Myths and Methodologies: Standardisation in human physiology research-should we control the controllables?

The premise of research in human physiology is to explore a multifaceted system whilst identifying one or a few outcomes of interest. Therefore, the control of potentially confounding variables requires careful thought regarding the extent of control and complexity of standardisation. One common factor to control prior to testing is diet, as food and fluid provision may deviate from participants' habitual diets, yet a self-report and replication method can be flawed by under-reporting. Researchers may also need to consider standardisation of physical activity, whether it be through familiarisation trials, wash-out periods, or guidance on levels of physical activity to be achieved before trials. In terms of pharmacological agents, the ethical implications of standardisation require researchers to carefully consider how medications, caffeine consumption and oral contraceptive prescriptions may affect the study. For research in females, it should be considered whether standardisation between- or within-participants in regards to menstrual cycle phase is most relevant. The timing of measurements relative to various other daily events is relevant to all physiological research and so it can be important to standardise when measurements are made. This review summarises the areas of standardisation which we hope will be considered useful to anyone involved in human physiology research, including when and how one can apply standardisation to various contexts.

Exposure to cigarette taxes as a teenager and the persistence of smoking into adulthood.

Are teenage and adult smoking causally related? Recent anti-tobacco policy is predicated on the assumption that preventing teenagers from smoking will ensure that fewer adults smoke, but direct evidence in support of this assumption is scant. Using data from three nationally representative sources and instrumenting for teenage smoking with cigarette taxes experienced at ages 14-17, we document a strong positive relationship between teenage and adult smoking: deterring 10 teenagers from smoking through raising cigarette taxes roughly translates into 5 fewer adult smokers. We conclude that efforts to reduce teenage smoking can have long-lasting consequences on smoking participation and, presumably, health.

Myths and Methodologies: Standardisation in Human Physiology Research-Should We Control the Controllables?

The premise of research in human physiology is to explore a multifaceted system whilst identifying one or a few outcomes of interest. Therefore, the control of potentially confounding variables requires careful thought regarding the extent of control and complexity of standardisation. One common factor to control prior to testing is diet, as food and fluid provision may deviate from participants' habitual diets, yet a self-report and replication method can be flawed by under-reporting. Researchers may also need to consider standardisation of physical activity, whether it be through familiarisation trials, wash-out periods, or guidance on levels of physical activity to be achieved before trials. In terms of pharmacological agents, the ethical implications of standardisation require researchers to carefully consider how medications, caffeine consumption and oral contraceptive prescriptions may affect the study. For research in females, it should be considered whether standardisation between- or within-participants in regards to menstrual cycle phase is most relevant. The timing of measurements relative to various other daily events is relevant to all physiological research and so it can be important to standardise when measurements are made. This review summarises the areas of standardisation which we hope will be considered useful to anyone involved in human physiology research, including when and how one can apply standardisation to various contexts.

Acyl-Ghrelin Attenuates Neurochemical and Motor Deficits in the 6-OHDA Model of Parkinson's Disease.

The feeding-related hormone, acyl-ghrelin, protects dopamine neurones in murine 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-based models of experimental Parkinson's disease (PD). However, the potential protective effect of acyl-ghrelin on substantia nigra pars compacta (SNpc) dopaminergic neurones and consequent behavioural correlates in the more widely used 6-hydroxydopamine (6-OHDA) rat medial forebrain bundle (MFB) lesion model of PD are unknown. To address this question, acyl-ghrelin levels were raised directly by mini-pump infusion for 7 days prior to unilateral injection of 6-OHDA into the MFB with assessment of amphetamine-induced rotations on days 27 and 35, and immunohistochemical analysis of dopaminergic neurone survival. Whilst acyl-ghrelin treatment was insufficient to elevate food intake or body weight, it attenuated amphetamine-induced circling behaviour and SNpc dopamine neurone loss induced by 6-OHDA. These data support the notion that elevating circulating acyl-ghrelin may be a valuable approach to slow or impair progression of neurone loss in PD.

Recreational marijuana laws and the misuse of prescription opioids: Evidence from National Survey on Drug Use and Health microdata.

Several studies have concluded that legalizing medical marijuana can reduce deaths from opioid overdoses. Drawing on micro data from the National Survey on Drug Use and Health, a survey uniquely suited to assessing patterns of substance use, we examine the relationship between recreational marijuana laws (RMLs) and the misuse of prescription opioids. Using a standard difference-in-differences (DD) regression model, we find that RML adoption reduces the likelihood of frequently misusing prescription opioids such as OxyContin, Percocet, and Vicodin. However, using a two-stage procedure designed to account for staggered treatment and dynamic effects, the DD estimate of relationship between RML adoption and the likelihood of frequently misusing prescription opioids becomes positive. Although event study estimates suggest that RML adoption leads to a decrease in the frequency of prescription opioid abuse, this effect appears to dissipate after only 2 or 3 years.

The phenomenon of summer diarrhea and its waning, 1910-1930.

During the first two decades of the 20th century, diarrheal deaths among American infants and children surged every summer. Although we still do not know what pathogen (or pathogens) caused this phenomenon, the consensus view is that it was eventually controlled through public health efforts at the municipal level. Using data from 26 major American cities for the period 1910-1930, we document the phenomenon of summer diarrhea and explore its dissipation. We find that water filtration is associated with a 15 percent reduction in diarrheal mortality among children under the age of two during the non-summer months, but does not seem to have had an effect on diarrheal mortality during the summer. In general, we find little evidence to suggest that public health interventions undertaken at the municipal level contributed to the dissipation of summer diarrhea.

Ghrelin-AMPK Signaling Mediates the Neuroprotective Effects of Calorie Restriction in Parkinson's Disease.

Calorie restriction (CR) is neuroprotective in Parkinson's disease (PD) although the mechanisms are unknown. In this study we hypothesized that elevated ghrelin, a gut hormone with neuroprotective properties, during CR prevents neurodegeneration in an 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD. CR attenuated the MPTP-induced loss of substantia nigra (SN) dopamine neurons and striatal dopamine turnover in ghrelin WT but not KO mice, demonstrating that ghrelin mediates CR's neuroprotective effect. CR elevated phosphorylated AMPK and ACC levels in the striatum of WT but not KO mice suggesting that AMPK is a target for ghrelin-induced neuroprotection. Indeed, exogenous ghrelin significantly increased pAMPK in the SN. Genetic deletion of AMPKß1 and 2 subunits only in dopamine neurons prevented ghrelin-induced AMPK phosphorylation and neuroprotection. Hence, ghrelin signaling through AMPK in SN dopamine neurons mediates CR's neuroprotective effects. We consider targeting AMPK in dopamine neurons may recapitulate neuroprotective effects of CR without requiring dietary intervention.

Migraine headache and labor market outcomes.

While migraine headache can be physically debilitating, no study has attempted to estimate its effects on labor market outcomes. Using data drawn from the National Longitudinal Study of Adolescent Health, we estimate the effect of being diagnosed with migraine headache on labor force participation, hours worked, and wages. Ordinary least squares (OLS) estimates suggest that migraines are associated with reduced labor force participation and lower wages among females. A negative association between migraine headache and the wages of female respondents is also obtained using an instrumental variables (IV) approach, although the IV estimates are imprecise relative to the OLS estimates.

Medical marijuana laws and suicides by gender and age.

OBJECTIVES: We estimated the association between legalizing medical marijuana and suicides. METHODS: We obtained state-level suicide data from the National Vital Statistics System's Mortality Detail Files for 1990-2007. We used regression analysis to examine the association between medical marijuana legalization and suicides per 100 000 population. RESULTS: After adjustment for economic conditions, state policies, and state-specific linear time trends, the association between legalizing medical marijuana and suicides was not statistically significant at the .05 level. However, legalization was associated with a 10.8% (95% confidence interval [CI] = -17.1%, -3.7%) and 9.4% (95% CI = -16.1%, -2.4%) reduction in the suicide rate of men aged 20 through 29 years and 30 through 39 years, respectively. Estimates for females were less precise and sensitive to model specification. CONCLUSIONS: Suicides among men aged 20 through 39 years fell after medical marijuana legalization compared with those in states that did not legalize. The negative relationship between legalization and suicides among young men is consistent with the hypothesis that marijuana can be used to cope with stressful life events. However, this relationship may be explained by alcohol consumption. The mechanism through which legalizing medical marijuana reduces suicides among young men remains a topic for future study.

National trends and outcomes of pediatric gastrostomy tube placement.

OBJECTIVES: National outcomes data regarding surgical gastrostomy tube (G-tube) and percutaneous endoscopic gastrostomy (PEG) tube procedures are lacking. Our objectives were to describe trends in G-tube and PEG procedures, examine regional variation, and compare outcomes. METHODS: This was a retrospective study using pediatric admissions during 1997, 2000, 2003, 2006, and 2009 from the Kids' Inpatient Database. Length of stay and cost were adjusted for demographics, complexity, setting, year, and infection or surgical complication. RESULTS: G-tubes were placed during 64,412 admissions, increasing from 16.6 procedures/100,000 US children in 1997 to 18.5 in 2009. Surgical gastrostomy rates increased by 19% (0.17 procedures/100,000/year, P < 0.002) and, among children <1 year, they increased by 32% (2.56 procedures/100,000/year, P < 0.01). PEG rates did not increase (0.02 procedures/100,000/year, P = 0.47) in the study years. The West had an 18% higher rate than the national average for surgical G-tubes and a 10% higher rate for PEGs. When the sole procedure during the admission was gastrostomy, the G-tube was associated with a 19% (confidence interval 9.7-57.5) longer length of stay, and a 25% higher cost (confidence interval 16.4-34.5) compared with PEG. CONCLUSIONS: Surgical gastrostomy insertion rates have increased whereas PEG rates have not, despite evidence of better severity-adjusted outcome measures for PEG tubes. Surgical gastrostomy insertion in children <1 year of age yielded the greatest increase, which may relate to a changing patient population; however, regional variation suggests that provider preference also plays a role. Our data underline the need for more robust collection and analysis of surgical outcomes to guide decision making.

The effects of becoming a physician on prescription drug use and mental health treatment.

There is evidence that physicians disproportionately suffer from substance use disorder and mental health problems. It is not clear, however, whether these phenomena are causal. We use data on Dutch medical school applicants to examine the effects of becoming a physician on prescription drug use and the receipt of treatment from a mental health facility. Leveraging variation from lottery outcomes that determine admission into medical schools, we find that becoming a physician increases the use of antidepressants, anxiolytics, opioids, and sedatives. Increases in the use of antidepressants, anxiolytics, and sedatives are larger among female physicians than among their male counterparts.

The effect of body weight on adolescent sexual activity.

Recent research suggests that overweight females suffer penalties in the labor and marriage markets, while overweight males do not. This study explores whether similar gender differences in the effect of body weight exist in what Cawley et al. (2006) labeled 'the adolescent sex market'. Drawing on data from the National Longitudinal Study of Adolescent Health, we use fixed effects and instrumental variables identification strategies to estimate the relationship between body weight and sexual activity. We find evidence that increased body weight lowers the probability that female adolescents become sexually active. In contrast, there is little evidence of a causal relationship between body weight and sexual activity for male adolescents.

Boys will be boys: are there gender differences in the effect of sexual abstinence on schooling?

A recent study by Sabia and Rees (2009) found that delaying first intercourse leads to a substantial increase in the probability that female students graduate high school. However, it is unclear whether the effect of abstinence extends to male students. Here we identify exogenous variation in the timing of first intercourse using a physical development index available for both females and males. Two-stage least squares estimates suggest that abstaining from sexual intercourse increases the probability that females graduate from high school, but has little effect on the educational attainment of males. This pattern of results is consistent with evidence from previous studies that males are less likely than females to suffer adverse psychological consequences from engaging in sexual intercourse at an early age.

Exercise and adolescent mental health: new evidence from longitudinal data.

BACKGROUND: Recent U.S. government recommendations state that increased physical activity can substantially improve adolescent psychological well-being. However, many of the studies upon which this conclusion is based did not adequately address the role of difficult-to-measure factors that could be correlated with both physical activity and adolescent mental health. AIM OF THE STUDY: The primary aim of this study is to estimate the effect of physical activity on adolescent psychological well-being controlling for the influence of unmeasured school- and individual-level confounding factors. METHODOLOGY: Drawing on data from the first two waves of the National Longitudinal Study of Adolescent Health, we use ordinary least squares to estimate the effect of physical activity and hours of inactivity on two psychometrically sound measures of psychological well-being (the Center for Epidemiologic Studies Depression scale and the Rosenberg Self-Esteem scale). Next, we add school fixed effects and then individual fixed effects to the estimating equation in order to control for difficult-to-measure factors that could be correlated with both physical activity and adolescent mental health. RESULTS: Ordinary least squares (OLS) estimates show that a higher frequency of moderate or physical exercise is associated with improved psychological well-being for adolescents. This result is robust to the inclusion of school fixed effects, but when we control for fixed individual heterogeneity by first-differencing the data, the estimated effects of physical activity on depression and self-esteem decline sharply, often becoming small in magnitude or statistically indistinguishable from zero. We conclude that OLS estimates of the effect of physical activity on emotional health may be biased upwards. LIMITATIONS: While the nationally representative panel data we use is a rich source of information on mental health, our measure of physical activity is limited in that it fails to capture total time spent exercising. Moreover, while our statistical approach controls for time-invariant unobservables, we cannot rule out the possibility of time-varying unobserved confounders. IMPLICATIONS: Although policy interventions designed to promote more exercise among youths may have important physical health benefits, our findings suggest that the short-run emotional benefits are likely small and concentrated at higher frequencies of physical activity. FUTURE RESEARCH: To enhance the internal validity of research in this area, future work could tackle the difficult challenge of identifying exogenous variation in physical exercise using a natural experiment approach.

Automated Quantification of Mitochondrial Fragmentation in an In Vitro Parkinson's Disease Model.

Neuronal mitochondrial fragmentation is a phenotype exhibited in models of neurodegeneration such as Parkinson's disease. Delineating the dysfunction in mitochondrial dynamics found in diseased states can aid our understanding of underlying mechanisms of disease progression and possibly identify novel therapeutic approaches. Advances in microscopy and the availability of intuitive open-access software have accelerated the rate of image acquisition and analysis, respectively. These developments allow routine biology researchers to rapidly turn hypotheses into results. In this protocol, we describe the utilization of cell culture techniques, high-content imaging (HCI), and the subsequent open-source image analysis pipeline for the quantification of mitochondrial fragmentation in the context of a rotenone-based in vitro Parkinson's disease model. © 2020 The Authors. Basic Protocol 1: SN4741 neuron culture and treatment in a rotenone-based model of Parkinson's disease Basic Protocol 2: Identification of cell nuclei, measurement of mitochondrial membrane potential, and measurement of mitochondrial fragmentation in mouse-derived midbrain dopaminergic neurons.

The affordable care act and ambulance response times.

This study contributes to the literature on the capacity challenges faced by health care providers after insurance expansions by examining the Affordable Care Act (ACA) and ambulance response times. Exploiting temporal and geographic variation in the implementation of the ACA as well as pre-treatment differences in uninsured rates, we estimate that the expansions of private and Medicaid coverage under the ACA combined to slow ambulance response times by an average of 24%. We conclude that, through extending coverage to individuals who, in its absence, would not have availed themselves of emergency medical services, the ACA added strain to emergency response systems.

Association of Ambulance Use in New York City With the Implementation of the Patient Protection and Affordable Care Act.

Importance: Expanding insurance coverage may be associated with overuse of medical care because newly insured patients are insulated from having to pay the full cost. Objective: To examine the use of ambulance transport before and after the rollout of the Patient Protection and Affordable Care Act (ACA) in New York City (NYC), New York. Design, Setting, and Participants: In this case-control study, the volume of ambulance dispatches in NYC for minor injuries before and after the rollout of the ACA was examined. Data were drawn from a census of all ambulance dispatches in NYC between January 1, 2013, and July 31, 2016. Ambulance dispatches for more severe injuries, which are more difficult to characterize as unnecessary, were used as the control group. Analyses were conducted from August 17, 2017, to May, 10, 2019. Main Outcome and Measures: The main outcome was the number of ambulance dispatches for minor injuries, defined per month per dispatch zone. The implementation of the ACA was measured using an indicator variable of 1 for dispatches starting January 1, 2014, and 0 for dispatches before January 1, 2014. The number of ambulance dispatches for injuries and major injuries was used to account for secular trends. Injury severity was classified by the dispatchers based on information from the 911 callers with a severity score on a scale of 1 to 8, where 1 is the most severe; minor injuries had a score of 7; injuries, 5; and major injuries, 3. Results: There were 4 787 180 ambulance dispatches in NYC during the study. After the 2014 expansion of insurance coverage under the ACA, there was an increase in ambulance dispatches for minor injuries compared with dispatches for more severe injuries. Compared with the preimplementation mean (SD) of 20.75 (14.24) minor injury dispatches per dispatch zone per month, there were 7.71 (95% CI, 1.23-14.19) additional minor injury dispatches per dispatch zone per month compared with dispatches for other types of injuries, an increase of 37.2%. Given that NYC has 31 dispatch zones, this increase is equivalent to approximately 239 additional dispatches per month or 2868 additional dispatches per year for minor injuries. Conclusions and Relevance: There was a significant increase in use of ambulance transport for minor injuries in NYC after the ACA insurance expansion, suggesting that the change in health insurance structure was associated with increased use of emergency medical services in nonemergent situations. Future reforms to the US health insurance system should take into account the potential for increased use of emergency medical services in nonemergent situations, which the literature suggests may lead to congestion and slower response times.

Calorie restriction activates new adult born olfactory-bulb neurones in a ghrelin-dependent manner but acyl-ghrelin does not enhance subventricular zone neurogenesis.

The ageing and degenerating brain show deficits in neural stem/progenitor cell (NSPC) plasticity that are accompanied by impairments in olfactory discrimination. Emerging evidence suggests that the gut hormone ghrelin plays an important role in protecting neurones, promoting synaptic plasticity and increasing hippocampal neurogenesis in the adult brain. In the present study, we investigated the role of ghrelin with respect to modulating adult subventricular zone (SVZ) NSPCs that give rise to new olfactory bulb (OB) neurones. We characterised the expression of the ghrelin receptor, growth hormone secretagogue receptor (GHSR), using an immunohistochemical approach in GHSR-eGFP reporter mice to show that GHSR is expressed in several regions, including the OB but not in the SVZ of the lateral ventricle. These data suggest that acyl-ghrelin does not mediate a direct effect on NSPC in the SVZ. Consistent with these findings, treatment with acyl-ghrelin or genetic silencing of GHSR did not alter NSPC proliferation within the SVZ. Similarly, using a bromodeoxyuridine pulse-chase approach, we show that peripheral treatment of adult rats with acyl-ghrelin did not increase the number of new adult-born neurones in the granule cell layer of the OB. These data demonstrate that acyl-ghrelin does not increase adult OB neurogenesis. Finally, we investigated whether elevating ghrelin indirectly, via calorie restriction (CR), regulated the activity of new adult-born cells in the OB. Overnight CR induced c-Fos expression in new adult-born OB cells but not in developmentally born cells, whereas neuronal activity was absent following re-feeding. These effects were not present in ghrelin-/- mice, suggesting that adult-born cells are uniquely sensitive to changes in ghrelin mediated by fasting and re-feeding. In summary, ghrelin does not promote neurogenesis in the SVZ and OB; however, new adult-born OB cells are activated by CR in a ghrelin-dependent manner.

The effect of adolescent virginity status on psychological well-being.

Although previous research has found that sexually active teens are more likely to suffer from depression, it is not clear whether this association is causal or spurious in nature. This study uses data from the National Longitudinal Study of Adolescent Health to examine whether virginity status affects self-esteem and depression. For males, fixed effects and instrumental variables (IV) estimates provide little evidence that sex is causally related to psychological well-being. In contrast, IV estimates indicate that sexually active female adolescents are at increased risk of exhibiting the symptoms of depression relative to their counterparts who are not sexually active. Comparing the psychological well-being of females who used contraception at last intercourse with that of virgins suggests that these effects may be ameliorated, but not eliminated, by contraceptive use.