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BACKGROUND In the gastrointestinal (GI) epithelium, enterochromaffin (EC) cells are enteroendocrine cells that use tryptophan hydroxylase 1 (TPH‐1) to produce serotonin (5‐hydroxytryptamine, 5‐HT). EC cells release 5‐HT in response chemical and mechanical stimuli. EC cell 5‐HT affects GI secretion, motility and sensation. Other neuroendocrine cells are electrically excitable, and several types use SCN3A ‐encoded voltage‐gated sodium channel Na V 1.3 for electrical excitability. It is unknown whether EC cells are electrically excitable. AIM To determine if the EC cells are electrically excitable. METHODS Murine TPH1‐CFP small bowel was dissociated, FAC sorted for TPH1‐CFP cells, and ion channel expression was analyzed by RNA‐seq. Murine TPH1‐CFP and human small bowel and colon were analyzed by immunohistochemistry (IHC). After dissociation, primary cultures from TPH1‐CFP mouse jejunum and colon were maintained for 72 hours. Scn3a expression in CFP+, CFP− cultured cells and media was analyzed by single cell polymerase chain reaction (PCR). Voltage‐ and current‐clamp experiments were performed on TPH1‐CFP cells at 24–48 hours. RESULTS RNA‐seq of FACS TPH1‐CFP EC cells from murine small bowel showed that Scn3a was the 3 rd most densely expressed ion channel gene, and no other ScnXa genes showed significant expression. Scn3a encodes a voltage‐gated sodium channel Na V 1.3. By IHC, Na V 1.3 is expressed in ~89–93% of the human and mouse small bowel and colon EC cells, which were defined as TPH1‐CFP+ for mouse and 5‐HT+ for human. Single cell PCR of CFP+ cells from primary cultures confirmed Scn3a expression. In voltage‐clamp, we found voltage‐gated fast activating and inactivating voltage‐gated currents in TPH1‐CFP EC cells from murine small bowel (78% of cells, −86±16 pA/pF, n=28), and colon (65% of cells, −66±12 pA/pF, n=14). These currents were abolished by Na + substitution by NMDG + and blocked by the Na V 1.3 inhibitor ICA‐121431 (IC 50 = 131±53 nM). Current‐clamped EC cells showed both elicited and spontaneous action potentials, also abolished by Na + substitution and Na V 1.3 block by ICA‐121431. Spontaneous current‐clamp activity showed action potential bursts of variable amplitude and frequency. CONCLUSIONS SCN3A ‐encoded voltage‐gated sodium channel Na V 1.3 is densely and specifically expressed by the EC cells of the human and mouse small and large bowel. In mouse EC cells, Na V 1.3 generates voltage‐gated sodium current which is critical for EC cell electrical excitability. Support or Funding Information NIH DK106456, DK84567, DK052766

Despite a staggering 100,000 heart valve replacements performed in the United States per year, current glutaraldehyde‐fixed xenogeneic bioprosthetic replacements are far from ideal. Although glutaraldehyde‐fixation effectively masks tissue antigenicity in the short term, chronic graft‐specific immune responses still commonly occur, limiting valve life expectancy. Additionally, glutaraldehyde‐fixation crosslinks extracellular matrix proteins preventing remodeling and growth in juvenile patients. A heart valve bioprosthesis formed from an unfixed xenogeneic ECM scaffold, in which antigenic burden has been eliminated or significantly reduced, has potential to overcome deficiencies of current heart valve replacements and improve patient outcomes. Based on protein chemistry principles of sequential, differential solubilization, we hypothesized that amidosulfobetaines would improve lipophilic antigen solubilization and subsequent removal from bovine pericardium following antigen removal (BP‐AR). Antigen removal (AR) was achieved by subjecting native BP samples to hydrophilic AR (48 h), followed by lipophilic AR (hydrophilic solution plus ASB‐14 or ASB‐16 at 0, 1, 2, 5, 8 and 0.1, 2, 3, 4, 5% respectively) (48 h), nuclease digestion (24 h) and washout (48 h). Residual protein was extracted from minced BP‐AR samples using 0.1% sodium dodecyl sulfate (SDS) (hydrophile extract) followed by 1% SDS (lipophile extract). Hydrophile and lipophile extracts ( n = 6 per group) were assessed for residual antigenicity using 1‐DE western blots assessed for IgG positivity using anti‐BP immune serum. Uniaxial tensile testing was performed on BP‐AR scaffolds to determine Young's modulus and Ultimate Tensile Strength. No significant change in scaffold residual hydrophile antigenicity was found between groups, with an average reduction in hydrophile antigenicity of 93.59% compared to untreated control tissue. Both ASB‐14 and ASB‐16 demonstrated statistically significant concentration dependent decreases in residual lipophile antigenicity of BP‐AR, which plateaued at 5% for ASB‐14 and 3% for ASB‐16 (79% and 92% lipophilic antigen removal respectively) ( p < 0.0001). Compared to native BP, there were no significant changes in Young's modulus and Ultimate Tensile Strength between BP‐AR scaffolds generated in the presence of ASB‐14 at 5% or ASB‐16 at 3% ( p < 0.6198) This study demonstrates that increasing amidosulfobetaine concentration increases removal of lipophilic antigens from candidate xenogeneic heart valve biomaterial. The extent to which the achieved reduction in biomaterial antigenicity equates to avoidance of in vivo recipient graft‐specific adaptive immune responses remains to be determined. Additionally, future studies are necessary to quantify ECM protein composition and histology of the resultant xenogeneic BP‐AR scaffolds. However, BP‐AR scaffolds generated using 5% ASB‐14 or 3% ASB‐16 represent promising xenogeneic heart valve prosthesis biomaterials. Support or Funding Information National Institutes of Health (NIH) R01HL115205

Adenosine triphosphate (ATP) is postulated to be a major exercise related vasodilating metabolite. In humans, ATP infusion mimics exercise hyperemia. However, it remains unknown whether ATP can evoke the prolonged vasodilation seen during exercise. Therefore we tested two hypotheses during a three‐hour intra‐arterial infusion of a high dose of ATP (20 μgrams ·100 mL forearm vol −1 ·min −1 ): (1) skeletal muscle blood flow would wane over time (tachyphalaxis); (2) exercise hyperemic responses during ATP administration would be blunted compared to baseline. Nine healthy young participants (25 ± 1 years) performed seven rhythmic handgrip exercise bouts (20% of maximum), two during saline (control) and five during 180 minutes of continuous ATP infusion. Forearm vascular conductance (FVC; ml min −1 (100 mmHg) −1 ) and percent (%) change from control were calculated. Five minutes of ATP infusion resulted in a 710% increase in FVC (4.8±0.3 vs 35.0±1.9 ml min −1 *100 mmHg −1 , P<0.05). FVC did not wane over time with values of 35.0±1.9 and 36±2.6 ml min −1 (100 mmHg) −1 (P>0.05) seen prior to the exercise bouts at 5 vs 150 minutes respectively. With superimposed exercise FVC increased from 35.0±1.9 to 49.6±2.2 ml min −1 *100 mmHg −1 at five minutes and 36.0±2.6 to 54.5±1.1 at 150 min (P<0.05). Our findings demonstrate (1) ATP vasodilation is sustained over time without tachyphalaxis; (2) vasodilator responses to exercise during high dose ATP infusion remain intact. This latter finding questions the extent to which ATP is obligatory for exercise hyperemia. Support or Funding Information This research was supported by National Institutes of Health Research Grants HL‐119337 and by CTSA UL1 TR000135. The Caywood Professorship via the Mayo Foundation also supported this research.

Delivery of biomolecules to the correct subcellular locales is critical for proper physiological function. To that end, we have previously determined that the type I and II TGFβ receptors (TβRI and TβRII, respectively) localize to the basolateral domain in polarized epithelia. While TβRII targeting was shown to be regulated by sequences between amino acids 529–538, the analogous region(s) within TβRI is unknown. To address that question, sequential cytoplasmic TβRI truncations and point mutations identified a targeting motif between residues 158 to 163 (VxxEED) required for basolateral TβRI expression. Further studies documented that receptor internalization, downregulation, direct recycling, or Smad signaling were unaffected by motif mutations which caused TβRI mislocalization. However, a 70 amino acid stretch containing the targeting motif was able to direct basolateral expression of the apically sorted Neurotrophin receptor (p75; extracellular and transmembrane regions) in a dominant manner. Lastly, co‐expression of apically targeted type I and type II TGFβ receptors mediated Smad3 signaling from the apical membrane of polarized epithelial cells. These later findings demonstrate that the absence of apical TGFβ signaling in normal epithelia reflects domain‐specific receptor expression and not an inability to couple with the signaling machinery. Support or Funding Information This work was supported by Public Health Service grants GM‐055816 and GM‐054200 from National Institutes of General Medical Sciences.

Proteases are ubiquitous enzymes that catalyze the hydrolysis of peptide bonds within protein substrates; they have served as key model enzymes for studying the molecular basis for catalytic power and specificity. Protease substrate specificity is most often defined in terms of linear sequence motifs that flank the cleavage site; however, the natural substrates of proteases are proteins with 3‐dimensional shapes and complex conformational dynamics that are not well represented by 1‐dimensional sequence alone. These structural and dynamical properties can impact recognition and binding of substrates by proteases, as well as the efficiency of catalysis itself. In this study, we explore the importance of substrate structure and dynamics for proteolysis using as our model the cleavage of the Kunitz‐BPTI family of canonical serine protease inhibitors by mesotrypsin. Bovine pancreatic trypsin inhibitor (BPTI), an archetypal serine protease inhibitor of the Kunitz family, has a high affinity substrate‐like interaction with trypsin, yet is cleaved many orders of magnitude slower than other peptide substrates. Mesotrypsin, a trypsin variant, has been shown to hydrolyze Kunitz family inhibitors at accelerated rates; this is especially true of human Kunitz domain inhibitors. Amyloid precursor protein inhibitor (APPI) and amyloid precursor like protein‐2 Kunitz domain (APLP2‐KD), two human Kunitz domain family members, are hydrolyzed by mesotrypsin several hundred times faster than BPTI. Here, we present a new crystal structure of a cleavage intermediate of APLP2‐KD bound to mesotrypsin, refined to 1.4 Å resolution. This newly solved structure reveals a dramatic substrate conformational change that we hypothesize is required during Kunitz domain cleavage. Using this structure along with published APPI and BPTI complexes, we have modeled acyl‐enzyme intermediates of mesotrypsin and we have carried out molecular dynamic simulations to explore the conformational transformation that allows the progression of the hydrolysis reaction. We identify dynamic features of the inhibitor scaffolds, located far from the enzyme binding site, that correlate with observed inhibitor cleavage rates and plausibly explain their differential propensity to undergo both conformational transformation and cleavage. Our findings implicate global scaffold flexibility as a determinant of proteolytic rate, as rigid substrates possessing stabilizing features that render them highly resistant to conformational change are proteolyzed more slowly than more flexible substrates of similar structure. Support or Funding Information NIH R01 CA154387 Mayo Graduate school

Leadership, among other non‐technical discipline‐independent skills, has been identified as an important parameter in promoting patient safety and quality improvement in healthcare. While literature suggests that early exposure to non‐technical skill development positively correlates with improved technical skill, incorporating this into medical school curricula remains challenging. For first‐year medical students, team‐based learning environments provide an opportunity to demonstrate and reflect on leadership roles when assessment of non‐technical skills is incorporated as a formal component of their final grade. At Mayo Medical School, 20% (subjective faculty (10%) and peer (10%)) of the final anatomy and histology grade is awarded for performance of non‐technical skills. As part of our quality improvement initiative, we analyzed five consecutive years of data regarding leadership feedback in order to better understand: (1) student interpretation of leadership (2) student level of confidence in displaying leadership skills (3) student ability to critically assess leadership skills. Quantitative data from self and peer assessment were compared with qualitative analysis of leader and peer reflections. We found that leader assertiveness or lack thereof was a common theme in written feedback to team leaders, being mentioned at least once in 69% of all groups. We also discovered that 71% of leaders gave themselves a score lower than the aggregate team member score on leadership metrics. Despite this, score assignment on several individual components of leadership including respect, integrity, responsibility, compassion, problem‐solving, commitment to excellence, and overall professionalism was highly congruent between leaders and team members; leader selfscores matched aggregate team scores within two points 90% of the time. These results suggest that first‐year medical students strongly associate a proper level of assertiveness with leadership skills, are less confident in their own leadership abilities than the peers they are leading, and tend to agree with peers on relative areas of leadership strengths and weaknesses. Better understanding of medical student perception of leadership will help to direct leadership skill discussion during faculty‐driven direct feedback sessions.

There is a paucity of human in vitro models available to study the effects of alcohol in hepatocellular carcinoma (HCC). HCC cell lines are often deficient in either alcohol dehydrogenase (ADH) or cytochrome P450 2E1 (CYP2E1). VL‐17a is currently the most widely studied model available. VL‐17a is derived from a HepG2 cell line engineered to overexpress mouse ADH and human CYP2E1. LH86 is a novel HCC cell line previously developed in our laboratory. Here we compare the LH86 cell line with the current model to identify a novel tool to study the effects of alcohol in HCC. The aim of this study is to evaluate the LH86 cell line as a novel human in vitro model to study the effects of alcohol in HCC. The cell lines were cultured with alcohol and gene expression and epigenomic changes were identified. The LH86 cell line was observed to have unique functional properties indicating that it is a potential novel tool to study the effects of alcohol in HCC. Support or Funding Information R01 AA19976 TO KR AND CL

Use of ultrasound as a diagnostic tool is fast becoming an expectation in clinical practice. With formal outcomes now being included as competencies in many residency programs, earlier exposure to use of ultrasound is being achieved through informal activities embedded within medical school curricula. Using ultrasound to learn anatomy is now standard practice for many courses in the United States. However, very few if any offer formal opportunities to assess student skill and anatomical knowledge at the same time. As part of the Mini Ultrasound Skills Institute (MUSI) in Anatomy, a component of the first year anatomy course, students were expected to complete a team‐based, hands‐on ultrasound skills assessment. Throughout the course, students received both didactic briefing sessions and hands‐on training on ultrasound basics, image interpretation, and scanning techniques. The ultrasound content was presented in parallel to anatomy and radiology curriculum. In the past, ultrasound knowledge was assessed via static images; however, due to the contextual nature of ultrasound interpretation, a live, hands‐on method was devised to assess student learning. At regular intervals during the course, students completed an ultrasound skills assessment. In their dissection teams of four, students completed four tasks: setup the machine, obtained, oriented, and labeled a desired image using a live model. Each student was given a unique role within this exercise. Students were graded on a rubric scale for each of the four components and given an overall team score. At the conclusion of the course, students completed a graded ultrasound objective structural practical examination (OSPE). Rotating through four stations, students completed the four tasks on four different structures. Students rotated through roles within their team as they moved between stations. OSPE grade was incorporated into their final laboratory practical grade. Preliminary data from formative OSPE assessments ‐‐‐‐showed an average score of 72% (±21%) with a minimum of 50%, maximum of 100%. Within the skill specific breakdown, students were very comfortable at labeling obtained images (100%) and had the most challenging time with either selecting the correct probe or setting up the scanner 40% (±51%). Overall, team‐based OSPE is an effective method to promote and evaluate ultrasound skills learned in an anatomy course.

Life long high fat (HF) diet has been shown to cause more hepatic lipid accumulation in offspring as compared to a post‐weaning HF diet. However, it is still unclear what the epigenetic mechanisms are. We hypothesize that a life long HF diet will cause greater hepatic lipid accumulation as compared to a post‐weaning HF diet. The objective of this study is to understand the epigenetic mechanisms of hepatic lipid accumulation as an outcome of life long HF diet. Timed‐pregnant Sprague‐Dawley rats were given either a control (C, 16% fat) or HF (HF, 45% fat) diet during gestation and lactation. Pups from both C and HF mothers were weaned onto the same HF diet, creating two offspring groups: C/HF and HF/HF. Rats were sacrificed at 12 weeks of age and the left lobe of the liver was used for further analysis. Liver histology showed greater hepatic fat accumulation in the HF/HF group. Gene expression data was measured for multiple genes in the following pathways: glycolysis, gluconeogenesis, fatty acid synthesis, and triacylglycerol synthesis. Only glycerol‐3‐phosphate acyltransferase (GPAM) and fatty acid synthase (FASN) showed higher levels of mRNA. DNA methylation of specific genes was analyzed based on data obtained from methyl‐DNA immunoprecipitation with high‐throughput sequencing. Differential methylation was observed for both GPAM and FASN. Our data suggests that life long HF diet increases lipid storage by increased hepatic GPAM and FASN gene expression in male rats, potentially through epigenetic regulation.