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Pancreatitis and pancreatic ductal adenocarcinoma (PDAC) are grave illnesses with high levels of morbidity and mortality. Intravital imaging (IVI) is a powerful technique for visualizing physiological processes in both health and disease. However, the application of IVI to the murine pancreas presents significant challenges, as it is a deep, compliant, visceral organ that is difficult to access, easily damaged and susceptible to motion artefacts. Existing imaging windows for stabilizing the pancreas during IVI have unfortunately shown poor stability for time-lapsed imaging on the minutes to hours scale, or are unable to accommodate both the healthy and tumour-bearing pancreata. To address these issues, we developed an improved stabilized window for intravital imaging of the pancreas (SWIP), which can be applied to not only the healthy pancreas but also to solid tumours like PDAC. Here, we validate the SWIP and use it to visualize a variety of processes for the first time, including (1) single-cell dynamics within the healthy pancreas, (2) transformation from healthy pancreas to acute pancreatitis induced by cerulein, and (3) the physiology of PDAC in both autochthonous and orthotopically injected models. SWIP can not only improve the imaging stability but also expand the application of IVI in both benign and malignant pancreas diseases.

Cyanuric acid (CYA) is used commercially for maintaining active chlorine to inactivate microbial and viral pathogens in swimming pools and hot tubs. Repeated CYA addition can cause a lack of available chlorine and adequate disinfection. Acceptable CYA levels can potentially be restored via cyanuric acid hydrolases (CAH), enzymes that hydrolyze CYA to biuret under mild conditions. Here we describe a previously unknown CAH enzyme from Pseudolabrys sp. Root1462 (CAH-PR), mined from public databases by bioinformatic analysis of potential CAH genes, which we show to be suitable in a cell-free form for industrial applications based upon favorable enzymatic and physical properties, combined with high-yield expression in aerobic cell culture. The kinetic parameters and modeled structure were similar to known CAH enzymes, but the new enzyme displayed a surprising thermal and storage stability. The new CAH enzyme was applied, following addition of inexpensive sodium sulfite, to hydrolyze CYA to biuret. At the desired endpoint, hypochlorite addition inactivated remaining enzyme and oxidized biuret to primarily dinitrogen and carbon dioxide gases. The mechanism of biuret oxidation with hypochlorite under conditions relevant to recreational pools is described., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

Purpose: We aimed to investigate genomic correlates underlying extremes of survivorship in metastatic colorectal cancer and their applicability in informing survival in distinct subsets of patients with metastatic colorectal cancer., Experimental Design: We examined differences in oncogenic somatic alterations between metastatic colorectal cancer cohorts demonstrating extremes of survivorship following complete metastasectomy: ≤2-year ( n = 17) and ≥10-year ( n = 18) survivors. Relevant genomic findings, and their association with overall survival (OS), were validated in two independent datasets of 935 stage IV and 443 resected stage I-IV patients., Results: In the extremes-of-survivorship cohort, significant co-occurrence of KRAS hotspot mutations and TP53 alterations was observed in ≤2-year survivors ( P < 0.001). When validating these findings in the independent cohort of 935 stage IV patients, incorporation of the cumulative effect of any oncogenic Ras/B-raf (i.e., either KRAS, NRAS , or BRAF ) and TP53 alteration generated three prognostic clusters: (i) TP53 -altered alone (median OS, 132 months); (ii) Ras/B-raf -altered alone (65 months) or Ras/B-raf - and TP53 pan-wild-type (60 months); and (iii) coaltered Ras/B-raf - TP53 (40 months; P < 0.0001). Coaltered Ras/B-raf - TP53 was independently associated with mortality (HR, 2.47; 95% confidence interval, 1.91-3.21; P < 0.001). This molecular profile predicted survival in the second independent cohort of 443 resected stage I-IV patients. Coaltered Ras/B-raf - TP53 was associated with worse OS in patients with liver ( n = 490) and lung ( n = 172) but not peritoneal surface ( n = 149) metastases. Moreover, coaltered Ras/B-raf - TP53 tumors were significantly more likely to involve extrahepatic metastatic sites with limited salvage options., Conclusions: Genomic analysis of extremes of survivorship following colorectal cancer metastasectomy identifies a prognostic role for coaltered Ras/B-raf - TP53 and its association with distinct patterns of colorectal cancer metastasis., (©2019 American Association for Cancer Research.)

The liver is the most common site of colorectal cancer metastasis. Fortunately, improvements have been made in the care of patients with colorectal liver metastasis (CRLM). Effective management of CRLM requires a multidisciplinary approach that is tailored to individuals in order to achieve long-term survival, and cure. Resection and systemic chemotherapy provides benefit in selected individuals. An adjunct to resection and/or systemic chemotherapy is the use of hepatic arterial infusion pump (HAIP) therapy. Many studies show HAIP provides benefit for select patients with CRLM. Added to the crucible of a multidisciplinary approach to managing CRLM is the ever growing understanding of tumor biology and genetic profiling. In this review, we discuss the outcomes of resection, systemic therapies and HAIP therapy for CRLM. We also discuss the impact of recent advances in genetic profiling and mutational analysis, namely mutation of KRAS and BRAF, for this disease.

Objectives: Morbidity after pancreaticoduodenectomy (PD) remains high. Computed tomography (CT) of intra-abdominal tissue has not been thoroughly evaluated to establish associations with the occurrence of complications after PD. The current study sought to determine whether differences in non-enhanced visceral attenuation predicted complications after PD., Methods: Outcomes in patients undergoing PD were analysed according to the Clavien system for classifying complications and the International Study Group on Pancreatic Fistula system for classifying postoperative pancreatic fistula (POPF). Preoperative non-enhanced CT scans were evaluated by a blinded investigator for attenuation of abdominal viscera and fat thickness. Data on pancreatic firmness and pancreatic duct size were collected. Univariate and multivariate analyses were performed., Results: A total of 134 patients underwent PD for malignant and benign disease. Rates of morbidity, mortality and POPF at 90 days were 61%, 4% and 23%, respectively. Patients with a body mass index of > 25 kg/m(2) had higher rates of POPF (P = 0.05) and complications (P < 0.01). In multivariate analysis, patients were more likely to develop any complication as CT attenuation decreased for paraspinus muscle (P < 0.01), spleen (P < 0.03) and liver (P = 0.01) parenchyma., Conclusions: Postoperative complications after PD remain prevalent. Decreased CT attenuation of abdominal viscera is an independent predictor of morbidity after PD and suggests a high-risk patient physiology for pancreatic resection., (© 2013 International Hepato-Pancreato-Biliary Association.)

Although gastrointestinal stromal tumors (GISTs) harboring activating KIT or platelet-derived growth factor receptor A (PDGFRA) mutations respond to treatment with targeted KIT/PDGFRA inhibitors such as imatinib mesylate, these treatments are rarely curative. Most often, a sizeable tumor cell subpopulation survives and remains quiescent for years, eventually resulting in acquired resistance and treatment failure. Here, we report that imatinib induces autophagy as a survival pathway in quiescent GIST cells. Inhibiting autophagy, using RNAi-mediated silencing of autophagy regulators (ATGs) or antimalarial lysosomotrophic agents, promotes the death of GIST cells both in vitro and in vivo. Thus, combining imatinib with autophagy inhibition represents a potentially valuable strategy to promote GIST cytotoxicity and to diminish both cellular quiescence and acquired resistance in GIST patients.

Purpose: Imatinib mesylate (imatinib) has revolutionized clinical outcomes of patients with advanced gastrointestinal stromal tumor (GIST). However, the degree of individual benefit varies, and little is known about prognostic factors for these patients. Importantly, selected patients may be treated with an approach to target both Kit and vascular endothelial growth factor receptor (VEGFR) expression., Experimental Design: Using tissue microarray technology, we analyzed 53 imatinib-naive GISTs for vascular endothelial growth factor (VEGF) expression from patients who then received imatinib. In multivariate analyses, we evaluated overall survival (OS) and progression-free survival (PFS) of these patients based on putative prognostic factors, including VEGF expression. In a separate study, 12 matched pre-imatinib and post-imatinib GIST patient specimens and two human GIST cell lines were assessed for VEGF production in response to imatinib., Results: Independent of kit genotype, patients with GIST expressing high VEGF had inferior median PFS (7.1 months versus 29 months, P = 0.42) and median OS (20 months versus not reached at >50 months; P = 0.02) compared with weak or nonexpressers of VEGF. Non-exon 11 kit mutation predicted inferior PFS but not OS. High mitotic rate was marginally predictive of improved OS. Imatinib resulted in decreased production of VEGF in only a subset of GIST patients (2 of 12) and both cell lines., Conclusions: We present a study to address the prognostic factors for patients with GIST in the imatinib era. We present a rationale to consider exploration of a front-line therapy of GIST with a regimen targeting both Kit and VEGFR based on the presence of tumor VEGF levels.

Background: Gastrointestinal stromal tumors (GISTs), the most common mesenchymal tumors of the gastrointestinal tract, are an example of a disease with an effective, molecularly targeted therapy., Methods: Published articles and author experience were used to comprehensively define the clinical features, biology, and state-of-the-art therapy of GISTs., Results: GISTs are thought to originate from the neoplastic transformation of the interstitial cells of Cajal, the intestinal pacemaker cells. GISTs commonly have mutations in the kit gene, resulting in a gain-of-function mutation and ligand-independent constitutive activation of the KIT receptor tyrosine kinase. Successful tyrosine kinase inhibitors target the aberrant pathways that are critical for tumor cell viability. The development of imatinib mesylate (formerly STI 571) in the treatment of metastatic GISTs represents a therapeutic breakthrough., Conclusions: Progress in the clinical diagnosis has led to an increased recognition of this disease as a distinct clinical entity. Treatment of metastatic GIST with imatinib has led to unprecedented improvements in progression-free and overall survival. The use of imatinib in the preoperative and postoperative treatment of GISTs is an area of intense investigation.

Bacillus amyloliquefaciens 1,3-1,4-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.73) was modified by the mechanism-based, affinity-labeling reagent [14C](3,4)-epoxybutyl beta-D-cellobioside. Following partial inactivation a completely inactivated enzyme preparation containing 1.1 mol of covalently bound inhibitor/mol of protein was obtained by chromatography on a cellulosic matrix. The inactivated enzyme was digested with endoproteinase Glu-C and radioactive peptides purified by reversed-phase high performance liquid chromatography (HPLC). The affinity label was esterified exclusively to the gamma-carboxylate of Glu105 in the sequence Gly-Thr-Pro-Trp-Asp-Glu-Ile-Asp-Ile-Glu109. The sequence motif Glu-(Ile/Leu)-Asp-Ile is found in many glucanases and xylanases and may therefore serve to identify the catalytic nucleophile in beta-glycanases, which otherwise exhibit a low degree of sequence identity. The esterification of Glu105 by the affinity label abolished endoproteinase Glu-C-mediated hydrolysis of the Glu-Ile106 peptide bond. Identification of phenylthiohydantoin-Glu105 during automated sequence analysis was not possible unless the affinity label was liberated by prior base hydrolysis. These observations formed the basis for the development of a highly sensitive approach for the identification of catalytic carboxylates in polysaccharide hydrolases employing non-radioactive inhibitors, comparative HPLC mapping, electrospray mass spectrometry, and Edman degradation.