A Histone Methylation-MAPK Signaling Axis Drives Durable Epithelial-Mesenchymal Transition in Hypoxic Pancreatic Cancer.

The tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) plays a key role in tumor progression and response to therapy. The dense PDAC stroma causes hypovascularity, which leads to hypoxia. Here, we showed that hypoxia drives long-lasting epithelial-mesenchymal transition (EMT) in PDAC primarily through a positive-feedback histone methylation-MAPK signaling axis. Transformed cells preferentially underwent EMT in hypoxic tumor regions in multiple model systems. Hypoxia drove a cell autonomous EMT in PDAC cells, which, unlike EMT in response to growth factors, could last for weeks. Furthermore, hypoxia reduced histone demethylase KDM2A activity, suppressed PP2 family phosphatase expression, and activated MAPKs to post-translationally stabilize histone methyltransferase NSD2, leading to an H3K36me2-dependent EMT in which hypoxia-inducible factors played only a supporting role. Hypoxia-driven EMT could be antagonized in vivo by combinations of MAPK inhibitors. Collectively, these results suggest that hypoxia promotes durable EMT in PDAC by inducing a histone methylation-MAPK axis that can be effectively targeted with multidrug therapies, providing a potential strategy for overcoming chemoresistance. SIGNIFICANCE: Integrated regulation of histone methylation and MAPK signaling by the low-oxygen environment of pancreatic cancer drives long-lasting EMT that promotes chemoresistance and shortens patient survival and that can be pharmacologically inhibited. See related commentary by Wirth and Schneider, p. 1739.

MCU-enriched dendritic mitochondria regulate plasticity in distinct hippocampal circuits.

Mitochondria are dynamic organelles that are morphologically and functionally diverse across cell types and subcellular compartments in order to meet unique energy demands. Mitochondrial dysfunction has been implicated in a wide variety of neurological disorders, including psychiatric disorders like schizophrenia and bipolar disorder. Despite it being well known that mitochondria are essential for synaptic transmission and synaptic plasticity, the mechanisms regulating mitochondria in support of normal synapse function are incompletely understood. The mitochondrial calcium uniporter (MCU) regulates calcium entry into the mitochondria, which in turn regulates the bioenergetics and distribution of mitochondria to active synapses. Evidence suggests that calcium influx via MCU couples neuronal activity to mitochondrial metabolism and ATP production, which would allow neurons to rapidly adapt to changing energy demands. Intriguingly, MCU is uniquely enriched in hippocampal CA2 distal dendrites relative to neighboring hippocampal CA1 or CA3 distal dendrites, however, the functional significance of this enrichment is not clear. Synapses from the entorhinal cortex layer II (ECII) onto CA2 distal dendrites readily express long term potentiation (LTP), unlike the LTP- resistant synapses from CA3 onto CA2 proximal dendrites, but the mechanisms underlying these different plasticity profiles are unknown. We hypothesized that enrichment of MCU near ECII-CA2 synapses promotes LTP in an otherwise plasticity-restricted cell type. Using a CA2-specific MCU knockout (cKO) mouse, we found that MCU is required for LTP at distal dendrite synapses but does not affect the lack of LTP at proximal dendrite synapses. Loss of LTP at ECII-CA2 synapses correlated with a trend for decreased spine density in CA2 distal dendrites of cKO mice compared to control (CTL) mice, which was predominantly seen in immature spines. Moreover, mitochondria were significantly smaller and more numerous across all dendritic layers of CA2 in cKO mice compared to CTL mice, suggesting an overall increase in mitochondrial fragmentation. Fragmented mitochondria might have functional changes, such as altered ATP production, that might explain a deficit in synaptic plasticity. Collectively, our data reveal that MCU regulates layer-specific forms of plasticity in CA2 dendrites, potentially by maintaining proper mitochondria morphology and distribution within dendrites. Differences in MCU expression across different cell types and circuits might be a general mechanism to tune the sensitivity of mitochondria to cytoplasmic calcium levels to power synaptic plasticity. MAIN TAKE HOME POINTS: The mitochondrial calcium uniporter (MCU) regulates plasticity selectively at synapses in CA2 distal dendrites.The MCU-cKO induced LTP deficit correlates with a trending reduction in spine density in CA2 distal dendrites.Loss of MCU in CA2 results in ultrastructural changes in dendritic mitochondria that suggest an increase in mitochondrial fragmentation. These ultrastructural changes could result in functional consequences, such as decreased ATP production, that could underlie the plasticity deficit.Dendritic mitochondrial fragmentation in MCU cKO occurred throughout the dendritic laminae, suggesting that MCU is dispensable for establishing layer-specific mitochondrial structural diversity.

Layer-specific mitochondrial diversity across hippocampal CA2 dendrites.

CA2 is an understudied subregion of the hippocampus that is critical for social memory. Previous studies identified multiple components of the mitochondrial calcium uniporter (MCU) complex as selectively enriched in CA2. The MCU complex regulates calcium entry into mitochondria, which in turn regulates mitochondrial transport and localization to active synapses. We found that MCU is strikingly enriched in CA2 distal apical dendrites, precisely where CA2 neurons receive entorhinal cortical input carrying social information. Furthermore, MCU-enriched mitochondria in CA2 distal dendrites are larger compared to mitochondria in CA2 proximal apical dendrites and neighboring CA1 apical dendrites, which was confirmed in CA2 with genetically labeled mitochondria and electron microscopy. MCU overexpression in neighboring CA1 led to a preferential localization of MCU in the proximal dendrites of CA1 compared to the distal dendrites, an effect not seen in CA2. Our findings demonstrate that mitochondria are molecularly and structurally diverse across hippocampal cell types and circuits, and suggest that MCU can be differentially localized within dendrites, possibly to meet local energy demands.

Model of severe malaria in young mice suggests unique response of CD4 T cells.

Severe malaria occurs most in young children but is poorly understood due to the absence of a developmentally-equivalent rodent model to study the pathogenesis of the disease. Though functional and quantitative deficiencies in innate response and a biased T helper 1 (Th1) response are reported in newborn pups, there is little information available about this intermediate stage of the adaptive immune system in murine neonates. To fill this gap in knowledge, we have developed a mouse model of severe malaria in young mice using 15-day old mice (pups) infected with Plasmodium chabaudi. We observe similar parasite growth pattern in pups and adults, with a 60% mortality and a decrease in the growth rate of the surviving young mice. Using a battery of behavioral assays, we observed neurological symptoms in pups that do not occur in infected wildtype adults. CD4+ T cells were activated and differentiated to an effector T cell (Teff) phenotype in both adult and pups. However, there were relatively fewer and less terminally differentiated pup CD4+ Teff than adult Teff. Interestingly, despite less activation, the pup Teff expressed higher T-bet than adults' cells. These data suggest that Th1 cells are functional in pups during Plasmodium infection but develop slowly.

Protein-retention expansion microscopy for visualizing subcellular organelles in fixed brain tissue.

BACKGROUND: Protein expansion microscopy (proExM) is a powerful technique that crosslinks proteins to a swellable hydrogel to physically expand and optically clear biological samples. The resulting increased resolution (~70 nm) and physical separation of labeled proteins make it an attractive tool for studying the localization of subcellular organelles in densely packed tissues, such as the brain. However, the digestion and expansion process greatly reduce fluorescence signals making it necessary to optimize ExM conditions per sample for specific end goals. NEW METHOD: Here we compare the staining and digestion conditions of existing proExM workflows to identify the optimal protocol for visualizing subcellular organelles (mitochondria and the Golgi apparatus) within reporter-labeled neurons in fixed mouse brain tissue. RESULTS: We found that immunostaining before proExM and using a proteinase K based digestion for 8 h consistently resulted in robust fluorescence retention for immunolabeled subcellular organelles and genetically-encoded reporters. COMPARISON WITH EXISTING METHODS: With these methods, we more accurately quantified mitochondria size and number and better visualized Golgi ultrastructure in individual CA2 neurons in the mouse hippocampus. CONCLUSIONS: This organelle optimized proExM protocol will be broadly useful for investigators interested in visualizing the spatial distribution of immunolabeled subcellular organelles in various reporter mouse lines, reducing effort, time and resources on the optimization process.

Robotic Buccal Mucosal Graft Ureteroplasty for Complex Ureteral Stricture.

OBJECTIVE: To demonstrate robot-assisted ureterolysis and buccal mucosal graft (BMG) ureteroplasty for the management of a complex, long recurrent ureteral stricture developing after ureterolysis, and also to demonstrate the use of near-infrared fluorescence (NIRF) imaging and intraoperative ureteroscopy during this procedure. METHODS: A 58-year-old man with a history of cabergoline treatment and a cardiac catheterization through the left groin presented with left flank pain and hydronephrosis. A computed tomography scan showed extensive fibrosis around the ureter and a ureteral stricture close to a tortuous left external iliac artery. A computed tomography-guided biopsy showed a benign fibrous tissue around the stricture with no increase in IgG4-expressing plasma cells. A robot-assisted ureterolysis with an omental wrap was performed. One year after the ureterolysis, the patient developed a recurrent ureteral stricture. Retrograde ureterogram showed a long, 6-cm stricture in the upper ureter. For the robotic ureteroplasty, the patient was placed in modified lateral position with port placement similar to the left pyeloplasty. Intraoperative flexible ureteroscopy and NIRF were used to define the distal extent of the stricture. For this, the ureteroscope was advanced until the stricture, and transilluminance of light from the ureteroscope was seen from the robotic camera using Firefly. Ureteral stricture was incised along its length over the ureteroscope. Two BMGs were harvested and sown together to obtain a longer graft. The graft was minimally defatted and brought in the abdomen through one of the ports. The composite graft was then sutured with 4-0 PDS as an onlay graft with the mucosal side facing toward the lumen of the ureter. Ureteroscopy was used to confirm patency, followed by stent placement. NIRF was used to confirm the viability of the ureter and the surrounding tissue. The omental flap was then harvested using a vessel sealer, fixed to the psoas fascia beneath the ureter, and then wrapped over the reconstructed ureter. The omental flap was also tacked to the side of the BMG with a suture to promote blood supply. RESULTS: The procedure was uncomplicated with an operative time of 280 minutes, an estimated blood loss of 75 mL, and an uneventful hospital stay. MAG3 Renal scan after 3 and 6 months of surgery showed no recurrence or obstruction. CONCLUSION: Despite the limitation of being a single case with only a 6-month follow-up, our report shows that robot-assisted BMG is a safe option for the reconstruction of long upper ureteral strictures. This procedure may be a less morbid alternative to an autotransplant and ileal ureter in these patients. However, outcomes need to be studied in a larger series with a longer follow-up.

Postoperative urinary retention in men is common after carotid endarterectomy and is associated with advanced age and prior urinary tract infection.

OBJECTIVE: This study was undertaken to analyze the occurrence of postoperative urinary retention (POUR) after carotid endarterectomy (CEA) and determine whether there are any associated modifiable risk factors. CEA was chosen to minimize the confounding effects of known risk factors for POUR, including immobilization, regional and severe pain, and neuroaxial anesthesia. METHODS: This was a retrospective record review of 186 male patients undergoing CEA between 2007 and 2011. Demographic, comorbidities, and operative characteristics were compared. Continuous variables are reported as median and interquartile range (IQR) and categoric variables as frequencies and proportions. Pearson χ(2) or Mann-Whitney U tests compared categoric and continuous variables, respectively. Logistic regression was used to examine univariate and multivariate odds of POUR. Multivariate analysis controlled for known predictors of urinary retention. Association with other complications was examined with the Pearson correlation coefficient. RESULTS: POUR occurred in 34 patients (18.3%). Median age and history of urinary tract infection (UTI) were significantly associated with POUR: median age was 73.0 years (IQR, 67-80 years) for those with POUR vs 69.5 years (IQR, 63-76 years) for those without (P = .047); 17.6% of patients with a history of UTI developed POUR vs 5.9% without (P = .023). These findings persisted on multivariate analysis controlling for known predictors of POUR (body mass index, history of diabetes, benign prostate hyperplasia, and prior prostate surgery): median age (odds ratio, 1.05; 95% confidence interval, 1-1.1) and history of UTI (odds ratio, 4.16; 95% confidence interval, 1.23-14.05; P = .022). The occurrence of POUR was significantly correlated with postoperative UTI: 18.8% with POUR vs 0.7% without (Pearson r = 0.369; P < .001). CONCLUSIONS: POUR requiring bladder catheterization after CEA predisposes patients to postoperative UTI and is more common in older patients and those with a history of UTI. CEA patients lack inherent risk factors for POUR and would be a useful population for prospective studies involving POUR.

Causes of hospital readmissions after urologic cancer surgery.

OBJECTIVES: The Hospital Readmissions Reduction Program mandates reimbursement reductions to hospitals with higher than expected rates of readmissions. We examine causes and predictors of readmissions following major procedures in urologic oncology. MATERIALS AND METHODS: Using the American College of Surgeons National Surgical Quality Improvement Program database, patients undergoing radical prostatectomy (RP), radical (RN) or partial nephrectomy (PN), and radical cystectomy (RC) during the year 2012 were abstracted. Rates of unplanned readmission within 30 days after surgery, as well as causes of readmission, were identified. Multivariable logistic regression models were fitted to examine the association between patient perioperative factors and odds of readmission. RESULTS: Overall, we observed a 5.5% unplanned 30-day readmission rate. Readmission rates for patients treated with RP, RN, PN, and RC were 4.1%, 5.2%, 4.5%, and 15.9%, respectively. For each procedure, approximately two-third of readmissions occurred within the first 10 days following hospital discharge. Commonest causes of readmission after RP included thromboembolic (13.6%), wound (12.2%), renal/genitourinary (12.2%), and gastrointestinal (11.8%); after RN, wound (12.9%) and gastrointestinal (12.9%); after PN, renal/genitourinary (19.6%), cardiovascular (9.8%), and bleeding/hematoma (9.8%); and after RC, renal/genitourinary (15.5%), wound (14.8%), and sepsis/infection (14.1%). RC was significantly associated with readmission. Patients undergoing open RP or PN were more likely to be readmitted relative to their minimally invasive counterparts (odds ratio = 1.53, 95% CI: 1.12-2.08, P = 0.007 and odds ratio = 2.51, 95% CI: 1.38-4.55, P = 0.003, respectively). CONCLUSIONS: Readmissions are relatively common following major urologic oncology procedures. Compared with RP, RN, or PN, RC patients experience the highest burden of readmission. Venous thromboembolism is a common modifiable cause of readmission following urologic cancer surgery. Minimally invasive approach is associated with decreased odds of readmission following RP and PN.

A Clinician's Guide to Avoiding and Managing Common Complications During and After Robot-assisted Laparoscopic Radical Prostatectomy.

CONTEXT: Robot-assisted radical prostatectomy (RARP) is on the advance globally, and it is essential for surgeons and patients to know the rates of perioperative complications. OBJECTIVE: To provide evidence-based clinical guidance on avoiding and managing common complications during and after RARP in the context of a comprehensive literature review. EVIDENCE ACQUISITION: In concordance with the Preferred Reporting Items for Systematic Reviews and Meta-analysis 2015 statement guidelines, a literature search of the PubMed database from August 1, 2011, to August 31, 2015, using the predefined search terms robot* AND radical prostatectomy, was conducted. The search resulted in 653 unique results that were subsequently uploaded to DistillerSR (Evidence Partners, Ottawa, Canada) for team-based screening and processing of references. EVIDENCE SYNTHESIS: Overall, 37 studies met the inclusion criteria and were included. Median rate of overall complication was 12.6% (range: 3.1-42%). Most of the complications were minor (Clavien-Dindo grades 1 and 2). Grade 3 complications comprised the bulk of the major complications with a median rate of 2.7%; grade IV and V complications were exceedingly rare in all reports. CONCLUSIONS: Despite continued adoption of the RARP technique globally, rates of overall complication remain low. Many of the complications experienced during and after RARP can be mitigated and prevented by experience and the implementation of safe techniques. PATIENT SUMMARY: Despite continued adoption of the robot-assisted radical prostatectomy (RARP) technique globally, rates of overall and major complications remain low at 12.6% and 2.7%, respectively. Complications can be minimized and successfully managed using established techniques. RARP is a safe and reproducible technique.

Impact of smoking on perioperative outcomes after major surgery.

BACKGROUND: To investigate the impact of smoking on perioperative outcomes in patients undergoing one of the 16 major cardiovascular, orthopedic, or oncologic surgical procedures. METHODS: We relied on the American College of Surgeons National Surgical Quality Improvement Program database (2005 to 2011). Procedure-specific multivariable logistic regression models assessed the association between smoking status (non, former, or current smokers) and risk of 30-day morbidity and mortality. RESULTS: Overall, 141,802 patients were identified. A total of 12.5%, 14.6%, and 14.9% of non, former, and current smokers, respectively, experienced at least one complication (P < .001). In multivariable models, current smokers had higher odds of overall, pulmonary, wound, and septic/shock complications following most cardiovascular and oncologic surgeries compared with nonsmokers. The odds of experiencing such adverse outcomes were significantly lower in former smokers compared with current smokers, but still higher compared with nonsmokers. CONCLUSIONS: The effect of smoking on perioperative outcomes is procedure dependent. Current and, even though mitigated, former smoking negatively influence outcomes following cardiovascular or oncologic procedures. Patients undergoing major procedures should be encouraged to discontinue tobacco smoking to achieve optimal procedural outcomes.

Robotic surgical skill acquisition: What one needs to know?

Robotic surgery has been eagerly adopted by patients and surgeons alike in the field of urology, over the last decade. However, there is a lack of standardization in training curricula and accreditation guidelines to ensure surgeon competence and patient safety. Accordingly, in this review, we aim to highlight 'who' needs to learn 'what' and 'how', to become competent in robotic surgery. We demonstrate that both novice and experienced open surgeons require supervision and mentoring during the initial phases of robotic surgery skill acquisition. The experienced open surgeons possess domain knowledge, however, need to acquire technical knowledge under supervision (either in simulated or clinical environment) to successfully transition to robotic surgery, whereas, novice surgeons need to acquire both domain as well as technical knowledge to become competent in robotic surgery. With regard to training curricula, a variety of training programs such as academic fellowships, mini-fellowships, and mentored skill courses exist, and cater to the needs and expectations of postgraduate surgeons adequately. Fellowships provide the most comprehensive training, however, may not be suitable to all surgeon-learners secondary to the long-term time commitment. For these surgeon-learners short-term courses such as the mini-fellowships or mentored skill courses might be more apt. Lastly, with regards to credentialing uniformity in criteria regarding accreditation is lacking but earnest efforts are underway. Currently, accreditation for competence in robotic surgery is institutional specific.