p38 MAPK activation and STIM1-Orai3 association mediate TRPC6 externalization.

Endothelial cell (EC) migration is critical for the repair of monolayer disruption following angioplasties, but migration is inhibited by lipid oxidation products, including lysophosphatidylcholine (lysoPC), which open canonical transient receptor potential 6 (TRPC6) channels. TRPC6 activation requires an increase in intracellular Ca2+ concentration ([Ca2+]i), the source of which is unknown. LysoPC can activate phospholipase A2 to release arachidonic acid (ArA). ArA can activate arachidonic acid-regulated calcium (ARC) channels that are formed by stromal interaction molecule 1 (STIM1) and Orai1 and Orai3 proteins. Both lysoPC and ArA can activate p38 mitogen-activated protein kinase (MAPK) that induces the phosphorylation required for STIM1-Orai3 association. This is accompanied by an increase in [Ca2+]i and TRPC6 externalization. The effect of lysoPC and ArA is not additive, suggesting activation of the same pathway. The increase in [Ca2+]i activates an Src kinase that leads to TRPC6 activation. Downregulation of Orai3 using siRNA blocks the lysoPC- or ArA-induced increase in [Ca2+]i and TRPC6 externalization and preserves EC migration. These data show that lysoPC induces activation of p38 MAPK, which leads to STIM1-Orai3 association and increased [Ca2+]i. This increase in [Ca2+]i activates an Src kinase leading to TRPC6 externalization, which initiates a cascade of events ending in cytoskeletal changes that disrupt EC migration. Blocking this pathway preserves EC migration in the presence of lipid oxidation products.NEW & NOTEWORTHY The major lysophospholipid component in oxidized LDL, lysophosphatidylcholine (lysoPC), can activate p38 MAP kinase, which in turn promotes externalization of Orai3 and STIM1-Orai3 association, suggesting involvement of arachidonic acid-regulated calcium (ARC) channels. The subsequent increase in intracellular calcium activates an Src kinase required for TRPC6 externalization. TRPC6 activation, which has been shown to inhibit endothelial cell migration, is blocked by p38 MAP kinase or Orai3 downregulation, and this partially preserves endothelial migration in lysoPC.

iPLA2 inhibition blocks LysoPC-induced TRPC6 externalization and promotes Re-endothelialization of carotid injuries in hypercholesterolemic mice.

Lipid oxidation products, including lysophosphatidylcholine (lysoPC), accumulate at the site of arterial injury after vascular interventions and hinder re-endothelization. LysoPC activates calcium-permeable channels, specifically canonical transient receptor potential 6 (TRPC6) channels that induce a sustained increase in intracellular calcium ion concentration [Ca2+]i and contribute to dysregulation of the endothelial cell (EC) cytoskeleton. Activation of TRPC6 leads to inhibition of EC migration in vitro and delayed re-endothelization of arterial injuries in vivo. Previously, we demonstrated the role of phospholipase A2 (PLA2), specifically calcium-independent PLA2 (iPLA2), in lysoPC-induced TRPC6 externalization and inhibition of EC migration in vitro. The ability of FKGK11, an iPLA2-specific pharmacological inhibitor, to block TRPC6 externalization and preserve EC migration was assessed in vitro and in a mouse model of carotid injury. Our data suggest that FKGK11 prevents lysoPC-induced PLA2 activity, blocks TRPC6 externalization, attenuates calcium influx, and partially preserves EC migration in vitro. Furthermore, FKGK11 promotes re-endothelization of an electrocautery carotid injury in hypercholesterolemic mice. FKGK11 has similar arterial healing effects in male and female mice on a high-fat diet. This study suggests that iPLA2 is a potential therapeutic target to attenuate calcium influx through TRPC6 channels and promote EC healing in cardiovascular patients undergoing angioplasty.

p85α regulatory subunit isoform controls PI3-kinase and TRPC6 membrane translocation.

Activation of phosphatidylinositol 3-kinase (PI3K) by lipid oxidation products, including lysophosphatidylcholine (lysoPC), increases the externalization of canonical transient receptor potential 6 (TRPC6) channels leading to a subsequent increase in intracellular calcium that contributes to cytoskeletal changes which inhibit endothelial cell (EC) migration in vitro and impair EC healing of arterial injuries in vivo. The PI3K p110α and p110δ catalytic subunit isoforms regulate lysoPC-induced TRPC6 externalization in vitro, but have many other functions. The goal of the current study is to identify the PI3K regulatory subunit isoform involved in TRPC6 externalization to potentially identify a more specific treatment regimen to improve EC migration and arterial healing, while minimizing off-target effects. Decreasing the p85α regulatory subunit isoform protein levels, but not the p85ß and p55γ regulatory subunit isoforms, with small interfering RNA inhibits lysoPC-induced translocation of the PI3K catalytic subunit to the plasma membrane, dramatically decreased phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production and TRPC6 externalization, and significantly improves EC migration in the presence of lysoPC. These results identify the important and specific role of p85α in controlling translocation of PI3K from the cytosol to the plasma membrane and PI3K-mediated TRPC externalization by oxidized lipids. Current PI3K inhibitors block the catalytic subunit, but our data suggest that the regulatory subunit is a novel therapeutic target to promote EC migration and healing after arterial injuries that occur with angioplasty.

Hypogastric artery luminal diameter predicts common-external iliac stent patency and major adverse limb events in patients with aortoiliac occlusive disease.

OBJECTIVE: Hypogastric coverage may be required for occlusive disease at the iliac arterial bifurcation. In this study, we sought to determine patency rates of common-external iliac artery (C-EIA) bare metal stents (BMS) spanning the hypogastric origin in patients with aortoiliac occlusive disease (AIOD). In addition, we sought to identify predictors of C-EIA BMS patency loss and major adverse limb events (MALE) in patients requiring hypogastric coverage. We hypothesized that worsening stenosis of the hypogastric origin would negatively influence C-EIA stent patency and freedom from MALE. METHODS: This is a single center, retrospective review of consecutive patients undergoing elective, endovascular treatment of aortoiliac disease (AIOD) between 2010 and 2018. Only patients with C-EIA BMS coverage of a patent IIA origin were included in the study. Hypogastric luminal diameter was determined from preoperative CT angiography. Analysis was performed using Kaplan-Meier survival analysis, univariable and multivariable logistic regression, and receiver operator characteristics (ROC). RESULTS: There were 236 patients (318 limbs) who were included in the study. AIOD was TASC C/D in 236/318 (74.2%) of cases. C-EIA stent primary patency was 86.5% (95% confidence interval: 81.1, 91.9) at 2 years and 79.7% (72.8, 86.7) at 4 years. Freedom from ipsilateral MALE was 77.0% (71.1, 82.9) at 2 years and 68.7% (61.3, 76.2) at 4 years. Luminal diameter of the hypogastric origin was most strongly associated with loss of C-EIA BMS primary patency in multivariable analysis (hazard ratio: 0.81, p = .02). Insulin-dependent diabetes, Rutherford's class IV or above, and stenosis of the hypogastric origin were significantly predictive of MALE in both univariable and multivariable analyses. In ROC analysis, luminal diameter of the hypogastric origin was superior to chance in prediction of C-EIA primary patency loss and MALE. Hypogastric diameter >4.5 mm had a negative predictive value of 0.94 for C-EIA primary patency loss and 0.83 for MALE. CONCLUSIONS: Patency rates of C-EIA BMS are high. Hypogastric luminal diameter is an important and potentially modifiable predictor of C-EIA BMS patency and MALE in patients with AIOD.

Nitrogen fixation and other biogeochemically important features of Atacama Desert giant horsetail plant microbiomes inferred from metagenomic contig analysis.

BACKGROUND AND AIMS: Canyon stream beds in the hyperarid Atacama Desert surprisingly harbour magnificent groves of endemic giant horsetail wetland plants, Equisetum xylochaetum. Our previous metagenomic study of eukaryotes closely associated with this plant indicated that the microbiome included prokaryotes that might likewise influence host success and environment. We explored this possibility by using the metagenomic sequence to characterize prokaryote taxa and functional genes present in the microbiome of E. xylochaetum sampled from remote sites differing in the degree of anthropogenic disturbance. We focused on biogeochemical functions known to be important in wetland ecosystems. METHODS: To ensure that analyses were conducted on microbes most closely associated with plants, we extracted DNA from well-washed plant organs whose microbial biofilms were revealed with scanning electron microscopy. To assess the benefits of longer sequences for taxonomic and gene classifications, results of analyses performed using contigs were compared with those obtained with unassembled reads. We employed methods widely used to estimate genomic coverage of single taxa for genomic analysis to infer relative abundances of taxa and functional genes. KEY RESULTS: Key functional bacterial genera (e.g. Hydrogenophaga, Sulfuritalea and Rhodoferax) inferred from taxonomic and functional gene analysis of contigs - but not unassembled reads - to occur on surfaces of (or within) plants at relatively high abundance (>50× genomic coverage) indicated roles in nitrogen, sulfur and other mineral cycling processes. Comparison between sites revealed impacts on biogeochemical functions, e.g. reduced levels of the nifH gene marker under disturbance. Vanadium nitrogenases were more important than molybdenum nitrogenases, indicated by both functional genes and taxa such as Rhodomicrobium and Phaeospirillum inferred from contigs but not unassembled reads. CONCLUSIONS: Our contig-based metagenomic analyses revealed that microbes performing key wetland biogeochemical functions occur as tightly adherent biofilms on the plant body, not just in water or sediments, and that disturbance reduces such functions, providing arguments for conservation efforts.

Microbiome and related structural features of Earth's most archaic plant indicate early plant symbiosis attributes.

Origin of earliest land plants from ancestral algae dramatically accelerated the evolution of Earth's terrestrial ecosystems, in which microbial symbioses have played key roles. Recent molecular diversification analyses identify the rare, geographically-limited moss Takakia as Earth's most archaic modern land plant. Despite occupying a phylogenetic position pivotal for understanding earliest plants, Takakia microbial associations are poorly known. Here, we describe symbiosis-related structural features and contig-based metagenomic data that illuminate the evolutionary transition from streptophyte algae to early embryophytes. We observed that T. lepidozioides shares with streptophyte algae secretion of microbe-harboring mucilage and bacterial taxa such as Rhizobium and genes indicating nitrogen fixation. We find that Takakia root-analogs produce lateral mucilage organs that are more complex than generally understood, having structural analogies to angiosperm lateral roots adapted for N-fixation symbioses, including presence of intracellular microbes. We also find structural and metagenomic evidence for mycorrhiza-like species of glomalean fungi (including Rhizophagus irregularis) not previously known for mosses, as well as ascomycete fungi (e.g. Rhizoscyphus ericae) that associate with other early-diverging plants. Because Takakia is the oldest known modern plant genus, this study of plants of a remote locale not strongly influenced by human activities may indicate microbiome features of early land plants.

Plastid Genome of Equisetum xylochaetum from the Atacama Desert, Chile and the Relationships of Equisetum Based on Frequently Used Plastid Genes and Network Analysis.

The modern pteridophyte genus Equisetum is the only survivor of Sphenopsida, an ancient clade known from the Devonian. This genus, of nearly worldwide distribution, comprises approximately 15 extant species. However, genomic information is limited. In this study, we assembled the complete chloroplast genome of the giant species Equisetum xylochaetum from a metagenomic sequence and compared the plastid genome structure and protein-coding regions with information available for two other Equisetum species using network analysis. Equisetum chloroplast genomes showed conserved traits of quadripartite structure, gene content, and gene order. Phylogenetic analysis based on plastome protein-coding regions corroborated previous reports that Equisetum is monophyletic, and that E. xylochaetum is more closely related to E. hyemale than to E. arvense. Single-gene phylogenetic estimation and haplotype analysis showed that E. xylochaetum belonged to the subgenus Hippochaete. Single-gene haplotype analysis revealed that E. arvense, E. hyemale, E. myriochaetum, and E. variegatum resolved more than one haplotype per species, suggesting the presence of a high diversity or a high mutation rate of the corresponding nucleotide sequence. Sequences from E. bogotense appeared as a distinct group of haplotypes representing the subgenus Paramochaete that diverged from Hippochaete and Equisetum. In addition, the taxa that were frequently located at the joint region of the map were E. scirpoides and E. pratense, suggesting the presence of some plastome characters among the Equiseum subgenera.

The pattern of apolipoprotein A-I lysine carbamylation reflects its lipidation state and the chemical environment within human atherosclerotic aorta.

Protein lysine carbamylation is an irreversible post-translational modification resulting in generation of homocitrulline (N-ε-carbamyllysine), which no longer possesses a charged ε-amino moiety. Two distinct pathways can promote protein carbamylation. One results from urea decomposition, forming an equilibrium mixture of cyanate (CNO-) and the reactive electrophile isocyanate. The second pathway involves myeloperoxidase (MPO)-catalyzed oxidation of thiocyanate (SCN-), yielding CNO- and isocyanate. Apolipoprotein A-I (apoA-I), the major protein constituent of high-density lipoprotein (HDL), is a known target for MPO-catalyzed modification in vivo, converting the cardioprotective lipoprotein into a proatherogenic and proapoptotic one. We hypothesized that monitoring site-specific carbamylation patterns of apoA-I recovered from human atherosclerotic aorta could provide insights into the chemical environment within the artery wall. To test this, we first mapped carbamyllysine obtained from in vitro carbamylation of apoA-I by both the urea-driven (nonenzymatic) and inflammatory-driven (enzymatic) pathways in lipid-poor and lipidated apoA-I (reconstituted HDL). Our results suggest that lysine residues within proximity of the known MPO-binding sites on HDL are preferentially targeted by the enzymatic (MPO) carbamylation pathway, whereas the nonenzymatic pathway leads to nearly uniform distribution of carbamylated lysine residues along the apoA-I polypeptide chain. Quantitative proteomic analyses of apoA-I from human aortic atheroma identified 16 of the 21 lysine residues as carbamylated and suggested that the majority of apoA-I carbamylation in vivo occurs on "lipid-poor" apoA-I forms via the nonenzymatic CNO- pathway. Monitoring patterns of apoA-I carbamylation recovered from arterial tissues can provide insights into both apoA-I structure and the chemical environment within human atheroma.

Does It Matter If Students (Dis)like School? Associations Between School Liking, Teacher and School Connectedness, and Exclusionary Discipline.

School liking is an important factor in student engagement, well-being, and academic achievement, but it is also potentially influenced by factors external to the individual, such as school culture, teacher support, and approaches to discipline. The present study employed a survey methodology to investigate the associations between school liking and disliking, teacher and school connectedness, and experiences of exclusionary discipline from the perspective of students themselves. Participants included 1,002 students (Grades 7-10) from three secondary schools serving disadvantaged communities. Results indicated clear differences between students who like and dislike school in terms of their preferred school activities and school disciplinary history, with students who disliked school experiencing overall lower school connectedness. Moreover, students who disliked school experienced less positive relationships with their teachers, and this was even more pronounced for students who had been previously suspended. The findings reveal key differences between students who do and do not like school, differences that may be masked by typical research approaches. This research indicates the need for more nuanced, student-informed approaches to inclusive school reform.

Spatial Variation of Cladophora Epiphytes in the Nan River, Thailand.

Cladophora is an algal genus known to be ecologically important. It provides habitats for microorganisms known to provide ecological services such as biosynthesis of cobalamin (vitamin B12) and nutrient cycling. Most knowledge of microbiomes was obtained from studies of lacustrine Cladophora species. However, whether lotic freshwater Cladophora microbiomes are as complex as the lentic ones or provide similar ecological services is not known. To illuminate these issues, we used amplicons of 16S rDNA, 18S rDNA, and ITS to investigate the taxonomy and diversity of the microorganisms associated with replicate Cladophora samples from three sites along the Nan River, Thailand. Results showed that the diversity of prokaryotic and eukaryotic members of Cladophora microbiomes collected from different sampling sites was statistically different. Fifty percent of the identifiable taxa were shared across sampling sites: these included organisms belonging to different trophic levels, decomposers, and heterotrophic bacteria. These heterogeneous assemblages of bacteria, by functional inference, have the potential to perform various ecological functions, i.e., cellulose degradation, cobalamin biosynthesis, fermentative hydrogen production, ammonium oxidation, amino acid fermentation, dissimilatory reduction of nitrate to ammonium, nitrite reduction, nitrate reduction, sulfur reduction, polyphosphate accumulation, denitrifying phosphorus-accumulation, and degradation of aromatic compounds. Results suggested that river populations of Cladophora provide ecologically important habitat for microorganisms that are key to nutrient cycling in lotic ecosystems.

Activation of the cytosolic calcium-independent phospholipase A2 ß isoform contributes to TRPC6 externalization via release of arachidonic acid.

During vascular interventions, oxidized low-density lipoprotein and lysophosphatidylcholine (lysoPC) accumulate at the site of arterial injury, inhibiting endothelial cell (EC) migration and arterial healing. LysoPC activates canonical transient receptor potential 6 (TRPC6) channels, leading to a prolonged increase in intracellular calcium ion concentration that inhibits EC migration. However, an initial increase in intracellular calcium ion concentration is required to activate TRPC6, and this mechanism remains elusive. We hypothesized that lysoPC activates the lipid-cleaving enzyme phospholipase A2 (PLA2), which releases arachidonic acid (AA) from the cellular membrane to open arachidonate-regulated calcium channels, allowing calcium influx that promotes externalization and activation of TRPC6 channels. The focus of this study was to identify the roles of calcium-dependent and/or calcium-independent PLA2 in lysoPC-induced TRPC6 externalization. We show that lysoPC induced PLA2 enzymatic activity and caused AA release in bovine aortic ECs. To identify the specific subgroup and the isoform(s) of PLA2 involved in lysoPC-induced TRPC6 activation, transient knockdown studies were performed in the human endothelial cell line EA.hy926 using siRNA to inhibit the expression of genes encoding cPLA2α, cPLA2γ, iPLA2ß, or iPLA2γ. Downregulation of the ß isoform of iPLA2 blocked lysoPC-induced release of AA from EC membranes and TRPC6 externalization, as well as preserved EC migration in the presence of lysoPC. We propose that blocking TRPC6 activation and promoting endothelial healing could improve the outcomes for patients undergoing cardiovascular interventions.

Fatal disseminated Anncaliia algerae myositis mimicking polymyositis in an immunocompromised patient.

We report the first New Zealand case of Anncaliia algerae myositis in a 55-year-old man with a history of psoriatic arthritis, treated with long-term immunosuppressive therapy. He resided in the city of Rotorua, which is famous for geothermal hot springs. A vastus lateralis muscle biopsy was performed to investigate the cause of an unexplained myositis. Light microscopy demonstrated a necrotizing myositis with scattered clusters of ovoid spores within the myocyte cytoplasm resembling microsporidia. DNA analysis by PCR and electron microscopy confirmed microsporidial myositis with features characteristic of A. algerae. Immunosuppressive drugs were stopped and the patient was treated with cholestyramine wash and albendazole. The patient deteriorated with involvement of bulbar and respiratory muscles requiring intensive care and ventilation. He died 3 weeks after diagnosis. Post-mortem examination of skeletal muscle from tongue and intercostal muscles also revealed numerous organisms confirming disseminated disease.

Collagen prolyl 4-hydroxylases modify tumor progression.

Collagen is the main component of the extracellular matrix. Hydroxylation of proline residues on collagen, catalyzed by collagen prolyl 4-hydroxylase (C-P4H), is essential for the stability of the collagen triple helix. Vertebrate C-P4H is an α2ß2 tetramer with three isoenzymes differing in the catalytic α-subunits, which are encoded by P4HA1, P4HA2, and P4HA3 genes. In contrast, ß-subunit is encoded by a single gene P4HB. The expressions of P4HAs and P4HB are regulated by multiple cellular factors, including cytokines, transcription factors, and microRNAs. P4HAs and P4HB are highly expressed in many tumors and participate in cancer progression. Several inhibitors of P4HAs and P4HB have been confirmed to have anti-tumor effects, suggesting that targeting C-P4H is a feasible strategy for cancer treatment. Here, we summarize recent progresses on the function and expression of regulatory mechanisms of C-P4H in cancer progression and point out the potential development of therapeutic strategies in targeting C-P4H in the future.

Inhibition of P110α and P110δ catalytic subunits of PI3 kinase reverses impaired arterial healing after injury in hypercholesterolemic male mice.

Endothelial cell (EC) migration is critical for healing arterial injuries, such as those that occur with angioplasty. Impaired re-endothelialization following arterial injury contributes to vessel thrombogenicity, intimal hyperplasia, and restenosis. Oxidized lipid products, including lysophosphatidylcholine (lysoPC), induce canonical transient receptor potential 6 (TRPC6) externalization leading to increased [Ca2+]i, activation of calpains, and alterations of the EC cytoskeletal structure that inhibit migration. The p110α and p110δ catalytic subunit isoforms of phosphatidylinositol 3-kinase (PI3K) regulate lysoPC-induced TRPC6 externalization in vitro. The goal of this study was to assess the in vivo relevance of those in vitro findings to arterial healing following a denuding injury in hypercholesterolemic mice treated with pharmacologic inhibitors of the p110α and p110δ isoforms of PI3K and a general PI3K inhibitor. Pharmacologic inhibition of the p110α or the p110δ isoform of PI3K partially preserves healing in hypercholesterolemic male mice, similar to a general PI3K inhibitor. Interestingly, the p110α, p110δ, and the general PI3K inhibitor do not improve arterial healing after injury in hypercholesterolemic female mice. These results indicate a potential new role for isoform-specific PI3K inhibitors in male patients following arterial injury/intervention. The results also identify significant sex differences in the response to PI3K inhibition in the cardiovascular system, where female sex generally has a cardioprotective effect. This study provides a foundation to investigate the mechanism for the sex differences in response to PI3K inhibition to develop a more generally applicable treatment option.

P110α and P110δ catalytic subunits of PI3 kinase regulate lysophosphatidylcholine-induced TRPC6 externalization.

Lipid oxidation products, including lysophosphatidylcholine (lysoPC) inhibit endothelial cell (EC) migration in vitro and impair EC healing of arterial injuries in vivo, in part by activating phosphatidylinositol 3-kinase (PI3K), which increases the externalization of canonical transient receptor potential 6 (TRPC6) channels and the subsequent increase in intracellular calcium. Inhibition of PI3K is a potential method to decrease TRPC6 activation and restore migration, but PI3K is involved in multiple intracellular signaling pathways and has multiple downstream effectors. The goal of this study is to identify the specific p110 catalytic subunit isoforms responsible for lysoPC-induced TRPC6 externalization to identify a target for intervention while minimizing impact on alternative signaling pathways. Down-regulation of the p110α and p110δ isoforms, but not the p110ß or p110γ isoforms, with small interfering RNA significantly decreased phosphatidylinositol (3,4,5)-trisphosphate production and TRPC6 externalization, and significantly improved EC migration in the presence of lysoPC. These results identify an additional role of p110α in EC and reveal for the first time a specific role of p110δ in EC, providing a foundation for subsequent in vivo studies to investigate the impact of p110 isoform inhibition on arterial healing after injury.

Shotgun metagenomics and microscopy indicate diverse cyanophytes, other bacteria, and microeukaryotes in the epimicrobiota of a northern Chilean wetland Nostoc (Cyanobacteria).

Prokaryotic Nostoc, one of the world's most conspicuous and widespread algal genera (similar to eukaryotic algae, plants, and animals) is known to support a microbiome that influences host ecological roles. Past taxonomic characterizations of surface microbiota (epimicrobiota) of free-living Nostoc sampled from freshwater systems employed 16S rRNA genes, typically amplicons. We compared taxa identified from 16S, 18S, 23S, and 28S rRNA gene sequences filtered from shotgun metagenomic sequence and used microscopy to illuminate epimicrobiota diversity for Nostoc sampled from a wetland in the northern Chilean Altiplano. Phylogenetic analysis and rRNA gene sequence abundance estimates indicated that the host was related to Nostoc punctiforme PCC 73102. Epimicrobiota were inferred to include 18 epicyanobacterial genera or uncultured taxa, six epieukaryotic algal genera, and 66 anoxygenic bacterial genera, all having average genomic coverage ≥90X. The epicyanobacteria Geitlerinemia, Oscillatoria, Phormidium, and an uncultured taxon were detected only by 16S rRNA gene; Gloeobacter and Pseudanabaena were detected using 16S and 23S; and Phormididesmis, Neosynechococcus, Symphothece, Aphanizomenon, Nodularia, Spirulina, Nodosilinea, Synechococcus, Cyanobium, and Anabaena (the latter corroborated by microscopy), plus two uncultured cyanobacterial taxa (JSC12, O77) were detected only by 23S rRNA gene sequences. Three chlamydomonad and two heterotrophic stramenopiles genera were inferred from 18S; the streptophyte green alga Chaetosphaeridium globosum was detected by microscopy and 28S rRNA genes, but not 18S rRNA genes. Overall, >60% of epimicrobial taxa were detected by markers other than 16S rRNA genes. Some algal taxa observed microscopically were not detected from sequence data. Results indicate that multiple taxonomic markers derived from metagenomic sequence data and microscopy increase epimicrobiota detection.

Superoxide-induced Type I collagen secretion depends on prolyl 4-hydroxylases.

Reactive oxygen species (ROS) including superoxide (O2•-) play an important role in a variety of diseases, including Alzheimer's Disease, cancer, and atherosclerosis. Early reports showed that O2•- is a stimulant for collagen synthesis. However, the mechanism remains incompletely understood. Here we showed that LY83583 (6-anilinoquinoline-5,8-quinone), a substance known to induce O2•- production by smooth muscle cell (SMC), increases Type I collagen secretion. This effect could be blocked by treating the cells with Tiron, a scavenger for O2•-. LY83583-induced Type I collagen secretion required P4HA1 and P4HA2. Knockout of either P4ha1 or P4ha2 greatly reduced LY83583-stimulated Type I collagen maturation whereas silencing of both P4ha1 and P4ha2 completely blocked LY83583-induced Type I collagen maturation. Although significantly more hydroxyproline on purified Type I collagen was detected from LY83583 treated mouse embryonic fibroblast (MEF) cells by mass spectrometry, the level of prolyl 4-hydroxylases was not altered. Thus, LY83583 might increase the enzymatic activity of prolyl 4-hydroxylases to increase Type I collagen maturation. In addition, we found that LY83583 activated prolyl 4-hydrolases differed from ascorbate-activated prolyl 4-hydroxylase in two aspects: (1) LY83583 activated both P4HA1 and P4HA2 involved in collagen maturation whereas ascorbate mainly stimulated P4HA1 in collagen maturation; (2) LY83583 did not induce N259 glycosylation on P4HA1 as ascorbate did. The mechanisms remain to be investigated.

Consumer Preference and Quality of Sachet Water Sold and Consumed in the Sunyani Municipality of Ghana.

Good quality water is odourless, colourless, and free from faecal pollution, and a satisfactory safe supply must be made available to consumers. The study assessed consumer preference and quality of sachet water sold and consumed in the Sunyani Municipality of Ghana. A cross-sectional study design was used, and data were collected using a structured questionnaire from a sample size of 500 respondents. A total of twenty (20) samples of sachet water commonly sold and consumed in the Sunyani Municipality were also collected and analyzed for bacteriological and physicochemical parameters. Out of these 20 samples, 10 samples of sachet water were collected from the production site and the other 10 samples from the market site. Results showed that respondents' choice of sachet water was based on several indicators. While 70% (350/500) of consumers indicated that sachet water had taste, 58% (290/500) of them were not enthused when the water had colour. Using smell as an indicator, 71% of consumers have had an experience with sachet water smell being questionable. Water taste (p < 0.001), colour of water (p < 0.001), smell (p < 0.001), and increase in cost (p < 0.001) were found as determinants of consumer preference. Consumers who said the colour of water informed their decision when buying sachet water were seven times more likely to have a preference for a particular brand (OR 7.057, CI: 4.474-11.133). Those who checked for expiry dates when buying water (OR 4.871, CI: 3.110-7.628) and those who looked out for Food and Drugs Authority stamp (OR 4.433, CI: 2.806-7.003) were both four times more likely to have a preference for a particular brand. Water quality tests also indicated that 30% of all water samples collected from the production site were contaminated with total coliforms and 50% of sachet water samples collected from the markets were also contaminated with coliforms. The same brands of sachet water that contained total coliforms from the production site were the same brands that contained total coliforms selected from the market. From this study, only total coliforms other than Escherichia coli were detected in the water samples analyzed. It was observed that the evaluated physicochemical parameters of the water analyzed were within the accepted WHO limits. It is recommended that consumers be educated about the indicators to look out for when buying sachet water and, also, that regulatory bodies be empowered to ban the sale of unwholesome brands of sachet water on the market.

Temperate-zone cultivation of Oedogonium in municipal wastewater effluent to produce cellulose and oxygen.

Cultivation of the filamentous chlorophyte Oedogonium in municipal wastewater effluent is known to improve water quality and yield lipid- and protein-rich biomass for industrial applications. Chlorophyte celluloses, whose molecular organization and physical traits differ from those of plants, represent yet another valuable extractive, and algal oxygen production is of economic value in wastewater treatment. Consequently, we explored cellulose and oxygen production from Oedogonium biomass batch-cultivated in treated secondary municipal wastewater effluent. We compared biomass, cellulose, and oxygen production outside and within an adjacent greenhouse, under differing dissolved CO2 and pH conditions, and during temperate-zone seasonal change from summer through fall. Overall production did not differ within or outside the greenhouse, but outside production was higher in summer and lower in fall as air temperatures declined. Batch cultivation offered advantages, but high levels of mixing and CO2 were essential to maintain neutral pH for optimal algal growth and oxygen production.