Unfolding simulations reveal the mechanism of extreme unfolding cooperativity in the kinetically stable alpha-lytic protease.

Kinetically stable proteins, those whose stability is derived from their slow unfolding kinetics and not thermodynamics, are examples of evolution's best attempts at suppressing unfolding. Especially in highly proteolytic environments, both partially and fully unfolded proteins face potential inactivation through degradation and/or aggregation, hence, slowing unfolding can greatly extend a protein's functional lifetime. The prokaryotic serine protease alpha-lytic protease (alphaLP) has done just that, as its unfolding is both very slow (t(1/2) approximately 1 year) and so cooperative that partial unfolding is negligible, providing a functional advantage over its thermodynamically stable homologs, such as trypsin. Previous studies have identified regions of the domain interface as critical to alphaLP unfolding, though a complete description of the unfolding pathway is missing. In order to identify the alphaLP unfolding pathway and the mechanism for its extreme cooperativity, we performed high temperature molecular dynamics unfolding simulations of both alphaLP and trypsin. The simulated alphaLP unfolding pathway produces a robust transition state ensemble consistent with prior biochemical experiments and clearly shows that unfolding proceeds through a preferential disruption of the domain interface. Through a novel method of calculating unfolding cooperativity, we show that alphaLP unfolds extremely cooperatively while trypsin unfolds gradually. Finally, by examining the behavior of both domain interfaces, we propose a model for the differential unfolding cooperativity of alphaLP and trypsin involving three key regions that differ between the kinetically stable and thermodynamically stable classes of serine proteases

Systems analysis of host-parasite interactions.

Parasitic diseases caused by protozoan pathogens lead to hundreds of thousands of deaths per year in addition to substantial suffering and socioeconomic decline for millions of people worldwide. The lack of effective vaccines coupled with the widespread emergence of drug-resistant parasites necessitates that the research community take an active role in understanding host-parasite infection biology in order to develop improved therapeutics. Recent advances in next-generation sequencing and the rapid development of publicly accessible genomic databases for many human pathogens have facilitated the application of systems biology to the study of host-parasite interactions. Over the past decade, these technologies have led to the discovery of many important biological processes governing parasitic disease. The integration and interpretation of high-throughput -omic data will undoubtedly generate extraordinary insight into host-parasite interaction networks essential to navigate the intricacies of these complex systems. As systems analysis continues to build the foundation for our understanding of host-parasite biology, this will provide the framework necessary to drive drug discovery research forward and accelerate the development of new antiparasitic therapies

Transcriptome-based reconstructions from the murine knockout suggest involvement of the urate transporter, URAT1 (slc22a12), in novel metabolic pathways.

URAT1 (slc22a12) was identified as the transporter responsible for renal reabsorption of the medically important compound, uric acid. However, subsequent studies have indicated that other transporters make contributions to this process, and that URAT1 transports other organic anions besides urate (including several in common with the closely related multi-specific renal organic anion transporters, OAT1 (slc22a6) and OAT3 (slc22a8)). These findings raise the possibility that urate transport is not the sole physiological function of URAT1. We previously characterized mice null for the murine ortholog of URAT1 (mURAT1; previously cloned as RST), finding a relatively modest decrement in urate reabsorptive capacity. Nevertheless, there were shifts in the plasma and urinary concentrations of multiple small molecules, suggesting significant metabolic changes in the knockouts. Although these molecules remain unidentified, here we have computationally delineated the biochemical networks consistent with transcriptomic data from the null mice. These analyses suggest alterations in the handling of not only urate but also other putative URAT1 substrates comprising intermediates in nucleotide, carbohydrate, and steroid metabolism. Moreover, the analyses indicate changes in multiple other pathways, including those relating to the metabolism of glycosaminoglycans, methionine, and coenzyme A, possibly reflecting downstream effects of URAT1 loss. Taken together with the available substrate and metabolomic data for the other OATs, our findings suggest that the transport and biochemical functions of URAT1 overlap those of OAT1 and OAT3, and could contribute to our understanding of the relationship between uric acid and the various metabolic disorders to which it has been linked

Alcohol use in fishing communities and men's willingness to participate in an alcohol, violence and HIV risk reduction intervention: qualitative findings from Rakai, Uganda.

Alcohol use, intimate partner violence (IPV) and HIV infection are associated, but few programmes and interventions have addressed their synergistic relationship or been evaluated for effectiveness and acceptability. This is a critical gap in populations with high rates of alcohol use, HIV and IPV, such as Uganda's fishing communities. This study examined drinking norms, barriers and facilitators to engagement in a risk reduction programme, and ideas for tailoring. Results showed that alcohol use is common in fishing villages. While men and women drink, gendered notions of femininity deem alcohol largely unacceptable for women. Plastic sachets of liquor were the most common alcoholic drink. Participants did not understand the definition of 'hazardous drinking', but recognised connections between drinking, violence and sexual risk-taking. The idea of an alcohol, IPV and HIV risk reduction intervention was supported, but barriers need to be addressed, including how best to help those uninterested in reducing their drinking, addressing normalisation of drinking and how best to inform those who truly need intervention. Intervention to people living with HIV around the time of diagnosis and treatment may be warranted. Study findings highlight the potential to integrate alcohol and IPV reduction programmes into an HIV service provision

Perceptions of Adolescent Pregnancy Among Teenage Girls in Rakai, Uganda.

The leading causes of death and disability among Ugandan female adolescents aged 15 to 19 years are pregnancy complications, unsafe abortions, and childbirth. Despite these statistics, our understanding of how girls perceive adolescent pregnancy is limited. This qualitative study explored the social and contextual factors shaping the perceptions of adolescent pregnancy and childbirth among a sample of 12 currently pregnant and 14 never pregnant girls living in the rural Rakai District of Uganda. Interviews were conducted to elicit perceived risk factors for pregnancy, associated community attitudes, and personal opinions on adolescent pregnancy. Findings indicate that notions of adolescent pregnancy are primarily influenced by perceptions of control over getting pregnant and readiness for childbearing. Premarital pregnancy was perceived as negative whereas postmarital pregnancy was regarded as positive. Greater understanding of the individual and contextual factors influencing perceptions can aid in development of salient, culturally appropriate policies and programs to mitigate unintended adolescent pregnancies

Hnrnph1 Is A Quantitative Trait Gene for Methamphetamine Sensitivity.

Psychostimulant addiction is a heritable substance use disorder; however its genetic basis is almost entirely unknown. Quantitative trait locus (QTL) mapping in mice offers a complementary approach to human genome-wide association studies and can facilitate environment control, statistical power, novel gene discovery, and neurobiological mechanisms. We used interval-specific congenic mouse lines carrying various segments of chromosome 11 from the DBA/2J strain on an isogenic C57BL/6J background to positionally clone a 206 kb QTL (50,185,512-50,391,845 bp) that was causally associated with a reduction in the locomotor stimulant response to methamphetamine (2 mg/kg, i.p.; DBA/2J < C57BL/6J)-a non-contingent, drug-induced behavior that is associated with stimulation of the dopaminergic reward circuitry. This chromosomal region contained only two protein coding genes-heterogeneous nuclear ribonucleoprotein, H1 (Hnrnph1) and RUN and FYVE domain-containing 1 (Rufy1). Transcriptome analysis via mRNA sequencing in the striatum implicated a neurobiological mechanism involving a reduction in mesolimbic innervation and striatal neurotransmission. For instance, Nr4a2 (nuclear receptor subfamily 4, group A, member 2), a transcription factor crucial for midbrain dopaminergic neuron development, exhibited a 2.1-fold decrease in expression (DBA/2J < C57BL/6J; p 4.2 x 10-15). Transcription activator-like effector nucleases (TALENs)-mediated introduction of frameshift deletions in the first coding exon of Hnrnph1, but not Rufy1, recapitulated the reduced methamphetamine behavioral response, thus identifying Hnrnph1 as a quantitative trait gene for methamphetamine sensitivity. These results define a novel contribution of Hnrnph1 to neurobehavioral dysfunction associated with dopaminergic neurotransmission. These findings could have implications for understanding the genetic basis of methamphetamine addiction in humans and the development of novel therapeutics for prevention and treatment of substance abuse and possibly other psychiatric disorders

The Tanzanian Trauma patients' Prehospital Experience: A Qualitative Interview-based Study.

We sought to characterise the prehospital experience of Tanzanian trauma patients, and identify barriers and facilitators to implement community-based emergency medical systems (EMS). Our study was conducted in the emergency department of an urban national referral hospital in Tanzania. A convenience sample of 34 adult trauma patients, or surrogate family members, presenting or referred to an urban referral emergency department in Tanzania for treatment of injury, participated in the study. Participation in semistructured, iteratively developed interviews until saturation of responses was reached. A grounded theory-based approach to qualitative analysis was used to identify recurrent themes. We characterised numerous deficiencies within the existing clinic-to-hospital referral network, including missed/delayed diagnoses, limited management capabilities at pre-referral facilities and interfacility transfer delays. Potential barriers to EMS implementation include patient financial limitations and lack of insurance, limited public infrastructure and resources, and the credibility of potential first aid responders. Potential facilitators of EMS include communities' tendency to pool resources, individuals' trust of other community members to be first aid responders, and faith in community leaders to organise EMS response. Participants expressed a strong desire to learn first aid. The composite themes generated by the data suggest that there are myriad structural, financial, institutional and cultural barriers to the implementation of a formal prehospital system. However, our analysis also revealed potential facilitators to a first-responder system that takes advantage of close-knit local communities and the trust of recognised leaders in society. The results suggest favourable acceptability for community-based response by trained lay people. There is significant opportunity for care improvements with short trainings and low-cost supply planning. Further research looking at the effects of delay on outcomes in this population is needed

Trauma admissions among street children at a tertiary care hospital in north-western Tanzania: a neglected public health problem

Background: Trauma among street children is an emerging but neglected public health problem in most low and middle income countries. This study was conducted to determine the incidence, etiological spectrum, injury characteristics and treatment outcome among street children and to identify the predictors of the outcome of these patients at Bugando Medical Centre in Mwanza, Tanzania.Methods: The study included street children aged <18 years. Routine investigations including haematological, biochemical and imaging were performed on admission. The severity of injury was determined using the Kampala Trauma Score II. Data on patient’s characteristics, circumstances of injury, injury characteristics, treatment offered, outcome variables, length of hospital stay and mortality were collected using a questionnaire.Results: A total of 342 street children (M: F = 6.8: 1) representing 11.5% of all paediatric injury patients were studied. The modal age group was 11-15 years (median = 12 years) accounting for 53.2% (n=182) of the patients. Assault was the most frequent (73.7%) cause of injury. More than three quarter of injuries occurred along the street. Most of patients (59.1%) presented late (>24 hours) after injury. Blunt injuries were the most common (76.0%) mechanism of injuries. Musculoskeletal (30.8%) and head (25.3%) were the most frequent body regions affected. Soft tissue injuries were the most common type of injuries affecting 322 (94.2%) cases. Majority of patients (96.5%) underwent surgical treatment of which wound debridement (97.6%) was the most common surgical procedure performed.  Complication rate was 39.5%. The median hospital stay was 6 days.  Mortality rate was 13.5% and it was significantly associated with injury-arrival time (OR =2.4, 95%CI (1.3-5.6), p = 0.002), severe injury (Kampala Trauma Score <6) (OR = 3.6, 95%CI (2.5-7.9), p = 0.001), severe head injuries (OR= 5.1, 95%CI (4.6 – 8.2), p =0.012) and surgical site infection.Conclusion: Trauma among street children is an emerging but neglected epidemic in Tanzania and contributes significantly to high morbidity and mortality. Assault was the most frequent cause of injury.  Urgent preventive measures targeting at reducing the occurrence of assault is necessary to reduce the incidence of trauma among street children in this region

Towards Robots that Influence Humans over Long-Term Interaction

When humans interact with robots influence is inevitable. Consider an autonomous car driving near a human: the speed and steering of the autonomous car will affect how the human drives. Prior works have developed frameworks that enable robots to influence humans towards desired behaviors. But while these approaches are effective in the short-term (i.e., the first few human-robot interactions), here we explore long-term influence (i.e., repeated interactions between the same human and robot). Our central insight is that humans are dynamic: people adapt to robots, and behaviors which are influential now may fall short once the human learns to anticipate the robot's actions. With this insight, we experimentally demonstrate that a prevalent game-theoretic formalism for generating influential robot behaviors becomes less effective over repeated interactions. Next, we propose three modifications to Stackelberg games that make the robot's policy both influential and unpredictable. We finally test these modifications across simulations and user studies: our results suggest that robots which purposely make their actions harder to anticipate are better able to maintain influence over long-term interaction. See videos here: https://youtu.be/ydO83cgjZ2