Preliminary investigation reveals novel pathological consequences of bluetongue virus-1 infection in the endocrine glands of pregnant Indian sheep.

Bluetongue virus (BTV), a major peril to the sheep industry, infects a wide range of the cells in the infected animals including mononuclear, dendritic and epithelial cells. However, little is known about its tropism for the secretory epithelial cells of endocrine glands and the pathogenesis it induces. The aim of the study was to assess the BTV load, antigen distribution in the tissue of the pituitary, thyroid as well as adrenal glands and associated histopathological consequences. BTV antigens were localized using immunohistochemistry in the thyroid's epithelial cells, zona fasciculata and zona reticularis cells and the anterior pituitary epithelial cells. The real-time PCR portrayed the high viral load in adrenals at 7th days postinoculation (DPI) and in thyroid and pituitary glands at 15th DPI. Serum examination revealed variation in the T-3 and T-4 of infected animals in comparison to the control group. Caspase-3 immunolocalization revealed BTV-1 induces apoptosis in the affected cells of endocrine gland of infected animals. Further, this study signifies the tropism of BTV in the novel sites (endocrine glands) of the host that might be one of the reasons for the poor performance of infected animals.

SLAM (CD150) receptor homologous peptides block the peste des petits ruminants virus entry into B95a cells.

The fusion of haemagglutinin-neuraminidase (HN) protein of peste des petits ruminant (PPR) virus with signaling lymphocyte activation molecules (SLAM) host cell receptor consequences the virus entry and multiplication inside the host cell. The use of synthetic SLAM homologous peptides (i.e., molecular decoy for HN protein of PPR virus) may check PPR infection at the preliminary stage. Hence, the predicted SLAM homologous peptides using bioinformatics tools were synthesized by solid phase chemistry with standard Merrifield's 9-fluorenylmethoxycarbonyl (Fmoc) chemistry and were purified by reverse phase high performance liquid chromatography. The secondary structures of synthesized peptides were elucidated by circular dichroism spectroscopy. The in vitro interactions of these peptides were studied through indirect Enzyme Linked Immuno Sorbent Assay (ELISA) and visual surface plasmon UV-visible spectroscopy. The SLAM homologous peptides were able to interact with the peste des petits ruminant virus (PPRV) with varying binding efficiency. The interaction of SLAM homologous peptide with the PPR virus was ascertained by the change in the plasmon color from red wine to purple during visual detection and also by bathochromic shift in absorbance spectra under UV-visible spectrophotometry. The cytotoxic and anti-PPRV effect of these peptides were also evaluated in B95a cell line using PPR virus (Sungri/96). The cytotoxic concentration 50 (CC50 ) value of each peptide was greater than 1000 µg mL-1 . The anti-PPRV efficiency of SLAM-22 was relatively high among SLAM homologous peptides, SLAM-22 at 25 µg mL-1 concentration showed a reduction of more than log10 3 virus titer by priming of B95a cell line while the use of SLAM-15 and Muco-17 at the same concentration dropped virus titer from log10 4.8 to log10 2.5 and log10 3.1 respectively. The concentration of SLAM homologous peptide (25 µg mL-1 ) to exert its anti-PPRV effect was much less than its CC50 level (>1000 µg mL-1 ). Therefore, the synthetic SLAM homologous peptides may prove to be better agents to target PPRV.

Nanoadjuvants: Promising Bioinspired and Biomimetic Approaches in Vaccine Innovation.

Adjuvants are the important part of vaccine manufacturing as they elicit the vaccination effect and enhance the durability of the immune response through controlled release. In light of this, nanoadjuvants have shown unique broad spectrum advantages. As nanoparticles (NPs) based vaccines are fast-acting and better in terms of safety and usability parameters as compared to traditional vaccines, they have attracted the attention of researchers. A vaccine nanocarrier is another interesting and promising area for the development of next-generation vaccines for prophylaxis. This review looks at the various nanoadjuvants and their structure-function relationships. It compiles the state-of-art literature on numerous nanoadjuvants to help domain researchers orient their understanding and extend their endeavors in vaccines research and development.

Development of Hemagglutinin-Neuraminidase Homologous Peptides as Novel Promising Therapeutic Agents Against Peste des Petits Ruminants Virus.

The lack of specific antiviral therapy and complications associated with the existing peste des petits ruminants (PPR) vaccines accentuates the search of novel antiviral blocking agents in order to curtail the PPR infection at initial level. The synthetic hemagglutinin-neuraminidase (HN) homologous peptides may compete with the natural HN protein of PPR virus for binding to signaling lymphocytic activation molecule (SLAM) receptor, consequently, may disrupt peste des petits ruminants virus (PPRV) at entry level. Therefore, insilico analysis, synthesis, purification and subsequent characterization of HN homologous peptides were conducted in this study. The HN homologous peptides were synthesized by means of solid phase chemistry and were purified by reversed-phase-high performance liquid chromatography. The mass as well as sequence of HN homologous peptides were assessed by mass spectroscopy while its secondary structure was elucidated by circular dichroism spectroscopy. The binding (interaction) efficacy of HN homologous peptides with PPRV antibodies was assessed via indirect enzyme linked immunosorbent assay, visual detection test (red wine to purple), bathochromic shift under UV-Vis spectrophotometry and lateral flow immunochromatographic strip test. The antiviral properties and cytotoxicity of these peptides were also assessed in B95a cell line with changes in cytopathic effect and titer of PPRV (Sungri/96). The presence of green fluorescein isothiocyanate over the B95a cell surface pointed towards the binding of HN homologous peptides with surface SLAM receptor. Moreover, the intact beta sheet configuration in water and lower cytotoxicity [cytotoxic concentration 50 (CC50) > 1000 µg/ml] of these peptides signifies its in vivo use. Among HN homologous peptides, the binding efficacy and antiviral properties of pep A was relatively high in comparison to pep B and Pep ppr peptides. The prerequisite concentration of HN homologous peptides (pep A = 12.5 µg/ml; pep B = 25 µg/ml; pep ppr = 25 µg/ml) to exemplify its antiviral effect was much lower than its CC50 level. Hence, this study signifies the therapeutic potential of synthetic HN homologous peptides.

Evaluation of immunological adjuvant activities of saponin rich fraction from the fruits of Asparagus adscendens Roxb. with less adverse reactions.

The hemolytic activity, in vitro as well as in vivo toxicity, and immunomodulatory potential of saponins-rich fraction of Asparagus adscendens Roxb. fruit (AA-SRF) have been assessed in this study in order to explore AA-SRF as an alternative safer adjuvant to standard Quil-A saponin. The AA-SRF showed lower hemolytic activity (HD50 = 301.01 ± 1.63 µg/ml) than Quil-A (HD50 = 17.15 ± 2.12 µg/ml). The sulforhodamine B assay also revealed that AA-SRF was less toxic to VERO cells (IC50≥200 ± 4.32 µg/ml) than Quil-A (IC50 = 60 ± 2.78 µg/ml). The AA-SRF did not lead to mortality in mice up to 1.6 mg and was much safer than Quil-A for in vivo use. Conversely, mice were subcutaneously immunized with OVA 100 µg alone or along with Alum (200 µg) or Quil-A (10 µg) or AA-SRF (50 µg/100 µg/200 µg) on days 0 and 14. The AA-SRF at 100 µg dose best supported the LPS/Con A primed splenocyte proliferation activity, elevated the serum OVA-specific total IgG antibody, IL-12, CD4 titer and upsurged CD3/CD19 expression in spleen as well as lymph node sections which in turn advocated its adjuvant potential. Thus, AA-SRF can be further studied for use as a safe alternative adjuvant in vaccines.

Effect of inhibition of Toll-like receptor 3 signaling on pathogenesis of rabies virus in mouse model.

Rabies is a zoonotic viral disease with inevitably fatal outcome. Toll-like receptor 3 (TLR3) could sense dsRNA viral infections, and implicated in pathogenesis of rabies and Negri bodies (NBs) formation. Present study was undertaken to elucidate the role of TLR3 in pathogenesis, NBs formation, and therapeutic potential of blocking TLR3/dsRNA interaction in rabies infection. Young Swiss albino mice were infected with 100 LD50 of street rabies virus (SRABV) intracerebrally (i/c) on day 0 and treated with 30 µg of CU CPT 4a (selective TLR3 inhibitor) i/c on 0, 3 and 5 days post-infection (DPI). Three mice each were sacrificed at 1, 3, 5, 7, 9, 11, and 13 DPI to study sequential pathological consequences through histopathology, Seller's staining, immunofluorescence, immunohistochemistry, TUNEL assay, flow cytometry, and viral and cytokine genes quantification by real-time PCR. CU CPT 4a inhibited TLR3 expression resulted in delayed development and decreased intensity of clinical signs and pathological lesions, low viral load, significantly reduced NBs formation, and increased survival time in SRABV-infected mice. These parameters suggested that TLR3 did influence the SRABV replication and NBs formation. Inhibition of TLR3 led to decreased expression of pro-inflammatory cytokines and interferons indicated an anti-inflammatory effect of CU CPT 4a during SRABV infection. Further, TLR3-inhibited group revealed normal CD4+/CD8+ T-cells ratio with less TUNEL-positive apoptotic cells indicated that immune cell kinetics are not affected during TLR3-inhibition. SRABV-infected and mock-treated mice were developed severe clinical signs and histopathological lesions, more NBs formation, high viral load, increased pro-inflammatory cytokines expression in brain, which were correlated with higher expression levels of TLR3. In conclusion, these data suggested that TLR3/dsRNA signaling pathway could play critical role in pathogenesis of SRABV infection in vivo and opens up new avenues of therapeutics.

Detection of porcine enteric picornaviruses from faecal samples of Indian pigs.

Porcine enteric picornaviruses often consequence diarrhoea and nervous complications in pig and pose enormous loss to pig farming. The present study expands the limited Indian data of porcine enteric picornaviruses which is needed for the early implementation of control measures and to check further outbreaks. A total of 398 porcine faecal samples from Uttar Pradesh, Madhya Pradesh, Chhattisgarh and Jharkhand state of India were screened for porcine teschovirus (PTV), porcine sapelovirus (PSV) and enterovirus G (EV-G) by reverse transcriptase-polymerase chain reaction (RT-PCR) using 5'UTR-specific primers. The prevalence of PTV, PSV and EV-G was found to be 12.81% (51/398), 5.77% (23/398) and 24.37% (97/398), respectively. EV-G was relatively higher in circulation in Indian pigs among all the included enteric picornaviruses. Conversely, the concurrent infection of more than one enteric picornavirus was also frequent. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-022-00756-0.

A comparative analysis of saponin-enriched fraction from Silene vulgaris (Moench) Garcke, Sapindus mukorossi (Gaertn) and Chlorophytum borivilianum (Santapau and Fernandes): an in vitro hemolytic and cytotoxicity evaluation.

To explore the newer saponin resources, in vitro toxicity of saponin-enriched fraction (SEF) extracted from Silene vulgaris(SV) was evaluated for first time and compared with in vitro toxicity of SEF extracted from Sapindus mukorossi (SM) and Chlorophytum borivilianum (CV). All extracted SEF from diverse resources were characterized by immersing TLC plates in 0.5% RBC suspension method, by ethanol: sulfuric acid method and by estimating hRst values. Each extracted SEF clearly portrayed specific pattern with varied hRst range. White spots against a pinkish-red background and greenish-black spots in case of immersion method and spraying method respectively were observed. After initial characterization, in vitro 0.5% sheep RBC lytic activities and VERO cell cytotoxic activities (via SRB assay) of each extracted SEF were also evaluated. Furthermore, SEF of SV showed very less hemolytic activity compared to SM and CB. The HD50 values for SV, SM, and CB were 736.7 ± 2.824, 18.0 ± 1.894, and 170.70 ± 2.783 µg/mL, respectively. SEF of SV (IC50 ≥ 200 µg/mL) was less toxic for VERO cell line than SEF of SM (IC50 = 150.8 µg/mL) and CB (IC50 = 137.1 µg/mL). Hence, the SEF of SV was found to be less toxic and can be used as a new and safer source of saponins.

Molecular and pathological screening of canine distemper virus in Asiatic lions, tigers, leopards, snow leopards, clouded leopards, leopard cats, jungle cats, civet cats, fishing cat, and jaguar of different states, India.

The present investigation was conducted to rule out canine distemper (CD) diseases in Indian wild felids (Asiatic lions, tigers, leopards, snow leopards, clouded leopards, leopard cats, jungle cats, civet cats, fishing cat, and jaguar). The collected samples were screened for CD virus (CDV) by histopathology (HP), immunohistochemistry (IHC) and reverse transcriptase-polymerase chain reaction (RT-PCR) targeting H gene and N gene. The HP and IHC of suspected samples portrayed that 22 [11 leopards, 6 lions, 3 tigers, 1 snow leopard and 1 civet cat] out of 129 (17.05%) wild felids were positive for CD. The major pathological consequences were observed in spleen, lung, kidney and brain. The syncytia and intranuclear as well as intracytoplasmic eosinophilic inclusion bodies were seen in CDV infected cells. Although the histopathological lesions in spleen were more specific and consistent, however, the severe demyelinated leukoencephalitis (usually expected in CD infected dog) was not observed in the brain of any Indian wild felids. Conversely, the CDV antigen has been portrayed via IHC in pancreatic islets of Langerhans of tiger species for the first time in this study. Moreover, the concurrent CD and babesiosis has also been observed in a lioness without a usual coffee-coloured urine. The N gene and H gene of CDV isolates were amplified, sequenced and subsequently constructed the phylogenetic tree. The phylogenetic analysis of H gene revealed that the CDV isolates from Indian lion formed separate clade with CDV isolates from Indian dog and Indian palm civet cat. Furthermore, two CDV isolates from Indian tigers formed clade with Onderstepoort vaccine strain and CDV isolates from dogs of Uttar Pradesh, USA and UK. Evidently, CDV is circulating in Indian wild felids and causing diseases in them.

Exploration of antigenic determinants in spike glycoprotein of SARS-CoV2 and identification of five salient potential epitopes.

Emerging pathogens have been an eternal threat to mankind. In a series of pandemics caused by notorious coronaviruses, a newly emerged SARS-CoV2 virus is creating panic among the world population. The unavailability of reliable theranostics insists the exploration of antigenic determinants in spike glycoprotein of SARS-CoV2. The four novel inserts ('70VSGTNGT76', '150KSWM153', 247SYLTPG252 and 674QTQTNSPRR682) in SARS-CoV2 spike protein were unraveled via multiple sequence alignment of spike proteins of SARS-CoV2, SARS-CoV, and MERS-CoV. The three-dimension (3D) modeling of the spike protein of the SARS-CoV2 and their interaction with the ACE2 receptor was delineated with the help of SWISS-MODEL and 3DLigandSite web servers. The predicted 3D model of SARS-CoV2 was further verified by SAVES, RAMPAGE, and ProSA-web tools. The potential B-cell immunogenic epitopes of SARS-CoV2 were predicted out by using various software viz. IEDB B-cell epitopes prediction tool, BepiPred linear epitope prediction tool, Emini Surface Accessibility Prediction tool, and Kolaskar-Tongaonkar antigenicity web tool. The five epitopes (i.e. '71SGTNGTKRFDN81, 247SYLTPG252, 634RVYST638, 675QTQTNSPRRARSV687, and 1054QSAPH1058) were selected as potent antigenic determinants. The quantum of information generated by this study will prove beneficial for the development of effective therapeutics, diagnostics, and multi-epitopic vaccines to combat this ongoing menace.

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

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

Non-infectious outer membrane vesicles derived from Brucella abortus S19Δper as an alternative acellular vaccine protects mice against virulent challenge.

The prime human and animal safety issues accentuate the search of promising newer alternative vaccine candidates to resolve complications associated with the live attenuated Brucella abortus strain19 (S19) vaccine. Outer membrane vesicles (OMVs S19 Δper) extracted from Brucella abortus S19Δper (S19Δper) as an alternative subunit vaccine candidate has been explored in the present study as OMVs are endowed with immunogenic molecules, including LPS and outer membrane proteins (OMPs) and do not cause infection by virtue of being an acellular entity. The LPS defective S19Δper released a higher amount of OMVs than its parent strain S19. Under transmission electron microscopy (TEM), OMVs were seen as nano-sized outward bulge from the surface of Brucella. Dynamic light scattering analysis of OMVs revealed that OMVs S19Δper showed the less polydispersity index (PDI) than OMVs S19 pointing towards relatively more homogenous OMVs populations. Both OMVs S19Δper and OMVs S19 with or without booster dose and S19 vaccine were used for immunization of mice and subsequently challenged with 2 × 105 CFU virulent Brucella abortus strain 544 (S544) to assess protective efficacy of vaccines. The less splenic weight index and less S544 count in OMVs immunized mice in comparison to unimmunized mice after S544 challenge clearly indicated good protective efficacy of OMVs. OMVs S19 Δper induced relatively high titer of IgG than OMVs S19 but conferred nearly equal protection against brucellosis. An ELISA based determination of IgG and its isotype response, Cytometric Bead Array (CBA) based quantitation of serum cytokines and FACS based enumeration of CD4+ and CD8+ T cells revealed high titer of IgG, production of both Th1 (IgG2a) and Th2 (IgG1) related antibodies, stimulation of IL-2, TNF (Th1) and IL-4, IL-6, IL-10 (Th2) cytokines, and induced T cell response suggested that OMVs S19Δper elicited Th1 and Th2 type immune response and ensured protection against S544 challenge in murine model.

Gold nanoparticle based immunochromatographic biosensor for rapid diagnosis of Mycobacterium avium subspecies paratuberculosis infection using recombinant protein.

Highly infectious and obvious withstand ability of Mycobacterium avium subspecies paratuberculosis (MAP) to environment as well as lack of on-site field diagnostic methods notably hampers the paratuberculosis (PTB) control. The existing intricacy, time-consuming and complicated diagnostic methods of PTB accentuate the development of novel and easy-to-perform on-site test. A gold nanoparticle (GNP) based lateral-flow assay (LFA) using MAP recombinant protein (44 kDa) has been developed for sensitive and specific detection of PTB in field conditions. The diagnostic sensitivity and specificity of the LFA for MAP specific antibodies was found approximately 84.2% and 83.3% in comparison to indirect enzyme-linked immunosorbent assay. Consequently, the newly developed GNP based LFA offers on-site and cost-effective method for the prompt diagnosis of PTB and precludes the time-consuming laboratory screening.

Development of novel iron-regulated Pasteurella multocida B: 2 bacterin and refinement of vaccine quality in terms of minimum variation in particle size and distribution vis-a-vis critical level of iron in media.

To enhance the qualitative bacterial biomass per unit of media and to overcome the limitations of the existing haemorrhagic septicaemia (HS) vaccines, a comprehensive study was undertaken encompassing the role of iron on the bacterial biomass of Pasteurella multocida B: 2 to vaccine development. Trypsin digested hydrochloric acid-treated sheep blood (THSB) as a novel iron rich supplement had been devised for the first time for augmenting the qualitative bacterial biomass per unit of media which was evident with growth kinetic study. The higher recovery of iron from THSB became evident via atomic absorbance spectrophotometry. The critical level of iron in the media as well as mode of iron supplementation showed a major impact on the outer membrane protein profile of P. multocida B:2 and variation in droplet size and particle-size distribution of formulated vaccine. Immune response study against iron-regulated bacterin adjuvanted with aluminum hydroxide gel in mouse model showed that 3% THSB supplementation of casein sucrose yeast (CSY) not only augmented the growth of P. multocida B:2 significantly but conferred highest pre-challenged ELISA IgG titer and protection against pasteurellosis. Thus, THSB supplementation of CSY can resolve existing up-scaling and immunogenic potential problems of HS vaccine production.

Peptidoglycan and Lipoteichoic Acid Induces Differential mRNA Response of Immune-Related Genes in PBMC of Crossbred, Tharparkar Cattle and Murrah Buffalo.

Subclinical mastitis, generally caused by Staphylococcus aureus, has a global economic impact all over the world. Hence, it needs to be resolved on higher priority which may be attained via. selection of mastitis resistant animals or inclusion of mastitis resistant trait into herd apart from management care. Diverse hosts with various genetic make-ups encounter pathogens in a diverse manner which in turn leads to contradicting outcome of the disease. Identification of species-wise or breed-wise differential expressed genes in response to S. aureus through relative evaluation of transcripts may be useful for judging the immuno-competency of a species or breed toward mastitis. The present study was undertaken to examine the stimulant effect of S. aureus peptidoglycan (PGN) and lipoteichoic acid (LTA) on Peripheral blood mononuclear cells (PBMC) harvested from blood samples of crossbred cattle, Tharparkar cattle, and Murrah buffaloes. After 6 h of in vitro stimulation qRT-PCR was used to measure the relative mRNA expression of TLR-2, TNF-α, IL-8, IFN-γ and IL-10 genes in stimulated and un-stimulated PBMC. The selected genes revealed significant differences in the pattern of immune response among crossbred cattle, Tharparkar cattle and Murrah buffalo in spite of the same stimulant dose.

Pathological and molecular investigation of porcine sapelovirus infection in naturally affected Indian pigs.

The aim of the present study was to pathological and molecular investigation of porcine sapelovirus (PSV) in naturally infected Indian pigs of various age groups. Eight samples (16%) out of 49 necropsied animals were positive for PSV on the basis of pathological and molecular investigation. Major lesions of PSV positive cases were thickening and clouding of meninges, congestion in brain, severe to moderate congestion in lungs along with froathy exudates in trachea, thickening of intestinal mucosa, especially mucosal folds of ileum. Microscopic lesions of PSV positive cases in CNS were perivascular cuffing, neuronophagia and focal gliosis. In lungs, interstitial pneumonia was noticed in all cases, and intestinal lesions comprised of sloughing of villi epithelium, moderate to severe congestion of blood vessels and infiltration of mononuclear cells mainly plasma cells in both large and small intestine. RT-PCR results of total cases examined for PSV were targeted for PSV 3D Polymerase, 5'UTR region and VP1 gene respectively. Genetic characterization was done on the basis of viral capsid protein 1 (VP1) gene of PSV. The sequencing and phylogenetic analysis of amplified VP1 gene product showed maximum identity 85-90% with South Korean, KJ821021.1 and Indian, KY053835.1 strain of PSV. Further explorative surveillance and epidemiological studies are suggested to find out the real impact of this economically important disease affecting pigs population of India.

Kaempferol alleviates palmitic acid-induced lipid stores, endoplasmic reticulum stress and pancreatic ß-cell dysfunction through AMPK/mTOR-mediated lipophagy.

Kaempferol, a natural flavonoid, has the beneficial effects of preserving pancreatic ß-cell mass and function, but its action on ß-cell lipid metabolism still remains elusive. Recently, autophagy has been reported to play a major role in lipid metabolism in various cell types, but its role in pancreatic ß-cell's lipid metabolism is rarely reported. Here, we investigated the role of kaempferol-induced autophagy in inhibition of lipid stores, ER stress and ß-cell dysfunction in palmitic acid-challenged RIN-5F cells and isolated pancreatic islets. The lipid-lowering effect of kaempferol was determined by Oil Red O staining, triglyceride assay, BODIPY labeling, RT-PCR and immunoblot analysis of PLIN2 (the lipid droplet coat protein) expression. Further, the involvement of AMPK/mTOR-mediated lipophagy was established by pharmacological and genetic inhibitors of autophagy and AMPK. The co-localization studies of lipid droplets with autophagosomes/lysosomes by BODIPY-MDC-LysoTracker co-staining, LC3/BODIPY labeling and LC3/PLIN2 double immunolabeling further strengthened the findings. Kaempferol treatment exhibited decreased lipid stores and increased co-localization of lipid droplets with autophagosomes and lysosomes in palmitic acid-challenged ß-cells. Moreover, inhibition of autophagy led to decreased co-localization and increased lipid droplets accumulation. Kaempferol-induced alleviation of ER stress and ß-cell dysfunctions was established by immunoblot analysis of CHOP-10 (a key mediator of cell death in response to ER stress) and insulin content/secretion analysis respectively. Together, these findings suggest that kaempferol prevents ectopic lipid accumulation and ER stress, thus restoring ß-cell function through AMPK-mediated lipophagy. The current data implies that kaempferol may be a potential therapeutic candidate to prevent obesity-linked diabetic complications.