Time-resolved cryo-EM using Spotiton.

We present an approach for preparing cryo-electron microscopy (cryo-EM) grids to study short-lived molecular states. Using piezoelectric dispensing, two independent streams of ~50-pl droplets of sample are deposited within 10 ms of each other onto the surface of a nanowire EM grid, and the mixing reaction stops when the grid is vitrified in liquid ethane ~100 ms later. We demonstrate this approach for four biological systems where short-lived states are of high interest.

Pharmacy deserts: More than where pharmacies are.

BACKGROUND: In the United States, geographic access is a major driver of health care disparities. Studies have shown that pharmacy deserts are prevalent in the United States, even in major metropolitan areas. However, one limitation often cited by these studies is the use of distance rather than travel time to define pharmacy deserts. OBJECTIVE: The aim of this study was to assess pharmacy deserts using travel time and to provide a more holistic approach by incorporating analysis of private vehicles and public transportation. METHODS: Pharmacy details were collected from the National Provider Identifier database and neighborhood characteristics from collected census data for the four largest U.S. cities. Pharmacy access was evaluated using open-source routing engines. We determined neighborhoods in pharmacy deserts using both distance and travel time analyses. Sensitivity analysis was performed to determine changes to pharmacy deserts based on small changes in travel time. RESULTS: Of 4654 neighborhoods identified in the four cities of interest, 670 (14.4%) neighborhoods were in pharmacy deserts based on distance. Despite accounting for 28.9% of all neighborhoods, predominantly white neighborhoods only accounted for 4.3% of pharmacy deserts. When evaluating pharmacy deserts by car and public transportation, predominantly white neighborhoods accounted for 2.3% and 1.7% of total pharmacy deserts, respectively. Finally, by reducing travel time from 15 minutes to 10 minutes, pharmacy deserts by car and public transportation increased by 105% and 199%, respectively. All but 3 of the new pharmacy deserts found in the sensitivity analysis were found in nonpredominantly white neighborhoods. CONCLUSION: Using travel time and incorporating modes of transportation, we found that disparities in pharmacy access are more than just where pharmacies are located geographically. There are additional layers of disparities, such as access to public transportation, that need to be addressed to reduce the number of pharmacy deserts.

funtrp: identifying protein positions for variation driven functional tuning.

Evaluating the impact of non-synonymous genetic variants is essential for uncovering disease associations and mechanisms of evolution. An in-depth understanding of sequence changes is also fundamental for synthetic protein design and stability assessments. However, the variant effect predictor performance gain observed in recent years has not kept up with the increased complexity of new methods. One likely reason for this might be that most approaches use similar sets of gene and protein features for modeling variant effects, often emphasizing sequence conservation. While high levels of conservation highlight residues essential for protein activity, much of the variation observable in vivo is arguably weaker in its impact, thus requiring evaluation at a higher level of resolution. Here, we describe functionNeutral/Toggle/Rheostatpredictor (funtrp), a novel computational method that categorizes protein positions based on the position-specific expected range of mutational impacts: Neutral (weak/no effects), Rheostat (function-tuning positions), or Toggle (on/off switches). We show that position types do not correlate strongly with familiar protein features such as conservation or protein disorder. We also find that position type distribution varies across different protein functions. Finally, we demonstrate that position types can improve performance of existing variant effect predictors and suggest a way forward for the development of new ones.

The copBL operon protects Staphylococcus aureus from copper toxicity: CopL is an extracellular membrane-associated copper-binding protein.

As complications associated with antibiotic resistance have intensified, copper (Cu) is attracting attention as an antimicrobial agent. Recent studies have shown that copper surfaces decrease microbial burden, and host macrophages use Cu to increase bacterial killing. Not surprisingly, microbes have evolved mechanisms to tightly control intracellular Cu pools and protect against Cu toxicity. Here, we identified two genes (copB and copL) encoded within the Staphylococcus aureus arginine-catabolic mobile element (ACME) that we hypothesized function in Cu homeostasis. Supporting this hypothesis, mutational inactivation of copB or copL increased copper sensitivity. We found that copBL are co-transcribed and that their transcription is increased during copper stress and in a strain in which csoR, encoding a Cu-responsive transcriptional repressor, was mutated. Moreover, copB displayed genetic synergy with copA, suggesting that CopB functions in Cu export. We further observed that CopL functions independently of CopB or CopA in Cu toxicity protection and that CopL from the S. aureus clone USA300 is a membrane-bound and surface-exposed lipoprotein that binds up to four Cu+ ions. Solution NMR structures of the homologous Bacillus subtilis CopL, together with phylogenetic analysis and chemical-shift perturbation experiments, identified conserved residues potentially involved in Cu+ coordination. The solution NMR structure also revealed a novel Cu-binding architecture. Of note, a CopL variant with defective Cu+ binding did not protect against Cu toxicity in vivo Taken together, these findings indicate that the ACME-encoded CopB and CopL proteins are additional factors utilized by the highly successful S. aureus USA300 clone to suppress copper toxicity.

Geographical affiliation with top 10 NIH-funded academic medical centers and differences between mortality from cardiovascular disease and cancer.

Community engagement and rapid translation of findings for the benefit of patients has been noted as a major criterion for NIH decisions regarding allocation of funds for research priorities. We aimed to examine whether the presence of top NIH-funded institutions resulted in a benefit on the cardiovascular and cancer mortality of their local population. METHODS AND RESULTS: Based on the annual NIH funding of every academic medical from 1995 through 2014, the top 10 funded institutes were identified and the counties where they were located constituted the index group. The comparison group was created by matching each index county to another county which lacks an NIH-funded institute based on sociodemographic characteristics. We compared temporal trends of age-standardized cardiovascular mortality between the index counties and matched counties and states. This analysis was repeated for cancer mortality as a sensitivity analysis. From 1980 through 2014, the annual cardiovascular mortality rates declined in all counties. In the index group, the average decline in cardiovascular mortality rate was 51.5 per 100,000 population (95% CI, 46.8-56.2), compared to 49.7 per 100,000 population (95% CI, 45.9-53.5) in the matched group (P = .27). Trends in cardiovascular mortality of the index counties were similar to the cardiovascular mortality trends of their respective states. Cancer mortality rates declined at higher rates in counties with top NIH-funded medical centers (P < .001). CONCLUSIONS: Cardiovascular mortality rates have decreased with no apparent incremental benefit for communities with top NIH-funded institutions, underscoring the need for an increased focus on implementation science in cardiovascular diseases.

Kinetics of human brown adipose tissue activation and deactivation.

Brown adipose tissue (BAT) has been identified as a potential target in the treatment and prevention of obesity and metabolic disease. The precise kinetics of BAT activation and the duration of stimulus required to recruit metabolically active BAT, and its subsequent deactivation, are not well-understood. In this clinical trial, 19 healthy adults (BMI: 23.7 ± 0.7 kg/m2, Age: 31.2 ± 2.8 year, 12 female) underwent three different cooling procedures to stimulate BAT glucose uptake, and active BAT volume was determined using 18F-Fluorodeoxyglucose (FDG) PET/CT imaging. We found that 20 min of pre-injection cooling produces activation similar to the standard 60 min (39.9 mL vs. 44.2 mL, p = 0.52), indicating that BAT activity approaches its peak function soon after the initiation of cooling. Furthermore, upon removal of cold exposure, active BAT volume declines (13.6 mL vs. 44.2 mL, p = 0.002), but the deactivation process persists even hours following cessation of cooling. Thus, the kinetics of human BAT thermogenesis are characterized by a rapid increase soon after cold stimulation but a more gradual decline after rewarming. These characteristics reinforce the feasibility of developing mild, short-duration cold exposure to activate BAT and treat obesity and metabolic disease.

Identification of Antibody Targets for Tuberculosis Serology using High-Density Nucleic Acid Programmable Protein Arrays.

Better and more diverse biomarkers for the development of simple point-of-care tests for active tuberculosis (TB), a clinically heterogeneous disease, are urgently needed. We generated a proteomic Mycobacterium tuberculosis (Mtb) High-Density Nucleic Acid Programmable Protein Array (HD-NAPPA) that used a novel multiplexed strategy for expedited high-throughput screening for antibody responses to the Mtb proteome. We screened sera from HIV uninfected and coinfected TB patients and controls (n = 120) from the US and South Africa (SA) using the multiplex HD-NAPPA for discovery, followed by deconvolution and validation through single protein HD-NAPPA with biologically independent samples (n = 124). We verified the top proteins with enzyme-linked immunosorbent assays (ELISA) using the original screening and validation samples (n = 244) and heretofore untested samples (n = 41). We identified 8 proteins with TB biomarker value; four (Rv0054, Rv0831c, Rv2031c and Rv0222) of these were previously identified in serology studies, and four (Rv0948c, Rv2853, Rv3405c, Rv3544c) were not known to elicit antibody responses. Using ELISA data, we created classifiers that could discriminate patients' TB status according to geography (US or SA) and HIV (HIV- or HIV+) status. With ROC curve analysis under cross validation, the classifiers performed with an AUC for US/HIV- at 0.807; US/HIV+ at 0.782; SA/HIV- at 0.868; and SA/HIV+ at 0.723. With this study we demonstrate a new platform for biomarker/antibody screening and delineate its utility to identify previously unknown immunoreactive proteins.

Spotiton: New features and applications.

We present an update describing new features and applications of Spotiton, a novel instrument for vitrifying samples for cryoEM. We have used Spotiton to prepare several test specimens that can be reconstructed using routine single particle analysis to ∼3 Šresolution, indicating that the process has no apparent deleterious effect on the sample integrity. The system is now in routine and continuous use in our lab and has been used to successfully vitrify a wide variety of samples.

A Contra Capture Protein Array Platform for Studying Post-translationally Modified (PTM) Auto-antigenomes.

Aberrant modifications of proteins occur during disease development and elicit disease-specific antibody responses. We have developed a protein array platform that enables the modification of many proteins in parallel and assesses their immunogenicity without the need to express, purify, and modify proteins individually. We used anticitrullinated protein antibodies (ACPAs) in rheumatoid arthritis (RA) as a model modification and profiled antibody responses to ∼190 citrullinated proteins in 20 RA patients. We observed unique antibody reactivity patterns in both clinical anticyclic citrullinated peptide assay positive (CCP+) and CCP- RA patients. At individual antigen levels, we detected antibodies against known citrullinated autoantigens and discovered and validated five novel antibodies against specific citrullinated antigens (osteopontin (SPP1), flap endonuclease (FEN1), insulin like growth factor binding protein 6 (IGFBP6), insulin like growth factor I (IGF1) and stanniocalcin-2 (STC2)) in RA patients. We also demonstrated the utility of our innovative array platform in the identification of immune-dominant epitope(s) for citrullinated antigens. We believe our platform will promote the study of post-translationally modified antigens at a breadth that has not been achieved before, by both identifying novel autoantigens and investigating their roles in disease development. The developed platforms can potentially be used to study many autoimmune disease-relevant modifications and their immunogenicity.

Multiple Surface Regions on the Niemann-Pick C2 Protein Facilitate Intracellular Cholesterol Transport.

The cholesterol storage disorder Niemann-Pick type C (NPC) disease is caused by defects in either of two late endosomal/lysosomal proteins, NPC1 and NPC2. NPC2 is a 16-kDa soluble protein that binds cholesterol in a 1:1 stoichiometry and can transfer cholesterol between membranes by a mechanism that involves protein-membrane interactions. To examine the structural basis of NPC2 function in cholesterol trafficking, a series of point mutations were generated across the surface of the protein. Several NPC2 mutants exhibited deficient sterol transport properties in a set of fluorescence-based assays. Notably, these mutants were also unable to promote egress of accumulated intracellular cholesterol from npc2(-/-) fibroblasts. The mutations mapped to several regions on the protein surface, suggesting that NPC2 can bind to more than one membrane simultaneously. Indeed, we have previously demonstrated that WT NPC2 promotes vesicle-vesicle interactions. These interactions were abrogated, however, by mutations causing defective sterol transfer properties. Molecular modeling shows that NPC2 is highly plastic, with several intense positively charged regions across the surface that could interact favorably with negatively charged membrane phospholipids. The point mutations generated in this study caused changes in NPC2 surface charge distribution with minimal conformational changes. The plasticity, coupled with membrane flexibility, probably allows for multiple cholesterol transfer routes. Thus, we hypothesize that, in part, NPC2 rapidly traffics cholesterol between closely appositioned membranes within the multilamellar interior of late endosomal/lysosomal proteins, ultimately effecting cholesterol egress from this compartment.

The A1 Subunit of Shiga Toxin 2 Has Higher Affinity for Ribosomes and Higher Catalytic Activity than the A1 Subunit of Shiga Toxin 1.

Shiga toxin (Stx)-producing Escherichia coli (STEC) infections can lead to life-threatening complications, including hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS), which is the most common cause of acute renal failure in children in the United States. Stx1 and Stx2 are AB5 toxins consisting of an enzymatically active A subunit associated with a pentamer of receptor binding B subunits. Epidemiological evidence suggests that Stx2-producing E. coli strains are more frequently associated with HUS than Stx1-producing strains. Several studies suggest that the B subunit plays a role in mediating toxicity. However, the role of the A subunits in the increased potency of Stx2 has not been fully investigated. Here, using purified A1 subunits, we show that Stx2A1 has a higher affinity for yeast and mammalian ribosomes than Stx1A1. Biacore analysis indicated that Stx2A1 has faster association and dissociation with ribosomes than Stx1A1. Analysis of ribosome depurination kinetics demonstrated that Stx2A1 depurinates yeast and mammalian ribosomes and an RNA stem-loop mimic of the sarcin/ricin loop (SRL) at a higher catalytic rate and is a more efficient enzyme than Stx1A1. Stx2A1 depurinated ribosomes at a higher level in vivo and was more cytotoxic than Stx1A1 in Saccharomyces cerevisiae. Stx2A1 depurinated ribosomes and inhibited translation at a significantly higher level than Stx1A1 in human cells. These results provide the first direct evidence that the higher affinity for ribosomes in combination with higher catalytic activity toward the SRL allows Stx2A1 to depurinate ribosomes, inhibit translation, and exhibit cytotoxicity at a significantly higher level than Stx1A1.

Neutral and weakly nonneutral sequence variants may define individuality.

Large-scale computational analyses of the growing wealth of genome-variation data consistently tell two distinct stories. The first is expected: coding variants reported in disease-related databases significantly alter the function of affected proteins. The second is surprising: the genomes of healthy individuals appear to carry many variants that are predicted to have some effect on function. As long as the complete experimental analysis of all human genome variants remains impossible, computational methods, such as PolyPhen, SNAP, and SIFT, might provide important insights. These methods capture the effects of particular variants very well and can highlight trends in populations of variants. Diseases are, arguably, extreme phenotypic variations and are often attributable to one or a few severely functionally disruptive variants. Our findings suggest a genomic basis of the different nondisease phenotypes. Prediction methods indicate that variants in seemingly healthy individuals tend to be neutral or weakly disruptive for protein molecular function. These variant effects are predicted to be largely either experimentally undetectable or are not deemed significant enough to be published. This may suggest that nondisease phenotypes arise through combinations of many variants whose effects are weakly nonneutral (damaging or enhancing) to the molecular protein function but fall within the wild-type range of overall physiological function.

A student proposal for ethical guidelines in anatomical education: Ethical and policy considerations.

The recent publication of a story regarding anatomical dissection in a medical school has revealed the need for increased attention to the ethical and policy aspects of anatomical education. While most of the attention devoted to these questions thus far has been focused on procedures before and after dissection, from the perspective of medical students, there are important considerations during the process of dissection itself. This proposal was developed by two third-year medical students through a review of the relevant published literature, reflection upon their personal experiences in anatomy courses in two separate institutions, and informal discussion of these topics with peers. The proposal is that basic ethical guidelines should be established and monitored by an independent committee tasked with reviewing them. The proposed guidelines include: First, a clear set of expectations about what the student is expected to learn with respect to anatomical knowledge and dissection technique; second, the establishment by schools or national bodies of minimal ethical standards regarding respectful behavior toward the donor bodies, and the communication of these standards to teachers and students involved in educational dissections; third, the use of materials that encourage students to view their donors with respect and ensure proper treatment of them; and fourth, the establishment of an oversight group (at each medical school and at national level) comprising students, faculty, community members, and staff, who will regularly review the anatomical education program and update these ethical guidelines as appropriate. While many of these proposals are already implemented in some anatomy departments, the establishment of clear guidelines at a national as well as a school-by-school level will permit students the freedom to participate fully in their education, knowing they have met the highest ethical standards as they prepare for a career as a humanistic physician.

Bioethics, Religion, and Public Policy: Intersections, Interactions, and Solutions.

Bioethics in America positions itself as a totalizing discipline, capable of providing guidance to any individual within the boundaries of a health or medical setting. Yet the religiously observant or those driven by spiritual values have not universally accepted decisions made by "secular" bioethics, and as a result, religious bioethical thinkers and adherents have developed frameworks and rich counter-narratives used to fend off encroachment by policies perceived as threatening. This article uses brain death in Jewish law, the case of Jahi McMath, and vaccination refusal to observe how the religious system of ethics is presently excluded from bioethics and its implications.

Antiviral antibody profiling by high-density protein arrays.

Viral infections elicit antiviral antibodies and have been associated with various chronic diseases. Detection of these antibodies can facilitate diagnosis, treatment of infection, and understanding of the mechanisms of virus-associated diseases. In this work, we assayed antiviral antibodies using a novel high-density nucleic acid programmable protein array (HD-NAPPA) platform. Individual viral proteins were expressed in situ directly from plasmids encoding proteins in an array of microscopic reaction chambers. Quality of protein display and serum response was assured by comparing intra- and inter-array correlation within or between printing batches with average correlation coefficients of 0.91 and 0.96, respectively. HD-NAPPA showed higher signal-to-background ratio compared with standard NAPPA on planar glass slides and ELISA. Antibody responses to 761 antigens from 25 different viruses were profiled among patients with juvenile idiopathic arthritis and type 1 diabetes. Common and unique antibody reactivity patterns were detected between patients and healthy controls. We believe HD-viral-NAPPA will enable the study of host-pathogen interactions at unprecedented dimensions and elucidate the role of pathogen infections in disease development.