Enzymology and structural enzymology of dye-decolorizing peroxidases and a primary study of encapsulin

The aim of this project is to provide a detailed comparative study in enzymology of dye-decolorising peroxidases, DyP, from Pseudomonas fluorescens and from Thermobifida fusca, a thermophile bacterium. Another objective is a primary study of encapsulin, a recently discovered icosahedral nanocompartment protein from Rhodococcus jostii Rha1. Three peroxidase genes from P. fluorescens and one from T. fusca were cloned, expressed, and their products were purified and the enzymes kinetically characterized with different substrates, lignin model compounds and lignocellulose. In addition, encapsulin has been purified, its assembly/disassembly under different pH conditions was studied and finally, its presence or absence in the extracellular fraction of R. jostii investigated. DyP type peroxidases from Gram-positive bacteria have been studied recently and showed oxidation activity toward Mn (II) and lignin model compounds. Gram-negative pseudomonads, also show activity for lignin oxidation and contain DyP-type peroxidase genes. P. fluorescens Pf-5 contains three DyP-type peroxidases (35, 40 and 47 kDa). In this study each of them was overexpressed in Escherichia coli, purified, and characterised by different substrates, Kraft lignin and lignocellulose. Each of the aforementioned enzymes shows activity for oxidation of most of the substrates, but the 35 kDa DyP1B and 40 kDa DyP2B enzymes show activity for oxidation of Mn (II). Only in the presence of Mn (II) and hydrogen peroxide, incubation of finely powdered lignocellulose with DyP1B leads to the release of a low molecular weight lignin fragment that was identified by mass spectrometry as a ß-aryl ether lignin dimer that contains one G unit and one H unit bearing a benzylic ketone. A mechanism for releasing of the -aryl ether lignin dimer fragment from the lignin molecule via oxidation is proposed. A DyP-type peroxidase enzyme from the thermophilic cellulose degrader Thermobifida fusca was investigated for its catalytic ability for lignin oxidation. TfuDyP was found to oxidise a ß-aryl ether lignin model compound, forming an oxidised tetramer. A crystal structure of TfuDyP was determined, to 1.7 Å resolution, which was found to contain a diatomic oxygen ligand bound to the heme centre, hydrogen-bonded to active site residues Asp-203 and Arg-315. For three amino acid residues present in distal heme pocket, site directed mutagenesis was performed and the effect of each mutation on enzyme activity was measured by three different substrates. Recently DyPB peroxidase from Rhodococcus jostii RHA1 has been recognised as a bacterial lignin peroxidase enzyme. The dypB gene is next to a gene that encodes an encapsulin protein that previously was shown in Thermotoga maritima to assemble and form into a nano-compartment comprised of 60-subunits. DyPB protein contains a C-terminal sequence motif that is supposed to lead the protein to the encapsulin nanocompartment. In this study, R. jostii RHA1 encapsulin gene was overexpressed in R. jostii RHA1, and the encapsulin protein was extracted as a high molecular weight native assembly (Mr >106 kDa). It was shown that by treatment of the purified nanocompartment at pH 3.0, it is able to be denatured to form a low molecular weight species and most importantly it is able to be re-assembled to form the native nanocompartment at pH 7.0. Dynamic light scattering showed that DyPB peroxidase in vitro could be assembled with encapsulin in a monomeric state to form an assembly of encapsulated DyPB in similar size and shape compared to the encapsulin-only nanocompartment. By using a nitrated lignin UV-Vis assay method, it was shown that the assembled complex of DyPB-encapsulin exhibited enhanced lignin degradation activity per mg DyPB present, compared with native DyPB. The stoichiometry of encapsulin/µmol DyPB in the assembled complex was measured, 8.6 mol encapsulin/mol DyPB, that was comparable to the predicted value of 10 obtained from the crystal structure

Protein engineering of Pseudomonas fluorescens peroxidase Dyp1B for oxidation of phenolic and polymeric lignin substrates

Directed evolution was applied to dye-decolourizing peroxidase Dyp1B from Pseudomonas fluorescens Pf-5, in order to enhance the activity for oxidation of phenolic and lignin substrates. Saturation mutagenesis was used to generate focused libraries at 7 active site residues in the vicinity of the heme cofactor, and the libraries were screened for activity towards 2,6-dichlorophenol. Mutants N193 L and H169 L were found to show 7–8 fold enhanced kcat/KM towards DCP, and replacements at Val205 and Ala209 also showed enhanced activity towards alkali Kraft lignin. Residues near the predicted Mn(II) binding site were also investigated by site-directed mutagenesis, and mutants S223 N and H127R showed 4-7-fold increased kcat/KM for Mn(II) oxidation. Mutant F128R also showed enhanced thermostability, compared to wild-type Dyp1B. Testing of mutants for low molecular weight product release from Protobind alkali lignin revealed that mutant H169 L showed enhanced product release, compared with WT enzyme, and the formation of three low molecular weight metabolites by this mutant was detected by reverse phase HPLC analysis

Periplasmic expression of Pseudomonas fluorescens peroxidase Dyp1B and site-directed mutant Dyp1B enzymes enhances polymeric lignin degradation activity in Pseudomonas putida KT2440

Expression of lignin-oxidising Pseudomonas fluorescens Dyp1B in the periplasm of Pseudomonas putida KT2440, using a tat fusion construct, was found to lead to enhanced whole cell activity for oxidation of DCP and polymeric lignin substrates. Four amino acid residues predicted to lie at the manganese ion binding site of Pseudomonas fluorescens peroxidase Dyp1B were investigated using site-directed mutagenesis. Mutants H127R and S223A showed 2-fold and 4-fold higher kcat for Mn(II) oxidation respectively, and mutant S223A showed 2-fold enhanced production of low molecular weight phenolic products from a polymeric soda lignin. The mutant Pfl Dyp1B genes were expressed as tat fusions to investigate their effect on lignin oxidation by P. putida KT2440

Sime Darby goes to Liberia.

This case study discusses the issues arising from Sime Darby Corporation being given a concession agreement to develop 220,000 hectares of land with palm and rubber plantations in Liberia for a period of 63 years. Sime Darby Bhd is a Malaysian multinational company with diverse interests. Its main focus is plantations with its major crops being oil palm and rubber. It has large plantations in Malaysia and Indonesia. With land becoming scarce in Southeast Asia, Sime Darby looked for opportunities in Africa. They found a partner in Liberia which needed foreign investment to revive its economy after two civil wars. While the opportunities looked great on papers, questions arose over the risks involved

Ore Genesis of the Abu Ghalaga Ferro-Ilmenite Ore Associated with Neoproterozoic Massive-Type Gabbros, South-Eastern Desert of Egypt: Evidence from Texture and Mineral Chemistry

Massif-type mafic intrusions (gabbro and anorthosite) are known for their considerable resources of vanadium-bearing iron–titanium oxide ores. Massive-type gabbroic and anorthosite rocks are frequently associated with magmatic rocks that have significant quantities of iron, titanium, and vanadium. The most promising intrusions that host Fe-Ti oxide ores are the gabbroic rocks in the south-eastern desert. The ilmenite ore deposits are hosted in arc gabbroic and anorthosite rocks. They are classified into three types, namely black ore, red ore, and disseminated ore. The black ilmenite ore is located at the deeper level, while the oxidized red ore is mainly located at or near the surface. Petrographically, the gabbro and ilmenite ores indicate a crystallization sequence of plagioclase, titaniferous pyroxene, and ilmenite. This reveals that the ilmenite is a magmatic deposit formed by the liquid gravity concentration of ilmenite following the crystallization of feldspar and pyroxene. Meanwhile, quartz, tremolite, zoisite, and opaque minerals are accessory minerals. The Fe-Ti ores are composed of ilmenite hosting exsolved hematite lamellae of variable sizes and shapes, gangue silicate minerals, and some sulfides. The X-ray diffraction (XRD) data reveal the presence of two mineral phases: ilmenite and hematite formed by the unmixing of the ferroilmenite homogeneous phase upon cooling. As a result, the ore is mostly made up of hemo-ilmenite. Using an electron microscope (SEM), as well as by observing the textures seen by the ore microscope, ilmenite is the dominant Fe-Ti oxide and contains voluminous hematite exsolved crystals. Under the scanning electron microscope, ilmenite contained intergrowths of hematite as a thin sandwich and lens shape. The formation of hematite lamellae indicates an oxidation process. Mineral chemistry-based investigations reveal late/post-magmatic activity at high temperatures. The examined ilmenite plots on the ferro-ilmenite line were created by continuous solid solution over 800 °C, whereas the analyzed magnetite and Ti-magnetite plot near the magnetite line and were formed by continuous solid solution exceeding 600 °C

Host mobility key management in dynamic secure group communication

The key management has a fundamental role in securing group communications taking place over vast and unprotected networks. It is concerned with the distribution and update of the keying materials whenever any changes occur in the group membership. Wireless mobile environments enable members to move freely within the networks, which causes more difficulty to design efficient and scalable key management protocols. This is partly because both member location dynamic and group membership dynamic must be managed concurrently, which may lead to significant rekeying overhead. This paper presents a hierarchical group key management scheme taking the mobility of members into consideration intended for wireless mobile environments. The proposed scheme supports the mobility of members across wireless mobile environments while remaining in the group session with minimum rekeying transmission overhead. Furthermore, the proposed scheme alleviates 1-affect-n phenomenon, single point of failure, and signaling load caused by moving members at the core network. Simulation results shows that the scheme surpasses other existing efforts in terms of communication overhead and affected members. The security requirements studies also show the backward and forward secrecy is preserved in the proposed scheme even though the members move between areas

Transcriptomic alterations in the heart of non-obese type 2 diabetic Goto-Kakizaki rats

BACKGROUND: There is a spectacular rise in the global prevalence of type 2 diabetes mellitus (T2DM) due to the worldwide obesity epidemic. However, a significant proportion of T2DM patients are non-obese and they also have an increased risk of cardiovascular diseases. As the Goto-Kakizaki (GK) rat is a well-known model of non-obese T2DM, the goal of this study was to investigate the effect of non-obese T2DM on cardiac alterations of the transcriptome in GK rats. METHODS: Fasting blood glucose, serum insulin and cholesterol levels were measured at 7, 11, and 15 weeks of age in male GK and control rats. Oral glucose tolerance test and pancreatic insulin level measurements were performed at 11 weeks of age. At week 15, total RNA was isolated from the myocardium and assayed by rat oligonucleotide microarray for 41,012 genes, and then expression of selected genes was confirmed by qRT-PCR. Gene ontology and protein-protein network analyses were performed to demonstrate potentially characteristic gene alterations and key genes in non-obese T2DM. RESULTS: Fasting blood glucose, serum insulin and cholesterol levels were significantly increased, glucose tolerance and insulin sensitivity were significantly impaired in GK rats as compared to controls. In hearts of GK rats, 204 genes showed significant up-regulation and 303 genes showed down-regulation as compared to controls according to microarray analysis. Genes with significantly altered expression in the heart due to non-obese T2DM includes functional clusters of metabolism (e.g. Cyp2e1, Akr1b10), signal transduction (e.g. Dpp4, Stat3), receptors and ion channels (e.g. Sln, Chrng), membrane and structural proteins (e.g. Tnni1, Mylk2, Col8a1, Adam33), cell growth and differentiation (e.g. Gpc3, Jund), immune response (e.g. C3, C4a), and others (e.g. Lrp8, Msln, Klkc1, Epn3). Gene ontology analysis revealed several significantly enriched functional inter-relationships between genes influenced by non-obese T2DM. Protein-protein interaction analysis demonstrated that Stat is a potential key gene influenced by non-obese T2DM. CONCLUSIONS: Non-obese T2DM alters cardiac gene expression profile. The altered genes may be involved in the development of cardiac pathologies and could be potential therapeutic targets in non-obese T2DM

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

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .)