Identification of a De Novo Peptide against Palmitoyl Acyltransferase 6 to Block Survivability and Infectivity of Leishmania donovani.

Palmitoylation is an essential post-translational modification in Leishmania donovani, catalyzed by enzymes called palmitoyl acyl transferases (PATs) and has an essential role in virulence. Due to the toxicity and promiscuity of known PAT inhibitors, identification of new molecules is needed. Herein, we identified a specific novel de novo peptide inhibitor, PS1, against the PAT6 Leishmania donovani palmitoyl acyl transferase (LdPAT6). To demonstrate specific inhibition of LdPAT6 by PS1, we employed a bacterial orthologue system and metabolic labeling-coupled click chemistry where both LdPAT6 and PS1 were coexpressed and displayed palmitoylation suppression. Furthermore, strong binding of the LdPAT6-DHHC domain with PS1 was observed through analysis using microscale thermophoresis, ELISA, and dot blot assay. PS1 specific to LdPAT6 showed significant growth inhibition in promastigotes and amastigotes by expressing low cytokines levels and invasion. This study reveals discovery of a novel de novo peptide against LdPAT6-DHHC which has potential to block survivability and infectivity of L. donovani.

Biological evaluation of novel side chain containing CQTrICh-analogs as antimalarials and their development as PfCDPK1 kinase inhibitors.

The rapid emergence of resistance to existing frontline antimalarial drugs emphasizes a need for the development of target-oriented molecules with novel modes of action. Given the importance of a plant-like Calcium-Dependent Protein Kinase 1 (PfCDPK1) as a stand-alone multistage signalling regulator of P. falciparum, we designed and synthesized 7-chloroquinoline-indole-chalcones tethered with a triazole (CQTrICh-analogs 7 (a-s) and 9) directed towards PfCDPK1. This was accomplished by reacting substituted 1-phenyl-3-(1-(prop-2-yn-1-yl)-1H-indol-3-yl) prop-2-en-1-one and 1-(prop-2-yn-1-yl)-1H-indole-3-carbaldehyde with 4-azido-7-chloroquinoline, respectively via a 'click' reaction. The selected CQTrICh-analogs: 7l and 7r inhibited the growth of chloroquine-sensitive 3D7 strain and -resistant RKL-9 isolate of Plasmodium falciparum, with IC50 values of 2.4 µM & 1.8 µM (7l), and 3.5 µM & 2.7 µM (7r), respectively, and showed no apparent hemolytic activity and cytotoxicity in mammalian cells. Intra-erythrocytic progression studies revealed that the active hybrids: 7l and 7r are effective against the mature stages of the parasite. 7l and 7r were found to stably interact with the catalytically active ATP-binding pocket of PfCDPK1 via energetically favourable H-bonds. The interaction was confirmed in vitro by microscale thermophoresis and kinase assays, which demonstrated that the active hybrids interact with PfCDPK1 and inhibit its kinase activity which is presumably responsible for the parasite growth inhibition. Interestingly, 7l and 7r showed no inhibitory effect on the human kinases, indicating their selectivity for the parasite kinase. We report the antiplasmodial potential of novel kinase-targeting bio-conjugates, a step towards developing pan-kinase inhibitors which is a prerequisite for multistage anti-malarial protection.

Epithelial homelessness: an atypical form of anoikis triggered by Leishmania interaction with epithelial cells.

Aim: Leishmaniasis is characterized by a spectrum of diseases with two main clinical forms, cutaneous and visceral, caused by Leishmania tropica and Leishmania donovani, respectively. Studying Leishmania's interaction with the epithelial barrier at the initial site of a bite is crucial to understanding the establishment of the disease. Materials & methods: To discern parasite-host epithelial interaction, we developed in vitro cellular models involving co-cultures of Leishmania and MDCK epithelial cells. Results: Both L. donovani-MDCK and L. tropica-MDCK co-culture models demonstrated a phenomenon known as atypical anoikis apoptosis, typically identified by distinctive 'flipping in' of cell membranes and disordered cytoskeletal frameworks. Conclusion: This study bridges the gap in the fundamental understanding of the intricate latticework involving vector-Leishmania-host and may inform drug development strategies.

Small parasites called Leishmania are passed to humans through the bites of sandflies. These parasites cause three deadly forms of disease: one that affects the organs, one that causes skin lesions and one that affects organ linings. This study looked at how Leishmania parasites behave when they enter through the skin. We found that when the parasites were in contact with cells, the cells changed their shape and lost contact with neighboring cells. This led to a type of cell death known as anoikis, a Greek term meaning 'homelessness'.

Host-Erythrocytic Sphingosine-1-Phosphate Regulates Plasmodium Histone Deacetylase Activity and Exhibits Epigenetic Control over Cell Death and Differentiation.

The evolution of resistance to practically all antimalarial drugs poses a challenge to the current malaria elimination and eradication efforts. Given that the epigenome of Plasmodium falciparum governs several crucial parasite functions, pharmaceutical interventions with transmission-blocking potential that target epigenetic molecular markers and regulatory mechanisms are likely to encounter drug resistance. In the malaria parasite, histone deacetylases (HDACs) are essential epigenetic modulators that regulate cellular transcriptional rearrangements, notably the molecular mechanisms underlying parasite proliferation and differentiation. We establish "lipid sequestration" as a mechanism by which sphingolipids, specifically Sphingosine-1-Phosphate (S1P) (a metabolic product of Sphingosine Kinase 1 [SphK-1]), regulate epigenetic reprogramming in the parasite by interacting with, and modulating, the histone-deacetylation activity of PfHDAC-1, thereby regulating Plasmodium pathogenesis. Furthermore, we demonstrate that altering host S1P levels with PF-543, a potent and selective Sphk-1 inhibitor, dysregulates PfHDAC-1 activity, resulting in a significant increase in the global histone acetylation signals and, consequently, transcriptional modulation of genes associated with gametocytogenesis, virulence, and proliferation. Our findings point to a hitherto unrecognized functional role for host S1P-mediated sphingolipid signaling in modulating PfHDAC-1's enzymatic activity and, as a result, the parasite's dynamic genome-wide transcriptional patterns. The epigenetic regulation of parasite proliferation and sexual differentiation offers a novel approach for developing host-targeted therapeutics to combat malaria resistance to conventional regimens. IMPORTANCE Sphingolipid is an 18-carbon amino-alcohol-containing lipid with a sphingosine backbone, which when phosphorylated by sphingosine kinase 1 (SphK-1), generates sphingosine-1-phosphate (S1P), an essential lipid signaling molecule. Dysregulation of S1P function has been observed in a variety of pathologies, including severe malaria. The malaria parasite Plasmodium acquires a host S1P pool for its growth and survival. Here, we describe the molecular attuning of histone deacetylase-1 (PfHDAC-1), a crucial epigenetic modulator that contributes to the establishment of epigenetic chromatin states and parasite survival, in response to S1P binding. Our findings highlight the host lipid-mediated epigenetic regulation of malaria parasite key genes.

Development of Mitochondria Targeting AIE-Active Cyclometalated Iridium Complexes as Potent Antimalarial Agents.

The emergence of resistance to conventional antimalarial treatments remains a major cause for concern. New drugs that target the distinct development stages of Plasmodium parasites are required to address this risk. Herein, water-soluble aggregation-induced emission active cyclometalated iridium(III) polypyridyl complexes (Ir1-Ir12) are developed for the elimination of malaria parasites. Remarkably, these complexes show potent antimalarial activity in low nanomolar range against 3D7 (chloroquine and artemisinin sensitive strain), RKL9 (chloroquine resistant strain), and R539T (artemisinin resistant strains) strains of Plasmodium falciparum with faster killing rate of malaria parasites. Concomitantly, these complexes exhibit efficient in vivo antimalarial activity against both the asexual and gametocyte stages of Plasmodium berghei malaria parasite, suggesting promising transmission-blocking potential. The complexes tend to localize into mitochondria of P. falciparum determined by image and cell-based assay. The mechanistic studies reveal that these complexes exert their antimalarial activity by increasing reactive oxygen species levels and disrupting its mitochondrial membrane potential. Furthermore, the mitochondrial-dependent antimalarial activity of these complexes is confirmed in yeast model. Thus, this study for the first time highlights the potential role of targeting P. falciparum mitochondria by iridium complexes in discovering and developing the next-generation antimalarial agents for treating multidrug resistant malaria parasites.

'Erythritol', a safe natural sweetener exhibits multi-stage anti-malarial activity by permeating into Plasmodium falciparum through aquaglyceroporin channel.

The increased resistance of human malaria parasite Plasmodium falciparum (Pf) to currently used drugs necessities the development of novel anti-malarials. Here, we examine the potential of erythritol, a sugar substitute for therapeutic intervention. Erythritol is a permeant of Plasmodium falciparum aquaglyceroporin (PfAQP) which is a multifunctional channel responsible for maintaining hydro-homeostasis. We show that erythritol effectively inhibited growth and progression of asexual blood stage malaria parasite, and effect invasion and egress processes. It also inhibited the liver stage (sporozoites) and transmission stage parasite (gametocytes) development. Interestingly, erythritol inhibited in vivo growth of malaria parasite in mouse experimental model. It was more effective in inhibiting parasite growth both in vivo and in vitro when tested together with a known anti-malarial 'artesunate'. Additionally, erythritol showed cytokine-modulating effect which suggests its direct effect on the host immune system. Ammonia detection assay demonstrated that erythritol uptake effects the amount of ammonia release across the parasite. Our functional complementation assays suggest that PfAQP expression in yeast mutant restores its growth in hyperosmotic conditions but showed reduced growth in the presence of erythritol. Osmotic lysis assay suggests that erythritol creates osmotic stress for killing the parasite. Overall, our data bestow erythritol as a promising lead compound with an attractive antimalarial profile and could possibly be combined with known drugs without losing its efficacy. We propose the use of erythritol based sweet candies for protection against malaria specially in children living in the endemic area.

The effects of autologous platelet concentrate on the healing of intra-bony defects: a randomized clinical trial.

PURPOSE: The present study aimed to evaluate the clinical and radiographic effects of autologous platelet concentrate (APC) on the healing of intra-bony defects filled with ß-tricalcium phosphate (ß-TCP) and covered with collagen membranes. SUBJECTS AND METHODS: This study included 30 defects of 14 systemically healthy subjects. All of them had, at least, two deep intra-bony, inter-proximal periodontal defects. Minimum probing pocket depth (PPD) was 6 mm. Clinical and imaging examination was performed both at baseline and at 3, 6, and 9 months after surgery. RESULTS: Both the test and control group revealed a significant reduction in all variables when compared with the base line. Mean reduction of the PPD in two groups at each follow-up time point showed no significant difference. Means of the clinical attachment gain of the same groups were significantly different (p<0.05). Mean gingival recession at 3 month was not significant. However, the means of gingival recession coverage of two groups were significantly different at 6 and 9 months (p<0.05 for both). CONCLUSION: Sites treated with APC are more likely to demonstrate more clinical attachment gain and recession coverage at the end of 9 month compared to those without APC.

Clinical Efficacy of Subgingivally Delivered 1.2 mg Simvastatin in the Treatment of Patients with Aggressive Periodontitis: A Randomized Controlled Clinical Trial.

Simvastatin (SMV) is a specific competitive inhibitor of 3-hydroxy-2-methylglutaryl coenzyme A reductase that promotes bone formation. The present clinical trial was designed to investigate the effectiveness of 1.2 mg SMV as a local drug delivery system and as an adjunct to scaling and root planing (SRP) in the treatment of aggressive periodontitis (AgP). A total of 68 intrabony defects from 24 patients with AgP were treated either with 1.2 mg SMV gel or placebo gel. The subjects were randomly assigned to SRP + placebo (group 1; n = 12) or SRP + SMV (group 2; n = 12). Clinical parameters were recorded at baseline and at 3 and 6 months and included bleeding index, Plaque Index, probing depth (PD), and clinical attachment level (CAL). At baseline and after 6 months, radiologic assessment of bone defect fill was done. The mean decrease in PD at 6 months was 1.14 ± 0.04 mm and 3.78 ± 0.62 mm in groups 1 and 2, respectively. Significant gain in mean CAL was found between the groups (P < .05). Furthermore, significantly greater mean percentage of bone fill was found in group 2 (34.01%) compared to group 1 (2.62%). Locally delivered SMV provides a comfortable method to improve clinical parameters and promotes bone formation.

Subgingivally Delivered 3% Satranidazole in the Treatment of Chronic Periodontitis among Smokers: A Randomized, Controlled Clinical Trial.

BACKGROUND: The present clinical trial was designed to investigate the effectiveness of subgingivally delivered satranidazole (SZ) gel as an adjunct to scaling and root planing (SRP) in the treatment of smokers with chronic periodontitis. METHODS: Sixty smoker subjects with probing depth (PD) ≥ 5 mm were selected. Thirty subjects each were randomly assigned to SRP + placebo and SRP + SZ. SZ or placebo (0.1 mL) was injected into the pocket using a syringe with a blunt cannula. The clinical outcomes evaluated were plaque index (PI), gingival index (GI), clinical attachment level (CAL) and PD at baseline, 1 month, 3 months and 6 months. RESULTS: At 6 months, SRP + SZ resulted in greater mean reduction (3.05 mm) in PD as compared to SRP + placebo (1.97 mm; p < 0.05) and also a greater mean CAL gain (2.89 mm) in SRP + SZ as compared to SRP + placebo (1.88 mm; p < 0.05). CONCLUSION: When compared to the placebo, the adjunctive use of 3% SZ resulted in significant improvement in clinical outcome in the treatment of chronic periodontitis among smokers.

Neonatal zygomycosis with gastric perforation.

Zygomycosis is a rare infection in neonates. The clinical presentation is non-specific and diagnosis most often is made at autopsy. Surgical debridement performed early improves survival. We report a case of neonatal zygomycosis with gastric perforation.

Omental cyst presenting as tubercular ascites.

Cystic lymphangiomas are uncommon congenital benign tumours of vascular origin with a lymphatic differentiation originating across various anatomical locations. Large intrabdominal cysts may mimic ascites. We report the case of a one-and-a-half-year-old male child with a giant cystic lymphangioma originating in the greater omentum presenting as tubercular ascites. This report aims to highlight the limitations of biochemical investigations such as ascitic adenosine deaminase (ADA) in differentiating the epidemiologically prevalent tubercular ascites from an intrabdominal cyst, especially in a resource-poor nation as ours, where invasive diagnostic procedures pose an economic burden.

Chemotherapy induced nail changes.

Anticancer chemotherapy is associated with a variety of nail changes. We present two children who developed different nail changes, while receiving almost similar antineoplastic drugs.

Celiac disease in a child with beta-thalassemia major: a need for improved screening and awareness.

Growth failure is one of the most common problems in children with thalassemia with multiple etiologies. We present a case of celiac disease, an underdiagnosed cause of growth failure in a child with beta-thalassemia major. A 10-year-old boy on a hypertransfusion regimen was referred for early onset growth failure. Serology for hepatitis B, hepatitis C, and HIV was negative. Serum zinc levels were normal. Thyroid function tests and growth hormone secretion, evaluated with clonidine stimulation test were normal. Malabsorption syndrome was suspected, even in the absence of gastrointestinal symptoms. Tissue transglutaminase were highly raised >300 IU/mL (normal values <15 U/L). Characteristic mucosal lesions on jejunal biopsy confirmed the diagnosis of celiac disease. Institution of a gluten-free diet resulted in rapid gain in weight and improvement in height velocity.