Empirical modeling of agricultural climate risk.

Effective policies for adaptation to climate change require understanding how impacts are related to exposures and vulnerability, the dimensions of the climate system that will change most and where human impacts will be most draconian, and the institutions best suited to respond. Here, we propose a simple method for more credibly pairing empirical statistical damage estimates derived from recent weather and outcome observations with projected future climate changes and proposed responses. We first analyze agricultural production and loan repayment data from Brazil to understand vulnerability to historical variation in the more predictable components of temperature and rainfall (trend and seasonality) as well as to shocks (both local and over larger spatial scales). This decomposed weather variation over the past two decades explains over 50% of the yield variation in major Brazilian crops and, critically, can be constructed in the same way for future climate projections. Combining our estimates with bias-corrected downscaled climate simulations for Brazil, we find increased variation in yields and revenues (including more bad years and worse outcomes) and higher agricultural loan default at midcentury. Results in this context point to two particularly acute dimensions of vulnerability: Intensified seasonality and local idiosyncratic shocks both contribute to worsening outcomes, along with a reduced capacity for spatially correlated ("covariate") shocks to ameliorate these effects through prices. These findings suggest that resilience strategies should focus on institutions such as water storage, financial services, and reinsurance.

Economic vulnerability, violence, and sexual risk factors for HIV among female sex workers in Tijuana, Mexico.

Economic vulnerability is often reported to underlie involvement in sex work among female sex workers (FSW), but may also create urgency in women's work, limiting women's negotiating power with clients and in turn, increasing their vulnerability for violence and HIV. This study assessed economic vulnerability in relation to violence and sexual risk behaviors for HIV among a sample of FSW in Tijuana, Mexico. FSW at least 18 years of age were recruited through venue-based sampling for a survey (n = 228) and in-depth interviews (n = 50) to investigate HIV risk factors in this region. Using crude and adjusted logistic regression models, we assessed lack of financial support from others as well as reports of financial hardship separately in relation to experiencing sexual violence (e.g. by clients, police, relationship partners, in the past 6 months), physical violence (past 6 months), STI diagnosis, and inconsistent condom use (past 30 days). Qualitative interviews (n = 50), conducted with a subsample of the survey participants, were also examined for related themes. FSW who reported no financial support were more likely to report sexual violence (OR = 2.1; 95% CI:1.1-4.2). FSW who reported financial hardship were more likely to experience sexual violence (OR = 1.9; 95% CI:1.1-3.6) and physical violence (OR = 1.9; 95% CI:1.1-3.6), as well as to report past 30-day inconsistent condom use (OR = 2.4; 95%CI: 1.3-4.6) and to test positive for an STI (OR = 1.9; 95% CI:1.1-3.4). Qualitative data substantiated these findings. Findings suggest that interventions to improve economic well-being may be useful to prevent the intersecting concerns of violence and HIV among FSW.

Splice correction therapies for familial hypercholesterolemic patients with low-density lipoprotein receptor mutations.

PURPOSE OF REVIEW: Antisense oligomers (ASOs) have been available for decades: however, only recently have these molecules been applied clinically. This review aims to discuss the possible development of antisense-mediated splice correction therapies as precision medicines for familial hypercholesterolemic patients carrying mutations that compromise normal splicing of the low-density lipoprotein receptor (LDLR) gene transcript. RECENT FINDINGS: Three antisense drugs are currently being assessed in ongoing clinical trials for dyslipidemias, aiming to lower the plasma concentrations of lipoproteins that lead to end-organ damage, principally coronary artery disease. Although a handful of drugs may be applicable to many patients with familial hypercholesterolemia (FH), mutation-specific personalised antisense drugs may be even more effective in selected patients. Currently, there is no therapy that effectively addresses mutations in the LDLR, the major cause of FH. Many mutations in the LDLR that disrupt normal pre-mRNA processing could be applicable to splice correction therapy to restore receptor activity. SUMMARY: Precision medicine could provide long-term economic and social benefits if they can be implemented effectively and sustainably. Many mutations found in the LDLR gene could be amendable to therapeutic splice correction and we should consider developing a therapeutic ASO platform for these mutations.

Removal of the Polyglutamine Repeat of Ataxin-3 by Redirecting pre-mRNA Processing.

Spinocerebellar ataxia type 3 (SCA3) is a devastating neurodegenerative disease for which there is currently no cure, nor effective treatment strategy. One of nine polyglutamine disorders known to date, SCA3 is clinically heterogeneous and the main feature is progressive ataxia, which in turn affects speech, balance and gait of the affected individual. SCA3 is caused by an expanded polyglutamine tract in the ataxin-3 protein, resulting in conformational changes that lead to toxic gain of function. The expanded glutamine tract is located at the 5' end of the penultimate exon (exon 10) of ATXN3 gene transcript. Other studies reported removal of the expanded glutamine tract using splice switching antisense oligonucleotides. Here, we describe improved efficiency in the removal of the toxic polyglutamine tract of ataxin-3 in vitro using phosphorodiamidate morpholino oligomers, when compared to antisense oligonucleotides composed of 2'-O-methyl modified bases on a phosphorothioate backbone. Significant downregulation of both the expanded and non-expanded protein was induced by the morpholino antisense oligomer, with a greater proportion of ataxin-3 protein missing the polyglutamine tract. With growing concerns over toxicity associated with long-term administration of phosphorothioate oligonucleotides, the use of a phosphorodiamidate morpholino oligomer may be preferable for clinical application. These results suggest that morpholino oligomers may provide greater therapeutic benefit for the treatment of spinocerebellar ataxia type 3, without toxic effects.

Systematic Approach to Developing Splice Modulating Antisense Oligonucleotides.

The process of pre-mRNA splicing is a common and fundamental step in the expression of most human genes. Alternative splicing, whereby different splice motifs and sites are recognised in a developmental and/or tissue-specific manner, contributes to genetic plasticity and diversity of gene expression. Redirecting pre-mRNA processing of various genes has now been validated as a viable clinical therapeutic strategy, providing treatments for Duchenne muscular dystrophy (inducing specific exon skipping) and spinal muscular atrophy (promoting exon retention). We have designed and evaluated over 5000 different antisense oligonucleotides to alter splicing of a variety of pre-mRNAs, from the longest known human pre-mRNA to shorter, exon-dense primary gene transcripts. Here, we present our guidelines for designing, evaluating and optimising splice switching antisense oligomers in vitro. These systematic approaches assess several critical factors such as the selection of target splicing motifs, choice of cells, various delivery reagents and crucial aspects of validating assays for the screening of antisense oligonucleotides composed of 2'-O-methyl modified bases on a phosphorothioate backbone.

In Vitro Validation of Phosphorodiamidate Morpholino Oligomers.

One of the crucial aspects of screening antisense oligonucleotides destined for therapeutic application is confidence that the antisense oligomer is delivered efficiently into cultured cells. Efficient delivery is particularly vital for antisense phosphorodiamidate morpholino oligomers, which have a neutral backbone, and are known to show poor gymnotic uptake. Here, we report several methods to deliver these oligomers into cultured cells. Although 4D-Nucleofector™ or Neon™ electroporation systems provide efficient delivery and use lower amounts of phosphorodiamidate morpholino oligomer, both systems are costly. We show that some readily available transfection reagents can be used to deliver phosphorodiamidate morpholino oligomers as efficiently as the electroporation systems. Among the transfection reagents tested, we recommend Lipofectamine 3000™ for delivering phosphorodiamidate morpholino oligomers into fibroblasts and Lipofectamine 3000™ or Lipofectamine 2000™ for myoblasts/myotubes. We also provide optimal programs for nucleofection into various cell lines using the P3 Primary Cell 4D-Nucleofector™ X Kit (Lonza), as well as antisense oligomers that redirect expression of ubiquitously expressed genes that may be used as positive treatments for human and murine cell transfections.

A comparison of carcass decomposition and associated insect succession onto burnt and unburnt pig carcasses.

The rate of decomposition and insect succession onto decomposing pig carcasses were investigated following burning of carcasses. Ten pig carcasses (40-45 kg) were exposed to insect activity during autumn (March-April) in Western Australia. Five replicates were burnt to a degree described by the Crow-Glassman Scale (CGS) level #2, while five carcasses were left unburnt as controls. Burning carcasses greatly accelerated decomposition in contrast to unburnt carcasses. Physical modifications following burning such as skin discolouration, splitting of abdominal tissue and leathery consolidation of skin eliminated evidence of bloat and altered microambient temperatures associated with carcasses throughout decomposition. Insect species identified on carcasses were consistent between treatment groups; however, a statistically significant difference in insect succession onto remains was evident between treatments (PERMANOVA F (1, 224) = 14.23, p < 0.01) during an 8-day period that corresponds with the wet stage of decomposition. Differences were noted in the arrival time of late colonisers (Coleoptera) and the development of colonising insects between treatment groups. Differences in the duration of decomposition stages and insect assemblages indicate that burning has an effect on both rate of decomposition and insect succession. The findings presented here provide baseline data for entomological casework involving burnt remains criminal investigations.

Down syndrome and DYRK1A overexpression: relationships and future therapeutic directions.

Down syndrome is a genetic-based disorder that results from the triplication of chromosome 21, leading to an overexpression of many triplicated genes, including the gene encoding Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase 1A (DYRK1A). This protein has been observed to regulate numerous cellular processes, including cell proliferation, cell functioning, differentiation, and apoptosis. Consequently, an overexpression of DYRK1A has been reported to result in cognitive impairment, a key phenotype of individuals with Down syndrome. Therefore, downregulating DYRK1A has been explored as a potential therapeutic strategy for Down syndrome, with promising results observed from in vivo mouse models and human clinical trials that administered epigallocatechin gallate. Current DYRK1A inhibitors target the protein function directly, which tends to exhibit low specificity and selectivity, making them unfeasible for clinical or research purposes. On the other hand, antisense oligonucleotides (ASOs) offer a more selective therapeutic strategy to downregulate DYRK1A expression at the gene transcript level. Advances in ASO research have led to the discovery of numerous chemical modifications that increase ASO potency, specificity, and stability. Recently, several ASOs have been approved by the U.S. Food and Drug Administration to address neuromuscular and neurological conditions, laying the foundation for future ASO therapeutics. The limitations of ASOs, including their high production cost and difficulty delivering to target tissues can be overcome by further advances in ASO design. DYRK1A targeted ASOs could be a viable therapeutic approach to improve the quality of life for individuals with Down syndrome and their families.

Effect of a cash transfer programme for schooling on prevalence of HIV and herpes simplex type 2 in Malawi: a cluster randomised trial.

BACKGROUND: Lack of education and an economic dependence on men are often suggested as important risk factors for HIV infection in women. We assessed the efficacy of a cash transfer programme to reduce the risk of sexually transmitted infections in young women. METHODS: In this cluster randomised trial, never-married women aged 13-22 years were recruited from 176 enumeration areas in the Zomba district of Malawi and randomly assigned with computer-generated random numbers by enumeration area (1:1) to receive cash payments (intervention group) or nothing (control group). Intervention enumeration areas were further randomly assigned with computer-generated random numbers to conditional (school attendance required to receive payment) and unconditional (no requirements to receive payment) groups. Participants in both intervention groups were randomly assigned by a lottery to receive monthly payments ranging from US$1 to $5, while their parents were independently assigned with computer-generated random numbers to receive $4-10. Behavioural risk assessments were done at baseline and 12 months; serology was tested at 18 months. Participants were not masked to treatment status but counsellors doing the serologic testing were. The primary outcomes were prevalence of HIV and herpes simplex virus 2 (HSV-2) at 18 months and were assessed by intention-to-treat analyses. The trial is registered, number NCT01333826. FINDINGS: 88 enumeration areas were assigned to receive the intervention and 88 as controls. For the 1289 individuals enrolled in school at baseline with complete interview and biomarker data, weighted HIV prevalence at 18 month follow-up was 1·2% (seven of 490 participants) in the combined intervention group versus 3·0% (17 of 799 participants) in the control group (adjusted odds ratio [OR] 0·36, 95% CI 0·14-0·91); weighted HSV-2 prevalence was 0·7% (five of 488 participants) versus 3·0% (27 of 796 participants; adjusted OR 0·24, 0·09-0·65). In the intervention group, we noted no difference between conditional versus unconditional intervention groups for weighted HIV prevalence (3/235 [1%] vs 4/255 [2%]) or weighted HSV-2 prevalence (4/233 [1%] vs 1/255 [<1%]). For individuals who had already dropped out of school at baseline, we detected no significant difference between intervention and control groups for weighted HIV prevalence (23/210 [10%] vs 17/207 [8%]) or weighted HSV-2 prevalence (17/211 [8%] vs 17/208 [8%]). INTERPRETATION: Cash transfer programmes can reduce HIV and HSV-2 infections in adolescent schoolgirls in low-income settings. Structural interventions that do not directly target sexual behaviour change can be important components of HIV prevention strategies. FUNDING: Global Development Network, Bill & Melinda Gates Foundation, National Bureau of Economic Research Africa Project, World Bank's Research Support Budget, and several World Bank trust funds (Gender Action Plan, Knowledge for Change Program, and Spanish Impact Evaluation fund).

Induced alternative splicing an opportunity to study PCSK9 protein isoforms at physiologically relevant concentrations.

Splice modulating antisense oligomers (AOs) are increasingly used to modulate RNA processing. While most are investigated for their use as therapeutics, AOs can also be used for basic research. This study examined their use to investigate internally and terminally truncated proprotein convertase subtilisin/kexin type 9 (PCSK9) protein isoforms. Previous studies have used plasmid or viral-vector-mediated protein overexpression to study different PCSK9 protein isoforms, creating an artificial environment within the cell. Here we designed and tested AOs to remove specific exons that encode for PCSK9 protein domains and produced protein isoforms at more physiologically relevant levels. We evaluated the isoforms' expression, secretion, and subsequent impact on the low-density lipoprotein (LDL) receptor and its activity in Huh-7 cells. We found that modifying the Cis-His-rich domain by targeting exons 10 or 11 negatively affected LDL receptor activity and hence did not enhance LDL uptake although the levels of LDL receptor were increased. On the other hand, removing the hinge region encoded by exon 8, or a portion of the prodomain encoded by exon 2, have the potential as therapeutics for hypercholesterolemia. Our findings expand the understanding of PCSK9 isoforms and their impact on the LDL receptor and its activity at physiologically relevant concentrations.

Neurodegenerative diseases: a hotbed for splicing defects and the potential therapies.

Precursor messenger RNA (pre-mRNA) splicing is a fundamental step in eukaryotic gene expression that systematically removes non-coding regions (introns) and ligates coding regions (exons) into a continuous message (mature mRNA). This process is highly regulated and can be highly flexible through a process known as alternative splicing, which allows for several transcripts to arise from a single gene, thereby greatly increasing genetic plasticity and the diversity of proteome. Alternative splicing is particularly prevalent in neuronal cells, where the splicing patterns are continuously changing to maintain cellular homeostasis and promote neurogenesis, migration and synaptic function. The continuous changes in splicing patterns and a high demand on many cis- and trans-splicing factors contribute to the susceptibility of neuronal tissues to splicing defects. The resultant neurodegenerative diseases are a large group of disorders defined by a gradual loss of neurons and a progressive impairment in neuronal function. Several of the most common neurodegenerative diseases involve some form of splicing defect(s), such as Alzheimer's disease, Parkinson's disease and spinal muscular atrophy. Our growing understanding of RNA splicing has led to the explosion of research in the field of splice-switching antisense oligonucleotide therapeutics. Here we review our current understanding of the effects alternative splicing has on neuronal differentiation, neuronal migration, synaptic maturation and regulation, as well as the impact on neurodegenerative diseases. We will also review the current landscape of splice-switching antisense oligonucleotides as a therapeutic strategy for a number of common neurodegenerative disorders.

Polyglutamine Ataxias: Our Current Molecular Understanding and What the Future Holds for Antisense Therapies.

Polyglutamine (polyQ) ataxias are a heterogenous group of neurological disorders all caused by an expanded CAG trinucleotide repeat located in the coding region of each unique causative gene. To date, polyQ ataxias encompass six disorders: spinocerebellar ataxia types 1, 2, 3, 6, 7, and 17 and account for a larger group of disorders simply known as polyglutamine disorders, which also includes Huntington's disease. These diseases are typically characterised by progressive ataxia, speech and swallowing difficulties, lack of coordination and gait, and are unfortunately fatal in nature, with the exception of SCA6. All the polyQ spinocerebellar ataxias have a hallmark feature of neuronal aggregations and share many common pathogenic mechanisms, such as mitochondrial dysfunction, impaired proteasomal function, and autophagy impairment. Currently, therapeutic options are limited, with no available treatments that slow or halt disease progression. Here, we discuss the common molecular and clinical presentations of polyQ spinocerebellar ataxias. We will also discuss the promising antisense oligonucleotide therapeutics being developed as treatments for these devastating diseases. With recent advancements and therapeutic approvals of various antisense therapies, it is envisioned that some of the studies reviewed may progress into clinical trials and beyond.

Induction of cryptic pre-mRNA splice-switching by antisense oligonucleotides.

Antisense oligomers (AOs) are increasingly being used to modulate RNA splicing in live cells, both for research and for the development of therapeutics. While the most common intended effect of these AOs is to induce skipping of whole exons, rare examples are emerging of AOs that induce skipping of only part of an exon, through activation of an internal cryptic splice site. In this report, we examined seven AO-induced cryptic splice sites in six genes. Five of these cryptic splice sites were discovered through our own experiments, and two originated from other published reports. We modelled the predicted effects of AO binding on the secondary structure of each of the RNA targets, and how these alterations would in turn affect the accessibility of the RNA to splice factors. We observed that a common predicted effect of AO binding was disruption of the exon definition signal within the exon's excluded segment.

Cost-effectiveness of hepatitis C virus (HCV) elimination strategies among people who inject drugs (PWID) in Tijuana, Mexico.

BACKGROUND AND AIMS: In Latin America, Mexico was first to launch a hepatitis C virus (HCV) elimination strategy, where people who inject drugs (PWID) are a main risk group for transmission. In Tijuana, HCV seroprevalence among PWID is > 90%, with minimal harm reduction (HR). We evaluated cost-effectiveness of strategies to achieve the incidence elimination target among PWID in Tijuana. METHODS: Modeling study using a dynamic, cost-effectiveness model of HCV transmission and progression among active and former PWID in Tijuana, to assess the cost-effectiveness of incidence elimination strategies from a health-care provider perspective. The model incorporated PWID transitions between HR stages (no HR, only opioid agonist therapy, only high coverage needle-syringe programs, both). Four strategies that could achieve the incidence target (80% reduction by 2030) were compared with the status quo (no intervention). The strategies incorporated the number of direct-acting anti-viral (DAA) treatments required with: (1) no HR scale-up, (2) HR scale-up from 2019 to 20% coverage among PWID, (3) HR to 40% coverage and (4) HR to 50% coverage. Costs (2019 US$) and health outcomes [disability-adjusted life years (DALYs)] were discounted 3% per year. Mean incremental cost-effectiveness ratios (ICER, $/DALY averted) were compared with one-time per capita gross domestic product (GDP) ($9698 in 2019) and purchasing power parity-adjusted per capita GDP ($4842-13 557) willingness-to-pay (WTP) thresholds. RESULTS: DAAs alone were the least costly elimination strategy [$173 million, 95% confidence interval (CI) = 126-238 million], but accrued fewer health benefits compared with strategies with HR. DAAs + 50% HR coverage among PWID averted the most DALYs but cost $265 million, 95% CI = 210-335 million). The optimal strategy was DAAs + 50% HR (ICER $6743/DALY averted compared to DAAs only) under the one-time per-capita GDP WTP ($9698). CONCLUSIONS: A combination of high-coverage harm reduction and hepatitis C virus treatment is the optimal cost-effective strategy to achieve the HCV incidence elimination goal in Mexico.

Cost and cost-effectiveness of a real-world HCV treatment program among HIV-infected individuals in Myanmar.

INTRODUCTION: Over half of those hepatitis C virus (HCV)/HIV coinfected live in low-income and middle-income countries, and many remain undiagnosed or untreated. In 2016, Médecins Sans Frontières (MSF) established a direct-acting antiviral (DAA) treatment programme for people HCV/HIV coinfected in Myanmar. The purpose of our study was to evaluate the real-world cost and cost-effectiveness of this programme, and potential cost-effectiveness if implemented by the Ministry of Health (MoH). METHODS: Costs (patient-level microcosting) and treatment outcomes were collected from the MSF prospective cohort study in Dawei, Myanmar. A Markov model was used to assess cost-effectiveness of the programme compared with no HCV treatment from a health provider perspective. Estimated lifetime and healthcare costs (in 2017 US$) and health outcomes (in disability-adjusted life-years (DALYs)) were simulated to calculate the incremental cost-effectiveness ratio (ICER), compared with a willingness-to-pay threshold of per capita Gross Domestic Product in Myanmar ($1250). We evaluated cost-effectiveness with updated quality-assured generic DAA prices and potential cost-effectiveness of a proposed simplified treatment protocol with updated DAA prices if implemented by the MoH. RESULTS: From November 2016 to October 2017, 122 with HIV/HCV-coinfected patients were treated with DAAs (46% with cirrhosis), 96% (n=117) achieved sustained virological response. Mean treatment costs were $1229 (without cirrhosis) and $1971 (with cirrhosis), with DAA drugs being the largest contributor to cost. Compared with no treatment, the program was cost-effective (ICER $634/DALY averted); more so with updated prices for quality-assured generic DAAs (ICER $488/DALY averted). A simplified treatment protocol delivered by the MoH could be cost-effective if associated with similar outcomes (ICER $316/DALY averted). CONCLUSIONS: Using MSF programme data, the DAA treatment programme for HCV among HIV-coinfected individuals is cost-effective in Myanmar, and even more so with updated DAA prices. A simplified treatment protocol could enhance cost-effectiveness if further rollout demonstrates it is not associated with worse treatment outcomes.

The short-term impacts of a schooling conditional cash transfer program on the sexual behavior of young women.

Recent evidence suggests that conditional cash transfer (CCT) programs for schooling are effective in raising school enrollment and attendance. However, there is also reason to believe that such programs can affect other outcomes, such as the sexual behavior of their young beneficiaries. Zomba Cash Transfer Program is a randomized ongoing CCT intervention targeting young women in Malawi that provides incentives (in the form of school fees and cash transfers) to current schoolgirls and recent dropouts to stay in or return to school. An average offer of US$10/month conditional on satisfactory school attendance - plus direct payment of secondary school fees - led to significant declines in early marriage, teenage pregnancy, and self-reported sexual activity among program beneficiaries after just one year of program implementation. For program beneficiaries who were out of school at baseline, the probability of getting married and becoming pregnant declined by more than 40 and 30%, respectively. In addition, the incidence of the onset of sexual activity was 38% lower among all program beneficiaries than the control group. Overall, these results suggest that CCT programs not only serve as useful tools for improving school attendance but may also reduce sexual activity, teen pregnancy, and early marriage.

Voter information campaigns and political accountability: Cumulative findings from a preregistered meta-analysis of coordinated trials.

Voters may be unable to hold politicians to account if they lack basic information about their representatives' performance. Civil society groups and international donors therefore advocate using voter information campaigns to improve democratic accountability. Yet, are these campaigns effective? Limited replication, measurement heterogeneity, and publication biases may undermine the reliability of published research. We implemented a new approach to cumulative learning, coordinating the design of seven randomized controlled trials to be fielded in six countries by independent research teams. Uncommon for multisite trials in the social sciences, we jointly preregistered a meta-analysis of results in advance of seeing the data. We find no evidence overall that typical, nonpartisan voter information campaigns shape voter behavior, although exploratory and subgroup analyses suggest conditions under which informational campaigns could be more effective. Such null estimated effects are too seldom published, yet they can be critical for scientific progress and cumulative, policy-relevant learning.

The heterogeneous effects of HIV testing.

An extensive multi-disciplinary literature examines the effects of learning one's HIV status on subsequent risky sexual behaviors. However, many of these studies rely on non-experimental designs; use self-reported outcome measures; or both. In this study, we investigate the effects of a randomly assigned home based HIV testing and counseling (HTC) intervention on risky sexual behaviors and schooling investments among school-age females in Malawi. We find no overall effects on HIV, Herpes Simplex Virus (HSV-2), or achievement test scores at follow-up. However, among the small group of individuals who tested positive for HIV, we find a large increase in the probability of HSV-2 infection, with this effect being stronger among those surprised by their test results. Similarly, those surprised by HIV-negative test results have significantly higher achievement test scores at follow-up, consistent with increased returns to investments in human capital.