Longitudinal wastewater sampling in buildings reveals temporal dynamics of metabolites.

Direct sampling of building wastewater has the potential to enable "precision public health" observations and interventions. Temporal sampling offers additional dynamic information that can be used to increase the informational content of individual metabolic "features", but few studies have focused on high-resolution sampling. Here, we sampled three spatially close buildings, revealing individual metabolomics features, retention time (rt) and mass-to-charge ratio (mz) pairs, that often possess similar stationary statistical properties, as expected from aggregate sampling. However, the temporal profiles of features-providing orthogonal information to physicochemical properties-illustrate that many possess different feature temporal dynamics (fTDs) across buildings, with large and unpredictable single day deviations from the mean. Internal to a building, numerous and seemingly unrelated features, with mz and rt differences up to hundreds of Daltons and seconds, display highly correlated fTDs, suggesting non-obvious feature relationships. Data-driven building classification achieves high sensitivity and specificity, and extracts building-identifying features found to possess unique dynamics. Analysis of fTDs from many short-duration samples allows for tailored community monitoring with applicability in public health studies.

Xenoprotein engineering via synthetic libraries.

Chemical methods have enabled the total synthesis of protein molecules of ever-increasing size and complexity. However, methods to engineer synthetic proteins comprising noncanonical amino acids have not kept pace, even though this capability would be a distinct advantage of the total synthesis approach to protein science. In this work, we report a platform for protein engineering based on the screening of synthetic one-bead one-compound protein libraries. Screening throughput approaching that of cell surface display was achieved by a combination of magnetic bead enrichment, flow cytometry analysis of on-bead screens, and high-throughput MS/MS-based sequencing of identified active compounds. Direct screening of a synthetic protein library by these methods resulted in the de novo discovery of mirror-image miniprotein-based binders to a ∼150-kDa protein target, a task that would be difficult or impossible by other means.

Leveraging Stochasticity for Open Loop and Model Predictive Control of Spatio-Temporal Systems.

Stochastic spatio-temporal processes are prevalent across domains ranging from the modeling of plasma, turbulence in fluids to the wave function of quantum systems. This letter studies a measure-theoretic description of such systems by describing them as evolutionary processes on Hilbert spaces, and in doing so, derives a framework for spatio-temporal manipulation from fundamental thermodynamic principles. This approach yields a variational optimization framework for controlling stochastic fields. The resulting scheme is applicable to a wide class of spatio-temporal processes and can be used for optimizing parameterized control policies. Our simulated experiments explore the application of two forms of this approach on four stochastic spatio-temporal processes, with results that suggest new perspectives and directions for studying stochastic control problems for spatio-temporal systems.

Primary Care Access to New Patient Appointments for California Medicaid Enrollees: A Simulated Patient Study.

PURPOSE: We undertook a study to evaluate variation in the availability of primary care new patient appointments for Medi-Cal (California Medicaid) enrollees in Northern California, and its relationship to emergency department (ED) use after Medicaid expansion. METHODS: We placed simulated calls by purported Medi-Cal enrollees to 581 primary care clinicians (PCCs) listed as accepting new patients in online directories of Medi-Cal managed care plans. Data from the California Health Interview Survey, Medi-Cal enrollment reports, and California hospital discharge records were used in analyses. We developed multilevel, mixed-effect models to evaluate variation in appointment access. Multiple linear regression was used to examine the relationship between primary care access and ED use by county. RESULTS: Availability of PCC new patient appointments to Medi-Cal enrollees lacking a PCC varied significantly across counties in the multilevel model, ranging from 77 enrollees (95% CI, 70-81) to 472 enrollees (95% CI, 378-628) per each available new patient appointment. Just 19% of PCCs had available appointments within the state-mandated 10 business days. Clinicians at Federally Qualified Health Centers had higher availability of new patient appointments (rate ratio = 1.56; 95% CI, 1.24-1.97). Counties with poorer PCC access had higher ED use by Medi-Cal enrollees. CONCLUSIONS: In contrast to findings from other states, access to primary care in Northern California was limited for new patient Medi-Cal enrollees and varied across counties, despite standard statewide reimbursement rates. Counties with more limited access to primary care new patient appointments had higher ED use by Medi-Cal enrollees.

Conformational Stabilization and Rapid Labeling of a 29-Residue Peptide by a Small Molecule Reaction Partner.

A 29-residue peptide (MP01), identified by in vitro selection for reactivity with a small molecule perfluoroaromatic, was modified and characterized using experimental and computational techniques, with the goal of understanding the molecular basis of its reactivity. These studies identified a six-amino acid point mutant (MP01-Gen4) that exhibited a reaction rate constant of 25.8 ± 1.8 M-1 s-1 at pH 7.4 and room temperature, approximately 2 orders of magnitude greater than that of its progenitor sequence and 3 orders of magnitude greater than background cysteine reactivity. MP01-Gen4 appeared to be conformationally dynamic and exhibited several properties reminiscent of larger protein molecules, including denaturant-sensitive structure and reactivity. We believe the majority of the reaction rate enhancement can be attributed to interaction of MP01-Gen4 with the perfluoroaromatic probe, which was found to stabilize a helical conformation of both MP01-Gen4 and nonreactive Cys-to-Ser or Cys-to-Ala variants. These findings demonstrate the ability of dynamic peptides to access proteinlike reaction mechanisms and the potential of perfluoroaromatic functionality to stabilize small peptide folds.

Studies on a landscape of perfluoroaromatic-reactive peptides.

We investigated 26 midsized peptides (∼30 amino acids in length) selected using mRNA display to perform a nucleophilic aromatic substitution reaction (SNAr). Analysis suggested a diverse set of reactive sequences with significant differences in primary sequence, secondary structure and even predicted tertiary structural features. Several of the sequences displayed rapid kinetics allowing for near complete labeling in under one hour. Rosetta ab initio structure prediction of these sequences suggested a landscape of structural features, ranging from beta-sheet-based sequences to those possessing more alpha-helical-like character. Circular dichroism spectroscopy confirmed elements of the structure predictions for the majority of peptides. This analysis additionally uncovered that several peptides underwent secondary structure alterations upon reaction. These results suggest a broad sequence and structural landscape of SNAr active peptides along with a potentially important feature of these biopolymers.

Balanced Permeability Index: A Multiparameter Index for Improved In Vitro Permeability.

The optimization of passive permeability is a key objective for orally available small molecule drug candidates. For drugs targeting the central nervous system (CNS), minimizing P-gp-mediated efflux is an additional important target for optimization. The physicochemical properties most strongly associated with high passive permeability and lower P-gp efflux are size, polarity, and lipophilicity. In this study, a new metric called the Balanced Permeability Index (BPI) was developed that combines these three properties. The BPI was found to be more effective than any single property in classifying molecules based on their permeability and efflux across a diverse range of chemicals and assays. BPI is easy to understand, allowing researchers to make decisions about which properties to prioritize during the drug development process.