Coping with access barriers to non-communicable disease medicines: qualitative patient interviews in eight counties in Kenya.

BACKGROUND: There is rich literature on barriers to medicines access for the treatment of non-communicable diseases (NCDs) in high-income countries. Less is known about low- and middle-income countries, in particular the differences in coping with medicines access barrier by household wealth and disease. The aim of this study was to compare the coping mechanisms of patients with the lack of availability and affordability of cardio-vascular diseases, diabetes and asthma medicines in Kenya. METHODS: This qualitative study was part of a larger mixed methods evaluation study conducted in eight counties of Kenya from 2016 to 2019. Forty-nine patient interviews at study end line explored their NCD journey, perceptions of availability, stockouts and affordability of NCD medicines, their enrollment in health insurance, and their relationship with the private chemists. Transcribed interviews were coded using Nvivo software. A two-step thematic approach was used, first conducting a priority coding which was followed by coding emerging and divergent themes. RESULTS: Overall, we found that patients across all disease types and wealth level faced frequent medicine stock-outs at health facilities. In the absence of NCD medicines at health facilities, patients coped by purchasing medicines from local chemists, switching health facilities, requesting a different prescription, admitting oneself to an inpatient facility, establishing connections with local staff to receive notifications of medicine stock, stocking up on medicines, utilizing social capital to retrieve medicines from larger cities and obtaining funds from a network of friends and family. Categorizing by disease revealed patterns in coping choices that were based on the course of the disease, severity of the symptoms and the direct and indirect costs incurred as a result of stockouts of NCD medicines. Categorizing by wealth highlight differences in households' capacity to cope with the unavailability and unaffordability of NCD medicines. CONCLUSIONS: The type of coping strategies to access barriers differ by NCD and wealth group. Although Kenya has made important strides to address NCD medicines access challenges, prioritizing enrollment of low wealth households in county health insurance programs and ensuring continuous availability of essential NCD medicines at public health facilities close to the patient homes could improve access.

Jeopardizing quality at the frontline of healthcare: prevalence and risk factors for disrespect and abuse during facility-based childbirth in Ethiopia.

Disrespect and abuse (D&A) experienced by women during facility-based childbirth has gained global recognition as a threat to eliminating preventable maternal mortality and morbidity. This study explored the frequency and associated factors of D&A in four rural health centres in Ethiopia. Experiences of women who delivered in these facilities were captured by direct observation of client-provider interaction (N = 193) and exit interview at time of discharge (N = 204). Incidence of D&A was observed in each facility, with failure to ask woman for preferred birth position most commonly observed [n = 162, 83.9%, 95% confidence interval (95% CI) 78.0-88.5%]. During exit interviews, 21.1% (n = 43, 95% CI 15.4-26.7%) of respondents reported at least one occurrence of D&A. Bivariate models using client characteristics and index birth experience showed that women's reporting of D&A was significantly associated with childbirth complications [odds ratio (OR) = 7.98, 95% CI 3.70, 17.22], weekend delivery (OR = 0.17, 95% CI 0.05, 0.63) and no previous delivery at the facility (OR = 3.20, 95% CI 1.27, 8.05). Facility-level fixed-effect models found that experience of complications (OR = 15.51, 95% CI 4.38, 54.94) and weekend delivery (OR = 0.05, 95% CI 0.01-0.32) remained significantly and most strongly associated with self-reported D&A. These data suggest that addressing D&A in health centres in Ethiopia will require a sustained effort to improve infrastructure, support the health workforce in rural settings, enforce professional standards and target interventions to improve women's experiences as part of quality of care initiatives.

Methods used in prevalence studies of disrespect and abuse during facility based childbirth: lessons learned.

BACKGROUND: Several recent studies have attempted to measure the prevalence of disrespect and abuse (D&A) of women during childbirth in health facilities. Variations in reported prevalence may be associated with differences in study instruments and data collection methods. This systematic review and comparative analysis of methods aims to aggregate and present lessons learned from published studies that quantified the prevalence of Disrespect and Abuse (D&A) during childbirth. METHODS: We conducted a systematic review of the literature in accordance with PRISMA (Preferred Reporting Items for Systematic Review and Meta-Analysis) guidelines. Five papers met criteria and were included for analysis. We developed an analytical framework depicting the basic elements of epidemiological methodology in prevalence studies and a table of common types of systematic error associated with each of them. We performed a head-to-head comparison of study methods for all five papers. Using these tools, an independent reviewer provided an analysis of the potential for systematic error in the reported prevalence estimates. RESULTS: Sampling techniques, eligibility criteria, categories of D&A selected for study, operational definitions of D&A, summary measures of D&A, and the mode, timing, and setting of data collection all varied in the five studies included in the review. These variations present opportunities for the introduction of biases - in particular selection, courtesy, and recall bias - and challenge the ability to draw comparisons across the studies' results. CONCLUSION: Our review underscores the need for caution in interpreting or comparing previously reported prevalence estimates of D&A during facility-based childbirth. The lack of standardized definitions, instruments, and study methods used to date in studies designed to quantify D&A in childbirth facilities introduced the potential for systematic error in reported prevalence estimates, and affected their generalizability and comparability. Chief among the lessons to emerge from comparing methods for measuring the prevalence of D&A is recognition of the tension between seeking prevalence measures that are reliable and generalizable, and attempting to avoid loss of validity in the context where the issue is being studied.

Combined serial analysis of gene expression and transcription factor binding site prediction identifies novel-candidate-target genes of Nr2e1 in neocortex development.

BACKGROUND: Nr2e1 (nuclear receptor subfamily 2, group e, member 1) encodes a transcription factor important in neocortex development. Previous work has shown that nuclear receptors can have hundreds of target genes, and bind more than 300 co-interacting proteins. However, recognition of the critical role of Nr2e1 in neural stem cells and neocortex development is relatively recent, thus the molecular mechanisms involved for this nuclear receptor are only beginning to be understood. Serial analysis of gene expression (SAGE), has given researchers both qualitative and quantitative information pertaining to biological processes. Thus, in this work, six LongSAGE mouse libraries were generated from laser microdissected tissue samples of dorsal VZ/SVZ (ventricular zone and subventricular zone) from the telencephalon of wild-type (Wt) and Nr2e1-null embryos at the critical development ages E13.5, E15.5, and E17.5. We then used a novel approach, implementing multiple computational methods followed by biological validation to further our understanding of Nr2e1 in neocortex development. RESULTS: In this work, we have generated a list of 1279 genes that are differentially expressed in response to altered Nr2e1 expression during in vivo neocortex development. We have refined this list to 64 candidate direct-targets of NR2E1. Our data suggested distinct roles for Nr2e1 during different neocortex developmental stages. Most importantly, our results suggest a possible novel pathway by which Nr2e1 regulates neurogenesis, which includes Lhx2 as one of the candidate direct-target genes, and SOX9 as a co-interactor. CONCLUSIONS: In conclusion, we have provided new candidate interacting partners and numerous well-developed testable hypotheses for understanding the pathways by which Nr2e1 functions to regulate neocortex development.

Effects of abaloparatide, a human parathyroid hormone-related peptide analog, on bone mineral density in postmenopausal women with osteoporosis.

CONTEXT: Abaloparatide is a novel synthetic peptide analog of parathyroid hormone-related protein (PTHrP) that is currently being developed as a potential anabolic agent in the treatment of postmenopausal osteoporosis. OBJECTIVE: This study sought to assess the effects of abaloparatide on bone mineral density (BMD) at the lumbar spine, total hip, and femoral neck in postmenopausal women with osteoporosis. DESIGN: Multi-center, multi-national, double-blind placebo controlled trial in which postmenopausal women were randomly assigned to receive 24 weeks of treatment with daily sc injections of placebo, abaloparatide, 20, 40, or 80 µg, or teriparatide, 20 µg. A 24-week extension was also performed in a subset of subjects. PARTICIPANTS: Postmenopausal women with osteoporosis (n = 222). MAIN OUTCOME MEASURES: BMD by dual-x-ray absorptiometry and biochemical markers of bone turnover. RESULTS: At 24 weeks, lumbar spine BMD increased by 2.9, 5.2, and 6.7% in the abaloparatide, 20-, 40-, and 80-µg groups, respectively, and 5.5% in the teriparatide group. The increases in the 40- and 80-µg abaloparatide groups and the teriparatide group were significantly greater than placebo (1.6%). Femoral neck BMD increased by 2.7, 2.2, and 3.1% in abaloparatide, 20-, 40-, and 80-µg groups, respectively, and 1.1% in the teriparatide group. The increase in femoral neck BMD with abaloparatide, 80 µg was significantly greater than placebo (0.8%). Total hip BMD increased by 1.4, 2.0, and 2.6% in the abaloparatide, 20-, 40-, and 80-µg groups, respectively. The total hip increases in the 40- and 80-µg abaloparatide groups were greater than both placebo (0.4%) and teriparatide (0.5%). CONCLUSIONS: Compared with placebo, 24 weeks of daily sc abaloparatide increases BMD of the lumbar spine, femoral neck, and total hip in a dose-dependent fashion. Moreover, the abaloparatide-induced BMD increases at the total hip are greater than with the marketed dose of teriparatide. These results support the further investigation of abaloparatide as an anabolic therapy in postmenopausal osteoporosis.

Targeted CNS Delivery Using Human MiniPromoters and Demonstrated Compatibility with Adeno-Associated Viral Vectors.

Critical for human gene therapy is the availability of small promoter tools to drive gene expression in a highly specific and reproducible manner. We tackled this challenge by developing human DNA MiniPromoters using computational biology and phylogenetic conservation. MiniPromoters were tested in mouse as single-copy knock-ins at the Hprt locus on the X Chromosome, and evaluated for lacZ reporter expression in CNS and non-CNS tissue. Eighteen novel MiniPromoters driving expression in mouse brain were identified, two MiniPromoters for driving pan-neuronal expression, and 17 MiniPromoters for the mouse eye. Key areas of therapeutic interest were represented in this set: the cerebral cortex, embryonic hypothalamus, spinal cord, bipolar and ganglion cells of the retina, and skeletal muscle. We also demonstrated that three retinal ganglion cell MiniPromoters exhibit similar cell-type specificity when delivered via adeno-associated virus (AAV) vectors intravitreally. We conclude that our methodology and characterization has resulted in desirable expression characteristics that are intrinsic to the MiniPromoter, not dictated by copy number effects or genomic location, and results in constructs predisposed to success in AAV. These MiniPromoters are immediately applicable for pre-clinical studies towards gene therapy in humans, and are publicly available to facilitate basic and clinical research, and human gene therapy.

Non-coding-regulatory regions of human brain genes delineated by bacterial artificial chromosome knock-in mice.

BACKGROUND: The next big challenge in human genetics is understanding the 98% of the genome that comprises non-coding DNA. Hidden in this DNA are sequences critical for gene regulation, and new experimental strategies are needed to understand the functional role of gene-regulation sequences in health and disease. In this study, we build upon our HuGX ('high-throughput human genes on the X chromosome') strategy to expand our understanding of human gene regulation in vivo. RESULTS: In all, ten human genes known to express in therapeutically important brain regions were chosen for study. For eight of these genes, human bacterial artificial chromosome clones were identified, retrofitted with a reporter, knocked single-copy into the Hprt locus in mouse embryonic stem cells, and mouse strains derived. Five of these human genes expressed in mouse, and all expressed in the adult brain region for which they were chosen. This defined the boundaries of the genomic DNA sufficient for brain expression, and refined our knowledge regarding the complexity of gene regulation. We also characterized for the first time the expression of human MAOA and NR2F2, two genes for which the mouse homologs have been extensively studied in the central nervous system (CNS), and AMOTL1 and NOV, for which roles in CNS have been unclear. CONCLUSIONS: We have demonstrated the use of the HuGX strategy to functionally delineate non-coding-regulatory regions of therapeutically important human brain genes. Our results also show that a careful investigation, using publicly available resources and bioinformatics, can lead to accurate predictions of gene expression.

Targeting of >1.5 Mb of human DNA into the mouse X chromosome reveals presence of cis-acting regulators of epigenetic silencing.

Regulatory sequences can influence the expression of flanking genes over long distances, and X chromosome inactivation is a classic example of cis-acting epigenetic gene regulation. Knock-ins directed to the Mus musculus Hprt locus offer a unique opportunity to analyze the spread of silencing into different human DNA sequences in the identical genomic environment. X chromosome inactivation of four knock-in constructs, including bacterial artificial chromosome (BAC) integrations of over 195 kb, was demonstrated by both the lack of expression from the inactive X chromosome in females with nonrandom X chromosome inactivation and promoter DNA methylation of the human transgene in females. We further utilized promoter DNA methylation to assess the inactivation status of 74 human reporter constructs comprising >1.5 Mb of DNA. Of the 47 genes examined, only the PHB gene showed female DNA hypomethylation approaching the level seen in males, and escape from X chromosome inactivation was verified by demonstration of expression from the inactive X chromosome. Integration of PHB resulted in lower DNA methylation of the flanking HPRT promoter in females, suggesting the action of a dominant cis-acting escape element. Female-specific DNA hypermethylation of CpG islands not associated with promoters implies a widespread imposition of DNA methylation during X chromosome inactivation; yet transgenes demonstrated differential capacities to accumulate DNA methylation when integrated into the identical location on the inactive X chromosome, suggesting additional cis-acting sequence effects. As only one of the human transgenes analyzed escaped X chromosome inactivation, we conclude that elements permitting ongoing expression from the inactive X are rare in the human genome.

A regulatory toolbox of MiniPromoters to drive selective expression in the brain.

The Pleiades Promoter Project integrates genomewide bioinformatics with large-scale knockin mouse production and histological examination of expression patterns to develop MiniPromoters and related tools designed to study and treat the brain by directed gene expression. Genes with brain expression patterns of interest are subjected to bioinformatic analysis to delineate candidate regulatory regions, which are then incorporated into a panel of compact human MiniPromoters to drive expression to brain regions and cell types of interest. Using single-copy, homologous-recombination "knockins" in embryonic stem cells, each MiniPromoter reporter is integrated immediately 5' of the Hprt locus in the mouse genome. MiniPromoter expression profiles are characterized in differentiation assays of the transgenic cells or in mouse brains following transgenic mouse production. Histological examination of adult brains, eyes, and spinal cords for reporter gene activity is coupled to costaining with cell-type-specific markers to define expression. The publicly available Pleiades MiniPromoter Project is a key resource to facilitate research on brain development and therapies.

Caspase-7 expanded function and intrinsic expression level underlies strain-specific brain phenotype of caspase-3-null mice.

Caspase-3-deficient mice of the 129S1/SvImJ (129) strain show severe brain development defects resulting in brain overgrowth and perinatal lethality, whereas on the C57BL/6J (B6) background, these mice develop normally. We therefore sought to identify the strain-dependent ameliorating gene. We biochemically isolated caspase-7 from B6-caspase-3-null (Casp3-/-) tissues as being the enzyme with caspase-3-like properties and capability of performing a caspase-3 surrogate function, apoptotic DNA fragmentation. Moreover, we show that, in contrast to the human enzymes, mouse caspase-7 is as efficient as caspase-3 at cleaving and thus inactivating ICAD (inhibitor of caspase-activated DNase), the inhibitor of apoptotic DNA fragmentation. Low levels of caspase-7 expression and activation correlate with lack of DNA fragmentation in 129-Casp3-/- apoptotic precursor neurons, whereas B6-Casp3-/- cells, which can fragment their DNA, show higher levels of caspase-7 expression and activation. The amount of caspase-7 activation in apoptotic precursor neurons is independent of the presence of caspase-3. Together, our findings demonstrate for the first time a strong correlation between caspase-7 activity, normal brain development, and apoptotic DNA fragmentation in Casp3-/- mice.

Retroposon compensatory mechanism hypothesis not supported: Zfa knockout mice are fertile.

It is hypothesized that autosomal retroposons compensate for the loss of their inactivated essential X-chromosome progenitors during spermatogenesis. Here we test this Retroposon Compensatory Mechanism (RCM) hypothesis using the Zfy gene family. The mouse autosomal retroposon Zfa is expressed in testes at the same developmental time points at which Zfx levels decline, which correspond to the time of male sex chromosome inactivation, suggesting that Zfa may compensate for the loss of Zfx during spermatogenesis. We examined the effect of Zfa-targeted mutagenesis on spermatogenesis in three genetically distinct mouse strains. Surprisingly, Zfa knockout mice showed no detectable fertility, sperm count, or testes morphology defects. We therefore conclude that Zfa is not an essential gene for spermatogenesis and fertility. This surprising finding now challenges the RCM hypothesis at least for the Zfy gene family. It also forces us to reevaluate the original data underpinning the RCM hypothesis for this family and to propose alternative hypotheses.