Retinal and renal observations on dogs with spontaneous diabetes mellitus are presented. Retinal vessels were prepared by the trypsin digestion, flat retinal technic and examined by light microscopy. Kidneys were examined by light and electron microscopy. Nondiabetic dogs of comparable ages served as controls. Retinal vessels in some diabetic subjects showed mural pericyte degeneration with “ghost” formation and zones of focal acellularity surrounded by aneurysms or hypercellular capillary shunts. These changes were indistinguishable from those observed in human diabetic retinas prepared by the same technic. Kidneys of the diabetic dogs showed a significantly greater incidence and severity of diffuse glomerulosclerosis and thickening of the basement membrane of the peripheral glomerular capillary loops than was found in the controls. Nodular glomerular lesions of Kimmelstiel and Wilson were not observed. Cataracts were noted in all diabetic animals with diabetes known to be six months in duration or longer but were rare in the controls. The pancreas was either described grossly by the contributing veterinarian or submitted for histologic examination in eight of the diabetic dogs. Five of these suggested possible pancreatitis and their diabetes may be secondary to pancreatic disease. The remaining three dogs with no evidence of pancreatitis are considered as primary (possibly hereditary) diabetes mellitus. Retinopathy and prominent diffuse glomerulosclerosis occurred in diabetic dogs with and without evidence of pancreatitis.
Transcription factors are proteins that recognize specific DNA sequences and affect local transcriptional processes. They are the primary means by which all organisms control specific gene expression. Understanding which DNA sequences a particular transcription factor recognizes provides important clues into the set of genes that they regulate and, through this, their potential biological functions. Insights may be gained through homology searches and genetic means. However, these approaches can be misleading, especially when comparing distantly related organisms or in cases of complicated transcriptional regulation. In this work, we used a biochemistry-based approach to determine the spectrum of DNA sequences specifically bound by the Thermus thermophilus HB8 TetR-family transcription factor TTHB023. The consensus sequence 5′–(a/c)Y(g/t)A(A/C)YGryCR(g/t)T(c/a)R(g/t)–3′ was found to have a nanomolar binding affinity with TTHB023. Analyzing the T. thermophilus HB8 genome, several TTHB023 consensus binding sites were mapped to the promoters of genes involved in fatty acid biosynthesis. Notably, some of these were not identified previously through genetic approaches, ostensibly given their potential co-regulation by the Thermus thermophilus HB8 TetR-family transcriptional repressor TTHA0167. Our investigation provides additional evidence supporting the usefulness of a biochemistry-based approach for characterizing putative transcription factors, especially in the case of cooperative regulation.
Advances in genomic sequencing have allowed the identification of a multitude of genes encoding putative transcriptional regulatory proteins. Lacking, often, is a fuller understanding of the biological roles played by these proteins, the genes they regulate or regulon. Conventionally this is achieved through a genetic approach involving putative transcription factor gene manipulation and observations of changes in an organism’s transcriptome. However, such an approach is not always feasible or can yield misleading findings. Here, we describe a biochemistry-centric approach, involving identification of preferred DNA-binding sequences for the Thermus thermophilus HB8 transcriptional repressor TTHA0973 using the selection method Restriction Endonuclease Protection, Selection and Amplification (REPSA), massively parallel sequencing, and bioinformatic analyses. We identified a consensus TTHA0973 recognition sequence of 5′–AACnAACGTTnGTT–3′ that exhibited nanomolar binding affinity. This sequence was mapped to several sites within the T. thermophilus HB8 genome, a subset of which corresponded to promoter regions regulating genes involved in phenylacetic acid degradation. These studies further demonstrate the utility of a biochemistry-centric approach for the facile identification of potential biological functions for orphan transcription factors in a variety of organisms.
Protein-DNA binding interactions are critical in several biological processes,especiallythe regulation of gene expression at the level of transcription initiation. An important technique for studyingthese interactions is the electrophoretic mobility shift assay (EMSA), whereby protein-DNA complexes are resolved on the basis of their mass:charge ratio using native polyacrylamidegel electrophoresis (nPAGE).Here we describe EMSA using PCR-generated, near infrared-fluorescent DNA probes,and IR fluorescence imaging to qualitatively and quantitatively study the interaction of transcriptional regulatory proteins fromthermophilic organisms with different DNAs. Direct imaging of IR fluorophore-labeled DNA probes is advantageous because itprovides high sensitivity (subnanomolar) without the need for intermediate staining steps or costly and problematic radiolabeled probes,thereby providing a more affordable and sensitive option to image protein-DNA on polyacrylamide gels by techniques such as EMSA.
The precise molecular mechanisms of diabetic retinopathy (DR) pathogenesis are unclear, and treatment options are limited. There is an urgent need to discover and develop novel therapeutic targets for the treatment of this disease. Glycosylation is a post-translational modification that plays a critical role in determining protein structure, function, and stability. Recent studies have found that serum glycoproteomic changes are associated with the presence or progression of several inflammatory diseases. However, very little is known about the glycoproteomic changes associated with DR. In this study, glycoproteomic profiling of the serum of diabetic patients with and without DR was performed. A total of 15 glycopeptides from 11 glycoproteins were found to be significantly altered (5 upregulated and 10 downregulated) within the serum glycoproteome of DR patients. These glycoproteins are known to be involved in the maintenance of the extracellular matrix and complement system through peptidolytic activity or regulation.
Background The COVID-19 pandemic has disrupted traditional resident recruitment practices, requiring virtual interviews and new forms of outreach. Social media, such as Twitter, is one tool programs can use to connect with applicants. This study sought to assess changes in Twitter use during the COVID-19 pandemic among general surgery programs. Methods Twitter and residency program websites were queried for public Twitter accounts related to general surgery residency programs. Publicly available tweets for available accounts were reviewed for all posts for the period March 15, 2019-November 25, 2020. Thematic analysis of each tweet was performed, and engagement was determined by likes and retweets on each tweet. Results The number of programs with active Twitter accounts increased after the onset of COVID-19 pandemic, as did the number of tweets, likes-, and retweets-per-tweet. There was a significant increase in the number of tweets regarding resident promotion, program promotion, and virtual event promotion. Tweets received more likes-per-tweet if the subject was program promotion and resident promotion than tweets regarding virtual events. All results were statistically significant ( P < .05). Discussion Twitter use and engagement with residency programs have increased significantly since pandemic onset. Engagement is highest for tweets regarding program and resident promotion as measured by likes-per-tweet and highest for program promotion and virtual events as measured by retweets-per-tweet. Given the nearly nationwide increase in Twitter engagement after pandemic onset, programs should consider the impact of Twitter as a means of communication with applicants and program branding.
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