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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]

• Designing genetically encoded fluorescence-based analyte biosensors is not trivial. • Many current design efforts take a nature-inspired, modular approach. • Individual sensor components can be engineered separately for desired properties. • Screening of large variant libraries is often required to optimize biosensors. Biosensors that selectively report on the presence of specific small molecule analytes have applications in many fields of research, medicine and biotechnology. Here, we review recent advances and emerging approaches in the design and optimisation of genetically encoded fluorescence-based small molecule biosensors. We discuss how natural sensory proteins can be exploited to produce novel biosensors and the strategies for optimizing ligand specificity and fluorescence readout. Finally, we provide insight into high-throughput sensor optimisation and discuss the challenges that are faced when designing novel biosensors. [ABSTRACT FROM AUTHOR]

Background: One approach to increasing the reach of syringe programs in rural areas could be through provision of syringes at community pharmacies. This study evaluated relationships between state-specific syringe policies, pharmacy, and pharmacist characteristics and pharmacists' nonprescription syringe dispensing behaviors in a 3- state Appalachian region at high risk for HIV and HCV transmission.Methods: We conducted a telephone census of community pharmacies in the Appalachian counties of North Carolina, Tennessee, and Virginia from April-June 2018. Behaviors studied included having ever sold syringes without a prescription, quantity of individuals to whom nonprescription syringes were dispensed in the past 30 days, having ever denied a request for nonprescription syringes, and past 30-day denial of nonprescription syringe requests. Behavioral intention and perceptions of legality were elicited.Results: A response rate of 52.3 % was achieved (N = 391). North Carolina pharmacists reported increased past 30-day dispensing, less denial of nonprescription syringe requests, and decreased justification for syringe dispensing (proof of medical need) as compared to Tennessee and Virginia pharmacists. Behavioral intention to dispense did not vary by state but did vary by political affiliation. Perceptions of syringe dispensing legality in NC were significantly different from those in TN and VA.Conclusions: Significant differences in pharmacists' perceptions and behaviors were noted across state lines with North Carolina pharmacists reporting more engagement in syringe dispensing as compared to pharmacists in Tennessee and Virginia. Policy allowing pharmacists to dispense syringes to people who inject drugs appears to foster some but not all pharmacist engagement in this harm reduction intervention. [ABSTRACT FROM AUTHOR]