Association of risk factors for high blood pressure across 46 low- and middle-income countries: A multi-country cross-sectional analysis.

Background: Despite acknowledging the influence of various lifestyle and metabolic risk factors on hypertension, it remains uncertain to identify the primary contributors and differentiate which modifiable risk factors mediate the causal effects of hypertension. We aimed to examine the hierarchical association of eight prominent lifestyle and metabolic risk factors, along with demographic variables, with hypertension in adults and to explore the mediating effects of modifiable metabolic risk factors on hypertension. Methods: A cross-sectional study was conducted in 46 low- and middle-income countries using the World Health Organization (WHO) STEPwise approach to noncommunicable disease risk factor surveillance from 2002 to 2020. In a sample of 179 535 non-pregnant adults, we assessed the weighted population-attributable risk percentages (PAR%) for hypertension associated with eight risk factors. Additionally, we investigated the mediating role of metabolic risk factors on the effects of lifestyle risk factors on hypertension. Results: After adjusting for the sample weight in each country, 26.7% of participants had hypertension. The prevalence of hypertension was highest in those aged ≥65 years, with obesity-associated hypertension (45.7%) exceeding the rates for overweight (32.2%) and non-overweight individuals (18.2%). These eight risk factors collectively explain 83.7% of the PAR% associated with hypertension adjusted for the communal variance. Among the modifiable factors, obesity contributed to a weighted PAR% of 38.2%, while sedentary behaviour and low physical activity combined accounted for a weighted PAR% of 3.1%. Overweight/obesity played a predominant mediating role in the correlation between lifestyle risk factors and systolic and diastolic blood pressure, with the indirect effect accounting for approximately 25-64% and 13-80% of the total effect, respectively. Conclusions: These findings offer new insights into the modified risk factors associated with hypertension in adults in low- and middle-income countries, highlighting the crucial role of maintaining a normal body weight for the effective prevention and management of hypertension.

Functional Studies of Genetic Variants Associated with Human Diseases in Notch Signaling-Related Genes Using Drosophila.

Rare variants in the many genes related to Notch signaling cause diverse Mendelian diseases that affect myriad organ systems. In addition, genome- and exome-wide association studies have linked common and rare variants in Notch-related genes to common diseases and phenotypic traits. Moreover, somatic mutations in these genes have been observed in many types of cancer, some of which are classified as oncogenic and others as tumor suppressive. While functional characterization of some of these variants has been performed through experimental studies, the number of "variants of unknown significance" identified in patients with diverse conditions keeps increasing as high-throughput sequencing technologies become more commonly used in the clinic. Furthermore, as disease gene discovery efforts identify rare variants in human genes that have yet to be linked to a disease, the demand for functional characterization of variants in these "genes of unknown significance" continues to increase. In this chapter, we describe a workflow to functionally characterize a rare variant in a Notch signaling related gene that was found to be associated with late-onset Alzheimer's disease. This pipeline involves informatic analysis of the variant of interest using diverse human and model organism databases, followed by in vivo experiments in the fruit fly Drosophila melanogaster. The protocol described here can be used to study variants that affect amino acids that are not conserved between human and fly. By "humanizing" the almondex gene in Drosophila with mutant alleles and heterologous genomic rescue constructs, a missense variant in TM2D3 (TM2 Domain Containing 3) was shown to be functionally damaging. This, and similar approaches, greatly facilitate functional interpretations of genetic variants in the human genome and propel personalized medicine.

TM2D genes regulate Notch signaling and neuronal function in Drosophila.

TM2 domain containing (TM2D) proteins are conserved in metazoans and encoded by three separate genes in each model organism species that has been sequenced. Rare variants in TM2D3 are associated with Alzheimer's disease (AD) and its fly ortholog almondex is required for embryonic Notch signaling. However, the functions of this gene family remain elusive. We knocked-out all three TM2D genes (almondex, CG11103/amaretto, CG10795/biscotti) in Drosophila and found that they share the same maternal-effect neurogenic defect. Triple null animals are not phenotypically worse than single nulls, suggesting these genes function together. Overexpression of the most conserved region of the TM2D proteins acts as a potent inhibitor of Notch signaling at the γ-secretase cleavage step. Lastly, Almondex is detected in the brain and its loss causes shortened lifespan accompanied by progressive motor and electrophysiological defects. The functional links between all three TM2D genes are likely to be evolutionarily conserved, suggesting that this entire gene family may be involved in AD.

Polyploid mitosis and depolyploidization promote chromosomal instability and tumor progression in a Notch-induced tumor model.

Ploidy variation is a cancer hallmark and is frequently associated with poor prognosis in high-grade cancers. Using a Drosophila solid-tumor model where oncogenic Notch drives tumorigenesis in a transition-zone microenvironment in the salivary gland imaginal ring, we find that the tumor-initiating cells normally undergo endoreplication to become polyploid. Upregulation of Notch signaling, however, induces these polyploid transition-zone cells to re-enter mitosis and undergo tumorigenesis. Growth and progression of the transition-zone tumor are fueled by a combination of polyploid mitosis, endoreplication, and depolyploidization. Both polyploid mitosis and depolyploidization are error prone, resulting in chromosomal copy-number variation and polyaneuploidy. Comparative RNA-seq and epistasis analysis reveal that the DNA-damage response genes, also active during meiosis, are upregulated in these tumors and are required for the ploidy-reduction division. Together, these findings suggest that polyploidy and associated cell-cycle variants are critical for increased tumor-cell heterogeneity and genome instability during cancer progression.

Post-Developmental Roles of Notch Signaling in the Nervous System.

Since its discovery in Drosophila, the Notch signaling pathway has been studied in numerous developmental contexts in diverse multicellular organisms. The role of Notch signaling in nervous system development has been extensively investigated by numerous scientists, partially because many of the core Notch signaling components were initially identified through their dramatic 'neurogenic' phenotype of developing fruit fly embryos. Components of the Notch signaling pathway continue to be expressed in mature neurons and glia cells, which is suggestive of a role in the post-developmental nervous system. The Notch pathway has been, so far, implicated in learning and memory, social behavior, addiction, and other complex behaviors using genetic model organisms including Drosophila and mice. Additionally, Notch signaling has been shown to play a modulatory role in several neurodegenerative disease model animals and in mediating neural toxicity of several environmental factors. In this paper, we summarize the knowledge pertaining to the post-developmental roles of Notch signaling in the nervous system with a focus on discoveries made using the fruit fly as a model system as well as relevant studies in C elegans, mouse, rat, and cellular models. Since components of this pathway have been implicated in the pathogenesis of numerous psychiatric and neurodegenerative disorders in human, understanding the role of Notch signaling in the mature brain using model organisms will likely provide novel insights into the mechanisms underlying these diseases.

Oncogenic Notch Triggers Neoplastic Tumorigenesis in a Transition-Zone-like Tissue Microenvironment.

During the initial stages of tumorigenesis, the tissue microenvironment where the pro-tumor cells reside plays a crucial role in determining the fate of these cells. Transition zones, where two types of epithelial cells meet, are high-risk sites for carcinogenesis, but the underlying mechanism remains largely unclear. Here, we show that persistent upregulation of Notch signaling induces neoplastic tumorigenesis in a transition zone between the salivary gland imaginal ring cells and the giant cells in Drosophila larvae. In this region, local endogenous JAK-STAT and JNK signaling creates a tissue microenvironment that is susceptible to oncogenic-Notch-induced tumorigenesis, whereas the rest of the salivary gland imaginal ring is refractory to Notch-induced tumor transformation. JNK signaling activates a matrix metalloprotease (MMP1) to promote Notch-induced tumorigenesis at the transition zone. These findings illustrate the significance of local endogenous inflammatory signaling in primary tumor formation.

Serrate/Notch Signaling Regulates the Size of the Progenitor Cell Pool in Drosophila Imaginal Rings.

Drosophila imaginal rings are larval tissues composed of progenitor cells that are essential for the formation of adult foreguts, hindguts, and salivary glands. Specified from subsets of ectoderm in the embryo, imaginal ring cells are kept quiescent until midsecond larval instar, and undergo rapid proliferation during the third instar to attain adequate numbers of cells that will replace apoptotic larval tissues for adult organ formation. Here, we show that Notch signaling is activated in all three imaginal rings from middle embryonic stage to early pupal stage, and that Notch signaling positively controls cell proliferation in all three imaginal rings during the third larval instar. Our mutant clonal analysis, knockdown, and gain-of-function studies indicate that canonical Notch pathway components are involved in regulating the proliferation of these progenitor cells. Both trans-activation and cis-inhibition between the ligand and receptor control Notch activation in the imaginal ring. Serrate (Ser) is the ligand provided from neighboring imaginal ring cells that trans-activates Notch signaling, whereas both Ser and Delta (Dl) could cis-inhibit Notch activity when the ligand and the receptor are in the same cell. In addition, we show that Notch signaling expressed in middle embryonic and first larval stages is required for the initial size of imaginal rings. Taken together, these findings indicate that imaginal rings are excellent in vivo models to decipher how progenitor cell number and proliferation are developmentally regulated, and that Notch signaling in these imaginal tissues is the primary growth-promoting signal that controls the size of the progenitor cell pool.

FOXO/Fringe is necessary for maintenance of the germline stem cell niche in response to insulin insufficiency.

The stem cell niche houses and regulates stem cells by providing both physical contact and local factors that regulate stem cell identity. The stem cell niche also plays a role in integrating niche-local and systemic signals, thereby ensuring that the balance of stem cells meets the needs of the organism. However, it is not clear how these signals are merged within the niche. Nutrient-sensing insulin/FOXO signaling has been previously shown to directly control Notch activation in the Drosophila female germline stem cell (GSC) niche, which maintains the niche and GSC identity. Here, we demonstrate that FOXO directly activates transcription of fringe, a gene encoding a glycosyltransferase that modulates Notch glycosylation. Fringe facilitates Notch inactivation in the GSC niche when insulin signaling is low. We also show that the Notch ligand predominantly involved is GSC niche-derived Delta. These results reveal that FOXO-mediated regulation of fringe links the insulin and Notch signaling pathways in the GSC niche in response to nutrition, and emphasize that stem cells are regulated by complex interactions between niche-local and systemic signals.

Excessive Dpp signaling induces cardial apoptosis through dTAK1 and dJNK during late embryogenesis of Drosophila.

BACKGROUND: To identify genes involved in the heart development of Drosophila, we found that embryos lacking raw function exhibited cardial phenotypes. raw was initially identified as a dorsal open group gene. The dorsal open phenotype was demonstrated to be resulted from the aberrant expression of decapentaplegic (dpp), a member of the tumor growth factor beta (TGF-ß), signaling pathway. Despite the role of dpp in pattering cardioblasts during early embryogenesis of Drosophila have been demonstrated, how mutation in raw and/or excessive dpp signaling involves in the differentiating heart of Drosophila has not been fully elaborated at late stages. RESULTS: We show that raw mutation produced a mild overspecification of cardial cells at stage 14, but these overproduced cells were mostly eliminated in late mutant embryos due to apoptosis. Aberrant dpp signaling is likely to contribute to the cardial phenotype found in raw mutants, because expression of dpp or constitutively activated thickven (tkvCA), the type I receptor of Dpp, induced a raw-like phenotype. Additionally, we show that dpp induced non-autonomous apoptosis through TGFß activated kinase 1 (TAK1), because mis-expression of a dominant negative form of Drosophila TAK1 (dTAK1DN) was able to suppress cell death in raw mutants or embryos overexpressing dpp. Importantly, we demonstrated that dpp induce its own expression through dTAK1, which also leads to the hyperactivation of Drosophila JNK (DJNK). The hyperactivated DJNK was attributed to be the cause of Dpp/DTAK1-induced apoptosis because overexpression of a dominant negative DJNK, basket (bskDN), suppressed cell death induced by Dpp or DTAK1. Moreover, targeted overexpression of the anti-apoptotic P35 protein, or a dominant negative proapoptotic P53 (P53DN) protein blocked Dpp/DTAK1-induced apoptosis, and rescued heart cells under the raw mutation background. CONCLUSIONS: We find that ectopic Dpp led to DJNK-dependent cardial apoptosis through the non-canonical TGF-ß pathway during late embryogenesis of Drosophila. This certainly will increase our understanding of the pathogenesis of cardiomyopathy, because haemodynamic overload can up-regulate TGF-ß and death of cardiomyocytes is observed in virtually every myocardial disease. Thus, our study may provide possible medical intervention for human cardiomyopathy.