Rationale and design of an ancillary study evaluating the effects of the cocoa flavanol and/or multivitamin interventions on falls and physical performance outcomes in the COcoa Supplement and Multivitamin Outcomes Study (COSMOS).

BACKGROUND: Falls and decreased physical function increase markedly with age and result in injury, hospitalization, and premature death. Emerging studies show potential benefits of supplemental cocoa extract on physical performance, including grip strength and walking speed in older adults. However, there are no large, long-term randomized controlled trials of effects of supplemental cocoa extract on falls, muscle performance, and/or fall-related injuries. METHODS: The COcoa Supplement and Multivitamin Outcomes Study (COSMOS) is a double-blind, placebo-controlled, 2 × 2 factorial trial investigating effects of supplementation with cocoa extract (500 mg/d, including 80 mg (-)-epicatechin) and/or a multivitamin on prevention of cardiovascular disease and cancer in 21,442 women (≥65 years) and men (≥60 years). COSMOS: Effects on Falls and Physical Performance is an ancillary study to COSMOS that will clarify effects of cocoa extract and/or multivitamin supplementation on falls, physical performance, and incident fracture outcomes in older adults. Injurious fall(s) resulting in healthcare utilization and recurrent falls were regularly assessed by follow-up questionnaires in the overall cohort. Incident fractures were also assessed by annual questionnaires. Circumstances surrounding falls and any fall-related injuries will be confirmed by medical record review. Effects of the interventions on 2-year changes in physical performance measures (grip strength, walking speed, and the Short Physical Performance Battery) will be tested in a clinic sub-cohort (n = 603). CONCLUSION: Results from this ancillary study will determine whether supplemental cocoa extract slows age-related declines in physical performance and decrease injurious and recurrent falls and fall-related injuries and fractures that are major public health problems in older adults.

Universal tumor screening for lynch syndrome: perspectives of patients regarding willingness and informed consent.

Aim: Lynch Syndrome is associated with a significant risk of colorectal carcinoma (CRC) and other cancers. Universal tumor screening is a strategy to identify high-risk individuals by testing all CRC tumors for molecular features suggestive of Lynch Syndrome. Patient interest in screening and preferences for consent have been underexplored. Methods: A postal survey was administered to CRC patients in a Canadian province. Results: Most patients (81.4%) were willing to have tumors tested if universal tumor screening were available and were willing to discuss test results with family members and healthcare professionals. The majority (62.6%) preferred informed consent be obtained prior to screening. Conclusion: Patients were supportive of universal screening. They expected consent to be obtained, contrary to current practice across Canada and elsewhere.

Reduced deoxyribonuclease enzyme activity in response to high postinjury mitochondrial DNA concentration provides a therapeutic target for Systemic Inflammatory Response Syndrome.

BACKGROUND: Cell-free mitochondrial DNA (mtDNA) is proinflammatory and has been detected in high concentrations in trauma patients' plasma. Deoxyribonuclease (DNAse) is the free plasma enzyme responsible for the digestion of extracellular DNA. The relationship between mtDNA and DNAse after major trauma is unknown. We hypothesized that DNAse activity would be elevated after injury and trauma surgery and would be associated with high concentrations of extracellular DNA. METHODS: Two-year prospective study was performed on 103 consecutive trauma patients (male, 81%; age, 38 years [interquartile range, 30-59 years]; injury severity score, 18 [interquartile range, 12-26 years]) who underwent standardized major orthopedic trauma surgical interventions. Blood was collected at five perioperative time points (preoperative, postoperative, 7 hours, 24 hours, and 3 days postoperatively). Healthy control subjects (n = 20) were also sampled. Cell-free mtDNA and nuclear DNA (nDNA) were measured using quantitative polymerase chain reaction. Deoxyribonuclease was also assayed in the same plasma samples. RESULTS: Increased levels of mtDNA (from preoperative 163 ± 86 ng/mL to 3 days 282 ± 201 ng/mL, p < 0.0001) and nDNA (from preoperative 28 ± 20 ng/mL to 3 days 37 ± 27 ng/mL, p < 0.05) were present in trauma patients at all perioperative time points compared with healthy controls (mtDNA: 4 ± 2 ng/mL; nDNA: 10 ± 5 ng/mL). Deoxyribonuclease activity was lower in the trauma cohort (from preoperative 0.06 ± 0.04U/mL to 3 days 0.08 ± 0.04U/mL, p < 0.0001) compared with healthy controls (DNAse: 0.17 ± 0.03U/mL). There was no correlation between DNAse and perioperative DNA concentrations. Elevated mtDNA (but not nDNA) correlated with the development of systemic inflammatory response syndrome (SIRS) (p = 0.026) but not multiple organ failure. CONCLUSIONS: The significant perioperative elevation in plasma-free mtDNA concentration is associated with the development of SIRS. The fact that increased cell-free DNA concentrations present with significantly lower than healthy control DNAse activity suggests a potential therapeutic opportunity with DNAse administration to modulate postinjury severe SIRS. LEVEL OF EVIDENCE: Prognostic/Epidemiological, level II.

Relationship between the cohesion of guest particles on the flow behaviour of interactive mixtures.

In this study, we aimed to investigate the effects cohesion of small surface-engineered guest binder particles on the flow behaviour of interactive mixtures. Polyvinylpyrrolidone (PVP) - a model pharmaceutical binder - was spray-dried with varying l-leucine feed concentrations to create small surface-engineered binder particles with varying cohesion. These spray-dried formulations were characterised by their particle size distribution, morphology and cohesion. Interactive mixtures were produced by blending these spray-dried formulations with paracetamol. The resultant blends were visualised under scanning electron microscope to confirm formation of interactive mixtures. Surface coverage of paracetamol by guest particles as well as the flow behaviour of these mixtures were examined. The flow performance of interactive mixtures was evaluated using measurements of conditioned bulk density, basic flowability energy, aeration energy and compressibility. With higher feed l-leucine concentrations, the surface roughness of small binder particles increased, while their cohesion decreased. Visual inspection of the SEM images of the blends indicated that the guest particles adhered to the surface of paracetamol resulting in effective formation of interactive mixtures. These images also showed that the low-cohesion guest particles were better de-agglomerated that consequently formed a more homogeneous interactive mixture with paracetamol compared with high-cohesion formulations. The flow performance of interactive mixtures changed as a function of the cohesion of the guest particles. Interactive mixtures with low-cohesion guest binder particles showed notably improved bulk flow performance compared with those containing high-cohesion guest binder particles. Thus, our study suggests that the cohesion of guest particles dictates the flow performance of interactive mixtures.

Adjuvant chemotherapy for stage III colon cancer: relative dose intensity and survival among veterans.

BACKGROUND: Given the paucity of information on dose intensity, the objective of this study is to describe the use of adjuvant chemotherapy for stage III colon cancer, focusing on relative dose intensity (RDI), overall survival (OS) and disease-free survival (DFS). METHODS: Retrospective cohort of 367 patients diagnosed with stage III colon cancer in 2003-2008 and treated at 19 VA medical centers. Kaplan-Meier curves summarize 5-year OS and 3-year DFS by chemotherapy regimen and RDI, and multivariable Cox proportional hazards regression was used to model these associations. RESULTS: 5-fluorouracil/leucovorin (FU/LV) was the most commonly initiated regimen in 2003 (94.4%) and 2004 (62.7%); in 2005-2008, a majority of patients (60%-74%) was started on an oxaliplatin-based regimen. Median RDI was 82.3%. Receipt of >70% RDI was associated with better 5-year OS (p < 0.001) and 3-year DFS (P = 0.009) than was receipt of ≤70% RDI, with 5-year OS rates of 66.3% and 50.5%, respectively and 3-year DFS rates of 66.1% and 52.7%, respectively. In the multivariable analysis of 5-year OS, oxaliplatin + 5-FU/LV (versus 5-FU/LV) (HR = 0.55; 95% CI = 0.34-0.91), >70% RDI at the first year (HR = 0.58; 95% CI = 0.37-0.89) and married status (HR = 0.66; 95% CI = 0.45-0.97) were associated with significantly decreased risk of death, while age ≥75 (versus 55-64) (HR = 2.06; 95% CI = 1.25-3.40), Charlson Comorbidity Index (HR = 1.17; 95% CI = 1.06-1.30), T4 tumor status (versus T1/T2) (HR = 5.88; 95% CI = 2.69-12.9), N2 node status (HR = 1.68; 95% CI = 1.12-2.50) and bowel obstruction (HR = 2.32, 95% CI = 1.36-3.95) were associated with significantly increased risk. Similar associations were observed for DFS. CONCLUSION: Patients with stage III colon cancer who received >70% RDI had improved 5-year OS. The association between RDI and survival needs to be examined in studies of adjuvant chemotherapy for colon cancer outside of the VA.

Cell necrosis-independent sustained mitochondrial and nuclear DNA release following trauma surgery.

BACKGROUND: Mitochondrial DNA (mtDNA), a potent proinflammatory damage-associated molecular pattern, is released in large titers following trauma. The effect of trauma surgery on mtDNA concentration is unknown. We hypothesized that mtDNA and nuclear DNA (nDNA) levels would increase proportionately with the magnitude of surgery and both would then decrease rapidly. METHODS: In this prospective pilot, plasma was sampled from 35 trauma patients requiring orthopedic surgical intervention at six perioperative time points. Healthy control subjects (n = 20) were sampled. DNA was extracted, and the mtDNA and nDNA were assessed using quantitative polymerase chain reaction. Markers of cell necrosis were also assayed (creatine kinase, lactate dehydrogenase, and aspartate aminotransferase). RESULTS: The free plasma mtDNA and nDNA levels (ng/mL) were increased in trauma patients compared with healthy controls at all time points (mtDNA: preoperative period, 108 [46-284]; postoperative period, 96 [29-200]; 7 hours postoperatively, 88 [43-178]; 24 hours, 79 [36-172]; 3 days, 136 [65-263]; 5 days, 166 [101-434] [healthy controls, 11 (5-19)]) (nDNA: preoperative period, 52 [25-130]; postoperative period, 100 [35-208]; 7 hours postoperatively, 75 [36-139]; 24 hours postoperatively, 85 [47-133]; 3 days, 79 [48-117]; 5 days, 99 [41-154] [healthy controls, 29 (16-54)]). Elevated DNA levels did not correlate with markers of cellular necrosis. mtDNA was significantly elevated compared with nDNA at preoperative period (p = 0.003), 3 days (p = 0.003), and 5 days (p = 0.0014). Preoperative mtDNA levels were greater with shorter time from injury to surgery (p = 0.0085). Postoperative mtDNA level negatively correlated with intraoperative crystalloid infusion (p = 0.0017). Major pelvic surgery (vs. minor) was associated with greater mtDNA release 5 days postoperatively (p < 0.05). CONCLUSION: This pilot of heterogeneous orthopedic trauma patients showed that the release of mtDNA and nDNA is sustained for 5 days following orthopedic trauma surgery. Postoperative, circulating DNA is not associated with markers of tissue necrosis but is associated with surgical invasiveness and is inversely related to intraoperative fluid administration. Sustained elevation of mtDNA levels could be of inflammatory origin and may contribute to postinjury dysfunctional inflammation. LEVEL OF EVIDENCE: Prospective study, level III.

Nucleotide excision repair deficiency in melanoma in response to UVA.

BACKGROUND: The causative link between UV exposure and melanoma development is well known, however the mechanistic relationship remains incompletely characterised. UVA and UVB components of sunlight are implicated in melanomagenesis; however the majority of studies have focused on the effects of UVB and UVC light. Interestingly, melanoma tumour sequencing has revealed an overrepresentation of mutations signature of unrepaired UV-induced DNA damage. Repair of UVA-induced DNA damage is thought to occur primarily through the Nucleotide Excision Repair (NER) pathway, which recognises and repairs damage either coupled to transcription (Transcription Coupled Repair; TCR), or through global genome scanning (Global Genome Repair; GGR). Current literature suggests NER is deficient in melanoma, however the cause of this remains unknown; and whether reduced NER activity in response to UVA may be involved in melanoma development remains uncharacterised. In this study we aimed to determine if melanoma cells exhibit reduced levels of NER activity in response to UVA. METHODS: Melanocyte and melanoma cell lines were UVA-irradiated, and DNA damage levels assessed by immunodetection of Cyclobutane Pyrimidine Dimer (CPD) and (6-4) Photoproduct [(6-4)PP] lesions. Expression of NER pathway components and p53 following UVA treatment was quantified by qPCR and western blot. RESULTS: UVA did not induce detectable induction of (6-4)PP lesions, consistent with previous studies. Repair of CPDs induced by UVA was initiated at 4 h and complete within 48 h in normal melanocytes, whereas repair initiation was delayed to 24 h and >40 % of lesions remained in melanoma cell lines at 48 h. This was coupled with a delayed and reduced induction of GGR component XPC in melanoma cells, independent of p53. CONCLUSION: These findings support that NER activity is reduced in melanoma cells due to deficient GGR. Further investigation into the role of NER in UVA-induced melanomagenesis is warranted and may have implications for melanoma treatment.

Mitochondrial DNA neutrophil extracellular traps are formed after trauma and subsequent surgery.

INTRODUCTION: Neutrophil extracellular traps (NETs) have not been demonstrated after trauma and subsequent surgery. Neutrophil extracellular traps are formed from pure mitochondrial DNA (mtDNA) under certain conditions, which is potently proinflammatory. We hypothesized that injury and orthopedic trauma surgery would induce NET production with mtDNA as a structural component. METHODS: Neutrophils were isolated 8 trauma patients requiring orthopedic surgery postinjury and up to 5 days postoperatively. Four healthy volunteers provided positive and negative controls. Total hip replacement patients acted as an uninjured surgical control group. Neutrophil extracellular traps were visualized with DNA (Hoechst 33342TM/Sytox Green/MitoSox/MitoTracker) stains using live cell fluorescence microscopy with downstream quantitative polymerase chain reaction analysis of DNA composition. RESULTS: Neutrophil extracellular traps were present after injury in all 8 trauma patients. They persisted for 5 days postoperatively. Delayed surgery resulted in NET resolution, but they reformed postoperatively. Total hip replacement patients developed NETs postoperatively, which resolved by day 5. Quantitative polymerase chain reaction analysis of NET-DNA composition revealed that NETs formed after injury and surgery were made of mtDNA with no detectable nuclear DNA component. CONCLUSIONS: Neutrophil extracellular traps formed after major trauma and subsequent surgery contain mtDNA and represent a novel marker of heightened innate immune activation. They could be considered when timing surgery after trauma to prevent systemic NET-induced inflammatory complications.

Dietary resveratrol supplementation normalizes gene expression in the hippocampus of streptozotocin-induced diabetic C57Bl/6 mice.

Diabetes is associated with cognitive impairment and brain aging, with alterations in hippocampal neurogenesis and synaptic plasticity implicated in these changes. As the prevalence of diabetes continues to rise, readily implemented strategies are increasingly needed in order to protect the brain's cognitive functions. One possibility is resveratrol (RES) (3,5,4- trihydroxystilbene), a polyphenol of the phytoalexin family that has been shown to be protective in a number of neuropathology paradigms. In the present study, we sought to determine whether dietary supplementation with RES has potential for the protection of cognitive functions in diabetes. Diabetes was induced using streptozotocin, and once stable, animals received AIN93G rodent diet supplemented with RES for 6 weeks. Genome-wide expression analysis was conducted on the hippocampus and genes of interest were confirmed by quantitative, real-time polymerase chain reaction. Genome-wide gene expression analysis of the hippocampus revealed that RES supplementation of the diabetic group resulted in 481 differentially expressed genes compared to non-supplemented diabetic mice. Intriguingly, gene expression that was previously found significantly altered in the hippocampus of diabetic mice, and that is implicated in neurogenesis and synaptic plasticity (Hdac4, Hat1, Wnt7a, ApoE), was normalized following RES supplementation. In addition, pathway analysis revealed Jak-Stat signaling was the most significantly enriched pathway. The Jak-Stat pathway induces a pro-inflammatory signaling cascade, and we found most genes involved in this cascade (e.g. Il15, Il22, Socs2, Socs5) had significantly lower expression following RES supplementation. These data indicate RES could be neuroprotective and beneficial for the maintenance of cognitive function in diabetes.

Fluoxetine prevents development of an early stress-related molecular signature in the rat infralimbic medial prefrontal cortex. Implications for depression?

BACKGROUND: Psychological stress, particularly in chronic form, can lead to mood and cognitive dysfunction and is a major risk factor in the development of depressive states. How stress affects the brain to cause psychopathologies is incompletely understood. We sought to characterise potential depression related mechanisms by analysing gene expression and molecular pathways in the infralimbic medial prefrontal cortex (ILmPFC), following a repeated psychological stress paradigm. The ILmPFC is thought to be involved in the processing of emotionally contextual information and in orchestrating the related autonomic responses, and it is one of the brain regions implicated in both stress responses and depression. RESULTS: Genome-wide microarray analysis of gene expression showed sub-chronic restraint stress resulted predominantly in a reduction in transcripts 24 hours after the last stress episode, with 239 genes significantly decreased, while just 24 genes had increased transcript abundance. Molecular pathway analysis using DAVID identified 8 pathways that were significantly enriched in the differentially expressed gene list, with genes belonging to the brain-derived neurotrophic factor - neurotrophin receptor tyrosine kinase 2 (BDNF-Ntrk2) pathway most enriched. Of the three intracellular signalling pathways that are downstream of Ntrk2, real-time quantitative PCR confirmed that only the PI3K-AKT-GSK3B and MAPK/ERK pathways were affected by sub-chronic stress, with the PLCγ pathway unaffected. Interestingly, chronic antidepressant treatment with the selective serotonin reuptake inhibitor, fluoxetine, prevented the stress-induced Ntrk2 and PI3K pathway changes, but it had no effect on the MAPK/ERK pathway. CONCLUSIONS: These findings indicate that abnormal BDNF-Ntrk2 signalling may manifest at a relatively early time point, and is consistent with a molecular signature of depression developing well before depression-like behaviours occur. Targeting this pathway prophylactically, particularly in depression-susceptible individuals, may be of therapeutic benefit.

The genetic landscape of the childhood cancer medulloblastoma.

Medulloblastoma (MB) is the most common malignant brain tumor of children. To identify the genetic alterations in this tumor type, we searched for copy number alterations using high-density microarrays and sequenced all known protein-coding genes and microRNA genes using Sanger sequencing in a set of 22 MBs. We found that, on average, each tumor had 11 gene alterations, fewer by a factor of 5 to 10 than in the adult solid tumors that have been sequenced to date. In addition to alterations in the Hedgehog and Wnt pathways, our analysis led to the discovery of genes not previously known to be altered in MBs. Most notably, inactivating mutations of the histone-lysine N-methyltransferase genes MLL2 or MLL3 were identified in 16% of MB patients. These results demonstrate key differences between the genetic landscapes of adult and childhood cancers, highlight dysregulation of developmental pathways as an important mechanism underlying MBs, and identify a role for a specific type of histone methylation in human tumorigenesis.

Improved RNA preservation for immunolabeling and laser microdissection.

Microdissection techniques have the potential to allow for transcriptome analyses in specific populations of cells that are isolated from heterogeneous tissues such as the nervous system and certain cancers. Problematically, RNA is not stable under the labeling conditions usually needed to identify the cells of interest for microdissection. We have developed an immunolabeling method that utilizes a high salt buffer to stabilize RNA during prolonged antibody incubations. We first assessed RNA integrity by three methods and found that tissue incubated in high salt buffer for at least 20 h yielded RNA of similar quality to that for RNA extracted from fresh-frozen tissue, which is considered highest quality. Notably, the integrity was superior to that for RNA extracted from tissue processed using rapid immunolabeling procedures (5 min total duration). We next established that high salt buffer was compatible with immunolabeling, as demonstrated by immunofluorescent detection of dopamine neurons in the brain. Finally, we laser microdissected dopamine neurons that were immunolabeled using high salt buffer and demonstrated that RNA integrity was preserved. Our described method yields high quality RNA from immunolabeled microdissected cells, an essential requirement for meaningful genomics investigations of normal and pathological cells isolated from complex tissues.

An integrated genomic analysis of human glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common and lethal type of brain cancer. To identify the genetic alterations in GBMs, we sequenced 20,661 protein coding genes, determined the presence of amplifications and deletions using high-density oligonucleotide arrays, and performed gene expression analyses using next-generation sequencing technologies in 22 human tumor samples. This comprehensive analysis led to the discovery of a variety of genes that were not known to be altered in GBMs. Most notably, we found recurrent mutations in the active site of isocitrate dehydrogenase 1 (IDH1) in 12% of GBM patients. Mutations in IDH1 occurred in a large fraction of young patients and in most patients with secondary GBMs and were associated with an increase in overall survival. These studies demonstrate the value of unbiased genomic analyses in the characterization of human brain cancer and identify a potentially useful genetic alteration for the classification and targeted therapy of GBMs.

The Alberta Bone and Joint Health Institute: creating sustainable accountability through collaboration, relevant measurement and timely feedback.

A robust accountability strategy is at the core of creating a safe, efficient, effective and sustainable system of healthcare. The commitment to be accountable must extend far beyond the providers of care to include every person involved in the funding, administration, delivery and support of patient care (both directly and indirectly). The Alberta Bone and Joint Health Institute has fostered a new system that will measure, analyze and give valuable feedback to all stakeholders in all three essential domains of system accountability: access, quality and cost. The Institute has employed four key strategies to create system accountability in a hip and knee pilot project: collaboration between stakeholders in defining goals and measures that matter to them; the use of "world's best evidence" to drive decisions and to establish goals and benchmarks to measure against; collection of useful data and its analysis to inform improvement decisions; and timely feedback of relevant data in domains of interest to stakeholders on system outputs in the key domains. While these strategies have not yet been proven to be effective in creating the desired "culture of accountability," they are having a significant clinical impact and do have potential to lead to that outcome.

Catecholamine and oxytocin cells respond to hypovolaemia as well as hypotension.

Medullary catecholamine and hypothalamic neurosecretory oxytocin cells are activated by hypotension, but previous studies have provided uncertain outcomes concerning their ability to respond to a purely hypovolaemic stimulus. In the present study, injections of PEG/water and pentolinium were used to elicit non-hypotensive, isosmotic hypovolaemia and isovolaemic, isosmotic hypotension, respectively, in conscious rats. Animals were sacrificed 2 h after treatment. Immunolabelling for Fos, tyrosine hydroxylase and oxytocin established that these two stimuli activate almost identical populations of catecholamine neurons in the ventrolateral and dorsomedial medulla, and very similar populations of oxytocin cells in the supraoptic and paraventricular nuclei of the hypothalamus.