Publications by authors named "Joshua Rosenthal"

78 Publications

A risk assessment tool for resumption of research activities during the COVID-19 pandemic for field trials in low resource settings.

BMC Med Res Methodol 2021 04 12;21(1):68. Epub 2021 Apr 12.

Division of International Epidemiology and Population Studies, Fogarty International Center, National Institutes of Health, Bethesda, USA.

Rationale: The spread of severe acute respiratory syndrome coronavirus-2 has suspended many non-COVID-19 related research activities. Where restarting research activities is permitted, investigators need to evaluate the risks and benefits of resuming data collection and adapt procedures to minimize risk.

Objectives: In the context of the multicountry Household Air Pollution Intervention (HAPIN) trial conducted in rural, low-resource settings, we developed a framework to assess the risk of each trial activity and to guide protective measures. Our goal is to maximize the integrity of reseach aims while minimizing infection risk based on the latest scientific understanding of the virus.

Methods: We drew on a combination of expert consultations, risk assessment frameworks, institutional guidance and literature to develop our framework. We then systematically graded clinical, behavioral, laboratory and field environmental health research activities in four countries for both adult and child subjects using this framework. National and local government recommendations provided the minimum safety guidelines for our work.

Results: Our framework assesses risk based on staff proximity to the participant, exposure time between staff and participants, and potential viral aerosolization while performing the activity. For each activity, one of four risk levels, from minimal to unacceptable, is assigned and guidance on protective measures is provided. Those activities that can potentially aerosolize the virus are deemed the highest risk.

Conclusions: By applying a systematic, procedure-specific approach to risk assessment for each trial activity, we were able to protect our participants and research team and to uphold our ability to deliver on the research commitments we have made to our staff, participants, local communities, and funders. This framework can be tailored to other research studies conducted in similar settings during the current pandemic, as well as potential future outbreaks with similar transmission dynamics. The trial is registered with clinicaltrials.gov NCT02944682 on October 26. 2016 .
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http://dx.doi.org/10.1186/s12874-021-01232-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8040756PMC
April 2021

A Risk Assessment Tool for Resumption of Research Activities During the COVID-19 Pandemic.

Res Sq 2020 Nov 12. Epub 2020 Nov 12.

National Institutes of Health.

Rationale: The spread of severe acute respiratory syndrome coronavirus-2 has suspended many non-COVID-19 related research activities. Where restarting research activities is permitted, investigators need to evaluate the risks and benefits of resuming data collection and adapt procedures to minimize risk.

Objectives: In the context of the multicountry Household Air Pollution Intervention (HAPIN) trial, we developed a framework to assess the risk of each trial activity and to guide protective measures. Our goal is to maximize integrity of reseach aims while minimizing infection risk based on the latest understanding of the virus.

Methods: We drew on a combination of expert consultations, risk assessment frameworks, institutional guidance and literature to develop our framework. We then systematically graded clinical, behavioral, laboratory and field environmental health research activities in four countries for both adult and child subjects using this framework.

Results: Our framework assesses risk based on staff proximity to the participant, exposure time between staff and participants, and potential aerosolization while performing the activity. One of of four risk levels, from minimal to unacceptable, is assigned and guidance on protective measures is provided. Those activities which can potentially aerosolize the virus are deemed the highest risk.

Conclusions: By applying a systematic, procedure-specific approach to risk assessment for each trial activity, we can compare trial activities using the same criteria. This approach allows us to protect our participants and research team and to uphold our ability to deliver on the research commitments we have made to our participants, local communities, and funders. The trial is registered with clinicaltrials.gov (NCT02944682).
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http://dx.doi.org/10.21203/rs.3.rs-103997/v1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7668754PMC
November 2020

Systems Science Approaches for Global Environmental Health Research: Enhancing Intervention Design and Implementation for Household Air Pollution (HAP) and Water, Sanitation, and Hygiene (WASH) Programs.

Environ Health Perspect 2020 10 9;128(10):105001. Epub 2020 Oct 9.

School of Social Work, Boston College, Boston, Massachusetts, USA.

Background: Two of the most important causes of global disease fall in the realm of environmental health: household air pollution (HAP) and poor water, sanitation, and hygiene (WASH) conditions. Interventions, such as clean cookstoves, household water treatment, and improved sanitation facilities, have great potential to yield reductions in disease burden. However, in recent trials and implementation efforts, interventions to improve HAP and WASH conditions have shown few of the desired health gains, raising fundamental questions about current approaches.

Objectives: We describe how the failure to consider the complex systems that characterize diverse real-world conditions may doom promising new approaches prematurely. We provide examples of the application of systems approaches, including system dynamics, network analysis, and agent-based modeling, to the global environmental health priorities of HAP and WASH research and programs. Finally, we offer suggestions on how to approach systems science.

Methods: Systems science applied to environmental health can address major challenges by ) enhancing understanding of existing system structures and behaviors that accelerate or impede aims; ) developing understanding and agreement on a problem among stakeholders; and ) guiding intervention and policy formulation. When employed in participatory processes that engage study populations, policy makers, and implementers, systems science helps ensure that research is responsive to local priorities and reflect real-world conditions. Systems approaches also help interpret unexpected outcomes by revealing emergent properties of the system due to interactions among variables, yielding complex behaviors and sometimes counterintuitive results.

Discussion: Systems science offers powerful and underused tools to accelerate our ability to identify barriers and facilitators to success in environmental health interventions. This approach is especially useful in the context of implementation research because it explicitly accounts for the interaction of processes occurring at multiple scales, across social and environmental dimensions, with a particular emphasis on linkages and feedback among these processes. https://doi.org/10.1289/EHP7010.
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http://dx.doi.org/10.1289/EHP7010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7546437PMC
October 2020

Designing a comprehensive behaviour change intervention to promote and monitor exclusive use of liquefied petroleum gas stoves for the Household Air Pollution Intervention Network (HAPIN) trial.

BMJ Open 2020 09 29;10(9):e037761. Epub 2020 Sep 29.

Department of International Health, Social and Behavioral Interventions, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, USA.

Introduction: Increasing use of cleaner fuels, such as liquefied petroleum gas (LPG), and abandonment of solid fuels is key to reducing household air pollution and realising potential health improvements in low-income countries. However, achieving exclusive LPG use in households unaccustomed to this type of fuel, used in combination with a new stove technology, requires substantial behaviour change. We conducted theory-grounded formative research to identify contextual factors influencing cooking fuel choice to guide the development of behavioural strategies for the Household Air Pollution Intervention Network (HAPIN) trial. The HAPIN trial will assess the impact of exclusive LPG use on air pollution exposure and health of pregnant women, older adult women, and infants under 1 year of age in Guatemala, India, Peru, and Rwanda.

Methods: Using the Capability, Opportunity, Motivation-Behaviour (COM-B) framework and Behaviour Change Wheel (BCW) to guide formative research, we conducted in-depth interviews, focus group discussions, observations, key informant interviews and pilot studies to identify key influencers of cooking behaviours in the four countries. We used these findings to develop behavioural strategies likely to achieve exclusive LPG use in the HAPIN trial.

Results: We identified nine potential influencers of exclusive LPG use, including perceived disadvantages of solid fuels, family preferences, cookware, traditional foods, non-food-related cooking, heating needs, LPG awareness, safety and cost and availability of fuel. Mapping formative findings onto the theoretical frameworks, behavioural strategies for achieving exclusive LPG use in each research site included free fuel deliveries, locally acceptable stoves and equipment, hands-on training and printed materials and videos emphasising relevant messages. In the HAPIN trial, we will monitor and reinforce exclusive LPG use through temperature data loggers, LPG fuel delivery tracking, in-home observations and behavioural reinforcement visits.

Conclusion: Our formative research and behavioural strategies can inform the development, implementation, monitoring and evaluation of theory-informed strategies to promote exclusive LPG use in future stove programmes and research studies.

Trial Registration Number: NCT02944682, Pre-results.
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http://dx.doi.org/10.1136/bmjopen-2020-037761DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7526279PMC
September 2020

Highly Efficient Knockout of a Squid Pigmentation Gene.

Curr Biol 2020 09 30;30(17):3484-3490.e4. Epub 2020 Jul 30.

The Eugene Bell Center, The Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543, USA. Electronic address:

Seminal studies using squid as a model led to breakthroughs in neurobiology. The squid giant axon and synapse, for example, laid the foundation for our current understanding of the action potential [1], ionic gradients across cells [2], voltage-dependent ion channels [3], molecular motors [4-7], and synaptic transmission [8-11]. Despite their anatomical advantages, the use of squid as a model receded over the past several decades as investigators turned to genetically tractable systems. Recently, however, two key advances have made it possible to develop techniques for the genetic manipulation of squid. The first is the CRISPR-Cas9 system for targeted gene disruption, a largely species-agnostic method [12, 13]. The second is the sequencing of genomes for several cephalopod species [14-16]. If made genetically tractable, squid and other cephalopods offer a wealth of biological novelties that could spur discovery. Within invertebrates, not only do they possess by far the largest brains, they also express the most sophisticated behaviors [17]. In this paper, we demonstrate efficient gene knockout in the squid Doryteuthis pealeii using CRISPR-Cas9. Ommochromes, the pigments found in squid retinas and chromatophores, are derivatives of tryptophan, and the first committed step in their synthesis is normally catalyzed by Tryptophan 2,3 Dioxygenase (TDO [18-20]). Knocking out TDO in squid embryos efficiently eliminated pigmentation. By precisely timing CRISPR-Cas9 delivery during early development, the degree of pigmentation could be finely controlled. Genotyping revealed knockout efficiencies routinely greater than 90%. This study represents a critical advancement toward making squid genetically tractable.
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http://dx.doi.org/10.1016/j.cub.2020.06.099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484294PMC
September 2020

Everybody Stacks: Lessons from household energy case studies to inform design principles for clean energy transitions.

Energy Policy 2020 Jun 8;141. Epub 2020 Apr 8.

Fogarty International Center, National Institutes of Health, Bethesda, MD, USA.

Stove stacking (concurrent use of multiple stoves and/or fuels) is a poorly quantified practice in regions where efforts to transition household energy to cleaner stoves/or fuels are on-going. Using biomass-burning stoves alongside clean stoves undermines health and environmental goals. This review synthesizes stove stacking data gathered from eleven case studies of clean cooking programs in low/middle-income country settings. Analyzed data are from ministry and program records, research studies, and informant interviews. Thematic analysis identify key drivers of stove stacking behavior in each setting. Significant (28%-100%) stacking with traditional cooking methods was observed in all cases. Reason for traditional fuel use includes: costs of clean fuel; mismatches between cooking technologies and household needs; and unreliable fuel supply. National household surveys often focus on 'primary' cookstoves and miss stove stacking data. Thus more attention should be paid to discontinuation of traditional stove use, not solely adoption of cleaner stoves/fuels. Future energy policies and programs should acknowledge the realities of stacking and incorporate strategies at the design stage to transition away from polluting stoves/fuels. Seven principles for clean cooking system program design and policy are presented, focused on a shift toward "cleaner stacking" that could yield household air pollution reductions approaching WHO targets.
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http://dx.doi.org/10.1016/j.enpol.2020.111468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7259482PMC
June 2020

Design and Rationale of the HAPIN Study: A Multicountry Randomized Controlled Trial to Assess the Effect of Liquefied Petroleum Gas Stove and Continuous Fuel Distribution.

Environ Health Perspect 2020 04 29;128(4):47008. Epub 2020 Apr 29.

Division of Epidemiology and Population Studies, Fogarty International Center, National Institutes of Health, Bethesda, Maryland, USA.

Background: Globally, nearly 3 billion people rely on solid fuels for cooking and heating, the vast majority residing in low- and middle-income countries (LMICs). The resulting household air pollution (HAP) is a leading environmental risk factor, accounting for an estimated 1.6 million premature deaths annually. Previous interventions of cleaner stoves have often failed to reduce exposure to levels that produce meaningful health improvements. There have been no multicountry field trials with liquefied petroleum gas (LPG) stoves, likely the cleanest scalable intervention.

Objective: This paper describes the design and methods of an ongoing randomized controlled trial (RCT) of LPG stove and fuel distribution in 3,200 households in 4 LMICs (India, Guatemala, Peru, and Rwanda).

Methods: We are enrolling 800 pregnant women at each of the 4 international research centers from households using biomass fuels. We are randomly assigning households to receive LPG stoves, an 18-month supply of free LPG, and behavioral reinforcements to the control arm. The mother is being followed along with her child until the child is 1 year old. Older adult women (40 to of age) living in the same households are also enrolled and followed during the same period. Primary health outcomes are low birth weight, severe pneumonia incidence, stunting in the child, and high blood pressure (BP) in the older adult woman. Secondary health outcomes are also being assessed. We are assessing stove and fuel use, conducting repeated personal and kitchen exposure assessments of fine particulate matter with aerodynamic diameter (), carbon monoxide (CO), and black carbon (BC), and collecting dried blood spots (DBS) and urinary samples for biomarker analysis. Enrollment and data collection began in May 2018 and will continue through August 2021. The trial is registered with ClinicalTrials.gov (NCT02944682).

Conclusions: This study will provide evidence to inform national and global policies on scaling up LPG stove use among vulnerable populations. https://doi.org/10.1289/EHP6407.
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http://dx.doi.org/10.1289/EHP6407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7228119PMC
April 2020

Air Pollutant Exposure and Stove Use Assessment Methods for the Household Air Pollution Intervention Network (HAPIN) Trial.

Environ Health Perspect 2020 04 29;128(4):47009. Epub 2020 Apr 29.

Gangarosa Department of Environmental Health, Rollins School of Public Health, Emory University, Atlanta, Georgia, USA.

Background: High quality personal exposure data is fundamental to understanding the health implications of household energy interventions, interpreting analyses across assigned study arms, and characterizing exposure-response relationships for household air pollution. This paper describes the exposure data collection for the Household Air Pollution Intervention Network (HAPIN), a multicountry randomized controlled trial of liquefied petroleum gas stoves and fuel among 3,200 households in India, Rwanda, Guatemala, and Peru.

Objectives: The primary objectives of the exposure assessment are to estimate the exposure contrast achieved following a clean fuel intervention and to provide data for analyses of exposure-response relationships across a range of personal exposures.

Methods: Exposure measurements are being conducted over the 3-y time frame of the field study. We are measuring fine particulate matter [PM  in aerodynamic diameter ()] with the Enhanced Children's MicroPEM™ (RTI International), carbon monoxide (CO) with the USB-EL-CO (Lascar Electronics), and black carbon with the OT21 transmissometer (Magee Scientific) in pregnant women, adult women, and children of age, primarily via multiple 24-h personal assessments (three, six, and three measurements, respectively) over the course of the 18-month follow-up period using lightweight monitors. For children we are using an indirect measurement approach, combining data from area monitors and locator devices worn by the child. For a subsample (up to 10%) of the study population, we are doubling the frequency of measurements in order to estimate the accuracy of subject-specific typical exposure estimates. In addition, we are conducting ambient air monitoring to help characterize potential contributions of exposure from background concentration. Stove use monitors (Geocene) are being used to assess compliance with the intervention, given that stove stacking (use of traditional stoves in addition to the intervention gas stove) may occur.

Conclusions: The tools and approaches being used for HAPIN to estimate personal exposures build on previous efforts and take advantage of new technologies. In addition to providing key personal exposure data for this study, we hope the application and learnings from our exposure assessment will help inform future efforts to characterize exposure to household air pollution and for other contexts. https://doi.org/10.1289/EHP6422.
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http://dx.doi.org/10.1289/EHP6422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7228125PMC
April 2020

Spatially regulated editing of genetic information within a neuron.

Nucleic Acids Res 2020 05;48(8):3999-4012

The Eugene Bell Center, Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02540, USA.

In eukaryotic cells, with the exception of the specialized genomes of mitochondria and plastids, all genetic information is sequestered within the nucleus. This arrangement imposes constraints on how the information can be tailored for different cellular regions, particularly in cells with complex morphologies like neurons. Although messenger RNAs (mRNAs), and the proteins that they encode, can be differentially sorted between cellular regions, the information itself does not change. RNA editing by adenosine deamination can alter the genome's blueprint by recoding mRNAs; however, this process too is thought to be restricted to the nucleus. In this work, we show that ADAR2 (adenosine deaminase that acts on RNA), an RNA editing enzyme, is expressed outside of the nucleus in squid neurons. Furthermore, purified axoplasm exhibits adenosine-to-inosine activity and can specifically edit adenosines in a known substrate. Finally, a transcriptome-wide analysis of RNA editing reveals that tens of thousands of editing sites (>70% of all sites) are edited more extensively in the squid giant axon than in its cell bodies. These results indicate that within a neuron RNA editing can recode genetic information in a region-specific manner.
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http://dx.doi.org/10.1093/nar/gkaa172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7192619PMC
May 2020

Adapting and Operationalizing the RE-AIM Framework for Implementation Science in Environmental Health: Clean Fuel Cooking Programs in Low Resource Countries.

Front Public Health 2019 20;7:389. Epub 2019 Dec 20.

Fogarty International Center, U.S. National Institutes of Health, Bethesda, MD, United States.

The use of models and frameworks to design and evaluate strategies to improve delivery of evidence-based interventions is a foundational element of implementation science. To date, however, evaluative implementation science frameworks such as (RE-AIM) have not been widely employed to examine environmental health interventions. We take advantage of a unique opportunity to utilize and iteratively adapt the RE-AIM framework to guide NIH-funded case studies of the implementation of clean cooking fuel programs in eleven low- and middle-income countries. We used existing literature and expert consultation to translate and iteratively adapt the RE-AIM framework across several stages of the NIH Clean Cooking Implementation Science case study project. Checklists and templates to guide investigators were developed at each stage. The RE-AIM framework facilitated identification of important emerging issues across this set of case studies, in particular highlighting the fact that data associated with certain important outcomes related to health and welfare are chronically lacking in clean fuel programs. Monitoring of these outcomes should be prioritized in future implementation efforts. As RE-AIM was not originally designed to evaluate household energy interventions, employing the framework required adaptation. Specific adaptations include the broadening of to encompass indicators of success toward any stated programmatic goal, and expansion of to include household-level uptake of technology. The RE-AIM implementation science framework proved to be a useful organizing schema for 11 case studies of clean fuel cooking programs, in particular highlighting areas requiring emphasis in future research and evaluation efforts. The iterative approach used here to adapt an implementation science framework to a specific programmatic goal may be of value to other multi-country program efforts, such as those led by international development agencies. The checklists and templates developed for this project are publicly available for others to use and/or further modify.
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http://dx.doi.org/10.3389/fpubh.2019.00389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6932973PMC
December 2019

Specialization for rapid excitation in fast squid tentacle muscle involves action potentials absent in slow arm muscle.

J Exp Biol 2020 02 12;223(Pt 3). Epub 2020 Feb 12.

Department of Biology, CB# 3280 Coker Hall, University of North Carolina, Chapel Hill, NC 27599, USA

An important aspect of the performance of many fast muscle fiber types is rapid excitation. Previous research on the cross-striated muscle fibers responsible for the rapid tentacle strike in squid has revealed the specializations responsible for high shortening velocity, but little is known about excitation of these fibers. Conventional whole-cell patch recordings were made from tentacle fibers and the slower obliquely striated muscle fibers of the arms. The fast-contracting tentacle fibers show an approximately 10-fold greater sodium conductance than that of the arm fibers and, unlike the arm fibers, the tentacle muscle fibers produce action potentials. hybridization using an antisense probe to the voltage-dependent sodium channel present in this squid genus shows prominent expression of sodium channel mRNA in tentacle fibers but undetectable expression in arm fibers. Production of action potentials by tentacle muscle fibers and their absence in arm fibers is likely responsible for the previously reported greater twitch-tetanus ratio in the tentacle versus the arm fibers. During the rapid tentacle strike, a few closely spaced action potentials would result in maximal activation of transverse tentacle muscle. Activation of the slower transverse muscle fibers in the arms would require summation of excitatory postsynaptic potentials over a longer time, allowing the precise modulation of force required for supporting slower movements of the arms.
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http://dx.doi.org/10.1242/jeb.218081DOI Listing
February 2020

Convergent and parallel evolution in a voltage-gated sodium channel underlies TTX-resistance in the Greater Blue-ringed Octopus: Hapalochlaena lunulata.

Toxicon 2019 Dec 21;170:77-84. Epub 2019 Sep 21.

Utah State University, Uintah Basin, Vernal, UT, USA. Electronic address:

The natural history and pharmacology of tetrodotoxin (TTX) has long intrigued biologists. This toxin has a remarkable distribution that spans two domains of life (Bacteria and Eukarya). Within Eukaryotes, TTX has only been identified in animals but is known to be present in over five-dozen species of phylogenetically distant Metazoans. Despite decades of work, the origin and biosynthetic pathways of TTX remain unresolved. Investigations in puffer fishes and salamanders have provided insights into the acquisition of auto-resistance to TTX through the evolution of voltage-gated sodium ion channels (VGSCs) that have reduced binding affinity for TTX. To date there have been no studies of these proteins in tetrodotoxic Blue-Ringed Octopuses. Here we report data demonstrating that the Greater Blue-ringed Octopus (Hapalochlaena lunulata) expresses a VGSC (HlNa1) gene with mutations that reduce the channel's TTX-binding affinity and likely render the organism TTX resistant. We identified three amino-acid substitutions in the TTX-binding site of HlNa1 that likely confer TTX-resistance to both the channel and the organism. These substitutions are associated with organismal TTX-resistance in other TTX-bearing taxa and are convergent with substitutions that have evolved in fish, salamanders, and some TTX-resistant invertebrates.
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http://dx.doi.org/10.1016/j.toxicon.2019.09.013DOI Listing
December 2019

Compensating control participants when the intervention is of significant value: experience in Guatemala, India, Peru and Rwanda.

BMJ Glob Health 2019 21;4(4):e001567. Epub 2019 Aug 21.

Department of International Health, Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA.

The Household Air Pollution Intervention Network (HAPIN) trial is a randomised controlled trial in Guatemala, India, Peru and Rwanda to assess the health impact of a clean cooking intervention in households using solid biomass for cooking. The HAPIN intervention-a liquefied petroleum gas (LPG) stove and 18-month supply of LPG-has significant value in these communities, irrespective of potential health benefits. For control households, it was necessary to develop a compensation strategy that would be comparable across four settings and would address concerns about differential loss to follow-up, fairness and potential effects on household economics. Each site developed slightly different, contextually appropriate compensation packages by combining a set of uniform principles with local community input. In Guatemala, control compensation consists of coupons equivalent to the LPG stove's value that can be redeemed for the participant's choice of household items, which could include an LPG stove. In Peru, control households receive several small items during the trial, plus the intervention stove and 1 month of fuel at the trial's conclusion. Rwandan participants are given small items during the trial and a choice of a solar kit, LPG stove and four fuel refills, or cash equivalent at the end. India is the only setting in which control participants receive the intervention (LPG stove and 18 months of fuel) at the trial's end while also being compensated for their time during the trial, in accordance with local ethics committee requirements. The approaches presented here could inform compensation strategy development in future multi-country trials.
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http://dx.doi.org/10.1136/bmjgh-2019-001567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6730613PMC
August 2019

Construction and Composition of the Squid Pen from .

Biol Bull 2019 08 8;237(1):1-15. Epub 2019 Jul 8.

The pen, or gladius, of the squid is an internalized shell. It serves as a site of attachment for important muscle groups and as a protective barrier for the visceral organs. The pen's durability and flexibility are derived from its unique composition of chitin and protein. We report the characterization of the structure, development, and composition of pens from . The nanofibrils of the polysaccharide β-chitin are arranged in an aligned configuration in only specific regions of the pen. Chitin is secreted early in development, enabling us to characterize the changes in pen morphology prior to hatching. The chitin and proteins are assembled in the shell sac surrounded by fluid that has a significantly different ionic composition from squid plasma. Two groups of proteins are associated with the pen: those on its surface and those embedded within the pen. Only 20 proteins are identified as embedded within the pen. Embedded proteins are classified into six groups, including chitin associated, protease, protease inhibitors, intracellular, extracellular matrix, and those that are unknown. The pen proteins share many conserved domains with proteins from other chitinous structures. We conclude that the pen is one of the least complex, load-bearing, chitin-rich structures currently known and is amenable to further studies to elucidate natural construction mechanisms using chitin and protein.
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http://dx.doi.org/10.1086/704209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7340512PMC
August 2019

An analysis of efforts to scale up clean household energy for cooking around the world.

Energy Sustain Dev 2018 Oct 4;46:1-10. Epub 2018 Jul 4.

Fogarty International Center, National Institutes of Health, Bethesda, MD, USA.

Approximately 3 billion people, most of whom live in Asia, Africa, and the Americas, rely on solid fuels (i.e. wood, crop wastes, dung, charcoal) and kerosene for their cooking needs. Exposure to household air pollution from burning these fuels is estimated to account for approximately 3 million premature deaths a year. Cleaner fuels - such as liquefied petroleum gas, biogas, electricity, and certain compressed biomass fuels - have the potential to alleviate much of this significant health burden. A wide variety of clean cooking intervention programs are being implemented around the world, but very few of these efforts have been analyzed to enable global learning. The Clean Cooking Implementation Science Network (ISN), supported by the U.S. National Institutes of Health (NIH) and partners, identified the need to augment the publicly available literature concerning what has worked well and in what context. The ISN has supported the development of a systematic set of case studies, contained in this Special Issue, examining clean cooking program rollouts in a variety of low- and middle-income settings around the world. We used the RE-AIM (reach, effectiveness, adaptation, implementation, maintenance) framework to coordinate and evaluate the case studies. This paper describes the clean cooking case studies project, introduces the individual studies contained herein, and proposes a general conceptual model to support future planning and evaluation of household energy programs.
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http://dx.doi.org/10.1016/j.esd.2018.06.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6419773PMC
October 2018

Dynamic pigmentary and structural coloration within cephalopod chromatophore organs.

Nat Commun 2019 03 1;10(1):1004. Epub 2019 Mar 1.

Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, 02115, USA.

Chromatophore organs in cephalopod skin are known to produce ultra-fast changes in appearance for camouflage and communication. Light-scattering pigment granules within chromatocytes have been presumed to be the sole source of coloration in these complex organs. We report the discovery of structural coloration emanating in precise register with expanded pigmented chromatocytes. Concurrently, using an annotated squid chromatophore proteome together with microscopy, we identify a likely biochemical component of this reflective coloration as reflectin proteins distributed in sheath cells that envelop each chromatocyte. Additionally, within the chromatocytes, where the pigment resides in nanostructured granules, we find the lens protein Ω- crystallin interfacing tightly with pigment molecules. These findings offer fresh perspectives on the intricate biophotonic interplay between pigmentary and structural coloration elements tightly co-located within the same dynamic flexible organ - a feature that may help inspire the development of new classes of engineered materials that change color and pattern.
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http://dx.doi.org/10.1038/s41467-019-08891-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397165PMC
March 2019

Current strategies for Site-Directed RNA Editing using ADARs.

Methods 2019 03 29;156:16-24. Epub 2018 Nov 29.

The Eugene Bell Center, Marine Biological Laboratory, Woods Hole, MA, USA. Electronic address:

Adenosine Deaminases that Act on RNA (ADARs) are a group of enzymes that catalyze the conversion of adenosines (A's) to inosines (I's) in a process known as RNA editing. Though ADARs can act on different types of RNA, editing events in coding regions of mRNA are of particular interest as I's base pair like guanosines (G's). Thus, every A-to-I change catalyzed by ADAR is read as an A-to-G change during translation, potentially altering protein sequence and function. This ability to re-code makes ADAR an attractive therapeutic tool to correct genetic mutations within mRNA. The main challenge in doing so is to re-direct ADAR's catalytic activity towards A's that are not naturally edited, a process termed Site-Directed RNA Editing (SDRE). Recently, a handful of labs have taken up this challenge and two basic strategies have emerged. The first involves redirecting endogenous ADAR to new sites by making editable structures using antisense RNA oligonucleotides. The second also utilizes antisense RNA oligonucleotides, but it uses them as guides to deliver the catalytic domain of engineered ADARs to new sites, much as CRISPR guides deliver Cas nucleases. In fact, despite the intense current focus on CRISPR-Cas9 genome editing, SDRE offers a number of distinct advantages. In the present review we will discuss these strategies in greater detail, focusing on the concepts on which they are based, how they were developed and tested, and their respective advantages and disadvantages. Though the precise and efficient re-direction of ADAR activity still remains a challenge, the systems that are being developed lay the foundation for SDRE as a powerful tool for transient genome editing.
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http://dx.doi.org/10.1016/j.ymeth.2018.11.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6814296PMC
March 2019

Noncommunicable Diseases in Low- and Middle-Income Countries: A Strategic Approach to Develop a Global Implementation Research Workforce.

Glob Heart 2018 06 30;13(2):131-137. Epub 2018 Jun 30.

Center for Translation Research and Implementation Science, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.

Globally, most of the burden from noncommunicable disease is now evident in low- and middle-income countries (LMICs). At the same time, many effective noncommunicable disease interventions are now available and recommended for implementation and scale-up across LMIC health systems-yet are not being widely implemented. Understanding optimal and sustainable implementation strategies for these interventions within the LMIC context will need locally led and conducted implementation research- a research capacity which currently is lacking. The National Institutes of Health institutes, centers, and offices work with the Fogarty International Center to support biomedical research and research training across the globe. The National Heart, Lung, and Blood Institutes' Center for Translation Research and Implementation Science has a strategic focus on implementation research in global health. The Center for Translation Research and Implementation Science is considering strategies for developing research capacity and skill sets to conduct this priority research along with National Institutes of Health institutes and centers and other key global institutions that highly value implementation research. Short-term and medium-term strategies will be needed along with building on current efforts and investments and considering new efforts to address gaps. Developing and sustaining this research workforce will present many challenges and require much effort, but the returns could be transformative in advancing the prevention, treatment, and control of noncommunicable diseases within LMICs.
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http://dx.doi.org/10.1016/j.gheart.2018.05.001DOI Listing
June 2018

Clean cooking and the SDGs: Integrated analytical approaches to guide energy interventions for health and environment goals.

Energy Sustain Dev 2018 Feb 8;42:152-159. Epub 2017 Dec 8.

Fogarty International Center, National Institutes of Health, USA.

Development and implementation of clean cooking technology for households in low and middle income countries (LMICs) offer enormous promise to advance at least five Sustainable Development Goals (SDGs): 3. Good health and well-being; 5. Gender equality; 7. Affordable and clean energy; 13. Climate action; 15. Life on land. Programs are being implemented around the world to introduce alternative cooking technologies, and we are well on the way to achieving the goal set by the Global Alliance for Clean Cookstoves to reach 100 million homes with cleaner and more efficient cooking methods by 2020. Despite evidence that household air pollution (HAP) from solid fuel combustion is responsible for 3-4 million early deaths per year, many cookstove programs are motivated and/or financed by climate change mitigation schemes and deploy alternative stoves that use solid fuels such as wood and charcoal. However, recent studies have demonstrated that improved biomass-burning stoves typically only incrementally improve air quality and yield modest or minimal health benefits. Likewise, their contributions to climate change mitigation and other SDGs may be limited. Evidence indicates that cleaner fuels, such as liquefied petroleum gas (LPG), ethanol and biogas, offer greater potential benefits not only to health, but also greater progress towards climate goals and other relevant SDGs. We present a modeled estimate of these potential gains for a diverse group of 40 LMICs. Our model suggests that cookstove programs using LPG stoves and fuel will yield greater reductions in both Disability Adjusted Life Years and Global Warming Commitment in these countries than those using improved biomass stoves. Cost and infrastructure requirements for clean fuels such as LPG are widely recognized constraints. In view of these constraints we present an analytical method to simultaneously consider health and climate needs at the national level for the same 40 countries in the context of estimated LPG expansion potentials. Comparative analyses integrating priorities across SDGs at the national and regional levels may guide more practical and effective household energy development choices going forward.
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http://dx.doi.org/10.1016/j.esd.2017.11.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5975963PMC
February 2018

International Approach to Environmental and Lung Health. A Perspective from the Fogarty International Center.

Ann Am Thorac Soc 2018 04;15(Suppl 2):S109-S113

Fogarty International Center, National Institutes of Health, Bethesda, Maryland.

The global burden of lung disease is substantial, accounting for an estimated 7.5 million deaths per year, approximately 14% of annual deaths worldwide. The prime illnesses include, in descending order, chronic obstructive pulmonary disease, lung cancer, tuberculosis, acute respiratory infections, asthma, and interstitial lung fibrosis. Key risk factors include smoking, both indoor and outdoor air pollution, and occupational exposures. Although the distribution of both the diseases and the risk factors varies greatly by age, geography, and setting, the greatest burden falls on populations living in low- and middle-income countries. Improvements in these metrics will require major public health interventions to curb smoking; improving air quality both in the community and the household; addressing the ever-present burden of infections, including tuberculosis, flu, and the many agents that cause acute respiratory disease; and identifying and protecting workers from the hazards of exposure to toxic substances. Although research over the years has identified many ways to reduce or prevent the enormous burden of disease, a huge gap exists between what we know and what we can do. This "implementation gap" is the greatest challenge we face in this field today. Research on how best to address and implement the changes needed will require not only biomedical advances to improve treatment but also social, economic, and policy research. We still need to elaborate more effective evidence-based policies and interventions to control tobacco use, address ambient and household air pollution, and improve the prevention and treatment of tuberculosis and acute respiratory infections with vaccines and drugs and reduce exposures to environmental and occupational hazards. Until these efforts receive greater prioritization, the burden of disease is unlikely to diminish a great deal more.
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http://dx.doi.org/10.1513/AnnalsATS.201708-685MGDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5946509PMC
April 2018

Abundant off-target edits from site-directed RNA editing can be reduced by nuclear localization of the editing enzyme.

RNA Biol 2018 01 13;15(1):104-114. Epub 2017 Nov 13.

a Eugene Bell Center for Regenerative Biology and Tissue Engineering , The Marine Biological Laboratory , Woods Hole , MA , USA.

Site-directed RNA editing (SDRE) is a general strategy for making targeted base changes in RNA molecules. Although the approach is relatively new, several groups, including our own, have been working on its development. The basic strategy has been to couple the catalytic domain of an adenosine (A) to inosine (I) RNA editing enzyme to a guide RNA that is used for targeting. Although highly efficient on-target editing has been reported, off-target events have not been rigorously quantified. In this report we target premature termination codons (PTCs) in messages encoding both a fluorescent reporter protein and the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein transiently transfected into human epithelial cells. We demonstrate that while on-target editing is efficient, off-target editing is extensive, both within the targeted message and across the entire transcriptome of the transfected cells. By redirecting the editing enzymes from the cytoplasm to the nucleus, off-target editing is reduced without compromising the on-target editing efficiency. The addition of the E488Q mutation to the editing enzymes, a common strategy for increasing on-target editing efficiency, causes a tremendous increase in off-target editing. These results underscore the need to reduce promiscuity in current approaches to SDRE.
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http://dx.doi.org/10.1080/15476286.2017.1387711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5786015PMC
January 2018

A-to-I RNA Editing in the Earliest-Diverging Eumetazoan Phyla.

Mol Biol Evol 2017 08;34(8):1890-1901

The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel.

The highly conserved ADAR enzymes, found in all multicellular metazoans, catalyze the editing of mRNA transcripts by the deamination of adenosines to inosines. This type of editing has two general outcomes: site specific editing, which frequently leads to recoding, and clustered editing, which is usually found in transcribed genomic repeats. Here, for the first time, we looked for both editing of isolated sites and clustered, non-specific sites in a basal metazoan, the coral Acropora millepora during spawning event, in order to reveal its editing pattern. We found that the coral editome resembles the mammalian one: it contains more than 500,000 sites, virtually all of which are clustered in non-coding regions that are enriched for predicted dsRNA structures. RNA editing levels were increased during spawning and increased further still in newly released gametes. This may suggest that editing plays a role in introducing variability in coral gametes.
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http://dx.doi.org/10.1093/molbev/msx125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5850803PMC
August 2017

Trade-off between Transcriptome Plasticity and Genome Evolution in Cephalopods.

Cell 2017 04;169(2):191-202.e11

Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University, Tel Aviv 69978, Israel; Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 69978, Israel. Electronic address:

RNA editing, a post-transcriptional process, allows the diversification of proteomes beyond the genomic blueprint; however it is infrequently used among animals for this purpose. Recent reports suggesting increased levels of RNA editing in squids thus raise the question of the nature and effects of these events. We here show that RNA editing is particularly common in behaviorally sophisticated coleoid cephalopods, with tens of thousands of evolutionarily conserved sites. Editing is enriched in the nervous system, affecting molecules pertinent for excitability and neuronal morphology. The genomic sequence flanking editing sites is highly conserved, suggesting that the process confers a selective advantage. Due to the large number of sites, the surrounding conservation greatly reduces the number of mutations and genomic polymorphisms in protein-coding regions. This trade-off between genome evolution and transcriptome plasticity highlights the importance of RNA recoding as a strategy for diversifying proteins, particularly those associated with neural function. PAPERCLIP.
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http://dx.doi.org/10.1016/j.cell.2017.03.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499236PMC
April 2017

Mutations underlying Episodic Ataxia type-1 antagonize Kv1.1 RNA editing.

Sci Rep 2017 02 20;7:41095. Epub 2017 Feb 20.

Department of Molecular Physiology &Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232, United States.

Adenosine-to-inosine RNA editing in transcripts encoding the voltage-gated potassium channel Kv1.1 converts an isoleucine to valine codon for amino acid 400, speeding channel recovery from inactivation. Numerous Kv1.1 mutations have been associated with the human disorder Episodic Ataxia Type-1 (EA1), characterized by stress-induced ataxia, myokymia, and increased prevalence of seizures. Three EA1 mutations, V404I, I407M, and V408A, are located within the RNA duplex structure required for RNA editing. Each mutation decreased RNA editing both in vitro and using an in vivo mouse model bearing the V408A allele. Editing of transcripts encoding mutant channels affects numerous biophysical properties including channel opening, closing, and inactivation. Thus EA1 symptoms could be influenced not only by the direct effects of the mutations on channel properties, but also by their influence on RNA editing. These studies provide the first evidence that mutations associated with human genetic disorders can affect cis-regulatory elements to alter RNA editing.
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http://dx.doi.org/10.1038/srep41095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5316942PMC
February 2017

An efficient system for selectively altering genetic information within mRNAs.

Nucleic Acids Res 2016 12 23;44(21):e157. Epub 2016 Aug 23.

Institute of Neurobiology, University of Puerto Rico Medical Sciences Campus, San Juan, PR 00901, USA

Site-directed RNA editing (SDRE) is a strategy to precisely alter genetic information within mRNAs. By linking the catalytic domain of the RNA editing enzyme ADAR to an antisense guide RNA, specific adenosines can be converted to inosines, biological mimics for guanosine. Previously, we showed that a genetically encoded iteration of SDRE could target adenosines expressed in human cells, but not efficiently. Here we developed a reporter assay to quantify editing, and used it to improve our strategy. By enhancing the linkage between ADAR's catalytic domain and the guide RNA, and by introducing a mutation in the catalytic domain, the efficiency of converting a U A: G premature termination codon (PTC) to tryptophan (U G: G) was improved from ∼11 % to ∼70 %. Other PTCs were edited, but less efficiently. Numerous off-target edits were identified in the targeted mRNA, but not in randomly selected endogenous messages. Off-target edits could be eliminated by reducing the amount of guide RNA with a reduction in on-target editing. The catalytic rate of SDRE was compared with those for human ADARs on various substrates and found to be within an order of magnitude of most. These data underscore the promise of site-directed RNA editing as a therapeutic or experimental tool.
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http://dx.doi.org/10.1093/nar/gkw738DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5137428PMC
December 2016

Transcriptome of the Caribbean stony coral Porites astreoides from three developmental stages.

Gigascience 2016 08 2;5(1):33. Epub 2016 Aug 2.

Marine Biological Laboratory, Woods Hole, Massachusetts, USA.

Background: Porites astreoides is a ubiquitous species of coral on modern Caribbean reefs that is resistant to increasing temperatures, overfishing, and other anthropogenic impacts that have threatened most other coral species. We assembled and annotated a transcriptome from this coral using Illumina sequences from three different developmental stages collected over several years: free-swimming larvae, newly settled larvae, and adults (>10 cm in diameter). This resource will aid understanding of coral calcification, larval settlement, and host-symbiont interactions.

Findings: A de novo transcriptome for the P. astreoides holobiont (coral plus algal symbiont) was assembled using 594 Mbp of raw Illumina sequencing data generated from five age-specific cDNA libraries. The new transcriptome consists of 867 255 transcript elements with an average length of 685 bases. The isolated P. astreoides assembly consists of 129 718 transcript elements with an average length of 811 bases, and the isolated Symbiodinium sp. assembly had 186 177 transcript elements with an average length of 1105 bases.

Conclusions: This contribution to coral transcriptome data provides a valuable resource for researchers studying the ontogeny of gene expression patterns within both the coral and its dinoflagellate symbiont.
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http://dx.doi.org/10.1186/s13742-016-0138-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4969664PMC
August 2016

Importance of the Voltage Dependence of Cardiac Na/K ATPase Isozymes.

Biophys J 2015 Nov;109(9):1852-62

Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas. Electronic address:

Cardiac cells express more than one isoform of the Na, K-ATPase (NKA), the heteromeric enzyme that creates the Na(+) and K(+) gradients across the plasmalemma. Cardiac isozymes contain one catalytic α-subunit isoform (α1, α2, or α3) associated with an auxiliary β-subunit isoform (β1 or β2). Past studies using biochemical approaches have revealed minor kinetic differences between isozymes formed by different α-β isoform combinations; these results make it difficult to understand the physiological requirement for multiple isoforms. In intact cells, however, NKA enzymes operate in a more complex environment, which includes a substantial transmembrane potential. We evaluated the voltage dependence of human cardiac NKA isozymes expressed in Xenopus oocytes, and of native NKA isozymes in rat ventricular myocytes, using normal mammalian physiological concentrations of Na(+)o and K(+)o. We demonstrate that although α1 and α3 pumps are functional at all physiologically relevant voltages, α2β1 pumps and α2β2 pumps are inhibited by ∼75% and ∼95%, respectively, at resting membrane potentials, and only activate appreciably upon depolarization. Furthermore, phospholemman (FXYD1) inhibits pump function without significantly altering the pump's voltage dependence. Our observations provide a simple explanation for the physiological relevance of the α2 subunit (∼20% of total α subunits in rat ventricle): they act as a reserve and are recruited into action for extra pumping during the long-lasting cardiac action potential, where most of the Na(+) entry occurs. This strong voltage dependence of α2 pumps also helps explain how cardiotonic steroids, which block NKA pumps, can be a beneficial treatment for heart failure: by only inhibiting the α2 pumps, they selectively reduce NKA activity during the cardiac action potential, leading to an increase in systolic Ca(2+), due to reduced extrusion through the Na/Ca exchanger, without affecting resting Na(+) and Ca(2+) concentrations.
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http://dx.doi.org/10.1016/j.bpj.2015.09.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4643266PMC
November 2015