Yeasts from sub-Antarctic region: biodiversity,enzymatic activities and their potential as oleaginous microorganisms

Various microbial groups are well known to produce a range of extracellular enzymes and other secondary metabolites. However, the occurrence and importance of investment in such activities have received relatively limited attention in studies of Antarctic soil microbiota. Sixty-one yeasts strains were isolated from King George Island, Antarctica which were characterized physiologically and identified at the molecular level using the D1/D2 region of rDNA. Fifty-eight yeasts (belonging to the genera Cryptococcus, Leucosporidiella, Rhodotorula, Guehomyces, Candida, Metschnikowia and Debaryomyces) were screened for extracellular amylolytic, proteolytic, esterasic, pectinolytic, inulolytic xylanolytic and cellulolytic activities at low and moderate temperatures. Esterase activity was the most common enzymatic activity expressed by the yeast isolates regardless the assay temperature and inulinase was the second most common enzymatic activity. No cellulolytic activity was detected. One yeast identified as Guehomyces pullulans (8E) showed significant activity across six of seven enzymes types tested. Twenty-eight yeast isolates were classified as oleaginous, being the isolate 8E the strain that accumulated the highest levels of saponifiable lipids (42 %).     

E1a gene expression blocks the ERK1/2 signaling pathway by promoting nuclear localization and MKP up-regulation: implication in v-H-Ras-induced senescence

In response to oncogenic signals, cells have developed safe mechanisms to avoid transformation through activation of a senescence program. Upon v-H-Ras overexpression, normal cells undergo senescence through several cellular processes, including activation of the ERK1/2 pathway. Interestingly, the E1a gene from adenovirus 5 has been shown to rescue cells from senescence by a yet unknown mechanism. We investigated whether E1a was able to interfere with the ERK1/2 signaling pathway to rescue cells from v-H-Ras-mediated senescence. Our results show that, E1a overexpression blocks v-H-Ras-mediated ERK1/2 activation by two different and concomitant mechanisms. E1a through its ability to interfere with PKB/Akt activation induces the down-regulation of the PEA15 protein, an ERK1/2 nuclear export factor, leading to nuclear accumulation of ERK1/2. In addition to this, we show that E1a increases the expression of the inducible ERK1/2 nuclear phosphatases (MAPK phosphatases) MKP1/DUSP1 and DUSP5, which leads to ERK1/2 dephosphorylation. We confirmed our observations in the human normal diploid fibroblasts IMR90, in which we could also show that an E1a mutant, unable to bind retinoblastoma protein (pRb), cannot rescue cells from v-H-Ras-induced senescence. In conclusion, E1a is able to rescue from Ras-induced senescence by affecting ERK1/2 localization and phosphorylation.     

Mnl2, a novel component of the ER associated protein degradation pathway

In eukaryotes, membrane and soluble proteins of the secretory pathway enter the endoplasmic reticulum (ER) after synthesis in an unfolded state. Directly after entry, most proteins are modified with glycans at suitable glycosylation sites and start to fold. A protein that cannot fold properly will be degraded in a process called ER associated degradation (ERAD). Failures in ERAD, either by loss of function or by premature degradation of proteins, are a cause of severe diseases. Therefore, the search for novel ERAD components to gain better insight in this process is of high importance. Carbohydrate trimming is a relevant process in ER quality control. In this work a novel putative yeast mannosidase encoded by the open reading frame YLR057W was identified and named Mnl2. Deletion of MNL2 diminished the degradation efficiency of misfolded CPY^* in the absence of the cognate mannosidase Mnl1, indicating a specific role in ERAD.     

Selective Cytotoxicity of 1,3,4-Thiadiazolium Mesoionic Derivatives on Hepatocarcinoma Cells (HepG2)

In this work, we evaluated the cytotoxicity of mesoionic 4-phenyl-5-(2-Y, 4-X or 4-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivatives (MI-J: X=OH, Y=H; MI-D: X=NO₂, Y=H; MI-4F: X=F, Y=H; MI-2,4diF: X=Y=F) on human hepatocellular carcinoma (HepG2), and non-tumor cells (rat hepatocytes) for comparison. MI-J, M-4F and MI-2,4diF reduced HepG2 viability by ~ 50% at 25 μM after 24-h treatment, whereas MI-D required a 50 μM concentration, as shown by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The cytotoxicity was confirmed with lactate dehydrogenase assay, of which activity was increased by 55, 24 and 16% for MI-J, MI-4F and MI-2,4diF respectively (at 25 μM after 24 h). To identify the death pathway related to cytotoxicity, the HepG2 cells treated by mesoionic compounds were labeled with both annexin V and PI, and analyzed by flow cytometry. All compounds increased the number of doubly-stained cells at 25 μM after 24 h: by 76% for MI-J, 25% for MI-4F and MI-2,4diF, and 11% for MI-D. It was also verified that increased DNA fragmentation occurred upon MI-J, MI-4F and MI-2,4diF treatments (by 12%, 9% and 8%, respectively, at 25 μM after 24 h). These compounds were only weakly, or not at all, transported by the main multidrug transporters, P-glycoprotein, ABCG2 and MRP1, and were able to slightly inhibit their drug-transport activity. It may be concluded that 1,3,4-thiadiazolium compounds, especially the hydroxy derivative MI-J, constitute promising candidates for future investigations on in-vivo treatment of hepatocellular carcinoma.     

A Field Evaluation of an External and Neutrally Buoyant Acoustic Transmitter for Juvenile Salmon: Implications for Estimating Hydroturbine Passage Survival

Turbine-passed fish are exposed to rapid decreases in pressure which can cause barotrauma. The presence of an implanted telemetry tag increases the likelihood of injury or death from exposure to pressure changes, thus potentially biasing studies evaluating survival of turbine-passed fish. Therefore, a neutrally buoyant externally attached tag was developed to eliminate this bias in turbine passage studies. This new tag was designed not to add excess mass in water or take up space in the coelom, having an effective tag burden of zero with the goal of reducing pressure related biases to turbine survival studies. To determine if this new tag affects fish performance or susceptibility to predation, it was evaluated in the field relative to internally implanted acoustic transmitters (JSATS; Juvenile Salmon Acoustic Telemetry System) used widely for survival studies of juvenile salmonids. Survival and travel time through the study reach was compared between fish with either tag type in an area of high predation in the Snake and Columbia rivers, Washington. An additional group of fish affixed with neutrally-buoyant dummy external tags were implanted with passive integrated transponder (PIT) tags and recovered further downstream to assess external tag retention and injury. There were no significant differences in survival to the first detection site, 12 river kilometers (rkm) downstream of release. Travel times were also similar between groups. Conversely, externally-tagged fish had reduced survival (or elevated tag loss) to the second detection site, 65 rkm downstream. In addition, the retention study revealed that tag loss was first observed in fish recaptured approximately 9 days after release. Results suggest that this new tag may be viable for short term (<8 days) single-dam turbine-passage studies and under these situations, may alleviate the turbine passage-related bias encountered when using internal tags, however further research is needed to confirm this.     

Fluorine‐Free Noble Salt Anion for High‐Performance All‐Solid‐State Lithium–Sulfur Batteries

Amongst post‐Li‐ion battery technologies, lithium–sulfur (Li–S) batteries have captured an immense interest as one of the most appealing devices from both the industrial and academia sectors. The replacement of conventional liquid electrolytes with solid polymer electrolytes (SPEs) enables not only a safer use of Li metal (Li°) anodes but also a flexible design in the shape of Li–S batteries. However, the practical implementation of SPEs‐based all‐solid‐state Li–S batteries (ASSLSBs) is largely hindered by the shuttling effect of the polysulfide intermediates and the formation of dendritic Li° during the battery operation. Herein, a fluorine‐free noble salt anion, tricyanomethanide [C(CN)₃^?, TCM^?], is proposed as a Li‐ion conducting salt for ASSLSBs. Compared to the widely used perfluorinated anions {e.g., bis(trifluoromethanesulfonyl)imide anion, [N(SO₂CF₃)₂)]^?, TFSI^?}, the LiTCM‐based electrolytes show decent ionic conductivity, good thermal stability, and sufficient anodic stability suiting the cell chemistry of ASSLSBs. In particular, the fluorine‐free solid electrolyte interphase layer originating from the decomposition of LiTCM exhibits a good mechanical integrity and Li‐ion conductivity, which allows the LiTCM‐based Li–S cells to be cycled with good rate capability and Coulombic efficiency. The LiTCM‐based electrolytes are believed to be the most promising candidates for building cost‐effective and high energy density ASSLSBs in the near future.     

GDP release preferentially occurs on the phosphate side in heterotrimeric G-proteins

After extra-cellular stimulation of G-Protein Coupled Receptors (GPCRs), GDP/GTP exchange appears as the key, rate limiting step of the intracellular activation cycle of heterotrimeric G-proteins. Despite the availability of a large number of X-ray structures, the mechanism of GDP release out of heterotrimeric G-proteins still remains unknown at the molecular level. Starting from the available X-ray structure, extensive unconstrained/constrained molecular dynamics simulations were performed on the complete membrane-anchored Gi heterotrimer complexed to GDP, for a total simulation time overcoming 500 ns. By combining Targeted Molecular Dynamics (TMD) and free energy profiles reconstruction by umbrella sampling, our data suggest that the release of GDP was much more favored on its phosphate side. Interestingly, upon the forced extraction of GDP on this side, the whole protein encountered large, collective motions in perfect agreement with those we described previously including a domain to domain motion between the two ras-like and helical sub-domains of G(α).     

Yeast aminopeptidase I is post-translationally sorted from the cytosol to the vacuole by a mechanism mediated by its bipartite N-terminal extension.

Transport of aminopeptidase I (API) to the vacuole appears to be insensitive to blockage of the secretory pathway. Here we show that the N-terminal extension of the 61 kDa precursor of API (pAPI) is proteolytically processed in two sequential steps. The first step involves proteinase A (PrA) and produces a 55 kDa unstable intermediate (iAPI). The second step involves proteinase B (PrB) and converts iAPI into the 50 kDa stable, mature enzyme (mAPI). Reversion of the cup1 growth phenotype by a pAPI-CUP1 chimera indicates that pAPI is transported to the vacuole by a post-translational mechanism. Deletion of the first 16 amino acids results in accumulation of the truncated protein in the cytosol, indicating that pAPI is actively transported to the vacuole. The chimera pAPI-myc, constructed by fusing a myc tag to the C-terminus of pAPI, was exploited to dissect the mechanism of pAPI transport. Cell fractionation studies show the presence of iAPI-myc and mAPI in a fraction of vacuoles purified by density centrifugation. This and the sequential conversion of pAPI-myc into iAPI-myc and mAPI lacking the myc tag is consistent with insertion of pAPI into the vacuolar membrane through its N-terminal extension. The specific mechanism of API sorting demonstrates a new pathway of protein transport in vacuolar biogenesis.