Immunogenicity and Neutralization Potential of Recombinant Chimeric Protein Comprising the Catalytic Region of Gp63 of Leishmania and LTB against Leishmania Donavani.

AIM: To study the inhibition potential of antibody against a recombinant chimera comprising of the catalytic epitope of gp63 of Leishmania donovani and B subunit of heat-labile enterotoxin [LTB] in the functional activity of L. donovani. BACKGROUND: Visceral leishmaniasis, caused by the protozoan parasite Leishmania donavani, is a major health problem and causes mortality in tropical regions. Protozoan proteases play a crucial role in the pathogenesis of the disease and in establishing infection by countering the host's innate immune responses, namely complement-mediated lysis and phagocytosis. A surface-bound metalloprotease [gp63] has been reported to be a major virulence factor resulting in the evasion of complement- mediated lysis, cleaving host extracellular and intracellular substrates, resulting in intra- phagolysosomal survival Method: The epitope corresponding to the catalytic motif of gp63 of Leishmania donovani has fused with the B subunit of heat-labile enterotoxin, which is known to be immunogenic. The chimera was cloned to a prokaryotic expression vector and purified using Ni NTA affinity chromatography. Antibodies were generated against the purified fusion protein and analyzed for its ability to bind to the gp63 catalytic motif peptide by ELISA. The effect of fusion protein antibody on the functional activity of gp63 was evaluated by assessing the effect of purified IgGs on the protease activity and complement-mediated lysis of L. donovani promastigotes in vitro. RESULTS: The present study reports that a recombinant chimera of the catalytic epitope of gp63 and B subunit of heat-labile enterotoxin [LTB] of E. coli, a potent adjuvant of humoral response can mount significant immune response towards the catalytic epitope. ELISA and Western blot analysis showed that the anti-fusion protein antiserum could recognize the native gp63. Also, it significantly inhibited the protease activity of promastigotes and subsequently increased complement-mediated lysis of the promastigotes in vitro. CONCLUSION: It could be concluded that the hybrid protein containing catalytic motif L. donovani gp63 protein and carrier protein [LTB] could elicit antibodies that could neutralise the functional activity of gp63 and thus could be a potential candidate for subunit leishmaniasis vaccine.

A genetic algorithm-based energy-aware multi-hop clustering scheme for heterogeneous wireless sensor networks.

Background: The energy-constrained heterogeneous nodes are the most challenging wireless sensor networks (WSNs) for developing energy-aware clustering schemes. Although various clustering approaches are proven to minimise energy consumption and delay and extend the network lifetime by selecting optimum cluster heads (CHs), it is still a crucial challenge. Methods: This article proposes a genetic algorithm-based energy-aware multi-hop clustering (GA-EMC) scheme for heterogeneous WSNs (HWSNs). In HWSNs, all the nodes have varying initial energy and typically have an energy consumption restriction. A genetic algorithm determines the optimal CHs and their positions in the network. The fitness of chromosomes is calculated in terms of distance, optimal CHs, and the node's residual energy. Multi-hop communication improves energy efficiency in HWSNs. The areas near the sink are deployed with more supernodes far away from the sink to solve the hot spot problem in WSNs near the sink node. Results: Simulation results proclaim that the GA-EMC scheme achieves a more extended network lifetime network stability and minimises delay than existing approaches in heterogeneous nature.

Scaling up a decentralized offline patient ID generation and matching algorithm to accelerate universal health coverage: Insights from a literature review and health facility survey in Nigeria.

Background: Quality of health service delivery data remains sub-optimal in many Low and middle-income countries (LMICs) despite over a decade of progress in digitization and Health Management Information Systems (HMIS) improvements. Identifying everyone residing in a country utilizing universal civil registration and/or national unique identification number systems especially for vulnerable patients seeking care within the care continuum is an essential part of pursuing universal health coverage (UHC). Many different strategies or candidate digital technologies exist for uniquely identifying and tracking patients within a health system, and the different strategies also have their advantages and trade-offs. The recent approval of Decentralized identifier (DID) core specification by World Wide Web Consortium (W3C) heralds the search for consensus on standard interoperable DID methods. Objective: This paper aims to: (1) assess how candidate Patient Identification Systems fit the digital Patient ID desirable attributes framework in literature; and (2) use insights from Nigeria to propose the scale-up of an offline, interoperable decentralized Patient ID generation and a matching model for addressing network reliability challenges of centralized electronic registries in LMICs. Methods: We combined: (i) systematic review of the literature to identify the characteristics of leading candidates for Patient ID systems, with (ii) review of policies and (iii) quantitative survey of 14 general hospitals in Nigeria's Federal Capital Territory to understand the model(s) of patient ID strategies currently implemented by public hospitals. Results: Evidence from the literature review and quantitative survey showed that no current Patient ID strategy in Nigeria simultaneously meets the six attributes of uniqueness, unchanging, uncontroversial, inexpensive, ubiquitous, and uncomplicated required for ensuring the reliability of unique patient identification systems and of the HMIS more generally. Conclusions: The findings are used to propose a model of algorithms for universal-offline Patient ID generation and matching models that is cost effective and can be easily scaled-up throughout Nigeria. The prototype has promise for generating and validating a universally unique Patient ID given a set of patient characteristics without a central rigid authority. The model can also help to fast-track the implementation of a Master Patient Index (MPI) and interoperability of existing digital health platforms in LMICs.

Standardizing Primary Health Care Referral Data Sets in Nigeria: Practitioners' Survey, Form Reviews, and Profiling of Fast Healthcare Interoperability Resources (FHIR).

BACKGROUND: Referral linkages are crucial for efficient functioning of primary health care (PHC) systems. Fast Healthcare Interoperability Resource (FHIR) is an open global standard that facilitates structuring of health information for coordinated exchange among stakeholders. OBJECTIVE: The objective of this study is to design FHIR profiles and present methodology and the profiled FHIR resource for Maternal and Child Health referral use cases in Ebonyi state, Nigeria-a typical low- and middle-income country (LMIC) setting. METHODS: Practicing doctors, midwives, and nurses were purposefully sampled and surveyed. Different referral forms were reviewed. The union of data sets from surveys and forms was aggregated and mapped to base patient FHIR resource elements, and extensions were created for data sets not in the core FHIR specification. This study also introduced FHIR and its relation to the World Health Organization's (WHO's) International Classification of Diseases. RESULTS: We found many different data elements from the referral forms and survey responses even in urban settings. The resulting FHIR standard profile is published on GitHub for adaptation or adoption as necessary to aid alignment with WHO recommendations. Understanding data sets used in health care and clinical practice for information sharing is crucial in properly standardizing information sharing, particularly during the management of COVID-19 and other infectious diseases. Development organizations and governments can use this methodology and profile to fast-track FHIR standards adoption for paper and electronic information sharing at PHC systems in LMICs. CONCLUSIONS: We presented our methodology for profiling the referral resource crucial for the standardized exchange of new and expectant moms' information. Using data from frontline providers and mapping to the FHIR profile helped contextualize the standardized profile.

Digital Health Solutions and State of Interoperability: Landscape Analysis of Sierra Leone.

BACKGROUND: The government and partners have invested heavily in the health information system (HIS) for service delivery, surveillance, reporting, and monitoring. Sierra Leone's government launched its first digital health strategy in 2018. In 2019, a broader national innovation and digital strategy was launched. The health pillar direction will use big data and artificial intelligence (AI) to improve health care in general and maternal and child health in particular. Understanding the number, distribution, and interoperability of digital health solutions is crucial for successful implementation strategies. OBJECTIVE: This paper presents the state of digital health solutions in Sierra Leone and how these solutions currently interoperate. This study further presents opportunities for big data and AI applications. METHODS: All the district health management teams, all digital health implementing organizations, and a stratified sample of 72 (out of 1284) health facilities were purposefully selected from all health districts and surveyed. RESULTS: The National Health Management Information System's (NHMIS's) aggregate reporting solution populated by health facility forms HF1 to HF9 was, by far, the most used tool. A health facility-based weekly aggregate electronic integrated disease surveillance and response solution was also widely used. Half of the health facilities had more than 2 digital health solutions in use. The different digital health software solutions do not share data among one another, though aggregate reporting data were sent as necessary. None of the respondents use any of the health care registries for patient, provider, health facility, or terminology identification. CONCLUSIONS: Many digital health solutions are currently used at health facilities in Sierra Leone. The government can leverage current investment in HIS from surveillance and reporting for using big data and AI for care. The vision of using big data for health care is achievable if stakeholders prioritize individualized and longitudinal patient data exchange using agreed use cases from national strategies. This study has shown evidence of distribution, types, and scale of digital health solutions in health facilities and opportunities for leveraging big data to fill critical gaps necessary to achieve the national digital health vision.

Metalloprotease Gp63-Targeting Novel Glycoside Exhibits Potential Antileishmanial Activity.

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.

Electricity, Computing Hardware, and Internet Infrastructures in Health Facilities in Sierra Leone: Field Mapping Study.

BACKGROUND: Years of health information system investment in many countries have facilitated service delivery, surveillance, reporting, and monitoring. Electricity, computing hardware, and internet networks are vital for health facility-based information systems. Availability of these infrastructures at health facilities is crucial for achieving national digital health visions. OBJECTIVE: The aim of this study was to gain insight into the state of computing hardware, electricity, and connectivity infrastructure at health facilities in Sierra Leone using a representative sample. METHODS: Stratified sampling of 72 (out of 1284) health facilities distributed in all districts of Sierra Leone was performed, factoring in the rural-urban divide, digital health activity, health facility type, and health facility ownership. Enumerators visited each health facility over a 2-week period. RESULTS: Among the 72 surveyed health facilities, 59 (82%) do not have institutionally provided internet. Among the 15 Maternal and Child Health Posts, as a type of primary health care unit (PHU), 9 (60%) use solar energy as their only electricity source and the other 6 (40%) have no electricity source. Similarly, among the 13 hospitals, 5 (38%) use a generator as a primary electricity source. All hospitals have at least one functional computer, although only 7 of the 13 hospitals have four or more functional computers. Similarly, only 2 of the 59 (3%) PHUs have one computer each, and 37 (63%) of the PHUs have one tablet device each. We consider this health care computing infrastructure mapping to be representative with a 95% confidence level within an 11% margin of error. Two-thirds of the PHUs have only alternate solar electricity, only 10 of the 72 surveyed health facilities have functional official internet, and most use suboptimal computing hardware. Overall, 43% of the surveyed health facilities believe that inadequate electricity is the biggest threat to digitization. Similarly, 16 (22%) of the 72 respondents stated that device theft is a primary hindrance to digitization. CONCLUSIONS: Electricity provision for off-electricity-grid health facilities using alternative and renewable energy sources is emerging. The current trend where GSM (Global System for Mobile Communication) service providers provide the internet to all health facilities may change to other promising alternatives. This study provides evidence of the critical infrastructure gaps in health facilities in Sierra Leone.

A Fast-Track Phenotypic Characterization of Plasmodium falciparum Vaccine Antigens through Lyse-Reseal Erythrocytes Mediated Delivery (LyRED) of RNA Interference for Targeted Translational Repression.

The minimal success of the malaria vaccine with available antigens indicates the need for intensive and accelerated research to identify and characterize new antigens that confer protection against infection, clinical manifestation, and even malaria transmission. Further, the genetic manipulation tools to characterize such antigens are very time-consuming and laborious due to the very low efficiency of transfection in the malaria parasite. Here, we report a human miRNA-mediated translational repression of antigens in Plasmodium falciparum as a fast-track method for understanding and validating their function. In this method, candidate miRNAs are designed based on favorable hybridization energy against a parasite gene, and miRNA mimics are delivered to the parasite by loading them as cargo in the erythrocytes by simple lyse-reseal method. Incubation of the miRNA loaded erythrocytes with purified mature trophozoites or schizonts results in the loaded erythrocytes' infection. The miRNA mimics are translocated to parasites, and the effect of miRNA-mediated translation repression can be monitored within 48-72 h post-invasion. Unlike other transfection based methods, this method is fast, reproducible, and robust. We call this method as lyse-reseal erythrocytes for delivery (LyRED) of miRNA, which is a rapid and straight-forward method providing an efficient alternative to the existing genetic tools for P. falciparum to characterize the function of antigens or genes. The identification of crucial antigens from the different stages of the Plasmodium falciparum life cycle by the miRNA targeting approach can fuel the development of efficacious subunit vaccines against malaria.

Plasmodium falciparum Antigen Expression in Leishmania Parasite: A Way Forward for Live Attenuated Vaccine Development.

Live attenuated vaccines (LAVs) are among the most critical interventions in modern medicine and have already proven their potential to save millions of lives. LAVs are always explored as potential vaccine candidates since they induce an immune response, which is as good as the wild type pathogen. For parasitic diseases, the efficacy of LAVs is still under investigation and needs extensive research to mark their presence in the field. In malaria, live attenuated sporozoites have been evaluated for a vaccine against the liver stage. This vaccine approach is limited due to the highly cumbersome technique of sporozoite isolation and related relapse issues. We have developed a novel vaccine against malaria by expressing Plasmodium falciparum antigens in Leishmania donovani promastigotes. These hybrid, recombinant L. donovani parasites mimicking P. falciparum parasite antigens were analyzed for their anti-malarial efficacy in preclinical studies. We demonstrate the potential of Leishmania spp. parasites in developing an important live vector vaccine against malaria for the induction of protective immune responses. Herein, we describe a method to express malaria parasite antigens in L. donovani promastigotes and analyze its potential for a vaccine against malaria. This methodology can be extended to live, attenuated Leishmania promastigotes parasites to develop LAV against malaria.

Defining Delayed Discharges of Inpatients and Their Impact in Acute Hospital Care: A Scoping Review.

BACKGROUND: With the ever-increasing demand on acute healthcare, the hospital discharge process and delayed discharges are considered relevant in achieving optimal performance in clinical settings. The purpose of this paper is to review the literature to identify conceptual and operational definitions of delayed discharges, identify causes and effects of delayed discharges, and also to explore the literature for interventions aimed at decreasing the impact (in terms of reducing the number/rate of delays) of delayed discharges in acute healthcare settings. METHODS: An extensive literature search yielded a total of 26 248 records. Sixty-four research articles were included in the scoping review after considering inclusion/exclusion criteria and the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) search strategy. The following databases were utilized: Cochrane, EBSCO, PubMed, PubMed Central, Medline, and Web of Science. The search was carried out between January 2017 and March 2020 and covered literature ranging from 1990 to 2019. Results were reviewed by authors for duplicates and filtered using the inclusion/ exclusion criteria. Tables were created to classify the chosen articles (n = 64), allowing us to organise findings and results. RESULTS: Conceptual and operational definitions were analysed. In turn, causes and effects of delayed discharges were extracted and represented in diagrammatic format, together with specific interventions used in acute healthcare settings to lessen the effect of delayed discharges. Operational definitions of delayed discharges were found to be more difficult to establish, particularly in the light of the vast number of different scenarios and workplace interventions uncovered in the literature. The main causes of delayed discharges were faulty organisational management, inadequate discharge planning, transfer of care problems, and age. The main effects were bed-blocking, A&E (Accident & Emergency) overcrowding, and financial implications. The main interventions included 'discharge before noon' initiative, 'discharge facilitation tools,' 'discharge delay tracking' mechanisms, and the role of general practitioners and social care staff. CONCLUSION: This paper fills a gap in the fragmented literature on delayed inpatient discharges by providing a researchbased perspective on conceptual and operational definitions, causes and effects, as well as interventions to minimize their impact. The findings and definitions are intended as points of reference for future research.

Formative Study of Mobile Phone Use for Family Planning Among Young People in Sierra Leone: Global Systematic Survey.

BACKGROUND: Teenage pregnancy remains high with low contraceptive prevalence among adolescents (aged 15-19 years) in Sierra Leone. Stakeholders leverage multiple strategies to address the challenge. Mobile technology is pervasive and presents an opportunity to reach young people with critical sexual reproductive health and family planning messages. OBJECTIVE: The objectives of this research study are to understand how mobile health (mHealth) is used for family planning, understand phone use habits among young people in Sierra Leone, and recommend strategies for mobile-enabled dissemination of family planning information at scale. METHODS: This formative research study was conducted using a systematic literature review and focus group discussions (FGDs). The literature survey assessed similar but existing interventions through a systematic search of 6 scholarly databases. Cross-sections of young people of both sexes and their support groups were engaged in 9 FGDs in an urban and a rural district in Sierra Leone. The FGD data were qualitatively analyzed using MAXQDA software (VERBI Software GmbH) to determine appropriate technology channels, content, and format for different user segments. RESULTS: Our systematic search results were categorized using Grading of Recommended Assessment and Evaluation (GRADE) into communication channels, audiovisual messaging format, purpose of the intervention, and message direction. The majority of reviewed articles report on SMS-based interventions. At the same time, most intervention purposes are for awareness and as helpful resources. Our survey did not find documented use of custom mHealth apps for family planning information dissemination. From the FGDs, more young people in Sierra Leone own basic mobile phones than those that have feature capablilities or are smartphone. Young people with smartphones use them mostly for WhatsApp and Facebook. Young people widely subscribe to the social media-only internet bundle, with the cost ranging from 1000 leones (US $0.11) to 1500 leones (US $0.16) daily. Pupils in both districts top-up their voice call and SMS credit every day between 1000 leones (US $0.11) and 5000 leones (US $0.52). CONCLUSIONS: mHealth has facilitated family planning information dissemination for demand creation around the world. Despite the widespread use of social and new media, SMS is the scalable channel to reach literate and semiliterate young people. We have cataloged mHealth for contraceptive research to show SMS followed by call center as widely used channels. Jingles are popular for audiovisual message formats, mostly delivered as either push or pull only message directions (not both). Interactive voice response and automated calls are best suited to reach nonliterate young people at scale.

Recombinant FimH, a fimbrial tip adhesin of Vibrio parahaemolyticus, elicits mixed T helper cell response and confers protection against Vibrio parahaemolyticus challenge in murine model.

Vibrio parahaemolyticus causes vibriosis in wide range of marine organisms, and is responsible for food borne illnesses in humans through consumption of contaminated uncooked/partially cooked seafood. Continued and widespread antibiotics usage to increase the productivity has led to antibiotics resistance development. This has necessitated the need to develop alternative methods to control its infection. Use of safe and effective vaccines against the virulence factors not only protects from infection, it also minimizes antibiotic usage. The colonization of V. parahaemolyticus in the host and disease development requires several adhesins present on the cell surface, and thereby make them attractive vaccine candidates. V. parahaemolyticus produces extracellular type 1 fimbriae that have been shown to play a role in adhesion, biofilm formation and virulence. FimH is one of the minor components of the type 1 fimbriae occurring on its very tip. Being present on the cell surface, it is highly immunogenic, and can be targeted as a potential vaccine candidate. The present study describes the immunogenic and vaccine potential of recombinant V. parahaemolyticus FimH (rVpFimH) expressed in E. coli. Immunization of BALB/c mice with the rVpFimH elicited a strong mixed immune response, T-cell memory (evidenced by antibody isotyping, cytokine profiling and T-cell proliferation assay), and agglutination positive antibodies. FACS analysis and immunogold labeling showed that the polyclonal anti-rVpFimH antibodies were able to recognize the FimH on V. parahaemolyticus cells. In vivo challenge of the rVpFimH-immunized mice with 2×LD50 dose of live bacteria showed one hundred percent survival. Thus, our findings clearly demonstrate the potential of FimH as an effective vaccine candidate against V. parahaemolyticus.

Toxin-antitoxin systems and their medical applications: current status and future perspective.

Almost all bacteria synthesize two types of toxins-one for its survival by regulating different cellular processes and another as a strategy to interact with host cells for pathogenesis. Usually, "bacterial toxins" are contemplated as virulence factors that harm the host organism. However, toxins produced by bacteria, as a survival strategy against the host, also hamper its cellular processes. To overcome this, the bacteria have evolved with the production of a molecule, referred to as antitoxin, to negate the deleterious effect of the toxin against itself. The toxin and antitoxins are encoded by a two-component toxin-antitoxin (TA) system. The antitoxin, a protein or RNA, sequesters the toxins of the TA system for neutralization within the bacterial cell. In this review, we have described different TA systems of bacteria and their potential medical and biotechnological applications. It is of interest to note that while bacterial toxin-antitoxin systems have been well studied, the TA system in unicellular eukaryotes, though predicted by the investigators, have never been paid the desired attention. In the present review, we have also touched upon the TA system of eukaryotes identified to date. KEY POINTS: Bacterial toxins harm the host and also affect the bacterial cellular processes. The antitoxin produced by bacteria protect it from the toxin's harmful effects. The toxin-antitoxin systems can be targeted for various medical applications.

Pathogenic Pore Forming Proteins of Plasmodium Triggers the Necrosis of Endothelial Cells Attributed to Malaria Severity.

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.

Recombinant outer membrane protein OmpC induces protective immunity against Aeromonashydrophila infection in Labeorohita.

Aeromonashydrophila is an opportunistic pathogen that causes enormous loss to aquaculture industry. The outer membrane proteins of Aeromonas help in bacterium-host interaction, and are considered to be potential vaccine candidates. In the present study, we evaluated immunogenicity and protective efficacy of recombinant OmpC (rOmpC) of A. hydrophila in Indian major carp, Labeorohita. The rOmpC-vaccinated fish produced specific anti-rOmpC antibodies with a significant antibody titer, and the antisera could specifically detect the rOmpC in the cell lysates of Escherichia coli expressing rOmpC and cross-react with different Aeromonas lysates, indicating the suitability of the anti-rOmpC antisera to detect Aeromonas infection. A significant increase was noted in ceruloplasmin level, myeloperoxidase and anti-protease activities in transient and temporal manner the sera of the rOmpC-immunized fish as compared to PBS-control fish. Higher agglutination- and hemolytic activity titers in the anti-rOmpC antisera indicate stimulation of innate immunity. Expression of immune-related genes comprising various acute phase proteins, cytokines and inflammatory response molecules were modulated in the head kidney of rOmpC-immunized L. rohita. While IgM, IL1ß, and TLR-22 were significantly up-regulated at early time points (3 h-72 h), the others showed a transient augmentation at both early and later time points (SOD, lysozymes C and G, NKEF-B, C3, CXCa and TNF-α) in the rOmpC-immunized L. rohita in comparison to PBS-injected controls. These data suggest that the rOmpC-induced immune response is temporally regulated to confer immunity. In vivo challenge of the rOmpC-immunized fish with A. hydrophila showed significantly greater survival when compared to PBS-injected control fish. Thus, our results highlight the immunomodulatory role of rOmpC and demonstrate its protective efficacy in L. rohita, along with the use of anti-rOmpC antisera in detecting Aeromonas infections.

Molecular docking analysis of Clostridium perfringens beta toxin model with potential inhibitors from the ZINC database.

Beta toxin from Clostridium perfringens after being secreted in gut is capable of causing necrotic enteritis in humans and several other animal species and does not respond to routinely used antibiotics. Therefore, there is a need to design an effective inhibitor for the Clostridium perfringens beta toxin (CPB) using cutting edge drug discovery technologies. Hence, potential CPB inhibitors were identified using computer aided screening of compounds from the ZINC database. Further, we document the molecular docking analysis of Clostridium perfringens beta toxin model (that revealed 4 binding pockets, A-D) with the identified potential inhibitors. We show that ZINC291192 [N-[(1-methylindol-3-yl) methyl eneamino]-7,10-dioxabicyclo[4.4.0]deca-2,4,11-triene-8- carboxamide] has optimal binding features with calculated binding energy of -10.38 kcal/mol and inhibition constant of 24.76 nM for further consideration.

Molecular docking analysis of P2X7 receptor with the beta toxin from Clostridium perfringens.

Clostridium perfringens beta-toxin (CPB) is linked to necrotic enteritis (over proliferation of bacteria) in several species showing cytotoxic effect on primary porcine endothelial and human precursor immune cells. P2X7 receptor on THP-1 cells is known to bind CPB. This is critical to understand the mechanism of pore formation for effective drug design. The structure of CPB and P2X7 receptor proteins were modeled using standard molecular modeling procedures (I-TASSER and Robetta server). This is followed by protein-protein docking (HADDOCK server) to study their molecular interaction. Interacting residues (19 residues from CPB and 21 residues from P2X7) were identified using the PISA server. Thus, we document the molecular docking analysis of P2X7 receptor with the beta toxin from Clostridium perfringens towards drug design and development of drugs to control necrotic enteritis.

Structural-functional characterization of recombinant Apolipoprotein A-I fromLabeo rohitademonstrates heat-resistant antimicrobial activity.

Apolipoprotein A-I is an anti-inflammatory, antioxidative, cardioprotective, anti-tumorigenic, and anti-diabetic in mammals. Apolipoprotein A-I also regulates innate immune defense mechanisms in vertebrates and invertebrates. Apolipoproteins A-I from mammals and several teleosts display antibacterial activities against Gram negative and Gram positive bacteria. The present study describes strategies to obtain high amounts of soluble purified recombinant Apolipoprotein A-I of Labeo rohita, an Indian major carp (rLrApoA-I). The study also reports its detailed structural and functional characterization i.e. antimicrobial activity against a number of important marine and fresh water bacterial pathogens. The rLrApoA-I was expressed in Escherichia coli BL21(DE3) pLysS expression host as a soluble protein under optimized conditions. The yield of purified rLrApoA-I was ~ 75 mg/L from soluble fraction using metal ion affinity chromatography. The authenticity of the rLrApoA-I was confirmed by MALDI-TOF-MS analysis. The secondary structure analysis showed rLrApoA-I to be predominantly alpha helical, an evolutionary conserved characteristic across mammals and teleosts. The purified rLrApoA-I exhibited antimicrobial activity as evident from inhibition of growth of a number of bacteria namely Aeromonas hydrophila, A. liquefaciens, A. culicicola, A. sobria, Vibrio harveyi, V. parahaemolyticus and Edwardsiella tarda in a dose-dependent manner. Minimum bactericidal concentration for A. liquefaciens, A. culicicola, and A. sobria, was determined to be 25 µg/ml or 0.81 µM whereas for A. hydrophila, E. tarda, V. parahaemolyticus and V. harveyi, it was determined to be 100 µg/ml or 3.23 µM. These data strongly suggest that recombinant ApoA-I from Labeo rohita could play a role in primary defense against fish pathogen. Further, at temperature ≥ 55 °C, though a loss in secondary structure was observed, no effect on its antibacterial activity was observed. This is of significance as the antibacterial activity is not likely to be lost even if the protein is subjected to high temperatures during transport.

Anonymity Preserving IoT-Based COVID-19 and Other Infectious Disease Contact Tracing Model.

Automated digital contact tracing is effective and efficient, and one of the non-pharmaceutical complementary approaches to mitigate and manage epidemics like Coronavirus disease 2019 (COVID-19). Despite the advantages of digital contact tracing, it is not widely used in the western world, including the US and Europe, due to strict privacy regulations and patient rights. We categorized the current approaches for contact tracing, namely: mobile service-provider-application, mobile network operators' call detail, citizen-application, and IoT-based. Current measures for infection control and tracing do not include animals and moving objects like cars despite evidence that these moving objects can be infection carriers. In this article, we designed and presented a novel privacy anonymous IoT model. We presented an RFID proof-of-concept for this model. Our model leverages blockchain's trust-oriented decentralization for on-chain data logging and retrieval. Our model solution will allow moving objects to receive or send notifications when they are close to a flagged, probable, or confirmed diseased case, or flagged place or object. We implemented and presented three prototype blockchain smart contracts for our model. We then simulated contract deployments and execution of functions. We presented the cost differentials. Our simulation results show less than one-second deployment and call time for smart contracts, though, in real life, it can be up to 25 seconds on Ethereum public blockchain. Our simulation results also show that it costs an average of $1.95 to deploy our prototype smart contracts, and an average of $0.34 to call our functions. Our model will make it easy to identify clusters of infection contacts and help deliver a notification for mass isolation while preserving individual privacy. Furthermore, it can be used to understand better human connectivity, model similar other infection spread network, and develop public policies to control the spread of COVID-19 while preparing for future epidemics.

A small bioactive glycoside inhibits epsilon toxin and prevents cell death.

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 ß-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.