Using Targeted Large Deletions and High-EfficiencyN-Ethyl-N-nitrosourea Mutagenesis for Functional Analyses of the Mammalian Genome

The Human Genome Project has generated nucleotide sequences from an estimated 80,000 to 100,000 genes, only a small fraction of which have a known role. Nucleotide sequence information alone is insufficient to predict gene function. One of the most powerful ways of revealing gene function, as demonstrated in bacteria, worms, yeast, and flies, is to generate mutations and characterize them at both the phenotypic and the molecular levels. Given the physiological and anatomical parallels between mouse and human, genotype–phenotype relationships established in mice can be extrapolated to human syndromes. A new method is described for functional genetic analyses in the mouse that uses loxP/Cre engineering to generate coat color-tagged large deletions. The haploid regions can then be dissected by mutagenesis withN-ethyl-N-nitrosourea in phenotype-driven screens to obtain functional information on genes in any desired region of the mouse genome.     

Point mutations in the melanocortin-4 receptor cause variable obesity in mice

Mutations in the melanocortin-4 receptor (MC4R) are associated with early-onset obesity in humans. Furthermore, a null Mc4r allele in mice leads to severe obesity due to hyperphagia and decreased energy expenditure. As part of independent N-ethyl- N-nitrosourea (ENU) mutagenesis screens, two obesity mutants, Fatboy and Southbeach, were isolated. Mapping revealed linkage to the melanocortin-4 receptor (Mc4r) and sequencing found single amino acid changes in Mc4r for each line. Expression of the mutant receptors in HEK 293 cells revealed defects in receptor signaling. The mutated Fatboy receptor (I194T) shows an increase in the effective concentration necessary for 50% of maximal signaling (EC₅₀) when stimulated with α-MSH. Based on competitive binding, I194T is expressed on the cell surface at lower levels than the nonmutated receptor. In contrast, Southbeach (L300P) displays minimal receptor signaling when stimulated with the natural ligand α-MSH or the synthetic agonist NDP-α-MSH. Cell surface binding is absent, which usually indicates a lack of cell surface expression. However, antibody binding to Flag-tagged receptors by flow cytometry analysis and immunofluorescence demonstrates that L300P is translocated to the plasma membrane at a level comparable to the wild-type receptor. These results indicate a correlation with remaining receptor activity and the severity of the obesity in the mice homozygous for the mutations. Southbeach has less receptor activity and becomes more obese. These mutants will serve as good models for the variability in phenotype in humans carrying mutations in the MC4R gene.     

Amyloid precursor protein revisited: neuron-specific expression and highly stable nature of soluble derivatives

APP processing and amyloid-β production play a central role in Alzheimer disease pathogenesis. APP has been considered a ubiquitously expressed protein. In addition to amyloid-β, α- or β-secretase-dependent cleavage of APP also generates soluble secreted APP (APPsα or APPsβ, respectively). Interestingly, APPsβ has been shown to be subject to further cleavage to create an N-APP fragment that binds to the DR6 death receptor and mediates axon pruning and degeneration under trophic factor withdrawal conditions. By performing APP immunocytochemical staining, we found that, unexpectedly, many antibodies yielded nonspecific staining in APP-null samples. Screening of a series of antibodies allowed us to identify a rabbit monoclonal antibody Y188 that is highly specific for APP and prompted us to re-examine the expression, localization, and stability of endogenous APP and APPsβ in wild-type and in APPsβ knock-in mice, respectively. In contrast to earlier studies, we found that APP is specifically expressed in neurons and that its expression cannot be detected in major types of glial cells under basal or neuroinflammatory conditions. Both APPsα and APPsβ are highly stable in the central nervous system (CNS) and do not undergo further cleavage with or without trophic factor support. Our results clarify several key questions with regard to the fundamental properties of APP and offer critical cellular insights into the pathophysiology of APP.     

Can Broader Diffusion of Value-Based Insurance Design Increase Benefits from US Health Care without Increasing Costs? Evidence from a Computer Simulation Model

Background

Evidence suggests that cost sharing (i.e.,copayments and deductibles) decreases health expenditures but also reduces essential care. Value-based insurance design (VBID) has been proposed to encourage essential care while controlling health expenditures. Our objective was to estimate the impact of broader diffusion of VBID on US health care benefits and costs.

Methods and Findings

We used a published computer simulation of costs and life expectancy gains from US health care to estimate the impact of broader diffusion of VBID. Two scenarios were analyzed: (1) applying VBID solely to pharmacy benefits and (2) applying VBID to both pharmacy benefits and other health care services (e.g., devices). We assumed that cost sharing would be eliminated for high-value services (<$100,000 per life-year), would remain unchanged for intermediate- or unknown-value services ($100,000–$300,000 per life-year or unknown), and would be increased for low-value services (>$300,000 per life-year). All costs are provided in 2003 US dollars. Our simulation estimated that approximately 60% of health expenditures in the US are spent on low-value services, 20% are spent on intermediate-value services, and 20% are spent on high-value services. Correspondingly, the vast majority (80%) of health expenditures would have cost sharing that is impacted by VBID. With prevailing patterns of cost sharing, health care conferred 4.70 life-years at a per-capita annual expenditure of US$5,688. Broader diffusion of VBID to pharmaceuticals increased the benefit conferred by health care by 0.03 to 0.05 additional life-years, without increasing costs and without increasing out-of-pocket payments. Broader diffusion of VBID to other health care services could increase the benefit conferred by health care by 0.24 to 0.44 additional life-years, also without increasing costs and without increasing overall out-of-pocket payments. Among those without health insurance, using cost saving from VBID to subsidize insurance coverage would increase the benefit conferred by health care by 1.21 life-years, a 31% increase.

Conclusion

Broader diffusion of VBID may amplify benefits from US health care without increasing health expenditures. Please see later in the article for the Editors'' Summary     

HIV Drug Resistance Early Warning Indicators in Namibia for Public Health Action

Background

HIV drug resistance (HIVDR) testing is not routinely available in many resource-limited settings, therefore antiretroviral therapy (ART) program and site factors known to be associated with emergence of HIVDR should be monitored to optimize the quality of patient care and minimize the emergence of preventable HIVDR.

Methods

In 2010, Namibia selected five World Health Organization Early Warning Indicators (EWIs) and scaled-up monitoring from 9 to 33 ART sites: ART prescribing practices, Patients lost to follow-up (LTFU) at 12 months, Patients switched to a second-line regimen at 12 months, On-time antiretroviral (ARV) drug pick-up, and ARV drug-supply continuity.

Results

Records allowed reporting on three of the five selected EWIs. 22 of 33 (67%) sites met the target of 100% initiated on appropriate first-line regimens. 17 of 33 (52%) sites met the target of ≤20% LTFU. 15 of 33 (45%) sites met the target of 0% switched to a second-line regimen.

Conclusions

EWI monitoring directly resulted in public health action which will optimize the quality of care, specifically the strengthening of ART record systems, engagement of ART sites, and operational research for improved adherence assessment and ART patient defaulter tracing.     

Age‐related Changes in the Circadian and Homeostatic Regulation of Human Sleep

The reduction of electroencephalographic (EEG) slow‐wave activity (SWA) (EEG power density between 0.75–4.5 Hz) and spindle frequency activity, together with an increase in involuntary awakenings during sleep, represent the hallmarks of human sleep alterations with age. It has been assumed that this decrease in non‐rapid eye movement (NREM) sleep consolidation reflects an age‐related attenuation of the sleep homeostatic drive. To test this hypothesis, we measured sleep EEG characteristics (i.e., SWA, sleep spindles) in healthy older volunteers in response to high (sleep deprivation protocol) and low sleep pressure (nap protocol) conditions. Despite the fact that the older volunteers had impaired sleep consolidation and reduced SWA levels, their relative SWA response to both high and low sleep pressure conditions was similar to that of younger persons. Only in frontal brain regions did we find an age‐related diminished SWA response to high sleep pressure. On the other hand, we have clear evidence that the circadian regulation of sleep during the 40 h nap protocol was changed such that the circadian arousal signal in the evening was weaker in the older study participants. More sleep occurred during the wake maintenance zone, and subjective sleepiness ratings in the late afternoon and evening were higher than in younger participants. In addition, we found a diminished melatonin secretion and a reduced circadian modulation of REM sleep and spindle frequency—the latter was phase‐advanced relative to the circadian melatonin profile. Therefore, we favor the hypothesis that age‐related changes in sleep are due to weaker circadian regulation of sleep and wakefulness. Our data suggest that manipulations of the circadian timing system, rather than the sleep homeostat, may offer a potential strategy to alleviate age‐related decrements in sleep and daytime alertness levels.     

Achieving global mortality reduction targets and universal health coverage: The impact of COVID-19

Wenhui Mao and coauthors discuss possible implications of the COVID-19 pandemic for health aspirations in low- and middle-income countries.

Summary points

• The Coronavirus Disease 2019 (COVID-19) pandemic threatens progress toward a “grand convergence” in global health—universal reduction in deaths from infections and maternal and child health conditions to low levels—and toward achieving universal health coverage (UHC).
• Our analysis suggests that COVID-19 will exacerbate the difficulty of achieving grand convergence targets for tuberculosis (TB), maternal mortality, and, probably, for under-5 mortality. HIV targets are likely to be met.
• By 2035, our analysis suggests that the public sectors of low-income countries (LICs) would only be able to finance about a third of the costs of a package of 120 essential non-COVID-19 health interventions through domestic sources, unless the country increases significantly the priority assigned to the health sector; lower middle-income countries (LMICs) would likewise only be able to finance a little less than half.
• The likelihood of getting back on track for reaching grand convergence and UHC will depend on (i) how quickly COVID-19 vaccines can be deployed in LICs and LMICs; (ii) how much additional public sector health financing can be mobilized from external and domestic sources; and (iii) whether countries can rapidly strengthen and focus their health delivery systems.

     

Increased risk of fragility fractures among HIV infected compared to uninfected male veterans

Background

HIV infection has been associated with an increased risk of fragility fracture. We explored whether or not this increased risk persisted in HIV infected and uninfected men when controlling for traditional fragility fracture risk factors.

Methodology/Principal Findings

Cox regression models were used to assess the association of HIV infection with the risk for incident hip, vertebral, or upper arm fracture in male Veterans enrolled in the Veterans Aging Cohort Study Virtual Cohort (VACS-VC). We calculated adjusted hazard ratios comparing HIV status and controlling for demographics and other established risk factors. The sample consisted of 119,318 men, 33% of whom were HIV infected (34% aged 50 years or older at baseline, and 55% black or Hispanic). Median body mass index (BMI) was lower in HIV infected compared with uninfected men (25 vs. 28 kg/m²; p<0.0001). Unadjusted risk for fracture was higher among HIV infected compared with uninfected men [HR: 1.32 (95% CI: 1.20, 1.47)]. After adjusting for demographics, comorbid disease, smoking and alcohol abuse, HIV infection remained associated with an increased fracture risk [HR: 1.24 (95% CI: 1.11, 1.39)]. However, adjusting for BMI attenuated this association [HR: 1.10 (95% CI: 0.97, 1.25)]. The only HIV-specific factor associated with fragility fracture was current protease inhibitor use [HR: 1.41 (95% CI: 1.16, 1.70)].

Conclusions/Significance

HIV infection is associated with fragility fracture risk. This risk is attenuated by BMI.