Estimates of Japanese Encephalitis mortality and morbidity: A systematic review and modeling analysis

BackgroundJapanese Encephalitis (JE) is known for its high case fatality ratio (CFR) and long-term neurological sequelae. Over the years, efforts in JE treatment and control might change the JE fatality risk. However, previous estimates were from 10 years ago, using data from cases in the 10 years before this. Estimating JE disease severity is challenging because data come from countries with different JE surveillance systems, diagnostic methods, and study designs. Without precise and timely JE disease severity estimates, there is continued uncertainty about the JE disease burden and the effect of JE vaccination.MethodologyWe performed a systematic review to collate age-stratified JE fatality and morbidity data. We used a stepwise model selection with BIC as the selection criteria to identify JE CFR drivers. We used stacked regression, to predict country-specific JE CFR from 1961 to 2030. JE morbidity estimates were grouped from similar study designs to estimate the proportion of JE survivors with long-term neurological sequelae.Principal findingsWe included 82 and 50 peer-reviewed journal articles published as of March 06 2021 for JE fatality and morbidity with 22 articles in both analyses. Results suggested overall JE CFR estimates of 26% (95% CI 22, 30) in 1961–1979, 20% (95% CI 17, 24) in 1980–1999, 14% (95% CI 11, 17) in 2000–2018, and 14% (95% CI 11, 17) in 2019–2030. Holding other variables constant, we found that JE fatality risk decreased over time (OR: 0.965; 95% CI: 0.947–0.983). Younger JE cases had a slightly higher JE fatality risk (OR: 1.012; 95% CI: 1.003–1.021). The odds of JE fatality in countries with JE vaccination is 0.802 (90% CI: 0.653–0.994; 95% CI: 0.62–1.033) times lower than the odds in countries without JE vaccination. Ten percentage increase in the percentage of rural population to the total population was associated with 15.35% (95% CI: 7.71, 22.57) decrease in JE fatality odds. Ten percentage increase in population growth rate is associated with 3.71% (90% CI: 0.23, 7.18; 95% CI: -0.4, 8.15) increase in JE fatality odds. Adjusting for the effect of year, rural population percent, age of JE cases, and population growth rate, we estimated that there was a higher odds of JE fatality in India compared to China. (OR: 5.46, 95% CI: 3.61–8.31). Using the prediction model we found that, in 2000–2018, Brunei, Pakistan, and Timor-Leste were predicted to have the highest JE CFR of 20%. Bangladesh, Guam, Pakistan, Philippines, and Vietnam had projected JE CFR over 20% for after 2018, whereas the projected JE CFRs were below 10% in China, Indonesia, Cambodia, Myanmar, Malaysia, and Thailand. For disability, we estimated that 36% (min-max 0–85) JE patients recovered fully at hospital discharge. One year after hospital discharge, 46% (min-max 0%-97%) JE survivors were estimated to live normally but 49% (min-max 3% - 86%)till had neurological sequelae.ConclusionJE CFR estimates were lower than 20% after 2000. Our study provides an updated estimation of CFR and proportion of JE cases with long-term neurological sequelae that could help to refine cost-benefit assessment for JE control and elimination programs.     

Phosphorylation of serine-15 of maize leaf sucrose synthase. Occurrence in vivo and possible regulatory significance.

Experiments were conducted to determine whether sucrose synthase (SuSy) was phosphorylated in the elongation zone of maize (Zea mays L.) leaves. The approximately 90-kD subunit of SuSy was 32P-labeled on seryl residue(s) when excised shoots were fed [32P]orthophosphate. Both isoforms of SuSy (the SS1 and SS2 proteins) were phosphorylated in vivo, and tryptic peptide-mapping analysis suggested a single, similar phosphorylation site in both proteins. A combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and automated Edman sequencing analysis unequivocally identified the phosphorylation site in the maize SS2 protein as serine-15. This site was phosphorylated in vitro by endogenous protein kinase(s) in a strictly Ca(2+)-dependent manner. A synthetic peptide, based on the phosphorylation site sequence, was used to identify and partially purify an endogenous Ca(2+)-dependent protein kinase(s) from the maize leaf elongation zone and expanding spinach leaves. Phosphorylation of SuSy in vitro selectively activates the cleavage reaction by increasing the apparent affinity of the enzyme for sucrose and UDP, suggesting that phosphorylation may be of regulatory significance. Conservation of the phosphorylation site, and the sequences surrounding it, among plant species suggests that phosphorylation of SuSy may be widespread, if not universal, in plants.     

Heterotrophy mitigates the response of the temperate coral Oculina arbuscula to temperature stress

Anthropogenic increases in atmospheric carbon dioxide concentration have caused global average sea surface temperature (SST) to increase by approximately 0.11°C per decade between 1971 and 2010 – a trend that is projected to continue through the 21st century. A multitude of research studies have demonstrated that increased SSTs compromise the coral holobiont (cnidarian host and its symbiotic algae) by reducing both host calcification and symbiont density, among other variables. However, we still do not fully understand the role of heterotrophy in the response of the coral holobiont to elevated temperature, particularly for temperate corals. Here, we conducted a pair of independent experiments to investigate the influence of heterotrophy on the response of the temperate scleractinian coral Oculina arbuscula to thermal stress. Colonies of O. arbuscula from Radio Island, North Carolina, were exposed to four feeding treatments (zero, low, moderate, and high concentrations of newly hatched Artemia sp. nauplii) across two independent temperature experiments (average annual SST (20°C) and average summer temperature (28°C) for the interval 2005–2012) to quantify the effects of heterotrophy on coral skeletal growth and symbiont density. Results suggest that heterotrophy mitigated both reduced skeletal growth and decreased symbiont density observed for unfed corals reared at 28°C. This study highlights the importance of heterotrophy in maintaining coral holobiont fitness under thermal stress and has important implications for the interpretation of coral response to climate change.     

Ultra-high resolution HLA genotyping and allele discovery by highly multiplexed cDNA amplicon pyrosequencing

ABSTRACT: BACKGROUND: High-resolution HLA genotyping is a critical diagnostic and research assay. Current methods rarely achieve unambiguous high-resolution typing without making population-specific frequency inferences due to a lack of locus coverage and difficulty in exon-phase matching. Achieving high-resolution typing is also becoming more challenging with traditional methods as the database of known HLA alleles increases. RESULTS: We designed a cDNA amplicon-based pyrosequencing method to capture 94% of the HLA class I open-reading-frame with only two amplicons per sample, and an analogous method for class II HLA genes, with a primary focus on sequencing the DRB loci. We present a novel Galaxy server-based analysis workflow for determining genotype. During assay validation, we performed two GS Junior sequencing runs to determine the accuracy of the HLA class I amplicons and DRB amplicon at different levels of multiplexing. When 116 amplicons were multiplexed, we unambiguously resolved 99%of class I alleles to four- or six-digit resolution, as well as 100% unambiguous DRB calls. The second experiment, with 271 multiplexed amplicons, missed some alleles, but generated high-resolution, concordant typing for 93% of class I alleles, and 96% for DRB1 alleles. In a third, preliminary experiment we attempted to sequence novel amplicons for other class II loci with mixed success. CONCLUSIONS: The presented assay is higher-throughput and higher-resolution than existing HLA genotyping methods, and suitable for allele discovery or large cohort sampling. The validated class I and DRB primers successfully generated unambiguously high-resolution genotypes, while further work is needed to validate additional class II genotyping amplicons.     

The metabolism of human mesenchymal stem cells during proliferation and differentiation

Human mesenchymal stem cells (MSCs) reside under hypoxic conditions in vivo, between 4% and 7% oxygen. Differentiation of MSCs under hypoxic conditions results in inhibited osteogenesis, while chondrogenesis is unaffected. The reasons for these results may be associated with the inherent metabolism of the cells. The present investigation measured the oxygen consumption, glucose consumption and lactate production of MSCs during proliferation and subsequent differentiation towards the osteogenic and chondrogenic lineages. MSCs expanded under normoxia had an oxygen consumption rate of ～98 fmol/cell/h, 75% of which was azide-sensitive, suggesting that these cells derive a significant proportion of ATP from oxidative phosphorylation in addition to glycolysis. By contrast, MSCs differentiated towards the chondrogenic lineage using pellet culture had significantly reduced oxygen consumption after 24 h in culture, falling to ～12 fmol/cell/h after 21 days, indicating a shift towards a predominantly glycolytic metabolism. By comparison, MSCs retained an oxygen consumption rate of ～98 fmol/cell/h over 21 days of osteogenic culture conditions, indicating that these cells had a more oxidative energy metabolism than the chondrogenic cultures. In conclusion, osteogenic and chondrogenic MSC cultures appear to adopt the balance of oxidative phosphorylation and glycolysis reported for the respective mature cell phenotypes. The addition of TGF-β to chondrogenic pellet cultures significantly enhanced glycosaminoglycan accumulation, but caused no significant effect on cellular oxygen consumption. Thus, the differences between the energy metabolism of chondrogenic and osteogenic cultures may be associated with the culture conditions and not necessarily their respective differentiation.     

Enzyme activities of aerobic lignocellulolytic bacteria isolated from wet tropical forest soils

Lignocellulolytic bacteria have promised to be a fruitful source of new enzymes for next-generation lignocellulosic biofuel production. Puerto Rican tropical forest soils were targeted because the resident microbes decompose biomass quickly and to near-completion. Isolates were initially screened based on growth on cellulose or lignin in minimal media. 75 Isolates were further tested for the following lignocellulolytic enzyme activities: phenol oxidase, peroxidase, β-d-glucosidase, cellobiohydrolase, β-xylopyranosidase, chitinase, CMCase, and xylanase. Cellulose-derived isolates possessed elevated β-d-glucosidase, CMCase, and cellobiohydrolase activity but depressed phenol oxidase and peroxidase activity, while the contrary was true of lignin isolates, suggesting that these bacteria are specialized to subsist on cellulose or lignin. Cellobiohydrolase and phenol oxidase activity rates could classify lignin and cellulose isolates with 61% accuracy, which demonstrates the utility of model degradation assays. Based on 16S rRNA gene sequencing, all isolates belonged to phyla dominant in the Puerto Rican soils, Proteobacteria, Firmicutes, and Actinobacteria, suggesting that many dominant taxa are capable of the rapid lignocellulose degradation characteristic of these soils. The isolated genera Aquitalea, Bacillus, Burkholderia, Cupriavidus, Gordonia, and Paenibacillus represent rarely or never before studied lignolytic or cellulolytic species and were undetected by metagenomic analysis of the soils. The study revealed a relationship between phylogeny and lignocellulose-degrading potential, supported by Kruskal–Wallis statistics which showed that enzyme activities of cultivated phyla and genera were different enough to be considered representatives of distinct populations. This can better inform future experiments and enzyme discovery efforts.     

Lipid composition and macromolecular crowding effects on CYP2J2‐mediated drug metabolism in nanodiscs

Lipid composition and macromolecular crowding are key external effectors of protein activity and stability whose role varies between different proteins. Therefore, it is imperative to study their effects on individual protein function. CYP2J2 is a membrane‐bound cytochrome P450 in the heart involved in the metabolism of fatty acids and xenobiotics. In order to facilitate this metabolism, cytochrome P450 reductase (CPR), transfers electrons to CYP2J2 from NADPH. Herein, we use nanodiscs to show that lipid composition of the membrane bilayer affects substrate metabolism of the CYP2J2‐CPR nanodisc (ND) system. Differential effects on both NADPH oxidation and substrate metabolism by CYP2J2‐CPR are dependent on the lipid composition. For instance, sphingomyelin containing nanodiscs produced more secondary substrate metabolites than discs of other lipid compositions, implying a possible conformational change leading to processive metabolism. Furthermore, we demonstrate that macromolecular crowding plays a role in the lipid‐solubilized CYP2J2‐CPR system by increasing the K_m and decreasing the V_max, and effect that is size‐dependent. Crowding also affects the CYP2J2‐CPR‐ND system by decreasing both the K_m and V_max for Dextran‐based macromolecular crowding agents, implying an increase in substrate affinity but a lack of metabolism. Finally, protein denaturation studies show that crowding agents destabilize CYP2J2, while the multidomain protein CPR is stabilized. Overall, these studies are the first report on the role of the surrounding lipid environment and macromolecular crowding in modulating enzymatic function of CYP2J2‐CPR membrane protein system.     

A digitonin-based procedure for the isolation of mitochondrial DNA from mammalian cells

A procedure for the isolation of closed circular mitochondrial DNA (mtDNA) from cells permeabilized with digitonin is described. Compared to the standard procedure in which cells are broken after osmotic swelling, the digitonin-based procedure is more consistent and results in higher mtDNA yields.