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ChatGPT and Bard (now known as Gemini) are becoming indispensable resources for researchers, academicians and diverse stakeholders within the academic landscape. At the same time, traditional digital tools such as scholarly databases continue to be widely used. Web of Science and Scopus are the most extensive academic databases and are generally regarded as consistently reliable scholarly research resources. With the increasing acceptance of artificial intelligence (AI) in academic writing, this study focuses on understanding the reliability of the new AI models compared to Scopus and Web of Science. The study includes a bibliometric analysis of green, sustainable and ecological buying behaviour, covering the period from 1 January 2011 to 21 May 2023. These results are used to compare the results from the AI and the traditional scholarly databases on several parameters. Overall, the findings suggest that AI models like ChatGPT and Bard are not yet reliable for academic writing tasks. It appears to be too early to depend on AI for such tasks.

IntroductionEnvironmental degradation poses a significant threat, making the comprehension of sustainable behaviour imperative for both environmental and business reasons. Embracing sustainable practices and reducing the unnecessary consumption of resources is essential in our current times. One can be sustainable by focusing on either buying sustainable or green products or by using fewer resources, i.e., by adopting curtailment behaviour. The purpose of this study is to determine various factors and their impact on the curtailment behaviour of youths.MethodsWe used an exploratory and descriptive research design. Responses were generated from 513 young respondents using a structured questionnaire developed based on the extant literature.ResultsThe structural model findings showed that social norms, eco-concern, and religiosity significantly and positively affect curtailment behaviour. However, we observed no significant influence of financial concern or government policy on curtailment behaviour. We also tested the moderation impact of government policy on the relationship between economic concern and curtailment behaviour. The findings suggest that the relationship between eco-concern and curtailment behaviour is stronger for consumers exhibiting higher commitment to government policy.DiscussionIt is imperative that politicians exercise critical thought and devise strategies to encourage more sustainable consumer behaviour. With the strains that our world is under now and in the future, we need to unite around a common goal: ensuring that our planet is sustainable for coming generations. The study findings are useful for academicians, marketers, and policymakers.

Summary: Malaria parasite invasion to host erythrocytes is mediated by multiple interactions between merozoite ligands and erythrocyte receptors that contribute toward the development of disease pathology. Here, we report a novel antigen Plasmodium prohibitin “PfPHB2” and identify its cognate partner “Hsp70A1A” in host erythrocyte that plays a crucial role in mediating host-parasite interaction during merozoite invasion. Using small interfering RNA (siRNA)- and glucosamine-6-phosphate riboswitch (glmS) ribozyme-mediated approach, we show that loss of Hsp70A1A in red blood cells (RBCs) or PfPHB2 in infected red blood cells (iRBCs), respectively, inhibit PfPHB2-Hsp70A1A interaction leading to invasion inhibition. Antibodies targeting PfPHB2 and monoclonal antibody therapeutics against Hsp70A1A efficiently block parasite invasion. Recombinant PfPHB2 binds to RBCs which is inhibited by anti-PfPHB2 antibody and monoclonal antibody against Hsp70A1A. The validation of PfPHB2 to serve as antigen is further supported by detection of anti-PfPHB2 antibody in patient sera. Overall, this study proposes PfPHB2 as vaccine candidate and highlights the use of monoclonal antibody therapeutics for future malaria treatment.

IntroductionThis study empirically investigates the attitude of tobacco and alcohol consumers towards health insurance purchase in India. The study aims to determine the factors which plays a significant role in determining the purchase intention of health insurance among tobacco and alcohol consumers.MethodsWe propose an extended theory of planned behavior (TPB) model comprising factors like attitude, subjective norms, perceived behavior control, perceived usefulness, perceived product risk, and intention to purchase. We collected responses from 420 tobacco and alcohol consumers through a Google Form link shared via different social media platforms. SPSS has been used to perform exploratory factor analysis, whereas AMOS has been used to validate the constructs, confirm the relationships among the variables, and analyze the data.ResultsThe analysis outcomes demonstrate that subjective norms, perceived product risk, and perceived behavioral control are the factors that have a positive and significant effect on health insurance purchase intention among consumers.DiscussionThis research offers valuable insights to the insurance sector, government officials, policymakers, and academicians. Insurance companies may consider the criteria analysed when creating policies to promote the expansion of the health insurance sector.

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy that is often associated with relapse and drug resistance after standard chemotherapy or targeted therapy, particularly in older patients. Hematopoietic stem cell transplants are looked upon as the ultimate salvage option with curative intent. Adoptive cell therapy using chimeric antigen receptors (CAR) has shown promise in B cell malignancies and is now being investigated in AML. Initial clinical trials have been disappointing in AML, and we review current strategies to improve efficacy for CAR approaches. The extensive number of clinical trials targeting different antigens likely reflects the genetic heterogeneity of AML. The limited number of patients reported in multiple early clinical studies makes it difficult to draw conclusions about CAR safety, but it does suggest that the efficacy of this approach in AML lags behind the success observed in B cell malignancies. There is a clear need not only to improve CAR design but also to identify targets in AML that show limited expression in normal myeloid lineage cells.

Treatment options in patients with recurrent endometrial carcinoma (EC) are limited and response rates to chemotherapy are poor. Here the authors report the results of a phase II trial of niraparib (PARP inhibitor) monotherapy or in combination with dostarlimab (anti-PD1) in recurrent EC.

Despite recent advances in the therapy of diffuse large B-cell lymphoma (DLBCL), many patients are still not cured. Therefore, new therapeutic strategies are needed. The anti-apoptotic B-cell lymphoma 2 (BCL2) gene is commonly dysregulated in DLBCL due to various mechanisms such as chromosomal translocation t(14;18)(q32;q21) and copy number alterations; however, targeting BCL-2 with the selective inhibitor, venetoclax, led to response in only a minority of patients. Thus, we sought to identify a rational combination partner of venetoclax to improve its activity against DLBCL cells. Utilizing a functional assay, dynamic BH3 profiling, we found that the DNA hypomethylating agent decitabine increased mitochondrial apoptotic priming and BCL-2 dependence in DLBCL cells. RNA-sequencing analysis revealed that decitabine suppressed the pro-survival PI3K-AKT pathway and altered the mitochondria membrane composition in DLBCL cell lines. Additionally, it induced a DNA damage response and increased BAX and BAK activities. The combination of decitabine and venetoclax synergistically suppressed proliferation of DLBCL cells both in vitro and in vivo in a DLBCL cell line-derived xenograft mouse model. Our study suggests that decitabine plus venetoclax is a promising combination to explore clinically in DLBCL.

Abstract Bromodomain-containing protein 9 (BRD9), an essential component of the SWI/SNF chromatin remodeling complex termed ncBAF, has been established as a therapeutic target in a subset of sarcomas and leukemias. Here, we used novel small molecule inhibitors and degraders along with RNA interference to assess the dependency on BRD9 in the context of diverse hematological malignancies, including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and multiple myeloma (MM) model systems. Following depletion of BRD9 protein, AML cells undergo terminal differentiation, whereas apoptosis was more prominent in ALL and MM. RNA-seq analysis of acute leukemia and MM cells revealed both unique and common signaling pathways affected by BRD9 degradation, with common pathways including those associated with regulation of inflammation, cell adhesion, DNA repair and cell cycle progression. Degradation of BRD9 potentiated the effects of several chemotherapeutic agents and targeted therapies against AML, ALL, and MM. Our findings support further development of therapeutic targeting of BRD9, alone or combined with other agents, as a novel strategy for acute leukemias and MM.

Summary: Cold shock proteins are characterized by the presence of one or more cold shock domains that bestow them with nucleic acid binding ability. Although cold shock proteins are well characterized in bacteria, plants and humans, there is no information on their existence and role in malaria parasite. Here, we have identified and delineated the function of a cold shock protein of Plasmodium falciparum (Pf) ‘PfCoSP’. We demonstrate that PfCoSP exhibits nucleic acid binding properties and regulates gene expression. PfCoSP promotes microtubule assembly by interacting with Pf α/β tubulin. We identified a human cold shock protein LIN28A inhibitor ‘LI71’ as a binding partner of PfCoSP which inhibited PfCoSP–DNA and α/β tubulin interactions and, also inhibited the development of asexual blood stages and gametocyte stage of malaria parasite. Because PfCoSP is essential for parasite survival, characterization of its interacting partners may form the basis for development of future anti-malarials.

Abstract The heterogeneous response of acute myeloid leukemia (AML) to current anti‐leukemic therapies is only partially explained by mutational heterogeneity. We previously identified GPR56 as a surface marker associated with poor outcome across genetic groups, which characterizes two leukemia stem cell (LSC)‐enriched compartments with different self‐renewal capacities. How these compartments self‐renew remained unclear. Here, we show that GPR56+ LSC compartments are promoted in a complex network involving epithelial‐to‐mesenchymal transition (EMT) regulators besides Rho, Wnt, and Hedgehog (Hh) signaling. Unexpectedly, Wnt pathway inhibition increased the more immature, slowly cycling GPR56+CD34+ fraction and Hh/EMT gene expression, while Wnt activation caused opposite effects. Our data suggest that the crucial role of GPR56 lies in its ability to co‐activate these opposing signals, thus ensuring the constant supply of both LSC subsets. We show that CDK7 inhibitors suppress both LSC‐enriched subsets in vivo and synergize with the Bcl‐2 inhibitor venetoclax. Our data establish reciprocal transition between LSC compartments as a novel concept underlying the poor outcome in GPR56high AML and propose combined CDK7 and Bcl‐2 inhibition as LSC‐directed therapy in this disease.

Introduction: The world is enveloped with the coronavirus disease 2019 (COVID-19) Pandemic with modern medicine and public health facing their most significant challenges ever posed. As the number of COVID-19 cases increased worldwide, an important issue of concern was the continuation of routine immunization services for children. This study has been conceptualized to assess the effect of the COVID-19 pandemic on infant Immunization. Materials and Methods: This study was a descriptive study conducted in an urban community amongst healthy children

Abstract Cytoskeletal structures of Apicomplexan parasites are important for parasite replication, motility, invasion to the host cell and survival. Apicortin, an Apicomplexan specific protein appears to be a crucial factor in maintaining stability of the parasite cytoskeletal assemblies. However, the function of apicortin, in terms of interaction with microtubules still remains elusive. Herein, we have attempted to elucidate the function of Plasmodium falciparum apicortin by monitoring its interaction with two main components of parasite microtubular structure, α-tubulin-I and β-tubulin through in silico and in vitro studies. Further, a p25 domain binding generic drug Tamoxifen (TMX), was used to disrupt PfApicortin-tubulin interactions which led to the inhibition in growth and progression of blood stage life cycle of P. falciparum.

Abstract Hijacking of host metabolic status by a pathogen for its regulated dissemination from the host is prerequisite for the propagation of infection. M. tuberculosis secretes an NAD+-glycohydrolase, TNT, to induce host necroptosis by hydrolyzing Nicotinamide adenine dinucleotide (NAD+). Herein, we expressed TNT in macrophages and erythrocytes; the host cells for M. tuberculosis and the malaria parasite respectively, and found that it reduced the NAD+ levels and thereby induced necroptosis and eryptosis resulting in premature dissemination of pathogen. Targeting TNT in M. tuberculosis or induced eryptosis in malaria parasite interferes with pathogen dissemination and reduction in the propagation of infection. Building upon our discovery that inhibition of pathogen-mediated host NAD+ modulation is a way forward for regulation of infection, we synthesized and screened some novel compounds that showed inhibition of NAD+-glycohydrolase activity and pathogen infection in the nanomolar range. Overall this study highlights the fundamental importance of pathogen-mediated modulation of host NAD+ homeostasis for its infection propagation and novel inhibitors as leads for host-targeted therapeutics.

Rationale: Clinicians encounter multiple alcohol-related illnesses in practice, and Marchiafava-Bignami disease is a rare and devastating entity among them. It is a toxic-demyelinating disease and seen in chronic alcoholics, although it may be occasionally observed in chronically malnourished teetotalers. The clinical presentations are diverse. The symptoms and signs are non-specific, and the onset can be acute or chronic. Patient’s concerns: A 45-year-old right-handed patient suffered from alcohol use disorder with multiple non-specific neuropsychiatric manifestations. Diagnosis: Marchiafava-Bignami disease. Interventions: Thiamine, folate, vitamin B12, and steroid therapy. Outcome: The patient’s behaviour was significantly improved but dysarthria and pyramidal signs persisted. He was left with permanent cognitive impairment. Lessons: Though prompt therapy may halt the demyelinating process in this disease, the treatment remains a challenge in clinical practice. The recognition of the neuro-radiologic features is crucial to establish an early diagnosis.

Visceral leishmaniasis (VL) and post kala-azar dermal leishmaniasis (PKDL) affect most of the poor populations worldwide. The current treatment modalities include liposomal formulation or deoxycholate salt of amphotericin B, which has been associated with various complications and severe side effects. Encouraged from the recent marked antimalarial effects from plant-derived glycosides, in this study, we have exploited a green chemistry-based approach to chemically synthesize a library of diverse glycoside derivatives (Gly1–12) and evaluated their inhibitory efficacy against the AG83 strain of Leishmania donovani. Among the synthesized glycosides, the in vitro inhibitory activity of Glycoside-2 (Gly2) (1.13 µM IC50 value) on L. donovani promastigote demonstrated maximum cytotoxicity with ~94% promastigote death as compared to amphotericin B that was taken as a positive control. The antiproliferative effect of Gly2 on promastigote encouraged us to analyze the structure–activity relationship of Gly2 with Gp63, a zinc metalloprotease that majorly localizes at the surface of the promastigote and has a role in its development and multiplication. The result demonstrated the exceptional binding affinity of Gly2 toward the catalytic domain of Gp63. These data were thereafter validated through cellular thermal shift assay in a physiologically relevant cellular environment. Mechanistically, reduced multiplication of promastigotes on treatment with Gly2 induces the destabilization of redox homeostasis in promastigotes by enhancing reactive oxygen species (ROS), coupled with depolarization of the mitochondrial membrane. Additionally, Gly2 displayed strong lethal effects on infectivity and multiplication of amastigote inside the macrophage in the amastigote–macrophage infection model in vitro as compared to amphotericin B treatment. Gp63 is also known to bestow protection against complement-mediated lysis of parasites. Interestingly, Gly2 treatment enhances the complement-mediated lysis of L. donovani promastigotes in serum physiological conditions. In addition, Gly2 was found to be equally effective against the clinical promastigote forms of PKDL strain (IC50 value of 1.97 µM); hence, it could target both VL and PKDL simultaneously. Taken together, this study reports the serendipitous discovery of Gly2 with potent antileishmanial activity and proves to be a novel chemotherapeutic prototype against VL and PKDL.

Introduction: From being declared as Public Health Emergency of International Concern and the transmission dynamics of COVID-19, outbreak control has been the cornerstone of combating COVID-19 cases within communities and clusters. An outbreak investigation of COVID-19 positive cases was conducted in a close community setting at an industrial warehouse which included working and residential areas in the premises, with the aim to break the chain of transmission rapidly and to formulate preventive strategy for dealing with other outbreaks in a community setting. Materials and Methods: This was a study where a rapid outbreak response was carried out after two individuals tested COVID-19 positive. Following the national and local guidelines, immediate decongestion followed by aggressive contact tracing, testing and isolating the cases were done. Results: After the rapid contact tracing exercise, contacts were identified, quarantined and tested. Daily monitoring of the contacts revealed six symptomatic confirmed cases and on testing, 19 asymptomatic contacts were positive. All confirmed cases were immediately admitted to the hospital. Additional two positive cases reported from family contacts. The mean age of the individuals was 46 years. There were 9 individuals with co-morbidities and two individuals developed acute respiratory distress syndrome and expired in the hospital. The secondary attack rate was calculated to be 17.51%. Conclusion: Prompt public health measures by rapid response team, creation of containment zone, contact tracing, early isolation along with daily influenza like illness screening and strict quarantine effectively helped in containment of the outbreak in the population under study.

Background: The clinical feature and radiographic appearance of orthokeratinized odontogenic keratocyst (OKC) are not characteristic, which may lead to the misdiagnosis. The radiographic appearance of OKC may range from a small unilocular radiolucency to a large multilocular radiolucency, resembling other odontogenic cysts and tumors. Aim: The aim was to illustrate the characteristic feature of OKC presented on the digital panoramic radiograph and cone-beam computed tomography (CBCT), which may provide great value for the differential diagnosis and the treatment planning and also to compare the various radiographic features of OKC in CBCT and digital panoramic radiograph. Materials and Methods: Digital panoramic and CBCT records of seven cases, with 11 lesions of OKC were analyzed retrospectively from the patients' database from our institute (2014 to 2016), which was histopathologically diagnosed as OKC. Results: The mean age of the patients was 24.1 years, and the female-to-male ratio was 3: 4. Six of 11 lesions were localized within the mandible, and five lesions were in the maxilla. Conclusion: The presurgical assessment with radiological information is extremely important for treatment planning, and CBCT provides us with an accurate and faster three-dimensional representation of a lesion at a lower dose and cost, but the role of panoramic radiograph cannot be refuted.

The characteristic features of Class III malocclusion are present at an early age, usually between 3 and 5 years of age. If left untreated, the Class III malocclusion or severe anterior crossbite may worsen, with the majority of these patients ultimately requiring orthognathic surgery as adults. The treatment in all developing Class III cases should be undertaken as soon as the abnormality is diagnosed to prevent it from becoming permanent and resulting in a full-blown Class III malocclusion. It provides more pleasing facial esthetics (the lip posture and facial appearance), thus improving the psychosocial development of a child. Various treatment options available for the management of developing Class III malocclusion are as follows: (1) functional appliances such as reverse twin block, Frankel appliance, and the removable mandibular retractor; (2) orthopedic appliances such as chin cap and face mask; and (3) orthodontic appliances, namely removable maxillary expansion plate or fixed orthodontic appliance with expanded upper arch wire. A treatment plan may be chosen depending on the amount of maxillary growth restriction, mandibular plane angle, the soft-tissue profile, and the age of the patient. This article discusses the various treatment options and the decision-making for different clinical cases based on the clinical features with a description of four different treatment approaches.

Ovarian hyperstimulation syndrome (OHSS) is a disorder that usually occurs with ovulation induction. It is rare in pregnant women and only a very few cases have been reported in spontaneous unstimulated conceptions. We report a case of spontaneous OHSS with primary hypothyroidism in a 19-year-old primigravida, where the imaging and laboratory findings confirmed the diagnosis of “Van Wyk–Grumbach syndrome” with pregnancy. This rare event in pregnancy needs attention, as early institution of thyroid hormone replacement therapy results in resolution of cysts and avoids unnecessary evaluation and surgical misadventures.

Protein kinases play a crucial role in cellular functions by adding phosphate group to the protein substrates. It is an indispensable post-translational modification that regulates intracellular signaling and key cellular processes. They thus serve as an excellent target for chemotherapeutic interventions. A vast repertoire of protein kinases is present in a cell with diverse substrates as well as phosphorylation sites. To study full kinome for its activity, there is an urgent need of designing a comprehensive, in vitro assay which itself is an impractical task. However, in this study, we have attempted to develop a robust assay that not only mimics the in vivo nature of the kinases but can also be used in a high throughput drug-screening platform. Herein, the Leishmania donovani parasites are lysed and the total protein content is extracted. This extracted proteome is further sub divided into two parts: one active fraction containing cellular kinases and the substrate is heat-denatured fraction that loses all the enzymatic activity but retains the potential phosphorylation sites. These fractions are then co-incubated in the presence of ATP to initiate the kinase reaction and the total kinase activity is measured using ADP-glo kinase assay. Overall, this method • Presents a simple and robust approach to understand the participation of kinases in signaling networks. • Presents a high-throughput platform for ex-vivo drug screening. Method name: Kinome survey, Keywords: High throughput kinase assay, Leishmania donovani, Cell lysate, Non-radioactive

Mature human erythrocytes contain a rich pool of microRNAs (miRNAs), which result from differentiation of the erythrocytes during the course of haematopoiesis. Recent studies have described the effect of erythrocytic miRNAs on the invasion and growth of the malaria parasite Plasmodium falciparum during the asexual blood stage of its life cycle. In this work, we have identified two erythrocytic miRNAs, miR-150-3p and miR-197-5p, that show favourable in silico hybridization with Plasmodium apicortin, a protein with putative microtubule-stabilizing properties. Co-expression of P. falciparum apicortin and these two miRNAs in a cell line model resulted in downregulation of apicortin at both the RNA and protein level. To create a disease model of erythrocytes containing miRNAs, chemically synthesized mimics of miR-150-3p and miR-197-5p were loaded into erythrocytes and subsequently used for invasion by the parasite. Growth of the parasite was hindered in miRNA-loaded erythrocytes, followed by impaired invasion; micronemal secretion was also reduced, especially in the case of miR-197-5p. Apicortin expression was found to be reduced in miRNA-loaded erythrocytes. To interpret the effect of downregulation of apicortin on parasite invasion to host erythrocytes, we investigated the secretion of the invasion-related microneme protein apical membrane antigen 1 (AMA1). AMA1 secretion was found to be reduced in miRNA-treated parasites. Overall, this study identifies apicortin as a novel target within the malaria parasite and establishes miR-197-5p as its miRNA inhibitor. This miRNA represents an unconventional nucleotide-based therapeutic and provides a new host factor-inspired strategy for the design of antimalarial molecular medicine. This article has an associated First Person interview with the first author of the paper.

The pore forming Plasmodium Perforin Like Proteins (PPLP), expressed in all stages of the parasite life cycle are critical for completion of the parasite life cycle. The high sequence similarity in the central Membrane Attack Complex/ Perforin (MACPF) domain among PLPs and their distinct functional overlaps define them as lucrative target for developing multi-stage antimalarial therapeutics. Herein, we evaluated the mechanism of Pan-active MACPF Domain (PMD), a centrally located and highly conserved region of PPLPs, and deciphered the inhibitory potential of specifically designed PMD inhibitors. The E. coli expressed rPMD interacts with erythrocyte membrane and form pores of ~10.5 nm height and ~24.3 nm diameter leading to hemoglobin release and dextran uptake. The treatment with PMD induced erythrocytes senescence which can be hypothesized to account for the physiological effect of disseminated PLPs in loss of circulating erythrocytes inducing malaria anemia. The anti-PMD inhibitors effectively blocked intraerythrocytic growth by suppressing invasion and egress processes and protected erythrocytes against rPMD induced senescence. Moreover, these inhibitors also blocked the hepatic stage and transmission stage parasite development suggesting multi-stage, transmission-blocking potential of these inhibitors. Concievably, our study has introduced a novel set of anti-PMD inhibitors with pan-inhibitory activity against all the PPLPs members which can be developed into potent cross-stage antimalarial therapeutics along with erythrocyte senescence protective potential to occlude PPLPs mediated anemia in severe malaria.

Opioid like morphine and codeine are used for the management of pain, but are associated with serious side-effects limiting their use. Wheat gluten proteins were assessed for the presence of opioid peptides on the basis of tyrosine and proline within their sequence. Eleven peptides were identified and occurrence of predicted sequences or their structural motifs were analysed using BIOPEP database and ranked using PeptideRanker. Based on higher peptide ranking, three sequences YPG, YYPG and YIPP were selected for determination of opioid activity by cAMP assay against µ and κ opioid receptors. Three peptides inhibited the production of cAMP to varied degree with EC50 values of YPG, YYPG and YIPP were 5.3 mM, 1.5 mM and 2.9 mM for µ-opioid receptor, and 1.9 mM, 1.2 mM and 3.2 mM for κ-opioid receptor, respectively. The study showed that in silico approach can be used for the prediction of opioid peptides from gluten.

Juvenile myelomonocytic leukemia (JMML) is an aggressive disease with limited options for treatment. Here, the authors analyse the DNA methylome and mutational profile of JMML to define three subgroups with unique molecular and clinical characteristics.

Fungal infections are very important infectious causes of granulomatous inflammation. Isolated subcutaneous fungal infections are uncommon and only seen in immunosuppressed patients or in those with other comorbidities. Such cases are usually mistaken as noninfectious benign lesions and fine-needle aspiration cytology (FNAC) can be used for an adequate diagnosis.

Clostridium perfringens epsilon toxin (Etx) is categorized as the third most lethal bioterrorism agent by the Centers for Disease Control and Prevention (CDC), with no therapeutic counter measures available for humans. Here, we have developed a high-affinity inhibitory compound by synthesizing and evaluating the structure activity relationship (SAR) of a library of diverse glycosides (numbered 1-12). SAR of glycoside-Etx heptamers revealed exceptionally strong H-bond interactions of glycoside-4 with a druggable pocket in the oligomerization and β-hairpin region of Etx. Analysis of its structure suggested that glycoside-4 might self-aggregate to form a robust micelle-like supra-molecular complex due to its linear side-chain architecture, which was authenticated by fluorescence spectroscopy. Further, this micelle hinders the Etx monomer-monomer interaction required for oligomerization, validated by both surface plasmon resonance (SPR) and immunoblotting. This phenomenon in turn leads to blockage of pore formation. Downstream evaluation revealed that glycoside-4 effectively blocked cell death of Etx-treated cultured primary cells and maintained cellular homeostasis via disrupting oligomerization, blocking pore formation, restoring calcium homeostasis, stabilizing the mitochondrial membrane and impairing high mobility group box 1 (HMGB1) translocation from nucleus to cytoplasm. Furthermore, a single dosage of glycoside-4 protected the Etx-challenged mice and restored normal function to multiple organs. This work reports for the first time a potent, nontoxic glycoside with strong ability to occlude toxin lethality, representing it as a bio-arm therapeutic against Etx-based biological threat.

Severe malaria caused by Plasmodium falciparum poses a major global health problem with high morbidity and mortality. P. falciparum harbors a family of pore-forming proteins (PFPs), known as perforin like proteins (PLPs), which are structurally equivalent to prokaryotic PFPs. These PLPs are secreted from the parasites and, they contribute to disease pathogenesis by interacting with host cells. The severe malaria pathogenesis is associated with the dysfunction of various barrier cells, including endothelial cells (EC). Several factors, including PLPs secreted by parasites, contribute to the host cell dysfunction. Herein, we have tested the hypothesis that PLPs mediate dysfunction of barrier cells and might have a role in disease pathogenesis. We analyzed various dysfunctions in barrier cells following rPLP2 exposure and demonstrate that it causes an increase in intracellular Ca2+ levels. Additionally, rPLP2 exposed barrier cells displayed features of cell death, including Annexin/PI positivity, depolarized the mitochondrial membrane potential, and ROS generation. We have further performed the time-lapse video microscopy of barrier cells and found that the treatment of rPLP2 triggers their membrane blebbing. The cytoplasmic localization of HMGB1, a marker of necrosis, further confirmed the necrotic type of cell death. This study highlights the role of parasite factor PLP in endothelial dysfunction and provides a rationale for the design of adjunct therapies against severe malaria.

Palmitoylation has been recently identified as an important post-translational rheostat for controlling protein function in eukaryotes. However, the molecular machinery underlying palmitoylation remains unclear in the neglected tropical parasite, Leishmania donovani. Herein, we have identified a catalog of 20 novel palmitoyl acyltransferases (PATs) and characterized the promastigote-specific PAT (LdPAT4) containing the canonical Asp-His-His-Cys (DHHC) domain. Immunofluorescence analysis using in-house generated LdPAT4-specific antibody demonstrated distinct expression of LdPAT4 in the flagellar pocket of promastigotes. Using metabolic labeling-coupled click chemistry method, the functionality of this recombinant enzyme could be authenticated in E. coli strain expressing LdPAT4-DHHC domain. This was evident by the cellular uptake of palmitic acid analogs, which could be successfully inhibited by 2-BMP, a PAT-specific inhibitor. Using CSS-Palm based in-silico proteomic analysis, we could predict up to 23 palmitoylated sites per protein in the promastigotes, and further identify distinctive palmitoylated protein clusters involved in microtubule assembly, flagella motility and vesicular trafficking. To highlight, proteins such as Flagellar Member proteins (FLAM1, FLAM5), Intraflagellar Transport proteins (IFT88), and flagellar motor assembly proteins including the Dynein family were found to be enriched. Furthermore, analysis of global palmitoylation in promastigotes using Acyl-biotin exchange purification identified a set of S-palmitoylated proteins overlapping with the in-silico proteomics data. The attenuation of palmitoylation using 2-BMP demonstrated several phenotypic alterations in the promastigotes including distorted morphology, reduced motility (flagellar loss or slow flagellar beating), and inefficient invasion of promastigotes to host macrophages. These analyses confirm the essential role of palmitoylation in promastigotes. In summary, the findings suggest that LdPAT4 acts as a functional acyltransferase that can regulate palmitoylation of proteins involved in parasite motility and invasion, thus, can serve as a potential target for designing chemotherapeutics in Visceral Leishmaniasis.