A single C-terminal residue controls SARS-CoV-2 spike trafficking and incorporation into VLPs.

The spike (S) protein of SARS-CoV-2 is delivered to the virion assembly site in the ER-Golgi Intermediate Compartment (ERGIC) from both the ER and cis-Golgi in infected cells. However, the relevance and modulatory mechanism of this bidirectional trafficking are unclear. Here, using structure-function analyses, we show that S incorporation into virus-like particles (VLP) and VLP fusogenicity are determined by coatomer-dependent S delivery from the cis-Golgi and restricted by S-coatomer dissociation. Although S mimicry of the host coatomer-binding dibasic motif ensures retrograde trafficking to the ERGIC, avoidance of the host-like C-terminal acidic residue is critical for S-coatomer dissociation and therefore incorporation into virions or export for cell-cell fusion. Because this C-terminal residue is the key determinant of SARS-CoV-2 assembly and fusogenicity, our work provides a framework for the export of S protein encoded in genetic vaccines for surface display and immune activation.

Transitioning diets: a mixed methods study on factors affecting inclusion of millets in the urban population.

BACKGROUND: The increasing health challenge in urban India has led to consumers to change their diet preferences by shifting away from staple cereals and making way for healthier foods such as nutri-cereals like millets and other diverse food groups. Taking the case of millets, this study seeks to uncover the exact drivers for this shift of consumers away from a traditional cereal dense diet to a nutritionally more diverse diet that includes nutri-cereal. We also look at deterrents that dissuade consumers from shifting to millets. METHOD: We use primary data by surveying respondents through interviews and focused group discussions and online questionnaires. A total of 20 personal consumer interviews and 4 focus group discussions having 8-12 members each were conducted to arrive at the measures for the study. We use logistic regression and Structural Equation Modeling for data analysis. Responses were obtained across major metropolitan cities and tier 2 cities of India thus ensuring representation of geographical, cultural and diet diversity. 875 participants' responses were analysed for results. RESULTS: Health reasons and social networks are the major drivers for shift to millets while lack of awareness, lack of easy availability, high prices, lack of branded products, family being averse to switching to millets and lack of attractive promotional cashbacks and discounts are major deterrents to trying out millets. CONCLUSIONS: Diet focussed interventions are urgently needed to curb rising diet related non communicable diseases. Government policies aimed at greater production of millets, running awareness campaigns on mass media and private sector initiatives aimed at generating better value added market offerings could lead the way.

Functional and Tableting Properties of Alkali-Isolated and Phosphorylated Barnyard Millet (Echinochloa esculenta) Starch.

The functional and tableting properties of barnyard millet starch (Echinochloa esculenta) were investigated in its native (alkali-treated) and chemically modified (phosphorylated) states. The grains were pulverized, soaked, and ground before filtration to separate starch and protein. Multiple NaOH treatments were performed. The starch was washed, neutralized, and dried. Sodium tripolyphosphate (STPP) and sodium sulfate were used to modify the starch, followed by maceration, washing, and drying to remove unreacted chemicals. The amylose content of alkali-treated barnyard millet starch increased by 19.96 ± 3.56% w/w. The amount of protein, the kind of starch used, and the size of the starch granules, all affected the ability of the starch granules to swell up. It was observed that alkali-extracted barnyard millet starch (AZS) has a swelling power of 194.3 ± 0.0064% w/w. The swelling capacity of treated starch was lesser as compared to the native alkali barnyard millet starch. Decrement in swelling power of phosphorylated starch was observed due to tightening of bonds in the molecular structure. The moisture content of the excipients may affect the overall stability of the formulation. The moisture content of the AZS was found to be 15.336 ± 1.012% w/w. Compared to AZS, cross-linked barnyard millet starch had a moisture content that was up to 20% lower than AZS. The Hausner ratio for phosphorylated starch was found to be 1.25, which indicates marked flow property. Similar morphologies could be seen in the alkali-isolated barnyard millet starch and the cross-linked/phosphorylated barnyard millet that was cross-linked using a mixture of sodium sulfate and sodium tripolyphosphate. The modest degree of substitution would have no effect on the surface morphology as shown by the scanning electron microscopic study. The crushing and compacting abilities of modified barnyard millet starch were also improved, but its friability and rate of disintegration were decreased. The whole study revealed that after cross-linking, barnyard millet had good tableting properties and it can be used as an excipient in drug delivery.

A new spline technique for the time fractional diffusion-wave equation.

The current research article proposes an approximate solution of the fractional diffusion wave equation (FDWE) by using a new collocation method based on the cubic B-splines. The fractional derivative in the time direction is considered in Caputo form. The theoretical research of the proposed algorithm is discussed with L ∞ and H 1 norms. The method presented in this article is found to be of order (∆t 3- α + h 4). The highlights of the current technique proposed in this article are as under:•The method is high-order collocation and uses a compact stencil. The error analysis is discussed to authenticate the order of convergence of the proposed numerical approximation.•The comparisons of errors with the already existing methods are done and observed that our method produces more accurate results than the methods presented in the literature.

Immunization with a self-assembling nanoparticle vaccine displaying EBV gH/gL protects humanized mice against lethal viral challenge.

Epstein-Barr virus (EBV) is a cancer-associated pathogen responsible for 165,000 deaths annually. EBV is also the etiological agent of infectious mononucleosis and is linked to multiple sclerosis and rheumatoid arthritis. Thus, an EBV vaccine would have a significant global health impact. EBV is orally transmitted and has tropism for epithelial and B cells. Therefore, a vaccine would need to prevent infection of both in the oral cavity. Passive transfer of monoclonal antibodies against the gH/gL glycoprotein complex prevent experimental EBV infection in humanized mice and rhesus macaques, suggesting that gH/gL is an attractive vaccine candidate. Here, we evaluate the immunogenicity of several gH/gL nanoparticle vaccines. All display superior immunogenicity relative to monomeric gH/gL. A nanoparticle displaying 60 copies of gH/gL elicits antibodies that protect against lethal EBV challenge in humanized mice, whereas antibodies elicited by monomeric gH/gL do not. These data motivate further development of gH/gL nanoparticle vaccines for EBV.

Improvements in Soil Physical, Chemical and Biological Properties at Natural Saline and Non-Saline Sites Under Different Management Practices.

Soil salinity is known to be a significant threat to food security for the increasing population, which is further aggravated under the climate change scenario. Indo-Gangetic plain (IGP) is one of the most productive in the world and is most affected by salinity. To understand the modifications in soil characteristics under different management practices followed to reclaim salinity affected land, the present study was conducted at variously reclaimed saline areas of three districts of Uttar Pradesh situated in IGP. Soil from six sites (electrical conductivity (EC) ranging from 0.89 to 10.28 mS) following different management practices, RJT (Rajatalab, rice-wheat +organic), BBN (Beerbhanpur, rice-wheat +inorganic), MZM (Mirzamurad, rice-mustard +organic), BRP (Baraipur, rice-wheat +organic), DHR (Dharahara, rice-fallow +organic) and SLM (Salempur, rice-wheat +inorganic) were assessed for physical, chemical and biological properties during the vegetative stage and after harvest of crops. Soil quality index (SQI) based on representative parameters obtained by principal component analysis and yield of crops were also calculated at saline and non-saline sites. The SLM site showed highest salinity followed by BRP, DHR, MZM, while BBN and RJT were non-saline. Total organic carbon, total nitrogen, microbial activity, and microbial biomass were low at saline compared to non-saline sites but were higher under organic matter amendment compared to inorganic. Activities of soil enzymes were negatively influenced while ß-glucosidase and alkaline phosphatase activities were enhanced under higher salinity. Organic amendments were more efficient in improving the soil properties along with SQI at saline soil resulting into a better yield in all crop combinations compared to inorganic amendments.

An extended motif in the SARS-CoV-2 spike modulates binding and release of host coatomer in retrograde trafficking.

ß-Coronaviruses such as SARS-CoV-2 hijack coatomer protein-I (COPI) for spike protein retrograde trafficking to the progeny assembly site in endoplasmic reticulum-Golgi intermediate compartment (ERGIC). However, limited residue-level details are available into how the spike interacts with COPI. Here we identify an extended COPI binding motif in the spike that encompasses the canonical K-x-H dibasic sequence. This motif demonstrates selectivity for αCOPI subunit. Guided by an in silico analysis of dibasic motifs in the human proteome, we employ mutagenesis and binding assays to show that the spike motif terminal residues are critical modulators of complex dissociation, which is essential for spike release in ERGIC. αCOPI residues critical for spike motif binding are elucidated by mutagenesis and crystallography and found to be conserved in the zoonotic reservoirs, bats, pangolins, camels, and in humans. Collectively, our investigation on the spike motif identifies key COPI binding determinants with implications for retrograde trafficking.

Structurally disordered C-terminal residues of GTP cyclohydrolase II are essential for its enzymatic activity.

GTP cyclohydrolase II (GCHII) is one of the rate limiting enzymes in riboflavin biosynthesis pathway and is shown to be a potential drug target for most of the pathogens. Previous biochemical and structural studies have identified the active site residues and elucidated the steps involved in the catalytic mechanism of GCHII. However, the last ∼20-25 C-terminal residues of GCHII remains unstructured in all the crystal structures determined to date and their role in the catalytic activity, if any, remains elusive. Therefore, to understand the role of these unstructured C-terminal residues, a series of C-terminal deletion mutants of GCHII from Helicobacter pylori (hGCHII) were generated and their catalytic activity was compared with its wild-type. Surprisingly, none of the C-terminal deletion mutants shows any enzymatic activity indicating that these are essential for GCHII function. To get additional insights for such loss of activity, homology models of full-length and deletion mutants of hGCHII in complex with GTP, Mg2+, and Zn2+ were generated and subjected to molecular dynamics simulation studies. The simulation studies show that a conserved histidine at 190th position from the unstructured C-terminal region of hGCHII interacts with α-phosphate of GTP. We propose that His-190 may play a role in the hydrolysis of pyrophosphate from GTP and in releasing the product, DARP. In summary, we demonstrate that the unstructured C-terminal residues of GCHII are important for its enzymatic activity and must be considered during rational drug designing. Communicated by Ramaswamy H. Sarma.

Assessment of physiological, biochemical and yield responses of wheat plants under natural saline and non-saline field conditions.

Soil salinity is a major threat to crop productivity all over the world including the Indo-Gangetic plain (IGP) region of India. Therefore, a field study was conducted for two consecutive years in wheat growing areas in IGP affected by salinity. Plants grown at a saline site (Salempur, SLM) and a non-saline site (Rajatalab, RJT), were analysed for selected biochemical, physiological and yield traits. Results showed that photosynthetic rate was not significantly affected, but transpiration rate and stomatal conductance declined at saline compared to non-saline site. Photosynthetic pigments increased during vegetative growth period, but decreased during reproductive stage at SLM site, while anthocyanin showed an opposite trend. Membrane damage, solute leakage, H2O2 and ·O2 - productions were intensified at saline site, SLM. Accumulation of osmolytes and antioxidants occurred in plants at saline compared to non-saline sites. K/Na and Ca/Na ratios in plants at SLM were reduced significantly compared to non-saline site, RJT. Biomass and yield also declined at SLM compared to RJT. Principle component and path analyses on the measured parameters clearly showed that defense strategies adopted by plants helped to maintain the photosynthetic rate but biomass and yield of wheat got compromised under high salinity.

Structurally related but genetically unrelated antibody lineages converge on an immunodominant HIV-1 Env neutralizing determinant following trimer immunization.

Understanding the molecular mechanisms by which antibodies target and neutralize the HIV-1 envelope glycoprotein (Env) is critical in guiding immunogen design and vaccine development aimed at eliciting cross-reactive neutralizing antibodies (NAbs). Here, we analyzed monoclonal antibodies (mAbs) isolated from non-human primates (NHPs) immunized with variants of a native flexibly linked (NFL) HIV-1 Env stabilized trimer derived from the tier 2 clade C 16055 strain. The antibodies displayed neutralizing activity against the autologous virus with potencies ranging from 0.005 to 3.68 µg/ml (IC50). Structural characterization using negative-stain EM and X-ray crystallography identified the variable region 2 (V2) of the 16055 NFL trimer to be the common epitope for these antibodies. The crystal structures revealed that the V2 segment adopts a ß-hairpin motif identical to that observed in the 16055 NFL crystal structure. These results depict how vaccine-induced antibodies derived from different clonal lineages penetrate through the glycan shield to recognize a hypervariable region within V2 (residues 184-186) that is unique to the 16055 strain. They also provide potential explanations for the potent autologous neutralization of these antibodies, confirming the immunodominance of this site and revealing that multiple angles of approach are permissible for affinity/avidity that results in potent neutralizing capacity. The structural analysis reveals that the most negatively charged paratope correlated with the potency of the mAbs. The atomic level information is of interest to both define the means of autologous neutralization elicited by different tier 2-based immunogens and facilitate trimer redesign to better target more conserved regions of V2 to potentially elicit cross-neutralizing HIV-1 antibodies.

The Structural Biology of Eastern Equine Encephalitis Virus, an Emerging Viral Threat.

Alphaviruses are arboviruses that cause arthritis and encephalitis in humans. Eastern Equine Encephalitis Virus (EEEV) is a mosquito-transmitted alphavirus that is implicated in severe encephalitis in humans with high mortality. However, limited insights are available into the fundamental biology of EEEV and residue-level details of its interactions with host proteins. In recent years, outbreaks of EEEV have been reported mainly in the United States, raising concerns about public safety. This review article summarizes recent advances in the structural biology of EEEV based mainly on single-particle cryogenic electron microscopy (cryoEM) structures. Together with functional analyses of EEEV and related alphaviruses, these structural investigations provide clues to how EEEV interacts with host proteins, which may open avenues for the development of therapeutics.

Withdrawal Notice: Fatal Outcomes from COVID-19 in Diabetes Patients and its Management: Impact of Diabetes and Other Comorbidities in COVID-19

The article has been withdrawn at the request of the authors and editor of the journal Current Diabetes Reviews, due to incoherent content.Bentham Science apologizes to the readers of the journal for any inconvenience this may have caused.The Bentham Editorial Policy on Article Withdrawal can be found at https://benthamscience.com/editorial-policies-main.php. BENTHAM SCIENCE DISCLAIMER: It is a condition of publication that manuscripts submitted to this journal have not been published and will not be simultaneously submitted or published elsewhere. Furthermore, any data, illustration, structure or table that has been published elsewhere must be reported, and copyright permission for reproduction must be obtained. Plagiarism is strictly forbidden, and by submit-ting the article for publication the authors agree that the publishers have the legal right to take appropriate action against the authors, if plagiarism or fabricated information is discovered. By submitting a manuscript, the authors agree that the copyright of their article is transferred to the publishers if and when the article is accepted for publication.

Protective antibodies against human parainfluenza virus type 3 infection.

Human parainfluenza virus type III (HPIV3) is a common respiratory pathogen that afflicts children and can be fatal in vulnerable populations, including the immunocompromised. There are currently no effective vaccines or therapeutics available, resulting in tens of thousands of hospitalizations per year. In an effort to discover a protective antibody against HPIV3, we screened the B cell repertoires from peripheral blood, tonsils, and spleen from healthy children and adults. These analyses yielded five monoclonal antibodies that potently neutralized HPIV3 in vitro. These HPIV3-neutralizing antibodies targeted two non-overlapping epitopes of the HPIV3 F protein, with most targeting the apex. Prophylactic administration of one of these antibodies, PI3-E12, resulted in potent protection against HPIV3 infection in cotton rats. Additionally, PI3-E12 could also be used therapeutically to suppress HPIV3 in immunocompromised animals. These results demonstrate the potential clinical utility of PI3-E12 for the prevention or treatment of HPIV3 in both immunocompetent and immunocompromised individuals.

Multimeric antibodies from antigen-specific human IgM+ memory B cells restrict Plasmodium parasites.

Multimeric immunoglobulin-like molecules arose early in vertebrate evolution, yet the unique contributions of multimeric IgM antibodies to infection control are not well understood. This is partially due to the difficulty of distinguishing low-affinity IgM, secreted rapidly by plasmablasts, from high-affinity antibodies derived from later-arising memory cells. We developed a pipeline to express B cell receptors (BCRs) from Plasmodium falciparum-specific IgM+ and IgG+ human memory B cells (MBCs) as both IgM and IgG molecules. BCRs from both subsets were somatically hypermutated and exhibited comparable monomeric affinity. Crystallization of one IgM+ MBC-derived antibody complexed with antigen defined a linear epitope within a conserved Plasmodium protein. In its physiological multimeric state, this antibody displayed exponentially higher antigen binding than a clonally identical IgG monomer, and more effectively inhibited P. falciparum invasion. Forced multimerization of this IgG significantly improved both antigen binding and parasite restriction, underscoring how avidity can alter antibody function. This work demonstrates the potential of high-avidity IgM in both therapeutics and vaccines.

Characterization of neutralizing antibodies from a SARS-CoV-2 infected individual.

B cells specific for the SARS-CoV-2 S envelope glycoprotein spike were isolated from a COVID-19-infected subject using a stabilized spike-derived ectodomain (S2P) twenty-one days post-infection. Forty-four S2P-specific monoclonal antibodies were generated, three of which bound to the receptor binding domain (RBD). The antibodies were minimally mutated from germline and were derived from different B cell lineages. Only two antibodies displayed neutralizing activity against SARS-CoV-2 pseudo-virus. The most potent antibody bound the RBD in a manner that prevented binding to the ACE2 receptor, while the other bound outside the RBD. Our study indicates that the majority of antibodies against the viral envelope spike that were generated during the first weeks of COVID-19 infection are non-neutralizing and target epitopes outside the RBD. Antibodies that disrupt the SARS-CoV-2 spike-ACE2 interaction can potently neutralize the virus without undergoing extensive maturation. Such antibodies have potential preventive/therapeutic potential and can serve as templates for vaccine-design.

Analysis of a SARS-CoV-2-Infected Individual Reveals Development of Potent Neutralizing Antibodies with Limited Somatic Mutation.

Antibody responses develop following SARS-CoV-2 infection, but little is known about their epitope specificities, clonality, binding affinities, epitopes, and neutralizing activity. We isolated B cells specific for the SARS-CoV-2 envelope glycoprotein spike (S) from a COVID-19-infected subject 21 days after the onset of clinical disease. 45 S-specific monoclonal antibodies were generated. They had undergone minimal somatic mutation with limited clonal expansion, and three bound the receptor-binding domain (RBD). Two antibodies neutralized SARS-CoV-2. The most potent antibody bound the RBD and prevented binding to the ACE2 receptor, while the other bound outside the RBD. Thus, most anti-S antibodies that were generated in this patient during the first weeks of COVID-19 infection were non-neutralizing and target epitopes outside the RBD. Antibodies that disrupt the SARS-CoV-2 S-ACE2 interaction can potently neutralize the virus without undergoing extensive maturation. Such antibodies have potential preventive and/or therapeutic potential and can serve as templates for vaccine design.

Extensive dissemination and intraclonal maturation of HIV Env vaccine-induced B cell responses.

Well-ordered HIV-1 envelope glycoprotein (Env) trimers are prioritized for clinical evaluation, and there is a need for an improved understanding about how elicited B cell responses evolve following immunization. To accomplish this, we prime-boosted rhesus macaques with clade C NFL trimers and identified 180 unique Ab lineages from ∼1,000 single-sorted Env-specific memory B cells. We traced all lineages in high-throughput heavy chain (HC) repertoire (Rep-seq) data generated from multiple immune compartments and time points and expressed several as monoclonal Abs (mAbs). Our results revealed broad dissemination and high levels of somatic hypermutation (SHM) of most lineages, including tier 2 virus neutralizing lineages, following boosting. SHM was highest in the Ab complementarity determining regions (CDRs) but also surprisingly high in the framework regions (FRs), especially FR3. Our results demonstrate the capacity of the immune system to affinity-mature large numbers of Env-specific B cell lineages simultaneously, supporting the use of regimens consisting of repeated boosts to improve each Ab, even those belonging to less expanded lineages.

Role of supplemental UV-B in changing the level of ozone toxicity in two cultivars of sunflower: growth, seed yield and oil quality.

Ultraviolet-B radiation (UV-B) is inherent part of solar spectrum and tropospheric ozone (O3) is a potent secondary air pollutant. Therefore the present study was conducted to evaluate the responses of Helianthus annuus L. cvs DRSF 108 and Sungold (sunflower) to supplemental UV-B (sUV-B; ambient + 7.2 kJ m-2 d-1) and elevated ozone (O3; ambient + 10 ppb), given singly and in combination under field conditions using open-top chambers. The individual and interactive effects of O3 and sUV-B induced varying changes in both the cultivars of sunflower ranging from ultrastructural variations to growth, biomass, yield and oil composition. Reduction in leaf area of Sungold acted as a protective feature which minimized the perception of sUV-B as well as uptake of O3 thus led to lesser carbon loss compared to DRSF 108. Number- and weight of heads plant-1 decreased although more in Sungold with decline of oil content. Both the stresses when given singly and combination induced rancidification of oil and thus made the oil less suitable for human consumption.

Comprehensive metabolite profiling in distinct chemotypes of Commiphora wightii.

Commiphora wightii (Arn.) Bhandari, known as guggul, produces a medicinally important gum resin which is used extensively by Ayurvedic physicians to treat various ailments. However, most of the studies on C. wightii have been limited to its gum resin. Comprehensive metabolic profiling of leaves, stem and gum resin samples was undertaken to analyse aqueous and non-aqueous metabolites from three distinct chemotypes (NBRI-101, NBRI-102 and NBRI-103) shortlisted from different agro-climatic zones. GC-MS, HPLC and NMR spectroscopy were used for comprehensive metabolomics. Multivariate analysis showed characteristic variation in quinic and citric acids, myo-inositol and glycine (aqueous metabolites) and 2,6-di-tert-butyl-phenol, trans-farnesol and guggulsterones (non-aqueous metabolites) amongst the three chemotypes. Quinic acid, citric acid and myo-ionositol were detected in substantial quantities from leaves and stem samples which provide opportunities for novel nutraceutical and pharmaceutical formulations. Quinic acid, from the leaves, was identified as a marker metabolite for early selection of high guggulsterones-yielding cultivars.

Heteropogon contortus BL-1 (Pilli Grass) and Elevated UV-B Radiation: The Role of Growth, Physiological, and Biochemical Traits in Determining Forage Productivity and Quality.

Exposure to elevated UV-B (eUV-B) is well known for its phytotoxicity, although studies made with UV-B exposure and its impact on grasses are limited especially from tropical countries including India. In this study, responses of a valuable grass species, Heteropogon contortus BL-1, were assessed under eUV-B (over ambient UV-B) at different growth stages. Damage caused by eUV-B was observed in the form of membrane damage and loss of pigments at early stages of growth, whereas tannin, phenol, and protein contents showed their increments at all the growth stages, to overcome the imposed stress. Reducing sugar showed its decline at all the growth stages, whereas starch and sucrose contents were higher mostly at later ages of plant growth. eUV-B caused a marked variations in anatomical structures with increase of mesophyll and spongy parenchymatous cells in leaves to reduce severity of irradiation, to maintain the growth and productivity. The study also highlights the significant negative influence of eUV-B on the growth of H. contortus BL-1 and its adaptive strategy to minimize the negative impacts. With the progression of age, plants although adopted to UV-B stress with maintenance of productivity, but palatability of forage was affected due to increment of tannin content.