Genetic characterization of outbred Sprague Dawley rats and utility for genome-wide association studies.

Sprague Dawley (SD) rats are among the most widely used outbred laboratory rat populations. Despite this, the genetic characteristics of SD rats have not been clearly described, and SD rats are rarely used for experiments aimed at exploring genotype-phenotype relationships. In order to use SD rats to perform a genome-wide association study (GWAS), we collected behavioral data from 4,625 SD rats that were predominantly obtained from two commercial vendors, Charles River Laboratories and Harlan Sprague Dawley Inc. Using double-digest genotyping-by-sequencing (ddGBS), we obtained dense, high-quality genotypes at 291,438 SNPs across 4,061 rats. This genetic data allowed us to characterize the variation present in Charles River vs. Harlan SD rats. We found that the two populations are highly diverged (FST > 0.4). Furthermore, even for rats obtained from the same vendor, there was strong population structure across breeding facilities and even between rooms at the same facility. We performed multiple separate GWAS by fitting a linear mixed model that accounted for population structure and using meta-analysis to jointly analyze all cohorts. Our study examined Pavlovian conditioned approach (PavCA) behavior, which assesses the propensity for rats to attribute incentive salience to reward-associated cues. We identified 46 significant associations for the various metrics used to define PavCA. The surprising degree of population structure among SD rats from different sources has important implications for their use in both genetic and non-genetic studies.

Effects of predictive and incentive value manipulation on sign- and goal-tracking behavior.

When a neutral stimulus is repeatedly paired with an appetitive reward, two different types of conditioned approach responses may develop: a sign-tracking response directed toward the neutral cue, or a goal-tracking response directed toward the location of impending reward delivery. Sign-tracking responses have been postulated to result from attribution of incentive value to conditioned cues, while goal-tracking reflects the assignment of only predictive value to the cue. We therefore hypothesized that sign-tracking rats would be more sensitive to manipulations of incentive value, while goal-tracking rats would be more responsive to changes in the predictive value of the cue. We tested sign- and goal-tracking before and after devaluation of a food reward using lithium chloride, and tested whether either response could be learned under negative contingency conditions that precluded any serendipitous reinforcement of the behavior that might support instrumental learning. We also tested the effects of blocking the predictive value of a cue using simultaneous presentation of a pre-conditioned cue. We found that sign-tracking was sensitive to outcome devaluation, while goal-tracking was not. We also confirmed that both responses are Pavlovian because they can be learned under negative contingency conditions. Goal-tracking was almost completely blocked by a pre-conditioned cue, while sign-tracking was much less sensitive to such interference. These results indicate that sign- and goal-tracking may follow different rules of reinforcement learning and suggest a need to revise current models of associative learning to account for these differences.

A Test-and-Not-Treat Strategy for Onchocerciasis Elimination in Loa loa-coendemic Areas: Cost Analysis of a Pilot in the Soa Health District, Cameroon.

BACKGROUND: Severe adverse events after treatment with ivermectin in individuals with high levels of Loa loa microfilariae in the blood preclude onchocerciasis elimination through community-directed treatment with ivermectin (CDTI) in Central Africa. We measured the cost of a community-based pilot using a test-and-not-treat (TaNT) strategy in the Soa health district in Cameroon. METHODS: Based on actual expenditures, we empirically estimated the economic cost of the Soa TaNT campaign, including financial costs and opportunity costs that will likely be borne by control programs and stakeholders in the future. In addition to the empirical analyses, we estimated base-case, less intensive, and more intensive resource use scenarios to explore how costs might differ if TaNT were implemented programmatically. RESULTS: The total costs of US$283 938 divided by total population, people tested, and people treated with 42% coverage were US$4.0, US$9.2, and US$9.5, respectively. In programmatic implementation, these costs (base-case estimates with less and more intensive scenarios) could be US$2.2 ($1.9-$3.6), US$5.2 ($4.5-$8.3), and US$5.4 ($4.6-$8.6), respectively. CONCLUSIONS: TaNT clearly provides a safe strategy for large-scale ivermectin treatment and overcomes a major obstacle to the elimination of onchocerciasis in areas coendemic for Loa loa. Although it is more expensive than standard CDTI, costs vary depending on the setting, the implementation choices made by the institutions involved, and the community participation rate. Research on the required duration of TaNT is needed to improve the affordability assessment, and more experience is needed to understand how to implement TaNT optimally.

Valuing the Unpaid Contribution of Community Health Volunteers to Mass Drug Administration Programs.

Community health volunteers (CHVs) are being used within a growing number of healthcare interventions, and they have become a cornerstone for the delivery of mass drug administration within many neglected tropical disease control programs. However, a greater understanding of the methods used to value the unpaid time CHVs contribute to healthcare programs is needed. We outline the two main approaches used to value CHVs' unpaid time (the opportunity cost and the replacement cost approaches). We found that for mass drug administration programs the estimates of the economic costs relating to the CHVs' unpaid time can be significant, with the averages of the different studies varying between US$0.05 and $0.16 per treatment. We estimated that the time donated by CHVs' to the African Programme for Onchocerciasis Control alone would be valued between US$60 and $90 million. There is a need for greater transparency and consistency in the methods used to value CHVs' unpaid time.

Sign-tracking behavior is difficult to extinguish and resistant to multiple cognitive enhancers.

The attribution of incentive-motivational value to drug-related cues underlies relapse and craving in drug addiction. One method of addiction treatment, cue-exposure therapy, utilizes repeated presentations of drug-related cues in the absence of drug (i.e., extinction learning); however, its efficacy has been limited due to an incomplete understanding of extinction and relapse processes after cues have been imbued with incentive-motivational value. To investigate this, we used a Pavlovian conditioned approach procedure to screen for rats that attribute incentive-motivational value to reward-related cues (sign-trackers; STs) or those that do not (goal-trackers; GTs). In Experiment 1, rats underwent Pavlovian extinction followed by reinstatement and spontaneous recovery tests. For comparison, a separate group of rats underwent PCA training followed by operant conditioning, extinction, and tests of reinstatement and spontaneous recovery. In Experiment 2, three cognitive enhancers (sodium butyrate, D-cycloserine, and fibroblast growth factor 2) were administered following extinction training to facilitate extinction learning. STs but not GTs displayed enduring resistance to Pavlovian, but not operant, extinction and were more susceptible to spontaneous recovery. In addition, none of the cognitive enhancers tested affected extinction learning. These results expand our understanding of extinction learning by demonstrating that there is individual variation in extinction and relapse processes and highlight potential difficulties in applying extinction-based therapies to drug addiction treatment in the clinic.

Econometric estimation of WHO-CHOICE country-specific costs for inpatient and outpatient health service delivery.

BACKGROUND: Policy makers require information on costs related to inpatient and outpatient health services to inform resource allocation decisions. METHODS: Country data sets were gathered in 2008-2010 through literature reviews, website searches and a public call for cost data. Multivariate regression analysis was used to explore the determinants of variability in unit costs using data from 30 countries. Two models were designed, with the inpatient and outpatient models drawing upon 3407 and 9028 observations respectively. Cost estimates are produced at country and regional level, with 95% confidence intervals. RESULTS: Inpatient costs across 30 countries are significantly associated with the type of hospital, ownership, as well as bed occupancy rate, average length of stay, and total number of inpatient admissions. Changes in outpatient costs are significantly associated with location, facility ownership and the level of care, as well as to the number of outpatient visits and visits per provider per day. CONCLUSIONS: These updated WHO-CHOICE service delivery unit costs are statistically robust and may be used by analysts as inputs for economic analysis. The models can predict country-specific unit costs at different capacity levels and in different settings.

Investment Success in Public Health: An Analysis of the Cost-Effectiveness and Cost-Benefit of the Global Programme to Eliminate Lymphatic Filariasis.

Background: It has been estimated that $154 million per year will be required during 2015-2020 to continue the Global Programme to Eliminate Lymphatic Filariasis (GPELF). In light of this, it is important to understand the program's current value. Here, we evaluate the cost-effectiveness and cost-benefit of the preventive chemotherapy that was provided under the GPELF between 2000 and 2014. In addition, we also investigate the potential cost-effectiveness of hydrocele surgery. Methods: Our economic evaluation of preventive chemotherapy was based on previously published health and economic impact estimates (between 2000 and 2014). The delivery costs of treatment were estimated using a model developed by the World Health Organization. We also developed a model to investigate the number of disability-adjusted life years (DALYs) averted by a hydrocelectomy and identified the cost threshold under which it would be considered cost-effective. Results: The projected cost-effectiveness and cost-benefit of preventive chemotherapy were very promising, and this was robust over a wide range of costs and assumptions. When the economic value of the donated drugs was not included, the GPELF would be classed as highly cost-effective. We projected that a typical hydrocelectomy would be classed as highly cost-effective if the surgery cost less than $66 and cost-effective if less than $398 (based on the World Bank's cost-effectiveness thresholds for low income countries). Conclusions: Both the preventive chemotherapy and hydrocele surgeries provided under the GPELF are incredibly cost-effective and offer a very good investment in public health.

Mathematical Modeling of Programmatic Requirements for Yaws Eradication.

Yaws is targeted for eradication by 2020. The mainstay of the eradication strategy is mass treatment followed by case finding. Modeling has been used to inform programmatic requirements for other neglected tropical diseases and could provide insights into yaws eradication. We developed a model of yaws transmission varying the coverage and number of rounds of treatment. The estimated number of cases arising from an index case (basic reproduction number [R0]) ranged from 1.08 to 3.32. To have 80% probability of achieving eradication, 8 rounds of treatment with 80% coverage were required at low estimates of R0 (1.45). This requirement increased to 95% at high estimates of R0 (2.47). Extending the treatment interval to 12 months increased requirements at all estimates of R0. At high estimates of R0 with 12 monthly rounds of treatment, no combination of variables achieved eradication. Models should be used to guide the scale-up of yaws eradication.

Thalamic mast cell activity is associated with sign-tracking behavior in rats.

Mast cells are resident immune cells in the thalamus that can degranulate and release hundreds of signaling molecules (i.e., monoamines, growth factors, and cytokines) both basally and in response to environmental stimuli. Interestingly, mast cell numbers in the brain show immense individual variation in both rodents and humans. We used a Pavlovian conditioned approach (PCA) procedure to examine whether mast cells are associated with individual variation in the attribution of incentive-motivational value to reward-related cues. During the PCA procedure, a lever response-independently predicts the delivery of a food pellet into a magazine, and over training sessions three conditioned responses (CRs) develop: sign-tracking (lever-directed CRs), goal-tracking (magazine-directed CRs), and an intermediate response (both CRs). In Experiment 1, we measured thalamic mast cell number/activation using toluidine blue and demonstrated that sign-trackers have increased degranulated (activated) but not granulated (inactive) mast cells. In Experiment 2, we infused the mast cell inhibitor, cromolyn (200µg/rat; i.c.v.), immediately before five daily PCA training sessions and demonstrated that mast cell inhibition selectively impairs the acquisition of sign-tracking behavior. Taken together, these results demonstrate that thalamic mast cells contribute to the attribution of incentive-motivational value to reward-related cues and suggest that mast cell inhibition may be a novel target for addiction treatment.

Sign-trackers have elevated myo-inositol in the nucleus accumbens and ventral hippocampus following Pavlovian conditioned approach.

Pavlovian conditioned approach (PCA) is a behavioral procedure that can be used to assess individual differences in the addiction vulnerability of drug-naïve rats and identify addiction vulnerability factors. Using proton magnetic resonance spectroscopy (1 H-MRS) ex vivo, we simultaneously analyzed concentrations of multiple neurochemicals throughout the mesocorticolimbic system 2 weeks after PCA training in order to identify potential vulnerability factors to addiction in drug-naïve rats for future investigations. Levels of myo-inositol (Ins), a 1 H-MRS-detectable marker of glial activity/proliferation, were increased in the nucleus accumbens (NAc) and ventral hippocampus, but not dorsal hippocampus or medial prefrontal cortex, of sign-trackers compared to goal-trackers or intermediate responders. In addition, Ins levels positively correlated with PCA behavior in the NAc and ventral hippocampus. Because the sign-tracker phenotype is associated with increased drug-seeking behavior, these results observed in drug-naïve rats suggest that alterations in glial activity/proliferation within these regions may represent an addiction vulnerability factor. Sign-tracking rats preferentially approach reward cues during Pavlovian conditioning, while goal-trackers instead approach the location of impending reward. Sign-trackers are also more prone to cue-induced drug-seeking behavior. We used magnetic resonance spectroscopy to show that myo-inositol levels are higher in the ventral hippocampus and nucleus accumbens of sign-trackers relative to goal-trackers. Thus, elevated myo-inositol may be a vulnerability factor for addiction.

Lesions of the ventral hippocampus attenuate the acquisition but not expression of sign-tracking behavior in rats.

Individual variation in the attribution of motivational salience to reward-related cues is believed to underlie addiction vulnerability. Pavlovian conditioned approach measures individual variation in motivational salience by identifying rats that are attracted to and motivated by reward cues (sign-trackers) or motivationally fixed on the reward itself (goal-trackers). Previously, it has been demonstrated that sign-trackers are more vulnerable to addiction-like behavior. Moreover, sign-trackers release more dopamine in the nucleus accumbens than goal-trackers in response to reward-related cues, and sign- but not goal-tracking behavior is dopamine-dependent. In the present study, we investigated whether the ventral hippocampus, a potent driver of dopaminergic activity in the nucleus accumbens, modulates the acquisition and expression of Pavlovian conditioned approach behavior. In Experiment 1, lesions of the ventral, but not dorsal or total hippocampus, decreased sign-tracking behavior. In Experiment 2, lesions of the ventral hippocampus did not affect the expression of sign- or goal-tracking behaviors nor conditioned reinforcement. In addition, temporary inactivation of the ventral subiculum, the main output pathway of the ventral hippocampus, did not affect the expression of sign- or goal-tracking behaviors. High-pressure liquid chromatography of nucleus accumbens tissue punches revealed that ventral hippocampal lesions decreased levels of homovanillic acid and the homovanillic acid/dopamine ratio (a marker of dopamine release and metabolism) in only sign-trackers, and decreased accumbal norepinephrine levels in both sign- and goal-trackers. These results suggest that the ventral hippocampus is important for the acquisition but not expression of sign-tracking behavior, possibly as a result of altered dopamine and norepinephrine in the nucleus accumbens. © 2016 Wiley Periodicals, Inc.

Down-regulation of the antisense mitochondrial non-coding RNAs (ncRNAs) is a unique vulnerability of cancer cells and a potential target for cancer therapy.

Hallmarks of cancer are fundamental principles involved in cancer progression. We propose an additional generalized hallmark of malignant transformation corresponding to the differential expression of a family of mitochondrial ncRNAs (ncmtRNAs) that comprises sense and antisense members, all of which contain stem-loop structures. Normal proliferating cells express sense (SncmtRNA) and antisense (ASncmtRNA) transcripts. In contrast, the ASncmtRNAs are down-regulated in tumor cells regardless of tissue of origin. Here we show that knockdown of the low copy number of the ASncmtRNAs in several tumor cell lines induces cell death by apoptosis without affecting the viability of normal cells. In addition, knockdown of ASncmtRNAs potentiates apoptotic cell death by inhibiting survivin expression, a member of the inhibitor of apoptosis (IAP) family. Down-regulation of survivin is at the translational level and is probably mediated by microRNAs generated by dicing of the double-stranded stem of the ASncmtRNAs, as suggested by evidence presented here, in which the ASncmtRNAs are bound to Dicer and knockdown of the ASncmtRNAs reduces reporter luciferase activity in a vector carrying the 3'-UTR of survivin mRNA. Taken together, down-regulation of the ASncmtRNAs constitutes a vulnerability or Achilles' heel of cancer cells, suggesting that the ASncmtRNAs are promising targets for cancer therapy.

Cost-effectiveness of a comprehensive programme for drug-resistant tuberculosis in China.

OBJECTIVE: To investigate the cost-effectiveness of a comprehensive programme for drug-resistant tuberculosis launched in four sites in China in 2011. METHODS: In 2011-2012, we reviewed the records of 172 patients with drug-resistant tuberculosis who enrolled in the comprehensive programme and we collected relevant administrative data from hospitals and China's public health agency. For comparison, we examined a cohort of 81 patients who were treated for drug-resistant tuberculosis in 2006-2009. We performed a cost-effectiveness analysis, from a societal perspective, that included probabilistic uncertainty. We measured early treatment outcomes based on three-month culture results and modelled longer-term outcomes to facilitate estimation of the comprehensive programme's cost per disability-adjusted life-year (DALY) averted. FINDINGS: The comprehensive programme cost 8837 United States dollars (US$) per patient treated. Low enrolment rates meant that some fixed costs were higher, per patient, than expected. Although the comprehensive programme appeared 30 times more costly than the previous one, it resulted in greater health benefits. The comprehensive programme, which cost US$ 639 (95% credible interval: 112 to 1322) per DALY averted, satisfied the World Health Organization's criterion for a very cost-effective intervention. CONCLUSION: The comprehensive programme, which included rapid screening, standardized care and financial protection, improved individual outcomes for MDR tuberculosis in a cost-effective manner. To support post-2015 global heath targets, the comprehensive programme should be expanded to non-residents and other areas of China.

Multidrug-resistant tuberculosis, Somalia, 2010-2011.

In a nationwide survey in 2011, multidrug-resistant tuberculosis (MDR TB) was found in 5.2% and 40.8% of patients with new and previously treated TB, respectively. These levels of drug resistance are among the highest ever documented in Africa and the Middle East. This finding presents a serious challenge for TB control in Somalia.

Xpert MTB/RIF for diagnosis of tuberculosis and drug-resistant tuberculosis: a cost and affordability analysis.

Xpert MTB/RIF is a rapid test to diagnose tuberculosis (TB) and rifampicin-resistant TB. Cost and affordability will influence its uptake. We assessed the cost, globally and in 36 high-burden countries, of two strategies for diagnosing TB and multidrug-resistant (MDR)-TB: Xpert with follow-on diagnostics, and conventional diagnostics. Costs were compared with funding available for TB care and control, and donor investments in HIV prevention and care. Using Xpert to diagnose MDR-TB would cost US$70-90 million per year globally and be lower cost than conventional diagnostics globally and in all high-burden countries. Diagnosing TB in HIV-positive people using Xpert would also cost US$90-101 million per year and be lower cost than conventional diagnostics globally and in 33 out of 36 high-burden countries. Testing everyone with TB signs and symptoms would cost US$434-468 million per year globally, much more than conventional diagnostics. However, in European countries, Brazil and South Africa, the cost would represent <10% of TB funding. Introducing Xpert to diagnose MDR-TB and to diagnose TB in HIV-positive people is warranted in many countries. Using it to test everyone with TB signs and symptoms is affordable in several middle-income countries, but financial viability in low-income countries requires large increases in TB funding and/or further price reductions.

Single prolonged stress disrupts retention of extinguished fear in rats.

Clinical research has linked post-traumatic stress disorder (PTSD) with deficits in fear extinction. However, it is not clear whether these deficits result from stress-related changes in the acquisition or retention of extinction or in the regulation of extinction memories by context, for example. In this study, we used the single prolonged stress (SPS) animal model of PTSD and fear conditioning procedures to examine the effects of prior traumatic stress on the acquisition, retention, and context-specificity of extinction. SPS administered one week prior to fear conditioning had no effect on the acquisition of fear conditioning or extinction but disrupted the retention of extinction memories for both contextual and cued fear. This SPS effect required a post-stress incubation period to manifest. The results demonstrate that SPS disrupts extinction retention, leading to enhanced fear renewal; further research is needed to identify the neurobiological processes through which SPS induces these effects.

Nicotinic and muscarinic acetylcholine receptor antagonism dose-dependently decreases sign- but not goal-tracking behavior in male rats.

RATIONALE: Acetylcholinergic antagonists have shown some promise in reducing addiction-related behaviors in both preclinical and clinical studies. However, the psychological mechanisms by which these drugs are able to affect addictive behavior remain unclear. A particular key process for the development of addiction is the attribution of incentive salience to reward-related cues, which can be specifically measured in animals using a Pavlovian conditioned approach procedure. When confronted with a lever that predicts food delivery, some rats engage with the lever directly (i.e., they sign track), indicating attribution of incentive-motivational properties to the lever itself. In contrast, others treat the lever as a predictive cue and approach the location of impending food delivery (i.e., they goal track), without treating the lever itself as a reward. OBJECTIVES: We tested whether systemic antagonism of the either nicotinic or muscarinic acetylcholine receptors would selectively affect sign- or goal-tracking behavior, indicating a selective effect on incentive salience attribution. METHODS: A total of 98 male Sprague Dawley rats were either given the muscarinic antagonist scopolamine (100, 50, or 10 µg/kg i.p.) or the nicotinic antagonist mecamylamine (0.3, 1.0, or 3 mg/kg i.p.) before being trained on a Pavlovian conditioned approach procedure. RESULTS: Scopolamine dose-dependently decreased sign tracking behavior and increased goal-tracking behavior. Mecamylamine reduced sign-tracking but did not affect goal-tracking behavior. CONCLUSIONS: Antagonism of either muscarinic or nicotinic acetylcholine receptors can reduce incentive sign-tracking behavior in male rats. This effect appears to be specifically due to a reduction in incentive salience attribution since goal-tracking either increased or was not affected by these manipulations.

Ukraine Trauma Project: the feasibility of introducing advanced trauma-care skills to frontline emergency medical services responders.

OBJECTIVES: To design, develop, deliver and assess a training initiative on haemorrhage control for emergency medical services (EMS) staff in Ukraine, in an active wartime setting. DESIGN: Using the Medical Research Council framework for complex interventions, a training programme was designed and developed in a collaboration between Irish and Ukrainian colleagues and delivered by experienced prehospital clinicians/educators. Feedback was gathered from participants. SETTING: The Russian invasion of Ukraine has caused large numbers of trauma patients with limited access to advanced prehospital emergency care. Ukrainian authorities requested support in delivering such care. PARTICIPANTS: Ukrainian EMS nominated clinical staff as trainees, in partnership with an educational institution in Kyiv. INTERVENTION: One day provider and train-the-trainer courses were developed and delivered, focused on early delivery of tranexamic acid (TXA), using intraosseous access (IO) in victims of wartime trauma. OUTCOME MEASURES: Safe organisation and delivery of courses, assessed knowledge and skills competence and self-reported satisfaction and pre/post confidence/competence. RESULTS: Two provider and one train-the-trainer courses and four equipment supply exercises were delivered for 89 EMS staff (doctors, nurses, paramedics); none had prior experience of IO or prehospital delivery of TXA. All participants were assessed as competent as providers and/or trainers. High levels of satisfaction and significantly improved self-assessed confidence and competence were reported. CONCLUSION: Rapid design and delivery of a training programme focused on an identified need for advanced care of trauma patients in a wartime setting has been possible. Training and immediate access to appropriate equipment was demonstrated. Evidence of frequency of use and safe, effective interventions has not been collected; such data are important for evaluation but difficult to collect in this setting. A high level of demand for this training now exists.

Inverse Modulation of Aurora Kinase A and Topoisomerase IIα in Normal and Tumor Breast Cells upon Knockdown of Mitochondrial ASncmtRNA.

Breast cancer is currently the most diagnosed form of cancer and the leading cause of death by cancer among females worldwide. We described the family of long non-coding mitochondrial RNAs (ncmtRNAs), comprised of sense (SncmtRNA) and antisense (ASncmtRNA) members. Knockdown of ASncmtRNAs using antisense oligonucleotides (ASOs) induces proliferative arrest and apoptotic death of tumor cells, but not normal cells, from various tissue origins. In order to study the mechanisms underlying this selectivity, in this study we performed RNAseq in MDA-MB-231 breast cancer cells transfected with ASncmtRNA-specific ASO or control-ASO, or left untransfected. Bioinformatic analysis yielded several differentially expressed cell-cycle-related genes, from which we selected Aurora kinase A (AURKA) and topoisomerase IIα (TOP2A) for RT-qPCR and western blot validation in MDA-MB-231 and MCF7 breast cancer cells, as well as normal breast epithelial cells (HMEC). We observed no clear differences regarding mRNA levels but both proteins were downregulated in tumor cells and upregulated in normal cells. Since these proteins play a role in genomic integrity, this inverse effect of ASncmtRNA knockdown could account for tumor cell downfall whilst protecting normal cells, suggesting this approach could be used for genomic protection under cancer treatment regimens or other scenarios.