An Expanded Heterologous GAL Promoter Collection for Diauxie-Inducible Expression in Saccharomyces cerevisiae.

The GAL promoters are applied in metabolic engineering and synthetic biology to control gene expression in the budding yeast Saccharomyces cerevisiae. In gal80Δ background strains, they show diauxie-inducible expression, a feature beneficial in metabolic pathway optimization. However, only a limited number of GAL promoters have been characterized and are available for engineering purposes. Multiple uses of the same promoters can result in genetic instability in engineered strains due to homologous recombination. Here, 11 GAL1/2 promoters from other Saccharomyces species were isolated and characterized in S. cerevisiae. They exhibited diauxie-inducible expression patterns with low strength in exponential growth phase and induction in the ethanol growth phase. These promoters represent an expansion to the collection of GAL promoters available for genetic engineering in S. cerevisiae, including an increased diversity of expression levels. This provides the capacity for increased numbers of genetic manipulations with a lower risk of genetic instability.

A biosensor-based framework to measure latent proteostasis capacity.

The pool of quality control proteins (QC) that maintains protein-folding homeostasis (proteostasis) is dynamic but can become depleted in human disease. A challenge has been in quantitatively defining the depth of the QC pool. With a new biosensor, flow cytometry-based methods and mathematical modeling we measure the QC capacity to act as holdases and suppress biosensor aggregation. The biosensor system comprises a series of barnase kernels with differing folding stability that engage primarily with HSP70 and HSP90 family proteins. Conditions of proteostasis stimulation and stress alter QC holdase activity and aggregation rates. The method reveals the HSP70 chaperone cycle to be rate limited by HSP70 holdase activity under normal conditions, but this is overcome by increasing levels of the BAG1 nucleotide exchange factor to HSPA1A or activation of the heat shock gene cluster by HSF1 overexpression. This scheme opens new paths for biosensors of disease and proteostasis systems.

A thiol probe for measuring unfolded protein load and proteostasis in cells.

When proteostasis becomes unbalanced, unfolded proteins can accumulate and aggregate. Here we report that the dye, tetraphenylethene maleimide (TPE-MI) can be used to measure cellular unfolded protein load. TPE-MI fluorescence is activated upon labelling free cysteine thiols, normally buried in the core of globular proteins that are exposed upon unfolding. Crucially TPE-MI does not become fluorescent when conjugated to soluble glutathione. We find that TPE-MI fluorescence is enhanced upon reaction with cellular proteomes under conditions promoting accumulation of unfolded proteins. TPE-MI reactivity can be used to track which proteins expose more cysteine residues under stress through proteomic analysis. We show that TPE-MI can report imbalances in proteostasis in induced pluripotent stem cell models of Huntington disease, as well as cells transfected with mutant Huntington exon 1 before the formation of visible aggregates. TPE-MI also detects protein damage following dihydroartemisinin treatment of the malaria parasites Plasmodium falciparum. TPE-MI therefore holds promise as a tool to probe proteostasis mechanisms in disease.Proteostasis is maintained through a number of molecular mechanisms, some of which function to protect the folded state of proteins. Here the authors demonstrate the use of TPE-MI in a fluorigenic dye assay for the quantitation of unfolded proteins that can be used to assess proteostasis on a cellular or proteome scale.

Chronic effects of atrazine exposure and recovery in freshwater benthic diatoms from two communities with different pollution histories.

Diffuse agricultural runoff into rivers can result in contamination with herbicides for prolonged periods of time. Chronic exposure to herbicides has the potential to alter toxic impacts in primary producers such as benthic diatoms. Determining how individual diatom taxa respond to herbicide exposure over varied exposure durations is essential for assessing herbicide impacts. This study investigated the responses of various benthic diatom taxa and effects at the community level over 12days of atrazine exposure. Diatom communities were collected from two sites with differing exposure histories; a relatively unpolluted site (Alligator Creek) and an agricultural stream (Barratta Creek) known to be polluted by atrazine and other herbicides. Diatom community composition and the proportion of healthy cells per taxon were assessed at 0, 2, 3, 6, 9 and 12days of atrazine exposure. Pollution history altered the response of the diatom community to atrazine exposure. In the Alligator Creek diatom community there was a shift in composition towards more tolerant taxa and the loss of sensitive taxa in atrazine exposed treatments. The sensitive taxon (Gomphonema truncatum) was consistently affected by atrazine toxicity. Conversely, the polluted Barratta Creek diatom community was not strongly affected by atrazine exposure. Our study shows that during chronic atrazine exposure some taxa demonstrated the ability to recover despite initial toxicity response. Recovery could be an important trait for understanding the ecological effect of herbicide exposure on diatom species in nature and in applied circumstances such as biomonitoring indices.

When proteostasis goes bad: Protein aggregation in the cell.

Protein aggregation is a hallmark of the major neurodegenerative diseases including Alzheimer's, Parkinson's, Huntington's and motor neuron and is a symptom of a breakdown in the management of proteome foldedness. Indeed, it is remarkable that under normal conditions cells can keep their proteome in a highly crowded and confined space without uncontrollable aggregation. Proteins pose a particular challenge relative to other classes of biomolecules because upon synthesis they must typically follow a complex folding pathway to reach their functional conformation (native state). Non-native conformations, including the unfolded nascent chain, are highly prone to aberrant interactions, leading to aggregation. Here we review recent advances in knowledge of proteostasis, approaches to monitor proteostasis and the impact that protein aggregation has on biology. We also include discussion of the outstanding challenges. © 2017 IUBMB Life, 69(2):49-54, 2017.

Choosing an out-of-hospital birth centre: Exploring women's decision-making experiences.

OBJECTIVE: the primary objective for this study was to explore women's experiences of choosing to plan a birth at an out-of-hospital birth centre. We sought to understand how women make the choice to plan for an out-of-hospital birth and the meaning that women ascribe to this decision-making process. DESIGN, SETTING, AND PARTICIPANTS: a qualitative phenomenological study was conducted in Winnipeg, Canada with a sample of seventeen post partum women who represent the socio-demographic characteristics of the actual users of the Birth Centre in Winnipeg. The women participated in semistructured interviews. Through a feminist perspective and using interpretative phenomenological analysis (IPA), each participant's experience of birthplace decision-making was explored. FINDINGS: six themes emerged through the analysis: (1) Making the decision in the context of relationships; (2) Exercising personal agency; (3) An expression of one's ideology; (4) Really thinking it through; (5) Fitting into the eligibility criteria; and (6) The psychology of the space. The findings suggested that a woman's sense of safety was related to each of these themes. KEY CONCLUSIONS AND IMPLICATIONS FOR PRACTICE: the birth centre decision-making experience has many similarities to the homebirth decision-making process. The visceral impact of the physical design of the facility plays an important role and differentiates the birth centre decision from other birth setting options. The concept of relational autonomy was emphasised in this study, in that women make the decision in the context of their relationships with their midwives and partners. The study has implications for midwifery practice and health-care policy related to: client education on birth settings, design of birth environments, validation of the birth centre concept, and upholding the women-centred midwifery model of care. The study highlighted the importance of increasing access to out-of-hospital birth centres.

How benthic diatoms within natural communities respond to eight common herbicides with different modes of action.

Herbicides are common pollutants of rivers in agricultural regions. These contaminants include various types of chemicals with different modes of toxic action. Herbicides can have toxic effects on freshwater benthic diatoms, the base of the aquatic food web. We examined the effects of (non-mixture) herbicide exposure to the health of diatoms for eight common herbicides with three different modes of action; the photosystem II (PSII) inhibitors: atrazine, simazine, hexazinone, tebuthiuron and diuron; two auxinic herbicides: MCPA and 2,4-D; and the EPSP synthase inhibitor: glyphosate. Benthic diatoms within riverine communities were exposed to each herbicide in rapid toxicity tests at concentrations of 50, 200 and 500µgL(-1). The most sensitive taxa were Gomphonema spp. and Encyonema gracilis. Navicula cryptotenella was the most tolerant to herbicide exposure. There was no significant effect of the different herbicide modes of action at the community level. Herbicide mode of action did not alter which taxa were most sensitive within the community and sensitivity rankings of the dominant diatom taxa were similar for each of the eight herbicides. The consistency of the results between herbicides suggests that freshwater benthic diatoms may be suitable in situ indicators for detecting the toxicity of herbicides with differing modes of action.

The influence of reduced light intensity on the response of benthic diatoms to herbicide exposure.

Herbicide pollution events in aquatic ecosystems often coincide with increased turbidity and reduced light intensity. It is therefore important to determine whether reduced light intensity can influence herbicide toxicity, especially to primary producers such as benthic diatoms. Benthic diatoms collected from 4 rivers were exposed to herbicides in 48 h rapid toxicity tests under high light (100 µmol m(-2) s(-1) ) and low light (20 µmol m(-2) s(-1) ) intensities. The effects of 2 herbicides (atrazine and glyphosate) were assessed on 26 freshwater benthic diatom taxa. There was no significant interaction of light and herbicide effects at the community level or on the majority (22 of 26) of benthic diatom taxa. This indicates that low light levels will likely have only a minor influence on the response of benthic diatoms to herbicides. Environ Toxicol Chem 2016;35:2252-2260. © 2016 SETAC.

Polyalanine expansions drive a shift into α-helical clusters without amyloid-fibril formation.

Polyglutamine (polyGln) expansions in nine human proteins result in neurological diseases and induce the proteins' tendency to form ß-rich amyloid fibrils and intracellular deposits. Less well known are at least nine other human diseases caused by polyalanine (polyAla)-expansion mutations in different proteins. The mechanisms of how polyAla aggregates under physiological conditions remain unclear and controversial. We show here that aggregation of polyAla is mechanistically dissimilar to that of polyGln and hence does not exhibit amyloid kinetics. PolyAla assembled spontaneously into α-helical clusters with diverse oligomeric states. Such clustering was pervasive in cells irrespective of visible aggregate formation, and it disrupted the normal physiological oligomeric state of two human proteins natively containing polyAla: ARX and SOX3. This self-assembly pattern indicates that polyAla expansions chronically disrupt protein behavior by imposing a deranged oligomeric status.

Determining the relative sensitivity of benthic diatoms to atrazine using rapid toxicity testing: a novel method.

Herbicides pose a potential threat to aquatic ecosystems, especially to phototrophic organisms such as benthic diatoms. Benthic diatoms may be a valuable indicator of the toxic impacts of herbicides in aquatic systems. However, this requires information on the herbicide sensitivity of a wide range of freshwater benthic diatom taxa. Unfortunately this information is only available for a limited number of species as current methods of developing new algae toxicity tests on individual taxa are lengthy and costly. To address this issue, we developed a new rapid toxicity test method to test natural benthic communities, from which the relative herbicide sensitivity of many individual taxa can be derived. This involved the collection of natural benthic communities from rocks in situ, which were placed directly into laboratory toxicity tests. Sensitivity data for several diatom genera in a 48 hour exposure toxicity test were produced, without the need for cultures or multiple site visits. After exposure to the highest treatment of atrazine (500 µg L(-1)) there were significant declines of healthy cells in the most sensitive genera: Gomphonema declined by 74%, Amphora by 62%, Cymbella by 54% and Ulnaria by 34% compared to control levels. In contrast, the genera, Eunotia, Achnanthidium and Navicula, had no statistically significant decline in cell health. This method can identify the diatom taxa most at risk of herbicide toxicity within the natural benthic diatom community. The rapid toxicity testing method presented is a simple and effective method to obtain sensitivity data for multiple taxa within a natural benthic diatom community in a relatively short period of time.

Tyrosine 416 is phosphorylated in the closed, repressed conformation of c-Src.

c-Src kinase activity is regulated by phosphorylation of Y527 and Y416. Y527 phosphorylation stabilizes a closed conformation, which suppresses kinase activity towards substrates, whereas phosphorylation at Y416 promotes an elevated kinase activity by stabilizing the activation loop in a manner permissive for substrate binding. Here we investigated the correlation of Y416 phosphorylation with c-Src activity when c-Src was locked into the open and closed conformations (by mutations Y527F and Q528E, P529E, G530I respectively). Consistent with prior findings, we found Y416 to be more greatly phosphorylated when c-Src was in an open, active conformation. However, we also observed an appreciable amount of Y416 was phosphorylated when c-Src was in a closed, repressed conformation under conditions by which c-Src was unable to phosphorylate substrate STAT3. The phosphorylation of Y416 in the closed conformation arose by autophosphorylation, since abolishing kinase activity by mutating the ATP binding site (K295M) prevented phosphorylation. Basal Y416 phosphorylation correlated positively with cellular levels of c-Src suggesting autophosphorylation depended on self-association. Using sedimentation velocity analysis on cell lysate with fluorescence detection optics, we confirmed that c-Src forms monomers and dimers, with the open conformation also forming a minor population of larger mass complexes. Collectively, our studies suggest a model by which dimerization of c-Src primes c-Src via Y416 phosphorylation to enable rapid potentiation of activity when Src adopts an open conformation. Once in the open conformation, c-Src can amplify the response by recruiting and phosphorylating substrates such as STAT3 and increasing the extent of autophosphorylation.