Gordonchu3828

Fluorescence microscopy is currently one of the more powerful and versatile techniques available for biological studies. With conventional biological immunofluorescence microscopy, caveolin-1 (CAV1) is visualized as numerous small dots, which are often distributed as a linear array or along the edge of the cell. Although its presence, as well as that of other proteins, can be detected by conventional immunofluorescence microscopy, those results do not clarify whether two different proteins exist in the plasma membrane of a specimen or how they are distributed two-dimensionally. Here, we describe an unroofing procedure that clearly reveals CAV1 localization in a single plane of the plasma membrane and also demonstrate a super-resolution structured illumination microscopy technique for observation of CAV1 in the plasma membrane.Recent evidence has indicated that caveolins are localized at the base of primary cilia, which are microtubule-based sensory organelles present on the cell surface, and that Caveolin-1 (CAV1) plays important roles in regulating ciliary membrane composition and function. Here we describe methods to analyze the localization and function of CAV1 in primary cilia of cultured mammalian cells. These include methods for culturing and transfecting mammalian cells with a CAV1-encoding plasmid or small interfering RNA (siRNA), analysis of mammalian cells by immunofluorescence microscopy (IFM) with antibodies against ciliary markers and CAV1, as well as methods for analyzing ciliary CAV1 function in siRNA-treated cells by IFM and cell-based signaling assays.Membrane topology information and views of membrane-embedded protein complexes promote our understanding of membrane organization and cell biological function involving membrane compartments. Freeze-fracturing of biological membranes offers both stunning views onto integral membrane proteins and perpendicular views over wide areas of the membrane at electron microscopical resolution. This information is directly assessable for 3D analyses and quantitative analyses of the distribution of components within the membrane if it were possible to specifically detect the components of interest in the membranes. Freeze-fracture replica immunolabeling (FRIL) achieves just that. In addition, FRIL preserves antigens in their genuine cellular context free of artifacts of chemical fixation, as FRIL uses chemically unfixed cellular samples that are rapidly cryofixed. In principle, the method is not limited to integral proteins spanning the membrane. Theoretically, all membrane components should be addressable as long as theron microscopy.Caveolae are small flask- or cup-shaped invaginations of the plasma membrane found in almost all vertebrate cells. Due to their small size (50-100 nm), transmission electron microscopy (TEM) has been the method of choice to study caveolae formation and ultrastructure and, more recently, to resolve the sub-caveolar localization of its protein components using novel protein labeling methods for TEM. This chapter describes a protocol for the selective visualization of caveolae and caveolar proteins by TEM, 3D tomography, and correlative light and electron microscopy (CLEM) using the peroxidase APEX2.Background Recent findings indicate a shift in the epidemiology of non-cardia gastric cancer in the USA. In particular, an uprising trend in incidence rates among non-Hispanic whites aged less then 50 years. Aim To examine secular trends in the incidence of non-cardia gastric cancer among adults aged less then 50 years in the USA by race/ethnicity and stage at diagnosis. Methods Age-adjusted incidence rates and trends in adults aged less then 50 years and ≥ 50 years were calculated using data from all 50 states in the National Program of Cancer Registries and the SEER program. We used joinpoint regression to compute average annual percent change (AAPC) in cancer incidence rates. Results Overall, we found an increasing trend of non-cardia gastric cancer among non-Hispanic whites aged less then 50 years between 2001 and 2014 (AAPC = 1.24, 95% CI 0.49, 1.99). However, among non-Hispanic whites aged less then 50 years, the rates of localized disease increased (AAPC = 5.28, 95% CI 3.94, 6.64), whereas the rates of distant stage non-cardia gastric cancer remained unchanged (AAPC = 0.68, 95% CI - 0.63, 2.00). Conversely, we found a significant increase in rates of distant stage non-cardia gastric cancer among Hispanics aged less then 50 years (AAPC = 1.78, 95% CI 0.66, 2.91). Non-cardia gastric cancer incidence rates decreased over the study period among non-Hispanic whites and Hispanics aged ≥ 50 years. Conclusion Given the rapid growth of the young Hispanic population in the USA, preventative strategies for non-cardia gastric cancer cannot neglect this population.Musculoskeletal pain and injuries (MSKPI) are common among gastroenterologists (GI) and GI fellows. Common areas of pain include the back, neck, hands/fingers, shoulders, and elbows. Although the prevalence of career-related pain and injuries among GIs is high, few endoscopists receive training in how to prevent MSKPI. We developed an ergonomics curriculum for our GI fellows that consisted of two modules that were led by physical therapists. Twelve out of 15 GI fellows, and one out of two hepatology fellows, participated in Module 1. Prior to the first module, 77% of participants reported pain in one or more body parts. Of those who reported pain, 100% of the fellows stated that this pain occurred during procedures, and 50% indicated this pain was performance-limiting. After completing Module 1, 100% of fellows reported that this was a valuable topic and 100% of the participants felt that this information would both help them feel and perform better. All fellows stated they had an immediate decrease in physical discomfort after engaging in the exercises that were included in Module 1. Eight fellows participated in Module 2. RG-7112 At the end of this module, 100% of fellows reported that this ergonomics training would likely help them to "physically perform better during procedures" and 100% of fellows indicated a reduction of physical discomfort (pain, aching) immediately after completing these exercises. Preliminary data indicated that this novel curriculum was perceived as valuable by GI fellows and that practicing these exercises reduced pain, particularly in the neck and the lower back.