A systematic review and narrative synthesis of health literacy interventions among Spanish speaking populations in the United States.

BACKGROUND: While many populations struggle with health literacy, those who speak Spanish preferentially or exclusively, including Hispanic, immigrant, or migrant populations, may face particular barriers, as they navigate a predominantly English-language healthcare system. This population also faces greater morbidity and mortality from treatable chronic diseases, such as hypertension and diabetes. The aim of this systematic review was to describe existing health literacy interventions for patients with a Spanish-language preference and present their effectiveness. METHODS: We carried out a systematic review where Web of Science, EMBASE, and PubMed were queried using MeSH terms to identify relevant literature. Included articles described patients with a Spanish-language preference participating in interventions to improve health literacy levels in the United States. Screening and data abstraction were conducted independently and in pairs. Risk of bias assessments were conducted using validated appraisal tools. RESULTS: A total of 2823 studies were identified, of which 62 met our eligibility criteria. The studies took place in a variety of community and clinical settings and used varied tools for measuring health literacy. Of the interventions, 28 consisted of in-person education and 27 implemented multimedia education, with 89% of studies in each category finding significant results. The remaining seven studies featured multimodal interventions, all of which achieved significant results. CONCLUSION: Successful strategies included the addition of liaison roles, such as promotores (Hispanic community health workers), and the use of multimedia fotonovelas (photo comics) with linguistic and cultural adaptations. In some cases, the external validity of the results was limited. Improving low health literacy in patients with a Spanish-language preference, a population with existing barriers to high quality of care, may help them better navigate health infrastructure and make informed decisions regarding their health. REGISTRATION: PROSPERO (available at https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021257655.t ).

Standards of proficiency for registered nurses-To what end? A critical analysis of contemporary mental health nursing within the United Kingdom context.

Against the backdrop of cultural and political ideals, this article highlights both the significance of mental health nursing in meeting population needs and the regulatory barriers that may be impeding its ability to adequately do so. Specifically, we consider how ambiguous notions of 'proficiency' in nurse education-prescribed by the regulator-impact the development of future mental health nurses and their mental health nursing identity. A key tension in mental health practice is the ethical-legal challenges posed by sanctioned powers to restrict patients' freedom at the same time as the desire (and obligation) to promote patients' self-determined recovery. The genericism of the UK's Future Nurse Standards do little to prepare mental health nurses to navigate the tensions that ensue. This has consequences for nurses and patients alike, as both risk experiencing the distress and dissonance that attends giving or receiving poor care. We argue that more needs to be done to enable mental health nurses to define and articulate the nuances of the profession as part of becoming critical, thoughtful and confident practitioners. Educators can contribute to this mission by aligning curriculum, pedagogy and assessment to create meaningful opportunities for mental health nursing students to engage with the complexities of mental health nursing practice. Without this, the credibility of the profession will continue to be questioned; its future uncertain.

Identifying LasR Quorum Sensors with Improved Signal Specificity by Mapping the Sequence-Function Landscape.

Programmable intercellular signaling using components of naturally occurring quorum sensing can allow for coordinated functions to be engineered in microbial consortia. LuxR-type transcriptional regulators are widely used for this purpose and are activated by homoserine lactone (HSL) signals. However, they often suffer from imperfect molecular discrimination of structurally similar HSLs, causing misregulation within engineered consortia containing multiple HSL signals. Here, we studied one such example, the regulator LasR from Pseudomonas aeruginosa. We elucidated its sequence-function relationship for ligand specificity using targeted protein engineering and multiplexed high-throughput biosensor screening. A pooled combinatorial saturation mutagenesis library (9,486 LasR DNA sequences) was created by mutating six residues in LasR's ß5 sheet with single, double, or triple amino acid substitutions. Sort-seq assays were performed in parallel using cognate and noncognate HSLs to quantify each corresponding sensor's response to each HSL signal, which identified hundreds of highly specific variants. Sensor variants identified were individually assayed and exhibited up to 60.6-fold (p = 0.0013) improved relative activation by the cognate signal compared to the wildtype. Interestingly, we uncovered prevalent mutational epistasis and previously unidentified residues contributing to signal specificity. The resulting sensors with negligible signal crosstalk could be broadly applied to engineer bacteria consortia.

Genetic Circuits for Feedback Control of Gamma-Aminobutyric Acid Biosynthesis in Probiotic Escherichia coli Nissle 1917.

Engineered microorganisms such as the probiotic strain Escherichia coli Nissle 1917 (EcN) offer a strategy to sense and modulate the concentration of metabolites or therapeutics in the gastrointestinal tract. Here, we present an approach to regulate the production of the depression-associated metabolite gamma-aminobutyric acid (GABA) in EcN using genetic circuits that implement negative feedback. We engineered EcN to produce GABA by overexpressing glutamate decarboxylase and applied an intracellular GABA biosensor to identify growth conditions that improve GABA biosynthesis. We next employed characterized genetically encoded NOT gates to construct genetic circuits with layered feedback to control the rate of GABA biosynthesis and the concentration of GABA produced. Looking ahead, this approach may be utilized to design feedback control of microbial metabolite biosynthesis to achieve designable smart microbes that act as living therapeutics.

Synthetic Homoserine Lactone Sensors for Gram-Positive Bacillus subtilis Using LuxR-Type Regulators.

A universal biochemical signal for bacterial cell-cell communication could facilitate programming dynamic responses in diverse bacterial consortia. However, the classical quorum sensing paradigm is that Gram-negative and Gram-positive bacteria generally communicate via homoserine lactones (HSLs) or oligopeptide molecular signals, respectively, to elicit population responses. Here, we create synthetic HSL sensors for Gram-positive Bacillus subtilis 168 using allosteric LuxR-type regulators (RpaR, LuxR, RhlR, and CinR) and synthetic promoters. Promoters were combinatorially designed from different sequence elements (-35, -16, -10, and transcriptional start regions). We quantified the effects of these combinatorial promoters on sensor activity and determined how regulator expression affects its activation, achieving up to 293-fold activation. Using the statistical design of experiments, we identified significant effects of promoter regions and pairwise interactions on sensor activity, which helped to understand the sequence-function relationships for synthetic promoter design. We present the first known set of functional HSL sensors (≥20-fold dynamic range) in B. subtilis for four different HSL chemical signals: p-coumaroyl-HSL, 3-oxohexanoyl-HSL, n-butyryl-HSL, and n-(3-hydroxytetradecanoyl)-HSL. This set of synthetic HSL sensors for a Gram-positive bacterium can pave the way for designable interspecies communication within microbial consortia.

Toolbox of Characterized Genetic Parts for Staphylococcus aureus.

Staphylococcus aureus is an important clinical bacterium prevalent in human-associated microbiomes and the cause of many diseases. However, S. aureus has been intractable to synthetic biology approaches due to limited characterized genetic parts for this nonmodel Gram-positive bacterium. Moreover, genetic manipulation of S. aureus has relied on cumbersome and inefficient cloning strategies. Here, we report the first standardized genetic parts toolbox for S. aureus, which includes characterized promoters, ribosome binding sites, terminators, and plasmid replicons from a variety of bacteria for precise control of gene expression. We established a standard relative expression unit (REU) for S. aureus using a plasmid reference and characterized genetic parts in standardized REUs using S. aureus ATCC 12600. We constructed promoter and terminator part plasmids that are compatible with an efficient Type IIS DNA assembly strategy to effectively build multipart DNA constructs. A library of 24 constitutive promoters was built and characterized in S. aureus, which showed a 380-fold activity range. This promoter library was also assayed in Bacillus subtilis (122-fold activity range) to demonstrate the transferability of the constitutive promoters between these Gram-positive bacteria. By applying an iterative design-build-test-learn cycle, we demonstrated the use of our toolbox for the rational design and engineering of a tetracycline sensor in S. aureus using the PXyl-TetO aTc-inducible promoter that achieved 25.8-fold induction. This toolbox greatly expands the growing number of genetic parts for Gram-positive bacteria and will allow researchers to leverage synthetic biology approaches to study and engineer cellular processes in S. aureus.

Algorithmic Programming of Sequential Logic and Genetic Circuits for Recording Biochemical Concentration in a Probiotic Bacterium.

Through the implementation of designable genetic circuits, engineered probiotic microorganisms could be used as noninvasive diagnostic tools for the gastrointestinal tract. For these living cells to report detected biomarkers or signals after exiting the gut, the genetic circuits must be able to record these signals by using genetically encoded memory. Complex memory register circuits could enable multiplex interrogation of biomarkers and signals. A theory-based approach to create genetic circuits containing memory, known as sequential logic circuits, was previously established for a model laboratory strain of Escherichia coli, yet how circuit component performance varies for nonmodel and clinically relevant bacterial strains is poorly understood. Here, we develop a scalable computational approach to design robust sequential logic circuits in probiotic strain Escherichia coli Nissle 1917 (EcN). In this work, we used TetR-family transcriptional repressors to build genetic logic gates that can be composed into sequential logic circuits, along with a set of engineered sensors relevant for use in the gut environment. Using standard methods, 16 genetic NOT gates and nine sensors were experimentally characterized in EcN. These data were used to design and predict the performance of circuit designs. We present a set of genetic circuits encoding both combinational logic and sequential logic and show that the circuit outputs are in close agreement with our quantitative predictions from the design algorithm. Furthermore, we demonstrate an analog-like concentration recording circuit that detects and reports three input concentration ranges of a biochemical signal using sequential logic.

Genetic circuits for feedback control of gamma-aminobutyric acid biosynthesis in probiotic Escherichia coli Nissle 1917.

Engineered microorganisms such as the probiotic strain Escherichia coli Nissle 1917 (EcN) offer a strategy to sense and modulate the concentration of metabolites or therapeutics in the gastrointestinal tract. Here, we present an approach to regulate production of the depression-associated metabolite gamma-aminobutyric acid (GABA) in EcN using genetic circuits that implement negative feedback. We engineered EcN to produce GABA by overexpressing glutamate decarboxylase (GadB) from E. coli and applied an intracellular GABA biosensor to identify growth conditions that improve GABA biosynthesis. We next employed characterized genetically-encoded NOT gates to construct genetic circuits with layered feedback to control the rate of GABA biosynthesis and the concentration of GABA produced. Looking ahead, this approach may be utilized to design feedback control of microbial metabolite biosynthesis to achieve designable smart microbes that act as living therapeutics.

Surveying the Genetic Design Space for Transcription Factor-Based Metabolite Biosensors: Synthetic Gamma-Aminobutyric Acid and Propionate Biosensors in E. coli Nissle 1917.

Engineered probiotic bacteria have been proposed as a next-generation strategy for noninvasively detecting biomarkers in the gastrointestinal tract and interrogating the gut-brain axis. A major challenge impeding the implementation of this strategy has been the difficulty to engineer the necessary whole-cell biosensors. Creation of transcription factor-based biosensors in a clinically-relevant strain often requires significant tuning of the genetic parts and gene expression to achieve the dynamic range and sensitivity required. Here, we propose an approach to efficiently engineer transcription-factor based metabolite biosensors that uses a design prototyping construct to quickly assay the gene expression design space and identify an optimal genetic design. We demonstrate this approach using the probiotic bacterium Escherichia coli Nissle 1917 (EcN) and two neuroactive gut metabolites: the neurotransmitter gamma-aminobutyric acid (GABA) and the short-chain fatty acid propionate. The EcN propionate sensor, utilizing the PrpR transcriptional activator from E. coli, has a large 59-fold dynamic range and >500-fold increased sensitivity that matches biologically-relevant concentrations. Our EcN GABA biosensor uses the GabR transcriptional repressor from Bacillus subtilis and a synthetic GabR-regulated promoter created in this study. This work reports the first known synthetic microbial whole-cell biosensor for GABA, which has an observed 138-fold activation in EcN at biologically-relevant concentrations. Using this rapid design prototyping approach, we engineer highly functional biosensors for specified in vivo metabolite concentrations that achieve a large dynamic range and high output promoter activity upon activation. This strategy may be broadly useful for accelerating the engineering of metabolite biosensors for living diagnostics and therapeutics.

The importance of pharmacokinetics and pharmacodynamics in antimicrobial drug development and their influence on the success of agents developed to combat resistant gram negative pathogens: A review.

A deep understanding of an antimicrobial's critical pharmacokinetic and pharmacodynamic properties is crucial towards optimizing its use in patients and bolstering the drug development program. With the growing threat of antimicrobial resistance and decline in antimicrobial development, the advancement of complex and rigorous pharmacokinetic and pharmacodynamic studies over a short time span has renewed confidence in the value of pharmacokinetic and pharmacodynamic studies and allowed it to become fundamental component of a robust drug development program with high chances of successful approval. In addition, recent guidance by various regulatory bodies have reinforced that a strong and dedicated focus on pharmacokinetics and pharmacodynamics throughout research and development lead to the use of an optimized dosing regimen in Phase 3 trials, improving the probability of drug approval. The objective of this review is to demonstrate the importance of pharmacokinetic and pharmacodynamic studies in the drug development decision-making process by highlighting the developments in pharmacokinetic and pharmacodynamic methods and discuss the role of pharmacokinetic and pharmacodynamic studies in antimicrobial successes and failures.

CRISPR-Based Approaches for Gene Regulation in Non-Model Bacteria.

CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) have become ubiquitous approaches to control gene expression in bacteria due to their simple design and effectiveness. By regulating transcription of a target gene(s), CRISPRi/a can dynamically engineer cellular metabolism, implement transcriptional regulation circuitry, or elucidate genotype-phenotype relationships from smaller targeted libraries up to whole genome-wide libraries. While CRISPRi/a has been primarily established in the model bacteria Escherichia coli and Bacillus subtilis, a growing numbering of studies have demonstrated the extension of these tools to other species of bacteria (here broadly referred to as non-model bacteria). In this mini-review, we discuss the challenges that contribute to the slower creation of CRISPRi/a tools in diverse, non-model bacteria and summarize the current state of these approaches across bacterial phyla. We find that despite the potential difficulties in establishing novel CRISPRi/a in non-model microbes, over 190 recent examples across eight bacterial phyla have been reported in the literature. Most studies have focused on tool development or used these CRISPRi/a approaches to interrogate gene function, with fewer examples applying CRISPRi/a gene regulation for metabolic engineering or high-throughput screens and selections. To date, most CRISPRi/a reports have been developed for common strains of non-model bacterial species, suggesting barriers remain to establish these genetic tools in undomesticated bacteria. More efficient and generalizable methods will help realize the immense potential of programmable CRISPR-based transcriptional control in diverse bacteria.

Synthesis, Biological Evaluation and Docking Studies of Ring-Opened Analogues of Ipomoeassin F.

The plant-derived macrocyclic resin glycoside ipomoeassin F (Ipom-F) binds to Sec61α and significantly disrupts multiple aspects of Sec61-mediated protein biogenesis at the endoplasmic reticulum, ultimately leading to cell death. However, extensive assessment of Ipom-F as a molecular tool and a therapeutic lead is hampered by its limited production scale, largely caused by intramolecular assembly of the macrocyclic ring. Here, using in vitro and/or in cellula biological assays to explore the first series of ring-opened analogues for the ipomoeassins, and indeed all resin glycosides, we provide clear evidence that macrocyclic integrity is not required for the cytotoxic inhibition of Sec61-dependent protein translocation by Ipom-F. Furthermore, our modeling suggests that open-chain analogues of Ipom-F can interact with multiple sites on the Sec61α subunit, most likely located at a previously identified binding site for mycolactone and/or the so-called lateral gate. Subsequent in silico-aided design led to the discovery of the stereochemically simplified analogue 3 as a potent, alternative lead compound that could be synthesized much more efficiently than Ipom-F and will accelerate future ipomoeassin research in chemical biology and drug discovery. Our work may also inspire further exploration of ring-opened analogues of other resin glycosides.

Effects of angiotensin II in the management of perioperative hypotension in kidney transplant recipients.

BACKGROUND: Due to the mechanisms of action of conventional catecholamine vasopressors, there is increased risk of renal allograft injury and adverse events in transplant recipients with fluid-refractory distributive shock during the perioperative period. As such, mechanistically alternative vasopressors like angiotensin II (ATII) may avoid these complications, but there is an absence of data supporting use in this population. METHODS: This was a single-center, single-arm, open-label, phase 4 study conducted as a 1-year pilot of 20 adult renal transplant recipients receiving ATII as their first continuous infusion vasopressor in the perioperative period. The study aim was to systematically assess the safety and hemodynamic effects of ATII. Safety was assessed based on the incidence of adverse events. Hemodynamic effect was assessed by the achievement of per protocol hemodynamic goals (i.e., SBP ≥120 mmHg) and the need for adjunct vasopressors. RESULTS: Most cases involved deceased donors (70%), with a corresponding mean (SD) cold ischemia time of 14.7 (8.6) h. Over a surgery duration of 5.3 (1.2) h, subjects received 3.2 (2.0) L of total volume resuscitation prior to ATII initiation. No adverse events were directly related to ATII administration. Throughout this period, ATII was utilized for a median of 1.0 (IQR, 1.5) h intraoperatively (N = 7), 26.5 (IQR, 84.8) h postoperatively (N = 4), and 63.8 (IQR, 57.8) h in subjects who required ATII both intra- and postoperatively (N = 9). Only one of the 20 patients needed adjunct continuous infusion vasopressors in addition to ATII. CONCLUSIONS: Based on the observations of this pilot study, ATII is a safe and effective vasopressor option for renal transplant recipients requiring perioperative hypotension reversal.

An alternative pathway for membrane protein biogenesis at the endoplasmic reticulum.

The heterotrimeric Sec61 complex is a major site for the biogenesis of transmembrane proteins (TMPs), accepting nascent TMP precursors that are targeted to the endoplasmic reticulum (ER) by the signal recognition particle (SRP). Unlike most single-spanning membrane proteins, the integration of type III TMPs is completely resistant to small molecule inhibitors of the Sec61 translocon. Using siRNA-mediated depletion of specific ER components, in combination with the potent Sec61 inhibitor ipomoeassin F (Ipom-F), we show that type III TMPs utilise a distinct pathway for membrane integration at the ER. Hence, following SRP-mediated delivery to the ER, type III TMPs can uniquely access the membrane insertase activity of the ER membrane complex (EMC) via a mechanism that is facilitated by the Sec61 translocon. This alternative EMC-mediated insertion pathway allows type III TMPs to bypass the Ipom-F-mediated blockade of membrane integration that is seen with obligate Sec61 clients.

Pharmacokinetics and dialytic clearance of apixaban during in vitro continuous renal replacement therapy.

BACKGROUND: To evaluate the transmembrane clearance (CLTM) of apixaban during modeled in vitro continuous renal replacement therapy (CRRT), assess protein binding and circuit adsorption, and provide initial dosing recommendations. METHODS: Apixaban was added to the CRRT circuit and serial pre-filter bovine blood samples were collected along with post-filter blood and effluent samples. All experiments were performed in duplicate using continuous veno-venous hemofiltration (CVVH) and hemodialysis (CVVHD) modes, with varying filter types, flow rates, and point of CVVH replacement fluid dilution. Concentrations of apixaban and urea were quantified via liquid chromatography-tandem mass spectrometry. Plasma pharmacokinetic parameters for apixaban were estimated via noncompartmental analysis. CLTM was calculated via the estimated area under the curve (AUC) and by the product of the sieving/saturation coefficient (SC/SA) and flow rate. Two and three-way analysis of variance (ANOVA) models were built to assess the effects of mode, filter type, flow rate, and point of dilution on CLTM by each method. Optimal doses were suggested by matching the AUC observed in vitro to the systemic exposure demonstrated in Phase 2/3 studies of apixaban. Linear regression was utilized to provide dosing estimations for flow rates from 0.5-5 L/h. RESULTS: Mean adsorption to the HF1400 and M150 filters differed significantly at 38 and 13%, respectively, while mean (± standard deviation, SD) percent protein binding was 70.81 ± 0.01%. Effect of CVVH point of dilution did not differ across filter types, although CLTM was consistently significantly higher during CRRT with the HF1400 filter compared to the M150. The three-way ANOVA demonstrated improved fit when CLTM values calculated by AUC were used (adjusted R2 0.87 vs. 0.52), and therefore, these values were used to generate optimal dosing recommendations. Linear regression revealed significant effects of filter type and flow rate on CLTM by AUC, suggesting doses of 2.5-7.5 mg twice daily (BID) may be needed for flow rates ranging from 0.5-5 L/h, respectively. CONCLUSION: For CRRT flow rates most commonly employed in clinical practice, the standard labeled 5 mg BID dose of apixaban is predicted to achieve target systemic exposure thresholds. The safety and efficacy of these proposed dosing regimens warrants further investigation in clinical studies.

Ring Expansion Leads to a More Potent Analogue of Ipomoeassin F.

Two new ring-size-varying analogues (2 and 3) of ipomoeassin F were synthesized and evaluated. Improved cytotoxicity (IC50: from 1.8 nM) and in vitro protein translocation inhibition (IC50: 35 nM) derived from ring expansion imply that the binding pocket of Sec61α (isoform 1) can accommodate further structural modifications, likely in the fatty acid portion. Streamlined preparation of the key diol intermediate 5 enabled gram-scale production, allowing us to establish that ipomoeassin F is biologically active in vivo (MTD: ∼3 mg/kg).

GEOS-S2S Version 2: The GMAO High Resolution Coupled Model and Assimilation System for Seasonal Prediction.

The Global Modeling and Assimilation Office (GMAO) has recently released a new version of the Goddard Earth Observing System (GEOS) Sub-seasonal to Seasonal prediction (S2S) system, GEOS-S2S-2, that represents a substantial improvement in performance and infrastructure over the previous system. The system is described here in detail, and results are presented from forecasts, climate equillibrium simulations and data assimilation experiments. The climate or equillibrium state of the atmosphere and ocean showed a substantial reduction in bias relative to GEOS-S2S-1. The GEOS-S2S-2 coupled reanalysis also showed substantial improvements, attributed to the assimilation of along-track Absolute Dynamic Topography. The forecast skill on subseasonal scales showed a much-improved prediction of the Madden-Julian Oscillation in GEOS-S2S-2, and on a seasonal scale the tropical Pacific forecasts show substantial improvement in the east and comparable skill to GEOS-S2S-1 in the central Pacific. GEOS-S2S-2 anomaly correlations of both land surface temperature and precipitation were comparable to GEOS-S2S-1, and showed substantially reduced root mean square error of surface temperature. The remaining issues described here are being addressed in the development of GEOS-S2S Version 3, and with that system GMAO will continue its tradition of maintaining a state of the art seasonal prediction system for use in evaluating the impact on seasonal and decadal forecasts of assimilating newly available satellite observations, as well as to evaluate additional sources of predictability in the earth system through the expanded coupling of the earth system model and assimilation components.

Cellular checkpoint control using programmable sequential logic.

Biological processes that require orderly progression, such as growth and differentiation, proceed via regulatory checkpoints where the cell waits for signals before continuing to the next state. Implementing such control would allow genetic engineers to divide complex tasks into stages. We present genetic circuits that encode sequential logic to instruct Escherichia coli to proceed through a linear or cyclical sequence of states. These are built with 11 set-reset latches, designed with repressor-based NOR gates, which can connect to each other and sensors. The performance of circuits with up to three latches and four sensors, including a gated D latch, closely match predictions made by using nonlinear dynamics. Checkpoint control is demonstrated by switching cells between multiple circuit states in response to external signals over days.