Sex differences in gout characteristics: tailoring care for women and men.

BACKGROUND: To characterize the differences between women and men with gout. METHODS: We analyzed a US national cohort of gout patients cared for by rheumatologists. RESULTS: Compared with the 1012 men with gout, women with gout (n = 262) were older (71 vs. 61 years, p < 0.001) and had a greater burden of comorbid conditions (p < 0.001 for hypertension, diabetes, renal disease and obesity). Risk factors for gout differed with women more often taking diuretics (p < 0.001), while men more frequently had dietary triggers (p < 0.05). CONCLUSIONS: The profiles of women and men with gout are markedly different, suggesting a need to tailor treatment recommendations.

Mortality from multiple sclerosis in British military personnel.

BACKGROUND: While analysing trends in occupational mortality in England and Wales, we noticed an unexpectedly elevated proportion of deaths from multiple sclerosis (MS) among men in the armed forces. AIMS: To document and explore possible explanations for the observed excess. METHODS: We analysed data on underlying cause of death and last full-time occupation for 3,688,916 deaths among men aged 20-74 years in England and Wales during 1979-2010, calculating proportional mortality ratios (PMRs) standardised for age. We compared PMRs for MS in the armed forces with those for each main social class, and in selected other occupations. We also compared PMRs for MS with those for motor neurone disease (MND). RESULTS: The overall PMR for MS in the armed forces during 1979-2010 was 243 (95%CI 203-288). The excess was apparent in each of three separate decades of study (PMRs, ranging from 220 to 259), and across the entire age range. PMRs for MS were not elevated to the same extent in comparator occupations, nor in any of the main social classes. There was no parallel increase in PMRs for MND. CONCLUSIONS: These findings suggest that the high proportional mortality from MS in British military personnel is unlikely to have occurred by chance, or as an artefact of the method of investigation. However, the only military cohort study with published results on MS does not support an increased risk. It would be useful to analyse data on MS from other established military cohorts, to check for evidence of a hazard.

A Daily Snack Containing Leafy Green Vegetables, Fruit, and Milk before and during Pregnancy Prevents Gestational Diabetes in a Randomized, Controlled Trial in Mumbai, India.

BACKGROUND: Prospective observational studies suggest that maternal diets rich in leafy green vegetables and fruit may help prevent gestational diabetes mellitus (GDM). OBJECTIVE: Our objective was to test whether increasing women's dietary intake of leafy green vegetables, fruit, and milk before conception and throughout pregnancy reduced their risk of GDM. METHODS: Project SARAS ("excellent") (2006-2012) was a nonblinded, individually randomized, controlled trial in women living in slums in the city of Mumbai, India. The interventions included a daily snack made from leafy green vegetables, fruit, and milk for the treatment group or low-micronutrient vegetables (e.g., potato and onion) for the control group, in addition to the usual diet. Results for the primary outcome, birth weight, have been reported. Women were invited to take an oral-glucose-tolerance test (OGTT) at 28-32 wk gestation to screen for GDM (WHO 1999 criteria). The prevalence of GDM was compared between the intervention and control groups, and Kernel density analysis was used to compare distributions of 120-min plasma glucose concentrations between groups. RESULTS: Of 6513 women randomly assigned, 2291 became pregnant; of these, 2028 reached a gestation of 28 wk, 1008 (50%) attended for an OGTT, and 100 (9.9%) had GDM. In an intention-to-treat analysis, the prevalence of GDM was reduced in the treatment group (7.3% compared with 12.4% in controls; OR: 0.56; 95% CI: 0.36, 0.86; P = 0.008). The reduction in GDM remained significant after adjusting for prepregnancy adiposity and fat or weight gain during pregnancy. Kernel density analysis showed that this was explained by the fact that fewer women in the treatment group had a 2-h glucose concentration in the range 7.5-10.0 mmol/L. CONCLUSIONS: In low-income settings, in which women have a low intake of micronutrient-rich foods, improving dietary micronutrient quality by increasing intake of leafy green vegetables, fruit, and/or milk may have an important protective effect against the development of GDM. This trial was registered at www.controlled-trials.com as ISRCTN62811278.

Trends in mortality from occupational hazards among men in England and Wales during 1979-2010.

OBJECTIVES: To monitor the impact of health and safety provisions and inform future preventive strategies, we investigated trends in mortality from established occupational hazards in England and Wales. METHODS: We analysed data from death certificates on underlying cause of death and last full-time occupation for 3 688 916 deaths among men aged 20-74 years in England and Wales during 1979-2010 (excluding 1981 when records were incomplete). Proportional mortality ratios (PMRs), standardised for age and social class, were calculated for occupations at risk of specified hazards. Observed and expected numbers of deaths for each hazard were summed across occupations, and the differences summarised as average annual excesses. RESULTS: Excess mortality declined substantially for most hazards. For example, the annual excess of deaths from chronic bronchitis and emphysema fell from 170.7 during 1979-1990 to 36.0 in 2001-2010, and that for deaths from injury and poisoning from 237.0 to 87.5. In many cases, the improvements were associated with falling PMRs (suggesting safer working practices), but they also reflected reductions in the numbers of men employed in more hazardous jobs, and declining mortality from some diseases across the whole population. Notable exceptions to the general improvement were diseases caused by asbestos, especially in some construction trades and sinonasal cancer in woodworkers. CONCLUSIONS: The highest priority for future prevention of work-related fatalities is the minority of occupational disorders for which excess mortality remains static or is increasing, in particular asbestos-related disease among certain occupations in the construction industry and sinonasal cancer in woodworkers.

Discontinuation of tumour necrosis factor inhibitors in patients with rheumatoid arthritis in low-disease activity: persistent benefits. Data from the Corrona registry.

BACKGROUND: There is increasing interest in discontinuing biological therapies for patients with rheumatoid arthritis (RA) achieving good clinical responses, provided patients maintain clinical benefit. METHODS: We assessed patients with RA from the Corrona registry who discontinued treatment with their first tumour necrosis factor inhibitor (TNFi) while in low-disease activity (LDA) or lower levels of disease activity. Patients were followed until they lost clinical benefit, defined as increased disease activity or change in RA medications. Duration of maintenance of clinical benefit was estimated using the Kaplan-Meier method. Cox proportional hazard models were assessed to identify factors related to maintenance of benefit. RESULTS: We identified 717 eligible patients with RA from 35,656 in the Corrona registry. At discontinuation, patients had a median RA duration of 8 years, mean clinical disease activity score of 4.3±0.11; 41.8% were using TNFi as monotherapy. 73.4% of patients maintained benefit for >12 months after discontinuing therapy and 42.2% did so through 24 months. Factors predictive of maintaining clinical benefit in multivariate analysis included lower disease activity, less pain and better functional status at the time of TNFi discontinuation. Among 301 patients initiating their first TNFi within the registry, faster responders (ie, those who achieved LDA in 4 months or less) did better than slower responders (HR 1.54 (95% CI 1.17 to 2.04)). RA disease duration did not affect maintenance of clinical benefit. CONCLUSIONS: Discontinuation of a first course of TNFi may be associated with persistent clinical benefit. Half of patients maintained response through 20 months. Several patient characteristics may help predict persistent benefit.

Electronic monitoring of oral therapies in ethnically diverse and economically disadvantaged patients with rheumatoid arthritis: consequences of low adherence.

OBJECTIVE: To quantify adherence to oral therapies in ethnically diverse and economically disadvantaged patients with rheumatoid arthritis (RA), using electronic medication monitoring, and to evaluate the clinical consequences of low adherence. METHODS: A total of 107 patients with RA enrolled in a 2-year prospective cohort study agreed to have their oral RA drug therapy intake electronically monitored using the Medication Event Monitoring System. Adherence to disease-modifying antirheumatic drugs (DMARDs) and prednisone was determined as the percentage of days (or weeks for methotrexate) on which the patient took the correct dose as prescribed by the physician. Patient outcomes were assessed, including function measured by the modified Health Assessment Questionnaire, disease activity measured by the Disease Activity Score in 28 joints (DAS28), health-related quality of life, and radiographic damage measured using the modified Sharp/van der Heijde scoring method. RESULTS: Adherence to the treatment regimen as determined by the percentage of correct doses was 64% for DMARDs and 70% for prednisone. Patients who had better mental health were statistically more likely to be adherent. Only 23 of the patients (21%) had an average adherence to DMARDs ≥80%. These patients showed significantly better mean DAS28 values across 2 years of followup than those who were less adherent (3.28 versus 4.09; P = 0.02). Radiographic scores were also worse in nonadherent patients at baseline and at 12 months. CONCLUSION: Only one-fifth of RA patients had an overall adherence to DMARDs of at least 80%. Less than two-thirds of the prescribed DMARD doses were correctly taken. Adherent patients had lower disease activity across the 2 years of followup.

Maternal mood, anxiety and mental health functioning after combined myo-inositol, probiotics, micronutrient supplementation from preconception: Findings from the NiPPeR RCT.

Observational studies have reported associations between nutrition during pregnancy and mental wellbeing. As secondary outcomes, the NiPPeR double-blind randomized trial in women planning conception investigated whether a myo-inositol, probiotics and enriched micronutrients formulation (intervention) taken preconception and throughout pregnancy could improve mental wellbeing during pregnancy and post-delivery, compared with a standard micronutrient supplement (control). Mood and anxiety symptoms were ascertained (Edinburgh Postnatal Depression Scale (EPDS), State-Trait Anxiety Inventory (STAI-state)) at preconception (baseline), 7, 28 and 34 weeks gestation, 3-weeks and 6-months post-delivery. EPDS>=13 was categorised as low mood; STAI-state>=45 as high anxiety. Change in mental health functioning was assessed as difference between preconception baseline and 6-month post-delivery 12-item Short-Form Health Survey (SF-12v2) mental component scores. Adjusting for site, ethnicity and baseline scores, there were no robust differences in EPDS and STAI-state scores between intervention and control groups across pregnancy (n = 630) and post-delivery (n = 532). Compared to controls, intervention group women averaged a 1.21 (95 %CI 0.04,2.39) higher change in SF-12v2 mental component score from preconception to 6-months post-delivery. Taking a myo-inositol, micronutrient and probiotic supplement during preconception/pregnancy had no effect on mood and anxiety, but there was evidence of a modest improvement in mental health functioning from preconception to 6-months post-delivery.

Evaluation of Preconception Dietary Patterns in Women Enrolled in a Multisite Study.

Background: Diet indices are widely used in nutritional research across communities but do not "capture" the full extent of diet variability across multiple countries. Empirically derived dietary patterns can provide additional information because they reflect combinations of foods potentially associated with health outcomes. Limited studies have evaluated preconception dietary patterns in heterogeneous populations. Objectives: In the multisite Nutritional Intervention Preconception and During Pregnancy to Maintain Healthy Glucose Metabolism and Offspring Health (NiPPeR) study, the secondary aims included: 1) derive pooled and site-specific preconception dietary patterns, and 2) evaluate these patterns using anthropometric measures and metabolic biomarkers. Methods: Women planning pregnancy (n = 1720) in the United Kingdom, Singapore, and New Zealand completed interviewer-administered harmonized FFQs and lifestyle questionnaires at recruitment. Across-cohort ("pooled") and site-specific dietary patterns were derived, and associations between dietary pattern scores and BMI, waist-to-hip ratio, plasma lipids, and glycemia assessed using multivariable linear regression, expressing results as SD change in outcome per SD change in dietary pattern score. Results: The pooled analysis identified 3 dietary patterns: "Vegetables/Fruits/Nuts" ("Healthy"), "Fried potatoes/Processed meat/Sweetened beverages" ("Less Healthy"), and "Fish/Poultry/Noodles/Rice" ("Mixed"). The "Healthy" and "Less Healthy" pooled pattern scores were highly correlated with their corresponding site-specific dietary pattern scores ("Healthy": ρ = 0.87-0.93; "Less Healthy": ρ = 0.65-0.88). Women with higher scores for the "Healthy" pooled pattern had a lower waist-to-hip ratio (standardized ß: -0.10; 95% CI: -0.18, -0.01); those with higher scores for the "Less Healthy" pooled pattern had a higher BMI (standardized ß: 0.17; 95% CI: 0.09, 0.24), higher LDL cholesterol (standardized ß: 0.10; 95% CI: 0.01, 0.19), and less optimal glucose profiles. However, we noted higher adherence to the "Healthy" pooled pattern with higher BMI. Conclusions: The "Healthy" and "Less Healthy" pooled patterns were comparable to the corresponding site-specific patterns. Although the associations between these patterns and objective anthropometric/metabolic measures were largely in the expected directions, future studies are required to confirm these findings.This trial is registered at clinicaltrials.gov (NCT02509988).

Assessment of naloxone as a therapeutic for inhaled carfentanil in the ferret.

Carfentanil is a powerful synthetic opioid that is approximately 100 times more potent than fentanyl and 10,000 times more potent than morphine. Carfentanil was originally intended to be used as a sedative for big game animals in a veterinary setting, but it is becoming increasingly recognized as a public health concern. We set out to investigate the effectiveness of naloxone against a potentially lethal dose of inhaled carfentanil in male ferrets. Ferrets were implanted with telemetry devices to study cardiac parameters and exposed to aerosolized carfentanil in a whole-body plethysmography chamber to record respiratory parameters. We observed profound respiratory depression in exposed animals, which led to apneic periods constituting 24-31 % of the exposure period. Concomitant with these apneic periods, we also observed cardiac abnormalities in the form of premature junctional contractions (PJCs). At our acute exposure dose, lethal in 3 % of our animals, naïve ferrets were unresponsive and incapacitated for a total of 126.1 ± 24.6 min. When administered intramuscularly at human equivalent doses (HEDs) of either 5 mg or 10 mg, naloxone significantly reduced the time that ferrets were incapacitated following exposure, although we observed no significant difference in the reduction of time that the animals were incapacitated between the treatment groups. Naloxone was able to quickly resolve the respiratory depression, significantly reducing the frequency of apneic periods in carfentanil-exposed ferrets. Our results suggest that naloxone, when administered via intramuscular injection following incapacitation, is a viable treatment against the effects of a potentially lethal dose of inhaled carfentanil.

Methylene blue and monosodium glutamate improve neurologic signs after fluoroacetate poisoning.

Fluoroacetate (FA) is a tasteless, odorless, water-soluble metabolic poison with severe toxicological effects. Characterized in the mid-1900s, it has been used as a rodenticide but is comparably lethal to all mammals. Many countries have restricted its use, and modern-day accidental human exposures are rare, but recently, concerns have been raised about its application as a chemical weapon with no known antidote. A combined treatment of methylene blue (MB), an antioxidant, and monosodium glutamate (MSG), a precursor of the citric acid cycle substrate alpha-ketoglutarate, has been recommended as an effective countermeasure; however, no peer-reviewed articles documenting the efficacy of this therapy have been published. Using a rodent model, we assessed the effects of MB and MSG on the neurologic, cardiac, and pulmonary systems. Transcriptomic analysis was used to elucidate inflammatory pathway activation and guide bioassays, which revealed the advantages and disadvantages of these candidate countermeasures. Results show that MB and MSG can reduce neurologic signs observed in rats exposed to sodium FA and improve some effects of intoxication. However, while this strategy resolved some signs of intoxication, ultimately it was unable to significantly reduce lethality.

Doctor-patient interactions in Mexican patients with rheumatic disease.

OBJECTIVE: To evaluate the characteristics of physician-patient communications in Mexican patients with rheumatic diseases. METHODS: We prospectively recruited 200 consecutive patients with chronic rheumatic diseases from a rheumatology clinic affiliated with a secondary care hospital in Mexico. All participants completed face-to-face interviews and surveys to determine their perceptions of the medical interaction with the physician at the clinic. Patients were assessed immediately before and after their visits. The Perceptions of Involvement in Care Scale was used to examine 3 factors: the doctor's facilitation of involvement, the level of information exchange, and the patient's participation in decision making. The Medical Outcomes Study scale was used to evaluate the patient satisfaction with the visit. A Patients' Trust in Physicians instrument was also administered. RESULTS: No differences in communication, satisfaction, or trust were observed across diseases. Most patients indicated they wanted to play a passive role, with the physicians making the decisions. However, concordance between preferred and actual roles was small (weighted kappa = 0.3), suggesting that not all patients played their preferred role. CONCLUSION: This is the first study to evaluate doctor-patient communication in Mexican patients with rheumatic diseases. In general, patients preferred to play a passive role in their medical interaction. In addition, they often did not attain their preferred communication style when interacting with their physicians. The effect of these findings in subsequent health outcomes is unknown but deserves further investigation.

Incorporation of Modified Amino Acids by Engineered Elongation Factors with Expanded Substrate Capabilities.

Noncanonical amino acid (ncAA) incorporation has led to significant advances in protein science and engineering. Traditionally, in vivo incorporation of ncAAs is achieved via amber codon suppression using an engineered orthogonal aminoacyl-tRNA synthetase:tRNA pair. However, as more complex protein products are targeted, researchers are identifying additional barriers limiting the scope of currently available ncAA systems. One barrier is elongation factor Tu (EF-Tu), a protein responsible for proofreading aa-tRNAs, which substantially restricts ncAA scope by limiting ncaa-tRNA delivery to the ribosome. Researchers have responded by engineering ncAA-compatible EF-Tus for key ncAAs. However, this approach fails to address the extent to which EF-Tu inhibits efficient ncAA incorporation. Here, we demonstrate an alternative strategy leveraging computational analysis to broaden EF-Tu's substrate specificity. Evolutionary analysis of EF-Tu and a naturally evolved specialized elongation factor, SelB, provide the opportunity to engineer EF-Tu by targeting amino acid residues that are associated with functional divergence between the two ancient paralogues. Employing amber codon suppression, in combination with mass spectrometry, we identified two EF-Tu variants with non-native substrate compatibility. Additionally, we present data showing these EF-Tu variants contribute to host organismal fitness, working cooperatively with components of native and engineered translation machinery. These results demonstrate the viability of our computational method and lend support to corresponding assumptions about molecular evolution. This work promotes enhanced polyspecific EF-Tu behavior as a viable strategy to expand ncAA scope and complements ongoing research emphasizing the importance of a comprehensive approach to further expand the genetic code.

Dietary micronutrient intakes among women of reproductive age in Mumbai slums.

OBJECTIVES: To (1) describe micronutrient intakes among women of reproductive age living in Mumbai slums; (2) assess the adequacy of these intakes compared with reference values; (3) identify important dietary sources of micronutrients. SUBJECTS/METHODS: Participants were 6426 non-pregnant women aged 16-39 years, registered in a randomised controlled trial of a food-based intervention set in the Bandra, Khar and Andheri areas of Mumbai, India. Cross-sectional quantified food frequency questionnaire (FFQ) data were collected. Vitamin (n = 9) and mineral (n = 6) intakes were calculated and analysed in relation to dietary reference values (DRVs). Important dietary sources were identified for each micronutrient. RESULTS: Median intakes of all micronutrients, except vitamin E, were below the FAO/WHO reference nutrient intake (RNI). Intakes of calcium, iron, vitamin A and folate were furthest from the RNI. For seven of the micronutrients, over half of the women had intakes below the lower reference nutrient intake (LRNI); this figure was over 75% for calcium and riboflavin. The majority of women (93%) had intakes below the EAR for 5 or more micronutrients, and 64% for 10 or more. Adolescents had lower intakes than women aged >19 years. Less than 1% of adult women and no adolescents met the EAR for all micronutrients. Animal source foods and micronutrient-rich fruit and vegetables were consumed infrequently. CONCLUSIONS: These women had low intakes of multiple micronutrients, increasing their risk of insufficiency. There is a need to determine the factors causing poor intakes, to direct interventions that improve diet quality and nutritional sufficiency.

Treatment adherence in patients with rheumatoid arthritis and systemic lupus erythematosus.

This study assessed self-reported adherence in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) from underserved healthcare settings. We conducted a cross-sectional survey of 102 ethnically diverse patients--70 with RA and 32 with SLE--attending rheumatology clinics at publicly funded hospitals in Houston, Texas; 43% were Hispanic, 32% African-American, and 25% White. Treatment adherence was evaluated using the compliance questionnaire rheumatology (CQR; 0, low adherence and 100, high adherence) and the questionnaire of the Adult AIDS Clinical Trials Group (AACTG). The patients were also asked how often they forgot to take their prescribed medications or discontinued them on their own. Mean patient age was 48.5 years; 75% were female, 32% were African-American, 43% Hispanic, and 25% White. Only one third reported never forgetting to take their medications; 40% reported having stopped their medications on their own because of side effects, and 20% because of lack of efficacy. Mean CQR score was 69.1 +/- 10.5, suggesting moderate adherence overall. Differences were also observed across ethnic groups: 23% of ethnic minority patients had problems taking their medications at specified times compared to 11% of Whites (p = 0.03). Lower education and side effects were associated with lower adherence. No differences were observed between RA and SLE patients. Many patients with RA and SLE report problems with treatment adherence. These appear to be more prevalent in African Americans and Hispanics than Whites; the impact of decreased adherence on outcomes could be significant and should be considered when treating patients with RA and SLE.

Coding Geriatric syndromes: How good are we?

High quality coding of hospital activity is important because the data is used for resource allocation and measuring performance. There is little information on the quality of coding of admissions of frail older people who have multiple diagnoses, co-morbidities and functional impairment. Presence or absence of four geriatric syndromes and eight medical conditions was noted on case note review (CNR). Discharge summaries (DS) and hospital coding (HC) were reviewed and compared with the CNR. Forty patients had at least one geriatric syndrome noted in the DS; 16 (40.0%) were captured by the HC. Of 57 patients with at least one medical condition noted in the DS, 52 (91.2%) were captured by the HC (p<0.0001 for difference in HC capture rates). We have demonstrated poor capture of information on geriatric syndromes compared to medical conditions in discharge summaries and hospital coding and propose a problem list bookmark approach to improve this.

Effect of a daily snack containing green leafy vegetables on women's fatty acid status: a randomized controlled trial in Mumbai, India.

BACKGROUND AND OBJECTIVES: There are few data on the fatty acid status of non-pregnant Indian women. Our objective was to investigate the effect of a snack containing green leafy vegetables (GLVs) on women's erythrocyte long chain polyunsaturated fatty acid status (LCPUFA). METHODS AND STUDY DESIGN: Non-pregnant women (n=222) aged 14-35 years from Mumbai slums were randomized to consume a snack containing GLVs, fruit and milk (treatment) or a control snack containing foods of low micronutrient content such as potato and onion, daily under observation. One treatment snack contained a mean (SD) of 54.1 (33.7) mg alpha-linolenic acid (ALA) and one control snack contained 4.1 (3.4) mg ALA. Blood was collected at baseline (0 weeks) and after 12 weeks of supplementation. Erythrocyte fatty acids were analyzed using gas chromatography and expressed as g/100g fatty acids. Plasma malondialdehyde, homocysteine, and erythrocyte superoxide dismutase and glutathione peroxidase were measured. The effect of the treatment on 12 week LCPUFA was assessed using ANCOVA models. RESULTS: Median (IQR) erythrocyte DHA in the treatment group increased from 1.50 (1.11, 2.03) at baseline to 1.86 (1.50, 2.43) (p<0.001) at 12 weeks, and fell in controls from 1.78 (1.37, 2.32) to 1.60 (1.32, 2.04) (p<0.001). The total n-3 fatty acids increased in the treatment group. There was no effect on malondialdehyde and antioxidant enzyme levels. Plasma homocysteine at 0 and 12 weeks was inversely associated with erythrocyte DHA at 12 weeks. CONCLUSION: Daily consumption of a snack containing GLV improved women's erythrocyte DHA levels without increasing oxidative stress.

Hospital admission for accidental pesticide poisoning among adults of working age in England, 1998-2003.

AIMS: To explore the circumstances associated with hospital admission of adults of working age for accidental pesticide poisoning in England. METHODS: From Hospital Episode Statistics we identified all adults aged 16-69 years who completed hospital admissions nationally for accidental pesticide poisoning during April 1998 to March 2003. Further information was obtained through a postal questionnaire completed by the treating consultant. RESULTS: Questionnaires were returned for 62% of 237 identified admissions, including 89 in which definite or possible accidental pesticide poisoning was confirmed. Fifty-four percent of episodes arose from an identifiable mishap, and 26% from unsatisfactory storage or transport of pesticides. Only two incidents resulted from exposure to spray drift. Three patients needed intensive care, but none died. DISCUSSION: Serious accidental pesticide poisoning is rare among adults in England. There is an important contribution from poor storage of pesticides. Non-occupational acute poisoning by spray drift is seldom if ever a cause of hospital admission.

Molecular Evolution Directs Protein Translation Using Unnatural Amino Acids.

Unnatural amino acids have in recent years established their importance in a wide range of fields, from pharmaceuticals to polymer science. Unnatural amino acids can increase the number of chemical groups within proteins and thus expand or enhance biological function. Our ability to utilize these important building blocks, however, has been limited by the inherent difficulty in incorporating these molecules into proteins. To address this challenge, researchers have examined how the canonical twenty amino acids are incorporated, regulated, and modified in nature. This review focuses on achievements and techniques used to engineer the ribosomal protein-translation machinery, including the introduction of orthogonal translation components, how directed evolution enhances the incorporation of unnatural amino acids, and the potential utility of ancient biomolecules for this process.

Engineering proteins by reconstructing evolutionary adaptive paths.

Reconstructing evolutionary adaptive paths (REAP) is a low-throughput technique used to design protein libraries that can be assayed for specific properties such as catalytic function or thermostability. This approach takes advantage of natural selection by using theoretical ancestral proteins as the foundation for library variants. REAP gives rise to smaller libraries but with a higher ratio of viable proteins than other high-throughput techniques. REAP uses analyses of ancestral sequences and signatures of functional divergence to modify extant protein sequences. This allows the experimenter to statistically evaluate which amino acid mutations in which sites within the protein are most likely to produce functional proteins having varied phenotypes.