Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB

The drug rapamycin has important uses in oncology, cardiology, and transplantation medicine, but its clinically relevant molecular effects are not understood. When bound to FKBP12, rapamycin interacts with and inhibits the kinase activity of a multiprotein complex composed of mTOR, mLST8, and raptor (mTORC1). The distinct complex of mTOR, mLST8, and rictor (mTORC2) does not interact with FKBP12-rapamycin and is not thought to be rapamycin sensitive. mTORC2 phosphorylates and activates Akt/PKB, a key regulator of cell survival. Here we show that rapamycin inhibits the assembly of mTORC2 and that, in many cell types, prolonged rapamycin treatment reduces the levels of mTORC2 below those needed to maintain Akt/PKB signaling. The proapoptotic and antitumor effects of rapamycin are suppressed in cells expressing an Akt/PKB mutant that is rapamycin resistant. Our work describes an unforeseen mechanism of action for rapamycin that suggests it can be used to inhibit Akt/PKB in certain cell types.     

Analysis of a spindle pole body mutant reveals a defect in biorientation and illuminates spindle forces

The spindle pole body (SPB) is the microtubule organizing center in Saccharomyces cerevisiae. An essential task of the SPB is to ensure assembly of the bipolar spindle, which requires a proper balancing of forces on the microtubules and chromosomes. The SPB component Spc110p connects the ends of the spindle microtubules to the core of the SPB. We previously reported the isolation of a mutant allele spc110-226 that causes broken spindles and SPB disintegration 30 min after spindle formation. By live cell imaging of mutant cells with green fluorescent protein (GFP)-Tub1p or Spc97p-GFP, we show that spc110-226 mutant cells have early defects in spindle assembly. Short spindles form but do not advance to the 1.5-microm stage and frequently collapse. Kinetochores are not arranged properly in the mutant cells. In 70% of the cells, no stable biorientation occurs and all kinetochores are associated with only one SPB. Examination of the SPB remnants by electron microscopy tomography and fluorescence microscopy revealed that the Spc110-226p/calmodulin complex is stripped off of the central plaque of the SPB and coalesces to from a nucleating structure in the nucleoplasm. The central plaque components Spc42p and Spc29p remain behind in the nuclear envelope. The delamination is likely due to a perturbed interaction between Spc42p and Spc110-226p as detected by fluorescence resonance energy transfer analysis. We suggest that the force exerted on the SPB by biorientation of the chromosomes pulls the Spc110-226p out of the SPB; removal of force exerted by coherence of the sister chromatids reduced fragmentation fourfold. Removal of the forces exerted by the cytoplasmic microtubules had no effect on fragmentation. Our results provide insights into the relative contributions of the kinetochore and cytoplasmic microtubules to the forces involved in formation of a bipolar spindle.     

Genotyping‐by‐sequencing and ecological niche modeling illuminate phylogeography,admixture, and Pleistocene range dynamics in quaking aspen (Populus tremuloides)

Populus tremuloides is the widest‐ranging tree species in North America and an ecologically important component of mesic forest ecosystems displaced by the Pleistocene glaciations. Using phylogeographic analyses of genome‐wide SNPs (34,796 SNPs, 183 individuals) and ecological niche modeling, we inferred population structure, ploidy levels, admixture, and Pleistocene range dynamics of P. tremuloides, and tested several historical biogeographical hypotheses. We found three genetic lineages located mainly in coastal–Cascades (cluster 1), east‐slope Cascades–Sierra Nevadas–Northern Rockies (cluster 2), and U.S. Rocky Mountains through southern Canadian (cluster 3) regions of the P. tremuloides range, with tree graph relationships of the form ((cluster 1, cluster 2), cluster 3). Populations consisted mainly of diploids (86%) but also small numbers of triploids (12%) and tetraploids (1%), and ploidy did not adversely affect our genetic inferences. The main vector of admixture was from cluster 3 into cluster 2, with the admixture zone trending northwest through the Rocky Mountains along a recognized phenotypic cline (Utah to Idaho). Clusters 1 and 2 provided strong support for the “stable‐edge hypothesis” that unglaciated southwestern populations persisted in situ since the last glaciation. By contrast, despite a lack of clinal genetic variation, cluster 3 exhibited “trailing‐edge” dynamics from niche suitability predictions signifying complete northward postglacial expansion. Results were also consistent with the “inland dispersal hypothesis” predicting postglacial assembly of Pacific Northwestern forest ecosystems, but rejected the hypothesis that Pacific‐coastal populations were colonized during outburst flooding from glacial Lake Missoula. Overall, congruent patterns between our phylogeographic and ecological niche modeling results and fossil pollen data demonstrate complex mixtures of stable‐edge, refugial locations, and postglacial expansion within P. tremuloides. These findings confirm and refine previous genetic studies, while strongly supporting a distinct Pacific‐coastal genetic lineage of quaking aspen.     

Studies toward the comprehension of fungal-macroalgae interaction in cold marine regions from a biotechnological perspective

In marine ecosystems, macroalgae are the habitat for several microorganisms, fungi being among them. In the Antarctic benthic coastal ecosystem, macroalgae play a key role in organic matter cycling. In this study, 13 different macroalgae from Potter Cove and surrounding areas were sampled and 48 fungal isolates were obtained from six species, four Rhodophyta Ballia callitricha, Gigartina skottsbergii, Neuroglossum delesseriae and Palmaria decipiens, and two Phaeophyceae: Adenocystis utricularis and Ascoseira mirabilis. Fungal isolates mostly belonged to the Ascomycota phylum (Antarctomyces, Cadophora, Cladosporium, Penicillium, Phialocephala, and Pseudogymnoascus) and only one to the phylum Mucoromycota. Two of the isolates could not be identified to genus level, implying that Antarctica is a source of probable novel fungal taxa with enormous bioprospecting and biotechnological potential. 73% of the fungal isolates were moderate eurypsychrophilic (they grew at 5–25 °C), 12.5% were eurypsychrophilic and grew in the whole range, 12.5% of the isolates were narrow eurypsychrophilic (growth at 15–25 °C), and Mucoromycota AUe4 was classified as stenopsychrophilic as it grew at 5–15 °C. Organic extracts of seven macroalgae from which no fungal growth was obtained (three red algae Georgiella confluens, Gymnogongrus turquetii, Plocamium cartlagineum, and four brown algae Desmarestia anceps, D. Antarctica, Desmarestia menziesii, Himantothallus grandifolius) were tested against representative fungi of the genera isolated in this work. All extracts presented fungal inhibition, those from Plocamium cartilagineum and G. turquetii showed the best results, and for most of these macroalgae, this represents the first report of antifungal activity and constitute a promising source of compounds for future evaluation.     

Enterococci as indicators of Lake Michigan recreational water quality: comparison of two methodologies and their impacts on public health regulatory events

The frequency of poor-water-quality advisories issued in Milwaukee and Racine, Wisconsin, in the absence of identifiable sources of contamination brought into question the reliability of the present indicator organism, Escherichia coli. Enteroccoci have been suggested as an alternative to E. coli for freshwater monitoring due to their direct correlation to swimmer-associated gastroenteritis. The purpose of this research was threefold: (i) to explore enterococci as an alternative to E. coli for monitoring freshwater Lake Michigan beaches, (ii) to evaluate the impact of the two indicators on regulatory decisions, and (iii) to compare membrane filtration m-enterococcus agar with indoxyl-beta-D-glucoside to a chemical substrate technique (Enterolert) for the recovery of enterococci. Recreational water samples from Milwaukee (n = 305) and Racine (n = 153) were analyzed for the enumeration of E. coli and enterococci using IDEXX Colilert-18 and Enterolert. Correlation between the indicators was low (R(2) = 0.60 and 0.69). Based on U.S. Environmental Protection Agency bacterial indicator threshold levels of risk for full body immersion, using enterococci would have resulted in 56 additional unsafe-recreational-water-quality advisories compared to the total from using E. coli and the substrate-based methods. A comparison of the two enterococcal methods (n = 124) yielded similar results (R(2) = 0.62). This was further confounded by the frequent inability to verify enterococci from those wells producing fluorescence by the defined substrate test using conventional microbiological methods. These results suggest that further research is necessary regarding the use of defined substrate technology interchangeably with the U.S. Environmental Protection Agency-approved membrane filtration test for the detection of enterococci from fresh surface water.     

Effect of Psychiatric Intervention in Attempted Suicide: A Controlled Study

All patients presenting at the casualty department of King''s College Hospital during the first six months of 1968 with deliberate self-poisoning or self-injury were followed up. Of 211 patients 204 (97%) were traced after a mean interval of 18 months (range one to two years). Despite official hospital policy, 22% had not been seen by a psychiatrist before discharge; these 44 untreated patients were compared with the remaining 160 who had received either brief (one or two interviews) or more prolonged psychiatric and social help.Subsequent suicidal attempts occurred significantly more often among untreated than among treated patients, prolonged treatment being associated with the best prognosis. The same trend was observed in respect of actual suicide, though the numbers were small and differences did not reach statistical significance. These findings held good when the untreated and treated groups were controlled for other variables which were found to be correlated with outcome. These results indicate that psychiatric intervention is associated with a significant reduction in subsequent suicidal behaviour.     

(+)- and (-)-N-allylnormetazocine binding sites in mouse brain: in vitro and in vivo characterization and regional distribution

In vivo and in vitro binding studies, both in whole brain and in selected areas, indicate that non-identical (+)- and (-)-NANM sites exist in the mouse brain, and each exhibits a different regional distribution. The in vivo binding of (+)-3H-NANM was found to be saturable at pharmacologically relevant doses, and represents a relatively small (10-22%) portion of total brain (+)-3H-NANM concentrations. The in vivo binding of (+)-3H-NANM was selectively displaced by (+)-NANM and PCP, and more sensitive to haloperidol and (+)-ketocyclazocine than the (-)-3H-NANM site. The in vivo binding of (-)-3H-NANM was selectively displaced by (-)-NANM, and more sensitive to naloxone and (-) ketocyclazocine than the (+)-3H-NANM site, and insensitive to PCP. This study indicates that the investigation of NANM binding sites is possible using in vivo binding techniques, and that each isomer apparently binds, in the mouse brain, to a single class of distinct sites.     

Quantitative real-time PCR and fluorescence in situ hybridization approaches for enumerating <Emphasis Type="Italic">Brevundimonas diminuta</Emphasis> in drinking water

Brevundimonas diminuta is a small Gram-negative bacterium used for validation of membranes and filters used in the pharmaceutical and drinking water treatment industries. Current assays are time consuming, nonselective, and may be subject to interference by competing indigenous microorganisms. The focus of this study is to develop rapid and specific enumeration methodologies for B. diminuta. Quantitative real-time polymerase chain reaction (qPCR) and fluorescence in situ hybridization (FISH) assays were developed based on the gyrB (1,166 bp) and rpoD (829 bp) gene sequences of B. diminuta ATCC 19146. Species-specific primers and probes were designed, and a 100–200 bp segment of each gene was targeted in the qPCR studies. For both the qPCR and FISH assays, an internal 25 bp sequence was selected for use as a TaqMan probe (labeled with 6-FAM and a Black Hole Quencher). Probe specificity studies, conducted against Gram-negative and Gram-positive reference strains as well as environmental strains, revealed high specificity of the primer/probe pairs to B. diminuta. Sensitivities of the qPCR reactions using purified genomic DNA from B. diminuta were determined to be 0.89 pg for rpoD and 8.9 pg for gyrB. The feasibility of using whole-cell B. diminuta suspensions directly with the rpoD qPCR protocol was also evaluated. The greatest sensitivity observed for B. diminuta was 1 × 10³ colony forming units (CFU) per mL when tryptic soy broth was used as the growth medium. When compared with direct microscopic enumeration using a 5′ 6-FAM FISH probe, traditional plating methods showed significant underestimation of B. diminuta concentration (P = 0.01) when this organism was cultivated in saline lactose broth. The results of this investigation demonstrate that qPCR and FISH are effective methods for rapid (<4 h) enumeration of B. diminuta and may be viable alternatives to plating when validating drinking water filtration systems.