The NuMA-related Mud protein binds Pins and regulates spindle orientation in Drosophila neuroblasts

Asymmetric cell division generates cell diversity during development and regulates stem-cell self-renewal in Drosophila and mammals. In Drosophila, neuroblasts align their spindle with a cortical Partner of Inscuteable (Pins)-G alpha i crescent to divide asymmetrically, but the link between cortical polarity and the mitotic spindle is poorly understood. Here, we show that Pins directly binds, and coimmunoprecipitates with, the NuMA-related Mushroom body defect (Mud) protein. Pins recruits Mud to the neuroblast apical cortex, and Mud is also strongly localized to centrosome/spindle poles, in a similar way to NuMA. In mud mutants, cortical polarity is normal, but the metaphase spindle frequently fails to align with the cortical polarity axis. When spindle orientation is orthogonal to cell polarity, symmetric division occurs. We propose that Mud is a functional orthologue of mammalian NuMA and Caenorhabditis elegans Lin-5, and that Mud coordinates spindle orientation with cortical polarity to promote asymmetric cell division.     

Ultrahigh resolution drug design. II. Atomic resolution structures of human aldose reductase holoenzyme complexed with Fidarestat and Minalrestat: implications for the binding of cyclic imide inhibitors

The X-ray structures of human aldose reductase holoenzyme in complex with the inhibitors Fidarestat (SNK-860) and Minalrestat (WAY-509) were determined at atomic resolutions of 0.92 A and 1.1 A, respectively. The hydantoin and succinimide moieties of the inhibitors interacted with the conserved anion-binding site located between the nicotinamide ring of the coenzyme and active site residues Tyr48, His110, and Trp111. Minalrestat's hydrophobic isoquinoline ring was bound in an adjacent pocket lined by residues Trp20, Phe122, and Trp219, with the bromo-fluorobenzyl group inside the "specificity" pocket. The interactions between Minalrestat's bromo-fluorobenzyl group and the enzyme include the stacking against the side-chain of Trp111 as well as hydrogen bonding distances with residues Leu300 and Thr113. The carbamoyl group in Fidarestat formed a hydrogen bond with the main-chain nitrogen atom of Leu300. The atomic resolution refinement allowed the positioning of hydrogen atoms and accurate determination of bond lengths of the inhibitors, coenzyme NADP+ and active-site residue His110. The 1'-position nitrogen atom in the hydantoin and succinimide moieties of Fidarestat and Minalrestat, respectively, form a hydrogen bond with the Nepsilon2 atom of His 110. For Fidarestat, the electron density indicated two possible positions for the H-atom in this bond. Furthermore, both native and anomalous difference maps indicated the replacement of a water molecule linked to His110 by a Cl-ion. These observations suggest a mechanism in which Fidarestat is bound protonated and becomes negatively charged by donating the proton to His110, which may have important implications on drug design.     

Crystal structure of the RNA polymerase domain of the West Nile virus non-structural protein 5

Viruses of the family Flaviviridae are important human and animal pathogens. Among them, the Flaviviruses dengue (DENV) and West Nile (WNV) cause regular outbreaks with fatal outcomes. The RNA-dependent RNA polymerase (RdRp) activity of the non-structural protein 5 (NS5) is a key activity for viral RNA replication. In this study, crystal structures of enzymatically active and inactive WNV RdRp domains were determined at 3.0- and 2.35-A resolution, respectively. The determined structures were shown to be mostly similar to the RdRps of the Flaviviridae members hepatitis C and bovine viral diarrhea virus, although with unique elements characteristic for the WNV RdRp. Using a reverse genetic system, residues involved in putative interactions between the RNA-cap methyltransferase (MTase) and the RdRp domain of Flavivirus NS5 were identified. This allowed us to propose a model for the structure of the full-length WNV NS5 by in silico docking of the WNV MTase domain (modeled from our previously determined structure of the DENV MTase domain) onto the RdRp domain. The Flavivirus RdRp domain structure determined here should facilitate both the design of anti-Flavivirus drugs and structure-function studies of the Flavivirus replication complex in which the multifunctional NS5 protein plays a central role.     

Cultivation of <Emphasis Type="Italic">Chlorella emersonii</Emphasis> with flue gas derived from a cement plant

The present study reviews the options of cultivating the green alga, Chlorella emersonii, under photoautotrophic conditions with flue gas derived from a cement plant. It was conducted in the Lafarge Perlmooser plant in Retznei, Austria, where stone coal and various surrogate fuels such as used tyres, plastics and meat-and-bone meal are incinerated for heating limestone. During 30 days of cultivation, flue gas had no visible adverse effects compared to the controls grown with pure CO₂. The semi-continuous cultivation with media recycling was performed in 5.5-L pH-stat photobioreactors. The essay using CO₂ from flue gas yielded a total of 2.00 g L⁻¹ microalgal dry mass and a CO₂ fixation of 3.25 g L⁻¹. In the control, a total of 2.06 g L⁻¹ dry mass was produced and 3.38 g L⁻¹ CO₂ was fixed. Mean growth rates were between 0.10 day⁻¹ (control) and 0.13 day⁻¹ (flue gas). No accumulation of flue gas residues was detected in the culture medium. At the end of the experiment, however, the concentration of lead was three times higher in algal biomass compared to the control, indicating that cultures aerated with this type of flue gas should not be used as food supplements or animal feed.     

Poor trail making test performance is directly associated with altered dual task prioritization in the elderly--baseline results from the TREND study

Background

Deterioration of executive functions in the elderly has been associated with impairments in walking performance. This may be caused by limited cognitive flexibility and working memory, but could also be caused by altered prioritization of simultaneously performed tasks. To disentangle these options we investigated the associations between Trail Making Test performance—which specifically measures cognitive flexibility and working memory—and dual task costs, a measure of prioritization.

Methodology and Principal Findings

Out of the TREND study (Tuebinger evaluation of Risk factors for Early detection of Neurodegenerative Disorders), 686 neurodegeneratively healthy, non-demented elderly aged 50 to 80 years were classified according to their Trail Making Test performance (delta TMT; TMT-B minus TMT-A). The subjects performed 20 m walks with habitual and maximum speed. Dual tasking performance was tested with walking at maximum speed, in combination with checking boxes on a clipboard, and subtracting serial 7 s at maximum speeds. As expected, the poor TMT group performed worse when subtracting serial 7 s under single and dual task conditions, and they walked more slowly when simultaneously subtracting serial 7 s, compared to the good TMT performers. In the walking when subtracting serial 7 s condition but not in the other 3 conditions, dual task costs were higher in the poor TMT performers (median 20%; range −6 to 58%) compared to the good performers (17%; −16 to 43%; p<0.001). To the contrary, the proportion of the poor TMT performance group that made calculation errors under the dual tasking situation was lower than under the single task situation, but higher in the good TMT performance group (poor performers, −1.6%; good performers, +3%; p = 0.035).

Conclusion

Under most challenging conditions, the elderly with poor TMT performance prioritize the cognitive task at the expense of walking velocity. This indicates that poor cognitive flexibility and working memory are directly associated with altered prioritization.     

Asymmetric ATP hydrolysis cycle of the heterodimeric multidrug ABC transport complex TmrAB from Thermus thermophilus

ATP-binding cassette (ABC) systems translocate a wide range of solutes across cellular membranes. The thermophilic gram-negative eubacterium Thermus thermophilus, a model organism for structural genomics and systems biology, discloses ～46 ABC proteins, which are largely uncharacterized. Here, we functionally analyzed the first two and only ABC half-transporters of the hyperthermophilic bacterium, TmrA and TmrB. The ABC system mediates uptake of the drug Hoechst 33342 in inside-out oriented vesicles that is inhibited by verapamil. TmrA and TmrB form a stable heterodimeric complex hydrolyzing ATP with a K(m) of 0.9 mm and k(cat) of 9 s(-1) at 68 °C. Two nucleotides can be trapped in the heterodimeric ABC complex either by vanadate or by mutation inhibiting ATP hydrolysis. Nucleotide trapping requires permissive temperatures, at which a conformational ATP switch is possible. We further demonstrate that the canonic glutamate 523 of TmrA is essential for rapid conversion of the ATP/ATP-bound complex into its ADP/ATP state, whereas the corresponding aspartate in TmrB (Asp-500) has only a regulatory role. Notably, exchange of this single noncanonic residue into a catalytic glutamate cannot rescue the function of the E523Q/D500E complex, implicating a built-in asymmetry of the complex. However, slow ATP hydrolysis in the newly generated canonic site (D500E) strictly depends on the formation of a posthydrolysis state in the consensus site, indicating an allosteric coupling of both active sites.     

Dual expression of mouse and rat VRL-1 in the dorsal root ganglion derived cell line F-11 and biochemical analysis of VRL-1 after heterologous expression.

The vanilloid-like TRP-channel VRL-1 (TRPV2) is a nonselective cation channel expressed by primary sensory neurons and non-neuronal tissues [Caterina, M.J., Rosen, T.A., Tominaga, M., Brake, A.J and Julius, D. (1999) Nature 398, 436-441]. It is one of the six members of the vanilloid-like TRP-channel family which is now termed the TRPV family [Montell, G., Birnbaumer, L., Flockerzi, V., Bindels, R.J., Brutford, E.A., Caterina, M.J., Clapham, D.E., Harteneck, C., Heller, S., Julius, D., Kojima, I., Mori, Y., Penner, R., Prawitt, D., Scharenberg, A.M., Schultz, G., Shimizu, N. and Zhu, M.X. (2002) Mol. Cell 2, 229-231]. As it is a temperature-gated channel, VRL-1 appears to be functionally related to VR1. In contrast to VR1, VRL-1 is activated at a higher temperature threshold and it does not respond to capsaicin or protons. Here we describe the expression of VRL-1 in the rat dorsal root ganglion-derived cell line F-11, a hybridoma of mouse neuroblastoma (N18TG2) and rat dorsal root ganglion cells. We found by RT-PCR that F-11 cells express not only the rat VRL-1, but also its mouse orthologue in a single cell. The F-11 parental cell line N18TG2 also expressed murine VRL-1. Due to its neuronal character, the DRG-derived F-11 cell line provides an experimental system for the study of VRL-1 biochemistry. However, one has to be aware that both the mouse and the rat protein are expressed simultaneously. Furthermore we cloned VRL-1 from rat brain and analyzed its glycosylation and localization in comparison to the endogenously expressed protein in F-11 cells. In contrast to the endogenous VRL-1 the overexpressed protein is glycosylated. Similar to VR1 the glycosylation is N-linked as shown by an deglycosylation assay. Immunofluorescence analysis of the endogenous VRL-1 in F-11 cells gives only weak signals in the cytoplasm whereas the overexpressed rat VRL-1 appears mainly at the plasma membrane.     

Genome editing of polyploid crops: prospects,achievements and bottlenecks

Plant breeding aims to develop improved crop varieties. Many crops have a polyploid and often highly heterozygous genome, which may make breeding of polyploid crops a real challenge. The efficiency of traditional breeding based on crossing and selection has been improved by using marker-assisted selection (MAS), and MAS is also being applied in polyploid crops, which helps e.g. for introgression breeding. However, methods such as random mutation breeding are difficult to apply in polyploid crops because there are multiple homoeologous copies (alleles) of each gene. Genome editing technology has revolutionized mutagenesis as it enables precisely selecting targets. The genome editing tool CRISPR/Cas is especially valuable for targeted mutagenesis in polyploids, as all alleles and/or copies of a gene can be targeted at once. Even multiple genes, each with multiple alleles, may be targeted simultaneously. In addition to targeted mutagenesis, targeted replacement of undesirable alleles by desired ones may become a promising application of genome editing for the improvement of polyploid crops, in the near future. Several examples of the application of genome editing for targeted mutagenesis are described here for a range of polyploid crops, and achievements and bottlenecks are highlighted.

     

Rat model of fractionated (2 Gy/day) 60 Gy irradiation of the liver: long-term effects

The liver is considered a radiosensitive organ. However, in rats, high single-dose irradiation (HDI) showed only mild effects. Consequences of fractionated irradiation (FI) in such an animal model have not been studied so far. Rats were exposed to selective liver FI (total dose 60 Gy, 2 Gy/day) or HDI (25 Gy) and were killed three months after the end of irradiation. To study acute effects, HDI-treated rats were additionally killed at several time points between 1 and 48 h. Three months after irradiation, no differences between FI and HDI treatment were found for macroscopically detectable small “scars” on the liver surface and for an increased number of neutrophil granulocytes distributed in the portal fields and through the liver parenchyma. As well, no changes in HE-stained tissues or clear signs of fibrosis were found around the portal vessels. Differences were seen for the number of bile ducts being increased in FI- but not in HDI-treated livers. Serum levels indicative of liver damage were determined for alkaline phosphatase (AP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltransferase (γGT) and lactate dehydrogenase (LDH). A significant increase of AP was detected only after FI while HDI led to the significant increases of AST and LDH serum levels. By performing RT-PCR, we detected up-regulation of matrix metalloproteinases, MMP-2, MMP-9, MMP-14, and of their inhibitors, TIMP-1, TIMP-2 and TIMP-3, shortly after HDI, but not at 3 month after FI or HDI. Overall, we saw punctual differences after FI and HDI, and a diffuse formation of small scars at the liver surface. Lack of “provisional clot”-formation and absence of recruitment of mononuclear phagocytes could be one explanation for scar formation as incomplete repair response to irradiation.