Neuropathogenesis of SARS-CoV-2 in human neuronal, microglial and glial cells.

Neurological complications, both acute and chronic, are reported commonly in COVID-19 affected individuals. In this context, the understanding of pathogenesis of SARS-CoV-2 in specific cells of central nervous system (CNS) origin is relevant. The present study explores infection biology of a clinical isolate of SARS-CoV-2 in human cell lines of neural origin such as the glioblastoma (U87-MG), neuroblastoma (SHSY5Y) and microglia (C20). Despite showing clear evidence of infection by immunofluorescence with an anti-spike protein antibody, all the three neural cell lines were observed to be highly restrictive to the replication of the infecting virus. While the U87-MG glioblastoma cells demonstrated no cytopathic effects and a low viral titre with no signs of replication, the SHSY5Y neuroblastoma cells exhibited cytopathic effects with bleb formation but no evidence of viable virus. The C20 microglial cells showed neither signs of cytopathic effects nor viable virus. Ultrastructural studies demonstrated intracellular virions in infected neural cells. The presence of lipid droplets in infected SHSY5Y cells suggested an impact on host cell metabolism. The decrease in viral RNA levels over time in all the neural cell lines suggested restricted viral replication. In conclusion, this study highlights the limited susceptibility of neural cells to SARS-CoV-2 infection. This reduced permissibility of neural cell lines to SARS-CoV-2 may point to their inherent lower expression of receptors that support viral entry in addition to the intracellular factors that potently inhibit viral replication. The study findings prompt further investigation into the mechanisms of SARS-CoV-2 infection of neural cells.

A single subset of dendritic cells controls the cytokine bias of natural killer T cell responses to diverse glycolipid antigens.

Many hematopoietic cell types express CD1d and are capable of presenting glycolipid antigens to invariant natural killer T cells (iNKT cells). However, the question of which cells are the principal presenters of glycolipid antigens in vivo remains controversial, and it has been suggested that this might vary depending on the structure of a particular glycolipid antigen. Here we have shown that a single type of cell, the CD8α(+) DEC-205(+) dendritic cell, was mainly responsible for capturing and presenting a variety of different glycolipid antigens, including multiple forms of α-galactosylceramide that stimulate widely divergent cytokine responses. After glycolipid presentation, these dendritic cells rapidly altered their expression of various costimulatory and coinhibitory molecules in a manner that was dependent on the structure of the antigen. These findings show flexibility in the outcome of two-way communication between CD8α(+) dendritic cells and iNKT cells, providing a mechanism for biasing toward either proinflammatory or anti-inflammatory responses.

Infection of human microglial cell line CHME-3 to study neuropathogenesis of chikungunya virus.

Chikungunya virus (CHIKV) infection, generally characterised by fever, rash and debilitating polyarthralgia, and/or arthritis, also causes complications of the central nervous system, including encephalitis. However, the role of microglial cells in the neuropathogenesis of CHIKV is poorly understood. The current study characterised the progression of CHIKV infection in the human microglial cell line CHME-3. The susceptibility of these cells to CHIKV and the viral replication kinetics were assessed during the early and late phases of infection. The cell viability was determined using the cell viability assay. Ultrastructural changes in CHIKV infected CHME-3 cells were assessed using transmission electron microscopy. The results showed that CHME-3 cells are susceptible to CHIKV infection and support viral replication with no significant loss in cell viability until 72 h post infection. Ultrastructural studies revealed the formation of cytopathic vacuoles-I (CPV-I) in the early stages and CPV-II in later stages with several virions organized along the membrane of CPV-II. Profuse vacuolation was observed in the later stages of infection. Abnormal giant mitochondria with altered cristae were observed in infected cells with an electron-dense matrix. The study establishes CHME-3 cells as a potential model for investigating the role of human microglial cells in neuropathogenicity of CHIKV.

Optimization of recombination of milk at different fat levels in a small volume universal disperser unit.

Recombined milk (RM) can be prepared by blending of butteroil, skim milk powder and water and used for variety of purposes in dairy industry. The present work was undertaken to investigate the feasibility of a custom designed and fabricated universal disperser unit for the preparation of recombined milk. Water, SMP and butteroil were used to manufacture the recombined milk. Three levels of fat viz 1.5%, 3.0% and 4.5% were evaluated to study the effect of fat content on the recombination efficiency of the universal disperser using a high shear saw tooth impeller to impart the required shear and disperse the fat in water-SMP matrix to form a stable emulsion. Two independent parameters i.e. temperature at three different levels (20, 35 and 50 °C) and impeller speed (10,000, 15,000 and 20,000 rpm) were selected for the study. All operations were carried out in heating cum process vessel having working capacity of 3 L. The efficiency of the operation was judged based on the dependent parameters namely, mixing time, mixing index, creaming index, power consumption and overall acceptability. Experiments were designed as per RSM in Design Expert V.10.0 software and results obtained were optimized and predicted solutions were compared with observed data. From the study, the optimal combinations for preparation of recombined milk were obtained as 1.5% Fat RM (17,820 rpm, 48 °C), 3.0% Fat RM (15,701 rpm, 48 °C) and 4.5% Fat RM (15,459 rpm, 48 °C).

Evaluation of vacuum impregnation as a novel approach for soaking of fried Gulabjamun balls.

Vacuum impregnation of sugar syrup into sub-baric fried Gulabjamun was evaluated as a technological approach to prepare a product of most acceptable quality. Sugar syrup concentrations (40, 50 and 60 °Brix) in combination with process time (2, 4 and 6 min) were analyzed for their effect on product quality in terms of its overall acceptability, expansion ratio, hardness, juiciness and sugar content. The effect of the process conditions on the five listed responses during the vacuum impregnation process was evaluated using response surface methodology and modelled using a second order polynomial equation. The optimum combination was obtained as soaking in a syrup of 55 °Brix for 5 min and was experimentally validated for its real time adequacy. The experimental values of the quality parameters thus obtained were found to be in close agreement with the predicted values.

Control of matting temperature during pressing of Paneer and its effect on Paneer quality.

An attempt was made to design, fabricate and evaluate a heat exchanger for controlling the matting temperature of Paneer during pressing. Based on preliminary investigations, the range of process parameters to be evaluated during the pressing of Paneer was selected as: pressure (2, 3, and 4 kg/cm2), matting temperature (63, 66 and 69 °C) for pressing time of 8, 10 and 12 min. Experiments were designed in a central composite design for 20 runs and the Paneer was evaluated for its moisture content, hardness, springiness, chewiness, cohesiveness, bulk density, porosity and sensory attributes. It was observed that with increasing pressure and matting temperature, the hardness of the product increased; this corresponded with reduced moisture content and porosity of the product. The springiness of the samples correlated linearly with increasing matting temperature and pressing time. Increasing the pressure during pressing resulted in poorer sensory scores. The process conditions for pressing of Paneer under controlled matting conditions were optimised using Response Surface Methodology for moisture content, overall acceptability, hardness and springiness of the pressed product. It was observed that the moisture content of Paneer pressed under controlled matting temperature was marginally lower than the control Paneer, the overall acceptability for the experimental samples was higher with superior body and texture scores.

Development of mathematical model for prediction of adulteration levels of cow ghee with vegetable fat using image analysis.

The present study was undertaken to develop a protocol for acquisition and analysis of images of ghee samples to derive mathematical parameters related to adulteration of cow ghee with vegetable fat and to develop a model to predict the adulteration levels. The images acquired using a flatbed scanner were quantified in terms of their pixel intensity, colour, morphological, textural and skeleton parameters using ImageJ software. The selected parameters were measured for images of pure cow ghee and compared with that obtained for ghee adulterated with 5%, 10%, 15% and 20% vegetable fat. The parameters were assessed for their ability to detect the fixed adulteration levels on a discrete scale was assessed using discriminant analysis and the adulteration levels of the samples were correctly classified to the extent of 92.2%. An equation for predicting adulteration levels on a continuous scale using regression analysis (adjusted R 2 value 0.94) was developed, tested and further validated using a fresh data set including a commercially popular market sample of ghee giving a good fit (R 2 value of 0.85).

Alterations in natural killer and dendritic cell subsets in individuals with HIV-associated neurotuberculosis.

One of the commonest HIV-associated opportunistic infections of the central nervous system is neurotuberculosis. Interaction between HIV, Mycobacterium tuberculosis and host immune system in co-infected individuals may result in altered frequencies of immune cells, thereby modulating dissemination and disease progression. We examined the frequencies of natural killer (NK) cell and dendritic cell (DC) subsets in HIV infected individuals with neurotuberculosis (HIVNTB) as compared to individuals with HIV associated systemic TB (HIVSTB), asymptomatic HIV, non-HIV NTB, non-HIV STB, and healthy controls. Peripheral blood mononuclear cells (PBMC) were stained with fluorochrome-conjugated monoclonal antibodies- Lineage cocktail (containing CD3, CD14, CD19, and CD20), HLA-DR, CD16, CD56, CD11c, and CD123, fixed with 2% paraformaldehyde and analyzed on the flow cytometer. The pDCs were significantly reduced in all HIV infected groups, with a marked reduction in HIVNTB cases as compared to healthy controls. While the CD56- CD16bt NK cell subset displayed a significant increase in frequency in all three HIV infected groups compared the three HIV negative groups, the CD56dim CD16bt subset was significantly lower in frequency in the HIVNTB compared to healthy controls. The decreased frequencies of plasmacytoid DCs and cytotoxic NK cells, which are crucial for innate immune defence against HIV, may result in ineffective virus control and lead to an exacerbated course of disease in HIVNTB individuals.

Kinetics and cellular site of glycolipid loading control the outcome of natural killer T cell activation.

CD1d-restricted natural killer T cells (NKT cells) possess a wide range of effector and regulatory activities that are related to their ability to secrete both T helper 1 (Th1) cell- and Th2 cell-type cytokines. We analyzed presentation of NKT cell activating alpha galactosylceramide (alphaGalCer) analogs that give predominantly Th2 cell-type cytokine responses to determine how ligand structure controls the outcome of NKT cell activation. Using a monoclonal antibody specific for alphaGalCer-CD1d complexes to visualize and quantitate glycolipid presentation, we found that Th2 cell-type cytokine-biasing ligands were characterized by rapid and direct loading of cell-surface CD1d proteins. Complexes formed by association of these Th2 cell-type cytokine-biasing alphaGalCer analogs with CD1d showed a distinctive exclusion from ganglioside-enriched, detergent-resistant plasma membrane microdomains of antigen-presenting cells. These findings help to explain how subtle alterations in glycolipid ligand structure can control the balance of proinflammatory and anti-inflammatory activities of NKT cells.

Solvent dependent morphology and 59Co internal field NMR study of Co-aggregates synthesized by a wet chemical method.

Different shapes of Co-aggregates were synthesized via reduction of a Co salt (CoCl2·6H2O) by chemical precipitation using glycerol, ethylene glycol and ethanol as solvents. The effect of solvent on the morphology, fcc or hcp phase-content and the magnetic properties of the synthesized samples were investigated. The Co-aggregates synthesized using glycerol have a dense spherical shape and high saturation magnetization (MS), whereas ethylene glycol leads to formation of flower-shaped spherical aggregates through loose packing of smaller plate-like particles which have a moderate MS value. When ethanol was used as a solvent, a dendritic (leaf like)-shape of the aggregates with the lowest MS value was obtained. The formation of the obtained morphology of the aggregates was explained based on the size of the solvent molecule, the viscosity of the solvent and the number of polar groups (-OH) present in the solvent molecules. The magnetic domain state and domain wall dynamics of all the Co-samples were investigated using 59Co Internal Field Nuclear Magnetic Resonance (IFNMR) spectroscopy at RT and at 77 K. Through the IFNMR spectroscopy, the presence of gain boundaries, single domain particles and multi-domain particles/aggregates with domain walls associated with fcc and hcp phases were identified and quantified. We observed that the use of ethanol facilitates formation of a higher amount of hcp phase in the sample than the use of glycerol or ethylene glycol.

Storage quality of shelled green peas under modified atmosphere packaging at different storage conditions.

Storage quality of shelled green peas (Pisum sativum var. sativum L) was investigated under modified atmosphere packaging (MAP: perforated and non perforated) compared to unsealed samples, respectively, at T1 (4 ± 1 °C and 94 ± 2 % RH) and T2 (10 ± 1 °C and 90 ± 2 % RH) for each sample and during period of storage (8, 16 and 24 days). Modified atmosphere (MA) was created using low density polyethylene (LDPE) film packages having 107 µm of film thickness and package size of 0.022 m(2). Quality parameters viz., weight loss (WL), total phenolic content (TPC), instrumental colour, ascorbic acid (AA) and sensory characteristics were evaluated during storage period. Weight loss was in the range of 0.18 to 3.54 (zero perforation at T1), 0.21 to 6.48(unsealed samples at T2) and 0.31 to 9.64 % (zero perforation at T1) after 8, 16 and 24 days of storage, respectively. Total phenolic content significantly increased to 102.47-161.54 mg/100 g from an initial value of 91.53 mg/100 g for all the samples and treatments studied. The MAP non perforated sample stored at T2 recorded maximum Hunter 'L' and '-a' colour values than all other samples. A significant decrease in AA content was observed in all the samples with maximum loss (53.77 %) in unsealed sample stored at T2, whereas MAP (3 perforations) sample stored at T1 retained maximum AA (90.50 %). Sensory quality analysis revealed that MAP (3 perforations) sample stored at T1 was in acceptable quality, with good appearance and overall acceptance. The study shows that shelled green peas can be stored in MAP with 3 perforations (0.4 mm dia) in the temperature range of 4 to 10 °C and 90-94 % RH to extend shelf life with marketable quality for 24 days.

Antioxidant activities of orange peel extract in ghee (butter oil) stored at different storage temperatures.

Antioxidant activities of butylatedhydroxyanisole (BHA) and orange peel powder extract in ghee stored at different storage temperatures (T1:6 ± 2 °C; T2: 32 ± 2 °C; T3:60 ± 2 °C) were evaluated during storage period of 21 days. Peroxide value (PV), thiobarbituric acid (TBA), radical scavenging activity (RSA) and free fatty acids (FFA) of ghee samples were analyzed during the study. PV, TBA and FFA of ghee samples increased significantly while radical scavenging activity (RSA) of ghee samples decreased significantly at accelerated temperature (T3) as compared to the temperatures at T1 and T2. Effect of storage temperature on development of peroxides and TBA of ghee samples was significantly higher than the effect of treatment and storage period while treatment had more significant effect on the change in FFA and RSA as compared to storage temperature and storage period. Ghee incorporated with orange peel extract (OPE) showed stronger activity in quenching DPPH radicals and least development of PV, TBA and FFA than ghee incorporated with BHA and control. The study revealed that orange peel could be a good natural source of antioxidants which can be used in fat rich food products like ghee to retard oxidative deterioration.

1,4-azaindole, a potential drug candidate for treatment of tuberculosis.

New therapeutic strategies against multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis are urgently required to combat the global tuberculosis (TB) threat. Toward this end, we previously reported the identification of 1,4-azaindoles, a promising class of compounds with potent antitubercular activity through noncovalent inhibition of decaprenylphosphoryl-ß-D-ribose 2'-epimerase (DprE1). Further, this series was optimized to improve its physicochemical properties and pharmacokinetics in mice. Here, we describe the short-listing of a potential clinical candidate, compound 2, that has potent cellular activity, drug-like properties, efficacy in mouse and rat chronic TB infection models, and minimal in vitro safety risks. We also demonstrate that the compounds, including compound 2, have no antagonistic activity with other anti-TB drugs. Moreover, compound 2 shows synergy with PA824 and TMC207 in vitro, and the synergy effect is translated in vivo with TMC207. The series is predicted to have a low clearance in humans, and the predicted human dose for compound 2 is ≤1 g/day. Altogether, our data suggest that a 1,4-azaindole (compound 2) is a promising candidate for the development of a novel anti-TB drug.

Thiazolopyridone ureas as DNA gyrase B inhibitors: optimization of antitubercular activity and efficacy.

Scaffold hopping from the thiazolopyridine ureas led to thiazolopyridone ureas with potent antitubercular activity acting through inhibition of DNA GyrB ATPase activity. Structural diversity was introduced, by extension of substituents from the thiazolopyridone N-4 position, to access hydrophobic interactions in the ribose pocket of the ATP binding region of GyrB. Further optimization of hydrogen bond interactions with arginines in site-2 of GyrB active site pocket led to potent inhibition of the enzyme (IC50 2 nM) along with potent cellular activity (MIC=0.1 µM) against Mycobacterium tuberculosis (Mtb). Efficacy was demonstrated in an acute mouse model of tuberculosis on oral administration.

Lipid and glycolipid antigens of CD1d-restricted natural killer T cells.

In spite of their relatively limited antigen receptor repertoire, CD1d-restricted NKT cells recognize a surprisingly diverse range of lipid and glycolipid antigens. Recent studies of natural and synthetic CD1d-presented antigens provide an increasingly detailed picture of how the specific structural features of these lipids and glycolipids influence their ability to be presented to NKT cells and stimulate their diverse immunologic functions. Particularly for synthetic analogues of alpha-galactosylceramides which have been the focus of intense recent investigation, it is becoming clear that the design of glycolipid antigens with the ability to precisely control the specific immunologic activities of NKT cells is likely to be feasible. The emerging details of the mechanisms underlying the structure-activity relationship of NKT cell antigens will assist greatly in the design and production of immunomodulatory agents for the precise manipulation of NKT cells and the many other components of the immune system that they influence.

Effect of modified atmosphere packaging and storage conditions on quality characteristics of cucumber.

Cucumbers (Cucumis sativus L.) stored in perforated modified atmosphere packaging (MAP) under cold room (4 ± 1 °C and 90 ± 2 % RH) and ambient condition (23-26 °C and 63-66 % RH) were evaluated for firmness, weight loss (WL), colour, chilling injury and sensory characteristics. The firmness of cucumbers was decreased to 0.333 and 0.326 N on 6th and 12th day of storage, respectively from initial value of 0.38 N. After 12 days of storage, the WL was in the range of 1.62-12.89 % whereas the cucumber stored under MAP having 2 perforations at 4 ± 1 °C and 90 ± 2 % RH recorded least WL of 1.62 %. The minimum change in colour (Hunter L, a and b values) was observed in the cucumber samples stored at cold room condition. The increase in 'b' values (yellowness) was more in the sample stored at ambient condition with unsealed sample registered highest 'b' values (35.82). On 12th day of storage, sensory quality evaluation revealed that samples stored under perforated MAP at 4 ± 1 °C and 90 ± 2 % RH were acceptable in condition with sensory score of 7.1 and 7.5. Chilling injury was severe in sample unsealed (4.4 chilling injury score) and slight to moderate chilling injury was observed in 2 and 4 perforated package samples stored under cold room condition. The study revealed that cucumber can be stored under MAP with 2 perforations at 4 ± 1 °C and 90 ± 2 % RH and ambient condition (23-26 °C and 63-66 % RH) for 12 and 6 days, respectively.

Effect of vacuum packaging and pomegranate peel extract on quality aspects of ground goat meat and nuggets.

The effect of vacuum packaging and pomegranate peel extract on ground goat meat and cooked nuggets during refrigerated storage (4 ± 1 °C) was evaluated. Three different treatments evaluated were: I). Aerobic packaging (AP); II) Vacuum packaging (VP) and III). Vacuum packaging along with 1 % pomegranate peel extract (VP + PPE). Results of quality evaluation showed that VP and VP + PPE maintained a more stable colour than AP. In all treatments, a significant (P < 0.05) increase in hardness and gumminess of nuggets was observed during the storage. But, VP nuggets showed minimum changes in texture parameters. TBARS values were significantly (P < 0.05) lower in VP and VP + PPE than AP. In ground meat, VP reduced the TBARS by 27 % and PPE reduced the TBARS by 41 %. In nuggets, TBARS was decreased by 17 % and 40 % in VP and VP + PPE respectively. Total plate counts were significantly higher (>log 7) in AP than VP meat and nuggets. Thus VP and PPE have a synergistic antioxidant effect and VP extended the refrigerated shelf life of goat meat and nuggets.

Development and performance evaluation of a garlic peeler.

Garlic peeling is a tedious, key, costly and time consuming unit operation in garlic processing. A power operated garlic peeler having a cylinder-concave mechanism was developed with an intention to reduce cost and time. Physical properties of garlic relevant for peeler development were identified and measured. The average length, width, thickness, geometric mean diameter, sphericity, weight of garlic segment and weight of 1,000 garlic segment were measured as 26.3 mm, 10.4 mm, 8.7 mm, 13.3 mm, 0.5, 1.8 g and 1,813 g, respectively. An experimental garlic peeler having cylinder covered with 10 mm thick rubber was fabricated and evaluated for its performance with crop-machine parameters viz., cylinder speed (29, 36 and 42 rpm), cylinder-concave clearance (8, 10 and 12 mm), moisture content (23.1, 27.7, 33.4 and 40.5 % wet basis) and concave mechanisms. Crop-machine parameters were optimized based peeling efficiency and they found to be cylinder speed of 36 rpm, cylinder-concave clearance of 10 mm, mild steel square (8 × 8 screen). Prototype garlic peeler was evaluated with the optimized crop-machine parameters. The peeling efficiency, yield of peeled garlic and unpeeled garlic, damage and peel separation were 86.6, 86.2, 4.7, 9.15 and 96 %, respectively with a machine throughput capacity of 27 kg/h and the energy requirement of 1.15 kw-h. Operation cost of the peeler was determined on the basis of fixed and variable cost and found to be INR 22.9/h. The developed garlic peeler saved INR 16.11/kg (94.99 %) and 1.63 (97 %) man hours in comparison to the hand peeling of garlic.

Evaluation of quality characteristics of chicken meat emulsion/nuggets prepared by using different equipment.

Chicken meat emulsions prepared using food processor (FP), an indigenous meat cutter (MC) and bowl chopper (BC) were evaluated for physicochemical, texture and electron microscopic studies (SEM). Product yield, emulsion stability, hydration properties and gel strength (N) were significantly (P < 0.05) higher in BC. Total fluid release (TFR), water release (WR) and fat release (FR) was lowest in BC. Significantly (P < 0.05) higher lightness (L) in BC and redness (a) in FP emulsion were observed. Higher firmness, gumminess, chewiness and cohesiveness were observed in BC emulsion. SEM studies revealed a dense and compact protein matrix characteristic of heat induced protein gels. All micrographs showed structures that are compatible with fat globules, muscle fiber, meat protein matrix and heat induced gel/protein matrix. Sensory evaluation showed no significant difference between three treatments for colour, flavour, texture and acceptability scores. Thus, food processor and indigenously developed meat cutter found suitable for producing a stable chicken meat emulsion required for indigenous meat products.

Chikungunya virus infection in human microglial C20 cells induces mitochondria-mediated apoptosis.

Introduction: Chikungunya virus (CHIKV) infection is associated with acute clinical manifestations and chronic joint inflammation. CHIKV has emerged as a significant causative agent of central nervous system (CNS) complications, including encephalitis and related sequelae. Microglial cells, crucial for immune responses and tissue repair in the CNS, play a vital role in the host response to viral infections, with their activation potentially leading to either protection or pathology. In this study, the infection biology of CHIKV in the C20 human microglial cell line was investigated. Methods: The permissiveness of C20 cells to CHIKV infection was assessed, and viral replication kinetics were compared to Vero E6 cells. Cytopathic effects of CHIKV infection on C20 cells were examined, along with ultrastructural changes using transmission electron microscopy. Additionally, apoptosis induction, mitochondrial membrane potential, and alterations in cell surface marker expression were evaluated by flow cytometry. Results: CHIKV infection demonstrated permissiveness in C20 cells, similar to Vero cells, resulting in robust viral replication and cytopathic effects. Ultrastructural analysis revealed viral replication, mature virion formation, and distinctive cytoplasmic and nuclear changes in infected C20 cells. CHIKV infection induced significant apoptosis in C20 cells, accompanied by mitochondrial membrane depolarization and altered expression of cell surface markers such as CD11c, CD14, and HLA-DR. Notably, decreased CD14 expression was observed in CHIKV-infected C20 cells. Discussion: The study findings suggest that CHIKV infection induces apoptosis in C20 microglial cells via the mitochondrial pathway, with significant alterations in cell surface marker expression, particularly CD14 that is linked with apoptosis induction. These observations provide valuable insights into the role of human microglial cells in the host response to CHIKV infection and contribute to the knowledge on the neuropathogenesis of this virus.