Leishmania protein KMP-11 modulates cholesterol transport and membrane fluidity to facilitate host cell invasion.

The first step of successful infection by any intracellular pathogen relies on its ability to invade its host cell membrane. However, the detailed structural and molecular understanding underlying lipid membrane modification during pathogenic invasion remains unclear. In this study, we show that a specific Leishmania donovani (LD) protein, KMP-11, forms oligomers that bridge LD and host macrophage (MΦ) membranes. This KMP-11 induced interaction between LD and MΦ depends on the variations in cholesterol (CHOL) and ergosterol (ERG) contents in their respective membranes. These variations are crucial for the subsequent steps of invasion, including (a) the initial attachment, (b) CHOL transport from MΦ to LD, and (c) detachment of LD from the initial point of contact through a liquid ordered (Lo) to liquid disordered (Ld) membrane-phase transition. To validate the importance of KMP-11, we generate KMP-11 depleted LD, which failed to attach and invade host MΦ. Through tryptophan-scanning mutagenesis and synthesized peptides, we develop a generalized mathematical model, which demonstrates that the hydrophobic moment and the symmetry sequence code at the membrane interacting protein domain are key factors in facilitating the membrane phase transition and, consequently, the host cell infection process by Leishmania parasites.

A mechanistic study on the tolerance of PAM distal end mismatch by SpCas9.

The therapeutic application of CRISPR-Cas9 is limited due to its off-target activity. To have a better understanding of this off-target effect, we focused on its mismatch-prone PAM distal end. The off-target activity of SpCas9 depends directly on the nature of mismatches, which in turn results in deviation of the active site of SpCas9 due to structural instability in the RNA-DNA duplex strand. In order to test the hypothesis, we designed an array of mismatched target sites at the PAM distal end and performed in vitro and cell line-based experiments, which showed a strong correlation for Cas9 activity. We found that target sites having multiple mismatches in the 18th to 15th position upstream of the PAM showed no to little activity. For further mechanistic validation, Molecular Dynamics simulations were performed, which revealed that certain mismatches showed elevated root mean square deviation values that can be attributed to conformational instability within the RNA-DNA duplex. Therefore, for successful prediction of the off-target effect of SpCas9, along with complementation-derived energy, the RNA-DNA duplex stability should be taken into account.

Antimony resistance mechanism in genetically different clinical isolates of Indian Kala-azar patients.

The central theme of this enterprise is to find common features, if any, displayed by genetically different antimony (Sb)-resistant viscerotropic Leishmania parasites to impart Sb resistance. In a limited number of clinical isolates (n = 3), we studied the breadth of variation in the following dimensions: (a) intracellular thiol content, (b) cell surface expression of glycan having N-acetyl-D-galactosaminyl residue as the terminal sugar, and (c) gene expression of thiol-synthesizing enzymes (CBS, MST, gamma-GCS, ODC, and TR), antimony-reducing enzymes (TDR and ACR2), and antimonial transporter genes (AQP1, MRPA, and PRP1). One of the isolates, T5, that was genotypically characterized as Leishmania tropica, caused Indian Kala-azar and was phenotypically Sb resistant (T5-LT-SSG-R), while the other two were Leishmania donovani, out of which one isolate, AG83, is antimony sensitive (AG83-LD-SSG-S) and the other isolate, T8, is Sb resistant (T8-LD-SSG-R). Our study showed that the Sb-resistant parasites, regardless of their genotype, showed significantly higher intracellular thiol compared with Sb-sensitive AG83-LD-SSG-S. Seemingly, T5-LT-SSG-R showed about 1.9-fold higher thiol content compared with T8-LD-SSG-R which essentially mirrored cell surface N-acetyl-D-galactosaminyl expression. Except TR, the expression of the remaining thiol-synthesizing genes was significantly higher in T8-LD-SSG-R and T5-LT-SSG-R than the sensitive one, and between the Sb-resistant parasites, the latter showed a significantly higher expression. Furthermore, the genes for Sb-reducing enzymes increased significantly in resistant parasites regardless of genotype compared with the sensitive one, and between two resistant parasites, there was hardly any difference in expression. Out of three antimony transporters, AQP1 was decreased with the concurrent increase in MRPA and PRP1 in resistant isolates when compared with the sensitive counterpart. Interestingly, no difference in expression of the above-mentioned transporters was noted between two Sb-resistant isolates. The enduring image that resonated from our study is that the genetically diverse Sb-resistant parasites showed enhanced thiol-synthesizing and antimony transporter gene expression than the sensitive counterpart to confer a resistant phenotype.

Glucogallin Attenuates the LPS-Induced Signaling in Macrophages and Protects Mice against Sepsis.

The anti-oxidant and anti-inflammatory effect of beta-glucogallin (BGG), a plant-derived natural product, was evaluated in both in vitro and in vivo studies. For the in vitro study, the ability of BGG pre-treatment to quench LPS-induced effects compared to LPS alone in macrophages was investigated. It was found that BGG pre-treatment showed a significant decrease in ROS, NO, superoxide, and pro-inflammatory cytokines (TNF-alpha, IL-4, IL-17, IL-1ß, and IL-6) and increased reduced glutathione coupled with the restoration of mitochondrial membrane potential. Gene profiling and further validation by qPCR showed that BGG pre-treatment downregulated the LPS-induced expression of c-Fos, Fas, MMP-9, iNOS, COX-2, MyD88, TRIF, TRAF6, TRAM, c-JUN, and NF-κB. We observed that BGG pre-treatment reduced nuclear translocation of LPS-activated NF-κB and thus reduced the subsequent expressions of NLRP3 and IL-1ß, indicating the ability of BGG to inhibit inflammasome formation. Molecular docking studies showed that BGG could bind at the active site of TLR4. Finally, in the LPS-driven sepsis mouse model, we showed that pre-treatment with BGG sustained toxic shock, as evident from their 100% survival. Our study clearly showed the therapeutic potential of BGG in toxic shock syndrome.

Detection of Leptomonas seymouri narna-like virus in serum samples of visceral leishmaniasis patients and its possible role in disease pathogenesis.

Kala-azar/Visceral Leishmaniasis (VL) caused by Leishmania donovani (LD) is often associated with Leptomonas seymouri (LS) co-infection in India. Leptomonas seymouri narna-like virus 1 (Lepsey NLV1) has been reported in multi-passaged laboratory isolates of VL samples which showed LD-LS co-infection. A pertinent question was whether this virus of LS is detectable in direct clinical samples. DNA from the serum of twenty-eight LD diagnosed patients was subjected to LD-specific and LS-specific PCR to reconfirm the presence of LD parasites and to detect LD-LS co-infections. RNA extracted from same samples was subjected to RT-PCR, qRT-PCR and sequencing using virus-specific primers to detect/identify and quantify the virus. The presence of the virus was confirmed in thirteen of eighteen (72%) recently collected VL and PKDL samples. Cytokine profiling showed significantly elevated IL-18 in only LD infected patients compared to the virus-positive LD and control samples. IL-18 is crucial for Th1 and macrophage activation which eventually clears the parasite. The Lepsey NLV1 interaction with the immune system results in reduced IL-18 which favors LD survival and increased parasitic burden. The study emphasizes the need to revisit LD pathogenesis in the light of the association and persistence of a protozoan virus in kala-azar and PKDL patients.

An insight into SARS-CoV-2 structure, pathogenesis, target hunting for drug development and vaccine initiatives.

SARS-CoV-2, the virus responsible for the COVID-19 pandemic, has been confirmed to be a new coronavirus having 79% and 50% similarity with SARS-CoV and MERS-CoV, respectively. For a better understanding of the features of the new virus SARS-CoV-2, we have discussed a possible correlation between some unique features of the genome of SARS-CoV-2 in relation to pathogenesis. We have also reviewed structural druggable viral and host targets for possible clinical application if any, as cases of reinfection and compromised protection have been noticed due to the emergence of new variants with increased infectivity even after vaccination. We have also discussed the types of vaccines that are being developed against SARS-CoV-2. In this review, we have tried to give a brief overview of the fundamental factors of COVID-19 research like basic virology, virus variants and the newly emerging techniques that can be applied to develop advanced treatment strategies for the management of COVID-19 disease.

A Short Review on Glucogallin and its Pharmacological Activities.

Plant derived natural products have multifaceted beneficial roles in human pathophysiology. Plant secondary metabolites have been used as an adjunct medicine for a long time and ß- Glucogallin is one such pharmaceutically important plant derived natural product. Β-glucogallin (1-O-galloyl-ß-d-glucopyranose), a plant-derived polyphenolic ester, is regarded as the primary metabolite in the biosynthesis of hydrolyzable tannins. It is majorly found in amla, pomegranate, strawberry etc. Owing to its free radical scavenging properties, ß-glucogallin (BG) is believed to protect against several diseases like diabetes and related complications like retinopathy, glaucoma, inflammation, hepatic damage, skin damage from UV, etc. Several semisynthetic derivatives of ß-Glucogallin are being developed, which have better pharmacokinetic and pharmacodynamic parameters than ß-glucogallin. Studies have shown the prophylactic role of ß-Glucogallin in developing defence mechanisms against the advent and progression of certain diseases. ß- glucogallin formulations have shown a positive effect as a neutraceutical. In this manuscript, we have discussed ß-glucogallin, its natural sources, biosynthetic pathways, its semi-synthetic derivatives, and the plethora of its pharmacological activities like antioxidant-antiinflammatory, antidiabetic, cataract-preventing, anti glaucoma, and UV protectant. We have also highlighted various biological pathways, which are modulated by ß-glucogallin. The manuscript will convey the importance of ß-glucogallin as a compound of natural origin, having multifaceted health benefits.

Wnt5A Signaling Blocks Progression of Experimental Visceral Leishmaniasis.

Visceral leishmaniasis, caused by L. donovani infection is fatal if left untreated. The intrinsic complexity of visceral leishmaniasis complicated further by the increasing emergence of drug resistant L. donovani strains warrants fresh investigations into host defense schemes that counter infections. Accordingly, in a mouse model of experimental visceral leishmaniasis we explored the utility of host Wnt5A in restraining L. donovani infection, using both antimony sensitive and antimony resistant L. donovani strains. We found that Wnt5A heterozygous (Wnt5A +/-) mice are more susceptible to L. donovani infection than their wild type (Wnt5A +/+) counterparts as depicted by the respective Leishman Donovan Units (LDU) enumerated from the liver and spleen harvested from infected mice. Higher LDU in Wnt5A +/- mice correlated with increased plasma gammaglobulin level, incidence of liver granuloma, and disorganization of splenic white pulp. Progression of infection in mice by both antimony sensitive and antimony resistant strains of L. donovani could be prevented by activation of Wnt5A signaling through intravenous administration of rWnt5A prior to L. donovani infection. Wnt5A mediated blockade of L. donovani infection correlated with the preservation of splenic macrophages and activated T cells, and a proinflammatory cytokine bias. Taken together our results indicate that while depletion of Wnt5A promotes susceptibility to visceral leishmaniasis, revamping Wnt5A signaling in the host is able to curb L. donovani infection irrespective of antimony sensitivity or resistance and mitigate the progression of disease.

A Review on CRISPR-mediated Epigenome Editing: A Future Directive for Therapeutic Management of Cancer.

Recent studies have shed light on the role of epigenetic marks in certain diseases like cancer, type II diabetes mellitus (T2DM), obesity, and cardiovascular dysfunction, to name a few. Epigenetic marks like DNA methylation and histone acetylation are randomly altered in the disease state. It has been seen that methylation of DNA and histones can result in down-regulation of gene expression, whereas histone acetylation, ubiquitination, and phosphorylation are linked to enhanced expression of genes. How can we precisely target such epigenetic aberrations to prevent the advent of diseases? The answer lies in the amalgamation of the efficient genome editing technique, CRISPR, with certain effector molecules that can alter the status of epigenetic marks as well as employ certain transcriptional activators or repressors. In this review, we have discussed the rationale of epigenetic editing as a therapeutic strategy and how CRISPR-Cas9 technology coupled with epigenetic effector tags can efficiently edit epigenetic targets. In the later part, we have discussed how certain epigenetic effectors are tagged with dCas9 to elicit epigenetic changes in cancer. Increased interest in exploring the epigenetic background of cancer and non-communicable diseases like type II diabetes mellitus and obesity accompanied with technological breakthroughs has made it possible to perform large-scale epigenome studies.

A Novel Bioimpedance-Based Detection of Miltefosine Susceptibility Among Clinical Leishmania donovani Isolates of the Indian Subcontinent Exhibiting Resistance to Multiple Drugs.

The extent of susceptibility towards miltefosine (Mil), amphotericin B (AmpB), and paromomycin (Paro) was measured among 19 clinical isolates of Leishmania donovani (LD). Thirteen of these clinical isolates were reported to exhibit low susceptibility towards sodium stibogluconate (SSG-R), while six of them were highly susceptible (SSG-S). The degree of clearance of amastigotes (EC50) for these predefined SSG-R- and SSG-S-infected macrophages was determined against Mil, AmpB, and Paro. Two out of the 13 SSG-R isolates (BHU575 and BHU814) showed low susceptibility towards all three drugs studied, while the rest of the 11 SSG-R isolates showed varying degrees of susceptibility either towards none or only towards individual drugs. Interestingly, all the SSG-S isolates showed high susceptibility towards Mil/AmpB/Paro. The total intracellular non-protein thiol content of the LD promastigotes, which have been previously reported to be positively co-related with EC50 towards SSG, was found to be independent from the degree of susceptibility towards Mil/AmpB/Paro. Impedance spectra analysis, which quantifies membrane resistance, revealed lower impedimetric values for all those isolates exhibiting low efficacy to Mil (Mil-R). Our analysis points out that while non-protein thiol content can be an attribute of SSG-R, lower impedimetric values can be linked with lower Mil susceptibility, although neither of these parameters seems to get influenced by the degree of susceptibility towards AmpB/Paro. Finally, a correlation analysis with established biological methods suggests that impedance spectral analysis can be used for the accurate determination of lower Mil susceptibility among LD isolates, which is further validated in the LD-infected in vivo hamster model.

CRISPR detectives against SARS-CoV-2: a major setback against COVID-19 blowout.

The emergence of SARS-CoV-2 has brought the world to a standstill, and till date, effective treatments and diagnostics against this idiosyncratic pathogen are lacking. As compared to the standard WHO/CDC qPCR detection method, which consumes several hours for detection, CRISPR-based SHERLOCK, DETECTR, and FELUDA have emerged as rapid diagnostic tools for the detection of the RNA genome of SARS-CoV-2 within an hour with 100% accuracy, specificity, and sensitivity. These attributes of CRISPR-based detection technologies have taken themselves one step ahead of available detection systems and are emerging as an inevitable tool for quick detection of the virus. Further, the discovery of Cas13s nucleases and their orthologs has opened a new corridor for exploitation of Cas13s as an antiviral therapy against SARS-CoV-2 and other viral diseases. One such approach is Prophylactic Antiviral CRISPR in huMAN cells (PACMAN), which needs a long haul to bring into therapy. The approval of SHERLOCK as the first CRISPR-based SARS-CoV-2 test kit by the FDA, for emergency diagnosis of COVID-19 patients, has given positive hope to scientists that sooner human trials of CRISPR-based therapy will be ratified. In this review, we have extensively reviewed the present CRISPR-based approaches, challenges, and future prospects in the light of diagnostics and therapeutics against SARS-CoV-2. KEY POINTS: • The discovery of Cas12 and Cas13 siblings allowed scientists to detect the viral genes. • Cas13d's identification aided scientists in precisely cleaving the SARS-CoV-2 ssRNA. • CRISPR-Cas system acts as "molecular detector and antiviral proctor."

Genome wide comparison of Leishmania donovani strains from Indian visceral leishmaniasis and para-kala-azar dermal leishmaniasis patients.

Visceral leishmaniasis (VL) or Kala-azar, primarily caused by Leishmania donovani, is a major health concern in many countries including India. Growing unresponsiveness among the parasites toward the available drugs is alarming, and so, it is necessary to decipher the underlying mechanism of such development for designing new therapeutics. Moreover, even after successful treatment, some VL patients develop apparently harmless skin lesions known as post-kala-azar dermal leishmaniasis (PKDL) which may serve as a reservoir of the parasite in the transmission cycle. Furthermore, recent reports of para-kala-azar dermal leishmaniasis (para-KDL) cases having PKDL manifestation with concomitant VL, emphasize the necessity of more attention to address complex nature of the parasite for eradicating the disease effectively. In the present study, whole genome sequencing is performed with sodium stibogluconate (SSG) sensitive and resistant L. donovani strains along with SSG sensitive para-KDL strains, derived from the clinical isolates of Indian patients to identify the genomic variations among them. Notably, the analyses of chromosome somy values and genome wide mutation profile in the coding regions reveal distinct clustering of the para-KDL strains with 24 genes being mutated uniquely in this group. Such distinguishing genomic changes among the para-KDL strains could be significant for the parasites to become dermatotropic. Overall, the study reveals a possible correlation of the development of SSG resistance and the transition towards the manifestation of PKDL with chromosome aneuploidy and non-synonymous genetic variations in the coding sequences of the L. donovani strains from Indian patients.

Modulation of Splenic B Cell Subsets during Experimental Leishmania donovani Infection in BALB/c Mice.

Sodium antimonials are one of the major and common drugs used against visceral form leishmaniasis (VL). However, the development of drug resistance makes it difficult to manage this disease. Current work investigates the modulation of splenic B cells during experimental infection with antimony-sensitive and -resistant Leishmania donovani infection. Here we phenotypically characterized splenic B cell subsets in BALB/c mice infected with antimony drug-sensitive and -resistant VL strains using flow-cytometry method. In the splenocytes we noticed increased number of Transitional T3 B cells and B1a B cells in drug-resistant VL strain infection. Besides, we also observed alteration in Follicular B cell population of antimony-resistant strain infected mice. Drug-resistant strain induced secretion of elevated level of IL-10 from B1a B cells and IL-6 from Transitional T3 B cell subsets in the splenocytes. Purified splenic B cells from antimony drug-resistant strain infected mice showed decrease in the Lyn kinase gene expression compared to sensitive strain infected and uninfected mice. The current study provides insight into changes in host splenic B-cell subsets during experimental infection with antimony-sensitive and -resistant L. donovani in murine model.

A mimotope attached to an ITIM-SHP-1 interaction inhibitory peptide boosts immune response and efficacy.

Antigen binding to the B-cell receptor initiates a downstream signalling pathway that contains both stimulatory and damping components. A malarial parasite-derived conformation-constrained peptide was conjugated to a signal-damping pathway inhibitor. Mice immunized with this antigen produced higher antibody levels which delayed parasitemia. This represents a new approach to antigen design.

Computational screening of FDA approved drugs of fungal origin that may interfere with SARS-CoV-2 spike protein activation, viral RNA replication, and post-translational modification: a multiple target approach.

Coronavirus spread is an emergency reported globally, and a specific treatment strategy for this significant health issue is not yet identified. COVID-19 is a highly contagious disease and needs to be controlled promptly as millions of deaths have been reported. Due to the absence of proficient restorative alternatives and preliminary clinical restrictions, FDA-approved medications can be a decent alternative to deal with the coronavirus malady (COVID-19). The present study aims to meet the imperative necessity of effective COVID-19 drug treatment with a computational multi-target drug repurposing approach. This study focused on screening the FDA-approved drugs derived from the fungal source and its derivatives against the SARS-CoV-2 targets. All the selected drugs showed good binding affinity towards these targets, and out of them, bromocriptine was found to be the best candidate after the screening on the COVID-19 targets. Further, bromocriptine is analyzed by molecular simulation and MM-PBSA study. These studies suggested that bromocriptine can be the best candidate for TMPRSS2, Main protease, and RdRp protein. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40203-021-00089-8.

Application of CRISPR/Cas System in the Metabolic Engineering of Small Molecules.

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated Cas protein technology area is rapidly growing technique for genome editing and modulation of transcription of several microbes. Successful engineering in microbes requires an emphasis on the aspect of efficiency and targeted aiming, which can be employed using CRISPR/Cas system. Hence, this type of system is used to modify the genome of several microbes such as yeast and bacteria. In recent years, CRISPR/Cas systems have been chosen for metabolic engineering in microbes due to their specificity, orthogonality, and efficacy. Therefore, we need to review the scheme which was acquired for the execution of the CRISPR/Cas system for the modification and metabolic engineering in yeast and bacteria. In this review, we highlighted the application of the CRISPR/Cas system which has been used for the production of small molecules in the microbial system that is chemically and biologically important.

Modification of Cas9, gRNA and PAM: Key to further regulate genome editing and its applications.

The discovery of CRISPR-Cas9 system has revolutionized the genome engineering research and has been established as a gold standard genome editing platform. This system has found its application in biochemical researches as well as in medical fields including disease diagnosis, development of therapeutics, etc. The enormous versatility of the CRISPR-Cas9 as a high throughput genome engineering platform, is derailed by its off-target activity. To overcome this, researchers from all over the globe have explored the system structurally and functionally and postulated several strategies to upgrade the system components including redesigning of Cas9 Nuclease and modification of guide RNA(gRNA) structure and customization of the protospacer adjacent motif. Here in this review, we portray the comprehensive overview of the strategies that has been adopted for redesigning the CRISPR-Cas9 system to enhance the efficiency and fidelity of the technology.

A review on potential of natural products in the management of COVID-19.

At the end of 2019, a life threatening viral infection (COVID-19) caused by a novel coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was reported. This virus has spread worldwide in a short duration and forced the world to face unprecedented life and economic loss. To date, there are no known specific drugs to combat this virus and the process for new drug development is lengthy. Most promising candidates, which emerged as potential leads, were abandoned in the later phases of clinical trials. Repurposing of already approved drugs for other therapeutic applications can be done only after extensive testing for safety and efficacy. With no definite therapeutics in the horizon, natural products are in extensive use arbitrarily as anti-viral agents and immune boosters. For ages it has been known that most natural products possess potent anti-viral activity and it is no different for SARS-CoV-2. It has been shown that natural products display inhibitory effects on MERS-CoV and SARS-CoV infections. In silico studies have shown that various natural products have strong binding affinity for and inhibitory action on the non-structural proteins of the virus, namely PLPRO, MPRO, and RdRp, and structural proteins such as spike (S) protein. Since the virus utilizes the transmembrane ACE2 receptor of the host cell, it also proves to be a valid target for drug development. In this review promising targets for drug development against SARS-CoV-2 and anti-viral activities of some of the known natural products are discussed.

Probing the molecular mechanism of aggressive infection by antimony resistant Leishmania donovani.

The disease visceral leishmaniasis (VL) or kala azar is caused by the protozoan parasite, Leishmania donovani (LD). For many decades the pentavalent antimonial drugs countered the successive epidemics of the disease in the Indian sub-continent and elsewhere. With time, antimony resistant LD (LDR) developed and the drug in turn lost its efficacy. Infection of mammals with LDR gives rise to aggressive infection as compared to its sensitive counterpart (LDS) coupled with higher surge of IL-10 and TGF-ß. The IL-10 causes upregulation of multidrug resistant protein-1 which causes efflux of antimonials from LDR infected cells. This is believed to be a key mechanism of antimony resistance. MicroRNAs (miRNAs) are tiny post-transcriptional regulators of gene expression in mammalian cells and in macrophage play a pivotal role in controlling the expression of cytokines involved in infection process. Therefore, a change in miRNA profiles of macrophages infected with LDS or LDR could explain the differential cytokine response observed. Interestingly, the outcome of LD infection is also governed by the critical balance of pro- and anti-inflammatory cytokines which is inturn regulated by miRNA-Ago2 or miRNP complex and its antagonist RNA binding protein HuR. Here Ago2 plays the fulcrum whose phosphorylation and de-phosphorylation dictates the process; which in turn is controlled by PP2A and HuR. LDS and LDR upregulate PP2A and downregulate HuR at different magnitude leading to various levels of anti-inflammatory to proinflammatory cytokine production and resulting pathology in the host. While ectopic HuR expression alone is sufficient to clear LDS infection, simultaneous upregulation of HuR and inhibition of PP2A is required to inhibit LDR mediated infection. Therefore, tampering with miRNA pathway could be a new strategy to control infection caused by LDR parasite.