6 research outputs found
In vitro reconstitution of Slm1 mediated TORC2 activation
No full textGrowth is a fundamental property of cells and is regulated in response to nutritional/environmental conditions a cell is experiencing. This regulation comes from signaling pathways that cells possess. TOR, an essential eukaryotic kinase, through TORC1 and TORC2 regulates cellular/organismal growth homeostasis. In S. cerevisiae, TORC2 is anchored at plasma membrane in MCTs. Increase in PM tension causes Slm1 to relocate from eisosomes to MCTs to activate TORC2. However, mechanism of how Slm1 activate TORC2 remains unknown. We identified that Slm1 BAR and PH domains are sufficient to perform its functions. We show that Slm1 BAR domain contributes to PI(4,5)P2 binding indirectly through dimerization which brings two PI(4,5)P2 binding PH domains in close vicinity. We showed that Slm1 oligomerizes via it's BAR domain that remodels membranes. We show that oligomerization together with PI(4,5)P2 binding are essential for Slm1 to activate TORC2. We provide a biochemical reconstitution of Slm1 mediated TORC2 activation
Cloning, expression, purification, and characterisation of the HEAT-repeat domain of TOR from the thermophilic eukaryote Chaetomium thermophilum
No full textInternational audienc
Reliability of Real-Time Video Smartphone for Assessing National Institutes of Health Stroke Scale in Stroke Patients: The Next Generation of Telestroke
No full textabstract: Telestroke networks reduce disparities in acute stroke care between metropolitan primary stroke centers and remote hospitals. Current technologies used to conduct remote patient assessments have very high start-up costs, yet they cannot consistently establish quality connection in a timely manner. Smartphgones can be used for high quality video teleconferencing (HQ-VTC). They are relatively inexpensive and widley used among healthcare providers. We aimed to study the reliability of HQ-VTC using smartphones for conducting the NIHSS. Two vascular neurologists (VNs) assessed 83 stroke patients with the NIHSS. The remote VN assessed patients using videoconferencing on a smartphone with the assistance of a bedside medical aide. The bedside VN rated patients ontemporaneously. Each VN was blinded to the other's NIHSS scores. We tested the inter-method agreement and physician satisfaction with the device. We demonstrated high total NIHSS score correlation between the methods (r=0.941, p<0.001). The mean total NIHSS scores for bedside and remote assessments were 7.3 plus or minus 7.9 and 6.7 plus or minus 7.6 with ranges of 0-30 and 0-37, respectively. Seven NIHSS categories had significantly high agreement beyond chance: LOC-questions, LOC-commands, visual fields, motor left arm, motor right arm, motor left leg, motor right leg; seven categories had moderate agreement: LOC-consciousness, best gaze, facial palsy, sensory, best language, dysarthria, extinction/inattention; one category had poor agreement: ataxia. There was high physician satisfaction with the device. The VNs rated 96% of the assessments as good or very good for "image quality," "sound quality," "ease of use," and "ability to assess subject using NIHSS," and 84% of the assesssments as good or very good for "reception in hospital." The smartphones with HQ-VTC is reliable, easy to use, and affordable for telestroke NIHSS administration. This device has high physician satisfaction. With the variety of smartphones and professional medical applications available today, the telestroke practitioner has all the tools necessary for fast clinical decision-makingby accessing electronic medial records, viewing images, and tracking patient vitals
Substrate recruitment mechanism by gram-negative type III, IV, and VI bacterial injectisomes
No full textBacteria use a wide arsenal of macromolecular substrates (DNA and proteins) to interact with or infect prokaryotic and eukaryotic cells. To do so, they utilize substrate-injecting secretion systems or injectisomes. However, prior to secretion, substrates must be recruited to specialized recruitment platforms and then handed over to the secretion apparatus for secretion. In this review, we provide an update on recent advances in substrate recruitment and delivery by gram-negative bacterial recruitment platforms associated with Type III, IV, and VI secretion systems
Substrate recruitment mechanism by gram-negative type III, IV, and VI bacterial injectisomes
No full textBacteria use a wide arsenal of macromolecular substrates (DNA and proteins) to interact with or infect prokaryotic and eukaryotic cells. To do so, they utilize substrate-injecting secretion systems or injectisomes. However, prior to secretion, substrates must be recruited to specialized recruitment platforms and then handed over to the secretion apparatus for secretion. In this review, we provide an update on recent advances in substrate recruitment and delivery by gram-negative bacterial recruitment platforms associated with Type III, IV, and VI secretion systems
Biochemical characterization of MmgB, a gene encoding a 3-Hydroxybutyryl-CoA dehydrogenase from bacillus subtilis 168 and genetic evidence for the methylcitric acid cycle in bacillus subtilis 168
No full textBacillus subtilis is an industrially important organism because of its ability to produce enzymes and antibiotics on a commercial scale. It is considered the Gram positive counterpart of E. coli in terms of its genetic and molecular biological accessibility. Also, it is a model organism for the study of sporulation, an example of prokaryotic cellular differentiation. Sporulation involves several groups of genes that encode apparent metabolic pathways, many of which remain uncharacterized at the biochemical level. The mmg (mother cell metabolic gene) operon in B. subtilis strain 168 is one of these groups, and is transcribed only during an early stage of sporulation. This operon contains mmgABC, which are similar to genes from fatty acid metabolism, and mmgDE and yqiQ, which encode homologs of enzymes involved in the 2-methylcitric acid cycle. The focus of this work will be on the biochemical characterization of the mmgB protein, which is similar by sequence to 3-hydroxyacyl-coenzyme A (CoA) dehydrogenase from a wide variety of organisms. So far, we successfully cloned and overexpressed mmgB, and purified the protein at a yield of 4.0 mg/liter of culture. A spectrophotometric assay and mass spectrometry showed that this enzyme indeed possesses 3-hydroxybutyryl-CoA dehydrogenase activity (E.C. 1.1.1.157) for the production of acetoacetyl-CoA. This oxidation specifically requires NADP+, and has an optimal pH of 9.8. We will describe these results including the steady-state kinetics that the enzyme follows. As previously said, the downstream 3 ORF's of the mmg operon - mmgD, mmgE and yqiQ are proposed to encode for the methylcitric acid cycle. Also, as a part of a master's thesis, it has been shown by the Reddick lab that mmgD encodes for a citrate/methylcitrate synthase with a substrate preference for propionyl-CoA over acetyl-CoA. This fact encouraged us even more with reference to the involvement of the mmg operon in the methylcitric acid cycle, a pathway for propionate metabolism. So, our goal was to create conditional knockout mutants of the mmg operon and study the growth characteristics of the organism by feeding studies using propionate as sole carbon source. Eventually, we would also want to perform some NMR analysis of the cultures of the mutants for the intermediates of methylcitric acid cycle/propionate metabolism
