Chikungunya Virus Vaccine Candidate Incorporating Synergistic Mutations Is Attenuated and Protects Against Virulent Virus Challenge.

BACKGROUND: Chikungunya virus (CHIKV) is an arbovirus that periodically emerges to cause large epidemics of arthritic disease. Although the robust immunity elicited by live-attenuated virus (LAV) vaccine candidates makes them attractive, CHIKV vaccine development has been hampered by a high threshold for acceptable adverse events. METHODS: We evaluated the vaccine potential of a recently described LAV, skeletal muscle-restricted virus (SKE), that exhibits diminished replication in skeletal muscle due to insertion of target sequences for skeletal muscle-specific miR-206. We also evaluated whether these target sequences could augment safety of an LAV encoding a known attenuating mutation, E2 G82R. Attenuation of viruses containing these mutations was compared with a double mutant, SKE G82R. RESULTS: SKE was attenuated in both immunodeficient and immunocompetent mice and induced a robust neutralizing antibody response, indicating its vaccine potential. However, only SKE G82R elicited diminished swelling in immunocompetent mice at early time points postinoculation, indicating that these mutations synergistically enhance safety of the vaccine candidate. CONCLUSIONS: These data suggest that restriction of LAV replication in skeletal muscle enhances tolerability of reactogenic vaccine candidates and may improve the rational design of CHIKV vaccines.

Elastic coupling power stroke mechanism of the F1-ATPase molecular motor.

The angular velocity profile of the 120° F1-ATPase power stroke was resolved as a function of temperature from 16.3 to 44.6 °C using a ΔµATP = -31.25 kBT at a time resolution of 10 µs. Angular velocities during the first 60° of the power stroke (phase 1) varied inversely with temperature, resulting in negative activation energies with a parabolic dependence. This is direct evidence that phase 1 rotation derives from elastic energy (spring constant, κ = 50 kBT·rad-2). Phase 2 of the power stroke had an enthalpic component indicating that additional energy input occurred to enable the γ-subunit to overcome energy stored by the spring after rotating beyond its 34° equilibrium position. The correlation between the probability distribution of ATP binding to the empty catalytic site and the negative Ea values of the power stroke during phase 1 suggests that this additional energy is derived from the binding of ATP to the empty catalytic site. A second torsion spring (κ = 150 kBT·rad-2; equilibrium position, 90°) was also evident that mitigated the enthalpic cost of phase 2 rotation. The maximum ΔGǂ was 22.6 kBT, and maximum efficiency was 72%. An elastic coupling mechanism is proposed that uses the coiled-coil domain of the γ-subunit rotor as a torsion spring during phase 1, and then as a crankshaft driven by ATP-binding-dependent conformational changes during phase 2 to drive the power stroke.

A framework for uncertainty and risk analysis in Total Maximum Daily Load applications.

In the United States, the computation of Total Maximum Daily Loads (TMDL) must include a Margin of Safety (MOS) to account for different sources of uncertainty. In practice however, TMDL studies rarely include an explicit uncertainty analysis and the estimation of the MOS is often subjective and even arbitrary. Such approaches are difficult to replicate and preclude the comparison of results between studies. To overcome these limitations, a Bayesian framework to compute TMDLs and MOSs including an explicit evaluation of uncertainty and risk is proposed in this investigation. The proposed framework uses the concept of Predictive Uncertainty to calculate a TMDL from an equation of allowable risk of non-compliance of a target water quality standard. The framework is illustrated in a synthetic example and in a real TMDL study for nutrients in Sawgrass Lake, Florida.

Anatomy of F1-ATPase powered rotation.

F1-ATPase, the catalytic complex of the ATP synthase, is a molecular motor that can consume ATP to drive rotation of the γ-subunit inside the ring of three αß-subunit heterodimers in 120° power strokes. To elucidate the mechanism of ATPase-powered rotation, we determined the angular velocity as a function of rotational position from single-molecule data collected at 200,000 frames per second with unprecedented signal-to-noise. Power stroke rotation is more complex than previously understood. This paper reports the unexpected discovery that a series of angular accelerations and decelerations occur during the power stroke. The decreases in angular velocity that occurred with the lower-affinity substrate ITP, which could not be explained by an increase in substrate-binding dwells, provides direct evidence that rotation depends on substrate binding affinity. The presence of elevated ADP concentrations not only increased dwells at 35° from the catalytic dwell consistent with competitive product inhibition but also decreased the angular velocity from 85° to 120°, indicating that ADP can remain bound to the catalytic site where product release occurs for the duration of the power stroke. The angular velocity profile also supports a model in which rotation is powered by Van der Waals repulsive forces during the final 85° of rotation, consistent with a transition from F1 structures 2HLD1 and 1H8E (Protein Data Bank).

Wetlands for Wastewater Treatment.

An update on the current research and development of the treatment technologies, which utilize natural processes or passive components in wastewater treatment, is provided in this paper. The main focus is on wetland systems and their applications in wastewater treatment (as an advanced treatment unit or decentralized system), nutrient and pollutant removal (metals, industrial and emerging pollutants including pharmaceutical compounds). A summary of studies involving the effects of vegetation, wetland design and modeling, hybrid and innovative systems, storm water treatment and pathogen removal is also included.

Wetlands for Wastewater Treatment.

This paper provides a review of the treatment technologies, which utilize natural processes or passive components in wastewater treatment. In particular, this paper primarily focuses on wetland systems and their applications in wastewater treatment (as an advanced treatment unit or decentralized system), nutrient and pollutant removal (single and multiple pollutants, and metals), and emerging pollutant removal (pharmaceuticals). A summary of studies involving the plant (vegetation) effects, wetland design and modeling, hybrid and innovative systems, storm water treatment and pathogen removal is also included.

Implementation of clopidogrel pharmacogenetic clinical decision support for a preemptive return of results program.

PURPOSE: To describe our experiences implementing and iterating CYP2C19 genotype-guided clopidogrel pharmacogenetic clinical decision support (CDS) tools over time in the setting of a large health system-wide, preemptive pharmacogenomics program. SUMMARY: Clopidogrel-treated patients who are genetically predicted cytochrome P450 isozyme 2C19 (CYP2C19) intermediate or poor metabolizers have an increased risk of atherothrombotic events, some of which can be life-threatening. The Clinical Pharmacogenetics Implementation Consortium provides guidance for the use of clopidogrel based on CYP2C19 genotype in patients with cardiovascular and cerebrovascular diseases. Our multidisciplinary team implemented an automated, interruptive alert that fires when clopidogrel is ordered or refilled for biobank participants with structured CYP2C19 intermediate or poor metabolizer genomic indicators in the electronic health record. The implementation began with a narrow cardiovascular indication and setting and was then scaled in 4 primary dimensions: (1) clinical indication; (2) availability across health-system locations; (3) care venue (e.g., inpatient vs outpatient); and (4) provider groups (eg, cardiology and neurology). We iterated our approach over time based on evolving clinical evidence and proactive strategies to optimize CDS maintenance and sustainability. A key facilitator of expansion was socialization of the broader pharmacogenomics initiative among our academic medical center community, accompanied by clinician acceptance of pharmacogenetic alerts in practice. CONCLUSION: A multidisciplinary collaboration is recommended to facilitate the use of CYP2C19 genotype-guided antiplatelet therapy in patients with cardiovascular and cerebrovascular diseases. Evolving clopidogrel pharmacogenetic evidence necessitates thoughtful iteration of implementation efforts and strategies to optimize long-term maintenance and sustainability.

Patient-Level Exposure to Actionable Pharmacogenomic Medications in a Nationally Representative Insurance Claims Database.

BACKGROUND: The prevalence of exposure to pharmacogenomic medications is well established but little is known about how long patients are exposed to these medications. AIM: Our objective was to describe the amount of exposure to actionable pharmacogenomic medications using patient-level measures among a large nationally representative population using an insurance claims database. METHODS: Our retrospective cohort study included adults (18+ years) from the IQVIA PharMetrics® Plus for Academics claims database with incident fills of 72 Clinical Pharmacogenetics Implementation Consortium level A, A/B, or B medications from January 2012 through September 2018. Patient-level outcomes included the proportion of days covered (PDC), number of fills, and average days supplied per fill over a 12-month period. RESULTS: Over 1 million fills of pharmacogenetic medications were identified for 605,355 unique patients. The mean PDC for all medications was 0.21 (SD 0.3), suggesting patients were exposed 21% (77 days) of the year. Medications with the highest PDC (0.55-0.89) included ivacaftor, tamoxifen, clopidogrel, HIV medications, transplant medications, and statins; with the exception of statins, these medications were initiated by fewer patients. Pharmacogenomic medications were filled an average of 2.8 times (SD 3.0, range 1-81) during the year following the medication's initiation, and the average days supplied for each fill was 22.3 days (SD 22.4, range 1-180 days). CONCLUSION: Patient characteristics associated with more medication exposure were male sex, older age, and comorbid chronic conditions. Prescription fill data provide patient-level exposure metrics that can further our understanding of pharmacogenomic medication utilization and help inform opportunities for pharmacogenomic testing.

Patients' perspectives of a pharmacist-provided clinical pharmacogenomics service.

Aim: To assess the perspectives and experiences of patients who participated in a pharmacist-provided clinical pharmacogenomics (PGx) service. Methods: We conducted individual semistructured interviews with 16 patients who received a pharmacist-provided PGx service. Qualitative data were analyzed to identify pertinent themes. Results: The major themes identified were: heterogeneity of patient PGx experiences and preferences, pharmacists as appropriate providers of PGx services, considerations regarding the use of PGx results in routine healthcare and perceived applications of PGx testing. Theme-derived considerations included the need to establish appropriate pre-genotyping expectations, individualize patient education, facilitate collaboration with patients' providers and sustainably update patients' PGx information over time. Conclusion: Patient-specific perspectives such as these are important to consider when providing clinical PGx services, with intention of optimizing patient experiences.

Household and climate factors influence Aedes aegypti presence in the arid city of Huaquillas, Ecuador.

Arboviruses transmitted by Aedes aegypti (e.g., dengue, chikungunya, Zika) are of major public health concern on the arid coastal border of Ecuador and Peru. This high transit border is a critical disease surveillance site due to human movement-associated risk of transmission. Local level studies are thus integral to capturing the dynamics and distribution of vector populations and social-ecological drivers of risk, to inform targeted public health interventions. Our study examines factors associated with household-level Ae. aegypti presence in Huaquillas, Ecuador, while accounting for spatial and temporal effects. From January to May of 2017, adult mosquitoes were collected from a cohort of households (n = 63) in clusters (n = 10), across the city of Huaquillas, using aspirator backpacks. Household surveys describing housing conditions, demographics, economics, travel, disease prevention, and city services were conducted by local enumerators. This study was conducted during the normal arbovirus transmission season (January-May), but during an exceptionally dry year. Household level Ae. aegypti presence peaked in February, and counts were highest in weeks with high temperatures and a week after increased rainfall. Univariate analyses with proportional odds logistic regression were used to explore household social-ecological variables and female Ae. aegypti presence. We found that homes were more likely to have Ae. aegypti when households had interruptions in piped water service. Ae. aegypti presence was less likely in households with septic systems. Based on our findings, infrastructure access and seasonal climate are important considerations for vector control in this city, and even in dry years, the arid environment of Huaquillas supports Ae. aegypti breeding habitat.

WASP 8: The Next Generation in the 50-year Evolution of USEPA's Water Quality Model.

The Water Quality Analysis Simulation Program (WASP) helps users interpret and predict water quality responses to natural phenomena and manmade pollution for various pollution management decisions. WASP is a dynamic compartment-modeling program for aquatic systems, including both the water column and the underlying benthos. WASP allows the user to investigate 1, 2 and 3 dimensional systems and a variety of pollutant types-including both conventional pollutants (e.g., dissolved oxygen, nutrients, phytoplankton, etc.) and toxic materials. WASP has capabilities of linking with hydrodynamic and watershed models which allows for multi-year analyses under varying meteorological and environmental conditions. WASP was originally developed by HydroScience, Inc. in 1970 and was later adapted by the US Environmental Protection Agency's Large Lakes Research Station (LLRS) for applications to the Great Lakes. The LLRS first publicly released the model in 1981. WASP has undergone continuous development since that time and this year will mark its 50th anniversary. This paper follows the development of WASP from its origin to the latest release of the model in 2020, documenting its evolution and present structure and capabilities.

Acute psychological stress increases serum circulating cell-free mitochondrial DNA.

Intrinsic biological mechanisms transduce psychological stress into physiological adaptation that requires energy, but the role of mitochondria and mitochondrial DNA (mtDNA) in this process has not been defined in humans. Here, we show that similar to physical injury, exposure to psychological stress increases serum circulating cell-free mtDNA (ccf-mtDNA) levels. Healthy midlife adults exposed on two separate occasions to a brief psychological challenge exhibited a 2-3-fold increase in ccf-mtDNA, with no change in ccf-nuclear DNA levels, establishing the magnitude and specificity for ccf-mtDNA reactivity. In cell-based studies, we show that glucocorticoid signaling - a consequence of psychological stress in humans - is sufficient to induce mtDNA extrusion in a time frame consistent with stress-induced ccf-mtDNA increase. Collectively, these findings provide evidence that acute psychological stress induces ccf-mtDNA and implicate neuroendocrine signaling as a potential trigger for ccf-mtDNA release. Further controlled work is needed to confirm that observed increases in ccf-mtDNA result from stress exposure and to determine the functional significance of this effect.

PINK1 attenuates mtDNA release in alveolar epithelial cells and TLR9 mediated profibrotic responses.

We have previously shown that endoplasmic reticulum stress (ER stress) represses the PTEN inducible kinase 1 (PINK1) in lung type II alveolar epithelial cells (AECII) reducing mitophagy and increasing the susceptibility to lung fibrosis. Although increased circulating mitochondrial DNA (mtDNA) has been reported in chronic lung diseases, the contribution of mitophagy in the modulation of mitochondrial DAMP release and activation of profibrotic responses is unknown. In this study, we show that ER stress and PINK1 deficiency in AECII led to mitochondrial stress with significant oxidation and damage of mtDNA and subsequent extracellular release. Extracellular mtDNA was recognized by TLR9 in AECII by an endocytic-dependent pathway. PINK1 deficiency-dependent mtDNA release promoted activation of TLR9 and triggered secretion of the profibrotic factor TGF-ß which was rescued by PINK1 overexpression. Enhanced mtDNA oxidation and damage were found in aging and IPF human lungs and, in concordance, levels of circulating mtDNA were significantly elevated in plasma and bronchoalveolar lavage (BAL) from patients with IPF. Free mtDNA was found elevated in other ILDs with low expression of PINK1 including hypersensitivity pneumonitis and autoimmune interstitial lung diseases. These results support a role for PINK1 mediated mitophagy in the attenuation of mitochondrial damage associated molecular patterns (DAMP) release and control of TGF-ß mediated profibrotic responses.

Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions.

Mitochondrial DNA (mtDNA) mutations are a common cause of primary mitochondrial disorders, and have also been implicated in a broad collection of conditions, including aging, neurodegeneration, and cancer. Prevalent among these pathogenic variants are mtDNA deletions, which show a strong bias for the loss of sequence in the major arc between, but not including, the heavy and light strand origins of replication. Because individual mtDNA deletions can accumulate focally, occur with multiple mixed breakpoints, and in the presence of normal mtDNA sequences, methods that detect broad-spectrum mutations with enhanced sensitivity and limited costs have both research and clinical applications. In this study, we evaluated semi-quantitative and digital PCR-based methods of mtDNA deletion detection using double-stranded reference templates or biological samples. Our aim was to describe key experimental assay parameters that will enable the analysis of low levels or small differences in mtDNA deletion load during disease progression, with limited false-positive detection. We determined that the digital PCR method significantly improved mtDNA deletion detection sensitivity through absolute quantitation, improved precision and reduced assay standard error.