Fast, accurate ranking of engineered proteins by target-binding propensity using structure modeling.

Deep-learning-based methods for protein structure prediction have achieved unprecedented accuracy, yet their utility in the engineering of protein-based binders remains constrained due to a gap between the ability to predict the structures of candidate proteins and the ability toprioritize proteins by their potential to bind to a target. To bridge this gap, we introduce Automated Pairwise Peptide-Receptor Analysis for Screening Engineered proteins (APPRAISE), a method for predicting the target-binding propensity of engineered proteins. After generating structural models of engineered proteins competing for binding to a target using an established structure prediction tool such as AlphaFold-Multimer or ESMFold, APPRAISE performs a rapid (under 1 CPU second per model) scoring analysis that takes into account biophysical and geometrical constraints. As proof-of-concept cases, we demonstrate that APPRAISE can accurately classify receptor-dependent vs. receptor-independent adeno-associated viral vectors and diverse classes of engineered proteins such as miniproteins targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike, nanobodies targeting a G-protein-coupled receptor, and peptides that specifically bind to transferrin receptor or programmed death-ligand 1 (PD-L1). APPRAISE is accessible through a web-based notebook interface using Google Colaboratory (https://tiny.cc/APPRAISE). With its accuracy, interpretability, and generalizability, APPRAISE promises to expand the utility of protein structure prediction and accelerate protein engineering for biomedical applications.

Adeno-associated viral vectors for functional intravenous gene transfer throughout the non-human primate brain.

Crossing the blood-brain barrier in primates is a major obstacle for gene delivery to the brain. Adeno-associated viruses (AAVs) promise robust, non-invasive gene delivery from the bloodstream to the brain. However, unlike in rodents, few neurotropic AAVs efficiently cross the blood-brain barrier in non-human primates. Here we report on AAV.CAP-Mac, an engineered variant identified by screening in adult marmosets and newborn macaques, which has improved delivery efficiency in the brains of multiple non-human primate species: marmoset, rhesus macaque and green monkey. CAP-Mac is neuron biased in infant Old World primates, exhibits broad tropism in adult rhesus macaques and is vasculature biased in adult marmosets. We demonstrate applications of a single, intravenous dose of CAP-Mac to deliver functional GCaMP for ex vivo calcium imaging across multiple brain areas, or a cocktail of fluorescent reporters for Brainbow-like labelling throughout the macaque brain, circumventing the need for germline manipulations in Old World primates. As such, CAP-Mac is shown to have potential for non-invasive systemic gene transfer in the brains of non-human primates.

Functional gene delivery to and across brain vasculature of systemic AAVs with endothelial-specific tropism in rodents and broad tropism in primates.

Delivering genes to and across the brain vasculature efficiently and specifically across species remains a critical challenge for addressing neurological diseases. We have evolved adeno-associated virus (AAV9) capsids into vectors that transduce brain endothelial cells specifically and efficiently following systemic administration in wild-type mice with diverse genetic backgrounds, and in rats. These AAVs also exhibit superior transduction of the CNS across non-human primates (marmosets and rhesus macaques), and in ex vivo human brain slices, although the endothelial tropism is not conserved across species. The capsid modifications translate from AAV9 to other serotypes such as AAV1 and AAV-DJ, enabling serotype switching for sequential AAV administration in mice. We demonstrate that the endothelial-specific mouse capsids can be used to genetically engineer the blood-brain barrier by transforming the mouse brain vasculature into a functional biofactory. We apply this approach to Hevin knockout mice, where AAV-X1-mediated ectopic expression of the synaptogenic protein Sparcl1/Hevin in brain endothelial cells rescued synaptic deficits.

Diminished Neuronal ESCRT-0 Function Exacerbates AMPA Receptor Derangement and Accelerates Prion-Induced Neurodegeneration.

Endolysosomal defects in neurons are central to the pathogenesis of prion and other neurodegenerative disorders. In prion disease, prion oligomers traffic through the multivesicular body (MVB) and are routed for degradation in lysosomes or for release in exosomes, yet how prions impact proteostatic pathways is unclear. We found that prion-affected human and mouse brain showed a marked reduction in Hrs and STAM1 (ESCRT-0), which route ubiquitinated membrane proteins from early endosomes into MVBs. To determine how the reduction in ESCRT-0 impacts prion conversion and cellular toxicity in vivo, we prion-challenged conditional knockout mice (male and female) having Hrs deleted from neurons, astrocytes, or microglia. The neuronal, but not astrocytic or microglial, Hrs-depleted mice showed a shortened survival and an acceleration in synaptic derangements, including an accumulation of ubiquitinated proteins, deregulation of phosphorylated AMPA and metabotropic glutamate receptors, and profoundly altered synaptic structure, all of which occurred later in the prion-infected control mice. Finally, we found that neuronal Hrs (nHrs) depletion increased surface levels of the cellular prion protein, PrPC, which may contribute to the rapidly advancing disease through neurotoxic signaling. Taken together, the reduced Hrs in the prion-affected brain hampers ubiquitinated protein clearance at the synapse, exacerbates postsynaptic glutamate receptor deregulation, and accelerates neurodegeneration.SIGNIFICANCE STATEMENT Prion diseases are rapidly progressive neurodegenerative disorders characterized by prion aggregate spread through the central nervous system. Early disease features include ubiquitinated protein accumulation and synapse loss. Here, we investigate how prion aggregates alter ubiquitinated protein clearance pathways (ESCRT) in mouse and human prion-infected brain, discovering a marked reduction in Hrs. Using a prion-infection mouse model with neuronal Hrs (nHrs) depleted, we show that low neuronal Hrs is detrimental and markedly shortens survival time while accelerating synaptic derangements, including ubiquitinated protein accumulation, indicating that Hrs loss exacerbates prion disease progression. Additionally, Hrs depletion increases the surface distribution of prion protein (PrPC), linked to aggregate-induced neurotoxic signaling, suggesting that Hrs loss in prion disease accelerates disease through enhancing PrPC-mediated neurotoxic signaling.

Primate-conserved carbonic anhydrase IV and murine-restricted LY6C1 enable blood-brain barrier crossing by engineered viral vectors.

The blood-brain barrier (BBB) presents a major challenge for delivering large molecules to study and treat the central nervous system. This is due in part to the scarcity of targets known to mediate BBB crossing. To identify novel targets, we leverage a panel of adeno-associated viruses (AAVs) previously identified through mechanism-agnostic directed evolution for improved BBB transcytosis. Screening potential cognate receptors for enhanced BBB crossing, we identify two targets: murine-restricted LY6C1 and widely conserved carbonic anhydrase IV (CA-IV). We apply AlphaFold-based in silico methods to generate capsid-receptor binding models to predict the affinity of AAVs for these identified receptors. Demonstrating how these tools can unlock target-focused engineering strategies, we create an enhanced LY6C1-binding vector, AAV-PHP.eC, that, unlike our prior PHP.eB, also works in Ly6a-deficient mouse strains such as BALB/cJ. Combined with structural insights from computational modeling, the identification of primate-conserved CA-IV enables the design of more specific and potent human brain-penetrant chemicals and biologicals, including gene delivery vectors.

Functional gene delivery to and across brain vasculature of systemic AAVs with endothelial-specific tropism in rodents and broad tropism in primates.

Delivering genes to and across the brain vasculature efficiently and specifically across species remains a critical challenge for addressing neurological diseases. We have evolved adeno-associated virus (AAV9) capsids into vectors that transduce brain endothelial cells specifically and efficiently following systemic administration in wild-type mice with diverse genetic backgrounds and rats. These AAVs also exhibit superior transduction of the CNS across non-human primates (marmosets and rhesus macaques), and ex vivo human brain slices although the endothelial tropism is not conserved across species. The capsid modifications translate from AAV9 to other serotypes such as AAV1 and AAV-DJ, enabling serotype switching for sequential AAV administration in mice. We demonstrate that the endothelial specific mouse capsids can be used to genetically engineer the blood-brain barrier by transforming the mouse brain vasculature into a functional biofactory. Vasculature-secreted Hevin (a synaptogenic protein) rescued synaptic deficits in a mouse model.

Principles of long-term fluids handling in paper-based wearables with capillary-evaporative transport.

We construct and investigate paper-based microfluidic devices, which model long-term fluid harvesting, transport, sensing, and analysis in new wearables for sweat analysis. Such devices can continuously wick fluid mimicking sweat and dispose of it on evaporation pads. We characterize and analyze how the action of capillarity and evaporation can cooperatively be used to transport and process sweat mimics containing dissolved salts and model analytes. The results point out that non-invasive osmotic extraction combined with paper microfluidics and evaporative disposal can enable sweat collection and monitoring for durations longer than 10 days. We model the fluid flow in the new capillary-evaporative devices and identify the parameters enabling their long-term operation. We show that the transport rates are sufficiently large to handle natural sweat rates, while we envision that such handling can be interfaced with osmotic harvesting of sweat, a concept that we demonstrated recently. Finally, we illustrate that the salt film deposited at the evaporation pad would eventually lead to cessation of the process but at the same time will preserve a record of analytes that may be used for long-term biomarker monitoring in sweat. These principles can be implemented in future platforms for wearable skin-interfacing assays or electronic biomarker monitors.

Immediate Closures and Violations Identified During Routine Inspections of Public Aquatic Facilities - Network for Aquatic Facility Inspection Surveillance, Five States, 2013.

PROBLEM/CONDITION: Aquatic facility-associated illness and injury in the United States include disease outbreaks of infectious or chemical etiology, drowning, and pool chemical-associated health events (e.g., respiratory distress or burns). These conditions affect persons of all ages, particularly young children, and can lead to disability or even death. A total of 650 aquatic facility-associated outbreaks have been reported to CDC for 1978-2012. During 1999-2010, drownings resulted in approximately 4,000 deaths each year in the United States. Drowning is the leading cause of injury deaths in children aged 1-4 years, and approximately half of fatal drownings in this age group occur in swimming pools. During 2003-2012, pool chemical-associated health events resulted in an estimated 3,000-5,000 visits to U.S. emergency departments each year, and approximately half of the patients were aged <18 years. In August 2014, CDC released the Model Aquatic Health Code (MAHC), national guidance that can be adopted voluntarily by state and local jurisdictions to minimize the risk for illness and injury at public aquatic facilities. REPORTING PERIOD COVERED: 2013. DESCRIPTION OF SYSTEM: The Network for Aquatic Facility Inspection Surveillance (NAFIS) was established by CDC in 2013. NAFIS receives aquatic facility inspection data collected by environmental health practitioners when assessing the operation and maintenance of public aquatic facilities. This report presents inspection data that were reported by 16 public health agencies in five states (Arizona, California, Florida, New York, and Texas) and focuses on 15 MAHC elements deemed critical to minimizing the risk for illness and injury associated with aquatic facilities (e.g., disinfection to prevent transmission of infectious pathogens, safety equipment to rescue distressed bathers, and pool chemical safety). Although these data (the first and most recent that are available) are not nationally representative, 15.7% of the estimated 309,000 U.S. public aquatic venues are located in the 16 reporting jurisdictions. RESULTS: During 2013, environmental health practitioners in the 16 reporting NAFIS jurisdictions conducted 84,187 routine inspections of 48,632 public aquatic venues. Of the 84,187 routine inspection records for individual aquatic venues, 78.5% (66,098) included data on immediate closure; 12.3% (8,118) of routine inspections resulted in immediate closure because of at least one identified violation that represented a serious threat to public health. Disinfectant concentration violations were identified during 11.9% (7,662/64,580) of routine inspections, representing risk for aquatic facility-associated outbreaks of infectious etiology. Safety equipment violations were identified during 12.7% (7,845/61,648) of routine inspections, representing risk for drowning. Pool chemical safety violations were identified during 4.6% (471/10,264) of routine inspections, representing risk for pool chemical-associated health events. INTERPRETATION: Routine inspections frequently resulted in immediate closure and identified violations of inspection items corresponding to 15 MAHC elements critical to protecting public health, highlighting the need to improve operation and maintenance of U.S. public aquatic facilities. These findings also underscore the public health function that code enforcement, conducted by environmental health practitioners, has in preventing illness and injury at public aquatic facilities. PUBLIC HEALTH ACTION: Findings from the routine analyses of aquatic facility inspection data can inform program planning, implementation, and evaluation. At the state and local level, these inspection data can be used to identify aquatic facilities and venues in need of more frequent inspections and to select topics to cover in training for aquatic facility operators. At the national level, these data can be used to evaluate whether the adoption of MAHC elements minimizes the risk for aquatic facility-associated illness and injury. These findings also can be used to prioritize revisions or updates to the MAHC. To optimize the collection and analysis of aquatic facility inspection data and thus application of findings, environmental health practitioners and epidemiologists need to collaborate extensively to identify public aquatic facility code elements deemed critical to protecting public health and determine the best way to assess and document compliance during inspections.

Ionoprinted Multi-Responsive Hydrogel Actuators.

We report multi-responsive and double-folding bilayer hydrogel sheet actuators, whose directional bending response is tuned by modulating the solvent quality and temperature and where locally crosslinked regions, induced by ionoprinting, enable the actuators to invert their bending axis. The sheets are made multi-responsive by combining two stimuli responsive gels that incur opposing and complementary swelling and shrinking responses to the same stimulus. The lower critical solution temperature (LCST) can be tuned to specific temperatures depending on the EtOH concentration, enabling the actuators to change direction isothermally. Higher EtOH concentrations cause upper critical solution temperature (UCST) behavior in the poly(N-isopropylacrylamide) (pNIPAAm) gel networks, which can induce an amplifying effect during bilayer bending. External ionoprints reliably and repeatedly invert the gel bilayer bending axis between water and EtOH. Placing the ionoprint at the gel/gel interface can lead to opposite shape conformations, but with no clear trend in the bending behavior. We hypothesize that this is due to the ionoprint passing through the neutral axis of the bilayer during shrinking in hot water. Finally, we demonstrate the ability of the actuators to achieve shapes unique to the specific external conditions towards developing more responsive and adaptive soft actuator devices.