Background Mixed lineage kinase domain-like protein (MLKL) is a necrosome component mediating programmed necrosis that may be an important determinant of cancer cell death. The goal of the current study was to evaluate the prognostic value of MLKL expression in patients with pancreatic adenocarcinoma (PAC). Methods Tissue from 80 patients was collected from a prospectively maintained database of patients with PAC who underwent pancreaticoduodenectomy between January 2000 and October 2008. Immunohistochemistry analysis was performed and scored using an established scoring system. Kaplan-Meier survival curves were generated for recurrence-free survival (RFS) and overall survival (OS) for all patients and for patients receiving adjuvant chemotherapy. MLKL scores were correlated with RFS and OS using univariate and multivariate Cox regression analyses incorporating clinically relevant covariates. Results The median age of the patients was 63 years and 53% were men. Low MLKL expression was associated with decreased OS (6 months vs 17 months; P=.006). In the subset of 59 patients who received adjuvant chemotherapy, low MLKL expression was associated with decreased RFS (5 months vs 15 months; P=.006) and decreased OS (6 months vs 19 months; P<.0001). On multivariate analysis, low MLKL expression was associated with poor OS in all patients (hazards ratio, 4.6 [95% confidence interval, 1.6-13.8]; P=.006) and in patients receiving adjuvant chemotherapy (hazards ratio, 8.1 [95% confidence interval, 2.2-29.2]; P=.002). Conclusions Low expression of MLKL is associated with decreased OS in patients with resected PAC and decreased RFS and OS in the subset of patients with resected PAC who receive adjuvant chemotherapy. The use of this biomarker in patients with PAC may provide important prognostic information.

Organophosphorus nerve agent (OPNA) adducts to butyrylcholinesterase (BChE) can be used to confirm exposure in humans. A highly accurate method to detect G-series and V-series OPNA adducts to BChE in 75 μL of filtered blood, serum, or plasma has been developed using immunomagnetic separation (IMS) coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS). The reported IMS method captures > 88% of the BChE in a specimen and corrects for matrix effects on peptide calibrators. The optimized method has been used to quantify baseline BChE levels (unadducted and OPNA-adducted) in a matched set of serum, plasma and whole blood (later processed in-house for plasma content) from 192 unexposed individuals to determine the interchangeability of the tested matrices. The results of these measurements demonstrate the ability to accurately measure BChE regardless of the format of the blood specimen received. Criteria for accepting or denying specimens were established through a series of sample stability and processing experiments. The results of these efforts are an optimized and rugged method that is transferrable to other laboratories and an increased understanding of the BChE biomarker in matrix.

Sirtuin 2 (SIRT2) is a sirtuin family deacetylase that directs acetylome signaling, protects genome integrity, and is a murine tumor suppressor. We show that SIRT2 directs replication stress responses by regulating the activity of cyclin-dependent kinase 9 (CDK9), a protein required for recovery from replication arrest. SIRT2 deficiency results in replication stress sensitivity, impairment in recovery from replication arrest, spontaneous accumulation of replication protein A to foci and chromatin, and a G2/M checkpoint deficit. SIRT2 interacts with and deacetylates CDK9 at lysine 48 in response to replication stress in a manner that is partially dependent on ataxia telangiectasia and Rad3 related (ATR) but not cyclin T or K, thereby stimulating CDK9 kinase activity and promoting recovery from replication arrest. Moreover, wild-type, but not acetylated CDK9, alleviates the replication stress response impairment of SIRT2 deficiency. Collectively, our results define a function for SIRT2 in regulating checkpoint pathways that respond to replication stress through deacetylation of CDK9, providing insight into how SIRT2 maintains genome integrity and a unique mechanism by which SIRT2 may function, at least in part, as a tumor suppressor protein.DNA damage | cell cycle checkpoint | genome maintenance M embers of the sirtuin NAD + -dependent deacetylase family regulate multiple biological processes, including genome maintenance, aging, tumorigenesis, differentiation, and metabolism (1-3). There are seven mammalian homologs of yeast silent information regulator 2 (Sir2). Whereas sirtuin 1 (SIRT1) and sirtuin 6 (SIRT6) have been linked directly to DNA repair or DNA damage response (DDR) signaling pathways (4-12), the role for other sirtuin family members, including SIRT2, is less clear. Mice deficient in Sirt2 develop breast, liver, and other cancers (13,14), and SIRT2 expression is decreased in human breast and liver cancers (14), suggesting that SIRT2 is a tumor suppressor protein (TSP). In support of a role for SIRT2 in the DDR, Sirt2 deficiency in mouse embryo fibroblasts (MEFs) results in a mitotic checkpoint deficit, increased DNA damage, and genomic instability (13). In addition, SIRT2 deficiency in chicken DT40 cells induces hypersensitivity to cisplatin (15). SIRT2 also deacetylates histone H3 lysine 56, a marker for packaging of newly replicated and repaired DNA into chromatin (16), implying that SIRT2 may function in the maintenance of genome integrity during DNA replication.The replication stress response (RSR) is a subset of the DDR signaling network that recognizes challenges to DNA replication and mobilizes diverse DNA repair and cell cycle checkpoint pathways. The RSR is critical for the prevention of cancer by acting as a barrier against genomic instability and tumorigenesis (17,18). At the apex of the RSR signaling cascade is the ataxiatelangiectasia and Rad3-related protein (ATR) checkpoint kinase (19). ATR function is essential to stabilize stalled replication forks and promote recovery. Disruption of ...

Both inflammation and oxidative injury are features of Alzheimer’s disease (AD), but the contribution of these intertwined phenomena to the loss of working memory in this disease is unclear. We tested the hypothesis that highly reactive γ-ketoaldehydes that are formed both by non-enzymatic free radical catalyzed lipid peroxidation and by cyclooxygenases may be causally linked to the development of memory impairment in AD. We found that levels of γ-ketoaldehyde protein adducts were increased in the hippocampus of brains obtained postmortem from patients with AD compared to age-matched controls, but that levels of γ-ketoaldehyde protein adducts in the cerebellum were not different in the two groups. Moreover, immunohistochemistry revealed that adducts localized to hippocampal pyramidal neurons. We tested the effect of an orally available γ-ketoaldehyde scavenger, salicylamine, on the development of spatial working memory deficits in hApoE4 targeted replacement mice, a mouse model of dementia. Long-term salicylamine supplementation did not significantly alter body weight or survival, but protected against the development of age-related deficits in spatial working memory in 12–14 month old ApoE4 mice. These findings suggest that γ-ketoaldehyde adduct formation is associated with damage to hippocampal neurons in patients with AD and can contribute to the pathogenesis of spatial working memory deficits in hApoE4 mice. These data provide a rational basis for future studies exploring whether γ-ketoaldehyde scavengers may mitigate the development of cognitive dysfunction in patients with AD