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1.  Crystallographic and Spectroscopic Snapshots Reveal a Dehydrogenase in Action 
Nature communications  2015;6:5935.
Aldehydes are ubiquitous intermediates in metabolic pathways, and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation which can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for ‘disarming’ the final aldehydic intermediate. Here we show crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, thiohemiacetal intermediate, a thioacyl intermediate, and an NAD+-bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site prior to an sp3-to-sp2 transition during enzyme-mediated oxidation.
doi:10.1038/ncomms6935
PMCID: PMC4286809  PMID: 25565451
2.  Cardiomyocyte-Specific Expression of CRNK, the C-terminal Domain of PYK2, Maintains Ventricular Function and Slows Ventricular Remodeling in a Mouse Model of Dilated Cardiomyopathy 
Rationale
Up-regulation and activation of PYK2, a member of the FAK family of protein tyrosine kinases, is involved in the pathogenesis of left ventricular (LV) remodeling and heart failure (HF). PYK2 activation can be prevented by CRNK, the C-terminal domain of PYK2. We previously demonstrated that adenoviral-mediated CRNK gene transfer improved survival and LV function, and slowed LV remodeling in a rat model of coronary artery ligation-induced HF.
Objective
We now interrogate whether cardiomyocyte-specific, transgenic CRNK expression prevents LV remodeling and HF in a mouse model of dilated cardiomyopathy (DCM) caused by constitutively active Protein Kinase Cε (caPKCε).
Methods and Results
Transgenic (TG; FVB/N background) mice were engineered to express rat CRNK under control of the α-myosin heavy chain promoter, and crossed with FVB/N mice with cardiomyocyte-specific expression of caPKCε to create double TG mice. LV structure, function, and gene expression was evaluated in all 4 groups (nonTG FVB/N; caPKCε(+/-); CRNK(+/-); and caPKCε x CRNK (PXC) double TG mice) at 1, 3, 6, 9 and 12mo of age. CRNK expression followed a Mendelian distribution, and CRNK mice developed and survived normally through 12mo. Cardiac structure, function and selected gene expression of CRNK mice were similar to nonTG littermates. CRNK had no effect on caPKCε expression and vice versa. PYK2 was up-regulated ~6-fold in caPKCε mice, who developed a non-hypertrophic, progressive DCM with reduced systolic (Contractility Index=151±5 vs. 90±4 sec-1) and diastolic (Tau=7.5±0.5 vs. 14.7±1.3 msec) function, and LV dilatation (LV Remodeling Index (LVRI)=4.2±0.1 vs. 6.0±0.3 for FVB/N vs. caPKCε mice, respectively; P<0.05 for each at 12mo). In double TG PXC mice, CRNK expression significantly prolonged survival, improved contractile function (Contractile Index=115±8 sec-1; Tau=9.5±1.0 msec), and reduced LV remodeling (LVRI=4.9±0.1).
Conclusions
Cardiomyocyte-specific expression of CRNK improves contractile function and slows LV remodeling in a mouse model of DCM.
doi:10.1016/j.yjmcc.2014.03.021
PMCID: PMC4064715  PMID: 24713463
Heart failure; gene expression; protein kinase C; echocardiography; focal adhesion kinase
3.  Focal Adhesion Signaling in Heart Failure 
Summary
In this brief review, recent evidence is presented to indicate a role for specific components of the cardiomyocyte costamere (and its related structure the focal adhesion complex of cultured cardiomyocytes) in initiating and sustaining the aberrant signal transduction that contributes to myocardial remodeling and the progression to heart failure (HF). Special attention is devoted to the focal adhesion kinase family of nonreceptor protein tyrosine kinases in bi-directional signal transduction during cardiac remodeling and HF progression. Finally, some speculations and directions for future study are provided for this rapidly developing field of research.
doi:10.1007/s00424-014-1456-8
PMCID: PMC4037338  PMID: 24515292
costameres; focal adhesion kinase; PYK2; talin; vinculin
4.  First Reported Case of Ehrlichia ewingii Involving Human Bone Marrow 
Journal of Clinical Microbiology  2014;52(11):4102-4104.
A 65-year-old female with a history of multiple tick bites presented with fever and pancytopenia. Intracytoplasmic rickettsial morulae were detected on peripheral smear and bone marrow biopsy specimens, and PCR amplified Ehrlichia ewingii DNA from both specimens. To our knowledge, this is the first report of E. ewingii infection of human bone marrow.
doi:10.1128/JCM.01670-14
PMCID: PMC4313272  PMID: 25187638
5.  PKCε-CREB-Nrf2 signalling induces HO-1 in the vascular endothelium and enhances resistance to inflammation and apoptosis 
Cardiovascular Research  2015;106(3):509-519.
Aims
Vascular injury leading to endothelial dysfunction is a characteristic feature of chronic renal disease, diabetes mellitus, and systemic inflammatory conditions, and predisposes to apoptosis and atherogenesis. Thus, endothelial dysfunction represents a potential therapeutic target for atherosclerosis prevention. The observation that activity of either protein kinase C epsilon (PKCε) or haem oxygenase-1 (HO-1) enhances endothelial cell (EC) resistance to inflammation and apoptosis led us to test the hypothesis that HO-1 is a downstream target of PKCε.
Methods and results
Expression of constitutively active PKCε in human EC significantly increased HO-1 mRNA and protein, whereas conversely aortas or cardiac EC from PKCε-deficient mice exhibited reduced HO-1 when compared with wild-type littermates. Angiotensin II activated PKCε and induced HO-1 via a PKCε-dependent pathway. PKCε activation significantly attenuated TNFα-induced intercellular adhesion molecule-1, and increased resistance to serum starvation-induced apoptosis. These responses were reversed by the HO antagonist zinc protoporphyrin IX. Phosphokinase antibody array analysis identified CREB1(Ser133) phosphorylation as a PKCε signalling intermediary, and cAMP response element-binding protein 1 (CREB1) siRNA abrogated PKCε-induced HO-1 up-regulation. Likewise, nuclear factor (erythroid-derived 2)-like 2 (Nrf2) was identified as a PKCε target using nuclear translocation and DNA-binding assays, and Nrf2 siRNA prevented PKCε-mediated HO-1 induction. Moreover, depletion of CREB1 inhibited PKCε-induced Nrf2 DNA binding, suggestive of transcriptional co-operation between CREB1 and Nrf2.
Conclusions
PKCε activity in the vascular endothelium regulates HO-1 via a pathway requiring CREB1 and Nrf2. Given the potent protective actions of HO-1, we propose that this mechanism is an important contributor to the emerging role of PKCε in the maintenance of endothelial homeostasis and resistance to injury.
doi:10.1093/cvr/cvv131
PMCID: PMC4431664  PMID: 25883219
Protein kinase C epsilon; Endothelium; Haem oxygenase-1; Inflammation; Apoptosis
6.  Competing Causes of Death and Medical Comorbidities Among Patients With Human Papillomavirus–Positive vs Human Papillomavirus–Negative Oropharyngeal Carcinoma and Impact on Adherence to Radiotherapy 
IMPORTANCE
Survival of patients with head and neck cancer can be affected by competing causes of mortality, as well as comorbidities that result in radiation treatment interruptions.
OBJECTIVE
To discern how differences in preexisting medical and psychosocial comorbidities potentially influence adherence to radiation therapy according to human papillomavirus (HPV) status.
DESIGN, SETTING, AND PARTICIPANTS
Retrospective analysis at a comprehensive cancer center of 162 consecutive patients with locally advanced squamous cell carcinoma of the oropharynx treated with primary chemoradiation (n = 95) or primary surgery followed by adjuvant radiation (n = 67). Immunostaining for p16 was used to determine HPV status.
MAIN OUTCOMES AND MEASURES
Difference in alcohol, tobacco, and marijuana use was compared between patients with HPV-positive and HPV-negative tumors, as well as the prevalence of the following comorbidities: diabetes mellitus, chronic obstructive pulmonary disease (COPD), anxiety disorder, and major depression. The number of total missed treatment days was analyzed as both a continuous and categorical variable.
RESULTS
Rates of self-reported heavy alcohol use (47% vs 16%; P = .02) and any marijuana use (47% vs 23%; P = .003) were significantly higher among HPV-negative patients. Fifty-four percent of HPV-positive patients self-identified as never smokers, compared with only 12% of HPV-negative patients (P < .001). HPV-negative patients had more missed treatment days (mean, 2.8 vs 1.7 days; P = .02), as well as an increased rate of at least 5 missed days (24% vs 10%; P = .04), and higher prevalences of COPD (12% vs 7%; P = .37) and anxiety disorder (12% vs 6%; P = .35).
CONCLUSIONS AND RELEVANCE
Pronounced differences exist in lifestyle habits between patients with HPV-negative and HPV-positive oropharyngeal cancer at diagnosis. These differences, as well as those of medical and psychosocial burden, may contribute to observed discrepancies in treatment adherence and need to be considered in outcomes reporting and clinical trial design.
doi:10.1001/jamaoto.2013.6732
PMCID: PMC4397215  PMID: 24526276
7.  Human Electroretinal Responses to Grating Patterns and Defocus Changes by Global Flash Multifocal Electroretinogram 
PLoS ONE  2015;10(4):e0123480.
The electrical response of the retina was examined as a function of retinal region, using stimuli of various spatial frequencies in the first experiment. In the second experiment, the regional response of the retina to defocus at high and low spatial frequencies was investigated. Twenty three subjects were recruited for global flash multifocal electroretinogram (mfERG) in experiment 1. Black and white gratings (printed on plastic transparent sheets) of four spatial frequencies (SF), 0.24, 1.2, 2.4 and 4.8 cycle per degree were presented in front of the mfERG stimulation. The amplitudes and implicit times of the direct (DC) and induced (IC) components of mfERG responses were pooled into six concentric rings for analysis. There was low amplitude DC at low SF, which increased with increasing SF, and which decreased with increasing eccentricity. The IC was high in amplitude at all SF and reduced in amplitude with increasing eccentricity. Our findings suggested that outer and inner retina had different characteristics in processing spatial details. In experiment 2, Twenty-three young adults were recruited for mfERG measurement. The retinal electrical responses for low (0.24cpd) and high (4.8cpd) SF under fully corrected conditions of short-term negative defocus (-2D) and short term positive defocus (+2D) conditions were measured. There was a sign-dependent response to defocus in the DC response, mainly in peripheral regions. The sign dependent response at low SF was more obvious than that at high SF, and was located more peripherally. The IC response showed no clear trends for either defocus condition. The human retina seems to have a decoding system for optical defocus, which was tuned for low spatial frequency, and was located in the retinal near periphery.
doi:10.1371/journal.pone.0123480
PMCID: PMC4395308  PMID: 25874564
8.  Personalized Oncogenomics: Clinical Experience with Malignant Peritoneal Mesothelioma Using Whole Genome Sequencing 
PLoS ONE  2015;10(3):e0119689.
Peritoneal mesothelioma is a rare and sometimes lethal malignancy that presents a clinical challenge for both diagnosis and management. Recent studies have led to a better understanding of the molecular biology of peritoneal mesothelioma. Translation of the emerging data into better treatments and outcome is needed. From two patients with peritoneal mesothelioma, we derived whole genome sequences, RNA expression profiles, and targeted deep sequencing data. Molecular data were made available for translation into a clinical treatment plan. Treatment responses and outcomes were later examined in the context of molecular findings. Molecular studies presented here provide the first reported whole genome sequences of peritoneal mesothelioma. Mutations in known mesothelioma-related genes NF2, CDKN2A, LATS2, amongst others, were identified. Activation of MET-related signaling pathways was demonstrated in both cases. A hypermutated phenotype was observed in one case (434 vs. 18 single nucleotide variants) and was associated with a favourable outcome despite sarcomatoid histology and multifocal disease. This study represents the first report of whole genome analyses of peritoneal mesothelioma, a key step in the understanding and treatment of this disease.
doi:10.1371/journal.pone.0119689
PMCID: PMC4370594  PMID: 25798586
9.  The Dilemma of Choosing a Reference Character for Measuring Sexual Size Dimorphism, Sexual Body Component Dimorphism, and Character Scaling: Cryptic Dimorphism and Allometry in the Scorpion Hadrurus arizonensis 
PLoS ONE  2015;10(3):e0120392.
Sexual differences in morphology, ranging from subtle to extravagant, occur commonly in many animal species. These differences can encompass overall body size (sexual size dimorphism, SSD) or the size and/or shape of specific body parts (sexual body component dimorphism, SBCD). Interacting forces of natural and sexual selection shape much of the expression of dimorphism we see, though non-adaptive processes may be involved. Differential scaling of individual features can result when selection favors either exaggerated (positive allometry) or reduced (negative allometry) size during growth. Studies of sexual dimorphism and character scaling rely on multivariate models that ideally use an unbiased reference character as an overall measure of body size. We explored several candidate reference characters in a cryptically dimorphic taxon, Hadrurus arizonensis. In this scorpion, essentially every body component among the 16 we examined could be interpreted as dimorphic, but identification of SSD and SBCD depended on which character was used as the reference (prosoma length, prosoma area, total length, principal component 1, or metasoma segment 1 width). Of these characters, discriminant function analysis suggested that metasoma segment 1 width was the most appropriate. The pattern of dimorphism in H. arizonensis mirrored that seen in other more obviously dimorphic scorpions, with static allometry trending towards isometry in most characters. Our findings are consistent with the conclusions of others that fecundity selection likely favors a larger prosoma in female scorpions, whereas sexual selection may favor other body parts being larger in males, especially the metasoma, pectines, and possibly the chela. For this scorpion and probably most other organisms, the choice of reference character profoundly affects interpretations of SSD, SBCD, and allometry. Thus, researchers need to broaden their consideration of an appropriate reference and exercise caution in interpreting findings. We highly recommend use of discriminant function analysis to identify the least-biased reference character.
doi:10.1371/journal.pone.0120392
PMCID: PMC4368692  PMID: 25793523
10.  Cancer risk in a cohort of patients with systemic lupus erythematosus (SLE) in California 
Cancer causes & control : CCC  2008;19(8):887-894.
Objective
We conducted a retrospective cohort study to examine cancer risk in a large cohort of systemic lupus erythematosus (SLE) patients in California.
Methods
The cohort consisted of individuals with SLE derived from statewide patient discharge data during the period 1991–2002. SLE patients were followed using cancer registry data to examine patterns of cancer development. Standardized incidence ratios (SlRs) and 95% CI were calculated to compare the observed to expected numbers of cancers based on age-, race-, and sex-specific incidence rates in the California population.
Results
The 30,478 SLE patients were observed for 157,969 person-years. A total of 1,273 cancers occurred within the observation interval. Overall cancer risk was significantly elevated (SIR=1.14, 95% CI=1.07–1.20). SLE patients had higher risks of vagina/vulva (SIR=3.27, 95% CI=2.41–4.31) and liver cancers (SIR=2.70, 95% CI=1.54–4.24). Elevated risks of lung, kidney, and thyroid cancers and several hematopoietic malignancies were also observed. Individuals had significantly lower risks of several screenable cancers, including breast, cervix, and prostate.
Conclusions
These data suggest that risks of several cancer types are elevated among SLE patients. Detailed studies of endogenous and exogenous factors that drive these associations are needed.
doi:10.1007/s10552-008-9151-8
PMCID: PMC4357418  PMID: 18386139
11.  Risk of cancer among rheumatoid arthritis patients in California 
Cancer causes & control : CCC  2009;20(6):1001-1010.
Objective
The objective of this retrospective cohort study was to evaluate cancer risk among rheumatoid arthritis (RA) patients in California.
Methods
The study cohort derived from statewide patient discharge records was followed via linkage with cancer registry data over the period 1991–2002. Age and sex adjusted standardized incidence ratios (SIRs) and 95% confidence intervals were calculated to compare observed to expected numbers of cancers based on age, race, and sex specific incidence rates in the California population.
Results
Among the 84,475 RA patients, who were observed for 405,540 person-years, 5,533 incident cancers were diagnosed during the observation interval. The risk of developing lymphohematopoietic cancer was significantly higher in the cohort for both sexes. Males had significantly higher risks of lung, liver, and esophageal cancer, but a lower risk of prostate cancer. Females were at significantly decreased risk for several cancers including breast, ovary, uterus, cervix, and melanoma, with the risk reduction ranging from 15 to 57% lower than the general population. Hispanics had increased risks of leukemia, vagina/vulva, lung, and liver cancers.
Conclusion
Studies investigating the mechanisms that underlie the reported associations between RA and specific cancer types are needed.
doi:10.1007/s10552-009-9298-y
PMCID: PMC4354853  PMID: 19184473
12.  Crystallographic and spectroscopic snapshots reveal a dehydrogenase in action 
Nature Communications  2015;6:5935.
Aldehydes are ubiquitous intermediates in metabolic pathways and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation that can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for ‘disarming’ the final aldehydic intermediate. Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, thiohemiacetal intermediate, a thioacyl intermediate and an NAD+-bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site before an sp3-to-sp2 transition during enzyme-mediated oxidation.
Aldehydes are common intermediates in enzymatic pathways, but their high reactivity can make them difficult to observe. Here, the authors study the mechanism of aldehyde deactivation in a dehydrogenase, showing a key E/Z isomerization and observing a thiohemiacetal intermediate by crystal structure analysis.
doi:10.1038/ncomms6935
PMCID: PMC4286809  PMID: 25565451
13.  Stress-Dependent Opioid and Adrenergic Modulation of Newly Retrieved Fear Memory 
Recent studies on the effect of stress on modulation of fear memory in our laboratory have uncovered endogenous opioid and adrenergic based modulation systems, working in concert, that limit the strengthening or weakening of newly acquired fear memory during consolidation under conditions of mild or intense stress, respectively. The present study sought to determine if similar stress-dependent modulation, mediated by endogenous opioid and adrenergic systems, occurs during reconsolidation of newly retrieved fear memory. Rats underwent contextual fear conditioning followed 24 hr later by reactivation of fear memory; a retention test was administered the next day. Stress was manipulated by varying duration of recall of fear memory during reactivation. In the first experiment, vehicle or the opioid-receptor blocker naloxone was administered immediately after varied durations (30 or 120 sec) of reactivation. The results indicate that 1) reactivation, in the absence of drug, has a marked effect on freezing behavior—as duration of reactivation increases from 30 to 120 sec, freezing behavior and presumably fearinduced stress increases and 2) naloxone, administered immediately after 30 sec (mild stress) or 120 sec (intense stress) of reactivation, enhances or impairs retention, respectively, the next day. In the second experiment, naloxone and the ß-adrenergic blocker propranolol were administered either separately or in combination immediately after 120 sec (intense stress) reactivation. The results indicate that separate administration of propranolol and naloxone impairs retention, while the combined administration fails to do so. Taken together the results of the two experiments are consistent with a protective mechanism, mediated by endogenous opioid and adrenergic systems working in concert, that limits enhancement and impairment of newly retrieved fear memory during reactivation in a stress-dependent manner.
doi:10.1016/j.nlm.2013.11.013
PMCID: PMC3943532  PMID: 24291724
reconsolidation; modulation; updating; stress; opioid; adrenergic; retrieval
14.  Controlled Trial of Transfusions for Silent Cerebral Infarcts in Sickle Cell Anemia 
The New England journal of medicine  2014;371(8):699-710.
BACKGROUND
Silent cerebral infarcts are the most common neurologic injury in children with sickle cell anemia and are associated with the recurrence of an infarct (stroke or silent cerebral infarct). We tested the hypothesis that the incidence of the recurrence of an infarct would be lower among children who underwent regular blood-transfusion therapy than among those who received standard care.
METHODS
In this randomized, single-blind clinical trial, we randomly assigned children with sickle cell anemia to receive regular blood transfusions (transfusion group) or standard care (observation group). Participants were between 5 and 15 years of age, with no history of stroke and with one or more silent cerebral infarcts on magnetic resonance imaging and a neurologic examination showing no abnormalities corresponding to these lesions. The primary end point was the recurrence of an infarct, defined as a stroke or a new or enlarged silent cerebral infarct.
RESULTS
A total of 196 children (mean age, 10 years) were randomly assigned to the observation or transfusion group and were followed for a median of 3 years. In the transfusion group, 6 of 99 children (6%) had an end-point event (1 had a stroke, and 5 had new or enlarged silent cerebral infarcts). In the observation group, 14 of 97 children (14%) had an end-point event (7 had strokes, and 7 had new or enlarged silent cerebral infarcts). The incidence of the primary end point in the transfusion and observation groups was 2.0 and 4.8 events, respectively, per 100 years at risk, corresponding to an incidence rate ratio of 0.41 (95% confidence interval, 0.12 to 0.99; P = 0.04).
CONCLUSIONS
Regular blood-transfusion therapy significantly reduced the incidence of the recurrence of cerebral infarct in children with sickle cell anemia. (Funded by the National Institute of Neurological Disorders and Stroke and others; Silent Cerebral Infarct Multi-Center Clinical Trial ClinicalTrials.gov number, NCT00072761, and Current Controlled Trials number, ISRCTN52713285.)
doi:10.1056/NEJMoa1401731
PMCID: PMC4195437  PMID: 25140956
15.  Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening 
An acoustic high-throughput screening method is described for harvesting protein crystals and combining the protein crystals with chemicals such as a fragment library.
Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s−1) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening.
doi:10.1107/S1399004714013728
PMCID: PMC4304690  PMID: 25615864
macromolecular crystallography; acoustic droplet ejection; crystal mounting; drug discovery; chemical biology; high-throughput screening
16.  An International Ki67 Reproducibility Study 
Background
In breast cancer, immunohistochemical assessment of proliferation using the marker Ki67 has potential use in both research and clinical management. However, lack of consistency across laboratories has limited Ki67’s value. A working group was assembled to devise a strategy to harmonize Ki67 analysis and increase scoring concordance. Toward that goal, we conducted a Ki67 reproducibility study.
Methods
Eight laboratories received 100 breast cancer cases arranged into 1-mm core tissue microarrays—one set stained by the participating laboratory and one set stained by the central laboratory, both using antibody MIB-1. Each laboratory scored Ki67 as percentage of positively stained invasive tumor cells using its own method. Six laboratories repeated scoring of 50 locally stained cases on 3 different days. Sources of variation were analyzed using random effects models with log2-transformed measurements. Reproducibility was quantified by intraclass correlation coefficient (ICC), and the approximate two-sided 95% confidence intervals (CIs) for the true intraclass correlation coefficients in these experiments were provided.
Results
Intralaboratory reproducibility was high (ICC = 0.94; 95% CI = 0.93 to 0.97). Interlaboratory reproducibility was only moderate (central staining: ICC = 0.71, 95% CI = 0.47 to 0.78; local staining: ICC = 0.59, 95% CI = 0.37 to 0.68). Geometric mean of Ki67 values for each laboratory across the 100 cases ranged 7.1% to 23.9% with central staining and 6.1% to 30.1% with local staining. Factors contributing to interlaboratory discordance included tumor region selection, counting method, and subjective assessment of staining positivity. Formal counting methods gave more consistent results than visual estimation.
Conclusions
Substantial variability in Ki67 scoring was observed among some of the world’s most experienced laboratories. Ki67 values and cutoffs for clinical decision-making cannot be transferred between laboratories without standardizing scoring methodology because analytical validity is limited.
doi:10.1093/jnci/djt306
PMCID: PMC3888090  PMID: 24203987
17.  Mitophagy Is Required for Acute Cardioprotection by Simvastatin 
Antioxidants & Redox Signaling  2014;21(14):1960-1973.
Abstract
Aims: We have shown that autophagy and mitophagy are required for preconditioning. While statin's cardioprotective effects are well known, the role of autophagy/mitophagy in statin-mediated cardioprotection is not. In this study, we used HL-1 cardiomyocytes and mice subjected to ischemia/reperfusion to elucidate the mechanism of statin-mediated cardioprotection. Results: HL-1 cardiomyocytes exposed to simvastatin for 24 h exhibited diminished protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling, increased activation of unc-51-like kinase 1, and upregulation of autophagy and mitophagy. Similar findings were obtained in hearts of mice given simvastatin. Mevalonate abolished simvastatin's effects on Akt/mTOR signaling and autophagy induction in HL-1 cells, indicating that the effects are mediated through inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Simvastatin-treated HL-1 cells exhibited mitochondrial translocation of Parkin and p62/SQSTM1, fission, and mitophagy. Because Parkin is required for mitophagy and is expressed in heart, we investigated the effect of simvastatin on infarct size in Parkin knockout mice. Simvastatin reduced infarct size in wild-type mice but showed no benefit in Parkin knockout mice. Inhibition of HMG-CoA reductase limits mevalonate availability for both cholesterol and coenzyme Q10 (CoQ) biosynthesis. CoQ supplementation had no effect on statin-induced Akt/mTOR dephosphorylation or macroautophagy in HL-1 cells, but it potently blocked mitophagy. Importantly, CoQ supplementation abolished statin-mediated cardioprotection in vivo. Innovation and Conclusion: Acute simvastatin treatment suppresses mTOR signaling and triggers Parkin-dependent mitophagy, the latter which is required for cardioprotection. Coadministration of CoQ with simvastatin impairs mitophagy and cardioprotection. These results raise the concern that CoQ may interfere with anti-ischemic benefits of statins mediated through stimulation of mitophagy. Antioxid. Redox Signal. 21, 1960–1973.
doi:10.1089/ars.2013.5416
PMCID: PMC4208607  PMID: 23901824
18.  Multipronged CD4 T cell effector and memory responses cooperate to provide potent immunity against respiratory virus 
Immunological reviews  2013;255(1):149-164.
Summary
Over the last decade, the known spectrum of CD4 T cell effect or subsets has become much broader and it has become clear that there are multiple dimensions by which subsets with a particular cytokine commitment can be further defined, including their stage of differentiation, their location and most importantly, their ability to carryout discrete functions. Here we focus on our studies that highlight the synergy among discrete subsets, especially those defined by helper and cytotoxic function, in mediating viral protection and on distinctions between CD4 T cell effectors located in spleen, draining lymph node, and in tissue sites of infection. What emerges is a surprising multiplicity of CD4 T cell functions that indicate a large arsenal of mechanisms by which CD4 T cells act to combat viruses.
doi:10.1111/imr.12088
PMCID: PMC4206082  PMID: 23947353
19.  Solvent minimization induces preferential orientation and crystal clustering in serial micro-crystallography on micro-meshes, in situ plates and on a movable crystal conveyor belt 
Journal of Synchrotron Radiation  2014;21(Pt 6):1231-1239.
Strategies are described for optimizing the signal-to-noise of diffraction data, and for combining data from multiple crystals. One challenge that must be overcome is the non-random orientation of crystals with respect to one another and with respect to the surface that supports them.
X-ray diffraction data were obtained at the National Synchrotron Light Source from insulin and lysozyme crystals that were densely deposited on three types of surfaces suitable for serial micro-crystallography: MiTeGen MicroMeshes™, Greiner Bio-One Ltd in situ micro-plates, and a moving kapton crystal conveyor belt that is used to deliver crystals directly into the X-ray beam. 6° wedges of data were taken from ∼100 crystals mounted on each material, and these individual data sets were merged to form nine complete data sets (six from insulin crystals and three from lysozyme crystals). Insulin crystals have a parallelepiped habit with an extended flat face that preferentially aligned with the mounting surfaces, impacting the data collection strategy and the design of the serial crystallography apparatus. Lysozyme crystals had a cuboidal habit and showed no preferential orientation. Preferential orientation occluded regions of reciprocal space when the X-ray beam was incident normal to the data-collection medium surface, requiring a second pass of data collection with the apparatus inclined away from the orthogonal. In addition, crystals measuring less than 20 µm were observed to clump together into clusters of crystals. Clustering required that the X-ray beam be adjusted to match the crystal size to prevent overlapping diffraction patterns. No additional problems were encountered with the serial crystallography strategy of combining small randomly oriented wedges of data from a large number of specimens. High-quality data able to support a realistic molecular replacement solution were readily obtained from both crystal types using all three serial crystallography strategies.
doi:10.1107/S1600577514017731
PMCID: PMC4211130  PMID: 25343789
in situ X-ray data collection; crystallography; acoustic droplet ejection; serial crystallography
20.  A Cell-Based High-Throughput Screen for Novel Chemical Inducers of Fetal Hemoglobin for Treatment of Hemoglobinopathies 
PLoS ONE  2014;9(9):e107006.
Decades of research have established that the most effective treatment for sickle cell disease (SCD) is increased fetal hemoglobin (HbF). Identification of a drug specific for inducing γ-globin expression in pediatric and adult patients, with minimal off-target effects, continues to be an elusive goal. One hurdle has been an assay amenable to a high-throughput screen (HTS) of chemicals that displays a robust γ-globin off-on switch to identify potential lead compounds. Assay systems developed in our labs to understand the mechanisms underlying the γ- to β-globin gene expression switch during development has allowed us to generate a cell-based assay that was adapted for a HTS of 121,035 compounds. Using chemical inducer of dimerization (CID)-dependent bone marrow cells (BMCs) derived from human γ-globin promoter-firefly luciferase β-globin promoter-Renilla luciferase β-globin yeast artificial chromosome (γ-luc β-luc β-YAC) transgenic mice, we were able to identify 232 lead chemical compounds that induced γ-globin 2-fold or higher, with minimal or no β-globin induction, minimal cytotoxicity and that did not directly influence the luciferase enzyme. Secondary assays in CID-dependent wild-type β-YAC BMCs and human primary erythroid progenitor cells confirmed the induction profiles of seven of the 232 hits that were cherry-picked for further analysis.
doi:10.1371/journal.pone.0107006
PMCID: PMC4165891  PMID: 25225870
21.  Acoustic methods for high-throughput protein crystal mounting at next-generation macromolecular crystallographic beamlines 
Journal of Synchrotron Radiation  2013;20(Pt 5):805-808.
A new system has been developed and tested at the National Synchrotron Light Source with the goal of enabling rapid protein crystal mounting at next-generation macromolecular crystallographic beamlines. The system uses an acoustic ejector to deposit nanoliter-volume droplets containing crystals onto an X-ray transparent conveyor belt, which then moves the droplets into position for cryo-cooling and data collection. The acoustic ejector is capable of operating at a rate of several hundred droplet ejections per second.
To take full advantage of advanced data collection techniques and high beam flux at next-generation macromolecular crystallography beamlines, rapid and reliable methods will be needed to mount and align many samples per second. One approach is to use an acoustic ejector to eject crystal-containing droplets onto a solid X-ray transparent surface, which can then be positioned and rotated for data collection. Proof-of-concept experiments were conducted at the National Synchrotron Light Source on thermolysin crystals acoustically ejected onto a polyimide ‘conveyor belt’. Small wedges of data were collected on each crystal, and a complete dataset was assembled from a well diffracting subset of these crystals. Future developments and implementation will focus on achieving ejection and translation of single droplets at a rate of over one hundred per second.
doi:10.1107/S0909049513020372
PMCID: PMC3747951  PMID: 23955046
acoustic droplet ejection; conveyor belt; crystal mounting; high throughput; X-ray diffraction; macromolecular crystallography
22.  Association of the family environment with behavioural and cognitive outcomes in children with chromosome 22q11.2 deletion syndrome 
Background
Children with 22q11.2 deletion syndrome (22q11DS) are at risk for social-behavioural and neurocognitive sequelae throughout development. The current study examined the impact of family environmental characteristics on social-behavioural and cognitive outcomes in this pediatric population.
Method
Guardians of children with 22q11DS were recruited through two medical genetics clinics. Con senting guardians were asked to complete several questionnaires regarding their child's social, emotional and behavioural functioning, as well as family social environment and parenting styles. Children with 22q11DS were asked to undergo a cognitive assessment, including IQ and achievement testing, and measures of attention, executive function and memory.
Results
Modest associations were found between aspects of the family social environment and parenting styles with social-behavioural and cognitive/academic outcomes. Regression models indicated that physical punishment, socioeconomic status, parental control and family organisation significantly predicted social-behavioural and cognitive outcomes in children with 22q11DS.
Conclusion
Characteristics of the family social environment and parenting approaches appear to be associated with functional outcomes of children with 22q11DS. Understanding the impact of environmental variables on developmental outcomes can be useful in determining more effective targets for intervention. This will be important in order to improve the quality of life of individuals affected by 22q11DS.
doi:10.1111/jir.12054
PMCID: PMC4086857  PMID: 23742203
chromosome 22q11.2 deletion syndrome; DiGeorge syndrome; family environment; parenting; social-behavioural functioning; velocardiofacial syndrome
23.  A Linear Relationship between Crystal Size and Fragment Binding Time Observed Crystallographically: Implications for Fragment Library Screening Using Acoustic Droplet Ejection 
PLoS ONE  2014;9(7):e101036.
High throughput screening technologies such as acoustic droplet ejection (ADE) greatly increase the rate at which X-ray diffraction data can be acquired from crystals. One promising high throughput screening application of ADE is to rapidly combine protein crystals with fragment libraries. In this approach, each fragment soaks into a protein crystal either directly on data collection media or on a moving conveyor belt which then delivers the crystals to the X-ray beam. By simultaneously handling multiple crystals combined with fragment specimens, these techniques relax the automounter duty-cycle bottleneck that currently prevents optimal exploitation of third generation synchrotrons. Two factors limit the speed and scope of projects that are suitable for fragment screening using techniques such as ADE. Firstly, in applications where the high throughput screening apparatus is located inside the X-ray station (such as the conveyor belt system described above), the speed of data acquisition is limited by the time required for each fragment to soak into its protein crystal. Secondly, in applications where crystals are combined with fragments directly on data acquisition media (including both of the ADE methods described above), the maximum time that fragments have to soak into crystals is limited by evaporative dehydration of the protein crystals during the fragment soak. Here we demonstrate that both of these problems can be minimized by using small crystals, because the soak time required for a fragment hit to attain high occupancy depends approximately linearly on crystal size.
doi:10.1371/journal.pone.0101036
PMCID: PMC4079544  PMID: 24988328
24.  Smooth muscle relaxation and activation of the large conductance Ca++ – activated K+ (BKCa) channel by novel oestrogens 
British Journal of Pharmacology  2013;169(5):1153-1165.
Background and Purpose
Oestrogens can interact directly with membrane receptors and channels and can activate vascular BKCa channels. We hypothesized that novel oestrogen derivatives could relax smooth muscle by an extracllular effect on the α and β1 subunits of the BKCa channel, rather than at an intracellular site.
Experimental Approach
We studied the effects of novel oestrogens on the tension of pre-contracted isolated rat aortic rings, and on the electrophysiological properties of HEK 293 cells expressing the hSloα or hSloα+β1 subunits. Two of the derivatives incorporated a quaternary ammonium side-chain making them membrane impermeable.
Key Results
Oestrone, oestrone oxime and Quat DME-oestradiol relaxed pre-contracted rat aorta, but only Quat DME-oestradiol-induced relaxation was iberiotoxin sensitive. However, only potassium currents recorded in HEK 293 cells over-expressing both hSloα and hSloβ1 were activated by oestrone, oestrone oxime and Quat DME-oestradiol.
Conclusion and Implications
The novel oestrogens were able to relax smooth muscle, but through different mechanisms. In particular, oestrone oxime required the presence of the endothelium to exert much of its effect, whilst Quat DME-oestradiol depended both on NO and BKCa channel activation. The activation of BKCa currents in HEK 293 cells expressing hSloα+β1 by Quat DME-oestradiol is consistent with an extracellular binding site between the two subunits. The binding site resides between the extracellular N terminal of the α subunit and the extracellular loop between TM1 and 2 of the β1 subunit. Membrane-impermeant Quat DME-oestradiol lacks an exchangeable hydrogen on the A ring obviating antioxidant activity.
doi:10.1111/bph.12211
PMCID: PMC3696336  PMID: 23586466
oestrogens; Ca++-activated K+ channel; β1 subunit
25.  Skeletal effects of zoledronic acid in an animal model of chronic kidney disease 
Summary
Bisphosphonates reduce skeletal loss and fracture risk, but their use has been limited in patients with chronic kidney disease. This study shows skeletal benefits of zoledronic acid in an animal model of chronic kidney disease.
Introduction
Bisphosphonates are routinely used to reduce fractures but limited data exists concerning their efficacy in non-dialysis chronic kidney disease. The goal of this study was to test the hypothesis that zoledronic acid produces similar skeletal effects in normal animals and those with kidney disease.
Methods
At 25 weeks of age, normal rats were treated with a single dose of saline vehicle or 100 µg/kg of zoledronic acid while animals with kidney disease (approximately 30 % of normal kidney function) were treated with vehicle, low dose (20 µg/kg), or high dose (100 µg/kg) zoledronic acid, or calcium gluconate (3 % in the drinking water). Skeletal properties were assessed 5 weeks later using micro-computed tomography, dynamic histomorphometry, and mechanical testing.
Results
Animals with kidney disease had significantly higher trabecular bone remodeling compared to normal animals. Zoledronic acid significantly suppressed remodeling in both normal and diseased animals yet the remodeling response to zoledronic acid was no different in normal and animals with kidney disease. Animals with kidney disease had significantly lower cortical bone biomechanical properties; these were partially normalized by treatment.
Conclusions
Based on these results, we conclude that zoledronic acid produces similar amounts of remodeling suppression in animals with high turnover kidney disease as it does in normal animals, and has positive effects on select biomechanical properties that are similar in normal animals and those with chronic kidney disease.
doi:10.1007/s00198-012-2103-x
PMCID: PMC4063946  PMID: 22907737
Anti-remodeling agents; Bisphosphonate; Bone mechanics; Remodeling suppression

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