The use of quantitative imaging to investigate regulators of membrane trafficking in Arabidopsis stomatal closure

Expansion of gene families facilitates robustness and evolvability of biological processes but impedes functional genetic dissection of signalling pathways. To address this, quantitative analysis of single cell responses can help characterize the redundancy within gene families. We developed high‐throughput quantitative imaging of stomatal closure, a response of plant guard cells, and performed a reverse genetic screen in a group of Arabidopsis mutants to five stimuli. Focussing on the intersection between guard cell signalling and the endomembrane system, we identified eight clusters based on the mutant stomatal responses. Mutants generally affected in stomatal closure were mostly in genes encoding SNARE and SCAMP membrane regulators. By contrast, mutants in RAB5 GTPase genes played specific roles in stomatal closure to microbial but not drought stress. Together with timed quantitative imaging of endosomes revealing sequential patterns in FLS2 trafficking, our imaging pipeline can resolve non‐redundant functions of the RAB5 GTPase gene family. Finally, we provide a valuable image‐based tool to dissect guard cell responses and outline a genetic framework of stomatal closure.      

High diversity of thermophilic cyanobacteria in Rupite hot spring identified by microscopy,cultivation, single-cell PCR and amplicon sequencing

Extremophiles - Genotypic and morphological diversity of cyanobacteria in the Rupite hot spring (Bulgaria) was investigated by means of optical microscopy, cultivation, single-cell PCR, and 16S...     

The highly diverged trypanosomal MICOS complex is organized in a nonessential integral membrane and an essential peripheral module

The mitochondrial contact site and cristae organization system (MICOS) mediates the formation of cristae, invaginations in the mitochondrial inner membrane. The highly diverged MICOS complex of the parasitic protist Trypanosoma brucei consists of nine subunits. Except for two Mic10‐like and a Mic60‐like protein, all subunits are specific for kinetoplastids. Here, we determined on a proteome‐wide scale how ablation of individual MICOS subunits affects the levels of the other subunits. The results reveal co‐regulation of TbMic10‐1, TbMic10‐2, TbMic16 and TbMic60, suggesting that these nonessential, integral inner membrane proteins form an interdependent network. Moreover, the ablation of TbMic34 and TbMic32 reveals another network consisting of the essential, intermembrane space‐localized TbMic20, TbMic32, TbMic34 and TbMic40, all of which are peripherally associated with the inner membrane. The downregulation of TbMic20, TbMic32 and TbMic34 also interferes with mitochondrial protein import and reduces the size of the TbMic10‐containing complexes. Thus, the diverged MICOS of trypanosomes contains two subcomplexes: a nonessential membrane‐integrated one, organized around the conserved Mic10 and Mic60, that mediates cristae formation, and an essential membrane‐peripheral one consisting of four kinetoplastid‐specific subunits, that is required for import of intermembrane space proteins.     

Lipidomic Analysis of α-Synuclein Neurotoxicity Identifies Stearoyl CoA Desaturase as a Target for Parkinson Treatment

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Redirecting the lipid metabolism of the yeast Starmerella bombicola from glycolipid to fatty acid production

Journal of Industrial Microbiology & Biotechnology - Free fatty acids are basic oleochemicals implemented in a range of applications including surfactants, lubricants, paints, plastics, and...     

Evaluation of a DC pulsed magnetic tracking system in neurosurgical navigation: technique, accuracies, and influencing factors]

Navigation systems are useful instruments in cranial neurosurgery. For specification of position, so-called sensor-based navigation techniques use: (a) a signal emitter that generates a defined electromagnetic field in the area of the operation site; and (b) small sensors that detect the position of various operating instruments in the electromagnetic field. For a long time, owing to a lack of clinical data and long-term studies, electromagnetic systems have been regarded as error-prone and imprecise. With the development of a pulsed direct current (DC) technique, precision levels can now be reached that are comparable with those of established optical and mechanical measuring procedures. However, it must be noted that the influence on the measuring accuracy within the operating field increases with increasing susceptibility of the various metals used in the operating theatre (titanium相似文献   

The role of arginine 310 in catalysis and substrate specificity in xanthine dehydrogenase from Rhodobacter capsulatus

The rapid reaction kinetics of wild-type xanthine dehydrogenase from Rhodobacter capsulatus and variants at Arg-310 in the active site have been characterized for a variety of purine substrates. With xanthine as substrate, k(red) (the limiting rate of enzyme reduction by substrate at high [S]) decreased approximately 20-fold in an R310K variant and 2 x 10(4)-fold in an R310M variant. Although Arg-310 lies on the opposite end of the substrate from the C-8 position that becomes hydroxylated, its interaction with substrate still contributed approximately 4.5 kcal/mol toward transition state stabilization. The other purines examined fell into two distinct groups: members of the first were effectively hydroxylated by the wild-type enzyme but were strongly affected by the exchange of Arg-310 to methionine (with a reduction in k(red) greater than 10(3)), whereas members of the second were much less effectively hydroxylated by wild-type enzyme but also much less significantly affected by the amino acid exchanges (with a reduction in k(red) less than 50-fold). The effect was such that the 4000-fold range in k(red) seen with wild-type enzyme was reduced to a mere 4-fold in the R310M variant. The data are consistent with a model in which "good" substrates are bound "correctly" in the active site in an orientation that allows Arg-310 to stabilize the transition state for the first step of the overall reaction via an electrostatic interaction at the C-6 position, thereby accelerating the reaction rate. On the other hand, "poor" substrates bound upside down relative to this "correct" orientation. In so doing, they are unable to avail themselves of the additional catalytic power provided by Arg-310 in wild-type enzyme but, for this reason, are significantly less affected by mutations at this position. The kinetic data thus provide a picture of the specific manner in which the physiological substrate xanthine is oriented in the active site relative to Arg-310 and how this residue is used catalytically to accelerate the reaction rate (rather than simply bind substrate) despite being remote from the position that is hydroxylated.     

Determinants of RNA quality from FFPE samples

The large archives of formalin-fixed paraffin-embedded (FFPE) tissue specimens that exist are a highly valuable source of sample material for molecular biological analysis, including gene expression profiling. However, current data on adverse effects of standard pathological practice on the usefulness of biomolecular analytes obtained from such archived specimens is largely anecdotal. Here, we present a systematic examination of the most relevant parameters for integrity and useability of RNA obtained from FFPE samples, including storage time and conditions, fixation time, and specimen size. The results are particularly relevant for any application relying on cDNA synthesis as an initial step of the procedure, such as RT-PCR, and microarray analysis.     

A novel technique to propagate primary human preadipocytes without loss of differentiation capacity

Objective: The study of human preadipocytes is hampered by the limited availability of adipose tissue and low yield of cell preparation. Proliferation of preadipocytes using common protocols, including fetal bovine serum (FBS), results in a markedly reduced differentiation capacity. Therefore, we were interested in developing an improved culture system that allows the proliferation of human preadipocytes without loss of differentiation capacity. Research Methods and Procedures: Adipose tissue samples were taken from subjects undergoing elective abdominal surgery. Cells were seeded at various densities and cultured using different formulations of proliferation media including factors such as fibroblast growth factor‐2 (basic fibroblast growth factor), epidermal growth factor, insulin, and FBS either alone or in combination. Cells were counted and induced to differentiate after confluence. After complete differentiation, cells were harvested, and glycerol‐3‐phosphate dehydrogenase activity was measured. Cells were subcultured for up to five passages. Results: The proliferation medium with 4 growth factors (PM4), consisting of 2.5% FBS, 10 ng/mL epidermal growth factor, 1 ng/mL basic fibroblast growth factor, and 8.7 µM insulin, resulted in lower doubling times at all seeding densities tested (0.05 × 10⁴ to 1.5 × 10⁴) compared with medium supplemented with 10% FBS. In contrast to cells in FBS medium, cells grown with PM4 medium retained full differentiation rate (glycerol‐3‐phosphate dehydrogenase activity, 493 ± 215 vs. 41 ± 17 mU/mg, p < 0.01). Differentiation capacity was fully retained at least for up to three passages in PM4 medium. Discussion: The use of PM4 medium results in substantial proliferation of human preadipocytes with preserved differentiation capacity. This novel technique represents a valuable tool for the study of human adipose tissue development and function starting from small samples.     

Site-directed mutagenesis of the active site loop of the rhodanese-like domain of the human molybdopterin synthase sulfurase MOCS3. Major differences in substrate specificity between eukaryotic and bacterial homologs

Sequence alignments of human molybdopterin synthase sulfurase, MOCS3, showed that the N-terminal domain is homologous to Escherichia coli MoeB, whereas the C-terminal domain is homologous to rhodanese-like proteins. Previous studies showed that the activity of the separately purified rhodanese-like domain of MOCS3 displayed 1000-fold lower activity in comparison to bovine rhodanese with thiosulfate as sulfur source. When the six amino acid active site loop of MOCS3 rhodanese-like domain was exchanged with the loop found in bovine rhodanese, thiosulfate:cyanide sulfurtransferase activity was increased 165-fold. Site-directed mutagenesis of each individual residue of the active site loop of the MOCS3 rhodanese-like domain showed that the charge of the last amino acid determines thiosulfate sulfurtransferase activity. Replacing Asp417 by threonine resulted in 90-fold increased activity, whereas replacing it by arginine increased the activity 470-fold. Using a fully defined in vitro system containing precursor Z, MOCS2A, E. coli MoaE, E. coli MoeB, Mg-ATP, MOCS3 rhodanese-like domain, and thiosulfate, it was shown that sulfur transfer to MOCS2A was also affected by the alterations, but not as drastically. Our studies revealed that in humans and most eukaryotes thiosulfate is not the physiologic sulfur donor for MOCS3, whereas in bacterial homologs, which have an arginine at the last position of the active site loop, thiosulfate can be used as a sulfur source for molybdenum cofactor biosynthesis. The phylogenetic analysis of MoeB homologs showed that eukaryotic homologs are of bacterial origin. Furthermore, it could be shown that an MoeB homolog named MoeZ, where the dual CXXC zinc-binding motif of the MoeB domain is not present, arose independently several times during evolution.