PTEN-induced kinase 1-induced dynamin-related protein 1 Ser637 phosphorylation reduces mitochondrial fission and protects against intestinal ischemia reperfusion injury.

BACKGROUND: Intestinal ischemia reperfusion (I/R) occurs in various diseases, such as trauma and intestinal transplantation. Excessive reactive oxygen species (ROS) accumulation and subsequent apoptotic cell death in intestinal epithelia are important causes of I/R injury. PTEN-induced putative kinase 1 (PINK1) and phosphorylation of dynamin-related protein 1 (DRP1) are critical regulators of ROS and apoptosis. However, the correlation of PINK1 and DRP1 and their function in intestinal I/R injury have not been investigated. Thus, examining the PINK1/DRP1 pathway may help to identify a protective strategy and improve the patient prognosis. AIM: To clarify the mechanism of the PINK1/DRP1 pathway in intestinal I/R injury. METHODS: Male C57BL/6 mice were used to generate an intestinal I/R model via superior mesenteric artery occlusion followed by reperfusion. Chiu's score was used to evaluate intestinal mucosa damage. The mitochondrial fission inhibitor mdivi-1 was administered by intraperitoneal injection. Caco-2 cells were incubated in vitro in hypoxia/reoxygenation conditions. Small interfering RNAs and overexpression plasmids were transfected to regulate PINK1 expression. The protein expression levels of PINK1, DRP1, p-DRP1 and cleaved caspase 3 were measured by Western blotting. Cell viability was evaluated using a Cell Counting Kit-8 assay and cell apoptosis was analyzed by TUNEL staining. Mitochondrial fission and ROS were tested by MitoTracker and MitoSOX respectively. RESULTS: Intestinal I/R and Caco-2 cell hypoxia/reoxygenation decreased the expression of PINK1 and p-DRP1 Ser637. Pretreatment with mdivi-1 inhibited mitochondrial fission, ROS generation, and apoptosis and ameliorated cell injury in intestinal I/R. Upon PINK1 knockdown or overexpression in vitro, we found that p-DRP1 Ser637 expression and DRP1 recruitment to the mitochondria were associated with PINK1. Furthermore, we verified the physical combination of PINK1 and p-DRP1 Ser637. CONCLUSION: PINK1 is correlated with mitochondrial fission and apoptosis by regulating DRP1 phosphorylation in intestinal I/R. These results suggest that the PINK1/DRP1 pathway is involved in intestinal I/R injury, and provide a new approach for prevention and treatment.

Two column classification of tibial plateau fractures; description, clinical application and reliability.

PURPOSE: In this era of life highly comminuted and multi planar tibial plateau fractures involving the posterior corners are more commonly seen and addressed in the literature than before. Among these several types have not been described in the currently used classification systems. In fact simple classification systems ignore several fracture types and leniently grouped the fractures with different mechanism, morphology, treatment modalities and prognosis in same category. On the other hand, more extensive nature classifications with detailed subdivisions are difficult to remember for clinicians. The clinical reliability of these classifications is another problem. All these issues demand the potential need of a new classification. The aim of this study was to describe a quadrant specific two column classification of tibial plateau fractures and to analyse its inter-observer and intra-observer reliability, clinical assessment and application. MATERIALS AND METHODS: From January 2009 to December 2015, 44 patients with tibial plateau fractures were studied retrospectively. The antero-posterior (AP), lateral X-rays and computed tomography (CT) with axial transverse, sagittal, coronal and three dimensional (3D) reconstruction images were performed for all the patients. All of the fractures were categorized according to quadrant specific two column classification and the traditional Schatzker's classification. The comparative analysis for inter-observer and intra-observer reliability of the new classification and the Schatzker's classification was conducted by four observers. RESULTS: Three cases didn't match any type in the Schatzker's classification. While on the other hand, all cases were classified by two column classification. The mean kappa values for inter-observer reliability by using the Schatzker's classification was 0.723 (range, 0.674-0.823), representing substantial agreement, whereas the mean kappa value was 0.939 (range: 0.897-0.974), representing almost perfect agreement according to two column classification. The mean kappa values for intra-observer reliability using the Schatzker's classification and two column classification were 0.789 (range: 0.590-0.864) and 0.955 (range:0.923-0.948) showing substantial agreement and almost perfect agreement. CONCLUSION: The quadrant specific two column classification is anatomically oriented, CT based and clinically valid. The different fracture types according to anatomic location are represented alphanumerically so that treatment matched to specific fracture type (quadrant specific anatomic fixation) for optimal outcomes. Furthermore, it demonstrates higher inter-observer and intra-observer reliability. This classification can be adopted to strengthen the traditional Schatzker's classification, particularly in the multi planar and posteriorly extended plateau fractures. It can be used as a reliable research tool. The database can be used to distinguish different fracture types, individual type incidences, specific treatment and also prognosis. Authors suggest a large multi-centre study.

Thyrotoxicosis presenting as syncope and rapid wide QRS tachycardia: A case report.

We report regarding a young lady who presented with recurrent syncopal episodes associated with palpitations, documented to be due to rapid wide QRS tachycardia, subsequently detected to have thyrotoxicosis and who responded promptly to appropriate medication to become free from arrhythmia.

Microarray Analysis of Differentially Expressed Profiles of Circular RNAs in a Mouse Model of Intestinal Ischemia/Reperfusion Injury with and Without Ischemic Postconditioning.

BACKGROUND/AIMS: Ischemic postconditioning (iPoC) represents a promising strategy to mitigate ischemia/reperfusion (I/R) injury of the intestine, yet the mechanisms of this treatment remain to be elucidated. Circular RNAs (circRNAs), a novel class of endogenous non-coding RNAs, have recently been recognized as important regulators of gene expression and pathological processes. Here, we aimed to investigate the expression patterns of circRNAs after intestinal I/R with and without iPoC and, furthermore, to explore the potential mechanisms of iPoC in relation to the differentially expressed circRNAs. METHODS: The global circRNA and mRNA expression profiles in mouse intestinal mucosa were initially screened by microarray (n = 3 per group) and quantitative real-time PCR was used to validate the expression pattern of circRNAs and mRNAs. Bioinformatics analysis including Gene ontology, KEGG pathway analysis, microRNA binding sites identification and circRNA-miRNA-mRNA network construction were utilized for in-depth mechanism exploration. RESULTS: There were 4 up- and 58 downregulated circRNAs as well as 322 up- and 199 downregulated mRNAs in the intestinal I/R group compared with the sham group, whereas compared with I/R, iPoC treatment significantly upregulated 12 circRNAs and 129 mRNAs and downregulated 21 circRNAs and 174 mRNAs. The expression levels of a randomly selected set of 6 circRNAs and 5 mRNAs were successfully validated by qRT-PCR. Through a systematic comparison of the direction of circRNA expression changes in all groups, we identified two circRNAs, circRNA_012412 and circRNA_016863, that may be closely associated with the protective mechanisms of iPoC. Finally, four possible circRNA_012412/circRNA_016863-miRNA-mRNA pathways were predicted, which may play important roles in endogenous protective signaling in iPoC. CONCLUSIONS: This study was the first to comprehensively delineate the expression profiles of circRNAs in a mouse model of intestinal I/R and iPoC and provides novel clues for understanding the mechanisms of iPoC against intestinal I/R injury.

miR-381-3p knockdown improves intestinal epithelial proliferation and barrier function after intestinal ischemia/reperfusion injury by targeting nurr1.

Impairment in gut barrier function induced by intestinal ischemia/reperfusion (I/R) injury is associated with high morbidity and mortality. Intestinal barrier function requires the tight coordination of epithelial migration, proliferation and differentiation. We previously observed that nuclear receptor-related protein 1 (nurr1)-mediated proliferative pathway was impaired in intestinal I/R injury. Here, we aimed to assess the effect of nurr1 on intestinal barrier function and to evaluate microRNA (miRNA)-nurr1-mediated restoration of intestinal barrier function in intestinal I/R injury. We induced an in vivo intestinal I/R injury mouse model by clamping and then releasing the superior mesenteric artery. We also performed an in vitro study in which we exposed Caco-2 and IEC-6 cells to hypoxia/reoxygenation (H/R) conditions to stimulate intestinal I/R injury. Our results demonstrated that nurr1 regulated intestinal epithelial development and barrier function after intestinal I/R injury. miR-381-3p, which directly suppressed nurr1 translation, was identified by microarray and bioinformatics analysis. miR-381-3p inhibition enhanced intestinal epithelial proliferation and barrier function in vitro and in vivo and also attenuated remote organ injury and improved survival. Importantly, nurr1 played an indispensable role in the protective effect of miR-381-3p inhibition. Collectively, these findings show that miR-381-3p inhibition mitigates intestinal I/R injury by enhancing nurr1-mediated intestinal epithelial proliferation and barrier function. This discovery may lead to the development of therapeutic interventions for intestinal I/R injury.

Biomechanical efficacy of AP, PA lag screws and posterior plating for fixation of posterior malleolar fractures: a three dimensional finite element study.

BACKGROUND: Clinically there are different fixation methods used for fixation of the posterior malleolar fractures (PMF), but the best treatment modality is still not clear. Few studies have concentrated on this issue, least of all using a biomechanical comparison. The purpose of this study was to carry out a computational comparative biomechanics of three different commonly used fixation constructs for the fixation of PMF by finite element analysis (FEA). METHODS: Computed tomography (CT) images were used to reconstruct three dimensional (3D) model of the tibia. Computer aided design (CAD) software was used to design 3D models of PMF. Finally, 3D models of PMF fixed with two antero-posterior (AP) lag screws, two postero-anterior (PA) lag screws and posterior plate were simulated through computational processing. Simulated loads of 500 N, 1000 N and 1500 N were applied to the PMF and proximal ends of the models were fixed in all degrees of freedom. Output results representing the model von Mises stress, relative fracture micro-motion and vertical displacement of the fracture fragment were analyzed. RESULTS: The mean vertical displacement value in the posterior plate group (0.52 mm) was lower than AP (0.68 mm) and PA (0.69 mm) lag groups. Statistically significant low amount of the relative micro-motion (P < 0.05) was observed in the posterior plate group. CONCLUSIONS: It was concluded that the posterior plate is biomechanically the most stable fixation method for fixation of PMF.

PKCζ phosphorylates TRAF2 to protect against intestinal ischemia-reperfusion-induced injury.

Intestinal ischemia-reperfusion (I/R) is a common clinical problem that occurs during various clinical pathological processes. Excessive apoptosis has an indispensable role in intestinal I/R injury. Tumor necrosis factor receptor-associated factor 2 (TRAF2) and PKCζ have an essential role in apoptosis. Here, we aimed to investigate the effects of PKCζ and TRAF2 and to explore the correlation between PKCζ and TRAF2 in intestinal I/R injury. Mice were subjected to intestinal I/R injury in vivo. In vitro experiments were conducted by treating Caco-2 cells with hypoxia/reoxygenation (H/R) stimulation to simulate intestinal I/R. Intestinal tissue samples and Caco-2 cells were examined using various approaches. Intestinal I/R induced the membrane translocation and phosphorylation of PKCζ. Pretreatment with the PKCζ activator phosphatidylcholine remarkably attenuated gut injury by suppressing apoptosis. H/R induced PKCζ to combine with TRAF2, which was phosphorylated by PKCζ at Ser55, but not at Ser11, under intestinal I/R or H/R conditions. In addition, TRAF2 Ser55 phosphorylation increased cell survival by inhibiting cell apoptosis in the H/R model. Mechanistically, TRAF2 Ser55 phosphorylation promoted NF-κB activation but suppressed c-Jun activation in Caco-2 cells under H/R conditions. The results of this study demonstrate that the PKCζ/TRAF2 pathway represents a novel protective mechanism against intestinal I/R injury. Therefore, the PKCζ/TRAF2 pathway is a novel target for potential treatments of intestinal I/R injury-related diseases.

Finite element analysis of the three different posterior malleolus fixation strategies in relation to different fracture sizes.

PURPOSE: Appropriate fixation method for the posterior malleolar fractures (PMF) according to the fracture size is still not clear. Aim of this study was to evaluate the outcomes of the different fixation methods used for fixation of PMF by finite element analysis (FEA) and to compare the effect of fixation constructs on the size of the fracture computationally. MATERIALS AND METHODS: Three dimensional model of the tibia was reconstructed from computed tomography (CT) images. PMF of 30%, 40% and 50% fragment sizes were simulated through computational processing. Two antero-posterior (AP) lag screws, two postero-anterior (PA) lag screws and posterior buttress plate were analysed for three different fracture volumes. The simulated loads of 350N and 700N were applied to the proximal tibial end. Models were fixed distally in all degrees of freedom. RESULTS: In single limb standing condition, the posterior plate group produced the lowest relative displacement (RD) among all the groups (0.01, 0.03 and 0.06mm). Further nodal analysis of the highest RD fracture group showed a higher mean displacement of 4.77mm and 4.23mm in AP and PA lag screws model (p=0.000). The amounts of stress subjected to these implants, 134.36MPa and 140.75MPa were also significantly lower (p=0.000). There was a negative correlation (p=0.021) between implant stress and the displacement which signifies a less stable fixation using AP and PA lag screws. CONCLUSION: Progressively increasing fracture size demands more stable fixation construct because RD increases significantly. Posterior buttress plate produces superior stability and lowest RD in PMF models irrespective of the fragment size.

Inhibition of p66Shc-mediated mitochondrial apoptosis via targeting prolyl-isomerase Pin1 attenuates intestinal ischemia/reperfusion injury in rats.

Intestinal epithelial oxidative stress and apoptosis constitute key pathogenic mechanisms underlying intestinal ischemia/reperfusion (I/R) injury. We previously reported that the adaptor 66 kDa isoform of the adaptor molecule ShcA (p66Shc)-mediated pro-apoptotic pathway was activated after intestinal I/R. However, the upstream regulators of the p66Shc pathway involved in intestinal I/R remain to be fully identified. Here, we focused on the role of a prolyl-isomerase, peptidyl-prolyl cis-trans isomerase (Pin1), in the regulation of p66Shc activity during intestinal I/R. Intestinal I/R was induced in rats by superior mesenteric artery (SMA) occlusion. Juglone (Pin1 inhibitor) or vehicle was injected intraperitoneally before I/R challenge. Caco-2 cells were exposed to hypoxia/reoxygenation (H/R) in vitro to simulate an in vivo I/R model. We found that p66Shc was significantly up-regulated in the I/R intestine and that this up-regulation resulted in the accumulation of intestinal mitochondrial reactive oxygen species (ROS) and massive epithelial apoptosis. Moreover, intestinal I/R resulted in elevated protein expression and enzyme activity of Pin1 as well as increased interaction between Pin1 and p66Shc. This Pin1 activation was responsible for the translocation of p66Shc to the mitochondria during intestinal I/R, as Pin1 suppression by juglone or siRNA markedly blunted p66Shc mitochondrial translocation and the subsequent ROS generation and cellular apoptosis. Additionally, Pin1 inhibition alleviated gut damage and secondary lung injury, leading to improvement of survival after I/R. Collectively, our findings demonstrate for the first time that Pin1 inhibition protects against intestinal I/R injury, which could be partially attributed to the p66Shc-mediated mitochondrial apoptosis pathway. This may represent a novel prophylactic target for intestinal I/R injury.