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Ribonucleotide reductases (RNRs) are used by all free-living organisms and many viruses to catalyze an essential step in the de novo biosynthesis of DNA precursors. RNRs are remarkably diverse by primary sequence and cofactor requirement, while sharing a conserved fold and radical-based mechanism for nucleotide reduction. Here, we structurally aligned the diverse RNR family by the conserved catalytic barrel to reconstruct the first large-scale phylogeny consisting of 6779 sequences that unites all extant classes of the RNR family and performed evo-velocity analysis to independently validate our evolutionary model. With a robust phylogeny in-hand, we uncovered a novel, phylogenetically distinct clade that is placed as ancestral to the classes I and II RNRs, which we have termed clade Ø. We employed small-angle X-ray scattering (SAXS), cryogenicelectron microscopy (cryo-EM), and AlphaFold2 to investigate a member of this clade from Synechococcus phage S-CBP4 and report the most minimal RNR architecture to-date. Based on our analyses, we propose an evolutionary model of diversification in the RNR family and delineate how our phylogeny can be used as a roadmap for targeted future study [ABSTRACT FROM AUTHOR]

In addition to its value in the study of molecular evolution, ancestral sequence reconstruction (ASR) has emerged as a useful methodology for engineering proteins with enhanced properties. Proteins generated by ASR often exhibit unique or improved activity, stability, and/or promiscuity, all of which are properties that are valued by protein engineers. Comparison between extant proteins and evolutionary intermediates generated by ASR also allows protein engineers to identify substitutions that have contributed to functional innovation or diversification within protein families. As ASR becomes more widely adopted as a protein engineering approach, it is important to understand the applications, limitations, and recent developments of this technique. This review highlights recent exemplifications of ASR, as well as technical aspects of the reconstruction process that are relevant to protein engineering. [Display omitted] • Proteins produced by ancestral sequence reconstruction (ASR) often exhibit high activity, stability, and evolvability. • ASR studies guide engineering by highlighting function-shifting substitutions. • ASR emphasizes fundamental drivers of, and constraints to, functional innovation. • Recent developments in ASR are making it more accessible to protein engineers. [ABSTRACT FROM AUTHOR]

Nutritional status can affect surgical patients in terms of stress response, healing time, and outcomes. Several abnormalities are known to have a high prevalence in the general population such as vitamin D deficiency (VDD) and subclinical hypothyroidism. We hypothesized that there will be elevated rates of nutritional deficiencies in preoperative patients which may adversely affect postoperative outcomes following pediatric otolaryngology surgery. IRB approval was obtained for a cross-sectional cohort study. Consecutive patients underwent nutritional evaluation when being scheduled for surgery including TSH, albumin and vitamin D. Demographic data, supplementation, and early complication rates were collected. 125 patients were included in the final cohort with adequate demographic distribution. Based on anthropometric data, 12% of our cohort was found to be undernourished, and 40% of our cohort with elevated BMI. However, there was no relationship found between Z-scores and complications. VDD was noted in 83/125 (66.4%) patients. Our cohort had increased rates of VDD in patients with elevated BMI and African American ethnicity. Thyroid hormone abnormalities were present in 12 patients. Mean serum albumin level was 4.29 in our cohort all within normal range. We did find increased risk of postoperative complications in patients with previously diagnosed comorbidities. (p=0.006). There is no current recommendation or consensus for nutritional assessment in preoperative pediatric patients. Our study did not show statistically significant correlation with z-scores, low vitamin D levels with supplementation, albumin, or TSH to postoperative complications. However, our patient cohort had higher than average rates of VDD compared to the many studies of the general pediatric population and significant negative correlation between vitamin D levels and z-scores. By early preoperative identification of VDD and supplementation with calciferol, we found no significant difference in complication rates in patients based on their initial vitamin D status. We suggest screening preoperative patients using z-score calculations and vitamin D levels based on individual patient risk factors including atrisk patient populations such as African American children, and obese children. [ABSTRACT FROM AUTHOR]

The ongoing COVID-19 pandemic has had great societal and health consequences. Despite the availability of vaccines, infection rates remain high due to immune evasive Omicron sublineages. Broad-spectrum antivirals are needed to safeguard against emerging variants and future pandemics. We used messenger RNA (mRNA) display under a reprogrammed genetic code to find a spike-targeting macrocyclic peptide that inhibits SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) Wuhan strain infection and pseudoviruses containing spike proteins of SARS-CoV-2 variants or related sarbecoviruses. Structural and bioinformatic analyses reveal a conserved binding pocket between the receptor-binding domain, N-terminal domain, and S2 region, distal to the angiotensin-converting enzyme 2 receptor-interaction site. Our data reveal a hitherto unexplored site of vulnerability in sarbecoviruses that peptides and potentially other drug-like molecules can target. [ABSTRACT FROM AUTHOR]

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second-generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor-binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that the neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies. [Display omitted] • Immunization with diverse sarbecorvirus RBDs induces cross-reactive antibodies • Cross-reactive antibodies use recurrent IGHV/IGKVs that mainly bind the class 4 epitope • Diverse sarbecovirus RBD responses vary in the dominance of class 4 epitope antibodies • Neutralizing class 4 antibodies are rare and extend the RBD beta-sheet main chain Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Burnett et al. immunized humanized mice with different diverse sarbecovirus RBDs to elicit antibodies targeting conserved sites. Non-neutralizing cross-reactive antibodies targeting the conserved class 4 epitope were readily elicited. Neutralizing ability was reserved only for antibodies binding this conserved supersite through an elongated CDRH3 that obstructed ACE2-RBD interactions. [ABSTRACT FROM AUTHOR]