Genetic basis of sorghum leaf width and its potential as a surrogate for transpiration efficiency.

KEY MESSAGE: Leaf width was correlated with plant-level transpiration efficiency and associated with 19 QTL in sorghum, suggesting it could be a surrogate for transpiration efficiency in large breeding program. Enhancing plant transpiration efficiency (TE) by reducing transpiration without compromising photosynthesis and yield is a desirable selection target in crop improvement programs. While narrow individual leaf width has been correlated with greater intrinsic water use efficiency in C4 species, the extent to which this translates to greater plant TE has not been investigated. The aims of this study were to evaluate the correlation of leaf width with TE at the whole-plant scale and investigate the genetic control of leaf width in sorghum. Two lysimetry experiments using 16 genotypes varying for stomatal conductance and three field trials using a large sorghum diversity panel (n = 701 lines) were conducted. Negative associations of leaf width with plant TE were found in the lysimetry experiments, suggesting narrow leaves may result in reduced plant transpiration without trade-offs in biomass accumulation. A wide range in width of the largest leaf was found in the sorghum diversity panel with consistent ranking among sorghum races, suggesting that environmental adaptation may have a role in modifying leaf width. Nineteen QTL were identified by genome-wide association studies on leaf width adjusted for flowering time. The QTL identified showed high levels of correspondence with those in maize and rice, suggesting similarities in the genetic control of leaf width across cereals. Three a priori candidate genes for leaf width, previously found to regulate dorsoventrality, were identified based on a 1-cM threshold. This study provides useful physiological and genetic insights for potential manipulation of leaf width to improve plant adaptation to diverse environments.

Genetically encoding phosphotyrosine and its nonhydrolyzable analog in bacteria.

Tyrosine phosphorylation is a common protein post-translational modification that plays a critical role in signal transduction and the regulation of many cellular processes. Using a propeptide strategy to increase cellular uptake of O-phosphotyrosine (pTyr) and its nonhydrolyzable analog 4-phosphomethyl-L-phenylalanine (Pmp), we identified an orthogonal aminoacyl-tRNA synthetase-tRNA pair that allows site-specific incorporation of both pTyr and Pmp into recombinant proteins in response to the amber stop codon in Escherichia coli in good yields. The X-ray structure of the synthetase reveals a reconfigured substrate-binding site, formed by nonconservative mutations and substantial local structural perturbations. We demonstrate the utility of this method by introducing Pmp into a putative phosphorylation site and determining the affinities of the individual variants for the substrate 3BP2. In summary, this work provides a useful recombinant tool to dissect the biological functions of tyrosine phosphorylation at specific sites in the proteome.

Enhancing protein stability with extended disulfide bonds.

Disulfide bonds play an important role in protein folding and stability. However, the cross-linking of sites within proteins by cysteine disulfides has significant distance and dihedral angle constraints. Here we report the genetic encoding of noncanonical amino acids containing long side-chain thiols that are readily incorporated into both bacterial and mammalian proteins in good yields and with excellent fidelity. These amino acids can pair with cysteines to afford extended disulfide bonds and allow cross-linking of more distant sites and distinct domains of proteins. To demonstrate this notion, we preformed growth-based selection experiments at nonpermissive temperatures using a library of random ß-lactamase mutants containing these noncanonical amino acids. A mutant enzyme that is cross-linked by one such extended disulfide bond and is stabilized by ∼9 °C was identified. This result indicates that an expanded set of building blocks beyond the canonical 20 amino acids can lead to proteins with improved properties by unique mechanisms, distinct from those possible through conventional mutagenesis schemes.

Recombinant thiopeptides containing noncanonical amino acids.

Thiopeptides are a subclass of ribosomally synthesized and posttranslationally modified peptides (RiPPs) with complex molecular architectures and an array of biological activities, including potent antimicrobial activity. Here we report the generation of thiopeptides containing noncanonical amino acids (ncAAs) by introducing orthogonal amber suppressor aminoacyl-tRNA synthetase/tRNA pairs into a thiocillin producer strain of Bacillus cereus .We demonstrate that thiopeptide variants containing ncAAs with bioorthogonal chemical reactivity can be further postbiosynthetically modified with biophysical probes, including fluorophores and photo-cross-linkers. This work allows the site-specific incorporation of ncAAs into thiopeptides to increase their structural diversity and probe their biological activity; similar approaches can likely be applied to other classes of RiPPs.

Bacterial Genome Containing Chimeric DNA-RNA Sequences.

Almost five decades ago Crick, Orgel, and others proposed the RNA world hypothesis. Subsequent studies have raised the possibility that RNA might be able to support both genotype and phenotype, and the function of RNA templates has been studied in terms of evolution, replication, and catalysis. Recently, we engineered strains of E. coli in which a large fraction of 2'-deoxycytidine in the genome is substituted with the modified base 5-hydroxymethyl-2'-deoxycytidine. We now report the generation of mutant strains derived from these engineered bacteria that show significant (∼40-50%) ribonucleotide content in their genome. We have begun to characterize the properties of these chimeric genomes and the corresponding strains to determine the circumstances under which E. coli can incorporate ribonucleotides into its genome and herein report our initial observations.

Progress toward a reduced phage genetic code.

All known living organisms use at least 20 amino acids as the basic building blocks of life. Efforts to reduce the number of building blocks in a replicating system to below the 20 canonical amino acids have not been successful to date. In this work, we use filamentous phage as a model system to investigate the feasibility of removing methionine (Met) from the proteome. We show that all 24 elongation Met sites in the M13 phage genome can be replaced by other canonical amino acids. Most of these changes involve substitution of methionine by leucine (Leu), but in some cases additional compensatory mutations are required. Combining Met substituted sites in the proteome generally led to lower viability/infectivity of the mutant phages, which remains the major challenge in eliminating all methionines from the phage proteome. To date a total of 15 (out of all 24) elongation Mets have been simultaneously deleted from the M13 proteome, providing a useful foundation for future efforts to minimize the genetic code.

Exploring the potential impact of an expanded genetic code on protein function.

With few exceptions, all living organisms encode the same 20 canonical amino acids; however, it remains an open question whether organisms with additional amino acids beyond the common 20 might have an evolutionary advantage. Here, we begin to test that notion by making a large library of mutant enzymes in which 10 structurally distinct noncanonical amino acids were substituted at single sites randomly throughout TEM-1 ß-lactamase. A screen for growth on the ß-lactam antibiotic cephalexin afforded a unique p-acrylamido-phenylalanine (AcrF) mutation at Val-216 that leads to an increase in catalytic efficiency by increasing kcat, but not significantly affecting KM. To understand the structural basis for this enhanced activity, we solved the X-ray crystal structures of the ligand-free mutant enzyme and of the deacylation-defective wild-type and mutant cephalexin acyl-enzyme intermediates. These structures show that the Val-216-AcrF mutation leads to conformational changes in key active site residues-both in the free enzyme and upon formation of the acyl-enzyme intermediate-that lower the free energy of activation of the substrate transacylation reaction. The functional changes induced by this mutation could not be reproduced by substitution of any of the 20 canonical amino acids for Val-216, indicating that an expanded genetic code may offer novel solutions to proteins as they evolve new activities.

Replacement of Thymidine by a Modified Base in the Escherichia coli Genome.

Prokaryotic and eukaryotic genomic DNA is comprised of the four building blocks A, G, C, and T. We have begun to explore the consequences of replacing a large fraction or all of a nucleoside in genomic DNA with a modified nucleoside. As a first step we have investigated the possibility of replacement of T by 2'-deoxy-5-(hydroxymethyl)uridine (5hmU) in the genomic DNA of Escherichia coli. Metabolic engineering with phage genes followed by random mutagenesis enabled us to achieve approximately 75% replacement of T by 5hmU in the E. coli genome and in plasmids.

Replacement of 2'-Deoxycytidine by 2'-Deoxycytidine Analogues in the E. coli Genome.

Several modified bases have been observed in the genomic DNA of bacteriophages, prokaryotes, and eukaryotes that play a role in restriction systems and/or epigenetic regulation. In our efforts to understand the consequences of replacing a large fraction of a canonical nucleoside with a modified nucleoside, we previously replaced around 75% of thymidine (T) with 5'-hydroxymethyl-2'-deoxyuridine (5hmU) in the Escherichia coli genome. In this study, we engineered the pyrimidine nucleotide biosynthetic pathway using T4 bacteriophage genes to achieve approximately 63% replacement of 2'-deoxycytidine (dC) with 5-hydroxymethyl-2'-deoxycytidine (5hmC) in the E. coli genome and approximately 71% replacement in plasmids. We further engineered the glucose metabolic pathway to transform the 5hmC into glucosyl-5-hydroxymethyl-2'-deoxycytidine (5-gmC) and achieved 20% 5-gmC in the genome and 45% 5-gmC in plasmid DNA.

Attenuation of High-Frequency (50-200 Hz) Thalamocortical Electroencephalographic Rhythms by Isoflurane in Rats Is More Pronounced for the Thalamus Than for the Cortex.

BACKGROUND: Thalamocortical electroencephalographic rhythms in gamma (30-80 Hz) and high-gamma (80-200 Hz) ranges have been linked to arousal and conscious processes. We have recently shown that propofol causes a concentration-dependent attenuation of the power of thalamocortical rhythms in the 50 to 200 Hz range and that this effect is far more pronounced for the thalamus. To determine whether similar attenuation occurs with other anesthetics, we characterized the concentration-effect relationship of the inhaled anesthetic isoflurane on the spectral power of these rhythms. METHODS: Local field potentials were recorded from the barrel cortex and ventroposteromedial thalamic nucleus in 9 chronically instrumented rats to measure spectral power in the gamma/high-gamma range (30-200 Hz). Rats were placed in an airtight chamber and isoflurane was administered at 0.75%, 1.1%, and 1.5% concentrations. Spectral power was assessed during baseline, at the 3 isoflurane concentrations after 30 minutes for equilibration, and during recovery over 4 frequency bands (30-50, 51-75, 76-125, and 126-200 Hz). Unconsciousness was defined as sustained loss of righting reflex. Multiple linear regression was used to model the change in power (after logarithmic transformation) as a function of concentration and recording site. P values were corrected for multiple comparisons. RESULTS: Unconsciousness occurred at the 1.1% concentration in all animals. Isoflurane caused a robust (P ≤ 0.008) linear concentration-dependent attenuation of cortical and thalamic power in the 30 to 200 Hz range. The concentration-effect slope for the thalamus was steeper than for the cortex in the 51 to 75 Hz (P = 0.029) and 76 to 200 Hz (P < 0.001) ranges but not for the 30 to 50 Hz range (P = 0.320). Comparison with our previously published propofol data showed that slope for cortical power was steeper with isoflurane than with propofol for all frequency bands (P = 0.033). For thalamic power, the slope differences between isoflurane and propofol were not statistically significant (0.087 ≤ P ≤ 0.599). CONCLUSIONS: Isoflurane causes a concentration-dependent attenuation of the power of thalamocortical rhythms in the 30 to 200 Hz range, and this effect is more pronounced for the thalamus than for the cortex for frequencies >50 Hz. In comparison with propofol, isoflurane caused a greater attenuation in the cortex, but the effects on the thalamus were similar. Isoflurane and propofol cause common alterations of fast thalamocortical rhythms that may constitute an electrophysiologic signature of the anesthetized state.

The sensory thalamus and cerebral motor cortex are affected concurrently during induction of anesthesia with propofol: a case series with intracranial electroencephalogram recordings.

PURPOSE: Brain imaging studies suggest that loss of consciousness induced by general anesthetics is associated with impairment of thalamic function. There is, however, limited information on the time course of these changes. We recently obtained intracranial electroencephalogram (EEG) recordings from the ventroposterolateral (VPL) nucleus of the thalamus and from the motor cortex during induction of anesthesia in three patients to study the time course of the alterations of cortical and thalamic function. CLINICAL FEATURES: The patients were American Society of Anesthesiologists physical status I-II males aged 33-57 yr with intractable central pain caused by brachial plexus injury (patient 1 and 2) or insular infarct (patient 3). Anesthesia was induced with propofol (2.5-3.1 mg·kg(-1) over 30-45 sec) followed, after loss of consciousness, by rocuronium for tracheal intubation. The data retained for analysis are from one minute before the start of propofol to 110 sec later during ventilation of the patients' lungs before tracheal intubation. Spectral analysis was used to measure absolute EEG power. Propofol caused significant increases of cortical and thalamic power in the delta to beta frequency bands (1-30 Hz). These increases of cortical and thalamic power occurred either concomitantly or within seconds of each other. Propofol also caused a decrease in cortical and thalamic high-gamma (62-200 Hz) power that also followed a similar time course. CONCLUSION: We conclude that induction of anesthesia with propofol in these patients was associated with concurrent alterations of cortical and sensory thalamic activity.

A Unitary Theory of Healing Through Touch.

Touch has been linked empirically to healing outcomes; therefore, developing theories about the dynamics between touch and healing is important for nursing. A practice-based theory is described within a Unitary Science perspective emerging from the findings of a qualitative descriptive study of the experiences of persons with advanced cancer receiving touch (massage and simple touch) during hospice care. Seventeen participants were interviewed. Through content analysis and retroductive constructivist theory development, healing through touch was described as a dynamic process cocreated by healer and healee, characterized by the simultaneous activities of sensing , reflecting , and connecting . Interpretation of findings from a unitary lens led to an overarching theme of touch as sanctuary and explication of theoretical alignment with the concepts of wholeness, awareness, and presence.

Mishel's uncertainty in illness theory: Informing nursing diagnoses and care planning.

PURPOSE: Patients with glioblastoma multiforme (GBM) face uncertainty during the illness trajectory that decreases their quality of life. Uncertainty manifests as an altered state of mind in patients with GBM and increases the burden of managing their diagnosis. The uncertainty in illness theory (UIT) can be used to guide nursing practice and create care plans to decrease this uncertainty. DATA SOURCES: The UIT and conceptual model were used to pair diagnoses from NANDA-I Nursing Diagnoses to form care plans for patients with GBM. METHODS: In a stepwise approach, concepts from the UIT were organized and cross-walked into an integrated theory-driven practice framework to enhance care planning. CONCLUSIONS: The UIT helps define and identify circumstances of uncertainty in patients experiencing illness, and Nursing Diagnoses provide engagement and outcomes for those uncertainty-based diagnoses. Some examples of the UIT guiding Nursing Diagnoses includes a patient's cognitive level guiding a Nursing Diagnosis care plan of Risk for Acute Confusion. Social Support can guide the care plan of Risk for Loneliness, and appraisal of uncertainty can guide a care plan for Ineffective Individual Coping. Using the UIT to guide theory-driven practice links interventions and outcomes in patients with GBM. IMPLICATIONS FOR PRACTICE: The UIT can guide NANDA-I Nursing Diagnoses to assist nurses in creating a nursing care plan for nursing engagement to decrease uncertainty and increase the quality of life in patients with GBM. Theory-driven research defines the application of the UIT in GBM and provides foundational knowledge for theory-driven practice to occur.

Theory Analysis: The Health Promotion Model and Motivation in Physical Activity.

Background: The health promotion model (HPM) depicts numerous factors impacting health-promoting behavior. Variables of the HPM including experiences, self-efficacy, and the influences affecting health choices portray a comprehensive picture of a person's values and barriers to health behavior change. Perceived harm of inaction is weighed against the perceived benefits of action within the HPM. Physical inactivity is a worldwide concern with deleterious consequences. Strategies are needed to increase physical activity participation to mitigate the impacts. The HPM has not been previously analyzed in the context of adult physical activity. Purpose: Analyze the HPM, apply it to the concept of adult motivation for physical activity, display the utility of theory application in practice, and demonstrate the vital role of nursing in connecting theory to practice. Methods: Theory analysis was conducted using Walker and Avant's method with a focus on motivation for physical activity among adults. Evaluation of the origins, meaning, logical adequacy, usefulness, generalizability, parsimony, and testability of the HPM adds depth to understanding the theory and its application in clinical practice. Results: The HPM is logical, generalizable, and has been broadly applied and tested. Adaptations were made to the HPM to reflect updated knowledge and for application to adult motivation for physical activity. Implications for practice and future research: Thorough evaluation of the HPM facilitates application to clinical practice to impact physical activity health behavior change. Examining motivation for physical activity through the lens of the HPM can guide nursing engagement and interventions to enhance behavior changes.

Nursing, Social Justice, and Health Inequities: A Critical Analysis of the Theory of Emancipatory Nursing Praxis.

Walter's theory of emancipatory nursing praxis (ENP) was created to explore nurses' social justice-related transformational learning process. We analyzed the theory within the context of stigma-related health inequities faced by transgender and gender-diverse (TGD) people. The theory of ENP provides a theoretical framework to incorporate social justice in nursing practice, research, education, and policy. We also determined the theory bridges the gap in understanding the role of nurses in advancing social justice through embodied actions and social activism. The theory of ENP underscores the moral, ethical, and professional obligations for nurses to abolish health inequities for TGD people.

Themes of Liminality: Stories From Cancer Family Caregiver Narratives.

Cancer family caregivers dwell in an evolving landscape of ambiguity and in-betweenness, as "betwixt and between," in a space of unknowing. This space of unknowing exists within a transformational threshold of liminality, between what was known prior to the cancer and what will be known and embodied in the future. Theoretically grounded in Unitary Caring Science, this study used narrative inquiry alongside photo elicitation to cocreate cancer caregiver narratives and identify themes of liminality. Four thematic threads of liminality were identified as woven within and across the cancer caregiver narratives; stories of paradox, time warp, life disruption, and waiting in silence.

Patient and Provider Perceptions of COVID-19-Driven Telehealth Use From Nurse-Led Care Models in Rural, Frontier, and Urban Colorado Communities.

The purpose of this study was to characterize the patient and provider engagement in the sudden telehealth implementation that occurred with the onset of the COVID-19 pandemic. Patients and providers from 3 nurse-led models of care (federally qualified health centers, nurse midwifery practices, and the Nurse-Family partnership program) in Colorado were surveyed. Data from the Patient Attitude toward Telehealth survey and Provider Perceptions about Telehealth were collected. Patient respondents (n = 308) who resided primarily in rural or frontier communities were female, white, and Hispanic. Patients in urban areas used telehealth more frequently than in rural or frontier areas (P < .001). Rural/Frontier patients had significantly lower attitude scores than urban patients across each of 5 domains assessed. Telehealth modality differed across location (P < .023), with video calls, used more frequently by urban providers, and phone calls used by rural/frontier providers. Our data highlight differences in telehealth access and attitudes across rurality. These findings may contribute to future policy while addressing barriers to telehealth access and delivery.

Sudden Shift to Telehealth in COVID-19: A Retrospective Cohort Study of Disparities in Use of Telehealth for Prenatal Care in a Large Midwifery Service.

INTRODUCTION: The coronavirus disease 2019 (COVID-19) pandemic created disruption in health care delivery, including a sudden transition to telehealth use in mid-March 2020. The purpose of this study was to examine changes in the mode of prenatal care visits and predictors of telehealth use (provider-patient messaging, telephone visits, and video visits) during the COVID-19 pandemic among those receiving care in a large, academic nurse-midwifery service. METHODS: We conducted a retrospective cohort study of those enrolled for prenatal care in 2 nurse-midwifery clinics between 2019 and 2021 (n = 3172). Use outcomes included number and type of encounter: in-person and telehealth (primary outcome). Comparisons were made in frequency and types of encounters before and during COVID-19. A negative binomial regression was fit on the outcome of telehealth encounter count, with race/ethnicity, age, language, parity, hypertension, diabetes, and depression as predictors. RESULTS: When comparing pre-COVID-19 (before March 2020) with during COVID-19 (after March 2020), overall encounters increased from 15.9 to 19.5 mean number of encounters per person (P < .001). The increase was driven by telehealth encounters; there were no significant differences for in-person prenatal visit counts before and during the pandemic period. Direct patient-provider messaging was the most common type of telehealth encounter. Predictors of telehealth encounters included English as primary language and diagnoses of diabetes or depression. DISCUSSION: No differences in the frequency of in-person prenatal care visits suggests that telehealth encounters led to more contact with midwives and did not replace in-person encounters. Spanish-speaking patients were least likely to use telehealth-delivered prenatal care during the pandemic; a small, but significant, proportion of patients had no or few telehealth encounters, and a significant proportion had high use of telehealth. Integration of telehealth in future delivery of prenatal care should consider questions of equity, patient and provider satisfaction, access, redundancies, and provider workload.

Directed metal (oxo) aliphatic C-H hydroxylations: overriding substrate bias.

The first general strategy for a directing effect on metal (oxo)-promoted C-H hydroxylations is described. Carboxylic acid moieties on the substrate overcome unfavorable electronic, steric, and stereoelectronic biases in C-H hydroxylations catalyzed by the non-heme iron complex Fe(PDP). In a demonstration of the power of this directing effect, C-H oxidation is diverted away from an electronically favored C-1 H abstraction/rearrangement pathway in the paclitaxel framework to enable installation of C-2 oxidation in the naturally occurring oxidation state and stereoconfiguration.