Improved Prediction of Stabilizing Mutations in Proteins by Incorporation of Mutational Effects on Ligand Binding.

While many computational methods accurately predict destabilizing mutations, identifying stabilizing mutations has remained a challenge, because of their relative rarity. We tested ΔΔG0 predictions from computational predictors such as Rosetta, ThermoMPNN, RaSP, and DeepDDG, using 82 mutants of the bacterial toxin CcdB as a test case. On this dataset, the best computational predictor is ThermoMPNN, which identifies stabilizing mutations with a precision of 68%. However, the average increase in Tm for these predicted mutations was only 1°C for CcdB, and predictions were poorer for a more challenging target, influenza neuraminidase. Using data from multiple previously described yeast surface display libraries and in vitro thermal stability measurements, we trained logistic regression models to identify stabilizing mutations with a precision of 90% and an average increase in Tm of 3°C for CcdB. When such libraries contain a population of mutants with significantly enhanced binding relative to the corresponding wild type, there is no benefit in using computational predictors. It is then possible to predict stabilizing mutations without any training, simply by examining the distribution of mutational binding scores. This avoids laborious steps of in vitro expression, purification, and stability characterization. When this is not the case, combining data from computational predictors with high-throughput experimental binding data enhances the prediction of stabilizing mutations. However, this requires training on stability data measured in vitro with known stabilized mutants. It is thus feasible to predict stabilizing mutations rapidly and accurately for any system of interest that can be subjected to a binding selection or screen.

Facing the wrath of enigmatic mutations: a review on the emergence of severe acute respiratory syndrome coronavirus 2 variants amid coronavirus disease-19 pandemic.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging respiratory virus responsible for the ongoing coronavirus disease 19 (COVID-19) pandemic. More than a year into this pandemic, the COVID-19 fatigue is still escalating and takes hold of the entire world population. Driven by the ongoing geographical expansion and upcoming mutations, the COVID-19 pandemic has taken a new shape in the form of emerging SARS-CoV-2 variants. These mutations in the viral spike (S) protein enhance the virulence of SARS-CoV-2 variants by improving viral infectivity, transmissibility and immune evasion abilities. Such variants have resulted in cluster outbreaks and fresh infection waves in various parts of the world with increased disease severity and poor clinical outcomes. Hence, the variants of SARS-CoV-2 pose a threat to human health and public safety. This review enlists the most recent updates regarding the presently characterized variants of SARS-CoV-2 recognized by the global regulatory health authorities (WHO, CDC). Based on the slender literature on SARS-CoV-2 variants, we collate information on the biological implications of these mutations on virus pathology. We also shed light on the efficacy of therapeutics and COVID-19 vaccines against the emerging SARS-CoV-2 variants.

Enhanced protective efficacy of a thermostable RBD-S2 vaccine formulation against SARS-CoV-2 and its variants.

With the rapid emergence of variants of concern (VOC), the efficacy of currently licensed vaccines has reduced drastically. VOC mutations largely occur in the S1 subunit of Spike. The S2 subunit of SARS-CoV-2 is conserved and thus more likely to elicit broadly reactive immune responses that could improve protection. However, the contribution of the S2 subunit in improving the overall efficacy of vaccines remains unclear. Therefore, we designed, and evaluated the immunogenicity and protective potential of a stabilized SARS-CoV-2 Receptor Binding Domain (RBD) fused to a stabilized S2. Immunogens were expressed as soluble proteins with approximately fivefold higher purified yield than the Spike ectodomain and formulated along with Squalene-in-water emulsion (SWE) adjuvant. Immunization with S2 alone failed to elicit a neutralizing immune response, but significantly reduced lung viral titers in mice challenged with the heterologous Beta variant. In hamsters, SWE-formulated RS2 (a genetic fusion of stabilized RBD with S2) showed enhanced immunogenicity and efficacy relative to corresponding RBD and Spike formulations. Despite being based on the ancestral Wuhan strain of SARS-CoV-2, RS2 elicited broad neutralization, including against Omicron variants (BA.1, BA.5 and BF.7), and the clade 1a WIV-1 and SARS-CoV-1 strains. RS2 elicited sera showed enhanced competition with both S2 directed and RBD Class 4 directed broadly neutralizing antibodies, relative to RBD and Spike elicited sera. When lyophilized, RS2 retained antigenicity and immunogenicity even after incubation at 37 °C for a month. The data collectively suggest that the RS2 immunogen is a promising modality to combat SARS-CoV-2 variants.

Protective Efficacy of Recombinant Influenza Hemagglutinin Ectodomain Fusions.

In current seasonal influenza vaccines, neutralizing antibody titers directed against the hemagglutinin surface protein are the primary correlate of protection. These vaccines are, therefore, quantitated in terms of their hemagglutinin content. Adding other influenza surface proteins, such as neuraminidase and M2e, to current quadrivalent influenza vaccines would likely enhance vaccine efficacy. However, this would come with increased manufacturing complexity and cost. To address this issue, as a proof of principle, we have designed genetic fusions of hemagglutinin ectodomains from H3 and H1 influenza A subtypes. These recombinant H1-H3 hemagglutinin ectodomain fusions could be transiently expressed at high yield in mammalian cell culture using Expi293F suspension cells. Fusions were trimeric, and as stable in solution as their individual trimeric counterparts. Furthermore, the H1-H3 fusion constructs were antigenically intact based on their reactivity with a set of conformation-specific monoclonal antibodies. H1-H3 hemagglutinin ectodomain fusion immunogens, when formulated with the MF59 equivalent adjuvant squalene-in-water emulsion (SWE), induced H1 and H3-specific humoral immune responses equivalent to those induced with an equimolar mixture of individually expressed H1 and H3 ectodomains. Mice immunized with these ectodomain fusions were protected against challenge with heterologous H1N1 (Bel/09) and H3N2 (X-31) mouse-adapted viruses with higher neutralizing antibody titers against the H1N1 virus. Use of such ectodomain-fused immunogens would reduce the number of components in a vaccine formulation and allow for the inclusion of other protective antigens to increase influenza vaccine efficacy.

Highly conserved epitopes of DENV structural and non-structural proteins: Candidates for universal vaccine targets.

Dengue is a severe emerging arthropod borne viral disease occurring globally. Around two fifths of the world's population, or up to 3.9 billion people, are at a risk of dengue infection. Infection induces a life-long protective immunity to the homologous serotype but confers only partial and transient protection against subsequent infection caused by other serotypes. Thus, there is a need for a vaccine which is capable of providing a life- long protection against all the serotypes of dengue virus. In our study, comparative genomics of Dengue virus (DENV) was conducted to explore potential candidates for novel vaccine targets. From our analysis we successfully found 100% conserved epitopes in Envelope protein (RCPTQGE); NS3 (SAAQRRGR, PGTSGSPI); NS4A (QRTPQDNQL); NS4B (LQAKATREAQKRA) and NS5 proteins (QRGSGQV) in all DENV serotypes. Some serotype specific conserved motifs were also found in NS1, NS5, Capsid, PrM and Envelope proteins. Using comparative genomics and immunoinformatics approach, we could find conserved epitopes which can be explored as peptide vaccine candidates to combat dengue worldwide. Serotype specific epitopes can also be exploited for rapid diagnostics. All ten proteins are explored to find the conserved epitopes in DENV serotypes, thus making it the most extensively studied viral genome so far.

Burden of household food insecurity in urban slum settings.

This study examined the burden of food insecurity in India's un-notified slums, using an SDG framework to identify correlates of food insecurity. A convenience sampling approach was employed in selecting 38 slums from 675 un-notified slums across four geographic zones. Ten percent of the households in each slum site were selected from each zone, and one household member was interviewed, based on their availability and fulfilment of the eligibility criteria. Eligible individuals included those aged 18 years and above, who were resident in the selected slums and provided consent. Individuals with mental or physical challenges were excluded. A total sample of 907 study participants were included. Results showed that 43% (n = 393) of the participants were food insecure. More than half were females (73%, n = 285), who had not completed any schooling (51%, n = 202). One-third (n = 128) resided in the Northern Region of Delhi. SDG-related predictors of food insecurity included: household educational level (SDG 4 Quality education) (p = 0.03), coverage of health service needs (SDG 3 Good health and well-being) (p = 0.0002), electricity needs (SDG 7 affordable and clean energy) (p<0.0001), and employment needs (SDG 8 Decent and economic growth) (p = 0.003). Having healthcare needs that were partially or fully met was equally associated with higher food insecurity: this could be attributed to high healthcare costs and the lack of federal subsidies in un-notified slums, collectively contributing to high out-of-pocket health costs. Failure to fully meet employment needs was also significantly associated with higher food insecurity. However, met needs for electricity, finance, women's safety and satisfactory family relationships, were associated with lower food insecurity. Household predictors of food insecurity included: number of household members, and the presence of physically disabled household members. Necessary interventions should include connecting food insecure households to existing social services such as India's Public Distribution System, and multi-sector partnerships to address the existing challenges.

Anti-apoptotic activity of caffeic acid, ellagic acid and ferulic acid in normal human peripheral blood mononuclear cells: a Bcl-2 independent mechanism.

Polyphenols have been shown to induce apoptosis in a variety of tumor cells including leukemia both in vitro and in vivo. However, their action on normal human peripheral blood mononuclear cells (PBMCs) during oxidative stress remains to be explored. In this study, we have evaluated the anti-apoptotic and radical scavenging activities of dietary phenolics, namely caffeic acid (CA), ellagic acid (EA) and ferulic acid (FA). H2O2-induced apoptosis in normal human PBMCs was assayed by phosphotidylserine externalization, nucleosomal damage and DNA fragmentation. Incubation of PBMCs with 5 mM H2O2 led to increased Annexin-V binding to externalized phosphatidyl serine (PS), an event of pre-apoptotic stage of the cell. Peripheral blood mononuclear cells pretreated with phenolics could resist H2O2-induced apoptotic damage. Caffeic acid (60 and 120 microM) and EA (100 and 200 microM) caused no change in externalization of PS, whereas FA (100 and 200 microM) increased externalization of PS in PBMCs treated with H2O2. The effects of phenolics were abolished to a large extent by culturing the PBMCs for 24 h after washing the phenolics from the medium. Inhibitory activities of these phenolics on lipid peroxidation were in the order of EA

Sleep-patterns, co-sleeping and parent's perception of sleep among school children: Comparison of domicile and gender.

This study was aimed at assessment of sleep schedule, pre-sleep behavior, co-sleeping and parent's perception of sleep of school going children. METHOD: Four schools each, from urban and rural area were included. Sleep patterns were assessed using the validated Hindi version of Childhood-Sleep-Habit-Questionnaire. Comparison was made between urban and rural group and between boys and girls. Interaction of gender, domicile and school-type was examined on the sleep patterns. RESULTS: This study included 831 school children with mean age of 8.9 years. Nearly half of the subjects were boys in this study. Urban children outnumbered those from rural area. Total sleep time on weekdays was 8.3 h that increased to 9.5 h on weekends. Rural children spent more time in sleep than urban children on weekdays and weekends. A higher proportion of urban children felt sleepy during the day. Television watching before bedtime was more common in urban settings. Room sharing was more common among rural children. Nearly 65% rural parents as compared to 77.5% urban parents reported that their child was sleeping sufficient enough. Gender did not affect sleep-schedule and parent's perception regarding their child's sleep. Interaction between gender, domicile and school-type did not have any significant effect on sleep patterns. CONCLUSION: Television watching before bedtime was more common among urban school children and they had shorter total sleep time. They had signs of sleep deprivation. Room sharing was more common among rural children. Despite longer sleep time, parents of rural children felt the need for more sleep.

Prevalence of Sleep Disorders Among Primary School Children.

OBJECTIVE: To screen symptoms of sleep disorders among primary school children. METHODS: Four schools from urban area and four rural schools were included in this study. Symptoms of sleep disorders were assessed using the validated Hindi version of Childhood Sleep Habit Questionnaire in 8-13 y old children. Comparison was made between urban and rural group and between boys and girls. Descriptive statistics was calculated. RESULTS: Mean age of the subjects included in this study was 8.9 ± 1.5 y. Boys and girls were equally distributed, however, rural sample was smaller. More than one awakening each night was found in 12.2 % children. In the whole group, prevalence of bed-wetting was 8.7 %, sleep-talking 20.9 %, sleep-walking 3.2 %, teeth grinding 15.4 % and night terrors 8.4 %. Daytime sleepiness was reported by 25.5 % and napping by 56.4 %. 17.3 % used to fall asleep in unusual circumstances and the teacher or the friend in 6.9 % students noticed it. Snoring was reported by 11.4 % children, and 6.3 % reportedly struggled to breathe during sleep. Domicile and gender did not affect prevalence of parasomnia, however, symptoms of sleep apnea were more frequent among rural children. Daytime sleepiness was more common among rural children as compared to urban. CONCLUSIONS: Symptoms of sleep disorders are prevalent among primary school children. Common disorders are parasomnia, sleep apnea and daytime sleepiness.

Gallic acid, an antioxidant, exhibits antiapoptotic potential in normal human lymphocytes: A Bcl-2 independent mechanism.

Oxidative stress, produced as a consequence of normal metabolism or induced by extraneous stimuli, has been proved to be a mediator of cell death. The inherent antioxidant defense system and exogenous antioxidants can help the body to combat this oxidative stress-induced cell death. In this study, we explored the antiapoptotic potential of gallic acid, a dietary phenolic having antioxidative and anticarcinogenic properties, in normal human peripheral blood lymphocytes (PBLs). Incubation of PBLs with 100 microM H2O2 for 1.5-2.0 h induced phosphatidyl serine externalisation, lipid peroxidation and high molecular weight DNA fragmentation. Pretreatment of lymphocytes with gallic acid for 18 h could effectively inhibit lipid peroxidation and apoptosis induced by oxidative stress. Treatment of PBLs with gallic acid failed to induce any change in the expression of Bcl-2, an antiapoptotic protein. It seems that the protection provided by gallic acid was due to its direct action in the scavenging of free radicals as it was found to be a stronger antiradical than trolox, a water- soluble analogue of vitamin E.

Giardia lamblia: expression of alkaline phosphatase activity in infected rat intestine.

Alkaline phosphatase (IAP) is a marker of intestinal microvillus membrane. Changes in IAP activity have been studied as a function of Giardia lamblia (G. lamblia) infection using rat as the experimental model. At day 11 and 15 post-infection, enzyme activity was reduced (p<0.01) compared to controls. The enzyme levels were essentially similar to control values by day 30 post-infection. Analysis of the enzyme activity in cell fractions across crypt-villus axis revealed a marked decrease in enzyme activity in the villus tip and mid villus regions but a considerable increase (p<0.01) in enzyme activity in the crypt base of 11 day post-infected animals compared to that in controls. The observed changes in IAP activity were confirmed by assaying the enzyme activity in acrylamide gels using bromo-chloro-indolyl phosphate staining under non-denaturing conditions. These findings indicate differential changes across the crypt-villus axis, but impaired alkaline phosphatase levels in G. lamblia infected rat intestine.