Reentrant DNA shells tune polyphosphate condensate size.

The ancient, inorganic biopolymer polyphosphate (polyP) occurs in all three domains of life and affects myriad cellular processes. An intriguing feature of polyP is its frequent proximity to chromatin, and in the case of many bacteria, its occurrence in the form of magnesium-enriched condensates embedded in the nucleoid, particularly in response to stress. The physical basis of the interaction between polyP and DNA, two fundamental anionic biopolymers, and the resulting effects on the organization of both the nucleoid and polyP condensates remain poorly understood. Given the essential role of magnesium ions in the coordination of polymeric phosphate species, we hypothesized that a minimal system of polyP, magnesium ions, and DNA (polyP-Mg2+-DNA) would capture key features of the interplay between the condensates and bacterial chromatin. We find that DNA can profoundly affect polyP-Mg2+ coacervation even at concentrations several orders of magnitude lower than found in the cell. The DNA forms shells around polyP-Mg2+ condensates and these shells show reentrant behavior, primarily forming in the concentration range close to polyP-Mg2+ charge neutralization. This surface association tunes both condensate size and DNA morphology in a manner dependent on DNA properties, including length and concentration. Our work identifies three components that could form the basis of a central and tunable interaction hub that interfaces with cellular interactors. These studies will inform future efforts to understand the basis of polyP granule composition and consolidation, as well as the potential capacity of these mesoscale assemblies to remodel chromatin in response to diverse stressors at different length and time scales.

Indole modulates cooperative protein-protein interactions in the flagellar motor.

Indole is a major component of the bacterial exometabolome, and the mechanisms for its wide-ranging effects on bacterial physiology are biomedically significant, although they remain poorly understood. Here, we determined how indole modulates the functions of a widely conserved motility apparatus, the bacterial flagellum. Our experiments in Escherichia coli revealed that indole influences the rotation rates and reversals in the flagellum's direction of rotation via multiple mechanisms. At concentrations higher than 1 mM, indole decreased the membrane potential to dissipate the power available for the rotation of the motor that operates the flagellum. Below 1 mM, indole did not dissipate the membrane potential. Instead, experiments and modeling indicated that indole weakens cooperative protein interactions within the flagellar complexes to inhibit motility. The metabolite also induced reversals in the rotational direction of the motor to promote a weak chemotactic response, even when the chemotaxis response regulator, CheY, was lacking. Experiments further revealed that indole does not require the transporter Mtr to cross the membrane and influence motor functions. Based on these findings, we propose that indole modulates intra- and inter-protein interactions in the cell to influence several physiological functions.

The Histone H1-Like Protein AlgP Facilitates Even Spacing of Polyphosphate Granules in Pseudomonas aeruginosa.

Synthesis of polyphosphate (polyP) is an ancient and universal stress and starvation response in bacteria. In many bacteria, polyP chains come together to form granular superstructures within cells. Some species appear to regulate polyP granule subcellular organization. Despite the critical role of polyP in starvation fitness, the composition of these structures, mechanism(s) underpinning their organization, and functional significance of such organization are poorly understood. We previously determined that granules become transiently evenly spaced on the cell's long axis during nitrogen starvation in the opportunistic human pathogen Pseudomonas aeruginosa. Here, we developed a granule-enrichment protocol to screen for polyP granule-localizing proteins. We identified AlgP as a protein that associates with polyP granules. We further discovered that AlgP is required for the even spacing of polyP granules. AlgP is a DNA-binding protein with a 154 amino acid C-terminal domain enriched in "KPAA" repeats and variants of this repeat, with an overall sequence composition similar to the C-terminal tail of eukaryotic histone H1. Granule size, number, and spacing are significantly perturbed in the absence of AlgP, or when AlgP is truncated to remove the C-terminus. The ΔalgP and algPΔCTD mutants have fewer, larger granules. We speculate that AlgP may contribute to spacing by tethering polyP granules to the chromosome, thereby inhibiting fusion with neighboring granules. Our discovery that AlgP facilitates granule spacing allows us for the first time to directly uncouple granule biogenesis from even spacing, and will inform future efforts to explore the functional significance of granule organization on fitness during starvation. IMPORTANCE The mechanisms underpinning polyP's pleiotropic effects on bacterial starvation physiology remain elusive. This simple polyanion's lack of protein binding specificity has impeded validation of bona fide polyP-binding proteins. However, polyP forms granule superstructures with spatial specificity. Our granule enrichment protocol identified a polyP granule-associated protein in Pseudomonas aeruginosa, AlgP. AlgP was originally reported as a regulator of alginate, an extracellular polysaccharide important in biofilm formation, including in cystic fibrosis (CF) chronic infections. AlgP's putative role in alginate biosynthesis has recently been called into question. We establish a distinct, previously unknown function for AlgP in modulating the subcellular organization of polyP, another polymer important for pathogenesis. In CF clinical isolates, the C-terminal repeat domain of AlgP is a hot spot for genetic rearrangements. Our finding that the C-terminus of AlgP is required for granule organization lays the groundwork for exploring the functional significance of these mutations in the evolutionary trajectory of chronic infections.

The Potential For Bias In Machine Learning And Opportunities For Health Insurers To Address It.

As the use of machine learning algorithms in health care continues to expand, there are growing concerns about equity, fairness, and bias in the ways in which machine learning models are developed and used in clinical and business decisions. We present a guide to the data ecosystem used by health insurers to highlight where bias can arise along machine learning pipelines. We suggest mechanisms for identifying and dealing with bias and discuss challenges and opportunities to increase fairness through analytics in the health insurance industry.

Protein expression-independent response of intensity-based pH-sensitive fluorophores in Escherichia coli.

Fluorescent proteins that modulate their emission intensities when protonated serve as excellent probes of the cytosolic pH. Since the total fluorescence output fluctuates significantly due to variations in the fluorophore levels in cells, eliminating the dependence of the signal on protein concentration is crucial. This is typically accomplished with the aid of ratiometric fluorescent proteins such as pHluorin. However, pHluorin is excited by blue light, which can complicate pH measurements by adversely impacting bacterial physiology. Here, we characterized the response of intensity-based, pH-sensitive fluorescent proteins that excite at longer wavelengths where the blue light effect is diminished. The pH-response was interpreted in terms of an analytical model that assumed two emission states for each fluorophore: a low intensity protonated state and a high intensity deprotonated state. The model suggested a scaling to eliminate the dependence of the signal on the expression levels as well as on the illumination and photon-detection settings. Experiments successfully confirmed the scaling predictions. Thus, the internal pH can be readily determined with intensity-based fluorophores with appropriate calibrations irrespective of the fluorophore concentration and the signal acquisition setup. The framework developed in this work improves the robustness of intensity-based fluorophores for internal pH measurements in E. coli, potentially extending their applications.

Biphasic chemotaxis of Escherichia coli to the microbiota metabolite indole.

Bacterial chemotaxis to prominent microbiota metabolites such as indole is important in the formation of microbial communities in the gastrointestinal (GI) tract. However, the basis of chemotaxis to indole is poorly understood. Here, we exposed Escherichia coli to a range of indole concentrations and measured the dynamic responses of individual flagellar motors to determine the chemotaxis response. Below 1 mM indole, a repellent-only response was observed. At 1 mM indole and higher, a time-dependent inversion from a repellent to an attractant response was observed. The repellent and attractant responses were mediated by the Tsr and Tar chemoreceptors, respectively. Also, the flagellar motor itself mediated a repellent response independent of the receptors. Chemotaxis assays revealed that receptor-mediated adaptation to indole caused a bipartite response-wild-type cells were attracted to regions of high indole concentration if they had previously adapted to indole but were otherwise repelled. We propose that indole spatially segregates cells based on their state of adaptation to repel invaders while recruiting beneficial resident bacteria to growing microbial communities within the GI tract.

Do payor-based outreach programs reduce medical cost and utilization?

There is growing interest in using predictive analytics to drive interventions that reduce avoidable healthcare utilization. This study evaluates the impact of such an intervention utilizing claims from 2013 to 2017 for high-risk Medicare Advantage patients with congestive heart failure. A predictive algorithm using clinical and nonclinical information produced a risk score ranking for health plan members in 10 separate waves between July 2013 and May 2015. Each wave was followed by an outreach intervention. The varying capacity for outreach across waves created a set of arbitrary intervention treatment cutoff points, separating treated and untreated members with very similar predicted risk scores. We estimate a difference-in-differences model to identify the effects of the intervention program among patients with a high score on care utilization. We find that enrollment in the intervention decreased the probability and number of hospitalizations (by 43% and 50%, respectively) and emergency room visits (10% and 14%, respectively), reduced the time until a primary care visit (8.2 days), and reduced total medical cost by $716 per month in the first 6 months following outreach.

A Skeptic's Guide to Bacterial Mechanosensing.

Surface sensing in bacteria is a precursor to the colonization of biotic and abiotic surfaces, and an important cause of drug resistance and virulence. As a motile bacterium approaches and adheres to a surface from the bulk fluid, the mechanical forces that act on it change. Bacteria are able to sense these changes in the mechanical load through a process termed mechanosensing. Bacterial mechanosensing has featured prominently in recent literature as playing a key role in surface sensing. However, the changes in mechanical loads on different parts of the cell at a surface vary in magnitudes as well as in signs. This confounds the determination of a causal relationship between the activation of specific mechanosensors and surface sensing. Here, we explain how contrasting mechanical stimuli arise on a surface-adherent cell and how known mechanosensors respond to these stimuli. The evidence for mechanosensing in select bacterial species is reinterpreted, with a focus on mechanosensitive molecular motors. We conclude with proposed criteria that bacterial mechanosensors must satisfy to successfully mediate surface sensing.

Molecular Mechanism for Attractant Signaling to DHMA by E. coli Tsr.

The attractant chemotaxis response of Escherichia coli to norepinephrine requires that it be converted to 3,4-dihydroxymandelic acid (DHMA) by the monoamine oxidase TynA and the aromatic aldehyde dehydrogenase FeaB. DHMA is sensed by the serine chemoreceptor Tsr, and the attractant response requires that at least one subunit of the periplasmic domain of the Tsr homodimer (pTsr) has an intact serine-binding site. DHMA that is generated in vivo by E. coli is expected to be a racemic mixture of the (R) and (S) enantiomers, so it has been unclear whether one or both chiral forms are active. Here, we used a combination of state-of-the-art tools in molecular docking and simulations, including an in-house simulation-based docking protocol, to investigate the binding properties of (R)-DHMA and (S)-DHMA to E. coli pTsr. Our studies computationally predicted that (R)-DHMA should promote a stronger attractant response than (S)-DHMA because of a consistently greater-magnitude piston-like pushdown of the pTsr α-helix 4 toward the membrane upon binding of (R)-DHMA than upon binding of (S)-DHMA. This displacement is caused primarily by interaction of DHMA with Tsr residue Thr156, which has been shown by genetic studies to be critical for the attractant response to L-serine and DHMA. These findings led us to separate the two chiral species and test their effectiveness as chemoattractants. Both the tethered cell and motility migration coefficient assays validated the prediction that (R)-DHMA is a stronger attractant than (S)-DHMA. Our study demonstrates that refined computational docking and simulation studies combined with experiments can be used to investigate situations in which subtle differences between ligands may lead to diverse chemotactic responses.

The impact of patient-centered medical homes on medication adherence?

Accreditation of providers helps resolve the pervasive information asymmetries in health care markets. However, meeting accreditation standards typically involves flexibility in implementation, leading to heterogeneity in performance. For example, the patient-centered medical home (PCMH) is a leading model for recognizing high-performing primary care practices. Flexibility in PCMH implementation allows for varying degrees of emphasis on processes designed to enhance medication adherence. To assess the impact of the PCMH on adherence, we combine 6 years of detailed patient claims data with a novel dataset containing detailed practice-level PCMH attributes. We study the effects of the number and configuration of adherence-relevant capabilities, using variation in the timing of PCMH adoption to estimate its impact. While PCMH adoption improved overall medication adherence, when combining claims data with the unique recognition data detailing what PCMH capabilities were adopted, we find that these gains are concentrated among patients in practices that adopted more adherence-relevant capabilities. Despite mixed evidence in the literature concerning costs and utilization, our results indicate that PCMH recognition improves medication adherence.

Viscous drag on the flagellum activates Bacillus subtilis entry into the K-state.

Bacillus subtilis flagella are not only required for locomotion but also act as sensors that monitor environmental changes. Although how the signal transmission takes place is poorly understood, it has been shown that flagella play an important role in surface sensing by transmitting a mechanical signal to control the DegS-DegU two-component system. Here we report a role for flagella in the regulation of the K-state, which enables transformability and antibiotic tolerance (persistence). Mutations impairing flagellar synthesis are inferred to increase DegU-P, which inhibits the expression of ComK, the master regulator for the K-state, and reduces transformability. Tellingly, both deletion of the flagellin gene and straight filament (hagA233V ) mutations increased DegU phosphorylation despite the fact that both mutants had wild type numbers of basal bodies and the flagellar motors were functional. We propose that higher viscous loads on flagellar motors result in lower DegU-P levels through an unknown signaling mechanism. This flagellar-load based mechanism ensures that cells in the motile subpopulation have a tenfold enhanced likelihood of entering the K-state and taking up DNA from the environment. Further, our results suggest that the developmental states of motility and competence are related and most commonly occur in the same epigenetic cell type.

Torque, but not FliL, regulates mechanosensitive flagellar motor-function.

The stator-complex in the bacterial flagellar motor is responsible for surface-sensing. It remodels in response to perturbations in viscous loads, recruiting additional stator-units as the load increases. Here, we tested a hypothesis that the amount of torque generated by each stator-unit modulates its association with the rotor. To do this, we measured stator-binding to the rotor in mutants in which motors reportedly develop lower torque compared to wildtype motors. First, we employed a strain lacking fliL. Contrary to earlier reports, measurements indicated that the torque generated by motors in the fliL strain was similar to that in the wildtype, at high loads. In these motors, stator-binding was unchanged. Next, experiments with a paralyzed strain indicated that the stator-binding was measurably weaker when motors were unable to generate torque. An analytical model was developed that incorporated an exponential dependence of the unit's dissociation rate on the force delivered to the rotor. The model provided accurate fits to measurements of stator-rotor binding over a wide range of loads. Based on these results, we propose that the binding of each stator-unit is enhanced by the force it develops. Furthermore, FliL does not play a significant role in motor function in E. coli.

Biophysical Characterization of Flagellar Motor Functions.

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel in response to a variety of environmental stimuli and are among the triggers for surface colonization and infections. The precise mechanisms by which motors remodel and promote cellular adaptation likely depend on key motor attributes. The photomultiplier-based bead-tracking technique presented here enables accurate biophysical characterization of motor functions, including adaptations in motor speeds and switch-dynamics. This approach offers the advantage of real-time tracking and the ability to probe motor behavior over extended durations. The protocols discussed can be readily extended to study flagellar motors in a variety of bacterial species.

Do patient-centered medical homes reduce emergency department visits?

OBJECTIVE: To assess whether adoption of the patient-centered medical home (PCMH) reduces emergency department (ED) utilization among patients with and without chronic illness. DATA SOURCES: Data from approximately 460,000 Independence Blue Cross patients enrolled in 280 primary care practices, all converting to PCMH status between 2008 and 2012. RESEARCH DESIGN: We estimate the effect of a practice becoming PCMH-certified on ED visits and costs using a difference-in-differences approach which exploits variation in the timing of PCMH certification, employing either practice or patient fixed effects. We analyzed patients with and without chronic illness across six chronic illness categories. PRINCIPAL FINDINGS: Among chronically ill patients, transition to PCMH status was associated with 5-8 percent reductions in ED utilization. This finding was robust to a number of specifications, including analyzing avoidable and weekend ED visits alone. The largest reductions in ED visits are concentrated among chronic patients with diabetes and hypertension. CONCLUSIONS: Adoption of the PCMH model was associated with lower ED utilization for chronically ill patients, but not for those without chronic illness. The effectiveness of the PCMH model varies by chronic condition. Analysis of weekend and avoidable ED visits suggests that reductions in ED utilization stem from better management of chronic illness rather than expanding access to primary care clinics.

Medical homes and cost and utilization among high-risk patients.

OBJECTIVES: Evaluate the effects of the patient-centered medical home (PCMH) model on medical costs and utilization in the nonpediatric population, particularly among high-risk patients. STUDY DESIGN: Longitudinal case-control design, comparing per member per month (PMPM) cost and utilization per 1000 patients for members enrolled in PCMH and non-PCMH practices from 2009 to 2011. METHODS: Commercial health maintenance organization members in nonpediatric practices that adopted the PCMH model in 2009 were matched to patients in nonpediatric practices that did not adopt the model until 2011 or later. Propensity score matching was used to identify a pool of similar controls, and difference-in-differences regression analysis was used to compare PCMH and non-PCMH patients relative to baseline. Analysis was conducted using the complete pool of matched patients (N = 6940 cases and 6940 controls), then using the 10% of patients with the highest DxCG risk scores (N = 654 cases and 734 controls). RESULTS: There were no significant cost or utilization differences for the overall population. Total cost decreased significantly more for the PCMH group than for controls in the high-risk group in years 1 and 2 (reductions of $107 and $75 PMPM), driven by lower inpatient costs. The PCMH group experienced a significantly greater reduction in inpatient admissions in all 3 years (61, 48, and 94 hospitalizations per 1000). CONCLUSIONS: PCMH practices had significantly reduced costs and utilization for the highest risk patients, particularly with respect to inpatient care. As high-risk members represent a high-cost group, the most benefit can be gained by targeting these members.

Patient-centered medical home impact on health plan members with diabetes.

OBJECTIVE: To compare costs and utilization for patients with diabetes enrolled in patient-centered medical home (PCMH) practices and non-PCMH practices. DESIGN: Commercial Health Maintenance Organization members with diabetes who enrolled between 2008 and 2011 in 26 Pennsylvania-based PCMH practices that were recognized by the National Committee for Quality Assurance in 2009 were compared with similar patients in 97 non-PCMH primary care practices. A difference-in-differences longitudinal research design was used to analyze differences between both groups on per-member, per-month costs and utilization. The statistical models controlled for baseline practice and patient-level characteristics through 2-step propensity score matching. The regression analysis on program effect further controlled for within-practice variation. Sensitivity analyses were also conducted on patients with type 1 and type 2 diabetes separately, and a third analysis was limited to diabetic patients enrolled in practices within Philadelphia. RESULTS: Adoption of the PCMH reduced overall medical costs for diabetic patients by 21% in year 1. This reduction was driven largely by inpatient costs, which fell by 44%. Reductions in emergency department visits, outpatient costs, and specialist visits were also seen in subsequent years among patients enrolled in PCMH practices. Additional sensitivity analyses indicated that adoption of the PCMH model yielded similar results when analyzing patients with type 2 diabetes as well as for diabetic patients enrolled in PCMH practices located within the city of Philadelphia. CONCLUSIONS: The cost of care for patients with diabetes can be reduced by securing care at a PCMH practice. Immediate results were seen in reduction of inpatient costs, which indicate that these patients enrolled in PCMH practices were using less costly inpatient services.