli FadR as a regulator of fatty acid metabolism is well known, the definitive functions of M. tuberculosis FadR proteins are still under investigation. An interesting question about the M. tuberculosis FadRs remains open: which one of these proteins is the functional homologue of E. coli FadR? To address this, we have applied two different approaches. The first one was the bioinformatics approach and the second one was the classical molecular genetic approach involving complementation studies. Surprisingly, the results of these two approaches did not agree. Among the five M. tuberculosis FadRs, Rv0494 shared the highest sequence similarity with FadRE. coli and Rv0586 was the second best match. However, only Rv0586, but not Rv0494, could complement E. coli ∆fadR, indicating that Rv0586 is the M. tuberculosis functional homologue of FadRE. coli. Further studies showed that both regulators, Rv0494 and Rv0586, show similar responsiveness to LCFA, and have conserved critical residues for DNA binding. However, analysis of the operator site indicated that the inter-palindromic distance required for DNA binding differs for the two regulators. The differences in the binding site selection helped in the success of Rv0586 binding to fadB upstream over Rv0494 and may have played a critical role in complementing E. coli ∆fadR. Further, for the first time, we report the lipid-responsive nature of Rv0586.

NKX6.1 induced pluripotent stem cell reporter lines for isolation and analysis of functionally relevant neuronal and pancreas populations.

Recent studies have reported significant advances in the differentiation of human pluripotent stem cells to clinically relevant cell types such as the insulin producing beta-like cells and motor neurons. However, many of the current differentiation protocols lead to heterogeneous cell cultures containing cell types other than the targeted cell fate. Genetically modified human pluripotent stem cells reporting the expression of specific genes are of great value for differentiation protocol optimization and for the purification of relevant cell populations from heterogeneous cell cultures. Here we present the generation of human induced pluripotent stem cell (iPSC) lines with a GFP reporter inserted in the endogenous NKX6.1 locus. Characterization of the reporter lines demonstrated faithful GFP labelling of NKX6.1 expression during pancreas and motor neuron differentiation. Cell sorting and gene expression profiling by RNA sequencing revealed that NKX6.1-positive cells from pancreatic differentiations closely resemble human beta cells. Furthermore, functional characterization of the isolated cells demonstrated that glucose-stimulated insulin secretion is mainly confined to the NKX6.1-positive cells. We expect that the NKX6.1-GFP iPSC lines and the results presented here will contribute to the further refinement of differentiation protocols and characterization of hPSC-derived beta cells and motor neurons for disease modelling and cell replacement therapies.

An IclR like protein from mycobacteria regulates leuCD operon and induces dormancy-like growth arrest in Mycobacterium smegmatis.

leuCD operon encodes isopropylmalate isomerase (IPMI), an essential enzyme in leucine biosynthesis. Leucine biosynthesis is one of the essential metabolic pathways for Mycobacterium tuberculosis survival inside the macrophage. In this study, we identified an IclR like transcription regulator, Rv2989 involved in regulation of leuCD expression. Further, we have shown that the Rv2989 binding site overlaps with the promoter region of leuCD, indicating its direct involvement in the regulation of this operon. Ectopic expression of Rv2989 in M. smegmatis induced growth arrest with significantly decreased levels of leuCD transcript. However, supplementation with leucine could not reverse the growth arrest, suggesting the involvement of Rv2989 in the regulation of other essential pathways. Growth-arrested cells were elongated, had lost acid fastness and accumulated lipid droplets similar to a dormancy-like state. In conclusion, the Rv2989 expression has pleiotropic effects on M. smegmatis. It negatively regulates leuCD operon and induces dormancy-like growth arrest.

Sex-related differences in hemato-biochemical indices of adult Vanaraja chickens during summer and winter seasons.

AIM: The objective of this study was to evaluate the changes in hemato-biochemical indices in male and female Vanaraja chickens under tropical environment during summer and winter season. MATERIALS AND METHODS: A total of 120 day-old sexed Vanaraja chicks were selected as experimental chickens and distributed equally in two groups having 60 female and 60 male chickens in each group, respectively. The experiment was continued for 8 weeks (56 days) and both male and female chickens were slaughtered by cervical dislocation method. All parameters were estimated at the end of the experiment in both seasons. RESULTS: Male had higher blood glucose, Ca and P level. Blood glucose level significantly (p<0.05) reduced in summer. Female had higher total protein, albumin, globulin, and albumin/globulin ratio. Alanine aminotransferase and aspartate aminotransferase enzyme concentration were significantly (p<0.05) higher in summer. Total erythrocyte count, total leukocyte count, hemoglobin (Hb), Hb/lymphocyte ratio, and packed cell volume were significantly (p<0.05) higher in males. Mean corpuscular volume and mean corpuscular Hb were significantly (p<0.05) higher in females. CONCLUSION: Sex of chickens had a significant (p<0.05) effect on different parameters whereas season had nonsignificant (p>0.05) effect in most of the observed parameters. Hence, Vanaraja chickens are adaptable to local tropical climate and can be reared efficiently as backyard poultry.

Studies of the macroscopic and microscopic morphology (hippocampus) of brain in Vencobb broiler.

AIM: The aim of this study was to study the anatomy of different parts of brain and histology of hippocampus of Vencobb broiler chicken. MATERIALS AND METHODS: A 12 adult experimental birds were sacrificed by cervical dislocation. After separation of the brain, gross anatomy features were studied. Brain tissue was fixed in 10% buffered neutral formalin for 2-3 days, and then routine dehydration process in ascending grades of ethyl alcohol was done. After xylene cleaning, paraffin impregnation was prepared. Paraffin blocks were cut, and slides were stained by Harris hematoxylin and eosin. Photography was carried out both under lower (×10) and higher (×40) magnifications. RESULTS: The brain structure (dorsal view) of Vencobb bird resembled the outline of a playing card symbol of a "spade." The brain subdivisions are cerebrum, cerebellum, and medulla oblongata. Cerebrum was devoid of usual convolutions (elevations), gyri, depressions (grooves), and sulci. The cerebral hemispheres were tightly apposed along a median sulcus called interhemispheric fissure and cerebrum and cerebellum were separated by a small transverse fissure. The olfactory bulb was small structures, and the pineal body was clearly visible. The optic lobes were partially hidden under cerebral hemispheres, but laterally, it was large, prominent rounded or spherical bodies of the midbrain. The hippocampal area appeared as dorso-medial protrusion. Different types of neurons were distinguished in the hippocampus were pyramidal neurons, pyramidal-like neurons, and multipolar neurons, etc. There was rich vascularization in the form of blood capillaries throughout the hippocampus. CONCLUSION: Cerebrum was pear shaped and largest part of the brain. Cerebrum hemisphere was smooth devoid of convolutions, gyri, and depressions, but in the surface of cerebellum, there was the presence of a number of transverse depression (grooves) and sulci subdividing into many folds. Olfactory bulb was poorly developed, whereas optic lobes were rounded and large. The exact boundary line of the hippocampus was not discernable. In hippocampus histology, two categories of neuron local circuit neurons and projection neurons, high vascularization and epididymal lining of lateral ventricle were observed. Hippocampal neurons were comparatively larger without any distinct layers. The afferent neurons projected to the medium septum.

Coordination between extrinsic extracellular matrix cues and intrinsic responses to orient the centrosome in polarizing cerebellar granule neurons.

Successful axon targeting during development is critically dependent on directionality of axon extension and requires coordination between the extrinsic cues that provide spatial information to the axon and the intrinsic responses that regulate structural specification of the axon during neuronal polarization. How these responses are coordinated is unclear but are known to involve aligning the centrosome with the base of the emerging axon. We have used a novel in vitro micropatterning assay that spatially segregates the extrinsic cues used by polarizing cerebellar granule cells to orient axon extension and used it to investigate the signaling mechanisms responsible for coordinating centrosome positioning with intrinsic responses. The results show that, when laminin and/or vitronectin are used as spatially restricted cues in association with substrate-associated sonic hedgehog, they are sufficient to induce cell cycle arrest, that laminin and vitronectin then induce integrin-mediated signaling that upregulates phosphoinositide-3 kinase and PKC function to produce phosphatidylinositol 3,4,5-trisphosphate (PIP3) that is associated with the centrosome, that this PIP3 can interact with PKC-phosphorylated growth-associated protein GAP-43, and that PKC-phosphorylated GAP-43 in turn is required for positioning Par6, Cdc42, and IQGAP1, all intrinsic response components, in proximity to the centrosome, such that, in the absence of GAP-43, they are mislocalized and microtubules are not oriented appropriately. We conclude from these results that GAP-43 plays an important role in coordinating extrinsic signaling and intrinsic responses in polarizing cerebellar granule neurons.

The AMPA receptor positive allosteric modulator, S18986, is neuroprotective against neonatal excitotoxic and inflammatory brain damage through BDNF synthesis.

Brain lesions induced in newborn mice by the glutamatergic agonists ibotenate (acting on NMDA and metabotropic receptors) or S-willardiine (acting on AMPA-kainate receptors) mimic some aspects of periventricular white matter lesions and neocortical grey matter damage observed in human neonates at risk for developing cerebral palsy. The neonatal mouse brain can be sensitized to excitotoxic damage by IL-1beta exposure similar to that observed in the human situation. Positive modulators of AMPA receptors have received increasing attention as potential neuroprotective agents in a number of neurodegenerative disorders of the adult. However whether they can also act as a neuroprotectant in neonatal brain damage has yet to be defined. Therefore the present study uses a well-defined rodent model of neonatal excitotoxic brain lesions to assess the neuroprotective effects of S18986, a positive allosteric modulator of AMPA receptors, as well as its mechanisms of action. In this model, S18986 provided a dose-dependent and long-lasting protection of developing white matter and cortical grey matter against an excitotoxic insult and also when this was combined with a sensitizing inflammatory insult. Neuroprotective effects of S18986 in cortical grey matter involved decreased necrotic and apoptotic cell death. S18986-induced neuroprotection against NMDA receptor-mediated brain lesions was blocked by inhibitors of ERK and PI3 kinase-Akt pathways. S18986 effects were abolished by a neutralizing anti-BDNF antibody and real time PCR confirmed the stimulation by S18986 of BDNF production in the neonatal brain. The present study provides strong experimental support for the role of S18986 as a candidate molecule for therapy in cases of excitotoxic perinatal brain lesions and identifies BDNF as a key mediator of this S18986-mediated neuroprotection.

NRSF downregulation induces neuronal differentiation in mouse embryonic stem cells.

Differentiation of embryonic stem (ES) cells into neurons is accompanied by global changes in transcriptional programs. One transcription factor that has been shown to be involved in neuronal differentiation is neuron restrictive silencing factor/RE1-silencing transcription factor (NRSF/REST). NRSF is a transcriptional repressor that silences the transcription of a large number of neuronal genes by binding to a 21-bp consensus DNA sequence, the RE1 binding site/neuron-restrictive silencer elements (RE1/NRSE), present in the regulatory regions of neuronal genes. The goal of the current study was to examine the role of NRSF during differentiation of ES cells into neurons. To do this, ShRNA construct was used to downregulate NRSF in undifferentiated ES cells. Our results show that although control ES cells required induction by retinoic acid (RA) to differentiate efficiently into neurons, downregulation of NRSF was sufficient to drive the ES cells down the neuronal lineage even in the absence of RA. This downregulation also led to increased expression of mature neuronal markers, and concomitantly decreased glial fibrillary acidic protein (GFAP) expression. The results suggest that NRSF downregulation increases the population of mature neurons at the expense of GFAP-positive cells.

GAP-43 is key to mitotic spindle control and centrosome-based polarization in neurons.

In neurons, the position of the centrosome during final mitosis marks the point of emergence of the future axon. However, the molecular underpinnings linking centrosome position to axon emergence are unknown. GAP-43 is a calmodulin-binding IQ motif protein that regulates neuronal cytoskeletal architecture by interacting with F-actin in a phosphorylation dependent manner. Here we show that GAP-43 is associated with the centrosome and plays a critical role in mitosis and acquisition of neuronal polarity in cerebellar granule neurons. In the absence of GAP-43, the centrosome position is delinked from process outgrowth and is only capable of mediating morphological polarization, however molecular specification of the axonal compartment does not take place. These results show that GAP-43 is required to link centrosome position to process outgrowth in order to generate neuronal polarity in cerebellar granule cells.

Neurospheres derived from human embryoid bodies treated with retinoic Acid show an increase in nestin and ngn2 expression that correlates with the proportion of tyrosine hydroxylase-positive cells.

In the central nervous system (CNS), generation of phenotypic diversity within the neuronal lineage is precisely regulated in a spatial and temporal fashion. Neural basic helix-loop-helix (bHLH) transcription factors are cell intrinsic factors that control commitment to neuronal lineage and play an important role in neuronal cell type specification. The ability to differentiate human embryonic stem (hES) cells into neurons provides a good model system to address human neuronal specification. Previous studies have shown neurogenin-2 (Ngn2) to be involved in the development of mesencephalic dopaminergic neurons. Toward the goal of correlating neuronal phenotype with early gene expression pattern, we have characterized the expression of Ngn2 during hES cell differentiation. Our results show that treatment of embryoid bodies (EBs) with retinoic acid (RA) leads to the greatest proportion of tyrosine hydroxylase (TH)-positive cells followed by vasoactive intestinal peptide (VIP)-treated EBs as compared to untreated EBs. This increase in the proportion of TH-positive neurons was correlated with the unique morphology of RA-treated aggregates and the spatial delocalization of the expression of Ngn2 within the EB. Neurospheres derived from RA-treated EBs contained many nestin-positive cells within regions that expressed Ngn2. We show that the extent of nestin-positive cells that arise from the region of Ngn2 expression is correlated with the appearance of TH-positive neurons. Our results show for the first time the expression of Ngn2 during the differentiation of hES cells.