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Objective: To investigate the relationship between hyperglycemia and adverse in-hospital outcomes in patients with and without known diabetes Research Design and Methods: The Diabetes IN-hospital: Glucose and Outcomes (DINGO) prospective cohort study was conducted over 26 weeks between Oct-20 and Mar-2020 at The Royal Melbourne Hospital. Admissions with ≥2 capillary glucose values and length of stay >24 hours were eligible. Stratification was by in-hospital hyperglycemia (IHH) , defined as ≥2 capillary glucose values of ≥11.1 mmol/L [≥200 mg/dL]. Results: We included 2,6admissions, of which 1,189 (46%) experienced in-hospital hyperglycemia. Following adjustment (covariates) , IHH was independently associated with in-hospital healthcare-associated infection (HAI) , acute kidney injury (AKI) , stroke, and mortality (see figure for incidence rate ratios [IRR]) . For each outcome the association was independent of diabetes status, except for mortality where the association was stronger in those without known diabetes. Patient-day mean glucose was elevated in those with HAI and AKI, on both whole-of-admission and prior-to-event calculations. Conclusions: In-hospital hyperglycemia is independently associated with in-hospital HAI and AKI in patients with and without known diabetes. Disclosure R.Barmanray: None. M.Kyi: None. L.J.Worth: None. P.G.Colman: None. T.N.Fazio: None. G.Rayman: None. S.Fourlanos: Advisory Panel; Pfizer Inc., Viatris Inc., Speaker's Bureau; AstraZeneca, Novo Nordisk. Funding Rowe Family Foundation Perpetual GrantAustralian Government Research Training Program Scholarship

Progressive impairment of proteostasis and accumulation of toxic misfolded proteins are associated with the cellular aging process. Here, we employed chronologically aged yeast cells to investigate how activation of the unfolded protein response (UPR) upon accumulation of misfolded proteins in the endoplasmic reticulum (ER) affects lifespan. We found that cells lacking a functional UPR display a significantly reduced chronological lifespan, which contrasts previous findings in models of replicative aging. We find exacerbated UPR activation in aged cells, indicating an increase in misfolded protein burden in the ER during the course of aging. We also observed that caloric restriction, which promotes longevity in various model organisms, extends lifespan of UPR-deficient strains. Similarly, aging in pH-buffered media extends lifespan, albeit independently of the UPR. Thus, our data support a role for caloric restriction and reduced acid stress in improving ER homeostasis during aging. Finally, we show that UPR-mediated upregulation of the ER chaperone Kar2 and functional ER-associated degradation (ERAD) are essential for proper aging. Our work documents the central role of secretory protein homeostasis in chronological aging in yeast and highlights that the requirement for a functional UPR can differ between post-mitotic and actively dividing eukaryotic cells.

Temozolomide (TEM) is an active treatment in metastatic neuroendocrine tumors (NETs). Patients affected by glioblastoma multiforme or advanced melanoma treated with TEM who have deficiency of OWe conducted a systematic review of the literature and meta-analysis, based on PRISMA methodology, searching in the main databases (PubMed, Embase, Scopus, Web of Science, Cochrane Library and clinical trial.gov) and the proceedings of the main international congresses, until April 26, 2021.Twelve out of 616 articles were selected for our analysis, regarding a total of 858 NET patients treated with TEM-based chemotherapy. The status of MGMT had been tested in 513 (60%) patients, using various methods. The pooled overall response rate (ORR) was higher in MGMT-deficient compared with MGMT-proficient NETs, with a risk difference of 0.31 (95% confidence interval, CI: 0.13-0.50; p 0.001; IOur meta-analysis suggested that MGMT status may be predictive of TEM efficacy. However, due to the high heterogeneity of the evaluated studies the risk of biases should be considered. On this hypothesis future homogeneous prospective studies are warranted.

Background Colorectal cancer is the 2nd leading cause of cancer death in Canada. In rapidly dividing tissues such as the intestine or colon, only long-lived, multipotent, self-renewing tissue stem cells have longevity to accumulate mutations and serve as the cellular origin of cancer. In the small intestine, genetic fate mapping studies have demonstrated that there are at least two principal stem cell pools: actively cycling, crypt base cells expressing Lgr5, and quiescent cells situated above the crypt base. Clevers and colleagues have previously shown that Lgr5-expressing cells can give rise to cancer upon mutation. Interestingly, when Lgr5+ stem cells are selectively “killed”, intestinal integrity remains intact and other stem cells restore homeostasis. To determine whether another stem cell population can give rise to cancer in the colon, we examined whether the atypical homeobox protein Hopx, marks stem cells in the colon and whether these cells can give rise to colon cancer. Aims In the present study, we aim to determine whether Hopx-expressing cells are colonic stem cells that contribute to gut healing and can give rise to colonic tumours following the loss of APC. Methods To determine whether Hopx expressing cells show stemness, we crossed Hopx-CreERT mice to R26-TdTomato reporter mice. We then conducted genetic lineage tracing studies in the colon during homeostasis and following dextran sodium sulphate (DSS)-induced colitis. To test the function of Hopx expressing cells, Hopx-CreERT mice were also crossed to R26DTR mice and treated with diphtheria toxin (DT) following tamoxifen. These mice were then exposed to either normal drinking water or DSS to determine the role of Hopx+ cells in colonic regeneration. To test whether Hopx expressing cells can serve as a cellular origin for colon cancer, Hopx-CreERT mice were crossed to Apcf/f (floxed) mice. Results Consistent with the labeling of a stem cell, following tamoxifen, Hopx+ cells expressing tdTomato expanded to lineage trace full colonic crypts within 7 days, and labelling was persistent for greater than 6 months. Interestingly, ablation of Hopx+ cells with DT did not alter weight, histological damage or survival during normal homeostasis, however, Hopx+ cell ablation in mice treated with DSS resulted in increased histological damage. Surprisingly, loss of APC in Hopx-expressing cells did not induce colonic adenomas even after 8 months following tamoxifen administration. Conclusions These findings prove that Hopx expressing cells identify a novel colonic stem cell pool that is redundant in homeostasis, but in the context of injury, are essential for epithelial regeneration. Interestingly, Hopx+ cells do not have the capacity to give rise to colorectal adenomas upon loss of the APC gene. Funding Agencies CIHR

BACKGROUND: Colorectal cancer (CRC) is the second leading cause of cancer death in Canada, with the major risk factor being chronic inflammation. As such, patients with inflammatory bowel disease (IBD) are at an increased risk of CRC. Despite the clear association between inflammation and cancer, the mechanism by which colitis leads to CRC is still not well understood. Our recent work has focused on a colonic epithelial cell known as the tuft cell that uniquely expresses the protein doublecortin-like kinase 1 (Dclk1). Using Cre-dependent lineage tracing of Dclk1-expressing cells, we showed that Dclk1 labels long-lived quiescent cells in the colon that serve as a cellular origin of CRC upon colonic inflammation. AIMS: In this study, we aim to explore the mechanism by which inflammation contributes to tuft cell cancer initiation. We hypothesized that colonic inflammatory insult leads to dedifferentiation of Dclk1+ tuft cells to a stem cell state susceptible to tumor initiation. METHODS: To generate tamoxifen-inducible Cre transgenic mice that allow for Dclk1(+) cell lineage tracing and cell-specific knock-out of the tumor suppressor adenomatous polyposis coli (APC), we first crossed our transgenic Dclk1-CreER(T2) mice to both ROSA26-tdTomato and APC(fl/fl) mice (Dclk1/APC(fl/fl)). To examine the role of dedifferentiation in colonic tumor initiation, these mice were further crossed to Lgr5-DTR-eGFP mice (Lgr5(DTR);Dclk1/APC(fl/fl)). These mice were given tamoxifen and DSS in order to induce tumorigenesis. Mice were administered diphtheria toxin (DT) for six weeks post DSS injury to ablate Lgr5-expressing cells and were compared to Lgr5(DTR)-negative control mice (Dclk1/APC(fl/fl)). RESULTS: Reverse transcription polymerase chain reaction (RT-PCR) analysis of mRNA levels revealed significantly reduced Lgr5, and increased RSPO1 and RSPO3 levels in colonic tissues of DSS-administered mice. Ablation of Lgr5(+) cells post DSS-colitis significantly reduced the number of colonic tumors in our Dclk1/APC(fl/fl) mouse model, with no significant difference in tumor size. Lgr5-expressing cells were readily seen throughout colonic tumors arising from Dclk1(+) cells. Interestingly, two weeks post DSS-induced colitis, rare Dclk1(+) cells that co-expressed Lgr5 were also detected. CONCLUSIONS: Our data prove that upon DSS-induced colonic injury, Dclk1(+) tuft cells express the stem cell marker Lgr5 prior to initiation of colonic tumorigenesis. These data suggest that dedifferentiation of Dclk1(+) cells plays an important role in colitis-associated CRC and provide insight into the molecular mechanism by which Dclk1-derived colonic tumors arise. FUNDING AGENCIES: CAG, CIHRCRS

Purpose Adjuvant chemotherapy improves survival of patients with gastric cancer. Intergroup Trial of Adjuvant Chemotherapy in Adenocarcinoma of the Stomach (ITACA-S) was a phase III study comparing sequential FOLFIRI followed by docetaxel/cisplatin versus 5-fluorouracil monotherapy. The intensive regimen was not superior in terms of disease-free survival (DFS) and overall survival (OS). Methods The treatment was to be started within 8 weeks from surgery. This analysis evaluates the impact of time from surgery to chemotherapy start (TSC) on outcomes. Results Out of 1,106 randomized, 1,072 patients without major violations of eligibility criteria and receiving at least one treatment cycle were analyzed. Median TSC was 50 days. Chemotherapy was interrupted in 201 (18.8%) cases, whereas it was completed without or with modifications in 277 (25.8%) and 594 (55.4%), respectively. At a median follow-up of 56.9 months, 513 progressions and 472 deaths occurred. A longer TSC was significantly associated with longer DFS (hazard ratio [HR] 0.95; 95% confidence interval [CI] 0.89-1.00; p = 0.05) and OS (HR 0.91; 95% CI 0.86-0.97; p = 0.004), after adjustment for treatment arm, age, sex, primary tumor site, number of resected nodes, and tumor stage. Better treatment compliance was associated with improved survival. Conclusions Our findings suggest that longer TSC had at least no detrimental effect on DFS and OS, whereas treatment completion had a protective effect. Our findings need to be confirmed prospectively.

During homeostasis, the colonic epithelium is replenished every 3–5 days by rapidly cycling Lgr5 (+) stem cells. However, various insults can lead to depletion of Lgr5 (+) stem cells, and colonic epithelium can be regenerated from Lgr5‐negative cells. While studies in the small intestine have addressed the lineage identity of the Lgr5‐negative regenerative cell population, in the colon this question has remained unanswered. Here, we set out to identify which cell(s) contribute to colonic regeneration by performing genetic fate‐mapping studies of progenitor populations in mice. First, using keratin‐19 (Krt19) to mark a heterogeneous population of cells, we found that Lgr5‐negative cells can regenerate colonic crypts and give rise to Lgr5 (+) stem cells. Notch1 (+) absorptive progenitor cells did not contribute to epithelial repair after injury, whereas Atoh1 (+) secretory progenitors did contribute to this process. Additionally, while colonic Atoh1 (+) cells contributed minimally to other lineages during homeostasis, they displayed plasticity and contributed to epithelial repair during injury, independent of Lgr5 (+) cells. Our findings suggest that promotion of secretory progenitor plasticity could enable gut healing in colitis.

A novel role for ATF3 is identified in acinar-to–duct cell metaplasia during pancreatic injury. ATF3 targets transcriptional regulators that affect acinar cell fate and reduces the severity of pancreatic injury. However, by doing so, ATF3 may also increase the susceptibility for progression to pancreatic adenocarcinoma., Pancreatitis is a debilitating disease of the exocrine pancreas that, under chronic conditions, is a major susceptibility factor for pancreatic ductal adenocarcinoma (PDAC). Although down-regulation of genes that promote the mature acinar cell fate is required to reduce injury associated with pancreatitis, the factors that promote this repression are unknown. Activating transcription factor 3 (ATF3) is a key mediator of the unfolded protein response, a pathway rapidly activated during pancreatic insult. Using chromatin immunoprecipitation followed by next-generation sequencing, we show that ATF3 is bound to the transcriptional regulatory regions of >30% of differentially expressed genes during the initiation of pancreatitis. Of importance, ATF3-dependent regulation of these genes was observed only upon induction of pancreatitis, with pathways involved in inflammation, acinar cell differentiation, and cell junctions being specifically targeted. Characterizing expression of transcription factors that affect acinar cell differentiation suggested that acinar cells lacking ATF3 maintain a mature cell phenotype during pancreatitis, a finding supported by maintenance of junctional proteins and polarity markers. As a result, Atf3–/– pancreatic tissue displayed increased tissue damage and inflammatory cell infiltration at early time points during injury but, at later time points, showed reduced acinar-to–duct cell metaplasia. Thus our results reveal a critical role for ATF3 as a key regulator of the acinar cell transcriptional response during injury and may provide a link between chronic pancreatitis and PDAC.

International audience; The Fourth Metronomic and Anti-angiogenic Therapy Meeting was held in Milan 24-25 June 2014. The meeting was a true translational meeting where researchers and clinicians shared their results, experiences, and insights in order to continue gathering useful evidence on metronomic approaches. Several speakers emphasised that exact mechanisms of action, best timing, and optimal dosage are still not well understood and that the field would learn a lot from ancillary studies performed during the clinical trials of metronomic chemotherapies. From the pre-clinical side, new research findings indicate additional possible mechanisms of actions of metronomic schedule on the immune and blood vessel compartments of the tumour micro-environment. New clinical results of metronomic chemotherapy were presented in particular in paediatric cancers [especially neuroblastoma and central nervous system (CNS) tumours], in angiosarcoma (together with beta-blockers), in hepatocellular carcinoma, in prostate cancer, and in breast cancer. The use of repurposed drugs such as metformin, celecoxib, or valproic acid in the metronomic regimen was reported and highlighted the potential of other candidate drugs to be repurposed. The clinical experiences from low- and middle-income countries with affordable regimens gave very encouraging results which will allow more patients to be effectively treated in economies where new drugs are not accessible. Looking at the impact of metronomic approaches that have been shown to be effective, it was admitted that those approaches were rarely used in clinical practice, in part because of the absence of commercial interest for companies. However, performing well-designed clinical trials of metronomic and repurposing approaches demonstrating substantial improvement, especially in populations with the greatest unmet needs, may be an easier solution than addressing the financial issue. Metronomics should always be seen as a chance to come up with new innovative affordable approaches and not as a cheap rescue strategy.

Several animal models have been developed to investigate the pathobiology of pancreatitis, but few studies have examined the effects that altered pancreatic gene expression have in these models. In this study, the sensitivity to secretagogue-induced pancreatitis was examined in a mouse line that has an altered acinar cell environment due to the targeted deletion of Mist1. Mist1 is an exocrine specific transcription factor important for the complete differentiation and function of pancreatic acinar cells. Mice lacking the Mist1 gene [ Mist1 knockout (KO) mice] exhibit cellular disorganization and functional defects in the exocrine pancreas but no gross morphological defects. Following the induction of pancreatitis with caerulein, a CCK analog, we observed elevated serum amylase levels, necrosis, and tissue damage in Mist1 KO mice, indicating increased pancreatic damage. There was also a delay in the regeneration of acinar tissue in Mist1 KO animals. Molecular profiling revealed an altered activation of stress response genes in Mist1 KO pancreatic tissue compared with wild-type (WT) tissue following the induction of pancreatitis. In particular, Western blot analysis for activating transcription factor 3 and phosphorylated eukaryotic initiation factor 2α (eIF2α), mediators of endoplasmic reticulum (ER) stress, indicated limited activation of this pathway in Mist1 KO animals compared with WT controls. Conversely, Mist1 KO pancreatic tissue exhibits increased expression of growth arrest and DNA damage inducible 34 protein, an inhibitor of eIF2α phosphorylation, before and after the induction of pancreatitis. These finding suggest that activation of the ER stress pathway is a protective event in the progression of pancreatitis and highlight the Mist1 KO mouse line as an important new model for studying the molecular events that contribute to the sensitivity to pancreatic injury.

// Teresa Peart 1, 2 , Yudith Ramos Valdes 1 , Rohann J. M. Correa 1, 3 , Elena Fazio 1, 2 , Monique Bertrand 1, 4, 5 , Jacob McGee 1, 4 , Michel Prefontaine 1, 4 , Akira Sugimoto 1, 4, 5 , Gabriel E. DiMattia 1, 3, 4, 5 , Trevor G. Shepherd 1, 2, 4, 5 1 Translational Ovarian Cancer Research Program, London Regional Cancer Program, London, Ontario, Canada 2 Departments of Anatomy & Cell Biology, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Ontario, Canada 3 Departments of Biochemistry, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Ontario, Canada 4 Departments of Obstetrics & Gynaecology, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Ontario, Canada 5 Departments of Oncology, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Ontario, Canada Correspondence to: Trevor G. Shepherd, e-mail: tshephe6@uwo.ca Keywords: ovarian cancer, spheroid, LKB1, AMPK, tumour cell dormancy Received: May 14, 2015 Accepted: May 23, 2015 Published: June 05, 2015 ABSTRACT Metastatic epithelial ovarian cancer (EOC) cells can form multicellular spheroids while in suspension and disperse directly throughout the peritoneum to seed secondary lesions. There is growing evidence that EOC spheroids are key mediators of metastasis, and they use specific intracellular signalling pathways to control cancer cell growth and metabolism for increased survival. Our laboratory discovered that AKT signalling is reduced during spheroid formation leading to cellular quiescence and autophagy, and these may be defining features of tumour cell dormancy. To further define the phenotype of EOC spheroids, we have initiated studies of the Liver kinase B1 (LKB1)-5′-AMP-activated protein kinase (AMPK) pathway as a master controller of the metabolic stress response. We demonstrate that activity of AMPK and its upstream kinase LKB1 are increased in quiescent EOC spheroids as compared with proliferating adherent EOC cells. We also show elevated AMPK activity in spheroids isolated directly from patient ascites. Functional studies reveal that treatment with the AMP mimetic AICAR or allosteric AMPK activator A-769662 led to a cytostatic response in proliferative adherent ovarian cancer cells, but they fail to elicit an effect in spheroids. Targeted knockdown of STK11 by RNAi to reduce LKB1 expression led to reduced viability and increased sensitivity to carboplatin treatment in spheroids only, a phenomenon which was AMPK-independent. Thus, our results demonstrate a direct impact of altered LKB1-AMPK signalling function in EOC. In addition, this is the first evidence in cancer cells demonstrating a pro-survival function for LKB1, a kinase traditionally thought to act as a tumour suppressor.

Mist1 is an exocrine-specific transcription factor that is necessary for the establishment of cell organization and function of pancreatic acinar cells. While Mist1 is not expressed in the endocrine pancreas, the disorganized phenotype of the exocrine component may affect endocrine function. Therefore, we examined endocrine tissue morphology and function in Mist1-knockout (Mist1KO) mice. Endocrine function was evaluated using a glucose-tolerance test on 2–10-month-old female mice and revealed a significant reduction in glucose-clearing ability in 10-month-old Mist1KO mice compared with wild-type mice. Immunohistochemical analysis of islet hormone expression indicated that the decreased endocrine function was not due to a decrease in insulin-, glucagon- or somatostatin-expressing cells. However, a decrease in the size of islets in 10-month-old Mist1KO mice was observed along with a decrease in Glut-2 protein accumulation. These results suggest that the islets in Mist1KO mice are functionally compromised, likely accounting for the decreased glucose tolerance. Based on these findings, we have identified that the loss of a regulatory gene in the exocrine compartment can affect the endocrine component, providing a possible link between susceptibility for various pancreatic diseases.

BACKGROUND: Colorectal cancer (CRC) is the 2(nd) leading cause of cancer death in Canada. A major risk factor for this disease is chronic inflammation. For this reason, patients with inflammatory bowel disease (IBD), such as Crohn’s disease or Ulcerative colitis, require frequent colon cancer screening. Despite the clear link between inflammation and cancer, the exact mechanism by which colitis leads to cancer is unknown. Our group has focused on a rare and ill-defined cell type in the gut known as a tuft cell that uniquely expresses doublecortin-like kinase-1 (Dclk1). Using a novel transgenic mouse model, we have previously shown that Dclk1+ tuft cells are quiescent and long-lived, and remain resistant to proliferation even upon mutation of the tumor suppressor APC. Interestingly, these cells become powerful cancer-initiating cells upon exposure to inflammation, but the mechanism by which inflammation leads to colonic tumors is not known. Intriguingly, Dclk1+ tuft cells express high levels of cyclooxygenase (COX)-1 and -2, the direct enzyme target of non-steroidal anti-inflammatory drugs (NSAIDs) which are known chemopreventative drugs in CRC. AIMS: In the present study, we aim to determine the effects of COX inhibition by NSAIDs on colitis-associated colorectal cancer. METHODS: Dclk1CreERT2/APC(flox/flox) mice were administered tamoxifen to induce an APC mutation in Dclk1-expressing cells. Mice were then exposed to the colitis-inducing agent dextran sodium sulfate (DSS), followed by daily treatment with oral NSAIDs or vehicle for the remainder of the experiment duration. The NSAIDs tested included Aspirin (non-selective COX inhibitor), celecoxib or rofecoxib (COX-2-selective inhibitors), or SC-560 (COX-1-selective inhibitor). Approximately 16 weeks post-tamoxifen, colonic tumour number and size were analyzed to determine the effect of these NSAIDs on tumour initiation and growth, respectively. Extent of inflammation was assessed by myeloperoxidase (MPO) activity and histology, and colonic tissue was taken for measurement of inflammatory mediators by qRT-PCR. RESULTS: Treatment with Aspirin and SC-560, but surprisingly, not celecoxib and rofecoxib, significantly reduced the number of colonic tumours. There was no significant difference in tumour size between vehicle and any of the NSAID-treated groups. Of note, the degree of colitis as assessed by MPO activity and histology was not significantly different between vehicle and NSAID-treated groups. CONCLUSIONS: These findings suggest a role for COX-mediated inflammation in colonic tumorigenesis arising from Dclk1+ tuft cells. Our results suggest that COX-1-selective, rather than COX-2-selective, NSAIDs may be useful for chemoprevention of CRC in patients with IBD. FUNDING AGENCIES: CIHR

BACKGROUND: The intestinal and colonic epithelia are rapidly renewed every 3 to 5 days. In the intestine, at least two principal stem cell pools, comprised of rapidly cycling crypt based columnar (CBC) Lgr5(+) cells and slower cycling Bmi1-expressing cells located above the crypt base, have been described. In the colon, however, we have recently identified an additional, spatially distinct, Lgr5-negative stem cell pool that is a subset of Krt19-positive cells. Recent studies have shown that colonic inflammation resulting from DSS-induced colitis leads to loss of distal colonic crypts associated with the rapid loss of Lgr5+ cells. Despite the transient loss of Lgr5+ stem cells, the colonic crypts regenerate and homeostasis is restored. AIMS: The goal of this study was to determine whether the Krt19+ colonic stem cell pool is responsible for colonic crypt regeneration during colitis. METHODS: To examine whether Lgr5 and/or Krt19 mark normal colonic stem cells that contribute to epithelial regeneration upon colonic injury, we crossed Lgr5-GFP-IRES-CreER or Krt19-BAC-CreER transgenic mice to the ROSA26r-Tdtomato reporter line. Mice were then treated with tamoxifen followed by water (control) or DSS (in the drinking water x 7 days) to induce colitis. Lgr5+ and Krt19+ cells were then studied in the context of normal homeostasis or following colonic injury following DSS injury. RESULTS: Our results demonstrated that Lgr5+ stem cells are sensitive to DSS-induced colonic injury. Similar DSS-induced colitis injury experiments were also carried out using Notch1-CreER and Math1-CreER transgenic mice crossed to ROSA26r-Tdtomato mice and revealed little to no lineage tracing from absorptive or secretory cells, respectively. In contrast, Krt19 marked long-lived cells above the crypt base that were resistant to DSS-induced colonic epithelial injury and gave rise to Lgr5(+) cells in the newly regenerated crypts. Moreover, in separate experiments using Lgr5-DTR-GFP;K19-BAC-CreER; ROSA26r-Tdtomato mice, diphtheria toxin induced ablation of Lgr5(+) cells demonstrated that Lgr5(+) stem cells are dispensable for colonic epithelial regeneration following DSS-induced colitis. CONCLUSIONS: Our data suggest that analogous to the small intestine, Krt19+ stem cells in the colon are similarly more resistant to epithelial injury than Lgr5+ stem cells. FUNDING AGENCIES: CIHR

The endoplasmic reticulum (ER) is the point of entry of proteins into the secretory pathway. Nascent peptides interact with the ER quality control machinery that ensures correct folding of the nascent proteins. Failure to properly fold proteins can lead to loss of protein function and cytotoxic aggregation of misfolded proteins that can lead to cell death. To cope with increases in the ER unfolded secretory protein burden, cells have evolved the Unfolded Protein Response (UPR). The UPR is the primary signaling pathway that monitors the state of the ER folding environment. When the unfolded protein burden overwhelms the capacity of the ER quality control machinery, a state termed ER stress, sensor proteins detect accumulation of misfolded peptides and trigger the UPR transcriptional response. The UPR, which is conserved from yeast to mammals, consists of an ensemble of complex signaling pathways that aims at adapting the ER to the new misfolded protein load. To determine how different factors impact the ER folding environment, various tools and assays have been developed. In this review, we discuss recent advances in live cell imaging reporters and model systems that enable researchers to monitor changes in the unfolded secretory protein burden and activation of the UPR and its associated signaling pathways.

Background: Some trial have demonstrated a benefit of adjuvant fluoropirimidine with or without platinum compounds compared with surgery alone. ITACA-S study was designed to evaluate whether a sequential treatment of FOLFIRI [irinotecan plus 5-fluorouracil/folinic acid (5-FU/LV)] followed by docetaxel plus cisplatin improves disease-free survival in comparison with 5-FU/LV in patients with radically resected gastric cancer. Patients and methods: Patients with resectable adenocarcinoma of the stomach or gastroesophageal junction were randomly assigned to either FOLFIRI (irinotecan 180 mg/m 2 day 1, LV 100 mg/m 2 as 2 h infusion and 5-FU 400 mg/m 2 as bolus, days 1 and 2 followed by 600 mg/m 2 /day as 22 h continuous infusion, q14 for four cycles) followed by docetaxel 75 mg/m 2 day 1, cisplatin 75 mg/m 2 day 1, q21 for three cycles (sequential arm) or De Gramont regimen (5-FU/LV arm). Results: From February 2005 to August 2009, 1106 patients were enrolled, and 1100 included in the analysis: 562 in the sequential arm and 538 in the 5-FU/LV arm. With a median follow-up of 57.4 months, 581 patients recurred or died (297 sequential arm and 284 5-FU/LV arm), and 483 died (243 and 240, respectively). No statistically significant difference was detected for both disease-free [hazard ratio (HR) 1.00; 95% confidence interval (CI): 0.85–1.17; P= 0.974] and overall survival (OS) (HR 0.98; 95% CI: 0.82–1.18; P= 0.865). Five-year disease-free and OS rates were 44.6% and 44.6%, 51.0% and 50.6% in the sequential and 5-FU/LV arm, respectively. Conclusions: A more intensive regimen failed to show any benefit in disease-free and OS versus monotherapy. Clinical trial registration: ClinicalTrials.gov Identifier: NCT01640782.

Recent genomics analysis of the high-grade serous subtype of epithelial ovarian cancer (EOC) show aberrations in the phosphatidylinositol 3-kinase (PI3K)/AKT pathway that result in upregulated signaling activity. Thus, the PI3K/AKT pathway represents a potential therapeutic target for aggressive high-grade EOC. We previously demonstrated that treatment of malignant ascites-derived primary human EOC cells and ovarian cancer cell lines with the allosteric AKT inhibitor Akti-1/2 induces a dormancy-like cytostatic response but does not reduce cell viability. In this report, we show that allosteric AKT inhibition in these cells induces cytoprotective autophagy. Inhibition of autophagy using chloroquine (CQ) alone or in combination with Akti-1/2 leads to a significant decrease in viable cell number. In fact, Akti-1/2 sensitizes EOC cells to CQ-induced cell death by exhibiting markedly reduced EC50 values in combination-treated cells compared with CQ alone. In addition, we evaluated the effects of the novel specific and potent autophagy inhibitor-1 (Spautin-1) and demonstrate that Spautin-1 inhibits autophagy in a Beclin-1-independent manner in primary EOC cells and cell lines. Multicellular EOC spheroids are highly sensitive to Akti-1/2 and CQ/Spautin-1 cotreatments, but resistant to each agent alone. Indeed, combination index analysis revealed strong synergy between Akti-1/2 and Spautin-1 when both agents were used to affect cell viability; Akti-1/2 and CQ cotreatment also displayed synergy in most samples. Taken together, we propose that combination AKT inhibition and autophagy blockade would prove efficacious to reduce residual EOC cells for supplying ovarian cancer recurrence. © The Author 2014. Published by Oxford University Press. All rights reserved.

People with severe mental illness (SMI) have a reduced life expectancy. A major cause of mortality is cardiovascular disease.The aims of this study were to document the prevalence of cardiovascular risk factors in people with SMI engaged in community psychiatric rehabilitation and compare prevalence rates to the general, and Aboriginal and Torres Strait Islander (ATSI) populations of Australia.A cross-sectional audit was conducted on patients receiving care from Melbourne's Inner-West Area Mental Health Service. Profiles were collected on: smoking status, body mass index, waist circumference, blood pressure, diabetic status and fasting lipid profiles. These were compared with the general and ATSI Australian populations.Complete data were available for 60 patients. Most were involuntary patients with a diagnosis of schizophrenia or schizoaffective disorder. Patients were more likely to smoke, be obese, have dyslipidaemia and the metabolic syndrome compared with the general and ATSI populations of Australia. Patients were more likely to have diabetes than the general population but had similar rates to the ATSI population. Patients had similar rates of hypertension to the general population but were less likely to be hypertensive compared with the ATSI population.Australians living with SMI have very high rates of cardiovascular risk factors, far in excess of the general Australian population and comparable with the ATSI population.

Engineers have experienced difficulty in applying standard project/construction management techniques (e.g., critical path method) to the reconstruction and maintenance of an operating railway. These problems are most acute with respect to work performed by company forces; however, they are encountered in contract construction, design, and developmental projects. Often this experience has led to the conclusion that standard methods are, in fact, inappropriate to the environment of an operating railway. The writers believe this conclusion to be erroneous. The problem lies in the misjudging of, or the inability to, define the problem. This in turn results in the improper application of a management method or in the application of an inappropriate method. The formation of commuter and regional rail agencies, which have a large public-funding basis and have initiated large rehabilitation projects, has sparked a renewed interest in utilizing the appropriate analytical techniques in managing railway engineering...

Background Stanniocalcin 2 (STC2) is a secreted protein activated by (PKR)-like Endoplasmic Reticulum Kinase (PERK) signalling under conditions of ER stress in vitro. Over-expression of STC2 in mice leads to a growth-restricted phenotype; however, the physiological function for STC2 has remained elusive. Given the relationship of STC2 to PERK signalling, the objective of this study was to examine the role of STC2 in PERK signalling in vivo. Results Since PERK signalling has both physiological and pathological roles in the pancreas, STC2 expression was assessed in mouse pancreata before and after induction of injury using a cerulein-induced pancreatitis (CIP) model. Increased Stc2 expression was identified within four hours of initiating pancreatic injury and correlated to increased activation of PERK signalling. To determine the effect of STC2 over-expression on PERK, mice systemically expressing human STC2 (STC2 Tg ) were examined. STC2 Tg pancreatic tissue exhibited normal pancreatic morphology, but altered activation of PERK signalling, including increases in Activating Transcription Factor (ATF) 4 accumulation and autophagy. Upon induction of pancreatic injury, STC2 Tg mice exhibited limited increases in circulating amylase levels and increased maintenance of cellular junctions. Conclusions This study links STC2 to the pathological activation of PERK in vivo, and suggests involvement of STC2 in responding to pancreatic acinar cell injury.

Background & Aims Fibroblast growth factor 21 (FGF21) acts as a hormonal regulator during fasting and is involved in lipid metabolism. Fgf21 gene expression is regulated by peroxisome proliferator-activated receptor (PPAR)-dependent pathways, which are enhanced during pancreatitis. Therefore, the aim of this study was to investigate FGF21's role in pancreatic injury. Methods Fgf21 expression was quantified during cerulein-induced pancreatitis (CIP) or following mechanical or thapsigargin-induced stress through Northern blot analysis, in situ hybridization, and quantitative reverse transcription polymerase chain reaction. FGF21 protein was quantified by Western blot analysis. Isolated acinar cells or AR42J acinar cells were treated with recombinant FGF21 protein, and extracellular regulated kinase 1/2 activation was examined. The severity of CIP was compared between wild-type mice and mice overexpressing FGF21 ( FGF21Tg ) or harboring a targeted deletion of Fgf21 ( Fgf21 −/− ). Results Acinar cell Fgf21 expression markedly increased during CIP and following injury in vitro. Purified FGF21 activated the extracellular regulated kinase 1/2 pathway in pancreatic acinar cells. The severity of CIP is inversely correlated to FGF21 expression because FGF21Tg mice exhibited decreased serum amylase and decreased pancreatic stellate cell activation, whereas Fgf21 −/− mice had increased serum amylase and tissue damage. The expression of Fgf21 was also inversely correlated to expression of Early growth response 1 , a proinflammatory and profibrotic transcription factor. Conclusions These studies suggest a novel function for Fgf21 as an immediate response gene protecting pancreatic acini from overt damage.

Streptozotocin (STZ), a pancreatic beta cell toxin, is used to induce diabetic conditions by targeting the Glut-2 transporter. We have recently identified decreased Glut-2 expression in beta cells of mice lacking the transcription factor Mist1 (Mist1(KO)). Given the loss in Glut-2 expression, we examined whether Mist1(KO) beta cells have an increased resistance to STZ. Mist1(KO) and wild-type (WT) female mice received a single 100 or 200 mg/kg injection of STZ, and resting glucose levels and islet morphology were assayed 3-7 days after injection. Ten-month-old Mist1(KO) mice have less beta cell damage when exposed to high levels of STZ while 2-month-old Mist1(KO) mice exhibit a dose-dependent resistance. Surprisingly, Mist1(KO) mice still have elevated fasting glucose levels when compared to WT mice. These results suggest that while Mist1(KO) islets have increased resistance to STZ, additional effects outside of beta cell loss alter blood glucose homeostasis.

Metastatic epithelial ovarian cancer (EOC) cells can form multicellular spheroids while in suspension and disperse throughout the peritoneum via ascites to seed secondary lesions. We hypothesize that EOC spheroids are key mediators of metastasis, which use specific signaling pathways to alter cell metabolism for increased survival. Our lab discovered that AKT signaling is reduced during spheroid formation leading to cellular quiescence and autophagy. Given the induction of quiescence and autophagy in EOC spheroids, we are studying the 5′-AMP-activated protein kinase (AMPK) pathway as a master controller of the metabolic stress response and dormant phenotype of EOC spheroids. We demonstrate AMPK activity and its upstream kinase LKB1 are increased in quiescent EOC spheroids compared with proliferating adherent EOC cells. We also show elevated AMPK activity in spheroids isolated directly from patient ascites. Targeted knockdown of STK11, encoding LKB1, reduces cell viability in ovarian cancer cell line spheroids; PRKAA1 (AMPKα1) knockdown has little to no effect on EOC cell or spheroid viability. Combination of STK11 knockdown with carboplatin treatment leads to a synergistic enhancement in EOC spheroid cell death. In contrast, AICAR treatment of proliferating adherent ovarian cancer cell lines and primary EOC cells induces AMPK activity and causes either cytostasis or cell death. In addition, AICAR treatment of spheroids during reattachment decreases the dispersion capacity of migrating EOC cells. These results offer a glimpse of the potential important contributions of LKB1-AMPK pathway in stress signaling related to EOC cell survival during metastasis. In addition, downstream effectors of upregulated LKB1-AMPK signalling may provide additional mechanisms by which EOC evades chemotherapy. It is foreseeable that these findings will allow us to identify new therapeutic targets within this pathway critical to EOC progression for ultimate translation to the clinic. Citation Format: Teresa Peart, Elena Fazio, Yudith Ramos-Valdes, Monique Bertrand, Jacob McGee, Michel Prefontaine, Akira Sugimoto, Gabriel E. DiMattia, Trevor G. Shepherd. The LKB1-AMPK pathway mediates the metabolic stress response of dormant ovarian cancer spheroids. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A47.

Epithelial ovarian cancer (EOC) metastasis proceeds by an intra-peritoneal route, involving detachment of cells from the primary tumor and dissemination throughout the peritoneal cavity as multicellular aggregates, or spheroids. Herein, we demonstrate that ascites-derived cells upregulate the process of macroautophagy (autophagy), a highly-conserved self-digestion mechanism that functions to mitigate cellular stresses. Both non-adherent culture of these cells to form in vitro spheroids as well as exposure to an allosteric AKT inhibitor stimulated this process, as evidenced by Western blot and immunofluorescence staining for the autophagy marker MAP1LC3B (LC3). Likewise, expression of an eGFP-labeled LC3 protein revealed a shift from diffuse to ‘punctate’ cytoplasmic localization, demonstrating the formation of intracellular autophagic vesicles (autophagosomes). Additionally, we performed transmission electron microscopy and autophagy flux assays, confirming that autophagic degradation proceeded to completion. To determine the role of autophagy upregulation, we attempted to block this process using siRNAs targeting critical autophagy-related (ATG) genes. Interestingly, we found that depletion of Beclin1/ATG6 had no effect on autophagy, despite its role as a canonical inducer of the process. Conversely, depletion of the autophagy regulators ATG5 & ATG7 efficiently blocked LC3 processing and autophagosome formation. Likewise, autophagy was also blocked pharmacologically using the classical inhibitor Chloroquine and the novel agent Spautin-1 (the first targeted autophagy inhibitor to be published). Autophagy blockade by any of these mechanisms reduced EOC cell viability in both adherent and spheroid cultures, suggesting that the upregulation of this process serves to promote cell survival. Moreover, when autophagy blockade was combined with AKT inhibition, a synergistic reduction in cell survival was observed based on combination index analysis. Taken together, this work describes spheroid formation as a stimulus for a non-canonical, Beclin1-independent autophagy program, but also reveals autophagy to be a promoter of tumor cell survival during ovarian cancer metastasis, particularly in cells subjected to AKT/mTOR inhibition. In light of these findings, we propose that clinical autophagy inhibition could enhance the effectiveness of current or investigational ovarian cancer therapies. Citation Format: Rohann Correa, Yudith Ramos-Valdes, Teresa Peart, Elena Fazio, Monique Bertrand, Jacob McGee, Michel Préfontaine, Akira Sugimoto, Trevor G. Shepherd, Gabriel E. DiMattia. Beclin1-independent autophagy promotes the survival of metastatic ovarian cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A45.

The problems encountered in the major reconstruction of an active passenger railway are examined. The requirements of the transportation department are for highly controlled construction operations; however, construction is most economical when the contractor is provided with maximum scheduling flexibility. These seemingly incompatible demands can only be accommodated by an integrated, i.e., construction and transportation, planning effort, similar to that initiated by Amtrak in the Northeast Corridor Improvement Project. Amtrak’s experience may prove useful where railway construction must be accomplished under critical train service constraints, particularly in the upgrading of rail lines into high-speed commuter or regional rail system.

AA. have tested a new drug (Ceftriaxone) on 40 children affected by upper and lower respiratory tract infectious diseases. As shown by results, this new drug has been remarkably effective and easy to use since it may be administered once in a day; moreover, the tested drug has not caused any kind of tissue or parenchymal involvement.