Projecting microbe characteristics along with phylogenies

Before probing blots for the presence of an antigen, the total composition of the transferred proteins can be determined by staining the nitrocellulose or polyvinylidene fluoride (PVDF) membrane. Staining for proteins is useful to determine the position of the non-prestained molecular weight markers or individual lanes on the gel and to ensure that efficient transfer has occurred. It can be also used to verify equal loading of the samples in the gel when a comparison of the protein of interest between the different samples is important. The conventional procedures such as Coomassie Blue and silver staining methods used for staining polyacrylamide gels are incompatible with immunoblotting. Ponceau S is the more common staining method in immunoblotting protocols because it is compatible with antibody-antigen binding, is cost efficient, and provides a good contrast between the stained bands and background. In this protocol, nitrocellulose or PVDF membrane is rinsed with ultrapure H2O after the transfer of proteins. Ponceau S dye is applied as an acidic aqueous solution, and the proteins on the membrane are stained with red color. The membrane is briefly destained with water and can be photographed or scanned to obtain the image of the total protein staining. Individual lane positions or the molecular weight standards can be marked with a pencil, if required. © 2020 Cold Spring Harbor Laboratory Press.BACKGROUND Keratometry is clinically important and is routinely performed as part of human ophthalmic examination. In veterinary ophthalmology, little is known about keratometry in dogs, and its practical application has been limited. The present study aimed to describe keratometry in some dog breeds popular in Japan using a handheld keratometer. METHODS Client-owned dogs of various signalment were enrolled prospectively in the keratometry examination. check details Interbreed variations in mean corneal curvatures (R1R2avg) and corneal astigmatism (Δ(R1-R2)) were evaluated statistically with respect to their bodyweight based on the data which fulfilled the predetermined inclusion criteria. P less then 0.05 was considered statistically significant. RESULTS On examination of 237 dogs from 16 different breeds, R1R2avg (mean±sd) ranged from 7.54±0.30 mm in Pomeranians to 9.28±0.19 mm in golden retrievers. Δ(R1-R2) (mean±sd) ranged from 0.22±0.11 mm in miniature schnauzers to 0.57±0.30 mm in French bulldogs. CONCLUSION The present study successfully described keratometry in 16 dog breeds. The study revealed considerable interbreed variations in both R1R2avg and Δ(R1-R2), which did not necessarily correlate with bodyweight. These results are useful both clinically in fitting contact lenses in the management of corneal diseases and non-clinically in optometric studies in dogs. © British Veterinary Association 2020. Re-use permitted under CC BY-NC. No commercial re-use. Published by BMJ.Microtubule associated serine threonine like kinase (MASTL), also known as Greatwall kinase (Gwl), has an important role in the regulation of mitosis. By inhibiting protein phosphatase 2A, it plays a crucial role in activating one of the most important mitotic kinases known as cyclin dependent kinase1 (CDK1). MASTL has been seen to be up regulated in various types of cancers and is also involved in tumor recurrence. It is activated by CDK1 through phosphorylations in the activation/T-loop but the complete mechanism of its activation is still unclear. Here we report that AKT phosphorylates MASTL at T299 residue which plays a critical role in its activation. Our results suggest that AKT increases CDK1 mediated phosphorylation and hence activity of MASTL which in turn promotes mitotic progression through PP2A inhibition. We also show that the oncogenic potential of AKT is augmented by MASTL activation as the AKT mediated proliferation in colorectal cell lines can be attenuated by inhibiting and/or silencing MASTL. In brief, we report that AKT has an important role in the progression of mitosis in mammalian cells and it does so through the phosphorylation and activation of MASTL. Copyright © 2020 American Society for Microbiology.The metabolic state of the brain can greatly impact neurologic function. Evidence of this includes the therapeutic benefit of ketogenic diet in neurologic diseases, including epilepsy. However, brain lipid bioenergetics remain largely uncharacterized. The existence, capacity and relevance for mitochondrial fatty acid β-oxidation (FAO) in the brain is highly controversial, with few genetic tools available to evaluate this question. We have provided evidence for the capacity of brain FAO using a pan-brain-specific, conditional knockout (KO) mouse incapable of FAO due to the loss of carnitine palmitoyltransferase 2 (CPT2B-/-), an obligate gene for FAO. Loss of CNS FAO did not result in gross neuroanatomical changes or systemic differences in metabolism. Loss of CPT2 in brain did not result in robustly impaired behavior. We demonstrate that the mammalian brain oxidizes a substantial quantity of long-chain fatty acids in vitro and in vivo by unbiased and targeted metabolomics. Loss of CNS FAO results in robust accumulation of long-chain acylcarnitines in the brain, suggesting that the mammalian brain is mobilizing fatty acids for their oxidation, irrespective of diet or metabolic state. Together, these data demonstrate that the mammalian brain is oxidizing fatty acids under normal circumstances with little influence from or on peripheral tissues. Copyright © 2020 American Society for Microbiology.Proteasomes are essential protease complexes that maintain cellular homeostasis, and aberrant proteasomal activity supports cancer development. The regulatory mechanisms and biological function of the ubiquitin-26S proteasome has been studied extensively, while those of the ubiquitin-independent 20S proteasome system remain obscure. Herein, we show that the CNC family transcription factor NRF3 specifically enhances 20S proteasome assembly in cancer cells and 20S proteasomes contribute to colorectal cancer development through ubiquitin-independent proteolysis of the tumor suppressor p53 and Rb proteins. The NRF3 gene is highly expressed in many cancer tissues and cell-lines, and is important for cancer cell growth. In cancer cells, NRF3 upregulates assembly of the 20S proteasome by directly inducing gene expression of the 20S proteasome maturation protein POMP Interestingly, NRF3 knockdown not only increases p53 and Rb protein levels but also increased p53 activities for tumor suppression, including cell-cycle arrest and induction of apoptosis.