Parkinson's-linked LRRK2-G2019S derails AMPAR trafficking, mobility, and composition in striatum with cell-type and subunit specificity.

Parkinson's disease (PD) is a multifactorial disease that affects multiple brain systems and circuits. While defined by motor symptoms caused by degeneration of brainstem dopamine neurons, debilitating non-motor abnormalities in fronto-striatal-based cognitive function are common, appear early, and are initially independent of dopamine. Young adult mice expressing the PD-associated G2019S missense mutation in Lrrk2 also exhibit deficits in fronto-striatal-based cognitive tasks. In mice and humans, cognitive functions require dynamic adjustments in glutamatergic synapse strength through cell-surface trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs), but it is unknown how LRRK2 mutation impacts dynamic features of AMPAR trafficking in striatal projection neurons (SPNs). Here, we used Lrrk2G2019S knockin mice to show that surface AMPAR subunit stoichiometry is altered biochemically and functionally in mutant SPNs in dorsomedial striatum to favor the incorporation of GluA1 over GluA2. GluA1-containing AMPARs were resistant to internalization from the cell surface, leaving an excessive accumulation of GluA1 on the surface within and outside synapses. This negatively impacted trafficking dynamics that normally support synapse strengthening, as GluA1-containing AMPARs failed to increase at synapses in response to a potentiating stimulus and showed significantly reduced surface mobility. Surface GluA2-containing AMPARs were expressed at normal levels in synapses, indicating subunit-selective impairment. Abnormal surface accumulation of GluA1 was independent of PKA activity and was limited to D1R SPNs. Since LRRK2 mutation is thought to be part of a common PD pathogenic pathway, our data suggest that sustained, striatal cell-type specific changes in AMPAR composition and trafficking contribute to cognitive or other impairments associated with PD.

Algorithms for seeding social networks can enhance the adoption of a public health intervention in urban India.

Targeting structurally influential individuals within social networks can enhance adoption of health interventions within populations. We tested the effectiveness of two algorithms to improve social contagion that do not require knowledge of the whole network structure. We mapped the social interactions of 2,491 women in 50 residential buildings (chawls) in Mumbai, India. The buildings, which are social units, were randomized to (1) targeting 20% of the women at random, (2) targeting friends of such randomly chosen women, (3) targeting pairs of people composed of randomly chosen women and a friend, or (4) no targeting. Both targeting algorithms, friendship nomination and pair targeting, enhanced adoption of a public health intervention related to the use of iron-fortified salt for anemia. In particular, the targeting of pairs of friends, which is relatively easily implementable in field settings, enhanced adoption of novel practices through both social influence and social reinforcement.

Bidirectional Neuronal Actuation by Uncaging with Violet and Green Light.

We have developed a photochemical protecting group that enables wavelength selective uncaging using green versus violet light. Change of the exocyclic oxygen of the laser dye coumarin-102 to sulfur, gave thio-coumarin-102, a new chromophore with an absorption ratio at 503/402 nm of 37. Photolysis of thio-coumarin-102 caged γ-aminobutyric acid was found to be highly wavelength selective on neurons, with normalized electrical responses >100-fold higher in the green versus violet channel. When partnered with coumarin-102 caged glutamate, we could use whole cell violet and green irradiation to fire and block neuronal action potentials with complete orthogonality. Localized irradiation of different dendritic segments, each connected to a neuronal cell body, in concert with 3-dimenional Ca2+ imaging, revealed that such inputs could function independently. Chemical signaling in living cells always involves a complex balance of multiple pathways, use of (thio)-coumarin-102 caged compounds will enable arbitrarily timed flashes of green and violet light to interrogate two independent pathways simultaneously.

Small circular interfering RNAs (sciRNAs) as a potent therapeutic platform for gene-silencing.

In order to achieve efficient therapeutic post-transcriptional gene-silencing mediated by the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) must be chemically modified. Several supra-RNA structures, with the potential to stabilize siRNAs metabolically have been evaluated for their ability to induce gene silencing, but all have limitations or have not been explored in therapeutically relevant contexts. Covalently closed circular RNA transcripts are prevalent in eukaryotes and have potential as biomarkers and disease targets, and circular RNA mimics are being explored for use as therapies. Here we report the synthesis and evaluation of small circular interfering RNAs (sciRNAs). To synthesize sciRNAs, a sense strand functionalized with the trivalent N-acetylgalactosamine (GalNAc) ligand and cyclized using 'click' chemistry was annealed to an antisense strand. This strategy was used for synthesis of small circles, but could also be used for synthesis of larger circular RNA mimics. We evaluated various sciRNA designs in vitro and in vivo. We observed improved metabolic stability of the sense strand upon circularization and off-target effects were eliminated. The 5'-(E)-vinylphosphonate modification of the antisense strand resulted in GalNAc-sciRNAs that are potent in vivo at therapeutically relevant doses. Physicochemical studies and NMR-based structural analysis, together with molecular modeling studies, shed light on the interactions of this novel class of siRNAs, which have a partial duplex character, with the RNAi machinery.

A two-step process for in silico screening to assess the performance of qRTPCR kits against variant strains of SARS-CoV-2.

BACKGROUND: Since inception of the COVID-19 pandemic, early detection and isolation of positive cases is one of the key strategies to restrict disease transmission. Real time reverse transcription polymerase chain reaction (qRTPCR) has been the mainstay of diagnosis. Most of the qRTPCR kits were designed against the target genes of original strain of SARS-CoV-2. However, with the emergence of variant strains of SARS-CoV-2, sensitivity of the qRTPCR assays has reportedly reduced. In view of this, it is critical to continuously monitor the performance of the qRTPCR kits in the backdrop of variant strains of SARS-CoV-2. Real world monitoring of assay performance is challenging. Therefore, we developed a two-step in-silico screening process for evaluating the performance of various qRTPCR kits used in India. RESULTS: We analysed 73 qRT-PCR kits marketed in India, against the two SARS-CoV-2 VoCs. Sequences of both Delta (B.1.617.2) and Omicron (B.1.1.529) VoCs submitted to GISAID within a specific timeframe were downloaded, clustered to identify unique sequences and aligned with primer and probe sequences. Results were analysed following a two-step screening process. Out of 73 kits analysed, seven were unsatisfactory for detection of both Delta and Omicron VoCs, 10 were unsatisfactory for Delta VoC whereas 2 were unsatisfactory for only Omicron VoC. CONCLUSION: Overall, we have developed a useful screening process for evaluating the performance of qRTPCR assays against Delta and Omicron VoCs of SARS-CoV-2 which can be used for detecting SARS-CoV-2 VoCs that may emerge in future and can also be redeployed for other evolving pathogens of public health importance.

Identification and validation of two alternatively spliced novel isoforms of human α-1-antichymotrypsin.

α-1-antichymotrypsin (ACT) is a serine proteinase inhibitor that controls the activity of proteases like chymotrypsin, cathepsin G and mast cell chymase. Familial variants of ACT results in liver and lung diseases, but it is also reported to be associated with several other disease conditions. ACT is mainly synthesized in the liver using four coding exons, namely E1, E2, E3 and E4 encoding a 423 amino acid protein that also includes a 23 amino acid signal peptide. It is found to be associated with amyloid plaques and is elevated during inflammatory response and modulates cytokine based signal transduction pathways, independent of its anti-protease activity. Therefore, the multispecificity of ACT and its non-inhibitory roles in diseased conditions warrants an assessment of possible existence of the other isoforms. Consequently, scanning of introns, 5' and 3' region of the ACT gene using computational tools like FGENESH and FEX did indicate the presence of coding regions. Using a combined approach of bioinformatics and molecular biology, we have found one novel exon located in the intronic region between exons E1 and E2, that splices with exon E2 and replaces N-terminal exon E1, generating an ACT isoform with a novel 151 base pair N-terminus. This isoform was found to lack the signal sequence and is smaller in size but its reactive centre loop remains intact. A truncated transcript was also confirmed with an extension of the E3 by a 12 nucleotide intronic region including a stop codon. Modelling studies show that due to removal of E4 this isoform lacks the RCL. Novel isoform ACT-N lacks E1 but has a conserved RCL. However, due to loss of strands of ß-sheet A, it may also be inactive, but with ability to bind the target proteases. The novel truncated ACT-T isoform lacks the RCL and may have a non-inhibitory role. These hypothesis will need further work for functional validation.

Unveiling the mechanisms and biosynthesis of a novel nickel-pincer enzyme.

The nickel-pincer nucleotide (NPN) coenzyme, a substituted pyridinium mononucleotide that tri-coordinates nickel, was first identified covalently attached to a lysine residue in the LarA protein of lactate racemase. Starting from nicotinic acid adenine dinucleotide, LarB carboxylates C5 of the pyridinium ring and hydrolyzes the phosphoanhydride, LarE converts the C3 and C5 carboxylates to thiocarboxylates, and LarC incorporates nickel to form a C-Ni and two S-Ni bonds, during the biosynthesis of this cofactor. LarB uses a novel carboxylation mechanism involving the transient formation of a cysteinyl-pyridinium adduct. Depending on the source of the enzyme, LarEs either catalyze a sacrificial sulfur transfer from a cysteinyl side chain resulting in the formation of dehydroalanine or they utilize a [4Fe-4S] cluster bound by three cysteine residues to accept and transfer a non-core sulfide atom. LarC is a CTP-dependent enzyme that cytidinylylates its substrate, adds nickel, then hydrolyzes the product to release NPN and CMP. Homologs of the four lar genes are widely distributed in microorganisms, with some species containing multiple copies of larA whereas others lack this gene, consistent with the cofactor serving other functions. Several LarA-like proteins were shown to catalyze racemase or epimerase activities using 2-hydroxyacid substrates other than lactic acid. Thus, lactate racemase is the founding member of a large family of NPN-containing enzymes.

Inter-laboratory testing as a strategy for external quality assessment for qualitative detection of SARS-CoV-2 by real-time RT-PCR testing in India.

To implement the strategy of test, track and treat to tackle the ongoing COVID-19 pandemic, the number of real-time RT-PCR-based testing laboratories was increased for diagnosis of SARS-CoV-2 in the country. To ensure reliability of the laboratory results, the Indian Council of Medical Research initiated external quality assessment (EQA) by deploying inter-laboratory quality control (ILQC) activity for these laboratories by nominating 34 quality control (QC) laboratories. This report presents the results of this activity for a period of September 2020 till November 2020. A total of 597 laboratories participated in this activity and 86 per cent of these scored ≥90 per cent concordance with QC laboratories. This ILQC activity showcased India's preparedness in quality diagnosis of SARS-CoV-2.

Investigating the pharmacodynamic durability of GalNAc-siRNA conjugates.

One hallmark of trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNAs is the remarkable durability of silencing that can persist for months in preclinical species and humans. Here, we investigated the underlying biology supporting this extended duration of pharmacological activity. We found that siRNA accumulation and stability in acidic intracellular compartments is critical for long-term activity. We show that functional siRNA can be liberated from these compartments and loaded into newly generated Argonaute 2 protein complexes weeks after dosing, enabling continuous RNAi activity over time. Identical siRNAs delivered in lipid nanoparticles or as GalNAc conjugates were dose-adjusted to achieve similar knockdown, but only GalNAc-siRNAs supported an extended duration of activity, illustrating the importance of receptor-mediated siRNA trafficking in the process. Taken together, we provide several lines of evidence that acidic intracellular compartments serve as a long-term depot for GalNAc-siRNA conjugates and are the major contributor to the extended duration of activity observed in vivo.

Clinico-Radiological-Pathological Correlation of Visual Loss in COVID-Associated Rhino-Orbito-Cerebral Mucormycosis.

PURPOSE: To correlate the clinical, radiological, and histopathological features in Covid-associated Rhino-orbito-cerebral mucormycosis cases presenting with acute visual loss. DESIGN: Cross-sectional study. METHODS: Covid-associated Rhino-orbito-cerebral mucormycosis cases with unilateral visual loss, planned for exenteration, underwent orbital and ophthalmological ocular examination. The available radiological sequences, doppler ultrasonography and histopathology findings were correlated with clinical manifestations. RESULTS: The median age was 51 years and the male: female ratio was 3:1. All except one presented with unilateral ophthalmoplegia. The ocular media were hazy in 2 eyes. In 8 eyes, retinal changes were suggestive of occlusion of CRA (6), combined occlusion of CRA and central retinal vein (1), and myopic degeneration with hypertensive retinopathy (1). The contralateral eye showed retinal ischemic changes in one patient. Radiological imaging showed orbital apex involvement in the 10 affected eyes and one contralateral eye. Ipsilateral cavernous sinus thrombosis, diffusion restriction on MRI of optic nerve, internal carotid artery narrowing/thrombosis, and cortical watershed infarcts were seen in 8, 4, 4, and 2 cases, respectively. The blood flow in CRA and ophthalmic artery was absent or reduced in all the 10 affected eyes and in 1 contralateral eye. On histopathology, orbital fat necrosis, fungal hyphae, acute inflammation, granuloma formation, ischemic thrombosis of ophthalmic artery was observed in 10 specimens. CRA was patent in 9 and thrombosed in 1 eye. Optic nerve was ischemic in 8 and viable in 2 eyes. CONCLUSION: Acute visual loss in ROCM cases is associated with orbital apex involvement and thrombotic ischemia of ophthalmic artery. Cessation of flow in CRA possibly occurs secondary to ophthalmic artery thrombosis.

Properties of Parallel Tetramolecular G-Quadruplex Carrying N-Acetylgalactosamine as Potential Enhancer for Oligonucleotide Delivery to Hepatocytes.

The development of oligonucleotide conjugates for in vivo targeting is one of the most exciting areas for oligonucleotide therapeutics. A major breakthrough in this field was the development of multifunctional GalNAc-oligonucleotides with high affinity to asialoglycoprotein receptors (ASGPR) that directed therapeutic oligonucleotides to hepatocytes. In the present study, we explore the use of G-rich sequences functionalized with one unit of GalNAc at the 3'-end for the formation of tetrameric GalNAc nanostructures upon formation of a parallel G-quadruplex. These compounds are expected to facilitate the synthetic protocols by providing the multifunctionality needed for the binding to ASGPR. To this end, several G-rich oligonucleotides carrying a TGGGGGGT sequence at the 3'-end functionalized with one molecule of N-acetylgalactosamine (GalNAc) were synthesized together with appropriate control sequences. The formation of a self-assembled parallel G-quadruplex was confirmed through various biophysical techniques such as circular dichroism, nuclear magnetic resonance, polyacrylamide electrophoresis and denaturation curves. Binding experiments to ASGPR show that the size and the relative position of the therapeutic cargo are critical for the binding of these nanostructures. The biological properties of the resulting parallel G-quadruplex were evaluated demonstrating the absence of the toxicity in cell lines. The internalization preferences of GalNAc-quadruplexes to hepatic cells were also demonstrated as well as the enhancement of the luciferase inhibition using the luciferase assay in HepG2 cell lines versus HeLa cells. All together, we demonstrate that tetramerization of G-rich oligonucleotide is a novel and simple route to obtain the beneficial effects of multivalent N-acetylgalactosamine functionalization.

Correction: Clua et al. Properties of Parallel Tetramolecular G-Quadruplex Carrying N-Acetylgalactosamine as Potential Enhancer for Oligonucleotide Delivery to Hepatocytes. Molecules 2022, 27, 3944.

In the original article [...].

Protective Effect of Methanolic Extract of Euglena tuba Against Dalton Lymphoma Induced Oxidative Stress in BALB/c Mice.

The identification and pharmacological validation of plant-based lead compounds for the cure of different diseases including cancer have always been globally strived. In addition to possessing numerous medicinal properties, many of the phytochemicals display antioxidant potential activities. Reactive oxygen species (ROS) causeoxidative stress leading to several severe diseases such as cancer. The antioxidants are substances that fight against ROS to protect the cells from their damaging effects. In the present study, the effects of methanol extract of Euglena tuba(ETME) have been evaluated for its antioxidant and antitumor potential against Dalton's lymphoma (DL) introduced in BALB/cmice. After 24 h of intraperitoneal inoculation of DL cells in mice, ETME (300 mg kg-1 body weight) was administered intraperitoneally upto18 alternative days. On the 18th day, the mice were sacrificed; the blood and tissues (liver and brain) were collected to determine the tumor growth parameters including morphological, behavioural, haematological profile, and antioxidant indices. The results indicated that ETME exhibited significant antioxidative and antitumor properties when compared with the data from DL bearing mice. The results from the present study indicated that ETME contained remarkable antitumor efficacy, which was mediated through amelioration of oxidative stress. The data suggested that ETME could be used as a potential natural anticancer agent.

Long-term impact of testicular torsion and its salvage on semen parameters and gonadal function.

Introduction: Testicular torsion is a urological emergency, and long-term outcomes of testicular torsion on infertility, hormonal function, and salvaged testicular size are unclear. Materials and Methods: We conducted an ambispective, observational study from January 2014 to December 2019. Baseline demographics, time of presentation, clinical features, and management details of all the patients of testicular torsion were recorded from the database. All the patients were followed up in the outpatient clinic for testicular size, hormone levels, semen analysis, and erectile function. Results: Of 85 patients, only 67 could be contacted and included in the final analysis. Group 1(orchiectomy) comprised 44 patients, and Group 2(salvage) had 23 patients. Follow-up duration ranged from 2 to 6 years and mean follow-up was 42 ± 12 months. The median time to presentation was significantly higher in Group 1 (48 hours) as compared to Group 2 (12 hours). The rate of testicular salvage did not vary with age of the patients. Doppler ultrasonography of the scrotum detected 92.5% of all cases of torsion. Antisperm antibody levels were within normal range in all patients. Approximately 47% of patients in the salvage group developed testicular atrophy on follow-up. Serum testosterone level was significantly lower in Group 1 and the subset of patients with testicular atrophy. Rest of the hormonal parameters, semen analysis, and erectile function were comparable between two groups. Conclusion: The time between onset and presentation is an important contributing factor in guiding testicular salvage. Even after salvage, many testes may atrophy on follow-up. Orchiectomy and testicular atrophy in the long term have negative impact on serum testosterone. The patients should be counseled for a long-term follow-up for the risk of testicular atrophy and low testosterone levels.

Checkerboard Analysis To Evaluate Synergistic Combinations of Existing Antifungal Drugs and Propylene Glycol Monocaprylate in Isolates from Recalcitrant Tinea Corporis and Cruris Patients Harboring Squalene Epoxidase Gene Mutation.

Recalcitrant dermatophytic infections of the glabrous skin (tinea corporis/cruris/faciei) pose a huge challenge to health care systems. Combinations of oral and topical drugs may potentially improve cure rates, but the same has never been objectively assessed for this condition in laboratory or clinical studies. The present study was undertaken with the aim of identifying synergistic combinations of oral and topical antifungals by testing clinical isolates obtained from patients with recalcitrant tinea corporis/cruris. Forty-two patients with tinea corporis/cruris who had failed oral antifungals or had relapsed within 4 weeks of apparent clinical cure were recruited. Twenty-one isolates were identified by sequencing (all belonging to the Trichophyton mentagrophytes/T. interdigitale species complex) and subjected to antifungal susceptibility testing (AFST) and squalene epoxidase (SQLE) gene mutation analysis. Finally, five isolates, four with underlying SQLE gene mutations and one wild-type strain, were chosen for checkerboard studies using various combinations of antifungal agents. Most isolates (n = 16) showed high MICs of terbinafine (TRB) (0.5 to >16 µg/ml), with SQLE gene mutations being present in all isolates with MICs of ≥0.5 µg/ml. Synergistic interactions were noted with combinations of itraconazole with luliconazole, TRB, and ketoconazole and propylene glycol monocaprylate (PGMC) with luliconazole and with the triple combination of PGMC with luliconazole and ketoconazole. In vitro synergistic interactions provide a sound scientific basis for the possible clinical use of antifungal combinations. Hence, these synergistic combinations may be tested for clinical utility in the wake of rising resistance among dermatophytic infections of the glabrous skin.

Inhibition of Plasmodium falciparum Lysyl-tRNA Synthetase via a Piperidine-Ring Scaffold Inspired Cladosporin Analogues.

Plasmodium falciparum lysyl-tRNA synthetase (PfKRS) represents a promising therapeutic anti-malarial target. Cladosporin was identified as a selective and potent PfKRS inhibitor but lacks metabolic stability. Here, we report chemical synthesis, biological evaluation and structural characterization of analogues where the tetrahydropyran (THP) frame of cladosporin is replaced with the piperidine ring bearing functional group variations. Thermal binding, enzymatic, kinetic and parasitic assays complemented with X-ray crystallography reveal compounds that are moderate in potency. Co-crystals of Cla-B and Cla-C with PfKRS reveal key atomic configurations that allow drug binding to and inhibition of the enzyme. Collectively these piperidine ring scaffold inhibitors lay a framework for further structural editing and functional modifications of the cladosporin scaffold to obtain a potent lead.

Formulation design, optimization and in vivo evaluation of oral co-encapsulated resveratrol-humic acid colloidal polymeric nanocarriers.

The study aims at formulation and optimization of resveratrol and humic acid co-encapsulated colloidal polymeric nanocarriers to improve stability, oral bioavailability, and antiradical activity of water-insoluble, resveratrol. The eudragit E100 polymeric material was used to fabricate resveratrol and humic acid co-encapsulated oral colloidal polymeric nanocarriers (Res-HA-co-CPNs) using emulsification-diffusion-evaporation method. Taguchi orthogonal array design was employed to check the effect of formulation factors on in vitro physicochemical characteristics. The optimized formulation was further evaluated for oral bioavailability as well as for antiradical potential. Optimized Res-HA-co-CPNs demonstrated spherical and smooth surface including mean particle size, 120.56 ± 18.8 nm; polydispersity index, 0.122; zeta potential, +38.25 mV; and entrapment efficiency, 82.37 ± 1.49%. Solid-state characterization confirmed the amorphous characteristic of optimized Res-HA-co-CPNs. In vitro release profile of Res-HA-co-CPNs showed sustained release behavior up to 48 h and CPNs were found to remain stable at the refrigerated condition for 6 months. In vivo pharmacokinetic studies revealed significant (p < 0.05) improvement of ∼62.76-fold in oral bioavailability. The radical-scavenging activity was found to be increased with time and after 72 h, it was analogous to pure Res. IC50 values were reported to be decreased with time. Henceforth, developed Res-HA-co-CPNs was proven to be a proficient dosage form to increase stability, oral bioavailability, and antiradical activity of resveratrol.HighlightsResveratrol-humic acid co-encapsulated colloidal polymeric nanocarriers (Res-HA-co-CPNs) were fabricated by emulsification-diffusion-evaporation method and optimized by Taguchi orthogonal array design.The Res-HA-co-CPNs revealed favorable mean particle size and percent encapsulation efficiency with a spherical and smooth surface.The Res-HA-co-CPNs showed diffusion-controlled release of Res and were found to be stable at the refrigerated condition for 6 months.The optimized Res-HA-co-CPNs demonstrated significantly (p < 0.05) higher oral bioavailability with respect to pure Res and PM.The optimized Res-HA-co-CPNs demonstrated higher radical-scavenging activity with respect to time.

Spontaneous subcapital femoral neck fracture complicating osteonecrosis of femoral head.

Spontaneous subcapital fracture (SSF) of femoral neck in pre-existent osteonecrosis of femoral head (ONFH) is a rare presentation. Only a few cases have been reported so far and majority of them have been reported to have unilateral hip involvement. We retrospectively reviewed clinical-radiological data of 10 patients (12 hips) with SSF complicating ONFH. All of them underwent uncemented total hip arthroplasty. All the available publications in the English language based medical literature were critically reviewed and results were summarized. The median age of presentation was 32 years (range : 24 years to 61 years). They were followed up for a mean duration of 25 months (range : 12 months to 59 months). The most common risk factor was corticosteroid consumption (7 out of 10 patients). All except one (modified Ficat and Arlet stage II) belonged to advanced stage of ONFH {stage III 3 patients (3 hips), stage IV 6 patients (8 hips)}. The mean time lag of ONFH to presentation was 22.3 months (range : 5 months to 60 months), and SSF to presentation was 13.8 days (range : 1 day to 28 days). Mean pre- operative Harris Hip Score was 10.8 (range : 8 to 14), which improved to 93 (range : 91 to 96) after total hip arthroplasty when last followed up (p<0.05). Corticosteroids induced ONFH has a propensity to develop SSF. This entity should find a place in existing classification system.

Cholera pulse vaccination: A possible elimination strategy for cholera in endemic countries.

Cholera is a diarrheal disease causing major health issue in developing countries where it is endemic and causes outbreaks. India ranks first with an estimated 675,188 number of cases and 20,256 number of deaths annually with one-third of its population at risk. The two broad approaches for cholera control are improving sanitation and vaccination. Now both live and killed oral vaccines are available. Live vaccines are advantageous in respect of intestinal colonization and rapid immune response and also lead to in vivo exposure of bacterial products leading to good immunological response against wild Vibrio cholerae infection. The three major delivery strategies which can be considered for the implementation of oral cholera vaccine are preemptive vaccination, reactive vaccinations, and National Immunization Program. We propose the use of cholera live oral vaccines for achieving control of this disease by repeated vaccination of the susceptible population in a series of pulses to control it from the entire population.