Collaborative study for the calibration of a replacement International Standard for tetanus toxoid for use in flocculation test.

Here we report the results of a study to establish a replacement WHO International Standard (IS) for tetanus toxoid for use in flocculation test. The standard was calibrated in flocculation units (Lf) against the 2nd IS using the Ramon flocculation method. At its 70th meeting in October 2019, WHO ECBS established the material (coded 16/302) as the 3rd WHO IS, with an assigned value of 970 Lf/ampoule from the results of seventeen laboratories across ten different countries. The study also provided an opportunity to assess the use of alternative methods for measuring Lf. Participants were asked to use an in-house Enzyme Linked Immunosorbent Assay (ELISA) developed at NIBSC, or other suitable in-house methods, to determine ELISA-specific Lf values (Lf-eq units are specific only for pre-calibration of antitoxin in the flocculation test) of 16/302 to compare to those of the flocculation test. Nine laboratories participated by performing the NIBSC ELISA, one laboratory performed flocculation by laser light-scattering following an in-house protocol, and three laboratories performed ELISA following in-house protocols. The results intimate that these alternative methods could be useful for monitoring consistency of production at different stages of vaccine manufacturing.

Inferring bona fide Differentially Expressed Genes and Their Variants Associated with Vitamin K Deficiency Using a Systems Genetics Approach.

Systems genetics is key for integrating a large number of variants associated with diseases. Vitamin K (VK) is one of the scarcely studied disease conditions. In this work, we ascertained the differentially expressed genes (DEGs) and variants associated with individual subpopulations of VK disease phenotypes, viz., myocardial infarction, renal failure and prostate cancer. We sought to ask whether or not any DEGs harbor pathogenic variants common in these conditions, attempt to bridge the gap in finding characteristic biomarkers and discuss the role of long noncoding RNAs (lncRNAs) in the biogenesis of VK deficiencies.

Whole genome sequencing of ASD 16 and ADT 43 to identify predominant grain size and starch associated alleles in rice.

BACKGROUND: The rice cultivars ASD 16 and ADT 43 are the most popular high-yielding Indica rice cultivars in southern India. Despite their popularity very little is known about their genetic basis due to lack of studies on the complete genome. In the current study, efforts were made to identify alleles and SNP markers that differentiate the two contrasting rice genotypes, ASD 16 and ADT 43 for grain shape and starch content. METHODS AND RESULTS: The complete genome of bold grain ASD 16 and slender grain ADT 43 were sequenced via Illumina's paired-end sequencing and the reads obtained were mapped to the Oryza sativa Indica Group cultivar 93-11 reference genome. The grain size of rice is controlled by Quantitative Trait Loci (QTL) that has a robust effect on grain yield and quality. To gain insight into genes that controlling grain size and starch content, an in-silico analysis was performed by taking into account of 72 grain elongation and starch biosynthesis genes. The identified alleles were further validated in the whole genome sequencing data of 32 bold grain and 25 slender grain varieties that were retrieved from the 3 K rice genome project. CONCLUSION: An "A to G" polymorphism leading to SER 74 PRO was identified at the CDS position 220 of the An-1 gene, encoding bHLH domain-containing protein that regulates awn formation and increase in grain length. The non-synonymous substitutions such as A545C variant leading PHE 182 CYS in ADP Glucose Pyrophosphorylase large subunit IV (AGPL4) and C3094G variant leading to VAL 1032 LEU in Starch synthase IIIb (OsSSIIIb) were also identified in the starch biosynthesis genes. These identified allelic variants may contribute to the crop improvement programs in rice.

Genetically detoxified tetanus toxin as a vaccine and conjugate carrier protein.

Tetanus toxoid (TTxd), developed over 100 years ago, is a clinically effective, legacy vaccine against tetanus. Due to the extreme potency of native tetanus toxin, manufacturing and regulatory efforts often focus on TTxd production, standardization, and safety, rather than product modernization. Recently, a genetically detoxified, full-length tetanus toxin protein (8MTT) was reported as a tetanus vaccine alternative to TTxd (Przedpelski et al. mBio, 2020). Here we describe the production of 8MTT in Gor/MetTM E. coli, a strain engineered to have an oxidative cytoplasm, allowing for the expression of soluble, disulfide-bonded proteins. The strain was also designed to efficiently cleave N-terminal methionine, the obligatory start amino acid for E. coli expressed proteins. 8MTT was purified as a soluble protein from the cytoplasm in a two-column protocol to > 99 % purity, yielding 0.5 g of purified 8MTT/liter of fermentation broth with low endotoxin contamination, and antigenic purity of 3500 Lf/mg protein nitrogen. Mouse immunizations showed 8MTT to be an immunogenic vaccine and effective as a carrier protein for peptide and polysaccharide conjugates. These studies validate 8MTT as commercially viable and, unlike the heterogenous tetanus toxoid, a uniform carrier protein for conjugate vaccines. The development of a recombinant, genetically detoxified toxin produced in E. coli aligns the tetanus vaccine with modern manufacturing, regulatory, standardization, and safety requirements.

The Omic Insights on Unfolding Saga of COVID-19.

The year 2019 has seen an emergence of the novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease of 2019 (COVID-19). Since the onset of the pandemic, biological and interdisciplinary research is being carried out across the world at a rapid pace to beat the pandemic. There is an increased need to comprehensively understand various aspects of the virus from detection to treatment options including drugs and vaccines for effective global management of the disease. In this review, we summarize the salient findings pertaining to SARS-CoV-2 biology, including symptoms, hosts, epidemiology, SARS-CoV-2 genome, and its emerging variants, viral diagnostics, host-pathogen interactions, alternative antiviral strategies and application of machine learning heuristics and artificial intelligence for effective management of COVID-19 and future pandemics.

Characterisation of tetanus monoclonal antibodies as a first step towards the development of an in vitro vaccine potency immunoassay.

Batch release testing for human and veterinary tetanus vaccines still relies heavily on methods that involve animals, particularly for potency testing. The quantity and quality of tetanus antigen present in these products is of utmost importance for product safety and clinical effect. Immunochemical methods that measure consistency of antigen content and quality, potentially as an indicator of potency, could be a better choice and negate the need for an in vivo potency test. These immunochemical methods require at least one well characterised monoclonal antibody (mAb) that is specific for the target antigen. In this paper we report the results of the comprehensive characterisation of a panel of mAbs against tetanus with a view to select antibodies that can be used for development of an in vitro potency immunoassay. We have assessed binding of the antibodies to native antigen (toxin), detoxified antigen (toxoid), adsorbed antigen and heat-altered antigen. Antibody function was determined using an in-house cell-based neutralisation assay to support prior in vivo potency data that was available for some, but not all, of the antibodies. In addition, antibody affinity was measured, and epitope competition analysis was performed to identify pairs of antibodies that could be deployed in a sandwich immunoassay format. Not all characterisation tests provided evidence of "superiority" of one mAb over another, but together the results from all characterisation studies allowed for selection of an antibody pair to be taken forward to assay development.

SiMa Cells for a Serotype Specific and Sensitive Cell-Based Neutralization Test for Botulinum Toxin A and E.

Botulinum toxins (BoNTs), of which there are seven serotypes, are among the most potent neurotoxins, with serotypes A, B and E causing human botulism. Antitoxins form the first line of treatment for botulism, and functional, highly sensitive in vitro methods for toxin neutralization are needed to replace the current in vivo methods used for determination of antitoxin potency. In this preliminary proof of concept study, we report the development of a neutralization test using the neuroblastoma SiMa cell line. The assay is serotype specific for either BoNT/A or BoNT/E, which both cleave unique sequences on SNAP-25 within SiMa cells. The end point is simple immunodetection of cleaved SNAP-25 from cell lysates with antibodies detecting only the newly exposed sequence on SNAP-25. Neutralizing antibodies prevent the toxin-induced cleavage of SNAP-25. The toxin neutralization assay, with an EC50 of ~2 mIU/mL determined with a standardized reference antiserum, is more sensitive than the mouse bioassays. Relevance was demonstrated with commercial and experimental antitoxins targeting different functional domains, and of known in vivo neutralizing activities. This is the first report describing a simple, specific, in vitro cell-based assay for the detection of neutralizing antibodies against BoNT/A and BoNT/E with a sensitivity exceeding that of the mouse bioassay.

Androgen-Induced Relaxation of Uterine Myocytes Is Mediated by Blockade of Both Ca(2+) Flux and MLC Phosphorylation.

CONTEXT: Uterine quiescence must be maintained until pregnancy reaches term. Premature activation of myometrial contractility leads to preterm labor and delivery. OBJECTIVE: To scrutinize the potential of androgens to relax the myometrium and the mechanism of their action. SAMPLES: A pregnancy-derived myometrial smooth muscle cell line (PHM1-41) and myometrial strips prepared from tissues obtained from pregnant women (lean, n = 9; obese, n = 6) undergoing elective cesarean section at term and from nonpregnant C57BL/6 mice (n=5) were each utilized. DESIGN: The contraction of collagen-embedded PHM1-41s and the stretch-induced contraction of human and murine myometrial strips were assessed after incubation with Testosterone (T), dihydrotestosterone (DHT), and T conjugated to BSA. Intracellular calcium ([Ca(2+)]) and phosphorylated myosin light chain concentrations were quantified in PHM1-41s using a Fluo-4 Ca(2+) assay and in-cell Westerns, respectively. SETTING: University research institute. RESULTS: DHT and T, but not T conjugated to BSA, impaired the contractile function of PHM1-41s and of human and murine myometrial strips. The response was rapid (observed within minutes), was sustainable for up to 48 hours, and was not abolished on knockdown of the androgen receptor. DHT (100 µm) reduced the amplitude of lean strip contraction to 2 ± 2% of the pretreatment value and T (100 µm) to 3.3 ± 1%. These values for obese strips were 15 ± 6.7% and 11 ± 6.7%, respectively. At the same doses, in murine strips, DHT reduced the amplitude to 4.8 ± 3% and T to 4.9 ± 3%. DHT (50 µm) pretreatment reduced the oxytocin-stimulated increase in [Ca(2+)] (P < .0001; n = 6) and phosphorylated myosin light chain (P < .05; n = 5) in PHM1-41s. CONCLUSION: Lipid-soluble androgens could be developed as tocolytic agents for the treatment of preterm labor.

Lipopolysaccharide promotes contraction of uterine myocytes via activation of Rho/ROCK signaling pathways.

Myometrial contraction is a central feature of labor. Although a link between infection and preterm labor is widely accepted, surprisingly little is known about the mechanisms coupling infection-induced inflammation to myocyte contractile machinery. This study explores the myocyte response to pathogen-derived ligands in vitro. The pregnant human myometrial cell line PHM1-41 and primary cultured uterine myocytes responded to Toll-like receptor (TLR) ligands, including the bacterial wall component LPS, which at 100 ng/ml increased contraction of cells embedded within collagen gels over 72 h compared to PBS. LPS-treated myocytes secreted inflammatory mediators, including prostaglandin F2α, the cytokines TNF-α and IL-6, and a range of chemokines. The contractile response to LPS required TLR4 signaling and was independent of prostaglandin synthesis. Neutralizing TNF-α had no effect on LPS-mediated contraction; however, the Rho-associated protein kinase (ROCK) inhibitors Y-27632 (10 µM) and GSK-269962 (50 nM) both abrogated the contractile response. The finding of LPS-mediated contraction was supported by a 1.38 ± 0.072-fold (mean±SE) increase in myosin light-chain phosphorylation 48 h post-treatment, assessed by in-cell Western blot analysis. Together, these data suggest that, in addition to modulating the local inflammatory environment, pathogen-derived ligands may directly promote myometrial contractility via Rho/ROCK signaling, thus contributing to preterm labor-mediated preterm birth.

Molecular regulators of resolution of inflammation: potential therapeutic targets in the reproductive system.

Inflammatory processes are central to reproductive events including ovulation, menstruation, implantation and labour, while inflammatory dysregulation is a feature of numerous reproductive pathologies. In recent years, there has been much research into the endogenous mechanisms by which inflammatory reactions are terminated and tissue homoeostasis is restored, a process termed resolution. The identification and characterisation of naturally occurring pro-resolution mediators including lipoxins and annexin A1 has prompted a shift in the field of anti-inflammation whereby resolution is now observed as an active process, triggered as part of a normal inflammatory response. This review will address the process of resolution, discuss available evidence for expression of pro-resolution factors in the reproductive tract and explore possible roles for resolution in physiological reproductive processes and associated pathologies.