Behavioral and Proteomic Studies Reveal Methylglyoxal Activate Pathways Associated with Alzheimer's Disease.

Diabetes is one of the major risk factors for Alzheimer's disease (AD) development. The role of elevated levels of glucose, methylglyoxal (MGO), and advanced glycation end products (AGEs) in the pathogenesis of AD is not well understood. In this pursuit, we studied the role of methylglyoxal in the pathogenesis of AD in rat models. The elevated plus-maze (EPM) behavioral study indicated that MGO induces anxiety. Treatment of telmisartan (RAGE expression inhibitor) and aminoguanidine (MGO quencher) attenuated MGO induced anxiety. Further, hippocampal proteomics demonstrated that MGO treated rats differentially regulate proteins involved in calcium homeostasis, mitochondrial functioning, and apoptosis, which may affect neurotransmission and neuronal plasticity. The hippocampal tau phosphorylation level was increased in MGO treated rats, which was reduced in the presence of aminoguanidine and telmisartan. The plasma fructosamine level was increased upon MGO treatment. Hippocampal histochemistry showed vascular degeneration and neuronal loss upon MGO treatment. This study provides mechanistic insight into the role of MGO in the diabetes-associated development of AD.

SARS-CoV-2 Kappa Variant Shows Pathogenicity in a Syrian Hamster Model.

Objectives: The emergence of SARS-CoV-2 lineage B.1.617 variants in India has been associated with a surge in the number of daily infections. We investigated the pathogenic potential of Kappa (B.1.617.1) variant in Syrian golden hamsters. Methods: Two groups of Syrian golden hamsters (18 each) were inoculated intranasally with SARS-CoV-2 isolates, B.1 (D614G) and Kappa variant, respectively. The animals were monitored daily for the clinical signs and body weight. Throat swab, nasal wash, and organ samples (lungs, nasal turbinate, trachea) were collected and screened using SARS-CoV-2-specific RT-qPCR. Histopathologic evaluation of the lung samples was performed. Results: The hamsters infected with the Kappa variant demonstrated increased body weight loss compared to the B.1 lineage isolate. The highest viral RNA load was observed in the nasal turbinate and lung specimens of animals infected with both variants. A significantly higher sgRNA load was observed in the nasal swabs (7 DPI), trachea (3 DPI), and lungs (3 DPI) of hamsters infected with the Kappa variant. Neutralizing antibody response generated in the B.1 lineage-infected hamster sera were comparable against both B.1 and Kappa variant in contrast to Kappa variant-infected hamsters, which showed lower titers against B.1 lineage isolate. Gross and microscopic evaluation of the lung specimens showed severe lung lesions in hamsters infected with Kappa variant compared to B.1. Conclusions: The study demonstrates pathogenicity of Kappa variant in hamsters evident with reduced body weight, high viral RNA load in lungs, and pronounced lung lesions. Both Kappa variant- and B.1-infected hamsters produced neutralizing antibodies against both variants studied.

Pathogenicity of SARS-CoV-2 Omicron (R346K) variant in Syrian hamsters and its cross-neutralization with different variants of concern.

BACKGROUND: SARS-CoV-2 Omicron variant is rampantly spreading across the globe. We assessed the pathogenicity and immune response generated by BA.1.1 sub-lineage of SARS-CoV-2 [Omicron (R346K) variant] in 5 to 6-week old Syrian hamsters and compared the observations with that of Delta variant infection. METHODS: Virus shedding, organ viral load, lung disease and immune response generated in hamsters were sequentially assessed. FINDINGS: The disease characteristics of the Omicron (R346K) variant were found to be similar to that of the Delta variant infection in hamsters like viral replication in the respiratory tract and interstitial pneumonia. The Omicron (R346K) infected hamsters demonstrated lesser body weight reduction and viral RNA load in the throat swab and nasal wash samples in comparison to the Delta variant infection. The viral load in the lungs and nasal turbinate samples and the lung disease severity of the Omicron (R346K) infected hamsters were found comparable with that of the Delta variant infected hamsters. Neutralizing antibody response against Omicron (R346K) variant was detected from day 5 and the cross-neutralization titre of the sera against other variants showed severe reduction ie., 7 fold reduction against Alpha and no titers against B.1, Beta and Delta. INTERPRETATION: This preliminary data shows that Omicron (R346K) variant infection can produce moderate to severe lung disease similar to that of the Delta variant and the neutralizing antibodies produced in response to Omicron (R346K) variant infection shows poor neutralizing ability against other co-circulating SARS-CoV-2 variants like Delta which necessitates caution as it may lead to increased cases of reinfection. FUNDING: This study was supported by Indian Council of Medical Research as an intramural grant (COVID-19) to ICMR-National Institute of Virology, Pune.

Protective Immunity of the Primary SARS-CoV-2 Infection Reduces Disease Severity Post Re-Infection with Delta Variants in Syrian Hamsters.

The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) Delta variant has evolved to become the dominant SARS-CoV-2 lineage with multiple sub-lineages and there are also reports of re-infections caused by this variant. We studied the disease characteristics induced by the Delta AY.1 variant and compared it with the Delta and B.1 variants in Syrian hamsters. We also assessed the potential of re-infection by these variants in Coronavirus disease 2019 recovered hamsters 3 months after initial infection. The variants produced disease characterized by high viral load in the respiratory tract and interstitial pneumonia. The Delta AY.1 variant produced mild disease in the hamster model and did not show any evidence of neutralization resistance due to the presence of the K417N mutation, as speculated. Re-infection with a high virus dose of the Delta and B.1 variants 3 months after B.1 variant infection resulted in reduced virus shedding, disease severity and increased neutralizing antibody levels in the re-infected hamsters. The reduction in viral load and lung disease after re-infection with the Delta AY.1 variant was not marked. Upper respiratory tract viral RNA loads remained similar after re-infection in all the groups. The present findings show that prior infection could not produce sterilizing immunity but that it can broaden the neutralizing response and reduce disease severity in case of reinfection.

Exposure to Atrazine through gestation and lactation period led to impaired sexual maturation and subfertility in F1 male rats with congenital deformities in F2 progeny.

Several scientific reports suggest perturbed reproductive and developmental defects associated with environmental exposure to Atrazine (ATR). ATR has been associated with altered endocrine and reproductive functioning in-vivo exposed during the critical window of development. Thus, the present study investigates the effect of ATR exposure on F1-F2 male progeny exposed through gestation and lactation. F0 dams administered with ATR at doses 2, 10, 70, and 100 mg/kg b. wt/day from gestation day 6 to postnatal day 21. The F1 male rats were monitored for sexual maturation and subjected to fertility assessment on PND75. Delayed testicular descent was observed in 10, 70, and 100 mg/kg b. wt/day ATR dose with significantly lower serum testosterone, sperm count, and motility with testicular defects in F1 male. Expression of Androgen receptor (AR), Estrogen receptors (ER α and ER ß), StAR, Aromatase, and INSL-3 were upregulated at all doses indicating estrogenic/anti-androgenic activity of ATR. Fertility assessment revealed subfertility in F1 males with high (%) pre- and post-implantation loss at 10, 70, and 100 mg/kg b. wt/day dose as compared to control. Further, F2 fetuses exhibited congenital disabilities viz. decreased weight, crown-rump length, and anogenital distance with several other morphological deformities. To conclude, ATR exerted estrogenic and/or anti-androgenic activity with fetotoxic effects through the male germline.

Antianemic activity of polyherbal formulation, Raktavardhak Kadha, against phenylhydrazine-induced anemia in rats.

BACKGROUND: Over two billion people around the world suffer from anemia. Majority of populations are using dietary supplements and herbal medicines for the management of the anemic conditions. Many polyherbal formulations such as RaktavardhakKadha (RK), are available in the Indian market as a nutritional supplement and herbal-based medicine for anemia. OBJECTIVES: The present study is aimed at investigating antianemic potential of polyherbal formulation, RK, against phenylhydrazine-induced anemia in rats. MATERIALS AND METHODS: RK was subjected to preliminary phytochemical analysis and iron estimation. Anemia was induced by phenylhydrazine administration (40 mg/kg, i.p.) for 2 consecutive days. Antianemic activity of RK was investigated at the dose of 1.8 ml/kg, twice daily for 12 days by estimating blood parameters and pathological changes in liver, heart, spleen and bone marrow. RESULTS: RK was found to contain saponins, steroids, flavonoids, tannins and phenolic compounds. Iron content was found to be 5 mg/100 ml in RK. Anemia induction by phenylhydrazine injections to rats caused significant decrease in red blood cells (RBCs), hemoglobin and hematocrit. These decreased levels of RBCs, hemoglobin and hematocrit in blood was significantly improved by the treatment with RK. Furthermore, RK restored pathological changes in liver, heart, spleen and bone marrow tissues near to normal. CONCLUSION: This study suggests antianemic activity of RK, which can be attributed to its iron content and ability to prevent hemolysis.

Immunogenicity and protective efficacy of inactivated SARS-CoV-2 vaccine candidate, BBV152 in rhesus macaques.

The COVID-19 pandemic is a global health crisis that poses a great challenge to the public health system of affected countries. Safe and effective vaccines are needed to overcome this crisis. Here, we develop and assess the protective efficacy and immunogenicity of an inactivated SARS-CoV-2 vaccine in rhesus macaques. Twenty macaques were divided into four groups of five animals each. One group was administered a placebo, while three groups were immunized with three different vaccine candidates of BBV152 at 0 and 14 days. All the macaques were challenged with SARS-CoV-2 fourteen days after the second dose. The protective response was observed with increasing SARS-CoV-2 specific IgG and neutralizing antibody titers from 3rd-week post-immunization. Viral clearance was observed from bronchoalveolar lavage fluid, nasal swab, throat swab and lung tissues at 7 days post-infection in the vaccinated groups. No evidence of pneumonia was observed by histopathological examination in vaccinated groups, unlike the placebo group which exhibited interstitial pneumonia and localization of viral antigen in the alveolar epithelium and macrophages by immunohistochemistry. This vaccine candidate BBV152 has completed Phase I/II (NCT04471519) clinical trials in India and is presently in phase III, data of this study substantiates the immunogenicity and protective efficacy of the vaccine candidates.

Immunogenicity and protective efficacy of BBV152, whole virion inactivated SARS- CoV-2 vaccine candidates in the Syrian hamster model.

The availability of a safe and effective vaccine would be the eventual measure to deal with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) threat. Here, we have assessed the immunogenicity and protective efficacy of inactivated SARS-CoV-2 vaccine candidates BBV152A, BBV152B, and BBV152C in Syrian hamsters. Three dose vaccination regimes with vaccine candidates induced significant titers of SARS-CoV-2-specific IgG and neutralizing antibodies. BBV152A and BBV152B vaccine candidates remarkably generated a quick and robust immune response. Post-SARS-CoV-2 infection, vaccinated hamsters did not show any histopathological changes in the lungs. The protection of the hamster was evident by the rapid clearance of the virus from lower respiratory tract, reduced virus load in upper respiratory tract, absence of lung pathology, and robust humoral immune response. These findings confirm the immunogenic potential of the vaccine candidates and further protection of hamsters challenged with SARS-CoV-2. Of the three candidates, BBV152A showed the better response.

Study of Kyasanur forest disease viremia, antibody kinetics, and virus infection in target organs of Macaca radiata.

The present manuscript deals with experimental infections of bonnet macaques (Macaca radiata) to study disease progression for better insights into the Kyasanur Forest Disease (KFD) pathogenesis and transmission. Experimentally, 10 monkeys were inoculated with KFD virus (KFDV) (high or low dose) and were regularly monitored and sampled for various body fluids and tissues at preset time points. We found that only 2 out of the 10 animals showed marked clinical signs becoming moribund, both in the low dose group, even though viremia, virus shedding in the secretions and excretions were evident in all inoculated monkeys. Anti-KFDV immunoglobulin (Ig)M antibody response was observed around a week after inoculation and anti-KFDV IgG antibody response after two weeks. Anaemia, leucopenia, thrombocytopenia, monocytosis, increase in average clotting time, and reduction in the serum protein levels were evident. The virus could be re-isolated from the skin during the viremic period. The persistence of viral RNA in the gastrointestinal tract and lymph nodes was seen up to 53 and 81 days respectively. Neuro-invasion was observed only in moribund macaques. Re-challenge with the virus after 21 days of initial inoculation in a monkey did not result in virus shedding or immune response boosting.

Efavirenz-loaded intranasal microemulsion for crossing blood-CNS interfaces in neuronal-AIDS: pharmacokinetic and in vivo safety evaluation.

Purpose: Development of delivery tool for the existing antiretroviral drugs against the neuronal-AIDS in itself is a big challenge because of blood-brain-barrier (BBB). Aim of present research is to formulate efavirenz (EFV) based mucoadhesive microemulsion (EMME) and investigates its efficiency through intranasal delivery.Methods: The EFV microemulsion (EME) was formulated by aqueous titration method. The formulation was screened for globule size, zeta potential and encapsulation efficiency. Bio-distribution of EFV was performed by gamma scintigraphy. Safety of optimized formulation was demonstrated using biochemical, hematological and histopathological data.Results: Experimental data demonstrate that optimized formulation showed significant size (19.04 nm), zeta potential (-32.2 mV) and entrapment efficiency (98.39%). The results of Cmax value suggested that intranasal (i.n.) 99mTc-EMME is able to improve the brain uptake of EFV around 2 folds more than i.n. 99mTC-EME and intravenous (i.v.) 99mTC-EME administrations. The drug targeting index (DTI= 10), drug targeting efficiency (DTE = 1000%) and direct transport percentage (DTP = 89%) were found highly significant for EMME (i.n.) than EME (i.n.). In vivo safety evaluation studies on experimental animals for biochemical, hematological and histopathological parameters remain unchanged.Conclusions: Hence, the intranasal delivery of EMME can be safe and effective tool in the treatment of neuronal-AIDS.

Gestational and lactational exposure to triclosan causes impaired fertility of F1 male offspring and developmental defects in F2 generation.

Triclosan (5-chloro-2-(2, 4-dichlorophenoxy) phenol, TCS), is a broad-spectrum antimicrobial agent and extensively used in household and daily daycare products. Recently, several reports have demonstrated the endocrine disruptive action of TCS to alter the testicular steroidogenesis. However, the gestational and lactational effects of TCS exposure on F1 offspring has not been studied. Present study aimed to investigate the effect of gestational and lactational exposure to TCS on F1 male progeny and its effect on fertility. Pregnant dams (F0) were administered with different doses of TCS (0.1, 4, 40 and 150 mg/kg b. wt./day) and Diethylstilbestrol (1 µg/kg b. wt./day), as a positive control daily by subcutaneous injection during Gestation Day 6 to Postnatal Day 21. Delayed testicular descent was observed at 150 mg/kg b. wt./day dose group. Dose-dependent decrease in testosterone level, sperm count and motility was observed. Significantly decreased expression of steroid hormone receptors (AR, ERα and ERß), StAR and aromatase were observed in F1 male rats; indicating its prolonged effect on spermatogenesis and steroidogenesis in adulthood and poor development in F2 fetuses. Further, gestational and lactational exposure to TCS has negative impact on the fertility of F1 male rats. The F1 male rats were found sub-fertile with increased (%) pre- and post-implantation loss (at 40 and 150 mg/kg b.wt./day dose) with a simultaneous decrease in litter size. The significant decrease in mean fetal weight and crown-rump length (CRL) of F2 fetuses were observed at 0.1, 4, 40 and 150 dose groups indicating impaired development of F2 fetuses caused by TCS exposure. Present study emphasizes for the first time that TCS exposure during the vulnerable developmental time point (gestation and lactation) adversely affects reproductive functions and fertility of F1 male rats, which were transmitted to F2 generations leading to reduced CRLs and weights of F2 fetuses.

n-Butylparaben exposure through gestation and lactation impairs spermatogenesis and steroidogenesis causing reduced fertility in the F1 generation male rats.

Parabens are class of preservatives used in vast majority of commercial products, and a potential Endocrine Disrupting Chemical (EDC). The present study was undertaken to delineate the effects of n-butylparaben on F1 male progeny exposed maternally through gestation and lactation via subcutaneous route. The F0 dams were given subcutaneous injections of n-butylparaben from gestation day (GD) 6 to postnatal day (PND) 21 with doses of 10, 100, 1000 mg/kg Bw/day in corn oil. The F1 male rats were monitored for pubertal development and sexual maturation; these were sacrificed on PND 30, 45 and 75. On PND 75, these F1 male rats were subjected for fertility assessment with unexposed female rats. A delayed testicular descent at 100 and 1000 mg/kg Bw dose and delayed preputial separation at 10 mg/kg Bw dose was observed in exposed F1 male rats. Decreased sperm count, motility and Daily Sperm Production was observed at 100 mg/kg Bw dose at PND 75. Interestingly, the sperm transit time in the epididymis was accelerated at this dose. Significant perturbed testicular expression of steroid receptors (ERα and ß, AR), INSL3 and StAR genes with increased T and LH levels indicates direct effect on spermatogenesis and steroidogenesis. These F1 generation adult rats were sub-fertile with increased (%) pre- and post-implantation loss at 100 and 1000 mg/kg Bw/day dose. This is the first report on n-butylparaben highlighting the involvement of testicular leydig cells with accelerated sperm transit time leading to reduced fertility in the maternally exposed F1 male rats through estrogenic/anti-androgenic action.

Mitochondrial cytochrome oxidase C subunit III (cox3) gene as a sensitive and specific target for molecular detection of Babesia gibsoni infection in dogs.

A PCR targeting mitochondrial cytochrome oxidase subunit III (cox3) for molecular detection of Babesia gibsoni infection in dogs has been developed in this study. Fifty blood samples from suspected clinical cases from dogs, brought to the veterinary college clinics, were examined for presence of B. gibsoni using conventional diagnosis by microscopic examination of Giemsa stained thin blood smears. In addition, species specific PCRs targeting ITS-1 region (BgITS-1 PCR) and nested PCR targeting 18S ribosomal RNA gene (Bg18SnPCR) were carried out. A 634 bp PCR fragment of B. gibsoni cox3 gene was amplified in positive samples from three geographical locations of Satara, Wai and Pune in Maharashtra state of India. From analysis of the sequence of the B. gibsoni cox3 gene, we found that the Indian isolate had 96-98% similarity to the isolate from Japan and China. Post sequencing, de-novo diagnostic primer pair for species specific amplification of 164 bp fragment of B. gibsonicox3 was designed and the PCR was standardized. The diagnostic results of de-novo Bgcox3 PCR were compared with BgITS-1 PCR and Bg18S nPCR. Thin blood smears detected 22% (11/50) samples positive for small form of Babesia species. The BgITS-1 PCR detected 25% samples (15/50) as positive and Bg18S nPCR detected 80% (40/50) B. gibsoni positive samples. The de-novo Bgcox3 PCR detected 66% (33/50) samples positive for B. gibsoni (at 95% CI). The analytical sensitivity of cox3 PCR was evaluated as 0.000003% parasitaemia or 09 parasites in 100  µl of blood. The de-novo diagnostic cox3 PCR did not cross react with control positive DNA from other haemoprotozoa and rickettsia like B. vogeli, Hepatozoon canis, Trypanosoma evansi, Ehrlichia canis and Anaplasma platys. Statistically, cox3 PCR had better diagnostic efficiency than ITS-1 PCR in terms of sensitivity (p = 0.0006). No statistically significant difference between results of cox3 PCR and 18S nPCR was observed (p = 0.1760). Kappa values estimated for each test pair showed fair to moderate agreement between the observations. Specificity of Bgcox3 PCR was 100% when compared with microscopy or BgITS-1 PCR. Sensitivity of Bgcox3 PCR was 100% when compared with that of Bg18S nPCR.

Zika virus Pathogenesis in Infant Mice after Natural Transmission by the Bite of Infected Mosquitoes.

OBJECTIVES: The objective of this study was to understand natural disease progression in infant CD1 mice after the bite of Aedes aegypti mosquitoes infected by the Zika virus (ZIKV, MR-766 strain). METHODS: A. aegypti mosquitoes were experimentally infected with ZIKV MR-766 strain via the oral feeding route. Infected mosquitoes were allowed to feed on infant CD1 mice. Sick mice were euthanized, and their organs were collected and subjected to real-time RT-PCR, histo-pathology, and immunohistochemistry. RESULTS: Clinical symptoms appeared in mice after 4-5 days of being bitten by mosquitoes, following which they were euthanized. Real-time RT-PCR analysis showed the presence of viral RNA in various organs such as the brain, liver, kidney, spleen, lungs, and intestines of the mice. The brain tissue specimens showed higher viral loads as determined by threshold values (Ct value) in the real-time RT-PCR assay. Histopathological and immunohistochemistry studies also revealed the presence of the virus and associated lesions in the brain, indicating that ZIKV shows tropism for neuronal tissue. CONCLUSIONS: This study demonstrates ZIKV pathogenesis in infant CD1 mice and that these mice are highly susceptible to natural infection with this ZIKV strain.