Export of functional Streptomyces coelicolor alditol oxidase to the periplasm or cell surface of Escherichia coli and its application in whole-cell biocatalysis

Streptomyces coelicolor A3(2) alditol oxidase (AldO) is a soluble monomeric flavoprotein in which the flavin cofactor is covalently linked to the polypeptide chain. AldO displays high reactivity towards different polyols such as xylitol and sorbitol. These characteristics make AldO industrially relevant, but full biotechnological exploitation of this enzyme is at present restricted by laborious and costly purification steps. To eliminate the need for enzyme purification, this study describes a whole-cell AldO biocatalyst system. To this end, we have directed AldO to the periplasm or cell surface of Escherichia coli. For periplasmic export, AldO was fused to endogenous E. coli signal sequences known to direct their passenger proteins into the SecB, signal recognition particle (SRP), or Twin-arginine translocation (Tat) pathway. In addition, AldO was fused to an ice nucleation protein (INP)-based anchoring motif for surface display. The results show that Tat-exported AldO and INP-surface-displayed AldO are active. The Tat-based system was successfully employed in converting xylitol by whole cells, whereas the use of the INP-based system was most likely restricted by lipopolysaccharide LPS in wild-type cells. It is anticipated that these whole-cell systems will be a valuable tool for further biological and industrial exploitation of AldO and other cofactor-containing enzymes.

The Aquatic Toxicity of Organic Compounds to Embryo-Larval Stages of Fish and Amphibians

Aquatic toxicity tests were conducted on 11 organic compounds considered hazardous to water resources. The toxicity of each compound was evaluated using embryo-larval stages of two to eight fish and amphibian species. Exposure was initiated at fertilization and maintained through 4 days posthatching. The animal test species exhibited varying degrees of sensitivity to the selected toxicants. Combined frequencies for mortality and teratogenesis at 4 days posthatching gave LC50 ranges of 3.66 to 8.25 mg/L for benzene, 1.16 to 22.42 mg/L for carbon tetrachloride, 0.11 to 1.20 mg/L for chlorobenzene, 2.03 to \u3e 68 mg/L for chloroform, 3.01 to 5.56 mg/L for 1,2-dichlorobenzene, 2.54 to .34 mg/L for 1,2-dichloroethane, 13.16 to \u3e 48 mg/L for methylene chloride, 0.002 to 0.64 mg/L for nitrobenzene, 0.04 to .32 mg/L for phenol, 0.02 to 0.85 mg/L for toluene, and 3.53 to 3.77 mg/L for m-xylene. The species which exhibited the greatest susceptibility to organic compounds were the rainbow trout, Rana pipiens, and Rana temporaria. The more sensitive amphibian species generally were those which normaly are restricted to aquatic or moist terrestrial habitats, whereas the more tolerant amphibians included those semi-aquatic and terrestrial species which appear to be more broadly adapted ecologically. Of the 11 test compounds, nitrobenzene, toluene, chlorobenzene, and phenol were the most toxic. The least toxic organics included dichloroethane and methylene chloride. For three chlorinated alkanes, including methylene chloride (CH2Cl2), chloroform (CHCl3), and carbon tetrachloride (CCl4), toxicity was found to 1ncrease with the degree of chlorination. Concerning several aromatic hydrocarbons, benzene always was found to be less toxic than its monosubstituted analogs. Toxicity of the 11 compounds was further evaluated by calculating toxicant concentrations which produced embryo-larval mortality and/or teratogenesis at frequencies of 10% (LC10) and 1% (LC1). The LC values, used to estimate toxicity thresholds, ranged from \u3c 0.l for nitrobenzene to 69.9 μg/L for methylene chloride. A limited number of toxicity tests were performed to determine whether embryo-larval bioassays are suitable to assess effects of transitory chemical exposures, such as those resulting from intermittent discharges or accidental spills of chemicals into water resources. Results indicated that Rana pipiens embryos were sufficiently sensitive to quantify effects produced by short-term exposures to chloroform. Animals tested during the earliest embryonic stage appeared to be less tolerant than organisms exposed later in development

Cortisol catabolism by lymphocytes of patients with chronic lymphocytic leukemia

A low rate of catabolism of cortisol by lymphocytes correlates with high sensitivity of the cells to the steroid and causes them to die at a greater rate than control samples. Since lymphocytes of patients with chronic lymphocytic leukemia respond to treatment with glucocorticosteroids and are cortisol sensitive, we attempted to see whether their capability to catabolize cortisol differs from that of normal lymphocytes. No difference was found between the two groups of cells with regard to the pattern of cortisol metabolites. However, the lymphocytes of the chronic lymphocytic leukemia groups showed a total cortisol catabolism per cell that was significantly lower than that of the control group. Patients with low lymphocyte count in peripheral blood showed a relatively higher cortisol metabolism by lymphocytes per cell than those with high counts.Key words: lymphocytes, cortisol, catabolism, chronic lymphocytic leukemia, morbidity. </jats:p

The microphthalmia-associated transcription factor Mitf interacts with beta-catenin to determine target gene expression.

Commitment to the melanocyte lineage is characterized by the onset of expression of the microphthalmia-associated transcription factor (Mitf). This transcription factor plays a fundamental role in melanocyte development and maintenance and seems to be crucial for the survival of malignant melanocytes. Furthermore, Mitf has been shown to be involved in cell cycle regulation and to play important functions in self-renewal and maintenance of melanocyte stem cells. Although little is known about how Mitf regulates these various processes, one possibility is that Mitf interacts with other regulators. Here we show that Mitf can interact directly with beta-catenin, the key mediator of the canonical Wnt signaling pathway. The Wnt signaling pathway plays a critical role in melanocyte development and is intimately involved in triggering melanocyte stem cell proliferation. Significantly, constitutive activation of this pathway is a feature of a number of cancers including malignant melanoma. Here we show that Mitf can redirect beta-catenin transcriptional activity away from canonical Wnt signaling-regulated genes toward Mitf-specific target promoters to activate transcription. Thus, by a feedback mechanism, Mitf can diversify the output of canonical Wnt signaling to enhance the repertoire of genes regulated by beta-catenin. Our results reveal a novel mechanism by which Wnt signaling and beta-catenin activate gene expression, with significant implications for our understanding of both melanocyte development and melanoma

Smart Mat for Respiratory Activity Detection: Study in a Clinical Setting

We discuss in this paper a study of a smart and unobtrusive mattress in a clinical setting on a population with cardiorespiratory problems. Up to recently, the vast majority of studies with unobtrusive sensors are done with healthy populations. The unobtrusive monitoring of the Respiratory Rate (RR) is essential for proposing better diagnoses. Thus, new industrial and research activity on smart mattresses is targeting respiratory rate in an Internet-of-Things (IoT) context. In our work, we are interested in the performances of a microbend fiber optic sensor (FOS) mattress on 81 subjects admitted in the Cardiac Intensive Care Unit (CICU) by estimating the RR from their ballistocardiograms (BCG). Our study proposes a new RR estimator, based on harmonic plus noise models (HNM) and compares it with known estimators such as MODWT and CLIE. The goal is to examine, using a more representative and bigger dataset, the performances of these methods and of the smart mattress in general. Results of applying these three estimators on the BCG show that MODWT is more accurate with an average mean absolute error (MAE) of 1.97 ± 2.12 BPM. However, the HNM estimator has space for improvements with estimation errors of 2.91 ± 4.07 BPM. The smart mattress works well within a standard RR range of 10–20 breaths-per-minute (BPM) but gets less accurate with a bigger range of estimation. These results highlight the need to test these sensors in much more realistic contexts

Trigger factor interacts with the signal peptide of nascent Tat substrates but does not play a critical role in tat-mediated export.

Twin-arginine translocation (Tat)-mediated protein transport across the bacterial cytoplasmic membrane occurs only after synthesis and folding of the substrate protein that contains a signal peptide with a characteristic twin-arginine motif. This implies that premature contact between the Tat signal peptide and the Tat translocon in the membrane must be prevented. We used site-specific photo-crosslinking to demonstrate that the signal peptide of nascent Tat proteins is in close proximity to the chaperone and peptidyl-prolyl isomerase trigger factor (TF). The contact with TF was strictly dependent on the context of the translating ribosome, started early in biogenesis when the nascent chain left the ribosome near L23, and persisted until the chain reached its full length. Despite this exclusive and prolonged contact, depletion or overexpression of TF had little effect on the kinetics and efficiency of the Tat export process

Holding-on: co-evolution between infant carrying and grasping behaviour in strepsirrhines

The origin and evolution of manual grasping remain poorly understood. The ability to cling requires important grasping abilities and is essential to survive in species where the young are carried in the fur. A previous study has suggested that this behaviour could be a pre-adaptation for the evolution of fine manipulative skills. In this study we tested the co-evolution between infant carrying in the fur and manual grasping abilities in the context of food manipulation. As strepsirrhines vary in the way infants are carried (mouth vs. fur), they are an excellent model to test this hypothesis. Data on food manipulation behaviour were collected for 21 species of strepsirrhines. Our results show that furcarrying species exhibited significantly more frequent manual grasping of food items. This study clearly illustrates the potential novel insights that a behaviour (infant carrying) that has previously been largely ignored in the discussion of the evolution of primate manipulation can bring.peerReviewe