Primordial aminoacyl-tRNA synthetases preferred minihelices to full-length tRNA.

Aminoacyl-tRNA synthetases (AARS) and tRNAs translate the genetic code in all living cells. Little is known about how their molecular ancestors began to enforce the coding rules for the expression of their own genes. Schimmel et al. proposed in 1993 that AARS catalytic domains began by reading an 'operational' code in the acceptor stems of tRNA minihelices. We show here that the enzymology of an AARS urzyme•TΨC-minihelix cognate pair is a rich in vitro realization of that idea. The TΨC-minihelixLeu is a very poor substrate for full-length Leucyl-tRNA synthetase. It is a superior RNA substrate for the corresponding urzyme, LeuAC. LeuAC active-site mutations shift the choice of both amino acid and RNA substrates. AARS urzyme•minihelix cognate pairs are thus small, pliant models for the ancestral decoding hardware. They are thus an ideal platform for detailed experimental study of the operational RNA code.

Base Pairing Promoted the Self-Organization of Genetic Coding, Catalysis, and Free-Energy Transduction.

How Nature discovered genetic coding is a largely ignored question, yet the answer is key to explaining the transition from biochemical building blocks to life. Other, related puzzles also fall inside the aegis enclosing the codes themselves. The peptide bond is unstable with respect to hydrolysis. So, it requires some form of chemical free energy to drive it. Amino acid activation and acyl transfer are also slow and must be catalyzed. All living things must thus also convert free energy and synchronize cellular chemistry. Most importantly, functional proteins occupy only small, isolated regions of sequence space. Nature evolved heritable symbolic data processing to seek out and use those sequences. That system has three parts: a memory of how amino acids behave in solution and inside proteins, a set of code keys to access that memory, and a scoring function. The code keys themselves are the genes for cognate pairs of tRNA and aminoacyl-tRNA synthetases, AARSs. The scoring function is the enzymatic specificity constant, kcat/kM, which measures both catalysis and specificity. The work described here deepens the evidence for and understanding of an unexpected consequence of ancestral bidirectional coding. Secondary structures occur in approximately the same places within antiparallel alignments of their gene products. However, the polar amino acids that define the molecular surface of one are reflected into core-defining non-polar side chains on the other. Proteins translated from base-paired coding strands fold up inside out. Bidirectional genes thus project an inverted structural duality into the proteome. I review how experimental data root the scoring functions responsible for the origins of coding and catalyzed activation of unfavorable chemical reactions in that duality.

HetMM: A Michaelis-Menten model for non-homogeneous enzyme mixtures.

The Michaelis-Menten model requires its reaction velocities to come from a preparation of homogeneous enzymes, with identical or near-identical catalytic activities. However, this condition is not always met. We introduce a kinetic model that relaxes this requirement, by assuming there are an unknown number of enzyme species drawn from a probability distribution whose standard deviation is estimated. Through simulation studies, we demonstrate the method accurately discriminates between homogeneous and heterogeneous data, even with moderate levels of experimental error. We applied this model to three homogeneous and three heterogeneous biological systems, showing that the standard and heterogeneous models outperform respectively. Lastly, we show that heterogeneity is not readily distinguished from negatively cooperative binding under the Hill model. These two distinct attributes-inequality in catalytic ability and interference between binding sites-yield similar Michaelis-Menten curves that are not readily resolved without further experimentation. Our user-friendly software package allows homogeneity testing and parameter estimation.

Genomic database furnishes a spontaneous example of a functional Class II glycyl-tRNA synthetase urzyme.

The chief barrier to studies of how genetic coding emerged is the lack of experimental models for ancestral aminoacyl-tRNA synthetases (AARS). We hypothesized that conserved core catalytic sites could represent such ancestors. That hypothesis enabled engineering functional "urzymes" from TrpRS, LeuRS, and HisRS. We describe here a fourth urzyme, GlyCA, detected in an open reading frame from the genomic record of the arctic fox, Vulpes lagopus. GlyCA is homologous to a bacterial heterotetrameric Class II GlyRS-B. Alphafold2 predicted that the N-terminal 81 amino acids would adopt a 3D structure nearly identical to the HisRS urzyme (HisCA1). We expressed and purified that N-terminal segment. Enzymatic characterization revealed a robust single-turnover burst size and a catalytic rate for ATP consumption well in excess of that previously published for HisCA1. Time-dependent aminoacylation of tRNAGly proceeds at a rate consistent with that observed for amino acid activation. In fact, GlyCA is actually 35 times more active in glycine activation by ATP than the full-length GlyRS-B α-subunit dimer. ATP-dependent activation of the 20 canonical amino acids favors Class II amino acids that complement those favored by HisCA and LeuAC. These properties reinforce the notion that urzymes represent the requisite ancestral catalytic activities to implement a reduced genetic coding alphabet.

Enzymic recognition of amino acids drove the evolution of primordial genetic codes.

How genetic information gained its exquisite control over chemical processes needed to build living cells remains an enigma. Today, the aminoacyl-tRNA synthetases (AARS) execute the genetic codes in all living systems. But how did the AARS that emerged over three billion years ago as low-specificity, protozymic forms then spawn the full range of highly-specific enzymes that distinguish between 22 diverse amino acids? A phylogenetic reconstruction of extant AARS genes, enhanced by analysing modular acquisitions, reveals six AARS with distinct bacterial, archaeal, eukaryotic, or organellar clades, resulting in a total of 36 families of AARS catalytic domains. Small structural modules that differentiate one AARS family from another played pivotal roles in discriminating between amino acid side chains, thereby expanding the genetic code and refining its precision. The resulting model shows a tendency for less elaborate enzymes, with simpler catalytic domains, to activate amino acids that were not synthesised until later in the evolution of the code. The most probable evolutionary route for an emergent amino acid type to establish a place in the code was by recruiting older, less specific AARS, rather than adapting contemporary lineages. This process, retrofunctionalisation, differs from previously described mechanisms through which amino acids would enter the code.

High-throughput thermal denaturation of tryptophanyl-tRNA synthetase combinatorial mutants reveals high-order energetic coupling determinants of conformational stability.

Landscape descriptions provide a framework for identifying functionally significant dynamic linkages in proteins but cannot supply details. Rate measurements of combinatorial mutations can implicate dynamic linkages in catalysis. A major difficulty is filtering dynamic linkages from the vastly more numerous static interactions that stabilize domain folding. The Geobacillus stearothermophilus (TrpRS) D1 switch is such a dynamic packing motif; it links domain movement to catalysis and specificity. We describe Thermofluor and far UV circular dichroism melting curves for all 16 D1 switch variants to determine their higher-order impact on unliganded TrpRS stability. A prominent transition at intermediate temperatures in TrpRS thermal denaturation is molten globule formation. Combinatorial analysis of thermal melting transcends the protein landscape in four significant respects: (i) bioinformatic methods identify dynamic linkages from coordinates of multiple conformational states. (ii) Relative mutant melting temperatures, δTM, are proportional to free energy changes. (iii) Structural analysis of thermal melting implicates unexpected coupling between the D1 switch packing and regions of high local frustration. Those segments develop molten globular characteristics at the point of greatest complementarity to the chemical transition state and are the first TrpRS structures to melt. (iv) Residue F37 stabilizes both native and molten globular states; its higher-order interactions modify the relative intrinsic impacts of mutations to other D1 switch residues from those estimated for single point mutants. The D1 switch is a central component of an escapement mechanism essential to free energy transduction. These conclusions begin to relate the escapement mechanism to differential TrpRS conformational stabilities.

Microcalorimetry reveals multi-state thermal denaturation of G. stearothermophilus tryptophanyl-tRNA synthetase.

Mechanistic studies of Geobacillus stearothermophilus tryptophanyl-tRNA synthetase (TrpRS) afford an unusually detailed description-the escapement mechanism-for the distinct steps coupling catalysis to domain motion, efficiently converting the free energy of ATP hydrolysis into biologically useful alternative forms of information and work. Further elucidation of the escapement mechanism requires understanding thermodynamic linkages between domain configuration and conformational stability. To that end, we compare experimental thermal melting of fully liganded and apo TrpRS with a computational simulation of the melting of its fully liganded form. The simulation also provides important structural cameos at successively higher temperatures, enabling more confident interpretation. Experimental and simulated melting both proceed through a succession of three transitions at successively higher temperature. The low-temperature transition occurs at approximately the growth temperature of the organism and so may be functionally relevant but remains too subtle to characterize structurally. Structural metrics from the simulation imply that the two higher-temperature transitions entail forming a molten globular state followed by unfolding of secondary structures. Ligands that stabilize the enzyme in a pre-transition (PreTS) state compress the temperature range over which these transitions occur and sharpen the transitions to the molten globule and fully denatured states, while broadening the low-temperature transition. The experimental enthalpy changes provide a key parameter necessary to convert changes in melting temperature of combinatorial mutants into mutationally induced conformational free energy changes. The TrpRS urzyme, an excerpted model representing an early ancestral form, containing virtually the entire catalytic apparatus, remains largely intact at the highest simulated temperatures.

Domain acquisition by class I aminoacyl-tRNA synthetase urzymes coordinated the catalytic functions of HVGH and KMSKS motifs.

Leucyl-tRNA synthetase (LeuRS) is a Class I aminoacyl-tRNA synthetase (aaRS) that synthesizes leucyl-tRNAleu for codon-directed protein synthesis. Two signature sequences, HxGH and KMSKS help stabilize transition-states for amino acid activation and tRNA aminoacylation by all Class I aaRS. Separate alanine mutants of each signature, together with the double mutant, behave in opposite ways in Pyrococcus horikoshii LeuRS and the 129-residue urzyme ancestral model generated from it (LeuAC). Free energy coupling terms, Δ(ΔG‡), for both reactions are large and favourable for LeuRS, but unfavourable for LeuAC. Single turnover assays with 32Pα-ATP show correspondingly different internal products. These results implicate domain motion in catalysis by full-length LeuRS. The distributed thermodynamic cycle of mutational changes authenticates LeuAC urzyme catalysis far more convincingly than do single point mutations. Most importantly, the evolutionary gain of function induced by acquiring the anticodon-binding (ABD) and multiple insertion modules in the catalytic domain appears to be to coordinate the catalytic function of the HxGH and KMSKS signature sequences. The implication that backbone elements of secondary structures achieve a major portion of the overall transition-state stabilization by LeuAC is also consistent with coevolution of the genetic code and metabolic pathways necessary to produce histidine and lysine sidechains.

Bladder tumor Resection Weight as a Prognostic Factor for Recurrence and Progression in Patients With High-Risk Non-Muscle Invasive Bladder Treated With BCG.

INTRODUCTION: Predicting outcomes of patients with high-risk non-muscle invasive bladder cancer (HR-NMIBC) is critical. Here, we evaluate whether bladder tumor resection weight might serve as a prognostic factor for recurrence and progression of HR-NMIBC patients treated with Bacillus Calmette-Guérin (BCG). METHODS: In this retrospective, single-centre study in the UK, we analysed a consecutive cohort of HR-NMIBC patients who have received adequate intravesical BCG immunotherapy between 2009 and 2019. Univariable and multivariable Cox proportional hazards models were used to assess the association of resection weight and established predictors with recurrence and progression. RESULTS: A total of 187 HR-NMIBC patients were analysed. The median resection weight was 1.4g (range: 0.2-28.5g). Within a median follow-up of 41 months, 58 (31%) tumors recurred and 19 (10%) progressed. Fifty-four patients (29%) died from any cause and 16 (9%) died from bladder cancer. Both the risk of recurrence (P = .007) and progression (P = .019) increased with rising resection weight. On the multivariable analysis, a resection weight of ≥ 2g and ≥ 3g conferred a 4.35-fold and a 9.03-fold increased risk of bladder cancer recurrence (P < .001) and progression (P < .001), respectively. The addition of resection weight improved the C index of multivariable standard prognostic models to a clinically significant extent (+ 3.8% for recurrence, + 4.3% for progression). CONCLUSION: In our HR-NMIBC patient cohort treated with BCG, bladder tumor resection weight was associated with disease recurrence and progression. Its addition improves discrimination of standard prognostic factors. Measurement may therefore be considered for routine clinical practice.

A Leucyl-tRNA Synthetase Urzyme: Authenticity of tRNA Synthetase Catalytic Activities and Promiscuous Phosphorylation of Leucyl-5'AMP.

Aminoacyl-tRNA synthetase (aaRS)/tRNA cognate pairs translate the genetic code by synthesizing specific aminoacyl-tRNAs that are assembled on messenger RNA by the ribosome. Deconstruction of the two distinct aaRS superfamilies (Classes) has provided conceptual and experimental models for their early evolution. Urzymes, containing ~120-130 amino acids excerpted from regions where genetic coding sequence complementarities have been identified, are key experimental models motivated by the proposal of a single bidirectional ancestral gene. Previous reports that Class I and Class II urzymes accelerate both amino acid activation and tRNA aminoacylation have not been extended to other synthetases. We describe a third urzyme (LeuAC) prepared from the Class IA Pyrococcus horikoshii leucyl-tRNA synthetase. We adduce multiple lines of evidence for the authenticity of its catalysis of both canonical reactions, amino acid activation and tRNALeu aminoacylation. Mutation of the three active-site lysine residues to alanine causes significant, but modest reduction in both amino acid activation and aminoacylation. LeuAC also catalyzes production of ADP, a non-canonical enzymatic function that has been overlooked since it first was described for several full-length aaRS in the 1970s. Structural data suggest that the LeuAC active site accommodates two ATP conformations that are prominent in water but rarely seen bound to proteins, accounting for successive, in situ phosphorylation of the bound leucyl-5'AMP phosphate, accounting for ADP production. This unusual ATP consumption regenerates the transition state for amino acid activation and suggests, in turn, that in the absence of the editing and anticodon-binding domains, LeuAC releases leu-5'AMP unusually slowly, relative to the two phosphorylation reactions.

Neonatal Abstinence Syndrome in North Carolina: Incidence and Characteristics among Infants and Mothers.

BACKGROUND Neonatal abstinence syndrome (NAS) is a complex disorder characterized by withdrawal symptoms secondary to in utero exposure to drugs capable of producing physical dependence. The objective of this study was to determine the incidence of NAS, as well as infant and maternal characteristics associated with NAS in North Carolina (NC).METHODS This retrospective, cross-sectional, observational study used the State Inpatient Database (SID) to compare the incidence rates of NAS for NC for the year 2016 to historical data (years 2000 to 2013). A multivariable logistic regression model including available covariates of interest was constructed.RESULTS Overall NAS incidence rate (IR) for NC was found to be 9.7 per 1,000 live births, a 32.3-fold increase since 2000 (IR=0.3 in 2000). The multivariable logistic regression model suggested race group (both black [OR 0.11; 95% CI: 0.08, 0.16] and 'other' [OR 0.43; 95% CI: 0.31, 0.61] vs white), ethnicity [OR 0.43; 95% CI: 0.31, 0.61], insurance group (both 'other/self-pay' [OR 0.35; 95% CI: 0.24, 0.52] and 'private insurance' [OR 0.07; 95% CI: 0.05, 0.10] vs Medicaid/Medicare), region (Piedmont [OR 0.62; 95% CI: 0.50, 0.79] vs Mountain), income quartile (both 4th [OR 0.45; 95% CI: 0.26, 0.79] and 3rd [OR 0.70; 95% CI: 0.50, 0.96] vs 1st), county population size (50k-249k [OR 0.54; 95% CI: 0.34, 0.86] vs ≥1 million), birth weight [OR 0.87; 95% CI: 0.78, 0.98], and length of stay [OR 1.23; 95% CI: 1.20, 1.26] as potentially important predictors of NAS.LIMITATIONS Only hospitals providing data to the SID for 2016 were included and ICD-9 codes, in use at the time of data collection, were used.CONCLUSIONS The incidence of NAS has increased in NC in 2016 compared to prior years spanning back to 2000. Specific infant and maternal characteristics including race, ethnicity, payer type, geographic region, county population, parental income status, birth weight, and length appear to be associated with an infant bearing the diagnosis of NAS.

Incorporation of near-real-time hospital occupancy data to improve hospitalization forecast accuracy during the COVID-19 pandemic.

Public health decision makers rely on hospitalization forecasts to inform COVID-19 pandemic planning and resource allocation. Hospitalization forecasts are most relevant when they are accurate, made available quickly, and updated frequently. We rapidly adapted an agent-based model (ABM) to provide weekly 30-day hospitalization forecasts (i.e., demand for intensive care unit [ICU] beds and non-ICU beds) by state and region in North Carolina for public health decision makers. The ABM was based on a synthetic population of North Carolina residents and included movement of agents (i.e., patients) among North Carolina hospitals, nursing homes, and the community. We assigned SARS-CoV-2 infection to agents using county-level compartmental models and determined agents' COVID-19 severity and probability of hospitalization using synthetic population characteristics (e.g., age, comorbidities). We generated weekly 30-day hospitalization forecasts during May-December 2020 and evaluated the impact of major model updates on statewide forecast accuracy under a SARS-CoV-2 effective reproduction number range of 1.0-1.2. Of the 21 forecasts included in the assessment, the average mean absolute percentage error (MAPE) was 7.8% for non-ICU beds and 23.6% for ICU beds. Among the major model updates, integration of near-real-time hospital occupancy data into the model had the largest impact on improving forecast accuracy, reducing the average MAPE for non-ICU beds from 6.6% to 3.9% and for ICU beds from 33.4% to 6.5%. Our results suggest that future pandemic hospitalization forecasting efforts should prioritize early inclusion of hospital occupancy data to maximize accuracy.

An Update on the Status of Vaccine Development for SARS-CoV-2 Including Variants. Practical Considerations for COVID-19 Special Populations.

The progress in the development of various vaccine platforms against SARS-CoV-2 have been rather remarkable owing to advancement in molecular and biologic sciences. Most of the current vaccines and those in development focus on targeting the viral spike proteins by generating antibodies of varying spectrum. These vaccines represent a variety of platforms including whole virus vaccines, viral vector vaccines, nucleic acid vaccines representing RNA, DNA, and their hybrid forms.The therapeutic efficacy of these vaccines varies owing to their pharmacodynamic individualities. COVID-19 variants are capable of inducing different pathologic responses and some of which may be resistant to antibodies generated by current vaccines. The current clinical use of these vaccines has been through emergency use authorization until recently. Moreover, the efficacy and safety of these vaccines have been tested in substantial numbers of individuals but studies in special populations that better reflect the global population are pending results. These specialized populations include young children, immunocompromised patients, pregnant individuals, and other specialized groups. Combination approaches, molecularly modified vaccination approaches, and vaccines conferring longer periods of immunity are being currently being investigated, as well as pharmacovigilance studies.The continual transformation of SARS-CoV-2 and its variants are of concern along with the breakthrough infections. These considerations pose new challenges for the development of vaccination platforms. For this purpose, booster doses, combination vaccine approaches, and other modalities are being discussed. This review provides an updated account of currently available vaccines and those in advanced development with reference to their composition and mechanisms of action.A discussion on the use of vaccines in special populations including immunocompromised patients, pregnant women and other specialized populations are also included.

Multidimensional Phylogenetic Metrics Identify Class I Aminoacyl-tRNA Synthetase Evolutionary Mosaicity and Inter-Modular Coupling.

The role of aminoacyl-tRNA synthetases (aaRS) in the emergence and evolution of genetic coding poses challenging questions concerning their provenance. We seek evidence about their ancestry from curated structure-based multiple sequence alignments of a structurally invariant "scaffold" shared by all 10 canonical Class I aaRS. Three uncorrelated phylogenetic metrics-mutation frequency, its uniformity, and row-by-row cladistic congruence-imply that the Class I scaffold is a mosaic assembled from successive genetic sources. Metrics for different modules vary in accordance with their presumed functionality. Sequences derived from the ATP- and amino acid- binding sites exhibit specific two-way coupling to those derived from Connecting Peptide 1, a third module whose metrics suggest later acquisition. The data help validate: (i) experimental fragmentations of the canonical Class I structure into three partitions that retain catalytic activities in proportion to their length; and (ii) evidence that the ancestral Class I aaRS gene also encoded a Class II ancestor in frame on the opposite strand. A 46-residue Class I "protozyme" roots the Class I tree prior to the adaptive radiation of the Rossmann dinucleotide binding fold that refined substrate discrimination. Such rooting implies near simultaneous emergence of genetic coding and the origin of the proteome, resolving a conundrum posed by previous inferences that Class I aaRS evolved after the genetic code had been implemented in an RNA world. Further, pinpointing discontinuous enhancements of aaRS fidelity establishes a timeline for the growth of coding from a binary amino acid alphabet.

Public Perceptions of Current COVID-19 Vaccinations. Results of a Pilot Survey.

INTRODUCTION: We conducted a cross-sectional survey as a part of an educational program in collaboration with the Global Thrombosis Forum (GTF), an affiliate of North American Thrombosis Forum (NATF), and Loyola University about public perceptions of COVID-19 and COVID-19 vaccinations in the US. In this study, we are reporting the results of this survey. MATERIALS AND METHODS: The survey, in the form of a questionnaire, has been developed by GTF and faculty members. A prepared questionnaire was sent to the members of the Georgia and Illinois communities. RESULTS: In our current study, the COVID-19 vaccine willingness rate was 94.5% and vaccination rate was 90.9%. In multivariate analysis believing to have enough information about the safety and efficacy of COVID-19 vaccines (OR: 3.730, 95% CI: 1.199-11.603, p: 0.023) and gender (OR: 0.123, 95% CI: 0.016-0.967, p: 0.046) were significant predictors for vaccine willingness. Previous COVID-19 infection (OR: 0.215, 95% CI: 0.061-0.758, p: 0.017), moderate and severe effects of COVID-19 pandemic on participant's life (OR: 4.631, 95% CI 1.681-12.760, p: 0.003) and believing to have enough information about the safety and efficacy of COVID-19 vaccines (OR: 4.119, 95% CI: 1.508-11.253, p: 0.006) were significant predictors for final vaccination status. CONCLUSION: In conclusion, currently vaccination remains one of the most effective tools in the fight against the COVID-19 pandemic. The vaccine hesitancy is a complex phenomenon that is driven by individuals' perceptions of safety, and efficiency of the vaccines. We must continue to educate the public and communities that vaccines are safe, that they are effective and that they are still required even after a COVID-19 infection.

The COVID-19 Pandemic and the Need for an Integrated and Equitable Approach: An International Expert Consensus Paper.

BACKGROUND: One year after the declaration of the coronavirus disease 2019 (COVID-19) pandemic by the World Health Organization (WHO) and despite the implementation of mandatory physical barriers and social distancing, humanity remains challenged by a long-lasting and devastating public health crisis. MANAGEMENT: Non-pharmacological interventions (NPIs) are efficient mitigation strategies. The success of these NPIs is dependent on the approval and commitment of the population. The launch of a mass vaccination program in many countries in late December 2020 with mRNA vaccines, adenovirus-based vaccines, and inactivated virus vaccines has generated hope for the end of the pandemic. CURRENT ISSUES: The continuous appearance of new pathogenic viral strains and the ability of vaccines to prevent infection and transmission raise important concerns as we try to achieve community immunity against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and its variants. The need of a second and even third generation of vaccines has already been acknowledged by the WHO and governments. PERSPECTIVES: There is a critical and urgent need for a balanced and integrated strategy for the management of the COVID-19 outbreaks organized on three axes: (1) Prevention of the SARS-CoV-2 infection, (2) Detection and early diagnosis of patients at risk of disease worsening, and (3) Anticipation of medical care (PDA). CONCLUSION: The "PDA strategy" integrated into state policy for the support and expansion of health systems and introduction of digital organizations (i.e., telemedicine, e-Health, artificial intelligence, and machine-learning technology) is of major importance for the preservation of citizens' health and life world-wide.

An Update on the Pathogenesis of COVID-19 and the Reportedly Rare Thrombotic Events Following Vaccination.

Today the coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has become a global health problem. After more than a year with the pandemic, although our knowledge has progressed on COVID-19, there are still many unknowns in virological, pathophysiological and immunological aspects. It is obvious that the most efficient solution to end this pandemic are safe and efficient vaccines. This manuscript summarizes the pathophysiological and thrombotic features of COVID-19 and the safety and efficacy of currently approved COVID-19 vaccines with an aim to clarify the recent concerns of thromboembolic events after COVID-19 vaccination. The influx of newer information is rapid, requiring periodic updates and objective assessment of the data on the pathogenesis of COVID-19 variants and the safety and efficacy of currently available vaccines.

The Practice Is the Curriculum.

The training family medicine residents receive will have a lasting impact on how they deliver care in the future. Evidence demonstrates an imprinting effect based on the training environment itself. Thus, residency training represents a critical time for establishing clinical experiences that embody core primary care principles and ensure excellent care delivery. This paper focuses on the clinical experience in the family medicine practice setting. We have used Starfield's four C's of primary care and added two more: cost and community, as the tools to achieve the triple aim. In reviewing the current state of residency programs across the country, we noted that there was a lack of measurement on how programs were performing when it came to the six C's. We will briefly describe some recent innovative collaboratives among residencies. Next, we examine the six C's of primary care in context of current care. These six C's inform our recommendations for residency training standards to create the family physicians of the future. The overarching theme of these recommendations is the need to measure and report on what we want to ultimately improve.

The Roots of Genetic Coding in Aminoacyl-tRNA Synthetase Duality.

Codon-dependent translation underlies genetics and phylogenetic inferences, but its origins pose two challenges. Prevailing narratives cannot account for the fact that aminoacyl-tRNA synthetases (aaRSs), which translate the genetic code, must collectively enforce the rules used to assemble themselves. Nor can they explain how specific assignments arose from rudimentary differentiation between ancestral aaRSs and corresponding transfer RNAs (tRNAs). Experimental deconstruction of the two aaRS superfamilies created new experimental tools with which to analyze the emergence of the code. Amino acid and tRNA substrate recognition are linked to phase transfer free energies of amino acids and arise largely from aaRS class-specific differences in secondary structure. Sensitivity to protein folding rules endowed ancestral aaRS-tRNA pairs with the feedback necessary to rapidly compare alternative genetic codes and coding sequences. These and other experimental data suggest that the aaRS bidirectional genetic ancestry stabilized the differentiation and interdependence required to initiate and elaborate the genetic coding table.