Sulfotransferase 1C2 Increases Mitochondrial Respiration by Converting Mitochondrial Membrane Cholesterol to Cholesterol Sulfate.

HYPOTHESIS: In this communication, we test the hypothesis that sulfotransferase 1C2 (SULT1C2, UniProt accession no. Q9WUW8) can modulate mitochondrial respiration by increasing state-III respiration. METHODS AND RESULTS: Using freshly isolated mitochondria, the addition of SULT1C2 and 3-phosphoadenosine 5 phosphosulfate (PAPS) results in an increased maximal respiratory capacity in response to the addition of succinate, ADP, and rotenone. Lipidomics and thin-layer chromatography of mitochondria treated with SULT1C2 and PAPS showed an increase in the level of cholesterol sulfate. Notably, adding cholesterol sulfate at nanomolar concentration to freshly isolated mitochondria also increases maximal respiratory capacity. In vivo studies utilizing gene delivery of SULT1C2 expression plasmids to kidneys result in increased mitochondrial membrane potential and confer resistance to ischemia/reperfusion injury. Mitochondria isolated from gene-transduced kidneys have elevated state-III respiration as compared with controls, thereby recapitulating results obtained with mitochondrial fractions treated with SULT1C2 and PAPS. CONCLUSION: SULT1C2 increases mitochondrial respiratory capacity by modifying cholesterol, resulting in increased membrane potential and maximal respiratory capacity. This finding uncovers a unique role of SULT1C2 in cellular physiology and extends the role of sulfotransferases in modulating cellular metabolism.

Margin ACcentuation for resectable Pancreatic cancer using Irreversible Electroporation - Results from the MACPIE-I study.

INTRODUCTION: Margin accentuation (MA) using Irreversible electroporation (IRE) offers an unique opportunity to reduce the R1 resections in resectable pancreatic cancer (RPC). This study aims to assess the rate of margin positivity using IRE for MA during pancreaticoduodenectomy (PD) for resectable pancreatic head tumours. MATERIALS AND METHODS: Following ethical approval, MA using IRE was carried out in 20 consecutive patients to posterior and superior mesenteric vein (SMV) margin, and the pancreatic neck, prior to the PD resection. The control group (non-IRE; n = 91) underwent PD without MA over the study period, March 2018 to March 2020. RESULTS: There was no difference between the two groups in terms of patients' age, gender, pre-op biliary drainage, site of malignancy or pre-operative TNM stage. The overall margin positive rate for IRE group was lesser (35.0%) when compared to non-IRE group (51.6%; p = 0.177), with significantly less posterior pancreatic margin positivity (5.0% vs. 25.3%; p = 0.046). When only treated margins (SMA margin excluded) were compared, the IRE group had significantly lower margin positive rates (20.0% vs. 51.6%; p = 0.013). There was no difference between the two groups in terms of intra- or post-operative complications. With a median follow-up of 15.6 months, the median DFS and OS for IRE and non-IRE groups were 17 and 18 months (p = 0.306) and 19 and 22 months (p = 0.227) respectively. CONCLUSION: Our pilot study confirms the safety of MA using IRE for RPC, with reduction in margin positivity. These results as a proof of concept are promising and need further validation with a randomised controlled trial.

Selective Phosphodiesterase 1 Inhibitor BTTQ Reduces Blood Pressure in Spontaneously Hypertensive and Dahl Salt Sensitive Rats: Role of Peripheral Vasodilation.

Regulation of the peripheral vascular resistance via modulating the vessel diameter has been considered as a main determinant of the arterial blood pressure. Phosphodiesterase enzymes (PDE1-11) hydrolyse cyclic nucleotides, which are key players controlling the vessel diameter and, thus, peripheral resistance. Here, we have tested and reported the effects of a novel selective PDE1 inhibitor (BTTQ) on the cardiovascular system. Normal Sprague Dawley, spontaneously hypertensive (SHR), and Dahl salt-sensitive rats were used to test in vivo the efficacy of the compound. Phosphodiesterase radiometric enzyme assay revealed that BTTQ inhibited all three isoforms of PDE1 in nanomolar concentration, while micromolar concentrations were needed to induce effective inhibition for other PDEs. The myography study conducted on mesenteric arteries revealed a potent vasodilatory effect of the drug, which was confirmed in vivo by an increase in the blood flow in the rat ear arteriols reflected by the rise in the temperature. Furthermore, BTTQ proved a high efficacy in lowering the blood pressure about 9, 36, and 24 mmHg in normal Sprague Dawley, SHR and, Dahl salt-sensitive rats, respectively, compared to the vehicle-treated group. Moreover, additional blood pressure lowering of about 22 mmHg could be achieved when BTTQ was administered on top of ACE inhibitor lisinopril, a current standard of care in the treatment of hypertension. Therefore, PDE1 inhibition induced efficient vasodilation that was accompanied by a significant reduction of blood pressure in different hypertensive rat models. Administration of BTTQ was also associated with increased heart rate in both models of hypertension as well as in the normotensive rats. Thus, PDE1 appears to be an attractive therapeutic target for the treatment of resistant hypertension, while tachycardia needs to be addressed by further compound structural optimization.

Kidney surveillance in the spotlight: contrast-induced acute kidney injury illuminated.

Acute kidney injury comprises a heterogeneous group of conditions characterized by a sudden decrease in renal function over hours to days. Contrast-induced acute kidney injury (CI-AKI) is caused by radiographic contrast agents used in diagnostic imaging. In the current issue of the JCI, Lau et al. use a mouse model of CI-AKI to study the role of resident and infiltrating phagocytes, recruited leukocytes, and tubular cells in the immune surveillance response to contrast agents. This study has the potential to provide innovative therapies for human CI-AKI.

Exogenous Gene Transmission of Isocitrate Dehydrogenase 2 Mimics Ischemic Preconditioning Protection.

Ischemic preconditioning confers organ-wide protection against subsequent ischemic stress. A substantial body of evidence underscores the importance of mitochondria adaptation as a critical component of cell protection from ischemia. To identify changes in mitochondria protein expression in response to ischemic preconditioning, we isolated mitochondria from ischemic preconditioned kidneys and sham-treated kidneys as a basis for comparison. The proteomic screen identified highly upregulated proteins, including NADP+-dependent isocitrate dehydrogenase 2 (IDH2), and we confirmed the ability of this protein to confer cellular protection from injury in murine S3 proximal tubule cells subjected to hypoxia. To further evaluate the role of IDH2 in cell protection, we performed detailed analysis of the effects of Idh2 gene delivery on kidney susceptibility to ischemia-reperfusion injury. Gene delivery of IDH2 before injury attenuated the injury-induced rise in serum creatinine (P<0.05) observed in controls and increased the mitochondria membrane potential (P<0.05), maximal respiratory capacity (P<0.05), and intracellular ATP levels (P<0.05) above those in controls. This communication shows that gene delivery of Idh2 can confer organ-wide protection against subsequent ischemia-reperfusion injury and mimics ischemic preconditioning.

Hydrodynamic Isotonic Fluid Delivery Ameliorates Moderate-to-Severe Ischemia-Reperfusion Injury in Rat Kidneys.

Highly aerobic organs like the kidney are innately susceptible to ischemia-reperfusion (I/R) injury, which can originate from sources including myocardial infarction, renal trauma, and transplant. Therapy is mainly supportive and depends on the cause(s) of damage. In the absence of hypervolemia, intravenous fluid delivery is frequently the first course of treatment but does not reverse established AKI. Evidence suggests that disrupting leukocyte adhesion may prevent the impairment of renal microvascular perfusion and the heightened inflammatory response that exacerbate ischemic renal injury. We investigated the therapeutic potential of hydrodynamic isotonic fluid delivery (HIFD) to the left renal vein 24 hours after inducing moderate-to-severe unilateral IRI in rats. HIFD significantly increased hydrostatic pressure within the renal vein. When conducted after established AKI, 24 hours after I/R injury, HIFD produced substantial and statistically significant decreases in serum creatinine levels compared with levels in animals given an equivalent volume of saline via peripheral infusion (P<0.05). Intravital confocal microscopy performed immediately after HIFD showed improved microvascular perfusion. Notably, HIFD also resulted in immediate enhancement of parenchymal labeling with the fluorescent dye Hoechst 33342. HIFD also associated with a significant reduction in the accumulation of renal leukocytes, including proinflammatory T cells. Additionally, HIFD significantly reduced peritubular capillary erythrocyte congestion and improved histologic scores of tubular injury 4 days after IRI. Taken together, these results indicate that HIFD performed after establishment of AKI rapidly restores microvascular perfusion and small molecule accessibility, with improvement in overall renal function.

A wandering path toward prevention for acute kidney injury.

Acute kidney injury (AKI) is a common cause of hospital-related mortality; therefore, strategies to either prevent or treat this complication are of great interest. In this issue of the JCI, Inoue, Abe, and colleagues have uncovered a targetable neuroimmunomodulatory mechanism that protects mice from ischemia-reperfusion injury (IRI) and subsequent AKI. Specifically, the authors demonstrate that vagus nerve stimulation (VNS) activates the cholinergic antiinflammatory pathway (CAP), resulting in activation of antiinflammatory effects via α7 nicotinic acetylcholine receptor-expressing splenic macrophages. Together, the results of this study have potential clinical implications in the prevention of AKI in at-risk individuals.

Severn School of Psychiatry education fellowships: a new way to promote educational practice and research.

This paper describes a model of training in leadership and project management skills for advanced trainees, using educational projects within the Severn School of Psychiatry. Fellowships lasting 1 year have been developed to enable trainees, working with a senior consultant trainer associated with the School of Psychiatry, to support important new educational initiatives. Linkage with the local university training and learning for health professionals research module has provided academic support for the trainees and the projects. Four examples for the first year of the programme are described and feedback from structured interviews with participants is presented. The development of the fellowships appears to have had wider benefits, in developing educational faculty in the School of Psychiatry and the trainees involved have had opportunities to extend their project management and leadership skills. The fellowship programme is continuing to develop, based on feedback from its first successful year.

Benign histology after pancreaticoduodenectomy for suspected malignancy. Lessons to be learned--a single centre experience.

UNLABELLED: The aim of the study was to perform a comprehensive analysis of patients with a benign final histology after pancreaticoduodenectomies (PD) for suspected pancreatic and periampullary cancer. MATERIAL AND METHODS: We searched the pathology database at the King's College Hospital for negative PD specimens submitted between January 2004-December 2010. Clinical, diagnostic, surgical,histopathological and outcome data were collected retrospectively. Pathology specimens and imaging results have been re-evaluated. A literature review was performed to identify factors affecting the incidence across centres. RESULTS: 469 PD were performed for presumed cancer. The incidence of benign disease encountered in this group was 7.25% (34/469). Autoimmune pancreatitis (AIP) was a finding in 26.47% (9/34) of cases. 17.65% of PD were complicated by a pancreatic leak and the overall mortality rate was 8.82%(3/34). Radiologists revised over 75% of pre-operative diagnoses. The incidence of benign disease was correlated with the overall centre experience and utilisation of CT imaging, but not ERCP or EUS. CONCLUSIONS: It is impossible with current diagnostics to entirely avoid cases of benign disease inpatients undergoing PD for suspected cancer. The mortality rate is higher in this group, but it is possible to avoid unnecessary procedures in experienced centres. AIP represents an important diagnostic entity, which should be actively pursued pre-operatively.

A systems life cycle approach to managing the radiology profession: an Australian perspective.

OBJECTIVE: Although the medical system has expanded considerably over the past two decades in almost all countries, so too has the demand for health care. The radiology specialisation may be an early system indicator, being especially sensitive to changes in supply and demand in both rural and urban environments. The question is whether the new policies of increasing the number of radiologists can be a proper long-term solution for the imbalance of workforce supply and demand or not. METHODS: Using system dynamics modelling, we present our integrated descriptive models for the supply and demand of Australian radiologists to find the actual gap. Followed by this, we pose a prescriptive model for the supply in order to lessen the identified imbalance between supply and demand. Our system dynamics models compare the demand and supply of Australian radiologists over 40 years between 2010 and 2050. RESULTS: The descriptive model shows that even if the radiology training program grows at a higher rate than the medical training growth rate and its own historical growth, the system will never be able to meet demand. The prescriptive model also indicates that although changing some influential factors (e.g the intake rate) reduces the level of imbalance, the system will still stay unstable during the study period. CONCLUSION: We posit that Australia may need to design a new system of radiology provision to meet future demands for high-quality medical radiation services. We also suggest some strategies, such as greater development of radiographers' role, are critical for enabling sustainable change over time. What is known about the topic? Long-term workforce planning for medical services at the national level has been very challenging for policy makers of the 21st century. The current demographic imbalance in the supply and demand of the Australian radiologist workforce makes it difficult to plan the effects of extra inflow of radiology students over time.

A regional teaching fellow community of practice.

BACKGROUND: Increasing numbers of clinical teaching fellows are responsible for a significant proportion of undergraduate teaching nationally. Developing a regional community of practice can help overcome the isolation of these posts, with potential benefits for all involved. CONTEXT: A community of practice relies on the mutual engagement of people in a similar situation working towards a common goal. Working together and sharing resources enables teaching fellows to make the most of their post, which ultimately benefits those that they are teaching. INNOVATION: We developed a regional clinical teaching fellow community of practice in Bristol in 2010/11. Our community has continued to develop since completing our posts as clinical teaching fellows, and has provided a platform for new communities to develop amongst the groups of subsequent teaching fellows coming through. We encourage all regions who have clinical teaching fellows to develop a regional community of practice IMPLICATIONS: We encourage all regions who have clinical teaching fellows to develop a regional community of practice. We also encourage trainees to join TASME (Trainees in the Association for the Study of Medical Education), a new national community of practice for trainees involved in medical education.

Improved outcome of severe acute pancreatitis in the intensive care unit.

Background. Severe acute pancreatitis (SAP) is associated with serious morbidity and mortality. Our objective was to describe the case mix, management, and outcome of patients with SAP receiving modern critical care in the Intensive Care Unit (ICU). Methods. Retrospective analysis of patients with SAP admitted to the ICU in a single tertiary care centre in the UK between January 2005 and December 2010. Results. Fifty SAP patients were admitted to ICU (62% male, mean age 51.7 (SD 14.8)). The most common aetiologies were alcohol (40%) and gallstones (30%). On admission to ICU, the median Acute Physiology and Chronic Health Evaluation (APACHE) II score was 17, the pancreatitis outcome prediction score was 8, and the median Computed Tomography Severity Index (CTSI) was 4. Forty patients (80%) tolerated enteral nutrition, and 46% received antibiotics for non-SAP reasons. Acute kidney injury was significantly more common among hospital nonsurvivors compared to survivors (100% versus 42%, P = 0.0001). ICU mortality and hospital mortality were 16% and 20%, respectively, and median lengths of stay in ICU and hospital were 13.5 and 30 days, respectively. Among hospital survivors, 27.5% developed diabetes mellitus and 5% needed long-term renal replacement therapy. Conclusions. The outcome of patients with SAP in ICU was better than previously reported but associated with a resource demanding hospital stay and long-term morbidity.

A method to facilitate and monitor expression of exogenous genes in the rat kidney using plasmid and viral vectors.

Gene therapy has been proposed as a novel alternative to treat kidney disease. This goal has been hindered by the inability to reliably deliver transgenes to target cells throughout the kidney, while minimizing injury. Since hydrodynamic forces have previously shown promising results, we optimized this approach and designed a method that utilizes retrograde renal vein injections to facilitate transgene expression in rat kidneys. We show, using intravital fluorescence two-photon microscopy, that fluorescent albumin and dextrans injected into the renal vein under defined conditions of hydrodynamic pressure distribute broadly throughout the kidney in live animals. We found injection parameters that result in no kidney injury as determined by intravital microscopy, histology, and serum creatinine measurements. Plasmids, baculovirus, and adenovirus vectors, designed to express EGFP, EGFP-actin, EGFP-occludin, EGFP-tubulin, tdTomato-H2B, or RFP-actin fusion proteins, were introduced into live kidneys in a similar fashion. Gene expression was then observed in live and ex vivo kidneys using two-photon imaging and confocal laser scanning microscopy. We recorded widespread fluorescent protein expression lasting more than 1 mo after introduction of transgenes. Plasmid and adenovirus vectors provided gene transfer efficiencies ranging from 50 to 90%, compared with 10-50% using baculovirus. Using plasmids and adenovirus, fluorescent protein expression was observed 1) in proximal and distal tubule epithelial cells; 2) within glomeruli; and 3) within the peritubular interstitium. In isolated kidneys, fluorescent protein expression was observed from the cortex to the papilla. These results provide a robust approach for gene delivery and the study of protein function in live mammal kidneys.

Utility of drain fluid amylase measurement on the first postoperative day after pancreaticoduodenectomy.

BACKGROUND: Early detection of pancreatic fistula (PF) may improve the outcome after pancreaticoduodenectomy, and exclusion of PF may allow earlier drain removal and accelerate recovery. The aim of the present study was to evaluate the relationship between drain fluid amylase on the first postoperative day (DFA(1)) and PF. PATIENTS AND METHODS: This work was designed as a prospective study and included patients undergoing pancreaticoduodenectomy in a single center. For each patient, DFA was measured on the first and fifth postoperative days, and PF was defined by drainage of amylase-rich fluid on the fifth postoperative day (DFA(5) >300 U/l). A cut-off value of DFA(1) was derived, which yielded sensitivity and negative predictive value of 100% for predicting a PF. RESULTS: A total of 70 patients (47% male) who underwent pancreaticoduodenectomy (Whipple procedure: 37; pylorus-preserving procedure: 33) between April 2009 and March 2010 were included. Nine of those patients developed a PF (grade A-2; B-5; C-2). There were two postoperative deaths (3%). The DFA(1) value significantly correlated with DFA(5) (Spearman rank coefficient 0.68; p < 0.0001). The median DFA(1) of patients with a PF (6,205; range 357-23,391) was significantly higher than in patients without a PF (69; range 5-5,180; p = 0.01; unpaired t test). No patient with a PF had a DFA(1) ≤350 U/l, compared to 48/61 patients (79%) without a PF. Using 350 U/l as a cut-off, a low DFA(1) excluded a PF with a sensitivity, specificity, positive and negative predictive values of 100, 79, 41, and 100%, respectively. CONCLUSIONS: Drain fluid amylase on the DFA(1) after pancreaticoduodenectomy stratifies patients according to likelihood of developing a PF.

Dishevelled-associated activator of morphogenesis 1 (Daam1) is required for heart morphogenesis.

Dishevelled-associated activator of morphogenesis 1 (Daam1), a member of the formin protein family, plays an important role in regulating the actin cytoskeleton via mediation of linear actin assembly. Previous functional studies of Daam1 in lower species suggest its essential role in Drosophila trachea formation and Xenopus gastrulation. However, its in vivo physiological function in mammalian systems is largely unknown. We have generated Daam1-deficient mice via gene-trap technology and found that Daam1 is highly expressed in developing murine organs, including the heart. Daam1-deficient mice exhibit embryonic and neonatal lethality and suffer multiple cardiac defects, including ventricular noncompaction, double outlet right ventricles and ventricular septal defects. In vivo genetic rescue experiments further confirm that the lethality of Daam1-deficient mice results from the inherent cardiac abnormalities. In-depth analyses have revealed that Daam1 is important for regulating filamentous actin assembly and organization, and consequently for cytoskeletal function in cardiomyocytes, which contributes to proper heart morphogenesis. Daam1 is also found to be important for proper cytoskeletal architecture and functionalities in embryonic fibroblasts. Biochemical analyses indicate that Daam1 does not regulate cytoskeletal organization through RhoA, Rac1 or Cdc42. Our study highlights a crucial role for Daam1 in regulating the actin cytoskeleton and tissue morphogenesis.

RhoA/Rho kinase (ROCK) alters fetuin-A uptake and regulates calcification in bovine vascular smooth muscle cells (BVSMC).

RhoA/Rho kinases (ROCK) play a critical role in vascular smooth muscle cell (VSMC) actin cytoskeleton organization, differentiation, and function and are implicated in the pathogenesis of cardiovascular disease. We have previously determined that an important step in the regulation of calcification is fetuin-A endocytosis, a process that is dependent on changes in the cytoskeleton, which, in turn, is known to be affected by the RhoA/ROCK signaling pathway. In the present study, bovine VSMC (BVSMC) were treated with the ROCK inhibitor Y-27632 or transfected with ROCK small interfering (si) RNA to knock down ROCK expression. Both conditions resulted in reduced actin stress fibers and increased Cy5-labeled fetuin-A uptake. Inhibition of ROCK by Y-27632 or siRNA also significantly increased BVSMC alkaline phosphatase (ALP) activity and calcification of BVSMC and rat aorta organ cultures. Cells were then incubated in calcification media in the presence or absence of Y-27632 and matrix vesicles (MV) isolated by collagenase digestion. These MV, isolated from BVSMC incubated with Y-27632, had increased ALP activity and increased ability of MV to subsequently calcify collagen by 66%. In contrast, activation of RhoA, which is upstream of ROCK, by transfecting plasmids encoding the dominant active Rho GTPase mutant (Rho-L63) led to decreased fetuin-A uptake and reduced calcification in BVSMC. These results demonstrate that the RhoA/ROCK signaling pathway is an important negative regulator of vascular calcification.

A possible variant of Bouveret's syndrome presenting as a duodenal stump obstruction by a gallstone after Roux-en-Y gastrectomy: a case report.

INTRODUCTION: Bouveret's syndrome is characterized by gastric outlet obstruction due to a gallstone in the duodenum, usually in association with a cholecystoduodenal fistula. CASE PRESENTATION: We report the case of a 69-year-old Caucasian man who developed duodenal stump obstruction due to an impacted gallstone after having previously undergone Roux-en-Y gastrectomy. CONCLUSIONS: Duodenal stump obstruction after Roux-en-Y gastrectomy is rare, and may be difficult to manage. Patients who present with upper gastrointestinal or pancreatobiliary pathology after previous gastric surgery should be managed in centres with the availability of appropriate endoscopic and surgical experience.

siRNA targeted to p53 attenuates ischemic and cisplatin-induced acute kidney injury.

Proximal tubule cells (PTCs), which are the primary site of kidney injury associated with ischemia or nephrotoxicity, are the site of oligonucleotide reabsorption within the kidney. We exploited this property to test the efficacy of siRNA targeted to p53, a pivotal protein in the apoptotic pathway, to prevent kidney injury. Naked synthetic siRNA to p53 injected intravenously 4 h after ischemic injury maximally protected both PTCs and kidney function. PTCs were the primary site for siRNA uptake within the kidney and body. Following glomerular filtration, endocytic uptake of Cy3-siRNA by PTCs was rapid and extensive, and significantly reduced ischemia-induced p53 upregulation. The duration of the siRNA effect in PTCs was 24 to 48 h, determined by levels of p53 mRNA and protein expression. Both Cy3 fluorescence and in situ hybridization of siRNA corroborated a short t(1/2) for siRNA. The extent of renoprotection, decrease in cellular p53 and attenuation of p53-mediated apoptosis by siRNA were dose- and time-dependent. Analysis of renal histology and apoptosis revealed improved injury scores in both cortical and corticomedullary regions. siRNA to p53 was also effective in a model of cisplatin-induced kidney injury. Taken together, these data indicate that rapid delivery of siRNA to proximal tubule cells follows intravenous administration. Targeting siRNA to p53 leads to a dose-dependent attenuation of apoptotic signaling, suggesting potential therapeutic benefit for ischemic and nephrotoxic kidney injury.