The Plan-Do-Study-Act (PDSA): An Iterative Approach to Optimize Residents Performance in the American Board of Surgery in-Training Exam (ABSITE).

INTRODUCTION: American Board of Surgery (ABS) In-Training Examination (ITE), or ABSITE, preparation requires an effective study approach. In 2014, the ABS announced the alignment of ABSITE to the SCORE® Curriculum. We hypothesized that implementing a Plan-Do-Study-Act (PDSA) approach would help surgery residents improve their performance on the ABSITE. METHOD: Over 20 years, in a single institution, residents' ABSITE performance was evaluated over 3 timeframes: Time A (2004-2013), no specific curriculum; Time B (2014-2019), an annual comprehensive ABSITE-simulated SCORE®-based multiple-choice exam (MCQ) was administered; and Time C (2020-2023), like Time B with the addition of the PDSA approach for those with less than 60% correct on the ABSITE-simulated SCORE®-based exam. At the beginning of the academic year, in July, all residents are encouraged to (1) initiate a study plan for the upcoming ABSITE using SCORE® guided by the published ABSITE outlines content topics (Plan), (2) take an ABSITE-simulated SCORE®-based exam in October (Do), (3) assess the results/scores (Study), and (4) identify appropriate next steps (Act). Correlational analysis was performed to evaluate the association between ABSITE scores and ABSITE-simulated SCORE®-based exam scores in Time B and Time C. The primary outcome was the change in the proportions of ABSITE scores <30th percentile. RESULTS: A total of 294 ABSITE scores of 94 residents (34 females and 60 males) were analyzed. We found stronger correlation between the correct percentage on ABSITE and ABSITE-simulated SCORE®-based exam scores in Time C (r = 0.73, p < 0.0001) compared to Time B (0.62, p < 0.0001). The percentage of residents with ABSITE scores lower than 30th percentile dropped significantly from 14.0% to 3.7% (p = 0.016). CONCLUSION: Implementing the Plan-Do-Study-Act (PDSA) approach using the SCORE® curriculum significantly enhances residents' performance on the ABSITE exam. Surgery residents are encouraged to use this approach and to utilize the SCORE-contents outlined by the ABS in their study plan.

Yeast Expressing a Phage Endolysin Reduces Endogenous Clostridium perfringens Ex Vivo in 21-Day-Old Broiler Chicken Intestinal Fluids.

The phage endolysin PlyCP41 when purified from Escherichia coli exhibits lytic activity against Clostridium perfringens (CP) in vitro. The anti-clostridial activity of PlyCP41 endolysin expressed in transgenic yeast (Saccharomyces cerevisiae) was verified in phosphate buffered saline via mixing experiments with cultured CP and transgenic yeast slurries followed by serial dilution plating and colony counts on tryptose sulfite cycloserine (CP indicator) plates. The transgenic yeast containing PlyCP41 resulted in a log10 4.5 reduction (99.997%; P < 0.01) of the cultured CP. In addition, this serial dilution plating assay was used to demonstrate that transgenic yeast slurries could reduce the endogenous CP content in fluids from three different gastrointestinal regions (proximal, medial, and distal) from 21-day-old broiler chickens. The transgenic yeast treatment of gut slurries resulted in a log 10 1.19, 4.53, and 1.28 reduction in proximal, medial, and distal gut slurries (90% to 99.99% of the endogenous CP; P < 0.01), respectively, compared to nontreatment controls. These results indicate that the phage endolysin PlyCP41 expressed in S. cerevisiae is effective at reducing the endogenous CP in gastrointestinal fluids of broiler chickens. Future studies will measure the anti-CP effect in vivo by administering transgenic yeast to broiler chickens in the feed.

Levadura que expresa una fago-endolisina reduce la presencia endógena de Clostridium perfringens Ex vivo en fluidos intestinales de pollos de engorde de 21 días. La fago endolisina PlyCP41, cuando se purifica a partir de Escherichia coli, exhibe actividad lítica contra Clostridium perfringens (Cp) in vitro. La actividad anticlostridial de la endolisina PlyCP41 expresada en levadura transgénica (Saccharomyces cerevisiae) se verificó en solución salina amortiguada con fosfato mediante experimentos de mezclas con cultivos de C. perfringens y suspensiones de levadura transgénica, seguido de cultivos de diluciones en serie y recuentos de colonias en placas de triptosa sulfito cicloserina (TSC; indicador para C. perfringens). La levadura transgénica que contenía PlyCP41 dio como resultado una reducción de log10 4.5 (99.997%; P <0.01) en el cultivo de C. perfringens. Además, este ensayo de dilución en serie en placas se utilizó para demostrar que las suspensiones de levadura transgénica podrían reducir el contenido de C. perfringens endógeno en fluidos de tres regiones gastrointestinales diferentes (proximal, medial y distal) de pollos de engorde de 21 días de edad. El tratamiento con levadura transgénica de las suspensiones intestinales dio como resultado una reducción de log10 de 1.19, 4.53 y 1.28 en las suspensiones intestinales proximal, medial y distal (90% a 99.99 % de C. perfringens endógena; P < 0.01), respectivamente, en comparación con los controles no tratados. Estos resultados indican que la fago-endolisina PlyCP41 expresada en S. cerevisiae es eficaz para reducir el contenido endógeno de C. perfringens en los fluidos gastrointestinales de pollos de engorde. Los estudios futuros medirán el efecto contra C. perfringens in vivo mediante la administración de levadura transgénica a pollos de engorde en el alimento.

Do Surgery Residents Prepare Enough for Surgical Cases?

IMPORTANCE: This study aimed to identify both modifiable and nonmodifiable factors that affect intraoperative-specific surgical education and performance, with an overall goal of increasing cognizance of such factors to improve surgical training. OBJECTIVE: To determine whether surgery residents prepare adequately for participation in surgical cases and to examine specific variables that affect resident preparation. DESIGN: This study is a retrospective survey-based study that included data from 1945 postoperative case evaluations completed by 59 different general surgery residents over a period of 8 years (2014-2022). SETTING: A Midwestern medical school's general surgery residency program. PARTICIPANTS: Fifty-nine general surgery residents at Western Michigan University's medical school; 50 attending surgeons and faculty with whom residents regularly operate. The sample was comprised of residents and attendings who voluntarily filled out postoperative performance surveys after elective cases. RESULTS: This retrospective survey-based study included postoperative evaluation data from 1945 procedures performed by 59 different residents and 50 attendings. Participants included 36 male residents, 23 female residents, 39 male attendings, and 11 female attendings. All included data were for elective cases. Self-reported preoperative communication was worst at the PGY1 level with positive correlation of improvement yearly (r = 0.30, p < 0.001). Positive correlation was seen between overall preparedness and case complexity (r = 0.25, p < 0.001). Positive correlation was seen between case complexity and resident perception of intraoperative teaching quality (r = 0.53, p < 0.001). Preoperative communication initiated by residents was significantly worse when the attending surgeon was female, regardless of resident gender (p < 0.001); this effect was particularly profound with male residents. Male residents overall rated themselves as more prepared compared to their female counterparts (11.13 ± 1.96 vs. 10.84 ± 2.03, p = 0.003). CONCLUSIONS AND RELEVANCE: There is a need to identify and address quantifiable gaps in communication between residents and faculty to optimize surgical education; one of the first steps is characterizing nonmodifiable factors that correlate with differences in pre-operative communication and case preparation.

The importance of USMLE step 2 on the screening and selection of applicants for general surgery residency positions.

Background: As announced by the Federation of State Medical Boards (FSMB) and National Board of Medical Examiners (NBME), the United States Medical Licensing Examination (USMLE) Step 1 score reporting has transitioned to pass/fail outcomes instead of the traditional numeric score after January 26, 2022. USMLE Step 1 scores have been used widely as a crucial tool in screening and selecting applicants for residency programs. This study aims to determine the role of USMLE Step 2 in the selection of applicants for general surgery residency. Methods: A retrospective study was conducted over six recruiting cycles from 2016 to 2021. The data from 334 interviewed applicants from one general surgery residency program were assessed. Data analyzed included USMLE Step 1 and Step 2 scores, applicant gender, Alpha Omega Alpha (AOA) status, letters of recommendation (LOR), and research/publications (RS). Results: Of the 334 interviewed applicants, 209 (62.6%) were male. The mean [SD] USMLE Step 1 and USMLE Step 2 C K (Clinical Knowledge) scores were 239.6 [±10.4] and 249.2 [±11.4], respectively. The mean (SD) LOR and RS scores were 4.24 [±0.4] and 3.9 [±0.7], respectively. A positive correlation was observed between USMLE Step 1 and USMLE Step 2 C K (Clinical Knowledge) scores (r = 0.60, p < .001), LOR scores (r = 0.24, p = .008), and AOA status (r = 0.19, p = .038). There was a negligible correlation between USMLE scores and applicant gender. Conclusion: Transitioning USMLE Step 1 to pass/fail will make the initial screening and selection process of applications challenging for residency programs. In the short term, USMLE Step 2 scores, LOR, and AOA status are important as screening assessments. Valid measures to ensure appropriate, equitable, and fair assessments are needed.

Resident Autonomy and Performance Independence in Surgical Training Are Time- and Skill-Dependent.

INTRODUCTION: Appropriate faculty supervision and conditional independence of residents during training are required for autonomous and independent postgraduate practice. However, there is a growing concern that competence for transition to independent practice is not universally met. We hypothesize that surgery residents play a significant and active role in achieving their own independent status. METHODS: Over seven academic years (July 2014 through June 2021), 46 surgeons supervised and intraoperatively assessed the performance of 51 residents using validated Objective Structured Assessment of Technical Skill (OSATS) and Zwisch Operative Autonomy (ROA) assessments. Resident readiness to perform procedures independently (RRI) was graded as yes, no, or not applicable. Data were analyzed using descriptive statistics with categorical variables reported as frequencies and percentages. RESULTS: A total of 1657 elective procedures were performed by residents supervised by faculty. Association between RRI and postgraduate year (PGY), OSATS scores, ROA, resident and faculty gender, and case complexity was analyzed. Results indicated positive correlation between RRI and summative OSATS score (r = 0.510, P < 0.001), PGY (r = 0.535, P < 0.001) and ROA (r = 0.473, P < 0.001). Percentage of overall RRI increased from 7% at PGY1 to 87.4% at PGY5. Meaningful autonomy ratings increased from 23.6% at PGY1 to 92.5% at PGY5. Variations in ratings was observed when considering case category and complexity. CONCLUSIONS: RRI increases with years of training with variation when considering the specialty/The Accreditation Council for Graduate Medical Education procedure category and the complexity of cases. Specialty fellowships are a viable option to address the gap in The Accreditation Council for Graduate Medical Education categories when residency alone cannot reach appropriate independence. Residents' technical skills play a crucial role in evaluating RRI and granting operative autonomy.

Effects of Feeding Varying Levels of DL-Methionine on Live Performance and Yield of Broiler Chickens.

This study was designed to evaluate the effects of dietary supplemental DL-methionine (MET) on live performance and meat yield for broilers raised to a common weight. A total of 1552 one-day old Ross 708, sexed broilers were randomly distributed to 32 pens resulting in eight treatments (TRT) of four replicates with 44 male or 53 female/pen. A randomized complete block with a 2 × 4 (sex × 4 MET levels 0, 0.5, 1, and 2 g/kg) factorial arrangement of TRT was used. A common weight of 2400 g was approached by day 46 (1 and 2 g MET/kg feed) and day 48 (0 and 0.5 g MET/kg feed). Supplementation of MET at 1, and 2 g/kg had a lower (p < 0.01) feed conversion ratio (FCR) at day 46/48 than broilers fed 0.5 g MET/kg. Broilers without supplemental MET had the worst (p < 0.01) feed conversion and average daily gain (ADG) at day 46/48. Birds fed 0 g MET/kg of feed had lower (p < 0.05) whole eviscerated carcass without giblets (WOG), yield than birds fed 2 g MET/kg of feed. Additionally, birds fed 0 g MET/kg of feed had lower (p < 0.05) breast fillet and tender percent yields than birds fed supplemental MET. Elimination of MET from organic broiler diets resulted in reduced ADG, breast fillet yield and feed efficiency of meat yield of broilers raised to day 46/48. Reduction in MET supplementation below current levels reduced the efficiency of meat production of organic broilers raised to day 46/48.

Vascular Anomalies Associated with Esophageal Atresia and Tracheoesophageal Fistula.

OBJECTIVE: To report the incidence of congenital vascular anomalies in a cohort of patients with esophageal atresia (EA) and tracheoesophageal fistula (TEF) while describing the clinical presentation, diagnosis, and consequences, and to evaluate the diagnostic value of esophagram in diagnosing an aberrant right subclavian artery (ARSA). METHODS: All patients born with EA/TEF between 2005 and 2013 were studied. Preoperative echocardiography reports, surgical descriptions of primary esophageal repair, and esophagrams were reviewed retrospectively. RESULTS: Of the 76 children born with EA/TEF included in this study, 14 (18%) had a vascular malformation. The incidence of a right aortic arch (RAA) was 6% (5 of 76), and that of an aberrant right subclavian artery (ARSA) was 12% (9 of 76). RAA was diagnosed in the neonatal period by echocardiography (4 of 5) or surgery (1 of 5), and ARSA was diagnosed by echocardiography (7 of 9) or later on the esophagram (2 of 9). Respiratory and/or digestive symptoms occurred in 9 of the 14 patients with vascular malformation. Both long-gap EA and severe cardiac malformations necessitating surgery were significantly associated with vascular anomalies (P<.05). The sensitivity of the esophagram for diagnosing ARSA was 66%, the specificity was 98%, the negative predictive value was 95%, and the positive predictive value was 85%. CONCLUSION: ARSA and RAA have an incidence of 12% and 6% respectively, in patients with EA/TEF. A computed tomography angioscan is recommended to rule out such malformations when stenting of the esophagus is indicated, before esophageal replacement surgery, and when prolonged (>2 weeks) use of a nasogastric tube is considered.

Divergent effects of muscarinic receptor subtype gene ablation on murine colon tumorigenesis reveals association of M3R and zinc finger protein 277 expression in colon neoplasia.

BACKGROUND: M3 and M1 subtype muscarinic receptors are co-expressed in normal and neoplastic intestinal epithelial cells. In mice, ablating Chrm3, the gene encoding M3R, robustly attenuates intestinal tumor formation. Here we investigated the effects of Chrm1 gene ablation, alone and in combination with Chrm3 ablation. METHODS: We used wild-type, Chrm1-/-, Chrm3-/- and combined Chrm1-/-/Chrm3-/- knockout (dual knockout) mice. Animals were treated with azoxymethane, an intestine-selective carcinogen. After 20 weeks, colon tumors were counted and analyzed histologically and by immunohistochemical staining. Tumor gene expression was analyzed using microarray and results validated by RT-PCR. Key findings were extended by analyzing gene and protein expression in human colon cancers and adjacent normal colon tissue. RESULTS: Azoxymethane-treated Chrm3-/- mice had fewer and smaller colon tumors than wild-type mice. Reductions in colon tumor number and size were not observed in Chrm1-/- or dual knockout mice. To gain genetic insight into these divergent phenotypes we used an unbiased microarray approach to compare gene expression in tumors from Chrm3-/- to those in wild-type mice. We detected altered expression of 430 genes, validated by quantitative RT-PCR for the top 14 up- and 14 down-regulated genes. Comparing expression of this 28-gene subset in tumors from wild-type, Chrm3-/-, Chrm1-/- and dual knockout mice revealed significantly reduced expression of Zfp277, encoding zinc finger protein 277, in tissue from M3R-deficient and dual knockout mice, and parallel changes in Zfp277 protein expression. Notably, mRNA and protein for ZNF277, the human analogue of Zfp277, were increased in human colon cancer compared to adjacent normal colon, along with parallel changes in expression of M3R. CONCLUSIONS: Our results identify a novel candidate mouse gene, Zfp277, whose expression pattern is compatible with a role in mediating divergent effects of Chrm3 and Chrm1 gene ablation on murine intestinal neoplasia. The biological importance of this observation is strengthened by finding increased expression of ZNF277 in human colon cancer with a parallel increase in M3R expression. The role of zinc finger protein 277 in colon cancer and its relationship to M3R expression and activation are worthy of further investigation.

Prevalence and differentiation of diseases in Maryland backyard flocks.

Several epidemiologic surveillance studies have implicated backyard flocks as a reservoir for poultry diseases; however, much debate still exists over the risk these small flocks pose. To evaluate this concern, the prevalence of Newcastle disease (ND), infectious laryngotracheitis (ILT), Mycoplasma gallisepticum (MG), and Salmonella was determined in 39 Maryland backyard flocks. Serum, tracheal, and cloacal swabs were randomly collected from 262 birds throughout nine counties in Maryland. Through PCR and ELISA analysis, disease prevalence and seroprevalence were determined in flocks, respectively, for the following: ND (0%, 23%); ILT (26%, 77%); MG (3%, 13%); and Salmonella (0%, not done). Vaccine status could not be accurately confirmed. Premise positives were further differentiated and identified by partial nucleotide sequencing. Screening of the 10 ILT premise positives showed that most were live attenuated vaccines: eight matched a tissue culture origin vaccine, one matched a chicken embryo origin (CEO) vaccine, and one was CEO related. The single MG-positive flock, also positive for the CEO-related sequence, was identified as the infectious S6 strain. The prevalence rates for these economically important poultry diseases ranged from none to relatively low, with the vast majority of sampled flocks presenting no clinical signs.

CANDIDATE GENES FOR LIMITING CHOLESTATIC INTESTINAL INJURY IDENTIFIED BY GENE EXPRESSION PROFILING.

The lack of bile flow from the liver into the intestine can have devastating complications including hepatic failure, sepsis and even death. This pathologic condition known as cholestasis can result from etiologies as diverse as total parenteral nutrition (TPN), hepatitis and pancreatic cancer. The intestinal injury associated with cholestasis has been shown to result in decreased intestinal resistance, increased bacterial translocation and increased endotoxemia. Anecdotal clinical evidence suggests a genetic predisposition to exaggerated injury. Recent animal research on two different strains of inbred mice demonstrating different rates of bacterial translocation with different mortality rates supports this premise. In this study, a microarray analysis of intestinal tissue following common bile duct ligation (CBDL) performed under general anesthesia on these same two strains of inbred mice was done with the goal of identifying the potential molecular mechanistic pathways responsible. Over 500 genes were increased more than 2.0 fold following CBDL. The most promising candidate genes included MUPs, Serpina1a and LCN-2. RT-PCR validated the microarray results for these candidate genes. In an in vitro experiment using differentiated intestinal epithelial cells, inhibition of MUP-1 by siRNA resulted in increased intestinal epithelial cell permeability. Diverse novel mechanisms involving the growth hormone pathway, the acute phase response and the innate immune response are thus potential avenues for limiting cholestatic intestinal injury. Changes in gene expression were at times found to be not only due to the CBDL but also due to the murine strain. Should further studies in cholestatic patients demonstrate inter-individual variability similar to what we have shown in mice, then a "personalized medicine" approach to cholestatic patients may become possible.

Gut microbiota, tight junction protein expression, intestinal resistance, bacterial translocation and mortality following cholestasis depend on the genetic background of the host.

Failure of the intestinal barrier is a characteristic feature of cholestasis. We have previously observed higher mortality in C57BL/6J compared with A/J mice following common bile duct ligation (CBDL). We hypothesized the alteration in gut barrier function following cholestasis would vary by genetic background. Following one week of CBDL, jejunal TEER was significantly reduced in each ligated mouse compared with their sham counterparts; moreover, jejunal TEER was significantly lower in both sham and ligated C57BL/6J compared with sham and ligated A/J mice, respectively. Bacterial translocation to mesenteric lymph nodes was significantly increased in C57BL/6J mice vs. A/J mice. Four of 15 C57BL/6J mice were bacteremic; whereas, none of the 17 A/J mice were. Jejunal IFN-γ mRNA expression was significantly elevated in C57BL/6J compared with A/J mice. Western blot analysis demonstrated a significant decrease in occludin protein expression in C57BL/6J compared with A/J mice following both sham operation and CBDL. Only C57BL/6J mice demonstrated a marked decrease in ZO-1 protein expression following CBDL compared with shams. Pyrosequencing of the 16S rRNA gene in fecal samples showed a dysbiosis only in C57BL/6J mice following CBDL when compared with shams. This study provides evidence of strain differences in gut microbiota, tight junction protein expression, intestinal resistance and bacterial translocation which supports the notion of a genetic predisposition to exaggerated injury following cholestasis.

Avian influenza seroprevalence and biosecurity risk factors in Maryland backyard poultry: a cross-sectional study.

Major implications on a country's economy, food source, and public health. With recent concern over the highly pathogenic avian influenza outbreaks around the world, government agencies are carefully monitoring and inspecting live bird markets, commercial flocks, and migratory bird populations. However, there remains limited surveillance of non-commercial poultry. Therefore, a cross-sectional study was conducted in backyard poultry flocks using a convenience sampling method across three regions of Maryland from July 2011 to August 2011. The objective of this study was to develop a better understanding of the ecology and epidemiology of avian influenza by investigating the prevalence and seroprevalence in this potentially vulnerable population and by evaluating biosecurity risk factors associated with positive findings. Serum, tracheal, and cloacal swabs were randomly collected from 262 birds among 39 registered premises. Analysis indicated bird and flock seroprevalence as 4.2% (11/262) and 23.1% (9/39), respectively. Based on RT-qPCR analysis, none of the samples were found to be positive for AI RNA and evidence of AI hemagglutinin subtypes H5, H7, or H9 were not detected. Although no statistically significant biosecurity associations were identified (p≤0.05), AI seroprevalence was positively associated with exposure to waterfowl, pest control, and location. AI seropositive flocks exposed to waterfowl were 3.14 times as likely to be AI seropositive than those not exposed (p = 0.15). AI seropositive flocks that did not use pest control were 2.5 times as likely to be AI seropositive compared to those that did and AI seropositive flocks located in the Northern region of Maryland were 2.8 times as likely to be AI seropositive than those that were located elsewhere.

Evaluation of Maryland backyard flocks and biosecurity practices.

Domesticated poultry are susceptible to infectious and zoonotic diseases and can serve as a transmission source to other bird and human populations. In recent years, the number of noncommercial poultry has been on the rise in the United States. To evaluate potential risks of this growing population, a descriptive epidemiologic survey was conducted among Maryland backyard flocks. Owner and flock demographics were characterized as well as management practices such as husbandry, human-to-bird interaction, bird exposure risks, poultry health status, and biosecurity. Data from the 41 returned questionnaires indicated a median flock size of 38 birds (range, 3-901). Chickens accounted for 86.5% of the reported birds overall. Just over half of the owners (51.2%) kept chickens only, with the remaining backyard flocks consisting of chickens, other gallinaceous species, waterfowl, or a combination. Of flocks with multiple species, 70.0% of owners did not keep them separate. Almost two thirds of owners (61.0%) had kept poultry for < or = 5 yr, with 44.0% of all flocks on free range. Over the past 2 yr, predation was the highest cause of specific mortality (57.1%) followed by disease (30.2%), unknown (8.7%), and injury (4.0%), and over half of owners (56.1%) reported signs of disease in their flock within the last 6 mo. Biosecurity practices were highly variable among flocks. Data from this study identified gaps in the disease prevention and biosecurity practices of backyard flocks. These results can be useful in developing educational extension and outreach programs as well as policies, in efforts to further mitigate the spread of diseases.

Induced expression of STIM1 sensitizes intestinal epithelial cells to apoptosis by modulating store-operated Ca2+ influx.

INTRODUCTION: Apoptosis plays a critical role in the maintenance of gut mucosal epithelial homeostasis and is tightly regulated by numerous factors including intracellular Ca(2+). Canonical transient receptor potential channel-1 (TRPC1) is expressed in intestinal epithelial cells (IECs) and functions as a store-operated Ca(2+) channel. We have recently demonstrated that increased TRPC1 activity sensitizes IECs to apoptosis, but the upstream signaling initiating TRPC1 activation remains elusive. The novel protein, stromal interaction molecule 1 (STIM1), is shown to act as a store Ca(2+) sensor, and it can rapidly translocate to the plasma membrane where it directly interacts with TRPC1. The current study determined whether STIM1 plays an important role in the regulation of IEC apoptosis by activating TRPC1 channel activity. METHODS: Studies were conducted in IEC-6 cells (derived from rat intestinal crypts) and stable TRPC1-transfected IECs (IEC-TRPC1). Apoptosis was induced by tumor necrosis factor-α (TNF-α)/cycloheximide (CHX), and intracellular free Ca(2+) concentration ([Ca(2+)](cyt)) was measured by fluorescence digital imaging analysis. Functions of STIM1 were investigated by specific siRNA (siSTIM1) and ectopic overexpression of the constitutively active STIM1 EF-hand mutants. RESULTS: Stable STIM1-transfected IEC-6 cells (IEC-STIM1) showed increased STIM1 protein expression (~5 fold) and displayed a sustained increase in Ca(2+) influx after Ca(2+) store depletion (~2 fold). Susceptibility of IEC-STIM1 cells to TNF-α/CHX-induced apoptosis increased significantly as measured by changes in morphological features, DNA fragmentation, and caspase-3 activity. Apoptotic cells were increased from ~20% in parental IEC-6 cells to ~40% in stable IEC-STIM1 cells 4 h after exposure to TNF-α/CHX (p<0.05). In addition, stable IEC-TRPC1 cells also exhibited an increased sensitivity to TNF-α/CHX-induced apoptosis, which was prevented by STIM1 silencing through siSTIM1 transfection. STIM1 silencing by siSTIM1 also decreased Ca(2+) influx after store depletion in cells overexpressing TRPC1. Levels of Ca(2+) influx due to store depletion were decreased by ~70% in STIM1-silenced populations. Similarly, exposure of IEC-STIM1 cells to Ca(2+)-free medium also blocked increased sensitivity to apoptosis. CONCLUSIONS: These results indicate that (1) STIM1 plays an important role in the regulation of IEC apoptosis by altering TRPC1 activity and (2) ectopic STIM1 expression sensitizes IECs to apoptosis through induction in TRPC1-mediated Ca(2+) influx.

Polyamines and Gut Mucosal Homeostasis.

The epithelium of gastrointestinal (GI) mucosa has the most rapid turnover rate of any tissue in the body and its integrity is preserved through the dynamic balance between cell migration, proliferation, growth arrest and apoptosis. To maintain tissue homeostasis of the GI mucosa, the rates of epithelial cell division and apoptosis must be highly regulated by various extracellular and intracellular factors including cellular polyamines. Natural polyamines spermidine, spermine and their precursor putrescine, are organic cations in eukaryotic cells and are implicated in the control of multiple signaling pathways and distinct cellular functions. Normal intestinal epithelial growth depends on the available supply of polyamines to the dividing cells in the crypts, and polyamines also regulate intestinal epithelial cell (IEC) apoptosis. Although the specific molecular processes controlled by polyamines remains to be fully defined, increasing evidence indicates that polyamines regulate intestinal epithelial integrity by modulating the expression of various growth-related genes. In this review, we will extrapolate the current state of scientific knowledge regarding the roles of polyamines in gut mucosal homeostasis and highlight progress in cellular and molecular mechanisms of polyamines and their potential clinical applications.

Polyamines regulate E-cadherin transcription through c-Myc modulating intestinal epithelial barrier function.

The integrity of the intestinal epithelial barrier depends on intercellular junctions that are highly regulated by numerous extracellular and intracellular factors. E-cadherin is found primarily at the adherens junctions in the intestinal mucosa and mediates strong cell-cell contacts that have a functional role in forming and regulating the epithelial barrier. Polyamines are necessary for E-cadherin expression, but the exact mechanism underlying polyamines remains elusive. The current study was performed to determine whether polyamines induce E-cadherin expression through the transcription factor c-Myc and whether polyamine-regulated E-cadherin plays a role in maintenance of the epithelial barrier integrity. Decreasing cellular polyamines reduced c-Myc and repressed E-cadherin transcription as indicated by a decrease in levels of E-cadherin promoter activity and its mRNA. Forced expression of the c-myc gene by infection with adenoviral vector containing c-Myc cDNA stimulated E-cadherin promoter activity and increased E-cadherin mRNA and protein levels in polyamine-deficient cells. Experiments using different E-cadherin promoter mutants revealed that induction of E-cadherin transcription by c-Myc was mediated through the E-Pal box located at the proximal region of the E-cadherin promoter. Decreased levels of E-cadherin in polyamine-deficient cells marginally increased basal levels of paracellular permeability but, remarkably, potentiated H(2)O(2)-induced epithelial barrier dysfunction. E-cadherin silencing by transfection with its specific small interfering RNA also increased vulnerability of the epithelial barrier to H(2)O(2). These results indicate that polyamines enhance E-cadherin transcription by activating c-Myc, thus promoting function of the epithelial barrier.