Dominant B cell-T cell epitopes instigated robust immune response in-silico against Scrub Typhus.

Scrub typhus, a potentially life-threatening infectious disease, is attributed to bacteria Orientia tsutsugamushi (O. tsutsugamushi). The transmission of this illness to humans occurs through the bite of infected chiggers, which are the larval forms of mites belonging to the genus Leptotrombidium. In this research, we developed a subunit vaccine specifically designed to target outer membrane proteins. Immunodominant cytotoxic T-lymphocytes (CTLs), B- lymphocytes (BCLs), and major histocompatibility complex (MHC)- II epitopes were identified using machine learning and bioinformatics approaches. These epitopes were arranged in different combinations with the help of suitable linkers like AAY, KK, GPGPG and adjuvant (cholera toxin B) that resulted in a vaccine construct. Physiochemical properties were assessed, where the predicted solubility (0.571) was higher than threshold value. Tertiary structure was predicted using I-TASSER web server and evaluated using Ramachandran plot (94 % residues in most favourable region) and z-score (-6.04), which had shown the structure to have good stability and residue arrangement. Molecular docking with immune receptors, Toll-like receptor (TLR)-2 and -4 showed good residue interaction with 13 and 5 hydrogen bonds respectively. Molecular dynamics simulations of receptor-ligand complex provided the idea about the strong interaction having 1.524751 × 10-5 eigenvalue. Amino acid sequence of vaccine was converted to nucleotide sequence and underwent codon optimization. The optimized codon sequence was used for in-silico cloning, which provided idea about the possibility of synthesis of vaccine using E. coli as host. Overall, this study provided a promising blueprint for a scrub typhus vaccine, although experimental validation is needed for confirmation. Furthermore, it is crucial to acknowledge that while bioinformatics provides valuable insights, in-vitro and in-vivo studies are imperative for a comprehensive evaluation of vaccine candidate. Thus, the integration of computational predictions with empirical research is essential to validate the efficacy, safety, and real-world applicability of the designed vaccine against Scrub Typhus. Nevertheless, the findings are good to carry forward for in-vitro and in-vivo investigations.

Super epitope dengue vaccine instigated serotype independent immune protection in-silico.

Dengue becomes the most common life-threatening infectious arbovirus disease globally, with prevalence in the tropical and subtropical areas. The major clinical features include dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS), a condition of hypovolemic shock. Four different serotypes of the dengue virus, known as dengue virus serotype (DENV)- 1, 2, 3 and 4 can infect humans. Only one vaccine is available in the market, named Dengvaxia by Sanofi Pasteur, but there is no desired outcome of this treatment due the antibody dependent enhancement (ADE) of the multiple dengue serotypes. As of now, there is no cure against dengue disease. Our goal in this work was to create a subunit vaccine based on several epitopes that would be effective against every serotype of the dengue virus. Here, computational methods like- immunoinformatics and bioinformatics were implemented to find out possible dominant epitopes. A total of 21 epitopes were chosen using various in-silico techniques from the expected 133 major histocompatibility complex (MHC)- I and major histocompatibility complex (MHC)- II epitopes, along with 95 B-cell epitopes which were greatly conserved. Immune stimulant, non-allergenic and non-toxic immunodominant epitopes (super epitopes) with a suitable adjuvant (Heparin-Binding Hemagglutinin Adhesin, HBHA) were used to construct the vaccine. Following the physicochemical analysis, vaccine construct was docked with Toll-like receptors (TLRs) to predict the immune stimulation. Consequently, the optimal docked complex that demonstrated the least amount of ligand-receptor complex deformability was used to conduct the molecular dynamics analysis. By following the codon optimization, the final vaccine molecule was administered into an expressing vector to perform in-silico cloning. The robust immune responses were generated in the in-silico immune simulation analysis. Hence, this study provides a hope to control the dengue infections. For validation of the immune outcomes, in-vitro as well as in-vivo investigations are essential.

Difficulty of Arch Reduction with Gillies Temporal Approach in a Coronoid Hyperplasia Case.

Introduction: Bilateral coronoid hyperplasia is an abnormal elongation of the mandibular coronoid process which is rare in occurrence and causes progressive but slow reduction in mouth opening. In case of reduction in zygomatic arch fracture by Gillies temporal approach, the presence of bilateral coronoid hyperplasia can be a possible factor for hindrance and improper reduction. Material and methods: We propose a technical note to overcome this hindrance caused by the coronoid hyperplasia and propose tips and tricks to successful reduction in zygomatic arch. Results: Adequate reduction in the zygomatic arch and pretrauma mouth opening was achieved. Conclusion: Manual repositioning of the mandible during Gillies temporal approach along with ultrasound guidance leads to a satisfactory outcome.

PTEN-induced kinase 1 (PINK1) and Parkin: Unlocking a mitochondrial quality control pathway linked to Parkinson's disease.

Dissection of the function of two Parkinson's disease-linked genes encoding the protein kinase, PTEN-induced kinase 1 (PINK1) and ubiquitin E3 ligase, Parkin, has illuminated a highly conserved mitochondrial quality control pathway found in nearly every cell type including neurons. Mitochondrial damage-induced activation of PINK1 stimulates phosphorylation-dependent activation of Parkin and ubiquitin-dependent elimination of mitochondria by autophagy (mitophagy). Structural, cell biological and neuronal studies are unravelling the key steps of PINK1/Parkin-dependent mitophagy and uncovering new insights into how the pathway is regulated. The emerging role for aberrant immune activation as a driver of dopaminergic neuron degeneration after loss of PINK1 and Parkin poses new exciting questions on cell-autonomous and noncell-autonomous mechanisms of PINK1/Parkin signalling in vivo.

Evaluation of transcutaneous electrical nerve stimulation as an adjunct therapy in trigeminal neuralgia - a randomized double-blind placebo-controlled clinical study.

BACKGROUND: Trigeminal neuralgia (TN) is a severe form of pain that affects the daily activities of a patient. Transcutaneous electrical nerve stimulation (TENS) therapy is an emerging option for the treatment of acute and chronic pain. The aim of this study was to evaluate the effect of TENS therapy as an adjunct to drug therapy for the treatment of TN. METHODS: A total of 52 patients diagnosed with TN according to the International Classification of Headache Disorders (version 3) were included. Each patient was randomized to either the TENS or placebo TENS groups. Intervention was given in continuous mode and 100-Hz frequency for 20 mins biweekly for 6 weeks. Parameters were measured at baseline, TENS completion and 3 months, 6 months, and 1 year of follow up. The parameters observed were mean carbamazepine dose, mean visual analog scale (VAS) score, mean present pain intensity (PPI) score, and functional outcome. Non-parametric analyses, one-way ANOVA and the Kruskal-Wallis test were applied for intragroup comparisons, while the Mann-Whitney U test and independent t-test were used for intergroup comparisons of variables. The chi-square test was applied to analyze categorical data. RESULTS: Compared to the placebo TENS group, the mean dose of carbamazepine in the TENS group was significantly reduced at TENS completion, as well as at 6 months and 1 year follow up. Changes in mean VAS score, mean PPI score, and functional outcome did not show significant differences between the groups (P>0.05). CONCLUSION: TENS therapy does not lead to any changes in pain levels but it may reduce the mean dose of carbamazepine when used as an adjunct treatment in patients with TN.

Uncovering the Cyclic AMP Signaling Pathway of the Protozoan Parasite Entamoeba histolytica and Understanding Its Role in Phagocytosis.

Second messenger signaling controls a surprisingly diverse range of processes in several eukaryotic pathogens. Molecular machinery and pathways involving these messengers thus hold tremendous opportunities for therapeutic interventions. Relative to Ca2+ signaling, the knowledge of a crucial second messenger cyclic AMP (cAMP) and its signaling pathway is very scant in the intestinal parasite Entamoeba histolytica. In the current study, mining the available genomic resources, we have for the first time identified the cAMP signal transduction pathway of E. histolytica. Three heptahelical proteins with variable G-protein-coupled receptor domains, heterotrimeric G-proteins (Gα, Gß, and Gγ subunits), soluble adenylyl cyclase, cyclase-associated protein, and enzyme carbonic anhydrase were identified in its genome. We could also identify several putative candidate genes for cAMP downstream effectors such as protein kinase A, A-kinase anchoring proteins, and exchange protein directly activated by the cAMP pathway. Using specific inhibitors against key identified targets, we could observe changes in the intracellular cAMP levels as well as defect in the rate of phagocytosis of red blood cells by the parasite E. histolytica. We thus strongly believe that characterization of some of these unexplored crucial signaling determinants will provide a paradigm shift in understanding the pathogenicity of this organism.

Ca2+-binding protein from Entamoeba histolytica (EhCaBP6) is a novel GTPase.

GTPases are molecular switches, which regulate a variety of cellular processes such as cell polarity, gene transcription, microtubule dynamics, cell-cycle etc. In this paper, we characterize a Ca2+-binding protein from Entamoeba histolytica (EhCaBP6) as a novel GTPase. We locate the active site for GTP hydrolysis within the C-terminal domain of EhCaBP6, although it requires full length protein for its complete range of activity. Using NMR studies, we observe that GTP binding induces conformational change in EhCaBP6. The identification of this novel and unusual Ca2+-dependent GTPase is important to elucidate the unconventional cell cycle of E. histolytica.

Surface Plasmon Resonance Analysis of the Protein-protein Binding Specificity Using Autolab ESPIRIT.

Direct protein-protein interactions are known to regulate a wide range of cellular activities. To understand these contacts one can employ various experimental methods like Dynamic Light Scattering (DLS), Fluorescence Resonance Energy Transfer (FRET), Isothermal titration calorimetry (ITC), Chemical crosslinking, Co-immunoprecipitation (Co-IP), Surface Plasmon Resonance (SPR) and many more. Among these, SPR stands out as a quick, label-free, reliable, and accurate quantitation technique. We have used SPR to elucidate the linkage between 14-3-3 Protein 3 (EhP3) and the actin cytoskeleton in the protist pathogen Entamoeba histolytica. It allowed us to screen EhP3 binding with several actin-binding/actin regulatory proteins (Coactosin, Actophorin, Twinfilin, Profilin, and Filamin). Our screening results suggested Coactosin as an important interacting partner of EhP3. A complete kinetic analysis indeed confirmed that EhCoactosin binds EhP3 with an affinity constant of 3 µM.

Natural Product Inspired Novel Indole based Chiral Scaffold Kills Human Malaria Parasites via Ionic Imbalance Mediated Cell Death.

Natural products offer an abundant source of diverse novel scaffolds that inspires development of next generation anti-malarials. With this vision, a library of scaffolds inspired by natural biologically active alkaloids was synthesized from chiral bicyclic lactams with steps/scaffold ratio of 1.7:1. On evaluation of library of scaffolds for their growth inhibitory effect against malaria parasite we found one scaffold with IC50 in low micro molar range. It inhibited parasite growth via disruption of Na+ homeostasis. P-type ATPase, PfATP4 is responsible for maintaining parasite Na+ homeostasis and is a good target for anti-malarials. Molecular docking with our scaffold showed that it fits well in the binding pocket of PfATP4. Moreover, inhibition of Na+-dependent ATPase activity by our potent scaffold suggests that it targets parasite by inhibiting PfATP4, leading to ionic imbalance. However how ionic imbalance attributes to parasite's death is unclear. We show that ionic imbalance caused by scaffold 7 induces autophagy that leads to onset of apoptosis in the parasite evident by the loss of mitochondrial membrane potential (ΔΨm) and DNA degradation. Our study provides a novel strategy for drug discovery and an insight into the molecular mechanism of ionic imbalance mediated death in malaria parasite.

Novel regulatory roles of PtdIns(4,5)P2 generating enzyme EhPIPKI in actin dynamics and phagocytosis of Entamoeba histolytica.

Motility and phagocytosis are the two important processes that are intricately linked to survival and virulence potential of the protist parasite Entamoeba histolytica. These processes primarily rely on actin-dependent pathways, and regulation of these pathways is critical for understanding the pathology of E. histolytica. Generally, phosphoinositides dynamics have not been explored in amoebic actin dynamics and particularly during phagocytosis in E. histolytica. We have explored the roles of PtdIns(4,5)P2 as well as the enzyme that produces this metabolite, EhPIPKI during phagocytosis. Immunofluorescence and live cell images showed enrichment of EhPIPKI in different stages of phagocytosis from initiation till the cups progressed towards closure. However, the enzyme was absent after phagosomes are pinched off from the membrane. Overexpression of a dominant negative mutant revealed a reduction in the formation of phagocytic cups and inhibition in the rate of engulfment of erythrocytes. Moreover, EhPIPKI binds directly to F and G-actin unlike PIPKs from other organisms. PtdIns(4,5)P2 , the product of the enzyme, also followed a similar distribution pattern during phagocytosis as determined by a GFP-tagged PH-domain from PLCδ, which specifically binds PtdIns(4,5)P2 in trophozoites. In summary, EhPIPKI regulates initiation of phagocytosis by regulating actin dynamics.

EhP3, a homolog of 14-3-3 family of protein participates in actin reorganization and phagocytosis in Entamoeba histolytica.

The highly conserved proteins of the 14-3-3 family are universal adaptors known to regulate an enormous range of cellular processes in eukaryotes. However, their biological functions remain largely uncharacterized in pathogenic protists comprising of several 14-3-3 protein isoforms. In this study, we report the role of 14-3-3 in coordinating cytoskeletal dynamics during phagocytosis in a professional phagocytic protist Entamoeba histolytica, the etiological agent of human amebiasis. There are three isoforms of 14-3-3 protein in amoeba and here we have investigated Eh14-3-3 Protein 3 (EhP3). Live and fixed cell imaging studies revealed the presence of this protein throughout the parasite phagocytosis process, with high rate of accumulation at the phagocytic cups and closed phagosomes. Conditional suppression of EhP3 expression caused significant defects in phagocytosis accompanied by extensive diminution of F-actin at the site of cup formation. Downregulated cells also exhibited defective recruitment of an F-actin stabilizing protein, EhCoactosin at the phagocytic cups. In addition, mass spectrometry based analysis further revealed a large group of EhP3-associated proteins, many of these proteins are known to regulate cytoskeletal architecture in E histolytica. The dynamics of these proteins may also be controlled by EhP3. Taken together, our findings strongly suggest that EhP3 is a novel and a key regulatory element of actin dynamics and phagocytosis in E. histolytica.

Calcium-dependent phosphorylation of Plasmodium falciparum serine repeat antigen 5 triggers merozoite egress.

The human malaria parasite Plasmodium falciparum proliferates in red blood cells following repeated cycles of invasion, multiplication, and egress. P. falciparum serine repeat antigen 5 (PfSERA5), a putative serine protease, plays an important role in merozoite egress. However, regulation of its activity leading to merozoite egress is poorly understood. In this study, we show that PfSERA5 undergoes phosphorylation prior to merozoite egress. Immunoprecipitation of parasite lysates using anti-PfSERA5 serum followed by MS analysis identified calcium-dependent protein kinase 1 (PfCDPK1) as an interacting kinase. Association of PfSERA5 with PfCDPK1 was corroborated by co-sedimentation, co-immunoprecipitation, and co-immunolocalization analyses. Interestingly, PfCDPK1 phosphorylated PfSERA5 in vitro in the presence of Ca2+ and enhanced its proteolytic activity. A PfCDPK1 inhibitor, purfalcamine, blocked the phosphorylation and activation of PfSERA5 both in vitroas well as in schizonts, which, in turn, blocked merozoite egress. Together, these results suggest that phosphorylation of PfSERA5 by PfCDPK1 following a rise in cytosolic Ca2+ levels activates its proteolytic activity to trigger merozoite egress.

Knee Injuries in Wrestlers: A Prospective Study from the Indian Subcontinent.

BACKGROUND: Wrestling is a very popular sport the world over and its popularity is rapidly increasing in India. However, due to its arduous nature it is associated with a high incidence of injuries. Out of all the injuries, those to the knee are one of the commonest injuries reported. OBJECTIVES: Our aim was to study the pattern of these injuries in the Indian wrestlers. METHODS: A prospective study was conducted involving 196 wrestlers who were followed up over a period of 2 years. Their knee injuries were studied by means of a structured questionnaire which they filled up with assistance from their athletic trainers. RESULTS: There were a total of 188 injuries in 121 wrestlers with overall injury rate of 5.13/1,000 athlete exposure. 35 wrestlers sustained 71 knee injuries (71/188; 37.77%). 71.83% injuries were new. More number of injuries occurred in competition (incidence density ratio = 20.7) and in attack position. There was a statistically significant association with age and duration of practice. No association was found between these injuries and style of wrestling, weight and height of wrestlers. Ligament sprains and muscular strains were the commonest injuries. CONCLUSIONS: Goal of any such study is to minimize the risk of injury in the young athlete by understanding the factors responsible and development of preventive programs. We hope to do just that with this first study involving Indian wrestlers.

Visualization and quantification of Plasmodium falciparum intraerythrocytic merozoites.

Malaria, a leading parasitic killer, is caused by Plasmodium spp. The pathology of the disease starts when Plasmodium merozoites infect erythrocytes to form rings, that matures through a large trophozoite form and develop into schizonts containing multiple merozoites. The number of intra-erythrocytic merozoites is a key-determining factor for multiplication rate of the parasite. Counting of intraerythrocytic merozoites by classical 2-D microscopy method is error prone due to insufficient representation of merozoite in one optical plane of a schizont. Here, we report an alternative 3-D microscopy based automated method for counting of intraerythrocytic merozoites in entire volume of schizont. This method offers a considerable amount of advantages in terms of both, ease and accuracy.

Structural insights into a key carotenogenesis related enzyme phytoene synthase of P. falciparum: a novel drug target for malaria.

Carotenoids represent a diverse group of pigments derived from the common isoprenoid precursors and fulfill a variety of critical functions in plants and animals. Phytoene synthase (PSY), a transferase enzyme that catalyzes the first specific step in carotenoid biosynthesis plays a central role in the regulation of a number of essential functions mediated via carotenoids. PSYs have been deeply investigated in plants, bacteria and algae however in apicomplexans it is poorly studied. In an effort to characterize PSY in apicomplexans especially the malaria parasite Plasmodium falciparum (P. falciparum), a detailed bioinformatics analysis is undertaken. We have analysed the Phylogenetic relationship of PSY also referred to as octaprenyl pyrophosphate synthase (OPPS) in P. falciparum with other taxonomic groups. Further, we in silico characterized the secondary and tertiary structures of P. falciparum PSY/OPPS and compared the tertiary structures with crystal structure of Thermotoga maritima (T. maritima) OPPS. Our results evidenced the resemblance of P. falciparum PSY with the active site of T. maritima OPPS. Interestingly, the comparative structural analysis revealed an unconserved unique loop in P. falciparum OPPS/PSY. Such structural insights might contribute novel accessory functions to the protein thus, offering potential drug targets.

A natural product based DOS library of hybrid systems.

Here we described a natural product inspired modular DOS strategy for the synthesis of a library of hybrid systems that are structurally and stereochemically disparate. The main scaffold is a pyrroloisoquinoline motif, that is synthesized from tandem Pictet-Spengler lactamization. The structural diversity is generated via "privileged scaffolds" that are attached at the appropriate site of the motif. Screening of the library compounds for their antiplasmodial activity against chloroquine sensitive 3D7 cells indicated few compounds with moderate activity (20-50 µM). A systematic comparison of structural intricacy between the library members and a natural product dataset obtained from ZINC(®) revealed comparable complexity.

Diversity-oriented synthesis and activity evaluation of substituted bicyclic lactams as anti-malarial against Plasmodium falciparum.

BACKGROUND: Malaria remains the world's most important devastating parasitic disease. Of the five species of Plasmodium known to infect and cause human malaria, Plasmodium falciparum is the most virulent and responsible for majority of the deaths caused by this disease. Mainstream drug therapy targets the asexual blood stage of the malaria parasite, as the disease symptoms are mainly associated with this stage. The prevalence of malaria parasite strains resistance to existing anti-malarial drugs has made the control of malaria even more challenging and hence the development of a new class of drugs is inevitable. METHODS: Screening against different drug resistant and sensitive strains of P. falciparum was performed for few bicyclic lactam-based motifs, exhibiting a broad spectrum of activity with low toxicity generated via a focussed library obtained from diversity oriented synthesis (DOS). The synthesis and screening was followed by an in vitro assessment of the possible cytotoxic effect of this class of compounds on malaria parasite. RESULTS: The central scaffold a chiral bicyclic lactam (A) and (A') which were synthesized from (R)-phenylalaninol, levulinic acid and 3-(2-nitrophenyl) levulinic acid respectively. The DOS library was generated from A and from A', by either direct substitution with o-nitrobenzylbromide at the carbon α- to the amide functionality or by conversion to fused pyrroloquinolines. Upon screening this diverse library for their anti-malarial activity, a dinitro/diamine substituted bicyclic lactam was found to demonstrate exceptional activity of >85% inhibition at 50 µM concentration across different strains of P. falciparum with no toxicity against mammalian cells. Also, loss of mitochondrial membrane potential, mitochondrial functionality and apoptosis was observed in parasite treated with diamine-substituted bicyclic lactams. CONCLUSIONS: This study unveils a DOS-mediated exploration of small molecules with novel structural motifs that culminates in identifying a potential lead molecule against malaria. In vitro investigations further reveal their cytocidal effect on malaria parasite growth. It is not the first time that DOS has been used as a strategy to identify therapeutic leads against malaria, but this study establishes the direct implications of DOS in scouting novel motifs with anti-malarial activity.

Cytotoxic effect of carotenoid phytonutrient lycopene on P. falciparum infected erythrocytes.

Resistance to almost all class of antimalarial drugs has emerged as one of the greatest challenges to malaria control. Strategies to limit the advent and spread of drug-resistant malaria include development of new drugs and its combination with existing drugs. In this work we provide a strong evidence for phytonutrient lycopene, a non-provitamin A carotenoid, to be effective against Plasmodium falciparum growth in vitro. Consistent with the previous findings in mammalian cells, lycopene's prooxidant activity promoted the production of reactive oxygen species (ROS) in P. falciparum. Also a significant loss of mitochondrial functionality and thus, the loss of the membrane potential was observed in lycopene treated schizonts. Taken together, our results indicated that the generation of ROS and loss of mitochondrial membrane potential accounted for lycopene's cytotoxicity against P. falciparum growth in vitro. These insights will help in the design of new treatment strategies to combat malaria.

Spectroscopic correlation and role of Azathioprine in long-term remission in patients of Hashimoto encephalopathy.

Hashimoto encephalopathy remains a Rubik's cube for the present generation of clinical research. Myriad presentations have been noted, and observations recorded in few subgroups of patients have gone on only to be trashed by a second group of patients with a completely different clinical profile. Steroids have been traditionally held to be the treatment for this condition, but long-term side effects associated with it limits its use. Although multiple drugs have been tried, yet there exists no data for their long-term efficacy in maintaining remission. No radiological findings have been consistently associated with this condition. We report the use of azathioprine in maintaining long-term remission in one such patient with Hashimoto encephalopathy and the presence of lactate peak in magnetic resonance spectroscopy of the patient, which showed dramatic regression with institution of immunosuppression.