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Introduction: The COVID-19 pandemic in Haiti led to increased challenges for a population concurrently dealing with natural and social disasters, poor quality health care, lack of clean running water, and inadequate housing. In 2022, the population of Haiti was 11.4 million. While half a million vaccines for COVID-19 were donated by the United States to the government of Haiti, less than 5% of the population agreed to be vaccinated. This resulted in thousands of unused doses that were diverted to other countries. The purpose of this study was to evaluate population characteristics related to vaccine uptake in order to inform future interventions to improve COVID-19 vaccine uptake as well as inform strategies to safeguard against future global health security threats., Methods: This was a mixed-methods, cross-sectional study conducted in the West Department of Haiti within peri-urban communes. Participants consisted of adults in this setting responding to a survey from June 15 to July 18, 2022. The survey assessed demographic information, household characteristics, religious beliefs, past vaccine use, and current COVID-19 vaccine status. Multivariate regression modeling was conducted to assess predictors of vaccine hesitancy. Qualitative focus group discussion participants were recruited and focus group discussions were conducted between August-September 2022 among community groups, religious leaders and health professionals to provide additional, community-level context on perceptions of the COVID-19 pandemic and vaccines., Results: A total of 1,923 respondents completed the survey; of which a majority were male (52.7%), between the age of 18-35 (58.5%), had a medical visit within the last year (63.0%) and received the COVID-19 vaccine (46.1%). Compared to those who had been COVID-19 vaccinated, participants who had not been vaccinated were more likely to be male (57.7% vs 46.8%, p < .0001), have completed secondary education (30.5% vs 16.6%, p < .001), unemployed (20.3% vs 7.3%, p < .0001) and had a medical visit 3 or more years ago (30.2% vs 11.2%, p < .0001). Unvaccinated COVID-19 participants were also more likely to have never received any other vaccine (36.1% vs22.5%, p < .0001), have a religious leader speak out against the vaccine (20.0% vs 13.1%, p < .0001), not believe in the effectiveness of the vaccine (51.2% vs 9.1%, p < .0001) and did not trust the healthcare worker administering the vaccine (35.2% vs 3.8%, p<0.0001)., Conclusion: These results show that targeted interventions to religious leaders and health care workers on how to engage with the community and share clearer messages around the COVID-19 vaccination may result in increased vaccine uptake. Results also shed light on how activities surrounding vaccinations can be tailored to meet client needs addressing the misinformation encountered to achieve greater health impact thereby safeguarding the population against future global health security threats., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Etienne-Mesubi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Distinguishing proton-coupled electron transfer (PCET) from uncoupled electron transfer (ET) in proteins can be challenging. A recent investigation [J. C. Koone, M. Simmang, D. L. Saenger, M. L. Hunsicker-Wang and B. F. Shaw, J. Am. Chem. Soc. , 145 , 16488-16497] reported that protein charge ladders and capillary electrophoresis can distinguish between single ET, PCET, and two-proton coupled ET (2PCET) by directly measuring the change in protein net charge upon reduction/oxidation (Δ Z ET ). The current study used similar methods to assess PCET in zinc-free, "double copper" superoxide dismutase-1 (4Cu-SOD1), where one copper is bound at the copper site of each monomer and one copper is bound at the bridging zinc site, resulting in a quasi-type III Cu center. At pH 7.4, the net charge ( Z ) of the 4Cu-SOD1 dimer was unaffected by reduction of all four Cu 2+ ions, i.e. , Δ Z 4ET = -0.09 ± 0.05 per dimer (-0.02 ± 0.01 per copper atom). These values suggest that PCET is taking place at all four Cu atoms of the homodimer. Molecular dynamics and Poisson-Boltzmann calculations suggest that a metal-coordinating histidine at the zinc site (His71) is the proton acceptor. These data show how ligands of a naturally occurring zinc site can help facilitate PCET when the right redox metal is bound.

The de novo design of miniprotein inhibitors has recently emerged as a new technology to create proteins that bind with high affinity to specific therapeutic targets. Their size, ease of expression, and apparent high stability makes them excellent candidates for a new class of protein drugs. However, beyond circular dichroism melts and hydrogen/deuterium exchange experiments, little is known about their dynamics, especially at the elevated temperatures they seemingly tolerate quite well. To address that and gain insight for future designs, we have focused on identifying unintended and previously overlooked heat-induced structural and chemical changes in a particularly stable model miniprotein, EHEE&#95;rd2&#95;0005. Nuclear magnetic resonance (NMR) studies suggest the presence of dynamics on multiple time and temperature scales. Transiently elevating the temperature results in spontaneous chemical deamidation visible in the NMR spectra, which we validate using both capillary electrophoresis and mass spectrometry (MS) experiments. High temperatures also result in greatly accelerated intrinsic rates of hydrogen exchange and signal loss in NMR heteronuclear single quantum coherence spectra from local unfolding. These losses are in excellent agreement with both room temperature hydrogen exchange experiments and hydrogen bond disruption in replica exchange molecular dynamics simulations. Our analysis reveals important principles for future miniprotein designs and the potential for high stability to result in long-lived alternate conformational states., (© 2024 The Protein Society.)

People with blindness have limited access to the high-resolution graphical data and imagery of science. Here, a lithophane codex is reported. Its pages display tactile and optical readouts for universal visualization of data by persons with or without eyesight. Prototype codices illustrated microscopy of butterfly chitin-from N -acetylglucosamine monomer to fibril, scale, and whole insect-and were given to high schoolers from the Texas School for the Blind and Visually Impaired. Lithophane graphics of Fischer-Spier esterification reactions and electron micrographs of biological cells were also 3D-printed, along with x-ray structures of proteins (as millimeter-scale 3D models). Students with blindness could visualize (describe, recall, distinguish) these systems-for the first time-at the same resolution as sighted peers (average accuracy = 88%). Tactile visualization occurred alongside laboratory training, synthesis, and mentoring by chemists with blindness, resulting in increased student interest and sense of belonging in science.

Programs aiming to reduce intimate partner violence (IPV) increasingly seek to shift social norms. Few interventions have been rigorously evaluated for their impact on norms and incidence of IPV, particularly in sub-Saharan Africa. Shifting norms at the community level and subsequent pathways to behavior change remain poorly understood. We assessed shifts in individual- and couple-level factors, social norms, and IPV from an 18-month community-based trial of the Masculinity, Faith, and Peace (MFP) program-a faith-based, norms-shifting approach-in Plateau state, Nigeria. This study was part of a community-based, mixed-methods, two-arm cluster randomized control trial (cRCT) to evaluate the MFP program. Quantitative surveys were conducted with women 18 to 35 years old ( n  = 350) and their male partners ( n  = 281). Respondents came from 10 Muslim and 10 Christian congregations. Social norms were measured based on results from factor analysis. Intent-to-treat analyses assessed intervention effects. Qualitative research in MFP congregations explored pathways of change. All forms of IPV reduced over time among MFP participants. Regression analyses showed a significant 61% reduction in odds of reporting experiencing any IPV among women, a 64% reduction among Christians, and a 44% reduction in MFP congregations compared to their respective controls. In addition to improvement in norms, we found significant intervention effects on individual attitudes toward IPV and gender roles, relationship quality, and community cohesion. Qualitative findings reinforce these pathways, suggesting that critical reflection and dialogue on existing norms and the focus on faith and religious texts were valued by participants and supported IPV reductions. This study demonstrates that a faith-based, norms-shifting intervention can significantly reduce IPV in a short time. There are several pathways through which MFP reduced IPV, including shifts in norms, attitudes, relationship quality, and community cohesion.

The degree to which redox-driven proton pumps regulate net charge during electron transfer (ΔZ ET ) remains undetermined due to difficulties in measuring the net charge of solvated proteins. Values of ΔZ ET can reflect reorganization energies or redox potentials associated with ET and can be used to distinguish ET from proton(s)-coupled electron transfer (PCET). Here, we synthesized protein "charge ladders" of a Rieske [2Fe-2S] subunit from Thermus thermophilus (trunc Tt Rp) and made 120 electrostatic measurements of ΔZ ET across pH. Across pH 5-10, trunc Tt Rp is suspected of transitioning from ET to PCET, and then to two proton-coupled ET (2PCET). Upon reduction, we found that trunc Tt Rp became more negative at pH 6.0 by one unit (ΔZ ET = -1.01 ± 0.14), consistent with single ET; was isoelectric at pH 8.8 (ΔZ ET = -0.01 ± 0.45), consistent with PCET; and became more positive at pH 10.6 (ΔZ ET = +1.37 ± 0.60), consistent with 2PCET. These ΔZ ET values are attributed to protonation of H154 and H134. Across pH, redox potentials of Tt Rp (measured previously) correlated with protonation energies of H154 and H134 and ΔZ ET for trunc Tt Rp, supporting a discrete proton pumping mechanism for Rieske proteins at the Fe-coordinating histidines.

The heterodimerization of wild-type (WT) Cu, Zn superoxide dismutase-1 (SOD1) and mutant SOD1 might be a critical step in the pathogenesis of SOD1-linked amyotrophic lateral sclerosis (ALS). Post-translational modifications that accelerate SOD1 heterodimerization remain unidentified. Here, we used capillary electrophoresis to quantify the effect of cysteine-111 oxidation on the rate and free energy of ALS mutant/WT SOD1 heterodimerization. The oxidation of Cys 111 -β-SH to sulfinic and sulfonic acid (by hydrogen peroxide) increased rates of heterodimerization (with unoxidized protein) by ∼3-fold. Cysteine oxidation drove the equilibrium free energy of SOD1 heterodimerization by up to ΔΔ G = -5.11 ± 0.36 kJ mol -1 . Molecular dynamics simulations suggested that this enhanced heterodimerization, between oxidized homodimers and unoxidized homodimers, was promoted by electrostatic repulsion between the two "dueling" Cys 111 -SO 2 - /SO 3 - , which point toward one another in the homodimeric state. Together, these results suggest that oxidation of Cys-111 promotes subunit exchange between oxidized homodimers and unoxidized homodimers, regardless of whether they are mutant or WT dimers.

The heterodimerization of WT Cu, Zn superoxide dismutase-1 (SOD1), and mutant SOD1 might be a critical step in the pathogenesis of SOD1-linked amyotrophic lateral sclerosis (ALS). Rates and free energies of heterodimerization (ΔG Het ) between WT and ALS-mutant SOD1 in mismatched metalation states-where one subunit is metalated and the other is not-have been difficult to obtain. Consequently, the hypothesis that under-metalated SOD1 might trigger misfolding of metalated SOD1 by "stealing" metal ions remains untested. This study used capillary zone electrophoresis and mass spectrometry to track heterodimerization and metal transfer between WT SOD1, ALS-variant SOD1 (E100K, E100G, D90A), and triply deamidated SOD1 (modeled with N26D/N131D/N139D substitutions). We determined that rates of subunit exchange between apo dimers and metalated dimers-expressed as time to reach 30% heterodimer-ranged from t 30%  = 67.75 ± 9.08 to 338.53 ± 26.95 min; free energies of heterodimerization ranged from ΔG Het  = -1.21 ± 0.31 to -3.06 ± 0.12 kJ/mol. Rates and ΔG Het values of partially metalated heterodimers were more similar to those of fully metalated heterodimers than apo heterodimers, and largely independent of which subunit (mutant or WT) was metal-replete or metal-free. Mass spectrometry and capillary electrophoresis demonstrated that mutant or WT 4Zn-SOD1 could transfer up to two equivalents of Zn 2+ to mutant or WT apo-SOD1 (at rates faster than the rate of heterodimerization). This result suggests that zinc-replete SOD1 can function as a chaperone to deliver Zn 2+ to apo-SOD1, and that WT apo-SOD1 might increase the toxicity of mutant SOD1 by stealing its Zn 2+ ., Competing Interests: Conflict of interest The authors declare no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

People who are blind do not have access to graphical data and imagery produced by science. This exclusion complicates learning and data sharing between sighted and blind persons. Because blind people use tactile senses to visualize data (and sighted people use eyesight), a single data format that can be easily visualized by both is needed. Here, we report that graphical data can be three-dimensionally printed into tactile graphics that glow with video-like resolution via the lithophane effect. Lithophane forms of gel electropherograms, micrographs, electronic and mass spectra, and textbook illustrations could be interpreted by touch or eyesight at ≥79% accuracy ( n = 360). The lithophane data format enables universal visualization of data by people regardless of their level of eyesight.

Background: Early adolescence is a critical period where social norms, attitudes, and behaviors around gender equality form. Social norms influence adolescent choices and behaviors and are reinforced by caregivers and community members, affecting girls' reproductive health and educational opportunities. Understanding how to shift these often-interconnected norms to delay child marriage, pregnancy and keep girls in school requires understanding of the structure and dynamics of family and community systems. The Senegalese and American non-governmental organization, the Grandmothers Project-Change through Culture, seeks to address these intertwined factors through innovative community change strategies that build on the specific structure and values of West African collectivist cultures., Methods: The Girls' Holistic Development approach in rural Vélingara, Senegal posits that by increasing recognition, knowledge and empowerment of elder community women and reinforcing intergenerational communication and decision-making, community members including girls will support and advocate on behalf of girls' interests and desires. We assessed the Girls Holistic Development approach using Realist Evaluation with a mixed-method, quasi-experimental design with a comparison population. We examined differences in intergenerational communication, decision-making and descriptive and injunctive norms related to early marriage, pregnancy and schooling., Results: After 18 months, intergenerational communication was more likely, grandmothers felt more valued in their communities, adolescent girls felt more supported with improved agency, and norms were shifting to support delayed marriage and pregnancy and keeping girls in school. Grandmothers in intervention villages were statistically significantly more likely to be perceived as influential decision-makers by both VYA girls and caregivers for marriage and schooling decisions compared to girls and caregivers in comparison villages., Conclusions: This realist evaluation demonstrated shift in social norms, particularly for VYA girls, in intervention villages favoring delaying girls' marriage, preventing early pregnancy and keeping girls in school along with increased support for and action by grandmothers to support girls and their well-being related to these same outcomes. These shifts represent greater community social cohesion on girl-child issues. This research helps explain the linkage between social norms and girls' reproductive health and education outcomes and demonstrates that normative shifts can lead to behavior change via collective community action mechanisms., (© 2021. The Author(s).)

Introduction: Skilled preceptors are crucial to the pharmacy profession as they contribute to the growth and development of student pharmacists and resident pharmacists. As a result, providing education and further growth for preceptors is vital. The purpose of this survey was to determine preceptor development topics of interest and preferred platforms for receiving education., Methods: A survey was conducted by the American Society of Health-System Pharmacists Section Advisory Group on Pharmacy Practice Experience Precepting. The survey was designed to identify preceptor needs based on experience and background. Professional development opportunities, tools needed to assist preceptors, and the preferred method of delivery were also determined., Results: Two hundred seventy-two pharmacists completed the entire 30 question survey. On demand webinars were identified as the preferred method of education delivery. A preceptor tip of the week email was selected as the most favored type of online education resource for preceptors. There was no major difference on the types of online resources for students that preceptors would use. A survey to assist in self-identifying areas for developmental improvement was favored by 81% of respondents., Conclusions: This needs assessment identified that preceptor development materials are in high demand for all surveyed topic areas provided via live and/or on demand webinars or other virtual means. Accessibility of resources should be highlighted in multiple forums in order to ensure the information reaches all preceptors., Competing Interests: Declaration of Competing Interest None., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Theory predicts that the net charge (Z) of a protein can be altered by the net charge of a neighboring protein as the two approach one another below the Debye length. This type of charge regulation suggests that a protein's charge and perhaps function might be affected by neighboring proteins without direct binding. Charge regulation during protein crowding has never been directly measured due to analytical challenges. Here, we show that lysine specific protein crosslinkers (NHS ester-Staudinger pairs) can be used to mimic crowding by linking two non-interacting proteins at a maximal distance of ~7.9 Å. The net charge of the regioisomeric dimers and preceding monomers can then be determined with lysine-acyl "protein charge ladders" and capillary electrophoresis. As a proof of concept, we covalently linked myoglobin (Z monomer  = -0.43 ± 0.01) and α-lactalbumin (Z monomer  = -4.63 ± 0.05). Amide hydrogen/deuterium exchange and circular dichroism spectroscopy demonstrated that crosslinking did not significantly alter the structure of either protein or result in direct binding (thus mimicking crowding). Ultimately, capillary electrophoretic analysis of the dimeric charge ladder detected a change in charge of ΔZ = -0.04 ± 0.09 upon crowding by this pair (Z dimer  = -5.10 ± 0.07). These small values of ΔZ are not necessarily general to protein crowding (qualitatively or quantitatively) but will vary per protein size, charge, and solvent conditions., (© 2021 The Protein Society.)

Repulsive electrostatic forces between prion-like proteins are a barrier against aggregation. In neuropharmacology, however, a prion's net charge (Z) is not a targeted parameter. Compounds that selectively boost prion Z remain unreported. Here, we synthesized compounds that amplified the negative charge of misfolded superoxide dismutase-1 (SOD1) by acetylating lysine-NH 3 + in amyloid-SOD1, without acetylating native-SOD1. Compounds resembled a "ball and chain" mace: a rigid amyloid-binding "handle" (benzothiazole, stilbene, or styrylpyridine); an aryl ester "ball"; and a triethylene glycol chain connecting ball to handle. At stoichiometric excess, compounds acetylated up to 9 of 11 lysine per misfolded subunit (ΔZ fibril =-8100 per 10 3 subunits). Acetylated amyloid-SOD1 seeded aggregation more slowly than unacetylated amyloid-SOD1 in vitro and organotypic spinal cord (these effects were partially due to compound binding). Compounds exhibited reactivity with other amyloid and non-amyloid proteins (e.g., fibrillar α-synuclein was peracetylated; serum albumin was partially acetylated; carbonic anhydrase was largely unacetylated)., (© 2021 Wiley-VCH GmbH.)

Handheld models help students visualize three-dimensional (3D) objects, especially students with blindness who use large 3D models to visualize imagery by hand. The mouth has finer tactile sensors than hand, which could improve visualization using microscopic models that are portable, inexpensive, and disposable. The mouth remains unused in tactile learning. Here, we created bite-size 3D models of protein molecules from "gummy bear" gelatin or nontoxic resin. Models were made as small as rice grain and could be coded with flavor and packaged like candy. Mouth, hands, and eyesight were tested at identifying specific structures. Students recognized structures by mouth at 85.59% accuracy, similar to recognition by eyesight using computer animation. Recall accuracy of structures was higher by mouth than hand for 40.91% of students, equal for 31.82%, and lower for 27.27%. The convenient use of entire packs of tiny, cheap, portable models can make 3D imagery more accessible to students., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

The degree by which metalloproteins partially regulate net charge (Z) upon electron transfer (ET) was recently measured for the first time using "protein charge ladders" of azurin, cytochrome c, and myoglobin [Angew. Chem. Int. Ed. 2018, 57(19), 5364-5368; Angew. Chem. 2018, 130, 5462-5466]. Here, we show that Cu, Zn superoxide dismutase (SOD1) is unique among proteins in its ability to resist changes in net charge upon single ET (e.g., ΔZ ET(SOD1) =0.05±0.08 per electron, compared to ΔZ ET(Cyt-c) =1.19±0.02). This total regulation of net charge by SOD1 is attributed to the protonation of the bridging histidine upon copper reduction, yielding redox centers that are isoelectric at both copper oxidation states. Charge regulation by SOD1 would prevent long range coulombic perturbations to residue pK a 's upon ET at copper, allowing SOD1's "electrostatic loop" to attract superoxide with equal affinity (at both redox states of copper) during diffusion-limited reduction and oxidation of superoxide., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Many patients with a self-reported penicillin allergy go on to tolerate beta-lactam antibiotics. Allergy specialists may be consulted to determine the nature and extent of the allergy. However, electronic allergy records must be appropriately updated such that recommendations are carried forward., Objective: To determine the percentage of patients who have their electronic allergy record updated after an allergy service consult (ASC)., Methods: This was a retrospective study of patients with at least 1 documented beta-lactam allergy and had an ASC during (inpatient) or prior to (outpatient) hospital admission at Northwestern Memorial Hospital and Prentice Women's Hospital in Chicago, Illinois., Results: Within the study period, a total of 26 526 patients were identified as having a documented antibiotic allergy, with 21 657 patients (81.6% of patients with allergies) having a listed beta-lactam allergy. Of these patients, 1689 (7.8%) patients were identified as having an ASC during or prior to admission, with 598 patients meeting inclusion criteria. Changes in the allergy record were recommended by the ASC for 62% (n = 371) of patients; however, the allergy record was updated after the ASC in 74.9% (n = 278) of patients., Conclusion: ASC recommendations to delabel a patient as beta-lactam allergic must result in updating the allergy record in order to optimize future treatment. Given the low proportion of allergy-labeled patients tested, programs outside formal ASCs should be considered.

The unseeded aggregation of superoxide dismutase-1 (SOD1) into amyloid-like fibrils occurs stochastically in vitro and in vivo , that is, isolated populations of SOD1 proteins (within microplate wells or living cells) self-assemble into amyloid at rates that span a probability distribution. This stochasticity has been attributed to variable degrees of monomer depletion by competing pathways of amorphous and fibrillar aggregation (inter alia). Here, microplate-based thioflavin-T (ThT) fluorescence assays were performed at high iteration (∼300) to establish whether this observed stochasticity persists when progenitor ("parent") SOD1 fibrils are used to seed the formation of multiple generations of progeny fibrils (daughter, granddaughter, and great-granddaughter fibrils). Populations of progenitor fibrils formed stochastically at different rates and fluorescence intensity, however, progeny fibrils formed at more similar rates regardless of the formation rate of the progenitor fibril. For example, populations of progenitor fibrils that formed with a lag time of ∼30 h or ∼15 h both produced progeny fibrils with lag times of ∼8 h. Likewise, populations of progenitor fibrils with high or low maximum fluorescence (e.g., ∼450 or ∼75 A.U.) both produced progeny fibrils with more similar maximum fluorescence (∼125 A.U.). The rate of propagation was found to be more dependent on monomer concentration than seed concentration. These results can be rationalized by classical rate laws for primary nucleation and monomer-dependent secondary nucleation. We also find that the seeding propensity of some "families" of in vitro grown fibrils exhibit a finite lifetime (similar to that observed in the seeding of small molecule crystals and colloids). The single biological takeaway of this study is that the concentration of native SOD1 in a cell can have a stronger effect on rates of seeded aggregation than the concentration of prion-like seed that infected the cell.

The "red reflex test" is used to screen children for leukocoria ("white eye") in a standard pediatric examination, but is ineffective at detecting many eye disorders. Leukocoria also presents in casual photographs. The clinical utility of screening photographs for leukocoria is unreported. Here, a free smartphone application (CRADLE: ComputeR-Assisted Detector of LEukocoria) was engineered to detect photographic leukocoria and is available for download under the name "White Eye Detector." This study determined the sensitivity, specificity, and accuracy of CRADLE by retrospectively analyzing 52,982 longitudinal photographs of children, collected by parents before enrollment in this study. The cohort included 20 children with retinoblastoma, Coats' disease, cataract, amblyopia, or hyperopia and 20 control children. For 80% of children with eye disorders, the application detected leukocoria in photographs taken before diagnosis by 1.3 years (95% confidence interval, 0.4 to 2.3 years). The CRADLE application allows parents to augment clinical leukocoria screening with photography., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Meeting the reproductive health needs of women in post-conflict settings is a global health priority. In the Democratic Republic of the Congo, social norms perpetuate gender-based violence and contribute to low contraceptive use and high fertility. The Masculinité, Famille, et Foi (MFF) intervention is working with communities in Kinshasa to create normative environments supportive of modern contraception access and use. Our analysis uses survey data collected from 900 men and women in 17 community groups prior to the MFF intervention. We aimed to measure the extent to which social norms influence intentions to use modern contraception. Using multiple items to assess social norms and reference groups related to family planning and gender equity, we identified four distinct social norms constructs through factor analysis. Through structural equation modeling, we found that social norms influence intentions to use modern contraception overall, but that normative influence varies by gender., Competing Interests: The authors have declared that no competing interests exist.

The net electrostatic charge (Z) of a folded protein in solution represents a bird's eye view of its surface potentials-including contributions from tightly bound metal, solvent, buffer, and cosolvent ions-and remains one of its most enigmatic properties. Few tools are available to the average biochemist to rapidly and accurately measure Z at pH≠pI. Tools that have been developed more recently seem to go unnoticed. Most scientists are content with this void and estimate the net charge of a protein from its amino acid sequence, using textbook values of pK a . Thus, Z remains unmeasured for nearly all folded proteins at pH≠pI. When marveling at all that has been learned from accurately measuring the other fundamental property of a protein-its mass-one wonders: what are we missing by not measuring the net charge of folded, solvated proteins? A few big questions immediately emerge in bioinorganic chemistry. When a single electron is transferred to a metalloprotein, does the net charge of the protein change by approximately one elementary unit of charge or does charge regulation dominate, that is, do the pK a values of most ionizable residues (or just a few residues) adjust in response to (or in concert with) electron transfer? Would the free energy of charge regulation (ΔΔG z ) account for most of the outer sphere reorganization energy associated with electron transfer? Or would ΔΔG z contribute more to the redox potential? And what about metal binding itself? When an apo-metalloprotein, bearing minimal net negative charge (e.g., Z=-2.0) binds one or more metal cations, is the net charge abolished or inverted to positive? Or do metalloproteins regulate net charge when coordinating metal ions? The author's group has recently dusted off a relatively obscure tool-the "protein charge ladder"-and used it to begin to answer these basic questions., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This article describes datasets for the FPwatch Project, a comprehensive facility-based family planning survey conducted by Population Services International in five countries in Africa and Asia from 2015 to 2017. Contents cover research design and background, methodology, sample selection, data collection, an overview of FPwatch indicators, and quality assurance measures taken. These datasets from the Democratic Republic of Congo, Ethiopia, India, Myanmar, and Nigeria complement other facility-based family planning surveys and are unique in their large-scale, standardized methodology, and comprehensive sampling approach. In addition, all datasets but Myanmar (private only) include both private and public facilities, a feature that gives a more complete picture of the family planning supply environment. Because of these factors, the data is well suited to inform global family planning efforts., (© 2018 The Authors Studies in Family Planning published by Wiley Periodicals, Inc. on behalf of Population Council.)

Interactions between superoxide dismutase-1 (SOD1) and lipid membranes might be directly involved in the toxicity and intercellular propagation of aggregated SOD1 in amyotrophic lateral sclerosis (ALS), but the chemical details of lipid-SOD1 interactions and their effects on SOD1 aggregation remain unclear. This paper determined the rate and mechanism of nucleation of fibrillar apo-SOD1 catalyzed by liposomal surfaces with identical hydrophobic chains (RCH 2 (O 2 C 18 H 33 ) 2 ), but headgroups of different net charge and hydrophobicity (i.e., R(CH 2 )N + (CH 3 ) 3 , RPO 4 - (CH 2 ) 2 N + (CH 3 ) 3 , and RPO 4 - ). Under semiquiescent conditions (within a 96 well microplate, without a gyrating bead), the aggregation of apo-SOD1 into thioflavin-T-positive (ThT(+)) amyloid fibrils did not occur over 120 h in the absence of liposomal surfaces. Anionic liposomes triggered aggregation of apo-SOD1 into ThT(+) amyloid fibrils; cationic liposomes catalyzed fibrillization but at slower rates and across a narrower lipid concentration; zwitterionic liposomes produced nonfibrillar (amorphous) aggregates. The inability of zwitterionic liposomes to catalyze fibrillization and the dependence of fibrillization rate on anionic lipid concentration suggests that membranes catalyze SOD1 fibrillization by a primary nucleation mechanism. Membrane-catalyzed fibrillization was also examined for eight ALS variants of apo-SOD1, including G37R, G93R, D90A, and E100G apo-SOD1 that nucleate slower than or equal to WT SOD1 in lipid-free, nonquiescent amyloid assays. All ALS variants (with one exception) nucleated faster than WT SOD1 in the presence of anionic liposomes, wherein the greatest acceleratory effects were observed among variants with lower net negative surface charge (G37R, G93R, D90A, E100G). The exception was H46R apo-SOD1, which did not form ThT(+) species.

Determining whether a protein regulates its net electrostatic charge during electron transfer (ET) will deepen our mechanistic understanding of how polypeptides tune rates and free energies of ET (e.g., by affecting reorganization energy, and/or redox potential). Charge regulation during ET has never been measured for proteins because few tools exist to measure the net charge of a folded protein in solution at different oxidation states. Herein, we used a niche analytical tool (protein charge ladders analyzed with capillary electrophoresis) to determine that the net charges of myoglobin, cytochrome c, and azurin change by 0.62±0.06, 1.19±0.02, and 0.51±0.04 units upon single ET. Computational analysis predicts that these fluctuations in charge arise from changes in the pK a  values of multiple non-coordinating residues (predominantly histidine) that involve between 0.42-0.90 eV. These results suggest that ionizable residues can tune the reactivity of redox centers by regulating the net charge of the entire protein-cofactor-solvent complex., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: In developing regions, an estimated 214 million women have an unmet need for family planning. Reaching Family Planning 2020 (FP2020) commitments will require a shift in modern contraceptive promotion, including improved access to long-acting reversible contraceptives (LARCs). Until now, a lack of market data limited understanding of the potential of LARCs to increase contraceptive access and choice., Methods: From 2015, the FPwatch Project conducted representative surveys in Ethiopia, Nigeria, and Democratic Republic of Congo (DRC) using a full census approach in selected administrative areas. In these areas, every public and private sector outlet with the potential to sell or distribute modern contraceptives was approached. In outlets with modern contraceptives, product audits and provider interviews assessed contraceptive market composition, market share, availability, price, and outlet readiness to perform services., Results: Fifty-four percent of outlets in Ethiopia had LARC commodities or services available at the time of the survey, versus 7% and 8% of outlets in Nigeria and DRC, respectively. When present, LARCs were usually available with at least two other methods (99%, 39%, and 84% of public health facilities in Ethiopia, Nigeria and DRC, respectively). Many public facilities had both implants and IUDs in stock (76%, 47%, and 53%, respectively). Lack of readiness to provide LARCs was mostly due to a lack of equipment, private room, or the commodity itself. Market share for implants in the public sector was 60%, 53%, and 37% of Couple Years of Protection (CYP) in Ethiopia, Nigeria, and DRC., Discussion: Limited availability of LARCs in Nigeria and DRC restricts contraceptive choice and makes it difficult for women to adopt and use modern contraception consistently. Brand-specific subsidies, task shifting, and promotion of methods that require less equipment and training are promising strategies for increasing uptake. Substantial government investment is required to improve availability and affordability. Investment in implants should be prioritized to make progress towards FP2020 commitments.

Background: An estimated 214 million women have unmet need for family planning in developing regions. Improved utilization of the private sector is key to achieving universal access to a range of safe and effective modern contraceptive methods stipulated by FP2020 and SDG commitments. Until now, a lack of market data has limited understanding of the private sector's role in increasing contraceptive coverage and choice., Methods: In 2015, the FPwatch Project conducted representative outlet surveys in Ethiopia, Nigeria, and DRC using a full census approach in selected administrative areas. Every public and private sector outlet with the potential to sell or distribute modern contraceptives was approached. In outlets with modern contraceptives, product audits and provider interviews assessed contraceptive market composition, availability, and price., Findings: Excluding general retailers, 96% of potential outlets in Ethiopia, 55% in Nigeria, and 41% in DRC had modern contraceptive methods available. In Ethiopia, 41% of modern contraceptive stocking outlets were in the private sector compared with approximately 80% in Nigeria and DRC where drug shops were dominant. Ninety-five percent of private sector outlets in Ethiopia had modern contraceptive methods available; 37% had three or more methods. In Nigeria and DRC, only 54% and 42% of private sector outlets stocked modern contraceptives with 5% and 4% stocking three or more methods, respectively. High prices in Nigeria and DRC create barriers to consumer access and choice., Discussion: There is a missed opportunity to provide modern contraception through the private sector, particularly drug shops. Subsidies and interventions, like social marketing and social franchising, could leverage the private sector's role in increasing access to a range of contraceptives. Achieving global FP2020 commitments depends on the expansion of national contraceptive policies that promote greater partnership and cooperation with the private sector and improvement of decisions around funding streams of countries with large populations and high unmet need like Ethiopia, Nigeria, and DRC.

The acylation of lysine residues in superoxide dismutase-1 (SOD1) has been previously shown to decrease its rate of nucleation and elongation into amyloid-like fibrils linked to amyotrophic lateral sclerosis. The chemical mechanism underlying this effect is unclear, i.e. hydrophobic/steric effects versus electrostatic effects. Moreover, the degree to which the acylation might alter the prion-like seeding of SOD1 in vivo has not been addressed. Here, we acylated a fraction of lysine residues in SOD1 with groups of variable hydrophobicity, charge, and conformational entropy. The effect of each acyl group on the rate of SOD1 fibril nucleation and elongation were quantified in vitro with thioflavin-T (ThT) fluorescence, and we performed 594 iterate aggregation assays to obtain statistically significant rates. The effect of the lysine acylation on the prion-like seeding of SOD1 was assayed in spinal cord extracts of transgenic mice expressing a G85R SOD1-yellow fluorescent protein construct. Acyl groups with >2 carboxylic acids diminished self-assembly into ThT-positive fibrils and instead promoted the self-assembly of ThT-negative fibrils and amorphous complexes. The addition of ThT-negative, acylated SOD1 fibrils to organotypic spinal cord failed to produce the SOD1 inclusion pathology that typically results from the addition of ThT-positive SOD1 fibrils. These results suggest that chemically increasing the net negative surface charge of SOD1 via acylation can block the prion-like propagation of oligomeric SOD1 in spinal cord., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Over 150 mutations in SOD1 (superoxide dismutase-1) cause amyotrophic lateral sclerosis (ALS), presumably by accelerating SOD1 amyloidogenesis. Like many nucleation processes, SOD1 fibrillization is stochastic (in vitro), which inhibits the determination of aggregation rates (and obscures whether rates correlate with patient phenotypes). Here, we diverged from classical chemical kinetics and used Kaplan-Meier estimators to quantify the probability of apo-SOD1 fibrillization (in vitro) from ∼10 3 replicate amyloid assays of wild-type (WT) SOD1 and nine ALS variants. The probability of apo-SOD1 fibrillization (expressed as a Hazard ratio) is increased by certain ALS-linked SOD1 mutations but is decreased or remains unchanged by other mutations. Despite this diversity, Hazard ratios of fibrillization correlated linearly with (and for three mutants, approximately equaled) Hazard ratios of patient survival (R 2 = 0.67; Pearson's r = 0.82). No correlation exists between Hazard ratios of fibrillization and age of initial onset of ALS (R 2 = 0.09). Thus, Hazard ratios of fibrillization might explain rates of disease progression but not onset. Classical kinetic metrics of fibrillization, i.e., mean lag time and propagation rate, did not correlate as strongly with phenotype (and ALS mutations did not uniformly accelerate mean rate of nucleation or propagation). A strong correlation was found, however, between mean ThT fluorescence at lag time and patient survival (R 2 = 0.93); oligomers of SOD1 with weaker fluorescence correlated with shorter survival. This study suggests that SOD1 mutations trigger ALS by altering a property of SOD1 or its oligomers other than the intrinsic rate of amyloid nucleation (e.g., oligomer stability; rates of intercellular propagation; affinity for membrane surfaces; and maturation rate).

Background: Ethiopia has experienced rapid improvements in its healthcare infrastructure, such as through the recent scale up of integrated community case management (iCCM) delivered by community-based health extension workers (HEWs) targeting children under the age of five. Despite notable improvements in child outcomes, the use of HEWs delivering iCCM remains very low. The aim of our study was to explain this phenomenon by examining care-seeking practices and treatment for sick children in two rural districts in the Oromia Region of Ethiopia., Methods: Using qualitative methods, we explored perceptions of child illness, influences on decision-making processes occurring over the course of a child's illness and caregiver perceptions of available community-based sources of child illness care. Sixteen focus group discussions (FGDs) and 40 in-depth interviews (IDIs) were held with mothers of children under age five. For additional perspective, 16 IDIs were conducted fathers and 22 IDIs with health extension workers and community health volunteers., Results: Caregivers often described the act of care-seeking for a sick child as a time of considerable uncertainty. In particular, mothers of sick children described the cultural, social and community-based resources available to minimize this uncertainty as well as constraints and strategies for accessing these resources in order to receive treatment for a sick child. The level of trust and familiarity were the most common dynamics noted as influencing care-seeking strategies; trust in biomedical and government providers was often low., Conclusions: Overall, our research highlights the multiple and dynamic influences on care-seeking for sick children in rural Ethiopia. An understanding of these influences is critical for the success of existing and future health interventions and continued improvement of child health in Ethiopia.

The chemical and physical mechanisms by which gyrating beads accelerate amyloid fibrillization in microtiter plate assays are unclear. Identifying these mechanisms will help optimize high-throughput screening assays for molecules and mutations that modulate aggregation and might explain why different research groups report different rates of aggregation for identical proteins. This article investigates how the rate of superoxide dismutase-1 (SOD1) fibrillization is affected by 12 different beads with a wide range of hydrophobicity, mass, stiffness, and topology but identical diameter. All assays were performed on D90A apo-SOD1, which is a stable and wild-type-like variant of SOD1. The most significant and uniform correlation between any material property of each bead and that bead's effect on SOD1 fibrillization rate was with regard to bead mass. A linear correlation existed between bead mass and rate of fibril elongation (R 2  = 0.7): heavier beads produced faster rates and shorter fibrils. Nucleation rates (lag time) also correlated with bead mass, but only for non-polymeric beads (i.e., glass, ceramic, metallic). The effect of bead mass on fibrillization correlated (R 2  = 0.96) with variations in buoyant forces and contact forces (between bead and microplate well), and was not an artifact of residual momentum during intermittent gyration. Hydrophobic effects were observed, but only for polymeric beads: lag times correlated negatively with contact angle of water and degree of protein adhesion (surface adhesion and hydrophobic effects were negligible for non-polymeric beads). These results demonstrate that contact forces (alone) explain kinetic variation among non-polymeric beads, whereas surface hydrophobicity and contact forces explain kinetic variation among polymeric beads. This study also establishes conditions for high-throughput amyloid assays of SOD1 that enable the control over fibril morphologies and produce eightfold faster lag times and fourfold less stochasticity than in previous studies., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Recent reports suggest that the nucleation and propagation of oligomeric superoxide dismutase-1 (SOD1) is effectively stochastic in vivo and in vitro. This perplexing kinetic variability-observed for other proteins and frequently attributed to experimental error-plagues attempts to discern how SOD1 mutations and post-translational modifications linked to amyotrophic lateral sclerosis (ALS) affect SOD1 aggregation. This study used microplate fluorescence spectroscopy and dynamic light scattering to measure rates of fibrillar and amorphous SOD1 aggregation at high iteration (ntotal = 1.2 × 10(3)). Rates of oligomerization were intrinsically irreproducible and populated continuous probability distributions. Modifying reaction conditions to mimic random and systematic experimental error could not account for kinetic outliers in standard assays, suggesting that stochasticity is not an experimental artifact, rather an intrinsic property of SOD1 oligomerization (presumably caused by competing pathways of oligomerization). Moreover, mean rates of fibrillar and amorphous nucleation were not uniformly increased by mutations that cause ALS; however, mutations did increase kinetic noise (variation) associated with nucleation and propagation. The stochastic aggregation of SOD1 provides a plausible statistical framework to rationalize how a pathogenic mutation can increase the probability of oligomer nucleation within a single cell, without increasing the mean rate of nucleation across an entire population of cells.

Background: In 2010, Ethiopia began scaling up the integrated community case management (iCCM) of childhood illness strategy throughout the country allowing health extension workers (HEWs) to treat children in rural health posts. After 2 years of iCCM scale up, utilization of HEWs remains low. Little is known about factors related to the use of health services in this setting. This research aimed to elicit perceptions and experiences of caregivers to better understand reasons for low utilization of iCCM services., Methods: A rapid ethnographic assessment was conducted in eight rural health post catchment areas in two zones: Jimma and West Hararghe. In total, 16 focus group discussions and 78 in-depth interviews were completed with mothers, fathers, HEWs and community health volunteers., Results: In spite of the HEW being a core component of iCCM, we found that the lack of availability of HEWs at the health post was one of the most common barriers to the utilization of iCCM services mentioned by caregivers. Financial and geographic challenges continue to influence caregiver decisions despite extension of free child health services in communities. Acceptability of HEWs was often low due to a perceived lack of sensitivity of HEWs and concerns about medicines given at the health post. Social networks acted both to facilitate and hinder use of HEWs. Many mothers stated a preference for using the health post, but some were unable to do so due to objections or alternative care-seeking preferences of gatekeepers, often mothers-in-law and husbands., Conclusion: Caregivers in Ethiopia face many challenges in using HEWs at the health post, potentially resulting in low demand for iCCM services. Efforts to minimize barriers to care seeking and to improve demand should be incorporated into the iCCM strategy in order to achieve reductions in child mortality and promote equity in access and child health outcomes., (© The Author 2015. Published by Oxford University Press in association with The London School of Hygiene and Tropical Medicine.)

The exchange of subunits between homodimeric mutant Cu, Zn superoxide dismutase (SOD1) and wild-type (WT) SOD1 is suspected to be a crucial step in the onset and progression of amyotrophic lateral sclerosis (ALS). The rate, mechanism, and ΔG of heterodimerization (ΔGHet) all remain undetermined, due to analytical challenges in measuring heterodimerization. This study used capillary zone electrophoresis to measure rates of heterodimerization and ΔGHet for seven ALS-variant apo-SOD1 proteins that are clinically diverse, producing mean survival times between 2 and 12 years (postdiagnosis). The ΔGHet of each ALS variant SOD1 correlated with patient survival time after diagnosis (R(2) = 0.98), with more favorable ΔGHet correlating with shorter survival by 4.8 years per kJ. Rates of heterodimerization did not correlate with survival time or age of disease onset. Metalation diminished the rate of subunit exchange by up to ∼38-fold but only altered ΔGHet by <1 kJ mol(-1). Medicinal targeting of heterodimer thermodynamics represents a plausible strategy for prolonging life in SOD1-linked ALS.

We conducted a cluster randomized trial of the effects of the integrated community case management of childhood illness (iCCM) strategy on careseeking for and coverage of correct treatment of suspected pneumonia, diarrhea, and malaria, and mortality among children aged 2-59 months in 31 districts of the Oromia region of Ethiopia. We conducted baseline and endline coverage and mortality surveys approximately 2 years apart, and assessed program strength after about 1 year of implementation. Results showed strong iCCM implementation, with iCCM-trained workers providing generally good quality of care. However, few sick children were taken to iCCM providers (average 16 per month). Difference in differences analyses revealed that careseeking for childhood illness was low and similar in both study arms at baseline and endline, and increased only marginally in intervention (22.9-25.7%) and comparison (23.3-29.3%) areas over the study period (P = 0.77). Mortality declined at similar rates in both study arms. Ethiopia's iCCM program did not generate levels of demand and utilization sufficient to achieve significant increases in intervention coverage and a resulting acceleration in reductions in child mortality. This evaluation has allowed Ethiopia to strengthen its strategic approaches to increasing population demand and use of iCCM services., (© The American Society of Tropical Medicine and Hygiene.)

A series of molecules capable of releasing of the strong organometallic Lewis acid B(C6F5)3 upon exposure to 254 nm light have been developed. These photo Lewis acid generators (PhLAGs) can now serve as photoinitiators for several important B(C6F5)3-catalyzed reactions. Herein is described the synthesis of the triphenylsulfonium and diphenyliodonium salts of carbamato- and hydridoborates, their establishment as PhLAGs, and studies aimed at defining the mechanism of borane release. Factors affecting these photolytic reactions and the application of this concept to photoinduced hydrosilylation reactions and construction of siloxane scaffolds are also discussed.

The monomerization of Cu, Zn superoxide dismutase (SOD1) is an early step along pathways of misfolding linked to amyotrophic lateral sclerosis (ALS). Monomerization requires the reversal of two post-translational modifications that are thermodynamically favorable: (i) dissociation of active-site metal ions and (ii) reduction of intramolecular disulfide bonds. This study found, using amide hydrogen/deuterium (H/D) exchange, capillary electrophoresis, and lysine-acetyl protein charge ladders, that ALS-linked A4V SOD1 rapidly monomerizes and partially unfolds in an external electric field (of physiological strength), without loss of metal ions, exposure to disulfide-reducing agents, or Joule heating. Voltage-induced monomerization was not observed for metal-free A4V SOD1, metal-free WT SOD1, or metal-loaded WT SOD1. Computational modeling suggested a mechanism for this counterintuitive effect: subunit macrodipoles of dimeric SOD1 are antiparallel and amplified 2-fold by metal coordination, which increases torque at the dimer interface as subunits rotate to align with the electric field.

Ethiopia has invested significant resources in integrated community case management (iCCM) of childhood illness. In Oromia Region, iCCM scale-up was phased in, allowing for comparisons between districts providing iCCM and routine services. We assessed the determinants of utilization of health extension workers (HEWs) delivering iCCM services at rural health posts by caregivers of sick, under-five children in a cross-sectional survey. We found low utilization of HEWs with only 9.3% of caregivers of a child sick with diarrhea, fever, and/or pneumonia in the previous 2 weeks taking their child to HEWs in both iCCM and routine areas. There was a higher likelihood of utilization of HEWs in iCCM areas (OR: 1.44; 95% CI: 0.97-2.12; P = 0.07), but this effect disappeared after accounting for confounders. In iCCM areas, maternal education, illness type, and distance were associated with utilization. Perceptions of illness severity and service quality were the primary reasons given for not utilizing the health post. Our findings suggest that though iCCM is reaching some vulnerable populations, there remain significant barriers to use of HEWs delivering iCCM services. Efforts for demand generation and minimization of remaining barriers are urgently needed for the sustained success of the iCCM strategy in Ethiopia., (© The American Society of Tropical Medicine and Hygiene.)

Although the magnitude of a protein's net charge (Z) can control its rate of self-assembly into amyloid, and its interactions with cellular membranes, the net charge of a protein is not viewed as a druggable parameter. This article demonstrates that aspirin (the quintessential acylating pharmacon) can inhibit the amyloidogenesis of superoxide dismutase (SOD1) by increasing the intrinsic net negative charge of the polypeptide, i.e., by acetylation (neutralization) of multiple lysines. The protective effects of acetylation were diminished (but not abolished) in 100 mM NaCl and were statistically significant: a total of 432 thioflavin-T amyloid assays were performed for all studied proteins. The acetylation of as few as three lysines by aspirin in A4V apo-SOD1-a variant that causes familial amyotrophic lateral sclerosis (ALS)-delayed amyloid nucleation by 38% and slowed amyloid propagation by twofold. Lysines in wild-type- and ALS-variant apo-SOD1 could also be peracetylated with aspirin after fibrillization, resulting in supercharged fibrils, with increases in formal net charge of ∼2 million units. Peracetylated SOD1 amyloid defibrillized at temperatures below unacetylated fibrils, and below the melting temperature of native Cu2,Zn2-SOD1 (e.g., fibril Tm = 84.49°C for acetylated D90A apo-SOD1 fibrils). Targeting the net charge of native or misfolded proteins with small molecules-analogous to how an enzyme's Km or Vmax are medicinally targeted-holds promise as a strategy in the design of therapies for diseases linked to protein self-assembly., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The functional and structural significance of the intrasubunit disulfide bond in copper-zinc superoxide dismutase (SOD1) was studied by characterizing mutant forms of human SOD1 (hSOD) and yeast SOD1 lacking the disulfide bond. We determined x-ray crystal structures of metal-bound and metal-deficient hC57S SOD1. C57S hSOD1 isolated from yeast contained four zinc ions per protein dimer and was structurally very similar to wild type. The addition of copper to this four-zinc protein gave properly reconstituted 2Cu,2Zn C57S hSOD, and its spectroscopic properties indicated that the coordination geometry of the copper was remarkably similar to that of holo wild type hSOD1. In contrast, the addition of copper and zinc ions to apo C57S human SOD1 failed to give proper reconstitution. Using pulse radiolysis, we determined SOD activities of yeast and human SOD1s lacking disulfide bonds and found that they were enzymatically active at ∼10% of the wild type rate. These results are contrary to earlier reports that the intrasubunit disulfide bonds in SOD1 are essential for SOD activity. Kinetic studies revealed further that the yeast mutant SOD1 had less ionic attraction for superoxide, possibly explaining the lower rates. Saccharomyces cerevisiae cells lacking the sod1 gene do not grow aerobically in the absence of lysine, but expression of C57S SOD1 increased growth to 30-50% of the growth of cells expressing wild type SOD1, supporting that C57S SOD1 retained a significant amount of activity., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

In this study, protein charge ladders and mass spectrometry were used to quantify how metal cations in the Hofmeister series (Na(+), K(+), Li(+), Mg(2+), and Ca(2+)) permute the effects of lysine acetylation on the rate of amide H/D exchange in a representative protein (myoglobin, Mb). The successive acetylation of up to 18 Lys-ε-NH3(+) groups in Mb caused a linear decrease in its global rate of amide H/D exchange (as measured by mass spectrometry), despite also decreasing the thermostability of Mb by >10 °C. The ability of a metal cation to screen kinetic electrostatic effects during H/D exchange-and to abolish the protective effect of acetylation against H/D exchange-was found to depend on the position of the cation in the Hofmeister series. Na(+) and K(+) cations did not fully equalize the rates of H/D exchange among each "rung" of the charge ladder, whereas Mg(2+) and Ca(2+) did equalize rates without eliminating the hydrophobic core of the protein (i.e., without unfolding Mb); Li(+) exhibited intermediate effects. The ability of Mg(2+) and Ca(2+) to completely screen electrostatic effects associated with the H/D exchange of charge isomers of Mb suggests that Mg(2+) or Ca(2+) (but not Na(+) or K(+)) can be used to quantify the magnitude by which electrostatic charge contributes to the observed rates of amide H/D exchange in proteins.

This article utilized "protein charge ladders"-chemical derivatives of proteins with similar structure, but systematically altered net charge-to quantify how missense mutations that cause amyotrophic lateral sclerosis (ALS) affect the net negative charge (Z) of superoxide dismutase-1 (SOD1) as a function of subcellular pH and Zn(2+) stoichiometry. Capillary electrophoresis revealed that the net charge of ALS-variant SOD1 can be different in sign and in magnitude-by up to 7.4 units per dimer at lysosomal pH-than values predicted from standard pKa values of amino acids and formal oxidation states of metal ions. At pH 7.4, the G85R, D90A, and G93R substitutions diminished the net negative charge of dimeric SOD1 by up to +2.29 units more than predicted; E100K lowered net charge by less than predicted. The binding of a single Zn(2+) to mutant SOD1 lowered its net charge by an additional +2.33 ± 0.01 to +3.18 ± 0.02 units, however, each protein regulated net charge when binding a second, third, or fourth Zn(2+) (ΔZ < 0.44 ± 0.07 per additional Zn(2+) ). Both metalated and apo-SOD1 regulated net charge across subcellular pH, without inverting from negative to positive at the theoretical pI. Differential scanning calorimetry, hydrogen-deuterium exchange, and inductively coupled plasma mass spectrometry confirmed that the structure, stability, and metal content of mutant proteins were not significantly affected by lysine acetylation. Measured values of net charge should be used when correlating the biophysical properties of a specific ALS-variant SOD1 protein with its observed aggregation propensity or clinical phenotype., (© 2014 The Protein Society.)

Background: Leukocoria is defined as a white reflection and its manifestation is symptomatic of several ocular pathologies, including retinoblastoma (Rb). Early detection of recurrent leukocoria is critical for improved patient outcomes and can be accomplished via the examination of recreational photography. To date, there exists a paucity of methods to automate leukocoria detection within such a dataset., Methods: This research explores a novel classification scheme that uses fuzzy logic theory to combine a number of classifiers that are experts in performing multichannel detection of leukocoria from recreational photography. The proposed scheme extracts features aided by the discrete cosine transform and the Karhunen-Loeve transformation., Results: The soft fusion of classifiers is significantly better than other methods of combining classifiers with p = 1.12 × 10-5. The proposed methodology performs at a 92% accuracy rate, with an 89% true positive rate, and an 11% false positive rate. Furthermore, the results produced by our methodology exhibit the lowest average variance., Conclusions: The proposed methodology overcomes non-ideal conditions of image acquisition, presenting a competent approach for the detection of leukocoria. Results suggest that recreational photography can be used in combination with the fusion of individual experts in multichannel classification and preprocessing tools such as the discrete cosine transform and the Karhunen-Loeve transformation.

The net charge of a folded protein is hypothesized to influence myriad biochemical processes (e.g., protein misfolding, electron transfer, molecular recognition); however, few tools exist for measuring net charge and this elusive property remains undetermined--at any pH--for nearly all proteins. This study used lysine-acetyl "protein charge ladders" and capillary electrophoresis to measure the net charge of superoxide dismutase-1 (SOD1)--whose aggregation causes amyotrophic lateral sclerosis (ALS)--as a function of coordinated metal ions and pH. The net negative charge of apo-SOD1 was similar to predicted values; however, the binding of a single Zn(2+) or Cu(2+) ion reduced the net negative charge by a greater magnitude than predicted (i.e., ~4 units, instead of 2), whereas the SOD1 protein underwent charge regulation upon binding 2-4 metal ions. From pH5 to pH8 (i.e., a range consistent with the multiple subcellular loci of SOD1), the holo-SOD1 protein underwent smaller fluctuations in net negative charge than predicted (i.e., ~3 units, instead of ~14) and did not undergo charge inversion at its isoelectric point (pI=5.3) but remained anionic. The regulation of SOD1 net charge along its pathways of metal binding, and across solvent pH, provides insight into its metal-induced maturation and enzymatic activity (which remains diffusion-limited across pH5-8). The anionic nature of holo-SOD1 across subcellular pH suggests that ~45 different ALS-linked mutations to SOD1 will reduce its net negative charge regardless of subcellular localization., (© 2013.)

Retinoblastoma is the most common primary intraocular tumor in children. The first sign that is often reported by parents is the appearance of recurrent leukocoria (i.e., "white eye") in recreational photographs. A quantitative definition or scale of leukocoria--as it appears during recreational photography--has not been established, and the amount of clinical information contained in a leukocoric image (collected by a parent) remains unknown. Moreover, the hypothesis that photographic leukocoria can be a sign of early stage retinoblastoma has not been tested for even a single patient. This study used commercially available software (Adobe Photoshop®) and standard color space conversion algorithms (operable in Microsoft Excel®) to quantify leukocoria in actual "baby pictures" of 9 children with retinoblastoma (that were collected by parents during recreational activities i.e., in nonclinical settings). One particular patient with bilateral retinoblastoma ("Patient Zero") was photographed >7, 000 times by his parents (who are authors of this study) over three years: from birth, through diagnosis, treatment, and remission. This large set of photographs allowed us to determine the longitudinal and lateral frequency of leukocoria throughout the patient's life. This study establishes: (i) that leukocoria can emerge at a low frequency in early-stage retinoblastoma and increase in frequency during disease progression, but decrease upon disease regression, (ii) that Hue, Saturation and Value (i.e., HSV color space) are suitable metrics for quantifying the intensity of retinoblastoma-linked leukocoria; (iii) that different sets of intraocular retinoblastoma tumors can produce distinct leukocoric reflections; and (iv) the Saturation-Value plane of HSV color space represents a convenient scale for quantifying and classifying pupillary reflections as they appear during recreational photography.

The reactivity of asparagine residues in Cu, Zn superoxide dismutase (SOD1) to deamidate to aspartate remains uncharacterized; its occurrence in SOD1 has not been investigated, and the biophysical effects of deamidation on SOD1 are unknown. Deamidation is, nonetheless, chemically equivalent to Asn-to-Asp missense mutations in SOD1 that cause amyotrophic lateral sclerosis (ALS). This study utilized computational methods to identify three asparagine residues in wild-type (WT) SOD1 (i.e., N26, N131, and N139) that are predicted to undergo significant deamidation (i.e., to >20%) on time scales comparable to the long lifetime (>1 year) of SOD1 in large motor neurons. Site-directed mutagenesis was used to successively substitute these asparagines with aspartate (to mimic deamidation) according to their predicted deamidation rate, yielding: N26D, N26D/N131D, and N26D/N131D/N139D SOD1. Differential scanning calorimetry demonstrated that the thermostability of N26D/N131D/N139D SOD1 is lower than WT SOD1 by ~2-8 °C (depending upon the state of metalation) and <3 °C lower than the ALS mutant N139D SOD1. The triply deamidated analog also aggregated into amyloid fibrils faster than WT SOD1 by ~2-fold (p < 0.008**) and at a rate identical to ALS mutant N139D SOD1 (p > 0.2). A total of 534 separate amyloid assays were performed to generate statistically significant comparisons of aggregation rates among WT and N/D SOD1 proteins. Capillary electrophoresis and mass spectrometry demonstrated that ~23% of N26 is deamidated to aspartate (iso-aspartate was undetectable) in a preparation of WT human SOD1 (isolated from erythrocytes) that has been used for decades by researchers as an analytical standard. The deamidation of asparagine--an analytically elusive, sub-Dalton modification--represents a plausible and overlooked mechanism by which WT SOD1 is converted to a neurotoxic isoform that has a similar structure, instability, and aggregation propensity as ALS mutant N139D SOD1.

The conserved nucleotide binding site (NBS), found in the Fab variable domain of all antibody isotypes, remains a not-so-widely known and under-utilized site. Here, we describe a UV photocrosslinking method (UV-NBS) that utilizes the NBS for site-specific covalent functionalization of antibodies, while preserving antibody activity. We identified a small molecule, indole-3-butyric acid (IBA), which has affinity for the NBS (K(d) = 1-8 μM) and can be photocrosslinked to antibodies upon UV energy exposure. By synthesizing their IBA conjugated versions, we have successfully photocrosslinked various types of functional ligands to antibodies at the NBS, including affinity tags (biotin), fluorescent molecules (FITC), peptides (iRGD), and chemotherapeutics (paclitaxel). An optimal UV exposure of 1-2 J/cm(2) yielded the most efficient photocrosslinking and resulted in 1-2 conjugations per antibody, while preserving the antigen binding activity and Fc related functions. Analysis of the photocrosslinked conjugates using western blotting, mass spectrometry, and computational docking simulations demonstrated that the photocrosslinking specifically takes place at the Y/F42 residue in framework region 2 of the antibody light chain. Taken together, the UV-NBS method provides a practical, site-specific, and chemically efficient method to functionalize antibodies with significant implications in diagnostic and therapeutic settings., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

This paper describes a biophysical investigation of residual mobility in complexes of bovine carbonic anhydrase II (BCA) and para-substituted benzenesulfonamide ligands with chains of 1-5 glycine subunits, and explains the previously observed increase in entropy of binding with chain length. The reported results represent the first experimental demonstration that BCA is not the rigid, static globulin that has been typically assumed, but experiences structural fluctuations upon binding ligands. NMR studies with (15)N-labeled ligands demonstrated that the first glycine subunit of the chain binds without stabilization or destabilization by the more distal subunits, and suggested that the other glycine subunits of the chain behave similarly. These data suggest that a model based on ligand mobility in the complex cannot explain the thermodynamic data. Hydrogen/deuterium exchange studies provided a global estimate of protein mobility and revealed that the number of exchanged hydrogens of BCA was higher when the protein was bound to a ligand with five glycine subunits than when bound to a ligand with only one subunit, and suggested a trend of increasing number of exchanged hydrogens with increasing chain length of the BCA-bound ligand, across the series. These data support the idea that the glycine chain destabilizes the structure of BCA in a length-dependent manner, causing an increase in BCA mobility. This study highlights the need to consider ligand-induced mobility of even "static" proteins in studies of protein-ligand binding, including rational ligand design approaches.

This paper describes the interaction between ubiquitin (UBI) and three sodium n-alkyl sulfates (SC(n)S) that have the same charge (Z = -1) but different hydrophobicity (n = 10, 12, or 14). Increasing the hydrophobicity of the n-alkyl sulfate resulted in (i) an increase in the number of distinct intermediates (that is, complexes of UBI and surfactant) that form along the pathway of unfolding, (ii) a decrease in the minimum concentrations of surfactant at which intermediates begin to form (i.e., a more negative ΔG(binding) of surfactant for UBI), and (iii) an increase in the number of surfactant molecules bound to UBI in each intermediate or complex. These results demonstrate that small changes in the hydrophobicity of a surfactant can significantly alter the binding interactions with a folded or unfolded cytosolic protein.

The amino acid substitution or post-translational modification of a cytosolic protein can cause unpredictable changes to its electrophoretic mobility during SDS-PAGE. This type of "gel shifting" has perplexed biochemists and biologists for decades. We identify a mechanism for "gel shifting" that predominates among a set of ALS (amyotrophic lateral sclerosis) mutant hSOD1 (superoxide dismutase) proteins, post-translationally modified hSOD1 proteins, and homologous SOD1 proteins from different organisms. By first comparing how 39 amino acid substitutions throughout hSOD1 affected SDS-PAGE migration, we found that substitutions that caused gel shifting occurred within a single polyacidic domain (residues ~80-101), and were nonisoelectric. Substitutions that decreased the net negative charge of domain 80-101 increased migration; only one substitution increased net negative charge and slowed migration. Capillary electrophoresis, circular dichroism, and size exclusion chromatography demonstrated that amino acid substitutions increase migration during SDS-PAGE by promoting the binding of three to four additional SDS molecules, without significantly altering the secondary structure or Stokes radius of hSOD1-SDS complexes. The high negative charge of domain 80-101 is required for SOD1 gel shifting: neutralizing the polyacidic domain (via chimeric mouse-human SOD1 fusion proteins) inhibited amino acid substitutions from causing gel shifting. These results demonstrate that the pattern of gel shifting for mutant cytosolic proteins can be used to: (i) identify domains in the primary structure that control interactions between denatured cytosolic proteins and SDS and (ii) identify a predominant chemical mechanism for the interaction (e.g., hydrophobic vs. electrostatic)., (Copyright © 2012 The Protein Society.)