Douglas J. Turner

Surgical Site Infection Prevention: The Importance of Operative Duration and Blood Transfusion—Results of the First American College of Surgeons–National Surgical Quality Improvement Program Best Practices Initiative

BACKGROUNDnSurgical site infections (SSI) continue to be a significant problem in surgery. The American College of Surgeons-National Surgical Quality Improvement Program (ACS-NSQIP) Best Practices Initiative compared process and structural characteristics among 117 private sector hospitals in an effort to define best practices aimed at preventing SSI.nnnSTUDY DESIGNnUsing standard NSQIP methodologies, we identified 20 low outlier and 13 high outlier hospitals for SSI using data from the ACS-NSQIP in 2006. Each hospital was administered a process of care survey, and site visits were conducted to five hospitals. Comparisons between the low and high outlier hospitals were made with regard to patient characteristics, operative variables, structural variables, and processes of care.nnnRESULTnHospitals that were high outliers for SSI had higher trainee-to-bed ratios (0.61 versus 0.25, p < 0.0001), and the operations took significantly longer (128.3+/-104.3 minutes versus 102.7+/-83.9 minutes, p < 0.001). Patients operated on at low outlier hospitals were less likely to present to the operating room anemic (4.9% versus 9.7%, p=0.007) or to receive a transfusion (5.1% versus 8.0%, p=0.03). In general, perioperative policies and practices were very similar between the low and high outlier hospitals, although low outlier hospitals were readily identified by site visitors. Overall, low outlier hospitals were smaller, efficient in the delivery of care, and experienced little operative staff turnover.nnnCONCLUSIONSnOur findings suggest that evidence-based SSI prevention practices do not easily distinguish well from poorly performing hospitals. But structural and process of care characteristics of hospitals were found to have a significant association with good results.

Akt Kinase Activation Blocks Apoptosis in Intestinal Epithelial Cells by Inhibiting Caspase-3 after Polyamine Depletion

Apoptosis plays a critical role in the maintenance of gut mucosal homeostasis and is regulated by numerous factors including polyamines. Although the exact roles of polyamines in apoptotic pathway are still unclear, inhibition of polyamine synthesis promotes the resistance of intestinal epithelial cells to apoptosis. Akt is a serine-threonine kinase that has been established as an important intracellular signaling in regulating cell survival. The current studies test the hypothesis that polyamines are involved in the control of Akt activity in normal intestinal epithelial cells (IEC-6 line) and that activated Akt mediates suppression of apoptosis following polyamine depletion. Depletion of cellular polyamines by α-difluoromethylornithine induced levels of phosphorylated Akt and increased Akt kinase activity, although it had no effect on expression of total Akt, pERK, p38, and Bcl-2 proteins. This activated Akt was associated with both decreased levels of active caspase-3 and increased resistance to tumor necrosis factor-α/cycloheximide-induced apoptosis. Inactivation of Akt by either treatment with LY294002 or ectopic expression of a dominant negative Akt mutant (DNMAkt) not only enhanced the caspase-3 activation in polyamine-deficient cells but also prevented the increased resistance to tumor necrosis factor-α/cycloheximide-induced apoptosis. Phosphorylation of glycogen synthase kinase-3, a downstream target of Akt, was also increased in α-difluoromethylornithine-treated cells, which was prevented by inactivation of Akt by LY294002 or DNMAkt overexpression. These results indicate that polyamine depletion induces the Akt activation mediating suppression of apoptosis via inhibition of caspase-3 in normal intestinal epithelial cells.

miR-503 represses CUG-binding protein 1 translation by recruiting CUGBP1 mRNA to processing bodies

This study shows that microRNA-503 interacts with the CUG-binding protein 1 (CUGBP1) mRNA and represses its translation by recruiting the CUGBP1 mRNA to processing bodies.

Regulation of Cyclin-Dependent Kinase 4 Translation through CUG-Binding Protein 1 and microRNA-222 by Polyamines

The findings presented indicate that polyamine-regulated CUG-binding protein 1 and microRNA-222 modulate cyclin-dependent kinase 4 (CDK4) translation at least in part by altering the recruitment of CDK4 mRNA to processing bodies.

Post-transcriptional regulation of MEK-1 by polyamines through the RNA-binding protein HuR modulating intestinal epithelial apoptosis.

MEK-1 [MAPK (mitogen-activated protein kinase) kinase-1] is an important signal transducing enzyme that is implicated in many aspects of cellular functions. In the present paper, we report that cellular polyamines regulate MEK-1 expression at the post-transcriptional level through the RNA-binding protein HuR (Hu-antigen R) in IECs (intestinal epithelial cells). Decreasing the levels of cellular polyamines by inhibiting ODC (ornithine decarboxylase) stabilized MEK-1 mRNA and promoted its translation through enhancement of the interaction between HuR and the 3-untranslated region of MEK-1 mRNA, whereas increasing polyamine levels by ectopic ODC overexpression destabilized the MEK-1 transcript and repressed its translation by reducing the abundance of HuR-MEK-1 mRNA complex; neither intervention changed MEK-1 gene transcription via its promoter. HuR silencing rendered the MEK-1 mRNA unstable and inhibited its translation, thus preventing increases in MEK-1 mRNA and protein in polyamine-deficient cells. Conversely, HuR overexpression increased MEK-1 mRNA stability and promoted its translation. Inhibition of MEK-1 expression by MEK-1 silencing or HuR silencing prevented the increased resistance of polyamine-deficient cells to apoptosis. Moreover, HuR overexpression did not protect against apoptosis if MEK-1 expression was silenced. These results indicate that polyamines destabilize the MEK-1 mRNA and repress its translation by inhibiting the association between HuR and the MEK-1 transcript. Our findings indicate that MEK-1 is a key effector of the HuR-elicited anti-apoptotic programme in IECs.

Induced JunD in intestinal epithelial cells represses CDK4 transcription through its proximal promoter region following polyamine depletion

Maintenance of intestinal epithelial integrity requires cellular polyamines that regulate expression of various genes involved in cell proliferation, growth arrest and apoptosis. In prior studies, depletion of cellular polyamines has been shown to stabilize JunD, a member of the AP-1 (activator protein-1) family of transcription factors, leading to inhibition of intestinal epithelial cell proliferation, but the exact downstream targets of induced JunD remain elusive. CDK4 (cyclin-dependent kinase 4) is essential for the G1- to S-phase transition during the cell cycle and its expression is primarily controlled at the transcriptional level. In the present study, we show that induced JunD in IECs (intestinal epithelial cells) is a transcriptional repressor of the CDK4 gene following polyamine depletion. Increased JunD in polyamine-deficient cells was associated with a significant inhibition of CDK4 transcription, as indicated by repression of CDK4-promoter activity and decreased levels of CDK4 mRNA and protein, all of which were prevented by using specific antisense JunD oligomers. Ectopic expression of the wild-type junD also repressed CDK4-promoter activity and decreased levels of CDK4 mRNA and protein without any effect on CDK2 expression. Gel shift and chromatin immunoprecipitation assays revealed that JunD bound to the proximal region of the CDK4-promoter in vitro as well as in vivo, while experiments using different CDK4-promoter mutants showed that transcriptional repression of CDK4 by JunD was mediated through an AP-1 binding site within this proximal sequence of the CDK4-promoter. These results indicate that induced JunD in IECs represses CDK4 transcription through its proximal promoter region following polyamine depletion.

Induced TRPC1 expression sensitizes intestinal epithelial cells to apoptosis by inhibiting NF-κB activation through Ca2+ influx

Apoptosis occurs within crypts and at the intestinal luminal surface and plays a critical role in mucosal homoeostasis. NF-kappaB (nuclear factor-kappaB) is the central regulator of the transcription of genes involved in apoptosis, and its activity is highly regulated in the intestinal mucosa. We have recently demonstrated that TRPC1 (transient receptor potential canonical-1) is expressed in IECs (intestinal epithelial cells) and functions as a Ca2+ permeable channel activated by Ca2+ store depletion. The present study tests the hypothesis that TRPC1 channels are implicated in the regulation of apoptosis by inhibiting NF-kappaB through the induction of TRPC1-mediated Ca2+ influx in the IEC-6 line. The expression of TRPC1 induced by stable transfection of IEC-6 cells with the wild-type TRPC1 gene (IEC-TRPC1 cells) increased Ca2+ influx after Ca2+ store depletion and repressed NF-kappaB transactivation, which was associated with an increase in susceptibility to apoptosis induced by exposure to TNFalpha (tumour necrosis factor-alpha) plus CHX (cycloheximide) (TNF-alpha/CHX), or STS (staurosporine). By contrast, the induction of endogenous NF-kappaB activity, by the depletion of cellular polyamines, promoted resistance to apoptosis, which was prevented by the ectopic expression of the IkappaBalpha super-repressor. Furthermore, inhibition of TRPC1 expression by transfection with siRNA (small interfering RNA) targeting TRPC1 (siTRPC1) decreased Ca2+ influx, increased NF-kappaB transactivation, and prevented the increased susceptibility of IEC-TRPC1 cells to apoptosis. Decreasing Ca2+ influx by exposure to a Ca2+-free medium also induced NF-kappaB activity and blocked the increased susceptibility to apoptosis of stable IEC-TRPC1 cells. These results indicate that induced TRPC1 expression sensitizes IECs to apoptosis by inhibiting NF-kappaB activity as a result of the stimulation of Ca2+ influx.

Inhibition of Smurf2 translation by miR-322/503 modulates TGF-β/Smad2 signaling and intestinal epithelial homeostasis

Smurf2 is an E3 ubiquitin ligase that regulates TGF-β/Smad signaling and is implicated in a wide variety of cellular responses. miR-322 and miR-503 repress Smurf2 translation and thus modulate TGF-β/Smad2 signaling and intestinal epithelial homeostasis.

Sphingosine-1-Phosphate Regulates the Expression of Adherens Junction Protein E-Cadherin and Enhances Intestinal Epithelial Cell Barrier Function

BackgroundThe regulation of intestinal barrier permeability is important in the maintenance of normal intestinal physiology. Sphingosine-1-phosphate (S1P) has been shown to play a pivotal role in enhancing barrier function in several non-intestinal tissues. The current study determined whether S1P regulated function of the intestinal epithelial barrier by altering expression of E-cadherin, an important protein in adherens junctions.MethodsStudies were performed upon cultured differentiated IECs (IEC-Cdx2L1 line) using standard techniques.ResultsS1P treatment significantly increased levels of E-cadherin protein and mRNA in intestinal epithelial cells (IECs) and also led to E-cadherin localizing strongly to the cell–cell border. S1P also improved the barrier function as indicated by a decrease in 14C-mannitol paracellular permeability and an increase in transepithelial electrical resistance (TEER) in vitro.ConclusionsThese results indicate that S1P increases levels of E-cadherin, both in cellular amounts and at the cell–cell junctions, and leads to improved barrier integrity in cultured intestinal epithelial cells.

The RNA-binding protein CUG-BP1 increases survivin expression in oesophageal cancer cells through enhanced mRNA stability.

Survivin, a member of the IAP (inhibitor of apoptosis protein) family, plays important roles in maintaining cellular homoeostasis and regulating cell-cycle progression. This IAP is overexpressed in oesophageal cancer cells, leading to uncontrolled cell growth and resistance to apoptosis. CUG-BP1 (CUG-binding protein 1) is an RNA-binding protein that regulates the stability and translational efficiency of target mRNAs. In the present paper, we report that CUG-BP1 is overexpressed in oesophageal cancer cell lines and human oesophageal cancer specimens. CUG-BP1 associates with the 3-untranslated region of survivin mRNA, thereby stabilizing the transcript and elevating its expression in oesophageal cancer cells. Our results show that overexpression of CUG-BP1xa0in oesophageal epithelial cells results in increased survivin mRNA stability and consequently survivin protein expression. Conversely, silencing CUG-BP1xa0in oesophageal cancer cells destabilizes survivin mRNA, lowering the level of survivin protein. In addition, we have found that altering CUG-BP1 expression modulates susceptibility to chemotherapy-induced apoptosis. Overexpression of CUG-BP1xa0in oesophageal epithelial cells increases resistance to apoptosis, whereas silencing CUG-BP1 makes oesophageal cancer cells more susceptible to chemotherapy-induced apoptosis. Co-transfection experiments with small interfering RNA directed against survivin suggest that the anti-apoptotic role for CUG-BP1 is not entirely dependent on its effect on survivin expression.