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1.  ‘Doing the hard yards’: carer and provider focus group perspectives of accessing Aboriginal childhood disability services 
Despite a high prevalence of disability, Aboriginal Australians access disability services in Australia less than non-Aboriginal Australians with a disability. The needs of Aboriginal children with disability are particularly poorly understood. They can endure long delays in treatment which can impact adversely on development. This study sought to ascertain the factors involved in accessing services and support for Aboriginal children with a disability.
Using the focus group method, two community forums, one for health and service providers and one for carers of Aboriginal children with a disability, were held at an Aboriginal Community Controlled Health Service (ACCHS) in the Sydney, metropolitan area of New South Wales, Australia. Framework analysis was applied to qualitative data to elucidate key issues relevant to the dimensions of access framework. Independent coding consistency checks were performed and consensus of analysis verified by the entire research team, several of whom represented the local Aboriginal community.
Seventeen health and social service providers representing local area government and non-government-funded health and social service organisations and five carers participated in two separate forums between September and October 2011. Lack of awareness of services and inadequate availability were prominent concerns in both groups despite geographic proximity to a major metropolitan area with significant health infrastructure. Carers noted racism, insufficient or non-existent services, and the need for an enhanced role of ACCHSs and AHWs in disability support services. Providers highlighted logistical barriers and cultural and historical issues that impacted on the effectiveness of mainstream services for Aboriginal people.
Despite dedicated disability services in an urban community, geographic proximity does not mitigate lack of awareness and availability of support. This paper has enumerated a number of considerations to address provision of disability services in an urban Australian Aboriginal community including building expertise and specialist capacity within Aboriginal Health Worker positions and services.
Increasing awareness of services, facilitating linkages and referrals, eliminating complexities to accessing support, and working with families and Aboriginal community organisations within a framework of resilience and empowerment to ensure a relevant and acceptable model are necessary steps to improving support and care for Aboriginal children with a disability.
PMCID: PMC3765087  PMID: 23958272
Childhood disability; Aboriginal and Torres Strait Islander peoples; Early intervention; Focus groups
2.  Chemical–Biological Fingerprinting: Probing the Properties of DNA Lesions Formed by Peroxynitrite 
Chemical research in toxicology  2007;20(11):1718-1729.
DNA-damaging agents usually produce a vast collection of lesions within the genome. Analysis of these lesions from the structural and biological viewpoints is often complicated by the reality that some of the lesions are chemically fragile, leading to an even larger set of secondary and tertiary products. In an effort to deconvolute complex DNA-damage spectra, a strategy is presented whereby an oligonucleotide containing a specific target for chemical reaction is allowed to react with a DNA-damaging agent. A large collection of HPLC-resolvable modified oligonucleotides is generated, and chromatographically distinct members of the set are then individually characterized using chemical, spectroscopic, biochemical, and genetic probes. The biological component of this “chemical–biological fingerprinting” tool is the use of polymerase bypass in vivo in cells having defined replication status and quantitative and qualitative patterns of lesion-directed mutagenesis, as key properties that complement physical analysis of modified DNA. This approach was applied to the complex product spectrum generated by peroxynitrite in the presence of CO2; peroxynitrite is a powerful oxidizing and nitrating agent generated as part of immune response. An oligonucleotide containing the primary oxidation product, 7,8-dihydro-8-oxoguanine (8-oxoGua), which is highly susceptible to further oxidation and/or nitration, was treated with peroxynitrite. Using mass spectrometry, coelution with authentic standards, sensitivity to piperidine, recognition and strand cleavage by the DNA repair enzyme MutM, and mutagenicity and genotoxicity in vivo, a matrix was created that defined the properties of the secondary DNA lesions formed when 3-morpholinosydnonimine (SIN-1) delivered a low, constant flux of peroxynitrite to an oligonucleotide containing 8-oxoGua. Two lesions were identified as the diastereomers of spiroiminodihydantoin (Sp), which had been observed previously in nucleoside-based experiments employing SIN-1. A third lesion, triazine, was tentatively identified. However, in addition to these lesions, a number of secondary lesions were generated that had chemical–biological fingerprints inconsistent with that of any known 8-oxoGua-derived lesion described to date. In vitro experiments showed that while some of these newly characterized secondary lesions were removed from DNA by MutM, others were in fact very poor substrates for this repair enzyme. These 8-oxoGua-derived lesions also showed varying degrees of sensitivity to piperidine. Furthermore, all of the secondary lesions observed in this work were potently mutagenic and genotoxic in Escherichia coli. Therefore, while 8-oxoGua itself is nontoxic and only mildly mutagenic in repair-proficient cells, peroxynitrite reveals the promutagenic potential and triggers the covert nature of this DNA lesion.
PMCID: PMC2848752  PMID: 17941698
3.  Dynamics of the Microbiota in Response to Host Infection 
PLoS ONE  2014;9(7):e95534.
Longitudinal studies of the microbiota are important for discovering changes in microbial communities that affect the host. The complexity of these ecosystems requires rigorous integrated experimental and computational methods to identify temporal signatures that promote physiologic or pathophysiologic responses in vivo. Employing a murine model of infectious colitis with the pathogen Citrobacter rodentium, we generated a 2-month time-series of 16S rDNA gene profiles, and quantitatively cultured commensals, from multiple intestinal sites in infected and uninfected mice. We developed a computational framework to discover time-varying signatures for individual taxa, and to automatically group signatures to identify microbial sub-communities within the larger gut ecosystem that demonstrate common behaviors. Application of this model to the 16S rDNA dataset revealed dynamic alterations in the microbiota at multiple levels of resolution, from effects on systems-level metrics to changes across anatomic sites for individual taxa and species. These analyses revealed unique, time-dependent microbial signatures associated with host responses at different stages of colitis. Signatures included a Mucispirillum OTU associated with early disruption of the colonic surface mucus layer, prior to the onset of symptomatic colitis, and members of the Clostridiales and Lactobacillales that increased with successful resolution of inflammation, after clearance of the pathogen. Quantitative culture data validated findings for predominant species, further refining and strengthening model predictions. These findings provide new insights into the complex behaviors found within host ecosystems, and define several time-dependent microbial signatures that may be leveraged in studies of other infectious or inflammatory conditions.
PMCID: PMC4094490  PMID: 25014551
4.  Activation of the Unfolded Protein Response Bypasses Trastuzumab-mediated Inhibition of the PI-3K Pathway 
Cancer letters  2012;329(2):236-242.
HER2-positive breast cancer initially responds to trastuzumab treatment, but over time, resistance develops and rapid cancer progression occurs, for which various explanations have been proposed. Here we tested the hypothesis that induction of the unfolded protein response (UPR) could override HER2 inhibition by trastuzumab, leading to the re-activation of growth signaling and the activation of the downstream target Lipocalin 2 (LCN2). Trastuzumab significantly inhibited the basal expression of LCN2 in HER2+ SKBr3 human breast cancer cells. The induction of the UPR completely abrogated trastuzumab-mediated LCN2 downregulation, and, in fact caused an increase in transcription and secretion of LCN2 over baseline. Reduction of the UPR using 4-phenyl butyric acid (PBA) a chemical chaperone that ameliorates ER stress, restored trastuzumab-mediated inhibition. Inhibition of the PI3K/AKT signaling pathway in trastuzumab-treated/UPR-induced SKBr3 cells partially reduced the upregulation of LCN2. These results suggest that the UPR is a possible way to override the effect of trastuzumab in HER2+ cancer cells.
PMCID: PMC4091625  PMID: 23200669
ER stress; Lipocalin 2; tumorigenesis
5.  Outcomes of Congenital Diaphragmatic Hernia in the modern era of management 
The Journal of pediatrics  2013;163(1):114-119.e1.
Objective To identify clinical factors associated with pulmonary hypertension and mortality in patients with congenital diaphragmatic hernia (CDH).
Study design A prospective cohort of neonates with a diaphragm defect identified at one of seven collaborating medical centers was studied. Echocardiograms were performed at one month and three months of age and analyzed at a central core by two cardiologists independently. Degree of pulmonary hypertension and survival were tested for association with clinical variables using Fischers exact test, chi-square and regression analysis.
Results 220 patients met inclusion criteria. Worse pulmonary hypertension measured at one month of life was associated with higher mortality. Other factors associated with mortality were need for extracorporeal membrane oxygenation (ECMO), patients inborn at the treating center and patients with a prenatal diagnosis of CDH. Interestingly, patients with right sided CDH did not have worse outcomes.
Conclusions Severity of pulmonary hypertension is associated with mortality in CDH. Other factors associated with mortality were birth weight, gestational age at birth, inborn status and need for ECMO.
PMCID: PMC3692597  PMID: 23375362
Congenital Diaphragmatic Hernia; Pulmonary Hypertension; Survival; Treatment
6.  Diet Influences Expression of Autoimmune Associated Genes and Disease Severity By Epigenetic Mechanisms in a Transgenic Lupus Model 
Arthritis and rheumatism  2013;65(7):1872-1881.
Lupus flares when genetically predisposed people encounter appropriate environmental agents. Current evidence indicates that the environment contributes by inhibiting T cell DNA methylation, causing overexpression of normally silenced genes. DNA methylation depends on both dietary transmethylation micronutrients and Erk-regulated DNA methyltransferase 1 (Dnmt1) levels. We used transgenic mice to study interactions between diet, Dnmt1 levels and genetic predisposition on the development and severity of lupus.
A doxycycline-inducible Erk defect was bred into lupus-resistant (C57BL/6) or lupus-susceptible (C57BL/6xSJL) mouse strains. Doxycycline treated mice were fed a standard commercial diet for eighteen weeks then switched to diets supplemented(MS) or restricted(MR) intransmethylation micronutrients. Disease severity was assessed by anti-dsDNA antibodies, proteinuria, hematuria and histopathology of kidney tissues. Pyrosequencing was used to determine micronutrient effects on DNA methylation.
Doxycycline induced modest levels of anti-dsDNA antibodies in C57BL/6 mice and higher levels in C57BL/6xSJL mice. Doxycycline-treated C57BL/6xSJL mice developed hematuria and glomerulonephritis on the MR and standard but not the MS diet. In contrast C57BL/6 mice developed kidney disease only on the MR diet. Decreasing Erk signaling and methyl donors also caused demethylation and overexpression of the CD40lg gene in female mice, consistent with demethylation of the second X chromosome. Both the dietary methyl donor content and duration of treatment influenced methylation and expression of the CD40lg gene.
Dietary micronutrients that affect DNA methylation can exacerbate or ameliorate SLE disease in this transgenic murine lupus model, and contribute to lupus susceptibility and severity through genetic/epigenetic interactions.
PMCID: PMC3735138  PMID: 23576011
Extracellular Receptor Kinase (Erk; Systemic Lupus Erythematosus (SLE; CD70; micronutrients; CD40L; KirL1
7.  Acquisition of Oral Microbes and Associated Systemic Responses of Newborn Nonhuman Primates 
The acquisition and development of the complex oral microbiome remain ill defined. While selected species of oral bacteria have been examined in relation to their initial colonization in neonates, a more detailed understanding of the dynamics of the microbiome has been developed only in adults. The current investigation used a nonhuman primate model to document the kinetics of colonization of the oral cavities of newborns and infants by a range of oral commensals and pathogens. Differences in colonization were evaluated in newborns from mothers who were maintained on an oral hygiene regimen pre- and postparturition with those displaying naturally acquired gingivitis/periodontitis. The results demonstrate distinct profiles of acquisition of selected oral bacteria, with the transmission of targeted pathogens, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, being passed on primarily from mothers with gingivitis/periodontitis. This colonization resulted in defined patterns of systemic antibody responses in the infants. The significant relative risk measures for infection with the pathogens, as well as the relationship of oral infection and blood serum antibody levels, were consistent with those of the newborns from mothers with gingivitis/periodontitis. These findings indicate that the early acquisition of potentially pathogenic oral bacterial species might impact the development of mucosal responses in the gingiva and may provide an enhanced risk for the development of periodontitis later in life.
PMCID: PMC3910915  PMID: 24173024
8.  Integrating EMR-Linked and In Vivo Functional Genetic Data to Identify New Genotype-Phenotype Associations 
PLoS ONE  2014;9(6):e100322.
The coupling of electronic medical records (EMR) with genetic data has created the potential for implementing reverse genetic approaches in humans, whereby the function of a gene is inferred from the shared pattern of morbidity among homozygotes of a genetic variant. We explored the feasibility of this approach to identify phenotypes associated with low frequency variants using Vanderbilt's EMR-based BioVU resource. We analyzed 1,658 low frequency non-synonymous SNPs (nsSNPs) with a minor allele frequency (MAF)<10% collected on 8,546 subjects. For each nsSNP, we identified diagnoses shared by at least 2 minor allele homozygotes and with an association p<0.05. The diagnoses were reviewed by a clinician to ascertain whether they may share a common mechanistic basis. While a number of biologically compelling clinical patterns of association were observed, the frequency of these associations was identical to that observed using genotype-permuted data sets, indicating that the associations were likely due to chance. To refine our analysis associations, we then restricted the analysis to 711 nsSNPs in genes with phenotypes in the On-line Mendelian Inheritance in Man (OMIM) or knock-out mouse phenotype databases. An initial comparison of the EMR diagnoses to the known in vivo functions of the gene identified 25 candidate nsSNPs, 19 of which had significant genotype-phenotype associations when tested using matched controls. Twleve of the 19 nsSNPs associations were confirmed by a detailed record review. Four of 12 nsSNP-phenotype associations were successfully replicated in an independent data set: thrombosis (F5,rs6031), seizures/convulsions (GPR98,rs13157270), macular degeneration (CNGB3,rs3735972), and GI bleeding (HGFAC,rs16844401). These analyses demonstrate the feasibility and challenges of using reverse genetics approaches to identify novel gene-phenotype associations in human subjects using low frequency variants. As increasing amounts of rare variant data are generated from modern genotyping and sequence platforms, model organism data may be an important tool to enable discovery.
PMCID: PMC4065041  PMID: 24949630
9.  A comparison of outcomes with coronary artery calcium scanning in Unselected Populations - The Multi-Ethnic Study of Atherosclerosis (MESA) and Heinz Nixdorf Recall Study (HNR) 
The Multi-Ethnic Study of Atherosclerosis (MESA) and the Heinz Nixdorf Recall Study (HNR)) differed in regards to informing physicians and patients of the results of their subclinical atherosclerosis.
This study investigates whether the association of coronary artery calcium (CAC) with incident non-fatal and fatal cardiovascular (CVD) events is different among these two large, population-based observational studies.
All Caucasian subjects aged 45–75 years, free of baseline cardiovascular disease were included (n=2232 in MESA, n=3119 HNR participants). We studied the association between CAC and event rates at 5 years, including hard cardiac events (MI, cardiac death, resuscitated cardiac arrest), and separately added revascularizations, and strokes (fatal and non-fatal) to determine adjusted hazard ratios (HR).
Both cohorts demonstrated very low CHD (including revascularization) rates with zero calcium (1.13 and 1.16% over 5 years in MESA and HNR respectively) and increasing significantly in both groups with CAC 100–399 (6.71 and 4.52% in MESA and HNR) and CAC >400 (12.5 and 13.54% in MESA and HNR respectively) and demonstrating strong independent predictive values for scores of 100–399 and >400, despite multivariable adjustment for risk factors. Risk factor adjusted five year revascularization rates were nearly identical for HNR and MESA, and generally low for both studies (1.4% [45/3119] for HNR and 1.9% [43/2232] for MESA) over 5 years.
Across two culturally diverse populations, CAC >400 is a strong predictor of events. High CAC did not determininistically result in revascularization and knowledge of CAC did not increase revascularizations.
PMCID: PMC3732186  PMID: 23849491
coronary artery calcification; subclinical atherosclerosis; Multi-Ethnic Study of Atherosclerosis (MESA); Heinz Nixdorf Recall Study (HNR)
10.  Mucopolysaccharidosis Type IIIA presents as a variant of Klüver–Bucy syndrome 
Mucopolysaccharidosis type IIIA (MPS IIIA) is a neurodegenerative disease with behavioral symptoms unique among the mucopolysaccharidoses. Children with MPS IIIA reportedly mouth things, explore novel environments almost continuously, disregard danger, and empathize/socialize and comply less with parents. These characteristics resemble Klüver–Bucy syndrome (K-Bs). To test the K-Bs hypothesis, 30 children with MPS IIIA were compared to 8 ‘post-transplant’ Mucopolysaccharidosis type IH patients in an experimental “Risk Room”. The room contained attractive and mildly frightening objects, exposure to a 92 dB startle noise triggered by contact with an attractive toy, mother’s return after a brief absence, and compliance with her clean-up directive. Children with MPS IIIA: 1) left mother sooner, 2) wandered more, 3) were more likely to approach frightening objects, 4) were less likely to respond to loud noise with whole body startle, 5) were less likely to avoid the toy associated with the startle noise, 6) interacted less with mother upon her return, and 7) complied less with her clean-up command. K-Bs is associated with loss of amygdala function. Brain MRIs of a subset of the children with MPS IIIA showed volume loss that was greater in the amygdala than the hippocampus; only amygdala loss correlated with reduced fearfulness. MPS IIIA may be the first identified pediatric disease presenting systematically as a K-Bs variant. If validated by further studies, the K-Bs hypothesis of MPS IIIA would provide important clinical and theoretical information for the guidance of families as well as markers for natural disease progression and treatment effects.
PMCID: PMC3931576  PMID: 23745734
amygdala; fear conditioning; orality; Sanfilippo syndrome; startle
11.  Molecular characterization of the transition to mid-life in Caenorhabditis elegans 
Age  2012;35(3):689-703.
We present an initial molecular characterization of a morphological transition between two early aging states. In previous work, an age score reflecting physiological age was developed using a machine classifier trained on images of worm populations at fixed chronological ages throughout their lifespan. The distribution of age scores identified three stable post-developmental states and transitions. The first transition occurs at day 5 post-hatching, where a significant percentage of the population exists in both state I and state II. The temperature dependence of the timing of this transition (Q10 ~ 1.17) is too low to be explained by a stepwise process with an enzymatic or chemical rate-limiting step, potentially implicating a more complex mechanism. Individual animals at day 5 were sorted into state I and state II groups using the machine classifier and analyzed by microarray expression profiling. Despite being isogenic, grown for the same amount of time, and indistinguishable by eye, these two morphological states were confirmed to be molecularly distinct by hierarchical clustering and principal component analysis of the microarray results. These molecular differences suggest that pharynx morphology reflects the aging state of the whole organism. Our expression profiling yielded a gene set that showed significant overlap with those from three previous age-related studies and identified several genes not previously implicated in aging. A highly represented group of genes unique to this study is involved in targeted ubiquitin-mediated proteolysis, including Skp1-related (SKR), F-box-containing, and BTB motif adaptors.
Electronic supplementary material
The online version of this article (doi:10.1007/s11357-012-9401-2) contains supplementary material, which is available to authorized users.
PMCID: PMC3636400  PMID: 22610697
Machine classifier; Biomarker of aging; Metastable aging state; Microarray analysis
12.  Agreement among RTOG Sarcoma Radiation Oncologists in Contouring Suspicious Peritumoral Edema for Pre-operative Radiotherapy of Soft Tissue Sarcoma of the Extremity 
Peritumoral edema may potentially harbor sarcoma cells. The extent of suspicious edema (SE) included in the treatment volume is subject to clinical judgment, balancing the risk of missing tumor cells with excess toxicity. Our goal was to determine variability in SE delineation by sarcoma radiation oncologists (RO).
Material and Method
Twelve expert ROs were provided with T1 Gadolinium and T2-weighted MR images of 10 patients with high-grade extremity soft tissue sarcoma. Gross tumor volume, CTV3cm (3cm longitudinal and 1.5cm radial margin) and CTV2cm (2cm longitudinal and 1cm radial margin) were contoured by a single observer. Suspicious peritumoral edema, defined as abnormal signal on T2 images, was independently delineated by all 12 ROs. Contouring agreement was analyzed using the Simultaneous Truth and Performance Level Estimation (STAPLE) algorithm and kappa statistics.
The mean volumes of GTV, CTV2cm and CTV3cm were respectively 130 cm3 (7–413 cm3), 280cm3 and 360cm3. The mean consensus volume computed using the STAPLE algorithm at 95% confidence interval was 188cm3 (24–565cm3) with a substantial overall agreement corrected for chance (mean kappa =0.71; range: 0.32–0.87). The minimum, maximum and mean volume of suspicious edema (excluding the GTV) were 4cm3, 182cm3 and 58 cm3 (representing a median of 29% of the GTV volume). The median volume of suspicious edema not included in the CTV2cm and in the CTV3cm was 5 cm3 and 0.3cm3 respectively. There were 3 large tumors with >30cm3 of suspicious edema not included in the CTV3cm volume.
Despite the fact that SE would empirically seem to be a more subjective volume, a substantial or near-perfect inter-observer agreement was observed in SE delineation in most cases with high-grade STS of the extremity. A median of 97% of the consensus SE is within the CTV2cm (99.8% within the CTV3cm). In a minority of cases, however, significant expansion of the CTVs is required to cover SE.
PMCID: PMC3646910  PMID: 23474110
14.  Comparison of Factors Associated with Carotid Intima-Media Thickness in the Multi-Ethnic Study of Atherosclerosis (MESA) and the Heinz Nixdorf Recall Study (HNR) 
The measurement of carotid intima-media thickness (CIMT) is a valid method to quantify levels of atherosclerosis. The present study was conducted to compare the strengths of associations between CIMT and cardiovascular risk factors in two different populations.
The Multi-Ethnic Study of Atherosclerosis (MESA) and the Heinz Nixdorf Recall Study (HNR) are two population-based prospective cohort studies of subclinical cardiovascular disease. All Caucasian subjects aged 45 to 75 years from these cohorts who were free of baseline cardiovascular disease (n = 2,820 in HNR, n = 2,270 in MESA) were combined. CIMT images were obtained using B-mode sonography at the right and left common carotid artery and measured 1 cm starting from the bulb.
In both studies, age, male sex, and systolic blood pressure showed the strongest association (P < .0001 for each) for a higher CIMT. The mean of mean far wall CIMT was slightly higher in MESA participants (0.71 vs 0.67 mm). Almost all significant variables were consistent between the two cohorts in both magnitude of association with CIMT and statistical significance, including age, sex, smoking, diabetes, cholesterol levels, and blood pressure. For example, the association with systolic blood pressure was (ΔSD = 0.011; 95% confidence interval, 0.0009 to 0.014) per mm Hg in MESA and (ΔSD = 0.010; 95% confidence interval, 0.005 to 0.021) per mm Hg in HNR. This consistency persisted throughout the traditional (Framingham) risk factors.
A comparison of the associations between traditional cardiovascular risk factors and CIMT across two culturally diverse populations showed remarkable consistency.
PMCID: PMC3694173  PMID: 23611058
Carotid intima-media thickness; Subclinical atherosclerosis; Multi-Ethnic Study of Atherosclerosis; MESA; Heinz Nixdorf Recall Study; HNR
15.  Results and Lessons Learned from a Nurse Practitioner-Guided Dementia Care Intervention for Primary Care Patients and Their Family Caregivers 
Older adults with dementia care needs often visit primary care physicians (PCPs), but PCP dementia care limitations are widely documented. This study tested the value of employing a nurse practitioner (NP) with geropsychiatric expertise to augment PCP care for newly and recently diagnosed patients and family caregivers. Twenty-one dyads received the NP intervention; 10 dyads were controls. Outcomes included patient neuropsychiatric symptom and quality of life changes, and caregiver depression, burden, and self-efficacy changes. Intervention acceptability by patients, caregivers, and PCPs was determined. No outcome differences were found; however, the NP intervention was deemed highly satisfactory by all stakeholders. Patients experienced no significant cognitive decline during their 12-month study period, helping explain why outcomes did not change. Given widespread acceptability, future tests of this PCP-enhancing intervention should include patients with more progressive cognitive decline at study entry. NPs with geropsychiatric expertise are ideal interventionists for this rapidly growing target population.
PMCID: PMC4040327  PMID: 24444453
16.  Meeting Report: Spontaneous Lesions and Diseases in Wild, Captive-Bred, and Zoo-Housed Nonhuman Primates and in Nonhuman Primate Species Used in Drug Safety Studies 
Veterinary pathology  2012;49(6):1057-1069.
The combination of loss of habitat, human population encroachment, and increased demand of select nonhuman primates for biomedical research has significantly affected populations. There remains a need for knowledge and expertise in understanding background findings as related to the age, source, strain, and disease status of nonhuman primates. In particular, for safety/biomedical studies, a broader understanding and documentation of lesions would help clarify background from drug-related findings. A workshop and a minisymposium on spontaneous lesions and diseases in nonhuman primates were sponsored by the concurrent Annual Meetings of the American College of Veterinary Pathologists and the American Society for Veterinary Clinical Pathology held December 3–4, 2011, in Nashville, Tennessee. The first session had presentations from Drs Lowenstine and Montali, pathologists with extensive experience in wild and zoo populations of nonhuman primates, which was followed by presentations of 20 unique case reports of rare or newly observed spontaneous lesions in nonhuman primates (see online files for access to digital whole-slide images corresponding to each case report at Seminars/2011/Primate%20Pathology/view.apml). The minisymposium was composed of 5 nonhuman-primate researchers (Drs Bradley, Cline, Sasseville, Miller, Hutto) who concentrated on background and spontaneous lesions in nonhuman primates used in drug safety studies. Cynomolgus and rhesus macaques were emphasized, with some material presented on common marmosets. Congenital, acquired, inflammatory, and neoplastic changes were highlighed with a focus on clinical, macroscopic, and histopathologic findings that could confound the interpretation of drug safety studies.
PMCID: PMC4034460  PMID: 23135296
amyloidosis; Aotus trivirgatus; Armillifer agkistrodontis; Aspergillus fumigatus; Brunner’s gland hyperplasia; bonobo; chimpanzee; Callithrix jacchus; cardiomyopathy; Cebus sp; Cercocebus torquatus torquatus; Cercopithecus nictitans; Chlorocebus aethiops; Chromobacterium violaceum; colitis; Cryptosporidium muris–like; cytomegalovirus; diabetic nephropathy; encephalitis; enteritis; ependymoblastoma; epitheliotropic lymphoma; gorilla; Gorilla beringei beringei; Helicobacter heilmannii–like; hyperadrenocorticism; intestinal carcinoma; lymphocytic choriomeningitis virus; Macaca fascicularis; Macaca mulatta; Macaca nemestrina; macaque; Mandrillus sphinx; marmoset; metapneumovirus; multiple myeloma; oncocytic adrenocortical carcinoma; orangutan; ovarian teratoma; Pan paniscus; Pan troglodytes; papillomavirus; Papio cynocephalus; pheochromocytoma; Plasmodium inui; pneumonia; Pongo pygmaeus; Saguinus oedipus; simian human immunodeficiency virus; simian immunodeficiency virus; squamous cell carcinoma; tamarin
17.  Do the benefits outweigh the side effects of colorectal cancer surveillance? A systematic review 
Most patients treated with curative intent for colorectal cancer (CRC) are included in a follow-up program involving periodic evaluations. The survival benefits of a follow-up program are well delineated, and previous meta-analyses have suggested an overall survival improvement of 5%-10% by intensive follow-up. However, in a recent randomized trial, there was no survival benefit when a minimal vs an intensive follow-up program was compared. Less is known about the potential side effects of follow-up. Well-known side effects of preventive programs are those of somatic complications caused by testing, negative psychological consequences of follow-up itself, and the downstream impact of false positive or false negative tests. Accordingly, the potential survival benefits of CRC follow-up must be weighed against these potential negatives. The present review compares the benefits and side effects of CRC follow-up, and we propose future areas for research.
PMCID: PMC4021326  PMID: 24834140
Colorectal cancer; Follow-up; Surveillance; False positive; Cancer survivorship
18.  Exome Sequencing Implicates an Increased Burden of Rare Potassium Channel Variants in the Risk of Drug Induced Long QT Syndrome 
To test the hypothesis that rare variants are associated with Drug-induced long QT syndrome (diLQTS) and torsade de pointes (TdP).
diLQTS is associated with the potentially fatal arrhythmia TdP. The contribution of rare genetic variants to the underlying genetic framework predisposing diLQTS has not been systematically examined.
We performed whole exome sequencing (WES) on 65 diLQTS cases and 148 drug-exposed controls of European descent. We employed rare variant analyses (variable threshold [VT] and sequence kernel association test [SKAT]) and gene-set analyses to identify genes enriched with rare amino-acid coding (AAC) variants associated with diLQTS. Significant associations were reanalyzed by comparing diLQTS cases to 515 ethnically matched controls from the NHLBI GO Exome Sequencing Project (ESP).
Rare variants in 7 genes were enriched in the diLQTS cases according to SKAT or VT compared to drug exposed controls (p<0.001). Of these, we replicated the diLQTS associations for KCNE1 and ACN9 using 515 ESP controls (p<0.05). A total of 37% of the diLQTS cases also had ≥1 rare AAC variant, as compared to 21% of controls (p=0.009), in a predefined set of seven congenital LQTS (cLQTS) genes encoding potassium channels or channel modulators (KCNE1,KCNE2,KCNH2,KCNJ2, KCNJ5,KCNQ1,AKAP9).
By combining WES with aggregated rare variant analyses, we implicate rare variants in KCNE1 and ACN9 as risk factors for diLQTS. Moreover, diLQTS cases were more burdened by rare AAC variants in cLQTS genes encoding potassium channel modulators, supporting the idea that multiple rare variants, notably across cLQTS genes, predispose to diLQTS.
PMCID: PMC4018823  PMID: 24561134
exome; torsade des pointes; long QT syndrome; genetics, adverse drug event
19.  A directional switch of integrin signaling and a new anti-thrombotic strategy 
Nature  2013;503(7474):10.1038/nature12613.
Integrins are critical in thrombosis and hemostasis1. Antagonists of the platelet integrin αIIbβ3 are potent anti-thrombotic drugs, but also have the life-threatening adverse effect of bleeding2,3. It is thus desirable to develop new antagonists that do not cause bleeding. Integrins transmit signals bidirectionally4,5. Inside-out signaling activates integrins via a talin-dependent mechanism6,7. Integrin ligation mediates thrombus formation and outside-in signaling8,9, which requires Gα13 and greatly expands thrombi. Here we show that Gα13 and talin bind to mutually exclusive, but distinct sites within the integrin β3 cytoplasmic domain in opposing waves. The first talin binding wave mediates inside-out signaling and also “ligand-induced integrin activation”, but is not required for outside-in signaling. Integrin ligation induces transient talin dissociation and Gα13 binding to an ExE motif, which selectively mediates outside-in signaling and platelet spreading. The second talin binding wave is associated with clot retraction. An ExE motif-based inhibitor of Gα13-integrin interaction selectively abolishes outside-in signaling without affecting integrin ligation, and suppresses occlusive arterial thrombosis without affecting bleeding time. Thus, we have discovered a novel mechanism for the directional switch of integrin signaling and, based on this mechanism, we designed a potent new anti-thrombotic that does not cause bleeding.
PMCID: PMC3823815  PMID: 24162846
20.  The University of Queensland study of physical and psychological outcomes for claimants with minor and moderate injuries following a road traffic crash (UQ SuPPORT): design and methods 
European Journal of Psychotraumatology  2014;5:10.3402/ejpt.v5.22612.
To date research investigating how mental health impacts physical recovery following a road traffic crash (RTC) has focused on cohorts with severe injuries. The UQ SuPPORT study aims to study the physical and psychological outcomes of claimants with minor injuries following an RTC under the Queensland common law compulsory insurance scheme.
This paper outlines the protocols of this study as a platform for future publications.
The 2-year longitudinal cohort study collected interview and survey data from claimants at 6, 12, and 24 months post-RTC. Measures used in the telephone interview included the DSM-IV Composite International Diagnostic Interview for posttraumatic stress disorder, generalized anxiety disorder, major depressive episode, panic attacks, agoraphobia; and self-reported disability (WHO-DAS-II). Quality of life (SF-36v2), alcohol use (AUDIT), social support (MSPSS), quality-adjusted life years (EQ-5D), and return to work outcomes were assessed via postal questionnaires.
A total of 382 claimants consented to participate at the beginning of the study, and these participants were approached at each wave. Retention was high (65%). The average age of participants at Wave 1 was 48.6 years, with 65% of the sample sustaining minor injuries (Injury Severity Score=1–3).
This study has collected a unique sample of data to investigate recovery patterns of claimants with minor injuries. Future publications will more fully assess the effects of the collected measures on recovery rates 2 years post-RTC.
PMCID: PMC4009486  PMID: 24799996
Posttraumatic stress; motor vehicle crash; longitudinal; physical recovery; minor injuries
22.  End-of-life cell cycle arrest contributes to stochasticity of yeast replicative aging 
FEMS yeast research  2013;13(3):267-276.
There is growing evidence that stochastic events play an important role in determining individual longevity. Studies in model organisms have demonstrated that genetically identical populations maintained under apparently equivalent environmental conditions display individual variation in lifespan that can be modeled by the Gompertz-Makeham law of mortality. Here we report that within genetically identical haploid and diploid wild type populations, shorter-lived cells tend to arrest in a budded state, while cells that arrest in an unbudded state are significantly longer-lived. This relationship is particularly notable in diploid BY4743 cells, where mother cells that arrest in a budded state have a shorter mean lifespan (25.6 vs. 35.6) and larger coefficient of variance with respect to individual lifespan (0.42 vs. 0.32) than cells that arrest in an unbudded state. Mutations that cause genomic instability tend to shorten lifespan and increase the proportion of the population that arrest in a budded state. These observations suggest that randomly occurring damage may contribute to stochasticity during replicative aging by causing a subset of the population to terminally arrest prematurely in the S or G2 phase of the cell cycle.
PMCID: PMC3960949  PMID: 23336757
replicative lifespan; longevity; yeast; senescence; DNA damage; sir2; cell cycle arrest
23.  Multiphasic Contrast-Enhanced MRI: Single-Slice Versus Volumetric Quantification of Tumor Enhancement for the Assessment of Renal Clear-Cell Carcinoma Fuhrman Grade 
To assess the association between clear-cell carcinoma pathology grade at nephrectomy and magnetic resonance imaging (MRI) tumor enhancement.
Materials and Methods
The Institutional Review Board approved this retrospective study and waived the informed consent requirement. In all, 32 patients underwent multiphase contrast-enhanced MRI prior to nephrectomy. MRI tumor enhancement was measured using two approaches: 1) the most enhancing portion of the tumor on a single slice and 2) volumetric analysis of enhancement in the entire tumor. Associations between pathological grade, tumor size, and enhancement were evaluated using the Kruskal–Wallis test and generalized logistic regression models.
No significant association between pathology grade and enhancement was found when measurements were made on a single slice. When measured in the entire tumor, significant associations were found between higher pathology grades and lower mean, median, top 10%, top 25%, and top 50% tumor enhancement (P < 0.001–0.002). On multivariate analysis the association between grade and enhancement remained significant (P = 0.041–0.043), but tumor size did not make an additional contribution beyond tumor enhancement alone in differentiating between tumor grades.
There is significant association between tumor grade and enhancement, but only when measured in the entire tumor and not on the most enhancing portion on a single slice.
PMCID: PMC3989993  PMID: 23152173
renal cell carcinoma; clear cell carcinoma; magnetic resonance imaging; genitourinary; oncology; aggressiveness
24.  Conserved Role of Intragenic DNA Methylation in Regulating Alternative Promoters 
Nature  2010;466(7303):253-257.
While the methylation of DNA in 5′ promoters suppresses gene expression, the role of DNA methylation in gene bodies is unclear1–5. In mammals, tissue- and cell type-specific methylation is present in a small percentage of 5′ CpG island (CGI) promoters, while a far greater proportion occurs across gene bodies, coinciding with highly conserved sequences5–10. Tissue-specific intragenic methylation might reduce,3 or, paradoxically, enhance transcription elongation efficiency1,2,4,5. Capped analysis of gene expression (CAGE) experiments also indicate that transcription commonly initiates within and between genes11–15. To investigate the role of intragenic methylation, we generated a map of DNA methylation from human brain encompassing 24.7 million of the 28 million CpG sites. From the dense, high-resolution coverage of CpG islands, the majority of methylated CpG islands were revealed to be in intragenic and intergenic regions, while less than 3% of CpG islands in 5′ promoters were methylated. The CpG islands in all three locations overlapped with RNA markers of transcription initiation, and unmethylated CpG islands also overlapped significantly with trimethylation of H3K4, a histone modification enriched at promoters16. The general and CpG-island-specific patterns of methylation are conserved in mouse tissues. An in-depth investigation of the human SHANK3 locus17,18 and its mouse homologue demonstrated that this tissue-specific DNA methylation regulates intragenic promoter activity in vitro and in vivo. These methylation-regulated, alternative transcripts are expressed in a tissue and cell type-specific manner, and are expressed differentially within a single cell type from distinct brain regions. These results support a major role for intragenic methylation in regulating cell context-specific alternative promoters in gene bodies.
PMCID: PMC3998662  PMID: 20613842
Intragenic DNA methylation; alternate promoters; comparative epigenomics; SHANK3
25.  Urinary ATP and visualization of intracellular bacteria: a superior diagnostic marker for recurrent UTI in renal transplant recipients? 
SpringerPlus  2014;3:200.
Renal transplant recipients (RTR) are highly susceptible to urinary tract infections (UTIs) with over 50% of patients having at least one UTI within the first year. Yet it is generally acknowledged that there is considerable insensitivity and inaccuracy in routine urinalysis when screening for UTIs. Thus a large number of transplant patients with genuine urine infections may go undiagnosed and develop chronic recalcitrant infections, which can be associated with graft loss and morbidity. Given a recent study demonstrating ATP is released by urothelial cells in response to bacteria exposure, possibly acting at metabotropic P2Y receptors mediating a proinflammatory response, we have investigated alternative, and possibly more appropriate, urinalysis techniques in a cohort of RTRs.
Mid-stream urine (MSU) samples were collected from 53 outpatient RTRs. Conventional leukocyte esterase and nitrite dipstick tests, and microscopic pyuria counts (in 1 μl), ATP concentration measurements, and identification of intracellular bacteria in shed urothelial cells, were performed on fresh unspun samples and compared to ‘gold-standard’ bacterial culture results.
Of the 53 RTRs, 22% were deemed to have a UTI by ‘gold-standard’ conventional bacteria culture, whereas 87%, 8% and 4% showed evidence of UTIs according to leukocyte esterase dipstick, nitrite dipstick, and a combination of both dipsticks, respectively. Intracellular bacteria were visualized in shed urothelial cells of 44% of RTRs, however only 1 of the 23 RTRs (44%) was deemed to have a UTI by conventional bacteria culture. A significant association of the ‘gold-standard’ test with urinary ATP concentration combined with visualization of intracellular bacteria in shed urothelial cells was determined using the Fisher’s exact test.
It is apparent that standard bedside tests for UTIs give variable results and that seemingly quiescent bacteria in urothelial cells are very common in RTRs and may represent a focus of subclinical infection. Furthermore, our results suggest urinary ATP concentration combined with detection of intracellular bacteria in shed urinary epithelial cells may be a sensitive means by which to detect ‘occult’ infection in RTRs.
PMCID: PMC4022969  PMID: 24839587
Intracellular bacteria; IBC; Pyuria; Urinary ATP; Bladder; Acridine orange stain

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