Displacement and performance of abutments in narrow-diameter implants with different internal connections.

STATEMENT OF PROBLEM: Displacement of abutments into conical connection implants during screw tightening may also occur during functional loading, creating unsettling forces that may cause loss of preload. A recent conical-hexagon connection with double friction fit (conical-hexagon connection) could prevent this axial displacement. PURPOSE: The purpose of this in vitro study was to measure the 3D axial displacement of abutments with a conical-hexagon connection or conical connection in narrow-diameter implants. Removal torque values (RTVs), preload efficiency, and survival after cyclic loading were also compared. MATERIAL AND METHODS: Narrow-diameter implants with a conical connection (Osseospeed EV, 3.0×13 mm-AST) and narrow-diameter implants with a conical-hexagon connection (Eztetic, 3.1×13 mm) were embedded in resin rods (G10) (n=6). Six titanium abutments per system were used, and their spatial relationship to the implant platforms after hand tightening was determined by using 3D digital image correlation. The abutments were tightened to the manufacturers' specified values, and the abutments' relative position was recorded again. The displacement of the abutment after tightening was calculated. The implants were subjected to cyclic loading (5×106 cycles at 2 Hz) under 200-N loads at a 30-degree angle. After cyclic loading, the RTVs of screws were measured and compared with those specified by the manufacturers to calculate preload efficiency. ANOVA was used to compare the differences in displacements after tightening and to compare differences in RTVs after cyclic loading across the groups (α=.05). RESULTS: The mean displacement in the U direction (X-axis) for the AST was -0.7 µm and -4.7 µm for ZIM, with no statistical difference (P=.73). The mean displacement in the V direction (Y-axis) for AST was -37.0 µm, and -150.0 µm for ZIM, with significant statistical difference (P<.001). The mean displacement in the W direction (Z-axis) for AST was -0.9 µm, and -23.0 µm for ZIM, with no statistical difference (P=.35). The survival of groups was similar (P=.058). During cyclic loading, 3 AST specimens fractured. After cyclic loading, mean RTV for AST was -8.77 Ncm, and -14.24 Ncm for ZIM, and these values were significantly different (P=.04). Preload efficiency was 28.1% for AST and 41.5% for ZIM. CONCLUSIONS: Greater abutment displacements were observed with the conical-hexagon connection, which required a higher torque, as specified by its manufacturer. The abutments displaced more in the V-axis in both implants. Only the conical connection implant (Ti Grade 4, commercially pure) had failures during cyclic loading, but the survival of the implants was similar. After cyclic loading, the abutment screws in both systems lost some of their torque value. The abutment screws of the conical-hexagon connection implant maintained preload more efficiently during cyclic loading than those of the conical connection implant.

Responding to Worldview Threats in the Classroom: An Exploratory Study of Preservice Teachers.

This study used two training sessions and two focus groups with 17 preservice teachers (aged 20-36) completing their first teaching practicum placement during their Bachelor of Education program at an urban research university in western Canada. The aim was to implement ideas from terror management theory (TMT) during their teaching practicum. Participants explored how to facilitate contentious issues so as to prevent defensive reactions when worldviews clash in the classroom. A dramaturgical analysis identified participant objectives, conflicts, tactics, attitudes, emotions, and subtexts as they explored how to anticipate and avoid worldview and self-esteem threat, navigate tense pedagogical spaces, build capacity for expressing uncomfortable emotions, and diffuse threat with humor. Because difficult emotions are central to teaching potentially polarizing content, participating preservice teachers explored when compensatory reactions might emerge and, as a result, developed their own emotional awareness-TMT became both an experience and a teachable theory.

Integrating GWAS and Transcriptomics to Identify the Molecular Underpinnings of Thermal Stress Responses in Drosophila melanogaster.

Thermal tolerance of an organism depends on both the ability to dynamically adjust to a thermal stress and preparatory developmental processes that enhance thermal resistance. However, the extent to which standing genetic variation in thermal tolerance alleles influence dynamic stress responses vs. preparatory processes is unknown. Here, using the model species Drosophila melanogaster, we used a combination of Genome Wide Association mapping (GWAS) and transcriptomic profiling to characterize whether genes associated with thermal tolerance are primarily involved in dynamic stress responses or preparatory processes that influence physiological condition at the time of thermal stress. To test our hypotheses, we measured the critical thermal minimum (CTmin) and critical thermal maximum (CTmax) of 100 lines of the Drosophila Genetic Reference Panel (DGRP) and used GWAS to identify loci that explain variation in thermal limits. We observed greater variation in lower thermal limits, with CTmin ranging from 1.81 to 8.60°C, while CTmax ranged from 38.74 to 40.64°C. We identified 151 and 99 distinct genes associated with CTmin and CTmax, respectively, and there was strong support that these genes are involved in both dynamic responses to thermal stress and preparatory processes that increase thermal resistance. Many of the genes identified by GWAS were involved in the direct transcriptional response to thermal stress (72/151 for cold; 59/99 for heat), and overall GWAS candidates were more likely to be differentially expressed than other genes. Further, several GWAS candidates were regulatory genes that may participate in the regulation of stress responses, and gene ontologies related to development and morphogenesis were enriched, suggesting many of these genes influence thermal tolerance through effects on development and physiological status. Overall, our results suggest that thermal tolerance alleles can influence both dynamic plastic responses to thermal stress and preparatory processes that improve thermal resistance. These results also have utility for directly comparing GWAS and transcriptomic approaches for identifying candidate genes associated with thermal tolerance.

Unravelling the Food-Health Nexus to Build Healthier Food Systems.

The urgent call to transform global food systems is well founded on the need to reduce the effects of food systems on human health, environment, peoples' rights, and creation of a just society. Unhealthy diets contribute significantly to the global disease burden and pose huge risks to morbidity and mortality. Efforts to transform diets are highly dependent on transformation of the food system. All countries are now affected by the various forms of malnutrition - undernutrition, overweight and obesity, micronutrient deficiencies - with progress often too slow and in some cases going into reverse. Concomitantly, the number of food insecure is increasing, and the prevalence of non-communicable disease is high. IPES-Food, in collaboration with the Global Alliance for the Future of Food, undertook a review of the scientific evidence covering a whole range of global health impacts associated with food systems. The review examined how food and farming systems affect human health, explored why the negative impacts are systematically reproduced and why we fail to prioritize them politically, and how we can build healthier food systems for all. Five categories of health impacts were examined: (i) occupational hazards; (ii) environmental contamination; (iii) contaminated, unsafe, and altered foods; (iv) unhealthy dietary patterns, and (v) food insecurity. The study confirmed that food systems affect health through multiple, interconnected pathways, generating severe human and economic costs. It also highlighted how prevailing power relations in the food system help to shape and sometimes obscure our understanding of the impacts. Five leverage points for building healthier food systems are recommended: (i) promotion of food systems thinking; (ii) reasserting scientific integrity and research as a public good; (iii) bringing the alternatives to light; (iv) adopting the precautionary principle, and (v) building integrated food policies under participatory governance.

Pseudomonas aeruginosa Ethanol Oxidation by AdhA in Low-Oxygen Environments.

Pseudomonas aeruginosa has a broad metabolic repertoire that facilitates its coexistence with different microbes. Many microbes secrete products that P. aeruginosa can then catabolize, including ethanol, a common fermentation product. Here, we show that under oxygen-limiting conditions P. aeruginosa utilizes AdhA, an NAD-linked alcohol dehydrogenase, as a previously undescribed means for ethanol catabolism. In a rich medium containing ethanol, AdhA, but not the previously described PQQ-linked alcohol dehydrogenase, ExaA, oxidizes ethanol and leads to the accumulation of acetate in culture supernatants. AdhA-dependent acetate accumulation and the accompanying decrease in pH promote P. aeruginosa survival in LB-grown stationary-phase cultures. The transcription of adhA is elevated by hypoxia and under anoxic conditions, and we show that it is regulated by the Anr transcription factor. We have shown that lasR mutants, which lack an important quorum sensing regulator, have higher levels of Anr-regulated transcripts under low-oxygen conditions than their wild-type counterparts. Here, we show that a lasR mutant, when grown with ethanol, has an even larger decrease in pH than the wild type (WT) that is dependent on both anr and adhA The large increase in AdhA activity is similar to that of a strain expressing a hyperactive Anr-D149A variant. Ethanol catabolism in P. aeruginosa by AdhA supports growth on ethanol as a sole carbon source and electron donor in oxygen-limited settings and in cells growing by denitrification under anoxic conditions. This is the first demonstration of a physiological role for AdhA in ethanol oxidation in P. aeruginosaIMPORTANCE Ethanol is a common product of microbial fermentation, and the Pseudomonas aeruginosa response to and utilization of ethanol are relevant to our understanding of its role in microbial communities. Here, we report that the putative alcohol dehydrogenase AdhA is responsible for ethanol catabolism and acetate accumulation under low-oxygen conditions and that it is regulated by Anr.

Profiling of Bacterial and Fungal Microbial Communities in Cystic Fibrosis Sputum Using RNA.

Here, we report an approach to detect diverse bacterial and fungal taxa in complex samples by direct analysis of community RNA in one step using NanoString probe sets. We designed rRNA-targeting probe sets to detect 42 bacterial and fungal genera or species common in cystic fibrosis (CF) sputum and demonstrated the taxon specificity of these probes, as well as a linear response over more than 3 logs of input RNA. Culture-based analyses correlated qualitatively with relative abundance data on bacterial and fungal taxa obtained by NanoString, and the analysis of serial samples demonstrated the use of this method to simultaneously detect bacteria and fungi and to detect microbes at low abundance without an amplification step. Compared at the genus level, the relative abundances of bacterial taxa detected by analysis of RNA correlated with the relative abundances of the same taxa as measured by sequencing of the V4V5 region of the 16S rRNA gene amplified from community DNA from the same sample. We propose that this method may complement other methods designed to understand dynamic microbial communities, may provide information on bacteria and fungi in the same sample with a single assay, and with further development, may provide quick and easily interpreted diagnostic information on diverse bacteria and fungi at the genus or species level.IMPORTANCE Here we demonstrate the use of an RNA-based analysis of specific taxa of interest, including bacteria and fungi, within microbial communities. This multiplex method may be useful as a means to identify samples with specific combinations of taxa and to gain information on how specific populations vary over time and space or in response to perturbation. A rapid means to measure bacterial and fungal populations may aid in the study of host response to changes in microbial communities.

Identification of the RNA recognition element of the RBPMS family of RNA-binding proteins and their transcriptome-wide mRNA targets.

Recent studies implicated the RNA-binding protein with multiple splicing (RBPMS) family of proteins in oocyte, retinal ganglion cell, heart, and gastrointestinal smooth muscle development. These RNA-binding proteins contain a single RNA recognition motif (RRM), and their targets and molecular function have not yet been identified. We defined transcriptome-wide RNA targets using photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) in HEK293 cells, revealing exonic mature and intronic pre-mRNA binding sites, in agreement with the nuclear and cytoplasmic localization of the proteins. Computational and biochemical approaches defined the RNA recognition element (RRE) as a tandem CAC trinucleotide motif separated by a variable spacer region. Similar to other mRNA-binding proteins, RBPMS family of proteins relocalized to cytoplasmic stress granules under oxidative stress conditions suggestive of a support function for mRNA localization in large and/or multinucleated cells where it is preferentially expressed.

Global target mRNA specification and regulation by the RNA-binding protein ZFP36.

BACKGROUND: ZFP36, also known as tristetraprolin or TTP, and ELAVL1, also known as HuR, are two disease-relevant RNA-binding proteins (RBPs) that both interact with AU-rich sequences but have antagonistic roles. While ELAVL1 binding has been profiled in several studies, the precise in vivo binding specificity of ZFP36 has not been investigated on a global scale. We determined ZFP36 binding preferences using cross-linking and immunoprecipitation in human embryonic kidney cells, and examined the combinatorial regulation of AU-rich elements by ZFP36 and ELAVL1. RESULTS: Targets bound and negatively regulated by ZFP36 include transcripts encoding proteins necessary for immune function and cancer, and transcripts encoding other RBPs. Using partial correlation analysis, we were able to quantify the association between ZFP36 binding sites and differential target RNA abundance upon ZFP36 overexpression independent of effects from confounding features. Genes with increased mRNA half-lives in ZFP36 knockout versus wild-type mouse cells were significantly enriched for our human ZFP36 targets. We identified thousands of overlapping ZFP36 and ELAVL1 binding sites, in 1,313 genes, and found that ZFP36 degrades transcripts through specific AU-rich sequences, representing a subset of the U-rich sequences ELAVL1 interacts with to stabilize transcripts. CONCLUSIONS: ZFP36-RNA target specificities in vivo are quantitatively similar to previously reported in vitro binding affinities. ZFP36 and ELAVL1 bind an overlapping spectrum of RNA sequences, yet with differential relative preferences that dictate combinatorial regulatory potential. Our findings and methodology delineate an approach to unravel in vivo combinatorial regulation by RNA-binding proteins.

Control of Candida albicans metabolism and biofilm formation by Pseudomonas aeruginosa phenazines.

Candida albicans has developmental programs that govern transitions between yeast and filamentous morphologies and between unattached and biofilm lifestyles. Here, we report that filamentation, intercellular adherence, and biofilm development were inhibited during interactions between Candida albicans and Pseudomonas aeruginosa through the action of P. aeruginosa-produced phenazines. While phenazines are toxic to C. albicans at millimolar concentrations, we found that lower concentrations of any of three different phenazines (pyocyanin, phenazine methosulfate, and phenazine-1-carboxylate) allowed growth but affected the development of C. albicans wrinkled colony biofilms and inhibited the fungal yeast-to-filament transition. Phenazines impaired C. albicans growth on nonfermentable carbon sources and led to increased production of fermentation products (ethanol, glycerol, and acetate) in glucose-containing medium, leading us to propose that phenazines specifically inhibited respiration. Methylene blue, another inhibitor of respiration, also prevented the formation of structured colony biofilms. The inhibition of filamentation and colony wrinkling was not solely due to lowered extracellular pH induced by fermentation. Compared to smooth, unstructured colonies, wrinkled colony biofilms had higher oxygen concentrations within the colony, and wrinkled regions of these colonies had higher levels of respiration. Together, our data suggest that the structure of the fungal biofilm promotes access to oxygen and enhances respiratory metabolism and that the perturbation of respiration by bacterial molecules such as phenazines or compounds with similar activities disrupts these pathways. These findings may suggest new ways to limit fungal biofilms in the context of disease. IMPORTANCE Many of the infections caused by Candida albicans, a major human opportunistic fungal pathogen, involve both morphological transitions and the formation of surface-associated biofilms. Through the study of C. albicans interactions with the bacterium Pseudomonas aeruginosa, which often coinfects with C. albicans, we have found that P. aeruginosa-produced phenazines modulate C. albicans metabolism and, through these metabolic effects, impact cellular morphology, cell-cell interactions, and biofilm formation. We suggest that the structure of C. albicans biofilms promotes access to oxygen and enhances respiratory metabolism and that the perturbation of respiration by phenazines inhibits biofilm development. Our findings not only provide insight into interactions between these species but also provide valuable insights into novel pathways that could lead to the development of new therapies to treat C. albicans infections.

Antifungal mechanisms by which a novel Pseudomonas aeruginosa phenazine toxin kills Candida albicans in biofilms.

Pseudomonas aeruginosa produces several phenazines including the recently described 5-methyl-phenazine-1-carboxylic acid (5MPCA), which exhibits a novel antibiotic activity towards pathogenic fungi such as Candida albicans. Here we characterize the unique antifungal mechanisms of 5MPCA using its analogue phenazine methosulphate (PMS). Like 5MPCA, PMS induced fungal red pigmentation and killing. Mass spectrometry analyses demonstrated that PMS can be covalently modified by amino acids, a process that yields red derivatives. Furthermore, soluble proteins from C. albicans grown with either PMS or P. aeruginosa were also red and demonstrated absorbance and fluorescence spectra similar to that of PMS covalently linked to either amino acids or proteins in vitro, suggesting that 5MPCA modification by protein amine groups occurs in vivo. The red-pigmented C. albicans soluble proteins were reduced by NADH and spontaneously oxidized by oxygen, a reaction that likely generates reactive oxygen species (ROS). Additional evidence indicated that ROS generation precedes 5MPCA-induced fungal death. Reducing conditions greatly enhanced PMS uptake by C. albicans and killing. Since 5MPCA was more toxic than other phenazines that are not modified, such as pyocyanin, we propose that the covalent binding of 5MPCA promotes its accumulation in target cells and contributes to its antifungal activity in mixed-species biofilms.

Effect of an oral iron chelator or iron-deficient diets on uroporphyria in a murine model of porphyria cutanea tarda.

UNLABELLED: Porphyria cutanea tarda is a liver disease characterized by elevated hepatic iron and excessive production of uroporphyrin (URO). Phlebotomy is an effective treatment that probably acts by reducing hepatic iron. Here we used Hfe(-/-) mice to compare the effects on hepatic URO accumulation of two different methods of hepatic iron depletion: iron chelation using deferiprone (L1) versus iron-deficient diets. Hfe(-/-) mice in a 129S6/SvEvTac background were fed 5-aminolevulinic acid (ALA), which results in hepatic URO accumulation, and increasing doses of L1 in the drinking water. Hepatic URO accumulation was completely prevented at low L1 doses, which partially depleted hepatic nonheme iron. By histological assessment, the decrease in hepatic URO accumulation was associated with greater depletion of iron from hepatocytes than from Kupffer cells. The L1 treatment had no effect on levels of hepatic cytochrome P4501A2 (CYP1A2). L1 also effectively decreased hepatic URO accumulation in C57BL/6 Hfe(-/-) mice treated with ALA and a CYP1A2 inducer. ALA-treated mice maintained on defined iron-deficient diets, rather than chow diets, did not develop uroporphyria, even when the animals were iron-supplemented either directly in the diet or by iron dextran injection. CONCLUSION: The results suggest that dietary factors other than iron are involved in the development of uroporphyria and that a modest depletion of hepatocyte iron by L1 is sufficient to prevent URO accumulation.

Effect of iron and ascorbate on uroporphyria in ascorbate-requiring mice as a model for porphyria cutanea tarda.

UNLABELLED: Excess hepatic iron is known to enhance both porphyria cutanea tarda (PCT) and experimental uroporphyria. Since previous studies have suggested a role for ascorbate (AA) in suppressing uroporphyria in AA-requiring rats (in the absence of excess iron), the present study investigated whether AA could suppress uroporphyria produced by excess hepatic iron. Hepatic URO accumulation was produced in AA-requiring Gulo(-/-) mice by treatment with 3,3',4,4',5-pentachlorbiphenyl, an inducer of CYP1A2, and 5-aminolevulinic acid. Mice were administered either sufficient AA (1000 ppm) in the drinking water to maintain near normal hepatic AA levels or a lower intake (75 ppm) that resulted in 70 % lower hepatic AA levels. The higher AA intake suppressed hepatic URO accumulation in the absence of administered iron, but not when iron dextran (300-500 mg Fe/kg) was administered. This effect of iron was not due to hepatic AA depletion since hepatic AA content was not decreased. The effect of iron to prevent AA suppression of hepatic URO accumulation was not observed until a high hepatic iron threshold was exceeded. At both low and high AA intakes, hepatic malondialdehyde (MDA), an indicator of oxidative stress, was increased three-fold by high doses of iron dextran. MDA was considerably increased even at low iron dextran doses, but without any increase in URO accumulation. The level of hepatic CYP1A2 was unaffected by either AA intake. CONCLUSION: In this mouse model of PCT, AA suppresses hepatic URO accumulation at low, but not high hepatic iron levels. These results may have implications for the management of PCT.

Effect of insulin and glucagon on accumulation of uroporphyrin and coproporphyrin from 5-aminolevulinate in hepatocyte cultures.

Primary cultures of chick embryo hepatocytes have been used to study the mechanisms by which various drugs and other chemicals cause accumulation of porphyrin intermediates of the heme pathway. When these cultures are incubated with the heme precursor, 5-aminolevulinic acid (ALA), there is a major accumulation of protoporphyrin. However, in the presence of ALA, addition of insulin caused a striking increase in accumulation of uroporphyrin I and coproporphyrin III, whereas addition of glucagon mainly caused an increase in uroporphyrin I. Treatment with both insulin and glucagon resulted in additive increases in uroporphyrin, but not coproporphyrin. Antioxidants abolished the uroporphyrin I accumulation and increased coproporphyrin III. Insulin caused an increase in uptake of ALA and an increase in porphobilinogen accumulation, suggesting that the accumulation of uroporphyrin I is due to increased flux through the heme pathway. Apparently, this increased flux could particularly affect the utilization of the intermediate hydroxymethylbilane, which would result in accumulation of uroporphyrin I.

Genetic factors influence ethanol-induced uroporphyria in Hfe(-/-) mice.

Two major risk factors for porphyria cutanea tarda (PCT) are alcohol consumption and homozygosity for the C282Y mutation in the hereditary hemochromatosis gene (HFE). We recently described an animal model for alcohol-induced uroporphyria, using Hfe(-/-) mice. In the present study we show that this effect is dependent on genetic background and ethanol dose. In the 129S6/SvEvTac (129) strain, treatment with 15% ethanol in the drinking water for 6.5 months produced an accumulation of hepatic uroporphyrin (URO) 4-fold higher than that observed with 10% ethanol, a 90% decrease in uroporphyrinogen decarboxylase activity (UROD), and further increased the activities of hepatic 5-aminolevulinate synthase (ALAS) and CYP1A2. Hepatic nonheme iron (NHFe) and hepatocyte iron staining were not further increased by 15% compared to 10% ethanol. Treatment of C57BL/6 Hfe(-/-) mice with 15% ethanol for 6.5 months did not increase hepatic URO. Although NHFe was increased by ethanol, the resulting level was only half that of ethanol-treated 129 Hfe(-/-) mice. ALAS induction was similar in both Hfe(-/-) strains. In wild-type 129 mice treated with ethanol for 6 to 7 months, administration of iron dextran increased hepatic URO accumulation and decreased UROD activity. In conclusion, this study demonstrates a strong effect of genetic background on ethanol-induced uroporphyria, which is probably due to a greater effect of ethanol on iron metabolism in the susceptible strain.