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Capsids of several RNA viruses are reported to have unconventional roles attributed to their subcellular trafficking property. The capsid of CHIKV is also found to localize in the nucleus, but the rationale is not yet clear. To understand the role of the nuclear-localized capsid, we examined the nucleic acid binding and cargo delivery activity of the CHIKV capsid. We used bacterially purified capsid protein to probe the binding affinity with CHIKV genome-specific and non-specific nucleic acids. We found that the capsid was able to bind non-specifically to different forms of nucleic acids. The successful transfection of GFP-tagged plasmid DNA by CHIKV capsid protein shows the DNA delivery ability of the protein. Further, we selected and investigated the DNA binding and cargo delivery activity of commercially synthesized Nuclear Localization Signal sequences (NLS 1 and NLS2) of capsid protein. Both peptides showed comparable DNA binding affinity, however, only the NLS1 peptide was capable of delivering plasmid DNA inside the cell. Furthermore, the cellular uptake study using the FITC-labelled NLS1 peptide was performed to highlight the membrane penetrating ability. Structural analysis was performed using circular dichroism and NMR spectroscopy to elucidate the transfection ability of the NLS1 peptides. Our findings suggest that the capsid of CHIKV might influence cellular trafficking in the infected cell via non-specific interactions. Our study also indicates the significance of NLS sequences in the multifunctionality of CHIKV capsid protein.
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Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Vírus Chikungunya/metabolismo , DNA/metabolismo , Sinais de Localização Nuclear , Sequência de Aminoácidos , Transporte Biológico , Modelos Moleculares , Domínios ProteicosRESUMO
Plasmodium falciparum leads to a virulent form of malaria. Progress has been achieved in understanding the mechanisms involved in the malarial infection, still there is no effective vaccine to prevent severe infection. An effective vaccine against malaria should be one which can induce immune responses against multiple epitopes in the context of predominantly occurring HLA alleles. In this study, an integrated approach was employed to identify promiscuous peptides of a well-defined sequence of erythrocyte binding antigen-175 and promiscuous peptides for HLA alleles were designed using bioinformatics tools. A peptide with 15 amino acids (ILAIAIYESRILKRK) was selected based on its high binding affinity score and synthesized. This promiscuous peptide was used as stimulating antigen in lymphoproliferative responses to evaluate the cellular immune response. It was observed this peptide evokes lymphoproliferative and cytokine responses in individuals naturally exposed to the malaria parasite. The intensity of PBMCs proliferation was observed to be higher in sera obtained from P. falciparum exposed as compared to unexposed healthy individuals, suggesting earlier recognition of peptide of this region by T cells. Furthermore, the binding mode of HLA-peptide complex and their interaction may lead to a rational and selective peptide-based vaccine candidate design approach which can be used as a malaria prophylaxis.
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Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/imunologia , Malária Falciparum/imunologia , Peptídeos/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Alelos , Sequência de Aminoácidos , Antígenos de Protozoários/metabolismo , Células Cultivadas , Desenho de Fármacos , Antígenos HLA/genética , Antígenos HLA/imunologia , Antígenos HLA/metabolismo , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-4/imunologia , Interleucina-4/metabolismo , Vacinas Antimaláricas/administração & dosagem , Malária Falciparum/parasitologia , Malária Falciparum/prevenção & controle , Simulação de Acoplamento Molecular , Peptídeos/química , Peptídeos/metabolismo , Plasmodium falciparum/metabolismo , Plasmodium falciparum/fisiologia , Ligação Proteica , Proteínas de Protozoários/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/parasitologiaRESUMO
Human lymphatic filariasis (LF) is a major cause of disability globally. The success of global elimination programmes for LF depends upon effectiveness of tools for diagnosis and treatment. In this study on stage-specific antigen detection in brugian filariasis, L3, adult worm (AW) and microfilarial antigenaemia were detected in around 90-95% of microfilariae carriers (MF group), 50-70% of adenolymphangitis (ADL) patients, 10-25% of chronic pathology (CP) patients and 10-15% of endemic normal (EN) controls. The sensitivity of the circulating filarial antigen (CFA) detection in serum samples from MF group was up to 95%. In sera from ADL patients, unexpectedly, less antigen reactivity was observed. In CP group all the CFA positive individuals were from CP grade I and II only and none from grade III or IV, suggesting that with chronicity the AWs lose fecundity and start to disintegrate and die. Amongst EN subject, 10-15% had CFA indicating that few of them harbour filarial AWs, thus they might not be truly immune as has been conventionally believed. The specificity for antigen detection was 100% when tested with sera from various other protozoan and non-filarial helminthic infections.
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Antígenos de Helmintos/sangue , Filariose Linfática/imunologia , Wuchereria bancrofti/crescimento & desenvolvimento , Wuchereria bancrofti/imunologia , Adulto , Animais , Doenças Assintomáticas , Dietilcarbamazina/uso terapêutico , Filariose Linfática/tratamento farmacológico , Filariose Linfática/parasitologia , Filariose Linfática/fisiopatologia , Feminino , Humanos , Índia , Estágios do Ciclo de Vida/imunologia , Masculino , Coelhos , Adulto JovemRESUMO
Chikungunya virus (CHIKV) causes persistent arthritis and neurological problems imposing a huge burden globally. The present study aims to understand the interaction mechanism of Chikungunya virus and CHIKV-capsid in Huh7 cells. The RNA-sequencing and qRT-PCR method was used for the transcript and gene profiles of CHIKV virus and CHIKV capsid alone. Transcriptional analysis showed capsid induced 1114 and 956 differentially expressed genes (DEGs) to be upregulated and downregulated respectively, while in virus, 933 genes were upregulated and 956 were downregulated. Total 202 DEGs were common in both capsid and virus; and nine were validated using qRT-PCR. Identified DEGs were found to be associated with metabolic pathways such as Diabetes, cardiac disease, and visual impairment. Further, knock-down study on one of the DEGs (MafA) responsible for insulin regulation showed low viral proteins expression suggesting a reduction in virus-infection. Thus, the study provides insight into the interplay of the virus-host factors assisting virus replication.
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Febre de Chikungunya , Vírus Chikungunya , Humanos , Capsídeo/metabolismo , Vírus Chikungunya/fisiologia , Replicação Viral , Proteínas do Capsídeo/metabolismo , Perfilação da Expressão Gênica , Redes e Vias Metabólicas/genéticaRESUMO
Malaria morbidity has various presentations and the focus now shifts to uncommon signs and symptoms of malaria infection such as cognitive impairment to address the morbidity when the mortality declines. About 50% of children admitted to hospitals due to malaria experience neurological complications due to factors like low blood sugar, inflammation, elevated pressure, decreased oxygen levels, and excitotoxicity. Malaria during pregnancy negatively also impacts children's cognitive, behavioral, and executive function leading to neurodevelopmental delay due to increased susceptibility which can significantly affect maternal and child health, leading to higher rates of underestimated factors like anxiety, depression, and PTSD. Despite having the world's second-largest tribal population, India's indigenous and tribal communities and their mental health are less explored and less understood. Western psychological tools and neurocognitive assessment tools are not universally applicable, thus necessitating the development of tailored tools to investigate psychological or neurocognitive impairment. This paper has illuminated the hidden mental health consequences of malaria infection, emphasizing the prevalence, nature, and implications of psychological distress among affected individuals. The findings underscore the importance of recognizing and addressing these psychological consequences in the holistic management and prevention of malaria and its mental health consequences.
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Probiotic supplements are suggested to promote human health by preventing pathogen colonization. However, the mechanistic bases for their efficacy in vivo are largely uncharacterized. Here using metabolomics and bacterial genetics, we show that the human oral probiotic Streptococcus salivarius K12 (SAL) produces salivabactin, an antibiotic that effectively inhibits pathogenic Streptococcus pyogenes (GAS) in vitro and in mice. However, prophylactic dosing with SAL enhanced GAS colonization in mice and ex vivo in human saliva. We showed that, on co-colonization, GAS responds to a SAL intercellular peptide signal that controls SAL salivabactin production. GAS produces a secreted protease, SpeB, that targets SAL-derived salivaricins and enhances GAS survival. Using this knowledge, we re-engineered probiotic SAL to prevent signal eavesdropping by GAS and potentiate SAL antimicrobials. This engineered probiotic demonstrated superior efficacy in preventing GAS colonization in vivo. Our findings show that knowledge of interspecies interactions can identify antibiotic- and probiotic-based strategies to combat infection.
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Probióticos , Infecções Estreptocócicas , Animais , Humanos , Camundongos , Antibacterianos , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes , SalivaRESUMO
INTRODUCTION: India's contribution to the malaria burden was highest in South-East Asia Region in 2021, accounting for 79% of the estimated malaria cases and 83% of malaria-related deaths. Intensified Malaria Control Programme supported by Global Funds to Fight against AIDS, Tuberculosis and Malaria has deployed crucial interventions to reduce the overall burden of malaria in India. Evaluation of utilisation of malaria elimination interventions by the community and assessment of the healthcare system is underway in eleven high malaria endemic states in India. Health system preparedness for malaria elimination, logistics, and supply chain management of diagnostic kits and anti-malarial drugs in addition to the knowledge, attitude and practice of the healthcare workers is also being assessed. METHODS AND ANALYSIS: The study is being undertaken in 11 malaria endemic states with a variable annual parasite incidence of malaria. In total, 47 districts (administrative unit of malaria control operations) covering 37 976 households are to be interviewed and assessed. We present here the protocol following which the study is being undertaken at the behest and approval of Ministry of Health and Family Welfare in India. ETHICS AND DISSEMINATION: No patients were involved in the study. Study findings will be shared with Institutional ethics board of National Institute for Malaria Research New Delhi (NIMR) in a timely, comprehensive, accurate, unbiased, unambiguous and transparent manner and to the National Vector-borne Disease (Malaria) Control Programme officers and the Community public who participated. Important findings will be communicated through community outreach meetings which are existing in the Health system. Results will be informed to study participants via local fieldwork supervised by District Malaria Officers. Also findings will be published in reputed journals based on Indian Council of Medical Research (ICMR) publication policy.The ICMR-NIMR ethics committee approved the study via letter No. NIMR/ECM/2023/Feb/14 dated 24 April 2023 for version 5. All standard ethical practices will be followed.
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Doenças Endêmicas , Malária , Humanos , Índia/epidemiologia , Malária/epidemiologia , Malária/prevenção & controle , Estudos Transversais , Projetos de Pesquisa , Antimaláricos/uso terapêutico , Conhecimentos, Atitudes e Prática em Saúde , Atenção à SaúdeRESUMO
Chikungunya virus; the pathogen for chikungunya febrile and arthritic disease, having 11.8 kb positive-sense RNA genome encodes polyproteins for structural and non-structural regions. The polyprotein (P1234) corresponding to the non-structural part from 5' end gets auto-cleaved by the action of nsP2 protease, which leads to the generation of individual functional enzymatic proteins like nsP4, nsP1, nsP2 and nsP3. Thus, nsP2 protein initiates viral replication. Targeting nsP2 to block virus replication has always been the foremost strategy to develop antivirals. Plant-based molecules are one of the top choices to develop as inhibitor due to their less toxicity and wide availability. Using a combination of receptor-based docking and MD simulations, we identified a flavanone glycoside- naringin, which binds to nsP2 protease at nM affinity. The biomolecular interaction between naringin and nsP2 was established through SPR. As discerned through FTIR and intrinsic fluorescence studies, upon binding with naringin, a global structural change in nsP2 occurs. This structural modulation in nsP2 due to binding of naringin is likely to interfere with the normal functioning of this enzyme during the viral life cycle. In conclusion, this report highlights the potential of naringin as an anti-viral agent against Chikungunya.
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Vírus Chikungunya/enzimologia , Cisteína Endopeptidases/química , Flavanonas/química , Simulação de Acoplamento Molecular , Sítios de Ligação , Cisteína Endopeptidases/metabolismo , Flavanonas/metabolismo , Ligação ProteicaRESUMO
The COVID-19 pandemic caused by SARS-CoV-2 has emerged as a global catastrophe. The virus requires main protease for processing the viral polyproteins PP1A and PP1AB translated from the viral RNA. In search of a quick, safe and successful therapeutic agent; we screened various clinically approved drugs for the in-vitro inhibitory effect on 3CLPro which may be able to halt virus replication. The methods used includes protease activity assay, fluorescence quenching, surface plasmon resonance (SPR), Thermofluor® Assay, Size exclusion chromatography and in-silico docking studies. We found that Teicoplanin as most effective drug with IC50 ~ 1.5 µM. Additionally, through fluorescence quenching Stern-Volmer quenching constant (KSV) for Teicoplanin was estimated as 2.5 × 105 L·mol-1, which suggests a relatively high affinity between Teicoplanin and 3CLPro protease. The SPR shows good interaction between Teicoplanin and 3CLPro with KD ~ 1.6 µM. Our results provide critical insights into the mechanism of action of Teicoplanin as a potential therapeutic against COVID-19. We found that Teicoplanin is about 10-20 fold more potent in inhibiting protease activity than other drugs in use, such as lopinavir, hydroxychloroquine, chloroquine, azithromycin, atazanavir etc. Therefore, Teicoplanin emerged as the best inhibitor among all drug molecules we screened against 3CLPro of SARS-CoV-2.
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Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/enzimologia , Reposicionamento de Medicamentos/métodos , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Sequência de Aminoácidos , Antivirais/química , Betacoronavirus/fisiologia , COVID-19 , Proteases 3C de Coronavírus , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Cisteína Endopeptidases , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Inibidores de Proteases/química , SARS-CoV-2 , Teicoplanina/química , Teicoplanina/farmacologia , Replicação Viral/efeitos dos fármacosRESUMO
Chikungunya has re-emerged as an epidemic with global distribution and high morbidity, necessitating the need for effective therapeutics. We utilized already approved drugs with a good safety profile used in other diseases for their new property of anti-chikungunya activity. It provides a base for a fast and efficient approach to bring a novel therapy from bench to bedside by the process of drug-repositioning. We utilized an in-silico drug screening with FDA approved molecule library to identify inhibitors of the chikungunya nsP2 protease, a multifunctional and essential non-structural protein required for virus replication. Telmisartan, an anti-hypertension drug, and the antibiotic novobiocin emerged among top hits on the screen. Further, SPR experiments revealed strong in-vitro binding of telmisartan and novobiocin to nsP2 protein. Additionally, small angle x-ray scattering suggested binding of molecules to nsP2 and post-binding compaction and retention of monomeric state in the protein-inhibitor complex. Protease activity measurement revealed that both compounds inhibited nsP2 protease activity with IC50 values in the low micromolar range. More importantly, plaque formation assays could show the effectiveness of these drugs in suppressing virus propagation in host cells. We propose novobiocin and telmisartan as potential inhibitors of chikungunya replication. Further research is required to establish the molecules as antivirals of clinical relevance against chikungunya.
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Antivirais/farmacologia , Febre de Chikungunya/virologia , Vírus Chikungunya/efeitos dos fármacos , Novobiocina/farmacologia , Telmisartan/farmacologia , Febre de Chikungunya/tratamento farmacológico , Vírus Chikungunya/genética , Vírus Chikungunya/fisiologia , Avaliação Pré-Clínica de Medicamentos , Humanos , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
OBJECTIVES: Parasitic infections such as toxoplasmosis, hydatidosis, and cysticercosis infect a large population worldwide. Toxoplasma gondii in the pancreas could damage the pancreatic cells. Hence, insulin secretion would be affected which leads to increased risk of diabetes. The present study was designed to investigate the cooccurrence of parasitic infections in patients with diabetes. METHODS: A total of 256 confirmed parasitic serum samples were included in the study, of which 95 were positive for toxoplasmosis, 87 for hydatidosis, and 74 for cysticercosis infection. These samples were then analyzed for hyperglycemia. RESULTS: In case of hydatidosis, the frequency of hyperglycemia was 14.94%, followed by cysticercosis (10.8%) and toxoplasmosis (8.42%). The frequency of insulin resistance was analyzed in these hyperglycemic samples. The coprevalence of diabetes was higher in case of hyperglycemic hydatidosis patient samples (11.49%), followed by cysticercosis (8.10%) while the coprevalence of diabetes was minimum (6.31%) in toxoplasmosis patients samples. Thus, the overall prevalence of diabetes with parasitic infections was 8.6%. CONCLUSION: The study suggested the coexistence of hyperglycemia in hydatidosis, cysticercosis, and toxoplasmosis infected samples. However, higher prevalence of high glucose level was detected among hydatidosis infected samples. Thus, suggested individuals infected with hydatidosis were more susceptible for diabetes in comparison to toxoplasmosis and cysticercosis.
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CONTEXT: T-cell hypo-responsiveness in microfilaria (Mf) carriers against the microfilarial stage antigen of Brugia malayi has been described, but no study has been carried out to assess antibody dynamics against stage-specific antigens. AIM: The work was carried out with the aim to assess stage-specific antibody responses against L3 and microfilarial stage antigens in brugian filariasis in an endemic area. SETTING AND DESIGN: Patients with different clinical spectra of brugian filariasis were recruited to evaluate antibody responses to brugian antigens. SUBJECTS AND METHODS: Serum samples were collected from patients with different clinical spectra and antibody response was evaluated for total immunoglobulin G (IgG), IgG isotypes (IgG1, IgG2, IgG3, IgG4) and immunoglobulin E (IgE) response to L3 and microfilarial stage by enzyme-linked immunosorbent assay. STATISTICAL ANALYSIS: Paired t-test and one-way analysis of variance were carried out to analyze the data. RESULTS: L3 and microfilarial stage antigens showed almost similar antibody responses in adenolymphangitis (ADL) and chronic pathology (CP) patients, however, diminished antibody response was observed with Mf stage antigen, especially with microfilaraemia. ADL patients had minimum antibody levels of all isotypes except IgG2 on day 0 which showed an increase subsequently, indicating suppression of antibody response during filarial fever. CP patients showed increase in IgE and decrease in IgG4 antibodies on day 365 indicating that these differences may be due to recent conversion into CP. CONCLUSION: A prominent hyporesponsiveness in microfilaraemic individuals against microfilarial stage, but not against the L3 stage of the same parasite was observed, concluding stage-specificity in humoral immune response in brugian filariasis.