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.

Factors influencing perceptions of processed baby foods and feeding practices among Indian mothers: a qualitative investigation.

OBJECTIVE: To explore how professionally qualified, working Indian mothers conceptualize healthy foods in general, perceive processed infant and baby foods available commercially and what feeding practices they actually follow with their children at home. DESIGN: Five focus groups with 8-12 participants were conducted around the participants' conceptualization of healthy food, their perceptions about commercially available processed baby and infant foods and their actual feeding practices that they routinely follow with their children. Discussion transcripts were analyzed using an inductive coding approach. SETTING: India. PARTICIPANTS: Fifty-one professionally qualified, working women with at least one child under 5 years of age. RESULTS: Participants agreed that fresh food is healthiest. They also had favorable opinions about processed infant and baby foods with regards to healthfulness, hygiene and safety. Healthy foods were largely conceptualized in relation to nutrient claims, ingredients and discernible health outcomes. They use cues, such as health claims, brand, price, package design and others to determine healthfulness of the product. Perception was heavily influenced by these extrinsic cues rather than by participants' own nutrition knowledge. Despite having the knowledge, most participants admitted to using these foods on account of factors such as their own inability to lactate, social pressure, lack of time and convenience. CONCLUSIONS: Most educated and well off people continue to use these products. While they rue the lack of stringent regulatory measures in India, they feel there is an urgent need to address this huge policy gap by way of legislation and regulation.

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.

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.

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.

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.

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.

Use of Ethylenediurea (EDU) in identifying indicator cultivars of Indian clover against ambient ozone.

Three clover (Trifolium alexandrium L.) cultivars (Bundel, Wardan and JHB-146) were assessed for their responses to ambient ozone (O3) with respect to growth, physiological and biochemical parameters at two rural sites (R1 and R2) using ethylenediurea (EDU). EDU solution (300ppm) was applied as soil drench, 10 days after germination (DAG) at an interval of 10 days up to 80 DAG. The average O3 concentrations were 52.76 and 60.86 ppb at R1 and R2 sites, respectively during the experimental period. Ambient O3 induced visible symptoms in all the cultivars at both the sites, with more at R2 site having high ambient O3 levels. Visible injury was observed first in non-EDU treated plants of Wardan at R2 site. Wardan also showed maximum reduction in leaf injury under EDU treatment at both the sites with more at R2. Under EDU treatment, better adaptation to ambient O3 at initial age of observation and higher acquisition of resources at later ages of observation at both the sites led to better physiological and biochemical adaptations in Wardan. Bundel retained more biomass in shoot as is reflected with higher shoot/root ratio and thus focused more on repair and defense. Shoot/root ratio of JHB-146 did not respond to EDU treatment and thus showed insignificant variations except at initial age of observation at R1 site. This study clearly suggests that Wardan and Bundel are sensitive to ambient O3 and can be used as bioindicator species in areas having higher O3 levels using EDU as a research tool.

Structural basis for competitive inhibition of 3,4-dihydroxy-2-butanone-4-phosphate synthase from Vibrio cholerae.

The riboflavin biosynthesis pathway has been shown to be essential in many pathogens and is absent in humans. Therefore, enzymes involved in riboflavin synthesis are considered as potential antibacterial drug targets. The enzyme 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) catalyzes one of the two committed steps in the riboflavin pathway and converts d-ribulose 5-phosphate (Ru5P) to l-3,4-dihydroxy-2-butanone 4-phosphate and formate. Moreover, DHBPS is shown to be indispensable for Mycobacterium, Salmonella, and Helicobacter species. Despite the essentiality of this enzyme in bacteria, no inhibitor has been identified hitherto. Here, we describe kinetic and crystal structure characterization of DHBPS from Vibrio cholerae (vDHBPS) with a competitive inhibitor 4-phospho-d-erythronohydroxamic acid (4PEH) at 1.86-Å resolution. In addition, we also report the structural characterization of vDHBPS in its apo form and in complex with its substrate and substrate plus metal ions at 1.96-, 1.59-, and 2.04-Å resolution, respectively. Comparison of these crystal structures suggests that 4PEH inhibits the catalytic activity of DHBPS as it is unable to form a proposed intermediate that is crucial for DHBPS activity. Furthermore, vDHBPS structures complexed with substrate and metal ions reveal that, unlike Candida albicans, binding of substrate to vDHBPS induces a conformational change from an open to closed conformation. Interestingly, the position of second metal ion, which is different from that of Methanococcus jannaschii, strongly supports an active role in the catalytic mechanism. Thus, the kinetic and structural characterization of vDHBPS reveals the molecular mechanism of inhibition shown by 4PEH and that it can be explored further for designing novel antibiotics.

Duodenal morphometry and small bowel permeability in children with portal hypertension.

OBJECTIVE: We prospectively studied children with portal hypertension (PHT) for portal hypertensive duodenopathy (PHTD) and small bowel intestinal permeability (SIP) with the objectives of defining histopathological parameters for PHTD and to find out whether any association existed among structural changes, SIP, and nutritional status. METHOD: SIP was assessed by using lactulose and mannitol sugar probes in 31 children with PHT (cirrhosis n = 15 and extrahepatic portal venous obstruction n = 16) and 15 healthy children as controls. Morphometric assessment from duodenal biopsies was done in children with PHT. SIP and morphometric parameters were correlated with nutritional status and dietary intake. RESULTS: Among children with PHT, 48% had PHTD defined as presence of villous atrophy (villous to crypt ratio < 2.5:1), dilated capillaries (capillary diameter > 16.8 µm, capillary area > 151 µm, capillary perimeter > 56 µm), and thickened muscularis mucosae (>22.2 µm). Lactulose excretion alone was increased in children with PHT as compared with healthy children (median %: 0.03, 0.02, and 0.01 for cirrhosis, extrahepatic portal venous obstruction, and controls, respectively [P < 0.01]) signifying increased paracellular permeability in PHT. Children with PHT had significantly lower z scores for height, weight, and triceps skin-fold thickness (<-2SD), whereas no differences were found in dietary intake between patients and controls. Increased SIP, nutritional compromise, and PHTD in our patients had no correlation. CONCLUSIONS: PHT is often associated with duodenopathy. SIP does occur as a result of increased paracellular permeability. Factors of increased SIP, undernutrition, and PHTD do not have correlation in childhood PHT.

Metabolomics and anti-inflammatory activity of Commiphora madagascariensis jacq. leaves extract using in vitro and in vivo models.

C. madagascariensis, an unexplored species of Burseraceae is used by local population for the management of inflammation and throat pain. The disease alleviation by this plant could be due to the presence of rich repository of active compounds with various pharmacological importances. In this study, therefore, the profiling of metabolites and isolation of active compounds of C. madagascariensis was performed. Furthermore, the ethanol, ethyl acetate extracts and a selected active compound was subjected for in vitro and in vivo anti-inflammatory activities. Metabolomic analysis identified and quantified 116 metabolites from leaves, young stem and gum-resins of C. madagascariensis (Burseraceae) followed by multivariate PCA analysis. NMR, GC-MS and HPLC were used to analyze primary and secondary metabolites. Subsequently, five main isolated compounds were identified as trimethoxy tetrahydrobenzo dioxolo isochromene (TTDI), butyl phenol, butyl propionate phenol, germacrone and ß-elemenone. Amongst them, TTDI was found to be a novel compound. Hence, a process was developed to obtain the enriched fraction of TTDI in ethanol and ethyl acetate extracts of leaves. Furthermore, TTDI and extracts were subjected for their in vitro anti-inflammatory activity in LPS sensitized murine splenocytes. The results showed that TTDI and both extracts significantly suppressed the levels of pro-inflammatorycytokines (TNF-α, IFN-γ). Interestingly, the suppression of pro-inflammatory cytokines was evenmore significant by the similar concentration of TTDI when compared with colchicine. However, the level of anti-inflammatory cytokine (IL-10) was found to be unchanged. Additionally, in vivo anti-inflammatory study revealed a significant reduction in carrageenan induced paw edema by TTDI and both the extracts. In the docking study, TTDI was more active than colchicine with strong binding affinity to COX-2, PLA2, and 5ß reductase. Our results highlighted that the presence of metabolites with medicinal and nutraceutical importance in C. madagascariensis, could provide opportunities for the development of a new plant-based therapeutics for inflammation.

Differential sensitivity of spinach and amaranthus to enhanced UV-B at varying soil nutrient levels: association with gas exchange, UV-B-absorbing compounds and membrane damage.

The metabolic reasons associated with differential sensitivity of C3 and C4 plant species to enhanced UV-B under varying soil nutrient levels are not well understood. In the present study, spinach (Spinacia oleracea L. var All Green), a C3 and amaranthus (Amaranthus tricolor L. var Pusa Badi Chaulai), a C4 plant were subjected to enhanced UV-B (280-315 nm; 7.2 kJ m(-2) day(-1)) over ambient under varying soil nutrient levels. The nutrient amendments were recommended Nitrogen (N), Phosphorus (P), Potassium (K), 1.5× recommended NPK, 1.5× recommended N and 1.5× recommended K. Enhanced UV-B negatively affected both the species at all nutrient levels, but the reductions varied with nutrient concentration and combinations. Reductions in photosynthetic rate, stomatal conductance and chlorophyll content were significantly more in spinach compared with amaranthus. The reduction in photosynthetic rate was maximum at 1.5× recommended K and minimum in 1.5× NPK amended plants. The oxidative damage to membranes measured in terms of malondialdehyde content was significantly higher in spinach compared with amaranthus. Enhanced UV-B reduced SOD activity in both the plants except in amaranthus at 1.5× recommended K. POX activity increased under enhanced UV-B at all nutrient levels in amaranthus, but only at 1.5× K in spinach. Amaranthus had significantly higher UV-B-absorbing compounds than spinach even under UV-B stress. Lowest reductions in yield and total biomass under enhanced UV-B compared with ambient were observed in amaranthus grown at 1.5× recommended NPK. Enhanced UV-B did not significantly change the nitrogen use efficiency in amaranthus at all NPK levels, but reduced in spinach except at 1.5× K. These findings suggest that the differential sensitivity of the test species under enhanced UV-B at varying nutrient levels is due to varying antioxidative and UV-B screening capacity, and their ability to utilize nutrients. Amaranthus tolerated enhanced UV-B stress more than spinach at all nutrient levels and 1.5× recommended NPK lowered the sensitivity maximally to enhanced UV-B with respect to photosynthesis, biomass and yield. PCA score has also confirmed the lower sensitivity of amaranthus compared with spinach with respect to the measured physiological and biochemical parameters.

Assessment of six Indian cultivars of mung bean against ozone by using foliar injury index and changes in carbon assimilation, gas exchange, chlorophyll fluorescence and photosynthetic pigments.

Six Indian cultivars of Vigna radiata L. (HUM-1, HUM-2, HUM-6, HUM-23, HUM-24 and HUM-26) were exposed with ambient and elevated (ambient + 10 ppb ozone (O3) for 6 h day(-1)) level of O3 in open top chambers. Ozone sensitivity was assessed by recording the magnitude of foliar visible injury and changes in various physiological parameters. All the six cultivars showed visible foliar symptoms due to O3, ranging 7.4 to 55.7 % injured leaf area. O3 significantly depressed total chlorophyll, photosynthetic rate (Ps), quantum yield (F(v)/F(m)) and total biomass although the extent of variation was cultivar specific. Cultivar HUM-1 showed maximum reduction in Ps and stomatal conductance. The fluorescence parameters also indicated maximum damage to PSII reaction centres of HUM-1. Injury percentage, chlorophyll loss, Ps, F(v)/F(m) and total biomass reduced least in HUM-23 depicting highest O3 resistance (R%).

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.

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 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.

Differential response of radish plants to supplemental ultraviolet-B radiation under varying NPK levels: chlorophyll fluorescence, gas exchange and antioxidants.

Current and projected increases in ultraviolet-B (UV-B; 280-315 nm) radiation may alter crop growth and yield by modifying the physiological and biochemical functions. This study was conducted to assess the possibility of alleviating the negative effects of supplemental UV-B (sUV-B; 7.2 kJ m⁻² day⁻¹; 280-315 nm) on radish (Raphanus sativus var Pusa Himani) by modifying soil nitrogen (N), phosphorus (P) and potassium (K) levels. The N, P and K treatments were recommended dose of N, P and K, 1.5 times recommended dose of N, P and K, 1.5 times recommended dose of N and 1.5 times recommended dose of K. Plants showed variations in their response to UV-B radiation under varying soil NPK levels. The minimum damaging effects of sUV-B on photosynthesis rate and stomatal conductance coupled with minimum reduction in chlorophyll content were recorded for plants grown at recommended dose of NPK. Flavonoids increased under sUV-B except in plants grown at 1.5 times recommended dose of N. Lipid peroxidation (LPO) also increased in response to sUV-B at all NPK levels with maximum at 1.5 times recommended dose of K and minimum at recommended dose of NPK. This study revealed that sUV-B radiation negatively affected the radish plants by reducing the photosynthetic efficiency and increasing LPO. The plants grown at 1.5 times recommended dose of NPK/N/K could not enhance antioxidative potential to the extent as recorded at recommended dose of NPK and hence showed more sensitivity to sUV-B.

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.

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.

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.