Comparative analysis of human chromosome 22 CES-DGCR syntenic regions in chimpanzee, baboon, bovine, mouse and zebrafish and expression profiling in zebrafish early developmental stages using whole mount in situ hybridization.

The final series of experiments were based on the earlier observation that 16 genes in the human chromosome 22 CES-DGCR region had reported expression but no detailed expression profiles while 6 others had no known expression profiles. Through the comparative sequencing and subsequent whole mount in situ studies reported in this dissertation, expression of these 22 genes was observed to occur during zebrafish development, mainly during early developmental stages followed by either decreased or no expression in later stages in the brain, ear, eyes, heart, pharyngeal arches, liver, and kidney, all organs related to anomalies resulting in phenotype observed in CES-DGCR patients. Therefore, the third major conclusions from this present work is that contrary to prior studies pointing to single gene alterations resulting in these diseases, it now is clear that both CES and DGCR are multigene-based diseases.**This dissertation is a compound document (contains both a paper copy and a CD as part of the dissertation). The CD requires the following system requirements: Adobe Acrobat.The majority of the amino acid substitutions in humans, chimpanzees, baboons and bovines are changes from hydrophilic to hydrophilic amino acids. The observed human and chimpanzee substitution rate was 1.2% and that between humans and baboons was 2.6%, with Ka/Ks ratios for human and chimpanzee at 0.44 and human and baboon at 0.48. Thus, the second major conclusion from this present work is that at least in the case of humans vs. primates, the genes are evolving by purifying selection.Comparative genomic analysis is a powerful tool that can illuminate the genomic sequence features that result in the changes that drive evolution. In this dissertation, the 4.5 Mb region proximal to the centromere of human chromosome 22 that encodes the contiguous Cat Eye Syndrome and DiGeorge-Velocardiofacial Syndrome (CES-DGCR/VCFS) Critical Regions and the orthologous regions from chimpanzee, baboon, cow, mice and zebrafish have been sequenced and compared. Overall the human and chimpanzee sequences were ∼ 98.5% identical and the human-baboon sequences were ∼ 92% identical at the nucleotide level. A high degree of conservation was observed in both the gene order and the coding region sequences for these synteny regions, with a lower degree of conservation in the intronic and intergenic regions. The conserved structural features likely represent conserved functional properties while the observed differences must be responsible for portions of the human and primate specific phenotypes. The region studied was slightly larger in humans than in chimpanzees and baboons, since the human lineage had a higher insertion frequency relative to the other primates (or the other primates have a higher deletion frequency compared to humans). By comparing the sequenced regions of the chimpanzee genome from three different individual chimpanzees, Clint (ch251), Donald (rp43) and Gon (ptb), the first major conclusion from this dissertation research is that these three chimpanzees differ from each other by ∼ 1.2%, almost as much as humans differ from chimpanzees

Lrp4 Modulates Extracellular Integration of Cell Signaling Pathways in Development

The extent to which cell signaling is integrated outside the cell is not currently appreciated. We show that a member of the low-density receptor-related protein family, Lrp4 modulates and integrates Bmp and canonical Wnt signalling during tooth morphogenesis by binding the secreted Bmp antagonist protein Wise. Mouse mutants of Lrp4 and Wise exhibit identical tooth phenotypes that include supernumerary incisors and molars, and fused molars. We propose that the Lrp4/Wise interaction acts as an extracellular integrator of epithelial-mesenchymal cell signaling. Wise, secreted from mesenchyme cells binds to BMP's and also to Lrp4 that is expressed on epithelial cells. This binding then results in the modulation of Wnt activity in the epithelial cells. Thus in this context Wise acts as an extracellular signaling molecule linking two signaling pathways. We further show that a downstream mediator of this integration is the Shh signaling pathway

Global burden of 369 diseases and injuries in 204 countries and territories, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019

Background: In an era of shifting global agendas and expanded emphasis on non-communicable diseases and injuries along with communicable diseases, sound evidence on trends by cause at the national level is essential. The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) provides a systematic scientific assessment of published, publicly available, and contributed data on incidence, prevalence, and mortality for a mutually exclusive and collectively exhaustive list of diseases and injuries. Methods: GBD estimates incidence, prevalence, mortality, years of life lost (YLLs), years lived with disability (YLDs), and disability-adjusted life-years (DALYs) due to 369 diseases and injuries, for two sexes, and for 204 countries and territories. Input data were extracted from censuses, household surveys, civil registration and vital statistics, disease registries, health service use, air pollution monitors, satellite imaging, disease notifications, and other sources. Cause-specific death rates and cause fractions were calculated using the Cause of Death Ensemble model and spatiotemporal Gaussian process regression. Cause-specific deaths were adjusted to match the total all-cause deaths calculated as part of the GBD population, fertility, and mortality estimates. Deaths were multiplied by standard life expectancy at each age to calculate YLLs. A Bayesian meta-regression modelling tool, DisMod-MR 2.1, was used to ensure consistency between incidence, prevalence, remission, excess mortality, and cause-specific mortality for most causes. Prevalence estimates were multiplied by disability weights for mutually exclusive sequelae of diseases and injuries to calculate YLDs. We considered results in the context of the Socio-demographic Index (SDI), a composite indicator of income per capita, years of schooling, and fertility rate in females younger than 25 years. Uncertainty intervals (UIs) were generated for every metric using the 25th and 975th ordered 1000 draw values of the posterior distribution. Findings: Global health has steadily improved over the past 30 years as measured by age-standardised DALY rates. After taking into account population growth and ageing, the absolute number of DALYs has remained stable. Since 2010, the pace of decline in global age-standardised DALY rates has accelerated in age groups younger than 50 years compared with the 1990–2010 time period, with the greatest annualised rate of decline occurring in the 0–9-year age group. Six infectious diseases were among the top ten causes of DALYs in children younger than 10 years in 2019: lower respiratory infections (ranked second), diarrhoeal diseases (third), malaria (fifth), meningitis (sixth), whooping cough (ninth), and sexually transmitted infections (which, in this age group, is fully accounted for by congenital syphilis; ranked tenth). In adolescents aged 10–24 years, three injury causes were among the top causes of DALYs: road injuries (ranked first), self-harm (third), and interpersonal violence (fifth). Five of the causes that were in the top ten for ages 10–24 years were also in the top ten in the 25–49-year age group: road injuries (ranked first), HIV/AIDS (second), low back pain (fourth), headache disorders (fifth), and depressive disorders (sixth). In 2019, ischaemic heart disease and stroke were the top-ranked causes of DALYs in both the 50–74-year and 75-years-and-older age groups. Since 1990, there has been a marked shift towards a greater proportion of burden due to YLDs from non-communicable diseases and injuries. In 2019, there were 11 countries where non-communicable disease and injury YLDs constituted more than half of all disease burden. Decreases in age-standardised DALY rates have accelerated over the past decade in countries at the lower end of the SDI range, while improvements have started to stagnate or even reverse in countries with higher SDI. Interpretation: As disability becomes an increasingly large component of disease burden and a larger component of health expenditure, greater research and developm nt investment is needed to identify new, more effective intervention strategies. With a rapidly ageing global population, the demands on health services to deal with disabling outcomes, which increase with age, will require policy makers to anticipate these changes. The mix of universal and more geographically specific influences on health reinforces the need for regular reporting on population health in detail and by underlying cause to help decision makers to identify success stories of disease control to emulate, as well as opportunities to improve. Funding: Bill & Melinda Gates Foundation. © 2020 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 licens

Global age-sex-specific fertility, mortality, healthy life expectancy (HALE), and population estimates in 204 countries and territories, 1950-2019 : a comprehensive demographic analysis for the Global Burden of Disease Study 2019

Background: Accurate and up-to-date assessment of demographic metrics is crucial for understanding a wide range of social, economic, and public health issues that affect populations worldwide. The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2019 produced updated and comprehensive demographic assessments of the key indicators of fertility, mortality, migration, and population for 204 countries and territories and selected subnational locations from 1950 to 2019. Methods: 8078 country-years of vital registration and sample registration data, 938 surveys, 349 censuses, and 238 other sources were identified and used to estimate age-specific fertility. Spatiotemporal Gaussian process regression (ST-GPR) was used to generate age-specific fertility rates for 5-year age groups between ages 15 and 49 years. With extensions to age groups 10–14 and 50–54 years, the total fertility rate (TFR) was then aggregated using the estimated age-specific fertility between ages 10 and 54 years. 7417 sources were used for under-5 mortality estimation and 7355 for adult mortality. ST-GPR was used to synthesise data sources after correction for known biases. Adult mortality was measured as the probability of death between ages 15 and 60 years based on vital registration, sample registration, and sibling histories, and was also estimated using ST-GPR. HIV-free life tables were then estimated using estimates of under-5 and adult mortality rates using a relational model life table system created for GBD, which closely tracks observed age-specific mortality rates from complete vital registration when available. Independent estimates of HIV-specific mortality generated by an epidemiological analysis of HIV prevalence surveys and antenatal clinic serosurveillance and other sources were incorporated into the estimates in countries with large epidemics. Annual and single-year age estimates of net migration and population for each country and territory were generated using a Bayesian hierarchical cohort component model that analysed estimated age-specific fertility and mortality rates along with 1250 censuses and 747 population registry years. We classified location-years into seven categories on the basis of the natural rate of increase in population (calculated by subtracting the crude death rate from the crude birth rate) and the net migration rate. We computed healthy life expectancy (HALE) using years lived with disability (YLDs) per capita, life tables, and standard demographic methods. Uncertainty was propagated throughout the demographic estimation process, including fertility, mortality, and population, with 1000 draw-level estimates produced for each metric. Findings: The global TFR decreased from 2·72 (95% uncertainty interval [UI] 2·66–2·79) in 2000 to 2·31 (2·17–2·46) in 2019. Global annual livebirths increased from 134·5 million (131·5–137·8) in 2000 to a peak of 139·6 million (133·0–146·9) in 2016. Global livebirths then declined to 135·3 million (127·2–144·1) in 2019. Of the 204 countries and territories included in this study, in 2019, 102 had a TFR lower than 2·1, which is considered a good approximation of replacement-level fertility. All countries in sub-Saharan Africa had TFRs above replacement level in 2019 and accounted for 27·1% (95% UI 26·4–27·8) of global livebirths. Global life expectancy at birth increased from 67·2 years (95% UI 66·8–67·6) in 2000 to 73·5 years (72·8–74·3) in 2019. The total number of deaths increased from 50·7 million (49·5–51·9) in 2000 to 56·5 million (53·7–59·2) in 2019. Under-5 deaths declined from 9·6 million (9·1–10·3) in 2000 to 5·0 million (4·3–6·0) in 2019. Global population increased by 25·7%, from 6·2 billion (6·0–6·3) in 2000 to 7·7 billion (7·5–8·0) in 2019. In 2019, 34 countries had negative natural rates of increase; in 17 of these, the population declined because immigration was not sufficient to counteract the negative rate of decline. Globally, HALE increased from 58·6 years (56·1–60·8) in 2000 to 63·5 years (60·8–66·1) in 2019. HALE increased in 202 of 204 countries and territories between 2000 and 2019

Bmp signalling in filiform tongue papillae development

OBJECTIVE: Tongue papillae are critical organs in mastication. There are four different types of tongue papillae; fungiform, circumvallate, foliate, and filiform papillae. Unlike the other three taste papillae, non-gustatory papillae, filiform papillae cover the entire dorsal surface of the tongue and are important structures for the mechanical stress of sucking. Filiform papillae are further classified into two subtypes with different morphologies, depending on their location on the dorsum of the tongue. The filiform papillae at the intermolar eminence have pointed tips, whereas filiform papillae with rounded tips are found in other regions (anterior tongue). It remains unknown how the shape of each type of filiform papillae are determined during their development. Bmp signalling pathway has been known to regulate mechanisms that determine the shapes of many ectodermal organs. The aim of this study was to investigate the role of Bmp signalling in filiform papillae development. DESIGN: Comparative in situ hybridization analysis of six Bmps (Bmp2–Bmp7) and two Bmpr genes (Bmpr1a and Bmpr1b) were carried out in filiform papillae development. We further examined tongue papillae in mice over-expressing Noggin under the keratin14 promoter (K14-Noggin). RESULTS: We identified a dynamic temporo-spatial expression of Bmps in filiform papillae development. The K14-Noggin mice showed pointed filiform papillae in regions of the tongue normally occupied by the rounded type. CONCLUSIONS: Bmp signalling thus regulates the shape of filiform papillae

Expression of Sox genes in tooth development

Members of the Sox gene family play roles in many biological processes including organogenesis. We carried out comparative in situ hybridization analysis of seventeen sox genes (Sox1-14, 17, 18, 21) during murine odontogenesis from the epithelial thickening to the cytodifferentiation stages. Localized expression of five Sox genes (Sox6, 9, 13, 14 and 21) was observed in tooth bud epithelium. Sox13 showed restricted expression in the primary enamel knots. At the early bell stage, three Sox genes (Sox8, 11, 17 and 21) were expressed in pre-ameloblasts, whereas two others (Sox5 and 18) showed expression in odontoblasts. Sox genes thus showed a dynamic spatio-temporal expression during tooth development

Correction: Lrp4 Modulates Extracellular Integration of Cell Signaling Pathways in Development

This paper describes a procedure for the synthesis and application of a citalopram-derived affinity resin in purifying the 5HT-reuptake system from human blood platelets. A two-step scheme has been developed for partial purification, based on wheat germ agglutinin-lectin (WGA) affinity and citalopram affinity chromatographies. Upon solubilization of the carrier with 1% digitonin, a 50-70-fold increase in specific [3H]imipramine binding activity with a 70% recovery could be accomplished through WGA-lectin chromatography. The WGA pool was then subjected to affinity chromatography on citalopram-agarose. At least 90% of the binding capacity adsorbed to the column. Specific elution using 10 µM citalopram resulted in a 22% recovery of binding activity. A 10000-fold overall purification was obtained by using this two-step procedure. Analysis of the fractions on SDS-PAGE after 125I labeling revealed specific elution of 78- and 55-kDa proteins concomitant with the appearance of [3H]imipramine binding activity. The pharmacological profile of the partially purified reuptake system correlated well with that derived from the crude membrane-bound reuptake system, suggesting a copurification of the 5HT binding activity and [3H] imipramine binding activity.