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Metastasis is the leading cause of cancer-related deaths. It is unclear how intratumor heterogeneity (ITH) contributes to metastasis and how metastatic cells adapt to distant tissue environments. The study of these adaptations is challenged by the limited access to patient material and a lack of experimental models that appropriately recapitulate ITH. To investigate metastatic cell adaptations and the contribution of ITH to metastasis, we analyzed single-cell transcriptomes of matched primary tumors and metastases from patient-derived xenograft models of breast cancer. We found profound transcriptional differences between the primary tumor and metastatic cells. Primary tumors upregulated several metabolic genes, whereas motility pathway genes were upregulated in micrometastases, and stress response signaling was upregulated during progression. Additionally, we identified primary tumor gene signatures that were associated with increased metastatic potential and correlated with patient outcomes. Immune-regulatory control pathways were enriched in poorly metastatic primary tumors, whereas genes involved in epithelial-mesenchymal transition were upregulated in highly metastatic tumors. We found that ITH was dominated by epithelial-mesenchymal plasticity (EMP), which presented as a dynamic continuum with intermediate EMP cell states characterized by specific genes such as CRYAB and S100A2. Elevated expression of an intermediate EMP signature correlated with worse patient outcomes. Our findings identified inhibition of the intermediate EMP cell state as a potential therapeutic target to block metastasis.

Background: Cervical arterial tortuosity is associated with adverse outcomes in Loeys-Dietz syndrome and other heritable aortopathies., Methods and Results: A method to assess tortuosity based on curvature of the vessel centerline in 3-dimensional space was developed. We measured cervical carotid tortuosity in 65 patients with Loeys-Dietz syndrome from baseline computed tomography angiogram/magnetic resonance angiogram and all serial images during follow-up. Relations between baseline carotid tortuosity, age, aortic root diameter, and its change over time were compared. Patients with unoperated aortic roots were assessed for clinical end point (type A aortic dissection or aortic root surgery during 4 years of follow-up). Logistic regression was performed to assess the likelihood of clinical end point according to baseline carotid tortuosity. Total absolute curvature at baseline was 11.13±5.76 and was relatively unchanged at 8 to 10 years (fold change: 0.026±0.298, P =1.00), whereas tortuosity index at baseline was 0.262±0.131, with greater variability at 8 to 10 years (fold change: 0.302±0.656, P =0.818). Baseline total absolute curvature correlated with aortic root diameter ( r =0.456, P =0.004) and was independently associated with aortic events during the 4-year follow-up (adjusted odds ratio [OR], 2.64 [95% CI, 1.02-6.85]). Baseline tortuosity index correlated with age ( r =0.532, P <0.001) and was not associated with events (adjusted OR, 1.88 [95% CI, 0.79-4.51]). Finally, baseline total absolute curvature had good discrimination of 4-year outcomes (area under the curve=0.724, P =0.014), which may be prognostic or predictive., Conclusions: Here we introduce cervical carotid tortuosity as a promising quantitative biomarker with validated, standardized characteristics. Specifically, we recommend the adoption of a curvature-based measure, total absolute curvature, for early detection or monitoring of disease progression in Loeys-Dietz syndrome.

Objectives: To study the incidence of Omicron infections in Malaysia and the exposures that could reduce the hazard of attaining Omicron infection., Methods: We used a multicenter, prospective cohort to study 482 healthcare workers vaccinated with two and three doses of BNT162b2 for SARS-CoV-2 infection during the Omicron-dominant period in Malaysia., Results: Between January 31 and July 31, 2022, the cumulative incidence was 44.6% (95% CI 40.2-49.1%), and the incidence rate was 3.33 (95% CI 2.91-3.80) per 1000 person-days. Our study found that protection against Omicron infection was significantly higher for persons with previous SARS-CoV-2 infection (hazard ratio [HR] 0.41, 95% CI 0.27-0.62) and persons with a more recent immunity event (<30 days [reference] vs >90 days, HR 3.82, 95%CI 1.34-10.90) from the beginning of the Omicron period., Conclusion: Pre-Omicron natural infection and a recent immunity event protect against future Omicron infections., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The evaluation of breakthrough infection and humoral immunity responses are important outcomes for vaccination policy for healthcare staff. This prospective cohort study collected blood samples at 5-time points; before primary vaccine doses, and at 2, 10 and 24 weeks after BNT162b2 vaccination from 551 HCWs, between March and October 2021. We investigated the association between anti-spike-1 protein receptor-binding domain (anti-S1-RBD) antibody geometric mean titre (GMT) and breakthrough infections. Two weeks post-vaccination, the GMT of anti-S1-RBD antibodies was measured at almost maximum detectable value (3115 BAU/ml [95% CI, 3051-3180]); it decreased to 1486 BAU/ml (95% CI, 1371-1610) at 10 weeks; and to 315 BAU/ml (95% CI, 283-349) at 24 weeks. Prior COVID-19 infection and age significantly affected the antibody titres. Fifty-six participants, none of whom were COVID-19 convalescents, had breakthrough infections between 10 and 24 weeks post-vaccination. Before breakthrough infections, the GMT was not different between the breakthrough and non-breakthrough individuals. After infection, the GMT was significantly higher in individuals with breakthrough infections (2038 BAU/ml [95%CI, 1547-2685]), specifically in symptomatic breakthroughs, compared to those without infection (254 BAU/ml [95%CI, 233-278]). A notable surge in breakthrough infections among healthcare workers coincided with the emergence of the Delta variant and when BNT162b2-elicited antibody responses waned in 10-24 weeks (i.e. approximately 3-6 months). Post-breakthrough, the antibody response was boosted in individuals with symptomatic presentations, but not asymptomatic individuals. The study finding supports administering booster vaccination for healthcare staff, including those who recovered from asymptomatic breakthrough infection.

Objective: To evaluate the impact of N-acetyl-cysteine (NAC) on amputation stump perfusion and healing in patients with critical limb-threatening ischemia (CLTI)., Background: Patients with CLTI are at increased risk of poor amputation site healing leading to increased procedure-associated morbidity., Methods: In a pilot, double-blind, placebo-controlled, randomized controlled trial, patients with CLTI undergoing major elective lower extremity amputation were randomized 1:1 to intravenous NAC (1200 mg twice-daily) or placebo for up to 5 days postoperatively. Primary outcomes were change in stump perfusion at postoperative day 3 (POD3) and POD5, and healing at POD30. Stumps were serially evaluated for wound healing, and tissue perfusion was evaluated using noninvasive laser-assisted fluorescent angiography., Results: Thirty-three patients were randomized to NAC (n = 16) or placebo (n = 17). Thirty-one patients were eligible for intent-to-treat analysis (NAC14; placebo17). Twenty patients (NAC7; placebo13) had amputation stump perfusion defects at POD0 and were considered high-risk for poor healing. Intent-to-treat analysis revealed no significant differences between treatment groups. Subgroup analysis of high-risk patients revealed differences in stump perfusion defect size (NAC-0.53-fold, placebo +0.71-fold; 95% confidence interval -2.11 to-0.35; P < 0.05) and healing (NAC [100%], placebo [46%]; P < 0.01) between study treatments., Conclusions: Postoperative NAC administration may improve amputation stump perfusion and healing in patients with CLTI and tissue perfusion defects at the time of amputation. Intraoperative laser-assisted fluorescent angiogra-phy may help surgeons identify high-risk patients with stump perfusion defects and provide early adjunctive interventions. Future studies can further explore the therapeutic benefits of NAC in the healing and perfusion of other surgical operative sites in high-risk individuals., Trial Registration: clinicaltrials.gov, Identifier: NCT03253328., Competing Interests: The authors report no conflicts of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Pharmaceuticals have extensive reciprocal interactions with the microbiome, but whether bacterial drug sensitivity and metabolism is driven by pathways conserved in host cells remains unclear. Here we show that anti-cancer fluoropyrimidine drugs inhibit the growth of gut bacterial strains from 6 phyla. In both Escherichia coli and mammalian cells, fluoropyrimidines disrupt pyrimidine metabolism. Proteobacteria and Firmicutes metabolized 5-fluorouracil to its inactive metabolite dihydrofluorouracil, mimicking the major host mechanism for drug clearance. The preTA operon was necessary and sufficient for 5-fluorouracil inactivation by E. coli, exhibited high catalytic efficiency for the reductive reaction, decreased the bioavailability and efficacy of oral fluoropyrimidine treatment in mice and was prevalent in the gut microbiomes of colorectal cancer patients. The conservation of both the targets and enzymes for metabolism of therapeutics across domains highlights the need to distinguish the relative contributions of human and microbial cells to drug efficacy and side-effect profiles., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Few patients with triple negative breast cancer (TNBC) benefit from immune checkpoint inhibitors with complete and durable remissions being quite rare. Oncogenes can regulate tumor immune infiltration, however whether oncogenes dictate diminished response to immunotherapy and whether these effects are reversible remains poorly understood. Here, we report that TNBCs with elevated MYC expression are resistant to immune checkpoint inhibitor therapy. Using mouse models and patient data, we show that MYC signaling is associated with low tumor cell PD-L1, low overall immune cell infiltration, and low tumor cell MHC-I expression. Restoring interferon signaling in the tumor increases MHC-I expression. By combining a TLR9 agonist and an agonistic antibody against OX40 with anti-PD-L1, mice experience tumor regression and are protected from new TNBC tumor outgrowth. Our findings demonstrate that MYC-dependent immune evasion is reversible and druggable, and when strategically targeted, may improve outcomes for patients treated with immune checkpoint inhibitors., (© 2022. The Author(s).)

Background: Post-traumatic headache (PTH) is a commonly experienced symptom after mild traumatic brain injury (mTBI). Blast injury- or blunt injury-related mechanisms for mTBI in veterans can also affect musculoskeletal structures in the neck, resulting in comorbid neck pain (NP). However, it is unknown whether the presence of comorbid NP may be associated with a different pattern of headache symptoms, physical functioning, or emotional functioning compared to those without comorbid NP. The purpose of this study is to examine the role of comorbid NP in veterans with mTBI and PTH., Design and Methods: This was a cross-sectional investigation of an existing dataset that included 33 veterans who met inclusion criteria for PTH after mTBI. Standardized measures of headache severity and frequency, insomnia, fatigue, mood disorders, and physical and emotional role function were compared between groups with and without comorbid NP., Results: The majority of participants with PTH reported comorbid NP (n = 22/33, 67%). Those with comorbid NP experienced more headache symptoms that were severe or incapacitating, as compared to mild or moderate for those without NP (φ = 0.343, P = .049); however, no differences in headache frequency (φ = 0.231, P = .231) or duration (φ = 0.129, P = .712) were observed. Participants with comorbid NP also reported greater insomnia (d = 1.16, P = .003) and fatigue (d = 0.868, P = .040) as well as lower physical functioning (d = 0.802, P = .036) and greater bodily pain (d = 0.762, P = .012). There were no differences in anxiety, depression, mental health, emotional role limitations, vitality, or social functioning between those with and without comorbid NP (d ≤ 0.656, P ≥ .079)., Conclusions: A majority of veterans with mTBI and PTH in our sample reported comorbid NP that was associated with greater headache symptom severity and physical limitations, but not with mood or emotional limitations. Preliminary findings from this small convenience sample indicate that routine assessment of comorbid NP and associated physical limitations should be considered in veterans with mTBI and PTH., (© The Association of Military Surgeons of the United States 2021. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The demand of thrombectomy treatment for acute ischemic stroke increased dramatically in the United States after the publication of 5 pivotal trials in 2015. The impact of call burden on career satisfaction and burnout in neurointerventionalists has not been explored., Methods: A qualitative approach was chosen to obtain rich, detailed accounts of physician experiences. From July 2018 to July 2019, we conducted in-depth, semistructured interviews (mean duration, 43 minutes) with 16 academic neurointerventionalists from various institutions in the United States. Participants were chosen from the Society of Neurointerventional Surgery to include multiple specialties, career stages, and geographic locations. Key themes/subthemes were organized into a conceptual model and shown by exemplary quotes., Results: Participants report a diverse range of previously uncharacterized ways in which stroke call responsibilities affect their lives. Unrealistic performance expectations, discipline-based inequalities, poor organizational workflow, limited opportunities for growth and advancement, and lack of control over call conditions are some of the key themes identified. Participants describe a need for interventions that address both the issues of workload stress (i.e., resources for stroke call and administrative work) and their ability to accomplish professional goals (i.e., flexible work arrangements, leadership autonomy support, and organizational value congruence)., Conclusions: The burden of stroke call has strong implications for career satisfaction and burnout in neurointerventionalists. This model can be used by hospitals, departments, and physician groups to better conceptualize conditions that facilitate burnout and attrition. Changes to hospital management practices and culture may be the most productive focal areas for intervention., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Targeting a tumor's metabolic dependencies is a clinically actionable therapeutic approach; however, identifying subtypes of tumors likely to respond remains difficult. The use of lipids as a nutrient source is of particular importance, especially in breast cancer. Imaging techniques offer the opportunity to quantify nutrient use in preclinical tumor models to guide development of new drugs that restrict uptake or utilization of these nutrients. We describe a fast and dynamic approach to image fatty acid uptake in vivo and demonstrate its relevance to study both tumor metabolic reprogramming directly, as well as the effectiveness of drugs targeting lipid metabolism. Specifically, we developed a quantitative optical approach to spatially and longitudinally map the kinetics of long-chain fatty acid uptake in in vivo murine models of breast cancer using a fluorescently labeled palmitate molecule, Bodipy FL c16. We chose intra-vital microscopy of mammary tumor windows to validate our approach in two orthotopic breast cancer models: a MYC-overexpressing, transgenic, triple-negative breast cancer (TNBC) model and a murine model of the 4T1 family. Following injection, Bodipy FL c16 fluorescence increased and reached its maximum after approximately 30 min, with the signal remaining stable during the 30-80 min post-injection period. We used the fluorescence at 60 min (Bodipy 60 ), the mid-point in the plateau region, as a summary parameter to quantify Bodipy FL c16 fluorescence in subsequent experiments. Using our imaging platform, we observed a two- to four-fold decrease in fatty acid uptake in response to the downregulation of the MYC oncogene, consistent with findings from in vitro metabolic assays. In contrast, our imaging studies report an increase in fatty acid uptake with tumor aggressiveness (6NR, 4T07, and 4T1), and uptake was significantly decreased after treatment with a fatty acid transport inhibitor, perphenazine, in both normal mammary pads and in the most aggressive 4T1 tumor model. Our approach fills an important gap between in vitro assays providing rich metabolic information at static time points and imaging approaches visualizing metabolism in whole organs at a reduced resolution.

The Pro47Ser variant of p53 (S47) exists in African-descent populations and is associated with increased cancer risk in humans and mice. Due to impaired repression of the cystine importer Slc7a11 , S47 cells show increased glutathione (GSH) accumulation compared to cells with wild -type p53. We show that mice containing the S47 variant display increased mTOR activity and oxidative metabolism, as well as larger size, improved metabolic efficiency, and signs of superior fitness. Mechanistically, we show that mTOR and its positive regulator Rheb display increased association in S47 cells; this is due to an altered redox state of GAPDH in S47 cells that inhibits its ability to bind and sequester Rheb. Compounds that decrease glutathione normalize GAPDH-Rheb complexes and mTOR activity in S47 cells. This study reveals a novel layer of regulation of mTOR by p53, and raises the possibility that this variant may have been selected for in early Africa., Competing Interests: KG, JL, SB, TB, MG, JL, WQ, CK, RA, JB, KW, QL, ZS, DG No competing interests declared, MM Senior Editor, eLife, (© 2020, Gnanapradeepan et al.)

Delayed cerebral ischemia (DCI) after aneurysmal subarachnoid hemorrhage (SAH) has been associated with numerous pathophysiological sequelae, including large artery vasospasm and microvascular thrombosis. The focus of this review is to provide an overview of experimental animal model studies and human autopsy studies that explore the temporal-spatial characterization and mechanism of microvascular platelet aggregation and thrombosis following SAH, as well as to critically assess experimental studies and clinical trials highlighting preventative therapeutic options against this highly morbid pathophysiological process. Upon review of the literature, we discovered that microvascular platelet aggregation and thrombosis occur after experimental SAH across multiple species and SAH induction techniques in a similar time frame to other components of DCI, occurring in the cerebral cortex and hippocampus across both hemispheres. We discuss the relationship of these findings to human autopsy studies. In the final section of this review, we highlight the important therapeutic options for targeting microvascular platelet aggregation and thrombosis, and emphasize why therapeutic targeting of this neurovascular pathology may improve patient care. We encourage ongoing research into the pathophysiology of SAH and DCI, especially in regard to microvascular platelet aggregation and thrombosis and the translation to randomized clinical trials.

Tumors that overexpress the MYC oncogene are frequently aneuploid, a state associated with highly aggressive cancers and tumor evolution. However, how MYC causes aneuploidy is not well understood. Here, we show that MYC overexpression induces mitotic spindle assembly defects and chromosomal instability (CIN) through effects on microtubule nucleation and organization. Attenuating MYC expression reverses mitotic defects, even in established tumor cell lines, indicating an ongoing role for MYC in CIN. MYC reprograms mitotic gene expression, and we identify TPX2 to be permissive for spindle assembly in MYC-high cells. TPX2 depletion blocks mitotic progression, induces cell death, and prevents tumor growth. Further elevating TPX2 expression reduces mitotic defects in MYC-high cells. MYC and TPX2 expression may be useful biomarkers to stratify patients for anti-mitotic therapies. Our studies implicate MYC as a regulator of mitosis and suggest that blocking MYC activity can attenuate the emergence of CIN and tumor evolution., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Importance: The Implicit Association Test (IAT) is a validated tool used to measure implicit biases, which are mental associations shaped by one's environment that influence interactions with others. Direct evidence of implicit gender biases about women in medicine has yet not been reported, but existing evidence is suggestive of subtle or hidden biases that affect women in medicine., Objectives: To use data from IATs to assess (1) how health care professionals associate men and women with career and family and (2) how surgeons associate men and women with surgery and family medicine., Design, Setting, and Participants: This data review and cross-sectional study collected data from January 1, 2006, through December 31, 2017, from self-identified health care professionals taking the Gender-Career IAT hosted by Project Implicit to explore bias among self-identified health care professionals. A novel Gender-Specialty IAT was also tested at a national surgical meeting in October 2017. All health care professionals who completed the Gender-Career IAT were eligible for the first analysis. Surgeons of any age, gender, title, and country of origin at the meeting were eligible to participate in the second analysis. Data were analyzed from January 1, 2018, through March 31, 2019., Main Outcomes and Measures: Measure of implicit bias derived from reaction times on the IATs and a measure of explicit bias asked directly to participants., Results: Almost 1 million IAT records from Project Implicit were reviewed, and 131 surgeons (64.9% men; mean [SD] age, 42.3 [11.5] years) were recruited to complete the Gender-Specialty IAT. Healthcare professionals (n = 42 991; 82.0% women; mean [SD] age, 32.7 [11.8] years) held implicit (mean [SD] D score, 0.41 [0.36]; Cohen d = 1.14) and explicit (mean [SD], 1.43 [1.85]; Cohen d = 0.77) biases associating men with career and women with family. Similarly, surgeons implicitly (mean [SD] D score, 0.28 [0.37]; Cohen d = 0.76) and explicitly (men: mean [SD], 1.27 [0.39]; Cohen d = 0.93; women: mean [SD], 0.73 [0.35]; Cohen d = 0.53) associated men with surgery and women with family medicine. There was broad evidence of consensus across social groups in implicit and explicit biases with one exception. Women in healthcare (mean [SD], 1.43 [1.86]; Cohen d = 0.77) and surgery (mean [SD], 0.73 [0.35]; Cohen d = 0.53) were less likely than men to explicitly associate men with career (B coefficient, -0.10; 95% CI, -0.15 to -0.04; P < .001) and surgery (B coefficient, -0.67; 95% CI, -1.21 to -0.13; P = .001) and women with family and family medicine., Conclusions and Relevance: The main contribution of this work is an estimate of the extent of implicit gender bias within surgery. On both the Gender-Career IAT and the novel Gender-Specialty IAT, respondents had a tendency to associate men with career and surgery and women with family and family medicine. Awareness of the existence of implicit biases is an important first step toward minimizing their potential effect.

Background: Axial spondyloarthritis (AxSpA) is a chronic disease which results in fatigue, pain, and reduced quality of life (QoL). Traditional Chinese medicine (TCM), especially acupuncture, has shown promise in managing pain. Although a TCM collaborative model of care (TCMCMC) has been studied in cancer, there are no randomized controlled trials investigating TCM in AxSpA. Therefore, we will conduct a pragmatic trial to determine the clinical effectiveness, safety, and cost-effectiveness of TCMCMC for patients with AxSpA. We define TCMCMC as standard TCM history taking and physical examination, acupuncture, and TCM non-pharmacological advice and communications with rheumatologists in addition to usual rheumatologic care. The purpose of this paper is to describe the rationale for and methodology of this trial., Methods/design: This pragmatic randomized controlled trial will recruit 160 patients who are diagnosed with AxSpA and have inadequate response to non-steroidal anti-inflammatory drugs (NSAIDs). Simple randomization to usual rheumatologic care or the intervention (TCMCMC) with a 1:1 allocation ratio will be used. Ten 30-min acupuncture sessions will be provided to patients assigned to the TCMCMC arm. All participants will continue to receive usual rheumatologic care. The primary endpoint - spinal pain - will be evaluated at week 6. Secondary endpoints include clinical, quality of life, and economic outcome measures. Patients will be followed up for up to 52 weeks, and adverse events will be documented., Discussion: This trial may provide evidence regarding the clinical effectiveness, safety, and cost-effectiveness of a TCMCMC for patients with AxSpA., Trial Registration: ClinicalTrials.gov, NCT03420404 . Registered on 14 February 2018.

Within the context of longitudinal medical care for adults, health care providers have a unique opportunity to inquire and respond to the traumatic life experiences affecting the health of their patients, as well as a responsibility to minimize retraumatizing these patients during medical encounters. While there is literature on screening women for intimate partner violence, and there is emerging data on pediatric screening for adverse life experiences, there is sparse literature on inquiry of broader trauma histories in adult medical settings. This lack of research on trauma inquiry results in an absence of guidelines for best practices, in turn making it challenging for policy makers, health care providers, and researchers to mitigate the adverse health outcomes caused by traumatic experiences and to provide equitable care to populations that experience a disproportionate burden of trauma. This state of the science summarizes current inquiry practices for patients who have experienced trauma, violence, and abuse. It places trauma inquiry within an anchoring framework of trauma-informed care principles, and emphasizes a focus on resilience. It then proposes best practices for trauma inquiry, which include tiered screening starting with broad trauma inquiry, proceeding to risk and safety assessment as indicated, and ending with connection to interventions.

The metabolite acetyl-coenzyme A (acetyl-CoA) is the required acetyl donor for lysine acetylation and thereby links metabolism, signaling, and epigenetics. Nutrient availability alters acetyl-CoA levels in cancer cells, correlating with changes in global histone acetylation and gene expression. However, the specific molecular mechanisms through which acetyl-CoA production impacts gene expression and its functional roles in promoting malignant phenotypes are poorly understood. Here, using histone H3 Lys27 acetylation (H3K27ac) ChIP-seq (chromatin immunoprecipitation [ChIP] coupled with next-generation sequencing) with normalization to an exogenous reference genome (ChIP-Rx), we found that changes in acetyl-CoA abundance trigger site-specific regulation of H3K27ac, correlating with gene expression as opposed to uniformly modulating this mark at all genes. Genes involved in integrin signaling and cell adhesion were identified as acetyl-CoA-responsive in glioblastoma cells, and we demonstrate that ATP citrate lyase (ACLY)-dependent acetyl-CoA production promotes cell migration and adhesion to the extracellular matrix. Mechanistically, the transcription factor NFAT1 (nuclear factor of activated T cells 1) was found to mediate acetyl-CoA-dependent gene regulation and cell adhesion. This occurs through modulation of Ca 2+ signals, triggering NFAT1 nuclear translocation when acetyl-CoA is abundant. The findings of this study thus establish that acetyl-CoA impacts H3K27ac at specific loci, correlating with gene expression, and that expression of cell adhesion genes are driven by acetyl-CoA in part through activation of Ca 2+ -NFAT signaling., (© 2018 Lee et al.; Published by Cold Spring Harbor Laboratory Press.)

The quantitative analysis of polyunsaturated fatty acyl (PUFA) chain oxidation products in tissue samples by mass spectrometry is hindered by the lack of durable internal standards for the large number of possible products. To address this problem in a study of oxidative PUFA degradation in Alzheimer's disease (AD) brain, uniformly 13 C-labeled arachidonic acid (ARA) was produced biosynthetically, and allowed to oxidize under controlled conditions into a mixture of U- 13 C-labeled ARA oxidation products. The components of this mixture were characterized with respect to their partitioning behavior during lipid extraction, their durability during saponification, trends in mouse brain tissue concentrations during post mortem intervals, and their overall suitability as internal standards for multiple-reaction monitoring tandem mass spectrometry. This mixture has now been used as a set of internal standards to determine the relative abundance of ARA and 54 non-stereospecific oxidation products in milligram samples of brain tissue. Many of these oxidation products were recovered from both healthy mouse and healthy human brain, although some of them were unique to each source, and some have not heretofore been described. The list of oxidation products detected in AD brain tissue was the same as in healthy human brain, although simple hydroxy-eicosanoids were significantly increased in AD brain. while more complex oxidation products were not. These results are consistent with an increased level of chemically-mediated oxidative ARA degradation in Alzheimer's disease. However, they also point to the existence of processes that selectively produce or eliminate specific oxidation products, and those processes may account for some of the inconsistencies in previously reported results., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Elevated O-GlcNAcylation is associated with disease states such as diabetes and cancer. O-GlcNAc transferase (OGT) is elevated in multiple cancers and inhibition of this enzyme genetically or pharmacologically inhibits oncogenesis. Here we show that O-GlcNAcylation modulates lipid metabolism in cancer cells. OGT regulates expression of the master lipid regulator the transcription factor sterol regulatory element binding protein 1 (SREBP-1) and its transcriptional targets both in cancer and lipogenic tissue. OGT regulates SREBP-1 protein expression via AMP-activated protein kinase (AMPK). SREBP-1 is critical for OGT-mediated regulation of cell survival and of lipid synthesis, as overexpression of SREBP-1 rescues lipogenic defects associated with OGT suppression, and tumor growth in vitro and in vivo. These results unravel a previously unidentified link between O-GlcNAcylation, lipid metabolism and the regulation of SREBP-1 in cancer and suggests a crucial role for O-GlcNAc signaling in transducing nutritional state to regulate lipid metabolism.

While maintaining the integrity of the genome and sustaining bioenergetics are both fundamental functions of the cell, potential crosstalk between metabolic and DNA repair pathways is poorly understood. Since histone acetylation plays important roles in DNA repair and is sensitive to the availability of acetyl coenzyme A (acetyl-CoA), we investigated a role for metabolic regulation of histone acetylation during the DNA damage response. In this study, we report that nuclear ATP-citrate lyase (ACLY) is phosphorylated at S455 downstream of ataxia telangiectasia mutated (ATM) and AKT following DNA damage. ACLY facilitates histone acetylation at double-strand break (DSB) sites, impairing 53BP1 localization and enabling BRCA1 recruitment and DNA repair by homologous recombination. ACLY phosphorylation and nuclear localization are necessary for its role in promoting BRCA1 recruitment. Upon PARP inhibition, ACLY silencing promotes genomic instability and cell death. Thus, the spatial and temporal control of acetyl-CoA production by ACLY participates in the mechanism of DNA repair pathway choice., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Arachidonic acid (ARA) is one of the most abundant polyunsaturated fatty acids (PUFAs) in the mammalian brain. Many enzymatically- and nonenzymatically-produced metabolic products have important and potent pharmacological properties. However, uniformly isotope labeled forms of ARA are not commercially available for studying the metabolic fates of ARA. This study describes a simple and efficient protocol for the biosynthesis of U- 13 C-ARA from U- 13 C-glucose, and U- 14 C-ARA from U- 14 C-glucose by Mortierella alpina. The protocols yield approximately 100nmol quantities of U- 13 C-ARA with an isotopic purity of 95% from a 500μl batch volume, and approximately 2μCi quantities of U- 14 C-ARA with an apparent specific activity in excess of 1200Ci/mol from a 250μl batch volume., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Mechanisms of metabolic flexibility enable cells to survive under stressful conditions and can thwart therapeutic responses. Acetyl-coenzyme A (CoA) plays central roles in energy production, lipid metabolism, and epigenomic modifications. Here, we show that, upon genetic deletion of Acly, the gene coding for ATP-citrate lyase (ACLY), cells remain viable and proliferate, although at an impaired rate. In the absence of ACLY, cells upregulate ACSS2 and utilize exogenous acetate to provide acetyl-CoA for de novo lipogenesis (DNL) and histone acetylation. A physiological level of acetate is sufficient for cell viability and abundant acetyl-CoA production, although histone acetylation levels remain low in ACLY-deficient cells unless supplemented with high levels of acetate. ACLY-deficient adipocytes accumulate lipid in vivo, exhibit increased acetyl-CoA and malonyl-CoA production from acetate, and display some differences in fatty acid content and synthesis. Together, these data indicate that engagement of acetate metabolism is a crucial, although partial, mechanism of compensation for ACLY deficiency., Competing Interests: Authors have no conflicts of interest to declare., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

In this study, the performance of a new most probable number (MPN) test (Pseudalert(®)/Quanti-Tray(®)) for the enumeration of Pseudomonas aeruginosa from hospital waters was compared with both international and national membrane filtration-based culture methods for P. aeruginosa: ISO 16266:2006 and UK The Microbiology of Drinking Water - Part 8 (MoDW Part 8), which both use Pseudomonas CN agar. The comparison based on the calculation of mean relative differences between the two methods was conducted according to ISO 17994:2014. Using both routine hospital water samples (80 from six laboratories) and artificially contaminated samples (192 from five laboratories), paired counts from each sample and the enumeration method were analysed. For routine samples, there were insufficient data for a conclusive assessment, but the data do indicate at least equivalent performance of Pseudalert(®)/Quanti-Tray(®). For the artificially contaminated samples, the data revealed higher counts of P. aeruginosa being recorded by Pseudalert(®)/Quanti-Tray(®). The Pseudalert(®)/Quanti-Tray(®) method does not require confirmation testing for atypical strains of P. aeruginosa, saving up to 6 days of additional analysis, and has the added advantage of providing confirmed counts within 24-28 hours incubation compared to 40-48 hours or longer for the ISO 16266 and MoDW Part 8 methods.

Replication stress induced by nucleotide deficiency plays an important role in cancer initiation. Replication stress in primary cells typically activates the cellular senescence tumor-suppression mechanism. Senescence bypass correlates with development of cancer, a disease characterized by metabolic reprogramming. However, the role of metabolic reprogramming in the cellular response to replication stress has been little explored. Here, we report that ataxia telangiectasia mutated (ATM) plays a central role in regulating the cellular response to replication stress by shifting cellular metabolism. ATM inactivation bypasses senescence induced by replication stress triggered by nucleotide deficiency. This was due to restoration of deoxyribonucleotide triphosphate (dNTP) levels through both upregulation of the pentose phosphate pathway via increased glucose-6-phosphate dehydrogenase (G6PD) activity and enhanced glucose and glutamine consumption. These phenotypes were mediated by a coordinated suppression of p53 and upregulation of c-MYC downstream of ATM inactivation. Our data indicate that ATM status couples replication stress and metabolic reprogramming during senescence., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

We studied the timing of occurrence of 1676 sporadic, community-acquired cases of Legionnaires' disease in England and Wales between 1993 and 2008, in relation to temperature, relative humidity, rainfall, windspeed and ultraviolet light using a fixed-stratum case-crossover approach. The analysis was conducted using conditional logistic regression, with consideration of appropriate lag periods. There was evidence of an association between the risk of Legionnaires' disease and temperature with an apparently long time lag of 1-9 weeks [odds of disease at 95th vs. 75th centiles: 3·91, 95% confidence interval (CI) 2·06-7·40], and with rainfall at short time lags (of 2-10 days) (odds of disease at 75th vs. 50th centiles: 1·78, 95% CI 1·50-2·13). There was some evidence that the risk of disease in relation to high temperatures was greater at high relative humidities. A higher risk of Legionnaires' disease may be indicated by preceding periods of warmer wetter weather.

Histone acetylation is sensitive to the availability of acetyl-CoA. However, the extent to which metabolic alterations in cancer cells impact tumor histone acetylation has been unclear. Here, we discuss our recent findings that oncogenic AKT1 activation regulates histone acetylation levels in tumors through regulation of acetyl-CoA metabolism.

Objective: Evaluate the Neonatal Screening Program (NSP) for congenital adrenal hyperplasia (CAH) of the Department of Health of the State of Santa Catarina (Secretaria de Estado da Saúde de Santa Catarina, SES/SC), and provide information to improve the program., Subjects and Methods: Descriptive, retrospective study of 748,395 children screened between January 2001 and December 2010. We analyzed the coverage of the NSP-SES/SC prevalence of CAH, child's age when the first sample for 17-hydroxyprogesterone (17OHP) measurement was collected, levels of 17OHP, mean age at treatment onset and main clinical manifestations., Results: The NSP-SES/SC covered 89% of the live newborns in the State. It diagnosed 50 cases of CAH, yielding an incidence of 1:14,967. Mean age at collection of the first sample was 7.3 days and mean level of 17OHP was 152.9 ng/mL. The most frequent manifestations were virilized genitalia with nonpalpable gonads, clitoromegaly and genital hyperpigmentation. In three girls, the genre established at birth was incorrect. The salt-wasting form was present in 74% of the cases. There was no occurrence of shock or death. Mean age at treatment onset in the salt-wasting form was 17.4 days compared with 54.9 days in those without the salt-wasting form of the disease. All children were treated with hydrocortisone, and those with salt-wasting CAH were also treated with fludrocortisone., Conclusions: The incidence of CAH was 1 case to 14,967 live newborns. Collection of the first sample occurred outside the recommended time, resulting in delays in treatment onset.

Histone acetylation plays important roles in gene regulation, DNA replication, and the response to DNA damage, and it is frequently deregulated in tumors. We postulated that tumor cell histone acetylation levels are determined in part by changes in acetyl coenzyme A (acetyl-CoA) availability mediated by oncogenic metabolic reprogramming. Here, we demonstrate that acetyl-CoA is dynamically regulated by glucose availability in cancer cells and that the ratio of acetyl-CoA:coenzyme A within the nucleus modulates global histone acetylation levels. In vivo, expression of oncogenic Kras or Akt stimulates histone acetylation changes that precede tumor development. Furthermore, we show that Akt's effects on histone acetylation are mediated through the metabolic enzyme ATP-citrate lyase and that pAkt(Ser473) levels correlate significantly with histone acetylation marks in human gliomas and prostate tumors. The data implicate acetyl-CoA metabolism as a key determinant of histone acetylation levels in cancer cells., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Glucocorticoids contribute to adipocyte differentiation by cooperating with transcription factors, such as CCAAT/enhancer-binding protein β (C/EBPβ), to stimulate transcription of the gene encoding peroxisome proliferator-activated receptor (PPARγ), a master regulator of adipogenesis. However, the mechanism of PPARγ gene regulation by glucocorticoids, the glucocorticoid receptor (GR), and its coregulators is poorly understood. Here we show that two GR binding regions (GBRs) in the mouse PPARγ gene were responsive to glucocorticoid, and treatment of 3T3-L1 preadipocytes with glucocorticoid alone induced GR occupancy and chromatin remodeling at PPARγ GBRs, which also contain binding sites for C/EBP and PPARγ proteins. GR recruited cell cycle and apoptosis regulator 1 (CCAR1), a transcription coregulator, to the PPARγ gene GBRs. Notably, CCAR1 was required for GR occupancy and chromatin remodeling at one of the PPARγ gene GBRs. Moreover, depletion of CCAR1 markedly suppressed differentiation of mouse mesenchymal stem cells and 3T3-L1 preadipocytes to mature adipocytes and decreased induction of PPARγ, C/EBPα, and C/EBPδ. Although CCAR1 was required for stimulation of several GR-regulated adipogenic genes in 3T3-L1 preadipocytes by glucocorticoid, it was not required for GR-activated transcription of certain anti-inflammatory genes in human A549 lung epithelial cells. Overall, our results highlighted the novel and specific roles of GR and CCAR1 in adipogenesis., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

As rates of obesity soar in the Unites States and around the world, cancer attributed to obesity has emerged as major threat to public health. The link between obesity and cancer can be attributed in part to the state of chronic inflammation that develops in obesity. Acetyl-CoA production and protein acetylation patterns are highly sensitive to metabolic state and are significantly altered in obesity. In this article, we explore the potential role of nutrient-sensitive lysine acetylation in regulating inflammatory processes in obesity-linked cancer.

Traumatic brain injury (TBI) is a global public health epidemic. In the US alone, more than 3 million people sustain a TBI annually. It is one of the most disabling injuries as it may cause motor and sensory deficits and lead to severe cognitive, emotional, and psychosocial impairment, crippling vital areas of higher functioning. Fueled by the recognition of TBI as the "signature injury" in our wounded soldiers in Iraq and Afghanistan, and its often devastating impact on athletes playing contact sports, interest in TBI and TBI research has increased dramatically. Unfortunately, despite increased awareness of its detrimental consequences, there has been little progress in developing effective TBI interventions. Recent evidence, however, strongly indicates that nutritional intervention may provide a unique opportunity to enhance the neuronal repair process after TBI. To date, two omega-3 fatty acids, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), have the most promising laboratory evidence for their neuro-restorative capacities in TBI. Although both animal models and human studies of brain injuries suggest they may provide benefits, there has been no clinical trial evaluating the effects of n-3 fatty acids on resilience to, or treatment, of TBI. This article reviews the known functions of n-3 fatty acids in the brain and their specific role in the cellular and biochemical pathways underlying neurotraumatic injury. We also highlight recent studies on the therapeutic impact of enhanced omega 3 intake in vivo, and how this may be a particularly promising approach to improving functional outcome in patients with TBI.

Songbirds communicate by learned vocalizations with concomitant changes in neurophysiological and genomic activities in discrete parts of the brain. Here, we tested a novel implementation of diffusive optical imaging (also known as diffuse optical imaging, DOI) for monitoring brain physiology associated with vocal signal perception. DOI noninvasively measures brain activity using red and near-infrared light delivered through optic fibers (optodes) resting on the scalp. DOI does not harm subjects, so it raises the possibility of repeatedly measuring brain activity and the effects of accumulated experience in the same subject over an entire life span, all while leaving tissue intact for further study. We developed a custom-made apparatus for interfacing optodes to the zebra finch (Taeniopygia guttata) head using 3D modeling software and rapid prototyping technology, and applied it to record responses to presentations of birdsong in isoflurane-anesthetized zebra finches. We discovered a subtle but significant difference between the hemoglobin spectra of zebra finches and mammals which has a major impact in how hemodynamic responses are interpreted in the zebra finch. Our measured responses to birdsong playback were robust, highly repeatable, and readily observed in single trials. Responses were complex in shape and closely paralleled responses described in mammals. They were localized to the caudal medial portion of the brain, consistent with response localization from prior gene expression, electrophysiological, and functional magnetic resonance imaging studies. These results define an approach for collecting neurophysiological data from songbirds that should be applicable to diverse species and adaptable for studies in awake behaving animals.

Background: The source of infection for most sporadic cases of Legionnaires' disease remains unknown. This study aims quantify the relationship between cases and wet cooling systems (WCS), a potential source of aerosolised legionella bacteria., Methods: The study analysed data on 1163 sporadic, community-acquired cases of Legionnaires' disease in England and Wales with onset between 1996 and 2006, and 11630 postcode controls randomly sampled in proportion to population size and matched on region, age group and sex. The relationship between risk of Legionnaires' disease and distance from a WCS was analysed by conditional logistic regression., Results: Cases and controls had a mean age of 56.3 years; 79.3% were male. Cases lived appreciably closer to WCS than their controls (mean distance of cases=2.11 km, controls=2.58 km; mean difference 0.47 km (95% CI 0.28 to 0.65)). The OR for disease within 1 km of a WCS compared with over 6 km (a distance taken to reflect background rates of Legionnaires' disease) was 1.59 (95% CI 1.26 to 2.01) when adjusted for socio-economic deprivation, and 1.33 (95% CI 1.04 to 1.71) when additionally adjusted for population density. The results suggest that residential proximity to a WCS may account for 19.6% of sporadic community-acquired cases., Conclusions: WCS may be an important source of sporadic, community-acquired cases of Legionnaires' disease, an observation that has important implications for health protection, especially given the likely increase in such systems as a component of strategies to improve energy efficiency in buildings.

Aims:   To perform an international trial to derive alert and action levels for the use of quantitative PCR (qPCR) in the monitoring of Legionella to determine the effectiveness of control measures against legionellae., Methods and Results:   Laboratories (7) participated from six countries. Legionellae were determined by culture and qPCR methods with comparable detection limits. Systems were monitored over ≥10 weeks. For cooling towers (232 samples), there was a significant difference between the log mean difference between qPCR (GU l(-1) ) and culture (CFU l(-1) ) for Legionella pneumophila (0·71) and for Legionella spp. (2·03). In hot and cold water (506 samples), the differences were less, 0·62 for Leg. pneumophila and 1·05 for Legionella spp. Results for individual systems depended on the nature of the system and its treatment. In cooling towers, Legionella spp. GU l(-1) always exceeded CFU l(-1) , and usually Legionella spp. were detected by qPCR when absent by culture. The pattern of results by qPCR for Leg. pneumophila followed the culture trend. In hot and cold water, culture and qPCR gave similar results, particularly for Leg. pneumophila. There were some marked exceptions with temperatures ≥50°C, or in the presence of supplementary biocides. Action and alert levels for qPCR were derived that gave results comparable to the application of the European Guidelines based on culture. Algorithms are proposed for the use of qPCR for routine monitoring., Conclusions:   Action and alert levels for qPCR can be adjusted to ensure public health is protected with the benefit that remedial actions can be validated earlier with only a small increase in the frequency of action being required., Significance and Impact of the Study:   This study confirms it is possible to derive guidelines on the use of qPCR for monitoring the control of legionellae with consequent improvement to response and public health protection., (© 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.)

Background: Nicotine dependence has been shown to represent a heritable condition, and several research groups have performed linkage analysis to identify genomic regions influencing this disorder though only a limited number of the findings have been replicated., Method: In the present study, a genome-wide linkage scan for nicotine dependence was conducted in a community sample of 950 probands and 1204 relatives recruited through the University of California, San Francisco (UCSF) Family Alcoholism Study. A modified version of the Semi-Structured Assessment for the Genetics of Alcoholism (SSAGA) with additional questions that probe nicotine use was used to derive DSM-IV nicotine dependence diagnoses., Results: A locus on chromosome 2q31.1 at 184 centiMorgans nearest to marker D2S2188 yielded a logarithm (base 10) of odds (LOD) score of 3.54 (point-wise empirical p=0.000012). Additional peaks of interest were identified on chromosomes 2q13, 4p15.33-31, 11q25 and 12p11.23-21. Follow-up analyses were conducted examining the contributions of individual nicotine dependence symptoms to the chromosome 2q31.1 linkage peak as well as examining the relationship of this chromosomal region to alcohol dependence., Conclusions: The present report suggests that chromosome 2q31.1 confers risk to the development of nicotine dependence and that this region influences a broad range of nicotine dependence symptoms rather than a specific facet of the disorder. Further, the results show that this region is not linked to alcohol dependence in this population, and thus may influence nicotine dependence specifically.

Ample data suggest that alcohol dependence represents a heritable condition, and several research groups have performed linkage analysis to identify genomic regions influencing this disorder. In the present study, a genome-wide linkage scan for alcohol dependence was conducted in a community sample of 565 probands and 1080 first-degree relatives recruited through the UCSF Family Alcoholism Study. The Semi-Structured Assessment for the Genetics of Alcoholism (SSAGA) was used to derive DSM-IV alcohol dependence diagnoses. Although no loci achieved genome-wide significance (i.e., LOD score > 3.0), several linkage peaks of interest (i.e., LOD score > 1.0) were identified. When the strict DSM-IV alcohol dependence diagnosis requiring the temporal clustering of symptoms served as the phenotype, linkage peaks were identified on chromosomes 1p36.31-p36.22, 2q37.3, 8q24.3, and 18p11.21-p11.2. When the temporal clustering of symptoms was not required, linkage peaks were again identified on chromosomes 1p36.31-p36.22 and 8q24.3 as well as novel loci on chromosomes 1p22.3, 2p24.3-p24.1, 9p24.1-p23, and 22q12.3-q13.1. Follow-up analyses were conducted by performing linkage analysis for the 12 alcohol dependence symptoms assessed by the SSAGA across the support intervals for the observed linkage peaks. These analyses demonstrated that different collections of symptoms often assessing distinct aspects of alcohol dependence (e.g., uncontrollable drinking and withdrawal vs. tolerance and drinking despite health problems) contributed to each linkage peak and often yielded LOD scores exceeding that reported for the alcohol dependence diagnosis. Such findings provide insight into how specific genomic regions may influence distinct aspects of alcohol dependence., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Glucocorticoids play important roles in the regulation of distinct aspects of adipocyte biology. Excess glucocorticoids in adipocytes are associated with metabolic disorders, including central obesity, insulin resistance and dyslipidemia. To understand the mechanisms underlying the glucocorticoid action in adipocytes, we used chromatin immunoprecipitation sequencing to isolate genome-wide glucocorticoid receptor (GR) binding regions (GBRs) in 3T3-L1 adipocytes. Furthermore, gene expression analyses were used to identify genes that were regulated by glucocorticoids. Overall, 274 glucocorticoid-regulated genes contain or locate nearby GBR. We found that many GBRs were located in or nearby genes involved in triglyceride (TG) synthesis (Scd-1, 2, 3, GPAT3, GPAT4, Agpat2, Lpin1), lipolysis (Lipe, Mgll), lipid transport (Cd36, Lrp-1, Vldlr, Slc27a2) and storage (S3-12). Gene expression analysis showed that except for Scd-3, the other 13 genes were induced in mouse inguinal fat upon 4-day glucocorticoid treatment. Reporter gene assays showed that except Agpat2, the other 12 glucocorticoid-regulated genes contain at least one GBR that can mediate hormone response. In agreement with the fact that glucocorticoids activated genes in both TG biosynthetic and lipolytic pathways, we confirmed that 4-day glucocorticoid treatment increased TG synthesis and lipolysis concomitantly in inguinal fat. Notably, we found that 9 of these 12 genes were induced in transgenic mice that have constant elevated plasma glucocorticoid levels. These results suggested that a similar mechanism was used to regulate TG homeostasis during chronic glucocorticoid treatment. In summary, our studies have identified molecular components in a glucocorticoid-controlled gene network involved in the regulation of TG homeostasis in adipocytes. Understanding the regulation of this gene network should provide important insight for future therapeutic developments for metabolic diseases.

Background: Glucocorticoids are potent anti-inflammatory agents commonly used to treat inflammatory diseases. They convey signals through the intracellular glucocorticoid receptor (GR), which upon binding to ligands, associates with genomic glucocorticoid response elements (GREs) to regulate transcription of associated genes. One mechanism by which glucocorticoids inhibit inflammation is through induction of the dual specificity phosphatase-1 (DUSP1, a.k.a. mitogen-activated protein kinase phosphatase-1, MKP-1) gene., Methodology/principal Findings: We found that glucocorticoids rapidly increased transcription of DUSP1 within 10 minutes in A549 human lung adenocarcinoma cells. Using chromatin immunoprecipitation (ChIP) scanning, we located a GR binding region between -1421 and -1118 upstream of the DUSP1 transcription start site. This region is active in a reporter system, and mutagenesis analyses identified a functional GRE located between -1337 and -1323. We found that glucocorticoids increased DNase I hypersensitivity, reduced nucleosome density, and increased histone H3 and H4 acetylation within genomic regions surrounding the GRE. ChIP experiments showed that p300 was recruited to the DUSP1 GRE, and RNA interference experiments demonstrated that reduction of p300 decreased glucocorticoid-stimulated DUSP1 gene expression and histone H3 hyperacetylation. Furthermore, overexpression of p300 potentiated glucocorticoid-stimulated activity of a reporter gene containing the DUSP1 GRE, and this coactivation effect was compromised when the histone acetyltransferase domain was mutated. ChIP-reChIP experiments using GR followed by p300 antibodies showed significant enrichment of the DUSP1 GRE upon glucocorticoid treatment, suggesting that GR and p300 are in the same protein complex recruited to the DUSP1 GRE., Conclusions/significance: Our studies identified a functional GRE for the DUSP1 gene. Moreover, the transcriptional activation of DUSP1 by glucocorticoids requires p300 and a rapid modification of the chromatin structure surrounding the GRE. Overall, understanding the mechanism of glucocorticoid-induced DUSP1 gene transcription could provide insights into therapeutic approaches against inflammatory diseases.

This study examined the impact of meteorological conditions on sporadic, community-acquired cases of Legionnaires' disease in England and Wales (2003-2006), with reference to the 2006 increase in cases. A case-crossover methodology compared each case with self-controlled data using a conditional logistic regression analysis. Effect modification by quarter and year was explored. In total, 674 cases were entered into the dataset and two meteorological variables were selected for study based on preliminary analyses: relative humidity during a case's incubation period, and temperature during the 10-14 weeks preceding onset. For the quarter July-September there was strong evidence to suggest a year, humidity and temperature interaction (Wald chi2=30.59, 3 d.f., P<0.0001). These findings have implications for future case numbers and resource requirements.

Clinical isolates of Legionella pneumophila, obtained from 167 patients, who acquired their illness in the community in England and Wales between January 2000 and March 2008, were compared with 276 environmental isolates of L. pneumophila obtained over the same period as part of the routine sampling of 'managed' water systems. The 443 isolates were typed by monoclonal antibody (mAb) subgrouping and the internationally standardised, seven-gene loci, sequence-based typing (SBT) scheme of the European Working Group for Legionella Infections (EWGLI). Of the clinical isolates, 97.6% were L. pneumophila serogroup (sgp) 1, compared with only 55.8% of environmental isolates (P = 0.0002); 91.6% were subgrouped as mAb3/1+ve, compared with only 8.3% of environmental isolates (P < 0.0001). The isolates were very diverse, with SBT identifying 111 sequence types (STs) (index of diversity [IOD] 0.954). Among the clinical isolates, 42 ST were seen, with one (ST47) accounting for 25.7% and three (ST47, ST37 and ST62) accounting for 46.1% of all isolates. Eighty-two STs were identified among the environmental isolates, with two (ST1 and ST79) accounting for 34.1% of these. Comparison of the STs seen among clinical and environmental isolates showed that there was very little overlap between the two populations (P < 0.0001), with common clinical strains found in the environment very infrequently: 0.4, 0.7 and 0% (ST47, ST37 and ST62, respectively), and common environmental strains rarely causing disease: 4.8 and 1.2% (ST1 and ST79, respectively). Combining phenotypic and genotypic data identified 144 phenons (IOD 0.970); 52 among clinical isolates and 101 among environmental isolates. The most abundant clinical strain, mAb 'Allentown' ST47, accounted for 22.8% of cases, but was only found once in the environment. Conversely, mAb 'Oxford/OLDA' ST1 was the most common environmental strain (17.0%), but only caused two infections. A review of the published data shows that mAb 'Allentown' ST47 is also an important cause of infection in France and possibly in the Netherlands. However, it was not found in a large study of German clinical isolates. This study confirms previous work showing that just a few strains of L. pneumophila cause the majority of community-acquired Legionella infection in England and Wales, and that these clinically significant strains are only rarely found in managed water systems. These data suggest that knowing which particular strain is present in an environment might be at least as important as knowing the quantity in which legionellae are present.

This paper provides one of the first assessments of the burden of both the public health investigation and the economic costs associated with an apparent outbreak of Legionnaires' disease (LD) in South East London. In addition to epidemiological, microbiological and environmental investigations, we collected data on the staff time and resources committed by the 11 main organizations responsible for managing the outbreak. Of the overall estimated costs of 455,856 pounds, only 14% (64,264 pounds) was spent on investigation and control of the outbreak compared with 86% (391,592 pounds) spent on the hospital treatment of the patients. The time and money spent on public health services in this investigation appear to represent good value for money considering the potential costs of a major outbreak, including the high case-fatality rate in LD generally and the high health-care costs. Further research is needed to determine optimum strategies for the cost-effective use of health system resources in investigations of LD. Whether the threshold for investigation of cases should be based on observed incidence rates or the cost-effectiveness of investigations, or both, should be debated further.

Eight cases of Legionnaires' disease were identified among the 215 German passengers after a cruise to the Nordic Sea in August 2003. An unmatched case-control study was conducted to identify risk factors and the source of infection. In total, eight passengers fulfilled the case definition, one of these died. Forty-two passengers served as controls. The attack rate was 4%. The mean age was 60 years for cases and 62 years for controls. Prolonged exposure to the spa pool seemed to be a risk factor of infection (OR 4.85, P=0.09). Legionella pneumophila serogroup 1, monoclonal antibody (mAb) subgroup 'Knoxville' was isolated from clinical and environmental samples. DNA sequence-based typing revealed that these isolates were indistinguishable from each other. The investigation showed the importance of an interdisciplinary approach of microbiology and epidemiology as not all sites on the ship that tested positive for L. pneumophila actually posed a relevant risk for the passengers.

During August 2006, there was a large increase in non-travel related legionella cases throughout England and in the Netherlands.

Helicobacter pylori is an important global human pathogen and there is growing evidence from PCR assays that contaminated drinking water might be a possible source of infection in some circumstances. There are no validated protocols for direct isolation but various culture media have been developed for possible environmental sampling. Our aim here was to investigate how inter-strain variation might affect the interpretation of results with such media. Two laboratory adapted reference strains and four recent clinical isolates were tested on four solid media and in ten liquid media. Considerable variation was found between strains in their ability to recover on the different media after stress exposure (suspension in sterile tap water). Generally, clinical isolates were less robust than the laboratory-adapted strains and, overall, the former required longer recovery times. Our findings highlighted the importance of using a range of isolates for evaluations, as examination of laboratory-adapted strains alone did not provide an accurate representation of the utility of media that may be used to recover H. pylori from water.