In silico study indicates antimalarials as direct inhibitors of SARS-CoV-2-RNA dependent RNA polymerase.

Coronavirus disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused a global pandemic. RNA-dependent RNA polymerase (RdRp) is the key component of the replication or transcription machinery of coronavirus. Therefore SARS-CoV-2-RdRp has been chosen as an important target for the development of antiviral drug(s). During the early pandemic of the COVID-19, chloroquine and hydroxychloroquine were suggested by the researchers for the prevention or treatment of SARS-CoV-2. In our study, the antimalarial compounds have been screened and docked against SARS-CoV-2-RdRp (PDB ID: 7BTF), and it was observed that the antimalarials chloroquine, hydroxychloroquine, and amodiaquine exhibit good affinity. Since the crystal structure of SARS-CoV-2-RdRp with its substrate is not available, poliovirus-RdRp crystal structure co-crystallized with its substrate ATP (PDB ID: 2ILY) was used as a reference structure. The superimposition of SARS-CoV-2-RdRp and poliovirus-RdRp structures showed that the active sites of both of the RdRps superimposed very well. The amino acid residues involved in the binding of ATP in the case of poliovirus-RdRp and residues involved in binding with the antimalarial compounds with SARS-CoV-2-RdRp were compared. In both cases, the conserved residues were found to be involved in establishing the interactions. The MMGBSA and molecular dynamic simulation studies were performed to strengthen our docking results. Further residues involved in binding of antimalarials with SARS-CoV-2-RdRp were compared with the residues involved in the SARS-CoV-2-RdRp complexed with remdesivir [PDB ID: 7BV2]. It was observed that co-crystallized remdesivir and docked antimalarials bind in the same pocket of SARS-CoV-2 -RdRp.Communicated by Ramaswamy H. Sarma.

Assessing L. donovani Skin Parasite Load: A Proof of Concept Study of a Microbiopsy Device in an Indian Setting.

Background: In the endgame of the elimination initiative of visceral leishmaniasis (VL) on the Indian subcontinent, one of the main questions remaining is whether asymptomatically infected individuals also contribute to transmission. We piloted a minimally invasive microbiopsy device that could help answer this question. While the potential of this device has been previously illustrated in Ethiopia, no such information is available for the setting of the Indian subcontinent. In this proof of concept study we aimed to assess 1) to what extent skin parasite load obtained with the new microbiopsy device correlates with disease status, 2) to what extent skin parasite load correlates with blood parasite load in the same subject, and 3) to what extent the skin parasite load obtained from different sampling sites on the body correlates with one another. Methods: We performed a pilot study in Bihar, India, including 29 VL patients, 28 PKDL patients, 94 asymptomatically infected individuals, 22 endemic controls (EC), and 28 non-endemic controls (NEC). Presence of infection with L. donovani in the blood was assessed using Direct Agglutination Test, rK39 ELISA, Whole Blood Analysis measuring IFN-γ and qPCR. A skin sample was collected with the microbiopsy device on two different locations on the body. PKDL patients provided a third skin sample from the edge of a PKDL lesion. Parasite load in the skin was measured by qPCR. Findings: We found a clear correlation between the skin parasite load obtained with the microbiopsy device and disease status, with both higher skin parasite loads and higher proportions of positive skin samples in VL and PKDL patients compared to asymptomatics, EC, and NEC. No clear correlation between skin parasite load and blood parasite load was found, but a moderate correlation was present between the skin parasite load in arm and neck samples. In addition, we found four positive skin samples among asymptomatic individuals, and 85% of PKDL lesions tested positive using this microbiopsy device. Conclusions: In line with previous pilot studies, our results from an Indian setting suggest that the microbiopsy device provides a promising tool to measure skin parasite load, and - if validated by xenodiagnosis studies - could facilitate much needed larger scale studies on infectiousness of human subgroups. In addition, we advocate further evaluation of this device as a diagnostic tool for PKDL.

IFN-γ+ CD4+T cell-driven prophylactic potential of recombinant LDBPK_252400 hypothetical protein of Leishmania donovani against visceral leishmaniasis.

Visceral leishmaniasis (VL) is a potentially fatal parasitic disease causing high morbidity and mortality in developing countries. Vaccination is considered the most effective and powerful tool for blocking transmission and control of diseases. However, no vaccine is available so far in the market for humans. In the present study, we characterized the hypothetical protein LDBPK_252400 of Leishmania donovani (LdHyP) and explored its prophylactic behavior as a potential vaccine candidate against VL. We found reduced hepato-splenomegaly along with more than 50% parasite reduction in spleen and liver after vaccination in mice. Protection in vaccinated mice after the antigen challenge correlated with the stimulation of antigen specific IFN-γ expressing CD4+T cell (~4.6 fold) and CD8+T cells (~2.1 fold) in vaccinated mice in compared to infected mice, even after 2-3 months of immunization. Importantly, antigen-mediated humoral immunity correlated with high antigen specific IgG2/IgG1 responses in vaccinated mice. In vitro re-stimulation of splenocytes with LdHyP enhances the expression of TNF-α, IFN-γ, IL-12 and IL-10 cytokines along with lower IL-4 cytokine and IL-10/IFN-γ ratio in vaccinated mice. Importantly, we observed ~3.5 fold high NO production through activated macrophages validates antigen mediated cellular immunity induction, which is critical in controlling infection progression. These findings suggest that immunization with LdHyP mount a very robust immunity (from IL-10 towards TFN-γ mediated responses) against L. donovani infection and could be explored further as a putative vaccine candidate against VL.

Nanodiagnostics in leishmaniasis: A new frontiers for early elimination.

Visceral leishmaniasis (VL) is still a major public health concern in developing countries having the highest outbreak and mortality potential. While the treatment of VL has greatly improved in recent times, the current diagnostic tools are limited for use in the post-elimination setting. Although conventional serological methods of detection are rapid, they can only differentiate between active disease in strict combination with clinical criteria, and thus are not sufficient enough to diagnose relapse patients. Therefore, there is a dire need for a portable, authentic, and reliable assay that does not require large space, specialized instrument facilities, or highly trained laboratory personnel and can be carried out in primary health care settings. Advances in the nanodiagnostic approaches have led to the expansion of new frontiers in the concerned area. The nanosized particles are blessed with an ability to interact one-on-one with the biomolecules because of their unique optical and physicochemical properties and high surface area to volume ratio. Biomolecular detection systems based on nanoparticles (NPs) are cost-effective, rapid, nongel, non-PCR, and nonculture based that provide fast, one-step, and reliable results with acceptable sensitivity and specificity. In this review, we discuss different NPs that are being used for the identification of molecular markers and other biomarkers, such as toxins and antigens associated with leishmaniasis. The most promising diagnostic approaches have been included in the article, and the ability of biomolecular recognition, advantages, and disadvantages have been discussed in detail to showcase the enormous potential of nanodiagnostics in human and veterinary medicine. This article is categorized under: Diagnostic Tools > Diagnostic Nanodevices Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease Diagnostic Tools > Biosensing.

Modified solid lipid nanoparticles encapsulated with Amphotericin B and Paromomycin: an effective oral combination against experimental murine visceral leishmaniasis.

The development of an effective oral therapeutics is an immediate need for the control and elimination of visceral leishmaniasis (VL). We exemplify the preparation and optimization of 2-hydroxypropyl-ß-cyclodextrin (HPCD) modified solid lipid nanoparticles (SLNs) based oral combinational cargo system of Amphotericin B (AmB) and Paromomycin (PM) against murine VL. The emulsion solvent evaporation method was employed to prepare HPCD modified dual drug-loaded solid lipid nanoparticles (m-DDSLNs). The optimized formulations have a mean particle size of 141 ± 3.2 nm, a polydispersity index of 0.248 ± 0.11 and entrapment efficiency for AmB and PM was found to be 96% and 90% respectively. The morphology of m-DDSLNs was confirmed by scanning electron microscopy and transmission electron microscopy. The developed formulations revealed a sustained drug release profile upto 57% (AmB) and 21.5% (PM) within 72 h and were stable at both 4 °C and 25 °C during short term stability studies performed for 2 months. Confocal laser scanning microscopy confirmed complete cellular internalization of SLNs within 24 h of incubation. In vitro cytotoxicity study against J774A.1 macrophage cells confirmed the safety and biocompatibility of the developed formulations. Further, m-DDSLNs did not induce any hepatic/renal toxicities in Swiss albino mice. The in vitro simulated study was performed to check the stability in simulated gastric fluids and simulated intestinal fluids and the release was found almost negligible. The in vitro anti-leishmanial activity of m-DDSLNs (1 µg/ml) has shown a maximum percentage of inhibition (96.22%) on intra-cellular amastigote growth of L. donovani. m-DDSLNs (20 mg/kg × 5 days, p.o.) has significantly (P < 0.01) reduced the liver parasite burden as compared to miltefosine (3 mg/kg × 5 days, p.o.) in L. donovani-infected BALB/c mice. This work suggests that the superiority of as-prepared m-DDSLNs as a promising approach towards the oral delivery of anti-leishmanial drugs.

Evaluation of Safety and Antileishmanial Efficacy of Amine Functionalized Carbon-Based Composite Nanoparticle Appended With Amphotericin B: An in vitro and Preclinical Study.

Visceral leishmaniasis (VL) has been a major health concern in the developing world, primarily affecting impoverished people. It is caused by a protozoan parasite Leishmania donovani and is characterized by immune dysfunction that can lead to deadly secondary infections. Several adverse side effects limit the existing treatment options; hence, the need of the hour is some drug option with high efficacy and no toxicity. To make targeted delivery of Amphotericin B (AmB), we have used amine-functionalized versions of carbon nanostructures, namely f-CNT and f-Graphene (f-Grap). The results with f-Grap-AmB, because of a much larger surface area, were expected to be better. However, the results obtained by us showed only marginal improvement (IC50 f-Grap-AmB; 0.0038 ± 0.00119 µg/mL). This is, in all likelihood, due to the agglomeration effect of f-Grap-AmB, which is invariably obtained with graphene. To resolve this issue, we have synthesized a graphene-CNT composite (graphene 70% and CNT 30% by weight). Because CNT is dispersed in between graphene sheets, the agglomeration effect is avoided, and our study suggests that the f-Composite-AmB (f-Comp-AmB) showed no toxicity against the murine J774A.1 macrophage cell line and did not induce any hepatic or renal toxicity in Swiss albino mice. The f-Comp-AmB also showed a remarkable elevation in the in vitro and in vivo antileishmanial efficacy in comparison to AmB and f-CNT-AmB or f-Grap-AmB in J774A.1 and Golden Syrian hamsters, respectively. Additionally, we have also observed that the percentage suppression of parasite replication in the spleen of the hamster was significantly higher in the f-Comp-AmB (97.79 ± 0.2375) treated group in comparison with the AmB (85.66 ± 1.164) treated group of hamsters. To conclude, f-Comp-AmB could be a safe and reliable therapeutic option over the other carbon-based nanoparticles (NPs), i.e., f-CNT-AmB, f-Grap-AmB, and conventional AmB.

Effect of Arsenic Stress on Expression Pattern of a Rice Specific miR156j at Various Developmental Stages and Their Allied Co-expression Target Networks.

In plants, arsenic (As) stress modulates metabolic cascades at various developmental stages by influencing the pattern of gene expressions mediated by small non-coding RNAs, especially Micro-RNAs, involved in the moderation of a myriad of cellular processes needed for plant adaptation upon oxidative stress. miR156j of miR156 gene family, involved mainly in the regulation of growth and development in plants. This study was designed to find out the role of arsenic toxicity on Osa-miR156j expression in all physiological growth stages. To better understand the functional role of Osa-miR156j in rice, we observed the expression in different developmental stages (seedlings, tillering and flowering) and various tissues of leaf, stem and root tissues (at 0, 24, 48, and 72 h) under 25 µM arsenite [As (III)] exposure. Additionally, using bioinformatic tools to target genes of Osa-miR156j and the potential co-expressed genes were explored at different development stages in the various tissues of rice under stress conditions. The expression of Osa-miR156j showed its temporal downregulation in various tissues in different developmental stages. Of note, the downregulation was more pronounced in root tissues at seedlings, tillering, and flowering stages during 0-72 h under arsenite exposure as compared to other tissues. Overall, the As stress altered the gene expression more prominently at seedlings developmental stage followed by flowering and tillering. Additionally, through the In silico approach, the target functions and presence of oxidative stress-responsive cis-acting regulatory elements/motifs also confirmed Osa-miR156j involvement in the regulation of arsenic stress in rice. The findings of this study demonstrate the prominent role of Osa-miR156j in rice under arsenite stress, which was found to modulate the metabolic activities in rice plants at different developmental stages, and thus it might be useful for the development of arsenic tolerant varieties.

Post kala-azar dermal leishmaniasis: A threat to elimination program.

Leishmaniasis remains a public health concern around the world that primarily affects poor folks of the developing world spanning across 98 countries with mortality of 0.2 million to 0.4 million annually. Post kala-azar dermal leishmaniasis (PKDL) is the late skin manifestation of visceral leishmaniasis (VL). It has been reported that about 2.5% to 20% of patients recovered from VL develop PKDL having stilted macular or nodular lesions with parasites. In the Indian subcontinent (ISC), it manifests a few months after recovery from VL, though in Africa it can occur simultaneously with VL or a little later. New cases of PKDL are also observed without prior VL in the ISC. These individuals with PKDL represent an important but largely neglected reservoir of infection that perpetuates anthroponotic Leishmania donovani transmission in the ISC and can jeopardize the VL elimination program as these cases can infect the sand flies and spread the endemic. Therefore, it becomes imperative to eradicate PKDL as a part of the VL elimination program. With the limited treatment options besides little knowledge on PKDL, this review stands out in focusing on different aspects that should be dealt for sustained VL elimination.

Epigenetic Aspects of Engineered Nanomaterials: Is the Collateral Damage Inevitable?

The extensive application of engineered nanomaterial (ENM) in various fields increases the possibilities of human exposure, thus imposing a huge risk of nanotoxicity. Hence, there is an urgent need for a detailed risk assessment of these ENMs in response to their toxicological profiling, predominantly in biomedical and biosensor settings. Numerous "toxico-omics" studies have been conducted on ENMs, however, a specific "risk assessment paradigm" dealing with the epigenetic modulations in humans owing to the exposure of these modern-day toxicants has not been defined yet. This review aims to address the critical aspects that are currently preventing the formation of a suitable risk assessment approach for/against ENM exposure and pointing out those researches, which may help to develop and implement effective guidance for nano-risk assessment. Literature relating to physicochemical characterization and toxicological behavior of ENMs were analyzed, and exposure assessment strategies were explored in order to extrapolate opportunities, challenges, and criticisms in the establishment of a baseline for the risk assessment paradigm of ENMs exposure. Various challenges, such as uncertainty in the relation of the physicochemical properties and ENM toxicity, the complexity of the dose-response relationships resulting in difficulty in its extrapolation and measurement of ENM exposure levels emerged as issues in the establishment of a traditional risk assessment. Such an appropriate risk assessment approach will provide adequate estimates of ENM exposure risks and will serve as a guideline for appropriate risk communication and management strategies aiming for the protection and the safety of humans.

Envisioning the innovations in nanomedicine to combat visceral leishmaniasis: for future theranostic application.

Visceral leishmaniasis (VL) is a life-threatening parasitic disease affecting impoverished people of the developing world; and much effort has been spent on the early case detection and treatment. However, current diagnostics and treatment options are not sufficient for appropriate surveillance in VL elimination setting. Hence, there is a dire need to develop highly sensitive diagnostics and less toxic effective treatments for proper management of cases and to achieve the sustained disease elimination. Although, promising results have been observed with nanomedicines in leishmaniasis; there are great challenges ahead especially in translating this to clinical setting. This review provides updated progress of nanomedicines in VL, and discussed how these innovations and future directions play vital role in achieving VL elimination.

Centrin-Deleted Leishmania donovani Parasites Help CD4+ T Cells to Acquire Th1 Phenotype and Multi-Functionality Through Downregulation of CD200-CD200R Immune Inhibitory Axis.

The protozoan parasite Leishmania has evolved several strategies to undermine host defense mechanisms by inducing Th2-type adaptive immunity and suppressing effector functions of Th1 phenotype. In our earlier studies, using centrin gene-deleted Leishmania (LdCen-/-) parasites as an immunogen, we have shown induction of an effective Th1-type immunity and robust memory responses that mediate protection against virulent challenge. However, role of inhibitory signals in Leishmania vaccine induced immunity in general, and LdCen-/- in particular has not been studied. Herein, we report that immunization with LdCen-/- parasites produces more functional Th1-type CD4+ T cells via downregulation of CD200-CD200R immune inhibitory axis compared to wild-type infection. We found that expression of CD200 and CD200R was significantly reduced in LdCen-/- infection compared to wild-type infection. Diminished CD200-CD200R signaling in LdCen-/- infection enabled proliferation of CD4+ T cells and resulted in the induction of pro-inflammatory cytokines and suppression of anti-inflammatory response. The effects of diminished CD200-CD200R signaling by LdCen-/- were most evident in the suppression of IL-10-producing CD4+ T cells that helped enhance more Th1 cytokine producing and multi-functional T cells compared to wild-type infection. In vivo blocking of CD200 expression with anti-CD200 treatment in wild-type infected mice limited Th2 response as indicated by reduction of IL-10-producing Tr1 cells and reduced parasite burden. On the other hand, treatment with anti-CD200 improved the LdCen-/- vaccine-induced multifunctional response and reduction in splenic parasite load upon challenge. Taken together, these studies demonstrate the role of CD200-CD200R signals in the protection induced by LdCen-/- parasites.

Limitations of Current Therapeutic Options, Possible Drug Targets and Scope of Natural Products in Control of Leishmaniasis.

Soon after the identification of Leishmania parasite as a causative agent, the pentavalent antimony compounds have been the mainstay to treat all forms of leishmaniasis. Due to growing incidences of antimony resistant parasites and unavailability of true antileishmanial compounds, few drugs like pentamidine (antimicrobial), amphotericin B (antifungal) or miltefosine (antitumor) are currently being used but these are associated with serious side effects. Unfortunately, the emergence of amphotericin B and miltefosine resistant parasites in clinical settings has further questioned their sustained use in leishmanial control. Moreover, the parameters of protective immunity are not well understood in leishmanial pathogenesis therefore, a vaccine candidate, either prophylactic or preventive, is still an unrealized goal. In addition, the emergence of insecticide resistance sand flies in disease endemic regions also stance a big threat for the current elimination strategies. Therefore, in lieu of the limited drug regimen and unavailability of a vaccine, the necessity of a true antileishmanial agent is always there. Although, leishmanial infections have been neglected for many decades but recent studies have identified potential drug targets that could be targeted to control the growth of parasites. In recent past many compounds derived from natural sources have also been shown to possess excellent antiparasitic potential; however, most of these studies are limited to primary evaluation and only a few have reached to clinical levels. In this review, we discuss the limitations of current drug regimen, explore possible drug targets of Leishmania species and summarize wide range of compounds isolated from various natural sources that are worth screening as antileishmanial drug candidates.

Leishmania donovani infection activates Toll-like receptor 2, 4 expressions and Transforming growth factor-beta mediated apoptosis in renal tissues

Abstract The present study was aimed to identify the underlying mechanisms of improper renal function in Leishmania donovani infection that causes VL. Mice (BALB/c) were infected with L. donovani and different parameters for proteinuria were assessed. The levels of superoxide anion (O2 -), hydrogen peroxide (H2O2), lipid peroxidation (MDA), inflammatory cytokines, and toll-like receptor (TLR) 2 and 4 expression were found significantly elevated at 60th day in these animals and declined at 90th day post infection. However, TGF-β and caspase 3 activities were higher at 90th day in comparison to 60th day post infection. These findings suggested that exacerbated inflammatory conditions correlate with abnormal renal functions in L. donovani infection, which is further augmented by activated TLRs expressions by circulating leishmanial antigens. Further, the increased levels of TGF-β and caspase 3 at 90th day suggested TGF-β mediated apoptotic cell death of renal and other cells during later stages of disease that may eventually result in release of host and parasitic factors in urine during visceral leishmaniasis.

Solute carrier protein family 11 member 1 (Slc11a1) activation efficiently inhibits Leishmania donovani survival in host macrophages.

Visceral leishmaniasis (kala-azar), a life threatening disease caused by L. donovani, is a latent threat to more than 147 million people living in disease endemic South East Asia region of the Indian subcontinent. The therapeutic option to control leishmanial infections are very limited, and at present comprise only two drugs, an antifungal amphotericin B and an antitumor miltefosine, which are also highly vulnerable for parasitic resistance. Therefore, identification and development of alternate control measures is an exigent requirement to control leishmanial infections. In this study, we report that functionally induced expression of solute carrier protein family 11 member 1 (Slc11a1), a transmembrane divalent cationic transporter recruited on the surface of phagolysosomes after phagocytosis of parasites, effectively inhibits Leishmania donovani growth in host macrophages. Further, the increased Slc11a1 functionality also resulted in increased production of NOx, TNF-α and IL-12 by activated macrophages. The findings of this study signify the importance of interplay between Slc11a1 expression and macrophages activation that can be effectively used to control of Leishmania growth and survival.

Leishmania donovani infection activates Toll-like receptor 2, 4 expressions and Transforming growth factor-beta mediated apoptosis in renal tissues.

The present study was aimed to identify the underlying mechanisms of improper renal function in Leishmania donovani infection that causes VL. Mice (BALB/c) were infected with L. donovani and different parameters for proteinuria were assessed. The levels of superoxide anion (O2-), hydrogen peroxide (H2O2), lipid peroxidation (MDA), inflammatory cytokines, and toll-like receptor (TLR) 2 and 4 expression were found significantly elevated at 60th day in these animals and declined at 90th day post infection. However, TGF-ß and caspase 3 activities were higher at 90th day in comparison to 60th day post infection. These findings suggested that exacerbated inflammatory conditions correlate with abnormal renal functions in L. donovani infection, which is further augmented by activated TLRs expressions by circulating leishmanial antigens. Further, the increased levels of TGF-ß and caspase 3 at 90th day suggested TGF-ß mediated apoptotic cell death of renal and other cells during later stages of disease that may eventually result in release of host and parasitic factors in urine during visceral leishmaniasis.

Corrigendum: Identification and Characterization of miRNAs in Response to Leishmania donovani Infection: Delineation of Their Roles in Macrophage Dysfunction.

[This corrects the article on p. 314 in vol. 8, PMID: 28303124.].

Identification and Characterization of miRNAs in Response to Leishmania donovani Infection: Delineation of Their Roles in Macrophage Dysfunction.

The outcome of Leishmania infection depends on parasite abilities to evade host immune response and its survival in hostile environment of host macrophages. Despite a wealth of gained crucial information, parasite strategies by which it dampens host macrophage functions remain poorly understood. Micro RNAs (miRNAs) are evolutionarily conserved class of endogenous 22-nucleotide small non-coding RNA gene products, described to participate in the regulation of almost every cellular process investigated so far. In this study, we identified 940 miRNAs in Leishmania donovani infected macrophages by de novo sequencing out of which levels of 85 miRNAs were found to be consistently modified by parasite infection. Herein, we report the functional characteristics of 10 miRNAs i.e., mir-3620, mir-6385, mir-6973a, mir-6996, mir-328, mir-8113, mir-3473f, mir-763, mir-6540, and mir-1264 that were differentially but constantly regulated in infected macrophages for their role in regulation of macrophage effector functions. The target gene prediction and biological interaction analysis revealed involvement of these miRNAs in various biological processes such as apoptosis inhibition, phagocytosis, drug response, and T cell phenotypic transitions. These findings could contribute for the better understanding of macrophages dysfunction and leishmanial pathogenesis. Further, the identified miRNAs could also be used as biomarker/s in diagnosis, prognosis, and therapeutics of Leishmania infection.