Ability of crassulacean acid metabolism plants to overcome interacting stresses in tropical environments

The characteristic high morphological and physiological plasticity of tropical CAM plants as adaptations to multistress locations is reviewed against the background of a realisation that tropical forests contain many more CAM species than do drier areas such as semi-desert

Global burden of 288 causes of death and life expectancy decomposition in 204 countries and territories and 811 subnational locations, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

BACKGROUND Regular, detailed reporting on population health by underlying cause of death is fundamental for public health decision making. Cause-specific estimates of mortality and the subsequent effects on life expectancy worldwide are valuable metrics to gauge progress in reducing mortality rates. These estimates are particularly important following large-scale mortality spikes, such as the COVID-19 pandemic. When systematically analysed, mortality rates and life expectancy allow comparisons of the consequences of causes of death globally and over time, providing a nuanced understanding of the effect of these causes on global populations. METHODS The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021 cause-of-death analysis estimated mortality and years of life lost (YLLs) from 288 causes of death by age-sex-location-year in 204 countries and territories and 811 subnational locations for each year from 1990 until 2021. The analysis used 56 604 data sources, including data from vital registration and verbal autopsy as well as surveys, censuses, surveillance systems, and cancer registries, among others. As with previous GBD rounds, cause-specific death rates for most causes were estimated using the Cause of Death Ensemble model-a modelling tool developed for GBD to assess the out-of-sample predictive validity of different statistical models and covariate permutations and combine those results to produce cause-specific mortality estimates-with alternative strategies adapted to model causes with insufficient data, substantial changes in reporting over the study period, or unusual epidemiology. YLLs were computed as the product of the number of deaths for each cause-age-sex-location-year and the standard life expectancy at each age. As part of the modelling process, uncertainty intervals (UIs) were generated using the 2·5th and 97·5th percentiles from a 1000-draw distribution for each metric. We decomposed life expectancy by cause of death, location, and year to show cause-specific effects on life expectancy from 1990 to 2021. We also used the coefficient of variation and the fraction of population affected by 90% of deaths to highlight concentrations of mortality. Findings are reported in counts and age-standardised rates. Methodological improvements for cause-of-death estimates in GBD 2021 include the expansion of under-5-years age group to include four new age groups, enhanced methods to account for stochastic variation of sparse data, and the inclusion of COVID-19 and other pandemic-related mortality-which includes excess mortality associated with the pandemic, excluding COVID-19, lower respiratory infections, measles, malaria, and pertussis. For this analysis, 199 new country-years of vital registration cause-of-death data, 5 country-years of surveillance data, 21 country-years of verbal autopsy data, and 94 country-years of other data types were added to those used in previous GBD rounds. FINDINGS The leading causes of age-standardised deaths globally were the same in 2019 as they were in 1990; in descending order, these were, ischaemic heart disease, stroke, chronic obstructive pulmonary disease, and lower respiratory infections. In 2021, however, COVID-19 replaced stroke as the second-leading age-standardised cause of death, with 94·0 deaths (95% UI 89·2-100·0) per 100 000 population. The COVID-19 pandemic shifted the rankings of the leading five causes, lowering stroke to the third-leading and chronic obstructive pulmonary disease to the fourth-leading position. In 2021, the highest age-standardised death rates from COVID-19 occurred in sub-Saharan Africa (271·0 deaths [250·1-290·7] per 100 000 population) and Latin America and the Caribbean (195·4 deaths [182·1-211·4] per 100 000 population). The lowest age-standardised death rates from COVID-19 were in the high-income super-region (48·1 deaths [47·4-48·8] per 100 000 population) and southeast Asia, east Asia, and Oceania (23·2 deaths [16·3-37·2] per 100 000 population). Globally, life expectancy steadily improved between 1990 and 2019 for 18 of the 22 investigated causes. Decomposition of global and regional life expectancy showed the positive effect that reductions in deaths from enteric infections, lower respiratory infections, stroke, and neonatal deaths, among others have contributed to improved survival over the study period. However, a net reduction of 1·6 years occurred in global life expectancy between 2019 and 2021, primarily due to increased death rates from COVID-19 and other pandemic-related mortality. Life expectancy was highly variable between super-regions over the study period, with southeast Asia, east Asia, and Oceania gaining 8·3 years (6·7-9·9) overall, while having the smallest reduction in life expectancy due to COVID-19 (0·4 years). The largest reduction in life expectancy due to COVID-19 occurred in Latin America and the Caribbean (3·6 years). Additionally, 53 of the 288 causes of death were highly concentrated in locations with less than 50% of the global population as of 2021, and these causes of death became progressively more concentrated since 1990, when only 44 causes showed this pattern. The concentration phenomenon is discussed heuristically with respect to enteric and lower respiratory infections, malaria, HIV/AIDS, neonatal disorders, tuberculosis, and measles. INTERPRETATION Long-standing gains in life expectancy and reductions in many of the leading causes of death have been disrupted by the COVID-19 pandemic, the adverse effects of which were spread unevenly among populations. Despite the pandemic, there has been continued progress in combatting several notable causes of death, leading to improved global life expectancy over the study period. Each of the seven GBD super-regions showed an overall improvement from 1990 and 2021, obscuring the negative effect in the years of the pandemic. Additionally, our findings regarding regional variation in causes of death driving increases in life expectancy hold clear policy utility. Analyses of shifting mortality trends reveal that several causes, once widespread globally, are now increasingly concentrated geographically. These changes in mortality concentration, alongside further investigation of changing risks, interventions, and relevant policy, present an important opportunity to deepen our understanding of mortality-reduction strategies. Examining patterns in mortality concentration might reveal areas where successful public health interventions have been implemented. Translating these successes to locations where certain causes of death remain entrenched can inform policies that work to improve life expectancy for people everywhere. FUNDING Bill & Melinda Gates Foundation

Nano-thin walled micro-compartments from transmembrane protein-polymer conjugates

The high interfacial activity of protein–polymer conjugates has inspired their use as stabilizers for Pickering emulsions, resulting in many interesting applications such as synthesis of templated micro-compartments and protocells or vehicles for drug and gene delivery. In this study we report, for the first time, the stabilization of Pickering emulsions with conjugates of a genetically modified transmembrane protein, ferric hydroxamate uptake protein component A (FhuA). The lysine residues of FhuA with open pore (FhuA ΔCVFtev) were modified to attach an initiator and consequently controlled radical polymerization (CRP) carried out via the grafting-from technique. The resulting conjugates of FhuA ΔCVFtev with poly(N-isopropylacrylamide) (PNIPAAm) and poly((2-dimethylamino)ethyl methacrylate) (PDMAEMA), the so-called building blocks based on transmembrane proteins (BBTP), have been shown to engender larger structures. The properties such as pH-responsivity, temperature-responsivity and interfacial activity of the BBTP were analyzed using UV-Vis spectrophotometry and pendant drop tensiometry. The BBTP were then utilized for the synthesis of highly stable Pickering emulsions, which could remain non-coalesced for well over a month. A new UV-crosslinkable monomer was synthesized and copolymerized with NIPAAm from the protein. The emulsion droplets, upon crosslinking of polymer chains, yielded micro-compartments. Fluorescence microscopy proved that these compartments are of micrometer scale, while cryo-scanning electron microscopy and scanning force microscopy analysis yielded a thickness in the range of 11.1 ± 0.6 to 38.0 ± 18.2 nm for the stabilizing layer of the conjugates. Such micro-compartments would prove to be beneficial in drug delivery applications, owing to the possibility of using the channel of the transmembrane protein as a gate and the smart polymer chains as trigger switches to tune the behavior of the capsules

FXR Controls the Tumor Suppressor NDRG2 and FXR Agonists Reduce Liver Tumor Growth and Metastasis in an Orthotopic Mouse Xenograft Model

<div><p>The farnesoid X receptor (FXR) is expressed predominantly in tissues exposed to high levels of bile acids and controls bile acid and lipid homeostasis. FXR^−/− mice develop hepatocellular carcinoma (HCC) and show an increased prevalence for intestinal malignancies, suggesting a role of FXR as a tumor suppressor in enterohepatic tissues. The N-myc downstream-regulated gene 2 (NDRG2) has been recognized as a tumor suppressor gene, which is downregulated in human hepatocellular carcinoma, colorectal carcinoma and many other malignancies.</p> <p>We show reduced NDRG2 mRNA in livers of FXR^−/− mice compared to wild type mice and both, FXR and NDRG2 mRNAs, are reduced in human HCC compared to normal liver. Gene reporter assays and Chromatin Immunoprecipitation data support that FXR directly controls NDRG2 transcription via IR1-type element(s) identified in the first introns of the human, mouse and rat NDRG2 genes. NDRG2 mRNA was induced by non-steroidal FXR agonists in livers of mice and the magnitude of induction of NDRG2 mRNA in three different human hepatoma cell lines was increased when ectopically expressing human FXR. Growth and metastasis of SK-Hep-1 cells was strongly reduced by non-steroidal FXR agonists in an orthotopic liver xenograft tumor model. Ectopic expression of FXR in SK-Hep1 cells reduced tumor growth and metastasis potential of corresponding cells and increased the anti-tumor efficacy of FXR agonists, which may be partly mediated via increased NDRG2 expression. FXR agonists may show a potential in the prevention and/or treatment of human hepatocellular carcinoma, a devastating malignancy with increasing prevalence and limited therapeutic options.</p> </div

Induction of NDRG2 in human hepatoma cell lines.

<p><b>A</b>, Ectopic expression of FXR in HepG2-FXR5 compared to HepG2 parental cells. Positions of bands representing FXR and GAPDH proteins in the western blots from aliquots of whole cell extracts are indicated. <b>B</b>, Dose dependent induction of NDRG2 mRNA in HepG2 and HepG2-FXR5 cells by the FXR agonist PX20350. HepG2 or HepG2-FXR5 cells were grown for 18 h in medium containing either DMSO, 0.5 µM of PX20350 or 0.5 µM of PX20606 in 96 well plates as indicated and relative fold induction of NDRG2 mRNA over the DMSO control was determined by RT-qPCR using the ΔΔCt method. <b>C</b>, Induction of NDRG2 protein by the FXR agonists Px20350 and PX20606 in HepG2-FXR5 cells. HepG2-FXR5 cells were grown to a density of around 60% and further cultivated for 18 h in medium supplemented with either DMSO as a vehicle, 0.5 µM PX20350 or 0.5 µM PX20606 as indicated and aliquots of whole cell extracts analysed by western blots. Relative positions of size marker protein bands and the position of NDRG2 and GAPDH proteins are indicated. <b>D</b>, Stable overproduction of FXR in HuH-7-37 compared to HuH-7 cells. Positions of bands representing FXR and GAPDH proteins analysed by western blots from aliquots of whole cell extracts are indicated. <b>E</b>, Dose dependent induction of NDRG2 mRNA in HuH7 and HuH7-37 cells by FXR agonist Px20350. HuH7 or HuH7-37 cells were grown for 18 h in medium containing either DMSO or increasing concentrations of PX20350 in DMSO in 96 well plates and relative fold induction over DMSO control of NDRG2 mRNA was determined by RT- qPCR using the ΔΔCt method. <b>F</b>, NDRG2 is a direct transcriptional target of FXR. HepG2-FXR5 cells, grown in quadruplicates in 12 well plates at a density of around 60%, were further incubated for 4 hours in growth medium supplemented with either DMSO, 0.5 µM PX20350 (PX) or 0.5 µM PX20350 together with 10 µM Cycloheximide (CHX). The relative fold induction of NDRG2- and SHP- mRNAs over DMSO as control were determined by RT- qPCR using the ΔΔCt method and a Student's t-test was performed (*** = p<0.001).</p

Number of animals with metastases in lymph nodes (A) and bone (B) and the total numbers of animals in each group surviving till day 56 (in parentheses) are shown.

<p>One animal died in each of the groups receiving Sorafenib, in the course of the experiment.</p

Identification of functional IR1 site(s) in NDRG2.

<p><b>A</b>, Schematic representation of the NDRG2 gene with the IR1 site(s) located in the first intron. Human, mouse and rat intronic sequences with IR1 elements (in bold) are shown below (IR1 elements start at positions +1560, +757 and +1394 respectively). A version of the human NDRG2 response element with mutated IR1 sites is shown below. <b>B</b>, Luciferase reporter assays from HEK 293 cells co-transfected with pTReX-Dest30-hFXRα2 and a luciferase reporter plasmid containing either one copy of the wild type IR1 element (IR1) or the mutated element (IR1^mut) from the human NDRG2 gene or 2 copies of the human IBABP-RE (IBABP). The fold induction in presence of 0.5 µM Px20350 (Px) versus DMSO control (D) of firefly luciferase activities (normalized against renilla luciferase) of triplicate experiments is shown. <b>C</b>, Amplification of a 196 bp DNA fragment encompassing the IR1 sequences from chromatin prepared by ChIP from HepG2 or HepG2-FLAG-FXR cells. PCR reactions from input DNA and DNA derived from the immunoprecipitation with the M2 anti-FLAG antibody are indicated <b>D</b>, Localization of murine FXRα and RXRα by ChIP-seq to a site corresponding to the predicted IR1 recognition element (star) in the first intron of the mouse Ndrg2 gene on chromosome 14 in mouse liver. The FXRα and RXRα UCSC genome browser tracks shown for the NDRG2 gene were retrieved from genome wide ChIP-seq datasets published by Thomas et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043044#pone.0043044-Thomas1" target="_blank">[22]</a> and Boergesen et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043044#pone.0043044-Boergesen1" target="_blank">[41]</a>, respectively. <b>E</b>, Chemical structures of FXR agonists. Among different structural changes, the unfavourable stilbene linker present in GW4064 was replaced by an amino-methylene linker in Px20350 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043044#pone.0043044-Abel1" target="_blank">[40]</a>. The further modified Px20606 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043044#pone.0043044-Kremoser1" target="_blank">[53]</a> yielded improved <i>in vivo</i> pharmakokinetic properties compared to Px20350. A characterization of FXR agonists in <i>in-vitro</i> assays is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043044#pone-0043044-t001" target="_blank">Table 1</a>.</p