BRITER: A BMP Responsive Osteoblast Reporter Cell Line

BACKGROUND: BMP signaling pathway is critical for vertebrate development and tissue homeostasis. High-throughput molecular genetic screening may reveal novel players regulating BMP signaling response while chemical genetic screening of BMP signaling modifiers may have clinical significance. It is therefore important to generate a cell-based tool to execute such screens. METHODOLOGY/PRINCIPAL FINDINGS: We have established a BMP responsive reporter cell line by stably integrating a BMP responsive dual luciferase reporter construct in the immortalized calvarial osteoblast cells isolated from tamoxifen inducible Bmp2; Bmp4 double conditional knockout mouse strain. This cell line, named BRITER (BMP Responsive Immortalized Reporter cell line), responds robustly, promptly and specifically to exogenously added BMP2 protein. The sensitivity to added BMP may be further increased by depleting the endogenous BMP2 and BMP4 proteins. CONCLUSION: As the dynamic range of the assay (for BMP responsiveness) is very high for BRITER and as it responds specifically and promptly to exogenously added BMP2 protein, BRITER may be used effectively for chemical or molecular genetic screening for BMP signaling modifiers. Identification of novel molecular players capable of influencing BMP signaling pathway may have clinical significance

Global age-sex-specific mortality, life expectancy, and population estimates in 204 countries and territories and 811 subnational locations, 1950–2021, and the impact of the COVID-19 pandemic: a comprehensive demographic analysis for the Global Burden of Disease Study 2021

Background: Estimates of demographic metrics are crucial to assess levels and trends of population health outcomes. The profound impact of the COVID-19 pandemic on populations worldwide has underscored the need for timely estimates to understand this unprecedented event within the context of long-term population health trends. The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021 provides new demographic estimates for 204 countries and territories and 811 additional subnational locations from 1950 to 2021, with a particular emphasis on changes in mortality and life expectancy that occurred during the 2020–21 COVID-19 pandemic period. Methods: 22 223 data sources from vital registration, sample registration, surveys, censuses, and other sources were used to estimate mortality, with a subset of these sources used exclusively to estimate excess mortality due to the COVID-19 pandemic. 2026 data sources were used for population estimation. Additional sources were used to estimate migration; the effects of the HIV epidemic; and demographic discontinuities due to conflicts, famines, natural disasters, and pandemics, which are used as inputs for estimating mortality and population. Spatiotemporal Gaussian process regression (ST-GPR) was used to generate under-5 mortality rates, which synthesised 30 763 location-years of vital registration and sample registration data, 1365 surveys and censuses, and 80 other sources. ST-GPR was also used to estimate adult mortality (between ages 15 and 59 years) based on information from 31 642 location-years of vital registration and sample registration data, 355 surveys and censuses, and 24 other sources. Estimates of child and adult mortality rates were then used to generate life tables with a relational model life table system. For countries with large HIV epidemics, life tables were adjusted using independent estimates of HIV-specific mortality generated via an epidemiological analysis of HIV prevalence surveys, antenatal clinic serosurveillance, and other data sources. Excess mortality due to the COVID-19 pandemic in 2020 and 2021 was determined by subtracting observed all-cause mortality (adjusted for late registration and mortality anomalies) from the mortality expected in the absence of the pandemic. Expected mortality was calculated based on historical trends using an ensemble of models. In location-years where all-cause mortality data were unavailable, we estimated excess mortality rates using a regression model with covariates pertaining to the pandemic. Population size was computed using a Bayesian hierarchical cohort component model. Life expectancy was calculated using age-specific mortality rates and standard demographic methods. Uncertainty intervals (UIs) were calculated for every metric using the 25th and 975th ordered values from a 1000-draw posterior distribution. Findings: Global all-cause mortality followed two distinct patterns over the study period: age-standardised mortality rates declined between 1950 and 2019 (a 62·8% [95% UI 60·5–65·1] decline), and increased during the COVID-19 pandemic period (2020–21; 5·1% [0·9–9·6] increase). In contrast with the overall reverse in mortality trends during the pandemic period, child mortality continued to decline, with 4·66 million (3·98–5·50) global deaths in children younger than 5 years in 2021 compared with 5·21 million (4·50–6·01) in 2019. An estimated 131 million (126–137) people died globally from all causes in 2020 and 2021 combined, of which 15·9 million (14·7–17·2) were due to the COVID-19 pandemic (measured by excess mortality, which includes deaths directly due to SARS-CoV-2 infection and those indirectly due to other social, economic, or behavioural changes associated with the pandemic). Excess mortality rates exceeded 150 deaths per 100 000 population during at least one year of the pandemic in 80 countries and territories, whereas 20 nations had a negative excess mortality rate in 2020 or 2021, indicating that all-cause mortality in these countries was lower during the pandemic than expected based on historical trends. Between 1950 and 2021, global life expectancy at birth increased by 22·7 years (20·8–24·8), from 49·0 years (46·7–51·3) to 71·7 years (70·9–72·5). Global life expectancy at birth declined by 1·6 years (1·0–2·2) between 2019 and 2021, reversing historical trends. An increase in life expectancy was only observed in 32 (15·7%) of 204 countries and territories between 2019 and 2021. The global population reached 7·89 billion (7·67–8·13) people in 2021, by which time 56 of 204 countries and territories had peaked and subsequently populations have declined. The largest proportion of population growth between 2020 and 2021 was in sub-Saharan Africa (39·5% [28·4–52·7]) and south Asia (26·3% [9·0–44·7]). From 2000 to 2021, the ratio of the population aged 65 years and older to the population aged younger than 15 years increased in 188 (92·2%) of 204 nations. Interpretation: Global adult mortality rates markedly increased during the COVID-19 pandemic in 2020 and 2021, reversing past decreasing trends, while child mortality rates continued to decline, albeit more slowly than in earlier years. Although COVID-19 had a substantial impact on many demographic indicators during the first 2 years of the pandemic, overall global health progress over the 72 years evaluated has been profound, with considerable improvements in mortality and life expectancy. Additionally, we observed a deceleration of global population growth since 2017, despite steady or increasing growth in lower-income countries, combined with a continued global shift of population age structures towards older ages. These demographic changes will likely present future challenges to health systems, economies, and societies. The comprehensive demographic estimates reported here will enable researchers, policy makers, health practitioners, and other key stakeholders to better understand and address the profound changes that have occurred in the global health landscape following the first 2 years of the COVID-19 pandemic, and longer-term trends beyond the pandemic

Creation of BRITER Cell Line.

<p>(<b>A</b>) Schematic showing steps involved in the creation of BRITER Cell Line. (<b>B</b>) Schematic showing critical genetic components of BRITER Cell Line. (<b>C</b>) BMP2 dependent FFLuc activity of BRITER Cell Line under four different conditions, namely “−BMP. −4-OHT”, “−BMP, +4-OHT”, “+BMP, +4-OHT” and “+BMP, −4-OHT”. (<b>D</b>) Anti-PSMAD 1/5/8 immunofluorescence in BRITER cell line under four different conditions: (<b>a</b>) −BMP−4-OHT, (<b>b</b>) −BMP+4-OHT, (<b>c</b>) +BMP+4-OHT and (<b>d</b>) +BMP−4-OHT. Scale bar 100 µm. (<b>E</b>) Quantification of Anti-PSMAD 1/5/8 immunofluorescence by Image J. (<b>F</b>) Western blot analysis of BRITER cell extracts cultured under indicated conditions with PSMAD 1/5/8 antibody. β-tubulin antibody has been used as loading control. Data shown are means ± SEM of three independent experiments carried out in triplicates.</p

Characterization of BRITER cell line.

<p>(<b>A</b>) RT-PCR analysis of BRITER cell line for osteblast specific marker genes: Osterix (lane 1), ColIAI (lane 2), BSP (lane 3), Runx2 (lane 4), Osteocalcin (lane 5), Alkaline Phosphatase (lane 6), Large T antigen (lane 7) and GAPDH (lane 8), respectively. (<b>B</b>) Quantification of ALP activity of BRITER cell extracts cultured under indicated conditions. (<b>C</b>) Alkaline Phosphatase staining (<b>a–d</b>). Alizarin red staining (<b>e–h</b>). von Kossa staining (<b>i–l</b>) of BRITER cell line cultured under following conditions “−BMP−4 OHT” (<b>a, e, i</b>), “−BMP+4-OHT” (<b>b, f, j</b>), “+BMP+4 OHT” (<b>c, g, k</b>) and “+BMP−4-OHT” (<b>d, h, l</b>). Scale bar 10 mm. Data shown is means ± SEM of experiments carried out in triplicates.</p

Figure 3

<p>(<b>A</b>) Time course analysis showing relative FFLuc activity in presence of 100 ng/ml (red line) and 10 ng/ml (blue line) BMP2 concentrations at different time points indicated. (<b>B</b>) Normalized FFLuc activity, (Inset showing absolute values of FFLuc activity) after 3 hours of stimulation with different BMP2 concentrations in presence (blue line) or absence (red line) of 4-OHT. (<b>C</b>) Relative luciferase activity after 3 hours of stimulation with different BMP2 concentrations in presence (blue line) or absence (red line) of 4-OHT. (<b>D</b>) Normalized relative luciferase activity after 3 hours of stimulation with different BMP2 concentrations in presence (blue line) or absence (red line) of 4-OHT. Data shown are means ± SEM of three independent experiments carried out in triplicates.</p

Comparison of BRITER with previously reported osteogenic BMP reporter cell Lines.

<p>Comparison of BRITER with previously reported osteogenic BMP reporter cell Lines.</p

Phosphate-induced activation of VEGFR2 leads to caspase-9-mediated apoptosis of hypertrophic chondrocytes

Summary: Low circulating phosphate (Pi) leads to rickets, characterized by expansion of the hypertrophic chondrocytes (HCs) in the growth plate due to impaired HC apoptosis. Studies in HCs demonstrate that Pi activates the Raf/MEK/ERK1/2 and mitochondrial apoptotic pathways. To determine how Pi activates these pathways, a small-molecule screen was undertaken to identify inhibitors of Pi-induced ERK1/2 phosphorylation in HCs. Vascular endothelial growth factor receptor 2 (VEGFR2) was identified as a target. In vitro studies in HCs demonstrate that VEGFR2 inhibitors block Pi-induced pERK1/2 and caspase-9 cleavage. Like Pi, rhVEGF activates ERK1/2 and caspase-9 in HCs and induces phosphorylation of VEGFR2, confirming that Pi activates this signaling pathway in HCs. Chondrocyte-specific depletion of VEGFR2 leads to an increase in HCs, impaired vascular invasion, and a decrease in HC apoptosis. Thus, these studies define a role for VEGFR2 in transducing Pi signals and mediating its effects on growth plate maturation

Sustainable Intensification of Rice Fallows with Oilseeds and Pulses: Effects on Soil Aggregation, Organic Carbon Dynamics, and Crop Productivity in Eastern Indo-Gangetic Plains

Climate-smart agriculture (CSA) practices are becoming increasingly important due to their better adaptability to harsh climatic conditions (in general) and the unpredictability of monsoons in India (in particular). Conventional rice cultivation (e.g., PTR) involves intensive tilling followed by intensive puddling in standing water that destroys the soil aggregation and depletes carbon pools. Therefore, alternative crop establishment methods need to be devised for the sustainability of system productivity, and the suitabilities of potential oilseeds and pulses need to be tested for cropping intensification in rice-fallow regions. Hence, an ongoing experiment (implemented in 2016) was evaluated to identify the appropriate CSA management practices in restoring soil C and physical health under diversified cropping systems in the rice-fallow system of eastern India. Six tillage and crop establishment methods along with residue management were kept as the main plots [zero-till-direct-seeded rice (ZTDSR), conventional-till-DSR (CT-DSR), puddled transplanted rice (PTR), ZTDSR with rice residue retentions (ZTDSRR+), CTDSR with rice residue retention (CTDSRR+), PTR with rice residue retention (PTRR+)] while five winter/post-rainy crops (oilseeds and pulses) were raised in a subplot. In the ZTDSRR+ production system, soil macro-aggregate (%), macro-aggregate-associated C, MWD, and GMD of aggregates increased by 60.1, 71.3, 42.1, and 17.1%, respectively, in comparison to conventional tillage practices (PTR). The carbon management index (CMI) was 58% more in the ZTDSRR+ production system compared to PTR. Among the winter crops, chickpeas recorded higher values of soil structural indices and C content. In the PTR production system, system productivity, in terms of rice equivalent yield, was comparable to ZTDSRR+. ZT with residue retention in rice followed by post-rainy/winter pulses led to higher C content and structural stability of the soil. Thus, CSA management practices can improve the crop productivity as well as soil health of rice-fallow production systems of eastern India and comparable agroecotypes of South Asia