A review of non-invasive samples and tools in kala-azar diagnosis and test of cure.

The recurrence of visceral leishmaniasis (VL), also called kala-azar (KA), in endemic regions of tropical countries like India, is primarily attributed to asymptomatic VL, post-kala azar dermal leishmaniasis (PKDL), and human immunodeficiency virus (HIV) co-infection. To effectively manage VL cases and elimination targets, an early and rapid diagnosis as well as accurate field surveillance is highly essential. The traditional sampling methods like bone marrow (BM), spleen, and lymph node (LN) tissue aspirations are invasive, painful, tedious, and prone to nosocomial infections, require skilled persons and hospital facilities, and are not feasible in rural areas. Therefore, there is an urgent requirement for the adoption of a patient-friendly, non-invasive, non-hospitalized sampling procedure that ensures an effective VL diagnosis. This review aims to meticulously evaluate the most recent scientific research that focuses on the precision, feasibility, and applicability of non-invasive sampling (NIS) and techniques for the diagnosis and test of cure of VL, particularly in resource-limited settings. Apart from that, the non-invasive techniques (NIT) that have shown promising results while monitoring VL treatment response and relapse are also reviewed. The limitations associated with NIT and possible improvements in this regard are discussed as well to improve the diagnosis and management of VL.

The application of MD simulation to lead identification, vaccine design, and structural studies in combat against leishmaniasis - A review.

Drug discovery, vaccine design, and protein interaction studies are rapidly moving toward the routine use of molecular dynamics simulations (MDS) and related methods. As a result of MDS, it is possible to gain insights into the dynamics and function of identified drug targets, antibody-antigen interactions, potential vaccine candidates, intrinsically disordered proteins, and essential proteins. The MDS appears to be used in all possible ways in combating diseases such as cancer, however, it has not been well documented as to how effectively it is applied to infectious diseases such as Leishmaniasis. As a result, this systematic review aims to survey the application of MDS in combating leishmaniasis. We have systematically collected articles that illustrate the implementation of MDS in drug discovery, vaccine development, and structural studies related to Leishmaniasis. Of all the articles reviewed, we identified that only a limited number of studies focused on the development of vaccines against Leishmaniasis through MDS. Also, the PCA and FEL studies were not carried out in most of the studies. These two were globally accepted utilities to understand the conformational changes and hence it is recommended that this analysis should be taken up in similar approaches in the future.

In silico screening, molecular dynamic simulations, and in vitro activity of selected natural compounds as an inhibitor of Leishmania donovani 3-mercaptopyruvate sulfurtransferase.

In Leishmania sp., the enzymes of de novo cysteine biosynthesis pathway require sulfide. Other organisms utilize sulfide through the sulfide reduction pathway, but Leishmania lacks the gene that encodes these enzymes. Hence, the major source of sulfide for Leishmania is believed to be from the action of 3-mercaptopyruvate sulfurtransferase (3MST) on 3-mercapto-pyruvate (3MP). There has been no effort reported in the past to screen inhibitors against L. donovani 3MST (Ld3MST). As a result, this study examines natural compounds that are potent against Ld3MST and validates it by in vitro activity and cytotoxicity tests. Initially, a library of ~ 5000 natural compounds was subjected to molecular docking approach for screening, and the best hit was validated using a long-term molecular dynamic simulation (MD). Among the docking results, quercetine-3-rutinoside (Rutin) was deemed the best hit. The results of the MD indicated that Rutin was highly capable of interacting with the varied active site segments, possibly blocking substrate access. Additionally, promastigotes and amastigotes were tested for Rutin activity and the IC50 was found to be 40.95 and 90.09 µM, respectively. Similarly, the cytotoxicity assay revealed that Rutin was not toxic even at a concentration of 819.00 µM to THP-1 cell lines. Additionally, the Ld3MST was cloned, purified, and evaluated for enzyme activity in the presence of Rutin. Reduction in the enzyme activity (~ 85%) was observed in the presence of ~ 40 µM Rutin. Thus, this study suggests that Rutin may act as a potent inhibitor of Ld3MST. With further in vivo investigations, Rutin could be a small molecule of choice for combating leishmaniasis.

Harnessing Fuzzy Rule Based System for Screening Major Histocompatibility Complex Class I Peptide Epitopes from the Whole Proteome: An Implementation on the Proteome of Leishmania donovani.

The development of peptide-based vaccines is enhanced by immunoinformatics, which predicts the patterns that B cells and T cells recognize. Although several tools are available for predicting the Major histocompatibility complex (MHC-I) binding peptides, the wide variants of human leucocyte antigen allele make it challenging to choose a peptide that will induce an immune response in a majority of people. In addition, for a peptide to be considered a potential vaccine candidate, factors such as T cell affinity, proteasome cleavage, and similarity to human proteins also play a major role. Identifying peptides that satisfy the earlier cited measures across the entire proteome is, therefore, challenging. Hence, the fuzzy inference system (FIS) is proposed to detect each peptide's potential as a vaccine candidate and assign it either a very high, high, moderate, or low ranking. The FIS includes input features from 6 modules (binding of 27 major alleles, T cell propensity, pro-inflammatory response, proteasome cleavage, transporter associated with antigen processing, and similarity with human peptide) and rules derived from an observation of features on positive samples. On validation of experimentally verified peptides, a balanced accuracy of ∼80% was achieved, with a Mathew's correlation coefficient score of 0.67 and an F-1 score of 0.74. In addition, the method was implemented on complete proteome of Leishmania donovani, which contains ∼4,800,000 peptides. Lastly, a searchable database of the ranked results of the L. donovani proteome was made and is available online (MHC-FIS-LdDB). It is hoped that this method will simplify the identification of potential MHC-I binding candidates from a large proteome.

Differential translational regulation of host exosomal proteins play key role in immunomodulation in antimony resistance in Visceral Leishmaniasis: A proteomic profiling study.

In host-pathogen interactions, exosomal secretions are crucial for cell to cell communication and have an established role in immunomodulation. Protozoans, including Leishmania, modulates their host vesicular secretions for better survival; although the role of exosomal secretions in unresponsive against sodium antimony gluconate (SAG) has never been documented. In this study, the exosomal proteome of RAW macrophages infected with either SAG responsive (SAGS) or SAG unresponsive (SAGR) L. donovani parasites has been compared with uninfected RAW macrophages. Proteins isolated from exosomes were labelled with iTRAQ reagents; followed by subsequent LC-TOF/-MS analysis. In total, 394 proteins (p < 0.05) were identified which were shared common among all sets. Highly differentially expressed proteins were sorted by log2 value -1 and +1 as down regulated and up regulated respectively which yielded 58 proteins in SAGR and 41 proteins during SAGS infection. Out of the 58 proteins identified during SAGR infection, 17 proteins were of immune modulatory function. Network visualization model and pathway analysis revealed the interactions among these proteins via different immunological pathways with reported involvement of some proteins in SAG resistance and host immune modulation. Hence, the differential abundance of immune pathway related proteins in exosomes of infected host during SAGR infection supports the immune modulatory strategy adopted by SAG resistant parasites for enhanced survival .

Exploring nod factor receptors activation process in chickpea by bridging modelling, docking and molecular dynamics simulations.

Plasma membrane-bound receptor proteins play crucial roles in the perception and further transmission of regulatory signals to modulate numerous developmental and metabolic events. Precise functioning and fine-tuning of Nod factor receptor (NFR) mediated signalling is a critical requirement for root nodule symbiosis. Here, we have identified, cloned and phylogenetically characterized chickpea NFR1 and NFR5, which are showing significant homology with other legume NFR receptors. Homology modelling and molecular dynamics simulations highlight the molecular structure of ligand binding ectodomains [EDs] and cytosolic kinase domains [KDs] of NFRs in chickpea. Our detailed structural analysis also revealed that both NFR1 and NFR5 share resemblance as well as dissimilarity in sequence, structure and substrate-binding pocket. Further, molecular docking simulations provide us adequate insights into the active site of receptors where the Nod factor (NF) binds. The outcome of this work sheds light on the binding specificity of NFs towards NFRs and thus may significantly contribute to the design of new strategies in improving root-nodule symbiosis towards meeting the agricultural demands.

Differentially modulated proteins associated with Leishmaniasis-a systematic review of in-vivo and in-vitro studies.

High-throughput proteomic technologies are widely used for understanding the disease mechanism, drug-resistant mechanism, and to identify drug targets and markers for diagnostics. Studies with proteomics applications, relating to Leishmaniasis, are being constantly reported in the literature. However, from such studies, a readily accessible knowledge of differentially modulated proteins associated with Leishmaniasis is lacking. Hence, we performed a systematic review concerning differentially modulated proteins (DMP) in Leishmania as well as host infected with Leishmania from the published articles between the years 2000 and 2019. This review is classified into five different sections, namely, DMP in the host after Leishmania infection, DMP between different strains of Leishmania, DMP in drug-resistant Leishmania, DMP in Leishmania under stress, and DMP in different life stages of Leishmania. A lot of consensuses could be observed among the DMP in drug-resistant and stressed Leishmania. In addition to the review, a database was constructed with the data collected in this study (protein accession ID, protein name, gene name, host organism, experimental conditions, fold change, and regulatory data). A total of 2635 records are available in the database. We believe this review and the database will help the researcher in understanding the disease better and provide information for the targeted proteomics study related to Leishmaniasis. Database availability: http://ldepdb.biomedinformri.com/ .

TrypInDB: A searchable online resource of small molecule inhibitors against Trypanosoma sp.

African Trypanosomiasis and American Trypanosomiasis are the diseases affecting more than thousands of people yearly and more than twenty-five million people risk acquiring the disease. The treatment for the disease is generally expensive, and most of the available drugs are of high-toxicity and cause fatal side-effects. Hence, there is a constant need for finding new treatment strategies for Trypanosomiasis. Combination therapy and repurposing or redesigning of existing inhibitors for new drugs are of high importance to address these hurdles, particularly the drug resistance. Hence, here we report TrypInDB, a searchable online resource of small molecule inhibitors having a varying degree of activity towards Trypanosoma sp. Information of about >14,000 small molecules from >700 published research articles was collected and made as an easy-to-search database. Four major sets of information were made available for each collected inhibitors viz., General information (activity values; source of the inhibitors; enzyme targets; etc.,), Structural information, Toxicity information, and Literature information. More than 25 different information about each inhibitor were collected or predicted and made accessible for searching. The database is designed to be queried easily with multiple-field filters with the provisions to perform sub-structure search and similar FDA approved drug searches. The database supports the easy export of queried records and structure in multiple formats. In addition, the TrypInDB is actively integrated into LeishInDB. We believe that the scope of TrypInDB permits the research community to exploit the available data for repurposing the inhibitors as well as for the investigation of new therapeutics. Database URL: http://trypindb.biomedinformri.com/.

Leishmania donovani infection induce differential miRNA expression in CD4+ T cells.

Visceral leishmaniasis is characterized by mixed production of Th1/2 cytokines and the disease is established by an enhanced level of Th2 cytokine. CD4+ T cells are main cell type which produces Th1/2 cytokine in the host upon Leishmania infection. However, the regulatory mechanism for Th1/2 production is not well understood. In this study, we co-cultured mice CD4+ T cells with Leishmania donovani infected and uninfected macrophage for the identification of dysregulated miRNAs in CD4+ T cells by next-generation sequencing. Here, we identified 604 and 613 known miRNAs in CD4+ T cells in control and infected samples respectively and a total of only 503 miRNAs were common in both groups. The expression analysis revealed that 112 miRNAs were up and 96 were down-regulated in infected groups, compared to uninfected control. Nineteen up-regulated and 17 down-regulated miRNAs were statistically significant (p < 0.05), which were validated by qPCR. Further, using insilco approach, we identified the gene targets of significant miRNAs on the basis of CD4+ T cell biology. Eleven up-regulated miRNAs and 9 down-regulated miRNAs were associated with the cellular immune responses and Th1/2 dichotomy upon Leishmania donovani infection. The up-regulated miRNAs targeted transcription factors that promote differentiation of CD4+ T cells towards Th1 phenotype. While down-regulated miRNAs targeted the transcription factors that facilitate differentiation of CD4+ T cells towards Th2 populations. The GO and pathway enrichment analysis also showed that the identified miRNAs target the pathway and genes related to CD4+ T cell biology which plays important role in Leishmania donovani infection.

Structural, molecular motions, and free-energy landscape of Leishmania sterol-14α-demethylase wild type and drug resistant mutant: a comparative molecular dynamics study.

Sterol-14α-demethylase (CYP51) is an ergosterol pathway enzyme crucial for the survival of infectious Leishmania parasite. Recent high-throughput metabolomics and whole genome sequencing study revealed amphotericin B resistance in Leishmania is indeed due to mutation in CYP51. The residue of mutation (asparagine 176) is conserved across the kinetoplastidae and not in yeast or humans, portraying its functional significance. In order to understand the possible cause for the resistance, knowledge of structural changes due to mutation is of high importance. To shed light on the structural changes of wild and mutant CYP51, we conducted comparative molecular dynamics simulation study. The active site, substrate biding cavity, substrate channel entrance (SCE), and cavity involving the mutated site were studied based on basic parameters and large concerted molecular motions derived from essential dynamics analyses of 100 ns simulation. Results indicated that mutant CYP51 is stable and less compact than the wild type. Correspondingly, the solvent accessible surface area (SASA) of the mutant was found to be increased, especially in active site and cavities not involving the mutation site. Free-energy landscape analysis disclosed mutant to have a rich conformational diversity than wild type, with various free-energy conformations of mutant having SASA greater than wild type with SCE open. More residues were found to interact with the mutant CYP51 upon docking of substrate to both the wild and mutant CYP51. These results indicate that, relative to wild type, the N176I mutation of CYP51 in Leishmania mexicana could possibly favor increased substrate binding efficiency.

In-silico screening and validation of high-affinity tetra-peptide inhibitor of Leishmania donovani O-acetyl serine sulfhydrylase (OASS).

OASS is a specific enzyme that helps Leishmania parasite to survive the oxidative stress condition in human macrophages. SAT C-terminal peptides in several organisms, including Leishmania, were reported to inhibit or reduce the activity of OASS. Small peptide and small molecules mimicking the SAT C-terminal residues are designed and tested for the inhibition of OASS in different organisms. Hence, in this study, all the possible tetra-peptide combinations were designed and screened based on the docking ability with Leishmania donovani OASS (Ld-OASS). The top ranked peptides were further validated for the stability using 50 ns molecular dynamic simulation. In order to identify the better binding capability of the peptides, the top peptides complexed with Ld-OASS were also subjected to molecular dynamic simulation. The docking and simulation results favored the peptide EWSI to possess greater advantage than previously reported peptide (DWSI) in binding with Ld-OASS active site. Also, screening of non-peptide inhibitor of Asinex Biodesign library based on the shape similarity of EWSI and DWSI was performed. The top similar molecules of each peptides were docked on to Ld-OASS active site and subsequently simulated for 20 ns. The results suggested that the ligand that shares high shape similarity with EWSI possess better binding capability than the ligand that shares high shape similarity with DWSI. This study revealed that the tetra-peptide EWSI had marginal advantage over DWSI in binding with Ld-OASS, thereby providing basis for defining a pharmacophoric scaffold for the design of peptidomimetic inhibitors as well as non-peptide inhibitors of Ld-OASS. Communicated by Ramaswamy H. Sarma.

Validation of NAD synthase inhibitors for inhibiting the cell viability of Leishmania donovani: In silico and in vitro approach.

NAD (nicotinamide adenine dinucleotide) synthase catalyses the biochemical synthesis of NAD, from nicotinic acid adenine dinucleotide (NAAD). NAD may be synthesized through the de novo pathways and/or the salvage pathways in cells. However, in Leishmania parasite, the synthesis of NAD solely depends on the salvage pathways. NAD synthetase is widely explored as a drug target in various microorganisms. In Bacillus anthracis, a group of sulphonamides 5599, 5617 and 5824 and complex amide 5833 were reported to have activity at micromolar range against NAD synthetase. Hence, in the present study, the same group of sulphonamides and complex amide were validated through in silico and in vitro studies for its efficiency towards Leishmania donovani NAD synthase. In silico study revealed the ligands 5824 and 5833 to have better docking score. Molecular dynamics simulation for a duration of 50 ns of all the ligand-protein complexes suggested that the complexes with the ligands 5824 and 5833 were stable and interacting. In vitro and ex vivo studies have shown that 5824 and 5833 inhibit the cell viability of the organism at a lower concentration than 5599 and 5617. Hence, with further in vivo validation, 5824 (or its synthetic analogues) and 5833 could be the choice that may work synergistically with other potential drugs in treating drug-resistant cases of leishmaniasis. Communicated by Ramaswamy H. Sarma.

Exploring microRNAs, Target mRNAs and their Functions in Leguminous Plant Arachis hypogaea.

BACKGROUND: MicroRNAs (miRNAs) are a class of small non-coding, endogenous RNAs that regulate gene expression at post-transcriptional level. In plants, miRNAs are usually of 18-24 nucleotide in length and play humongous role by aiding in development, growth, defense, biotic and abiotic stress responses, etc. Objective: Arachis hypogaea is an economically important oil seed crop and human dietary source cultivated mostly in tropical and subtropical regions. In the present study, an initiative was taken to uncover miRNAs, their targets and functions in this important plant species. METHOD: Comparative genomics strategy coupled with bioinformatics approaches was deployed for the identification of miRNAs, their corresponding targets and functions by exploiting biological databases and tools. RESULTS: The study was able to identify 34 conserved miRNA candidates, belonging to 17 miRNA families, contributed by 23 and 3 precursor miRNAs from A. hypogaea Expressed Sequence Tags (EST) and Genome Survey Sequences (GSS), respectively. As well, 495 EST and 917 unigene sequences were predicted as targets for the identified miRNAs. Herein, psRNAtarget server and TargetFinder tool were used to predict unigene targets, whereas comparative genomics strategy was used for identifying EST targets. Functional annotation of the identified targets revealed that the identified miRNAs regulate mRNAs that participate in key biological and metabolic processes. Pathway enrichment analysis using KEGG database also revealed that they regulate important metabolic pathways including antibiotic biosynthesis, biosynthesis of unsaturated fatty acids, amino acids metabolism and flavonoid biosynthesis. CONCLUSION: The outcome of the study would aid experimental biologists to focus on these miRNAs to facilitate improved crop development and yield.

LeishInDB: A web-accessible resource for small molecule inhibitors against Leishmania sp.

Despite the availability of drugs to treat Leishmaniasis, various other factors including drug resistance and adverse side effects encourage the researchers to search for new strategies and alternatives for treating Leishmaniasis. Repurposing and devising combination therapy with the existing small molecules would serve as an alternative strategy to address the issue, especially the drug resistance. Hence, here we report LeishInDB, a web-accessible resource of small molecule inhibitors having a varying degree of activity towards Leishmania sp. The database includes searchable information of >7000 small molecules collected from >600 literature. The comprehensive information of inhibitors mainly include the activity details (IC50, EC50, Ki, binding energy etc., if any); information on species and form of Leishmania the inhibitor is active against; and the details about their protein target (actively linked to TriTrypDB). In addition, chemical properties including the log P-value, number of rotatable bonds, number of hydrogen bond donors and acceptors, molecular weight, 2D/3D structural information etc., were also included. Toxicity prediction for each molecule was performed using admetSAR and their corresponding results were available to perform the filtered search. In addition, facility to perform sub-structure search, facility to perform the dynamic search on various fields, and facility to download all the structure of molecules that match the search criteria were also included. We believe that the scope of LeishInDB allows the researchers to utilize the available information for repurposing the inhibitors as well as for the investigation of new therapeutics. Database URL:http://leishindb.biomedinformri.com/.

Recent progress in drug targets and inhibitors towards combating leishmaniasis.

Lesihmaniasis is one of the major neglected tropical disease caused by the parasite of the genus Leishmania. The disease has more than one clinical forms and the visceral form is considered fatal. With the lack of potential vaccine, chemotherapy is the major treatment source considered for the control of the disease in the infected people. Drugs including amphotericin B and miltefosine are widely used for the treatment, however, development of resistance by the parasite towards the administered drug and high-toxicity of the drug are of major concern. Hence, more attention has been shown on identifying new targets, effective inhibitors, and better drug delivery system against the disease. This review deals with recent studies on drug targets and exploring their essentiality for the survival of Leishmania. Further, new inhibitors for those targets, novel anti-leishmanial peptides and vaccines against leishmaniasis were discussed. We believe that this pool of information will ease the researchers to gain knowledge and help in choosing right targets and design of new inhibitors against Leishmaniasis.

Allosteric site-mediated active site inhibition of PBP2a using Quercetin 3-O-rutinoside and its combination.

Recent crystallographic study revealed the involvement of allosteric site in active site inhibition of penicillin binding protein (PBP2a), where one molecule of Ceftaroline (Cef) binds to the allosteric site of PBP2a and paved way for the other molecule (Cef) to bind at the active site. Though Cef has the potency to inhibit the PBP2a, its adverse side effects are of major concern. Previous studies have reported the antibacterial property of Quercetin derivatives, a group of natural compounds. Hence, the present study aims to evaluate the effect of Quercetin 3-o-rutinoside (Rut) in allosteric site-mediated active site inhibition of PBP2a. The molecular docking studies between allosteric site and ligands (Rut, Que, and Cef) revealed a better binding efficiency (G-score) of Rut (-7.790318) and Cef (-6.194946) with respect to Que (-5.079284). Molecular dynamic (MD) simulation studies showed significant changes at the active site in the presence of ligands (Rut and Cef) at allosteric site. Four different combinations of Rut and Cef were docked and their G-scores ranged between -6.320 and -8.623. MD studies revealed the stability of the key residue (Ser403) with Rut being at both sites, compared to other complexes. Morphological analysis through electron microscopy confirmed that combination of Rut and Cefixime was able to disturb the bacterial cell membrane in a similar fashion to that of Rut and Cefixime alone. The results of this study indicate that the affinity of Rut at both sites were equally good, with further validations Rut could be considered as an alternative for inhibiting MRSA growth.

Mammalian Mitochondrial ncRNA Database.

UNLABELLED: Mammalian Mitochondrial ncRNA is a web-based database, which provides specific information on non-coding RNA in mammals. This database includes easy searching, comparing with BLAST and retrieving information on predicted structure and its function about mammalian ncRNAs. AVAILABILITY: The database is available for free at http://www.iitm.ac.in/bioinfo/mmndb/.

Quercetin 3-O-rutinoside mediated inhibition of PBP2a: computational and experimental evidence to its anti-MRSA activity.

The PBP2a is a cell wall synthesizing protein, which causes resistivity in methicillin resistant Staphylococcus aureus (MRSA) from ß-lactam antibiotics but it is susceptible to 5th generation cephalosporin, ceftobiprole. Ceftobiprole inhibits the growth of MRSA by targeting the PBP2a-mediated cell wall synthesis, but it is reported to have adverse side effects. Due to this, there is a constant need to develop natural alternatives, which are generally free from adverse side effects. Hence in this study, in silico based docking analysis was performed with 37 quercetin derivatives towards PBP2a inhibition and their efficiencies were compared with ß-lactam antibiotic, ceftobiprole. The docking studies suggested that quercetin 3-O-rutinoside (ZINC5280805) interacted efficiently with PBP2a, attaining the highest LibDock score (187.32) compared to other quercetin derivatives. The structural stability and dynamics of the identified lead with PBP2a were validated through molecular dynamics simulation. Simulation results such as RMSD, RMSF, and Rg values indicated that the stability of quercetin 3-O-rutinoside with PBP2a was better, with respect to the un-ligated PBP2a. Furthermore, the quercetin 3-O-rutinoside was subjected to an antibacterial susceptibility test and found to have antibacterial activity at 500, 700, and 900 µM concentration. Also, morphological changes in the bacterial colony and bacterial surface were observed using a scanning electron microscope, when MRSA was treated with 900 µM concentration of quercetin 3-O-rutinoside. Collectively, results from this study suggest that the quercetin 3-O-rutinoside has the capability to inhibit PBP2a and hence could be used as an alternative or in combination with other drugs in treating MRSA infection.